IfOL, THE AMERICAN JOURNAL OF PHYSIOLOGY V^OLUME LI I I" BALTIMORE, MD. 1920 V A,- CONTENTS No. 1. May 1. 1920 Gastric Response to Foods. X. The Psychic Secretiox of Gastric Juice in Normal Men. Raymowi J. Miller, Olaf Bergeim, Martin E. Rehfuss and Philip B. Haivk 1 Gastric Response to Foods. XI. The Influence of Tea. Coffee and Cocoa upon Digestion. Raymond J. Miller, Olaf Bergeim. Martin E. Rehfuss and Philip B. Hawk 2S I. Rapid Blood Plasma Protein Depletion and the Curve of Regen- eration. H. P. Smith. A. E. Belt and G. H. Whipple 54 II. Shock as a Manifest.ation of Tissue Injury Following Rapid Plasma Protein Depletion. The St.vbilizing Value of Plasma Proteins. G. H. Whipple, H. P. Smith and A. E. Belt 72 III. F.actors Concerned in the Perfusion of Living Org.^ns and Tissues. Artificial Solutions Substituted for Blood Serum and THE Resulting Injury' to Parenchyma Cells. A. E. Belt, H. P. Smith and G. H. Whipple 101 The Seasonal Variation in the Growth of Boston School Children. W. T. Porter 121 Cert.un Changes Noted in Ergographic Response as a Result of Tobacco-Smoking. Theophile Raphael 132 Determination of the Capillary Blood Pressure in Man with the ^SIicro-Capill.'vry' Tonometer. C. S. Danzer and D. R. Hooker 130 A Plethy'smographic Study" of Shock and Stammering in a Trephined Stammerer. Samuel D. Robbins lt>S The Chemical Constitution of Adenine Nucleotide and of Yeast Nucleic Acid. Walter Jones 193 The Action of Boiled Pancreas Extract on Yeast Nucleic Acid. Walter Jones 203 No. 2. June 1, 1920 A Study of Forced Respiration: Experimental Production of Tetany. Satnuel B. Grant and Alfred Goldman 209 A Study' of the Carbohy'drate Tolerance in Eck Fistula and Hypophy- SECTOMIZED AnIMALS (POSTERIOR LoBE REMOVAL;. LiVER FUNCTION IN THE Met.\bolism OF SuGARS. Conrad Jacobson 233 The Gastric Response to Foods. XII. The Response of the Human Stomach to Pies, Cakes and Puddings. Raymond J. Miller, Harry L. Fowler, Olaf Bergeim, Martin E. Rehfuss and Philip B. Hawk 245 Segmental Activity- in the Heart of the Limulus. D. J. Edwards 276 iii IV CONTENTS The Arterial Pressure Curve as IxFLrEXCED by the Occlusion of Certain Vascular Areas and by Histamine. D. J. Edwards 284 The Artificial Production of Monsters Demonstrating Localized Defects as the Result of Injury from X-Rays. W. M. Baldwin.. 296 The Relation of the Epinephrin Output of the Adrenals to Changes in the Rate of the Denervated Heart. G. X. Stewart and J. M. Rogoff -"^04 The Effect of Acids, Alkalies and Salts on Catalase Production. W. E. Surge 364 On the Permeability of the Placenta for Adrenalin in the Pregnant Rabbit and Albino Rat. Yoshitaka Shimidzu 377 The Character of the Sympathetic Innervation to the Retractor Penis Muscle of the Dog. Charles W. Edmunds 395 Energy Expenditure in Household Tasks. C. F. Langworthy and H. G. Barott 400 Xo. 3. July 1, 1920 Amplification of Action Currents with the Electron Tube in Record- ing with the String Gala'anometer. Alexander Forbes and Cath- arine Thacher 409 Varations in the Respiratory Dead Air Space Due to Changes in the Depth of Breathing. R. G. Pearce and D. H. Hoover 472 The Sub-Arachnoid and Intra-Arterial Administration of Sodium Bicarbonate and Other Electrolytes. J. B. Collip 483 The Influence of Internal Secretions on Blood Pressure and the Formation of Bile. Ardrey W. Downs 498 Gastrin Studies. V. Chemical Studies on Gastrin Bodies. F. C. Koch, A. B. Luckhardt and R. W. Keeton 508 Essentials in ^Measuring Epinephrin Output with Further Obser- vations ON Its Relation to the Rate of the Denervated Heart. G. N. Stewart and J. M. Rogoff 521 The Effect of Splenectomy upon Growth in the Young. Samuel Chester Henn 562 The Importance of Vagal and Splanchnic Afferent Impulses on the Onset and Course of Tetanl\ Parathyropriva. Waller Lincoln Palmer 581 Index 591 /3 THE AMERICAN Journal of Physiology VOL. 52 MAY 1, 1920 No. 1 GASTRIC RESPONSE TO FOODSi X. The Psychic Secretion of Gastric Juice in Normal Men RAYMOND J. MILLER, OLAF BERGEIM, MARTIN E. REHFUSS ani> PHILIP B. HAWK From the Laboratory of Physiological Chemistry, Jefferson Medical Collegey Philadelphia Received for publication February 24, 1920 As early as 1852 it was shown by Bidder and Schmidt (1) that the mere sight of food called forth the secretion of gastric juice in the dog. It remained, however, for Pavlov (2), some forty years later, to estab- lish more definitely the character of the so-called "appetite" or "psychic" secretion of the gastric juice. He pointed out that such a secretion was the normal initiator of gastric digestion in dogs, and might be induced by the sight, smell, taste, mastication or thought of food, or even through the stimulation of appetite by the presence of solid matter in the stomach. This was further emphasized by the fact that very appetizing foods, such as meats, induced a greater secretion in "sham feeding" experiments than milk or bread, which were not so greatly relished by the animals. The results of experiments on man have been by no means so con- clusive. Thus Carlson (3) was able to point out probable sources of" error in most of the earlier investigations. This author concluded from the work of Homborg (4) and his own experiments that in spite of apparent evidence in the literature to the contrary, what Pavlov found true for animals was also true for man, namely, that gastric secretion was not induced by the chewing of indifferent substances nor by the taste or smell of chemical substances not arousing the appetite sensation. 1 The expenses of this investigation were defrayed by funds furnished by Mrs. M. H. Henderson, The Curtis Publishing Company and Dr. L. M. Halsey. 1 THE AMERICAN JOURNAL OF PHTSIOLOGT, VOL. 52, NO. 1 2 MILLER, BERGEIM, REHFUSS AND HAWK Accepting the view that the initiation of gastric secretion is de- pendent upon arousing or augmenting the appetite sensation, it becomes of immediate interest to determine whether the thought, sight, smell or taste of food exerts the greatest influence in this direction as well as to estimate the absolute importance of the appetite secretion as a factor in gastric digestion. Carlson found that no secretion could be induced in his gastric fistula subject by the thought of food, and that the secretion produced by- seeing or smelling food was relatively slight and inconstant, the signifi- cant appetite secretion being that induced by the tasting and chewing of good food. Luckhardt (5), on the contrary, employing a Rehfuss stomach tube and using as a subject a completely normal man, found under good experimental conditions that the combined sight and smell of food markedly increased the flow of gastric juice. Pavlov believed the appetite secretion to be of very great import- ance in initiating gastric digestion. This is discounted by Carlson, who found in his human subject, as well as in cats and dogs, that the continuous secretion of the stomach served a similar purpose, and that elimination of the appetite secretion did not cause indigestion. That the unpalatable condition of a food need not necessarily influ- ence its ultimate digestion and utilization in the alimentary tracts of normal men was indicated by work carried out in the laboratory of Atwater, who found that nauseating meat, the so-called "embalmed beef" fed to soldiers in the Spanish-American war, was still well utilized. That the economy of man is well served under such circumstances must remain doubtful. In this connection it is also necessary to consider psychic or other influences tending to inhibit the development of the appetite secretion ; bearing in mind that emotional excitement may destroy the motor (6) as well as the secretory (7) activities of the stomach. The present paper is a contribution toward determining the relative importance of the various factors involved in the appetite stimulation of gastric secretion, as well as toward estimating the influence of the appetizing or unappetizing character of a meal and of the mental attitude of the subject upon the gastric response and the ultimate digestion of food. Following the appetite stimulation in each case, the stomach was emptied at regular intervals, using a Rehfuss stomach tube, the volume of secretion determined, its free and total acidity, pepsin and amino- acid nitrogen estimated according to procedures previously described (8). PSYCHIC SECRETION OF THE GASTRIC JUICE 3 The sight psychic secretion and the effect of smell. Experiments were carried out to determine whether the sight of food alone, or the sight and odor of food combined gave rise to the production of any gastric secretion, as well as to compare the effects on such secretion of the sight of foods prepared in an unpleasing manner. A breakfast table was set, in a small well-lighted laboratory, with clean linen, bright chinaware, and the following foods were served in a pleasant manner and at One time: ham and eggs, oranges, shredded wheat, bread and butter, hot coffee, cream and sugar. Subjects were brought to this room after any residua had been removed and the level of continuous secretion established. The nose was kept lightly but effectively clamped throughout. In figure 1 are charted the results obtained with a subject who was a laboratory helper, accustomed to eating lunch at the laboratory. Total volumes of secretion removed are charted with total and free acidities, amino-acid nitrogen values and a so-called total actual acidity which indicates the product of the volume by the total acidity and represents the total amount of acid in the gastric samples. It will be noted that this subject showed a marked response to the sight of food as regards the volume of gastric juice secreted and the "total actual" acidity. The continuous secretion in this case was high and a greater augmentation by psychic stimulation might perhaps be expected. Under similar conditions another subject (fig. 2) showed a lower and less acid continued secretion. Sight augmented the volume but slightly, although the acidity was distinctly increased. Somewhat less accustomed to eating in the laboratory were the subjects of the experiments charted in figures 3, 4 and 5, which were carried out in exactly the same way except that clamps were not used. The distinct but not voluminous secretion in these cases may be attributed mainly to the effect of sight as the odor of the meal was not pronounced and odor was found to have little influence on psychic secretion in these subjects. The subjects were, of course, led to believe they would actually receive the food. To determine whether any elaboration in setting was requisite for the stimulation of secretion by sight, the meal was simplified to a simple half grape fruit served in the usual way. The odor being imperceptible, the noses were not clamped. The results are charted in figures 6 and 7. A stimulation of secretion was brought about by the sight of the food in both cases. It is also probable that some of the psychic secretion may leave the stomach during the intervals of the experiment with the 4 MILLEK, BERGEIM, REHFUSS AND HAWK increase in gastric tonus and produce a stimulation of the pancreatic secretion. The effect of allowing subjects to seat themselves at a breakfast table prepared in an unpleasing manner was also tried out on six sub- jects. The same table was used as in the preceding experiments, and the same foods were served. However, the ham and eggs were scorched ; the shredded wheat biscuits and bread roughly broken ; the coffee and milk weak and diluted; the butter soft; sugar lumpy and dark; the oranges partly squeezed; the dishes generally somewhat greasy and with an appearance of dirtiness induced by the use of charcoal. Newspapers were used in place of linen. The noses of the first two subjects were clamped, of the others free. The results of these experiments are charted in figures 8, 9, 10, 11, 12 and 13. In only one of these cases (see fig. 12) was any secretion induced above the level of the continuous secretion. It is evident, therefore, that food served in an unpleasant manner will not give rise to an appetite secretion under ordinary conditions, although custom and degree of hunger will naturally influence the conception of an appetizing food. Breakfasts served in a pleasant manner and with appetizing foods were set before each of the six subjects just mentioned from 15 to 30 minutes after they had been presented with a view^ of a breakfast of the opposite and discouraging character and which had evoked no psychic gastric response. The results are plotted in the same charts as the preceding tests (figs. 8, 9, 10, 11, 12 and 13). It will be noted that in the first two cases a marked appetite secretion followed the presentation of the second meal, this being a sight effect as noses of these subjects were clamped. However, the other four sub- jects did not show a psychic secretion under these conditions. The subjects showing a response w^ere accustomed to eating in the laboratory and may have felt that they would not be expected to actually partake of the disagreeable food. The other subjects having no knowledge of the character of the test might well be more strongly repulsed by the first meal, this effect being carried over for the period of 15 to 30 minutes until the palatable meal was set before them. A secondary effect might also be their suspicion that since they w^ere not permitted to partake of the first meal they might not have a chance at the second, although the contrary view was impressed upon them. The psychic secretion and the odor of food. The influence of the odor of food alone on psychic secretion was tried out on seven subjects (see PSYCHIC SECRETION OF THE GASTRIC JUICE 5 figs. 14 to 20). The odor of frying beefsteak was used as a stimulus, the odor being pleasant, strong and unmistakably that of an appetizing food of common consumption. Subjects were blindfolded and the ears were muffled in order to exclude the influence of the sight of the steak and of hearing it fried. Subjects inhaled liberally the fumes arising from the frying steak. As in our other tests, the subjects had had no food for fourteen hours. Three of the subjects showed no increase in the volume of secretion under the influence of these odors. The four others showed some increase, but in no case was the secretion voluminous. It would appear from these tests that odor was considerably less important than sight in inducing the appetite secretion, at least in man. These results are supported by the experiments previously mentioned, in which com- bined sight and odor of food brought forth no greater secretion than sight alone. Odor may have an influence on the motor activity' of the stomach and may be of importance in animals with a more highly developed sense of smell. There may very well also be considerable differences in individuals of the human species. The psychic secretion and the tasting and chewing of food. The sub- jects of the preceding tests on the influence of odor were permitted to rest for half an hour to reestablish the level of continued secretion which had in most cases been little affected. They were then per- mitted to chew for five minutes portions of tenderloin steak with strict caution to swallow none of the pieces, this possibility being checked by careful examinations of the gastric contents. In all cases the sub- jects remained blindfolded. In the first four cases the noses of the subjects were also clamped so that none of the vapors could be inhaled by that channel. The results are charted in figures 14, 15, 16 and 17, and show no distinct infiuence of tasting and chewing meat under these conditions upon the secretion of gastric juice. In one case the volume of gastric contents was somewhat increased, but the low acidity shows that very little acid gastric juice could have been secreted. Ap- parently the taste and chewing of food in the absence of sight or odor produced no marked psychic secretion. In three other cases the same procedure of chewing and tasting beef- steak was carried out, but the noses of the subjects were undamped, sight, however, being excluded (see figs. 18, 19 and 20). As illustrated in these cases the influence of the combined tasting, chewing and smelling of food on the secretion of appetite gastric juice was very pronounced and was much greater than that of smell alone. 6 MILLEE, BERGEIM, REHFL'SS AND HAWK The influence of the sound or thought of food. Subjects were blind- folded and had their noses clamped to exclude the sight and smell of food. They were seated before a frying pan in which a steak was being broiled with plainly audible sputtering and sizzling. They were told what a fine, juicy steak was being prepared for them and a general attempt made to keep their attention on the subject of appetizing meats. The results are charted in figures 21 and 22. In one case the result was negative; in the other case a distinct stimulation of secretion resulted. The variation must be ascribed to individual differences. After one-half to three-quarters of an hour rest, the nose clips were removed and the subjects permitted to smell as well as hear the sputtering of frying steak. Results are plotted on the same charts and show that in one case a very slight rise in secretion took place. In the other case a definite stimulation occurred, although the earlier level for hearing and thought of food was not surpassed. In one case the subject was permitted to smell feces of a repulsive odor fifteen minutes after smelling steak. Any psychic secretion appears to have been depressed to the level of the continuous secretion but not below this. Experiment 23 gives a comparsion of the psychic effects of: a, the sight of a frying steak (ears not stoppered); h, sight and smell; and c, taste of the same food. A distinct stimulation was produced by the sight of the food. One-half hour later the sight and odor of similar food produced a very similar stimulation. After a further interval of 15 minutes the taste of the food gave a lesser stimulation than sight or sight and smell had previously done. A summary of some of the results obtained on two of our subjects in so far as volumes and "total actual acidities" of appetite secretions were concerned, is given in figures 24 and 25. They must, of course, be considered in connection with details of individual experiments. They serve, however, to emphasize the important role of the sight of food as a stimulus to the appetite. The influence of palatahility or unpalatahility of a meal on its gastric digestion. Two subjects were given uniform meals prepared and served in the ordinary manner. On a later day they were given the same foods prepared in as unpalatable a manner as possible without altering their chemical composition. The meal used consisted of: cream of wheat, 100 gm.; sugar, 10 gm.; milk, 35 cc; coffee, 100 cc; graham crackers, 50 gm.; oranges, 50 gm.; water, 100 cc. On the second experimental day these foods were all mixed together in a conglomerate PSYCHIC SECRETION OF THE GASTRIC JUICE 7 mass, discolored with small amounts of burnt crackers and charcoal, and the atmosphere at the table saturated with the repulsive odors of valeric and butyric acids. The first of these subjects was of a nervous temperament and from his statements and manner was judged to be easily influenced or dis- turbed by the character and preparation of his food. In fact he posi- tively refused on one occasion to continue eating a meal of the second type mentioned above, although urged to do so in the interest of science. The results on this subject (see figs. 26 and 27) show no delay or inhibition of the acid response of the stomach, although the evacua- tion time was somewhat prolonged. The second subject was accustomed to eating in a laboratory, was of a phlegmatic temperament, claiming and appearing to be very little disturbed by the appearance of food or the condition in which it was served. The results on this subject are given in figures 28 and 29. The unpalatable food showed a rapid, though not quite so rapid, development of acidity and a few minutes quicker evacuation. The first subject was also tried out with a palatable meal 50 minutes after he had violently refused one which he believed to be contami- nated. The result is charted in figure 30. The development of acidity was even more rapid than in the case where the meal was given under normal conditions. If any depression of psychic secretion was carried over through this interval, there were no signs of it. The first subject was also given a meal of unpalatable character similar to the ones already described, but prepared by himself and hence known by him to be innocuous. The results as charted in figure 31 show a rapid development of acidity and quick evacuation. Some information with regard to the gastric response to foods un- palatable in appearance, odor and taste was obtained by experiments on the feeding of Chinese preserved duck eggs called "pidan." These eggs have dark greenish yolks and yellow-brown "whites" of a firm, gelatinous consistency and possess distinct odors of ammonia and hydrogen sulphide. One subject disliked these eggs but did not know what they were and was not especially prejudiced against them. The other subject, "Don," was of a nervous type, and just as he finished eating the eggs he was told in a joking manner by one of the laboratory wits that they were of prehistoric Chinese origin. The subject became clearly suspicious that something had been given him that was not entirely fresh. The results of this test as compared with similar tests on boiled duck eggs and on raw white and yolk of egg are given in 8 MILLER, BERGEIM, REHFUSS AND HAWK figure 32. They show a depression of gastric secretion after "pidan" lasting for an hour and a quarter, the aciditj' then rising rapidly to normal figures. This delay may have been due to inhibition of appe- tite secretion, gastric activity being finally aroused through chemical stimulation following the solvent action of the slow continued secretion. The failure of this unappetizing food to arouse the secretory or motor activities of the stomach to a normal response is indicated also by our results on the first subject mentioned above. The curves are given in figure 33 and show that while raw hens' eggs gave an acid response of over 100 in the first hour and left in 2j hours, the preserved eggs at no time gave acidities of over 30 and remained in the stomach 4| hours. It appears that the unappetizing character of these eggs led to a delayed acid response and slow evacuation, perhaps complicated by their failure to show some early digestion with consequent chemical stimulation. Influence of prejudice against a food on its digestihility in the stomach. It is very common to find people who have a prejudice against certain foods generally classed among the most wholesome articles of diet. Certain cases maj- be due to a food anaphylaxis or sensitivity. Others may be due to defective gastric or intestinal digestion or other causes. Undoubtedly many have no foundation and are the results of wrongly placing the blame of certain digestive disturbances. One of our subjects, "Ham," had a strong prejudice against eggs in any form and had not eaten them for years. He was with difficulty persuaded to take eggs prepared in several different waj's. The results ■of these tests are plotted in figure 34, and show that eggs were digested by this subject in a perfectly normal manner, at least as far as the stomach was concerned. Neither did untoward sjanptoms of any kind develop. Influence of newspaper reading on gastric digestion. Subjects were permitted to read newspapers throughout the course of a meal of palatable foods, the same test meal as used in previous experiments. The gastric responses of two subjects who read newspapers and re- sponses of same subjects with no reading but with usual table talk are charted in figures 26, 28, 35 and 36. No distinct influence of news- paper reading was noted. Responses were quite normal in all cases. The slight differences in acid development and evacuation time were in one case favorable and in the other case unfavorable to newspaper reading. Influence of the unpalatable character of a diet on its idtimate utiliza- tion by the human body. Smith, Holder and Hawk found (9) in a me- tabolism experiment on a normal man that where a uniform diet of a PSYCHIC SECRETION OF THE GASTRIC JUICE 9 palatable character was given for several days, followed b}^ a period in which the same foods were jumbled together in dirty dishes and served amidst ill-smelling and otherwise unpleasant surroundings, that the nitrogen utilization in the first case was 86.7 per cent and in the second, 85.6 per cent. The nitrogen balance showed a retention in the first period of 3.0 per cent and in the second, of 6.4 per cent. This in spite of the fact that the subject was onh' with difficulty persuaded to eat the unpalatable food and that another subject who was given the same kind of food became nauseated and could not continiie. Influence of anxiety on gastric digestion (10). The study of the influence of emotional strain on digestion in man offers some difficulties due to the fact that the emotions cannot be readity controlled, nor are the subjects of extreme emotion readily amenable to experimentation. We were, however, able to obtain an interesting illustration of the profound effect of mental anxiety on gastric digestion in the case of one of our subjects. The man was a first-year medical student who had previously served as a subject of gastric tests. He was given 100 grams of fried chicken on the morning of an important examination in chemistry and was asked to write out his answers during the course of the test. He was plainly worried over the outcome of the exami- nation and of his j^ear's work. The effect upon gastric digestion was the prolonging of the evacuation time to 6j hours. The intra-gastric acidit}' remained in the neighborhood of 90 for 3 hours. The normal digestion curve for fried chicken on this subject was obtained a week later under the best mental conditions. The time required was 4j hours and the maximum acidity about 65. It is not at all surprising that worry aggravates a condition of gastric ulcer. An interesting experiment on the digestion of milk in the human stomach may be cited in this connection (11). It was found that in the stomach of one of our subjects milk would not curdle. The test was carried out at the end of the year immediately before the final examinations. The subject was one of the most brilliant students in his class and had worked hard. We made several tests on this student and in every case milk left his stomach rapidly and without curdling. He digested all other food normally. The next fall, upon his return to college, we made another milk test upon him and found that his stomach curdled milk in a normal manner. At this time he was in fine physical condition, having had a long, pleasant vacation, whereas in the spring he was in a highly nervous state as a result of his hard study. This serves to illustrate the influence which rigid and prolonged mental application may exert upon the stomach in certain types of individuals. 10 MILLER, BERGEIM, REHFUSS AND HAWK SUMMARY AND CONCLUSIONS The sight alone of a table well set with nourishing foods was found to give rise to a distinct secretion of gastric juice in normal men. The sight of a half grape fruit only resulted likewise in an appetite secre- tion. The sight of the same foods illy prepared and poorly served resulted in no stimulation of appetite secretion. The service of a well prepared meal half an hour after the service of a poorly prepared one- gave in some instances a distinct secretion, in others not. The odor alone of frying meat produced in some cases no appetite secretion, in others a slight secretion. Odor alone produced less stimulation than sight alone. The tasting and chewing of food in the absence of smell or sight produced no marked psychic secretion. The combined influence of the tasting, chewing and smelling of food was pronounced and much greater than that of smell alone. The sound and thought alone of a frying steak gave rise to a gastric secretion. The influence of smell with hearing produced little ad- ditional effect. Evil odors depressed secretion to the level of the continuous secretion. In consecutive tests the sight of food, with and without odor, pro- duced similar degrees of stimulation, while taste alone had less effect. Mixed meals consisting of nourishing ingredients but very un- pleasantly prepared and served gave rise in the case of a phlegmatic individual to no distinct delay in the development of intra-gastric acidity or in evacuation. A more susceptible individual showed a slight delay in evacuation time, but none in acid response. Chinese preserved eggs, unpalatable to our subjects in appearance, odor, taste and belief in their unwholesome character led to delayed acid response and evacuation. In one case the normal acid level was ultimately attained due to chemical stimulation. In one subject a strong prejudice against eggs was found not to result in any abnormal gastric response when eggs were eaten. The ultimate utilization of the protein of a diet prepared in a most unpalatable manner was not found to be appreciably less than that of the same diet served under the best conditions. Newspaper reading during the course of a meal could not be shown to have any distinct influence on gastric digestion. Anxiety and mental strain were found to markedly delay gastric digestion. The authors desire to thank for their cooperation the students of Jefferson Aledical College who acted as subjects of these tests. PSYCHIC SECRETION OF THE GASTRIC JUICE 11 BIBLIOGRAPHY (1) Bidder and Schmidt: Die verdauungssafte, etc., 1852. (2) Pavlov: The work of the digestive glands, 2nd English Ed., Philadelphia, 1910. (3; Carlson: The control of hunger in health and disease, Chicago, 1916, pages 236-240. (4) Homborg: Skand. Arch. f. Physiol. 1904, xv, 209. (5) Luckh.\rdt: Report to Amer. Physiol. Soc, December, 1917, and private communication to one of the authors. (6) Cannon: The mechanical factors of digestion, London, 1911. (7) BiCKEL AND Sasaki: Deutsch. med. Wochenschr., 1905, xxxi, 1829. (8) Fishback, Smith, Bergeim, Lichtenthaeler, Rehfuss and Hawk: This Journal, 1919, xlix, 174. (9) Smith, Holder and Hawk: Reported Soc. Exper. Biol. Med., February 18, 1920; Science, in press. (10) Miller. Bergeim and Hawk: Reported Soc. Exper. Biol. Med., February 18, 1920; Science, in press. (11) Lichtenthaeler, Bergeim and Hawk: L^'npublished data. Fig. 2. Ca^3..3^-0'3m /'hour' ^ Fig. 3. Fig. 4. ^ Ca^e... 3(0- Uho. ^ 1-hour B.^DeL. Ca3e.-Z9-md.Cyi. Fig. 5. V / I hour. Fig. 6. Fig. 7. 13 Oase^ 7-Q-o Cose 3/-Ph£l/r, 5 1 ( \ Q / ^ / '4 ^/ V tit tf. nhour' Fig. 11. Qa3e...^Z-SujQ Case.- 3^-LJai. •vS d QJ r © Q) -0 -0 4^. ^ ti Cose,. ZX-Sok Cade,-Z4-UJat. Cose^Zh-OBrxL. /}hour Z Fig. 20. Ca^e,^ q-rrn 'Z-f^L. j-hour Fig. 23. i s ^. 2 00 c8. CO 00 ^5 C29®,- /^. ^ Fig. 24. 21 ^ g g 3 I 3 *0 § 00 Case,- Qo. •:. ^ 1^ I 1 a C^ 1 Fig. 25. Oa9e,./3-Qo POeaL , liaLa-tabL& I hour Fig. 26. I §1 § s.- ^ s Co3e..JS-CrO. PO&aL; unfiaiahQ-bLe,. S ^ ^ § I. hour Fig. 27. Case.- /4- -POL Meat,, /■hour Fig. 2S. I o" a § ^ ^ o Q VS o ft) PleaL, un/naLa-hahl&. ^ '■hour Fig. 29. T ^ § Q ^ ^. S 1^ c ft § CaQe.-/7-Qo- I. hour Fig. 30. s Co^e.-IS-Pli Pleat, unfKxLa-hahU ^ I. hour %. Fig. 31. ^ Cose,- 7^0/7 -39 yfaiM Lolks (Uo) ■■ -4-5' ■■ TfaLo CJhc^eS . (L/hy " ~^<^J;Jp^i^d.^'=>''l'^d. TurH-eij £<^^^ (Bo) ' j-\ x — ' \ ^ ' \ \ V rhour Fig. 32. 25 -j^ Ca'se,. Tjhm - /oe Chinese T^cdan^. {Po) ^^ / / \ ^ Q8 // V^'^ /'hour Fig. 33. § Case,. HdX- 4-7 ■■ fresh Tr^ed ti^e^^ /•■ \ - BZ ■■ yi-ona-ae chocL. I ■■ \r S3 ■ Shurl^edL £$^9 r Fig. 34. Dhoor s I ^ ^ ^ g Q ^ Case,- /Q~Qo. Plea L, fna La-ira ble /■hour i' Fig. 35. Pleat, halatable , ■ s s § ^ § ^ • The expenses of this investigation were defrayed by grants from Mrs. M. H. Henderson, The Curtis Publishing Company and Dr. L. M. Halsey. 28 GASTRIC RESPONSE TO TEA, COFFEE AXD COCOA 29 lumps of sugar per liter were given. In the case of cream approxi- mately 15 cc. of a 20 per cent cream were used. No milk or added sugar were used in preparing the cocoa, which was made up according to the directions on the container. As soon as the meal was finished the subjects were made to swallow the Rehfuss stomach tubes. From 5 to 8 cc. of the stomach contents were aspirated at 15-minute intervals for 1| hours. The subjects were then allowed to attend a class and returned 1^ hours later (after a total experimental period of 3 hours). Then two samples were with- drawn at 10-minute intervals, and finally the stomach was completely emptied, this being checked by a lavage of 150 cc. of water. Total and free acidities were determined by titration and pepsin by the Mett method. The volumes of contents at the end of the 3-hour, period were measured. The pulse rate was also determined at inter- vals and subjects reported any unusual sjinptoms. Tests were carried out in which the following beverages were added to the uniform test meals: (1) Cold water, 1 liter, 15°C. (2) Hot coffee, plain, 1 liter, 45-50°C. (3) Hot coffee, with sugar, 1 liter, 45-50°C. (4) Hot coffee, with cream, 1 liter, 45-50°C. (5) Hot coffee, cream and sugar, | liter, 45-50°C. (6) Cold tea, plain, 1 hter, 13°C. (7) Hot tea, plain, 1 liter, 50°C. (8) Hot cocoa, plain, 1 liter, 57°C. (9) Hot cocoa, plain, ^ liter, 57°C. Thirty-seven experiments were carried out on four different subjects, duplicates being made on each subject with the basal diet alone. The results are charted in figures 1 to 34. The results with regard to evac- uation time are summarized in figure 34. INFLUENCE OF BEVERAGES ON THE ACID RESPONSE OF THE STOMACH Influence of cold water on the acid response. The diet alone contain- ing, as it does, considerable meat gives rise to the rapid development of a relatively high acidity, much of which is represented by combined acid (figs. 1, 10, 19 and 26). As no hquid whatever was taken with the meals in these cases and in the early part of digestion the absorp- tion of gastric juice by the meat was pronounced, a uniform mixture 30 MILLEE, BERGEIM, REHFUSS AXD HAWK was not at once obtained in the stomach; and the samples withdrawn varied somewhat with the exact location of the tip in the stomach. For this reason the duplicate curves do not coincide, in spite of the fact that digestion follows the same course and is concluded after the same lapse of time. Where a moderate amount of liquid is present or the combining power of the food less, it is possible to sample uniformly and to obtain very similar curves with the same food eaten at different times. For this reason the experiments on different beverages are, with regard to acid response, more comparable with each other than with the diet lacking liquid. The curves of acidity, especially total and combined acidity, appeared to rise somewhat more quickly and to a generall}^ higher level with the •diet alone than with the added liter of cold water. As the evacuation time was practically unaffected and the acidities still high following the high water ingestion, the water cannot be said to have hindered digestion and must have given rise to a considerable stimulation of acid secretion or been rapidly emptied in large measure, or more probably both. In consideration of the results of these tests it must be constantly borne in mind that unusual amounts of the various beverages were given. This was intentionally done in order that any untoward in- fluences might be accentuated. Influence of coffee on the acid response. Hot coffee, plain, that is without cream or sugar and at a temperature of 45 to 50°C. was given to four subjects. The acid responses are charted in figures 3, 12, 21 and 27. In the first three cases a liter of the coffee was taken; in case 27, however, only half a hter. The charts show in a general way that the acid responses were very similar to the results obtained with cold water, and not very different from those obtained with the basic meal by itself, although not attaining quite the heights of total acidity given by the latter. There was no distinct indication that the coffee in- hibited the secretion of the gastric juice. Sugar was added to the coffee in two cases (see figs. 4 and 22). Comparisons of the acid responses with those of the same individuals taking plain coffee (see figs. 3 and 21) show that the sugar depressed considerably the acid response. It has been clearly demonstrated in this laboratory that sugar depresses gastric secretion; and it is, there- fore, improbable that the low acid values in the earlier period of diges- tion in the cases where sugar was added to the coffee, are due merely to the slower evacuation of the l^everage. GASTRIC RESPONSE TO TEA, COFFEE AND COCOA 31 The addition of moderate amounts of cream (15 cc. of 20 per cent cream to the Hter) was tried out in two cases (compare figs. 12 and 13 and figs, 27 and 28). It will be noted that the acid response was not demonstrably influenced b}^ such addition. Hot coffee with both cream and sugar was given in four cases (see figs. 5, 14 and 29). The results indicate that with the addition of cream and sugar the acid response was higher than where sugar alone was used, and more nearly approached that of the plain coffee. This may perhaps be explained by the fact that the coffee was more appe- tizing with additions of cream and sugar, provoking a more marked appetite secretion and also leading to more rapid evacuation. Influence of tea on the acid response. One liter of cold tea (12° to 13°C.) was given during the course of the standard meal to each of these subjects (see figs. 6, 15 and 30). If the acid responses are com- pared with those of the same subjects to cold water or plain hot coffee, it will be noted that they are very similar, although one subject showed a slightly higher curve with tea than with water or coffee. Hot tea (50°C.) was also given to three subjects (see figs. 7, 16, 24 and 31). With slight variations, generally in favor of the hot tea, the results obtained were not distinctly different from those with water, coffee or cold tea. Influence of cocoa on the acid response. That cocoa when taken in amounts of 1 liter at a meal has a depressing action on the develop- ment of gastric acidit}" is clearly apparent from the results on our four subjects as charted in figures 8, 17, 25 and 32. In each case the intragastric acidity rises slowly as compared with either cold water, hot coffee or hot or cold tea. This may be due to several causes. Un- doubtedly the sugar content depresses secretion and delays evacuation as sugar solutions have been clearly shown to do. The higher fat eon- tent of cocoa may play a part as well as the presence of other con- stituents. In three cases the amount of cocoa drunk was reduced to one-half hter. The depressing action of cocoa on the development of acidity may be noted in these cases (see figs. 9, 18 and 33). In general this depression is about equal to that of one liter of tea or coffee. 32 MILLER, BERGEIM, REHFUSS AND HAWK INFLUENCE OF WATER, TEA, COFFEE AND COCOA ON THE EVACUATION TIME OF THE NORMAL HUMAN STOMACH The influence of the beverages studied on the evacuation time of the stomach is summarized in figure 34, which gives the volumes of the gastric contents at the time of complete removal (approximately 3 hours after ingestion). The evacuation of the stomach was not ap- preciably delayed by the drinking during the meal of 1 liter of cold water; hot coffee, plain; hot coffee with cream; hot coffee with cream and sugar; cold tea, or hot tea; nor were there any distinct differences between the evacuation periods of any of these beverages. The ad- dition of sugar alone to coffee did, however, delay evacuation distinctly in both cases. Most striking, however, was the pronounced delay in evacuation caused by cocoa, this being noted in every case, even where half amounts only were given. For example, in one case the gastric contents at three and one-half hours measured, following the meal alone, 124 cc; following hot coffee with cream and sugar, 110 cc; and after cocoa, 465 cc. Temperature appeared to have little influence on evacuation • time, meals including cold tea leaving the stomach in the same time as those including equal volumes of hot tea; nor did cold water delay evacuation. INFLUENCE OF TEA, COFFEE AND COCOA ON THE PEPTIC RESPONSE Peptic activities were determined by the Mett method. In figure 35 are illustrated the values obtained after the uniform meals had been given with water, tea, coffee and cocoa respectively. The only strik- ing differences in the development of peptic activity noted were the low values found after the ingestion of coffee with sugar and especially after the ingestion of cocoa. These low values may be due to the de- pression of the secretory mechanism by the sugar and other substances present in cocoa as well as to retention of the ingested beverages. INFLUENCE OF TEA, COFFEE AND COCOA ON THE PULSE RATE The pulse rate was also followed in each case, although not as sys- tematicall,y as would be necessary for exact comparisons of the effects of the different beverages upon the heart beat. Tea, and particularly coffee, did, however, bring about in liter quantities a pronounced ac- celeration of the heart beat in the first half-hour, one subject showing an GASTRIC RESPONSE TO TEA, COFFEE AND COCOA 33 increase to 150 and 160 beats per minute after hot tea and coffee re- spectively. There was some evidence that coffee brought about a more rapid and pronounced acceleration of the heart beat than the other beverages. From its slow evacuation, cocoa might be expected to produce a less rapid stimulation. An attempt (2) has been made to account for the more rapid action of coffee as compared with tea by showing that in coffee the alkaloid exists as caffeotannic acid soluble in cold water and not readily precipitated, while tea contains a caffeine tannate soluble in hot but not cold water and which is readily precipi- tated by acid (hence presumably by the gastric juice), going readily back in solution in an alkaline medium (such as that of the pancreatic juice). While coffee is usually about a 6 per cent decoction and tea about 1| per cent, it must be borne in mind that tea contains 3 to 4 per cent of caffeine and coffee about 1 per cent. Other systemic effects of tea, coffee and cocoa. Tea and coffee in the amounts taken gave rise in our subjects to nervousness, vasomotor relaxation, sweating, tremors, headaches, dizziness and sleeplessness, in some cases to a marked degree. Three of our subjects were unac- customed to the use of coffee, and the fourth never drank more than one cup at a meal. It is, therefore, probable that the symptoms noted were of a somewhat more aggravated character than would be found in the case of persons habitually drinking much tea or coffee. There is no question, however, that tea and coffee may have a marked effect upon the circulation, and that they are in no wise to be considered as beverages to be used in an unrestricted manner. Cocoa did not give rise to nervous or vasomotor symptoms to any- thing like the same extent as tea and coffee. This may in part have been due to slow absorption. In all cases there was a feeling of fulness after cocoa and a lack of hunger, which may be readily explained by the prolonged retention of this beverage in the stomach. DIURESIS AFTER TEA AND COFFEE DRINKING The urine secretion of our subjects was measured during the period immediately following the ingestion of the test meals with tea, coffee and cocoa. The volumes of urine eliminated are indicated by charts 36, 37 and 38. As much as 866 cc. of urine was excreted within an hour and a half after 1 liter of tea was given, and the values after coffee drinking were of the same order. It is clear, therefore, that these bev- erages left the stomach quickly, were rapidly absorbed, and the excess THE AMERICAN- JOURNAL OF PHYSIOLOGY, VOL. 52, NO. 1 34 MILLER, BERGEIM, REHFUSS AND HAWK water soon eliminated. Coffee with sugar resulted in less rapid elim- ination, and the secretion after cocoa was low in volume, as might be expected from the delayed gastric evacuation. The specific gravities of the urines during the period of most rapid elimination varied from 1.004 to 1.001 or less, indicating a very dilute secretion. SUMMARY AXD CONCLUSIONS A study was made of the influence of water, tea, coffee and cocoa upon the gastric digestion of a uniform mixed meal as measured by the acid responses and evacuation times. Evacuation of the stomach was not appreciably delayed by the drinking of 1 liter of cold water, cold or hot tea, hot coffee, either plain, with cream or with cream and sugar. The addition of sugar alone to coffee delayed evacuation. Cocoa in 1 hter quantities markedly delayed evacuation. To a less extent this was true of half-liter volumes. One hter quantities of water, hot or cold tea, hot coffee, plain or with cream, delayed somewhat the rise of the level of intragastric acidity as compared with the basal meal alone. As high acidities and normal evacuation were, however, attained these beverages must have stimu- lated gastric secretion, been rapidly evacuated, or more probably both. Coffee with sugar alone delayed the development of gastric acidity. Coffee with sugar and cream had less effect. Cocoa delayed distinctly the development of intragastric acidity. One liter quantities of tea and coffee gave rise to marked acceleration of the heart beat, to vasomotor relaxation, tremors and other nervous symptoms. Cocoa did not produce these effects but brought about a feehng of fulness at the stomach. Urine secretion during the first 90 minutes after tea or coffee inges- tion varied from 550 to 866 cc, after cocoa from 125 to 372 cc. The authors desire to thank the students who kindly served as the subjects of these tests. BIBLIOGRAPHY (1) Penzoldt: Deutsch. Arch. klin. Med., 1893, xiv, 535. (2) Lancet, 1911, i, 46; ii, 1573. § 3 Q ^ Fig. 1 I ? Case.- ff-CcL /race. I. hour 1 — Fig. 2 35 Case,- Z4 -Co. 5 I ■hour' Fig. 3 I /3a cc , 8^ Cose.- 49 -Ca Titeh ouoirh / L Hc-t Coffee, Suq/gCr. ZS'O.cc. remot/ol. 36 I. hour — t: Fio. 5 //Qcc. , remoiAx.L i Case.- 44 -Ch Uc&i Lui-bh /^. Cold lea./ 3^ I. hour JT Fig. 6 1 50.CC , remoi^C. 37 3^ Case..2.8-Cci s /5Z.CC I remoutLo I. hour Z Fig. 7 S Case.-3;i-Gx D^et u^tM lU Cocoa, syc. o < cc 5 c -J /.hoi. Fig. 8 A65.CC. remoi/al. 38 ^ § n ^ ^ ^ a Case,. 40 -Ca J)oei ujlih '1% 4- Hoh Qoc/oci Case.- S3 -Ql JJie-t fiLone. no. cc , remot/ac. (0 ^ /:houf Fig. 10 39 ^ ^ # o cc ^ Case,- /8-Ql JDce^ ujo^h /L Cold bJa-ter, I S"\ 10. cc t^moL/bC I. hour 1 — Fig. 11 CO 5 Q Case, %S-QL JDiei oulih I L. HoiCoffee,lnL(iLnA5"-50X^. 3.CC , remo{/aL. I hour Fig. 12 40 ^0 §^ Case.- 49-Ql. IJiei; uuihh 1 1^. Ho^ Cofj/^ee. Cream. C /O. cc. , remoutxL I. hour _OL. Fig. 13 s o Case,- lil-Q^ yr y,^^ Tlleh LuUh 'h L, Ho-bCoffe&,C/eam c^^^an / / / remoiAxL. I. hour FiQ. 14 41 s JDoG-b uuihh /4 Cold le-a, 1^X2 LhoL Fig. 15 zacc. , 8 S 2 I 8 Case,.Z9-G/ ~Die^ uj'ahh 1 1^ TeahohSO'C. 2Zcc , I hour ~~7r- FiG. 16 42 §g Case,- 33-Gl 8 5 jDlet uuL-bh I U Cocoa, 51°C /hour % Fig. 17 t%^ . 5:34 § Cas>e,.4/-G/ o JDcet ujohh '/%L Cocoa Hoi. /45: cc , remoi/a L 3. 43 ^ Case.- /9-7?i, 8 /.hoL J Fig. 20 44 /S'cc remcMiL ^ § Case..Z6- o Tlue-t uiu-hh/L Hoh Coffee,/ilacn , 45°- 50°C remOWdi I. hour Fig. 21 Ca'se..50-~Rl JDle-t u^lih /^ HotCoff'ee^uqar. Fig. 22 45 remoi/ai \0 ^ Case,- 36-~Hi remojxL I houf Fig. 23 I S Cose,. 30-7?o 6:gc I. he or Z Fig. 24 46 §£ Case.. 34 -Til JDtet uuc-thiL Cocoa, 57°C S ZIQCC , renioiul I Case.- 22- Jo. JDie-h /^Lone. § ^ o 24. cc. , rewoi/ac. I.houi^ Fig. 26 47 ^ § Case.. I'f-Jo DoehouUh y^4 ^o-i;Coffe&,fnlaon,45°-5CrC. o CO ^ 3 cc I hour' Z Fig. 27 ^ ■g o" ^ 8 Case,- 51 -Jo. JOoeir lulU) '/z 4 HoirCoffee.. Qream. ex /.hour Fig. 28 48 1^ s Ccise,-39-L/o JDcei uuohh '/%l^ Hoi Coffee, Cream and Sugar. I. hour Fig. 29 /Z.CC. , remoi/ai I Case.- AJ-Jo X)oe-ir uji^h '/% L CoLd Tea /ZV. I .hour — jr Fig. 30 49 ;23. cc. , rewotAt. THE AMERICAN JOURNAL OF PHTSIOLOGT, VOL. 52, NO. 1 1^ s Case, 31-Jo T)i^uul-hh 'kJ^ Hob lea,^0°C 5 Q / lOcc , remoi/aL ihOL Z Fig. 31 3 o CO Ca^e.-A3-Jo. TJi-e-b ujo-hh I L^. Ho-b Cocoa IdQcc. , remoi/uL 50 Case,. 3S- Sj So Case,. ~Ri. %3 /l/o U/a-bsr / •■ hlatn CoffGe.. ,, ^ / " ■ -Jk / " " Qococu IS. ze. sro. Be. 3o. 34. (keijy hot Coffee Cream cind •- nionui^es. Fig. 36 52 GO /OO /.00-3 Urine. Secre-&con / $, Q) /oo Fig. 37 Urine Secre-huon . Case.. Ca /ooaS:°'-"^:>jooo\ /- / A.ooi /.004 Fig. 3S 53 I. RAPID BLOOD PLASMA PROTEIN DEPLETION AND THE CURVE OF REGENERATION H. P. SMITH, A. E. BELT and G. H. WHIPPLE From the George Williams Hooper Foundation for Medical Research, University of California Medical School, Saji Francisco Received for publication February 25, 1920 The published work of Kerr, Hurwitz and Whipple (1) brings out several facts about the blood serum proteins which may be mentioned briefly before we go on to a consideration of the experiments given below. The stability of the serum protein concentration is truly re- markable and obviously of some importance to the body physiology. The normal level is quite constant and considerable deviations from this normal base line are not well tolerated by the body; in fact, pro- found shock may result. WTien the serum proteins are depleted or washed out by the technique employed the repair or regeneration of these proteins is a slow process requiring from 5 to 10 days, depend- ing upon the amount removed and other factors. It is as difficult to reconstruct these proteins as it is for the body to repair and replace liver cells following an extensive liver injury. It appears that the liver is especially concerned in the normal regeneration of blood serum proteins. Fasting does not prevent serum protein regeneration, therefore it is possible for the body to release these substances or to construct serum proteins from its own protein end products. There is no evidence that increased nitrogen breakdown is responsible for this regeneration of serum protein. The experiments of Kerr, Hurwitz and "Whipple were different as to method of blood serum depletion when compared with the experi- ments given below. These earlier experiments were mostly done by interval bleedings followed in each instance by a return of the washed red corpuscles suspended in modified Locke's solution. Under such cir- cumstances a dog was bled 100 to 200 cc. and after washing by centri- fugalization the same red cells were returned intravenously in Locke's solution. This procedure was repeated several times during the day until the serum protein depletion was carried to a minimum figure. 54 REGENERATION OF PROTEINS OF BLOOD PLASMA 55 This method has been used by Abel, Rowntree and Turner (2) and called "plasmapharesis." It is obvious that this experimental pro- cedure introduced . wide fluctuations in blood volume and it was sus- pected that the shock which resulted was to be explained by the repeated hemorrhages and infusions. We shall use the term plasma depletion or plasmapharesis to indicate a removal of plasma proteins by means of repeated hemorrhage followed or accompaiiied by the replacement of like amounts of red cells sus- pended in a protein-free fluid. The plasma depletion in our experi- ments was effected by a method first introduced by IMorawitz (3); bleeding and the removal of whole blood was simultaneous with the replacement of the red cell Locke's solution mixture The inflow and outflow volume was at all times constant and obviated any fluctua- tion in blood volume. All aseptic precautions were taken in manipu- lation, washing and final preparation of the red cell mixtures. The red cell mixtures were introduced at bodj^ temperature and the dog was kept warm during the experiment. The method employed in these experiments enables an investigator to reduce the blood plasma proteins from the normal level of 5 to 6 per cent to a very low level of 1.5 to 0.9 per cent. This can be done in a matter of minutes (2 to 10 minutes) leaving the animal uninfluenced by the large and numerous fluctuations in blood volume and oxygen-carry- ing capacity of the blood which undoubtedly occur in the method used by Kerr, Hurwitz and Whipple. In addition it facilitates observa- tions of that portion of the curve of protein regeneration immediately following a large single depletion and permits observations on an uninterrupted regeneration curve. METHOD The animals used were sound young dogs maintained on a mixed diet. In most cases no food was given the animal for a period of 12 hours pre- ceding the experiment. Free access to water obtained. Under com- plete ether anesthesia and with aseptic precautions an incision was made either into the region of the femoral vessels or of the large vessels of the neck. The artery and vein were exposed and clamped. Into each was introduced a vasehne-coated cannula pointing toward the heart. Plasma removal was effected by withdrawing through the cannula placed in the artery large quantities of blood. This blood was allowed to flow into a graduated bottle. Simultaneouslj", a suspension 56 H. p. SMITH, A. E. BELT AND G. H. WHIPPLE of washed corpuscles warmed to body temperature was injected under pressure through the venous cannula. This suspension was delivered from a flask which was also graduated. The graduations were used to permit a comparison of the inflow with outflow to be made at any- time during the exchange. In this way inflow and outflow were ob- served and kept equal at all times. In order to maintain an even sus- pension of the injection mass, the latter was frequently shaken. The fluid injected consisted of washed dog corpuscles suspended in a modified Locke's solution in the ratio of three parts packed corpuscles to two parts by volume of the saline mixture. The composition of the Locke's solution was: sodium chloride, 0.9 per cent; potassium chloride, 0.042 per cent; sodium bicarbonate, 0.02 per cent. The corpuscles in all cases were obtained from the blood of healthy dogs. This blood was drawn into sodium oxalate, centrifugalized, and the plasma removed from the sedimented corpuscles. The corpuscles were then washed twice in the modified Locke's solution by resuspension and centrifugali- zation. Aseptic precautions were observed in all these manipulations. The exchange was effected in a period ranging in different animals from 2 to 25 minutes. The amount of fluid withdrawn varied in indi- vidual experiments from 60 per cent to 195 per cent of the animal's blood volume. The amount of blood sunultaneously injected corre- sponded within a few cubic centimeters to the amount withdrawn. The blood volume was estimated as 10 cc. per 100 grams of body weight. In a number of experiments the actual blood volume was kindly deter- mined for us by Dr. C. W. Hooper, using a dye method recently de- scribed. This paper (4) shows that the blood volume of active normal dogs as determined b}^ the dye method is approximately 10 cc. per 100 grams of body weight. At the end of the operative procedure the cannulae were withdrawn and the vessels ligated. Vaseline was applied liberally to the wound. In a few instances it was necessary to make a single suture through the subcutaneous tissues at the site of operation. In practically every experiment the wound healed quickly with little or no suppuration. Samples of blood were collected through the arterial cannula at the beginning and at the end of the exchange and again 15 minutes later. Subsequent Samples were taken from the jugular vein by means of a needle and syringe. Oh each of these occasions two samples were with- drawn. One of these was drawn into a 15 cc. hematocrit tube contain- ing 3 cc. of 1 per cent sodium oxalate. The other sample was drawn into a plain heavy-walled glass test tube and allowed to clot. Both EEGENERATION OF PROTEINS OF BLOOD PLASMA 57 of these samples were then centrifugaHzed at a high rate of speed. The serum from the clotted sample was then used for estimation of serum albumins, serum globulins and the non-protein fraction by the refractometric method of Robertson (5). Percentage corpuscles read- ings were made from the oxalated sample, correction being made for the amount of oxalate solution present. The plasma from this sample was also used for the determination of fibrin. This was carried out according to the method of Cullen and Van Slyke (6). This method consists in diluting 5 cc. of plasma in 100 cc. of salt solution. To this mixture 1.5 cc. of a 2.4 per cent solution of CaCl2 was added to supply calcium and to promote clotting, and a Kjeldahl done on the mass of fibrin obtained. The clinical condition of the animal was closel}^ observed. The rectal temperature, rate of respiration, pulse, diarrhea and vomitus, as well as the general appearance of the animal, were noted. In cases in which death resulted, careful autopsies were performed at once. The clinical condition of the animal will be made the subject of a sub- sequent paper with a discussion of the peculiar type of shock which may develop under these conditions. EXPERIMENTAL OBSERVATIONS This paper in general deals with the recovery experiments but in certain tables we include many of the lethal shock experiments (tables 12, 13 and 14). For the sake of comparison we give in table 1 a type experiment which was followed promptly by fatal shock. Many of these experiments will be found in the next paper of this series and in that place the general discussion of this peculiar shock will be pre- sented. It will be noted in table 1 that the reduction of total proteins, albumin and globulin is pretty uniform and is a fall to approximately one-third of normal. The emergency increase of protein during the 15 minutes following the plasma depletion is not as marked as usual (see table 2). There is no further increment of serum protein in the hour following this 15-minute sample and this may be explained in part by the profound shock. The content of red cells in whole blood as shown by the hematocrit is lower than usual and the fall which appears imme- diately after plasmapharesis would indicate the use of a red cell mix- ture containing fewer red cells than intended. This factor does not complicate the remaining experiments and we believe has no signifi- cance. 58 H. P. SMITH, A. E. BELT AND G. H. WHIPPLE Experiment 104- (See table 1). 122 per cent exchange. Dog 18-48. Young female bull dog. Weight 16.9 pounds. Blood volume on November 20, 1917 (by dye method) was 941 cc. November 20, 1917. Under ether anesthesia 940 cc. blood were withdrawn from the left carotid artery. Simultaneously 1000 cc. of blood corpuscle suspension were injected into the left external jugular vein. The duration of the exchange was 10.5 minutes. Animal showed almost immediately a great fall in pulse pressure and arterial tension. Profound depression with forced irregular respira- tion developed in about an hour. Death 2 hours after the exchange. The autopsy findings are uniform in all fatal experiments and will be described in detail in the following paper. TABLE 1 122 per cent blood volume exchange; dog 18-48; experiment 104 TIME BLOOD SERUM PER READINGS IN CENT FIBRIN IN PER CENT HEMATO- CRIT RED CELL PER CENT REMARKS Total protein Albu- min Globu- lin Non- protein Before exchange 5.7 4.2 1.5 2.0 0.25 47 Immediately after. . . 1.9 1.4 0.5 1.6 0.12 35 Considerable 15 minutes after 2.3 1.9 0.4 1.7 0.11 38 hemolysis Considerable 1 hour and 20 min- hemolysis utes 2.4 2.0 0.4 2.1 30 Fatal shock Table 2 gives the results of an experiment which contrasts with this lethal shock experiment (table 1). The second experiment presents an even large blood exchange in plasmapharesis but this dog is not dis- turbed by the procedure. It will be noted that there are marked indi- vidual differences in dogs as to their tolerance to this plasma depletion. Any given dog will show a considerable uniformity of reaction to a unit exchange but' must be standardized to ascertain this reaction. Table 2 also shows a fall of total protein, albumin and globulin to about one-third normal following the plasmapharesis. We wish to call atten- tion to the emergency increase in blood proteins which appears within 15 minutes. This is a characteristic reaction which obtains in practi- cally all experiments (tables 11 and 12). The increase in serum pro- teins during the next 24 hours is veiy marked and exceeds 1 per cent protein — equivalent to more than 20 per cent of the total protein re- placed in the blood serum. Subsequent regeneration of protein in the serum is slow and requires several days for complete recovery. REGEXERATIOX OF PROTEINS OF BLOOD PLASMA 59 Experiment 69. (See table 2). 170 per cent exchange. Dog 18-9. Young female bull dog. Weight 14 pounds. Blood volume on July 19, 1917 (by dye method) was 772 cc. August 2, 1917. Under ether anesthesia 1081 cc. blood were withdra\sTi from the right femoral artery. Simultaneously 1081 cc. of blood corpuscle suspen- sion were injected into the right femoral vein. The duration of exchange was 12 minutes. Following the exchange the temperature fell about 2 degrees, but returned to the original level within 2 hours. Pulse and respiration were fair at all times. A much smaller volume exchange is shown in table 3, yet consider- able shock resulted. It will be seen that the level of total proteins, albumin and globulin falls to approximately one-half of normal cor- responding to the smaller exchange volume (90 per cent). There is a TABLE 2 170 -per cent blood volume exchange; dog 18-9; experiment 69 BLOOD SEBUM READINGS IN PER CENT TIME Total protein Albumin Globulin Non- protein Before exchange Immediately after 15 minutes after 2d dav 5.6 2.0 2.9 4.2 4.5 4.8 3.8 1.3 2.0 3.1 2.5 3.2 1.8 0.7 0.9 1.1 2.0 1.6 1.7 1.4 1.3 1.9 2.1 1.7 Moderate hemolysis Moderate hemolysis. No shock Slight hemolysis. 3d day Normal 4th day moderate increase in serum proteins during the 15-minute period and less than usual during the first 24 hours following plasmapharesis. We cannot explain satisfactorily the remarkable drop in red cell hemato- crit which is present after 11 hours and persists many daj'S. Possibly the red cells used for infusion in this experiment had been seriouslj^ injured and went to pieces in the circulation. In confirmation of this suggestion we note the presence of hemolysis in blood samples taken on the first four days following the experiment. It may be suspected that an hemolysin was present in this dog's blood but there is reason- able doubt whether hemolj^sins actually do occur in the dog in sufficient amount to destro}' large numbers of homologous red cells. Experiment 103. (See table 3). 91 per cent exchange. Dog 18-66. Young female bull mongrel. Weight 17 pounds. 60 H. P. SMITH, A. E. BELT AND G. H. WHIPPLE November 16, 1919. Under ether anesthesia 700 cc. blood were withdrawn from the right femoral artery. Simultaneously 700 cc. of blood corpuscle sus- pension were injected into the right femoral vein. The duration of exchange was 9 minutes. Following the exchange the temperature showed little or no alteration from the original level. The pulse was regular but poor in tension for a number of hours. One-half cubic centimeter of adrenalin subcutaneously was given 4 hours after the operation. After 24 hours the animal was in excel- lent condition. In all these experiments the washing out of plasma proteins is accom- plished by a rapid exchange. The bleeding and simultaneous infusion of the red cell mixture occupies only a few minutes, the Imiits being TABLE 3 91 per cent blood volume exchange; dog 18-66; experiment 103 Before exchange.. Immediately after 15 minutes after.. 3 hours 11 hours 2d day 3d day 5th day 6th day 8th day 10th dav 12th day BLOOD 8ERUS PER HEADINGS IN CENT FIBRIN IN HEMATO- CRIT Total Albu- Globu- Non- PER CENT PER CENT protein min lin protein 6.2 4.5 1.7 2.0 0.42 49 3.2 2.2 1.0 1.5 49 3.8 2.9 0.9 1.6 0.21 56 4.3 3.0 1.3 1.7 0.58 50 4.0 2.9 1.1 2.3 0.47 33 4.3 2.8 1.5 2.6 0.48 35 4.8 3.7 1.1 2.5 0.47 29 4.5 3.1 1.4 3.0 0.41 27 5.3 3.9 1.4 2.3 5.4 3.8 1.6 2.9 0.45 32 5.7 4.1 1.6 2.0 0.42 32 5.4 3.9 1.5 2.1 0..5'3 32 Moderate shock Dog nomal. Hemolysis Hemolysis , Hemolysis 2 to 25 minutes. Within these Hmits the speed of exchange, whether 2 minutes or 25 minutes, seems to make little difference. To make this point clear we may contrast tables 4 and 5. The first of these two ex- periments done on the same animal (table 4) shows the reaction follow- ing an exchange of 100 per cent done in 14 minutes. There was definite shock but a rapid recovery. The second experiment done on this dog after an interval of 2 weeks to insure complete recover}^, shows the reaction following a very rapid 100 per cent exchange which was com- pleted within 2 minutes. There was if anything less shock on this occa- sion than after the first exchange. It is interesting to note how closely REGENEKATION OF PROTEINS OF BLOOD PLASMA 61 the curves of total protein, albumin and globulin in the two experi- ments coincide. The prompt rise in the 15-minute interval is identical and the initial fall corresponds to the other experiments discussed. Experiment 93. (See table 4). 99 per cent exchange. Dog 18-48. Young female bull dog. Weight 13.3 pounds. October 4, 1917. Under ether anesthesia 600 cc. blood were withdrawn from the right femoral artery. Simultaneously 700 cc. of blood corpuscle suspension TABLE 4 99 per cent blood volume exchange; dog 18-48; experiment 93 TIME BLOOD SEBUM PER READINGS IN CENT FIBRIN IN PER CENT HEMATO- CRIT RED CELL PER CENT REMARKS Total protein Albu- min Globu- lin Non- protein Before exchange Immediately after. . . 15 minutes after 9§ hours 4.5 2.0 3.3 3.9 4.0 4.4 3.2 1.3 2.6 3.0 3.1 3.3 1.3 0.7 0.7 0.9 0.9 1.1 2.2 1.6 1.5 1.8 1.9 1.7 0.26 0.16 0.15 0.22 0.18 0.30 0.39 58 58 58 46 54 42 Definite shock 2d day Normal 3d day 4th day TABLE 5 104 per cent blood volume exchange; dog 18-48; experiment 96 BLOOD SERUM READINGS IN FEB CENT TIME Total protein Albumin Globulin Non- protein Before exchange Immediately after 15 minutes after 10 hours 6.4 2.1 3.2 3.9 4.5 3.5 1.3 2.2 2.7 2.6 2.9 0.8 1.0 1.2 1.9 1.7 1.3 1.6 1.6 1.9 Slight shock 2d day Normal were injected into the right femoral vein. The duration of exchange was 14 minutes. Animal showed definite signs of intoxication after about an hour following the exchange, with some bloody feces after about 5^ hours. After 24 hours animal appeared to have recovered completely. Experiment 96. (See table 5). 104 per cent exchange. Dog 18-48. Young female bull dog. Weight 13.8 pounds. "Plasmapha- resis," 99 per cent exchange with a duration of 14 minutes, done on October 4 (exper. 93, table 4). Showed definite signs of intoxication. 62 H. P. SMITH, A. E. BELT AND G. H. WHIPPLE October 18, 1917. Under ether anesthesia 650 cc. blood were withdra^ATi from the left femoral artery. Simultaneously 650 cc. of blood corpuscle suspension were injected into the left femoral vein. The duration of exchange was 2 minutes. Immediately following the exchange there was a slight transient fall in tem- perature. There was no immediate alteration of the pulse; however after about an hour the pulse was of poor volume and the animal appeared decidedly dull. Bloody feces were noted at this time. After 24 hours the animal appeared to be in good condition. Table 6 gives valuable data concerning the speed of exchange in its relation to shock and the curve of protein regeneration. In this ex- periment the blood volume exchange of 75 per cent was completed in 2.5 minutes. There was no shock and we may compare the previous exchanges done on this same animal (Sept. 12, 1917, exper. 84, 80 per TABLE 6 75 per cent blood volume exchange; dog 18-35; experiment 94 Before exchange.. Immediately after 15 minutes after. . 3 J hours 6| hours 9J hours 2d day 3d day 4th day BLOOD SERUM PER READINGS IN CENT FIBRIN IN PER CENT HEMATO- CRIT RED CELL PER CENT Total Albu- Globu- Non- protein min lin protein 5.4 3.8 1.6 1.9 0.21 40 3.0 2.2 0.8 1.5 0.19 48 3.7 2.7 1.0 1.8 0.23 54 4.6 3.4 1.2 1.8 0.26 61 4.7 3.5 1.2 1.8 0.30 44 4.1 2.9 1.2 1.5 4.9 3.6 1.3 1.9 0.44 4.4 3.1 1.3 2.8 4.6 2.4 2.2 2.4 REMARKS No shock Normal cent exchange, shock very shght, time 7 minutes; October 3, 1917, exper. 92, 74 per cent exchange, no shock, time 14 minutes). The curve of serum protein depletion and regeneration is similar to other experiments. The small per cent exchange lowers the total protein to 3.0 per cent and the emergency increase is definite within 15 min- utes, giving a rise to 3.7 per cent. It appears that the emergency re- action by which a considerable amount of serum protein is thrown into the blood stream, can be called out by a large or small exchange using this method. Experiment 94. (See table 6). 75 per cent exchange. Dog 18-35. Young female bull dog. Weight 16 pounds. "Plasmapharesis," 80 per cent exchange, done in 7 minutes on September 12, 1917, showing very REGENERATION OF PROTEINS OF BLOOD PLASMA 63 slight shock (exper. 84) ; another 14 per cent exchange done in 14 minutes on October 3, 1917, showing no shock (exper. 92). Blood volume on October 9, 1917 (by dye method) was 671 cc. October 11, 1917. Under ether anesthesia 545 cc. blood were withdrawn from the right carotid artery. Simultaneously 545 cc. of blood corpuscle suspension were injected into the right external jugular vein. The duration of the exchange was 2§ minutes. Animal showed a transient drop in temperature of 3 degrees mmediately following the exchange. Animal showed little or no signs of ntoxication. Table 7 shows a remarkably prompt return to normal after a large exchange (109 per cent). The total proteins fell to a level of 50 per cent normal which is a normal reaction. We cannot explain the fig- ures, which appear to show a pecuhar reaction on the part of the albu- min and globulin fractions. These peculiar reactions will appear at TABLE 7 109 per cent blood volume exchange; dog 18-20; experiment 89 Before exchange. . . Immediately after. 15 minutes after. . . 4| hours 2d day BLOOD SERCM PER RE.\DINGS IN CEXT . FIBRIN IN PER CENT HEMATO- CRIT RED CELL PER CENT Total protein Albu- min Globu- lin Non- protein 5.0 2.5 3.0 4.2 5.1 3.6 0.9 1.5 2.2 2.9 1.4 1.6 1.5 2.0 2.2 1.9 1.4 1.6 1.7 2.1 0.35 0.12 0.11 0.13 50 60 73 73 41 REM.\RKS Very slight shock Normal times in spite of every care used in the method, but we are inclined to suspect technical errors as in part responsible. The return of the total protein to normal within 24 hours is unusual and would indicate an unusually large emergency reserve. The peculiar rise in red cell hematocrit will be found in this experiment and in a few subsequent experiments. That it appears in the 15-minute and 4-hour samples but not in the sample taken immediateh^ after the exchange is very perplexing. There is no severe shock to account for any withdrawal of fluid from the blood. We have no convincing explanation to offer. Experiment 89. (See table 7). 109 per cent exchange. Dog 18-20. Young female bull dog. Weight 16.1 pounds. September 20, 1917. Under ether anesthesia 800 cc. blood were withdrawn from the femoral artery. Simultaneously 800 cc. of blood corpuscle suspension were injected into the femoral vein. The duration of the exchange was 14§ 64 H. P. SMITH, A. E. BELT AND G. H. WHIPPLE minutes. Animal showed little or no depression. The temperature fell 3 de- grees, however, and the animal shivered considerably for 8 or 9 hours. In good condition after 24 hours. Table 8 shows another typical experiment giving the usual curve of blood proteins following plasmapharesis of moderate amount (90 per cent). The fibrin curve is given in this experiment and we believe this illustrates the usual reaction on the part of this plasma globulin. The method used gives certain opportunities of error when small amounts of plasma are analj^zed for fibrin. More work in this field has been completed by Mr. Foster in this laboratory and will soon be pubhshed. We do not wish to put too much emphasis on these figures. TABLE 8 90 per cent blood volume exchange; dog 18-68; experiment 105 Before exchange.. Immediately after 15 minutes after. . 2\ hours 5 hours 2d day 4th day 6th day BLOOD SERUM PER READINGS IN CENT FIBRIN IN PER CENT Total protein Albu- min Globu- lin Non- protein 5.5 4.0 1.5 2.2 0.42 2.8 2.1 0.7 1.7 0.22 3.5 2.8 0.7 1.8 0.27 4.6 3.3 1.3 1.8 0.30 0.19 4.8 3.8 1.0 2.0 0.56 4.9 3.4 1.5 1.3 0.42 5.4 3.7 1.7 2.1 0.49 Moderate shock Normal Experiment 105. (See table 8). 90 per cent exchange. Dog 18-68. Young female bull dog. Weight 15.3 pounds. November 21, 1917. Under ether anesthesia 623 cc. of blood were withdrawn from the right femoral artery. Simultaneously 623 cc. of blood corpuscle sus- pension were injected into the right femoral vein. The duration of the exchange was 9 minutes. The animal showed moderate depression and slight decrease in pulse volume for several hours. Tables 9 and 10 give figures to show the low level of serum pro- teins which may be effected by very large blood volume exchanges (159 and 195 per cent). The usual normal dog will not tolerate such large exchanges without exhibiting profound and often fatal shock. These two dogs were unusually resistant to this experimental procedure and give us the opportunity to study the reaction following such large REGENERATION OF PROTEINS OF BLOOD PLASMA 65 exchanges uncomplicated by shock or notable hemolysis. The low level of total proteins is to be expected and one experiment (table 10) reaches the minimum figure for total protein (0.9 per cent). We have no observation in any of our experiments to show a lower level of pro- tein in the blood stream. The protein regeneration is very rapid in the 15-minute period as well as in the following 24 hours, indicating con- siderable emergency reserve material. TABLE 9 159 per cent blood volume exchange; dog 17-215; experiment 67 BLOOD ( 3ERUM READINGS IN PER CENT Total protein Albumin Globulin Non- protein Before exchange Immediately after 15 minutes after 2d day 6.2 1.3 2.2 4.1 4.3 5.2 3.6 0.3 1.2 2.7 2.1 2.9 2.6 1.0 1.0 1.4 2.2 2.3 1.7 1.6 1.5 1.4 2.0 1.7 Slight hemolysis Slight hemolysis. Slight shock Normal 3d day 4th day TABLE 10 195 per cent blood volume exchange; dog 17-232; experiment 70 Before exchange.. Immediately after 15 minutes after.. 2d day 3d day BLOOD SERUM READINGS IN PER CENT Total protein Albumin Globulin Non- protein 6.3 0.9 1.4 4.3 5.5 3.7 0.3 0.7 2.3 3.8 1.6 0.6 0.7 2.0 1.7 1.9 1.5 1.7 1.5 1.9 Very slight shock Normal Experiment 67. (See table 9). 159 per cent exchange. Dog 17-215. Adult female fox terrier. Weight 15.25 pounds. Blood volume on July 19, 1917 (by dye method) was 858 co. July 31, 1917. Under ether anesthesia 1105 cc. of blood were withdrawn from the right femoral artery. Simultaneously 1105 cc. of blood corpuscle sus- pension were injected into the right femoral vein. The duration of the exchange was 10 minutes. There was slight decrease in force of pulse beat for about an hour. Animal showed little sign of depression thereafter. Experiment 70. (See table 10). 195 per cent exchange. Dog 17-232. Young female coach dog. Weight 13.5 pounds. Blood volume on July 19, 1917 (by dye method) was 714 cc. THE AMERICAN JOURN.\L OF PHYSIOLOGY, VOL. 52, NO. 1 66 H, P. SMITH, A. E. BELT AND G. H. WHIPPLE August 3, 1917. Under ether anesthesia 1200 cc. blood were withdrawn from the right femoral artery. Simultaneously 1200 cc. of blood corpuscle suspen- sion were injected into the right femoral vein. The duration of the exchange was 10 minutes. The pulse pressure was poor for about an hour following the exchange. TABLE 11 o < n cs a n s a S •A TOTAL PROTEIN o K P< b O » <: PER CENT PROTEIN REGAINED O U GO HEil.^^TOCRIT BLOOD (per CENT cells) o X < H z a » m c 0! 1 "o S to « a) U5 >> o" 1 3 C "i s o A C es Ji X £ S, o pa (V <; a) si ■si >> •s •a o § GO 67 60 5.3 2.8 3.9 2.5 1.1 74 92 5.5 3.7 3.8 1.8 0.1 50 58 58 74 87 6.2 3.3 3.9 2.9 0.6 +++ 58 58 55 75 94 5.4 3.0 3.7 4.9 2.4 0.7 1.9 40 54 54 75 90 5.3 2.9 3.4 4.3 2.4 0.5 1.4 ++ 48 54 .55 38 80 84 5.6 3.1 3.1 6.6 2.5 0.0 3.5 + 50 63 58 33 80 82 5.6 2.4 4.4 3.2 2.0 50 44 84 74 4.9 2.6 2.3 * 89 101 5.5 2.1 2.4 3.4 0.2 +++ 60 41 53 90 105 5.5 2.8 3.5 4.8 2.7 0.7 2.0 ++ 43 49 52 91 103 6.2 3.2 3.8 4.3 3.0 0.6 1.1 ++ 49 49 56 35 94 62 4.9 1.8 2.8 4.8 3.1 1.0 3.0 ++ 96 61 5.5 2.7 3.6 2.8 0.9 98 64 6.3 3.0 2.8 3.3 -0.2 + 99 81 4.5 1.2 2.5 4.1 3.3 1.3 2.9 ++ 99 93 4.5 2.0 3.3 4.0 2.5 1.3 2.0 + 58 58 58 42 100 100 5.6 3.0 2.9 4.6 2.6 -0.1 1.6 + 46 62 64 36 Averages 5.4 2.7 3.3 4.8 2.7 0.6 2.1 50 55 56 38 Shock readings: + means slight shock; ++ means moderate to severe shock; 4- + + means lethal shock. * Death from overdose of ether. A summary of certain factors in many plasma depletion experiments will be found in tables 11 and 12 and the average figures give much in- teresting information. The averages of the experiments which show 100 per cent or less of blood volume exchange (table 11) show an iden- tical emergency increase in the blood serum proteins. The two tables are practically in accord and we note that the average replacement of serum protein during the 15 minutes following the plasmapharesis REGENERATION OF PROTEINS OF BLOOD PLASMA 67 amounts to 0.5 to 0.7 per cent protein — which is an increase of 10 to 14 per cent of the total proteins. The increase during the 24 hours fol- lowing the plasma depletion is considerable and amounts to 2.0 per cent protein which is an increase of 40 per cent total protein, figuring 5.0 protein per cent as the normal for a healthy dog. Further analysis of the blood cell hematocrit figures is of interest. It is unfortunate that we did not obtain hematocrit readings in all our experiments. It is clear that the normal hematocrit before the ex- TABLE 12 z HEMATOCRIT (PER ;ent E a TOTAL PEOTEI^ PROTEIN REGAINED BLOOD cells) & (, ■ a , I, H z So a ^ dJ to C S m H d 03 m % m z o X £ o o C C .J3 II a T3 ■a . a o O OS a < 3 C s 3 O m < o o W Si n as T3 T3 C O 0. K H i .2 « 6 "o o T3 M a < o 1 13 S U5 -0 c o 1 3 a E to a 3 O a o c c3 -a i v2 > a o 73 74 0.21 0.16 0.15 0.28 0.05 -0.01 0.12 50 58 58 74 87 0.18 0.08 0.15 0.10 0.07 + + + 58 58 55 75 94 0.21 0.19 0.23 0.44 0.02 0.03 0.25 40 48 54 75 90 0.25 0.15 0.20 0.35 0.10 0.05 0.20 ++ 48 54 55 38 80 84 0.17 0.06 0.03 0.40 0.11 -0.03 0.34 + 50 63 58 33 80 82 0.17 0.08 0.13 0.33 0.09 0.05 0.25 50 44 84 74 0.36 0.15 0.21 * 89 101 0.24 0.11 0.11 0.13 0.00 +++ 60 41 53 90 105 0.42 0.22 0.27 0.56 0.20 0.05 0.34 ++ 43 49 52 91 103 0.42 0.19 0.21 0.48 0.23 0.02 0.29 ++ 49 49 56 35 99 93 0.26 0.16 0.15 0.30 0.10 -0.01 0.14 + 58 58 58 42 99 81 0.32 0.19 ++ 100 100 0.25 0.16 0.16 0.45 0.09 0.00 0.29 + 46 62 64 36 102 80 0.42 0.26 +++ 104 96 0.50 0.13 0.14 0.40 0.37 0.01 0.27 + 52 66 38 108 98 0.25 0.12 0.11 0.13 -0.01 +++ 33 44 53 109 89 0.35 0.12 0.11 0.23 -0.01 + 50 60 73 41 110 83 0.75 0.18 0.30 0.44 0.57 0.12 0.26 + 46 122 104 0.50 0.13 0.14 0.40 0.37 0.01 0.27 +++ 52 66 38 191 77 0.44 0.13 0.10 0.31 -0.03 +++ Ave r- a ges.. 0.33 0.14 0.17 0.40 0.19 0.02 0.25 49 55 57 39 Shock readings: + means slight shock; ++ means moderate to severe shock; + + + means lethal shock. * Death from overdose of ether. been submitted to considerable manipulation in the necessary washing previous to the injection. Dogs' corpuscles too are notoriously fragile. We may assume for the present at any rate that many of the red cells which were introduced had been seriously injured and went to REGENERATION OF PROTEINS OF BLOOD PLASMA 69 pieces in the blood stream during the 24 hours following the blood exchange. We can review the fibrin analyses in table 13 and at once a decided difference appears when we compare the serum protein curve with that of the plasma globulin, fibrinogen. The exchange of blood re- duces the fibrin content to about the same level — that is, we can wash out the same percentage of fibrinogen by the usual plasmapharesis as we do in the case of the serum proteins. The fibrin content is reduced to a little less than one-half normal — from 0.33 to 0.14. During the 15 minutes following the plasma depletion there is no emergency reac- tion on the part of the fibrin as is so constant for the serum proteins. During the next 24 hours the fibrin is restored completely to normal. This may mean that there is no emergency reserve of the fibrin as it can be produced so rapidly in the body in any emergency. We know of many other facts which point to com-plete dissociation of fibrin and other blood proteins as to production and repair and general usefulness in the body economy. DISCUSSION A theoretical consideration of the factors involved in this protein replacement is difficult at this time. It may be claimed that this in- crease represents, in part at least, not a true increase in the quantity of circulating serum protein, but is the result of the escape from the cir- culation of fluid poor in protein material. However, if any consider- able escape of fluid from the circulation were to occur, one would expect to note a rise in the percentage of red cells in the circulating medium. That no sufficient change in the cell-plasma ratio does occur can be seen from the hematocrit figures given in tables 11 and 12. On the other hand, it may be that the figures represent an actual influx of pro- tein into the circulating medium. Such an influx could conceivably come from some tissue or organ which serves as a storehouse for this type of protein material. Seitz (7) thinks that the liver acts as such a storehouse. Earlier work in this laboratory by Kerr, Hurwitz and Whipple (1) shows a lack of reserve production of serum proteins after plasmapharesis in the Eck fistula dog. This indicates that liver insufficiency may impair the normal emergency reproduction of blood proteins. Of particular interest is the rather remarkable increase in the 15 minutes immediately following the end of the experimental depletion. 70 H. p. SMITH, A. E. BELT AND G. H. WHIPPLE This, if blood volume changes be excluded, appears to be truly a throwing in of readj'-formed materials. AATiile the depletion curve of the fibrin fraction of the plasma pro- teins brought about by our experimental procedure compares closely with the curve of depletion of the serum proteins, still a distinctly dif- ferent type of curve of fibrin repletion is revealed. A fairly typical expermient is presented in table 8. This point may also be studied by an examination of the results given in table 13. In these tables it may be seen that the rapid rise immediately following the procedure of depletion which is typical of the serum proteins is absent or at least negligible in the case of fibrin. However, the body seems to be able to supply large amounts of this protein in a space of 24 hours, for as the summary in table 13 shows, the fibrin on the day following the ex- change is already as high as the original figure, or, as occurs in some cases, even higher^ When such an over-production does occur the level usually returns to normal in one to two days. This lack of correspondence between the regeneration figures for serum proteins and for fibrin protein in the period of initial regenera- tion, we believe furnishes additional evidence against a theory which would account for all changes in protein concentration in this period by the loss from the circulation of fluids poor in protein. For, in such a case, the concentration of the proteins might be expected to occur to practically the same degree in each. That this does not occur tends to strengthen the evidence given by the hematocrit figures. It may be pointed out that the curve of serum protein regeneration is very different for this type of experiment when compared to the experiments of Kerr, Hm'witz and Whipple. We beheve that these differences are to be explained wholly by the differences in the experi- mental depletion of the serum protein. Kerr, Hurwitz and Whipple used interval depletions of smaller amounts but repeated many times during a single day. In this manner they undoubtedly removed much of the large emergency reserve which is so conspicuous in the 24-hour regeneration in the experiments tabulated above. There- fore Kerr, Hurwitz and Whipple observed a curve of protein regen- eration which was much more prolonged before a return to normal was observed. These experiments supplement the earlier ones and strengthen their conclusions. REGENERATION OF PROTEINS OF BLOOD PLASMA 71 SUMMARY A rapid depletion of serum proteins is brought about in these experi- ments by the introduction of normal red blood cells suspended in a modified Locke's solution, care being taken to keep equivalent the volume of blood removed from the artery and the volume of red blood cell suspension simultaneously injected into the vein. The serum protein depletion is roughly proportional to the size of this exchange and it is noteworthy that the rapid depletion of the total serum proteins can rarely be carried below 1 .0 per cent without causing a fatal reaction. An increase in serum protein concentration (serum protein replace- ment) begins immediately following the exchange or plasmapharesis. The increase is very rapid during the first 15 minutes following the ex- change. The increase in serum proteins is more gradual thereafter during the first 24 hours and still more sluggish during the next few days. The normal level may be reached in 2 to 7 days. The rapid replacement of serum proteins during the first 15 minutes following the exchange indicates some reserve supply of this material perhaps held in the body cells. The emergency supply is evidently small and the production of other similar material is difficult and requires time. The blood fibrin reacts in a different fashion. The same initial fall is not followed by a rapid rise in the first 15 minutes. The recov- ery however is complete within 24 hours and probably earlier than this. Fibrin is a very labile protein as compared with the serum albumin and globuhn. Blood volume fluctuations are probably very httle concerned in these experimental results. The red blood cell hematocrit ratio shows but little change during the period of initial reaction. BIBLIOGRAPHY (1) Kerr, Hurwitz and Whipple: This Journal, 1918, xlvii, 356, 370, 379. (2) Abel, Rowntree and Turner: Journ. Pharm. Exper. Therap., 1914, v, 625. (3) MoRAWiTz: Beitr. z. chem. Physiol, u. Pathol., 1906. vii, 153. (4) Hooper, Smith, Belt and Whipple: This Journal, 1920, li, 205. (5) Robertson: Journ. Biol. Chem., 1915, xxii, 233. (6) CuLLEN AND Van Sltke : Proc. Soc. Exper. Biol, and Med., 1916, xiii, 197 (7) Seitz: Arch, gesammt. Physiol., 1906, cxi, 309. II. SHOCK AS A IMANIFESTATIOX OF TISSUE INJURY FOLLOWING RAPID PLASMA PROTEIN DEPLETION The Stabilizing Value of Plasma Proteins G. H. WHIPPLE, H. P. SMITH and A. E. BELT From the George Williams Hooper Foundation for Medical Research, University of California Medical School, San Francisco Received for publication Februarj^ 25, 1920 In the preceding communication we have estabUshed the curve of serum protein regeneration following a single rapid replacement of whole blood with a red cell Locke's solution mixture. This plasma depletion (plasmapharesis) washes out more or less of the blood pro- teins and lowers the concentration of the blood proteins in the circu- lating blood. In the preceding article we have submitted manj^ ex- periments which are associated with little or no "shock," using this term in the familiar chnical sense. In this paper we wish to discuss more particularly those cases which are associated with severe or lethal shock. A number of such experiments are given in detail below. This intoxication associated with plasmapharesis has been noted by the earlier workers: Morawitz (1), Abel, Rowntree and Turner (2) and Kerr, Hurwitz and Whipple (3). A varietj^ of explanations has been given. The 'physiological value of the serum proteins is admittedly httle understood and we believe our experiments throw some light on this point. Published work from this laboratory (3) indicates that the serum proteins cannot be concerned with the nutrition of the body cells and the constant exchange between food protein and bodj^ pro- tein. The experiments outlined below suggest rather strongly that one important function of these proteins is their "stabilizing value." The stahilizing value of the blood serum proteins is brought out with especial emphasis by two experiments (tables 19 and 20). The dog is bled large amounts from the femoral artery while simultane- ously equal amounts of a washed red cell, dialyzed serum mixture are injected into the femoral vein. No shock followed an exchange of 72 RELATION OF BLOOD PROTEINS TO SHOCK 73 large size which would surely have been fatal if the dialyzed serum had been replaced by Locke's solution as in the standard plasma- pharesis. During the dialj^sis of the serum it underwent consider- able dilution while the dialyzable substances were being removed, but this dilute dialyzed serum was still able to protect the body cells against the shock which develops if the blood proteins are too much diluted as in the routine plasma depletion. We believe that this furnishes the last bit of evidence to show that the blood serum pro- teins make up an essential part of the environmental complex of the body cells. Too great a dilution of these substances invariably results in profound injury of certain cells and a reaction identical with "clinical shock." When these protein substances are suddenly washed out of the blood serum there is a certain amount of similar material thrown in as an emergency reserve. If the depletion is too severe the body cells are injured bj?" the very persistence of this abnormal condition and the condition of "shock" supervenes. Further it is evident that certain body cells are more sensitive than others to changes in the serum protein content — for example, liver cells. That these facts have some significance in relation to the general problem of clinical shock is at once evident. Other experiments (3) already cited give proof that the simple plasma depletion with more or less clinical shock is associated with a certain amount of cell injury, as shown by the rise in urinary nitrogen in the two days following the exchange. There is neither gross nor histological evidence of cell necrosis, but this increase in nitrogen must come from body protein. This is further evidence for actual cell injury as an essential part of the clinical complex nayned "shock." It may be noted also that when once the clinical picture of "shock" is established in these experiments we have been unable to save the animal by any of the familiar clinical measures, even by infusion of whole blood. The essential injury in these experiments is cell proto- plasm injury induced by a sudden change in the colloidal solution which forms the normal environment of these cells. This may be a new type of cell injury but it may help us to understand the more complex cell injury which is probably responsible for "surgical shock." 74 G. H. WHIPPLE, H. P. SMITH AND A. E. BELT EXPERIMENTAL OBSERVATIONS The various experimental methods have been described in detail in the preceding communication. To save repetition we may refer to some of the experiments detailed in the first paper of this series. The experiments given below are only types which illustrate a characteristic reaction and usually represent groups of similar experiments. The first two tabulated experiments (tables 14 and 15) illustrate the reaction which was so common in the experiments of paper I of this series. In addition these two experiments done on the same dog at an interval of three weeks show that this procedure (plasmapha- resis) does not sensitize a dog to any subsequent repetition of this procedure. This shock so exactly resembles the anaphylactic shock in dogs that it seemed necessary to exclude this possibility. Other experiments giving the same negative results need not be instanced. TABLE 14 80 per cent blood volume exchange; very slight shock; dog 18-35; experiment -84 Before exchange. . Immediately after 15 minutes after. . 4 hours 8 hours 2nd day BLOOD SERUM READINGS IN PER CENT FIBRIN IN PER CENT Total protein Albumin Globulin Non- protein 5.6 3.1 3.1 4.1 6.1 6.6 4.6 2.5 2.9 3.3 4.9 5.5 1.0 0.6 0.2 0.8 1.2 1.1 2.4 2.8 2.6 2.5 2.5 2.7 0.17 0.06 0.03 0.06 0.16 0.40 HEMATO- CRIT RED CELL PER CENT 50 63 58 58 45 33 The curve of protein regeneration during the eight hours following this plasma depletion is beautifully shown in both experiments. The emergency reserve was sufficient to replace all the serum proteins removed (table 14) but it is noted that the total drop in serum protein was but 2.5 per cent total protein. Experiment 84. (See table 14). 80 per cent exchange. Dog 18-85. Female bull pup. Weight 15 pounds. Appears to be in excellent condition. September 12. Under ether anesthesia 545 cc. of blood were withdrawn from the right femoral artery. Simultaneously and at the same rate 545 cc. of Locke's corpuscle suspension were injected into the right femoral vein. The exchange was effected in 7 minutes. There was a fall in rectal temperature of about 1°C. following the exchange. No definite sign of intoxication was noted except for a slight amount of vomiting 2 hours following the exchange. The animal appeared to be in good condition on the 2nd day. RELATION OF BLOOD PROTEINS TO SHOCK 75 Experiment 92. (See table 15). 74 per cent exchange. Dog 18-35. Female bull pup. Weight 16.25 pounds. On September 12 an 80 per cent exchange was effected in 7 minutes without any decided signs of in- toxication (see table 14). October 3. Dog seems to be in excellent condition. Under ether anesthesia 545 cc. of blood were withdrawn from the left femoral artery. Simultaneously and at the same rate 545 cc. of Locke's corpuscle suspension were injected into the left femoral vein. The exchange was effected in 14 minutes. There was practically no alteration in rectal temperature and at no time were there any signs of intoxication. Experiment 61. No clinical shock. 96 per cent exchange. Dog 18-7. Female terrier pup. Weight 11 pounds. Estimated blood volume (by dye method) 530 cc. July 19. Under ether anesthesia 480 cc. of blood were withdrawn from the right femoral artery. Simultaneously and at the same rate 480 cc. of Locke-corpuscle suspension were injected into the right femoral vein. The duration of the ex- change was 12 minutes. There was practically no disturbance in rectal temper- TABLE 15 74 per cent blood volume exchange; no clinical shock; dog 18-35; experiment 92 BLOOD SERUM HEADINGS IN PER CENT FIBRIN IN PER CENT HEMATO- CRIT RED TIME Total protein Albumin Globulin Non- protein CELL PER CENT Before exchange Immediately after 15 minutes after 2| hours 6j hours 5.5 3.7 3.8 4.3 4.6 4.0 2.7 2.9 3.4 3.5 1.5 1.0 0.9 0.9 1.1 1.8 1.7 1.8 1.8 1.8 0.21 0.16 0.15 0.18 0.21 0.28 50 58 58 48 48 2nd day ature. There was a slight amount of drowsiness for several hours. Otherndse no disturbance was noted. The total serum proteins fell from the initial value of 5.5 per cent at the beginning of the exchange to a level of 2.7 per cent at the end of the exchange. Fifteen minutes later a value 3.6 per cent was found. No sam- ples were taken subsequently. At no time was there any decided alteration in the albumin-globulin ratio. The next group of experiments illustrates the fatal shock which may develop following an exchange of blood equal to 100 per cent blood volume or more. From these and other experiments it is ob\dous that the body can supply an emergency reserve of serum proteins even during the period of profound shock which precedes death (2 to 5 hours). Moreover the ratio of albumin and globulin is not especially disturbed as is so frequently seen in severe intoxication due to bac- terial invasion. 76 G. H. WHIPPLE, H, P. SMITH AND A. E. BELT The clinical and anatomical pictures described in this condition of shock following plasma depletion are veiy constant and resemble in the dog the reaction observed in fatal anaphylaxis. The fall in blood pressure may be delayed several minutes — sometimes 30 minutes after completion of the exchange — but the fall in temperature is prompt. At times there may be a subsequent rise in temperature even in fatal intoxication, but often the loss of temperature control is complete and rectal temperatures of 30°C. may be recorded. Gastro-intestinal dis- turbance is the rule. Vomiting and diarrhea are seen early, sometimes within 30 minutes, and persist. This watery, blood-tinged diarrhea is common in fatal cases. Mucus may be very abundant in certain cases, even occasionally when recovery takes place following a severe intoxi- cation. The dull lethargic appearance with clinical prostration is very typical of this type of shock. This picture corresponds closely with the surgical condition of "shock" associated with intoxication (for example, intestinal obstruction) or hemorrhages or prolonged op- erative manipulation. The autopsy findings also are very uniform. For these the descrip- tion of a single case will suffice. Blood removed from the heart at autopsy or from the veins at intervals before death may show de- layed coagulation but this is not uniform. The fibrin content is low because this plasma protein like the serum proteins has been washed out by the exchange. The liver, spleen and kidneys show engorge- ment, usually most marked in spleen and liver. The thorax, heart and lungs are negative. The stomach may be pale or slightly injected. The entire small intestine shows congestion of its mucosa often more marked in the upper tract. The mucosa may be velvety, purpUsh red and coated with thick creamy mucous. All degrees of congestion are found. The lumen contains a thin, watery, blood-tinged fluid in which more or less mucus is present. The colon shows the same material and a mottled congested mucosa. It will be noted that this picture of shock is almost identical with that produced by large' doses of adrenalin, clamping of aorta or vena cava and trauma of the intestines, recentlj^ studied and described by Erlanger (5). Experiment 98. (See table 16). 108 per cent exchange. Dog 18-20. Female bull-terrier pup. Weight 19.4 pounds. On September 20 an exchange of 109 per cent in 14J minutes produced moderately severe shock. October 31. Under ether anesthesia 950 cc. of blood were withdrawn from the left femoral artery. Simultaneously and at about the same rate 1000 cc. of Locke's corpuscle suspension were injected into the left femoral vein. The duration of RELATION OF BLOOD PROTEIXS TO SHOCK 77 the exchange was 12^ minutes. The rectal temperature fell about 1°C. during the exchange. Subsequently there was a fall of 1° more, when death occurred. The arterial tension was fairly good at the end of the exchange but became quite poor in the course of the next 15 minutes. It remained poor until death. Deep respiration developed in the course of the first hour following the exchange. No marked signs of depression or loss of power of attention appeared for about 2 hours after the exchange. The condition then became rapidly worse and death occurred 1 hour later. Autopsy shows swollen congested spleen. The liver is deep red, the lobulation is obscure. The mucosa of the entire intestinal tract is congested. There is a considerable excess of mucus. The other organs are negative. Blood drawn from the heart at time of autopsy when placed in a test tube clots in 25 minutes; that which is left in contact with the tissues clots in 10 minutes. The clot formed is quite flabby. Experiment 101. (See table 17). 89 per cent exchange. Dog 18-5. Young male terrier. Weight 18.5 pounds. On July 18 an exchange of 67 per cent produced a very mild grade of shock. TABLE 16 108 per cent blood volume exchange; fatal shock; dog 18-20; experiment 98 Before exchange . . Immediately after 15 minutes after. . 2 hours BLOOD SERUM READINGS IN PER CENT FIBRIN IN HEMATO- CRIT RED Total Al- Globu- Non- PER CENT protein bumin lin protein 6.3 4.4 1.9 2.1 0.25 33 2.2 1.3 0.9 1.4 0.12 44 2.7 1.7 1.0 1.6 0.11 53 3.6 2.5 1.1 2.1 0.12 42 Death November 8. Under ether anesthesia 750 cc. of blood were withdrawn from the left femoral artery. Simultaneously 750 cc. of Locke's corpuscle suspension were injected into the left femoral vein. The duration of the exchange was 11 minutes. The rectal temperature fell 2°C. as a result of the exchange but returned subse- quently to the original level of slightly above 40°. The arterial tension was good at the end of the exchange but became poor within the course of 30 minutes. Def- inite signs of general depression or "shock" appeared within an hour following the end of the exchange. The power of attention was completely lost 2 hours later. Death occurred 5 hours after the exchange. Autopsy: The thymus is somewhat larger than normal. Otherwise the thor- acic organs are negative. The spleen is moderately enlarged and congested. The liver is negative except for pronounced indistinctness of lobulation. The mucosa of the duodenum is slighth' reddened, but no excess of mucus is found in the lumen. Experiment 76. Fatal shock. 178 per cent exchange. Dog 18-7. Female terrier pup. Weight 10.5 pounds. A 96 per cent exchange was carried out on July 19 (see exper. 61) with practi- cally no sign of shock. 78 G. H, WHIPPLE, H. P. SMITH AXD A. E. BELT On July 26 an exchange of 118 per cent was effected with very slight reaction. Immediately following this second exchange an injection of phosphorus was given. Xo definite injury was noted (see exper. 65, table 24). August 16. Animal appeared to be in excellent condition. Under ether anes- thesia 850 cc. of blood were withdrawn from the right carotid artery. Simul- taneously and at about the same rate 900 cc. of Locke's corpuscle suspension were injected into the right external jugular vein. The duration of the exchange was 7 minutes. Within a few minutes definite signs of shock appeared. The pulse rapidly diminished in volume, the respiration became irregular and the rectal temperature fell steadily from the original of 38.5°C. to 36.1° at the time of death, l\ hours following the exchange. Autopsy: The thoracic organs are negative. The spleen, liver and kidneys show moderate congestion. The upper part of the small intestines shows marked thickening and congestion of the mucosa. Thin bloody fluid is present in consid- erable quantities within the lumen of the intestines. The mucosa of the large intestine is slightly congested. The pancreas is decidedly swollen by interlob- ular edema. There is a considerable amount of hemolysis. TABLE 17 89 per cent blood volume exchange; fatal shock; Dog 18-5; experiment 101 Before exchange. . Immediately after 15 minutes after . . 3 hours 5 hours BLOOD SERUM READINGS IN PER CENT FIBRIN IN HEMATO- CRIT RED Total Al- Globu- Non- PER CENT protein bumin lin protein 5.5 4.4 1.1 2.2 0.24 60 2.1 1.7 0.4 1.7 0.11 41 2.4 1.8 0.6 2.2 0.11 0.13 53 54 3.6 2.6 1.0 2.2 0.21 56 Death The refractometric estimation of serum proteins was not carried out. Nitro- gen estimation by the Kjeldahl method showed that the total plasma proteins decreased as a result of the exchange from 4.9 per cent to 2.0 per cent. The fibrin content of the plasma fell from a level of 0.44 per cent to a level of 0.19 per cent as a result of the exchange. The value at the end of 15 minutes was 0.15 per cent, and 0.24 per cent at autopsy. From a perusal of many experiments in this paper it is evident that there are wide individual variations in the susceptibility of different dogs to the plasma depletion. But each individual dog will usually react with considerable uniformity to a repeated plasmapharesis of unit volume if sufficient time is allowed between experiments for com- plete recovery. This is noted in tables 14 and 15 which give data from two experifnents performed on the same animal at 3 weeks interval. If the second or succeeding exchanges are larger in amount RELATION OF BLOOD PROTEINS TO SHOCK 79 we may expect to record increasing degrees of intoxication and finally severe or fatal shock. This fact is illustrated by the preceding experi- ment (no. 76) in which two previous plasma depletions had no ill effects. The first one was an exchange of only 96 per cent with no signs of intoxication. The second exchange was slightly larger (118 per cent) and caused a shght intoxication. The final exchange of 178 per cent caused a prompt and fatal intoxication with the char- acteristic post-mortem findings described in fatal shock. The substitution of serum for Locke's solution in plasma depletion (fibrinpharesis) Experiment 323. (See table 18). 144 per cent exchange. Dog 19-74. Adult female mongrel terrier. Weight 16 pounds. August 2. Under ether anesthesia 1050 cc. of blood were withdrawn from the right femoral artery. Simultaneously and at the same rate 1100 cc. of a serum corpuscle mixture were injected into the right femoral vein. This serum corpus- cle mixture consisted of 550 cc. of packed dog corpuscles washed twice with sterile TABLE 18 i44 V^''' <^^^^ blood volume exchange; substitution of serum for Locke's solution in plasma depletion; no clinical shock ; dog 19-74; experiment 323 TIME TOTAL SERUM PBOTEINS PER CENT HEMATOCRIT RED CELL PER CENT FIBRIN IN PER CENT Before exchange 6.3 5.4 5.8 5.3 58 49 54 50 45 0.60 Immediatelj' after 0.08 15 minutes after 3 hours ... 0.30 24 hours 0.50 calcium-free Locke's solution in the customary way, to which was added an equal amount of serum. The serum for this purpose was obtained by drawing into large centrifuge tubes blood from normal dogs. After the process of clotting was completed the tubes were centrifugalized and the supernatant serum with- drawn. The duration of the exchange was 6 minutes. Ether anesthesia lasted 1 hour. The rectal temperature fell 2J°C. as a result of the procedure but re- turned to the original level within the space of about 2 hours. The arterial ten- sion was good at the end of the exchange. There was at no time any definite im- pairment in the quality of the pulse. The animal showed no signs of shock. August 3. Dog in excellent condition. The substitution of serum for Locke's solution in the plasma depletion (fibrinpharesis) Experiment 324. 176 per cent exchange. Female bull-terrier pup (3 months old). Weight 15 pounds. Aug^ist 5. Under ether anesthesia 1200 cc. of blood were withdrawn from the right femoral artery. Simultaneously and at the same rate 1275 cc. of serum corpuscle suspension made up as described in experiment 323 were injected into the right femoral vein. The duration of the exchange was 15 minutes. Ether 80 G. H. WHIPPLE, H. P. SMITH AND A. E. BELT anesthesia lasted 40 minutes. The rectal temperature was depressed about 3°C. for a periof of about 2 hours. The animal remained quiet for a period of 1 hour following the exchange, the power of attention being, however, good at all times. At the end of this time the animal was in excellent condition. The hematocrit values fluctuated but slightly as a result of the experimental exchange. The fibrin content of the plasma fell from its normal level of 0.43 to 0.30 per cent 3 hours after the exchange. The reading after 24 hours was 0.65 per cent. The two preceding experiments (table 18, expers. 323 and 324) bring out several important facts. The experimental manipulation of the red cells and the actual exchange of one mass of red cells for another are not responsible for the intoxication. In these two experi- ments we employed washed red cells from normal dogs prepared exactly as described for other experiments. These cells were sus- pended not in Locke's solution but in the proper amount of fresh nor- mal dog serum. These large exchanges then did not wash out any serum proteins but did remove much of the fibrin. These experiments serve as good controls of the operative procedures. These large ex- changes gave no evidence of any resultant intoxication. The last one especially (exper. 324) was a particularly large exchange (176 per cent) and done upon a young dog. Our experience shows that young animals as compared with adults are more sensitive to the shock of plasma depletion. One hundred and fifty per cent exchange using washed corpuscles suspended in dia- lyzed seruju Experiment 327. (See table 19). One thousand cubic centimeters of blood were drawn from normal dogs, poured immediately into large centrifuge tubes and allowed to clot. The clot formed in each tube was freed from the side of the tube and the tube centrifugalized. The supernatant serum was removed. Three hundred cubic centimeters of this serum were then placed in 15 celloidin sacs which were then immersed in 5,000 cc. of Locke's solution containing no calcium or glucose and made about 10 per cent more concentrated than normal in order that the increased osmotic pressure might in part overcome the tendency of the serum proteins to dilute themselves by attraction of water from the surrounding fluid. After dialysis had proceeded for 4 hours the modified Locke's solution was replaced by 10,000 cc. more of fresh solution of the same constitution. Dialysis was then continued for 11 hours, at the end of which time the serum contained in the celloidin sacs had increased from 300 cc. to 450 cc. To 400 cc. of the dialyzed serum 600 cc. of dog corpuscles twice washed with calcium-free Locke's solution in the ordinary way were added. The mixture was strained and heated to 38°C. Under ether anesthesia the entire 1000 cc. of the serum corpuscle mixture were injected into the right femoral vein of a normal short-haired bull pup weighing RELATION OF BLOOD PROTEINS TO SHOCK 81 12.5 pounds. Simultaneously and with moderate fluctuations in the rate of flow, 850 cc. of blood were withdrawn from the right femoral artery. Thirty-five minutes were consumed in effecting the exchange. The animal showed but little alteration in body temperature as a result of the exchange. Consciousness re- turned shortly after the discontinuance of the anesthetic. The animal was some- what quiet for a period of about 45 minutes. Subsequently he was bright and apparently in very good condition. One hundred and ninety-nine per cent exchange using washed corpuscles suspended in dialyzed serum Experiment 329. (See table 20). Nine hundred cubic centimeters of blood were drawn from normal dogs, poured immediately into large centrifuge tubes and allowed to clot. The clot formed in each tube was freed from the side of the tube and .the tube centrifugalized. The supernatant serum was removed. Three hundred and fifty cubic centimeters of this serum were then placed in 18 cel- TABLE 19 150 per cent blood volume exchange using washed corpuscles suspended in dialyzed serum; experiment 327 SAMPLE BLOOD SERUM HEADINGS IN PER CENT Total protein Albumin Globulin Non-protein Of serum of perfusate : Before dialysis After dialysis 6.2 3.3 5.7 3.8 4.5 3.8 2.5 3.0 2.1 2.3 2.4 0.8 2.7 1.7 2.2 1.7 1.2 Of dog perfused: Before exchange Immediately after 4 hours after 1.9 1.9 2.0 loidin sacs which were then immersed in 4000 cc. of Locke's solution containing no calcium or glucose, and made about 10 per cent more concentrated than nor- mal. After dialysis had proceeded for 5 hours the modified Locke's solution was replaced by 9000 cc. of fresh modified Locke's solution. Dialysis was then con- tinued for 10 hours, at the end of which time the serum contained within the celloidin sacs had increased from 350 cc. to 450 cc. To 400 cc. of the dialyzed serum 600 cc. of dog corpuscles twice washed in calcium-free Locke's solution in the ordinary manner, were added. The mixture was strained and warmed to 38°C. A normal short-haired black female mongrel terrier (no. 20-62), weighing 10.2 pounds, was anesthetized with ether and the entire corpuscle suspension was injected into the right femoral vein. Simultaneously and at the same rate 925 cc. of blood were withdrawn from the right femoral artery. The exchange was effected in 10 minutes. The temperature fell to 34.7°C. immediately following the exchange but under the influence of the heat-pad returned to 37.5°C. within a space of about 1| hour. The animal regained consciousness within about 30 THE AMERICAN JOURNAL OP PHYSIOLOGY, VOL. 52, NO. 1 82 G. H. WHIPPLE, H. P. SMITH AND A. E. BELT minutes following the exchange and was rather quiet for another 30 minutes, but thereafter appeared to be quite normal. The pulse was at no time markedly depressed. The two experiments, tables 19 and 20, confirm the two preceding experiments (table 18) using fresh dog's serum. Suspension of washed red blood cells in fresh dialj^zed dog serum (tables 19 and 20) gives a mixture which can be used in almost unlimited amounts to exchange with whole blood by the method adopted. This exchange is associ- ated with no chnical shock. There is a slight lowering in the concen- tration of blood serum protein and of course the fibrinogen is almost completely washed out of the blood. This fibrinogen, however, can be reproduced rapidly and gives no clinical reaction as its normal content is reestablished in the blood in a few hours. TABLE 20 199 per cent blood volume exchange using ivashed corpuscles suspended in dialyzed serum; experiment 329 BLOOD SERUM RE.4.DINGS IN PER CENT HEMATO- CRIT RED CELL, PER CENT UREA NITROGEN PER 100 CC. NON- PROTEIN Total protein Al- bumin Globu- lin Non- protein PER 100 CC. Serum of perfusate : Before dialysis After dialj'sis Of dog perfused: Before exchange Immediately after .... 6.7 5.1 5.9 4.9 4.0 3.5 4.3 3.5 2.7 1.6 1.6 1.4 1.5 1.0 1.6 1.6 45.1 57.4 mgm. 20 2 mgm. 40 16 It appears from these experiments that the essential factor respon- sible for the "shock" is the dilution of the serum proteins which is effected by the plasma depletion. The body cells cannot tolerate this diluted medium which for them is an abnormal environment. Protoplasmic injury is readily proved and if this injuiy is too extensive we note a familiar sequence of events which ends with fatal "shock." One may point out the narrow line which delimits a mild injury due to this plasma dilution from a severe or lethal injury and at times the' reaction almost approaches the "all or none law." The change in urinary nitrogen following a moderate reaction and plasma depletion may be almost zero but following a severe or almost fatal shock due to plasma depletion we may observe a rise in urinary nitrogen on the day following which amounts to 100 to 200 per cent increase over nor- RELATION OF BLOOD PROTEINS TO SHOCK 83 mal. This indicates a serious injury of protein substance in the body. In a fatal plasmapharesis we may note a rapid increase in the blood non-protein nitrogen which may show over 100 per cent rise within 3 to 4 hours. PLASMAPHARESIS COMPLICATED BY KNOWN TISSUE INJURY In the large table 21 are collected a number of experiments to show that the presence of injured liver cells will predispose an animal to severe or lethal shock following a control or standard plasmapharesis. The control experiments show little or no shock following the plasma depletion of a given volume. But the same exchange performed after chloroform or phosphorus usually results in fatal shock. These experiments are in contrast to those in table 28, which presents the results of plasma depletion associated with cell injury of the kidney, pancreas and intestine. Injured cells of these organs do not modify the reaction following a standard plasmapharesis. The three following experiments (tables 22, 23 and 24) illustrate in detail the reaction which follows plasmapharesis when preceded by chloroform anesthesia to insure a certain amount of liver necrosis and injury. The first of this group (table 22) gives a control plasmaphare- sis to prove that the plasma depletion alone was not responsible. The amount of liver injury was not extreme and could be tolerated by any normal animal with no clinical reaction. Note other experi- ments with controls in table 21. The emergency reaction which makes possible a rapid replacement of the washed out serum proteins shows in all these experiments. The presence of the injured liver and the development of fatal shock does not modify the usual reaction by which a considerable amount of serum proteins is thrown into the circulation. This may suggest that this reaction is not purely a functional reflex but perhaps a physical phe- nomenon in which we see a simple exchange of protein between body cells and the circulating blood plasma — a simple washing out of a given substance related to the serum proteins which is normally pres- ent in certain body cells. 84 G. H. WHIPPLE, H. P. SMITH AND A. E. BELT TABLE 21 lAver injury predisposes to fatal shock after plasma depletion DOG NUMBER 17-212 17-212 18-6 18-6 18-9 18-9 17-215 17-215 17-233 17-233 18-34 18-34 18-7 18-7 18-66 18-66 18-68 18-68 18-68 BLOOD VOLUME EX- CHANGE Chloroform (1 hour) Chloroform (I5 hour) Chloroform (1| hour) Phosphorus (17.5 mgm.) Phosphorus (14 mgm.) Phosphorus (5.2 mgm.) Phosphorus (11 mgm.) Hydrazine (140 mgm.) Hydrazine (100 mgm.) Hydrazine (100 mgm.) 141 144 118 175 170 198 159 140 10 67 118 75 77 96 118 91 82 90 88 95 5^ 15 6 61 12 7 9 9 9 9 7 None Fatal Slight Fatal Moderate Severe Slight Fatal BLOOD 8EKUM PROTEINS IN PER CENT a u H 5.5 5.5 7.1 6.6 5.6 4.8 6.2 4.9 Slight Fatal Moderate Fatal None Moderate Moderate Fatal Moderate Moderate None 4.9 5.3 5.6 5.5 6.5 6.2 5.7 5.5 4.9 5.2 2.6 2.1 4.2 1.7 2.0 2.0 1.3 1.2 0.7 2.9 3.1 2.7 1.4 3.2 4.2 2.8 2.6 2.3 E? 2.5 3.4 2.5 2.9 2.4 2.2 1.2 2.1 3.4 3.5 3.6 3.2 3.8 3.5 3.3 3.1 3.9 4.8 4.2 4.0 4.1 REMARKS Drug given 48 hours previ- ously m Drug given 40 hours previ- ously 4.3 3.7 4.3 4.8 4.6 Drug given 5 hours later Poison given in every experiment 18 to 26 hours before plasmapharesis unless othervvise noted. RELATION OF BLOOD PROTEINS TO SHOCK 85 Plasmapharesis before and after chloroform Experiment 71. (See table 22). 198 per cent exchange. Dog 18-9. Female bull-terrier pup. Weight 12 pounds. August 2. Plasmapharesis, 170 per cent exchange in 12 minutes. Little if any intoxication. August 4. Chloroform anesthesia for 1^ hour, undergoing recovery without clinical signs of injury. August 6. Animal appears to be in excellent condition. Under ether anesthesia 1081 cc. of blood were withdrawn from the left femoral artery. At the same time and at the same rate 1081 cc. of Locke's corpuscle suspension were injected into the left femoral vein. The exchange was effected in a space of 10 minutes. There was a steady fall in blood pressure and in the volume of the pulse. An extreme grade of depression was present within a half-hour and the heart beat was barely palpable 3 hours after the exchange. The rectal temperature had fallen at this time to a level of 30°C. From this point on slow but gradual improvement was noted. Eventually complete recovery occurred. TABLE 22 198 per cent blood volume exchange; plasmapharesis following chloroform; dog 18-9; experiment 71 Before exchange . . Immediately after 15 minutes after. . 2nd day 3rd day BLOOD SERUM READINGS IN PER CENT Total protein Albumin Globulin Non- protein 4.8 3.2 1.6 1.7 2.0 1.2 0.8 1.3 2.4 1.5 0.9 1.6 4.0 2.4 1.6 2.3 4.9 3.1 1.8 2.0 REMARKS Profound shock Good recovery Plasmapharesis following chloroform Experiment 72. (See table 23). 175 per cent exchange. Dog 18-6. Young-adult female Dachshund. Weight 13.4 pounds. Blood volume on July 1 (by dye method) was 761 cc. On July 24 a 118 per cent exchange was performed in 8 minutes with little or no shock. Atigust 7. Chloroform anesthesia for 1^ hour. August 8. Under ether anesthesia 1065 cc. of blood were withdrawn from the left femoral artery. Simultaneously an equal quantity of Locke's corpuscle suspension was injected into the left femoral vein. The duration of the exchange was 12 minutes. The rectal temperature showed little immediate alteration as a result of the exchange. However a gradual fall in temperature soon appeared, the level of 36.4°C. being reached at the time of death. 1§ hour later. The ar- terial pulse became slow and weak almost at once following the exchange. The respiration was gasping in character within 15 minutes following the exchange 86 G. H. WHIPPLE, H. P. SMITH AND A. E. BELT and a profound degree of depression existed. The condition gradually became worse and death occurred 1^ hour following the exchange. Autopsy: The blood drawn from the heart shows no tendency to clot within the space of 24 hours. Even such blood when placed in contact with fresh tissues shows no tendency to clot. The thoracic organs are negative. The spleen is somewhat enlarged and the Malpighian bodies are approximately twice their normal size. The pancreas is slightly congested. The liver is congested. A considerable amount of necrosis due to chloroform injury is seen in the centers of the lobules. Histological examination shows a fairly extensive central hyaline necrosis involving about one-half of each liver lobule. There is some fatty de- TABLE 23 175 per cent blood volume exchange; plasmapharesis following chloroform; dog 18-6; experiment 72 BLOOD SERUM READINGS IN PER CENT Total protein Albumin Globulin Non- protein Before exchange 6.6 1.7 2.5 3.6 0.6 1.5 3.0 1.1 1.0 1.7 1.6 1.6 Immediately after 15 minutes after I5 hours after Death TABLE 24 1^. per cent blood volume exchange; plasmapharesis following chloroform; dog 17-212; experiment 78 Before exchange . . Immediately after 15 minutes after . . 2 hours BLOOD SERUM READINGS IN PER CENT FIBRIN IN PER CENT HEMO- GLOBIN PER CENT (SAHLl) Total protein Al- bumin Globu- lin Non- protein 5.5 3.1 2.4 1.6 0.33 97 2.1 1.4 0.7 1.5 0.13 92 3.4 2.4 1.0 1.3 0.18 92 5.5 3.6 1.9 1.7 0.12 93 REMARKS Death generation of the liver cells in the mid-zone of each lobule. This lesion could be tolerated by a dog with few if any clinical sjonptoms. The kidneys show con- siderable engorgement of the medulla. The mucosa of the entire gastro-intes- tinal tract is pink and a considerable amount of mucus is contained in the lumen. Plasmapharesis following chloroform Experiment 78. (See table 24). 144 per cent exchange. Dog 17-212. Young adult female spaniel. Weight 14.5 pounds. Blood vol- ume on July 19 (bj^ dye method) was 632 cc. RELATIOX OF BLOOD PROTEINS TO SHOCK 87 An exchange of 94 per cent was performed on July 23, and another of 141 per cent on August 1, with practically no signs of shock in either case. August 21. Chloroform anesthesia for 1 hour. August 22. Animal appears to be in excellent condition. Under ether anes- thesia 950 cc. of blood were withdrawal from the left carotid artery. Simulta- neously 950 cc. of Locke's corpuscle suspension were injected into the left external jugular vein. The duration of the exchange was 13 minutes. The rectal tempera- ture gradually fell about 4°C. from the normal level in the 2 hours following the exchange. Fluid blood-stained feces were noted at the end of the first hour follow- ing the exchange. The dog went into profound shock and died 2 hours following the exchange. Autopsy: The thoracic organs are negative. The spleen and kidneys show considerable congestion. The liver is large and congested. In gross there is evidence of chloroform injury and histological sections show an ^arly stage of chloroform necrosis which involves liver cells in the centers of lobules. This injury is slight in degree and by itself would give no clinical reaction in the dog. The mucosa of the stomach and small intestines is thickened and dark red in color. A considerable excess of mucus and fluid material is present in the intestinal lumen. The following experiment (table 25, exper. 95) is complete in that a control plasmapharesis causes only a little intoxication. The dose of phosphorus is less than one-half a lethal dose and would be toler- ated by a normal dog without clinical sjonptoms. The combined phosphorus injury and a second plasmapharesis causes a typical lethal shock. It may be noted that the hematocrit figures which are complete for this experiment show no evidences of any definite change in red cell plasma ratio. The same observation holds in the chloroform experi- ments. When we review all these shock experiments and compare them with duplicate experiments in which no shock appears we cannot assign any of these reactions to a process of concentration of the blood. In certain experiments there is a rise in cell hematocrit taken 15 min- utes and 1 to 4 hours after the exchange. But the same rise is noted at the YQYY end of the exchange and the correct explanation we believe is to be found in the red cell mixture introduced. This red cell mix- ture contains more red cells per cubic centimeter than the blood of the dog under observation. There is a constant fall of hematocrit on the 2nd day but we believe this is to be explained by the disintegration of the red cells which have been injured in the routine process of washing in Locke's solution. The second phosphorus experiment (no. 73) is given in table 21. The control plasmapharesis caused no reaction but the same exchange 88 G. H. WHIPPLE, H. P. SMITH AND A. E. BELT preceded by a small dose of phosphorus was fatal in 2 hours. In an- other experiment the plasmapharesis was followed by a large dose of phosphorus. The intent was to follow the curve of protein regenera- tion as influenced by this drug which causes such characteristic liver injury. Plasmapharesis follomng phosphorus Experiment 95. (See table 25). 77 per cent exchange. Dog 18-34. Female bull pup. Weight 17.1 pounds. Blood volume (by dye method) was 805 cc. September 5. The usual plasmapharesis with 80 per cent exchange was com- pleted in 4 minutes without the production of shock. September 26. Plasmapharesis with 75 per cent exchange was carried out in 6 minutes. There was a certain amount of clinical depression, but no serious shock. TABLE 25 77 per cent blood volume exchange; plasmapharesis following phosphorus; dog 18-34; experiment 95 TIME BLOOD SERUM READINGS IN PER CENT FIBRIN IN PER CENT HEMA- TOCRIT RED CELL PER CENT REMARKS Total protein Al- bumin Globu- lin Non- protein Before exchange Immediately after. . . . 15 minutes after 3 hours 5.6 3.1 3.5 3.6 4.0 4.2 2.3 2.6 2.6 2.9 1.4 0.8 0.9 1.0 1.1 2.0 1,3 1.6 1,7 1.5 0.16 0.26 0.33 0.30 0.29 55 54 58 54 54 6 hours Profound shock October 16. Phosphorus, 5.2 mgm. in olive oil, given subcutaneously. October 17. Animal appears to be in excellent condition. Under ether anes- thesia 600 cc. of blood were withdra%\Ti from the right carotid artery. Simul- taneously 600 cc. of Locke's corpuscle suspension were injected into the right external jugular vein. The duration of the exchange was 6.5 minutes. The arterial tension was fair at the end of the exchange, but was very poor at the end of another half-hour. Although showing a considerable amount of pros- tration, the animal was conscious for several hours. The animal was in pro- found shock at the end of 6 hours, and was found dead 12 hours after the ex- change. The bodj' was still somewhat warm, but rigor mortis was fairly well developed. Autopsy: The tissues at the root of the lungs and about the smaller bronchi within the lung are somewhat edematous. The spleen is practically normal. The liver is quite pale and slightly translucent. Its lobulation is indistinct. Kidneys show slight congestion along the cortico-medullary line. The stomach is negative. The mucosa of the small intestine is thickened and moderately congej5ted. A considerable amount of mucus is found in the lumen. RELATION OF BLOOD PROTEINS TO SHOCK 89 Histological sections: The liver shows very little evidence of cell injury. There are a few pale nuclei, but the fatty change so common in the cell proto- plasm after large doses of phosphorus is absent. A slight increase in the leuco- cytes in the liver capillaries is noted. Spleen, pancreas, lung and intestines are negative. Plasmapharesis following phosphorus Experiment 73. 140 per cent exchange. Dog 17-215. Young adult female fox-terrier. Weight 17.4 pounds. Blood volume on July 19 (by dye method) was 858 cc. July 31. An exchange, 159 per cent, was performed in 10 minutes, causing no definite signs of shock. August 7. Phosphorus, 17.5 mgm. in olive oil, was given subcutaneously. Aiigiist 9. Under ether anesthesia 1105 cc. of blood were withdrawTi from the left femoral artery. Simultaneously 1105 cc. of Locke's corpuscle suspension were injected into the left femoral vein. The duration of the exchange was 8 minutes. Profound depression was in evidence almost immediately. There was a very marked weakening in the pulse. Bloody feces appeared within an hour following the exchange. Death followed the exchange by 2 hours. There was a gradual fall in rectal temperature of 4°C. during the course of the experi- ment. The total blood serum proteins fell from 4.9 per cent to 1.2 per cent as a result of this exchange. Other figures are not available because of loss of material. Autopsy: Blood drawn from the heart immediately after death does not clot even on the addition of tissue juices. The thoracic organs are negative. The spleen is dark red and enlarged to about twice the normal size. The Malpighian bodies are large, distinct and opalescent. The liver is somewhat enlarged. The centers of the hepatic lobules are dull red while the peripheral portions are yellowish. The stomach shows distention of the superficial veins and moderate engorgement of its mucosa. The mucosa of the duodenum and upper portion of the jejunum is markedly engorged. The mucosa of the lower portion of the small intestine is but slightly reddened, w^hile the large intestine is negative. The pyramids of the kidneys are slightly engorged. A few scars are seen in the cortex. Histological sections: Liver shows early changes in cell protoplasm, especially small fat droplets. This dose of phosphorus should give a severe but not lethal liver injury. The injury at this stage is very inconspicuous. There is a not- able interstitial edema of the pancreas. Other organs are negative. Plasmapharesis followed by phosphorus Experiment 66. (See table 26). 118 per cent exchange. Dog 18-7. Young adult female mongrel terrier. Weight 10.9 pounds. Blood volume (by dye method) was 530 cc. July 19. An exchange, 96 per cent, performed in 12 minutes caused no shock. Jtily 26. LTnder ether anesthesia 583 cc. of blood were withdrawn from the femoral artery. Simultaneously 583 cc. of Locke's corpuscle suspension were 90 G. H. WHIPPLE, H. P. SMITH AND A. E. BELT injected into the femoral vein. The duration of the exchange was 7 minutes. With the exception of a fall of about 1°C. in rectal temperature there was little obvious disturbance as a result of the exchange. About 5 hours after the ex- change 11 mgm. of phosphorus dissolved in olive oil were injected subcutaneously. On the following day the animal appeared rather quiet, but not otherwise dis- turbed. The food was not eaten for several days and on August 1 the dog weighed 9.25 pounds. Complete recovery occurred several days later. TABLE 26 118 per cent blood volume exchange; plasmapharesis followed by phosphorus ; dog 18-7; experiment 65 Before exchange. . Immediately after 15 minutes after . . 2nd day 3rd day 5th day 10th day BLOOD SERUM HEADINGS IN PEB CENT Total protein Albumin Globulin Non-protein 6.5 5.7 0.8 2.0 1.4 0.3 1.1 1.5 3.2 1.8 1.4 1.7 3.7 2.6 1.1 2.5 3.8 2.4 1.4 2.3 5.1 2.0 3.1 1.5 4.3 2.0 2.3 1.8 TABLE 27 88 per cent blood volume exchange; plasmapharesis following hydrazine sulfate; dog 18-68; experiment 108 Before exchange . . Immediately after 15 minutes after . . 3^ hours 2nd day 3rd day 6th day BLOOD SEBUM BEADINGS IN PER CENT Total protein 4.9 2.6 3.3 3.7 4.6 4.7 5.2 Albumin Globulin 3.2 1.7 2.3 2.6 3.2 3.3 2.8 1.7 0.9 1.0 1.1 1.4 1.4 2.4 Non- protein 1.7 1.6 1.4 1.9 1.7 1.6 2.3 FIBRIN IN PER CENT 0.31 0.26 0.31 0.40 HEMATO- CRIT RED CELL PER CENT 27 42 56 41 33 Plasmapharesis folloicing hydrazine sulfate Experiment 108. (See table 27). 88 per cent exchange. Dog 18-68. Female mongrel bull pup. Weight 14.3 pounds. November 21. An exchange of 90 per cent performed in 9 minutes caused a moderate grade of shock. November 27. Hydrazine sulfate, 100 mgm., injected subcutaneously. November 28. Under ether anesthesia 575 cc. of blood were withdra-mi from the left femoral artery. Simultaneously 575 cc. of Locke's corpuscle suspension were injected into the left femoral vein. The duration of the exchange was 9 RELATION OF BLOOD PROTEINS TO SHOCK 91 minutes. There were at no time any definite signs of depression. The arterial tension remained moderately good throughout. There was a fall in rectal tem- perature of about 1°C. during the exchange. There was, however, a prompt return — in fact to a point slightly above the original temperature for a period of several hours, after which the temperature returned to the normal level. The preceding experiment (table 27) gives some evidence that hydrazine sulfate as a liver poison differs somewhat when compared with chloroform or phosphorus. This dog (18-68) showed no less reac- tion to the control plasmapharesis than to the same exchange preceded by hydrazine sulfate. In another experiment, however, (table 21, exper. 109) we see the familiar realction with fatal shock due to a com- bined plasmapharesis and hydrazine poisoning. The control of the plasmapharesis showed a definite but not severe intoxication. The preceding table (table 28) lists the reactions which follow a plasmapharesis combined with cell injuries of various other organs and tissues. The control plasma depletion on the same dog is given in each experiment. When the remarkable reaction and fatal shock were noted in the phosphorus and chloroform experiments we sus- pected at once that any cell injury might render the experimental ani- mal more sensitive to the shock of plasmapharesis. The experiments in table 28, however, show that such is not the case. The kidney epithelium was injured by administration subcutaneously of uranium nitrate in suitable dosage. Two experiments show identical reactions in the control plasma depletion as in the plasmapharesis fol- lowing the administration of uranium nitrate. One experiment (dog 18-35) shows a fatal reaction but there are many unusual features which we cannot explain — see table 30 below for details. Pancreas injury is represented by only a single experiment but this is very clean-cut. The pancreas was injured by the injection of bile into its main duct. The control exchange gives the same negative reaction as the plasma depletion preceded by the acute pancreatitis. The Roentgen-ray is able to cause a specific and extensive in j jury to the lymphatic tissue but especially to the epithelium of the small intestine as has been shown by the work of Hall and Whipple (6). This injury and consequent intoxication develops to its maximum on the 4th day following an exposure over the abdomen. A plasmaphare- sis done 24 hours after X-ray exposure gives the same reaction as in the control period. This shows that even the extensive injury which in a fatal case of X-ray intoxication involves the greater part of the epithelium of the small intestine does not modify the shock of plasma- pharesis. This is in striking contrast to the liver injury. 92 G, H. WHIPPLE, H. P. SMITH AND A. E. BELT TABLE 28 Kidney, pancreas and intestinal epithelium injury does not predispose to shock after plasma depletion POISO>f BLOOD VOLUME EXCHANGE SHOCK BLOOD SERUM PBOTEIN8 IN PER CENT DOG NUMBEB u CD 3 a a ffl V Si o| c " m S .2 §3 1^ 2 3 O t. 18-35 74 14 None 5.5 3.7 3.8 18-35 Uranium (5 mgm.) 72 14 Fatal 6.3 3.9 4.6 5.8 18-48 104 2 Slight 6.4 2.1 3.2 4.5 18-48 Uranium (6 mgm.) 77 4 None 6.1 3.1 4.1 4.6 18-66 91 9 Moderate 6.2 3.2 3.8 4.3 18-66 Uranium (8 mgm.) 96 lU Moderate 5.1 2.9 3.2 4.8 18-65 100 13 Slight 5.6 3.0 2.9 4.6 18-65 Pancreatitis 94 15 None 5.3 3.3 3.9 4.8 18-68 90 9 Moderate 5.5 2.8 3.5 4.8 18-68 X-ray* (175 M.A.M.) 90 7 Moderate 5.9 3.2 3.9 4.7 18-65 100 13 Slight 5.6 3.0 2.9 4.6 18-65 X-ray (200 M.A.M.) 105 9 None 5.7 2.9 3.3 4.7 Injury given in every experiment 20 to 24 hours before plasmapharesis with exception noted : * X-ray given 45 hours before plasmapharesis. TABLE 29 77 per cent blood volume exchange; plasmapharesis following uranium nitrate; dog 18-48; experiment 99 BLOOD SEBUM BEADING8 IN PEB CENT FIBBIN IN PEB CENT HEMATO- CRIT Total protein Albumin Globulin Non- protein BED CELL PEB CENT Before exchange Immediately after 15 minutes after 2 hours 6.1 3.1 4.1 4.0 4.7 4.6 5.2 5.1 5.1 5.2 3.4 1.9 2.6 2.6 2.9 3.0 3.4 3.4 3.4 3.5 2.7 1.2 1.5 1.4 1.8 1.6 1.8 1.7 1.7 1.7 1.7 1.4 1.5 1.5 1.8 2.0 2.2 2.2 2.1 2.4 0.25 0.25 0.32 0.45 0.42 0.29 0.33 61 36 6 hours 29 2nd day 41 6th day 41 8th day 46 9th day 11th day 43 RELATION OF BLOOD PROTEINS TO SHOCK 93 Plasmapharesis following uranium nitrate Experiment 99. (See table 29). 77 per cent exchange. Dog 18-^8. Female bull pup. Weight 14.3 pounds. October 4- Exchange of 99 per cent performed in 12 minutes with the pro- duction of but slight grade of shock. October 18. An exchange of 104 per cent in 2 minutes was performed with very little shock. October 31. Uranium nitrate, 6 mgm., given subcutaneously. November 1. Animal seems to be in excellent condition. Under ether anes- thesia 500 cc. of blood were withdrawn from the right carotid artery. Simul- taneously 500 cc. of Locke's corpuscle suspension were injected into the right external jugular vein. The duration of the exchange was 14 minutes. At no time during the experiment was there any definite sign of shock. The arterial tension was good at the end of the exchange but shortly thereafter fell slightly for a period of several hours. The rectal temperature fell nearly 2°C. during the exchange but returned to normal in the course of several hours. Plasmapharesis before and after uranium nitrate Experiment 97. (See table 30). 72 per cent exchange. Dog 18-35. Female bull pup. Weight 16.7 pounds. September 12. An exchange of 80 per cent was performed in 7 minutes, causing slight intoxication. October 3. An exchange of 73. per cent was effected in 14 minutes, with no signs of intoxication. October 22. An aqueous solution of 10.4 mgm. uranium nitrate was injected subcutaneously. October 23. Animal appears to be in good general condition. Under ether anesthesia 545 cc. of blood were withdrawn from the left carotid artery. Simul- taneously and with no great variation in rate 545 cc. of Locke's corpuscle sus- pension were injected into the left jugular vein. The duration of the exchange was 13 minutes. The rectal temperature fell from 39.5 to 37°C. within a space of 4 hours. Drowsiness soon appeared, the pulse diminished in volume after about 3 hours and was decidedly poor several hours later. Bloody feces were first noted about 11 hours following the exchange. The next morning the condition was worse and a considerable amount of bloody feces had been passed. The animal was suffering from convulsive attacks. Thfe temperature was 37°C. Death occurred about 20 hours following the exchange. Autopsy: The tissues are definitely jaundiced. The thoracic organs are essentially negative. The spleen is large, the edges rounded, and on section presents definite congestion. The liver shows only indistinct lobulation. The kidneys are definitely engorged. The stomach is negative except for a slight amount of engorgement of the mucosa. The intestinal mucosa is decidedly engorged with blood, the condition being more marked in the lower portion of the gut. Histological sectio7is: Kidneys show much epithelial degeneration and necrosis involving particularly the convoluted tubules. There are numerous hyaline, hemoglobin and blood casts in the collecting tubules. Other organs present nothing of interest. 94 G. H. WHIPPLE, H. P. SMITH AND A. E. BELT The preceding experiments (tables 29 and 30) are in conflict. The first one (exper. 99) shows a negative reaction when a standard plasma depletion is combined with kidney injury due to uranium nitrate. The other experiment (exper. 97) shows a fatal reaction but it is atypi- cal. The shock did not develop quite as usual and the dog seemed about to recover. When the shock of plasmapharesis is tolerated for 12 hours the dog usually recovers and appears normal and active within 24 hours. This dog on the day after the experiment developed con- vulsions and died. There was jaundice and at autopsy signs of blood destruction. The histological sections give evidence of considerable epithelial injury in the secreting tubules of the kidney. That this kidney injury played a part in the late death is highly probable but TABLE 30 72 per cent blood volume exchange; plasmapharesis following uranium nitrate; dog 18-35; experiment 97 Before exchange . . Immediately after 15 minutes after. . 2^ hours 5 hours 2nd day BLOOD SERUM READINGS IN PER CENT FIBRIN IN HEMATO- CRIT Total Albu- Globu- Non- PER CENT RED CELL PER CENT protein min lin protein 6.3 4.7 1.6 1.7 0.32 22 3.9 3.0 0.9 1.5 0.14 25 4.6 3.5 1.1 1.4 0.16 42 5.1 3.7 1.4 1.4 0.19 38 5.2 3.9 1.3 1.4 37 5.8 4.0 1.8 1.8 0.32 30 REMARKS Death it is clear that the shock of plasmapharesis was atypical. In view of the other experiments we do not attach too much importance to a single atypical experiment which appears to be at variance with the general type reaction. Acute pancreatitis followed by plasmapharesis Experiment 102. (See table 31). 94 per cent exchange. Dog 18-66. Female bull pujj. Weight 17 pounds. November 7. An exchange of 100 per cent effected in 13 minutes caused a very moderate grade of shock. November 13. Under ether anesthesia laparotomy was performed and 10 cc. of sterile bile injected bj^ means of a hypodermic needle into the pancreatic duct. The wound was closed. It is known that this will cause an intense diffuse hemorrhagic pancreatitis. RELATION OF BLOOD PROTEINS TO SHOCK 95 November 14. The animal is lively and apparently in quite good condition. Under ether anesthesia 730 cc. of blood were withdrawn from the left femoral artery. Simultaneously 730 cc. of Locke's corpuscle suspension were injected into the left femoral vein. Seven minutes were consumed in effecting the ex- change. At no time was there any definite evidence of intoxication. Refer to experiment 106, table 33, for autopsy. The preceding experiment (table 31) is complete and supports the two following experiments with X-raj^ injury. This pancreas was severely injured by an injection of bile into the pancreatic duct. We have frequently produced an acute hemorrhagic pancreatitis in this wa}'' and the injur}^ may be sufficient to produce lethal intoxication by itself. That extensive injury was done this pancreas is afforded by TABLE 31 94 per cent blood volume exchange; acute pancreatitis followed by plasmapharesis ; dog 18-65; experiment 102 Before exchange . . Immediately after 15 minutes after. . 3 hours 8 hours 2nd day 3rd day 4th day 6th day 7th day BLOOD SERUM READINGS IN PER CENT FIBRIN IN PER CENT Total protein Albumin Globulin Non- protein 5.3 3.3 3.9 4.6 4.3 4.8 4.6 4.0 5.0 5.0 3.6 2.5 2.9 3.1 2.9 2.9 2.6 2.8 2.8 3.7 1.7 0.8 1.0 1.5 1.4 1.9 2.0 1.2 2.2 1.3 2.2 1.6 1.4 1.4 2.2 2.2 2.2 2.8 2.9 2.7 0.49 0.29 0.22 0.40 0.34 0.29 0.25 0.35 0.31 0.40 HEMATO- CRIT RED CELL PER CENT 36 54 58 53 41 40 35 32 27 29 examination of the autopsy record of this dog (exper. 106, table 33 below) which shows a scarred, indurated pancreas speckled with old fat necroses. Yet these injured pancreas cells did not modify the reaction following a controlled plasma depletion. Plasmapharesis following sublethal X-ray exposure Experiment HI. (See table 32). 90 per cent exchange. Dog 18-68. Female bull pup. Weight 14 pounds. November 21. An exchange of 90 per cent effected in 9 minutes caused a very moderate grade of shock. December 10. X-ray exposure over abdomen in 4 quadrants, 2 mm. aluminum filter, 175 milliampere minutes, with 9 inch spark gap. Distance from target to skin is 10 inches. 96 G. H. WHIPPLE, H. P. SMITH AND A. E. BELT December 12. Animal seems to be in excellent condition. Under ether anes- thesia 575 cc. of blood were \vithdra^^^l from the left carotid artery. Simul- taneously 575 cc. of Locke's corpuscle suspension were injected into the left external jugular vein. The duration of the exchange was 7 minutes. There was but a very slight amount of depression. The arterial tension was good at the end of the exchange, but became of poorer quality in the course of the next 2 hours. The animal walked about occasionally. The rectal temperature fell about 1°C. during the course of the experiment. Lethal dose of X-ray followed by plasmapharesis Ex-periment 106. (See table 33). 105 per cent exchange. Dog 18-65. Female bull pup. Weight 16.8 pounds. November 7. An exchange of 100 per cent effected in 13 minutes caused a very moderate grade of shock. November 14. An exchange of 94 per cent following acute experimental pan- creatitis produced no definite signs of intoxication (see exper. 102, table 31). November 21. X-ray exposure over abdomen in 4 quadrants, 2 mm. aluminum filter, 200 milliampere minutes, with 9 inch spark gap. Distance from target to skin is 10 inches. November 22. The animal appears to be in good condition. Under ether anesthesia 800 cc. of blood were withdra^^^l from the right carotid artery. Si- multaneously 800 cc. of Locke's corpuscle suspension were injected into the right external jugular vein. The duration of the exchange was 9 minutes. The ani- mal showed a very slight amount of depression as a result of the exchange. The pulse remained fair throughout. There was but slight depression of the rectal temperature (1°C.). November 23. The animal seems a bit weak. Has vomited material containing some intestinal worms. November 24. The dog appears somewhat better. November 25. Quite weak. Mucous, blood-tinged feces. Some vomiting. Refuses food. November 26. Death occurred in the afternoon. Autopsy performed at once. Autopsy: Thoracic organs negative. Blood drawn from the heart clots nor- mally. Blood urea nitrogen is 32.5 mgm. per 100 cc. Spleen is small, light red, with an increase in fibrous tissue. Liver is pale and anemic. Pancreas: lower arm is hard, shrunken and scarred; the result of the pancreatitis described in experiment 102, table 31. Many old fat necroses are present. The upper arm is scarred but appears more nearly normal. Kidneys and adrenals are negative. Gastro-intestinal tract shows only a few scattered patches of congastion. Histological sections: The pancreas shows extensive fibrosis — the result of the preceding acute injury. The small intestine shows much epithelial injury in its deep crypts. There is some evidence of epithelial regeneration as well as degen- eration. This epithelial injury we believe to be the immediate cause of death. Other organs present nothing of interest for this experiment. The two preceding experiments (tables 32 and 33) show the influ- ence of X-ray injury of the body cells upon a standard plasmapharesis. RELATION OF BLOOD PROTEINS TO SHOCK 97 The first experiment (table 32) shows the result of a sublethal exposure to the X-rays. The reaction to the plasma depletion in a control exchange is not modified. The second experiment (table 33) shows a reaction recently de- scribed in some detail by Hall and Whipple (6). This reaction is due TABLE 32 90 per cent blood volume exchange; plasmapharesis following sublethal x-ray expo- sure; dog 18-68; experiment 111 Before exchange. . Immediately after 15 minutes after. . 3 hours 5? hours 2nd day 4th day BLOOD SERUM READINGS IN PER CENT Total protein Albumin Globulin Non- protein 5.9 4.2 1.7 2.2 3.2 2.6 0.6 1.7 3.9 3.0 0.9 1.8 4.4 3.1 1.3 1.9 4.5 3.3 1.2 1.9 4.7 3.3 1.4 2.7 4.5 3.0 1.5 2.6 No shock Dog normal TABLE 33 105 per cent blood volume exchange; lethal dose of x-ray followed by plasmapharesis; dog 18-65; experiment 106 Before exchange . . Immediately after 15 minutes after . . 4 hours 7 hours 2nd day 3rd day 4th day 5th day BLOOD SERUM READINGS IN PER CENT FIBRIN IN PER CENT HEMA- TOCRIT RED CELL PER CENT Total pro- tein Albu- min Globu- lin Non- pro- tein 5.7 3.7 2.0 2.1 0.45 33 2.9 2.3 0.6 1.7 0.22 44 3.3 2.6 0.7 1.7 0.27 34 4.1 2.8 1.3 1.8 0.28 37 4.2 3.4 0.8 1.5 4.7 3.5 1.2 2.1 0.47 26 3.9 2.1 1.8 3.0 25 4.0 1.7 2.3 3.8 0.72 30 5.9 3.0 2.9 3.1 0.95 Slight shock X-ray intoxication Death to a lethal dose of the X-ray — in this instance 200 milliampere min- utes, 90 kilo volts, given over the abdomen. Death on the 4th day with the usual blood-tinged feces and prostration is the usual reaction in these animals given a lethal exposure of the X-ray. Details of this reaction and the post-mortem findings may be found in the publica- THE AMERICAN JOURNAL OF PHYSIOLOGY, VOL. 52, NO. 1 98 G, H. WHIPPLE, H. P. SMITH AND A. E. BELT tion just noted. The control plasmapharesis which was done 24 hours after the X-ray exposure did not give an}^ symptoms of intoxication and this reaction due to the plasma depletion was not modified by the presence of a great amount of injured epithelium of the small intes- tine. We have many experiments to show that on the second day after X-ray exposure epithelial injury and necrosis can be made out histologically in the small intestine. These cells will undergo rapid autolysis under a variety of conditions and it is quite remarkable that the plasmapharesis should not be modified by this great mass of injured epithelial cells. DISCUSSION In some earlier experiments, Kerr, Hurwitz and Whipple (3), it was noted that the presence of liver injury or liver cell necrosis made a given animal much more vulnerable to the injury and consequent shock which followed a given plasma depletion. Using single rapid de- pletion by the method described in the experiments cited above similar results were observed. A number of such experiments are given in table 21 above and it will be noted that the control experiment in every case shows little or no shock following plasmapharesis, but an identical procedure if associated with slight liver injury was almost always fatal. There is apparently little or no difference in this respect between the liver injurj^ due to chloroform and that due to phosphorus. The liver injury due to hydrazine sulfate was not studied in a sufficient number of experiments. EiThe interesting fact stands out that a trifling injury due to phos- phorus or chloroform can be tolerated by a dog with no clinical reac- tion. But if at this time (24 to 48 hours after administration of the chloroform or phosphorus) we perform a plasmapharesis of small volume which was previously tolerated by the same dog with little or no in- toxication, we immediately precipitate severe or fatal shock. The combination of slight liver injury and a moderate exchange (plasmapha- resis) will result fatally in almost all cases. How may we explain this observation? There are many possibilities but we favor the following explanation. The chloroform or phosphorus causes an injury -to many liver cells and these cells are more susceptible to other injurious agents than are the normal liver cells. A sudden change in the protein con- tent of the blood which bathes these injured cells will react more un- favorably upon them than upon the healthy and more resistant nor- mal liver cells. These damaged (phosphorus) and then shocked (plas- RELATION OF BLOOD PROTEINS TO SHOCK 99 mapharesis) liver cells form substance;^ which are taken up by the blood and carried to all the living cells of the body. If these poison- ous substances are sufl&cient in amount we observe the development of lethal shock. We may not assume simple intensive injury and paralysis of the liver cells alone because it is known that the body can tolerate complete ablation of the liver cells for a period of 5 to 7 hours (7). WTien we produce an intensive form of shock (plasmapharesis) we may observe death within 1.5 hour. This cannot be explained by any local reaction limited strictly to the liver cells. We observe in other experiments (table 28) that cell injury of other organs (kidney, pancreas and intestine) does not modifj'- the familiar reaction following a moderate exchange. The control and poisoning experiments give similar reactions. This indicates a peculiar relation of the liver cells to the shock reaction associated with plasma depletion. SUMMARY Bleeding a dog from a large artery and a simultaneous replacement of a red blood cell Locke's solution mixture may be called "plasma depletion" or "plasmapharesis." This procedure will rapidly wash out large amounts of plasma proteins and cause a precipitous fall in the blood plasma protein concentration. The reaction following such procedures may be minimal or it may be lethal. In general the larger the exchange the greater the proba- biHty of lethal shock. Repeated plasma depletions carried out at intervals of daj^s or weeks on the same animal will give uniform reac- tions if the volume exchange and other experimental factors are constant. "Plasmapharesis" may be performed with washed red cells suspended in normal dog serum or fresh dialyzed dog serum. When we replace the Locke's solution in the red cell mixture by dog serum we remove completely the toxic effect of the plasma depletion. This gives con- trol for the experimental procedure but, more important, gives strong indication that the hlood serum -proteins are stabilizing or protective factors. They are essential environmental factors of the circulating blood in its relation to the body cells. This may be the most important function of these plasma colloids. The presence of injured cells of the kidney, pancreas or intestine does not seriously modify the expected reaction following a uniform plasmapharesis. 100 G. H, WHIPPLE, H. P. SMITH AXD A. E. BELT The presence of injured liver cells (chloroform, phosphorus) does profoundly modify the expected reaction following a unit plasmapharesis. A fatal shock reaction is almost constant following even a moderate plasma depletion preceded by hver injury. This would indicate that the liver cells are particularly concerned in the peculiar shock reaction which may follow plasmapharesis and lowering of the blood plasma protein values. It may be that this type of "shock" is not unlike the common "surgical shock." The evidence in our experiments gives strong support to the theory that in "shock" there is a primary cell injury which precedes the familiar clinical reaction. BIBLIOGRAPHY (1) MoRAWiTz: Beitr. z. chem. Physiol, u. Path., 1906, vii, 153. (2) Abel, Rowntree and Turner: Journ. Pharm. Exper. Therap., 1914, v, 625. (3) Kerr, Hurwitz and Whipple: This Journal, 1918, xlvii, 356, 370, 379. (4) Kerr, Hurwitz and Whipple: This Journal, 1918, xlvii, 356. (5) Erlanger and Gasser: This Journal, 1919, xlix, 151. (6) Hall axd Whipple: Amer. Journ. Med. Sci., 1919, clvii, 453. (7) Whipple and Hooper: Jour. Exper. Med., 1913, xvii, 612. III. FACTORS CONCERNED IN THE PERFUSION OF LIVING ORGANS AND TISSUES Artificial Solutions Substituted for Blood Serum and the Resulting Injury to Parenchyma Cells A. E. BELT, H. P. SMITH axd G. H. WHIPPLE From the George Williams Hooper Foundatio7i for Medical Research, University of California Medical School, San Francisco Received for publication February 25, 1920 These experiments were undertaken for the purpose of investigating the formation of the serum proteins in the body. The source of the serum proteins is still a mystery in spite of indirect evidence brought out by Kerr, Hurwitz and Whipple (1) to show that the liver is concerned in the regeneration of new serum proteins as well as in the mainten- ance of the normal serum protein concentration in the circulating blood. It seems too that other organs or tissues as well as the liver must be able under emergency conditions to produce certain amounts of new serum protein. It would appear that the normal wear and tear of serum proteins must be slight as these substances can be formed only with so much difficulty when the normal level has been greatly lowered. Theoretically, perfusion of organs should offer an ideal method of solving these problems. Using a mixture of red cells and whole serum or diluted sermn or modified Locke's solution, the investigator should be able to perfuse satisfactorily the various organs or combinations of organs and tissues. A simple determination of the protein values be- fore and after such a procedure would then give the desired information and enable one to say whether a certain organ did or did not contribute any serum proteins. Information as to whether serum proteins are used by or destroyed in these organs might also be made available. We have been able to convince ourselves that the present methods are not satisfactory to permit of the solution of this serum protein problem just outlined above. One of the conclusions which has been forced upon us is that much of the older experimentation in the field of organ perfusion is of little or no value as regards deductions made from 101 102 A. E. BELT, H. P. SMITH AND G. H. WHIPPLE such experiments which postulated a hving organ or organ cells. When parenchjTnatous organs are perfused with Locke's solution or some mod- ification of this solution with or without red cells, we wish to suggest the probability that the research worker is dealing not with normal cells but with cells which have been injured or destroyed by contact with the perfusate. The investigator is then perfusing a dying or dead autolysing tissue or organ. Deductions drawn from such experi- ments must be cautious and proper allowance in every case made for this profound injury of the parenchyma cells. The "stabilizing value" of the blood serum proteins and the injury done the various body cells by contact with diluted plasma have been emphasized in the preceding communication. We may also note the observation of Guthrie (2) to the effect that organ transplantation is a failure if the transplanted organ is washed out with normal saline be- fore the blood flow through the organ is reestablished. The trans- planted organ does not resume its function and we may assume that its cells were definitely injured bj^ the short period of contact with the salt solution. The gradual slowing of the perfusion flow through any given organ is a familiar observation and we believe it may be due in part to actual injury of cells, including the endothelium. The simultaneous develop- ment of edema is a part of the same general reaction. From many of our earlier experiments we gained the impression that perfusion or plasmapharesis with the use of Locke's solution inflicted a destructive injury upon certain body cells and that this injury of pro- tein was responsible for the fatal intoxication and death. Our con- ception was somewhat as follows: the initial injury might damage irreparably liver cells (or other body cells) and from these injured organ cells were derived poisonous substances (protein split products) sufficient to cause death. It has been pointed out that complete par- alysis of all liver cells could not explain this phenomenon as ablation of the liver can be tolerated for 5 to 7 hours, while the shock following a large plasmapharesis may cause death within 2 hours. Postulating the presence in the body of some poisonous substance referable to the perfusion or plasmapharesis, we have attempted in many of our perfusion experiments to demonstrate the presence of a poison in the perfusate at the end of any given experiment. In only one experiment (exper. 5) have we evidence for the presence of any poison under these conditions of perfusion. But we have some evi- dence (expers. 17 and 18) to show that a known poison of protein origin FACTORS CONCERNED IN PERFUSION OF LIVING ORGANS 103 added to a given non-toxic perfusate ma}' be in part removed within 20 to 30 minutes' continuance of perfusion. This is evidence that a poison of colloid nature may be removed from the circulation in such experi- ments — so the absence of a poison in our perfusates does not negative the possibility of poison being formed by the injured cells and contrib- uted to the blood stream or perfusate. We have further been able to show that an enormous dose of proteose-like, toxic material may be wholly removed from the blood stream within a period of 5 minutes after intravenous injection in a normal dog. We have discussed the results of the experiments given below in the light of the plasmapharesis experiments given in the two preceding papers. It may be noted that these perfusion experiments were done before the plasma depletion experiments. The e\adence which may be taken from our perfusion experiments is not as definite as that obtained in the later plasmapharesis experiments but these data are all in har- mony. A most important fact is that physiological perfusion of organs is very difficult and slight modification of the blood plasma may have profound effect upon body cells. DEVELOPMENT OF PERFUSION METHODS The notion of artificial perfusion was long ago expressed by Le Gal- lois (3). He maintained that by artificial perfusion life might be kept up in any portion of the animal even though separated from the rest of the body. It remained, however, for other workers actually to under- take such experiments. In 1828 Kay (4) show^ed that artificial per- fusion with blood was capable of restoring irritabihty to djang muscle. Artificial perfusion of kidneys was first attempted in 1849 by LobeU (5). The work of Brown-Sequard (6) done several years later showed the necessity of oxygenation of the blood used as a perfusate. The oxygenated blood was forced through the arteries by means of a syr- inge. In this manner he perfused various regions including the iso- lated head. He found that he was able in this manner to maintain certain evidences of reflex nervous activity provided the perfusion was commenced promptly after decapitation. Ludwig and Schmidt (7) in 1868 described an apparatus by means of which blood could be forced under constant pressure from a reservoir. Improvements in aeration of the perfusion medium were made by Schroder (8), Fry and Gruber (9) devised an artificial lung by means of which the aeration of the per- fusate could be accompHshed without interrupting the flow of blood 104 A. E. BELT, H. P. SMITH AND G. H. WHIPPLE to the region being perfused. Although fluctuation in the pressure suppHed to the perfusion medium occurred in the work of the earher investigators using the sjTinge injection method, the distinct beneficial effects of such variations in pressure were first recognized by Ludwig and Schmidt (7). Fry and Gruber (9) attached the piston of a syringe supplying the arterial pressure to a motor-driven wheel thus creating by mechanical means a pulsatile pressure. Hamel (10) emphasized the need for pulsatile pressure. He devised an apparatus in which the movements of a pendulum periodically interrupted the flow of the per- fusate to the tissues, thus converting a constant pressure into an inter- mittent one. Jacob] (11) devised an elaborate perfusion apparatus in which pulsatile pressure was created by periodic and forcible compres- sion of a rubber balloon placed in the arterial side of the circuit. He used the principle of aerating the blood by forcing a mixture of air and venous blood through a stretch of tubing at the end of which the blood and air were separated by gravitation. In a later paper Jacob] (12) described a method by means of which the blood was aerated by per- fusion through a lung in which respiration was artificially maintained. In this manner he avoided the direct mixing of the blood with the air. In 1903 Brodie (13) published an account of an apparatus which has subsequently been used by several investigators. With it he was able to perfuse an organ with the use of no other blood than that obtained from the animal itself— a considerable advantage over many of the types previously emploj^ed. To create pulsatile pressure he suggests that a fairly distensible piece of rubber tubing placed in the arterial side be rhythmically compressed by a wooden arm. Other forms of perfusion apparatus have been described by Hoff- mann (14), Richards and Drinker (15), Friedmann (16), Mandel (17) and Kingsbury (18). Pulse pressure as a necessary factor in the mechanics has been re- cently reemphasized by Hooker (19), whose apparatus was employed in our experiments. His apparatus can be adjusted in such a manner as to furnish a pulse curve identical in form to that produced in a normal intact animal. Aeration is effected. In addition to the purely mechanical methods of perfusion another slightl}^ different procedure has been employed by some. As far back as 1881 Martin (20) attempted to study the activity of the heart by diverting all of the blood issuing from the aorta back into the right auricle. A heart-lung preparation was thus effected, the circulation being successfully excluded from the rest of the body. This procedure FACTORS CONCERNED IN PERFUSION OF LIVING ORGANS 105 or modifications thereof have been used by many workers since that time. In 1914 Bainbridge and Evans (21) susbtituted this Hving prep- aration for the artificial perfusion machine. The organ to be perfused received its blood directly from the aorta of the preparation. The venous blood issuing from the vein was returned to the right side of the heart. The perfused tissue thus received blood aerated by the lungs and under pulsatile pressure supplied by the heart itself. It should be remarked that in this form of perfusion the study of the per- fused organ is complicated by the metabolism of the heart and lungs themselves. EXPERIMENTAL OBSERVATIONS All perfusion experiments were performed by use of the apparatus designed and described in detail by Hooker (19), (22). Through his courtesy we were able to obtain this machine which was made after the model of his original apparatus. We take this opportunity to ac- knowledge our appreciation for valuable assistance on the part of Dr. D. R. Hooker. The general experimental procedures are covered by the brief description in individual experiments. All experiments were done on dogs under complete ether anesthesia. In all perfusion ex- periments the dog was placed upon a warm pad to keep up the body temperature. Perfusion of hind legs with Locke's solution Experiment 5. Male bull pup. Weight 4.8 kilos. Under ether anesthesia cannulae were inserted and the hind legs were per- fused for i hour with Locke's solution. The temperature of the perfusate varied between 30° and 40°C. The perfusion pressure was between 100 and 110 mm. mercury. The pulse pressure was between 20 and 30 mm. mercury. Pulse rate 130 a minute. The 240 cc. of perfusate recovered at the end of the perfusion con- tained 77,000 red blood cells per cubic millimeter. Hemolysis was moderate in amount. To test the toxicity of this perfusate the cells were removed by centrifugali- zation and 100 cc. of the supernatant fluid were injected intravenously into a normal dog. A rise of 1.4° in temperature with vomiting and diarrhea was noted. The pulse was not markedly altered. The perfusate was therefore moderately toxic. Ten cubic centimeters of the centrifugalized perfusate were also injected intraperitoneally into a 100 gram rat. Slight toxicity was evident. Perfusion of hind legs with red corpuscles suspended in Locke's solution Experiment 7. Male collie mongrel. Weight 16 pounds. The hind legs were perfused with a suspension of blood corpuscles in Locke's 106 A. E. BELT, H. P. SMITH AND G. H. WHIPPLE solution. The temperature of the perfusate varied between 32° and 38°C. The mean pressure was 50 mm. mercury. The pulse rate was 130 and the rate of flow 180 cc. a minute. Autopsy shows irregular petechial hemorrhages in the muscles and fascia of the hind legs. Slight hemolysis was noted in the 250 cc. of perfusate recovered at the end of perfusion. One hundred and sixty-one cubic centimeters of the centrifugalized end-product were injected intravenously into a normal 13.75 pound dog. A temperature rise of 0.4° was observed. No vomiting or diarrhea occurred. Twentj^ cubic centimeters of this perfusate were also injected intraperitoneally into a rat weighing 100 grams. There were no evidences of toxicity from the use of this perfusate. These two experiments (expers. 5 and 7) give little information con- cerning the actual perfusion conditions but supply data concerning the production of a poison by the Locke's perfusion. There is shght posi- tive evidence for a toxic reaction in experiment 5 but a negative reaction in experiment 7. In general we have no distinctly positive evidence that this destructive perfusion of body tissues will give demonstrable evidence of a toxic element in the perfusate. As stated above this may be explained by the capacity of the body cells to remove such poisons from circulating fluids. Perfusion below diaphragm with red corpuscles suspended in modified Locke's solution Experiment 9. Normal female black and white pup. Weight 6.75 pounds. The oxalated blood from a normal dog was centrifugalized and the corpuscles washed in gelatin-Locke's solution minus calcium by mixture and recentrifugali- zation. The red cells were then suspended in gelatin -Locke's minus calcium in the ratio of packed corpuscles 1 to solution 5. This mixture was used as the perfusion medium. Under ether anesthesia the animal was bled. Cannulae, were inserted and the perfusate forced through the aorta and recovered from the right auricle, thus perfusing the area below the diaphragm. The temperature of the perfu- sate varied between 35° and 38°C. The mean pressure was maintained at 50 to 80 mm. mercurj^; the pulse pressure between 10 and 15 mm. mercury. The pulse rate was 130 a minute. The flow was excellent. Perfusion began 20 minutes after bleeding and was continued for 1 hour. The autopsy was delayed for a few hours after the completion of the per- fusion. The muscles of the hind legs were pale and showed very little edema. No hemorrhages were present in the muscles and connective tissue. The liver was normal except for some air bubbles in its vessels. The capsule of the kid- neys stripped readily. Hyperemia was seen at the cortico-meduUary boundary. Suprarenals were negative. The IjTnph nodes of the mesentery were normal. FACTORS CONCERNED IN PERFUSION OF LIVING ORGANS 107 The pancreas showed a considerable amount of edema. The spleen was dark red except for a transverse light band possibly caused by block from emboli. The intestines were filled with red mucoid material. The mucosa was velvety, swollen and deep purplish-red in color. Histological sections of liver and intes- tine give no evidence of tissue abnormality. On centrifugalization a sample of perfusate taken 10 minutes after beginning of perfusion was light pink. A sample taken at the end of 40 minutes was some- what deeper in color, while a sample taketi at the end of perfusion was dark red. One hundred and fifty cubic centimeters of a centrifugalized sample taken after 10 minutes of perfusion were injected into a small normal dog. The ani- mal vomited once. There was a rise of 1°C. in temperature. The pulse remained good. No marked symptoms of intoxication were present. Eight cubic centimeters of the 10-minute sample were injected intraperitone- ally and 2 cc. were injected subcutaneously into a 75-gram rat. No toxic action was noted. This test was repeated by injecting 5 cc. intraperitoneally into a 38-gram rat. No toxic action was noted. The same amount of the 40-minute perfusate sample was injected intraperitoneally into a 62-gram rat. No toxic action. Perfusion below diaphragm with red blood cells in modified Locke's solution Ex-periment 11. Female shepherd pup. Weight 2280 grams. In preparation of the perfusate blood corpuscles were obtained from the blood of a normal dog bled several hours previously into a 1 per cent sodium oxalate solution. The red cells were centrifugalized and washed in Rous' gelatin-Locke's solution minus calcium. The packed cells were then suspended in a similarly prepared calcium-free gelatin-Locke's solution in the ratio of one part of corpuscles to five of the saline mixture. Under ether anesthesia the animal was bled and the cannulae were arranged to perfuse all of the tissues below the diaphragm. Ten minutes were consumed in arranging the cannulae. The temperature of the perfusate was between 35° and 38°C. The pulse pressure was about 10 mm. mercury. Due to clots in the gauze two stops were necessitated over a period of 5 minutes each. One occurred soon after the beginning of perfusion and one some minutes later. The dura- tion of the perfusion was 1 hour. During this period the animal increased 760 grams in weight. Autopsy showed about 75 cc. of pale bloody fluid in the abdominal cavity. Marked edema was present about the pancreas and throughout the mesentery. Hemorrhagic spots were observed over the surface of the kidney and stomach, about the ovary and throughout the muscles and fascia of the hind legs. The liver on section was translucent. On section the kidney showed indefinite dark hemorrhagic spots up to 0.5 cm. in diameter. The whole organ was dark and congested. The stomach contents were normal. Mucus and bloody fluid were present in the small intestine. Congestion and bloody intestinal contents were more prominent in the lower part of the small intestine. The perfusate showed a moderate grade of hemolysis before perfusion but less after 15 minutes of perfusion. Moderate hemolysis existed at the end of the perfusion. Bacteriological cultures showed 500 bacteria per cubic centimeter in samples taken at end of perfusion. 108 A. E. BELT, H, P. SMITH AND G. H. WHIPPLE Two hundred and eight cubic centimeters of the perfusion fluid obtained at the end of the perfusion were injected intravenously into a normal small dog. With the exception of a temperature rise of 1.5° and some shivering there were no signs of intoxication. The two experiments (expers, 9 and 11) show the results of a perfu- sion of all the organs and tissues below the diaphragm by a red cell Locke's solution mixture. It is to be noted especially that there is marked edema of retroperitoneal tissues and the pancreas. This edema is invariably present in considerable amount except when whole de- fibrinated blood is used as perfusate. We accept the edema as one indication of tissue or ceil injury. The same is true of hemorrhagic areas and ecchymoses, but some of them may be due to emboli. The marked congestion of the intestinal mucosa with the escape of blood- tinged fluid and mucus is also a valuable index of injury. This is a familiar reaction noted in dogs dead from anaphylaxis or large doses of proteose or from surgical shock. The perfusate in both these experi- ments contained no poisonous substance for normal dogs and white rats. Hemolysis is always present in slight or moderate degree in all our experiments. We are inclined to explain a part of this hemolysis by the cell injury in organs or tissues and this cell injury reacts unfavorably upon the red cells with resulting hemolysis. We realize that the dog's red corpuscles are most fragile and that the red cells are subjected to much mechanical injury in these experiments. Other observers may choose to explain all this hemolysis upon a purely traumatic basis. Perfusion below diaphragm ivith diluted defibrinated blood Experiment IS. Male bull pup. Weight 2150 grams. Two parts of defibrinated blood obtained from a normal dog were diluted with one part of gelatin-Locke's solution. The animal was anesthetized with ether, the thorax opened and the cannulae inserted in such a way that the per- fusion medium was forced into the aorta just above the diaphragm and the blood received from the inferior vena cava just below the heart. In this way the entire region below the diaphragm was perfused. The temperature of the perfusion medium was maintained at about 32° to 38°C. The systolic pressure varied from 95 to 120 mm. mercury with a pulse pressure of 20 mm. mercury. The return flow was accidentally occluded for a few seconds at the beginning of the experiment. The perfusion lasted 1 hour. Examination of the region perfused showed hemorrhagic streaks in the dia- phragm and gall bladder. Numerous small areas of hemorrhage accompanied by a considerable amount of edema existed about both kidneys. The liver showed considerable congestion and edema. The lobules of the pancreas were FACTORS CONCERNED IN PERFUSION OF LIVING ORGANS 109 distinctly separated by edema. The spleen was quite dark. The stomach showed considerable engorgement with sub-mucous hemorrhages. Externally the small intestines were spotted by numerous small subserous hemorrhages. The intestinal mucosa showed diffuse congestion while the lumen contained a little dark mucus. The colon was more nearly normal in appearance; however, a few small sub-serous hemorrhages were seen. The hind legs showed no hemor- rhage or edema. There was a weight increase of 400 grams. The perfusate showed slight hemolysis before perfusion and a moderate grade of hemolysis at the end of perfusion. The perfusate obtained at the end of per- fusion contained 39 mgm. of non-protein nitrogen and 16 mgm. of urea-nitrogen per 100 cc. TABLE 34 Perfusion below the diaphragm with defibrinated blood. Experiment 16 TIME COLOR OF CENTRIFUGAL- IZED PERFUSATE CARBON DIOXIDE CAPACITY PER 100 CC. HYDROGEN ION CONCENTRA- TION REMARKS Before Pale pink Rose Rose Deep rose 38.5 15.7 26.1 12.8 7.5 7.3 7.4 After 20 minutes After 40 minutes More blood added At the end Perfusion below the diaphragm with defibrinated blood Experiment 16. Normal male bull pup. Weight 4.2 pounds. Under ether anesthesia cannulae were inserted for perfusion below the dia- phragm. This region was perfused for 30 minutes with pure defibrinated blood at a rate of 100 cc. per minute. The cannulae were then shifted to the lower ab- dominal vessels and the hind legs perfused at a rate of 25 cc. a minute for 60 min- utes, with the same perfusate. The perfusate was aerated with pure oxygen and was maintained at a temperature varying from 36° to 39°C. The pulse rate was 141 a minute. Intestinal peristalsis was quite conspicuous at the beginning of perfusion but was less noticeable after the perfusion had been in progress for about 5 minutes. During this period the abdominal wall was very sensitive to touch and contracted violently when touched. At autopsy a large amount of clear straw-colored fluid was noted in the abdomi- nal cavity. The animal had gained 200 grams in weight as a result of the per- fusion. The liver was slightly translucent although apparently normal. The spleen was somewhat congested. The pancreas showed no edema. The kid- neys were slightly congested in the pyramidal areas. The stomach was normal. The duodenum was likewise normal but the ileum showed a mucosa congested and dark red with grey mucoid material in the lumen. The hind legs showed no edema and were quite dry in appearance. A few petechial hemorrhages appeared in the fascia. Histological sections: Pancreas and kidney are normal. The spleen and liver show capillary congestion but normal parenchyma cells. The stomach and small intestine are normal except for slight congestion of the ileum. 110 A. E. BELT, H. P. SMITH AND G. H. WHIPPLE In these two experiments (expers. 13 and 16) we used diluted or whole defibrinated blood. The general autopsy picture following the use of whole blood is almost normal and the lack of the edema we believe is to be explained on this ground. Even in the last experiment we note the development of ascites which of course indicates circulatory ab- normality. There is further a distinct acidosis to be explained by inadequate aeration of the blood. Perfusion with whole defibrinated blood — toxic proteose added Experiment 17. Adult male poodle dog. Weight 10.25 pounds. Under ether anesthesia the animal was bled. The arterial cannula was placed just above the bifurcation of the iliacs. The venous cannula was inserted in the inferior vena cava just above the renal veins. Thirty minutes were con- sumed in bleeding, arranging the cannula and starting the perfusion flow. The hind legs were perfused for 32 minutes with defibrinated blood. To the 500 cc. of perfusate then remaining in the apparatus 100 cc. of a proteose solution (lethal dose is 2 cc. per pound body weight, adult dog) were added and the perfusion continued for 12 minutes. The perfusate was warmed to a temperature of 35° to 38°C. The rate of flow was 80 cc. per minute until near the end of the experi- ment, when it decreased to 50 cc. per minute. The aeration of the perfusate was excellent. The proteose solution was prepared as described elsewhere (23) from the material of the obstructed intestine. Autopsy of the perfused dog showed slight icterus of the tissues of the hind legs but no hemorrhages or edema. A few small hemorrhages were found in the right testicle. The pelvic organs were negative. At the end of perfusion there were obtained 300 cc. of. perfusate for analysis. Of this were injected into a 14-pound normal pup, 227 cc, which represented 38 cc. of the original proteose. This would be a fatal dose for an adult dog weigh- ing 19 pounds, provided no proteose had been lost. Pups are more susceptible to proteose intoxication than adults. This dog therefore received a theoretical dose of one and one-half times its lethal dose, assuming that no proteose was lost from the perfusate during the perfusion. The injection of 227 cc. of perfusate caused death in 3 hours with the clinical picture of acute proteose intoxication. Diarrhea and vomiting appeared within I hour after injection and continued until death. There was an initial rise in temperature followed by a drop to .36.8°C. half an hour before death. Autopsy findings showed exquisite splanchnic engorgement especially marked in the mucosa of the small intestines, which was a velvety purplish-red coated with mucus — described and pictured elsewhere (24). The results of examination of the perfusate are given in the table below (table 35). FACTORS CONCERNED IN PERFUSION OF LIVING ORGANS 111 Perfusion with whole defihrinated blood — toxic proteose added Experiment 18. Under ether anesthesia a small female mongrel dog was bled. Cannulae were inserted preparatory to perfusing all of the body below the dia- phragm. Defihrinated blood warmed to 35° was perfused through this region under a mean pressure of 110 mm. of mercury. The pulse pressure varied between 5 and 15 mm. of mercury. The pulse rate was 120 per minute. Aeration of the perfusate was not quite as satisfactory as usual. Actual perfusion commenced 30 minutes after bleeding the animal. After the flow has been maintained for 20 minutes 100 cc. of proteose solution were added to the 250 cc. of defihrinated blood then remaining in the apparatus. Perfusion was continued for 15 min- utes. The rate of flow was 150 cc. a minute at first, but gradually decreased to 85 cc. a minute toward the end of the experiment. Marked intestinal peri- stalsis was present during the first few minutes of perfusion; blood-tinged feces later. Autopsy showed a moderate quantitj' of pale blood-tinged fluid in the peri- toneal cavity. The animal had gained 180 grams in weight during the experi- ment. The liver showed edema and small hemorrhages throughout. Numerous small hemorrhages as well as several larger ones were seen in the wall of the gall bladder. The pancreas was negative except for one small hemorrhage. The mesenteric honphatics contained blood-stained fluid. The adrenals contained several small hemorrhagic areas. The kidneys were negative. The spleen was small, dark and translucent. The stomach showed one fairly large but no small hemorrhages. The mucosa of the duodenum was engorged. The intestinal lumen contained an excess of thin bloody fluid with little mucus. It is pos- sible that some of the hemorrhages noted above are to be explained by short periods of high blood pressure during the periods of perfusion. As was stated above, 100 cc. of the proteose solution (lethal dose is 2 cc. per pound bod)' weight, adult dog) were added to the 250 cc. of perfusate then in circulation and the perfusion continued for 15 minutes. Of the final perfusate 77 cc. were injected intravenously into a normal 11-pound pup (no. 17-181). It is evident that these 77 cc. contained 22 cc. of the original proteose solution pro- vided none of this toxic material had been removed during perfusion by the tis- sues of the animal perfused. The reaction of the animal might be expected therefore to be lethal, if no proteose was removed during the perfusion through the tissues of the first dog. The reaction to this intravenous injection was typical for a sublethal toxic dose of proteose. There was vomiting and diarrhea for 2 hours and much prostration. Recovery was evident in 3 hours and the dog was normal in a few more hours. It appears that some of the proteose had been removed as the amount given was more than a lethal dose for a pup of 11 pounds body weight. The final perfusate was further examined and sho-«-n to contain definite amounts of hemoglobin (hemolysis). Bacteriological examination (plates) showed 40,000 colonies per cubic centimeter of the perfusate. The non-protein nitrogen at beginning of perfusion was 34.7 mgm. per 100 cc. of perfusate and at the end was 35.8 mgm. 112 A. E. BELT, H. P. SMITH AND G. H. WHIPPLE In these two experiments (expers. 17 and 18) the perfusion was done with whole defibrinated blood to insure a minimum injury of the per- fused tissues. After the initial perfusion a standardized toxic solution was added to the perfusate and again circulated as before. The per- fusate at the end of the experiment was tested on a normal dog and in this way it was demonstrated that some of the poison had been removed. It is eas3" to show that large doses of this toxic proteose are rapidly re- moved from the circulation of a dog. Following intravenous injection of large amounts of toxic proteose it is possible to demonstrate its presence for 2 to 3 minutes in the blood stream but not after 5 minutes. The presence of bacteria as noted in this and other experiments will not seriously disturb the reaction. If anything, their presence will increase the toxicity of the perfusate mixture. These bacteria probably gain TABLE 35 Perfusion with whole defibrinated blood — toxic proteose added. Experiment 17 TIME HEMOLYSIS PER CENT BLOOD CELLS UREA NITROGEN NON- PROTEIN NITROGEN HYDROGEN ION CARBON DIOXIDE CAPACITY Before perfusion After one-half hour. . . . On adding proteose. . . . At end of perfusion. . . . Moderate Moderate Moderate Slight 47 50 47 45 mgm. per 100 cc. 19 16 18 20 mgm. per 100 cc. 26 31 30 89 7.5 7.4 7.4 7.4 16 16 19 13 entrance in part through the intestinal tract and in part are added by the manipulation of the perfusate. Efforts were made to preserve the circulating machinerj' in as near a sterile condition as possible but there are many possibilities for introducing contamination. The "proteose solution" used in these experiments is prepared from material obtained from obstructed intestines or closed intestinal loops in dogs. The material is precipitated by five volumes of alcohol, th« precipitate dissolved in water and the protein removed by boiling in dilute acetic acid solution. A second precipitation with alcohol is often employed. The final solution is an opalescent fluid which con- tains proteose-like materials. This fluid material is then standardized by intravenous injection in normal dogs, as has been described else- where (23). FACTORS CONCERNED IN PERFUSION OF LIVING ORGANS 113 Large infusion of Locke's solution into portal vein Experiment 21. Dog 17-125. Normal adult mongrel, black and tan. Weight 43 pounds. Under ether anesthesia" and with sterile precautions the abdomen was opened and the hepatic-pancreatico-duodenal and pancreatico-duodenal arteries were ligated. Sterilized calcium-free Locke's solution, 1750 cc, was warmed to ap- proximately 36°C. and injected at a rate of 55 cc. a minute into the portal system through a small venous branch in the mesentery. The abdomen was closed. The animal showed no severe reaction for some time but died 3 days later. Autopsy performed several hours later shows considerable post-mortem change. Dark red softened areas containing bubbles of gas are seen scattered throughout the liver. The serosa of the intestines is quite red. The mucosa shows moderate engorgement and is covered by a buttery exudate. The coil of intestine to which the perfused vein was distributed differs in no way from the other parts of the small intestine. Kidneys are negative. The total non-pro- tein nitrogen of the blood shows but slight alteration from the original value as a result of the experimental procedure. Histological sections: The picture is somewhat confused by post-mortem changes but it is clear that there are scattered areas of liver cell necrosis which are ante-mortem and presumably related to the experimental procedure. These areas include many liver lobules and present a uniform necrosis with scattered leucocytes between the liver cell strands. Bile ducts and blood vessels are nor- mal and no evidence of vascular thrombosis is observed in any sections. Other sections show normal liver parenchyma. Other tissues present nothing of importance. Large infusion of Locke's solution into splenic artery Experiment 23. Dog 17-200. Young male collie. Weight 29.5 pounds. Normal except for a slight attack of distemper. Under ether anesthesia and with sterile precautions the abdomen was opened. A cannula was inserted into the splenic artery in such a manner that the upper arm of the pancreas, a part of the duodenum and stomach, as well as the liver, were perfused by 2000 cc. of sterile calcium-free Locke's solution injected at a rate of 50 cc. a minute. The saline was injected at room temperature. Splen- ectomy followed the perfusion. The pancreas showed moderate edema at the end of the experiment, but the dog was not severely shocked. Later a state of intoxication slowly developed and at the end of 36 hours death was imminent. The animal was killed by ether. Autopsy performed at once shows a little blood-stained peritoneal fluid. The liver shows a moderate grade of cloudy swelling. The subserous tissues of the gall bladder are thick and edematous. A number of sub-serous ecchymoses are scattered over the small intestine. Several hemorrhagic areas are present in the mucosa of the small intestine. Hemorrhages and fat necrosis are rather pro- nounced in the upper arm of the pancreas. Histological selections: Organs are normal with the exception of the pancreas and liver. The pancreas shows extensive hemorrhagic necrosis and much nec- THE AMEBICAN JOURNAL OF PHYSIOLOGY, VOL. 52, NO 1. 114 A. E. BELT, H. P. SMITH AXD G. H. WHIPPLE rosis of fat and gland parenchyma. There are no thrombus masses noted in any of the vessels. The head of the pancreas is essentially normal. The liver shows scattered clumps of polymorphonuclear leucocytes and evidence of injury to small clusters of liver cells in various portions of the liver lobules. Large infusion of Locke's solution into the portal vein Experiment 24. Dog 17-203. Adult male mongrel. Weight 29.5 pounds. Slight distemper. Under ether anesthesia the abdomen was opened and the hepatic artery clamped. One of the larger splenic veins was isolated, a cannula inserted and 1500 cc. of sterile calcium-free Locke's solution warmed approximately to body temperature were injected into the portal system over a period of 18 minutes. The clamps were then removed from the hepatic artery. Splenectomy was per- formed and the abdomen was closed. The temperature rose to 40°C. shortly after the operation and the animal vomited once; otherwise no clinical disturbance was noted. Complete recovery ensued. The animal was killed 7 days later. The autopsy was negative. No alteration could be made out in the liver. To obviate the mechanical difficulties inherent in organ perfusion and to get information concerning the direct effect of Locke's solution upon tissue and organ cells we performed a number of experiments of which experiments 21, 23 and 24 are examples. The first experiment (exper. 21) shows a fatal reaction following a large infusion in the portal vein after ligation of the branches of the hepatic artery to limit the blood flow through the liver. We have explained this reaction as due in part to injury of the liver cells by contact with the mixtures of Locke's solution and blood. It is known that ligation of the hepatic artery will cause no disturbance in the dog. It seems hard to account for these areas of hver necrosis except as due to the action of the portal blood diluted by the large infusion of Locke's solution into the portal vein. It is to be noted (exper. 24) that a similar experiment was tol- erated without ob%dous liver injury but the occlusion of the hepatic artery in this experiment was onl}^ temporary. The pancreas necrosis was surely caused by the perfusion of the splenic artery (exper. 23). It may be objected that perfusion in this way against the arterial stream will cut off the tissues from oxygen bj- wash- ing away all available red cells. We are inclined to believe that ar- terial collaterals which are numerous in this region will insure the presence of the necessary number of oxygen-carrying red cells. This objection cannot appl^- to experiment 21. FACTORS CONCERNED IN PERFUSION OF LIVING ORGANS 115 DISCUSSION In attempting an analysis of our own experiments we wish to draw freely on the published work of other investigators. We wish to keep constantly in the reader's mind that physiological perfusion of an}^ organ is a matter of extreme difficulty and often great confusion is introduced by such methods which are intended to simplify the study of organ function. By physiological perfusion we mean a perfusion adequate to maintain the organ in its normal physiological activity. In the first place let us inquire what criteria of tissue abnormality we have. What sort of evidence is going to lead us to pass judgment concerning the physiological condition of tissues? There are certain conditions under which we may ascertain what is going on in the tissues by a direct observation of the functional activity of the part perfused. For example, in perfusing the kidney the quantity and quality of the urine secreted furnishes some evidence concerning the condition of that organ. The reduction within the organ of oxyhemoglobin to hemo- globin was noted by the earliest observers and is indicative of meta- bolic activity of some nature on the part of the perfused tissues. The nature of the heart beat is indicative of the condition of the perfused heart, although Magnus (25) has shown that if such an inert substance as hydrogen gas be perfused through the coronary arteries heart beats will be stimulated. Sollmann (26) showed that the same result fol- lowed perfusion with cottonseed or paraffin oil. In such cases the per- fusion fluid cannot be thought of as being a nutrient fluid; on the other hand it is not altogether impossible that, as was suggested by Sollmann, the heart beats may be stimulated by purely mechanical factors. For these reasons we must not hastily conclude that, because the gross mechanical movements simulate those occurring in the intact animal, the preparation is in fact an example of normal physiological activity. A criterion as to the condition of the perfused medulla is furnished by observing whether the medulla continues to maintain its normal control over the heart and muscles of respiration. In addition to direct observation of the functional activity of an organ perfused, we have still other kinds of evidence which help us to judge concerning the condition of the tissue. Thus the rate of flow through the vessels is in some cases a valuable indicator for it is a general rule that tissue injury brings about in some way or other a decrease in the rate of flow through the part. We have, in addition, the still more crude signs of tissue injury: edema, congestion and hem- orrhage. 116 A. E. BELT, H, P. SMITH AND G. H. WHIPPLE Of the factors in the procedure of perfusion whose variations might bring about injury to the part, the following may be mentioned as being perhaps the most important: aeration of the perfusion medium, composition of the perfusion medium, interruptions in continuity of flow, temperature of the perfusion medium when it enters the perfused organ, mean pressure and pulse pressure. It should be realized that a perfusion experiment is no better than its weakest point. If any of the above factors react in such a way as to cause injury to an organ, perfection of the other factors will not rem- edy the defect. It is also conceivable that when several organs are being perfused simultaneously, injury to one organ or tissue may react injuriously on others. Concerning the effect on the tissues of composition of the perfusate, the literature contains many references to condition of the tissues as shown by functional activity. The injurious effects of foreign blood have been known since the time of Provost and Dumas (27) when this fact first began to be recognized through the failure of foreign blood to act normally after transfusion. Though repeatedly shown to be harm- ful in its effects on tissues of another species, foreign blood has been used in perfusions even as late as Brodie (13) who says that ox, sheep or horse blood cannot be used in the perfusion of organs taken from dogs. He finds that as soon as foreign blood is supplied to the perfused heart the beat becomes irregular. The heart next goes into fibrillary twitchings and cannot be recovered from this state even with the ani- mal's own blood. Although defibrinated blood had been used in the transfusion experi- ments of Provost and Dumas (27) without the observation of harmful results, Magendie found it incapable of carrying on the normal function of the circulating medium after transfusion. In a series of experiments in 1822 (28) and again in 1838 (29) he presented experimental data to show that the lack of fibrin, reduced through repeated bleeding, de- fibrination and reinjection of the defibrinated blood gives rise to a serous and bloody transudate into the lungs and intestine with the death of the animal. The weight of Magendie's name behind such a statement did much to discredit defibrination in the eyes of other workers, but eventually Bischoff (30), Goll (31), Polli (32), Panum (33), Ponfick (34), and many others began to turn the weight of experimental evidence against a belief in the extreme toxicity of defibrinated blood when used in trans- fusions between animals of the same species FACTORS CONCERNED IN PERFUSION OF LIVING ORGANS 117 More recently, by means of perfusion experiments, Stevens and Lee (35) and Brodie (13) present evidence of slight vasoconstriction due to the use of defibrinated blood as a perfusion medium. Their work again points to an injurious action of defibrinated blood when substi- tuted for the normal circulating medium. However, Stevens and Lee believe that the slight vascular contraction which they note can be readily counteracted with pharmacological agents. In 1903 Pfaff and Vejux-Tyrode (36) found defibrinated blood def- initely injurious to the kidney of the dog. Quantities of from f to ^g of the total blood were withdrawn from the carotid artery, whipped, filtered and reinjected into the jugular vein. The repetition of this procedure resulted in the appearance of albumin, hemoglobin and red blood cells in the urine and finally cessation of secretion. However, a rapid return to normal was effected in these animals by bleeding fol- lowed by direct transfusion of whole blood from a normal dog. It would seem that the kidney may be unusually sensitive to this procedure. It is certain that in those of our animals which underwent quite com- plete defibrination (37) — (see expers. 323 and 324) — there were no clinically evidenced signs of injury or toxic manifestations. This may also be said of those experiments of \Miipple and Goodpasture (38) in which quite complete defibrination was also effected. The importance of a physiological balance of the normal inorganic salts of the blood is generally recognized. Solutions containing ab- normal quantities of these salts have been shown to be toxic to the per- fused heart. Hooker (22), (39) showed that in perfusion of the res- piratory center a balance of potassium and calcium is essential for a normal function of the preparation. As was early shown by several investigators and more recently reemphasized by Hooker (19) and br- others, and as we have found in our experiments, saline solution has the property of setting up such a condition in the tissues that the rate of flow gradually decreases. The effects of variations in composition of perfusion media may also be manifest from the morphological side. Brodie (13) shows that edema results from the use of foreign blood. Hamel (10) shows that edema results in organs perfused with saline under pulsatile pressure. Similarly the kidney when perfused with Locke's solution exhibits more edema then when perfused with defibrinated blood (19). These re- sults are entirely in accord with our experience. Perfusion with pure Locke's solution almost invariably produces an extreme grade of edema. Dog's defibrinated blood diluted with Locke's solution produced less, 118 A. E. BELT, H. P. SMITH AXD G. H. WHIPPLE and pure defibrmated blood produces very little demonstrable edema. It is difficult to explain just why dilution with Locke's solution should produce edema. In several of our experiments we carried out hydrogen- ion and carbon dioxide determinations on the perfusate and it is inter- esting to note that in cases of marked edema there was a rise in the hydrogen ion concentration and a fall in the carbon dioxide capacity. Whether the edema is the result of the acidosis or not, there still re- mains the question as to what is the cause of the decrease in the buffer substances. Perhaps it maj' be attributable in part at least to insuf- ficient oxidation in the tissues. Poor oxidation may result from in- sufficient oxygen-carrj'ing capacity of the perfusate or from inadequate aeration of the perfusate in the artificial lung, or may result from a decreased rate of flow through the anhnal and a stagnation of the blood in the tissues with consequent asphjTda. A factor which has been shown to be of considerable importance in causing tissue injury is that of loss of time in establishing the artificial perfusion after interrupting the normal relations. !Most of the earlier workers paid but scant attention to this phase of the problem. In man}' experiments several hours elapsed before any attempt was made to reestablish the flow. Grube (40) mentions that in perfusion of the hver with defibrinated blood to which glucose had been added, the gly- cogen content of the liver rises, but only in case the circulation is very promptly reestabhshed. The deleterious effect on the kidney of tem- porary anemia is weU known. IVIomentarj^ compression of the renal vessels may cause a cessation of secretion for many minutes. The effect of compression of the cerebral vessels has been known since very early limes. The duration of such anemia necessary to produce irreparable damage was long ago studied by Astley Cooper (41). Signs of activity can be restored to the brain of the isolated head provided only that the perfusion is promptly commenced (6), (42), (43) and (44). Skel- etal muscle is capable of surviving much longer periods of anemia than is the case of brain or kidney. IMunk (45) holds that in perfusion of the kidney if the flow is not promptly commenced the vessels become nar- rowed, the perfusion flow rendered difficult and a delay in the formation and flow of urinary fluid occurs. Recently Bainbridge and Evans (21) have succeeded in perfusing the kidney without any interruption what- soever in the continuity of flow. A great deal has been said in the literature concerning the value of pulsatile pressure. As has already been noted, Ludwig and Schmidt (7) observed that with constant pressure the rate of outflow from the FACTORS COXCERXED IX PERFUSION OF LIVING ORGANS 119 perfused tissue decreases, but that recovery occurs if the flow is halted for a while. Similar observations were subsequently made by many workers. The recognition of the importance of this factor is evidenced by the numerous forms of apparatus devised to accompUsh this end. Hooker (19) holds that in the perfused kidney the amount of urinary filtrate formed varies directly with the magnitude of the pulse pressure. The amount of proteins in the urinary filtrate varies inversely with the magnitude of the pulse pressure. The rate of blood flow through the organ varies directly with the magnitude of the pulse pressure. Recently Gesell (46) has been able in the intact animal to abohsh almost completely the pulse pressure in the renal arteries without interfering with the normal mean blood pressure. The result of this alteration was an immediate and practically complete cessation of urinary secre- tion. SUMMARY Physiological perfusion of organs is a matter of great difficulty. Much of the work done with organ perfusion is of little value because a proper appreciation of the limitations of the method does not obtain among laboratory workers. The use of physiological sahne, Locke's solution or various modified solutions with or without red blood corpuscles does not permit of physiological perfusion of organs. The contact of these salt solutions with the tissue cells will result in profound injury or actual cell destruc- tion. Any deduction made from experiments of this nature must be limited by these facts just outlined. BIBLIOGRAPHY (1) Kerr, Hurwitz and Whipple: This Journal, 1918, xlvii, 356, 370, 379. (2) Guthrie: Arch. Int. Med., 1910, v, 232. (3) Le Gallois: Experiments on the principle of life (Transl. by X. C. and J. G. Nancrede), Philadelphia, 1813. (4) Kay: Journ. des progres d. sci. et inst. Medic, 1828, x, 67; xi, 18 (cited by Brown-Sequard, see (6)). (5) Lobell: Diss. Marburg, 1849 (cited by Jacobj, see (11)). (6) Brown-Sequard: Journ. de la Physiol, de I'Homme et des Animaux, 1858, i, 95, 353. (7) LuDWiG AND Schmidt: Leipziger Ber., 1868, xx, 12. (8) VON Schroder: Arch, exper. Path. u. Pharm., 1882, xv, 364. (9) VON Fry and Gruber: Arch. Anat. u. Physiol. (Physiol. Abth.), 1885, 519. (10) Hamel: Zeitschr. Biol., 1888, xx\', 474. (11) Jacobj: Arch, exper. Path. u. Pharm., 1890, xxvi, 388. 120 A. E. BELT, H. P. SMITH AND G. H. WHIPPLE (12) Jacobj: Arch, exper. Path. u. Pharm., 1895, xxxvi, 330. (13) Brodie: Journ., Physiol., 1903, xxix, 266. (14) Hoffmann: Arch, gesammt. Physiol., 1903, c, 242, 249. (15) Richards and Drinker: Journ. Pharm. Exper. Therap., 1915, vii, 467. (16) Friedmann: Biochem. Zeitschr., 1910, xxvii, 87. (17) Mandel: Zeitschr. f. biol. Technik u. Methodik, 1908, i, 44. (18) Kingsbury: Journ. Biol. Chem., 1916, xxviii, 167. (19) Hooker: This Journal, 1910. xxvii, 24. (20) Martin: Studies from the Biol. Laby., Johns Hopkins Univ., 1881, ii, 119. Reprint in Memoirs from the Biol. Laby., Johns Hopkins Univ., 1895, iii, 1. (21) Bainbridge and Evans: Journ. Physiol., 1914, xlviii, 278. (22) Hooker: This Journal, 1915, xxviii, 200. (23) Whipple and Cooke: Journ. Exper. Med., 1917, xxv, 461. (24) Whipple, Stone and Bernheim: Journ. Exper. Med., 1913, xvii, 286. (25) Magnus: Arch, exper. Path. u. Pharm., xlvii, 200. (26) Sollmann: This Journal, 1906, xv, 121. (27) Prevost and Dumas: Ann. de Chimie, 1821, xviii, 294. (28) Magendie: Journ. de Physiol., 1822, ii, 338 (cited by Jullien, Transfusion du Sang, 1875). (29) Magendie : Legons sur les Phenomenes Physiques de la Vie, 1838, ii. (30) Bischoff: Arch. f. Anat. Physiol, u. Wissensch. Med., 1835, 347. (31) Goll: Zeitschr. f. rat. Med., 1854, iv, 78. (32) PoLLi: Arch. gen. de Med., Oct. and Nov., 1854 (cited by Jullien, Trans- fusion du Sang, 1875). (33) Panum: Arch. path. Anat. u. Physiol., 1864, xxix, 241. (34) Ponfick: Arch. f. Path. Anat. u. Physiol., 1875, Ixii, 273. (35) Stevens and Lee: Studies from the Biol. Laby., Johns Hopkins Univ., iii, 99 (cited by Pfaff and Vejux-Tyrode, see (36)). (36) Pfaff and Vejux-Tyrode: Arch. f. exper. Path. u. Pharm., 1903, xlix, 324. (37) Whipple, Smith and Belt: This Journal, 1920, Iii, 72. (38) Whipple and Goodpasture : This Journal, 1914, xxxiii, 50, 70. (39) Hooker: Journ. Pharm. Exper. Therap., 1913, iv, 443. (40) Grube: Journ. Physiol., 1903, xxix, 276. (41) Cooper: Guy's Hospital Repts., 1836, i, 457. (42) Laborde: Cited by Hayem and Barriere (see (43)). (43) Hayem and Barriers: Arch, de Physiol., 1887, x, 1. (44) Guthrie, Pike and Stewart: This Journal, 1906, xvii, 344. (4.5) Munk: Arch. path. Anat. u. Physiol., 1887, cvii, 291. (46) Gesell: This Journal, 1913, xxxii, 70. THE SEASONAL VARIATION IN THE GROWTH OF BOSTON SCHOOL CHILDREN W. T. PORTER From the Laboratory of Comparative Physiology in the Harvard Medical School Received for publication February 28, 1920 In 1892 I secured the measurement of weight and height, and other physical dimensions, from 34,500 boys and girls in the pubhc schools of St. Louis. In this investigation, as in similar studies upon which our present standards are chiefly based, all the measurements were col- lected at one time, once for all. The children were then distributed by sex and age, and the median weight and height, etc., determined for each year. This is tlie "generalizing" method. Its economies are great. Thus, in the St. Louis investigation, a little more than a mil- lion data were harvested in eleven weeks, and work in the class room was interrupted for only seven half-hour periods. Far different is the "individualizing" method. This procedure de- mands the measurement of the same child again and again, throughout its period of growth. The measurements must begin at the earliest age with many thousand children, lest death and desertion so thin the J ranks that the survivors will be too few for safe statistical treatment. The individuaHzing method demands, therefore, a formidable expendi- ture in time and effort through many years. Toilsome as this task may be, it cannot be foregone. For the generalizing method conceals a grave flaw; it does not give the grow^th of the individual child. ^ With this in mind, I asked the Boston School Committee, in 1909, to measure the height and weight of several thousand of the youngest children and to repeat the measurements monthly in the same children throughout their school life. The measurements were made by the school nurses, under the direction of Dr. T. F. Harrington, and after his lamented death they were continued by his successor, Dr. W. H. 1 This defect in the generalizing method and the contrasting advantages of the individualizing method were discussed by me in the Quarterly Publications of the American Statistical Association, December, 1893. 121 122 W. T. PORTER Devine. In this land of criticism, in which our minds are often occu- pied with the real, and still more often with the imaginary defects of our public institutions, the patient, laborious collection of these meas- urements, month after month, year after year, is a monument of devo- tion in which we may all take pride. II These measurements of the weights and heights of Boston children throughout their school life were completed in June, 1919. The sta- tistical analysis began the following month. The first step was to distribute the weights according to age in months. For example, all boys 110 months of age were placed in one group, and the median weight for that group was calculated. It will be observed that the principle of this first distribution is still that of the generalizing method. This principle must be clearly apprehended. In the generalizing method the individual measurements are distributed into groups, by which distribution the personal character of each individual is lost — the individual becomes merely a statistical unit. Whether the meas- urements so distribuited have been made monthly, or yearly, or whether all the children are measured but once and the measurements distrib- uted by years of age, is immaterial; the essential mark of the general- izing method is the loss of personal identity — the transformation of the boy or girl into a statistical unit. In this present investigation the first distribution of boys' weights, described above, is therefore a distribution by the generalizing method. Its fruits are given in table 1, which records the median weight of boys at each month of age from the 60th to the 176th month, inclusive. In this table, the months are not calendar months, but months of age. For example, the weight opposite 110 months is the median weight of 1226 boys born in 1904, 1905 and 1906. Of these, 33 were born in Oc- tober, 1904; 71 in January, 1905; and 10 in April, 1906. The October boys reached 110 months of age in December, 1913; the January boys in March, 1914; and the April boys in June, 1915. When the data in table 1 are plotted, there results the curve shown in figure 1. This curve of monthly growth is in principle that also ob- tained when the median value is calculated for each year, and these annual values are connected by a line — the curve of growth, old style. These curves rise steadily and smoothly in an unbroken line. Such curves have given rise to much loose thought; for a curve ob- tained by the generalizing method is a statistical and not a personal SEASONAL VARIATION IX GROWTH OF CHILDREN 123 TABLE 1 Weights at each month of age from 60 through 176. Boys horn in 1904, 1905 and 1906 MONTHS POUNDS MONTHS POUNDS MONTHS POUNDS MONTHS POUNDS MONTHS POUNDS 60 42.75 72 44.00 84 47.68 96 52.48 108 57.74 1 41.10 3 44.25 85 48.30 7 52.96 9 58.34 2 41.30 4 44.35 6 48.78 8 53.74 110 58.64 3 42.00 75 45.22 7 49.29 9 53.90 1 59.51 4 41.44 6 44.97 8 49.80 100 54.41 2 60.01 65 42.26 7 45.65 9 50.09 1 54.91 3 60.54 6 42.31 78 45.67 90 50.56 2 55.27 4 60.94 7 42.06 9 46.09 1 50.73 3 55.87 115 61.58 8 41.98 80 46.34 2 51.25 4 55.83 6 61.56 9 42.56 1 47.03 3 51.32 105 56.27 7 61.83 70 43.17 2 47.18 4 51.85 6 56.76 8 62.36 1 44.53 3 47.47 95 52.22 7 57.23 9 62.95 120 63.42 132 69.42 144 75.21 156 82.19 168 92.75 1 64.22 3 70.17 145 76.23 7 83.56 9 93.13 2 64.96 4 70.90 6 76.44 8 84.43 170 93.88 3 65.14 135 71.60 7 77.48 9 85.94 1 97.00 4 65.69 6 72.85 8 78.17 160 86.66 2 92.67 125 66.46 7 72.92 9 78.55 1 87.56 3 94.00 6 66.97 8 73.13 150 79.82 2 88.47 4 96.50 7 67.49 9 73.67 1 80.24 3 89.44 175 96.00 8 67.67 140 73.72 2 80.22 4 90.17 6 95.50 9 68.31 1 74.50 3 80.96 165 91.58 130 68.49 2 74.43 4 80.98 6 92.25 1 68.97 3 74.90 155 81.53 7 91.19 90 80 70 60 50 40 i 1 y X ^^ ■^ \ i \ i j ^ i ^ ^^^ ! \ 60 70 80 90 100 110 120 130 140 150 160 170 Fig. 1. The median weight of boys at each month of age from the 60th to the 176th month, inclusive (table 1). In this figure the months are not calendar months but months of age. The ordinates are pounds. 124 W. T. PORTER phenomenon. This will be clear, if we consider the personal history of two boys, whom we will call John and James. At age six, John is 30 per cent above, and James is 30 per cent below the average. But John's family fall upon evil days; illness and poverty pursue them. The family of his comrade prosper; James spends his vacations in the country, where he has a horse to ride; fresh air and good food do their accustomed work. At age sixteen, James and John have exchanged places; James has risen to 30 per cent above the average and John has fallen to 30 per cent below it. Their personal fortunes have altered; but they are still the same distance from the average. The inhuman average is unmoved by the deplorable fate of John and the happy success of James — to the statistical average, John and James are not persons, but statistical units. Thus the generalizing method per se gives no accurate information as to the growth of the individual child. Nor can this defect be removed by measuring repeatedly the same children throughout their period of growth, either monthly or at any other interval, so long as the resulting measurements are treated as statistical units. Thus, table 1 and figure 1 give accurately the monthly increase in the statistical median value, and th'ey give with accuracy the relation between the size of any individual child and the size of other children of the same age; but they do not give, with certainty, the increase in weight of any child. They are not standards of growth, but merely standards of relative size. Ill The extinction of the individual is not the only indictment which can be brought against the generalizing method. This method, still so much employed, is blind to the possibility of seasonal growth. Consider the case of Hyman Katz, drawn at random from the mass of children whose growth histories are now before us. Hjniian Katz was born in August, 1905. In his ninth year, he gained four pounds between September and January, whereas he did not gain any weight, to speak of, between February and June. The case of Hyman Katz is the starting point of a series of interesting and important observations - It will be seen that Hyman Katz is not an exception, save that he is a somewhat extreme illustration of a law binding on other boys and girls. Examine table 3 which deals with all boys and girls born in August, 1905. Again the growth in weight is much larger in the second half SEASONAL VARIATIOX IX GROWTH OF CHILDREN 125 TABLE 2 Growth in pounds of Hyman Katz 1914-16 MONTH WEIGHT GAIN IN WEIGHT MONTH WEIGHT GAIN IN WEIGHT 1914 pounds pounds 191B pounds pounds September 56.50 January 60.50 0.37 October 57.00 0.50 February 60.50 November 58.25 1.25 March 60.00 -0.50 December 60.13 1.88 April 60.00 May 60.50 0.50 June 61.00 0.50 TABLE 3 Gain in weight of boys and girls born in August, 1905 BOYS GIBL8 September to January February to June September to January February to June pounds pounds pounds pounds 1912-13 3.34 +0.75 1.88 0.12 1913-14 2.17 -0.17 2.50 0.65 1914-15 2.85 +0.67 2.96 0.13 1915-16 1.29 +0.81 1.63 0.71 1917-18 5.85 -0.07 3.19 0.81 1918-19 4.88 +2.90 4.63 1.83 Average 3.40 0.82 2.79 0.71 Ratio 3.40 0.82 4.1 1 2.79 0.71 3.9 1 The year 1916-17 is omitted because the schools were closed in September, 1916, on accoimt of an epidemic. 126 W. T. PORTER of the year than in the first half. This conclusion is supported by the examination of the weights of boys born in all the months of 1905, when these weights are distributed by the months of the year. In table 4 the median weight of all boys born in 1905 is given for each month of the year, and in table 5, the increase in the median weight is recorded. Seasonal growth is again demonstrated. The data in table 4 are reproduced in figure 2. 90 80 70 60 50 40 1911 1912 1913 1914 1915 1916 1917 1918 1919 Fig. 2. The weights of boys born in 1905, distributed by months of the year. The ordinates are pounds. (See table 4.) Fig. 2 is the true curve of growth in weight; it shows the seasonal variations. Compare this figure with Fig. 1, the curve of growth "old style." in which the seasonal variations are lost. We have dealt thus far with the absolute increase in weight. It is desirable to examine also the percentile increase, i.e., the absolute in- crease of each month divided by the weight at the beginning of the month. As would be expected, the seasonal variation again appears. The average total percentile gain in the first five months of the years 1913, 1914, 1917 and 1918 is 1.89; whereas in the last five months of those years, the average total percentile gain is 6.61. SEASONAL VARIATION IN GROWTH OF CHILDREN 127 TABLE 4 The weights of boys born in 1905, distributed by months of the year TEAR ■< B < < n S 5 < >< < s s z es a a S a n o n S m > o a m n S a o pounds pounds pounds pounds pounds pounds pounds pounds pounds pounds 1911 43.00 42.23 42.93 42.83 42.48 42.46 46.20 45.04 45.21 45.31 1912 46.82 46.70 46.62 47.03 46.99 47.03 48.98 49.22 49.98 50.38 1913 50.94 51.49 51.73 52.18 52.05 51.97 53.31 54.16 55.00 55.37 1914 56.45 56.73 57.19 57.38 56.99 56.82 58.34 59.38 60.05 60.79 1915 61.50 62.08 62.38 62.48 62.50 62.72 64.94 65.02 65.66 66.47 1916 67.09 67.88 68.34 68.58 68.32 68.53 71.50 72.18 72.89 1917 73.39 73.81 74.21 74.43 74.81 74.79 76.49 77.32 78.27 79.23 1918 80.17 79.88 81.13 81.38 80.85 81.06 84.29 87.00 87.13 88.00 1919 89.93 90.27 91.17 92.05 91.83 91.55 In table 4, the median weight of all boys born in 1905 is given for each month of the year without regard to the month of age. The seasonal gro'n'th is thereby shown. Thus, in the five months from January to June, 1914, the gain was less than J pound, but in the five months from June to November, 1914, the gain was 3 J pounds. TABLE 5 The monthly increase in weight of boys born in 1905 TEAK >- < •< « c k o 1 = o y B, S < z s «-> o < S K m n s Big D m n s g go o c h BS cs a Ed n 2 s a o > a a z o a « B < S a z u < 1911 1912 1913 1914 1915 1916 1917 1918 -0.77 -0.12 +0.55 +0.28 +0.58 +0.79 +0.42 -0.29 +0.70 -0.08 +0.24 +0.46 +0.30 +0.46 +0.40 +1.25 -0.10 +0.41 +0.45 +0.19 +0.10 +0.24 +0.22 +0.25 -0.35 -0.04 -0.13 -0.39 +0.02 -0.26 +0.38 -0.53 -0.02 +0.04 -0.08 -0.17 +0.22 +0.21 -0.02 +0.21 +3.74 +1.95 +1.34 +1.42 +2.22 +1.70 +3.23 +0.24 +0.85 +1.04 +0.08 +0.83 +2.71 +0.17 +0.76 +0.84 +0.67 +0.64 +0.68 +0.95 +0.13 +0.10 +0.40 +0.37 +0.74 +0.81 +0.71 +0.96 +0.87 +1.51 +0.56 +1.08 +0.71 +0.62 +0.50 +0.94 +1.93 Average . . . +0.18 +0.47 +0.22 -0.16 +0.05 +2.23 +0.96 +0.61 +0.63 +0.98 For convenience we may divide the total of 2.23 pounds from June to Sep- tember into 0.74 pound for each of the three months. 128 W. T. PORTER The errors caused by neglecting the seasonal growth are strikingly shown by comparing a series of weights chosen from table 1 and table 4. Let us take from table 1, twelve w^eights, beginning with 56.76 pounds, and from table 4, twelve weights, beginning with 56.73 pounds. FBOM TABLE 1— BOYS' WEIGHTS DISTRIBUTED BY FROM TABLE 4 — BOTS' WEIGHTS DISTRIBUTED BT MONTHS OF AGE MONTHS OF YEAR pounds pounds 56.76 56.73 57.23 57.19 57.74 57.38 58.34 56.99 58.64 56.82 59.51 * 60.01 * 60.54 58.34 60.94 59.38 61.58 60.05 61.56 60.79 61.83 61.50 * July and August; the vacation months. In these two series, the beginning and the end weights are almost the same, but in the middle of the twelve months the divergence is 2 pounds or more. Fig. 3. The ordinates are pounds and the abscissae are months. The lower curve records boys' weights distributed by months of the year (table 4) ; the upper curve records boys' weights distributed by months of age (table 1). SEASONAL VAKIATION IN GEOWTH OF CHILDREN 129 In figure 3, the two series are shown graphically. The enquirer will wish to know how the marked seasonal variation in weight is so completely masked in table 1 and figure 1, and in the upper curve of figure 3, examples of the statistical method hitherto in use. The answer is that in the method hitherto in use — in which the only criterion is the month of age — the month of age for half the boys will fall in a season of rapid gro\\i;h, while for the other half the same month of age will fall in a season of slow growth. Compare during the months of age from 106 to 118 the growth in weight of boys born in August, 1905, with that of boys born in February, 1905 (table 6). TABLE 6 AGE IN MONTHS SEASON WEIGHT AGE IN MONTHS SEASON WEIGHT Boys born in August, 1905 112 106 December, 1914 June, 1914 pounds 59.96 55.25 118 112 June, 1915 December, 1914 pounds 61.56 59.96 4.71 1.60 Boys horn in February, 1905 112 106 June, 1914 December, 1913 55.75 54.50 118 112 December, 1914 June, 1914 61.17 55.75 1.25 5.42 It is clear that if the age in months is alone considered, the period of rapid growth in boys born in Februar}^ will coincide with the period of slower growth in boys born in August. The two periods will compen- sate each other if the February and the August boj's are treated en masse, as in table 1 and figure 1, and in the standards hitherto so largely used to determine the growth of school children.^ IV Rather than accept a dictum so far-reaching, the reader may here suggest that my entire contention rests on a palpable error — a failure to take into account the heavier garments worn in winter. Table 5 2 My data do not yet show any seasonal variation in the height of school children. THE AMERICAN JOURNAL OF PHTSIOLOGY, VOL. 52, NO. 1 130 W. T. PORTER shows a net increase of 0.76 pounds in the first five months of the year (+0.18, 0.47, 0.22, 0.05, —0.16), whereas the increase in the last five months of the year is 3.92 pounds (+0.74, 0.96, 0.61, 0.63, 0.98). But it will be urged that in May, which falls in the first five months, the winter clothing is laid aside; and in October, which falls in the last five months, it is again put on. A careful examination of the data will show that the seasonal dif- ferences in growth cannot be explained by seasonal differences in the weight of garments worn. Consider the following groups, taken from table 5. INCREASE FROM INCREASE FROM INCREASE FROM pounds pounds pounds Januarj^-February 0. 18 June-September. 2.23 October-November. . . . 0.61 February-March. 0.47 November-December . . . 0.63 March- April 0.22 December- January .. 0.98 Average 0.29 0.74 0.74 Obviously, the second group is composed of summer months and the third group of winter months. The first group requires, perhaps, a word of explanation. In Boston the month of April offers the promise but not the reality of spring. The average temperature rises from 40° on April 1 to 51° on April 30. In 1919, the maximum temperature on April 16 was 42° and on April 25 it was 38°. In only three days of April was the maximum temperature above 61°. It is unlikely that materially lighter clothing is worn during April. For our present purpose April, therefore, should be classed with the winter months. The figures just presented indicate that clothing is, relatively, a negligible factor. Were clothing an important factor, the weights for June to September would show a decrease, as compared with those for January to April, and the weights for October to January would show a considerable increase as compared with those for June to September. No such fiuctuations are apparent. Observe now the figures from March to June (table 5). Increase in weight Pounds March to April +0.22 April to May —0. 16 May to June +0. 05 SEASONAL VARIATION IN GROWTH OF CHILDREN 131 If a change from winter to summer clothing be invoked to explain the low average of growth from January to April, just discussed, it cannot be used again to explain the failure to grow from April to May, and a third time to explain the failure to grow from May to June. It will doubtless be found, taking the average of eight or nine years, that the change from winter to summer clothing is made within a short period and bears a definite relation to the temperature curve. Finally, too much emphasis must not be laid upon hypothetical dif- ferences in summer and winter clothing. Careful enquiry shows that the indoor clothing of Boston public school children does not change during the school year as much as might be supposed. Such changes cannot explain away the periodic growth demonstrated in this investi- gation. With every reasonable allowance for error, it seems impos- sible to deny a seasonal variation in the weights of school children. In figure 2 the seasonal curve departs from the present standard curve by fully 2 pounds, a difference almost three times the average total growth for the first five months of that year. Such deviations justify two deductions: 1. To determine the normal growth in weight, the child must be weighed once a month, or oftener. If the child is weighed less often, the seasonal variation will be missed. 2. True curves of growth in weight demand that the monthly weights be distributed according to the months of the year, and not according to the months or years of age, as is the present custom.^ ' A substantial part of the cost of this study has been paid by the Permanent Charity Fund, whose assistance is gratefully acknowledged. I am much indebted also to the Department of Education in Harvard University for the loan of Mr. L. A. Maverick during the summer of 1919. CERTAIN CHANGES NOTED IN ERGOGRAPHIC RESPONSE AS A RESULT OF TOBACCO-SMOKING THEOPHILE RAPHAEL From the Physiological Laboratory of the University of Michigan Medical School^ Ann Arbor, Michigan Received for publication March 1, 1920 This report is based upon material secured in the course of an investi- gation undertaken with a view to ascertaining the effect of tobacco- smoking upon voluntary muscular work, as determined through ergo- graphic observation. The study was suggested by Lombard's (1), (2) findings and the procedure followed was essentially that utilized by him (q.v.). In the summer of 1915 when, for a month, I had the opportunity of association with Lombard in connection with an ergographic study (3) upon which he was engaged at the time, I noted that my own tracings were uniformly free from those periodic recoveries, after the first loss of power, which were consistently characteristic of Lombard's records (1). These recoveries, or returns of power, failed to manifest them- selves in my curves in spite of any subjective awareness, or even de- sire, and continued physical endeavor. The records showed through- out only a gradual falling of& of performance registry (fig. 1) from maximal contraction to zero. After smoking, Lombard noted that the initial loss of power came more quicklj^, to be followed, however, as in his non-smoking records, by the characteristic periods. In the spring of 1917 I found it possible, personallj^, to undertake a short stud}^ of the effect of smoking upon ergographic response, expect- ing to encounter, if anything, only a diminution in total amount of work performed. Greatly to my surprise, I noted the consistent appearance of periods practically identical with those characteristic of Lombard's smoking and non-smoking records. Since that time it has been possible to elaborate the results somewhat and, in spite of their apparent slen- derness, they were deemed worthj^ of report. Prior to the time of undertaking this study, I had been only an occasional smoker, just able to tolerate one or two mild cigarettes with- 132 EFFECT OF TOBACCO OX ERGOGRAPHIC RESPONSE 133 out suffering any of the acute tobacco effects. The specific procedure followed in this work was, as indicated, that employed by Lombard (1). The ergographic records were taken every second hour, daily, from about approximately 9 a.m. to 7 p.m., care being taken to look away from the record as it was being taken and, as far as possible, to avoid 5 P.M. 7 P.M. Figs. 1-6 all suggestive influence. After a week of preliminary work to secure adaptation to the schedule and to overcome initial practice effect, the actual smoking records were begun; i.e., a cigarette of medium strength was smoked just prior to the moment of beginning a record, at the ex- piration of the two-hour interval. Effort was suspended after the first loss of power. 134 T. RAPHAEL At first, aside from an apparently marked decrease in the extent of the record, no essential change was noted. On the third day of the test, however, it was found that, if effort were continued after the first loss of power, it was possible, in contrast to the results of previous trials, to continue long beyond this point, the curve then showing distinct per- iodicity (figs. 2, 3 and 4), seemingly' identical with that so manifest in Lombard's curves. This finding, it might be well to note, was extremely unexpected and wholly unlooked for. It was found, in addition, that this effect would persist for some hours without additional smoking (fig. 5). When electrical stimulation was applied directly to the muscles by means of the induction current, essentiall}' as described by Lombard in his Laboratory Manual (4), the weight having been materially dimin- ished, the record showed both with and without the use of tobacco a distinctly unbroken character, marked l)y no period formation whatso- ever, thus indicating the causal locus of the periodicity to be further central than the muscles or the nerve endings. The same findings were noted on nerve trunk stimulation also, thus in both respects corrobor- ating Lombard's personal observations. Four other subjects, only one of whom had previously smoked to any degree, were obtained. Although these subjects were untrained and the schedule much less thorough, the findings seemed to bear out in a general way those determinable in my own records. It might be well to indicate, at this point, that these results seem to be at variance with observations made by Hough (5) in tests carried out upon himself, utilizing a somewhat different apparatus. It appears, therefore, that the essential cause of the periodicity is central nervous system fatigue, or depression, induced in this instance by tobacco products absorbed in the act of smoking. On this basis it might not be impossible to account for Lombard's periodicity, for which, thus far, apparently no satisfactory explanation has been afforded. He reports having already smoked for a number of years when the periods were first noted and has since observed that although, as indicated, the first loss of power comes more quickly on smoking days, the periods always appear sooner or later, and have apparently the same character at all times. In view of the fact that the periods are present constantly, even when smoking had been dropped for a period as long as a month, it is not inconceivable that the long-continued use of tobacco may have, in this case, exercised some more or less permanent change in the sub- stance of the central nervous system, specifically concerned in the mech- EFFECT OF TOBACCO ON ERGOGRAPHIC RESPONSE 135 anism of ergographic response. On the other hand, it is equally pos- sible that the periods might have occurred even without smoking, had the central nervous system been originally of a type especially readily susceptible to fatigue. Lombard is unable to state whether any of his eight subjects, two of whom manifested periods, were smokers. In the same way it is conceivable that other central nervous system depressants may produce similar changes in ergographic response. It is distinctly unfortunate that time and opportunity did not permit more extensive investigation, and with a larger series of subjects, both smokers and non-smokers, and it is primarily in the hope of stimulating further work that this brief note is submitted. SUMMARY On the basis of the results obtained in this study, it appears that under the influence of tobacco-smoking, a distinct periodicity may be demonstrated in the curve of ergographic response, arising apparently as a result of central nervous system fatigue or depression. BIBLIOGRAPHY (1) Lombard: Journ. Physiol., 1892, xiii, 1. (2) Lombard: Amer. Journ. Pyschol., 1890, 1. (3) Lombard: This Journal, 1916, xl, 132. (4) Lombard : Laboratory work in physiology, Ann Arbor, 1914. (6) Hough: This Journal, 1901, v, 240. DETERMINATION OF THE CAPILLARY BLOOD PRESSURE IN MAN WITH THE ]\IICRO-CAPILLARY TONOMETER C. S. DANZER AND D. R. HOOKER From the Physiological Laboratory of the Johns Hopkins University Received for publication March 8, 1920 Physiologists have long appreciated the prime significance of the capillary circulation and in recent times clinicians are coming more and more to realize that this part of the circulatory bed is of importance in connection with purely medical problems. The cardio-vascular sys- tem functions for the distribution of the blood but the effective changes pertaining to the nutriinent of the tissues occur in the capillary bed. Here the metabolic interchange takes place; food is delivered to and waste is largely withdrawn from the active cells. It is, therefore, im- portant to know the pressure under which the blood is delivered to the capillaries in different conditions of health and disease and to correlate this knowledge with the functional activity in other parts of the vascu- lar bed. At the present time no method is adapted to such a study either in experimental animals or in the human being. The fundamental observations of Roy and Brown (1) in 1878 on the capillary circulation in the frog were never developed for application to the circulation in the mammal because the method w^as inadequate to this purpose. Quite recently Cannon (2) has emphasized the signif- icance of the capillary bed in traumatic shock, and Dale and Laidlaw (3) and Dale and Richards (4) in histamine shock. These authors con- ceive that in shock the capillary walls are injured by circulating poisons so that they lose tone with the result that a large volume of blood is pooled in the capillary area. This pooUng of the blood in the capil- laries accounts for the primary fall of arterial pressure, which is further accentuated by a transudation of plasma through the injured capillary wall so that there results an actual decrease in circulating blood volume. These two factors are regarded as the basal cause of the circulatory phenomena of shock. Lombard (5) showed a number of years ago that if a drop of oil be placed upon the skin it is possible to see the underlying capillaries, and 136 CAPILLARY BLOOD PRESSURE IX MAX 137 he sought with this information to develop a method of determining the pressure of the blood in these vessels. For this purpose he used a small glass chamber, the floor of which was made of gold beaters' skin with a small hole in the center. The chamber was filled with glycerine and brought into contact with the skin so that it was possible with a microscope to observe through the glass roof and a small opening in the membranous floor the effects which were developed in the under- lying capillaries when the pressure on that area of skin was elevated by forcing more glycerine into the chamber. Although Lombard made a number of observations with this instrument, the technique was so difficult that it was not possible of wide application. With Lombard's experience in mind, we have developed a method which we believe is applicable to the study of capillary blood pressure both in animals and in man, and the purpose of this paper is to describe our method and to give the results which we have thus far obtained with its use. There are some twenty-five articles in the literature bearing upon the determination of the capillary blood pressure obtained by the use of various methods, and the results reported in these articles vary from 7 mm. to 70.5 mm. Hg. for the normal capillary blood pressure in man. It is obvious that if the results by the several methods vary to this extent, they cannot be considered as trustworthy. It is not surprising, therefore, that Friedenthal (6) in a recent review of the subject, reaches the conclusion that capillary blood pressure determinations can be of little practical significance. They are, in fact, of less value than in- ferential deductions drawn from the values of arterial and venous pres- sure. Inferential deductions, on the other hand, are certainly not free from objections, as is shown by the following. Thus Fick (7) thought that pressure in the capillaries is almost as high as in the arteries, and that the greatest fall in pressure occurs in the small veins. His conclusions are based on theoretical considerations. Following Poioselle it is generally beheved that the principal loss of energy occurs in the capillaries because the blood channel is narrowest in the capillary area. On the other hand Campbell (8) has assumed that the greatest fall in pressure occurs in the small arteries and Levy (9) came to the same conclusion as the result of mathematical calcu- lations. Goldmann (10) believes that the principal loss of energy occurs in the arteries. The lumina of the smallest arteries are not much greater than those of the capillaries. The velocity is much greater in the former, however. The result is the great fall of pressure in the small arteries. 138 C. S. DANZER AND D. R. HOOKER Bargolomez (11) has thrown some hght on this unportant question by the following experiments. He cannulated the smallest possible branches of the arteries (middle and posterior auricular arteries) and also the corresponding venous tributaries, and recorded their pressures monometrically. To facilitate the work he dilated the vessels (appli- cation of heat) and introduced needle cannulas. Then he allowed the vessels to return to their original caliber before starting his experiment. Thus he found that the normal blood pressure fall in the capillaries is very slight (about 4 mm. Hg. in the rabbit's ear). He concludes that the greatest fall in blood pressure (90 per cent) occurs normally in the arteries of moderate caliber. It is perhaps as striking to the reader as to ourselves that a trust- worthy method of actually determining the pressure in the capillaries is wanting. Let us for a moment review the previous methods em- ployed for this purpose, The majority of them rest upon the principle, introduced by von Kries (12), that the paling of the skin is dependent upon the pressure and amount of blood in the superficial capillaries, von Kries emplo3^ed a small glass plate of known size and applied it to the surface of the skin and the amount of pressure necessary to cause the skin to pale was interpreted as a criterion of the capillary blood pressure. We shall show that this criterion is inadequate in that the paling of the skin is not necessarily accompanied by a cessation of flow in the capillaries and we believe that it is due rather to an empty- ing of one or more of the venous plexuses lying in the dermis. Spalteholtz (13) has published drawings of a reconstruction model of the blood vessels of the skin. These show that there are three venous plexuses in the cutis and one in the subcuticular layer. We believe that as increasing degrees of pressure are applied to the skin, these plexuses are successively more or less emptied of their contained blood with the result that the skin develops varying degrees of pallor. Our observations make it clear that even extreme grades of pallor are not necessarily associated with a cessation of blood flow in the super- ficial capillaries. It follows, therefore, that pallor of the skin cannot properly be regarded as evidence of capillary collapse. The principle of skin pallor underlying the method of von Kries was likewise employed by von Basch (14), by von Recklinghausen (15) and by Easier (16). von Basch sealed a small glass capsule on to the skin and determined the pressure necessary to cause paling of the under- lying area. CAPILLARY BLOOD PRESSURE IN MAN 139 von Recklinghausen's method was essentially the same. It is that which he employed for the determination of the venous blood pressure. Easier used an instrument which he called an ochrometer; this consisted of two closed chambers with glass roofs and floors of gold beaters' skin which was sufficiently translucent to indicate any changes in color in the underlying skin. Two fingers were used, one to observe the degree of pallor and the other for the control. The microscopic fields were brought up to a single eye piece by means of prisms. One of the chambers was then inflated, causing the membranous floor to press upon the skin until the first evidence of pallor, as determined by the control finger, was observed. von Kries, using his method, gives as the normal capillary pressure 37.7 mm. Hg. This determination was made when the hand was 490 mm. below the crown, a position which we assumed to be approximately at heart level. He likewise reports the capillary blood pressure in the ear as being 20 mm. Hg., and in the mucous membrane of the rabbit's mouth as 33 mm. Hg. Hough and Ballantyne (17) employed the method of von Kries to study the effect of temperature on the capillary blood pressure. Their readings for the normal ranged from 40 to 50 mm. Hg. at a room tem- perature of 20-21°C. When the temperature of the surrounding air was reduced to 6°C., the capillary pressure was 65 mm. Hg., and when the temperature was raised to 26°, it was from 50 to 55 mm. Hg. From our own observations we judge that the temperature effects noted by these authors are associated not with actual changes in capillary blood pressure but rather with the behavior of the superficial venous plexus. We believe a significant factor contributing to the pallor of the skin is a decreased amount of blood in this plexus. If the latter is relatively empty and the skin is correspondingly pale, it will require a greater skin pressure to develop a further paling. The paling under these cir- cumstances would then be due to the pressure emptying the deeper- lying plexuses. The resistance to compression of the skin vessels in- creases as the vessels compressed lie deeper and deeper in the several layers of the skin. It follows, therefore, that when the skin is cold and consequently pale, a greater pressure will be required to produce a further paling than would be the case if the skin were of a normal color. Hough and Ballantyne also report a heightened capillary pressure asso- ciated with arterial constriction, hence they thought that capillary pres- sure was dependent on some other factor than that of the arterial tone. These findings like their findings in connection with the effects of tem- 140 C. S. DANZER AND D. R. HOOKER perature do not agree with our results, and we believe that the latter, as in the case of the former, are associated with the inadequacy of the criterion employed. Natanson (18) has published two papers using the von Kries method. He took for his standard the complete blanching of the skin and studied, among other things, the effect of mass compression of the arm on the capillary pressure in the hand. Using the criterion of complete blanch- ing of the skin, he found the normal capillary pressure to be 70.5 mm. Hg. The highest capillary pressures were observed when the con- stricting band exerted a pressure of 42.5 mm. Hg. When the constrict- ing pressure was raised to 52.3 mm. Hg., capillary pressure fell. He assumes that at this pressure both the arteries and veins were being compressed and draws the conclusion that the capillaries cannot be filled with blood except under the influence of arterial pressure. If the latter fails, the capillaries become bloodless and collapse through the effect of a relative rise of tissue tension. We have not been able to confirm the latter observation, and believe that here again the inade- quacy of the criterion employed by Natanson is sufficient to explain his results. Schiller (19) and also Rotermund (20) made use of von Fries's method slightly modified by the addition of Fick's ophthalmotonometer (21). This addition served merely to make the pressure readings easy since they could be read off on a scale calibrated against a spring. Schiller found that the highest capillary pressure (about 40 mm. Hg.) occurred when the temperature of water applied to the skin was approximately that of the skin, namely, 35°C. Rotermund, using the same method, found the capillary pressure on the skin of the forehead when the subject was in a recumbent position, averaged about 26.8 mm. Hg. He also applied this method to study the influence of age, nutritional state, dj^spnea, nephritis and arterio- sclerosis on capillary blood pressure. von Basch applied his glass capsule method to a study of the capillary blood pressure in human beings and upon experimental animals. The criterion was essentially the same as that employed in the von Kries method. He found that the capillary blood pressure in the rabbit's ear did not differ materially from that of human subjects. In the rabbit's ear the pressure ranged from 21 to 25 mm. Hg., and in the healthy human being it was between 25 and 30 mm. Hg. In human beings he observed that a low capillary blood pressure was frequently associated with high arterial tension, but at times the converse of this CAPILLARY BLOOD PRESSURE IN MAN 141 was true, namely, that a high capillary pressure was associated with a low arterial tension. His inference from these observations led him to the belief that the capillary pressure is independent of the arterial pressure. When the chest of an experimental animal was compressed, there occurred a rise in capillary pressure accompanied by a moderate fall in arterial pressure. He interpreted this result as indicating that the rise of capillary pressure followed venous stasis. Similarly in man chest compression produced a moderate rise of capillary pressure with a corresponding fall of arterial pressure. The fact that the capillary pressure rose less in man than in the experimental animal suggested that the venous stasis was less extreme. He also cut the cervical sjon- pathetic nerve in the rabbit and found an increase in capillary pressure associated with the slight fall of arterial pressure. This pointed to a dilatation of the arterioles of the rabbit's ear and was confirmed by direct inspection. The injection of strychnine caused a rise of arterial pressure with a fall of capillary pressure; in other words, the converse of the above experiment. This was interpreted to mean that a constriction of the arterioles had occurred. von Basch formulated the following hypothesis: that the degree of capillary pressure rise may serve to differentiate between venous stasis and arteriolar dilatation. While the former raises capillary pressure markedly without altering arterial blood pressure, a condition of ar- teriolar dilatation produces a moderate increase of capillary pressure with a corresponding fall in arterial blood pressure. He applied these results clinically and obtained data which he regarded as of distinct value in both diagnosis and treatment. Using Hooker's (22) capsule with the criterion of paling, Briscoe (23), in a study of the Raynaud phenomena, in cases of "irritable heart," found that when the hand was cyanotic the capillary pressure was 36.9 cm. of water as compared with the normal controls which gave a pres- sure of 23.5 cm. of water. In individuals subject to vasomotor changes when the color of the hand was normal, the capillary pressure was 25.3 cm. of water, and that when the same hand was blue the capillary pres- sure was increased to 33.3 cm. of water. von Recklinghausen gives as the normal capillary pressure in the finger tip with the hand at heart level, a reading of 52.5 mm. Hg. Basler's ochrometer has been used by Basler himself and by a number of other observers. The normal capillary pressure as given by Basler is about 7 mm. Hg. Goldmann, using this method, concludes 142 C. S. DANZER AND D. R. HOOKER that the normal capillary pressure is about 8.5 mm. Hg. A moderate rise in external temperature causes no appreciable change in the cap- illary pressure, but if the external temperature be raised 10°C. or more, the capillary pressure is increased. Landerer (24), using Basler's ochrometer, found that the normal capillary blood pressure ranged from 17 to 25 mm. Hg. During a cold bath the capillary pressure fell while the arterial pressure rose. In a warm bath the capillary pressure was unchanged while the arterial pressure fell. Landerer, Krauss (25), Friedenthal and others have used this method in cUnical cases. Krauss studied the capillary pressure in circulatory disturbances (valvular disease, myocardial disease, venous stasis), pulmonary dis- eases (emphysema, chronic bronchitis, tuberculosis), status asthenicus, severe anemia, carcinoma and cachexia. Besides the ochrometer he also used an apparatus of his own (a microscopic method) and also Weiss' blood method. He obtained some ver}^ interesting results. In brief, they confirmed the older observations of von Basch and Roter- mund and agreed with those of Landerer. The foregoing methods are all based upon the principle of paling of the skin originally introduced by von Kries. A decidedly different method has been employed by Easier and by Weiss. Weiss 's (26) method consisted of pricking the skin and observing the pressure necessary to stop the exudation of blood through the wound. The finger tip was enclosed in a chamber containing fluid in which the pressure could be raised. It was necessary, of course, to insure that bleeding continue after the pressure was removed. Easier (27) pricked the volar surface of the middle finger with a needle ; he then sealed a piece of rubber tubing onto the finger (by apply- ing a hot rod against the edge of the rubber) . This formed a little chamber in the center of which was a bleeding spot. The chamber was filled with hirudinized saline solution, and closed with a roof. Now the pressure of the exuded blood was registered manometrically. His re- corder was of the lever type and rather delicate. He named this apparatus the "Hautmanometer," The determinations made with this apparatus agreed quite closely with those of the ochrometer. Both of these blood methods are faulty because it is not possible to know the depth of the wound, since the skin thickness is so variable and consequently the size or the depth of the vessels punctured is a matter of chance. CAPILLARY BLOOD PRESSURE IN MAN 143 The theoretical objections to the various methods above discussed together with the fact emphasized by Friedenthal that the readings obtained by these methods vary by more than 100 per cent indicate clearly that they have little or no practical apphcation. In order to be sure that the readings accurately represent the pressure in the capil- laries themselves, a criterion similar to that employed by Roy and Brown, namely, the visual determination of the point at which the capillary flow ceases, is essential. Using a tonometer similar to that which we have employed and the criterion of cessation of corpuscular flow, these authors obtained pressure readings in the capillaries and venules of the web of the frog's foot of 7.3 to 11 mm. Hg. Lapinski (28) employed the Roy-Brown method to study the effect of nerve section on the capillary pressure in the frog. He obtained no very clear-cut results; however, his determinations of normal capil- lary blood pressure in small frogs was between 15 and 44 mm. Hg. and in large frogs between 30 and 60 mm. Hg. Natanson, in his studies on the effect of mass ligature, controlled his observations on man by experiments on the frog. He found the normal capillary pressure varied from 12 to 24 mm. Hg. in the different capil- laries of the frog's web. That such a criterion may be applied in the case of the human sub- ject is indicated by the observation of Weiss (29) who, using oil on the skin as suggested by Lombard, first observed corpuscular flow in the human capillaries. We have developed an instrument appUcable to man and experimental animals which permits of the accurate use of this criterion. If a drop of oil be placed on the skin of the hand and the area so treated be examined with the microscope under a strong light, as, for example, a 40 Watt electric bulb placed close to the microscope objec- tive, one may readily see the capillary tufts scattered through the field. Most of these tufts come up from the deeper parts of the dermis, fold over and return so that only a small part of the capillary loop can be brought into focus. Under such conditions it is scarcely ever possible to see the corpuscular flow. Occasionally a capillary may be found which, arising from the depths of the dermis, turns at right angles and takes an horizontal course for a short distance, and in such a case, under favorable conditions, the corpuscular flow may be observed. Such capillaries are so rare, however, on the general surface of the skin, that they do not serve for pressure determinations. In the skin overlying the matrix of the finger nail however, many capillaries take such an 144 C. S. DAXZER AND D. R. HOOKER horizontal course and in a single microscopic field a number of these vessels may be brought into focus at one time. Here one frequently sees corpuscular flow without offering any resistance to the blood stream. If a constricting pressure be applied to the upper arm the corpuscular stream may be seen in manj- of the capillaries without difficult}-. A drawing of the picture seen is given in figure 1. The sharply outlined capillaries are seen as red threads lying on a pink ground. The picture is very much clarified if the skin be first scrubbed with soap and water and afterwards thoroughly dried. This procedure removes the loose epidermis and softens the tissues. The methods based on this principle introduced bj- Lombard are the Krauss method, Basler's kapillar-tonometer (30) and Kylin's method (31). In Krauss's method the capillaries are visualized through a simple lens (magnifying lOX). Krauss takes as his criterion the disappear- ance of the capillaries. "We have emploj^ed such a method but have not succeeded in making the capillaries disappear at pressures correspond- ing to those of Krauss. We believe that the paling of the skin (which occurs at very low pressures) and the minute size of the capillaries as seen at such a low magnification may give the impression of a dis- appearance of the capillaries when even slight pressure is applied. When controlling this method with our own (in which a magnification of at least 68 is used) we have been able to see distinctly capillaries which were hazy and almost invisible with a magnification of ten. Concerning Basler's tonometer we may say that except for the fact that the gold beaters' skin has no circular opening in its center, it is almost identical with that of Lombard. The glycerine reservoir and the fact that the chamber is filled with glycerine, is common to both methods. An added difficulty in the method of Easier may be the collection of air bubbles within the chamber which will disturb the clarity of vision. In our experiments in the early part of 1919 we used chambers which were almost the counterpart of Basler's, but have found them difficult to work with and for reasons of practicability have discarded them. Basler's criterion is the disappearance of the capillaries. We believe this to be incorrect in principle and difficult of execution in the majority of instances. In the publication dealing with this method he gives no results obtained by its use. Kylin's method, which was presented at the Ninth Nordiske Congress of Internal Medicine held at Copenhagen, August, 1919, depends on the visualization of the capillaries. L'nfortunately we have been unable to Capillary blood pressure in man 145 obtain any exact information about his method, his criterion or his results. Fig. 1. A drawing of the finger tip to show the area of skin at the base of the finger nail which is used in the determination of the capillary blood pressure. The circle overlying this area represents the miscroscopic field when the skin is observed under a drop of oil with the aid of a strong light. The capillaries are slightly diagrammatic: a single focal plane will not bring them all into focus at one time. THE AMERICAN JOURN-\L OF PHYSIOLOGY, VOL. 52, NO. 1 14() C. S, DAXZER AND D. R. HOOKER The micro-cnpiUary tonometer. The instrument which we have used is shown in figure 2. It consists essentially of two adjusting devices. Screw 1 permits of raising and lowering the finger rest so that the area of skin under observation may be brought into an horizontal plane. Screw 2 permits of adjusting the pressure capsule in suitable contact with the skin. The finger rests on plate 3, which rocks so that the finger tip maj^ assume a comfortable position. The instrument is placed on a microscope stage and when the forearm is supported with a comfortable rest, the subject is sufficiently comfortable, so that there Fig. 2. The micro-capillary tonometer The instrument is of such a size that it rests on the microscope stage. Description in text. is no movement or tremor of the finger. The pressure capsule is shown in the insert figure, which represents a cross section along the line AB. It consists of a brass ring, 4, into the upper surface of which is sealed a thin glass plate, 5. Through the latter passes tube 6, which serves for the inflow and egress of air. To the lower surface of plate 4 is attached a brass ring, 7, threaded on the outside to receive the collar, 8. The latter screws air-tight against the washer, 9. Over the brass collar 8 is tied the gold beaters' skin, 10. The metal tube, 6, is con- nected through a rubber tube with a rubl^er ball of about 200 cc. capacit}', which maj^ be compressed between two plates by means of CAPILLARY BLOOD PRESSURE IN MAN 147 an adjusted screw, a device similar to that which is ordinarily used for calibrating the Hiirthle manometer. A bypass on the tube connecting the capsule with the rubber bulb leads to a single arm mercury man- ometer, by means of which the pressure in the chamber may be determined. After the finger has been brought to a comfortable position on the finger-rest with the area for study in the horizontal plane, the pressure capsule is lowered until the gold beaters' skin comes softly in contact with the upper surface of the finger. The gold beaters' skin is tied on suf- ficiently loosely so that when the air is forced into the capsule it exerts pressure upon the skin without loss of pressure due to the tension on the membrane. Preparation of membrane. The successful use of this device depends upon obtaining gold beaters' skin which is free of porous openings, is entirely soft and pliable and is transparent. We have found that gold beaters' skin prepared according to the following directions fulfills these requirements. The membrane is first washed in tepid water to remove all powder and dust which may be adherent to the surface. This cleansing is facilitated by rubbing both surfaces of the membrane with the ball of the finger or with a piece of absorbent cotton. It is then rinsed several times in clean water, after which it is ready for use. The brass collar, 8, in figure 2 is now removed from the instrument and screwed down over an obturator which, presenting through the collar, has an oval contour such that when the membrane is tied into position it will lie somewhat as shown in the detail. With the collar in position on the obturator the membrane is laid over the top and tied securely with several turns of silk thread. The collar is now returned to the instrument, when it may be tested to see whether or not the attached membrane is perfect. If it stands a pressure of 50 mm. Hg. without leak, it is suitable for further treatment. The collar with the attached membrane is now drained free of water and placed in pure glycerine for 24 hours, after which it is removed, drained free of glycerine and placed in castor oil. The preparation should be left in castor oil for at least 24 hours and may be left in the oil indefinitely. It is thus possible to prepare a number of mem- branes at one time and to keep them in castor oil until such time as they may be required. When a membrane is required for use, the ring to which it is attached is removed from the oil and screwed into position on the instrument, a test being again made to be sure that the chamber is air-tight. If a THE AMERICAN JOURNAL OF PHYSIOLOGY, VOL. 52, NO. 1 148 C. S. DANZER AND D. R, HOOKER leakage occurs at this point it is probably due to an imperfection in the washer. If now the adjustment to the finger be made and the area of skin beneath the member be observed with the microscope, it will be seen that the membrane is entirely transparent so that there is no difficulty in observing the underlying capillaries. Failure to see the capillary field plainly indicates that the membrane is clouded, in which case it should be discarded. This condition is not likely to be found when using a new membrane. A membrane, however, which has been used repeatedly and exposed to air and dirt is liable in the course of time (weeks) to deteriorate and if there is any difficulty in obtaining clear vision of the field it is probable that the transparency of the mem- brane is at fault. When the instrument is not in constant use it is advisable to return the membrane to castor oil. With reasonable c^re a membrane should last indefinitely. A second technical requirement is that the skin should be clean and free from moisture. It should be scrubbed lightly with soap and water and thoroughly dried. The oil on the skin which makes the underlying capillaries visible probably serves by its intimate penetration of the epidermal layer to do away with light reflection fr'om the uneven sur- face. If then moisture intervenes at the oil-skin boundary, the field is less distinct. It sometimes happens in prolonged observations that the clarity of the field is lost. This may be due to the excretion of sweat. If the oil be wiped off and the finger be thoroughly dried a fresh drop of oil will make the field as clear as before. In using the instrument we have found it desirable to employ a microscope giving a magnification of approximately 70 X. The essen- tial point is to magnify enough to readily visualize the movement of the red cells when the stream is slowed without undue loss of definition and depth of focus. Leitz objective 3 and ocular 1 fulfill these require- ments. To use such a lens combination with a rather short working distance, the skin with the overlying oiled membrane must be brovight quite close to the glass roof of the chamber. Care must be exercised, therefore, that in manipulation the membrane shall not touch the glass. If this occurs one must dismount the collar and clean the glass, other- wise the capillary picture will be clouded. It is of distinct practical help to one using the instrument for the first time, to first become fam- iliar with the location and appearance of the capillary bed under the microscope. When this is done it is a simple matter to locate the proper field with the chamber in position. CAPILLARY BLOOD PRESSURE IN MAN 149 We have experimented with chambers of different diameters but have found that this is a matter of no consequence in obtaining correct read- ings and we now use for routine observations a chamber of such a size that the brass collar is 25 mm. in diameter. In order to insure that the pressure within the chamber is all transmitted to the underlying skin, it is advisable to make several determinations of the capillary- pressure with the chamber at slightly different vertical positions. The position of the chamber which gives the lowest capillary pressure read- ing is the correct one. With a little practice one makes this adjustment readily and there is no necessity for such preliminary observations. The purpose is, of course, to insure that none of the pressure is lost in the resistance offered by the membrane itseK. Corpuscular flow. With the chamber in position over the finger, when there is no pressure exerted on the underlying skin, the picture is frequently disturbed by reflections of light from the folds of the mem- brane. This disturbing feature is at once removed if a pressure of 2 or 3 mm. be applied, a procedure which has the effect of smoothing out the membrane over the surface under observation. When the area is first observed it is uncommon to obtain any indication of corpuscular flow. If now the pressure within the chamber be slowly raised by com- pression of the rubber bulb, the corpuscular flow becomes evident and the red cells are seen streaming slowiy through the capillary. As the pressure is further raised this corpuscular flow becomes slower and slower until there is no further continuous forward movement of the corpuscles. At this time one frequently sees a to-and-fro movement of the corpuscles without progress. At first thought this phenomenon would seem to be associated with a transmitted pulsation from the underlying vessels. It may however be due to rhythmic contractility of the capillary endothelium. As the pressure is further raised, this to-and-fro movement of the corpuscles ceases. Now a further increase in the pressure acting on the capillaries may cause the corpuscles to travel in a reversed direction, that is to say, from the venous side toward the arterial side. We are able to offer no satisfactory explanation of this fact at the present time. If now the pressure be slowly lowered the reversed flow ceases and the corpuscles again stand still. Then the to-and-fro movement of the corpuscles is seen, and finally with a further lowering of the pressure the corpuscles stream forward in the normal direction. The pressure at this instant is taken to represent the cap- illary blood pressure and our reading is therefore made at this point. 150 C. S. DANZER AND D. R. HOOKER The pressures at which the above events occur in one and the same capillary are depicted in figure 3 and again for a second subject in figure 4. The plotted lines in each figure represent several observations on a single capillary. Note that the skin pales in both subjects at a pressure of about 8 mm. Hg., while the corpuscular stream is not slowed until the pressure has been raised to 18 mm. Hg. in one case (fig. 3) and 27 30 it 3i ^f Xf 39 XX a; iO If n « If 13 n II w \ 7 U f H 3 Z Sue., C.SD Corpijscular Corpuscular OLOWINC C? _ _ UscILUATIONaCf RPiy5CL.LAR0£Clt,LATIO« AUING StREAI rEAOfDrt^OwNcrRCGRESS SrA&MATIONNoPROCRtJsDErAPEDrLowSLOwSrREAn STREAM Fig. 3. To illustrate the pressures at which the several events in capillary blood flow occur. The figures along the abscissa represent pressure in milli- meters of mercury. The ordinates divide the chart according to the observed behavior of the corpuscles with an increasing and then with a decreasing pressure.- All the observations were made on one capillary, the several plotted lines repre- senting single observations. mm. Hg. in the other (fig. 4). The slowing of the corpuscular stream undoubtedly represents the condition when the pressure without ap- proximates the pressure within the capillary. It is clear, therefore, that paling of the skin is a wholly inadequate criterion of capillary blood pressure. These figures indicate further that repeated observations of the same capillary agree closely in the pressures at which the events under discussion occur. Experience has taught us, however, that the CAPILLARY BLOOD PRESSURE IN MAN 151 slowing of the corpuscular stream which occurs with a rising pressure is much less readily appreciated than is the quickening of the stream which occurs with a falling pressure. Consequently we have found it expedient to raise the pressure until the forward movement of the cor- puscles ceases and then to lower it, giving close attention to the beha- vior of the corpuscles. As the pressure falls the corpuscles at first progress slowly and with a further slight lowering of the pressure there is a sudden sharp acceleration which serves as a sharp criterion and P/u.iwr, CoRfVSCUUR CoRPlJSCULAI? Slowing of 0sciiLAtrDR^OftP«auLftR0scia.A-nQNg Rapid SlBEAIi. SrSEAd Fig. 4. See figure 3. one which is easily recognized. The pressure then represents what we have taken to be the capillary blood pressure. During the procedure above outlined, the capillaries are sometimes emptied of red blood corpuscles, but more often this is not the case, that is to say, when the pressure applied to the skin is sufficient to stop completely the corpuscular flow, the cells are still present in the capillary loop. This observation goes to show that the assumption made by von Kries and other earlier observers that the paling of the 152 C. S. DANZER AND D, R. HOOKER skin is associated with an emptying of the Imnina of the capillaries is incorrect. Furthermore, one may readily appreciate that the paling of the skin associated with the application of pressure becomes very extreme even before the corpuscular flow is distinctly slowed. Obser- vations of this kind have led us to the conviction that the criteria used by most other observers are wholly inadequate to represent true cap- illary blood pressure. It frequently happens that when one obtains a focus of the capillary field, one or more of the capillaries will stand out conspicuous and large. Such capillaries represent vessels in which the corpuscular flow is stag- nated. The individual corpuscles in such a capillar}- cannot be made out; apparently they are thickly packed together. The pressure may be raised very considerably without changing in an\^ way the appear- ance of such a vessel. This fact accords with the original observations of Roy and Brown which have been abundantly confirmed by later observers, that all the capillaries in a given vascular bed are not neces- sarily functioning at the same time. The idea that not all of the capillaries are functioning at all times is indeed not a new one. Worm-]Muller (32) in 1873 in studying the influence of blood volume on the arterial pressure comes to the follow- ing conclusion: "Under normal conditions (most Hkely in every part of the body) a large number of empty or poorly filled capillaries stand ready to respond to the needs of the blood stream." He also spoke of the dilatation of the capillaries resulting from the transfusion of blood. He found on post-mortem examination that the arteries and veins were not overfilled after transfusion. He therefore assumed that the blood collected in the capillaries. Heubner (33) in 1907 saw many new capillaries open up as the re- sult of the injection of gold sodium chloride into frogs, rabbits, cats and dogs. The experiments on the frogs are described in greatest detail. In these he looked at the frog's mesenterj^ microscopically and injected an amount equivalent to 0.25 mgm. of metallic gold and in one-half to one minute saw the animal practically bleeding into its capillaries. He estimates that the number of . capillaries visible after the injection is three to four times as many as before. Concerning Dale and Laidlaw's work little can be added to what is so generally known about their researches on "Histamine Shock." By a series of ingenious experiments these workers have attempted to prove a, the active functioning of the capillaries; h, the independence of the capillaries from the rest of the vascular system; c, the capillo- CAPILLARY BLOOD PRESSURE IX MAX 153 dilating effect of histamine. They were led to these problems by the discrepancy between the effect of histamine in the intact animal and on the excised arterial or uterine strip. In the former it lowered the blood pressure and in the latter it increased the tone of involuntary muscle strips. Dale and his co-workers explain the marked fall in arterial blood pressure which occurs in the intact animal and which cannot be due to a relaxation of arterial tone since histamine causes the isolated artery to contract, as due to a specific dilator action of hista- mine on the capillaries. According!}^ the capillary beds throughout the body are flooded and the circulating blood is insufficient to fill these areas and at the same time sustain the arterial pressure in spite of a concomitant increase of arterial tone. AYhereas the experiments are very suggestive the crucial experiment, namely the effect of hista- mine on the capillaries as observed directlj^ (through the microscope) is as yet lacking. Krogh (34) has thrown considerable light on this subject by counting the number of capillaries in a definite area of muscle tissue in the rest- ing state and immediately after exercise. He examined both fresh and fixed tissues. He found that the working muscle showed many more capillaries than the resting tissue. Hence, the conclusion that many new capillaries open up when necessary (e.g., in the working state). It is obvious perhaps that we avoid the term capillary collapse. We have done this because of a good deal of experimental evidence (partic- ularly that of Roy and Brown) which indicates that capillaries do not collapse when compressed. WTiy then do we not take capillary empty- ing (i.e., when corpuscles travel back from capillaries into the arterioles) as the criterion? The reason is that in a number of capillaries it is necessary to raise the pressure acting upon them much above that which will cause the corpuscles to stagnate. Hence the readings would be much too high. This is due to the fact that blood is piling up in the patent capillaries as each neighboring one is emptied of its contents (von Recklinghausen). We believe also that as the externally appKed pressure rises the arterioles and capillaries are being simultaneously compressed. On this account pressure determination in a great many capillaries maj^ be impossible with the methods of Lombard, Krauss or Easier. Krauss himself admits this difficulty. The impression that we have received from Doctor Lombard personally concerning some of the drawbacks of his method are to the same effect. 154 C. S. DANZER AXD D. R. HOOKER o 00 1-4 s lO I-H h' PL, Pi ^ -t> s t-l < K LI lO J: Ha" ^ O ■. CI t^ l^ o t^ s s o 1 T 1 + T 1 1 7 + 7 7 H "« o o IC O o a; >o o o 00 o LO G £ o cc CO »c tC o lO lO LO CO a S t— ( T-H i-H T-H -H I-H 1—i f-H t-i^ ^ S "7^" ^ ^ ^ 7~ a r-Ki ^'U -cl« «(M »-!M GO cc T— t O o o 00 CO •^ o o ""^ '"' '^ s ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ~ i-O o lO •o lC LO lO LO LO LO LO a o 77 (M C-] o zi ^ ° -5" CO o o "3 o o o o s o CO !5° s '3 > (I > 2 "o Q o CD ID -^ CC '5 en .2 '3 3 « u S3 •s a CO O c3 &, O -(J to o ^1 -5 3 .2 cc -3 'o 1q 3 e3 TJ o o o o »o o o »o o q O o yj, fO Ol o C^l lO 00 l>. o CO d lO c<» C^l CO C^l O O r-H ^ (^^ iM CM c^ l>- o oc LO rH .-( ^ (M <* Tt< «D 00 00 1 1 1 1 + + + + + QO ?5 T— 1 00 ^i; c-1 (M ?^[ oo iO~ ^3~ CO g[ CO ^~* CO ^ (M o t^ '*" o Lo' lO t^ o CO ^ lO C^" o" CO c^i '-' lO -H ^H C^l r o (M !M C^l - -V ^ ^ ^ ^ ^ ^ — 1 - C^ iM C^ (M ^ ^ ^ ^ -V ^ ^ ^ ^ o lo r^ -M lO (M ■<* CO Oi lO to M CO Ol ^ C^l —1 ^H '"' ■"■ r-H Tt< CC CO 00 CI 7 7 + 1 + o •o lO 1^ lO ^ ^0 >-o to C2 CO t-- M< o ~^ ^ ^ ^ ^ ^ >o LT lO kO o t^ M > > 156 C. S. DANZER AND D. R. HOOKER Because some capillaries exhibit no corpuscular flow and because there is an appreciable variation in the pressure in several capillaries in a given area, we have found it expedient to make pressure determina- tions in five or more capillaries and to average the results. This will minimize the possibility of striking capillaries of extreme pressures. The majority of our determinations, therefore, represent such an average. Capillary blood pressure in normal individuals. Twenty-five adults and six children were studied. All of the determinations were made with the subjects in the sitting posture, with the hand a little below the heart level. The average capillary pressure in our series was 22.2 mm. Hg. The ages of our subjects ranged from 8 to 47 years. Of the sub- jects studied the lowest average capillary pressure was 17.5 mm. Hg., the highest was 26.5 mm. Hg. These results are given in table 1. Four of our cases (A, B, C, and D in table 1) cannot be grouped with the remaining ones, because of the existence of infections (colds) in two of them and on account of technical difficulties in the other two which occurred at the inception of the work. In some of the cases the distance betwen the level of the hand and the heart level was determined. Like von Kries, von Recklinghausen and Goldmann, we found that capillary pressure varies with the dis- tance between the hand and the heart level. The difference in the pres- sure readings taken when the hand is at heart level and when the hand is above or below the heart level does not, however, correspond to the hydrostatic differrence in these positions. This is clearly brought out by the following table given by von Kries (35). HAND CHANGED PROM HEART LEVEL DIFFERENCE IN CAP. PRESS. DIFFERENCE TO CAPILLARY PRESSURE HYDROSTATIC DIFFERENCE mm. Hg. per cent 205 mm. below heart level 65 33 285 mm. below heart level 116 40 350 mm. below heart level 225 64 The hydrostatic factor is thus a disturbing element in the determi- nation of capillary blood pressure at the present tune. It-is therefore advisable that determinations of this pressure should be made at heart level although the inconvenience of such a procedure is considerable. Our readings unfortunately do not conform to this specification, never- theless the range between the determinations on different subjects is remarkablv small. In the case of children our readings run appreciably CAPILLAEY BLOOD PRESSURE IX AL\X 157 lower than in adults but when correction is made for the hydrostatic factor the results correspond closely with those obtained in older indi- viduals. Similarly the results in the group of adults show no relation- ship between age and capillary pressure. We have not noted in the table whether the subjects were men or women because here also we found the sex factor to be of no significance. On the whole, therefore, the data which we have thus far collected point to a very remarkable constancy in the capillary pressure in normal individuals of both sexes regardless of their ages. Krauss also found that the capillar}^ pressure in children was practically the same as in adults. Concerning the readings obtained in the individual capillaries, it may be seen that they run within fairh' close limits. There are, however, capillaries whose pressure determinations are definitely outside of these limits. We do not average these extreme ones with the rest. The capillaries of higher pressure may be the ones Ijdng deeper down in the skin or those of larger bore. Possibly they represent the piled-up blood in the patent capillaries resulting from the emptjdng of the neigh- boring capillaries, as suggested by von Recklinghausen. We discard very low readings when they occur in biit one or two capillaries. Theo- retically these are the most accurate. For practical reasons, however, we have arbitrarily eliminated these infrequent (low pressure) capillaries and have adopted instead another criterion which is quite as accur- ate as the former, but more easily carried out. That series of six or more capillaries of the lowest pressure is taken, in which the difference between the extreme capillaries of the series is not more than 6 or 7 (less is preferable). We warn against averaging capillaries of very low or very high pressures with the series. Let us take a concrete example. ( 12l „ > eliminate — too low 19 21 25 20 23 23 32 eliminate — too high 20 38 "1 f eliminate — too high Capillary pressure in separate capillaries. . . . 158 C. S. DANZER AND D. R. HOOKER Thus the series consists of. 19 21 25 20 23 23 151 -— = 21* mm. Hg. = average Our figures for normal capillary blood pressure (with the subject in sitting posture, hand below heart level) accord with those of Lombard (18 to 22 mm. Hg.), Landerer and Krauss. The latter two investigators (using Basler's ochrometer) obtained readings varying from 17 to 25 mm. Hg. on the normal individuals. In our table we have also put down the difference between the actual and the ideal weight of the subject with the idea of noting the effect of over- or under-nutrition on the capillary pressure. Thus far we have nothing definite to say on this subject. The effect of temperature on capillary pressure. Cold. Cold towels with pieces of ice placed between the layers were wrapped around the arm. The cold was not directly applied over the capillary area, hence the results are not to be interpreted as the direct effect of cold on the capillaries themselves but rather as a reflex effect. The duration of the application was 15 minutes. The results are graphically presented in figure 5. Two neighboring capillaries represented by the continuous and the broken lines were studied. Immediately on the application of cold there was a definite drop in the capillary blood pressure. When the cold was removed a very prompt rise of capillary blood pressure, which was maintained for about 13 minutes, occurred. Then there was a marked drop to a point slightly above normal, then another rise and finally a fall. The occurrences after the removal of cold represent the reaction after such a procedure. It is striking how closely the curves of the individual capillaries follow each other. Only at one point do they diverge. The explanation for this would seem to be a change in the lumen of one of the capillaries independent of that of the arteriole since both capillaries were in all probability supplied by the same arteriole. Heat. Heat was studied by means of an electric pad which was wrapped around the arm and kept on for 26 minutes. We made observations on six neighboring capillaries. The electric pad was set so as to create moderate heat. The effect produced by the application CAPILLARY BLOOD PRESSURE IN MAX 159 of heat is also shown in figure 5. Almost immediately there was a rise in capillary blood pressure which was maintained throughout the period during which heat was applied. Upon the removal of the pad the pres- sure in the capillaries fell to a point below the normal value. Soon the pressure average in the six capillaries observed was as before the experi- ment. One is amazed at the striking parallelism of the pressure curves of the whole group of capillaries. Only here and there does a disparity become evident. For example, in the last phase two capillaries show a fall in pressure while two others show a simultaneous rise in pressure, Ttx. % ^ ^5, ^'w»«^ I t 1 > * ^ t, ^ $r m i 13 \ y « \ / H V A / 7 \ ae \\ / \ ; m I'V / \ 1 2S 1' * -'' \ /'*♦ \ 1 > r / \ \ 3i l{ \\ y \ \ l/l "'-~. V %!■ ' i \ » _yf/ / V\' Zt M \ //// 23 \ \ /// n \ \ ('// , \^ %f \ f V i / 4 1 xo --— "7 )^^:^ 19 ^ ^-v / i^.-^'' Afv t1 \ »^ Nj; / '\ -J "i^- lb \ ' >.i / t ' \ a- « — '. t Ice 4-Towa] Remcvcd 1) / 1% CtU. l\ffV^ Cict * c eiecir c Pdifnlcierite HeAt) Applied.. lA S.bj = T>R.M ii/n/.i Fig. 5. To show the effect of cooling and warming the fore-arm on the capillary blood pressure in the finger. Two capillaries were followed in the application of cold and six in the application of heat. although the average in the whole series is practically normal. Here again we must assume a contraction or dilatation of the capillaries themselves. There were occasions when we felt that the diameter of the capillaries underwent changes in the course of our observations, yet most of our evidence supporting the idea of capillaiy contractihty is indirect and sometimes necessarily assumed. In the heat experiment the electric pad was not placed directly over the capillaries but over the forearm and hand. Here again the results are due, therefore, to a reflex effect. 160 C. S. DANZER AXD D. R. HOOKER In these experiments the capillary pressure responded to the appli- cation of heat and cold as if the stimulus produced vasodilatation and vasoconstriction of the feeding arterioles. We assume this to be a re- flex effect from the spinal centers. Krogh (36), however, is reported to have shown that contraction of the capillaries may be evoked by reflex stimulation after the sensory nerves have been cocainized, an observation which indicates an axon reflex. In our experiments, how- ver, the stimulus was applied at some distance from the field of obser- vation so that it seems improbable that the results are due to axon reflexes. The suddenness with which the capillary pressure changes, particularly after removal of the stimulus, excludes the hj^pothesis that the results might be due to differences in the temperature of the blood. It would be possible to differentiate between a true reflex and an axon reflex effect if the observations were repeated on a subject with impaired cutaneous sensibility. The figure (fig. 5) shows a remarkable over-compensation of the capillary pressure after the stimulus is removed. Within three min- utes after the cold was removed the pressure rose from 17 to 32 mm. Hg. and remained at this high level for a period of 15 minutes, after which it began to fall. Thirty minutes later it was still above the original value. A similar though less striking effect is seen to fol- low the application of heat. These changes in capillary pressure un- doubtedly occur when the temperature of the surface of the whole body is altered. We have no notes indicating a definite change in the caliber of the capillaries under such conditions although, of course, when the capillary pressure was low the corpuscular stream was fre- quently seen without the application of external pressure. Our observations correspond pretty well with those of Goldmaim. The relative crudeness of his method, however, prevented him from investigating it in such detail as has been done here. His readings (taken with Basler's ochrometer) were definitelj'" lower than ours. Hough and Ballantjme, who studied the effect of temperature on capillary pressure by means of the von Kries method, found that when the temperature of the external air was reduced from 20°C. to 6°C. the capillary pressure rose from 40 or 50 mm. Hg. to 65 mm. Hg. This was probabty due to the constriction of the more superficial vessels and to the fact that the first noticeable color change in the skin was produced when the deeper vessels were compressed. This again illustrates the danger of working with a method depending on skin pallor. This error is clearly the result of the fallacy of the method. CAPILLARY BLOOD PRESSURE IN MAN 161 When the temperature of the external air was raised to 26°C. the capil- lary pressure was 50 or 55. In other words, very little (if any) change occurred. Here again the inadequacy of the method becomes evident. Schiller also studied the effect of temperature on capillary pressure by means of the modified von Kries method (with Fick's ophthal- motonometer) but his results are difficult to interpret because of the reasons given above. Briefly they are as follows: the highest capillary pressure (40 mm. Hg.) occurred when the temperature of the externally applied water was nearest to that of the skin (30 to 35°C.). Landerer (working with Basler's ochrometer) found that the capillary pressure fell during a cold bath, while it was unchanged in a warm bath. Krauss (using his own apparatus, modified after Lombard) found that « S i if n A If )t,Y.*,6 'O'tftreTir Cfl(^ IX II Y ' / X ^ s^hy-. K.R.H eff«t at Posr„«e le / \ \ "/•i/i'l F.-ijcr at l4€»rT Level Frr,,,r a.^ hMff i.«»«I. Fig. 6. Effect of posture on capillary blood pressure. Finger was at heart level throughout so that hydrostatic factor is excluded. The numbers at the left indicate pressure in millimeters of mercury. the application of ice to his arm produced a fall of capillary pressure amounting to 20 mm. water and a rise of arterial pressure of 15 to 20 mm. Hg. The effect of posture. The effect of posture on the capillary blood pressure is shown in figure 6. The hand was held at heart level in these observations in order to eliminate the hydrostatic effect. Hence our results represent the true postural effect. It will be seen that the lowest pressure occurs in the horizontal position, the highest in the vertical, and the capillary pressure in the sitting posture is midway between the two. Here again we see in the figure the individuality of some capillaries. There are two pairs of capillaries which differ from each other, yet the individual members of each couple act in close harmony as is evidenced by the parallelism of their pressure curves. Again the contractility of the capillaries suggests itself. 162 C. S. DANZER AND D, R. HOOKER From this experiment it may be seen that capillary pressure deter- minations will vary according to the posture of the subject during the examination, although the hand be maintained at heart level. This has been impressed rather strongly on us very recently in connection with our clinical work on the bed cases in the hospital. In a number of these patients (in whom the capillary pressure was probably nor- mal) readings ranging from 13 to 15 mm. Hg. were commonly obtained. These, of course, are a good deal lower than our original figures, which were made with the subject in the sitting position with the hand slightly below the heart level. \ s.,b; = f'B l.if-ifo isr 3 Oo 3.0^ X3 VAi.ih{.va Tirpei-. /Mjllcr Etfr- Fig. 7 Fig. 8 Fig. 7. To show the variations in pressure in individual capillaries from day to day. While the pressure in an individual capillary may thus alter from day to day and from hour to hour, the average pressure in a group of capillaries is remarkably constant. See tables 2, 3 and 4. Fig. 8. To show the effect of changes in intrathoracic pressure on the capillary pressure. Two observations on raising the intrathoracic pressure (left) and two on lowering the intrathoracic pressure (right) are given. The following represents pressures in a series of capillaries studied on two consecutive days. Diurnal variations. Hans Friedenthal saj^s that capillar}^ blood pressure varies by many hundreds per cent during the same 24 hours. In our experience this has never occurred. We have found on the con- trary that the average pressure of a series of capillaries varies within relatively narrow limits from hour to hour, or even day to day. Some capillaries which we have followed in this manner have shown practi- cally constant pressures, while others have shown ver}^ great variations. CAPILLARY BLOOD PRESSURE IX MAN 163 This tendency for the pressure to vary in an individual capillary from da}^ to day is illustrated in figure 7. The figure represents the pressures found in three different capillaries on four successive days. However much the pressure may fluctuate in an individual capillary there is nevertheless a remarkable uniformity in the results obtained when the pressures in a number of capillaries are averaged. This fact is well seen in the accompanjdng tables (tables 2, 3 and 4) dealing with the diurnal variations in capillary pressure. So far as our pres- ent observations g-o, therefore, there is practically no variation in the TABLE 2 Determination of the pressure in a single capillary at different pzriods of the day 10:30 a.m 15.00 mm. Hg. (Readings taken by Dr. H.) 1:00 p.m 15.00 mm. Hg. (Rgadings taken by C. S. D.) Lunch 2:00 p.m 14.16 mm. Hg. (Readings taken by C. S. D.) 2:45 p.m 14.50 mm. Hg. (Readings taken by C. S. D.) 3:00 p.m 15.17 mm. Hg. (Readings taken by C . S. D.) TABLE 3 Determination of the pressure in four individual capillaries mads on two days DATE TIME CAPILLARY A CAPILLARY B CAPILLARY C CAPILLARY D December 6 10:30 a.m. 27 27 29 f 9:15 a.m. 23 23 22 1 11:15 am. 24 23 22 12:30 p.m. 25 24 22 December 7 ■ 2:00 p.m. 26 23 3:30 p.m. 24 24 4:30 p.m. 24 24 24 5:00 p.m. 24 22 average values for a given group of capillaries at different times of day and on different days. The effect of intrathoracic pressure on capillary pressure. Disturb- ances of intrathoracic pressure affect both the arterial (37) and venous (38) blood pressure. We have perfoniied one experiment to study its effect on the capillary pressure. Our results, given in figure 8, show that forced expiration with the glottis closed (Valsalva experiment) causes a rise, while forced inspiration with the glottis closed (Miiller experiment) causes a fall in capillary pressure. The curves are prac- tically mirror pictures of one another. Since a decreased intrathoracic TABLE 4 Determinatwn of the pressure in a group of capillaries studied on two consecutive days. Note the constancy of the pressure DATE Hg. PRESSURE .WERAGE PRESSURE mm. Hg. mm. Hg. 23.5 28.0 November 5 1 24.0 a. m Ring finger -1 1 27.0 25.0 264 i 28.0 [ 30.0 26.0 27.0 Small finger 25.0 23.0 22.0 22.0 22.0 23.0 19.0 21.0 23.0 24.0 24.0 24 ]). Ill Middle finger { 22.0 22.0 23 22.0 22.0 23.0 25.0 26.0 > 27.0 ( 23.0 25.0 November 6 26.0 27 a. m Ring finger 28.0 30.0 30.0 / 23.0 24.0 p. m Small finger 22.0 27.0 24 23.0 The pressure in the capillaries of the ring finger is practically constant for the two days; likewise the pressure in the small finger capillaries. 164 CAPILLARY BLOOD PRESSURE IN MAN 165 negative pressure lowers the arterial and raises the venous and capil- lary pressures, while an increased intrathoracic negative pressure pro- duces the opposite circulatory conditions, it follows that capillary blood pressure is more closely dependent upon venous than it is upon arterial pressure. von Basch (39), who reports results similar to the above, has also emphasized the association of capillary with venous pressure. With these observations in mind, a systematic study of capillary pressure in cases of cardiac insufficiency which determines a high venous pressure and in cases of emphysema, asthma, pleural effusions, etc., which determine disturbances of intrathoracic pressure, should prove fruitful. The effect of venous compressioji on capillary pressure. A rubber band was applied around the little finger and the pressure in one and the same capillary was taken with varying degrees of compression. Very marked compression which closes both the arteries and veins reduces the capillary pressure to zero. Moderate compression, however, (which compresses the veins only) raises capillary pressure (18 mm. Hg.); when the rubber band is removed the pressure returns to normal. Normal L5 .0 mm. Hg. Rubber band firmly applied Rubber band loosely applied 18.0 mm. Hg. Rubber band removed 15.5 mm. Hg. Hence we see that increased venous pressure (produced by compres- sion) increases the pressure in the capillaries. The effects of venous compression were previously studied by von Basch and later by Krauss. Both of these observers concluded that a high capillary pressure re- sulted from the compression of its veins, von Basch even went as far as to formulate an hypothesis which was to the effect that capillary pressure was an index of the amount of venous stasis. SUMMARY A method for the study of capillary blood pressure is presented which differs from all of the previous methods in the criterion at which the readings are taken. It depends on the production of stagnation of the flow of corpuscles in the capillaries. With this method capillary pressure in man can be determined in the fingers and in the toes. For the study of capillary pressure in animals (cat, dog, rabbit) the shaved ear should be used. Our appa- ratus readily adapts itself for such study. THE AMERICAN JOURNAL OF PHYSIOLOGY, VOL. 52, NO. 1 166 C. S. DANZER AND D. R, HOOKER We have studied the capillary blood pressure in normal individuals (sitting posture). The average pressure was 22.2 mm. Hg, The study on the effect of temperature showed that cold lowers and heat raises capillary blood pressure. Posture was found to vary capillary pressure although the hand was at heart level at all times. It was lowest in the recumbent, highest in the standing and midway between in the sitting posture. The diurnal variation, contrary to previous opinions, is very slight. Increased intrathoracic pressure raises capillary pressure. Dimin- ished intrathoracic pressure lowers capillary pressure. Venous compression causes an increased capillary blood pressure. We believe that the prevalent conception that the redness of the skin is due to its capillaries and that pallor means capillary empty- ing, is erroneous. While undoubtedl}^ contributing somewhat to the color of the skin, nevertheless the role of the capillaries is rather slight. The venous plexuses principally contribute to the color of the skin. The collapse of these plexuses is the principal cause of skin pallor resulting from compression. The simplicity and the accuracy of our method gives us hope that it may find a place in the laboratory and in the clinic. We propose as the name of our apparatus, the "micro-capillary tonometer."^ BIBLIOGRAPHY (1) Roy and Browx: Arch. f. Anat. u. Physiol. (Physiol. Abt.), 1878, 158; Journ. Physiol., 1879, ii, 323. (2) Cannon: .lourn. Amer. Med. Assoc, 1918, Ixix, 611. Cannon, Fraser and Hooper: Journ. Amer. Med. Assoc, 1918, Ixx, 527. (3) Dale and Laidlaw: Journ. Physiol., 1919, lii, 355. (4) Dale and Richards: Journ. Plwsiol., 1918, lii, 110. (5) Lomb.\rd: This Journal, 1912, xxix, 335. (6) Friedenthal: Zeitschr. f. Exper. Path. u. Therap., 1917, Ixxix, 222. (7) Fick: Pfliiger's Arch., 1888, xlii, 482. (8) Campbell: Lancet, 1894, i, 594. (9) Levy: Pfluger's Arch., 1897, Ixv, 447. (10) Goldmann: Pfluger's Arch., 1914, clix, 51. (11) Bogomolez: Pfluger's Arch., 1911, cxli, 118. (12) V. Kries: Ludwig's Arbeiten, 1875, x, 69. (13) Spalteholtz: Arch. f. Anat. u. Physiol. (Anat. Abt.), 1893, 1. (14) V. Basch: Wiener klin. Rundschau, 1900, xiv, 549. (15) V. Recklinghausen: Arch. f. Exper. Path. u. Pharm., 1906, Iv, 463. (16) Basler: Pfluger's Arch., 1912, cxliii, 393. I We are indebted to Mr. Klett of the Klett Mfg. Co., 402 East 46th Street, New York City, for practical assistance in developing the apparatus. I CAPILLARY BLOOD PRESSURE IN MAN 167 (17) Hough and Ballantyne: Boston Journ. Med. Sci., 1899, iii, 330. (18) Nat ANSON : (a) Pfiuger's Arch., 1886, xxxix, 386. (b) Inaugural Dissertation, Konigsberg, 1886, 14. (19) Schiller: Zentralbl. f. Physiol., 1911, xxiv, 391. (20) Rotermuxd: Inaugural Dissertation, Marburg, 1904, 10. (21) Fick: Pfliiger's Arch., 1888, xlii, 86. (22) Hooker: This Journal, 1914, xxxv, 73. (23) Briscoe: Heart, 1918, ii, 35. (24) Landerer: Zeitschr. f. klin. Med., 1913, Ixxviii, 91. (25) Krauss: Sammlung. klin. Vortrage, 1914. Innere Med. Nr. 237/239, 315. (26) Weiss: Zentralbl. f. Physiol., 1914, xxviii, 375. (27) Basler: Pfliiger's Arch., 1914, clvii, 345; Miinch. med. Wochenschr., 1913, Ix, 1972. (28) Lapinski: Arch. f. Anat. u. Physiol. (Physiol. Supplement), 1899, 476. (29) Weiss: Deutsch. Arch. f. klin. Med., 1916, cxix, 1. (30) Basler: Pfliiger's Arch., 1919, clxxiii, 389. (31) Kylix: Ninth Nordiske Cong. Int. Med., Copenhagen, August, 1919. Quoted from Journ. Amer. Med. Assoc, 1919. (32) Worm-Muller: Ludwig's Arbeiten, iii; Ber. d. Sachs. Gesellsch. d. Wis- sensch., Leipzig Math. Phys. Klin., 1873, 573, 649. (33) Heubner: Arch. f. Exper. Path. u. Pharm., 1907, Ivi, 370. (34) Krogh: Journ. Physiol., 1919, Iii, 457. (35) V. Kries: Ber. d. Sachs. Gesellsch. d. Wissensch., 1875, xxvii, 149. (36) Krogh: Journ. Physiol., 1919, liii. Quoted from Baj-liss, Science Progress, 1919, xiv, 272. (37) Dawson: This Journal, 1916, xl, 139. (38) Hooker: This Journal, 1914, xxxv, 73. (39) V. Basch: Internat. Beitr. z. innere Med., 1903, i, 65. A PLETHYSMOGRAPHIC STUDY OF SHOCK AND STAMMER- ING IN A TREPHINED STAMMERER SMIUEL D. ROBBINS From the Psychological Laboratory, Harvard University, Cambridge, I\Iassachusetts^ Received for publication March 12, 1920 The experimental work here reported was done in January, 1920. The purpose of the experiment was to test the organic reactions accom- panjdng stammering, with special reference to Dr. C. S. Bluemel's cerebral congestion theory of stammering, and to determine whether stammering, like shock, is accompanied by congestion in the brain, and hence by increased intracranial pressure, as was conjectured by me in the theoretical conclusions to "A plethj^smographic study of shock and stammering" published in the April, 1919, number of this Journal. Subject. The subject who took part in this experiment was born in Telsburg, Norway, November 14, 1875, and came to America in 1893. He went to school for nine years in Norway. He speaks Norwegian, Swedish and Danish as well as English, and used to speak Spanish; he stammers equally in all languages. He is a laborer of low intelligence; he was a sailor for five years, a rigger for ten years, a pipe fitter for four years and a pile driver for two years. He attained a total score of 21, rating D, in Group Exam- ination Alpha used in the United States Army, corresponding to a mental age of eleven j^ears. His verbal imagery was the least vivid of any of the subjects tested in (5): his auditory was 1.03 compared with the average, 2.2: his kinesthetic was but 0.13 compared with the average, 0.8; and his visual but 0.27 compared with the average, for stammerers, 1.2. All types of his non-verbal imagery were vivid ex- cept the kinesthetic. The subject spoke without hesitancy until, at the age of eight, he was so terrified on the ice by a boy who impersonated a bear that he was rendered speechless for two hours and has stammered ever since. ^ The subject's salary and expenses were paid from a grant of $100 from the Committee on Grants of the American Association for the Advancement of Science. 168 EFFECT OF STAMMERING ON BRAIN VOLUME 169 His stammering was increased at the age of thirty when he was shot by a robber, the bullet splintering the skull bone over the right eye and necessitating the trephine described by Doctor Cobb. The skin of the noticeable dip or hollow covering the trephine was loose and free in its movements and the pulsations were often conspicuous. As far as can be learned, the subject inherited no tendency to stam- mer. He sings normally and reads and speaks without hesitancy when alone. He frequently stammers on words beginning with H, L and R and sometimes repeats the first letters of certain words without con- tortions, but often avoids this stammering by using synonyms for the words he wishes to speak. He is not nervous or excitable, and is not apparently embarrassed or sensitive about his stammering. Pneumo- grams traced while the kjanograph was running at high speed showed that he breathes correctly before he speaks, but continues to speak after his lungs are empt3^ In short, the physical aspect of his stam- mering is far more prominent than the mental aspect, and the im- pediment in his speech, which is of a common type, would not be difficult to correct in a j^ounger man who was willing to w^ork con- scientiously. The subject was examined at the Massachusetts General Hospital by Dr. Stanley Cobb on January 8, 1920. His report follows: Complaint: Stammering. Past History: Smallpox at 21. Gonorrhea at 23 and 24. At about this time he also had a hard chancre for which he was treated with pills and inunctions for three months. He does not know of any other sickness except that of August 30, 1905, when he^eceived a gunshot wound in the right forehead which perforated the skull. He was taken to the Long Island College Hospital and operated on. Evidently the bullet was removed and a small trephine hole left open. Physical Examination: Cranial nerves: Smell normal. Ocular movements are normal. No squint or difficulty in convergence. Muscles of mastication strong and equal. The sensation on the face is normal. There is no facial weakness. Hearing tests show slight deafness in the left ear. The Rrnne test if referred to the left. The defect seems to be in the air conduction apparatus. The drum appears normal. Taste is normal. The pulse is regular and slow (see special report in experiments). No weakness of the sternomastoid or trapezius muscles. Tongue protrudes in midline. The fundi show normal vessels. In the right eye the disk is sharp but in the left the margins are slightly hazy. Visual acuity is within normal limits. He dod^ not wear glasses. Reflexes: Biceps, triceps, knee and ankle jerks are all equal and if anything slightly depressed. The superficial reflexes of the abdomen and scrotum are slightly more active than usual and equal on the two sides. Motor system: The muscle groups are strong and symmetrical. There is 170 SAMUEL D. ROBBINS no disturbance of gait, no ataxia, asjmergia or aphonia. There is a slight fine tremor of the extended fingers. Rjlomberg test normal. Sensory sj'stem: There is no disturbance of touch, pain or discrimination on any part of the body. Endocrin system: Thyroid not enlarged. Bony development normal. The distribution of the hair on the body and face is normal. The testicles are in normal position and well developed. SjTupathetic system : The pupils react to light and accommodation. They are round and equal. Skin reactions are slight. There is no dermographia. Skull: In the right fronto-temporal region 5 cm. from the midline is a defect in the bone irregularly circular in shape, its widest diameter being 2.5 cm. and its narrowest 2 cm. This pulsates visibly and when the patient leans over it is seen to bulge slightl}\ Respiratory system: Lungs are clear. The nose, throat and tonsils are negative. Cardiovascular system: The heart is not enlarged and the sounds are clear and without murmurs. The peripheral arteries are palpable but not thickened. The pulse is full. Blood pressure 110 80 (see special experiments) . The aortic second sound is exaggerated and somewhat louder than the pulmonic second. Alimentary system: The teeth are in fairly good condition except for three bad roots. The abdomen is level, soft and tympanitic. There are no masses felt. Blood: No anemia. Wassermann reaction negative. Mental status: General behavior: Normal, quiet and cooperative. Stream of talk is slow but pertinent. Mood: No depression or elation of spirits. Special preoccupations : No worries, imagmations, delusions or hallucinations. Orientation: Accurate for time, place and person. Memory: Accurate for remote and recent events. General Information: He knows the dates, names of the principal government oflBcials, etc., but shows no interest in the affairs of the countrj^. Speech: There is a marked stammering, especially when embarrassed by the presence of a stranger. The sticking mainly occurs on the hard consonants. There is no aphasia, apraxia or astereognosis. Diagnosis: An individual of the mentally dull type, probably not to be classed as a real defective, who has sustained a bullet wound of the right frontal region. There is no evidence of any brain injury. Apparatus. The apparatus used in this experiment included the same Zimmerman k\Tnograph, Sumner pneumograph, finger plethys- mograph, piston recorder and the tA^o electromagnets described in (6), pages 293 to 302, a less sensitive tambour than the one used in my earher work, a second piston recorder like the first, and two forms of brain plethysmograph. EFFECT OF STAMMERING ON BRAIN VOLUME 171 The first form of brain plethy sinograph, which I will call the rubber plethysmograph, consisted of a hard rubber cup having an edge of soft rubber. A metal tube led out through the top side of the cup, and a rubber tube connected this with a syringe and piston recorder as in my earlier experiment. This plethysmograph was held in place by a single bandage passed around the subject's head from front to back. As the pressure of this bandage gave the subject a severe headache, most of the experiments were performed with the second form of brain plethys- mograph, consisting of a glass funnel 4.7 cm. in inside diameter, which was cemented to the subject's forehead with collodion and removed with ether. This glass plethysmograph had two other advantages over the rubber one : it was not pressed against the forehead by bandages whose pressure varied with head movements, and an observer could detect sudden pulsations within the trephine which registered on the drum so much like movements, that in the records where the rubber plethysmograph was used I mistook them for movements and called in expert witnesses while the records were being traced to affirm that these abrupt rises were not due to movement in the large majority of cases. With the apparatus described in (6), page 300, it was found that a rise of 1 mm. on the records always denoted an increase in volume of the brain of 3.0 cu, mm. when the rubber plethj^smograph was used, and of 2.5 cu. mm. when the glass plethysmograph was used. Procedure. The procedure and arrangement of apparatus was prac- tically the same as described in (6), pages 302 to 306, except that the kymograph was run at higher speed to show the pulse. ' As five tracings were being made at the same time, it was very difficult to readjust one writing needle without throwing another out of adjustment. Being most interested in the brain tracing, I neglected the other writing need- les when the one tracing the brain volume needed attention. For this reason, the needle which traced the finger volume and which moved freely only when it just touched the drum was frequently pressed too tightly against the drum to make a true tracing. The vasomotor reactions studied in (6), where this dehcate pressure was kept constant, are far more reHable than those in this study. As the pressure of the needle tracing the finger plethysmograms could not be kept uniform, no attempt was made to measure the finger ple- thysmograms; these were recorded simply as increases or decreases. To determine what route the writing needle connected with the brain plethysmograph would have taken had the subject done no mental or 172 SAMUEL D. BOBBINS physical work and been given no stimulus, I placed the celluloid tri- angle along the crests of the highest pulses in the troughs of the Traube- Hering waves of the rest period immediately preceding and of that immediately following the period of work or disturbance. As the Traube-Hering waves were never large in these rest periods, and as the pulses at their troughs were much more uniform than those at their crests, this gave a fairly accurate reference Kne, though of course mea- surements made from it were necessarily approximate compared with those made from as accurate a reference line as was determined for the finger plethysmograms in (6). The lowest point on each plethysmo- gram, like the highest, was always considered to be the crest of a pulse at anj^ phase of a Traube-Hering wave. About half of the curves had to be discarded because of very shght leaks which developed in the brain plethysmograph when movements made by the subject loosened the bandage of the rubber plethysmo- graph or weakened some spot in the collodion which held on the glass plethysmograph; the discarded curves confirmed those retained as to rises and falls, but did not admit of accurate measurement. If the brain plethysmogram was reUable, the curve was retained, no matter how imperfect the finger plethysmogram. The subject was seated in a large Morris chair so inclined that the trephine was nearly horizontal when he laid his head back in the chair; this put the least strain on the collodion which held on the glass plethysmograph. An observer, seated close to the subject in most of these experiments, pressed a key whenever the subject moved his head, thereby enabUng me to determine which abrupt rises in the brain plethj^smograms were due to a rapid change in the brain's volume. In expermients arranged to study the effect of movements, it was found that small movements showed little if at all in the records, whereas big or quick movements were noticeable, raising the head or turning it to the right appearing to give a rise, lowering the head or turning it to the left appearing to give a fall. Curves containing head movements after which the writ- ing needle did not return to normal were discarded. Table 1 contains a brief summary of ni}' results. The percentages of increases, decreases, no changes and complex reactions are given for both brain and finger volume: + denotes increase in v^olume; — de- notes decrease in volume; denotes no change in volmue; +( — ) de- notes a prolonged increase followed by a short decrease; and so on. Rise in millimeters was measured to the highest point of the brain EFFECT OF STAMMERING ON BRAIN VOLUME 173 plethysmogram from the path its writing needle would have traced had the subject's mind been a blank. This normal path was assumed to pass through the crest of the highest pulse at the trough of each Traube-Hering wave. The column entitled "Increase in height of brain pulse" gives the number of times greater in height the average pulse during the performance of an assigned task was than the normal pulse in the rest periods immediately preceding and immediately follow- ing the period of activity. The maximum pulse referred to in the next column is the highest brain pulse traced during the period of activity. t gives the time in seconds from the beginning of an assigned task to the time when the recording needle connected with the brain plethj^smo- graph first attained its maximum rise. T gives the time in seconds from this maximum rise to the time when this recording needle first returned to normal, that is, to the path it would have taken had the subject's mind remained a blank. Or, if the time of maximum rise occurred while the subject was performing an assigned task, T was measured from the instant of completion of the task instead of from the end of t. Every period of stammering while reading or speaking showed marked increase in brain volume usually accompanied by a greatly increased pulse for at least part of the period (see figs. 4, 5 and 6). Six periods of reading, averaging 77 seconds in length, gave rises of from 23 to 88 mm., averaging 49 mm. The maxmium height of pulse registered dur- ing each reading period ranged from 1.7 to 5.0 times the normal size of pulse before and after the period of stammering, averaging 3.3 times the normal pulse. Twelve periods of talking, averaging 95 seconds in length, gave rises of from 32 to 70 mm., averaging 53 mm., and maxi- mum pulse of from 1.4 to 7.0 times the size of the normal pulse, averag- ing 4.2 tmies the normal pulse. Thus there appeared to be slightly greater cerebral congestion during talking than during reading. The rise for these reading and talking periods, taken together, ranged from 23 to 88 mm., averaging 52 mm.; and the maximum pulse for the same periods ranged from 1.4 to 7.0 times the size of the normal pulse, aver- aging 3.9 times the normal pulse. The maximum rise occurred on the average near the middle of the period in both reading and speaking. The brain volume and pulse became normal in from 20 to 46 seconds, averaging 33 seconds, after the end of the reading periods, and in from to 82 seconds, averaging 37 seconds, after the end of the talking periods. 174 SAMUEL D. ROBBINS lO 00 (N lO lO TjH &H CO i-H CO lO lO "5 ji p2 .2 .s ^ ^ ■13 ^ TS •M -d -d a 13 C '>H § § w § H ^ •i S o lO q O o q §«:< t^ (N 00 ci >*" d s2 s 1—1 S£§ b z ^ CJ C5 lO CO -* »o W5 o s ^ ago CO (N CO 1—1 ci t^ CO ■2 S i< a 5sg •M Tjl lO o CO (N 00 to ^ m |i i-H (N I— 1 y-l 1—1 C6 S W , J* fes (M CO o> TtH 05 »-( ^^ »o »o 1—1 1—1 (M e S si ^ CO CO o o ■ Oi PQ li (M »o 1 ^ c CO t^ r- ^88 TtH CO CO GO » ^ ^ + t>. 1— 1 T-( j 2 a «o + 1 + + + ° 1 + (N (M CO 05 r^ CO t^ 05 Tt< O CO 1> J". g + 1— I 1 1 + CO CO IC (N 1-1 ° + 1 lO CO rH + ° 1 + Eg 0) 55. + + + CO e ' . > — . — - ^' O K « oc 1-H CO t^ 00 CO ^; a n u o o • a I ^ • ft -kJ ^ go it b a bl ■Si •§2 D O O 1 z ) 4< o n bO PI o a bO -3 i ^ "S K £ .a ■»s u V ■< n •4-9 73 o3 g s ^ I ^ ^ ^ J3 1 cc P5 tf a O Ph f EFFECT OF STAMMERING ON BRAIN VOLUME 175 "2 "2 e^ T-4 + + CO o ^' d + + + + 00 n LO GO CO 1> LO 00 lO oo 1-H 1-H '-' o CO ■* CO UO o o o ^ o l-H '"' CO ^ C 7 + + S3 g 3 CO CO 1 ^ 00 CO S 8 g CO 7 GO CO o S CO o 00 IC o o + + + <0 a m as 03 (U (U o a a o 176 • SAMUEL D. ROBBINS The finger plethysmograms gave about an equal number of vaso- dilatations and vasoconstrictions in both reading and speaking, two +, one — , one -\ — , one — \-, one — | — , and ten no change; finger move- ments made it impossible to determine the other reactions. It is important to determine what part of the increased intracranial pressure is due to stammering and what is due to the ordinary mental and physical work of reading or speaking. The subject read aloud in the room by himself without hesitancy; but there were the usual dis- turbances in breathing and the employment of superfluous effort which are usual in stammering whether reading alone or in public; and the single record (see fig. 3) in which the results were not injured by move- ment or by the needles' leaving the drum when no experimenter was in the room gave a maximum rise of 53 mm. in the middle of the 140 second reading period, an average pulse 1.8 times the normal pulse before and after reading, and a maximum pulse 2.5 times the normal, 25 mm. compared with 10 mm. The volume and pulse returned to normal more quickly, however, than after a period of severe stammering, this being accomplished in about 18 seconds. The finger plethysmo- grams showed marked vasodilatation. This single record indicates that a stammerer may have increased intracranial pressure while read- ing aloud or speaking whether he is stammering or not, but that this intracranial pressure is highest during periods of severe stammering. A more satisfactory comparison would be that of the subject reading aloud a given passage and reading aloud another from the same book after he was cured. I was, fortunately, able to teach him in five days to read without hesitancy to any person who came into the room, and believe a comparison of records obtained while he was thus reading normally with the above stammering reading records will be a fair comparison of reactions to stammering and normal speech. He read aloud in this way in six periods free from movements in periods averag- ing 65 seconds in length (see fig. 7). Every time the subject breathed he raised his head, causing a distinct rise on the brain plethysmogram. A wave was thus caused in the curve which might be termed an in- direct breathing curve. This curve was neglected in making measure- ments of changes in brain volume. One record gave no change in brain volume, one gave — 6, + to 0, —6, and four gave rises from 12 to 20 mm., averaging 15 mm. The average pulse during these periods varied greatly and could be determined in only two records, in one increasing from 5 to 20 mm. and in the other decreasing from 12 to 8 mm. In two of the records the maximum pulse did not increase at all, EFFECT OF STAMMERING OX BRAIN VOLUME 177 and in another it was as high as 8 times the normal, averaging 3.3 times the normal pulse. The greatest rise occurred in the middle of the per- iod having the greatest average pulse and near the end of the other 5 periods. The time of recovery was 24 seconds in one, 40 seconds in another, and in doubt in 4. The finger plethysmograms showed a rise in two and no change in four of the records. There appears, there- fore, to be much greater intracranial pressure in stammering reading than in normal reading, the average rise being 49 mm. compared with 15 mm. To determine how much of the rise in normal reading was due to the mental work of the reading itself, I had the subject read silently seven passages in periods averaging 46 seconds (see fig. 2) . The brain trac- ing showed no change in two, H h in two, and + in three; where there was a + in any part of a reaction, it ranged from 12 to 29 mm., averag- ing 20 mm., and will therefore account for the rise in normal reading aloud. The average pulse was about the same as the normal pulse, and the maximum pulse ranged from 1.2 to 2.0, averaging 1.4 times the normal. Hence the mental work of reading does not account for the increased pulse during normal reading; on the other hand, it will be seen that the physical work does account for this. The greatest rise occurred near the middle of the reading period and the time of recovery averaged 55 seconds, as it did in the case of other kinds of mental work. The finger plethysmograms showed one +, one H — , and four no changes. Other kinds of mental work, including checking additions, multi- plications and divisions (see fig. 2), and counting the number of E's on a page, gave reactions similar to silent reading in eight 1 to 3 minute periods. The brain volume remained the same in one, 0+ in another, (+)( — )+ in'another, — h in another, and + in four, the rise varying from to 40 mm", and averaging 19 mm. The average pulse during periods of mental work was the same as the normal in four tracings, was lower in one, and was higher in three, as much as 3 times higher in one case; the maximum pulse was from 1.2 to 4.0, averaging 2.5 times the normal, being greater than that in reading silently. The maximum rise occurred near the end of the period of mental work, and the time of recovery ranged from 15 to 99 seconds, averaging 55 seconds. The finger plethysmograms showed no change in two curves, + in three, -\ — in one; and two were in doubt. Periods of physical work on the ergograph used by Anderson and de- scribed in (1) page 42, gave six clear cut results (see fig. 1). There was 178 SAMUEL D. EOBBINS one fatigue period which lasted 184 seconds; the other test periods lasted one minute each. There was a rise of the brain plethysmogram with increased pulse in five periods, (+)(-)0 with larger pulse in the sixth. The rises ranged from 19 to 25 mm., averaging 21 mm., includ- ing the increased pulse which averaged 16 mm., about half of which is above the line drawn through the center of the pulse. The average pulse during physical work was from 2.4 to 8.0 times the normal, aver- aging 5.4 times the normal, and remained nearly uniform throughout the period of work, the maximum ranging from 1.2 to 1.7 and averaging 1.4 times the average pulse during the period of work. In the case of the long fatigue period the maximum rise occurred near the end; and in the short periods its position varied. The time of recovery ranged from 18 to 72 seconds, averaging 54 seconds. The finger plethysmo- grams gave + in five of the curves and ( — )+ in the other: in this one the brief fall was due, no doubt, to the increased mental activity due to the change of task. Phj^sical work, therefore, is accompanied by slight increase in both brain and finger volume, and by marked increase in brain pulse. It seemed well, also, to study the brain volume during increased intrathoracic pressure, for this occurs with the muscular spasms which accompany stammering. The subject was asked, therefore, to clear his throat and also to bear down as if straining at the stool. In both cases the immediate rise was so great and so abrupt that I thought it was caused by a movement of the head until I saw through the glass plethysmograph that the skin over the trephine flattened out instantly at these times and that the subject did not move. Both showed a brief fall in brain volmne after a marked rise, then a slow return to normal, the high pulse gradually decreasing (see fig. 9). The rise varied from 52 to 82 mm., averaging 68 mm. The maximum pulse averaged 1.7 times the normal in the clearing of the throat, and 11.5 times the normal in the bearing down, being 23 times the normal in one case, and even higher in discarded records where the piston of the recorder came out of its barrel. The time of recovery averaged 16 seconds for the clearing of the throat, and 36 seconds for the bearing down. In one record chewing chocolate gave a rise in brain volume of 64 mm., the average pulse during chewing increasing from the normal of 9 to 20, and the maximum pulse to 45. This rise was not due to the pleasant sensation of the chocolate, as there was no appreciable rise during the half -minute he held this on his tongue before chewing; or EFFECT OF STAMMERING ON BRAIN VOLUME 179 to the opening and closing of the mouth per se, as this gave a fall of 7 mm. and a low pulse when the subject opened his mouth every time he inhaled and closed it every time he exhaled. It must have been due to the physical work of chewing, and might be expected in any stammerer who forces words with his jaws. A comparison of different kinds of breathing was made to learn what effect these had upon intracranial volume. Holding a deep breath caused a fall at first, then a rise until the subject breathed. Twelve seconds after the subject had kept his lungs emptj^ for fourteen seconds, a maximum rise of 38 mm. occurred with maximum pulse nearly double the normal. Two records of sniffing (see fig. 8) gave rises of 58 and 60 mm. with average pulse during the period 6 times the normal in the first and twice the normal in the second. The time of recovery was 40 seconds in the first and 33 seconds in the second. There was no change in finger volmne. The actions of clearing the throat and sniffing both tend to fix the diaphragm and thus cause increase of intrathoracic pressure. Deep breathing, on the other hand, had quite a different effect (see fig. 8). Three periods gave (+) — , one +, and one — in the brain tracing, and one — and two no change in the finger tracing (two were obscured by movement). The single instance where the change in brain volume was + throughout was but 5 mm.; the four decreases ranged from 10 mm. to 32 mm., averaging 16 mm. The average pulse during this deep breathing either remained the same or decreased, the decrease ranging from 1.7 to 3 times as low as that of normal, averaging 1.8 times as low. The greatest decrease occurred at the end of each period. In one record I had the subject read aloud normally, then breathe deeply, then read normally again, and then read silently, without pausing between these periods. The curve showed little change for these different periods of mental and physical work. The volume remained about 12 mm. above normal throughout the first normal reading pei'iod, averaged 10 mm. during the period of deep breathing, rose from 10 to 13 mm. during the second period of normal reading, and kept at 12 mm. during the period of silent reading, the pulse being higher for the silent reading period than for the other periods. Fear of stammering was also compared with shock as in my earlier study, but in spite of my many efforts to get the subject to live over again an experience in which he feared that he would stammer, I was able to cultivate this emotion only four times. On the most successful 180 SAMUEL D. ROBBIXS occasion (see fig. 5), I told him that a lady to whom my pupils found it most difficult to speak would enter the room in a few minutes and ask him some questions; she entered at the psychological moment. The four above mentioned brain plethysmograms gave rises of 15, 19, 33, and 45 mm. with increases in pulse, the average pulse increasing 4 times above the normal in the 33 mm. rise in figure 5. No change in finger volume could be detected in any of these curves. My subject's reactions to shock confirmed those of Shepard, (7) and (8), discounting the fact that my subject was less emotional (see fig. 10). There was no change in brain volume in two of the ten reac- tions to the quick stimulus of a single loud noise, and an increase in the other eight ranging from 9 to 38 mm., averaging 20 mm. The maximum pulse ranged from no increase to an increase of 8.3 times the normal pulse, averaging 3.8 tunes the normal pulse. The maxi- mum rise occurred from 5 to 18 seconds after the stimulus (30 seconds in the case of one stimulus whose length was in doubt), averaging 15 seconds. The time of recovery ranged from 14 to 34 seconds, averag- ing 23 seconds. The finger plethj^smograms showed a decrease in one case, no change in six cases, and movements in the other three; this proves my assertion that this subject Avas far less emotional than the subjects in mj'- earlier experiments (6). Now and then I noted a decided rise in a rest period and asked my subject to tell me at the end of the experiment whether he was thinking of something pleasant or unpleasant just then; I found a reason for ever}^ such rise. In one case something exciting flashed in his mind for 4 seconds causing a rise of 12 mm. A very pleasant emotion for which he reported he was miles away caused a rise of 23 mm. (see fig. 1) and a maximum pulse 13 times the normal; the finger volume also increased in this case. I w^as unsuccessful in getting hmi to cultivate emotions at will, so had to study emotions by retrospective reports in this way. In one case I had an assistant make a loud noise behind the subject in the middle of a speaking period and found that this made him stam- mer worse and greath^ increased the brain volume; this noise startled the subject so that he moved enough to make a reference line inaccurate, but not enough to spoil the record. So far as I know, there has been no previous work on a trephined stammerer. A comparison of my results with those of Berger, INIosso, Shepard and Weber upon normal speakers, summarized under "brain volume" at the bottom of table 1 (6, p. 289), shows that my results agree in every case with those of Mosso and Shepard, and agree with EFFECT OF STAMMERING ON BRAIN VOLUME 181 those of Berger and Weber in the reactions to stimiih and to mental and physical work. A few readings of the subjects systolic and diastolic blood pressure were taken at intervals while the experiments were in progress. The subject's normal blood pressure averaged 110/80. When asked a sud- den question and put completely off his guard, Doctor Cobb found the subject's blood pressure to be 145/108. Bearing down sent the blood pressure up to 125 104, slight stammering to 122/?, physical work on the ergograph to 118/?. Increase in brain volume would seem, there- fore, to have a high correlation with increase in blood pressure. SUMMARY 1. Pronounced shock, fear of stammering and emotions of every kind always brought about increase in brain volume accompanied by in- creased size of pulse. 2. Mental work was accompanied by slight congestion in the brain in a majority of cases with litxle if any increase in pulse; physical work was accompanied by slightly more congestion in the brain with marked increase in size of pulse. Physical work was accompanied by slight vasodilatation in the finger; mental work, including silent reading, by vasodilatation in the finger in 38 per cent of the curves, by vasodilata- tion followed by vasoconstriction in 16 per cent, and by no change in 46 per cent. 3. Sniffing was accompanied by marked increase in brain volume with increased size of pulse; deep breathing by decrease with slightly decreased size of pulse. 4. Change of task v/as accompanied by increase in brain volume with increased pulse and was responsible for many temporary rises. 5. Normal reading aloud was accompanied by slightly less increase in brain volume than was silent reading. 6. Stammering was accompanied by much more marked increase in brain volume than could be accounted for by either the physical or mental work used in normal speech. 7. When the stammerer read as normal speakers do, there was a return of the brain volume to normal ; it is reasonable to conclude that increase in brain volume is an important factor in the production of stammering. 8. In order to correct stammering, both the fear of stammering and the abnormal muscular contractions which usually accompany stammering must be eliminated. THE AMERICAN JOURNAL OF PHYSrOLOGY, VOL. 52, NO. 1 182 SAMUEL D. ROBBINS BIBLIOGRAPHY The following works have been specifically referred to in the text. For a complete bibliography, see (6), p. 323. (1) Anderson: Circulatory reactions during physical and mental work, 1917. (This has not been published, but may be consulted in the Widener Library of Harvard University.) (2) Berger: Uber die Korperlichen Ausserungen psychischer Zustande, Jena, 1904-7. (3) Blitemel: Stammering and cognate defects of speech, New York, 1913. (4) Mosso: Uber den Kreislauf d. Blutes im mench. Gehirn, 1881. (5) RoBBixs: Psychol. Rev., 1920, xxvii, 38. (6) RoBBixs: This Journal, 1919, xlviii, 285. (7) Shepard: Amer. Joum. Psychol., 1906, xvii, 522. (8) Shepard: The circulation and sleep, New York, 1914. (9) Weber: Der Einfluss psychischer Vorgange auf den Korper, Berlin, 1910. EXPLANATION OF FIGURES The top line in all figures is the time line, the second line is the stimulus line, the third line is the thoracic pneumogram, the fourth line is the finger plethysmo- gram and the bottom line is the brain plethysmogram. Each notch in the time line represents two seconds unless otherwise stated. A notch on the stimulus line indicates when a stimulus was given or when the subject began or stopped reading or speaking, and will be exi^lained in the de- scription of each record. The top of the pneumogram indicates empty lungs and the bottom full lungs, just the reverse of the notation in my former monograph (6). The top of each plethysmogram indicates vasodilatation, the bottom vasoconstriction. The following curves are all typical reactions; I have avoided reproducing extremes or abnormal curves. If any reader wishes to see my other curves, he is invited to examine those in mv album at the Bostom Stammerers' Institute. Q« -a +^ -o «" ^ -t^ bc a ^ o « s w . - o t; a S3 O 5 oj bC « S -1^ ^ "7 .S o3 -fj -(^ ~ *■ S ^- fi 03 _C 3 bC ^ >>^ ^ « a; 9 fi s:S ^^^ 'S c3 fi C +f •p O fi - O o s s - 03 3 ^ . 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At W a shrill whistle was blowTi unexpectedly. The clock stopped running for a few minutes at the beginning of this curve. Note that the brain volume increased soon after the whistle was blown and that the finger volume remained constant. Two-fifths natural size. 192 THE CHEMICAL CONSTITUTION OF ADENINE NUCLEO- TIDE AND OF YEAST NUCLEIC ACID WALTER JONES From the Laboratory of Physiological Chemistry, Johns Hopkins Medical School Received for publication March 12, 1920 I. A COMPARISON OF THE RATE AT WHICH PHOSPHORIC ACID IS SET FREE FROM YEAST NUCLEIC ACID WITH THE RATE AT WHICH PHOSPHORIC ACID IS SET FREE FROM THE INDIVIDUAL NUCLEOTIDES After an extended study of the rate at which phosphoric acid is spht from yeast nucleic acid by hydrolysis with mineral acid, Jones and Riley^ concluded that the nucleic acid liberates its phosphoric acid by two widely different laws: and they predicted that if the individual nucleotides were ever prepared from j-east nucleic acid and examined in this respect, each of the purine nucleotides would be found to obey one of these laws and each of the pyrimidine nucleotides would obey the other. Various preparations of mixed nucleotides were afterwards obtained and their conduct justified this assumption since each nucleo- tide preparation was found to set free its phosphoric acid in accordance with the proportion of purine and pyrimidine material that it con- tained.- Finalh' Jones and Kennedy^ found that the nucleotide groups are burned from yeast nucleic acid by potassium permanganate in a definite order, adenine nucleotide being the last to go; so that it was possible to obtain pure adenine nucleotide in this way. The substance consists of characteristic long transparent needles of the composition C10H14N5PO7.H2O and its possession furnished an excellent opportunity to ascertain the rate at which phosphoric acid is set free from a purine nucleotide. This rate was found to be the same as that of the pre^aously determined rate for guanine nucleotide,* ' Journ. Biol. Chem., 1916, xxiv, i. 2 Jones and Germann: Journ. Biol. Chem., 1916, xxv, 100. For the analytical data see Jones and Read, Journ. Biol. Chem., 1917, xxix, 123. 3 Journ. Pharm., 1918, xii, 253; 1919, xiii, 45. ^ Jones and Read: Journ. Biol. Chem., 1917, xxxi, 337. 193 194 WALTER JONES both being the rate that Jones and Riley^ had predicted for purine nucleotides. This relation is expressed diagrammatically in figures 1 and 2, which are constructed with the same system of coordination and the same linear units. Abscissae represent time and ordinates represent weights of Fig. 1. The two curves at the top show the rate at which phosphoric acid is set free from the purine nucleotides. The straight line shows the rate for the pyrimidine nucleotides. hours I ■S- 3 4- Fig. 2. The upper curve is a fusion of 3 with 1 or 2. The lower curve shows the rate at which phosphoric acid is set free from yeast nucleic acid. phosphoric acid (expressed in terms of ammonium magnesium phos- phate). In order to avoid confusion, the curves in each diagram have different origins placed vertically above one another.^ ' Journ. Biol. Chem., 1916, xxiv, i. ^ For analytical data see end of the article. YEAST NUCLEIC ACID 195 The upper curve of figure 1 was constructed from experimental data obtained with guanine nucleotide three years ago by Jones and Read. 7 The lower curve of figure 1 was similarly constructed from experi- mental data obtained more recently by Jones and Kennedy^ with adenine nucleotide. The two curves are practically identical and may be called the law for purine nucleotides. The straight Hne of figure 1 was drawn from experimental data obtained by Jones and Read^ with a mixture of the two pyrimidine nucleotides. It shows the very slow regular rate which may be called the law for pyrimidine nucleotides. The upper curve of figure 2 was constructed by fusing the pyrimidine curve with either one of the purine curves of figure 1. The lower curve of figure 2 was constructed from experimental data obtained with yeast nucleic acid, and it practically coincides with the lower curve. This coincidence points to a very definite conclusion. If it be granted that the rates are expressions of phosphoric acid linkages, then the phosphoric acid linkage of yeast nucleic acid must coincide with the phosphoric acid linkages of its four component nucleotides. HO. 0=P-0 • CoHsOs • C5H4X5O Guanine Nucleotide^" HOv 0=P-0 • CsHsOs • C4H4N3O Cytosine Nucleotide^i HOv 0=P-0 • CsHsOa • C0H4X5 Adenine Nucleotides^ ^ Journ. Biol. Chem., 1917, xxxi, 337. » Journ. Pharm., 1918, xii, 253; 1919, xiii, 45. 9 Journ. Biol. Chem., 1917, xxxi, 39. 1" Jones and Richards: Journ. Biol. Chem., 1914, xvii, 71. " Thannhauser and Dorfmiiller: Ber. d. d. chem. Gesellsch., 1918, li, 467. Zeitschr. f. physiol. Chem., 1919, ciiii, 65. 12 Jones and Kennedy: Journ. Pharm., 1918, xii, 253; xiii, 45. 196 WALTER JONES HOx 0=P - O • CsHsOs • C4H3N2O2 Uracil Xucleotide^' If yeast nucleic acid is a chemical combination of the four nucleo- tides, then in this union the phosphoric acid groups of the nucleotides must not be disturbed. No additional phosphoric acid linkages can be introduced. The four nucleotides possess together eight replace- able hj^drogen atoms; so also, yeast nucleic acid must contain eight replaceable hydrogen atoms. Therefore the nucleotide linkages of yeast nucleic acid cannot be through the phosphoric acid groups: the linkages cannot involve any one of the four phosphoric acid groups.^'* HOx HOx 0=P - O • CsHsOa • C5H4X5 0=P - O • C5H7O2 • C5H4N6 I HQ/ I O 1 HO. 1 0=P - O • CsHsOs • C5H4X5O 0=P - O • C5H7O2 • CsHiXsO Two nucleotides united through Two nucleotides united but not their phosphoric acid groups through their phosphoric acid groups Until very recently it was conceded by everyone that if the nucleotide linkage of yeast nucleic acid is not through its phosphoric acid groups then it is naturalh' through the carboh^'drate groups. That question is taken up in the following section. II. A COMPARISON OF THE RATE AT WHICH THE PURINES ARE SET FREE FROM YEAST NUCLEIC ACID WITH THE RATE AT W^HICH THE PURINES ARE SET FREE FROM THE INDIVIDUAL PURINE NUCLEOTIDES At the time the phosphoric acid studies were made with guanine nucleotide and adenine nucleotide, the rates at which they set free their guanine and adenine were ascertained. It is exceedingly rapid and the same for both purines and for yeast nucleic acid. The rapidity " Levene: Journ. Biol. Chem., 1920, xli, 1. " The argument here used is essentially that which was employed for the same purpose by Jones and Read (Journ. Biol. Chem., 1917, xxix, 123). Their argument is today as sound as it was when they wrote. YEAST NUCLEIC ACID 197 is such that the time required to dissolve the nucleotide in the hydro- lytic agent has to be considered and in the case of yea.st nucleic acid the other products of hydrolysis make the determination of the liber- ated purines very difficult. But one can conclude from the data without hesitation that the purines are set free from nucleic acid and from the individual purine nucleotides with the same rapidity. Again, the exceeding slowness with which the pja-imidines are split by hydrolysis from yeast nucleic acid makes quantitative work impos- sible. But the same slowness characterizes the pyrimidine nucleotides. Therefore the argument which was used above to show that tha nucleotide linkages of j^ast nucleic acid do not involve the phosphoric acid groups, may nov\^ be used to show that the nucleotide linkages do not involve the purine groups nor probably the pyrimidine groups. This leaves the carbohj'drate groups and indicates the formula: HOx 0=P - O • C0H7O2 • C5H4N6 HQ/ I O HO. I 0=P-0 • CsHeO • C4H4N3O HO^ I O HOx I 0=P - O • CsHeO • C4H.N2O2 HO^ i O HO. I 0=P-0 ■ CSH7O2 • C5H4NSO HO^ III. A COMPARISON OF THE RATE AT WHICH PHOSPHORIC ACID IS SET FREE FROM ADENINE NUCLEOTIDE WITH THE RATE AT WHICH ADENINE IS SET FREE FROM ADENINE NUCLEOTIDE The longest known nucleotide (though not a nucleotide of yeast nucleic acid) is the substance that Liebig^^ discovered in meat extract and called inosinic acid. It was afterwards shown contemporaneously by Bauer^^ and by Neuberg and Brahn^^ that inosinic acid is com- 1* Liebig's Annalen, 1847, Ixii, 317. i« Hofmeister's Beitr., 1907, x, 345. " Biochem. Zeitschr., 1907, v, 439; Ber. d. d. chem. Gesellsch., 1908, xli, 3376. THE AMERICAN JOURNAL OF PHYSIOLOGY, VOL. 52, NO 2 198 WALTER JONES posed of the groups of three substances, viz., phosphoric acid, pentose and hypoxanthine. Hence inosinic acid maj- have any one of three structures, i.e., anj^ one of the three groups may be the central group connecting the other two. HOv^ 0=P— O : pentose : hypoxanthine HO (1) O II Pentose : O— P — O : hypoxanthine I OH (2) ^OH Pentose : hypoxanthine : O— P=0 ^OH (3) Liebig knew that inosinic acid is a dibasic acid. This excludes formula (2). Haiser^^ found that by acid hydrolysis inosinic acid loses its hypoxanthine much more rapidly than its pentose.^^ This excludes formula (3) and leaves the correct formula (1) : H0\ 0=P-0 • CsHsOs • C0H3N4O HO^ This method may be used to find the gross structure of am^ purine nucleotide (but obviously not of a pj^rimidine nucleotide) and was ap- plied to guanine nucleotide by Jones and Read^" who found that the substance is a dibasic acid that forms a dibrucine salt and that, by acid hydrolysis, the nucleotide loses its guanine very much more rap- idly than its phosphoric acid. These t^vo facts necessitate the fol- lowing arrangement of the three groups: HOx 0=P-0 • CsHsOa • C5H4N5O HO^ »' Monatshcfte f. Chem., 1895, xvi, 190. ^^ Haiser mistook the pentose for trioxj'valeric acid, the formula C5H10O;,. =0 .Journ. Biol. Chem., 1917, xxxi, 337. Both substances have YEAST NUCLEIC ACID 199 The curves of figure 3 are constructed from experimental data ob- tained with adenine nucleotide. The upper curve expresses the rate for adenine and the lower curve the rate for phosphoric acid. In five minutes three times as much adenine is set free as phosphoric acid: or, the liberation of adenine is nearly complete in thirty minutes, that of phosphoric acid only after two hours. rtours , 2 J •*■ Fig. 3. The upper curve shows the rate at which adenine is set free from adenine nucleotide. The lower curve shows the slower rate for phosphoric acid. Adenine nucleotide forms a dibrucine salt and conducts itseK toward alkalis like a dibasic acid. Hence its groups must be arranged as indicated in the formula HOk 0=P-0 • CsHsOs • C6H4N6O IV. THE ACIDITY OF ADENINE NUCLEOTIDE In the article that follows it will be assumed that the nucleotides conduct themselves like acids toward indicators and alkalis. One cannot work long with the nucleotides without knowing that this is true of them all. When a lead salt is decomposed with sulphuretted hydrogen the filtrate from the lead sulphide is strongly acid to litmus. As the matter is of considerable importance, however, an exact exami- nation of adenine nucleotide was made. A weighed portion of the nucleotide (50 mgm.) was covered with water and titrated with 0.104 N sodium hydroxide using phenolph- thalein for the indicator; 2.69 cc. were required. A second portion of 50 mgm. was titrated using methyl orange for the indicator. Half as much alkali was required. 200 WALTER JONES STANDARD ALKALI USED FOR 50 MGM. OF ADENINE NUCLEOTIDE THEORETICALLT REQUIRED FOR Using phenolphthalein Using methyl orange Two equivalents of hydrogen One equivalent of hydrogen cc. 2.69 cc. 1.35 cc. 2.68 cc. 1.31 When free phosphoric acid is titrated with sodium hydroxide using methyl orange as an indicator exactly one equivalent of hydrogen is neutrahzed; but if phenolphthahen is used as an indicator, twice as much alkaH is required, which of course corresponds exactly to two equivalents of hj^drogen. Adenine nucleotide therefore conducts itself toward alkahs exactly hke free phosphoric acid. This is what one would expect a substance of its structure to do. HOv 0=P-OH phosphoric acid H0\ 0==P-OR adenine nucleotide Experimental Weighed portions of adenine nucleotide, placed in flasks provided with condensing tubes and treated with twenty parts of 5 per cent suKuric acid, were heated in a boiling water bath. At various inter- vals from 15 minutes to 3 hours, a flask was removed from the water bath and both free phosphoric acid and free adenine were quantita- tively determined as follows. The hot fluid was made alkaline with ammonia and treated with a slight excess of magnesia mixture. After standing 5 hours, the crystalline magnesium ammonium phosphate was filtered off, allowed to dry in the air and weighed. The weight was divided by the weight of the nucleotide used in the experiment to obtain the weight per gram of nucleotide, so that the results of various experiments could be dhectly compared with one another. It was also found convenient not to calculate the corresponding amount of phos- phorus but to express the phosphoric acid throughout in terms of mag- nesium ammonium phosphate (MgNH4P04.6H20). The ammoniacal filtrate containing adenine was treated with a solu- tion of silver nitrate in ammonia and the precipitated adenine-silver YEAST NUCLEIC ACID 201 compound was filtered off, thoroughly washed, suspended in water and decomposed with hydrochloric acid. After filtration from silver chloride, the acid fluid was evaporated to dryness (with the usual pre- cautions) for the expulsion of all but a trace of free hydrochloric acid, and a solution of the residue in a little warm water was treated with a slight excess of picric acid. The precipitated crystalUne adenine picrate was filtered off, allowed to dry in the air and weighed. This weight was divided by the weight of the nucleotide used in the experi- ment and the corresponding amount of adenine was calculated. In addition, the total amount of phosphoric acid obtainable from the nucleotide after completely burning was determined and, for com- parison, was expressed in terms of magnesium ammonium phosphate per gram of nucleotide. The results are given in table 1. Results obtained in a similar way by Jones and Read with guanine nucleotide are given in table 2, and for comparison the results obtained with yeast nucleic acid by Jones and Riley are given in table 3.^^ TABLE 1 Adenine micleotide NUCLEO- TIME OF HTDROLTSIS MAGNESIUM AMMONIUM PHOSPH.VTE MgNHlP04. 6H2O ADENINE PICR.^TE CALCULA- TED ADENINE PER CENT OF TIDE USED Obtained Per gm. of nucleotide Per cent of theo- retical (0.671) Obtained Per gm. of nucleotide RETICAL (0.3700) 0.2161 5 min. 0.0304 0.1409 21.0 0.1253 0.5800 0.2146 58.0 0.6204 15 min. 0.1915 0.3087 46.0 0.5569 0.8976 0.3330 90.0 0.6167 30 min. 0.2566 0.4160 62.0 0.5980 0.9696 0.3587 97.2 0.6302 1 hr. 0.3552 0.5637 84.0 0.6285 0.9973 0.3700 100.0 0.6111 2 hrs. 0.4084 0.6683 99.5 0.6017 0.9844 0.3652 98.7 0.6123 3 hrs. 0.4088 0.6678 99.7 0.6154 1.0051 0.3729 100.8 0.3033 4 hrs. 0.2023 0.6669 99.4 0.1114 0.9930 0.3674 99.3 0.3247 Total 0.2159 0.6646 99.5 0.3103 Total 0.2060 0.6639 98.9 Theoretical 0.6710 100.0 1.000 0.370 100.0 '' Two values are the results of later experiments. 202 WALTER JONES TABLE 2 Guanine nucleotides^ NUCLEOTIDE TIME OF HYDROLYSIS MAGNESIUM AMMONIUM PHOSPHATE MgNH4P04. 6H2O GUANINE Obtained Per gm. of nucleotide Per cent of total Obtained Per gm. of nucleotide Per cent of total 0.4234 5 mill. 0.0499 0.1178 18.8 0.0835 0.1972 50.0 0.5030 15 min. 0.1348 0.2680 42.8 0.1747 0.3473 88.1 0.5000 30 min. 0.2004 0.4008 64.0 0.1901 0.3802 96.3 0.3487 1 hr. 0.1881 0.5395 86.2 0.1396 0.4002 101.5 0.3360 2 hrs. 0.2017 0.6000 95.8 0.1310 0.3899 98.9 0.3448 3 hrs. 0.2158 0.6259 100.0 0.1360 0.3944 100.0 0.4U2 4 hrs. 0.2539 0.6174 98.6 0.1617 0.3932 99.8 TABLE 3 Yeast nucleic acid^^ TIME MAGNESIUM AM.MONIUM PHOSPHATE MgNH4P04. 6H2O NUCLEIC ACID USED Obtained Per gram From pyrimi- dine nu- cleotides I Per cent of half the total (0.295) From purine nu- cleotides II Per cent of half the total Sum of I and II 1.0031 15 min. 0.1374 0.137 0.003 0.85 0.134 40.4 41.25 1.0001 30 min. 0.2090 0.209 0.005 1.70 0.204 61.3 63.0 0.8642 1 hr. 0.2315 0.269 0.010 3.40 0.259 87.8 91.2 0.9837 2 hrs. 0.3125 0.318 0.020 6.80 0.298 101.0 107.8 0.9251 3 hrs. 0.3038 0.329 0.030 10.20 0.299 101.0 111.2 1.0305 4 hrs. 0.3488 0.338 0.040 13.60 0.298 101.0 114.6 0.8333 5 hrs. 0.2876 0.345 0.050 17.00 0.295 100.0 117.0 0.9927 6 hrs. 0.3565 0.359 0.060 20.40 0.299 101.0 121.4 0.8179 Total 0.5112 0.590 half th e total = 0.295 TABLE 4 Pyrimidine nucleotides^* NUCLEOTIDES USED TIME OF HYDROLYSIS MAGNESIUM AMMONIUM PHOSPHATE MgNH4P04. 6H2O Obtained Per gm. of nucleotides Per cent of total 1.0362 0.3587 3 hours Total 0.0647 0.220 0.0624 0.624 10.0 22 Table of Jones and Read. Journ. Biol. Chem., 1917, xxxi, 337. The four- hour period is added. " Data taken from article of Jones and Riley, Journ. Biol. Chem., 1916, xxiv, i, except 15-minute and 30-minute periods. ** From data of Jones and Read, Journ. Biol. Chem., 1917, xxxi, 39. THE ACTION OF BOILED PANCREAS EXTRACT ON YEAST NUCLEIC ACID WALTER JONES From the Laboratory of Physiological Chemistry, Johns Hophins Medical School Received for publication March 12, 1920 The earliest investigations of nucleic acid had shown that by mild acid hydrolysis, the purines are set free with part of the phosphoric acid and part of the carbohydrate; but that violent hydrolytic processes are required to set free the pyrimidines with the remainder of the phos- phoric acid and carbohydrate. A great amount of ingenuity was there- fore not required to formulate a hypothetical structure for nucleic acid. The substance must be composed of four complexes, all of which con- tain a group of phosphoric acid and a group of carbohydrate but each complex contains a different one of the four nitrogenous groups. Change the word "complex" to the word "nucleotide" and the above becomes essentially the modern nucleotide theory of the constitution of yeast nucleic acid. But one important matter concerning the constitution of yeast nu- cleic acid was not touched upon by the early investigators. At what points are the nucleotides joined together to forai j^east nucleic acid or, in other words, what is the mode of nucleotide linkage in yeast nu- cleic acid? Without any experimental evidence it was finally agreed that the nucleotide linkage is through the phosphoric acid groups and this assumption remained undisturbed until Jones, Germann and Read furnished the experimental evidence to show that this mode of nucleo- tide linkage is not correct. The principal object of the present paper is to describe an experiment which proves this in a very simple and strik- ing way. Pig's pancreas contains a variety of active agents (ferments) which decompose nucleic and further act on its decomposition products. When an aqueous extract of pancreas is boiled, all of these active agents are destroyed but one, viz., the one that decomposes yeast nucleic acid into its nucleotides. 203 204 WALTER JONES An aqueous extract of pig's pancreas was boiled and filtered. Yeast nucleic acid was added to the clear extract and the mixture was allowed to digest for 20 hours at 40°, when the nucleic acid had disappeared^ and a mixture of the four nucleotides could be isolated. This ferment (if it is a ferment) acts rather rapidly at first but more slowly afterwards. It is exceedingly more active at 40° than at 20° and exhibits about the same activity whether the solution be amphoteric, faintly alkaline or acid to litmus. It is not present in the liver nor the spleen and does not decompose thymus nucleic acid.^ In the decompo- sition of yeast nucleic acid by this ferment neither phosphoric acid nor purine bases are set free and deaminization does not occur. But when nucleic acid is converted into nucleotides by this active agent there is not the slightest change in the acidity of the solution. Yeast nucleic acid is represented below by two formulas which differ from one another in only one respect. Formula I has its nucleotide link- ages through the phosphoric acid groups; formula II has its nucleotide linkages through the carbohydrate groups. If formula I is correct, the conversion of nucleic acid into its nucleo- tides should be attended by a marked increase in acidity: but if formula II is correct, there should be no increase in acidity. If formula I cor- rectly represents the structure of nucleic acid, then the increased acidity due to the decomposition of 2 grams of nucleic acid into its nucleotides should require about 8 cc. of 0.1 N sodium hydroxide for neutralization. But as a matter of fact it was not possible to demonstrate any change in acidity with sensitive indicators when 2 grams of nucleic acid was decomposed into its nucleotides. This is a crucial experiment that decides against the phosphoric acid linkage and, I think, in favor of the carbohydrate linkage since the other possibilities are only of academic interest. I cannot feel responsible for, nor even interested in the impossibility of two carbohydrate groups uniting with one another, and presume that such a union will be conceded without any proof if the other conceivable possibilities are excluded. 1 The merest trace of nucleic acid can be detected in such a solution by the ad- dition of sulphuric or hydrochloric acid. ^ This is curious. An active agent is present in animal pancreas which is specifically adapted to plant nucleic acid. It suggests evolutionary matters. YEAST NUCLEIC ACID 205 H0\ 0=P- 1 I -0 • CsHgOs • C6H4N6O HO. O^P- HO^ -0- II C6H702 • 1 C6H4N6O H H OH i" HO. 0:^P- HO/ 1 OH = P- 1 -0 • CtHgOa • C4H4N3O -0 • CeHeO • 1 C4H4N3O H HO. O^P- HQ/ i H OH l" OH = P- 1 -0 • CfiHgOa • C6H4N6 -0 1 C6H4NJ H HO. O^P- HO^ i H OH i OH 0=P- HO^ -OCsHgOa • C4H3N2O2 -0 • CbHvOj -C4H3N2O2 i i HO. HO. O^P- HO^ HO^ -O'CsHsOs • C6H4N6O -0 • CsHgOa • CBH4N6O HO. O^P- HO^ -O-CsHsOs ■ C4H4N3O HO. O^P- HO^ -0 • CfiHgOs • C4H4N3O H0\ HO. O^P- HO/ O^P- HO^ -O-CsHsO, • C5H4N6 -0 • CeHsOj • C5H4N6 HO. O^P- HO^ -O-CfiHsOa • C4H3N2O2 HO. O^P- HO^ -0 • CsHsOa • C4HSN2O2 EXPERIMENTAL A mixture of 2 kilos of carefully trimmed and ground pig's pancreas, 2 liters of water and 30 cc. of chloroform was allowed to digest for 12 hours at the room temperature in a tightly closed vessel with frequent and violent agitation. After the tissue had by this means become thoroughly penetrated with chloroform, the mixture was placed in a thermostat and allowed to digest at 40° for 2 days, when it was cooled and filtered. The clear, pale j^ellow filtrate was then boiled, filtered from a small coagulum and after cooling was preserved with chloroform for use in the following experiments. The data given are selected from a large amount of similar data. a. 50 cc. of boiled extract + 0.250 gm. yeast nucleic acid' * In all experiments the digesting material was preserved with chloroform. 206 WALTER JONES 6. 50 cc. of boiled extract + 0.375 gm. yeast nucleic acid c. 50 cc. of boiled extract + 0.500 gm. yeast nucleic acid d. 50 cc. of boiled extract + 0.750 gm. yeast nucleic acid e. 50 cc. of boiled extract + 1.000 gm. yeast nucleic acid /. 50 cc. of boiled extract + 1.250 gm. yeast nucleic acid g. 50 cc. of boiled extract + 1.500 gm. yeast nucleic acid All were digested at 40°. After 24 hours a, h, c, d and e gave no cloud with H2SO4; a and h gave nothing with HCl, but c, d and e gave a slight cloud. After 48 hours a, h and c gave nothing with HCl; d and e only an opalescence;/ and g a faint cloud. a. 50 cc. of boiled extract + 0.750 gm. yeast nucleic acid at 20° 6. 50 cc. of boiled extract + 0.750 gm. yeast nucleic acid at 40° After 12 hours sulphuric acid gave a dense precipitate with a but nothing with h. a. 25 cc. of boiled extract + 0.375 gm. yeast nucleic acid 6. 25 cc. of boiled extract + 0.375 gm. yeast nucleic acid c. 25 cc. of boiled extract + 0.375 gm. yeast nucleic acid d. 25 cc. of boiled extract + 0.375 gm. yeast nucleic acid a was made amphoteric to litmus; h was made faintly alkaline; c was markedly alkaline; (i was left acid. All behaved about alike. After 20 hours digestion at 40° all gave a faint cloud with H2SO4 but nothing after 36 hours. a. 50 cc. of boiled pancreas extract + 0.750 gm. yeast nucleic acid h. 50 cc. of boiled pancreas extract + 0.250 gm. thjTnus nucleic acid c. 50 cc. of boiled spleen extract + 0.250 gm. yeast nucleic acid d. 50 cc. of boiled liver extract + 0.250 gm. yeast nucleic acid e. 50 cc. of phosphate mixture (pH = 6.4) + 0.250 gm. yeast nucleic acid. Digested at 40°. After 19 hours a failed to give a cloud with H2SO4 but h, c, d and e all gave dense precipitates with H2SO4 even after 48 hours. Three cubic centimeters of boiled pancreas extract were diluted with 10 cc. of water and treated with 4 drops of a solution of brom-cresol purple. On comparison of the color with a set of standard colors its acidity (pH) was found to be between 6.0 and 6.4 (about 6.2). A larger quantity of the extract was then titrated with 0.1 N sodium hy- droxide to an acidity (pH) of 6.4. Changes in the acidity of such a solution can easily be detected with brom-cresol purple. One and one-half gram of yeast nucleic acid were dissolved in 75 cc. of the above extract and the solution was titrated with 0.1 N sodium hydroxide to an acidity of 6.4, i.e., until the color which it gave with 4 drops of brom-cresol purple exactly matched the color similarly given by the extract. The extract and the solution were then digested at YEAST NUCLEIC ACID 207 40° for 48 hours. The nucleic acid had entirely disappeared and the color produced by brom-cresol purple with the solution exactly matched the color produced with the extract. The acidity of both solution and extract had not changed from 6.4. A trace of 0.1 N hydrochloric acid caused a very perceptible change in the acidity of the solution. This experiment confirms many less accurate ones that were made. In the tests for the influence of acidity on the activity of the ferment no faintly alkaline solution ever became amphoteric to litmus and no amphoteric solution ever became acid. In fact, no change at all in the tint given to litmus was ever noticed as digestion proceeded. 50 cc. boiled pancreas extract 50 cc. boiled pancreas extract + 0.750 gm. yeast nucleic acid The two solutions were digested at 40° for 48 hours, when the nucleic acid had entirely disappeared. Each solution was made alkaline with ammonia and treated at the boiling point with an excess of magnesia mixture, when perfectly white crystalline ammonium magnesium phosphate was precipitated. This was filtered the next day, allowed to dry and weighed. MgNH4P04.6H20 from the experiment 0.5474 MgNH4P04.6H20 from the blank 0.5469 An experiment was made with boiled extract which involved 125 gm. of yeast nucleic acid. After the nucleic acid had disappeared by diges- tion at 40° the product was heated to boiling and treated with neutral lead acetate as long as the reagent gave a precipitate in the hot fluid. This precipitate which consists principally of lead phosphate was fil- tered off with a pump and the pale yellow filtrate was treated in the warm with more lead acetate. At first no precipitate was produced but after the addition of a sufficient excess of the reagent a copious granular precipitate was thrown down. After cooling, the precipitated lead salts of the nucleotides were filtered off, washed, suspended in warm water and decomposed with sulphuretted hydrogen. The filtrate from lead sulphide was evaporated at 45° under diminished pressure and the nucleotides were thrown out and dried with absolute alcohol. This product is undoubtedly identical with the mixture of nucleotides for- merly obtained by Jones and Richards^ from yeast nucleic acid by treat- ment with an extract of pig's pancreas that had previously been di- gested for a long time at 40°. It contains all four nucleotides and forms a mixture of crystalline brucine salts. As the separation of the nucleo- tides from one another is foreign to the principal point of this paper, the matter will be taken up later in a separate article. * Journ. Biol. Chem., 1915, xx, 25. THE AMERICAN Journal of Physiology VOL. 52 JUNE 1, 1920 No. 2 A STUDY OF FORCED RESPIRATION: EXPERIMENTAL PRO- DUCTION OF TETANY SAMUEL B. GRANT and ALFRED GOLDMAN From the Physiological Department of Washington University Received for publication March 15, 1920 INTRODUCTION Tetany is a disease which has been subjected to a rather large amount of experimental study in recent years, the fact that its symptoms may be so readily produced by parathyroidectomy in animals, and the ques- tion of the relation of parathyroid tetany to idiopathic tetany in man, making it a particularly inviting field for experimental work. In the course of the work about to be described, it was found that all the essential symptoms of tetany could be produced in the human subject by forced respiration. It was noted during the course of a series of experiments in which the subject was required to go through a period of increased respiration, that the urine collected at the end of this period was cloudy, and that the cloudiness was due to the fact that the urine was alkaline and con- tained precipitated phosphates. In explanation of these observations it was surmised that the overventilation, by washing out carbon dioxide of the blood, tended to make the blood more alkaline, and that in order to maintain a constant hydrogen ion concentration in the body, alkali was excreted by way of the kidneys. The present investigation was undertaken with the idea of determining this question. METHODS The experiments were carried out on hunian subjects according to the following plan. During the period of forced respiration the subject, with thoracic and abdominal pneumographs attached, was lying on his 209 TBE AMERICAN JOURNAL OF PHYSIOLOGY. VOL. 52, NO 2 210 SAMUEL B. GRANT AND ALFRED GOLDMAN back, and breathed as deeply as possible in time with a metronome at the rate of about 14 per minute. This was continued until symptoms of tetany developed, usually for from 15 to 60 minutes. The pneumo- graphs recorded the depth of respiration on smoked paper, within sight of the subject, who was thus better able to maintain the deep breathing. In the later experiments, when the subjects had become accustomed to forcing respiration, the pneiunographs were discarded. The metronome, however, was used throughout to prevent the rate of breathing from becoming involuntarily too slow. The alveolar carbon dioxide tension was measured at the beginning and at the end of each experiment, the Plesch (1) bag method of col- lecting the air and the Marriott (2) colorimetric method of determining carbon dioxide tension being used. In some of the experiments the alveolar carbon dioxide tension was determined at intervals throughout the period of forced respiration, and afterwards until it had returned to normal. The urine secreted during a measured period of time just before the experiment was collected, and also that for the period during which the deep breathing occurred, and in some cases at fixed intervals subse- quently until the urine had returned to normal. Each specimen was analyzed as to hydrogen ion concentration, titratable acidity and ammonia content. The hydrogen ion concentration was determined by the method of Henderson and Palmer (3). The acidity was titrated with 0.1 N NaOH, using phenolphthalein as the indicator. Ammonia was determined by the Folin permutit (4) method. Twenty cubic centuneters of blood were drawn with a syringe from the veins at the elbow just before and at the end of forced respiration, and the hydrogen ion concentration, carbon dioxide capacity and cal- cium content were determined. The hj^drogen ion concentration was determined by the dialyzing and colorimetric method of Levy, Rown- tree and Marriott (5). Samples of blood immediately upon being col- lected and before coming in contact with the air, were put into the col- lodion sacs and dialyzed. Specimens of oxalated whole blood were also used in several cases. The carbon dioxide capacity of the blood was determined by the method of Van Slyke (6), and calcium was deter- mined by the Lyman (7) method, both whole blood and serum being used. TETANY FOLLOWING FORCED RESPIRATION IN MAN 211 RESULTS Tetany in forced respiration. Sjonptoms of tetany developed in each of twenty-four experiments, with the exception of the one control. The diagnosis was based on the presence of carpopedal spasm, Chvostek's sign, Trousseau's sign, Erb's sign of increased electrical irritability, and in one instance a tetanic convulsion. Attention was drawn to this condition in the first experiment. The subject, S. G., breathed as deeply as possible for sixteen minutes, at the rate of 15 per minute. About ten minutes after the start his fingers began to tingle. At the end of fifteen minutes the muscles of his face felt stiff, and there was some difficulty in articulation. In the second experiment, A. G., subject, tingling in the fingers was noted. When at the end of forty minutes an attempt was made to determine the blood pressure, inflation of the arm band caused the hand imme- diately to go into a tj^pical spasm of tetany. The fingers were flexed at the metacarpophalangeal joints; the second and third phalanges remained extended at the phalangeal joints; all the fingers, especially the thiunb, were adducted and the hand was flexed at the wrist. The spasm could be voluntarily overcome by active motions, but as soon as these ceased the hand returned to the former position. Generally the spasm relaxed almost immediately after cessation of the forced breath- ing, but sometimes it persisted for as long as three minutes. In sub- sequent experiments the other signs and symptoms of tetany noted above were brought out. Two subjects were used throughout the course of the experiments, and it was found that they differed somewhat in their reaction. Sub- ject S. G. developed, after breathing deeply for about ten minutes, spasticity of the facial muscles. Chvostek's sign for tetany could then be easily ehcited, i.e., tapping a branch of the facial nerve caused spasmodic contraction of the facial muscles. As the deep breathing continued this spasticity increased, until finally the muscles of the face, especially those about the mouth, contracted spontaneously, causing puckering of the Hps and decided difiiculty in articulation. In the meantime the hands had begun to tingle and feel numb, and finally, after the spasm of the face was well marked, they spontaneously went into the typical spasm described above. Chvostek's sign remained positive after forced respiration had ceased, and a sHght reaction could be obtained at least twenty minutes after the end of the experiment. In subject A. G. the facial phenomenon was not nearly so marked, but the spasm of the hands was greater. He often noted a sHght headache 212 SAMUEL B. GRANT AND ALFRED GOLDMAN soon after deep breathing started. In about fifteen minutes tingling of the hands began, and this gradually became more intense until the hands suddenly went into a very marked spasm. Trousseau's sign could usually be obtained several minutes before the onset of the spon- taneous spasm. When the facial nerve was tapped only a slight twitching of the muscles occurred and this was usually not obtained until the carpal spasm was intense, and not always then. No spon- taneous facial spasm occurred as in subject S. G. Spasm of the feet took place in two experiments in which A. G. was subject. The feet were extended, the first phalanges of the toes flexed and the second and third extended. Tingling in the abdominal and thoracic muscles was noted by both subjects on several occasions, when the period of deep breathing was prolonged. The electrical irritability of the muscles of the forearm, or Erb's sign, was tested in two experiments, and in both it was found that con- traction occurred with a much smaller current at the end of deep respi- ration than before. The indifferent electrode was fastened to the upper part of the right arm, and the stimulating electrode was applied over the ulnar nerve just above the elbow, the same point of stimulation being used before and after forced breathing. The number of milli- amperes of current necessary to cause contraction with the cathodal opening, cathodal closing, anodal opening and anodal closing stimuli before and at the end of deep respiration in experiment 18 were as fol- lows: Experiment 18 C c c c c A O C A C C Before deep respiration 5.5 2.5 0.5 2.5 1.25 3.5 0.5 2.0 CC TETANY At end of deep respiration 1.25 1.25 In experiment 5 they were : Before deep respiration 1.8 At end of deep respiration 0.+ A complete tetanic convulsion occurred in experiment 18, in which A. G. was subject. No note of the exact rate of respiration was made but it was faster than usual. After breathing very deeply for thirty minutes he suddenly went into a complete tetanic convulsion. At the onset he involuntarily gave a loud, high-pitched scream, probably due TETANY FOLLOWING FORCED RESPIRATION IN MAN 213 to contraction of the muscles of respiration and forcing of air out through contracted vocal cords. The entire body was rigid, all the muscles being contracted in tetanic spasm. The back was arched some- what, and all extremities extended completely. Relaxation occurred within thirty seconds, and there was no further spasm. There were no ill effects following the convulsion. The usual sequence of symptoms in the experiments is given in the following chart of a typical experiment, together with some of the experimental data. Experiment 11. A. G., subject TIME Before ^ minute 10th minute 13th minute 16th minute 17th minute 20th minute 21st minute 22d minute 23d minute 25th minute Alveolar CO2 tension, 40 mm. Urine pH = 4.9 Blood pH = 7.4 Plasma CO2 capacity, 63.6 vol. per cent Starts deep breathing — 12 per minute Slight headache Tingling in hands Trousseau's sign obtained. Slight Chvostek Both hands tingle markedly Spasm of both hands Alveolar CO2 tension 20 mm. Blood collected pH = 7.5 Plasma bicarbonate 45.8 vol. per cent Spasm of feet. No facial spasm. Stops deep respiration Urine collected. pH = 7.9 Changes in the blood, urine and alveolar air. Decided changes took place in the alveolar air, blood and urine as a result of the forced respi- ration. Alevolar carbon dioxide tension fell, the blood became slightly more alkaline, the urine became decidedly alkaline, the plasma bicar- bonate was reduced, the ammonia excretion was diminished, and there was a slight increase in the calcium content of the blood. Alveolar carbon dioxide. In fifteen experiments in which the alveolar carbon dioxide tension was measured it fell from an average of 42 mm. before to 21 mm. of mercury at the end of the period of deep breathing (see table 1). The fall was rapid at first and then became slower. With the termination of the period of forced breathing the tension rose at first rapidly and then more and more slowly, to almost reach normal in twenty minutes (see figs. 1, 2, 3, 4). 214 SAMUEL B. GRANT AND ALFRED GOLDMAN In experiment 17 the alveolar air was collected both by the Hal- dane (8) method, which gives alveolar air as determined by the tension of the gases in arterial blood, and the Plesch method, which gives alveolar air at the tension the gases are contained in venous blood. It was found that the difference in carbon dioxide tension as deter- 1 TABLE 1 ALVEOLAR COj TENSION LENGTH OF PERIOD OF DEEP RMPI- RATION Before deep respiration At end of deep respiration minutes 1 40 22 16 S. G. 2 35 18 50 A. G. 3 38 18 32 A. G. 4 43 22 15 S. G. 5 40 22 25 A. G. 6 40 25 25 S. G. 8 45 22 25 S. G. 9 41 24 26 A. G. 10 45 22 20 S. G. 11 40 20 23 A. G. 12 45 25 20 S. G. 13 37 20 28 A. G. 14 45 21 37 A. G. 15 45 20 23 S. G. 16 45 22 23 S. G. Average 41.8 21 25.9 TABLE 2 TIME AFTER START OF ALVEOLAR CO2 TENSION (MM.Hg.) DIFFERENCE IN TENSION Plesch Haldatne miniites mm. mm. mm. 45 37 8 5.5 31 23 8 11.5 26 19 7 16.0 23 18 5 21.5 22 18 4 mined by these two methods was less at the termination of the period of exaggerated respiration than it had been at the beginning, as shown in table 2. Hydrogen ion concentration of the blood. This was determined in five experiments, and the average fall was from pH 7.41 before to pH 7.57 i TETANY FOLLOWING FORCED RESPIRATION IN MAN 215 at the end of deep breathing (see table 3). These determinations were made on fresh blood directly from the vein, before clotting occurred. In three experiments the hydrogen ion concentration of oxalated blood was determined. Loss of carbon dioxide to the air could not be so readily prevented when this was done, but the blood was placed in a narrow test tube from the syringe, corked, gently mixed with oxalate crystals, and then dialyzed at once in the deep narrow collodion sacs. The results were as in table 4. The hydrogen ion concentration of blood may be calculated from the carbon dioxide capacity of the plasma TABLE 3 pH OF BLOOD LENGTH OF PERIOD OF DEEP RESPI- RATION Before deep respiration At end of deep respiration minutes 9 7.40 7.60 26 A. G. 10 7.45 7.65 20 S. G. 11 7.40 7.50 23 A. G. 12 7.35 7.60 20 S. G. 13 7.45 7.50 28 A. G. Average 7.41 7.57 23.5 TABLE 4 pH OF OXALATED BLOOD LENGTH OF PERIOD OF DEEP RESPI- RATION Before deep respiration At end of deep respiration SUBJECT 11 12 13 7.45 7.35 7.35 7.65 7.60 7.50 minutes 23 20 28 A. G. S. G. A. G. and the alveolar carbon dioxide tension, using Hasselbach's (9) formula. This was done in three experiments in which the required data had been obtained, and in each of these the blood was shown to have be- come more alkaline during deep respiration. In experiment 2 pH rose from 7.44 to 7.61, in experiment 12 from 7.34 to 7.48, and in ex- periment 13 from 7.40 to 7.55. Plasma bicarbonate. The carbon dioxide combining power of the plasma was determined in four experiments. It fell from an average of 59.5 v.p.c. before to 44.9 v.p.c. at the end of deep respiration. The greatest fall was 17.8, the smallest 13.0 (see table 5). 216 SAMUEL B, GRANT AND ALFRED GOLDMAN TABLE 5 CO2 CAPACITY OF PLASMA LENGTH OF PERIOD OF DEEP RESPI- RATION Before At end of deep respiration 11 12 13 21 v.p.c. 63.6 56.0 52.9 65.6 v.p.c. 45.8 43.0 38.7 52.2 minutes 22 17 27 21 A. G. S. G. A. G. S. G. Average 59.5 44.9 21.75 Hydrogen ion concentration of urine. The urine specimen collected after forced respiration was alkaline to litmus in twelve of thirteen ex- periments. The determination of the hydrogen ion concentration showed, in the average of thirteen experiments, pH 5.2 in the speci- mens voided before the period of exaggerated respiration, and pH 7.4 in the specimens voided after this period (see table 6) . TABLE 6 pH OF URINE LENGTH OF PERIOD OF DEEP RESPI- RATION Before deep respiration After deep respiration minutes 1 6.4 6.9 16 S. G. 2 4.7 7.1 50 A. G. 3 5.5 7.4 32 A. G. 4 4.8 7.3 15 S. G. 5 4.7 7.9 25 A. G. 6 5.0 7.1 25 S. G. 8 4.7 7.7 25 S. G. 9 5.5 7.2 26 A. G. 10 5.2 7.8 20 S. G. 11 4.9 7.9 23 A. G. 12 4.8 7.1 20 S. G. 13 5.8 7.7 28 A. G. 16 5.7 7.2 23 S. G Average 5.2 7.4 25.2 Titratahle acidity of urine. In twelve experiments the acidity of the urine as titrated with 0.1 N sodium hydroxide, using phenolphthalein as the indicator, fell from an average of 7.54 cc. 0.1 N acid to 2.23 cc. per half -hour (see table 7). I TETAfNY FOLLOWING FORCED RESPIRATION IN MAN 217 TABLE 7 TITRATED ACID LENGTH or PERIOD OF DEEP RESPI- RATION Of before urine; ec. 0.1 N per ^ hour Of after urine; cc. 0.1 N per J hour SUBJECT cc. 0.1 N cc.O.tN minutes 2 10.93 2.80 60 A. G. 3 8.70 1.75 32 A. G. 4 9.37 1.68 15 S. G. 6 10.54 2.36 25 A. G. 6 10.05 1.46 25 S. G. 8 4.59 0.75 25 S. G. 9 6.34 2.14 26 A. G. 10 3.53 1.24 20 S. G. 11 5.40 1.67 23 A. G. 12 5.80 2.07 20 S. G. 13 7.88 1.74 28 A. G. 16 9.72 3.05 23 S. G. Average 7.54 2.23 26 Ammonia excretion. The ammonia content of the urine was deter- mined in twelve experiments. The average decrease was from 13.18 mgm. ammonia nitrogen per half-hour before, to 5.57 mgm. ammonia nitrogen after forced respiration (see table 8) . TABLE 8 AMMONIA LENGTH OF PERIOD OF DEEP RE8PIR.\TION EXPERIMENT In before urine; mgm. NHs nitrogen per half-hour In after urine; mgm. NHs nitrogen per half-hour SUBJECT mgni. Tngm. minutes 2 16.45 7.85 50 A. G. 3 13.40 4.74 32 A. G. 4 14.20 5.32 15 S. G. 5 16.50 5.85 25 A. G. 6 9.00 3.90 25 S. G. 8 11.74 4.28 25 S. G. 9 17.40 10.13 26 A. G. 10 8.00 3.93 20 S. G. 11 12.00 7.20 23 A. G. 12 10.20 4.50 20 S. G. 13 14.25 4.13 28 A. G. 16 15.00 5.00 23 S. G. Average 13.18 5.57 26 218 SAMUEL B. GRANT AND ALFRED GOLDMAN Calcium content of blood. Calcium was determined in whole blood in one experiment and in serum in three experiments, and in each case a small but definite increase in calcium content was found in the blood collected at the end of the period of deep respiration over that found in the blood before deep respiration. All determinations were run in duplicate, some in triplicate, and a blank was run in each case. The figures are given in table 9. TABLE 9 Ca CONTENT OF BLOOD LENGTH OF PERIOD OF DEEP RESPIRATION EXPERIMENT Before deep respiration At end of deep respiration mgm. per 100 cc. mgm. per 100 cc. minutes 18 (Whole blood) 19 (Serum) 7.00 11.81 7.29 13.19 67 51 A. G. S. G. 20 (Serum) 12.75 13.31 62 A. G. 21 (Serum) 12.84 13.44 21 S. G. The blood taken from the veins at the end of deep respiration had a much brighter red color than normal venous blood. Control experiments. It was necessary to show, a, that the intake of food was not influencing the results; and h, that the results could not be attributed to any factor other than the o'verventilation. In experi- ments 9, 10, 11 and 12 the subjects omitted breakfast on the morning of the experiment. There was no appreciable difference between the results in these experiments and any of the others except that the titratable acidity of the control urine specimens was less than in the other experiments. In experiment 7 means were taken to prevent the subject from low- ering the carbon dioxide content of the alveolar air. He breathed through a tube connected to a fifteen-liter bottle, while the observer at the same time breathed through a second tube connected to the bottom of the bottle. The resulting high carbon dioxide content of the air in the bottle prevented the subject from washing out the carbon dioxide from his blood, in spite of the forced respiration. Under these condi- tions the alveolar carbon dioxide tension was slightly higher at the end of the experiment than it had been before. The acidity and ammonia content of the urine increased sHghtly, rather than decreasing as in the other experiments, and the hydrogen ion concentration of the urine remained about the same. The figures are given in table 10. TETANY FOLLOWING FORCED RESPIRATION IN MAN 219 No symptoms of tetany occurred in this experiment. The subject became very slightly cyanosed, and after the experiment was dyspnoeic for a few minutes. Breakfast was omitted on the morning of the ex- periment. TABLE 10 A. G. subject. Deep breathing 17 minutes tJBINE pH URINE ACID (CC. O.I N) NHs NITBOGEN (mgM.) ALVEOLAR COj Before After Before After Before After Before After 4.6 4.8 7.79 8.27 11.2 13.3 38 40 Four of the experiments may now be taken up in detail in order to illustrate more concisely the exact method of procedure and the uni- formity of the results. TIME Experiment 12. S. G., subject Alveolar CO2 tension =45 mm. Urine pH=4.8 minutes Blood pH = 7.35 Plasma CO2 capacity=56 v.p.c. 10 minutes Starts forced respiration 20 minutes Chvostek's sign obtained. Alveolar CO2, 28 mm. Tin- gling in fingers 23 minutes Spasm of facial muscles — Chvostek's sign marked. Trousseau's sign positive 27 minutes Blood collected pH = 7.60 Plasma CO2 capacity = 43 v.p.c. 30 minutes Alveolar CO2 tension = 25 mm. Stops forced respiration 40 minutes Urine collected pH = 7.1 45 minutes Alveolar CO2 tension = 33 mm. 55 minutes Alveolar CO2 tension = 37 mm. 1 hour 10 minutes Urine collected pH = 5.0 1 hour 15 minutes Alveolar CO2 tension = 39 mm. 2 hours 10 minutes Urine collected pH = 5.0 2 hours 20 minutes Alveolar CO2 tension = 40 mm. The graph of this experiment (fig. 1) shows the synchronous changes in alveolar air, blood and urine. Time in minutes is plotted on the horizontal axis, and alveolar carbon dioxide tension in millimeters of mercury, plasma carbon dioxide capacity in volume per cent, hydrogen 220 SAMUEL B. GRANT AND ALFRED GOLDMAN 10 20 30 40 50 1 10 20 30 40 50 2 : 20 50 MM __JJ 1 1 I 1 1 1 1 1 1 1 i 1 1 1 40 30 ~ ^*-*^^,X^LVEOLAR COg TENSION ~ 60 50 N.CO2 CAPACITY N^ OF BLOOD PLASMA — 40 — ^^ Ph8 • PH^Or BLOOD — 7 Ph ofurine 6 — 5 ^^^ 13 12 \ cc .In acid ^/zy/y. / y 1 . T RO' AF II 10 Zv/y/ ^/ T - EU DR. aI eH. D . 1 T Y A . M^ V Oc' N R 1 E" AT 9 8 7 ^ y^ y\Xy\y\y^y 6 /// y/y^ 1 5 /// yC>Cx 4 S^^Sl $$S656566< 3 2 % 1 • N. 1 Xk\ " Fig. 1. Experiment 12. Forced respiration begins at 10 minutes and ends at 30 minutes. TETANY FOLLOWING FORCED RESPIRATION IN MAN 221 ion concentration of blood and urine in terms of pH, and ammonia excretion and titrated acidity of urine per half-hour in cc. .IN solutions on the perpendicular axis. Deep breathing starts at 10 and ends at 30 minutes. As the alveolar CO2 tension falls, the CO2 capacity of the plasma falls, hydrogen ion concentration of blood and urine fall, excretion of ammonia decreases, and titrated acidity of urine falls. After deep breathing ceases, alveolar CO2 tension rises, and correspond- ing changes occur in the urine. Experiment IS. A. G., subject TIME minute Alevolar CO2 tension =37 mm. Urine pH = 5.8 Blood pH = 7.45 Plasma CO2 capacity = 52.9 v.p.c. 10 minutes Starts forced respiration 25 minutes Slight headache 30 minutes Alveolar CO2 tension = 23 mm. 31 minutes Tingling in right foot. No Chvostek 32 minutes Hands tingle 33 minutes Tingling increased 34 minutes Trousseau's sign markedly positive 35 minutes Both hands in spontaneous spasm 36 minutes Slight spasm of feet 37 minutes Blood drawn pH = 7.50 Plasma CO2 capacity = 38.7 v.p.c. Spasms of hand more intense 38 minutes Stops forced respiration 40 minutes Alveolar CO2 tension = 20 mm. 42 minutes Urine collected pH = 7.7 Temporary dizziness and trembling of hands on standing up after forced respiration 45 minutes Alveolar CO2 tension =28 mm. 50 minutes Alveolar CO2 tension = 32 mm. 1 hour Alveolar CO2 tension = 37 mm. 1 hour 23 minutes Urine collected pH = 6.9 1 hour 50 minutes Urine collected pH = 5.0 In the graph of this experiment (fig. 2) the same changes are shown as in figure 1, with the exception that the titrated acidity of the urine is not charted. Deep breathing starts at 10 and ends at 38 minutes. The third specimen of urine after forced breathing is more acid than the control urine specimen. 222 SAMUEL B. GRANT AND ALFRED GOLDMAN 10 20 30 40 50 1 10 20 30 40 H^ I r I \ J \ r r r Fig. 2. Experiment 13. Forced respiration begins at 10 minutes and ends at 38 minutes. TETANY FOLLOWING FORCED RESPIRATION IN MAN 223 Experiment 16. S. G., subject 30 minutes Urine collected pH=5.0 1 hour Urine collected pH = 5.7 1 hour 5 minutes Alveolar CO2 tension = 45 mm. 1 hour 7 minutes Starts forced respiration 1 hour 12 minutes Alveolar CO2 tension = 31 mm. 1 hour 18 minutes Alveolar CO2 tension = 26 mm. 1 hour 22 minutes Tingling in hands 1 hour 23 minutes Marked tingling in hands. Alveolar CO2 tension = =23 mm. 1 hour 26 minutes Carpal spasm 1 hour 28 minutes Alveolar CO2 tension = 22 mm. 1 hour 30 minutes Stops forced respiration 1 Urine collected pH = 7.2 2 hours Urine collected pH=6.8 2 hours 45 minutes Urine collected pH = 5.8 3 hours 45 minutes Urine collected pH = 4.8 4 hours 15 minutes Urine collected pH=5.5 Experiment 4- S. G., subject TIME 20 minutes Alveolar C02 = 43 mm. 25 minutes Urine collected pH = 4.8 30 minutes Starts forced respiraton 38 minutes Tingling and numbness in hands 40 minutes Headache 41 minutes Rigidity of facial muscles. Chvostek markedly positive, right and left 42 minutes Carpal spasm both hands 44 minutes Abdominal and thoracic muscles begin to tingle. No spasticity felt by observer 45 minutes Stops forced respiration Aveolar CO2 tension = 22 mm. 55 minutes Urine collected pH = 7.3 Chvostek weakly positive Alveolar CO2 tension = 32 mm. 1 hour 7 minutes Chvostek negative 1 hour 15 minutes Alveolar CO2 tension = 40 mm. 1 hour 25 minutes Urine collected pH = 4.8 1 hour 35 minutes Alveolar CO2 tension = 43 mm. 2 hour 25 minutes Urine collected pH = 4.7 3 hour 25 minutes Urine collected pH = 5.2 224 SAMUEL B. GRANT AND ALFRED GOLDMAN 10 20 30 40 50 1 10 20 30 40 50 2 10 20 30 40 50 3 K) I I I I I I I I I I I I I I I I I Fig. 3. Experiment 16. Forced respiration begins at 30 minutes and ends at 53 minutes. TETANY FOLLOWING FORCED RESPIRATION IN MAN 225 Figure 3 is a graph of experiment 16. Deep breathing starts at 30 minutes and ends at 53 minutes. The fall in alveolar CO2 tension is very marked at first and then gradually becomes smaller as the low 50 MM 40 10 20 30 40 50 1 10 20 30 40 50 ?-W O 30 LVEOLAR CO2 TENSION Ph of urine Fig. 4. Experiment 4. Forced respiration begins at 30 minutes and ends at 45 minutes. level is reached. • The urine pH reaches its control level 1 hour and 15 minutes after the forced respiration, while the specimen collected 2 hours 15 minutes after is more acid than the control one. The other changes are similar to those shown in figures 1 and 2. THE AMERICAN JOURNAL OF PHYSIOLOGY, VOL. 52, NO. 2 226 SAMUEL B. GRAXT AND ALFRED GOLDMAX The changes in experiment 16 (fig. 4) are similar to those described in the previous ones. The deep breathing begins at 30 and ends at 45 minutes. The return of the alveolar CO2 tension to the control level is shown, 60 minutes elapsing before this occurs. The ammonia does not reach the control level in 2 hours and 30 minutes. DISCUSSION Forced respiration, by washing out carbon dioxide from the alveoli, reduces the carbon dioxide content of the blood and so tends to make the blood more alkaline than normal. This reduction of carbon dioxide XT r^r\ in the blood disturbs the . .-- l^ _,^ ratio upon which the hydrogen ion JN aH' OU3 concentration depends, and on the reestablishinent of this ratio, NaH CO3 passes out of the blood plasma into the tissues, and a con- siderable portion is excreted by way of the kidneys. Apparently, COo may be washed out of the blood more rapidly by overventilation, than NaH CO3 is decreased by excretion or other means, for on this assump- tion alone is it possible to account for the increased alkalinity actually found in blood during deep respiration. The diminution in ammonia formation is a further evidence of the effort of the body to compensate for the increased alkalinitj^ of the blood. The lowered urine acidity is due partly to the increased alkali excretion, but probably largely to a retention of acid radicals. In view of these considerations it seems logical to conclude that there is a true condition of "alkalosis" in the bodj^ during overventilation; the body fluids are actually more alkaline than normal. It is realized that the term " alkalosis " is a poor one, but we are using it strictly in the sense of an actual change in hydrogen ion concentration. In this con- dition of alkalosis we have an actual reduction of the "alkaline re- serve," due to the elimination of NaHCOs from the body fluids in an effort to maintain a constant hydrogen ion concentration. The coex- istence of a more alkaline blood and a reduced alkaline reserve is another example of the inadequateness of the terms "alkalosis" and "acidosis." The question now arises, is it possible to establish an etiological re- lationship between this condition of alkalosis and the tetany which develops during forced respiration? This may be discussed in con- nection with the views as to the cause of tetanj^ that are held at the present time, the two principal ones of which are, first, that it is due to a calcium deficiency in the blood; and second, that it is due to a disturbance of the acid-base equilibrium in the bod3\ TETANY FOLLOWING FORCED RESPIRATION IN MAN 227 The literature contains some references to this problem. Thus, Wilson, Stearns and Thurlow (10) and Wilson, Stearns and Janney (11) have found that in parathyroidectomized dogs, just before an attack of tetany, a period of "alkalosis" develops, which is neutralized by the acid substances that are produced during tetany. They base their conclusion that an "alkalosis" obtains on the study of the dissociation constant of the oxj^hemoglobin, the CO2 content of alveolar air, the hydrogen ion concentration of blood, the ammonia content of urine and the total acidity and hydrogen ion concentration of urine. In- jections of acid and also of calciimi salts relieve the tetany. They state that possibly part of the beneficial action of the calcium salts may be due to a relative increase in acid radicals caused by their administration. This might be brought about by the formation of Ca3(P04)2 from the carbonates, thus liberating HCl. McCann (12) finds after parathyroidectomy (and following operations on the stomach which exclude the acid secreted from the duodenum) that there is a marked increase in the CO2 combining power of the plasma, coincident with the development of tetany and concludes that tetany is a condition of alkalosis in which a disproportion between the rates of secretion of acids and alkahes by the gastro-intestinal tract may be a factor. He points out that clinicalty gastric tetany is most apt to occur in those cases in which there is some pyloric obstruction, so that the acid gastric secretion does not pass into the duodenum to excite the alkaline intestinal secretion, and that this might be expected to leave the blood with an excess of basic substances. MacCalkmi (13) and his coworkers in a paper just pubhshed state that when the pylorus of a dog is occluded, and only distilled water is introduced into the duodenum, the gastric mucosa continues to secrete hydrochloric acid. At the same time the alkali reserve increases and tetany-like sjTuptoms appear. This can be prevented by the introduction into the duodenum of sodium chloride or the s>Tnptoms may be made to disappear b}^ the intravenous injection of sodium chloride. Injection of hydrochloric acid is not so successful. Calcium determinations by a dialyzing method showed no change in the calcium content of plasma. The authors think that possibly the disturbed equilibriimi of acids and bases in itself is the cause of the symptoms. Cases of tetany following therapeutic administration of sodium bi- carbonate have been reported. Rowland and ^Marriott (14) cite three cases in children, all of which were associated with a marked diminution of the calcium content of the serum. Harrop (15) reports a case in an 228 SAMTEL B. GRAXT AND ALFRED GOLDMAN adult who developed tetan}' after the intravenous injection of sixty- grams of soditiin bicarbonate. There was no associated diminution in calcium. Binger (16) has shown that when sodium phosphate in sufficient quantity and at the right reaction is injected into dogs intravenously, SATnptoms of tetany result, associated with a marked decrease in the calcium content of the serum. But if the solution injected has a hydro- gen ion concentration greater than 10"^, no tetany occurs even though there is a dkninution in calcium. Ammonia poisoning produces symptoms strikingly similar to those of parathyroid tetany. Marfori (17) found after intravenous injections of the carbonate, lactate and tartrate of ammonia in dogs, tremors, muscle twitchings, even tetany and opisthotonus, and irregular respi- ration, vomiting, etc. MacCallum and Voegtlin (18) found increased ammonia content of the blood in parathyroid tetany as did also Carlson and Jacobson (19) in their earlier work. Later, however, using im- proved methods, Carlson and Jacobson could find no change in ammonia content of blood in parathj-roidectomized dogs. The fact that increased respiration is associated with changes in the reaction of the urine had been previously noted b}^ Hasselbach (20). He found that when the alveolar CO2 tension was raised a few milli- meters by the influence of morphine on the respiratory center, the urine became verj' slightly more acid, and that when the alveolar CO2 tension was decreased by stimulation of respiration by Hght baths, the urine became barely perceptibly more alkahne. After the present work had been started, Leathes (21) pviblished a paper in which it was stated that increasing the respiration voluntarily for from one-half to one hour increased the alkalinity per cent of the urine from about 35 per cent to about 90 per cent. There was no men- tion made of symptoms of tetany or other changes during deep respi- ration. Yandell Henderson and Haggard (22) found a great reduction of CO2 capacit}^ of blood plasma of dogs in ether hyperpnoea and in ex- cessive pulmonary ventilation by artificial respiration, together with a concomitant fall in blood pressure. They state that when the CO2 capacity of plasma falls below 33 to 36 v.p.c, the process becomes irre- versible, and the animal dies in shock. The lowest point to which CO2 capacity of plasma fell in our work, in those experiments in which it was determined, was 43 v.p.c. Sj^mptoms of tetany are not mentioned in their paper. TETANY FOLLOWING FORCED RESPIRATION IN MAN 229 Stein (23) in discussing tetany as a sequel of gynecological operations cites fifteen cases in which it occurred. In four of these the symptoms of tetany occurred during the operation, and disappeared quickly with- out recurrence later. In three cases the tetany occurred during light ether anesthesia, one of these as narcosis was being induced, while the patient was still conscious. In the fourth case tetany occurred after the removal of eight liters of fluid from the peritoneal cavity by para- centesis, and lasted for fifteen minutes. It seems not unlikely that tetany in these instances may have been due to overventilation. McClendon (24) has studied the effect of hydrogen and hydroxyl ions on the pulsation rate of the jelly fish and the heart of the conch, and finds that the activity may be inhibited by increasing H-ions in the surrounding fluid or perfusing Kquid, as the case may be, and augmented by increasing the OH-ions. Biedermann (25) found that the height- ened excitability of voluntary muscle produced by NaCl solution is greatly increased by the addition of NaoCOs, the muscle then contracting rhythmically and exhibiting a striking increase of response to artificial stimuli. Strong solutions of NaS04 and dilute NaOH act in a similar manner but to a less degree. He seems to attribute this to a specific effect of the sodium ion, but it may be that part of the action is due to the alkalinity of the solutions in the case of Na2C0:< and NaOH. Recently a number of investigators have come to regard tetany as being due to a decrease in the calcium content of the blood. Thus MacCallum and Voegtlin (18) and MacCallum and Vogel (26) have shown that in the tetany of parathyroidectomized dogs there is a marked diminution of blood calcium. Howland and Marriott (27) have found that in infantile tetany the calcium content of the serum is greatly reduced, the average being about 5.6 mgm. per 100 cc. compared to 10 or 11 mgm. in the normal child. The administration of calcium promptly relieved the symptoms of tetany. On the other hand we have found in tetany of forced respiration no diminution of calcium content of the blood or serum. There is, as a matter of fact, a shght increase in calcium in every instance in blood specimens taken at the onset of tetany over those obtained under normal conditions of respiration. This may not necessarily mean that calcium plays no definite part in the production of the tetany of increased respiration. Thus Barille (Wells, 28) states that a decrease of CO2 in the blood results in precipi- tation of calcium. And Marriott (29) has shown that when artificial blood is brought into equilibrium with CO2 at 30 mm. Hg. tension, calcium is precipitated in a perceptible cloud. The possibility must 230 SAMUEL B. GRANT AND ALFRED GOLDMAN therefore be considered that when the CO2 of blood is reduced by over- ventilation a portion of the calcium is "precipitated" or in some way rendered inactive, though still present in the circulating blood. On the basis of this supposition the increase of calcium which we have found might be interpreted as being due to a compensatory mechanism on the part of the body to make up for a loss of active calcium. The calcium in our subjects was not precipitated in the blood, however, for centrifuging the serum specimens did not affect the analysis. It is conceivable, however, that the calcium is "precipitated" as very minute particles which are held in solution by the colloids of the serum, in which case centrifuging might have no effect on the calcium present. A fur- ther attempt to determine whether calcium plays a part in the produc- tion of tetany of overventilation will be made in a future investigation. It may not be inappropriate to refer in this connection to the hypoth- esis advanced by Stoeltzner (30) attributing tetany to an accumulation of calcium in the blood. He found support for his views in some obser- vations on the electrical reactions of peripheral nerves in infants after the administration of calcium. But these observations of Stoeltzner's have failed of confirmation. Tetany occurs under so many circimistances that it probably cannot be attributed to a single etiological factor. It is more Ukely that it must be regarded as a symptom complex, and possibly any condition that heightens the irritability of peripheral nerves may cause it. Obser- vation seems to indicate that alkalosis may be such a cause, and it probably is the underlying factor in the tetany that develops as a result of forced respiration. SUMMARY 1. Forced respiration causes symptoms of tetany to occur in the human subject; these include carpopedal spasm, Chvostek's sign. Trousseau's sign, Erb's sign, and in one instance a tetanic convulsion. 2. As a result of the fall of alveolar CO2 tension produced by over- ventilation, there is a reduction in the hydrogen ion concentration of the blood, a reduction of the CO2 capacity of plasma, a change in the reaction of the urine to the alkaline side, a decreased excretion of am- monia, and a slight increase in the calcium content of the serum. 3. The underlying factor in the tetany of forced respiration is the alkalosis. TETANY FOLLOWING FORCED RESPIRATION IN MAN 231 We wish to express our deep appreciation of the invaluable help and interest in this work given us by Dr. Joseph Erlanger. We are also indebted to Dr. P. A, Shaffer and Dr. W. McK. Marriott for their kindly interest throughout the work. Note. After the completion of this paper, several important refer- ences bearing upon our results were found in a journal which had been temporarily lost from the library. H. M. Vernon (31) in some obser- vations on the production of prolonged apnoea in man says, "I found that after about 6 minutes of forced breathing the muscles of my hands passed into a condition of tonic rigidity, and for the first one and one- half minutes of the subsequent apnoea they were completely paralyzed. My other muscles were apparently unaffected." In no case in our experiments were the hands paralyzed, for we have always been able to voluntarily overcome the spasm, and this is also true of the spasm of idiopathic tetany. Yandell Henderson (32) had a large number of men undergo voluntary hyperpnoea for short periods of time (usually 45 to 90 seconds) and makes the following statement. "Several of the subjects after vigorous hyperpnoea for 1 or 2 minutes, experienced in varying degrees the phenomenon mentioned by Vernon. . . . Even more common in our experience is a prickling sensation in the legs and arms, or in some cases in the entire body and face, somewhat similar to a hand or foot 'asleep.'" In one case, after vigorous hyperpnoea for two minutes a "shivering fit" came on, similar to that seen in a chill, and involving apparently all the muscles. Hill and Flack (33), in determining the effect of oxygen inhalation on muscular work, had men undergo voluntary hyperpnoea for short periods. They observed feehngs of numbness in the limbs, with a spastic state of the hands. One subject felt "twitching of the facial muscles." In another the "mouth was felt pursed up in the form of an O, so that speaking became difficult," and the muscles surrounding the eyes seemed contracted, so that the eyes were difficult to open fully. Apparently none of these investigators realized that the phenomena observed in forced breathing are identical with those seen in tetany, and no effort was made to determine the factors upon which they depend. 232 ' SAMUEL B. GRANT AND ALFRED GOLDMAN BIBLIOGRAPHY (1) Plesch: Zeitschr. p. expev. Path. u. Therap., 1909, vi, 380. (2) Marriott: Journ. Amer. Med. Assoc, 1916, Ixvi, 1594. (3) Henderson and Palmer: Journ. Biol. Chem., 1912, xiii, 393. (4) FoLiN and Bell: Journ. Biol. Chem., 1917, xxix, 329. (5) Levy, Rowntree and Marriott: Arch. Int. Med., 1915, xvi, 389. (6) Van Slyke: Journ. Biol. Chem., 1917, xxx, 347. (7) Lyman: Journ. Biol. Chem., 1917, xxix, 169. (8) Haldane and Priestley: Journ. Physiol., 1905, xxxii, 225. (9) Hasselbalch: Biochem. Zeitschr., 1916, Ixxviii, 112. (10) Wilson, Stearns and Thurlow: Journ. Biol. Chem., 1915, xxiii, 89. (11) Wilson, Stearns and Janney: Journ. Biol. Chem., 1915, xxiii, 123. (12) McCann: Journ. Biol. Chem., 1918, xxxv, 553. (13) MacCalltjm et al: Bull. Johns Hopkins Hosp., 1920, xxxi, 1. (14) HowLAND AND Marriott: Quart. Journ. Med., 1918, xl_. 289. (15) Harrop: Bull. Johns Hopkins Hosp., 1919, xxx, 62. (16) Binger: Journ. Pharm. Exper. Therap., 1917, x, 105. (17) Marfori: Arch. f. exper. Path. u. Pharm., 1893, xxxiii, 71. (18) MacCallum and Voegtlin: Journ. Exper. Med., 1909, xi, 118. (19) Carlson and Jacobson: This Journal, 1910, xxv, 403; 1911, xxviii, 133. (20) Hasselbalch: Biochem. Zeitschr., 1912, xlvi, 403. (21) Leathes: Brit. Med. Journ., 1919, no. 3056, 165. (22) Henderson and Haggard: Journ. Biol. Chem., 1918, xxxiii, 333. (23) Stein: Interstate Med. Journ., 1916, xxiii, 1078. (24) McClendon: Journ. Biol. Chem., 1916, xxviii, 135. (25) Biedermann: Electro-physiology, New York, 1898, 104 (26) MacCallum and Vogel: Journ. Exper. Med., 1913, xviii, 618. (27) HowLAND and Marriott: Quart. Journ. Med., 1918, ii, 289. (28) Wells: Chemical pathologj', Philadelphia and London, 1918, 443. (29) Marriott: Personal communication. (30) Stoeltzner: Jahrb. f. Kinderh., 1906, Ixiii, 661. (31) Vernon: Journ. Physiol., 1909, xxxviii, xx. (32) Henderson: This Journal, 1909, xxv, 310. (33) Hill and Flack: Journ. Physiol., 1910, xl, 360. A STUDY OF THE CARBOHYDRATE TOLERANCE IN ECK FISTULA AND HYPOPHYSECTOIMIZED ANIMALS (POSTERIOR LOBE RE:M0VAL) Liver Fuxctiox ix the ^Ietabolism of Sugars CONRAD JACOBSON From the Peter Bent Brigham Hospital, Boston, Mass., and the Hunterian Labo- ratory of Surgical Research, Johns Hopkins Medical School, Baltimore, Md. Received for publication March 17, 1920 In the course of some studies in the Hunterian Laboraton- on the sugar tolerance of animals deprived of the hypophyseal gland (1) it was shown that there was a temporar}- lowering of the assimilation limit for saccharose (the sugar which was primarily used for reasons then given), often with spontaneous mellituria — usually dextro-rotatorj-, occasionally laevo-rotatorj- — followed by a subsequent rise to above normal. It was observed at the same time that this artificialh' raised tolerance as well as the normal tolerance of animals (cat, dog and rabbit) could be lowered occasionally to the point of spontaneous glj'- cosuria by the administration, intravenously or by mouth, of hypophys- eal posterior lobe extracts. It was assumed and has been since proven that the administration of the extract had a glycogenolytic effect and was accompanied by a hyperglycemia. In the further investigation of this subject, some efforts were made to determine the source of this gly- cogen. This was first attempted by staining methods — Best's Carmine of the liver — before and after discharge of the glj'cogen either by Ber- nard's Piqure, by stimulation of the hypophysis or b}- giving hypo- physeal or adrenal extracts. Though a lessening in the glycogen con- tent of the cells could be demonstrated, the cells were never found entirely free from glycogen and the method had to be abandoned. Marked fatty changes in the liver had also been noted in some of the hypophysectomized animals and the suggestion arose that these changes might produce modifications in the storage capacity of the liver for glycogen and for carbohydrate metabolism. It was therefore decided to use an Eck fistula animal and to note what effect the shunting of the 233 234 CONRAD JACOBSON liver from the portal circulation would have upon the sugar tolerance both before and after hypophysectomy. The Eck fistula operation was first successfully performed on animals by von Eck in 1877 (2) and repeated with equal success by Stolnikow in 1882 (3). It consisted essentially of making an anastomosis of the portal vein to the vena cava with subsequent ligation and more re- cently, double ligation and transection of the portal vein near the liver hilus. In such a condition the blood supply of the liver is maintained by the intact hepatic arterj^, but the blood coming from spleen, pancreas, stomach and intestines, carrying with it the products of gastro-intestinal and splenic activity, is carried into the vena cava instead of to the liver and thus directly into the venous circulation. The purpose of the origi- nal operation was a clinical one, that of furnishing drainage for the abdominal fluid, ascites, incident to a cirrhosis of the liver, and neither of these two original investigators did anj^ reliable work on changes in the metabolism. The operation has never proven successful when applied to human subjects both because of the operative difficulties and of the danger of subsequent toxic effects. It has, however, on repeated occasions been of extreme value in physiological investigations regarding carbohydrate metabolism and liver function. In 1893 Hahn, Massen, Nencki and Pawlow (4), working with Eck fistula dogs, showed that such animals fed on a meat diet developed marked toxic effects, usually ending in the death of the animal. There was noted a definite decrease in the urinary output of uric acid and ammonia and a large increase in carbamate. On injecting the latter substance into normal and Eck fistula animals, they were able to pro- duce similar toxic effects and concluded that accumulation of carbamate was the causal factor of the toxic symptoms. Rothberger and Winter- berg in 1895 (5) verified the work of Pawlow et al, but could not satisfy themselves that carbamate was the toxic agent, concluding indefi- nitely that the toxic effect was caused by some substance which was removed by the liver under normal conditions but which, under the altered conditions of Eck fistula, accumulated in the blood. Nencki, Pawlow and Zaleski in 1896 (6) from experimental evidence concluded that ammonia was the cause of the toxic symptoms and not the car- bamate. However in 1897 Nencki and Pawlow (7), by further experi- mentation with Eck fistula animals in which large portions of the liver were either removed or thrown out of function by ligation of the hepatic artery, concluded that the extreme toxic symptoms were far out of pro- portion to the increase of ammonia and could not consider the latter LIVER FUXCTIOX IX METABOLISM OF SUGARS 235 as the main causal factor. Popelski in 1898 (8) showed that the Eck fistula dog had a low carbohydrate tolerance and that an alimentary glycosuria resulted when such an animal was fed large quantities of glucose. Sachs in 1898 (9), working with frogs whose livers had been extirpated, showed that they had a lower tolerance for laevulose than normal intact control animals, a finding which could not be confirmed with dextrose, galactose or arabinose. In 1901 Strauss (10), working with Sachs's findings as a rationale, introduced the laevulose test for hepatic insufficiency. In 1902 Schultz and Miller (11) had the oppor- tunity of closely observing a clinical case of portal embolus at the liver hilus and were unable to find anj^ definite untoward s\Tnptoms except that of ascites, reporting no pathological findings in urine. In 1907 Filippi (12) showed that there was a lowered tolerance for carbohydrates in dogs subsequent to Eck fistula operation. He showed that such animals possess an increased muscle glycogen content characteristic of over-nutrition and a low liver glycogen content characteristic of inanition. He inferred that the muscles could form glycogen inde- pendently and that the liver was neither specific for nor indispensable for carbohydrate metabolism. Hawk in 1908 (13), from feeding experi- ments, showed that quite extreme toxic effects were produced by feeding meat extracts (especially Liebig's extracts of meat) in conjunction with the meat diet. He was, however, unable to produce any such toxic symptoms by the introduction of carbamate nor was he able to produce glycosuria on carbohydrate feeding. Fischler in 1910-11 (14) noted the acute degeneration in the liver and fat necrosis following Eck fistula such as could be produced by pancreatic injur3^ These could be pre- vented by preliminary injections of trj'psin. The meat intoxication, according to his view, was caused bj- an alkalosis due most probably to ammonia unneutrahzed by the liver and this condition could be reheved by acids. Michaud in 1911 (15) showed that Eck fistula prevented adrenal glycosuria but that when such an animal is fed sugar by mouth, the blood sugar either remains normal or increases within normal limits, as in any intact animal. In 1911 Voegthn and Bernheim (16) showed that the bile pigments and bile acids were decreased in an Eck fistula dog to such an extent that the ligation of the common duct was not followed by jaundice and by only a trace of bile in the urine. They noted also the occasional attacks of convulsions, somewhat similar to those occurring in tetany, produced by meat feeding and showed that these could be prevented by feeding plenty of calcium in the form of bone. They inferred that the Eck fistula condition was compatible 236 CONRAD JACOBSON with life if care was used in feeding. In 1911 Towles and Voegtlin (17) showed that there was no essential difference in the metabolism of creatin and creatinin in a normal and an Eck fistula dog and concluded that the liver is not an organ of prime importance in the metabolism of these substances and that the creatinin could hardly be held account- able for the toxic symptoms evoked on meat feeding. In thus excluding the portal circulation from the liver and diverting the portal stream directly into the systemic venous circulation, there is seen to be a re- duction in the liver function: a, in the synthesis of the urea from ammonia salts; h, in the production of bile salts and bile acids; and c, in the lowered carbohydrate tolerance. It is with this latter feature that this paper is mainly concerned. Conduct of experiments. Normal healthy dogs were used throughout the experiment — good-sized animals and those only w^ho proved adapt- able to sugar feeding on the pre-operative tolerance determinations. To have comparable results, weighed quantities from the same lot of sugar were used on all the animals. Sugar feedings were under as uni- form conditions as possible, — namely, one sugar feeding a day, on empty stomach, 4 to 5 hours after meals, with definite amounts of water for dissolving the sugar and avoidance of all food or drugs with the feed- ings. Sugar in the urine was tested for and determined by Fehling, Nylander, fermentation and the polariscopic methods. The animals were fed on a mixed diet of boiled meat and bread, and were kept in cages during the tests. The urine was collected from beneath the cages. The Eck fistula operation consisted of making a typical lateral anas- tomosis between the portal vein and the inferior vena cava using small enterostomy clamps ( Jerusalem- Jeger) and silk for the sutures. On completion of the anastomosis, the portal vein was doubly ligated and transected between the ligatures, near the liver hilus.^ Posterior 1 Following Eck fistula operation, the animals remained apparently well and in all outward respects normal. There is an early but decreasing tendency to loss of weight and at first bounteous feedings are necessary to maintain the general body weight. When fed on a meat diet for a great length of time or by the addi- tion of meat extract to a diet for several days, convulsive attacks somewhat suggestive of tetany are likely to occur. These are characterized by disorien- tation, indifference to surroundings, muscular twitchings, paralysis of the limbs, rigidity of back, ataxia and fine tremors. These may last as long as 48 hours and may lead to the death of the animal. The attacks are usually preceded by a period during which the animal seems below par, may be dull and drowsy or may be unusually restless or nervous. At this stage the acute attack may occasionally LIVER FUNCTION IN METABOLISM OF SUGARS 237 lobe hypophysectomy was performed by the intracranial method de- scribed by Crowe, Gushing and Homans (18). This consists of con- tralateral temporal openings through bone and dura which allows the brain to be lifted and dislocated upwards through one decompression opening. The posterior lobe is then enucleated from the dangling gland from below, through the temporal opening of the opposite side. Protocols Animal A. Well-nourished, normal male dog, weight 11.2 kilos April 10-16. Pre-operative cane sugar tolerance, 130-140 grams April 17-28. Eck fi.stula operation — uneventful convalescence April 28-May 15. Post-operative sugar tolerance. Weight 10.8 kilos Cane sugar, 14 grams. Laevo sugar in urine Glucose, 55-60 grams Laevulose, 7-8 grams May 25-June 3. 3nd post-operative sugar tolerance Cane sugar, 14-25 grams Glucose, 55-60 grams Laevulose, 7-8 grams June 18-25. 3rd post-operative sugar tolerance. Weight 10.8 kilos Cane sugar, 14-15 grams Glucose, 55-60 grams Laevulose, 7-8 grams. Mild convulsive attacks July 7-15. ith post-operative sugar tolerance. Weight 10.2 kilos Cane sugar, 14-15 grams Glucose, 55-60 grams Laevulose, 7-8 grams be aborted by feeding the animal plenty of bone, a substance for which he seems particularly desirous. Injections of calcium chloride or feedings of calcium lactate do not seem to have any effect when the acute attacks have already begun. After the attack has worn away the animal seems to be in normal condition again. One of the animals died during a severe second attack while the other two dogs had two and five attacks respectively. If care is taken to keep the animal on a mixed diet and excess of meat is avoided and if there is plenty of cal- cium in the form of bone, there seems to be no reason why an Eck fistula animal should not live for a long period of time. The animals reported here were ob- served for 139. 340 and 408 days respectively after the Eck fi.stula operation; the two latter finally succumbed to conditions unassociated with the fistula operation. On autopsy the only pathological findings of interest are those associated with the liver. This organ is small, hard, firm and rather tougher than normal. The gall bladder and bile ducts appear to be normal. On section, the liver lobules seem to be decidedly smaller than normal with central atrophy and some fatty degeneration, conditions which have been produced experimentally by a reduction of the blood supply of the liver itself. 238 CONRAD JACOBSON July 17-30. Hypophysectomy — posterior lobe removal — ether Urinary output from 1520 cc. to normal in 5 days. No permanent poly- uria Slight trace of sugar in the first specimen; no permanent glycosuria August 3-10. 1st post-operative tolerance. Weight 11.2 kilos Cane sugar, 14-15 grams Glucose, 45-50 grams Laevulose, 7-8 grams August 18-24. 2nd post-operative tolerance Cane sugar, 14-15 grams Glucose, 50-55 grams Laevulose, 7-8 grams September 6-13. 3rd post-operative tolerance. Weight 11.3 kilos Cane sugar, 14-15 grams Glucose, 55-60 grams Laevulose, 7-8 grams September 20-28. 4th post-operative tolerance Cane sugar, 14-15 grams Glucose, 60-70 grams Laevulose, 7-8 grams October 5-13. 5th post-operative tolerance. Weight 10.8 kilos Cane sugar, 14-15 grams Glucose, 65-70 grams Laevulose, 7-8 grams October 20-26. 6th post-operative tolerance Cane sugar, 14-15 grams Glucose, 65-70 grams Laevulose. 7-8 grams November 6-15. 7th post-operative tolerance Cane sugar, 14-15 grams Glucose, 65-70 grams Laevulose, 7-8 grams. Mild convulsions November 24-30. 8th post-operative tolerance. Weight 10.8 kilos Cane sugar, 14-15 grams Glucose, 65-70 grams Laevulose, 7-8 grams Summary of animal A NORMAL TOLERANCE AFTER ECK FISTULA AFTER HYPO- PHT8ECTOMT Cane sugar grams 130-140 grams 14-15 55-60 7-8 grams 14-15 Glucose 65-70 Laevulose 7-8 Animal used for pancreatectomy — died May 24 of pneumonia. LIVER FUNCTION IX METABOLISM OF SUGARS 239 In this, the first of the Eck fistula animals in good condition, the saccharose tests even in small amounts gave sugar which proved to be laevo-rotatory. As the saccharose is necessarily broken up into dex- trose and laevulose in the process of its absorption and utiHzation in the body, it seemed necessary to resort to separate tests for the tolerance of the two sugars before the operation. It showed also that the assimi- lation tests under the circumstances with a complex sugar like saccha- rose, such as had been used, was capable of misinterpretation; for with hepatic insufficiency there would always be a low assimilation limit and a laevo-rotatory substance in the urine whereas the glucose tolerance itself might be little modified. Animal B. Well-nourished, normal male dog, weight 13.3 kilos May 11-June 6. Pre-operative sugar tolerance. Weight 13.3 kilos Cane sugar, 170-175 grams Glucose, 110-115 grams Laevulose, 85-90 grams June 6-19. Eck fistula operation — uneventful convalescence June 19- July 10. Post-operative sugar tolerance Cane sugar, 10-15 grams Laevulose, 7-8 grams Glucose, 75-80 grams. July 12-22. Hypophysectomy^posterior lobe removal — ether. Weight 12. S kilos Urinary output 1360 cc. to normal in 7 days. No permanent polyuria No glycosuria July 23-August 2. 1st post-operative tolerance Laevulose, 8-9 grams Cane sugar, 10-15 grams Glucose, 70-80 grams August 10-18. 2nd post-operative tolerance. Weight 12.6 kilos Laevulose, 8-9 grams Cane sugar, 10-15 grams Glucose, 70-80 grams August 28-September 8. 3rd post-operative tolerance Laevulose, 8-9 grams Cane sugar, 10-15 grams Glucose, lOO-llO grams September 17-24. 4th post-operative tolerance. Weight 12.7 kilos Laevulose, 8-9 grams. 1st series of convulsions Cane sugar, 10-15 grams Glucose, 100-110 grams October 4-10. olh post-operative tolerance Laevulose. 8-9 grams Cane sugar, 10-15 grams Glucose, 100-110 grams 240 CONRAD JACOBSON October 17-22. 6th post-operative tolerance. Weight 12.9 kilos Laevulose, 8-9 grams Cane sugar, 10-15 grams Glucose, 100-110 grams. 2nd convulsive attack Animal died on October 24 — 2nd series of convulsions Summary of animal B NORMAL TOLERANCE AFTER ECK FISTULA AFTER HYPO- PHY8ECT0MY Cane sugar grams 170-175 110-115 85-90 grams 10-15 75-80 8-9 grams 10-15 Glucose . 100-110 Laevulose 8-9 Animal C. Well-nourished, normal male dog, weight 14.6 kilos October 12-November 1. Pre-operative sugar tolerance Cane sugar, 170-175 grams Glucose, 120 grams Laevulose, 90 grams. Weight 16 kilos November 2-10. Eck fistula operation — uneventful convalescence November 10-December 1. Post-operative sugar tolerance Cane sugar, 20-25 grams Laevulose, 7-8 grams Glucose, 100 grams. Weight 12.7 kilos December 2-12. Hypophysectomy — posterior lobe removal — -ether Urinary output 1970 cc. to normal in 10 days. No permanent polyuria No gh^cosuria December 12-Januarj' 1. 1st post-operative tolerance Laevulose, 7-8 grams Glucose, 80-90 grams Cane sugar, 20-25 grams. Weight 12.7 kilos January 12-19. 2nd post-operative tolerajice Laevulo.se, 7-8 grams Glucose, 80-90 grams Cane sugar, 20-25 grams. 1st attack of convulsions January 29-February 6. 3rd post-operative tolerance Laevulose, 7-8 grams Ciuie sugar, 20-25 grams Glucose, 80-90 grams. Weight 12.9 kilos. 2nd attack of convulsions February 17-24. 4th post-operative tolerance Laevulose, 7-8 grams Cane sugar, 20-25 grams Glucose, 80-90 grams March 14-23. 5th post-operative tolerance Laevulose, 7-8 grams Cane sugar, 20-25 grams Glucose, 90-100 grams. Weight 13 kilos LIVER FUXCTIOX IX METABOLISM OF SUGARS 241 April 9-20. 6th post-operative tolerance Laevxilose, 7-8 grams Cane sugar, 20-25 grams Glucose, 110-120 grams. 3rd attack of convulsions May 15-26. 7th post-operative tolerance Laevulose, 7-8 grams Cane sugar, 20-25 grams Glucose, 140-150 grams June 8-14. 8th post-operative tolerance Laevulose, 7-8 grams Cane sugar, 20-25 grams Glucose, 140-150 grams. Weight 13.1 kilos July 6-14. 9th post-operative tolerance Laevulose, 7-8 grams Cane sugar, 20-25 grams Glucose, 140-150 grams. 4th attack of convulsions July 28-August 4. 10th post-operative tolerance Laevulose, 7-8 grams Cane sugar, 20-25 grams Glucose, 140-150 grams. Weight 13.3 kilos August 11-16. 11th post-operative tolerance Laevulose, 7-8 grams Cane sugar, 20-25 grams Glucose, 140-150 grams August 24-30. 12th post-operative tolerance Laevulose, 7-8 grams Cane sugar, 20-25 grams Glucose, 140-150 grams. Weight 13.3 kilos Summary of animal C NORMAL TOLER.\NCE AFTER ECK FISTULA AFTER HTPO- PHYSECTOMT Cane sugar Glucose grams 170-175 120-130 90-95 grams 20-25 90-100 7-8 grams 20-25 140-150 Laevulose 7-8 Animal used for pancreatectomy — died December 15. The tolerance of an animal for sugar is the lowest dose of that sugar, following the ingestion of which, sugar just appears in the urine — the threshold dose producing glycosuria. It is dependent upon a number of factors such as the variety of sugar, the general state and condition of the animal, the manner and method of administering the sugar. This threshold dose is an indefinite amount varj'ing between rather wide limits. Of the several methods of introducing sugar, — intra- THE AMERICAN JOURNAL OF PHY.SIOLOGY, VOL. 52, NO. 2 242 COXRAD JACOBSON venous, subcutaneous and alimentary,— the two former give the lower values and the latter/ the most frequently used, the highest values. For alimentary glycosuria the absorption rate from the gastro-intestinal tract must exceed the storage and utilization power of the tissues so that a hyperglycemia is produced with a blood sugar content high enough to overcome the normal impermeability of the kidney. It is evident that anj^thing that concerns the speed of absorption, the utili- zation rate, the storage power of the tissue and the permeability of the kidney will result in changes in the tolerance. It is also evident that small amoimts of sugar, through rapid absorption, will produce an abrupt rise in the blood sugar content with resultant gtycosuria while larger amounts with slower absorption may be unaccompanied by the appearance of the sugar in the urine. The rate of absorption of sugar from the gastro-intestinal tract is not a constant factor and, as such, influences markedly the amount of sugar brought to the blood and tissues for utilization. The higher the tolerance, the more inaccurate the determination is and the greater the limits of variance. With the lower tolerance sugars, the determination is less difficult and the limits more readily defined. In many cases it is extremely difficult, and in some animals impossible, to obtain the tolerance with high tolerance sugars on accovmt of the intolerance of stomach and intestinal tract for the huge and abnormal masses required for one feeding. The tol- erance is usually stated in grams per kilo of body weight. This is open to grave error as young animals have a much lower tolerance per kilo than adult animals and obesity, as is well known, is not accompanied by a proportionate increase in the sugar tolerance. The indefiniteness of alimentar}' sugar tolerances is readily seen when one peruses the literature for tolerances determined by different investigators. Hof- ineister's (19) tolerances for glucose in dogs ranged from 1.3 to 5.8 grams with 2 to 3 grams per kilo as the average. This is an unusually low tolerance, in fact much lower than that after intravenous admin- istration and is rather out of the question. Pflueger (20) found some 11 to 16 grams per kilo. That the method of administration had a definite effect upon the tolerance is seen by his figures of 11 to 12 grams when the dextrose was given with meat stew and of less than 8 grams per kilo when it was given with soup. Schlessinger (21) found the tolerance to be about 10 to 11 grams per kilo and that small young animals had a lower tolerance per kilo than the larger adult animals. Boeri and De Andreis (22) found values of 4 to 6 grams for fasting animals and 10 to 13 grams for well-nourished animals. Pratt and LIVER FUNCTION IN METABOLISM OF SUGARS 243 Spooner (23) found values of 11.5 grams for dogs. Quarta (24) found an average of 4 grams for male and 10 grams for female dogs. Sex has been found to have no appreciable effect upon the sugar tolerance and his value of 4 grams per kilo is entirely too low. Filippi's values were 8 to 10 grams for fasting animals and he lays great stress upon the fact that to obtain comparable results in alimentary feeding, sugar should be given under certain definite conditions. The tolerance values given for laevulose are few in number. Quai'ta gave the value as between 3 to 4 grams per kilo and de Filippi as about 1.5 gram. The tolerance for saccharose shows the same wide variation. Hoeppe-Seyler (25) sets it at 20 to 30 grams per kilo, Hofmeister at 3.6 grams and de Filippi at about 4 grams per kilo. The osmotic irritation, the gastric and intestinal intolerance for large and abnormal masses of sugar, the lack of any control over the varying rates of absorption, make the alimentary method, though practically the only feasible one, far from an ideal one for investigative purposes. With an adaptable animal, under uniform conditions, comparative results on the same animal may however furnish data of some value. Table 1 gives the tolerances obtained in this investigation. The normal values differ considerably from those given by de Filippi. The saccharose tolerances agree fairly closely with those obtained by Goetsch, Gushing and Jacobson who found in their series of animals weighing 9 to 11 kilos, a tolerance of 9 to 13 grams per kilo of body weight. Tolerance after Eck fistula operation. Following Eck fistula opera- tion there are marked changes in the carbohydrate tolerance ascertained by alimentary feeding. Subsequent to this surgical procedure there is usually a marked loss in body weight, and some of the post-operative tolerances giv^en in the above table are undoubtedly high since they are calculated upon the lowered body weight. The glucose tolerance is modified to a less degree than the laevulose or cane sugar tolerance. This seems to indicate that the muscle and the body tissue generally can manage very well the storage and utilization of the glucose that is shunted to them by the Eck fistula operation and leads one to infer that the liver is an organ not absolutely necessary for the utilization of that sugar. The most marked changes occur in the laevulose tolerance. In an Eck fistula animal a small amount of laevulose ingested is followed by a laevulosuria, there being almost an intolerance for that particular sugar. Muscular tissue can undoubtedly utilize a small amount of 244 CONRAD JACOBSON laevulose as is evidenced by muscle perfusion experiments. It is questionable whether the muscles can change laevulose into glycogen and to any large extent thus utilize it. It seems quite probable that the liver acts as an organ of storage for the laevulose and alters it in some manner or other to make it more utilizable by the tissues. The liver evidently is indispensable for laevulose metabolism, TABLE 1 Sugar tolerance; grams per kilogram of body weight AFTER ECK FISTULA AFTER HTPOPHTSECTOMT Dog A Weight . Cane sugar. Glucose. . . Laevulose. . 11.4 kilos 12.0 grams 11.4-10.2 kilos 1.4 grams 5.6 grams 0.7 gram 10.8 kilos 1.3 grams 6.2 grams 0.7 gram Dog B Weight . Cane sugar. Glucose. . . Laevulose. . 13.3 kilos 13.3 grams 8.4 grams 6.5 grams 13.3-12.3 kilos 12.3-12.9 kilos 0.9 gram 5.9 grams 0.6 gram 1.0 gram 8.0 grams 0.6 gram Dog C Weight . Cane sugar. Glucose. . . Laevulose . . 14.6 kilos 11.7 grams 8.2 grams 6.2 grams 16.0-12.5 kilos 12.7-13.3 kilos 2.0 grams 8.0 grams 0.6 grams 1.7 gram* 11.9 grams 0.6 gram * As this animal had lost considerable weight after the Eck fistula operation these values figured on the body weight are as a consequence rather high. The tolerance changes shown by saccharose are similar to those shown by laevulose. This sugar is broken down into dextrose and laevulose in the process of assimilation and the split products exert their respective effects upon the tolerance. Small doses of saccharose given to an Eck fistula animal are followed b}^ a laevulosuria. These changes in tolerance subsequent to Eck fistula operation in the dog, though the absolute tolerance values are quite different, are similar to those reported by de Filippi and are given here as confirmation of his work. LIVER FUNCTION IN METABOLISM OF SUGARS 245 Tolerance changes following subsequent hypophysectomy. Following hypophysectomy in an Eck fistula animal the following changes were noted : 1. Transient glycosuria. In only one of the three cases was there a glycosuria following the operation. This was of a temporary char- acter and appeared only in the first specimen of urine voided. There was no persistent or permanent glycosuria. Such a slight transient gtycosuria was also found in three out of ten cases reported by Goetsch, Gushing and Jacobson, in their series of posterior lobe hypophysectomy. It is questionable whether this shght occasional gtycosuria is due to the removal of the gland or incidental to the operation itself. 2. Polyuria. A definite post-operative polyuria was noted in all the three cases. Following operation there was a high urinary output of 1560, 1360 and 1970 cc. per day, gradually decreasing in amount so that the normal pre-operative urinary output was reached in 5 to 7 and 8 days respectively. The fluid intake on these days was increased also in proportionate amounts. There was no permanent or persistent polyuria as the result of the posterior lobe removal. 3. Increased carbohydrate tolerance. There was a moderate but definite increase in the tolerance for glucose after posterior lobe removal in all of the three animals, while the cane sugar and laevulose values remained essentially umnodified at their low levels. This augmenta- tion of glucose tolerance came on extremely late, however, when com- pared to the augmentation following hypophysectomy in normal ani- mals. In the series of hypophysectomies described bj^ Gushing, Goetsch and Jacobson there was a transient lowering of carbohydrate tolerance followed by augmentation of tolerance reaching its height or greatest value from 9 to 21 days after the posterior lobe removal. In animal A there is a post-operative lowering of tolerance for 51 days, augmentation of tolerance during 28 days, making 79 days in all before the high tolerance was reached. No change in tolerance was noted subsequently during 51 days of observation. In animal B there was no definite post-operative lowering of tolerance, the tolerance reaching its highest value 58 days after operation with no change for 44 daj^s subsequent observation. Animal G showed a low post-operative tolerance for 111 days, augmentation during 84 days or 195 days before the high value was reached. There was no change in tolerance on 96 days of further observation. The conclusion is quite evident that this aug- mented tolerance, indicative of increased storage and utilization ca- pacity for converted carbohydrates, is the result of some extra-hepatic 246 CONRAD JACOBSON function. It is most probably associated with the glycogenic and glyco- genolytic power of the muscle tissue itself. Whatever function or capacity the liver has in this direction is readily assimaed by the muscles themselves when the liver is removed from the portal circulation. CONCLUSIONS 1. Eck fistula animals have an extremely low tolerance for laevulose. The liver is evidently essential for laevulose metabolism. The function of converting laevulose into glycogen is possessed by the liver; this function is permanently lost when the portal blood is diverted into the vena cava by Eck fistula. 2. Glucose tolerance is only slightly modified in Eck fistula animals. The liver is not entirely essential for glucose metabolism. The muscles undoubtedly perform well the functions of glycogenesis and glyco- genolysis when the liver is shunted out of the portal circulation. 3. The glycogenic capacity of muscle is increased following posterior lobe removal in an Eck fistula animal as in an intact animal. The augmentation of tolerance is, however, considerably slower. No aug- mentation of laevulose tolerance is noted. BIBLIOGRAPHY (1) GoETSCH, Gushing and Jacobson: Johns Hopkins Hosp. Bull., 1911, xxii, 165. (2) VON Eck: Militar-med. Journ., 1877, Iv, 130. (3) Stolnikow: Arch. f. d. gesammt. Physiol., 1882, xxviii, 255. (4) Hahn, Massen, Nencki .and Pawlow: Arch. f. exper. Path. u. Pharm., 1893, xxxii, 161. (5) RoTHBERGER AND Winterberg: Zcitschr. f. exper. Path. u. Therap., 1904, i, 312. (6) Nencki, Pawlow and Zaleski: Arch. f. exper. Path. u. Pharm., 1896, xxxvii, 26. (7) Nencki and Pawlow: Arch. f. exper. Path. u. Pharm., 1897, xxxviii, 215. (8) PoPELSKi: Bolnitsch gaz. Botkina, St. Petersburg, 1897, vii, 1787. (9) Sachs: Zeitschr. f. klin. Med., 1899, xxxviii, 87. (10) Strauss: Deutsch. med. Wochenschr.. 1901, 757, 786. (11) ScHULz and Miller: Deutsch. Arch. f. klin. Med., 1903, Ixxvi, 544. (12) DE FiLippi: Zeitschr. f. Biol., 1907, xl, 511; 1, 38. (13) Hawk: This Journal, 1908, xxi, 259. (14) Fischler: Deutsch. Arch. f. klin. Med., 1910, c, 329; 1911, ciii, 156; civ, 300. (15) Michaud: Verh. d. Kong. f. inn. Med., 1911, 560. (16) VoEGTLiN andBernheim: Journ. Pharm. Exper. Therap., 1911, ii, 455. LIVER FUNCTION IN METABOLISM OF SUGARS 247 (17) TowLES AND Voegtlin: Journ. Biol. Chem., 1911, x, 479. (18) Crowe, Gushing and Homans: Johns Hopkins Hosp. Bull., 1910, xxi, 127. (19) Hoffmeister: Arch. f. exper. Path. u. Pharm., 18SS, xxv, 240. (20) Pflueger: Pfluegers Arch., 1908, cxxiv, 1. (21) Schlessinger: Wiener klin. Wochenschr., 1902, xxx, 768. (22) Boeri and de Andreis: Policlin. V. Med., 1898, 477. (23) Pratt and Spooner: Arch. Int. Med., 1911, vii, 665. (24) Quarta: Zeitschr. f. Biol., 1907, xl, 511. (25) Hoeppe-Seyler: Virchow's Arch., 1856, x, 144. THE GASTRIC RESPONSE TO FOODS^ XII. The Response of the Human Stomach to Pies, Cakes and Puddings RAYMOND J. MILLER, HARRY L. FOWLER, OLAF BERGEIM, MARTIN E. REHFUSS AND PHILIP B. HAWK From the Laboratory of Physiological Chemistry of Jefferson Medical College, Philadelphia Received for publication March 20, 1920 Among the preparations classed under the heads of pies, cakes and puddings are a great variet}' of food products, the majority of which are commonly used as desserts although, in this era of quick lunches, pies, doughnuts, cinnamon buns, etc., frequently usurp the position in the diet of more substantial foods. For this misuse there can only be the excuse of necessity. However, the pies, cakes and puddings in- clude many highly nutritious and most pleasing foods for whose proper preparation the capable cook may well be praised. In spite of their very wide use there is a general impression that pies, cakes and puddings as a class are rather indigestible. The blame for this may rest in part on the diversitj^ of cooking, in part on the depress- ing effect on the appetite of these foods if very sweet and eaten between meals, and in part to their being frequenth^ crammed into an already well-filled stomach. Little study has been made of the response of the stomach to these classes of foods. Some experiments were made b}^ Beaumont (1), although amounts were not controlled. This author found in his sub- ject that rice left the stomach in 1 hour; sago, in If hour; tapioca, in 2 hours; gelatin, in 2| hours; sponge cake, in 2| hours, and apple dumpling in 3 hours. Direct comparison with our results cannot be made ; but, as an instance, where Beaumont found rice to leave in less than half the time required for gelatin we found the gelatin to leave sooner than any other pudding. Our average time for cake was also distinctly longer than found by Beamuont. ^The expense of this investigation was defrayed by contributions from Mrs. M. H. Henderson, The Curtis Publishing Company and Dr. L. M. Halsey. 248 GASTRIC RESPONSE TO PASTRY AND PUDDINGS 249 Our experiments on gastric response were carried out according to a procedure previously described (2). The subjects of the test were nor- mal medical students who were given the food preparations in Heu of breakfast about 8 o'clock in the morning. Residumns were not removed. About 90 experiments were carried out. The acid responses and evacuation times of 81 of these are charted in figures 1 to 28 and summarized in tables 1, 2 and 3. RESPONSE OF THE HUMAN STOMACH TO PIES Fruit pies. Among the fruit pies studied were apple, peach, cherry, pumpkin and raisin pies. Apple pie was tried out on four subjects (see figs. 1, 3, 4 and 6). This pie was evacuated in from 2 hours to 2f hours, or an average emptying time of 2| hours. Fairly high acidities were developed within an hour to an hour and a half, the highest value attained varying from 83 to 102, with an average of 94. Further, most of this acidity was due to free HCl, showing that these pies have but a slight combining power for acid, and that in spite of this high free acidity gastric evacuation was not delayed. Peach pie was given to one subject (see fig. 1) who had previously received apple pie. The acid curves for the two pies were practically identical, an acidity of 98 being developed in an hour and a half with peach pie. This latter pie, however, left the stomach half an hour sooner than apple pie. Cherry pie was tried out in two cases (see figs. 7 and 8). High acidi- ties, due mainly to free HCl, were developed in both cases. The evacu- ation time of one subject was 3 hours as compared with 2f hours for apple pie. Not quite so high an acidity was developed on cherry pie, perhaps because its higher sugar content depressed secretion slightly, an effect we have shown sugar solutions to produce (3) . Raisin pie was given to one subject (see fig. 10). It was evacuated quite rapidly (in 2j hours) or in the same time as required for rhubarb pie, and more rapidty than mince or pumpkin pie. A moderate acidity (with little combined acid) was developed, as might be expected from a pie fairly high in sugar but containing little protein. Rhubarb pie in general character and gastric response may be classed with the fruit pies. Two subjects were given this pie (see figs. 9 and 11). It was one of the most readily evacuated pies, leaving the stomach in the same time as raisin pie {2\ hours) and more rapidly than cherry pie. The acid responses were high, averaging 101 at the maximum, nearly all of this being due to free HCl. 250 MILLER, fo"«t:,er, bergeevi, rehftjss and hawk TABLE 1 The response of the human stomach to pies 1-Son 2-Wil 3-Son 4-Wil 5-Son 6-Wil 7-Son 8-Rud 9-Rud 10-Rud 11-Wil 12-Mil 13-Kar 14-Lea lo-Mil 16-Wil 17-Rud 18-Son KIND OF PREPARATION Pumpkin Pumpkin Mince Mince Lemon meringue . Lemon meringue . Raisin Cocoanut custard . Custard pie Cherry Cherry Peach Apple Apple Apple Apple Rhubarb Rhubarb TYPE OF INDIVIDUAL Rapid Slow Rapid Slow Rapid Slow Rapid Rapid Rapid Rapid Slow Rapid Rapid Rapid Rapid Slow Rapid Rapid Average EVACUA- TION TIME 2:45 2:00 2:45 3:15 2:30 2:30 2:15 2:15 2:30 2:45 3:00 2:00 2:00 2:30 2:30 2:45 2:00 2:15 2:27 HIGHEST TOTAL ACID ITT 99.5 102.5 107.5 105.0 74.0 95.0 94.5 107.0 84.5 99.0 95.0 98.5 97.0 83.0 95.0 102.5 106.0 95.0 90.0 Pie combinations 19-Rud 20-Lea 21-Wil Cherry (with ice cream) Apple (with ice cream) Apple (with cheese) . . . Rapid Rapid Slow Average , 3:15 2:30 3:00 ii:oo 115.0 69.5 113.5 99.0 Pie crust vs. insides 22-Lea 23-Mil 24-Wil 25-Son 26-Far 27-Mil 28-Rud 29-Wil Pie crust Pie crust Pie crust Pie crust Insides (apple) . . . Insides (peach). . . Insides (rhubarb) Insides (apple) . . , Rapid Rapid Slow Rapid Slow Rapid Rapid Slow Pie crust, average . Insides, average . . 2:45 2:45 3:15 2:45 3:15 1:30 2:15 2:45 2:52 2:26 66.5 92.5 102.5 98.5 93.0 103.5 118.0 99.0 90.0 103.0 GASTRIC RESPONSE TO PASTRY AND PUDDINGS 251 Pumpkin pie differs somewhat in character from the fruit pies men- tioned, but the acid response was not distinctly different. The acid combining power was sHghtly greater for pumpkin pie. A subject of TABLE 2 The response of the human stomach to cakes 1-Far 2-Mur 3-Owe 4-Far 5-Mur 6-Owe 7-Gol 8-Mur 9-Gol 10-Mur 11-Mur 12-Cor 13-Lot 14^Lot 15-Spe 16-Cor 17-Far I&-G0I 19-Lot 20-Owe 21-Far 22-Mur 23-Spe 24-Owe 25-Rud 26-Rud 27-Ree 2&-Spe 29-Owe KIND OF PREPARATION Angels' food Angels' food Angels' food Devils' food Devils' food Devils' food Fruit cake, fresh Fruit cake, fresh Fruit cake, old Fruit cake, old Ladj^ fingers Crullers Crullers Doughnuts Doughnuts Cream puffs Ginger snaps Spiced cookies Spiced cookies Chocolate laj-er cake .... Mary Ann cookies Cinnamon bun Cinnamon bun Ginger bread Ginger bread Short cake, strawberry . . . Short cake, strawberry . . . Bread with peanut butter . Bread with corn syrup. . . . TYPE OF INDIVIDUAL Slow Rapid Rapid Slow Rapid Rapid Rapid Rapid Rapid Rapid Rapid Slow Rapid Rapid Slow Slow Slow Rapid Rapid Rapid Slow Rapid Slow Rapid Rapid Rapid Slow Slow Rapid Average . EVACUA- TION TIME 4:30 3:45 2:45 4:00 3:00 2:45 3:15 3:15 3:30 3:30 3:15 4:15 2:15 2:30 3:00 2:30 3:30 2:15 2:15 2:30 2:45 2:30 2:45 2:15 3:00 3:00 3:15 3:15 2:15 3:02 HIGHEST TOTAL ACIDITY 97.5 100.0 99.0 92.0 83.0 73.5 73.0 80.5 78.0 77.5 75.5 88.0 91.5 83.0 106.0 107.0 101.5 89.0 104.0 26.5 70.5 72.0 127.5 65.0 106.0 124.5 130.0 120.5 55.0 90.0 the slo.w emptying tj^pe required 2f hours as compared with 2| hours for raisin and rhubarb pies (see fig. 10). A subject of the rapid empty- ing type (see fig. 7) required only 2 hours for pumpkin pie as compared with 3 hours for cherry and 2f hours for apple pie. 252 MILLER, FOWLER, BERGEIM, REHFUSS AND HAWK Mince, custard and lemon meringue pies. The mince, custard and meringue pies are higher in protein than simple fruit pies and might, therefore, be expected to undergo greater change in the stomach. Mince pies are generally considered to be the least easily digested of pies, due to their content of meat and spices. This is borne out by our experiments (see figs. 7 and 10). Mince pie required 3j hours to leave TABLE 3 The response of the human stomach to puddings 1-Dal 2-Gle 3-Dal 4-Spe 5-Dal 6-Spe 7-Kar 8-Spe 9-Lea 10-Mil 11-Lea 12-Mil 13-Gle 14-Dal 15-Gle 16-Kar 17-Dal 18-Gol 19-Kar 20-McD 21-McD 22-Col 23-Kar KIND OF PREPARATION Corn starch Chocolate Chocolate Brown Bettj' . . . . Brown Betty . . . . Cabinet Cabinet Rice Rice Rice with raisins . Cup custard Cup custard Plum Plum Bread Bread Bread Bread Tapioca Apple tapioca . . . Gelatine Gelatine TYPE OF EVACUA- INDIVIDUAL TIONTIMB Indian Rapid Slow Rapid Slow Rapid Slow Rapid Slow Rapid Rapid Rapid Rapid Slow Rapid Slow Rapid Rapid Rapid Rapid Slow Slow Rapid Rapid Average . 2:00 2:30 2:00 2:00 2:15 2:45 2:00 2:45 2:00 2:00 1:45 2:00 4:15 2:15 4:30 2:15 2:00 1:30 1:45 2:30 2:15 2:00 1:30 HIGHEST TOTAL ACIDITT 2:18 113.5 92.5 110.0 91.0 69.5 110.0 87.0 82.0 67.0 101.0 76.5 107.0 112.0 113.0 96.5 79.5 108.0 96.0 104.0 74.0 106.5 61.5 62.0 92.0 the stomach of an individual who required 2f hours for apple pie and 3 hours for cherry pie. The other subject required 2f hours for mince and 21 hours for either raisin or rhubarb pie. The highest acid re- sponse was given with mince pie, and the acid combining power was also markedly greater than for simple fruit pies. One experiment each was made on plain custard and cocoanut cus- tard pie, the same subject receiving both (see fig. 8). The plain custard GASTRIC RESPONSE TO PASTRY AND PUDDINGS 253 required 15 minutes longer to digest and developed a higher acidity due probably to the slightly higher protein content. The high combined acidities of both would be expected from their known content of milk and egg proteins. The custards required a longer digestion than rhu- barb pie but were by no means handled with difficulty inasmuch as cherry pie took 15 minutes longer than either. Related in composition to the custards are the lemon meringues which, when properly made, are fairly high in eggs. They leave the stomach in about the same time as the custards (2| hours) and show a less rapid development of acidity than other pies (figs. 7 and 10). COMPARATIVE RESPONSE OF THE STOMACH TO PIE CRUST, CONTENTS AND WHOLE PIES The popular opinion that pie crusts are the least digestible portions of ordinary pies would also appear probable from their known com- positions, possessing as they do most of the protein and fat of many pies, especially fruit pies. The absolute digestibility of pie crusts must, of course, depend a good deal on their texture and composition. The response of the stomach to pie contents would be expected to approxi- mate that of fruit sauces or puddings of similar composition. Thus we have found (4) that fruits in general leave the stomach very rapidly with the development of considerable free but little combined acidity. Pie crust alone was given in 100-gram portions to four subjects (figs. 2, 5, 6 and 11). This was evacuated in from 2f to 3j hours. In the case of one subject the contents of a peach pie left the stomach in 1| hour as compared with 2f hours for the crust. For apple pie and crust the required periods were 2f and 83 hours respectively. Pie crust thus remains in the stomach distinctly longer than an equal weight of the contents of apple or peach pie. The acid responses of the two were not so different, although crust usually gave a slower rise with a more sustained curve. The whole pie naturally is intermediate between pie crust and con- tents in the response it evokes. Thus subject "Mil" (figs. 1 and 2) retained peach pie 2 hours and contents 1^ hour. Another subject, "Rud" (fig. 9) required a few minutes longer for the rhubarb alone than for the pie, while a third subject, "Wil," took 2f hours in each case (fig. 6). Thus these fruit pies require little longer to digest in the stomach than the interiors alone. 254 MILLER, FOWLER, BERGEIM, REHFUSS AND HAWK If crust is compared with whole pie, we find in the case of "Mil" the pies requiring 2 to 2| hours and crust 2f hours; in the case of "Son" 2 J hours for rhubarb pie and 2f hours for crust alone. Sub- ject " Wil" required 2§ to 3j hours for pies (mince being the slowest) and 3j hours for crust. Crust thus takes somewhat longer to digest in the stomach than the contents of all but the least digestible pies. It will be seen, therefore, that the response of the stomach to whole pies approximates that of the pie contents, in spite of the fact that the crust alone requires a distinctty longer digestion period. We did not, however, find that crust was in any sense indigestible, although 100 grams of it were given at a time. PIE WITH ICE CREAM AND WITH CHEESE To the usual 100-gram portions of apple and cherry pie were added in each of two cases 50 grams of vanilla ice cream. The responses are charted in figures 4, 5 and 8. The cherry pie alone required 2j hours. With ice cream it remained in the stomach half an hour longer. Apple pie, however, showed the same evacuation time (2| hours) in each case. In one case the total acidity was slightly higher after adding ice cream; in the other the reverse was true. It is clear, therefore, that ice cream, though greath' increasing the food value of the dish, need not necessarih^ increase the burden of the stomach to any marked extent. Twent}' grams of cream cheese were added to apple pie in one case and compared with apple pie alone (see fig. 6), It will be noted that the acid responses in the two cases do not differ greatly, although nat- urally cheese increases the acid combining power of the food ingested. The addition of the cheese delayed evacuation about a quarter of an hour, and led to a shght secretion of gastric juice after the stomach was otherwise empty. THE RESPONSE OF THE HUMAN STOMACH^TO CAKES Among the cakes and related products tested out by us were angels' food, devils' food, fruit cake, chocolate layer cake, lady fingers, straw- berry short cake, crullers, doughnuts, cream puffs, cinnamon buns, ginger bread and cookies of different kinds. One hundred grams were given in each case. GASTRIC RESPONSE TO PASTRY AND PUDDINGS 255 Angels' food, devils' food, fruit cake and short cake. Angels' food was given to three men. A few days later an equal amount of devils' food was given to each of the same three subjects for comparison (see figs. 12, 13 and 15). It was found that angels' food cake remained on the average 20 minutes longer in the stomach than devils' food, the evacu- ation time of these cakes ranging from 2f to 4^ hours. This may well be related to the greater protein content of the angels' food which is particularly high in eggs. The distinctly higher acidities developed in the stomach in the case of angels' food cake, and the much more pro- nounced acid combining power must also be due to the high protein content of this cake as compared with devils' food which, however, is higher in calories due to its sugar and chocolate content. Chocolate layer cake (fig. 15) left the stomach of a subject of the rapid-emptying type in 2| hours or a few minutes sooner than devils' food cake. The acidity developed was low, due perhaps in part to the depressing action of the sugar icing on secretion. Old and fresh fruit cake were compared, using two subjects on each (see figs. 13, 14 and 17). It is a popular belief that old fruit cake is much more easily digested than the same cake in the fresh condition. Portions of freshly baked cake were given to the men, and three weeks later the same subjects were given other portions of the cake which had been kept in tin at room temperature for that period. In each case it was found that while the fresh cake required 3i hours for gastric digestion the older cake required 3| hours. The acid re- sponses were practically identical on the fresh and older cake. No distinct difference in gastric response to fresh and old fruit cake could, therefore, be detected. Two subjects were given strawberry shortcake (75 grams cake and 25 grams of berries) without cream. This cake left the stomach in moderate time (3 to 3j hours) and gave rise to the development of high intragastric acidities, in part probably due to the acidity of the berries themselves. Lady fingers were given to one subject (see fig. 13). The evacuation time of these was 3| hours as compared with 3 hours for devils' food and 3j hours for fresh fruit cake. Doughnuts, crullers, ginger bread, cinnamon buns and cream puffs. Ginger bread was given to two subjects (see figs. 9 and 16). It re- mained in the stomach from 2j to 3 hours or a few minutes less than strawberry shortcake or chocolate layer cake and a shorter time than devils' food or angels' food cake. Ginger bread brought about a mod- 256 MILLER, FOWLER, BERGEIM, REHFUSS AND HAWK erate stimulation of acid secretion with, in one case, a rather slow devel- opment. Evidently the ginger did not induce any increased secretion of gastric juice. Cinnamon buns were tested out on two subjects (figs. 14 and 21), and required from 2| to 2f hours. In one subject these buns left the stomach sooner than fruit cake, angels' or devils' food cake or lady fingers. The other subject evacuated cinnamon buns a few minutes sooner than doughnuts. In acid response the buns were found to follow the general trend of the cakes just mentioned, not attaining, however, to the level of angels' food cake. Neither is the acid response as high as that of bread with peanut butter. Probably the sugar of the buns depresses secretion somewhat. Bread with peanut butter (100 grams bread and 25 grams of peanut butter) remained in the stomach 3j hours as compared with 2f hours for cinnamon buns. The acidity developed was considerably higher in the case of bread with peanut butter (see fig. 22). Bread with corn syrup (80 grams of bread and 20 grams corn syrup) left the stomach in 2j hours (fig. 16) or in the same time as ginger bread and a httle sooner than chocolate layer cake or devils' food. The acidity developed was only moderate, being sHghtly lower than for ginger bread, the syrup probably depressing secretion. Cream puffs were given to one subject (fig. 19). They developed an acidity of over 100 and left in 2| hours or much sooner than crullers. Crullers and doughnuts were compared on one subject (fig. 20). The doughnuts remained in the stomach 15 minutes longer than the crullers or 2| hours. The maximmn acidities developed were about the same in each case, but the acid development was slower in the case of crullers due, perhaps, to their higher sugar and fat content. One subject required 4j hours for crullers. His digestion did not, how- ever, appear to be entirely normal on the day of this test. A third sub- ject required for doughnuts a digestion tune of 3 hours as compared with 2f hours for cinnamon buns (see fig. 21). It seems, therefore, that doughnuts are handled a little less readily than crullers, but that neither of these are unusually difficult for the normal stomach to handle. The response of the stomach to cookies. Spiced cookies, ginger snaps and Mary Ann cookies were studied in a few cases. The Mary Ann cookies remained in the stomach of a subject of the slow-emptjang type for 2 1 hours as compared with 4 hours for devils' food and 4^ hours for angels' food cake, and this in spite of the higher content of the cookies in dry matter. Ginger snaps required in this subject | hour GASTRIC RESPONSE TO PASTRY AND PUDDINGS 257 longer than the Mary Anns. A high acidity was developed on ginger snaps, but this was very slow in development, indicating that the ginger depressed gastric secretion. Spiced cookies left the stomach in 2j hours in both cases where they were tried out. They remained the same time as crullers but not so long as doughnuts. They left an hour sooner than fruit cake. Fairly high acidities were developed by these cookies. It appears, therefore, that cookies, in spite of their high content of dry matter, leave the stomach sooner than many cakes, and in really equivalent amounts would be handled more readily by the stomach. This may in part be due to their more granular texture. THE DIGESTION OF PUDDINGS IN THE HUMAN STOMACH The following puddings were studied : vanilla corn starch, chocolate corn starch, tapioca, apple tapioca, gelatin, Indian pudding, bread pudding. Brown Betty, cabinet, rice, rice with raisins, cup custard and plum pudding. One hundred grams of pudding were given in each case. Starch and tapioca puddings. Five experiments were carried out on corn starch and tapioca puddings (see figs. 22, 23, 24 and 26). Vanilla and chocolate corn starch and plain tapioca and apple tapioca were tested. All of these puddings left the stomach in from 2 to 2^ hours. Apple tapioca was evacuated 15 minutes sooner than the plain pudding. It developed a higher acidity with less combined hydrochloric acid. The starch puddings left the stomach rather quickly and developed a moderately high acidity (91 to 110). Cereal puddings, cabinet, Brown Betty, bread pudding, rice pudding and Indian pudding. Bread pudding quickly developed in the stomach a high total and moderate combined acidity and was rapidly digested, leaving the stomach in from 1\ to 2 hours in the case of subjects of the rapid-emptying type (see figs. 17, 24 and 27). Brown Betty pud- ding remained in the stomach 2\ hours as compared with 2 hours for bread pudding. A subject of the slow-emptying type required 2\ hours for this pudding (figs. 22 and 24). Cabinet pudding left the stomach of a subject of the rapid type in 2 hours as compared with If hours for bread pudding (see fig. 27). A subject of the slow type (see fig. 22) required 2f hours or the same time as for Brown Betty. Cabinet developed a lower acidity than bread pudding. TeE AMERICAN JOUBNAi OF PHTSIOLOGT, VOL. 52, NO. 2 258 MILLER, FOWLER, BERGEIM, REHFUSS AND HAWK Indian pudding gave a response practically identical with that of bread pudding, leaving the stomach in 2 hours and developing a high acid point of 113 at an hour and a half, the high protein content of this pudding being undoubtedly responsible for the high combined acidities found (see fig. 25). Rice pudding was tried both with and without raisins (see figs. 1 and 4). The acid responses were in each case practically the same. The evacuation times on plain rice pudding were in each case 2 hours. Rice pudding with raisins required in one case 2 hours and in the other only If hours. The combined acidities were practically the same in each case. The rice puddings are thus among the more easily digested puddings. Gelatin and plum puddings and cup custards. A widely used gelatin dessert of strawberry flavor was given to each of two men (see figs. 27 and 28). It left their stomachs very rapidly (in 1^ to 2 hours) and gave rise to very little stimulation of acid secretion. The low com- bined acidities also indicate that this product leaves the stomach too soon and with too little stimulatory effect to be markedly altered in the stomach or to throw any considerable burden upon it. Plum pudding was found to be one of the less readilj^ digested pud- dings (see figs. 23 and 27). Its relatively high food value must, how- ever, be borne in mind. A subject of the rapid-emptying type required 2 J hours to evacuate this food as compared with 1| hours for gelatin and If hours for bread pudding. A subject of slow-emptying type required 4| hours for plum as compared with 2^ hours for corn starch pudding. Moderately high total and combined acidities were devel- oped. Cup custards remained in the stomach about the same time as Brown Betty pudding and a few minutes longer than bread pudding, corn starch or Indian puddings, but not so long as plum pudding (see figs. 23 and 24). Being very high in protein, custards would be expected to show a rapid gastric stimulation with much combined acidity. This was found to be the case. COMPARATIVE RESPONSES OF THE STOMACH TO PIES, CAKES AND PUDDINGS From tables 1, 2 and 3 the average acid responses and evacuation times of all subjects on pies, cakes and puddings may be obtained. They show that the grand average evacuation times were for puddings, GASTRIC RESPONSE TO PASTRY AND PUDDINGS 259 pies and cakes, 2:18, 2:27 and 3:02, respectively. The average highest total acidity was practically 90 in each of the three cases. Perhaps a more significant comparison may be obtained from table 4, in which are summarized the results from six subjects, each of whom besides receiving a number of pies or cakes or puddings was also given certain foods belonging to one of the other two classes. It will be seen that here also pies left the stomach sooner than cakes, and puddings still more rapidly. TABLE i Gol Spe Kar Lea Mil Rud Average EVACUATION TIME AND ACIDITIES Pudd ings Pies Cakes 1:30 96.0 3:00 80.2 . 2:30 94.3 3:00 118.0 1:52 83.1 2:00 97.0 1:67 71.5 2:30 76.0 2:00 104.0 2:23 100.5 2:41 101.4 3:00 115.0 2:00 89.8 2:24 93.7 3:00 104.4 SUMMARY AND CONCLUSIONS A study was made of the acid responses and evacuation times of nearly fifty pies, cakes and puddings in the normal human stomach. The average evacuation time on puddings for all subjects was 2 hours and 18 minutes as against 2 hours and 27 minutes for pies and 3 hours and 2 minutes for cakes. Averaging the highest total acidities values w^ere obtained for puddings of 92, for pies of 90 and for cakes of 90. Direct comparisons of the three types of foods on the same individuals indicated also that pies were handled more readily than cakes, and pudding somewhat more readity than either. Fruit pies, such as apple, pumpkin, raisin and peach, left the stomach in from 2 to 2f hours and developed a moderately high acidity (90 to 100) . Most of the acidity is due to free HCl, the acid combining powers of these pies being low. Cherry pie, high in sugar, remained in the stomach a few minutes longer than the above. Rhubarb pie was treated in the same way as fruit pies, lea\dng the stomach in from 2 to 2 J hours. Custard pies left the stomach in moderate time (2^ to 2| hours) and possessed a fairly high acid combining power. Lemon meringue 260 MILLER, FOWLER, BERGEIM, REHFUSS AND HAWK showed a similar response except that the acidity developed more slowly. Mince pies required a rather long time to leave the stomach (2f to 3j hours) and developed high total and combined acidities. Pie crusts alone remained in the stomach distinctly longer than most whole pies or the contents alone of such pies, and gave a lower but more sustained acid curve. On the other hand, differences in evacuation time of whole pies and the contents of such pies were usually very sUght so that pies with crust, if properly made, could by no means be classified as difficult for the stomach to handle. The addition of 50 grams of ice cream to a small piece of pie did not increase the burden of the stomach to any marked extent. The addi- tion of 20 grams of cheese to apple pie increased the digestion time only a few minutes. Angels' food cake remained distinctly longer in the stomach than devils' food cake and developed a higher total and combined acidity. Chocolate layer cake left the stomach in moderate time, acid secre- tion being depressed by the sugar of this cake. Fresh and old fruit cakes showed ahnost identical acid responses and evacuation times in the human stomach. Strawberry' shortcakes left the stomach in moderate time (3 to 3| hours) and developed high intragastric acidities. Lad}^ fingers left the stomach in 3 hours or in about the same time as other cakes. Ginger bread evoked a rather slow acid response and left the stomach in moderate time. Cinnamon buns left the stomach sooner than most cakes but with a similar acid response. Bread with peanut butter remained in the stomach longer than cinnamon buns and developed a higher acidit^^ Bread with corn syrup left the stomach in 2J hours, the sja-up de- pressing secretion somewhat. Doughnuts remained in the stomach a few minutes longer than crullers. Acidities developed a little more slowly in the case of crullers, due perhaps to the sugar and fat content. These fried cakes required a digestion time but little longer than the average for cakes. Cookies, in spite of their high content of diy matter, were found to leave the stomach sooner than most cakes, probably on account of their granular texture. Ginger cookies required a little longer than those less highly flavored, and the acid development was less rapid. GASTRIC RESPONSE TO PASTRY AND PUDDINGS 261 Chocolate corn starch pudding, rice pudding with or without raisins, Indian pudding, bread pudding and gelatin left the stomachs of indi- viduals of the rapid-emptying type very quickly (in 1| to 2 hours). Rice pudding with raisins left a little sooner than plain rice pudding. Cabinet pudding, Brown Betty pudding, cup custard and apple tapioca required only a few minutes longer. Plain tapioca remained in the stomach a little longer than apple tapioca. Plum pudding left the stomach slowly. The highest total and combined acidities were caused by Indian pud- ding, bread pudding and cup custard, all of these being high in protein. The other puddings developed moderate acidities except gelatin, which produced little acid stimulation and left the stomach very quickly. BIBLIOGRAPHY (1) Beaumont: Physiology of digestion, 2nd ed., Burlington, Vt., 1847. (2) FiSHBACK, Smith, Bergeim, Lichtenthaeler, Rehfuss and Hawk: This Journal, 1919, xlix, 174. (3) Miller, Bergeim and Hawk : Unpublished data. (4) Smith, Fishback, Bergeim and Hawk: Unpublished data. §- ^ ^ 39- •^LUL-thl^o-l^i.n^JT^ I hour i Fig. 1 § 8 ^ Case,. -So A7 o^ 9 mince ^e (P^n) \Z. Z6 Lemon PQeronque ^^t-?) ^ 49 T>umf^Hcn 7=1© JF^') Fig. 10 266 s^ CaS'e,-Son 8 5 /.hour 1 Fin. 11 t- ^8- §J ^- 30. flnqel Tood d7(ye. '^pitd^(yenier Luoi^h -^^-^^^i^J^^ ^'^^ ^- W \ r^l V I hour — T~ Fig. 22 272 4- I- ^ V Q . 2-3 Choc r> La±& HuddLnq lUi} Case. T)al ~^ I hour Fig. 24 273 ^1 § § Q ^ (hse,. 7^i (fiA- Sj^ni-u/ijecryXoe Cf^eam Sundae 79 indurnHj-t/dimg /'hour 2 Fig. 25 T 11 lahiQcoTPuMonq ~R/ I -hour i T Fig. 26 274 1^ Q2Se,- fiar 6>Z ~PLum ~Pudd'cnq I v. \tfibune-t •• . 36 SvrriLU/ berry QeCi I hour Fig. 27 t H # «§!- Ocxse.. QoL 35" Si:rcLujherrLj Qelebin(J^ 5- / hour At Fia. 28 275 T SEGMENTAL ACTIVITY IN THE HEART OF THE LIMULUS D. J. EDWARDS From the Physiological Laboratory, Cornell University Medical College, New York City Received for publication March 22, 1920 In the structural make-up of the Limulus heart the arrangement of the muscular elements into a long tube with well-defined segments, and the grouping of the nervous elements into a median ganglion and lateral nerves, are features which have a significant bearing upon its functional activity. Attention is especiallj^ directed to these features, since it appears from the work of Carlson (1) and others that activit}^ in this heart is initiated and conditioned by this intrinsic nervous system, and that contraction in all segments is practically simultaneous. These considerations have suggested the present study, which is an analysis of the contractile process in the different parts of the muscular tube, and of the sequence of activity in the different segments in an attempt to discover the exact temporal relations of the different parts. In order to make a detailed study of the contraction event it is neces- sary to employ instruments of unquestionable efficiency. The short- comings of the suspension method of recording heart action are too well known to need repeating; it suffices to note that records made from the heart of this form by arranging in various ways, so that a thread hooked into one of the lateral arteries may be attached to a cumbersome record- ing lever, gives a very inadequate representation of the contraction. Small myocardiographs recording by means of Frank capsules were used in the experiments here described, and the arrangement of the apparatus was essentially the same as described by Wiggers (2) and used for analyzing the contraction process in mammalian auricle. The method of adapting it to the Limulus heart was as follows : The myo- cardiograph arms were adjusted to a given segment so as toi exactly span the width of the heart at the end of diastole. A fine silk thread was then passed through the superficial layer of the heart at the lateral borders and the arms of the myocardiograph tied thereto. In this manner there was slight traction on the heart until it began to contract. 276.' SEGMENTAL ACTIVITY IX LIMULUS HEART 277 Moreover, there was very little resistance offered to the shortening as the myocardiograph and also the Frank capsule were covered with very thin rubber membrane. The record obtained is that of the mid-segmental shortening, the part of the heart that normally exhibits greater contractility than the inter-segmental portions where the ostea are located and in the anterior part the lateral arteries are attached. The myogram of different segments. In considering the relation of the heart of the Limulus to its circulatory system it will be noted that the five anterior segments are each connected with outgoing pathways for the blood. If one reasons from analogy to the mammalian heart, a greater contractility should mark the myogram of these parts as compared with that of the posterior segments. Fig. 1. Simultaneous myograms from heart segments 2, S2, and 5, So, showing differences in the contour of the curve from these parts. A comparison of the myogram from the two ends of the heart, for example, segments 2 and 8, shows differences in the contour, but the curve from segment 5, which has lateral openings, does not exhibit significant difference from that of segment 6, which has no such open- ings. It appears, therefore, that specialization in contractiUty, such as is shown in the ventricle of the mammalian heart, is not sharply delimited in the heart of the Limulus to that part from which the circu- lating fluid is outgoing. The contraction amplitude is nearly always greatest in segment 2. The myogram of this segment, in figure 1, shows a small primary rise, h to c, upon which is superposed the main contraction, c to d. The upstroke of the principal rise is rapid and the summit is relatively THE AMERICAN JOURNAL OF PHYSIOLOGY, VOL. 52, NO. ? 278 D. J. EDWARDS pointed, indicating that the state of maximal contraction occurs in all of the elements of this segment at j^ractically the same instant. All of these features are exhibited in the myogram of segment 3, except- ing that the degree of contraction is usually a Httle less. The myograms of 4 to 8 all show a smooth rovmded contour and, barring differences in the amplitude, the curves from these different segments are practically superimposable. The shortening process in these segments develops slowly, as is shown fairly typically in the myogram of segment 5, figure 1, and moreover the maximal shortening is reached gradually, giving the curve a flattened summit. These features indicate that the con- traction takes place in a wave-like manner; some of the elements at the beginning of systole are contracting w'hile others are quiescent, while at the end of systole some are undergoing relaxation while others are still contracting. This form of contraction is suggestive of the type found in the mammalian auricle (2). In the vigorously acting heart the myograms of segments 2 and 3 are characterized by a slowly developing state of tension preceding the beginning of contraction. This is shown in the myogram of segment 2, figure 1, in the part of the curve from a to b. The diastolic phase con- tinues so that the curve passes somewhat below the level at the begin- ning of systole. Then follows the state of developing tonus during which the curve gradually swings back. In a slowly beating heart the curve reaches the level before the next systole begins, but with a more rapid rhythm the tonus increase extends right into the next systole. These presystolic tonus changes have not been found in hearts exposed for an hour or more to experimental procedures; they have also not been exhibited in the records from the posterior segments. The sequence of segmental activity. Inasmuch as the experimental evidence for the Limulus heart favors the view that the impulse is initiated in the median ganglion, the precise time of activity in different segments is a matter of some interest. A casual inspection of a pulsat- ing heart gives the impression of a simultaneous event throughout the nine segments. Carlson (3) states that 'Svhen an empty heart has been beating for several hours, or till nearly exhausted and the rate of the pul- sations is in consequence much reduced, it can be made out .... that the contractions start in the posterior third of the heart and travel anteriorly This is evidently the case in the fresh and vigor- ous heart." In a later communication the same w^-iter (4), discussing the degree of automatism of different parts of this heart, attributes this function primarily to the middle third, in a less degree to the posterior third, and least in the segments of the anterior third. SEGMENTAL ACTIVITY IX LIMULUS HEART 279 From the foregoing statements it is e\adent that the question of segmental sequence can be answered only by exact measurements of initial activity in different segments. This has been done by simul- taneous myograms of selected parts of segments, and by myograms related to the wave of excitation as detected by a string galvanometer. The usual procedure of taking simultaneous myograms was to attach one myocardiograph to segment 2 and a second to segment 3, then after a short interval records were taken. One myocardiograph was then changed to the next segment and the process repeated, and in a similar manner records were taken from all segments. Usually only one series was taken from a heart and sometimes only a few segments were com- pared, but each record was taken in duplicate and the mean of these was used in making up the data for that experiment. In general the results show that the middle segments, that is, segments 4 and 5, exhibit activity in a given cycle earlier than the posterior or anterior segments. A comparison of the beginning of contraction of segment 5 and of segment 2 in sixteen experiments shows that segment 5 preceded by an average interval of 0.046 second. In nine experiments comparing segment 2 with segment 4 there is shown an average time interval of 0.047 second, with segment 4 preceding. This indicates that the beginning of contraction in segments 4 and 5 is practically simultaneous and that it normally precedes the contraction of segment 2. On the other hand, a comparison of segment 2 with segment 6, for example, shows in eight experiments an average difference of 0.023 second, indicating that while segment 6 slightly precedes the beginning of contraction of segment 2, it follows by an average inten^al of 0.024 second the contraction of segment 4. A comparison of the initial activity in segments 7 and 8 by this method has given less satisfactory results because of the usually low degree of contractility which makes it difficult to determine precisely the beginning of shortening. It is possible, therefore, to make only a general statement concerning them. In six experiments comparing the beginning contraction of segments 2 and 7, it is shown that segment 7 precedes that of segment 2 in four instances and follows it in the remaining two. In a similar way, the data for seven experiments comparing segments 2 and 8 show that the contraction of segment 8 preceded the contraction of segment 2 in three instances and followed it in the remaining four. This indicates that the contraction of the posterior segments normally takes place at practically the same time as the anterior ones. 280 D. J. EDWARDS In order to test the matter of segmental sequence in another way, the excitation process of different segments was taken with a string galvanometer and in turn related to the myogram of a particular seg- X IC Fig. 2. Electrocardiogram recorded from segments 2, /; 4, II; 6, III, and 8, IV , to show time relation of excitation in these parts to the contraction of seg- ments 2 and 5. Position of points indicated by P. Ordinates in bottom portion of curves designate time intervals of 0.20 second. ment. The current was led off by placing one electrode on part of the dorsal muscle and the other beneath the heart so that the segment under observation rested upon it. SEGMENTAL ACTIVITY IX LIMULUS HEART 281 The characteristic form of the electrocardiogram for this heart has been very well described by Hoffmann (5); little attention, therefore, was given to this feature. In the experiments reported here the begin- ning of deflection was taken as the index of initial activity, and in figure 2 is shown a series of records which illustrate the time relations for different segments in a fairly typical way by this method. Curve / shows that excitation in segment 2 precedes the beginning of shortening in this segment b}- 0.08 second, and by 0.05 second the initial recorded shortening in segment 5. In curve // of this figure it is shown that excitation of segment 4 precedes the contraction of segment 5 by 0.09 second. In a similar manner it is determined from curve /// that excitation in segment 6 precedes the contraction of segment 5 by 0.06 second, and from curve IV, that excitation in segment 8 precedes contraction of segment 5 by onh' about 0.03 second. Relating these data, it appears, therefore, that the excitation of the median segments takes place about 0.04 second before excitation of the anterior segments and about 0.03 second before excitation of the posterior segments. In this connection it is interesting to note that the distance separating the electrode contact on segment 4 and that on segment 2 was 29 mm., and the time difference in the sequence of activity of these segments, as related to the myogram of segment 5, was 0.04 second. It is evident from these data that the rate of conduction was approximately 72 cm. per second, a figure that is significant in view of the average rate of 40 cm. per second for the conduction rate in the Limulus heart nerves, as obtained by Carlson (6) with less sensitive methods. Mention should be made of the condition met with in some experi- ments in which the anterior segments showed unmistakably an earlier state of activit}' than the middle segments. We have in figure 3, for example, a record of contractions of segments 3 and 5 showing the contraction of section 3 preceding by 0.05 second. This proved to be a constant feature of the contraction of this particular heart, since other records in this experiment comparing segments 1 and 6, and 1 and 5, show the same general result. Of the thirty-three experiments in which records have been made this phenomenon has been observed to occur with a degree of constancy in only four hearts. It is significant, how- ever, that records taken from the same heart late in the experiment, after a considerable period of exposure, show initial activity in the median segments. A careful study of initial activity in the different segments of the Limulus heart makes quite evident that the distribution of the excita- 282 D. J. EDWARDS tioii process is a rapid event, and that it normally encounters no marked areas of resistance which slow the excitation wave in its spread from the middle region of the heart. Garrey (7) has very clearly demon- strated, however, that this heart is susceptible to the usual types of blocking influences. Moreover, it is a fact well known to all who have worked with this heart that an adequate stimulus appHed to any part starts a contraction wave which passes in either direction. The evidence presented here supports the conclusion, I believe, that normally the contractions of the middle segments precede those of both anterior and posterior segments. The initial tonus changes often Fig. 3. Myograms from heart segments 3, . 5. EDWARDS The records, when viewed as a whole for the seven experiments of the series and the successive stages in each compared, show moderate changes after about 3 hours of the alternate occlusion and deocclusion. All of the experiments were continued for approximately 4 hours. The final contour of the pressure curve usually takes the form of a marked primary oscillation followed immediately by a sharp decline of the curve replacing the systolic plateau of the normal curve. These features are quite characteristically shown in the three segments reproduced in figure 1, in which corresponding points in the curves are indicated by similar letters. These curves were taken as follows: I at the outset of the experiment; // at the end of 2 hours and 20 minutes during which the alternate 20-minute occlusion and 5-minute release of two femorals c e d ck f a Fig. 1. Segments of the optical arterial curves during the progress of successive occlusion and deocclusion of the veins of three extremities. and one brachial veins were carried out; /// at the end of 3 hours and 40 minutes of alternate occlusion and release. The change in mean blood pressure taking place in this period is slight, but significant in view of the fact that the directional change is downwards and that the optical curve is indicative of lowered peripheral resistance. The following table will assist in bringing out this feature of the blood pressure change and also the time of appearance of a marked change in the form of the pressure curve. This table shows that averaging the interval before a marked change was apparent in the contour of the pressure curve there was required about 3| hours of alternate clamping of the veins or ligating of the extremities and that much longer time was necessary to give unmistakable signs of decreased peripheral resistance. Definite indications of circulatory failure and PULSE CURVE AFTER HISTAMINE AND VASCULAR OCCLUSION 287 a shock-like condition were shown, however, in only one instance, that of experiment 8, but the data for this experiment give ground for the view that the occlusion effects per se played a relatively less part than trauma of the sensory nerves from application of the ligatures. There were marked sensory signs with each application of the ligatures and mean blood pressure in the early part of the experiment rose from 172 to 208 mm. Hg.; then occurred a fairly sudden break and a fall in press- ure. The optical curve does not give indication of increased resistance as the main cause of this rise; it is probably due, therefore, to an in- creased cardiac output. Thus, however viewed, the alternate occlusion and deocclusion of the circulation of three extremities fails to effect changes in the dynamics of a more pronounced character than have been shown to occur in the TABLE 1 BLOOD PRESSURE LENGTH OF ONSET OF STATE OF ARTE- EXPERIMENT EXPERIMENTAL PERIOD PRESSURE CURVE RIAL RESISTANCE At the start At the finish AT FINISH hours minutes hours minutes 1 106 110 5 50 3 45 Decreased 2 74* 118 3 45 3 15 Decreased 8 172 8S 4 30 3 25 Very low 10 132 114 3 2 10 Decreased 11 152 132 3 50 3 20 Decreased 14 166 112 5 3 45 Decreased 15 126 112 4 No change initial stages of circulatory failure of shock (2) and a period well over 3 hours is required for this change to appear. Clamping of the inferior cava: The moderate degree of change in the pressure curve resulting from occulsion of the circulation of the extremities suggested extending the procedure to include larger vascular areas. It is interesting to notice in this connection that the minute volume flow to the posterior extremities, as calculated from the flow in the femoral vein (7), is 45 cc. There are no data available concerning the supply to the anterior extremity but it is usually assumed to be approximately equal to that of the posterior extremity. The three extremities, therefore, would represent a volume flow of 135 cc. per minute. On the other hand, it has been shown that the kidneys have a minute volume flow of 195 cc. for the two organs (8). These data represent average normal values and at best are only approximate, 288 D. J. EDWARDS but they serve to indicate that the clamping of the cava affects a many times greater vascular field than was included under the experiments on three extremities. The pressure changes following cava obstruction indicate in a striking way that there is a greatly diminished venous return which causes some cardiac embarrassment. There is a reduction in the amplitude of the curves with a retardation in the gradient of the upstroke and the end of systole is not sharply marked by a distinct incisura. With the first deocclusion of the cava there is evident in the pressure curve a resumption of the main characteristics of the original, but after the second period of clamping Uttle tendency to recovery is shown; further- more, there is indicated a marked relaxation in peripheral tone. The total time of occlusion of the inferior cava necessary to give these indi- cations of a break in the dynamics was never more than 1 hour. It may have been somewhat less since the periods of occlusion were from 25 to 30 minutes. The results of one experiment in which the clamp was applied just posterior to the inlet of the renal veins is of interest, since in this manner the kidney circuit is left intact. The blood pressure fell about 30 mm. Hg. when the clamp was on but showed practicall}" complete recovery each time it was removed. This was repeated four times, covering a period of 2 hours and 10 minutes, and at the end of this time the optical curve showed the characteristics of initial failure. There is suggested, therefore, a degree of proportionality between the time of appearance of these circulator}^ changes which influence the form of the pressure curve and the extent of the vascular field occluded Occlusion of the inferior mesenteric vein: The inferior mesenteric vein drains an entirely visceral area in contrast to the conditions considered above. IVIoreover, it represents a vascular field as indicated b}^ the figure for blood flow of 164 cc. per minute (9) quite comparable to that studied under occlusion of the extremities. The outstanding features in the pressure curve during the progress of this type of occlusion are the relatively sudden appearance of the change in the contour of the curve and the slight indication of progressive development after this initial change. The first period of clamping was about 40 minutes and the i:)ressure curve shows (fig. 2, //) at the end of this time a sharp primary oscillation (a-5) without plateau (6-c) and with low diastolic limb (e-f). Each record was taken at the end of the 5-minute period of deocclusion. Segments III to TV, while differing somewhat in amj^litude, show slight changes in their essential PULSE CURVE AFTER HISTAMINE AND VASCULAR OCCLUSION 289 characteristics and give evidence of a comparatively fixed state of dilatation after the first period of stasis. Furthermore, these succeeding periods of occlusion show only moderate depression in blood pressure and nearly identical recoveries during the periods of deocclusion. These changes lend support to the view that the cardiac mechanism is not markedly affected by the degree of reduction in the venous return. The significant features in these results appear, therefore, as the rapid initial decrease in blood pressure with comparatively small changes from the alternate periods of occlusion and deocclusion, and the shght tendency to a progressive type of change in the pressure curve. Many factors are concerned proliably in the production of the effects fol- lowing occlusion of the venous return from such vascular areas, but the i,^e '^ ^ :2Z" i Ic M JJ- \ { i \ \ c 3 c Fig. 2. Segments of optical arterial curves during the progress of successive occlusion and deocclusion of the inferior mesenteric vein. following warrant special consideration: (a) Mechanical distention of the blocked venous system; (6) a toxic action of products formed in the stagnant areas which exert further injurious effects when periodically flushed into the circulation; and (c) the damaging of the vascular walls with accompanying alterations in permeability. The present experi- ments do not permit of direct conclusions concerning which of these factors plays the essential role in the production of the effects studied. Indirectly they give evidence, however, in favor of the view of mechani- cal dilatation as the main cause of these primary changes, since it has been shown that occlusion of an intestinal area which affords by virtue of its plastic character a favorable site for mechanical stretching of its vascular walls, is immediately affected and the change appears almost in full magnitude at the outset. The occlusion of a peripheral 290 D. J. EDWARDS field of comparahle vascular magnitude, which presents a comparatively rigid vascular area, gives results essentially different in that corre- sponding changes in the pressure curve appear much later in the course of events. Thus far, no account has l)een taken of the possible toxic effects of substances formed during the periods of stagnation. In order to throw light directly upon the probable degree of influence which substances of this character exert the experiments ])el()w with histamine were performed. Successive injections of histamine: The results of Dale and Laidlaw (10) show quite conclusively that the administration of histamine may produce a condition simulating traumatic shock. This phenomenon h i c e e d Fig. 3. Segments of optical arterial curves at certain stages during the blood pressure change from injection of 0.15 mgm. histamine. The numbering of seg- ments corresponds to positions indicated on figure 4. they assign to increased capaciousness through the relaxation of capillary tone from the toxic action of this substance. In their experiments cats were used for the most part and a dosage of a milligram per kilo body weight was found necessary to produce circulatory collapse. In the present experiments on dogs it was sought to test not only the immediate effects but also the accunuilative action of histamine on the dynamics of the circulation; accordingly, the injections were, usually 0.04 or 0.05 mgm. (the ergamine of Burroughs, Wellcome & Co.) repeated at 10-minute intervals over a period of 3 to -4 hours. Such a dosage never fails to give at each injection a fall of over 50 per cent in blood pressure at the start. The recovery is rapid at first and usually complete in less than 3 minutes but as the experiment progresses and f PULSE CURVE AFTER HISTAMINE AND VASCULAR OCCLUSION 291 also following larger injections a somewhat longer time is required before the pressure retvu'ns. In figure 3 are shown portions of the optical records taken during the changes produced by the injection of 0.15 mgm. histamine. These curves, taken in connection with the tracing of the blood pressure change shown in figure 4, portray the immediate effects of this substance upon the pressure relations. Coincident with the fall in mean blood pressure the optical curve shows (fig. 3, segment 77) a large primary oscillation uui'MmmmimiitiumuiimummiiuimituM^^^ Fig. 4. Tracing showing blood pressure change as related to optical curves in figure 3. Numerals correspond to segments in that figure. The intervals A, B and C correspond to 2 minutes, 5 minutes and 5 minutes, respectively. Time curve indicates seconds. (a-6), indicative of a collapsed state of the arterial sj^stem (c-rf), and a flattened diastolic limb {e-f), signifying a low state of peripheral resist- ance. There is a rapid restoration of peripheral tone indicated in segment 777, e-j, by the immediate rise in this portion of the curve. This change in resistance suffices to restore mean blood pressure in a short time but it will be seen from segment 7 V that the recovery of the dynamic relations is complete before mean blood pressure has reached its original level. The extent to which cardiac output is influenced by 292 D. J. EDWARDS these changes does not appear from the present data. Further experi- ments deahng with this feature are in progress. Turning now to the influence of the successive injections, it was demonstrated at the outset that small doses, e.g., 0.01 mgm. per injec- tion, have little accumulative effect. With individual injections in amounts of 0.05 mgm. it has been possible generally to produce in dogs of about 8 kilos signs of initial circulatory failure after a total of 0.5 to 0.7 mgm. had been injected in the manner indicated. The stages in the failing dynamics are very well l3rought out in figure 5. Segment / was taken at the outset of the experiment and serves as a basis for comparison; segment // was taken after seven 0.04 mgm. injections had been given; and segment ///, after thirteen such injections. Close ~ 2zr ''' f Fig. 5. Segments I, II, III and IV, optical arterial curve during progress of successive injections of histamine. Segment V, taken 25 minutes following injection of 5 cc. adrenalin (1 : 5000). Description in text. inspection of these curves shows a sharper contour in II and III and a slight diminution in the height of the primary oscillation. These features are significant in that they anticipate the more marked changes characteristic of failing peripheral resistance and that they take place without significant changes in mean blood pressure. At this stage of the experiment the dosage was increased. There followed two 0.2 mgm. injections at the usual 10-minute intervals and one injection of 0.5 mgm. ^\liereas the smaller amounts had usually caused a fall in blood pressure to 50 to 60 mm. Hg., the latter dose depressed the press- ure to 36 mm. Hg. Segment IV was taken 20 minutes following the 0.5 mgm. injection and at a time when blood pressure had practically recovered. It will be seen that the introduction of the larger amounts augmented the features already noted and that the curve now exhibits PULSE CURVE AFTER HISTAMINE AND VASCULAR OCCLUSION 293 a certain likeness to that shown under the occlusion of the veins to the extremities and characteristic of initial failure. These results present quite typically the effects on the optical curve of repeated injections of histamine. It is significant to note, also, that all of the experiments were continued for a period of about 4 hours without a single instance of circulatory collapse appearing, notwith- standing injections at the conclusion of some of the experiments of a milHgram of histamine following closely upon successive doses only slightly less, with a total given during the experiment amounting to. 3 mgm. or more. These observations lend support to the view that the dilatation resulting from the amounts of histamine used, and adminis- tered in the manner indicated, causes only moderate djmamic changes. Furthermore, it appears that the change is one that may be due to the reduced resistance from simple capillary dilatation. With the possi- bility in mind that a moderate vasoconstriction might counteract the effects of the periodic histamine dilatations and thereby restore the failing dynamics, injections of 5 cc. of adrenalin (1:5000) were given at the rate of about 1 cc. per minute. The usual constrictor response followed but passed off in ten to fifteen minutes and a vascular equih- brium was again estabhshed. The pressure relations at this stage are very well shown in segment V of figure 5, taken 25 minutes following the administration of the adrenalin. It shows that, notwithstanding a good state of peripheral resistance signified by the height and slope of the diastolic limb, e-f of the curve, there is a diminished distention of the arterial system evidenced by the increased amphtude and sharp contour of the primary oscillation, a-h. The marked vasoconstriction of the adrenalin appears to have the effect, therefore, of augmenting the condition started by the histamine. In this connection it is interesting to consider briefly the possible mechan- ism of action of adrenahn wherebj^ it accentuates the dilatation initi- ated by histamine. Hartman and McPhedran (11) state that large doses of adrenahn dilate the intestinal vessels. On the other hand Erlanger and Gasser (12), while not able to agree with this conclusion, point out that portal pressure is greatly increased by giving large amounts. The results presented above give unmistakable e\'idence of a decrease in peripheral resistance after repeated small doses of histamine. It is well to bear in mind that several factors may contribute to this end, important of which are decreased arterial tone, dilatation of the capillary areas and decreased viscosity of the blood. While it is not possible THE AMERICAN JOURNAL OF PHYSIOLOGY, VOL. 52, NO. 2 294 D. J. EDWARDS to state with certainty regarding which of these factors plays the greater role in the reduction of peripheral resistance with histamine, it is reason- able to assume that the action of adrenalin described above is due to a further dilatation of the weakest link in the vascular circuit. The usual absence of reduction in arterial tone from adrenalin, and also the excellent results of Dale and Richards (13), indicating that his- tamine is a capillary dilatant; support the conclusion that the capillary system is the part less resistant and is the area which compensates for the intense constriction of the adrenalin. The data at hand do not indicate whether this effect is mainlj^ on the intestinal area or is gener- ally present in the capillary blood-bed of all organs. The observation of a great increase in portal pressure lends support to the view that the area supplied by this vessel may play the larger part in these results. One point still remains to be discussed; that is, the bearing of the results with histamine upon the effects following occlusion of different, vascular areas. The optical curves show features in common, each presenting changes indicative of initial failure of normal dynamic relations. It might appear, therefore, that the occlusion effects were caused by metabolic products with histamine — like action formed during the periods of stasis. There seems little doubt that such prod- ucts may contribute to the effects of occlusion, but that they are not the main cause is evidenced, I believe, by the relatively large amounts of histamine necessary to initiate failure changes and by the observa- tion recorded above that during the periods of reestablished circulation through the occluded part there are never indications of a dilating sub- stance being carried into the general circulation. SUMMARY 1. Alternate periods of occlusion and release of the circulation of three extremities, when continued for about 4 hours, produce changes in the optical pressure curve comparable to those shown in initial circulatory failure. There appears to be little difference whether the ■occlusion is produced bj^ a, ligating all of the limb structures except the main arterial supply; or b, clamping the principal venous channels from these organs. 2. Clamping the inferior cava anterior to the renal veins shows indications in the pressure curve of cardiac embarrassment from di- minished venous return. Two 30-minute periods of occlusion of this vein interrupted by 5-minute intervals of reestablished flow produce PULSE CUBVE AFTER HISTAMINE AND VASCULAR OCCLUSION 295 marked changes in the dynamic relations. By lessening the vascular area blocked, the time of appearance of these changes is somewhat delayed. 3. Blocking the venous return from a visceral area brings on a failure type of pressure curve much sooner than occlusion of a peripheral area of comparable vascularity. 4. The observations of a slow, progressive development of the occlu- sion effects of peripheral structures, the rapid onset with little evidence of progressive development for visceral areas, and the character of the changes portrayed in the pressure records, support the interpretation that the cause is essentially one of mechanical distention of the blood- bed in the occluded parts. 5. The optical pressure curves during the blood pressure change from a small dose of histamine show a sudden decrease in the peripheral resistance with a greatly diminished arterial distention, and an immedi- ate recovery of peripheral tone. Injections of 0.04 to 0.05 mgm. repeated at 10-minute intervals produce after many such doses a small degree of decrease in peripheral resistance. Larger doses serve to accentuate these initial effects of the periodic dilatations. 6. Adrenalin augments the failure changes initiated by the repeated histamine injections. BIBLIOGRAPHY (1) WiGGERs: Modern aspects of the ciculation in health and disease, Philadel- phia, 1915; Arch. Int. Med., 1915, xv, 77; This Journal, 1914, xxxiii, 382. (2) WiGGERs: This Journal, 1917, xlv, 485. (3) Edwards: This Journal, 1914, xxxv, 15. (4) Roy and Brown: Journ. Physiol, 1879, ii, 323. (5) Mann: This Journal, 1918, xlvii, 248. (6) Erlanger and Gasser: This Journal, 1919, xlix, 151. (7) Burton-Opitz : This Journal, 1903, ix, 161. (8) Burton-Opitz: Arch. f. d. gesammt. Phj^sioL, 1908, cxxiii, 553. (9) Burton-Opitz: Arch. f. d. gesammt. Physiol., 1908, cxxiv, 495. (10) Dale and Laidlaw: Journ. Physiol., 1918, lii, 355. (11) Hartman and McPhedran: This Journal, 1917, xliii, 311. (12) Erlanger and Gasser: This Journal, 1919, xlix, 345. (13) Dale and Richards: Journ. Physiol., 1918, lii. 110 THE ARTIFICIAL PRODUCTION OF MONSTERS DEMON- STRATING LOCALIZED DEFECTS AS THE RESULT OF INJURY FROM X-RAYS W. M. BALDWIN From the Union University {Albany) Medical College Received for publication March 22, 1920 In a recent paper (1919)^ the author has detailed the results of a series of experiments upon frogs' ova where, by the use of X-rays generally apphed to the surface of the ovum, a uniform defect was produced in the embrj^os. Inasmuch as a fixed quantity of energy was concentrated upon the ovum in the early stages of its development, practically all of the embryos presented the same gross and micro- scopic structural defects. In this present series of experiments, how- ever, the X-ray energy was permitted to act upon a relatively small amount of the egg substance through the utihzation of a perforated lead screen interposed between the tube and the specimen. The energy was derived from a large Coolidge tube which carried a milU- amperage of 50 at 50 kilovolts. The interposed lead diaphragm meas- ured about 24 mm. in thickness, through which a hole 0.3 mm. in diameter had been bored. Fertilized eggs of the frog, in a develop- mental stage no later than the two-celled, were placed behind the hole in the diaphragm in line with the center of the target of the tube and at a distance of 22.5 cm. from it. By this arrangement a maxi- mum effect of X-ray energy upon a relatively small amount of the pro- toplasmic mass of the egg was brought about. An effort was made to so place the eggs that the energy entered at a point in the animal hemisphere and emerged at a corresponding point in the vegetable hemisphere. A columnar mass of protoplasm, 0.3 mm. in diameter, extending from the upper to the lower surface of the L7 mm. ovum, was subjected thereby to a maximimi amount of direct radiant energy. In each instance an exposure varying from two to four minutes was 1 The artificial production of monsters conforming to a definite type by means of X-Rays. Anat. Rec, xvii, no. 3, November, 1919. A bibliography list is appended. 296 PRODUCTIOX OF MONSTERS BY X-RAY 297 made. The eggs were then transferred to a specimen jar containing 1000 cc. of tap water and permitted to develop at room temperature, the water being changed frequenth'. It was ascertained after repeated experimentation first, that the amount of X-ray energy passing through the thickness of the 24 mm. screen at the distance given, and under the conditions of exposure men- tioned, exercised no injurious effect whatever upon normal ova. Like- wise, it was demonstrated that the secondary radiation arising from the edges of the hole in the diaphragm was so small in amount as to be a negligible factor. The effects recorded in these experiments were owing apparenth^ therefore, to the direct action of the X-raj^ energy upon the protoplasm of the egg using this term in its broadest sense, in the restricted region mentioned. A study of the external features of the developed embryos reveals but little in comparison with the microscopic findings. In some instances it may be detected that the development of the eye or of the ear or both upon the same side is defective. Occasionally, as well, the gill tuft upon one side is absent. Furthermore, a lateral curvature of the body at the junction of the head with the trunk or at the level of the trunk, or at the level of the junction of the trunk with the tail may be observed, but this curvature is no greater than that which occurs with many normal tadpoles which are fixed together in large numbers. Some show an asymmetrical position of the dorsal fin at the level of several adjacent segments of the trunk. On the other hand, the color of the epidermis is normal throughout. The length, breadth and thickness of the tadpoles have not suffered to any appre- ciable degree over the controls. A study of the serial sections of the tadpoles demonstrates, however, a peculiarly abnormal condition of the anlagen of several segments of the trunk and neck regions. As the sections are traced in sequence from the head to the tail, at a level beginning ordinarily at the optic chiasm and extending caudally to the region of the stomach, a marked difference is to be observed when the corresponding portions of the embryo to the right and to the left of the notochord are compared. Upon first glance the impression gained is that of unusual asymmetry of the embryo in which the neural tube, the notochord and the enteron have left their customary median position and migrated together toward one or the other side. The space ordinarily occupied by the mesodermal tissue and its derivatives is considerably smaller in area upon the side toward which the notochord has migrated than upon the 298 W. M. BALDWIN opposite side. At such levels the neural tube and the notochord, while occupying the real median plane of the embryo so far as anlagen are concerned, actually lie nearer one lateral trunk surface than the other by reason chiefly of the above mentioned inequality of development of the mesodermal derivatives. This inequality of mass distribution is the result of a retardation of development of certain consecutive hemi- segments of the embryos. In no instance is this phenomenon restricted to one segment. Most often it involves those located immediately caudal to the point of origin of the optic stalk from the brain, and usually the defect extends as far caudally as the stomach. A close microscopic examination of the cells in this defective area demonstrates the point that the various organ anlagen are not absent, but the processes of differentiation in the cells constituting them have been so greatly checked by the X-ray energj^ in contrast with those of the corresponding anlagen of the opposite half, that they present an appearance of the greatest degree of developmental retardation. The individual cells present certain features of both cytoplasm and of nuc- leus, moreover, which are unmistakable indications of deviations from the normal morphological characters of these cells. The abrupt shifting of the neural tube, of the notochord and of the enteron from their normal median position in the mass of the embryo gives rise to the appearance of a lateral flexure of the trunk at the level of injury. In the caudal portion of the affected area a simi- larly abrupt reverse flexure brings these structures back to their normal median position. A singular feature of the abnormal embrj^os consists in the absence of any indication whatever of inflammatory reaction to injury in the affected area. Similarly the absence of ex- truded cells and of protoplasmic degenerations, involving either nucleus or cell body, may be interpreted as indicating a type of injury not of the most severe grade, as that noted by the author in his previous paper. The cytoplasm of the cells in the affected area possesses a large, more-coarsely granular appearance than is normal. Pigment granules which, in the later stages of development, are more completely restricted to the epidermal cells, are to be observed in those cells the substance of which lay in the path of the rays through the ovum. Their presence in numbers enables one to mark out readily the direction of this path in the sectioned embryo. The nucleus of these cells presents, in the main, two general departures from normal; first, the chromatin is clumped either into large, deeply-staining masses restricted for the PRODUCTION OF MONSTERS BY X-RAY 299 most part to the periphery of the nucleus, leaving the center in contrast comparatively clear; or, secondly, the chromatin is more finely gran- ular, more uniformly distributed, but invariably much less intensely staining than normal. It might be inferred that these two features represent different degrees of the same type of injury. There is no difficulty encountered, however, in readily differentiating between normal and rayed cells through these features. Furthermore, the contrasted appearance of these cells to normal cells in these partly- rayed specimens is all the more readily appreciated since both types may be seen in the same microscopic field. In the previous paper on type embryos, the presence of nuclear and of protoplasmic detritus in the body cavities, such as the enteron, neurocele, optic vesicle, etc., was interpreted, as had been done by Hertwig previously with radium embryos, as a certain indication of such a degree of severe injury as to eliminate these cells from partici- pation in the subsequent developmental stages of the embryo. Sucli features in this present series are, however, as was mentioned above, entirely absent. The injury produced by the rays was not sufficiently- intense, apparently, to destroy the vitality of any of the cells. There ensued, on the contrary, a temporary suspension of differentiation fol- lowed by a lengthened tempo in the developmental rate of these affected cells. The absence of a reactive inflammation is, at present, difficult of interpretation unless we assume that possibly the damage wrought by the rays has been wholly intracellular. As the different systems of the embryo are taken up in order and studied through the serial sections, several facts assume significant importance dependent upon the degree of reaction to X-ray influence. The ectoderm in the areas not in the path of the X-rays appears normal in thickness and in the disposition of its cells. Where the pencil of rays has passed through the embryo, however, those ectodermal cells in line with it depart from the normal in structure most markedly. In this area the entire thickness of the ectoderm in general is markedly increased. The individual cells are larger and the nuclei more deeply^ staining. The cell borders and the nuclear outlines are, however, distinct. This increase in thickness is, however, not uniform through- out the area affected, there being produced as a result an unevenness in the external surface of the ectoderm which amounts to a corrugation or wrinkling of this layer without involving, however, the basement membrane. In many instances the ridges so produced are hollowed out through the presence of channels which run a greater or less dis- 300 W. M. BALDWIN tance in a direction generally parallel to the underlying surface of the ectoderm. This reaction to X-ray influence resembles strongly that referred to by the author in his previous paper and noted as well by Hertwig in his study of radium embryos. There is no evidence of actual death or of desquamation of any of the ectodermal cells, neither are there indications of inflammatory reaction. In no specimen has the thickness of the Isijer been reduced. As the brain and spinal cord are followed through the sections, there is to be noted at the level of injury a marked dwarfing of the brain vesicle upon the affected side. What appears to be a compensatory hypertrophy of the vesicle wall upon the unaffected side has resulted in a shifting of the median ventral furrow in the floor of the neurocele toward the affected side. This accentuates the appearance of asym- metry. The neuroblasts upon the unrayed side are normal in size, in shape and in the appearance of their nuclear and cytoplasmic material. Stratification ordinarily has progressed to such a degree that the ex- ternal fiber layer ma}^ be distinctly differentiated from the nerve-cell layer. Upon the affected half of the vesicle there is a remarkable dwarfing of the neuroblast layer. The size of the individual cells is remarkably reduced, the nucleus is small and but faintly staining, and the protoplasm is but slightly granular. The sectional cross-area of the cell-mass constituting the floor of the vesicle is less than one-half that of the unaffected side. The dorsal median ridge in the roof of the brain occupies, however, its normal position. But very few bits of extruded nuclear or protoplasmic material are to be found in the neuro- cele. As the tube is traced cephalically or caudally, it is found to make a sharp, almost right-angled flexure away from the affected hemiseg- mental area, this flexure being so sharp when seen in the cross-sections as to appear as almost a longitudinal section. The purpose of these bends is naturally to bring the tube back into its normal median posi- tion in the unaffected segments of the body. Dependent upon the level of the segments involved, either the optic vesicle or the otic vesicle, or both, upon the same side may be involved in the injury. Where the former has been encountered by the X-rays the cells constituting the optic stalk and the vesicle demonstrate the same abnormal features of nuclear and of protoplasmic structure as those presented by the neuroblasts upon the affected side of the brain. Stratification of the lining cells is absent; the lens is ordinarily not formed. These same features may be detected as well when the otic vesicle is involved in the injury. Where, however, a greater amount PRODUCTION OF MONSTERS BY X-RAY 301 of X-ray energy has acted upon these two organs, they are then entirely absent. The optic vesicle may be reduced to a mere stump attached to the ventral surface of the brain. There is no indication given through the thickening of the ectoderm of the formation of a lens. As was noted in the brain cavity, there is but little indication of the pres- ence of extruded cells or of nuclei in these two vesicles. The shifting of the notochord from its normal position toward the affected side is brought about by the same abrupt angularity of flexure as was noted in the neural tube. By reason, however, of a greater degree of migration, its normal ventral relationship to the tube is somewhat altered. There is nothing, however, to be noted as ab- normal either in the appearance of its cells or in their arrangement. Since the injury to these embryos is restricted to the neck and cephalic segments, the only portion of the enteron demonstrating abnormalities is the pharynx. In those segments where the injury has been severe but half of the pharyngeal wall is present. No effort has been made by the organism apparently to remedy the defect through the completion of the defective half of the tube-wall. The lining cells upon the affected half remain small, spherular and isolated. The number of them is greatly reduced and an orderly stratified grouping is completely absent. The general arrangement of these cells corre- sponds to that found in a very early stage of development. Differen- tiation of these cells, as was true also of the neuroblasts and of the cells of the optic vesicle, has been practically completely suspended. Upon the unaffected side, however, the pharyngeal wall is distinctly clothed by well-differentiated cells corresponding to the period of devel- opment. The contrast between these two halves of the pharyngeal wall is one of the most striking features presented by the embryos. Similarly, the pronephros in the affected area is reduced greatly in the number of its tubules and in their size. The individual cells lining the walls of these tubules present features corresponding to those seen in the affected half of the pharynx, i.e., irregularity in arrangement together with spherulation and isolation of the cellular elements. Cephalic to and caudal to the affected area the muscle segments are normally symmetrical in point of differentiation and of growth. In the affected segments, however, the myoblasts may be sharply con- trasted from the standpoint of differentiation. The pigmentation of these cells, present normally only in early stages of development, is retained and, as well, the spherular, isolated, nonndifferentiated char- acters of the cells are distinctive. Upon the unaffected side the myo- 302 W. M. BALDWIN fibrillae may be readily distinguished. The myoblasts, which at this stage are elongated, have assumed their definitive arrangement with respect to the long axis of the embryo. These cells are sharply con- trasted developmentally with those of the affected side. The cross- sectional area of the affected half of any myomere approaches ordinarily but less than half that of the unaffected side. But few or no abnormal features may be discerned either in the heart or the pericardium. The great vessels passing through the rayed seg- ments, on the other hand, demonstrate certain abnormal features. Most prominent among these is the retarded, embryonic condition of the lining endothelial cells and the undifferentiated condition of the tunics of the vessel walls. In only the largest of these have they made their appearance. Moreover the smaller blood vessels are entirely absent. The cellular elements which should assist in the formation of them remain small, spherular and isolated. There are no coordinated attempts at organization. As a direct result of these defects the gill tufts upon the affected side, normally dependent for their size chiefly upon the presence of these blood vessels, owing to their almost complete absence are markedly dwarfed or completely absent. There is a great difference in size between the mesenchymal mass upon the affected half when compared with that upon the unaffected half of the same segments. That upon the affected half is much smaller owing to the defective size, shape and number of its cellular elements. The affected cells retain their earlier, embryological, spherular features. Isolation of the individual cells is most marked. The absence of mitotic figures in these cells is characteristic. Development of them has been strongly inhibited. There is no indication, however, of an edematous condition which might be anticipated in view of the absence of small blood vessels, thereby bringing about a separation of these cells as was found to be the case where the heart was markedly affected when the whole ovum was rayed. Microscopic study of these mesen- chjrmal cells shows a lack of features of specialization in their pro- toplasm. It is significant that evidences of reparative reaction to the injury sustained by the cells are entirely wanting. Assuming that the posi- tion of the neural tube and of the notochord indicates the median plane of the body, the cross-sectional area of the affected hemi-seg- ments is reduced to less than half that of the normal segments. The . several factors responsible for this condition are to be found, as was PRODUCTION OF MONSTERS BY X-RAY 303 noted above, in the undifferentiated condition and lack of growth of the myomeres, the pharynx, the blood vessels, the otic and optic ves- icles and in the connective tissue-forming cells themselves. These specimens demonstrate, therefore, the possibility of the pro- duction of localized injury by means of X-ray energy. This localiza- tion is sharply focused upon certain regions but involves all cells within the rayed area. This may be marked out most distinctly from the unaffected area. It is significant, in addition, that the injury received by the ovum at the two-cell stage should be transmitted through so many cell generations without any greater manifest effort at restitu- tion. Sharp localization of organ-forming substances at this early stage of development is brought out as well by the experiments. The nature of the change whether physical or chemical is of less importance from the embryological standpoint than is the demonstration of sym- metrical distribution of these substances at so early a developmental stage. A consideration of the nature of the change wrought, whether an upset in the phase of an emulsion globule- or a distinct oxidation or reduction of protein, lipin or carbohydrate is less striking than the phenomenon of the transmission and distribution to subsequent cell- generations of this same altered sub^stance. Here again this feature is overshadowed by the manifest inability either of the cells or of the organism as a whole to rectify the damage done. The absence of cell extrusions, of protoplasmic and of nuclear detritus, all argue for an invariably uniform quantity of energy applied to the embryos, and so regulated as to avoid these evidences of more severe injury as were noted in the previous paper. The absence of blood-cellular features of inflammatory reaction may be referred to two possible sources, due either to a destruction of the anlagen of these elements but for which no definite evidence exists, however, or because of the intra-cellular and non -destructive character of the alteration produced. THE RELATION OF THE EPINEPHRIN OUTPUT OF THE ADRENALS TO CHANGES IN THE RATE OF THE DENERVATED HEART G. N. STEWART and J. M. ROGOFF From the H. K. Gushing Laboratory of Experimental Medicine, Western Reserve University Received for publication March 26, 1920 INTRODUCTION It is well known that adrenalin causes acceleration of the heart after section of the vagi and excision of the stellate ganglia, v. Anrep (1) showed in the dog that stimulation of the peripheral end of the splanch- nic nerve caused acceleration, associated with the peculiar features of the splanchnic blood pressure curve which Elliott (2) explains as due to the augmented epinephrin output known to be elicited by stimula- tion of that nerve. Pearlman and Vincent (3), working with the heart only partially isolated from the central nervous system by section of the vagi, have confirmed Elliott's interpretation of the peculiar form of the blood pressure curve caused by splanchnic stimulation and observed manifest augmentation of the heart. We have ourselves shown (in the cat) that the epinephrin given off at the normal average rate under the conditions of our experiments causes acceleration when the adrenal vein blood collected in a cava pocket for a minute or two is released. The acceleration begins a little time after release of the pocket, the length of the interval depending upon the state of the circulation, and is coincident or nearly so with the dilatation of the pupil if the superior cervical ganglion has been previously excised. The beginning of the dilatation has been seen to coincide approxi- mately with the "dip" associated by Elliott with the action of the liberated epinephrin (4). Also when the splanchnic is stimulated with the adrenal veins open it can sometimes be seen that about the same time as the reactions of the eye (sensitized by removal of the superior cervical ganglion) appear, the denervated heart begins to become accelerated. From all this it seems clear that epinephrin 304 EPINEPHRIN OUTPUT AND RATE OF DENERVATED HEART 305 liberated from the adrenal in response to stimulation of the splanchnic plays a part in the acceleration of the heart. We shall show later on that the whole acceleration is not due to the epinephrin, but that other factors are involved, since a good acceleration, even as great as that obtained with intact adrenals, can be observed when they have been removed or when discharge of epinephrin has been prevented in other ways. It will be pointed out in discussing these results that, with such a reaction as acceleration of the heart, there is no inconsistency in attributing a share in the reaction to epinephrin and yet asserting that sometimes as great a maximum acceleration may be attained in its absence as when it is being given off. It is possible that the position of the maximum acceleration or of the beginning of the acceleration on the blood pressure curve may be shifted when epinephrin action is excluded. But however this may be, it will, we believe, be evident when our results have been displayed that it would be a very unprom- ising venture to attempt to found a method of estimating the rate of output of epinephrin upon such a reaction, even under conditions, as in splanchnic stimulation, in which it is known that an increase in the epinephrin output can contribute to it. Where adrenal blood is col- lected in a cava pocket and then released there is no question that the acceleration is produced by the epinephrin and practically by that alone, because the experiment has been so simplified that only one factor is acting, namely the admission into the circulation of the epi- nephrin-containing blood. But even here the reaction is of such a char- acter that it could hardly lend itself to anything like an exact assay of the epinephrin in the blood from the pocket. Nevertheless in a recent paper Cannon (5) has described experi- ments on cats in which, from the acceleration of the so-called denervated heart caused by stimulation of the central end of the sciatic nerve, by asphyxia and by emotional excitement, he professes to prove that these conditions produce a marked increase in the rate of output of epi- nephrin from the adrenals. Apparently admitting that the catheter method (6) is a difficult one to obtain positive results with, he intro- duces this as a relatively simple method which can be carried out by ''any competent experimenter," and he states that the results of these experiments confirm "in every particular" his previous conclusions as to the influence of emotional excitement, asphyxia and sensory stimula- tion upon the adrenal secretion. We have never quarreled with the catheter method because of its difficulty, but because it cannot yield the data necessary to determine the rate of output of epinephrin or to 306 G. N. STEWART AND J. M. ROGOFF measure the changes in that rate. We shall take another occasion to point out again the reasons why we cannot accept Cannon's con- clusions based on experiments with the catheter method. The tech- nique of obtaining the cava blood is surely not beyond the reach of "any competent experimenter" in physiolog}^, and the assaying of the epinephrin in the blood would be easy if such concentrations existed there as, by implication, we must conclude that Cannon assumed to exist during or after the action of the factors studied by him. The denervated heart reaction now adopted by Cannon "as an indi- cator of adrenal secretion" is also easy enough to carry out. But it labors under even more serious defects, when employed as a quantita- tive method of measuring the epinephrin output and of estimating changes in the rate of output, than does the catheter method. For in the latter an attempt was at any rate made to obtain blood containing a portion of the epinephrin given off by the adrenals and to test it by a method of bio-assay which, if properly applied, does permit the epinephrin concentration in the sample of blood to be estimated. When Doctor Cannon stimulates the central end of the sciatic numer- ous reflex effects may be caused, the consequences of which upon the rate of the heart cannot be easily controlled. Among these the vaso- motor reflex changes are conspicuous, and it is obvious that in this way great variations may be produced in the pressure in the cavities of the heart, the rate of blood flow through the coronary system and, therefore, the amount of epinephrin passing through the coronaries, any of which may lead to an acceleration of the heart beat without any change having occurred in the rate of output of epinephrin. Cannon, however, states that after removal of the adrenals or their ligation en masse or after removal of one adrenal and section of the splanchnic of the other side, acceleration of the heart is no longer caused by sciatic stimulation. In one experiment he obtained some acceleration after division of both splanchnics in the thorax, but much less than before. He concludes that the acceleration following sciatic stimulation is due entirely to a reflexly increased output of epinephrin. And obviously assuming that no other factors are involved, he states that "comparisons of the increased rate due to sciatic stimulation with effects of adrenalin (quantitated as base) injected intravenously indi- cate that the range of reflex adrenal secretion lies between 0.001 and 0.005 mgm. per kgm. per minute, i.e., from 5 to 25 times the amount regarded by Stewart and Rogoff as the normal output." In view of our own results proving conclusively that epinephrin, if a factor, cannot EPINEPHRIN OUTPUT AND RATE OF DENERVATED HEART 307 be the sole factor in the heart reaction eHcited by stimulation of the sciatic, it would scarcely be worth while to spend much time in exam- ining such data. But it may be pointed out that Cannon does not state by what control experiments he has established a quantitative relation between the maximum acceleration reached and the dose of adrenalin, and not, for instance, between the dose of adrenalin and the duration of the acceleration or the total surplus number of beats in the period of acceleration. Also if such data are to have real quanti- tative value the adrenalin ought to be administered while the blood pressure and therefore the coronary blood flow are increased to approx- imately the same extent as during stimulation of the sciatic. As regards Cannon's statement that the reaction is abolished by removal of the adrenals, it must be noted that, if it were granted that the only change caused by removal of the adrenals or section of the splanchnics is the suppression of the epinephrin output, his result would simply show that the acceleration previously obtained had been due essentially to epinephrin. It would not show that any increase had occurred in the rate of output, unless it were demonstrated that a redistribution of the blood, due to the vascular reactions evoked by the stimulation and necessarily associated with the passage through the coronary circulation of an increased proportion of the epinephrin already being given off, was insufficient to account for the reaction. But it cannot be granted that the operations practised to eliminate the epinephrin output have no other consequences. It is astonishing with what indifference both splanchnics are cut merely in order to interfere with the epinephrin output, as if all or most of the splanchnic fibers innervated the adrenals. The same is true of the removal of the adrenals. Some writers seem to assume that it is practically impos- sible to injure any important nerves when the adrenals are removed or tied off and that all the consequences which follow their removal are necessarily due to the loss of epinephrin. Gley and Quinquaud (7) have pointed out that the opposite is the case. In the dog according to them it is practically impossible to remove the adrenals without severely injuring the splanchnics. They say, however, that with their large experience they have succeeded in operating on the dog also, so as to eliminate the adrenals without injury to the splanchnics. Pearlman and Vincent (3) take exception to Gley's statement that the difference in the splanchnic blood pressure curve observed by v. Anrep (1) in dogs after and before removal of the adrenals is due to injury to nerve fibers, and no doubt in Vincent's hands the operation is as little harmful as it 308 G. N. STEWART AND J. M. ROGOFF is possible for it to be. If the operation can be more easity done in the cat, it still needs care and experience to reduce this cause of error to a minimum, particularly in the case of the right adrenal, the liga- tion of which is liable to injure the splanchnic. For this and other reasons the removal of the adrenals, as frequently done in acute experiments, may be attended with a marked drop in the blood pres- sure not related to loss of function of the glands. It must be remem- bered that in experiments on the denervated heart, the adrenals are removed after a considerable preliminary operation and the extrinsic regulatory nerves of the heart are eliminated. This may make it more difficult to excise or tie off the adrenals without a fall of blood pressure than in experiments on normal animals, such as those of Young and Lehman (8), of Hoskins and McClure (9) and of Bazett (10). The diminution in the heart rate remarked by Cannon, and apparently attributed by hhn entirely to the loss of epinephrin, is according to our observations not unrelated to the fall of pressure, although since there is reason to believe that the epinephrin liberated at the ordinary rate may affect the heart, some part of the slowing may be due to loss of epinephrin. Cannon has not given any data by which one can judge how great the change of blood pressure was in his experiments, but as he injected gum salt solution in one of them the fall may be assumed to have been sometimes considerable. Now, whatever inter- pretation one chooses to put upon the heart acceleration produced by sciatic stimulation, a reflex or more than one reflex action must be essentially involved in it. Any operation which impairs the conduc- tivity of the reflex arcs must, therefore, tend to diminish or aboHsh the effect. And if a negative result obtained after removal of a certain organ is attributed solely to the specific effect which the operator intended to produce, and not at all to the general effects which he did produce, then, of course, positive results elicited before the operation in any way whatever will seem to depend entirety upon the specific activity of the organ removed. In what has been said above we desire to state distinctly that we do not imply that Doctor Cannon did not remove the adrenals sldlfuUy, and with full knowledge of the importance for his control experiment of injuring the nerves in the vicinity as little as possible and of main- taining the animal in a good general condition. All we know is that we obtained positive results after elimination of the adrenals where he obtained completely negative results. It is clearly the positive results which are significant for the decision of the question of the relation of EPIXEPHRIN OUTPUT AND RATE OF DEXERVATED HEART 309 the epinephrin output to the heart acceleration caused by sciatic stimulation and not the negative ones. Our experiments were all made on cats. The greater number of them were performed two years ago. Only a brief notice of a portion of them has been published (11). They were mentioned, so far as could be done in the few minutes allotted to us, in the discussion of Doctor Cannon's paper at the meeting of the American Physiological Society last December. We studied the effect upon the acceleration produced by stimulation of sensory nerves of eliminating the epineph- rin secretion: a. By clipping the adrenal veins, either with or without simultaneous ligation of the renal vessels. b. By removing one adrenal (the right) and denervating the other and allowing the animal to recover completely from the operation. In a number of these animals the denervated adrenal was also removed in the final experiment, and the heart reactions obtained before and after its removal compared. c. By removing one adrenal (almost always the right) and allowing an interval for the animal to recover, before performing the experiment upon sensory nerve stimulation with removal of the other adrenal. In this way it was supposed that the condition of the animal after removal of the remaining adrenal would be better than if both were removed at the time of the experiment. d. By removing both adrenals during the experiment on the heart reaction. In all cases in excising the glands every precaution was taken to avoid injur\' to nerves, by making a careful dissection between the capsule of the gland and the cortex, tying the vessels with fine ligatures, EXPERIMENTS IX W^HICH THE ADREXAL VEIXS WERE CLIPPED OFF OR TIED In principle this is the most satisfactory way of eliminating the epine- phrin output, since no other organ than the adrenals is interfered with and there is no damage to important nerves and no injurious fall of blood pressure. The procedure has been extensivel}' emplo^^ed by Gley. V. Anrep (1) also used it in some of his experiments on splanchnic stimulation and satisfied himself that the effects attributed by him to epinephrin were not obtained when the suprarenal vein was clipped and the corresponding splanchnic stimulated. And Pearlman and Vincent (3) state that they have usually obtained quite satisfactory^ THE AMERICAN JOLRXAL OF PHYSIOLOGY, VOI>. 52, NO. 2 310 G. N. STEWART AND J. M. ROGOFF results by simply clamping and unclamping the adrenal veins. Can- non takes exception to clipping because it does not eliminate the heart reaction to sciatic stimulation or asphyxia which he interprets as indi- cating increased epinephrin output, and he, therefore, assumes that there must be leakage of epinephrin by anastomotic venous channels. He quotes, for instance, an experiment in which before ligation of the adrenal veins asphyxia of a certain duration caused, an acceleration in the heart rate of 40 beats. After ligation of the veins the accelera- tion was precisely the same. We should have thought the only pos- sible interpretation of such a result would have been that epinephrin had nothing to do with the reaction, in the particular experiment, at any rate. For who will believe that after the adrenal veins were tied just as much epinephrin passed out of the adrenals by some difficult collateral path, in the minute for which asphyxia was maintained, as would have passed out by the adrenal veins plus these hypothetical anastomotic channels? And how can the heart acceleration be a quantitative reaction for epinephrin if blocking the adrenal veins does not at least diminish the reaction? When a reaction known to be caused by epinephrin is studied the result is quite different. For example, we sometimes observe a small dilatation of the pupil (after previous removal of the superior cervical ganglion) on stimulation of the peripheral end of a splanchnic nerve, with the corresponding or even with both adrenal veins clipped. But this is much smaller than the reaction obtained before or after by similar stimulation with the veins open, and usually on release of the clips there is an additional and greater reaction indicating that epinephrin had been pent up in the adrenal veins by the clips. We suggest that the reason why Can- non gets such positive results with the adrenal veins tied, and must have recourse to removal or ligation of the adrenals to obtain negative results, is that in tying the veins he does not inflict such injury as causes a general deterioration of the animal incompatible with a posi- tive heart reaction, whereas in removing the adrenals he appears to -do so. In our experiments with clipping of the adrenal veins we compared the heart acceleration and rise of blood pressure caused by stimulation of the sciatic when the blood flow from the glands and, therefore, the epinephrin output were going on unhindered, with the acceleration and blood pressure rise obtained when the flow from one or both adrenals was obstructed. It was supposed that if the epinephrin is an impor- tant factor in the heart reaction, the reaction would be distinctly EPINEPHRIN OUTPUT AND RATE OF DENERVATED HEART 311 smaller with the adrenal veins clipped off, that is to say, provided that the reaction can be used at all as a quantitative test for epi- nephrin. The protocols show that this expectation was not realized. If the epinephrin liberated from the adrenals is an appreciable factor, it is not easy to disentangle its influence from that of the other factors which can affect the heart rate. It must be remembered that even if it were clearly demonstrated that the acceleration caused by sciatic stimulation was diminished by interference with the output of epi- nephrin, the other potential factors, such as rise of blood pressure, not being interfered with, this would only prove that epinephrin takes a share in the reaction, not that its rate of output is increased by the stimulation. The peripheral end of a splanchnic nerve was also stimulated with the corresponding, or both adrenal veins clipped or open, in order to compare the effect of a procedure which is known to increase the epi- nephrin output on the heart rate with the effect of sciatic stimulation. It will be seen that it may be difficult to demonstrate, by comparing the maximum acceleration caused with the adrenal veins clipped and open, that the epinephrin undoubtedly liberated by stimulation of the splanchnic takes any sensible share in the heart reaction. However, this is rather an illustration of the deficiencies of such a reaction as a quantitative test for epinephrin than a proof that the epinephrin liberated with the adrenal veins open is without effect upon the rate of the heart. As already mentioned, w^hen the epinephrin is allowed to accumulate in a cava pocket or even when the epinephrin pent up in the adrenal vessels by clipping of the adrenal veins is released a dis- tinct acceleration is produced, and it may be assumed that the epineph- rin coming steadily off from the adrenals, without being accumulated, will tend to exert a similar action, especially when the amount of epinephrin passing per unit of time through the coronaries, or its con- centration, is abruptly increased by the vasomotor changes associated with splanchnic or sciatic stimulation. It seems, however, improbable that with such a reaction as acceleration of the heart the acceleration produced by simultaneous action of two factors, singly effective, should be the sum of the separate effects, whether the reaction is measured by the maximum acceleration attained, or by the duration of the acceleration or by the total surplus number of beats. It seems more likely that when the heart is keyed up to a certain point by the action of one factor, whether this acts upon a local accelerator mechanism or not, it will break loose, so to speak, from the relative stability of rate 312 G. N. STEWART AND J. M. ROGOFF imposed upon it by removal of its extrinsic nerves, and execute a run of quicker beats, the duration and maximum acceleration of which maj^ have no simple relation to the absolute magnitude of the exciting influence, and which may not be greatly modified by a concomitantly acting influence, itself capable of independently producing a similar reaction. The results of these experiments are illustrated by some protocols. Where it is mentioned that a nerve was stimulated with a vein clipped, the clip was applied, unless otherwise stated, a few seconds before the stimulation and was removed as soon as the portion of the curve to be used for counting the heart rate had been completed. When it is simply mentioned that a nerve was stimulated, without any reference to clipping, it is implied that the veins were open. Under "rate" is always given the number of heart beats per minute; under "pressure" the blood pressure in millimeters of mercury. Protocol. Cat 175; male; weight, 2.2 kgm. 11:30 a.m. Urethane, 5 gm. by stomach tube. 1:10-1:48 p.m. Cut vago-sympathetics and excised stellate ganglia;^ pre- pared central end of left sciatic for stimulation. Rate 1 :50 p.m. Before stimulation of sciatic 188 25 seconds after beginning stimulation 210 1:55 p.m. Before stimulation of ^ciatic 187 20 seconds after beginning stimulation 202 40 seconds after beginning stimulation 212 Now opened abdomen and purposely manipulated intestines 2:03 p.m. Before stimulation of sciatic 178 30 seconds after beginning stimulation 212 2: 10 p.m. Before stimulation of sciatic 186 30 seconds after beginning stimulation 214 2:14 p.m. Before stimulation of sciatic; left adrenal vein clipped 184 30 seconds after beginning stimulation 206 2:16 p.m. Prepared peripheral end of left splanchnic in abdomen 2:20 p.m. Before stimulation of splanchnic; left adrenal vein clipped.. . . 184 15 seconds after beginning stimulation 192 30 seconds after beginning stimulation 208 45 seconds after beginning stimulation 224 * With the cat in the supine position an incision is made in the axilla and a part of the second rib exposed by separation of overlying muscle. A portion of the rib anterior to the angle is carefully separated from its periosteum and resected. The lower part of the stellate ganglion is usually thus exposed and the excision of the ganglion easily completed without injury to the pleura. In all the experiments, except when otherwise stated, the period of stimula- tion of the sciatic was 30 to 40 seconds. When the heart rate is given after a certain number of seconds from beginning of stimulation this means that a suffi- cient portion of the curve beginning at that point was counted. The corre- sponding blood pressure was that at the end of the portion counted. EPINEPHRIN OUTPUT AND RATE OF DENERVATED HEART 313 2:26 p.m. Before stimulation of splanchnic; left adrenal vein clipped 180 15 seconds after beginning stimulation 196 30 seconds after beginning stimulation 208 45 seconds after beginning stimulation 220 . 60 seconds after beginning stimulation 220 2:32 p.m. Before stimulation of splanchnic 182 During first 15 seconds of stimulation 180 During second 15 seconds of stimulation 200 During third 15 seconds of stimulation 204 During fourth 15 seconds of stimulation 216 2:47 p.m. Before stimulation of sciatic; both adrenal veins clipped 184 During first 15 seconds of stimulation 188 During second 15 seconds of stimulation 188 During third 15 seconds of stimulation 200 During fourth 15 seconds of stimulation 204 2:53 p.m. Before stimulation of sciatic 176 During first 15 seconds of stimulation 180 During second 15 seconds of stimulation 196 During third 15 seconds of stimulation 208 During fourth 15 seconds of stimulation 216 3:05 p.m. Before stimulation of sciatic; both adrenal veins clipped 188 During first 15 seconds of stimulation 188 During second 15 seconds of stimulation 196 During third 15 seconds of stimulation 204 During fourth 15 seconds of stimulation 204 Rate Pressure 3:45 p.m. Before stimulation of sciatic 176 82 24 seconds after beginning stimulation 203 140 3:50 p.m. Before stimulation of splanchnic; left adrenal vein clipped 174 58 28 seconds after beginning stimulation 215 166 3:57 p.m. Before stimulation of splanchnic; both adrenal veins clipped 185 62 24 seconds after beginning stimulation 206 131 40 seconds after beginning stimulation 221 4:03 p.m. Before stimulation of sciatic 174 46 12 seconds after beginning stimulation 197 118 40 seconds after beginning stimulation 200 4:10 p.m. Before stimulation of sciatic; both adrenal veins clipped 189 52 15 seconds after beginning stimulation 200 112 30 seconds after beginning stimulation 204 4:15 p.m. Before stimulation of splanchnic 184 55 20 seconds after beginning stimulation 209 130 Excised left adrenal 4:30 p.m. Before stimulation of splanchnic 215 70 22 seconds after beginning stimulation 229 90 314 G. N. STEWART AND J. M. ROGOFF Some of the curves from these experiments are used in the next paper to demonstrate that the acceleration of the heart on stimulation of the central end of the sciatic, contrary to Cannon's statement, is easily obtainable after the abdomen has been opened. The protocols themselves and other protocols coming later in the present paper also afford abundant evidence that the statement is baseless. In figure 1 portions of the blood pressure curve from cat 175 before and during stimulation of the left splanchnic {A and B, taken at 3:50 p.m.) are Fig. 1. Parts of blood pressure tracings from cat 175. A, before and B, a portion commencing 28 seconds after beginning of splanchnic stimulation with corresponding adrenal vein clipped; C, before and D, 22 seconds after beginning of splanchnic stimulation with corresponding adrenal excised. In all figures line of zero pressure corresponds with time trace; time in seconds; numbers above time trace represent heart rate per minute. Reduced to four-fifths. reproduced, and corresponding portions after removal of the adrenal (C and D, taken at 4:30 p.m.). The maximum acceleration in B was 41 beats per minute; the maximum in D, 12 to 14 beats as compared with a maximum acceleration of 25 beats in the curve taken with the last splanchnic stimulation prior to removal of the adrenal. It is impossible to know how much, if any part, of the difference is due to the lack of epinephrin from the left adrenal when the curve C D was being written, since the stimulation was much less effective in rais- ing the blood pressure, as shown in figure 2. The much reduced curves (black on white) are not intended for counting. EPINEPHRIN OUTPUT AND RATE OF DENERVATED HEART 315 A ^* tif^f^ 3 „ ii n. .iii / i i?4i i l i ■ I I > ^ tii I I ■■'■'■ 2/S c ZZ9 _ ■ '■■' ^ ' " ■ " Ill MIL ^'.^. Fig. 2. Blood pressure curves from cat 175. A, sciatic stimulation; B, splanchnic stimulation with corresponding adrenal vein clipped; C, splanchnic stimulation with corresponding adrenal excised, 40 minutes after B. Reduced to one-half. '^ VvvA^^-WW'/t •'•'Vw, ■'^'^'Kvv'V^^v>/V^vv^,^/w,vw\.,^,vv\aaA^\ v-^^'^Hvwv'^. %;^^V«'^W^^^A/^''^^'^'^^^'%Ay^'^H Fig. 3. Parts of blood pressure tracings from cat 237. A, before and B, a portion commencing 33 seconds after beginning of sciatic stimulation; C, before and D, 40 seconds after beginning of sciatic stimulation with both adrenal veins clipped. Reduced to three-fourths. 316 G. N. STEWART AND J. M. ROGOFF In figure 3 portions of the curve from cat 237 are given, A and B taken respectively before and during stimulation of the sciatic with the adrenal veins open and C and D with the adrenal veins clipped. The curves were practically parallel and the acceleration of the heart about the same in both (about 18 beats per minute), in the portions shown in the figure. The maximum acceleration with stimulation after clipping was 21, and without clipping 20 beats per minute. It may be remarked that not only had the abdomen been opened but the renal arteries and veins had been tied 40 minutes before these curves were obtained. Protocol. Cat 177; male; weight, 3.6 kgm. 2:05 p.m. Under urethane (6 gm.) cut vago-sympathetics; excised stellate ganglia and prepared central end of left sciatic for stimulation. Rate Pressure 2:25 p.m. Before sciatic stimulation 185 132 20 seconds after beginning stimulation 228 154 2:30 p.m. Opened abdomen; prepared peripheral end of left splanchnic 2:32 p.m. Before sciatic stimulation 173 105 24 seconds after beginning stimulation 200 126 49 seconds after beginning stimulation 216 2:35 p.m. Before stimulation of left splanchnic; left adrenal vein clipped 167 108 12 seconds after beginning stimulation 191 24 seconds after beginning stimulation 215 140 45 seconds after beginning stimulation 211 110 2:40 p.m. Before stimulation of sciatic; both adrenal veins clipped 181 80 23 seconds after beginning stimulation 190 37 seconds after beginning stimulation 197 106 2:45 p.m. Before stimulation of left splanchnic; left adrenal vein clipped 171 90 17 seconds after beginning stimulation 194 22 seconds after beginning stimulation 206 124 2:50 p.m. Before stimulation of left splanchnic; both adrenal veins clipped 191 100 20 seconds after beginning stimulation 213 128 2:55 p.m. Beforestimulationof sciatic; left adrenal vein clipped. . 184 94 28 seconds after beginning of stimulation 192 107 3:00 p.m. Before stimulation of sciatic; both adrenal veins clipped 167 78 23 seconds after beginning stimulation 173 98 3:10 p.m. Before stimulation of left splanchnic; left adrenal vein clipped 160 74 18 seconds after beginning stimulation 194 92 EPINEPHRIN OUTPUT AND RATE OF DEXERVATED HEART 317 3:15 p.m. Before stimulation of left splanchnic; both adrenal veins clipped 160 82 23 seconds after beginning stimulation 167 42 seconds after beginning stimulation 185 55 seconds after beginning stimulation 200 106 3:20 p.m. Before stimulation of left splanchnic; left adrenal vein clipped 165 72 20 seconds after beginning stimulation 183 82 3:30 p.m. Before stimulation of left splanchnic; both adrenal veins clipped 166 68 25 seconds after beginning stimulation 186 86 3:35 p.m. Before stimulation of left splanchnic; left adrenal vein clipped 173 76 20 seconds after beginning stimulation ISO 90 4:10 p.m. Before stimulation of peripheral end of right splanch- nic in thorax, with left adrenal vein clipped 170 30 15 seconds after beginning stimulation 205 78 In cat 177, in which a good acceleration of the heart was obtained with sciatic stimulation after opening the abdomen and section of one splanchnic nerve, the reaction from the sciatic after repeated splanch- nic and sciatic stimulations and repeated clipping of the adrenal veins was distinctly diminished toward the end of the experiment. Stimula- tion of the peripheral end of the splanchnic continued to give a good acceleration of the heart when the reaction, as reflexly elicited by stim- ulation of the central end of the sciatic, together with the reflex rise of blood pressure, was being exhausted. In comparing the acceleration accompanying a given rise of blood pressure caused by sciatic, with that accompanying a similar rise of pressure caused by splanchnic stimulation it generally appeared that the splanchnic acceleration was the greater. This, of course, would be consistent with the view that the epinephrin secretion in response to direct splanchnic stimulation may play a substantial part in the reac- tion. It would appear probable that any share of the epinephrin in the acceleration both when the output is increased, as by splanchnic stimulation, and when, without an actual increase in the output, more epinephrin is sent through the coronary circulation in response to a rise of blood pressure caused by sensory nerve stimulation, must vary with the state of the heart and may, therefore, be expected to vary in different animals and in the same animal at different periods of an experiment. 318 G. N. STEWART AND J. M. ROGOFF Protocol. Cat 179; male; weight, 2.2 kgm. Under urethane (5 gm.) cut vago- sympathetics; excised stellate ganglia; prepared central end of left sciatic for stimulation. Rate Pressure 4:00 p.m. Before sciatic stimulation 169 80 25 seconds after beginning stimulation 210 130 4:05 p.m. Prepared peripheral end of left splanchnic (extraperi- toneally) 4:10 p.m. Before stimulation of left splanchnic; left adrenal vein clipped 168 70 20 seconds after beginning stimulation 182 94 4:13 p.m. Before sciatic stimulation 173 81 32 seconds after beginning stimulation 204 113 4:17 p.m. Before stimulation of left splanchnic; left adrenal vein clipped 177 73 20 seconds after beginning stimulation 180 98 4:20 p.m. Before stimulation of left splanchnic 170 80 24 seconds after beginning stimulation 183 109 4:25 p.m. Beforestimulationof sciatic; left adrenal vein clipped. . 170 76 23 seconds after beginning stimulation 180 106 4:28 p.m. Before stimulation of left splanchnic; left adrenal vein clipped :... 170 71 23 seconds after beginning stimulation 180 100 4:30 p.m. Opened abdomen; manipulated intestines 4:32 p.m. Before sciatic stimulation 171 66 25 seconds after beginning stimulation 196 102 4:35 p.m. Before stimulation of sciatic; both adrenal veins clipped 173 64 20 seconds after beginning stimulation 192 95 4:40 p.m. Before stimulation of left splanchnic with both ad- renal veins clipped 173 70 21 seconds after beginning stimulation 179 103 4:45 p.m. Before stimulation of splanchnic 171 62 25 seconds after beginning stimulation 179 90 4:48 p.m. Cut right splanchnic 4:50 p.m. Before sciatic stimulation 160 44 23 seconds after beginning stimulation 161 56 The protocol of cat 179 shows again quite clearly good heart reac- tions with sciatic stimulation after opening the abdomen and section of one splanchnic. The practical disappearance of the reaction after section of the remaining splanchnic, when the blood pressure fell to 44 mm. of mercury is well shown. But is it likely that this disappear- ance is due solely to the suppression of the epinephrin secretion when a little earlier in the experiment a decided reaction was obtained by stimulation of the sciatic with both adrenal veins clipped and after section of the left splanchnic? Here the blood pressure reaction was a EPINEPHRIN OUTPUT AND RATE OF DENERVATED HEART 319 good one, the pressure rising from 64 to 95 mm. of mercury. But after the section of the second splanchnic only a trifling rise of pressure was caused by sciatic stimulation. The absence of any substantial rise of pressure and the depressing influence of the lowered blood pressure on the paths for any other reflexes than the vascular reflexes which may be concerned in the reaction offer a more probable explanation. In a number of cats one superior cervical ganglion (the left) had been previously excised, so that it was possible to compare the acceleration of the heart caused by stimulation of the sciatic or splanchnic with the eye reactions. The following is a typical protocol. Protocol. Cat 190; female; weight, 2.31 kgm. Left superior cervical ganglion excised 21 days previously. Under urethane (5 gm.) cut vago-sympathetics; excised stellate ganglia; prepared central end of left sciatic for stimulation. Rate Pressure 1 :05 p.m. Before stimulation of sciatic^ 253 123 35 seconds after beginning stimulation 259 164 1:10 p.m. Before sciatic stimulation^. 229 15 seconds after beginning stimulation 237 1:15 p.m. Prepared peripheral end of left splanchnic (extraperi- toneally 1:35 p.m. Before stimulation of left splanchnic 231 80 10 seconds after beginning stimulation 268 155 Very good pupil and nictitating reactions in 9 seconds 1:40 p.m. Before stimulation of left splanchnic with left adrenal vein clipped 223 64 20 seconds after beginning stimulation 280 150 Small pupil and nictitating reactions in 18.4 seconds 1:50 p.m. Before stimulation of left splanchnic with left adrenal vein clipped 227 70 20 seconds after beginning stimulation 263 48 seconds after beginning stimulation 278 130 Very small eye reactions in 24.8 seconds 2:15 p.m. Excised left adrenal 2:20 p.m. Before stimulation of left splanchnic 219 72 25 seconds after beginning stimulation 227 100 Doubtful, if any eye reactions '^ Both pupils dilate instantaneously, the left becoming much wider than the right, both returning to previous state very soon after stimulation of the sciatic is stopped. With a prolonged or strong stimulation the left nictitating may slowly retract. This reaction is distinctly different from that obtained with splanchnic stimulation (when the adrenal veins are free) which affects only the dener- Vated eye and occurs after a distinct latent period. 320 G. N. STEWART AND J. M. ROGOFF 2 :30 p.m. Before stimulation of sciatic 223 80 22 seconds after beginning stimulation 262 168 2:35 p.m. Opened abdomen; prepared peripheral end of right splanchnic 2:40 p.m. Before stimulation of right splanchnic 210 45 20 seconds after beginning stimulation 285 105 2:55 p.m. Before clipping aorta just above diaphragm 249 48 After clipping aorta just above diaphragm 260 ' 146 It is to be remarked that when the left splanchnic was stimulated with the left adrenal vein clipped, the eye reactions were not com- pletely abolished but were much diminished and appeared after a much longer delay than when the splanchnic was stimulated with the vein open. Complete loss of the eye reactions on stimulating the peripheral end of a splanchnic nerve maj^ be seen when the corresponding adrenal vein has been clipped off, although good reactions were being obtained with the vein open, or the result may be what was found in cat 190, This result is compatible with the view that some epinephrin may find its way into the blood stream by a collateral route, as suggested by Cow (12) (the renal vessels were not tied in this experiment*) but may also be due solely to a change in the concentration or quantity of epi- nephrin from the other adrenal passing through the coronary circulation, associated with the vasomotor effects of the splanchnic stimulation. The observation that in this experiment after excision of the left adrenal stimulation of the left splanchnic produced only a doubtful, if any, pupil reaction does not enable us to decide against the latter view. For the vasomotor effect (rise of blood pressure) and the heart accelera- tion were also much reduced, possibly owing to some injury to the splanchnic in removal of the adrenal. The fact that the eye reactions, while still present, were greatly diminished by the clipping of the adrenal vein and that it took a much greater rime for them to appear while the acceleration of the heart was not af all diminished, shows clearly that the latter reaction cannot be a quantitative test for epi- nephrin. With sciatic stimulation the pupil reaction is complicated bj'' the fact that both pupils dilate immediately after stimulation, of course through the nervous system. Although the sensitized pupil widens more than the other, this reaction is different from the typical epinephrin reaction yielded, for instance, by splanchnic stimulation. The return of the pupils to their previous size on stoppage of the stim- *In making a cava pocket we generally tie the renal vein at the hilus and also at its entrance into the cava. EPINEPHRIN OUTPUT AND RATE OF DENERVATED HEART 321 ulation begins at once and is accomplished more quickly after sciatic stimulation than when a true epinephrin reaction has been induced by splanchnic stimulation. Further, the pupil reaction elicited by stimu- lation of the sciatic after interference with the epinephrin output by removal of one adrenal and section of the nerves of the other, or after removal of both adrenals, has the same characters as that seen with intact adrenals. A nictitating reaction was not obtained with sciatic stimulations which caused marked acceleration of the heart. Only with quite strong stimulation of the sciatic was there any retraction of the nictitating. This also is different from the genuine epinephrin eye reactions. The observations on the eye reactions, then, are inconsist- ent with the idea that stimulation of the sciatic causes a marked increase in the output of epinephrin, which in its turn causes the observed acceleration of the heart. It may further be asked how an acceleration (see protocol of cat 190) caused by sciatic stimulation, which is much smaller with both splanchnics intact at the beginning of the experiment than later on when one splanchnic has been divided and the corresponding adrenal removed, can be considered a quanti- tative reaction for the rate of output of epinephrin. It will be seen in another section of the paper that it is common, or indeed the rule, to obtain as large a reaction on stimulation of the sciatic in animals from which one adrenal has been removed and the animal allowed to recover as in animals with both adrenals intact. Another cat (191) from which one superior cervical ganglion had been previously removed yielded results so similar to those of cat 190 that the protocol need not be reproduced. The only difference was that sciatic stimulation, as usual, gave a good acceleration at the beginning of the experiment, from 240 to a maximum of 295 beats per minute, the blood pressure rising from 124 to 182 mm. of mercury. With a second stimulation of the sciatic the heart rate increased from 256 to 275, the blood pressure rising from 112 to 150 mm. The eye reactions were the same as in cat 190. The abdomen was now opened and the peripheral end of the left splanchnic stimulated. The pulse rate rose from 243 to 270 beats, the blood pressure from 90 to 144 mm. of mercury. Ex- cellent pupil and nictitating reactions were obtained in 9 seconds. The left adrenal vein was then clipped and the left splanchnic again stimu- lated. The heart rate increased from 237 to 262 beats per minute and the blood pressure from 110 to 150 mm. of mercury. Small pupil and nictitating reactions were observed in 20.4 seconds. 322 G. N. STEWART AND J. M. ROGOFF Protocol. Cat 195; female; weight, 2.4 kgm. Left superior cervical ganglion excised 14 days previously. Under urethane (5.5 gm.) prepared peripheral end of left splanchnic for stimulation in abdomen, tied lumbar veins just before they cross the adrenals; prepared central end of left sciatic for stimulation. Rate Pressure 2:55 p.m. Before stimulation of left splanchnic 202 78 12 seconds after beginning stimulation , 216 96 Very good pupil and nictitating reactions in 12.8 seconds 3:00 p.m. Before stimulation of left splanchnic; left adrenal veiii clipped 186 80 19 seconds after beginning stimulation 211 108 No eye reactions 3:05 p.m. Before stimulation of left splanchnic; left adrenal vein clipped 178 71 14 seconds after beginning stimulation 181 78 3:20 p.m. Cut vago-sympathetics and excised stellate ganglia 3:30 p.m. Before stimulation of sciatic^ 145 52 20 seconds after beginning stimulation 169 94 3:35 p.m. Before weaker stimulation of sciatic 152 63 54 seconds after beginning stimulation 151 63 3:40 p.m. Prepared peripheral end of right splanchnic in thorax 3:42 p.m. Before stimulation of right splanchnic 146 35 40 seconds after beginning stimulation 192 76 Good eye reactions in about 40 seconds 3:45 p.m. Before stimulation of right splanchnic; right adrenal vein clipped 157 44 20 seconds after beginning stimulation 184 30 seconds after beginning stimulation 207 82 No eye reactions 3:50 p.m. Before stimulation of right splanchnic; both adrenal veins clipped 150 44 40 seconds after beginning stimulation 199 84 No eye reactions 3:55 p.m. Before stimulation of right splanchnic 146 44 30 seconds after beginning stimulation 200 68 Good eye reactions occurred in 30-35 seconds In the above experiment (cat 195) the abdomen was opened, the lumbar vein tied just before it crosses the left adrenal and the left splanchnic stimulated several times before the vago-sympathetics were cut and the stellate ganglia excised. The central end of the sciatic was then stimulated and caused an acceleration of 24 beats and a rise of blood pressure from 52 to 94 mm. of mercury, showing again that Cannon's statement that the heart reaction is scarcely ever ob- tained with sciatic stimulation after opening the abdomen is unfounded. * Eye reactions with sciatic stimulation as described in protocol of cat 190, p. 319. EPINEPHRIN OUTPUT AND RATE OF DENERVATED HEART 323 Of course when a weak enough stimulus was employed (5 minutes there- after) the heart rate remained unchanged, but so did the blood pres- sure. Precisely the same result was obtained with stimulation of the peripheral end of the splanchnic, a stimulus which failed to cause any appreciable rise of blood pressure (as at 3: 05 p.m.) also caused little if any acceleration of the heart. Whether the corresponding adrenal vein was open or clipped seemed to have no influence on the maximum acceleration. Indeed in the first splanchnic stimulation (at 2: 55 p.m.) with the vein open the maximum acceleration was only 14 beats, whereas in the next stimulation, with the vein cHpped, it was 25 beats per minute. Yet the eye reactions, which were very good in the first case, were abolished in the second. The eye reactions are universally admitted to be due to epinephrin, when elicited by stimulation of the peripheral end of the splanchnic. How is it possible to believe that when they are negative, while the heart acceleration is even greater than when they were strongly positive, the acceleration of the heart is a specific reaction for epinephrin? In another cat (196), in which the left superior cervical ganglion had been excised 20 days previously, the vago-sympathetics were cut, the stellate ganglia excised and the abdomen opened under urethane at the beginning of the experiment. The lumbar veins, just before crossing the adrenals, and the renal arteries and veins were tied on both sides and both adrenal veins prepared for clipping. The peripheral end of the left sympathetic in the thorax was prepared for stimulation and the central end of a sciatic. Stimulation of the left s>Tnpathetic with the adrenal veins open gave a good pupil reaction in 8 seconds. The heart rate, which was 212 before stimulation, reached a maximum of 255 beats per minute (counting from 16 seconds after beginning of stimulation). The blood pressure rose from 72 to 150 mm. of mercury. The left s>Tnpathetic was now stimulated with the left adrenal vein cHpped. There was ho pupil reaction. The heart rate increased from 201 to a maximum of 260 beats per minute (counting from 16 seconds after the beginning of stimulation) and the blood pressure from 94 to 134 mm. of mercury. Then the left s>Tnpathetic was again stimu- lated with both adrenal veins clipped. There was no pupil reaction but the heart rate increased from 200 to a maximum of 245 beats a minute and the blood pressure from 58 to 107 mm. of mercury. Is not this again quite inconsistent with the view that the heart accelera- tion constitutes a quantitative reaction by which the output of epi- nephrin can be determined? 324 G. N. STEWART AND J. M. ROGOFF The next protocol (from cat 200) illustrates the general parallelism between the acceleration of the heart elicited by sciatic stimulation and the rise of blood pressure, and the absence of any demonstrable influence of clipping of the adrenal veins on either the maximum accel- eration or the increase of blood pressure. Protocol. Cat 200; female; weight, 1.47 kgm. Left superior cervical ganglion excised 16 daj^s previously. Under urethane anesthesia cut vago-sympathetics; excised stellate ganglia; prepared central end of sciatic for stimulation. Rate Pressure 10:00 a.m. Before sciatic stimulation' 210 211 84 84 Counts of successive portions of the curve after begin- 215 102 ning of stimulation (with progressive increase in < 230 112 strength) 230 128 .244 128 10:05 a.m. Before sciatic stimulation 214 100 Counts of successive portions of the curve with stimu- lation as above '224 240 240 117 126 118 10:10 a.m. Prepared adrenal veins for clipping (extraperitonealljO 10:15 a.m. Before sciatic stimulation 217 223 234 .248 84 Successive counts after beginning of stimulation as above 100 110 110 10:20 a.m. Before sciatic stimulation; both adrenal veins clipped. 206 '212 70 88 Successive counts after beginning of stimulation as 222 89 above 223 225 88 86 Just after release of adrenal veins 228 245 95 15 seconds after release of adrenal veins 104 10:25 a.m. Before sciatic stimulation 214 '216 80 88 Successive counts as above during stimulation < 239 104 [253 117 10:30 a.m. Before sciatic stimulation; both adrenal veins clipped. 215 77 During stimulation as above >, „„„ 84 98 Just after release of adrenal veins 236 239 96 12 seconds after release of adrenal veins 100 In the last experiment (cat 193) to be quoted in this section of the paper, only the vago-sympathetics were cut at the beginning of the experiment, and the abdomen was opened. ^ The effects on the ej'e were those described in the footnote to protocol of cat 190, p. 319. EPINEPHRIN OUTPUT AND RATE OF DENERVATED HEART 325 Protocol. Cat 193; male; weight, 2.7 kgm. Left superior cervical ganglion excised 10 days previously. Under urethane (5 gm.) opened abdomen; prepared peripheral end of left splanchnic for stimulation; cut vago-sympathetics. Rate Pressure 12:08 p.m. Before stimulation of splanchnic; left adrenal vein clipped 157 98 11 seconds after beginning stimulation 175 18 seconds after beginning stimulation 180 145 No eye reactions during stimulation; after release of clip good pupil and nictitating reactions in 9.2 seconds 12:10 p.m. Before stimulation of splanchnic 162 96 10 seconds after beginning stimulation 209 136 Good pupil and nictitating reactions in 11.5 seconds 12:15 p.m. Before stimulation of splanchnic; left adrenal vein clipped 172 120 17 seconds after beginning stimulation 190 162 No eye reactions during stimulation; on release of clip good pupil and nictitating reactions in 8.2 seconds 12:20 p.m. Excised stellate ganglia 12:40 p.m. Before stimulation of splanchnic; left adrenal vein clipped 185 114 18 seconds after beginning stimulation 201 144 No eye reactions during stimulation; on release of clip good pupil and nictitating reactions in 11 seconds 12:50 p.m. Stimulated splanchnic with left adrenal vein and coeliac and superior mesenteric arteries clipped. Before stimulation 203 134- 17 seconds after beginning stimulation 202 136 No eye reactions during stimulation; on release of adrenal vein good pupil and nictitating reactions in 10.6 seconds Before removal of adrenal clip 202 121 12 seconds after release of adrenal vein 238 128 Removed clips from coeliac and superior mesenteric 1:00 p.m. Prepared central end of sciatic for stimulation 1 :02 p.m. Before stimulation of sciatic 174 78 11 seconds after beginning stimulation 184 102 20 seconds after beginning stimulation 194 34 seconds after beginning stimulation 200 104 1 : 20 p.m. Before stimulation of sciatic 184 68 16 seconds after beginning stimulation 200 108 The effects of stimulating the splanchnic, with the corresponding adrenal vein open and clipped, were not obviously different from those in the other experiments, where the stellate ganglia had been excised at the beginning, nor did removal of the stellate ganglia cause any essential change. The failure of the heart reaction with splanchnic THE AMERICAN JOURNAL OF PHTSIOLGGT, VOL. 52, NO. 2 326 G. N. STEWART AND J. M. ROGOFF stimulation when the coeliac and superior mesenteric arteries were clipped is clearly associated with the absence of a rise of pressure when the splanchnic area is thus eliminated. On removal of the clip from the adrenal vein the usual good eye reactions were obtained and also an acceleration of the heart due to release of the epinephrin pent up in the adrenal vessels. It may be tedious to point out again that an hour and a half after the abdomen had been opened, with one splanchnic cut, after all the clip- ping and unclipping of the adrenal veins and of the coeliac and superior mesenteric arteries (and a good many observations actually made have been omitted from the protocol to save space) stimulation of the sciatic still gave a good acceleration of the heart, although, according to Can- non, no reaction ought to have been obtained. In view of such facts what becomes of Doctor Cannon's assertion, unsupported by any evidence that when we collect adrenal vein blood from a cava pocket, the "peculiar" conditions of our experiments do not permit o.f the demonstration that with stimulatipn of sensory nerves there is a vast outpouring of epinephrin from the adrenals, a demonstration which he obtains by a misinterpretation of a heart reaction equally well elicited whether the adrenal veins are open or clipped off, whether the eye (sensitized by removal of the superior cervical ganglion) is giving negative or positive reactions for epinephrin? Since, however. Doctor Cannon maintains that the mere clipping or tying of the adrenal veins need not interfere in the least with the passage of epinephrin to the heart, and has even convinced himself that adrenalin, injected into the lumbar vein crossing the adrenal after it has been ligated on each side of the gland, passes so* freely into the circulation that it causes a large rise of blood pressure, evidence of another kind will now be given that the acceleration of the heart relied upon by him to prove a markedly augmented rate of epinephrin output when the sciatic nerve is stimu- lated has no such significance. EXPERIMENTS ON ANIMALS WITH ONE ADRENAL REMOVED AND THE NERVES OF THE OTHER CUT We have previously shown (13) that after this operation the epinephrin output is either greatly reduced or abolished, within the limits of sensitiveness of the test objects (rabbit's intestine and uterus segments) employed to detect and estimate it. It was there- fore of interest to see whether the acceleration of the heart would EPINEPHRIN OUTPUT AND RATE OF DENERVATED HEART 327 be obtained in these animals by stimulation of sensory nerves. The result was positive in all the cats, a good acceleration being ehcited by stimulation of the sciatic. For example, in cat 201, a female weighing 2.575 kgm., the right adrenal was removed, the nerves of the left including the splanchnic cut. A portion of the left semilunar ganglion was removed. The left superior cervical ganglion was excised at the same time. Eighteen days thereafter, the animal being in good condi- tion, the vago-sympathetics were cut and the stellate ganglia removed under urethane and the central end of the left sciatic nerve prepared for stimulation. A blood pressure tracing was taken as usual from" the right carotid. Stimulation of the sciatic caused the heart rate to increase from 177 before stimulation to a maximum rate of 211 beats per minute. The blood pressure increased from 54 mm. before stimu- ^^r\r\/^\r\/\j-^^ v/'V^- N/^^'X.^'^^-'^"^'"^-"^ -t ' ^ t B 2// t—t 1 ' 1 1 1 ' 1 1 1 1 1 1 1 1 1 1 1 t 1 1 1 1 1 -1— (-1 III Fig. 4. Parts of blood pressure tracings from cat 201. A, before and B, a portion commencing 20 seconds after beginning of sciatic stimulation. Reduced to three-fifths. lation to a maximum of 132 mm. of mercury. The acceleration and the blood pressure increased together. Successive portions of the curve starting from the point of stimulation yielded the following heart rates and blood pressures 180 (72,) 193 (97), 202 (107), 209 (118), 209 (132). The blood pressures are in parentheses. A sample of the curve is reproduced in figure 4. The effect of sciatic stimulation on the pupils did not differ materially from that already described in cats in which the epinephrin output had not been interfered with (see foot- note to protocol of cat 190). The nictitating membrane did not move. The abdomen was afterwards opened, a short cava pocket made and adrenal vein blood collected, which was assayed on rabbit segments. It was shown that the epinephrin output could not have been more than one-fiftieth of the average output per kilogram per minute, under 328 G. N. STEWART AND J. M. ROGOFF the conditions of our experiments. The prehminary operation had, therefore, effectively severed the epinephrin-secretory nerves of the left adrenal. In spite of this and also in spite of the fact that one splanchnic had been divided, and with both splanchnics intact a greater reflex rise of blood pressure might have been obtained, a maximum acceleration of the heart of 32 to 34 beats was elicited by stimulation of the sciatic. According to Cannon, the whole of the acceleration must have been due to a great outpouring of epinephrin from the left adrenal, reflexly stimulated along efferent nerve paths which had been divided 18 days before, and which certainly had not regenerated in that time. In another cat (202), a female weighing 2.545 kgm., the right adrenal was removed and the nerves of the left cut. The left superior cervical ganglion was excised at the same time. Eighteen days thereafter the heart was denervated under urethane and blood pressure tracings taken from the right carotid. Stimulation of the central end of the sciatic caused an acceleration of the heart rate from 145 per minute before stimulation to 161. The blood pressure rose from 92 to 134 mm. of mercury. Successive portions of the curve, from the beginning of stimulation, yielded the following heart rates and blood pressures: 152 (100), 155 (110), 157 (130), 160 (126), 161 (134). The pupil reactions were as previously described in the footnote to the protocol of cat 190. There was no movement of the nictitating membrane. After the sciatic stimulations the abdomen was opened, and 3 speci- mens of adrenal vein blood collected — the first, about 1 gram (dis- carded), the second, 2.9 grams in 4 minutes (0.75 gm. per minute), the third 5.1 grams in 9 minutes (0.57 gm. per minute). No evidence was obtained with rabbit segments that the blood contained any epinephrin. It was shown that the third specimen could not have contained 1:70,000,000 epinephrin, i.e., the output could not have been 0.000007 mgm. per minute for the cat, or 0.0000027 mgm. per kilogram per minute. In other words it could not have been one- hundredth of the average normal output, and there was no evidence that any epinephrin was being given off. The interval after the pre- liminary operation was quite short in this animal, 8 days, which pos- sibly may be an unfavorable condition for obtaining a large heart reaction, although we have no evidence as to this. In the next experiment (cat 215) the animal was allowed to survive for 2 months. EPINEPHRIN OUTPUT AND RATE OF DENERVATED HEART 329 Protocol. Cat 215; female; weight 2.38 kgm. May 17, 1918. Right adrenal excised, left denervated and right superior cervical ganglion excised. July 15, 1918. Under urethane (5 gm.) cut vago-sympathetics, excised stel- late ganglia, prepared central end of left sciatic for stimulation. Rate Pressure 11:15 a.m. Before sciatic stimulation for 38 seconds (8 cm.)^ 219 134 During first 14 seconds of stimulation 232 162 During next 13 seconds of stimulation 238 170 During next 13 seconds 244 172 During next 20 seconds 228 147 11:20 a.m. Before sciatic stimulation for 33 seconds (6 cm.) 222 144 During first 14 seconds of stimulation 240 180 During next 14 seconds of stimulation 248 196 During next 15 seconds 249 180 11:25 a.m. Before sciatic stimulation for 26 seconds (6.5 cm.) 219 152 During first 8 seconds of stimulation 226 178 During next 7 seconds of stimulation 230 182 During next 12 seconds 246 190 11:30 a.m. Before sciatic stimulation for 40 seconds (6 cm.) 224 148 During first 13 seconds of stimulation 239 177 During next 14 seconds of stimulation 252 180 During next 13 seconds of stimulation 251 182 During next 10 seconds 248 168 11:35 a.m. Before sciatic stimulation for 37 seconds (4 cm.) 222 148 During first 12 seconds of stimulation 239 173 During next 11 seconds of stimulation 244 180 During next 1 1 seconds of stimulation 252 180 During next 14 seconds 249 168 Later on the abdomen was opened and adrenal vein blood collected. Assays of the adrenal blood released from a cava pocket were also made by the pupil reaction. The protocol shows that stimulation of the sciatic caused good acceleration of the heart with corresponding changes in the blood pressure. The rabbit intestine assay of the adrenal blood demon- strated that a substantial output of epinephrin was still going on. The concentration of the second specimen was about 1 : 6,500,000 and that of the third specimen about 1:3,750,000, corresponding in either case to an output of 0.00008 mgm. per minute for the cat, or 0.00003 mgm. per kilogram per minute, one-seventh or one-eighth of the aver- age normal output. It is impossible to say whether an unusually large proportion of the secretorj^ fibers of the left adrenal had escaped * Eye reactions, as described in footnote to protocol of cat 190, p. 319. The distance between the coils is given in brackets. 330 G. N. STEWART AND J. M. ROGOFF section at the preliminary- operation, or whether some regeneration had occurred. In any case the innervation of the one adrenal remain- ing must still have been seriously crippled. Yet the heart reaction on which Cannon relies as an "indicator of adrenal secretion" was as well obtained with sciatic stimulation as in normal animals, corre- sponding with the general excellent condition of the cat two months after the primary operation. In two cats operated on in the same way but only a relatively short time before (8 days and 7 days), the experiment was completed by excising the remaining (already denervated) adrenal. It was not thought advisable in these cats to complicate the experiment by at- tempting to estimate the residual epinephrin output, if any, on the adrenal vein blood. But it was shown that the epinephrin store of the adrenal constituted a full load, a good indication that protection of the gland from depletion during the experiment was relatively complete. Protocol. Cat 444; male; weight, 2.9 kgm. Right adrenal excised and left denervated 8 days, left superior cervical ganglion excised 14 days previously. Under ether cut vago-sympathetics, excised stellate ganglia, prepared central end of right sciatic for stimulation. Rate Pressure 10:40 a.m. Before sciatic stimulation (8 cm.) 133 132 2 seconds after beginning stimulation 130 128 30 seconds after beginning stimulation 132 130 10:45 a.m. Before sciatic stimulation (5 cm.) 133 112 6 seconds after beginning stimulation 140 40 seconds after beginning stimulation 152 92 60 seconds after beginning stimulation 145 106 10:50 a.m. Before sciatic stimulation (8 cm.) 127 108 3 seconds after beginning sttoulation 132 30 seconds after beginning stimulation 136 100 45 seconds after beginning stimulation 131 104 10:55 a.m. Before sciatic stimulation (6 cm.) 121 120 5 seconds after beginning stimulation 130 126 11:10 a.m. Excised left adrenal (extraperitoneally) 11:15 a.m. Before sciatic stimulation (8 cm.) 130 103 3 seconds after beginning stimulation 133 96 23 seconds after beginning stimulation 140 60 seconds after beginning stimulation 144 114 11:48 a.m. Opened abdomen, tied coeliac axis and superior mes- enteric artery 11:50 a.m. Before sciatic stimulation (6 cm.) 139 74 8 seconds after beginning stimulation 144 74 3 seconds after stopping stimulation 142 72 40 seconds after stopping stimulation 134 70 EPINEPHRIN OUTPUT AND RATE OF DENERVATED HEART 331 11:58 a.m. Before sciatic stimulation (4 cm.) 123 72 2 seconds after beginning stimulation 130 77 15 seconds after beginning stimulation 133 80 40 seconds after beginning stimulation 137 81 60 seconds after beginning stimulation 138 78 12:05 p.m. Before sciatic stimulation (8 cm.) 130 76 2 seconds after beginning stimulation 133 80 30 seconds after beginning stimulation 135 82 In cat 444 the greatest acceleration caused by sciatic stimulation before excision of the remaining adrenal was 19 beats. With repeated stimulations the acceleration effect declined. The last stimulation before excision gave only 9 beats. A weaker stimulation just after excision gave a maximum acceleration of 14 beats, and another stronger stimulation gave a maximum acceleration of 15 beats. Even after the abdomen was opened and the superior mesenteric artery and coeliac axis tied, a maximum acceleration of 15 beats was obtained in the absence of the adrenals. In cat 441, also, while the greatest acceleration before excision of the remaining adrehal (17 beats) was not reached afterwards, the acceleration immediately after excision was the same as that obtained just before it (9 beats), although the stimulation in the latter case was the stronger. Protocol. Cat 441; female; weight, 1.66 kgm. Right adrenal excised and left denervated 7 days previously. Under ether cut vago-sympathetics, excised stellate ganglia, prepared central end of right sciatic for stimulation. Rate Pressure 10:55 a.m. Before sciatic stimulation (10 cm.) 219 100 6 seconds after beginning stimulation 229 116 20 seconds after beginning stimulation 228 10:58 a.m. Before sciatic stimulation (8 cm.) 217 115 6 seconds after beginning stimulation 223 122 11:02 a.m. Before sciatic stimulation (6 cm.) 208 106 10 seconds after beginning stimulation 215 118 30 seconds after beginning S'cimulation 213 11:06 a.m. Before sciatic stimulation (6 cm.) 211 109 5 seconds after beginning stimulation 217 18 seconds after beginning stimulation 228 120 28 seconds after beginning stimulation 227 11:10 a.m. Before sciatic stimulation (10 cm.) 207 113 5 seconds after beginning stimulation 215 116 11:15 a.m. Before sciatic stimulation (8 cm.) 206 109 5 seconds after beginning stimulation 222 139 332 G. N. STEWABT AND J. M. ROGOFF Rate Pressure 11:20 a.m. Before sciatic stimulation (7 cm.) 216 116 5 seconds after beginning stimulation 224 148 11:25 a.m. Before sciatic stimulation (6 cm.) 212 98 5 seconds after beginning stimulation 221 119 11:40 a.m. Excised left adrenal (lumbar route) 11:42 a.m. Before sciatic stimulation (8 cm.) 214 86 5 seconds after beginning stimulation 220 20 seconds after beginning stimulation 223 114 11:45 a.m. Before sciatic stimulation (10 cm.) 211 84 5 seconds after beginning stimulation 214 100 23 seconds after beginning stimulation 215 11:51a.m. Before sciatic stimulation (9 cm.) 210 70 6 seconds after beginning stimulation 214 88 11:55 a.m. Before sciatic stimulation (7 cm.) 208 70 6 seconds after beginning stimulation 210 84 11:58 a.m. Before sciatic stimulation (8 cm.) 211 68 16 seconds after beginning stimulation 218 97 12:00 m. Prepared central end of left sciatic for stimulation 12:05 p.m. Before stimulation of left sciatic (9 cm.) 214 68 30 seconds after beginning stimulation 217 72 12:10 p.m. Before stimulation of right sciatic (10 cm.). ., 215 68 5 seconds after beginning stimulation 216 76 12:15 p.m. Before stimulation of right sciatic (8 cm.) 236 55 5 seconds after beginning stimulation 242 62 11 seconds after beginning stimulation 245 The left adrenal weighed 0.226 gm. and contained 0.23 mgm. of epinephrin at end of experiment. In the last cat (450) of this series to be mentioned a longer period (34 days) was allowed to elapse between the primary operation and the experiment, in order that the animal might have more fully recovered. Protocol. Cat 450; male; weight, 2.96 kgm. Right adrenal excised and left denervated (with section of left splanchnic as usual) 34 days previously. Under urethane (5 gm.) cut vago-sympathetics; excised stellate ganglia; prepared central end of left sciatic for stimulation. Rate Pressure 11:10 a.m. Before sciatic stimulation (10 cm.) 173 102 During first 18 seconds of stimulation 178 114 During next 20 seconds of stimulation 182 114 Just after end of stimulation 185 30 seconds after end of stimulation 180 11:14 a.m. Before sciatic stimulation (9 cm.) 174 100 During first 12 seconds of stimulation 178 During next 16 seconds of stimulation 183 133 Just after end of stimulation 191 126 20 seconds after end of stimulation 189 129 EPINEPHRIN OUTPUT AND RATE OF DENERVATED HEART 333 Rate Pressure 11:18 a.m. Before sciatic stimulation (8 cm.) 171 97 During first 16 seconds of stimulation 178 During next 17 seconds of stimulation 187 134 Just after end of stimulation 192 134 20 seconds after end of stimulation 190 11:40 a.m. Excised left adrenal (extraperitoneally) 11:43 a.m. Before sciatic stimulation (9 cm.) 174 94 During first 15 seconds of stimulation 179 130 During next 20 seconds of stimulation 189 127 11:48 a.m. Before sciatic stimulation (8 cm.) 174 103 During first 14 seconds of stimulation 178 132 During next 20 seconds of stimulation 191 144 Just after end of stimulation 200 150 15 seconds after end of stimulation 198 128 35 seconds after end of stimulation 194 127 11:58 a.m. Before sciatic stimulation (7 cm.) 177 96 During first 17 seconds of stimulation 181 128 During next 20 seconds of stimulation 192 138 Just after end of stimulation 200 122 20 seconds after end of stimulation 195 120 12:10 p.m. Prepared peripheral end of right splanchnic (in thorax) 12:12 p.m. Before splanchnic stimulation (10 cm.) 167 70 During first 20 seconds of stimulation 176 96 During next 20 seconds of stimulation 184 104 Just after end of stimulation 186 92 12:15 p.m. Before splanchnic stimulation (8 cm.) 177 61 During first 20 seconds of stimulation 175 70 During next 20 seconds of stimulation 185 90 Just after end of stimulation 189 86 12:21p.m. Before splanchnic stimulation (7 cm.) 178 60 During first 16 seconds of stimulation 179 93 During next 20 seconds of stimulation 186 108 Just after end of stimulation 187 96 12:26 p.m. Before sciatic stimulation (7 cm.) 179 62 During first 20 seconds of stimulation 182 83 During next 20 seconds of stimulation 180 72 12:35 p.m. Cut left splanchnic in thorax 12:36 p.m. Before sciatic stimulation (7 cm.) 181 63 During first 18 seconds of stimulation 182 82 During next 20 seconds of stimulation 181 78 Just after end of stimulation 184 74 1:01p.m. Before splanchnic stimulation (7 cm.) 183 46 During first 16 seconds of stimulation 184 78 During next 20 seconds of stimulation 196 88 Just after end of stimulation 208 74 15 seconds after end of stimulation 209 65 22 seconds after end of stimulation 212 60 334 G. N. STEWART AND J. M. ROGOFF It will be seen from the protocol that a good acceleration was obtained on sciatic stimulation before removal of the remaining (already dener- vated) adrenal, (12 beats, 17 beats, 21 beats per minute in successive observations with different strength of stimulation). After removal of the adrenal the accelerations obtained were fully as great as before (15 beats, 26 beats, 23 beats per minute). When, however, in the absence of the adrenals, the right splanchnic was cut in the thorax sciatic stimulation caused practically no acceleration, and the rise of pressure was, of course, much reduced (fig. 5). This was not due to any change in the condition of the animal which rendered direct stimu- lation of the splanchnic less effective, for the rise of pressure and the acceleration on stimulating the peripheral end of the right splanchnic ^^-' • ■ . /7.y : A ni Fig. 5. Blood pressure curves from cat 450. A, sciatic stimulation before and B, after excision of remaining (already denervated) adrenal; C, after sec- tion of remaining splanchnic. Reduced to three-fifths. was quite as great as before the last sciatic stimulation (as much as 29 beats per minute), (fig. 6). The failure of the heart reaction with sciatic stimulation after section of the right splanchnic (the left had been divided at the primary operation) is precisely what Cannon describes as occurring after removal of the adrenals. But in this animal the removal of the remaining adrenal did not affect the heart reaction, while subsequent section of the remaining splanchnic abol- ished it. This is incompatible with Cannon's interpretation of the failure of the reaction after section of the splanchnics as due entirely to interference with the epinephrin output. In the present experi- ment, to be logical, he would have to. attribute the result to the loss of something coming from the liver (sugar?) or from the intestines, mobil- ized reflexly through the splanchnic. EPINEPHRIN OUTPUT AND RATE OF DENERVATED HEART 335 C CD O ^ > cS o3 to o O ■^ -fj U-H -1^ O 3 O -»^ 2 3 X o oS o3 c3 m .3 o Q^ m CO « -►i ^ 3 '^ e a o o lO io -u m c3 _3 w 3 ^ g o -►J CO M-H t4 aj « o bO > S ^ o t-l u -i-i ■Jl t> u Xi 3 -t^ i/2 CO '$ -3 a o3 336 G. N. STEWART AND J. M. ROGOFF EXPERIMENTS WITH AN INTERVAL BETWEEN REMOVAL OF THE TWO ADRENALS To avoid division of one splanchnic and to make the first operation less severe than the ordinary operation for suppression of the epi- nephrin output, while making the second operation less severe than the removal of both adrenals at one time, a number of experiments were performed in which one adrenal was removed, and then, after an interval of 3 to 13 days, the observations on sciatic stimulation with denervation of the heart and excision of the remaining adrenal were made. In cat 440, for example, the experiment was performed 6 days after the removal of the right adrenal. Protocol. Cat 440; male; weight, 2.31 kgm. Right adrenal excised 6 days previously. Under ether cut vago-sympathetics, excised stellate ganglia, pre- pared central end of right sciatic for stimulation. Rate Pressure 10:45 a.m. Before sciatic stimulation (10 cm.) 226 134 5 seconds after beginning stimulation 256 168 10:49 a.m. Before sciatic stimulation (8 cm.) 220 128 6 seconds after beginning stimulation 253 171 15 seconds after end of stimulation 264 156 11:00 a.m. Excised left adrenal (extraperitoneally) 11:02 a.m. Before sciatic stimulation (8 cm.) 212 124 5 seconds after beginning stimulation 242 15 seconds after beginning stimulation 255 156 23 seconds after beginning stimulation 250 156 37 seconds after beginning stimulation 228 138 60 seconds after beginning stimulation 218 134 11:05 a.m. Before sciatic stimulation (6 cm.) 213 119 6 seconds after beginning stimulation 252 154 28 seconds after beginning stimulation 247 11:09 a.m. Before sciatic stimulation (4 cm.) 212 118 5 seconds after beginning stimulation 230 133 22 seconds after beginning stimulation 248 153 11:20 a.m. Before sciatic stimulation (8 cm.) 221 112 6 seconds after beginning stimulation 236 130 22 seconds after beginning stimulation 247 140 11:25 a.m. Before sciatic stimulation (6 cm.) 230 118 8 seconds after beginning stimulation 241 130 11:28 a.m. Before sciatic stimulation (4 cm.) 225 133 7 seconds after beginning stimulation 231 146 43 seconds after beginning stimulation 227 130 11:45 a.m. Exposed cord in midcervical region 11 :50 a.m 208 90 EPINEPHRIN OUTPUT AND RATE OF DENERVATED HEART 337 Rate Pressure 11:55 a.m. Before total asyphxia (for 45 seconds) 207 84 3 seconds after beginning asphyxia 204 10 seconds after beginning asphyxia 225 88 55 seconds after beginning asphyxia 210 88 12:05 a.m. 3 minutes after transection of cord between 4th and 5th cervical segments 193 51 As will be seen from the protocol, excellent heart reactions were obtained both before and after the removal of the remaining adrenal in cat 440. It is to be particularly noted that excision of the left adrenal left the blood pressure and heart rate practically unchanged. Fig. 7. Parts of blood pressure tracings from cat 440. A, before and B, a portion commencing 15 seconds after beginning of sciatic stimulation, before excision of remaining adrenal; C, before and D, 15 seconds after beginning of sciatic stimulation, after excision of remaining adrenal. Zero line moved up 41 mm. The maximum acceleration for the last sciatic stimulation prior to removal of the left adrenal was 33 beats per minute, and for the first sciatic stimulation after removal of the adrenal 42 beats, the maximum pulse rate reached being practically identical in the two cases (253, 250) . Samples of the curves used in counting the pulse rate are given in figure 7. The greater portion of each of the two curves, much reduced, is reproduced in figure 8, to show that excision of the second adrenal has not in any way essentially changed the vascular reaction. Here was a cat, then, without adrenals in which the acceleration pro- duced by stimulation of the sciatic was actually greater than that 338 G. N. STEWART AND J, M. ROGOFF produced by a similar stimulation while one adrenal was still intact. Yet, according to Cannon, the last acceleration, like the jfirst, must have been due solely to augmented epinephrin output from the adrenals. It should be noted that the exposure of the cord in the midcervical region led to a drop of blood pressure to 90 mm. of mercury and a corresponding slowing of the heart to 208 beats a minute. Subsequent transection of the cord caused the blood pressure to fall to 51 mm. of mercury and the heart rate to 193. It must be remembered that the adrenals were out, and the decrease in the heart rate can have nothing to do with absence of adrenal epinephrin. Cannon has emphasized the fact that after removal of the adrenals the pulse rate drops. Our observations show that this is always true provided the blood pressure ^XZO ZS3 U^^ ^^.t,,. ■ ■''■■''■ l''l''* .........llJ Fig. 8. Blood pressure curves from cat 440. A, sciatic stimulation before and B, after removal of remaining adrenal. Reduced to one-half. falls decidedly. But if the pressure is maintained there is not neces- sarily any sensible slowing of the heart. The diminution in the pulse rate is, therefore, no index of the previous rate of output of epinephrin. This statement of observed facts is made without prejudice to the question whether the epinephrin liberated at the ordinary rate, under our experimental conditions, is capable of exerting an influence upon the heart, including its rate. We have l^rought forward some evidence that there is such an influence. But it may not be obvious after sup- pression of the epinephrin output when the blood pressure is well maintained. In cat 440 repeated stimulation of the sciatic caused some exhaustion of the heart reaction after removal of the last adrenal. But this, of course, is true of all reflexes, and in the case of the heart reaction can occur also with intact adrenals. EPINEPHRIN OUTPUT AND RATE OF DENERVATED HEART 339 In the next experiment (cat 443) an example is given in which the heart acceleration elicited by sciatic stimulation was small from the beginning, but quite as good Reactions were obtained after removal of the second adrenal as before (greatest acceleration before removal of the adrenal, 7 beats in one observation and 9 beats in another; after removal, 10 beats in one observation and 11 beats in another). It will be noted that as the blood pressure continued to fall progressively the heart rate diminished also, and this was not related to the adrenalec- tomy. Thus half an hour after removal of the adrenal, the pulse rate was 160 and the blood pressure 76. When the blood pressure had fallen to 50 the heart rate was 147. Before the removal of the adrenal the pulse rate was 180, 179 and 200 in 3 observations. After the adrenalectomy it was 182, the blood pressure being 83 mm. of mer- cury instead of 110 mm. at the last observation. As in other pro- tocols counts of the heart rate at different parts of the curve and the results of stimulation of different strengths are given in order to show that the maximum accelerations quoted were really the maximum obtainable, under our conditions, in each animal. Protocol. Cat 443; female; weight, 1.53 kgm. Right adrenal excised 7 days, left superior cervical ganglion excised 13 days previously. Under ether cut vago-sympathetics, excised stellate ganglia, prepared central end of left sciatic for stimulation. Rate Pressure 10:55 a.m. Before sciatic stimulation (10 cm.)' 180 116 6 seconds after beginning stimulation 187 148 10:59 a.m. Before sciatic stimulation (9 cm.) 179 106 6 seconds after beginning stimulation 183 128 11:04 a.m. Before sciatic stimulation (9 cm.) 200 110 20 seconds after beginning stimulation 209 122 45 seconds after beginning stimulation 205 116 11 : 15 a.m. Excised left adrenal (extraperitoneally) 11:16 a.m. Before sciatic stimulation (10 cm.) 182 83 5 seconds after beginning stimulation 180 85 11:20 a.m. Prepared central end of right sciatic 11:27 a.m. Before stimulation of left sciatic (8 cm.) 165 80 23 seconds after beginning stimulation 168 100 11:43 a.m. Before stimulation of right sciatic (6 cm.) 161 75 2 seconds after beginning stimulation 160 6 seconds after beginning stimulation 164 17 seconds after beginning stimulation 170 94 3 seconds after end of stimulation 171 ' The eye reactions were the same as described in footnote to protocol of cat 190, p. 319. 340 G. N. STEWART AND J. M. ROGOFF Rate Pressure 11:46 a.m. Before sciatic stimulation (6 cm.) 160 76 10 seconds after beginning stimulation 169 98 6 seconds after end of stimulation 170 11:50 a.m. Before stimulation of left sciatic (6 cm.) 155 62 6 seconds after beginning stimulation 157 70 12:07 p.m. Before stimulation of left sciatic (6 cm.) 147 50 7 seconds after beginning stimulation 151 30 seconds after beginning stimulation 153 56 In both of the experiments hitherto cited in this section (cats 440 and 443) the blood pressure after removal of the second adrenal remained good, practically unchanged in the first cat and only moderately low- ered in the other. In the next experiment (cat 438) a considerable fall of pressure accompanied the operation for removal of the second adrenal. There is no reason to attribute this to the fact that the operation was done by the abdominal route. A similar fall of pressure occurred in cat 439 after removal of the second adrenal extraperi- toneally by the lumbar route. Protocol. Cat 438; female, weight 1.91 kgm. Right adrenal excised 3 days previously. Under urethane (3 gm.) cut vago-sympathetics, excised stellate ganglia, prepared central end of left sciatic for stimulation. Rate Pressure 12:08 p.m. Before sciatic stimulation (6 cm. to 4 cm.) 189 122 After increasing strength of stimulus (5 cm.) 194 After increasing strength of stimulus (4 cm.) 200 At end of stimulation 213 154 12:30 p.m. Before sciatic stimulation (6-4 cm.) 206 110 4 seconds after beginning stimulation 220 140 After increasing strength of stimulus (4 cm.) 237 178 12:45 p.m. Opened abdomen, excised left adrenal 12:50 p.m. Before sciatic stimulation (4 cm.) 130 40 During first 15 seconds stimulation 138 After increasing strength of stimulus 143 52 After further increasing strength of stimulus 150 64 1:03 p.m. Before sciatic stimulation (6-4 cm.) 161 5 seconds after beginning stimulation 170 1:10 p.m. Before sciatic stimulation (6-4 cm.) 161 66 5 seconds after beginning stimulation 162 30 seconds after beginning stimulation 166 83 1 : 15 p.m. Before sciatic stimulation (4 cm.) 171 60 10 seconds after beginning stimulation 170 74 1 :20 p.m. Before total asphyxia for 1 minute 175 64 Counting from end of asphyxia 225 64 EPINEPHRIN OUTPUT AND RATE OF DENERVATED HEART 341 In cat 438 it should be noted that with the fall of pressure from well over 100 mm. to 40 mm. of mercury, associated with the adrenalec- tomy, the pulse rate diminished from 206 before the previous sciatic stimulation to 130. To attribute this diminution of 76 beats per minute entirely to the lack of the epinephrin from the second adrenal is, we believe, unwarranted. As the pressure gradually rose later on in the experiment, be it remembered in the absence of the adrenals, the pulse rate increased also to 161, 171 and 175 with pressures of 60 to 66. In spite of the rather low blood pressure which, however, showed a tendency to improve, a fair acceleration of the heart was elicited by stimulation of the sciatic after removal of the second adrenal (as much as 20 beats per minute, compared with 24 beats and 31 beats in two observations before the adrenalectomy^- Protocol. Cat 439; male; weight, 1.93 kgm. Right adrenal excised 5 days previously. Under urethane (3 gm.) cut vago-sympathetics, excised stellate ganglia and prepared central end of left sciatic for stimulation. Rate Pressure 11:30 a.m. Before sciatic stimulation (10 cm.) 210 125 4 seconds after beginning stimulation 217 146 After increasing strength of stimulus (8 cm.) 234 160 11:40 a.m. Before sciatic stimulation (6 cm.) 200 100- 3 seconds after beginning stimulation 222 142 11 :50 a.m. Excised left adrenal (extraper itoneally) 11:53 a.m. Before sciatic stimulation (6 cm.) 178 54 15 seconds after beginning stimulation 181 68 After increasing strength of stimulus (5 cm.) 180 66 12:00 m. Before sciatic stimulation (6 cm.) 179 56 6 seconds after beginning stimulation 183 72 12:10 p.m. Prepared central end of right sciatic 12:12 p.m. Before stimulation of right sciatic (7 cm.) 180 40 5 seconds after beginning stimulation 183 58 After increasing strength of stimulus (3 cm.) 183 49 12:20 p.m. Before sciatic stimulation (6 cm.) 177 40 5 seconds after beginning stimulation 182 44 After end of stimulation . 182 40 12:25 p.m. Before intravenous injection of Ringer 173 20 Immediately after injection of 100 cc. Ringer 188 46 30 seconds after end of injection 190 68 In cat 439 although the blood pressure, immediately after removal of the second adrenal, was a little higher than in cat 438, it tended to grow progressively worse, sinking at last to 20 mm. of mercur}', whereas the opposite tendency was seen in cat 438. Only very trifling accel- erations of the heart were caused by sciatic stimulation after the adre- THE AMERICAN JOUBNAL OF PHYSIOLOGY, VOL. 52, NO. 2 342 G. N. STEWART AND J. M. ROGOFF nalectomy, although before fair reactions were obtained (as much as 24 beats in one observation and 22 beats per minute in another) . The changes of blood pressure produced by stimulation of the sciatic were also small. Since the heart reaction depends upon a reflex or reflexes, as already pointed out, it necessarily fails or is diminished when the reflex arcs have deteriorated under the influence of a low blood pres- sure and a poor blood flow. Toward the end of the experiment an injection of Ringer's solution raised the blood pressure from 20 to 68 nam, and the pulse rate from 173 to 190. Epinephrin, of course, could have nothing to do with this acceleration. That the relative failure of the heart reaction on sciatic stimulation after removal of the second adrenal was not dependent upon the impossiblity of a reflex increase in the epinephrin output but upon other, probably circulatory condi- tions, is indicated in the next experiment (cat 446), the last to be cited in this section. Protocol. Cat 446; female; weight, 1.67 kgm. Right adrenal excised 6 days previously. Under urethane cut vago-sympathetics, excised stellate ganglia, prepared central end of right sciatic for stimulation. Rate Pressure 11:00 a.m. Before sciatic stimulation (8 cm.) 185 115 10 seconds after beginning stimulation 200 152 20 seconds after beginning stimulation 203 30 seconds after beginning stimulation 204 123 11:02 a.m. Before sciatic stimulation (9 cm.) 185 104 10 seconds after beginning stimulation 200 130 32 seconds after beginning stmiulation 199 113 11 :20 a.m. Excised left adrenal (extraperitoneally) 11:22 a.m. Before sciatic stimulation (9 cm.) 148 57 10 seconds after beginning stimulation 150 64 34 seconds after beginning stimulation 150 61 11:30 a.m. Intravenous injection of 100 cc. Ringer 11:50 a.m. Before sciatic stimulation (7 cm.) 164 80 10 seconds after beginning stimulation 174 25 seconds after beginning stimulation 178 115 40 seconds after beginning stimulation 173 11:55 a.m. Before sciatic stimulation (8 cm.) 161 76 4 seconds after beginning stimulation 163 22 seconds after beginning stimulation 168 97 40 seconds after beginning stimulation 162 82 12:03 p.m. Before sciatic stimulation (6 cm.) 162 72 5 seconds after beginning stimulation 170 88 23 seconds after beginning stimulation 171 42 seconds after beginning stimulation 169 70 12:06 p.m. Opened abdomen, tied renal arteries and veins EPINEPHRIN OUTPUT AND RATE OF DENERVATED HEART 343 Rate Pressure 12:11 p.m. Clipped abdominal aorta and stimulated sciatic; before stimulation (7 cm.) 171 74 10 seconds after beginning stimulation 173 35 seconds after beginning stimulation 176 83 After removal of clip 172 53 12:20 p.m. Intravenous injection of 50 cc. Ringer 12:25 p.m. Clipped abdominal aorta and stimulated sciatic; before stimulation (7 cm.) 170 90 10 seconds after beginning stimulation 179 28 seconds after beginning stimulation 176 96 After release of aorta 174 81 12:30 p.m. Prepared central end of left sciatic for stimulation. . . . 12:32 p.m. Clipped abdominal aorta and stimulated left sciatic; before stimulation (7 cm.) 173 88 10 seconds after beginning stimulation 180 30 seconds after beginning stimulation 185 110 45 seconds after beginning stimulation 180 88 10 seconds after release of aorta 174 63 12:35 p.m. Clipped abdominal aorta and stimulated left sciatic; before stimulation (6 cm.) 163 70 10 seconds after beginning stimulation 169 78 31 seconds after beginning stimulation 171 60 10 seconds after release of aorta 166 45 After removal of the second adrenal the blood pressure fell from over 100 mm, of mercury to 57 mm. and the pulse rate dropped from 185 to 147 beats per minute. Sciatic stimulation caused practically no acceleration of the heart (3 beats per minute as compared with 15 beats before the adrenalectomy) and very little rise of blood pressure (7 mm.). The pressure went on falling and Ringer's solution was injected, which brought the blood pressure up to 80 mm. Stimulation of the sciatic nerve now caused an acceleration of 14 beats per minute and a rise of blood pressure of 35 mm, of mercury. Another stimula- tion caused an acceleration of 12 beats when the blood pressure had been raised to 88 mm. of mercury by temporary clipping of the abdom- inal aorta, the clip being put on and the pressure allowed to become constant, which only required a fraction of a minute, before the begin- ning of stimulation. Samples of portions of the tracings used for counting the pulse rate are given in figures 9 and 10, and the whole curves (much reduced) from which these portions were taken, in figure 11. 344 G. N. STEWART AND J. M. ROGOFF "Nyv-^v, ''''''''V/'Xv/*^''w%^>^^ A 1^5 Fig. 9. Parts of blood pressure tracings from cat 446. A, before and B, a portion commencing 19 seconds after beginning of sciatic stimulation. Reduced to four-fifths. Fig. 10. Parts of blood pressure tracings from cat 446. A, before and B, a portion commencing 16 seconds after beginning of sciatic stimulation, after ex- cision of remaining adrenal; C, before and D, 23 seconds after beginning of sciatic stimulation, after intravenous injection of Ringer. Reduced to four- fifths. EPINEPHRIN OUTPUT AND RATE OF DENERVATED HEART 345 EXPERIMENTS IN WHICH BOTH ADRENALS WERE REMOVED AT ONE TIME It seems probable that in most of the cats with one adrenal removed some time before the experiment the interval was too short for the full advantage of this procedure to be obtained, if there is an advantage. The results were, nevertheless, decisive as regards the question at issue. To check the matter a series of observations was made in which both adrenals were removed, as carefully as possible, at the time of the experiment on the denervated heart reaction. The protocol of cat 449 is cited as an example of experiments in which the adrenals were extir- pated after opening the abdomen. ~ ' '' I '' I ' 1 1 1 1 1 1 1. 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 [ I A Jlf ,111 X06 , 20 f B '"" /T y """"""""' * ' ^ V'f tf* *' ' ' • '/ 'f f • • • , Fig. 11. Blood pressure curves from cat 446. A, sciatic stimulation before and B, after excision of remaining adrenal; C, after intravenous injection of Ringer. Reduced to one-half. Protocol. Cat 449; male; weight, 3.38 kgm. Under urethane (6 gm.) cut vago- sympathetics, excised stellate ganglia, prepared central end of left sciatic for stimulation. Rate Pressure 12:00 m. Before sciatic stimulation (8 cm.) 285 125 10 seconds after beginning stimulation 293 170 25 seconds after beginning stimulation 294 12 : 12 p.m. Before sciatic stimulation (6 cm.) 256 110 15 seconds after beginning stimulation 280 147 40 seconds after beginning stimulation 283 130 12:35 p.m. Opened abdomen, excised both adrenals 12:36 p.m. Before sciatic stimulation (6 cm.) 228 70 12 seconds after beginning stimulation 238 124 35 seconds after beginning stimulation 238 90 346 G. N. STEWART AND J. M. ROGOFF Rate Pressure 12:50 p.m. Before sciatic stimulation (5 cm.) 223 74 15 seconds after beginning stimulation 241 127 30 seconds after beginning stimulation 238 89 1:05 p.m. Before sciatic stimulation (7 cm.) 222 73 10 seconds after beginning stimulation 237 130 30 seconds after beginning stimulation 234 92 1:15 p.m. Prepared peripheral end of left splanchnic in thorax for stimulation. 1 :20 p.m. Before splanchnic stimulation (9 cm.) 226 58 10 seconds after beginning stimulation 231 111 30 seconds after beginning stimulation 223 69 1:25 p.m. Before splanchnic stimulation (7 cm.) 221 59 8 seconds after beginning stimulation 229 110 1 :40 p.m. Before sciatic stimulation (6 cm.) 221 52 10 seconds after beginning stimulation 231 92 26 seconds after beginning stimulation 230 1 :45 p.m. Before sciatic stimulation (6 cm.) 223 52 12 seconds after beginning stimulation 232 98 40 seconds after beginning stimulation 233 64 Two sciatic stimulations were made before extirpation of the adre- nals. The first yielded a maximum acceleration of 9 beats only, but the initial heart rate was unusually great. In the second stimulation the maximum acceleration was 24 to 27 beats per minute, the initial rate being decidedly lower than in the first observation. After removal of the adrenals, accelerations of 10, 18 and 15 beats were obtained in 3 successive sciatic stimulations, and even at the end of the experiment, when the blood pressure had fallen considerably, an acceleration of 10 beats was gotten. It is scarcely necessary to point out that it would be futile to try to determine from such figures whether epinephrin was taking any sensible share in the reaction before the adrenalectomy, and if so, how much. For the maximum acceleration of which a given heart is capable at different stages in an experiment, under the influ- ence of the changes produced by stimulation of the sciatic other than any possible change in the epinephrin output, must vary with the condition of the heart, and this with the blood flow on which its nutri- tion depends. Stimulation of the peripheral end of a splanchnic nerve after removal of both adrenals caused also distinct acceleration, as much as 8 to 10 beats per minute, in this experiment although, of course, some further fall of blood pressure had been caused by division of the nerve. Portions of the curves used for counting the heart rate in the last sciatic stimulation prior to removal of the adrenals and the second stimulation after ''s'^z3 2V/ I I ' I ' ■ ' A ZS6 %20 '■''''''■''■''■''''■ .■■.... , . , , ■ .1. Fig. 13. Blood pressure curves from cat 449. A, sciatic stimulation before and B, after excision of both adrenals. Reduced to one-half. 348 G. N. STEWART AND J. M. ROGOFF of the greater part of the two curves in figure 13. It will be seen that neither the blood pressure reaction nor the acceleration was essentially modified by the absence of the adrenals. The blood pressure, although decidedly lower after the adrenalectomy, was still good (70 to 80 mm. of mercury, as compared with 110 mm. before the operation). In the next experiment (cat 448) the adrenals were removed extra- peritoneally by the lumbar route. The results were practically the same as in cat 449, in which the abdomen had been opened. Protocol. Cat 448; male; weight, 3.29 kgm. Under urethane (6 gm. in two doses) cut vago-sympathetics, excised stellate ganglia, prepared central end of left sciatic for stimulation. Rate Pressure 11 :43 a.m. Before sciatic stimulation (10 cm.) 217 132 10 seconds after beginning stimulation 226 158 30 seconds after beginning stimulation 232 144 11:45 a.m. Before sciatic stimulation (8 cm.) 210 135 10 seconds after beginning stimulation 234 170 30 seconds after beginning stimulation 242 149 12:20 p.m. Extirpated both adrenals (extraperitoneally) 12:25 p.m. Before sciatic stimulation (8 cm.) 165 90 6 seconds after beginning stimulation 171 103 25 seconds after beginning stimulation 170 12:32 p.m. Before sciatic stimulation (7 cm.) 164 87 10 seconds after beginning stimulation 181 132 26 seconds after beginning stimulation 184 128 35 seconds after beginning stimulation 182 118 12:40 p.m. Sciatic now stimulated twice with an interval of only 2 minutes, to fatigue the reaction, and then at 12:50 p.m. Before sciatic stimulation (6 cm.) 173 92 10 seconds after beginning stimulation 181 126 35 seconds after beginning stimulation 183 1:03 p.m. Sciatic again stimulated twice in rapid succession and then at 1 : 14 p.m. Before sciatic stimulation (6 cm.) 171 76 10 seconds after beginning stimulation 175 107 40 seconds after beginning stimulation 177 1:20 p.m. Opened abdomen, tied renal arteries and veins 1 :24 p.m. Before sciatic stimulation (6 cm.) 174 88 4 seconds after beginning stimulation 181 110 30 seconds after beginning stimulation 181 98 1:33 p.m. Stimulated sciatic with abdominal aorta clipped. Before clipping aorta 170 77 Before sciatic stimulation 177 100 10 seconds after beginning stimulation 184 30 seconds after beginning stimulation 186 114 After release of aorta 184 30 seconds after release of aort a ■ 185 86 EPINEPHRIN OUTPUT AND RATE OF DENERVATED HEART 349 1:37 p.m. Stimulated sciatic with abdominal aorta and vena cava clipped ; Rate Pressure Before stimulation (7 cm. ) 174 76 8 seconds after beginning stimulation 180 30 seconds after beginning stimulation 182 100 10 seconds after removal of clips from aorta and cava. 176 68 2:21 p.m. Before sciatic stimulation (7 cm.) 159 47 10 seconds after beginning stimulation 159 60 Just after end of stimulation 158 2:25 p.m. Before sciatic stimulation (4 cm.) 155 43 10 seconds after beginning stimulation 161 55 40 seconds after beginning stimulation 161 Accelerations as great as 20 beats per minute were obtained after removal of the adrenals. Before removal the maximum acceleration seen in two observations with different strengths of stimulus was 15 and 32 beats respectively. The blood pressure fell from 135 to 90 mm. of mercury after the adrenalectomy. When the sciatic was then repeatedly stimulated with only short intervals between the successive stimulations, the heart reaction, as estimated bj^ the maximum accel- eration, diminished but about the same absolute rate (180 to 183) was reached at the height of the acceleration. Later on in the experiment, when the abdomen had been opened and the renal vessels tied, an acceleration of 7 beats per minute was caused by clipping the abdom- inal aorta just above the bifurcation, as is done in the collection of blood from the cava pocket. This acceleration was accompanied by a rise of blood pressure from. 77 to 100 mm. of mercury. When the sciatic was now stimulated with the aorta still chpped (the cHp was put on only a short time before stimulation of the sciatic so that the excitation of the nerve should not be interfered with) a further accel- eration of 9 beats a minute occurred. Sciatic stimulation gave a similar acceleration with both abdominal aorta and cava cUpped, as in making the pocket. At the end of the experiment, when the blood pressure had fallen to 40 to 50 mm. of mercurj", no sensible accelera- tion, or with stronger stimulation only a small one, could be elicited through the sciatic and the rise of blood pressure was also small. But it would surely be absurd to attribute this belated failure of a reaction which had been well obtained after the adrenalectomy to the absence of a reflexly excited outpouring of epinephrin. As to varying the strength of stimulation, it should be stated that we did not consider there would be any point in merely comparing the reaction obtained before and after the removal of the adrenals with 350 G. N. STEWART AND J, M. ROGOFF practically the same nominal strength of stimulus, since the excitability of the reflex mechanisms cannot be counted upon to remain the same, especially where considerable changes of blood pressure have occurred. What we tried to do in all the experiments was to elicit as large a reaction as possible, both before and after elimination of the adrenals, using for this purpose the strength of stimulation which seemed most effective. The strongest stimuli were not generalty the best. Not infrequently we found that stimuli of the same strength as gave the maximum acceleration before removal of the adrenals did so after their removal also. But sometimes it was necessary to increase the stim- Fig. 14. Parts of blood pressure tracings from cat 448. A, before and B, a portion commencing 26 seconds after beginning of sciatic stimulation, after excision of both adrenals. Reduced to four-fifths. ulus. This is mentioned because if the same strength of stimulus is employed before and after removal of the adrenals, a small, or even no acceleration might be obtained after the operation, which would not mean that the reaction had disappeared because of the loss of the adrenals, but that the excitability of the mechanisms concerned in it had diminished. The same is, of course, true of the vasomotor reflex, which we took as an indicator of effective stimulation. Portions of a curve from cat 448, showing acceleration on sciatic stimulation after removal of the adrenals, are reproduced in figure 14, and the greater part of the curve on a reduced scale in figure 15. EPINEPHRIN OUTPUT AND RATE OF DENERVATED HEART 351 Protocol. Cat 437; female; weight, 1.61 kgm. Left superior cervical ganglion excised one month previously. Under urethane (3 gm.) cut vago-sympathetics, excised stellate ganglia, prepared central end of left sciatic for stimulation. Rate Pressure 11 :46 a.m. Before sciatic stimulation^ 260 116 5 seconds after beginning stimulation 274 150 11:50 a.m. Opened abdomen, tied off renal arteries and veins 11 :55 a.m. Before sciatic stimulation 253 60 10 seconds after beginning stimulation 264 84 12:00 m. Before stimulation of sciatic with abdominal aorta clipped 250 64 2 seconds after beginning stimulation 270 88 15 seconds after beginning stimulation 273 12:05 p.m. Before stimulation of sciatic with abdominal aorta and cava clipped 255 60 4 seconds after beginning stimulation 278 84 12:10 p.m. Excised both adrenals 12:11p.m. Before sciatic stimulation 203 44 4 seconds after beginning stimulation 232 59 2:15 p.m. Before sciatic stimulation 214 42 10 seconds after beginning stimulation 225 53 J •^/V*^^^^*^^^ yV^^" /^'^ . (g/^ Fig. 15. Blood pressure curve from cat 448. Sciatic stimulation after excision of both adrenals. Reduced to one-half. In cat 437, in which a superior cervical ganglion had been excised a month previously and in which, therefore, eye reactions were also available, the adrenalectomy was performed at a different stage of the experiment, and after the abdomen had been opened for some time and observations made on the results of sciatic stimulation with the abdom- inal aorta and cava clipped, as in the collection of blood from the adrenals. The demonstration that the heart reaction is obtained after removal of the adrenals was, if anything, more striking than in many of the other experiments. Sciatic stimulation yielded a maximum ^ The eye reactions were those described in the footnote to protocol of cat 190, p. 319. 352 G. N. STEWART AND J. M. ROGOFF acceleration of 14 beats per minute before the abdomen was opened. After opening the abdomen and tying off the renal vessels, the blood pressure had fallen to 60 mm. of mercury as compared with 116 mm. at the beginning of the experiment. An acceleration of 11 beats was given by stimulation of the sciatic. The abdominal aorta was now clipped and a short interval allowed for any effect on blood pressure and heart rate to develop, and then the sciatic was stimulated. The heart rate compared with that immediate^ before stimulation was increased by 23 beats per minute. An equal acceleration was given in another observation, in which the abdominal aorta and inferior cava (above the junction of the iliac veins) were clipped in the same way shortly before stimulation. In such observations the clips were not removed till the portion of the curve to be used for counting the heart beats had been completed. Both adrenals were then excised and there- after an acceleration of 29 beats was caused by stimulation of the sciatic. It is surety impossible to reconcile the results of such an experiment with Cannon's statements: a, that after opening the abdo- men it is very rare to obtain any acceleration of the heart by sciatic stimulation; h, that preparing the cava pocket for collection of adrenal blood, as practised by Biedl, Hoskins and McClure (14), ourselves and other investigators, renders it impossible to detect the great increase in epinephrin output which according to him is indicated by the heart reaction; and c, that removal of the adrenals abohshes the reaction because it is due entirely to a reflexly excited increase in the secretion of epinephrin. The last experiment to which we shall allude (cat 447) was one in which sciatic stimulation caused only a small acceleration at the begin- ning of the experiment, accompanied by insignificant changes of blood pressure. The cat was only half grown, but it is not known whether this had anything to do with the relatively small effects. The reaction was not essentially modified b}^ removal of the adrenals, or subse- quently bj^ clipping the abdominal aorta. The blood pressure was diminished from about 100 mm. to little over 50 mm. of mercury after the adrenalectomy, and the heart rate was also diminished from over 200 to 175 beats a minute. As the blood pressure fell still lower toward the end of the experiment, the heart rate diminished further to 160 beats a minute. EPINEPHRIN OUTPUT AND RATE OF DENERVATED HEART 353 Protocol. Cat 447; female; weight, 1.24 kgm. Under ether cut vago-sym- pathetics; excised stellate ganglia; prepared central end of left sciatic for stimulation. Rate Pressure 11 :00 a.m. Before sciatic stimulation (8 cm.) 203 115 Just after end of stimulation 218 110 11:02 a.m. Before sciatic stimulation (10 cm.) 210 100 12 seconds after beginning stimulation 207 100 11:05 a.m. Before sciatic stimulation (9 cm.) 205 110 10 seconds after beginning stimulation 209 112 11:11a.m. Before sciatic stimulation (7 cm.) 206 110 12 seconds after beginning stimulation 209 118 20 seconds after beginning stimulation 212 11:18 a.m. Before sciatic stimulation (9 cm.) 202 104 15 seconds after beginning stimulation 208 108 11:40 a.m. Opened abdomen, excised both adrenals 11 :43 a.m. Before sciatic stimulation (7 cm.) 175 53 15 seconds after beginning stimulation 181 63 40 seconds after beginning stimulation 184 11 :46 a.m. Before sciatic stimulation (9 cm.) 179 55 15 seconds after beginning stimulation 182 55 30 seconds after beginning stimulation 183 55 11 :48 a.m. Before sciatic stimulation (5 cm.) 172 54 Just after beginning stimulation 177 10 seconds after beginning stimulation 172 54 11:53 a.m. Before stimulation of sciatic with abdominal aorta clipped (8 cm.) 172 55 6 seconds after beginning stimulation 171 59 After end of stimulation 179 12:10 p.m. Before stimulation of sciatic with abdominal aorta clipped (7 cm.) 160 49 6 seconds after beginning stimulation 160 51 25 seconds after beginning stimulation 166 12 : 15 p.m. Before sciatic stimulation (8 cm. ) 163 35 10 seconds after beginning stimulation 166 48 30 seconds after beginning stimulation 164 The slowing of the denervated heart after interference with the epinephrin output. It is not easy to demonstrate conclusively that elimination of the epinephrin output of the adrenals causes a slowing of the dener- vated heart, indicating that the ordinary output, under the conditions of our experiments, is capable of exerting an influence upon the heart. For when the elimination of the epinephrin output is brought about by removal of the adrenals in an acute experiment, the result may be complicated by a slowing associated with a fall of blood pressure. Nevertheless, as stated previously (15), "evidence of a relation of the 354 G. N. STEWART AND J. M. ROGOFF normal epinephrin output to the heart rate seems to be afforded by a comparison of the effects produced in cats on the rate by excision of the stellate ganglia after previous section of the vagi when the adrenal epinephrin is normally entering the circulation and in the absence of epinephrin." This is illustrated in table 1. Although the number of animals is small, the results are suggestive. In the three cats which had been subjected to the adrenal operation mentioned there was a decided diminution in the heart rate after removal of the ganglia. In cat 235 the rate was unusually slow TABLE 1 BEFORE AFTER REMOVAL OF 8TELLATE8 CAT Rate Pres- sure Rate Pres- sure 231 216 176 1 (a)152 (b)164 104 82 233 193 87 168 76 235 142 106 1 (a)144 (b)129 90 78 234 266 152 255 122 236 250 184 1 (a)249 (b)233 168 118 237 192 126 1 (a)187 (b)178 110 104 198 172 120 188 114 195 181 78 145 52 REMARKS 33 days after adrenal operation (b) 10 minutes later than (a) 36 days after adrenal operation 34 days after adrenal operation (b) 2 minutes later than (a) (b) 7 minutes later than (a) (b) 4 minutes later than (a) f Stellates removed an hour after beginning , of experiment after repeated splanchnic stimulation In the first three cats the right adrenal had been removed and the nerves of the left cut. The last 5 cats were normal animals. All were anesthetized with urethane. (142 beats per minute) before the excision of the stellate ganglion and immediately after excision of the second ganglion it was 144. But it at once began to fall and in 2 minutes was 129. An acceleration is not infrequently seen during the operation for removal of the ganglia, due it may be supposed to reflex stimulation of the accelerantes or direct mechanical stimulation or possibly to indirect effects produced through vasomotor changes. The full effect of the elimination of the accel- erator nerves may then not be seen for a minute or two. In cat 231 the epinephrin output as estimated on adrenal vein blood by assay on rabbit intestine segments could not have been one-twenty- EPINEPHRIN OUTPUT AND RATE OF DENERVATED HEART 355 fifth of the normal average. In cat 233 it could not have been more than one-twentieth of the normal average (intestine and pupil assay), and in cat 235 it could not have been one-three-hundredth of the nor- mal; even the adrenal blood sample with the slowest flow (0.6 gm. per minute) was shown by the intestine segment test to have a smaller concentration than 1 : 300,000,000 adrenalin. In the five normal cats in table 1 only in one was a decided slowing of the heart observed after excision of the stellate ganglia and this was associated with a marked fall of blood pressure. TABLE 2 CAT BEFORE AFTER REMOVAL OF REMAINING ADRENAL Days after operation Rate Pressure Rate Pressure 441 219 100 214 86 7 444 131 141 137 138 8 450 173 102 174 94 34 438 195 104 1 (a)195* (b)130 74 40 3 439 210 125 178 54 5 44D 226 134 212 124 6 443 180 116 182 83 7 445 161 128 143 86 5 446 185 115 147 164 57 80t 6 In the first three cats the right adrenal had been excised and the nerves of the left cut. In cat 445 the left and in the others the right adrenal had been re- moved. Cats 440, 441, 443, 444, 445 were etherized, the others were under urethane. * (a) immediately and (b) 4 to 5 minutes after removal of adrenal. The blood pressure was falling continuously during this time. t After injection of Ringer's solution. In table 2 are shown the heart rates and blood pressures before and after removal of the remaining adrenal from three cats in which the right adrenal had been excised and the nerves of the left cut, and in seven cats from which one adrenal had been removed at a pre\'ious operation. The vago-sympathetics had been cut and the stellate ganglia excised at the beginning of the experiment. In the three cats whose remaining adrenal had been denervated no diminution in the pulse rate was caused by excision of the gland. As already remarked, the fact that the blood pressure was practically unaltered by removal 356 G. N. STEWART AND J. M. ROGOFF of the pre\'ioiisly denervated adrenal in these cats makes it difficult to estimate the influence of the suppression of the epinephrin output from the gland upon the result. In four out of the other seven cats a sub- stantial slowing of the heart was found after removal of the remaining adrenal. In another of the seven cats (438) although the rate was unchanged immediately after removal of the remaining adrenal the blood pressure was steadily falling and 5 minutes later the pulse rate was only 130. In table 3 are given the heart rates and blood pressures in five normal cats before and after removal of both adrenals at one operation, the vago-sympathetics having been cut and the stellate ganglia excised at the beginning of the experiment. In every case there was a diminu- TABLE 3 CAT BEFORE AFTER REMOVAL OF BOTH ADRENALS ANESTHETIC Rate Pressure Rate Pressure 447 203 115 175 53 Ether 448 218 132 165 90 Ether 449 285 125 228 70 Urethane 437 256 116 203 44 Urethane 195* 146 44 131 38 Urethane * In cat 195 the adrenals were removed about an hour after removal of stel- lates. The rate just before removal of the adrenals ^yas 145. tion in the heart rate, but this again was in every case associated with a fall of blood pressure. Table 4 shows the heart rates and blood pressures in fourteen nor- mal cats after section of the vago-sympathetics and excision of the stellate ganglia. In three dogs anesthetized with morphine and ether the pulse rates after denervation of the heart were 106, 153 and 173, beats per minute, with blood pressures of 92, 72 and 38 mm. of mercury respectivel^^ That different anesthetics may affect the pulse rate of the denervated heart differently may be assumed, e.g., chloroform diminishes the rate in dogs according to v. Anrep (1). So far as can be judged from a relatively' small number of observations in our own experiments, the difference between ether and urethane was not conspicuous. EPrNEPHRIN OUTPUT AND RATE OF DEXERVATED HEART 357 If all the results on the cats are brought together, some further suggestive points seem to emerge. Thus, in twelve out of twenty^ normal eats with both adrenals intact the pulse rate was over 200 a minute after denervation of the heart. In two out of seven cats with one adrenal previously removed and in onlj^ two out of nine cats with one adrenal previously removed and the nerves of the other cut, was the pulse rate over 200 after denervation of the heart. The average pulse rate of the twenty cats with both adrenals intact, after denerva- tion of the heart, was 209 beats per minute; the average for the nine cats whose epinephrin output had been previously interfered with by excision of one adrenal and section of the nerves of the other 170 beats TABLE 4 CAT RATE PRESSURE CAT BATE PRESSURE 201 177 54 190 250 123 202 145 92 191 240 124 215 219 134 196 212 72 175 188 198 233 60 176 82* 199 137 113 176 160 200 210 84 177 185 132 436 203 56 179 169 80 * 2 hours after the first observation. In cats 201, 202 and 215 the right adrenal had been excised and the nerves of the left cut 18 days, 8 days and 59 days respectively before the operation, the others were normal cats. All were anesthetized with urethane, cat 436 more deeply than the rest. per minute; and the average for the seven cats from which one adrenal had been previously removed 184 beats per minute. A possible influ- ence of the previous operation as such, apart from interference with epinephrin output, where the interval was only a few days is not ex- cluded, but is not discernible in the tables. ^ In 4 additional normal cats the heart rates and blood pressures after de- nervation of the heart, but before adrenalectomy, were 266 (214), 284 (145), 265 (145), and 150 (66). In the last cat the abdomen had been opened before the heart was denervated. After removal or ligation of both adrenals the corre- sponding numbers were 222 (114), 283 (102), 255 (130), and 143 (57). In a fifth cat the rate after denervation of the heart w^as 248 and the blood pressure 150 mm. of mercury. THE .AMERICAN" JOURN.\L OF PHTSIOLOGT, VOL. 52, NO. 2 358 G. N. STEWART AND J. M. ROGOFF DISCUSSION AND SUMMARY It has been shown by us that the acceleration of the heart caused by stimulation of the central end of the sciatic is in no way a reaction by which the rate of output of epinephrin from the adrenals can be esti- mated, or changes in that rate demonstrated, as claimed by Cannon. This is proved by the following facts : a. Clipping of the adrenal veins has no demonstrable influence upon the occurrence and magnitude of the heart reaction caused by sciatic stimulation, although it markedly diminishes or abolishes reactions which are known to be genuine reactions for epinephrin, such as the dilatation of the pupil following stimulation of the peripheral end of a splanchnic nerve. b. Acceleration of the denervated heart on sciatic stimulation is well obtained in cats which have been allowed to survive after removal of one adrenal and section of the nerves of the other, an operation which is known to abolish or greatly diminish the epinephrin output. In such cats the reaction is still elicited after the remaining adrenal has been removed. c. Good acceleration of the heart can be elicited by stimulation of the sciatic in cats from which both adrenals have been removed, either in two operations with an interval for recovery interposed, or at one operation. d. When tlie reaction disappears after removal of the adrenals this is not because of the absence of increased epinephrin discharge on stimulation of the sciatic but for other reasons, such as deterioration in the condition of the animal (fall of blood pressure, etc.) which interferes with the reflex or reflexes necessarily involved in the reaction or with the capacit}^ of the heart to markedh^ accelerate its beat. e. Contrary to Cannon's statement, the reaction is well obtained after opening the abdomen. It can be elicited after ligation of the renal vessels, abdominal aorta and inferior cava, as practised in forming a cava pocket for collection of adrenal vein blood. If the reaction indi- cates a greatly increased output of epinephrin reflexly induced by stimulation of the sciatic, as assumed ])y Cannon, we could not have failed to detect the increase by the direct method of collecting adrenal vein blood and assaying its epinephrin content on rabbit segments. But our results were negative (16). As regards the real mechanism of the acceleration of the denervated heart caused by sciatic stimulation, we desire to point out, once for EPINEPHRIX OUTPUT AND RATE OF DEXERVATED HEART 359 all, that the onus of explaining this probably complex indirect reaction, which Cannon erroneously interprets as indicating increased epinephrin secretion, does not rest upon us at all. It is for Doctor Cannon to exclude, if he can, bj' control experiments, other possible factors in the reaction which he attributes solely to epinephrin. Our position is simply this. We have investigated the influence of stimulation of the sciatic and brachial nerves upon the rate of epinephrin output by a direct method, correct in principle and free from ambiguity, and have obtained negative results. Doctor Cannon states that by means of an indirect method (the denen'ated heart reaction) he obtains a positive result. We show that this reaction cannot yield any information as to the rate of epinephrin output from the adrenals or as to changes in that rate, since it is obtainable when the epinephrin output of the adrenals is aboHshed. And here we are entitled to rest our case, not. of course, claiming that sensory stimulation can?wt increase the epineplirin out- put, but that no increase has hitherto been proved. However, certain fairly obvious suggestions may be made as to factors which may play a part in the heart reaction under discussion, a reaction probably made up of more than one component. One is the larger amount of epinephrin sent through the coronarj- circulation and perhaps the greater concentration of it, owing to the vasomotor changes produced by stimulation of the sciatic (11), (15). It may be pointed out that even if Cannon's statement that the reaction cannot be obtained in the absence of the adrenals had been found correct, that of itself would only have sho^\Ti that epinephrin is essentially concerned. For it might be due to the redistribution of the epinephrin without any increase in the rate of output. Cannon attempts to invalidate this suggestion by an experiment in which he prevents a rise of pressure in the carotid during sciatic stimulation by compression of the chest, and yet obtains an acceleration of the heart. Now this is a quite complex experiment, and Doctor Cannon is doing several other things which may affect the heart besides keeping the pressure in the aorta constant. One of the things he is doing is impeding the venous return. The epinephrin, even if its rate of output remains unchanged, must, therefore, be diluted with a smaller proportion of indilTerent blood in the right heart. Blood with a greater con- centration of epinephrin must accordingly be passing through the coronaries, and as the concentration will increase in the same measure as the slowing in the venous return necessary to prevent rise of pressure, approximately the same amount of epinephrin will pass through the coronaries per unit of time as with a similar sciatic stimulation without compression of the chest. If then the ordinary output of epinephrin was a factor in the acceleration without com- pression it may be expected to exert the same influence during compression. In other words, an increase in the amount of epinephrin passing per unit of time through the coronary circulation during sciatic stimulation is not prevented by compressing the chest, so as to keep the blood pressure in the aorta from rising, 360 G. N. STEWART AND J. M. ROGOFF even if no increase in the rate of output of epinephrin has occurred, and the experiment is without significance for the question at issue. That no further acceleration occurred when the blood pressure rose after releasing the chest, is also just as intelligible, so far as epinephrin is a factor, on the assumption that the epinephrin output was not increased as on the assumption that it was increased by stimulation of the sciatic. For if the epinephrin concentration remained unchanged and the coronary blood flow was increased on decompressing the chest, an increased amount of epinephrin would pass through the coronaries per minute whether the output had been augmented by stimulation of the sciatic or not. The fact is, however, that with the increase in the venous inflow to the heart the concentration of epinephrin in the blood of the right heart must be proportionally diminished. But why, in any case, should a further increase in the heart rate have been expected, since the acceleration was already 36 beats a minute, the same as without compression of the chest? As there is no possi- bility that epinephrin is the sole factor in the heart reaction, as elicited reflexly, the point need not be labored. Whatever other factors are ordinarily concerned in the reaction, apart from the rise of arterial blood pressure, may be expected to act as well during compression of the chest as before. The possibility that a special factor, the gross interference with the mechanism of the heart, especially the filling and pressure of the right side and with the respiration might exert an influence in this experiment is not excluded. We ourselves made two experiments (cats 198, 199) in which the rise of pres- sure on sciatic stimulation was largely prevented by hemorrhage, controlled by a mercury valve. The animals were anesthetized with urethane. In cat 198 before sciatic stimulation the pulse was 225, the blood pressure 78; during stimu- lation the pulse rate rose to 253 and the blood pressure to 192 mm. of mercury. In the next observation the pulse was 233 and the blood pressure 60 before stimu- lation. During stimulation the blood pressure was prevented from rising beyond 90 mm. of mercury, and the pulse rate increased only to 242 beats per minute. Blood (mixed with salt solution) was reinjected. Before stimulation of the sciatic the pulse rate was 272, the pressure 96. During stimulation the pulse rate rose to 286 with a pressure of 168 mm. of mercury. The sciatic was now stimu- lated while the pressure was prevented, by hemorrhage, from changing (it rose from 88 to 92). The pulse rate before stimulation was 258 and during stimula- tion 259. This experiment might seem to show that the acceleration was largely prevented by keeping the blood pressure from rising. But in the other cat a different result was obtained. In cat 199 the following pulse rates and blood pressures, the latter in parentheses, were recorded. Before stimulation 137 (113), during stimulation 215 (196). Before stimulation 130 (108), during stimulation with hemorrhage 193 (116-90). Before stimulation 144 (100), during stimula- tion 206 (162). Before stimulation 145 (102), during stimulation with hemorrhage 194 (115). Only these two experiments were made. For on reflection it was seen that this method also could not lead to any definite conclusion. If the epinephrin were the only factor in the acceleration, its concentration in the blood coming to the heart would increase as the mass of the circulating blood diminished, even provided that no increase were taking place in the rate of output. Besides the direct accelerating action of epinephrin upon the heart there is another way in which the normal output of epinephrin may possibly play a part EPINEPHRIN OUTPUT AND RATE OF DENERVATED HEART 361 in the acceleration caused by stimulation of the sciatic, by sensitizing the heart to the action of other factors, such as a rise of blood pressure, which in the absence of epinephrin might not be so effective. In this connection we recall the obser- vation of V. Anrep (1) on the influence of adrenalin upon the power of the heart to adapt itself by changes in its tone to changes in the arterial pressure. Whatever share epinephrin may take in the acceleration reaction it cannot be the only factor and is probably not the most important one, since excellent heart reactions can be obtained in the absence of the adrenals, provided that good vascular reflexes, as evidenced by the change of blood pressure, are elicited. The most obvious of the changes caused by stimulation of the central end of the sciatic, the rise of blood pressure, is the one which seems to be most intimately related to the heart acceleration. Since this relation is seen after, as before, elimination of the adrenals, the most direct suggestion is that the better blood flow through the coronary circulation is an important factor in the acceleration, either by raising the nutritive condition and the excitability of the mechanism in which the beat originates or by acting upon a local accelerator mechanism. An action of the increased blood pressure as such is not excluded. Indeed, long ago Johansson (17) pointed out that the acceleration of the heart (after section of the vago-sympathetics and excision of the stellate ganglia) caused by stimulation of the peripheral end of a splanchnic nerve and of the cut cervical cord, was dependent on the abruptness of the rise of pressure. At the time of Johansson's work, nothing was known of the secretory innervation of the adrenals, and it might be asked whether the whole acceleration in his experiments was not due to increased epinephrin output. This question, in our opinion, must be answered in the negative. For stimulation of the cord with one splanchnic cut produced in general a much greater acceleration than stimulation of one splanchnic, without any obvious reason why it should have caused a greater liberation of epinephrin, and this greater acceleration was accompanied by a larger rise of blood pressure. Further the dogs were curarized and curara (18) depresses the conductivity of the efferent epinephrin secretory path. We do not, however, know how much importance should be attached to this, as our work with curara was done on cats and was concerned only with the spontaneous liberation of epinephrin. It is significant that the accelerations observed by him, taken in relation to the blood pressure changes, are of the same order of magnitude as those obtained by us with splanchnic and sciatic stimulation, whether with the adrenal veins clipped or in the absence of the adrenals. From our o^n observations, recorded in the preceding pages, it cannot be doubted that a rise of pressure, caused by stimulation either of the sciatic or the peripheral end of the splanchnic nerve, is associated with an acceleration of the denervated heart, unrelated to any immediate action of epinephrin. The maximum acceleration, as in Johansson's observations, may not be reached till the blood pressure has again begun to decline. We agree with Johansson and with Lehndorff (19) that the manner in which the rise of pressure is produced is important as regards its action upon the heart. The latter observer remarks that raising the pressure by compressing the aorta seldom causes an acceleration, and that when acceleration is caused it is only slight. He was never able by inducing an asphyxial rise of pressure to elicit 362 G. N. STEWART AND J. M. ROGOFF the characteristic changes in the heart's action which he found on splanchnic stimulation. Guthrie and Pike (20) also found compression of the aorta ineffi- cient. We have seen a definite acceleration, e.g., in cat 190 (see protocol), clip- ping the aorta in the thorax increased the pulse rate from 249 to 260 beats a minute but this was a much smaller acceleration than that caused by splanchnic stimulation just before, although the increase of arterial pressure was greater when the aorta was clipped. In other cases we have seen no acceleration when the aorta was clipped in the thorax. Clipping of the abdominal aorta has been sometimes seen to cause some acceleration with a moderate rise of pressure in the absence of the adrenals. The character of the blood sent to the heart is, not the same when the thoracic as when the abdominal aorta at the bifurcation is clipped, the liver and other viscera not being interfered with in the latter case. Apart altogether from the possible effect of epinephrin when the splanchnic is stimulated, the mechanical conditions under which the heart works are very- different when the aorta is clipped and when the central end of the sciatic or the peripheral end of the splanchnic is stimulated, particularly as regards the venous inflow. Cannon seems to criticise us for quoting the experiments of Guthrie and Pike on the influence of increased pressure of the perfusion fluid in accelerating the excised heart, as if this represented the sum total of our knowledge. We quoted this work, which was done in the laboratory of one of us, in a footnote to another paper (11), to show, and we believe it does show, that under certain conditions the heart, deprived of extrinsic innervation, can respond to changes of blood pressure by very considerable changes in frequency. When we stated that there is nothing strange about an increase in the rate of the denervated heart in sihi when the central end of the sciatic or the peripheral end of the splanchnic is stimulated, and went on to say that it is obviously dependent upon the better blood flow through the coronary vessels, we had before us much of the evidence given on preceding pages that marked acceleration could be produced by stimulation of both of these nerves, in the absence of any possible output of epinephrin from the adrenals, and that the acceleration was associated with a rise of arterial pressure. Of the results of other observers on the heart in situ, we had in mind particularly the elaborate paper of Johansson (17) already referred to. The experiments of Martin and of Knowlton and Starling on the heart-lung preparation, which Cannon cites, were familiar to us, but we saw no point in quoting in a footnote observations which threw no light upon the acceler- ations we were obtaining by stimulation of the sciatic, under conditions which eliminated the liberation of epinephrin from the adrenals. Of course, the vaso- motor reactions expressed in the rise of blood pressure under discussion might possibly have other actions upon the denervated heart, in addition to their most obvious action, the increased blood flow through the coronaries. It has been pointed out by various writers that it is not the same thing, as regards the heart rate, whether changes are induced in the arterial or in the venous pressure. In the case of sciatic stimulation it might still be argued that the rise of pres- sure is only a sign that the stimulus is effective for some other reflex or reflexes on which the acceleration depends essentially. This is theoretically true, but any reflex which affects the composition of the blood, as sciatic stimulation may do (by causing hyperpnoea, increased reflex muscular action, possibly increased EPINEPHRIN OUTPUT AND RATE OF DEXERVATED HEART 363 mobilization of sugar or other changes in the liver through the splanchnics) , must affect the heart concomitantly with the vasomotor reflex which increases the blood flow through the coronaries. In any case, the experiments on stimu- lation of the splanchnic in the absence of epinephrin output indicate the rise of arterial pressure as the most obvious of the factors associated with the heart reaction studied. In two cats (one normal, 447, and one in which the epinephrin output had been interfered with, 444, both etherized), we have seen an acceleration, when* sciatic stimulation elicited no rise of pressure either before or after eliminatiom of the adrenals. But then there was evidence of effective stimulation, not only in the increased respiratory movements and respiratory blood pressure waves but also in a small depression of the blood pressure, succeeded by some rise after stoppage of the stimulation. In such a case the question presents itself, whether it is certain that in every individual the heart is completely severed from the central nervous system by section of the vagi and excision of the stellate ganglia. We repeat that, having shown, as we believe, that Cannon's supposed proof, by means of the heart reaction, of augmented epinephrin output through stimu- lation of the sciatic is illusory, we do not consider that the explanation of the mechanism of the reaction is our concern. We have simply made some suggestions. Nor do we judge it necessary to discuss in this paper his supposed proof of the augmenting action of asphyxia and of emotional excitement upon the output. Clearh' all his conclusions upon this matter stand or fall together. It may be mentioned, however, that we have observed acceleration of the heart, induced by asphyxia, after removal of the adrenals. BIBLIOGRAPHY (1) V. Anrep: Journ. Physiol., 1912, xlv, 307. (2) Elliott: Journ. Physiol., 1912, xliv, 374. (3) Pearlman and Vincent: Endocrinology, 1919, iii, 121. (4) Stewart and Rogoff: Journ. Pharm. Exper. Therap., 1916, viii 479. (5) Cannon: This Journal, 1919, 1, 399. (6) Cannon and de la Paz: This Journal, 1911, xxviii, 64. (7) Gley and Quinquaud: Compt. rend., 1916, clxii, 86. (8) YooNG and Lehman: Journ. Physiol., 1908, xxxvii, p. liv. (9) HosKiNS AND McClure: This Journal, 1912, xxx, 192. (10) Bazett: Journ. Physiol., 1920, liii, 320. (11) Stewart and Rogoff: This Journal, 1918, xlvi, 96. (12) Cow: Journ. Physiol., 1914, xlviii, 443. (13) Stewart and Rogoff: Journ. Pharm. Exper. Therap., 1917, x, 1. (14) HosKiNS AND McClure: Arch. Int. Med., 1912, x, 343. (15) Stewart and Rogoff: Journ. Pharm. Exper. Therap., 1919, xiii, 95. (16) Stewart and Rogoff: Journ. Exper. Med., 1917, xxvi, 637. (17) Johansson: Arch. f. Anat. u. Physiol., 1891, 103. (18) Stewart and Rogoff: Journ. Pharm. Exper. Therap., 1919, xiv, 351. (19) Lehndorff: Arch. f. .\nat. u. Physiol., 1908, 362. (20) Guthrie and Pike: This Journal, 1907, xviii, 14. THE EFFECT OF ACIDS, ALKALIES AND SALTS ON CATALASE PRODUCTION W. E. BURGE From the Physiological Laboratory of the University of Illinois Received for publication March 27, 1920 The acids used were hydrochloric, propionic, acetic and butyric; the alkahes or alkahne salts, sodium carbonate, sodium phosphate, ammonium carbonate and sodium acetate; the neutral salts, disodium phosphate, ammonium chloride; and the acid salt, monosodium phos- phate. The animals were rabbits and dogs. The method of adminis- tration and the amounts of the substances used will be given in the description of the experiments. The catalase of the blood was deter- mined before as well as at intervals after introducing the materials into the alimentary tract of the animal. The determinations were made by adding 1 cc. of blood to hydrogen peroxide in a bottle and the amount of ox^'gen liberated in 10 minutes was taken as a measure of the catalase content of the cubic centimeter of blood. The dog's blood was used undiluted while the rabbit's blood was diluted 1 to 5 with 0.9 per cent sodium chloride. The effect on the catalase of the jugular blood of the introduction of the acids, alkahes and salts into the ali- mentary tract of rabbits and dogs is shown in figure 1. The figures along the ordinates represent percentage increase or decrease in catalase and those along the abscissae time in minutes. It may be seen under acids that the introduction of 1.5 gram per kilo of acetic acid dissolved in 75 cc. of water per kilo into the stomach of rabbits increased the catalase of the blood 22 per cent in one rabbit and 12 per cent in another in 90 minutes; proprionic acid increased the blood catalase 16 per cent; butyric, 10 per cent; that 1.5 gram per kilo of hydrochloric acid dissolved in 75 cc. of water per kilo decreased the blood catalase 7 per cent in one rabbit, 9 per cent in another rabbit; and that 3 grams per kilo of hydrochloric acid decreased the catalase 28 per cent. While these percentages are in most cases small, at the same time they represent large differences in the amount of oxj^gen liberated by the blood from hydrogen peroxide. An increase or decrease 3U EFFECT OF ACID, ALKALI AND SALT ON BLOOD CATALASE 365 of 20 per cent in catalase, for example, represents an increase or decrease of about 100 cc. of oxygen liberated. It may be seen further in figure 1 under carbonates, phosphates and sodium acetate that the introduction of these substances into the upper part of the intestine of dogs produced an increase in the catalase of the blood of the jugular. The amounts of these salts used were 10 grams per kilo dissolved in 75 cc. of water per kilo, with the exception of ^30 U-l 1 5 CO 20 b^io a U-l ^20 AC\D5 CARBONATES PHOSPHATES. 50DIUM ACETATE /\z\ 26 // Z6 / zz A 22 / J ^ 16 ^ 18 dk /./^ IZ lA f// r •; 14 c f , ^'° /^ W / ^S4 p f ^^''' / /^ r / p 7 9 \> \ \ \ 2S 30 60 90 30 60 90 JO 60 90 TIME IN MINUTES 30 60 90 Fig. 1. Curves showing effect in vivo of the substances named in the chart on the catalase content of the jugular blood. ammonium carbonate, 0.9 gram per kilo of this salt being used. After opening the abdominal wall of dogs while under ether, the materials were injected into the upper part of the small intestine. The wound was sewed up and the ether discontinued after the introduction of the substances. The question that arises in this connection is, how is the increase as well as the decrease in catalase, observed in the preceding experi- 366 W. E. BURGE a|t : / f \ 1 h 1 a~ <; 1 » lad ra 9 11 'i^. r 1 i i s -- ^ lati cu 9 )1 =; i S S^ ■— ' — 1 S^ ^ t4 J ■"' )0I ki ^ - 1' « I i \ ° '\ § s N \ M isd ffi 19 31 Ul r SJi « K / 1 / t; ' '• ^S s ^ -1 — ^^ ~^"- « •< kJ »UJ 3 6-0 %^ S -J o -J K 13 S S.1 f-- - \ ^ b 1 =:j ii, W • cu. ( 1 < j 2 < 5 ■o s - -~; li^- V ■ su s i •V ! = = -J O Q! 5S ■^. V - \ "^ Iti sj 5^ V M ■•^ £)! 1 o >- ' i 3S o \ \ 1 !H' ^g s^ <■ yii %^ u S s a. - § 1 ( 5 t ■~-~^ "- -':. '-^ .,„^ 'i^ ~-- •^ ^ w "B -1 S a O as S / ft \' SI f" -S ^ ^ i^. M 1 c US s-i T Sq _i> <1) 03 (U > o r^ u « -M ,^ n D t3 r. C o o o •"^ -u •n ns ^ ^ (1) +i ^ o o fl T3 QJ *^^ a> ^ ^ h 3 eri -) QJ o +3 s Z y o lO ^ -t^ ^ t3 F -»J %< n =! o o o h O « ^ o CJ o C u. > 03 > ;3 fl (Tt a O •^H 3 -tJ fl C 3 o 3 o n o J !? o s c3 A 0) o s a 0) o m 'd > o r» o OJ o 3 -Q > ^ 8 O (M' o -M 1/) ■^ c 03 c OJ ■o -! \^ c o J3 O S u -tj 03 EFFECT OF ACID, ALKALI AND SALT OX BLOOD CATALASE 367 ments, brought about. We (1) had already' found that the ingestion of food, for example, produced an increase in catalase by stimulating the alimentary glands, particularly the liver, to an increased output of this enz3'me, and that narcotics produced a decrease in catalase by decreasing its output from the Uver and by direct destruction. So we naturally turned to the aUmentary glands, particularly the liver, in looking for an explanation of the change in the amount of the blood catalase observed in the preceding experiments. After opening the abdominal wall of a dog while under ether, catalase determinations were made using 1 cc. of blood from the liver, portal and jugular veins. The material to be studied was then introduced into the intestine and catalase determinations were made at fixed intervals. The results of the determinations are given in figure 2. The figures along the ordinate indicate amounts of oxygen hberated from hydrogen peroxide in 10 minutes by 1 cc. of blood, and the figures along the abscissae time in minutes. The continuous line curves were constructed from data obtained from the blood of the Kver, the dash fine curves from the blood of the portal, and the dotted line curves from the blood of the jugular vein. Under acetic it may be seen that previous to the introduction into the intestine of a dog of 1.5 gram per kilo of acetic acid, dissolved in 75 cc. of water per kilo, 1 cc. of blood from the liver liberated 103 cc. of oxygen in 10 minutes from hydrogen peroxide; 1 cc. of blood from the portal liberated 104 cc. of oxygen, and 1 cc. from the jugular 101 cc. of oxygen. Fifteen minutes after the introduction of the acetic acid, the liver blood liberated 130 cc. of oxygen, the portal blood 122 cc. and the jugular blood 118 cc. Similarly, it may be seen that 90 minutes after the introduction of the acetic acid the Uver blood liberated 128 cc. of oxygen, the portal blood 125 cc. and the jugular 124 cc. By comparing these figures it may be seen that the hver blood, particularly during the first fifteen minutes after the introduction of the acetic acid, liberated more ox^'gen from hydrogen peroxide than did the portal blood, and that this blood in turn liberated more oxj^gen than did the jugular blood. This is taken to mean that the acetic acid was stimulating the ahmentaiy glands, particularly the liver, to an increased output of catalase. It may be seen further that proprionic, butyric and hydrochloric acids, ammoniimi chloride, glycocoll, ammo- nium carbonate, sodimn carbonate and the phosphates produced an increase in catalase in a similar manner, namely, by stimulating the hver to an increased production of this enzjTne. 368 W. E. BURGE The question that arises in this connection is, does the increase or decrease in the amount of oxygen Uberated in the preceding experiments represent an actual increase or decrease in catalase, or does it smiply represent a change in catatytic activity brought about by a decrease in the acidity of the hydrogen peroxide. So far as I have been able to find, Jacobson (2) was the first to show that catalytic activity is greatly decreased by acids. This work has been repeated and con- firmed by several investigators (Loevenhart (3) ; Issajew (4) ; Senter (5) ; Winternitz and Rogers (6); Mendel and Leavonworth (7); Bodansky (8)) and extended to include the effect of acid salts, alkalies and alkaUne salts on catalytic activity. The description of a simple experiment will suffice to show the effect of acids and alkaHes on catalytic activity. If catalase determinations are made using equal quantities (1 cc.) of blood and neutral, slightly acid and sUghtly alkaline hydrogen peroxide, it will be found that the amount of oxygen liberated from the neutral and slight!}^ alkahne hydrogen peroxide will be the same, while that fiber ated from the sUghtly acid hydrogen peroxide will be less. By increasing the acidity of the hydrogen peroxide, the amount of oxygen fiberated will be decreased more and more until the action of the catalase is completely inhibited, and no oxygen is liberated. By the addition of an alkali (sodium hydroxide) to the acid hydrogen peroxide, the amount of oxygen liberated will be increased until a certain maximum is reached. Further addition of alkali at this point will begin to decrease the amount of oxygen fiberated. It should be mentioned in this con- nection that alkafies are not nearly so effective in decreasing catalytic activity as the acids. Hence the amount of oxygen fiberated from acid hydrogen peroxide can be increased by simply neutralizing the acid of the hydrogen peroxide by means of an alkali, or it can be decreased by making the hydrogen peroxide more acid. Since in our experiments acids and alkalies were used, it might be thought by some that what we have been observing is not an increase or decrease in catalase, but only a change in catalytic activitj^ due to an increase or decrease in the acidity of the hydrogen peroxide. The following experiments were carried out in an attempt to clear up this phase of the problem. It should be said in this connection that most of these experiments had already been performed time and again in connection with our previous work on catalase, but they were not thought of sufficient importance to publish. Twenty-five cubic centimeters of blood were taken from the external jugular of a dog and defibrinated. It was found that defibrination had EFFECT OF ACID, ALKALI AND SALT ON BLOOD CATALASE 369 little or no effect on the catalase content of the blood. The abdominal wall of the dog was opened while the dog was under ether and 10 grams per kilo of sodium carbonate dissolved in 75 cc. of water per kilo were introduced into the upper part of the small intestine. The wound was then sewed up and the ether discontinued. Ninety minutes later 25 cc. more of blood were drawn from the jugular and defibrinated. The blood drawn before the introduction of the carbonate will be referred to hereafter as normal blood, while that drawn 90 minutes after the introduction of the carbonate will be referred to as carbonate blood. The catalase content of 1 cc of the normal blood and of the carbonate blood was determined using sHghtly acid hydrogen peroxide, and it was found that the carbonate blood liberated about 30 per cent more oxygen than the normal blood. If this increase in the amount of oxygen liberated by the carbonate blood be due to the neutralization of the acid of the peroxide, thus removing the inhibiting effect of the acid, then the difference in the amount of oxygen liberated should be greatly decreased or disappear when neutral or shghtly alkaline hydrogen peroxide is used. Catalase determinations of this same sample of defibrinated dog's blood were accordingly made, using neutral and slightly alkaUne hydrogen peroxide. It was found that the carbonate blood liberated about 24 per cent more oxygen than the normal blood. This experiment would seem to show that only a very small per cent (6 per cent) of the increase in oxygen liberated by the carbonate blood was due to the neutraUzing effect of the carbonate blood, while the remaining 24 per cent was due to an actual increase in catalase. Further experiments bearing on this point of the following kind were carried out. Two cubic centimeters of blood were taken from the jugular of a dog. The catalase content of 1 cc. was determined immedi- ately, while the other cubic centimeter was shaken with 10 cc. of 0.9 per cent of sodium chloride and placed in a water bath at 80°C. until the enzyme was destroj^ed. This tube was shaken at intervals to prevent the formation of a firm coagulum. Ten grams per kilo of sodium carbonate were introduced into the intestine of this animal and 90 minutes later another 2 cc. of blood were drawn from the jugular. The catalase of 1 cc. of this was determined immediately, while the other cubic centimeter was added to salt solution and heated at 80°C. for the same time that the control had been. The amount of oxygen liberated from hydrogen peroxide by the cubic centimeter of normal blood was 120 cc, while the carbonate blood liberated 168 cc. The heated cubic centimeter of normal blood as well as the heated cubic 370 W. E. BURGE centimeter of carbonate blood was added to slightly acid hydrogen peroxide. One cubic centimeter of blood from the same sample was added to each of these bottles and the amount of oxygen liberated was determined. Slightly more oxygen was liberated from the peroxide containing the heated carbonate blood than from that containing the heated normal blood. This result was the same as was obtained in the preceding experiment in that it shows that the neutralizing effect of the carbonate blood is responsible for a very small part of the increase in ox;\'gen liberated by the blood after the introduction of sodium carbonate into the alimentary tract when slightly acid hydrogen peroxide is used. It should be mentioned in this connection that shghtly acid hydrogen peroxide was not used in the experiments on the effect of acids, alkalies and salts reported in this paper, but neutral or shghtly alkaline hydrogen peroxide. Ten cubic centimeters of dog's blood were drawn from the jugular before as well as 90 minutes after the introduction of sodium carbonate into the alimentary tract and defibrinated. The carbonate blood Hberated about 18 per cent more oxygen than the normal blood. Both of these samples were introduced into dialyzing tubes made of collodion and dialyzed against 2 liters of 0.9 per cent sodium chloride for 18 hours. At the end of this time these two samples were removed from the dialyzing tubes and made equal in amount by the addition of 0.9 per cent sodium chloride. Catalase determinations were made using 1 cc. from each of these samples, and it was found that dialyzing decreased very slightly the difference in the catalytic activity of the two samples. It had been previously found that catalase did not dialyze through these membranes to any appreciable extent. If the increase in the catalytic activity of the carbonate blood were due to the presence of the alkali in the blood, it would seem that dialyzing should have done away with the difference in activity between the normal and the car- bonate blood or greatly decreased it. It might be objected, however, that the carbonate in the blood was in some way fixed and rendered non-dialyzable. Hence, the following experiments were carried out to determine if this objection were vahd. Ten cubic centimeters of blood were drawn from the jugular before as well as after the introduction of 10 grams per kilo of sodium carbonate into the intestine of a dog. It was found that the carbonate blood liberated 16 per cent more oxygen than the normal blood from neutral hydrogen peroxide. These two samples of blood were ashed at a temperature too low to decompose sodium carbonate, and the ash of EFFECT OF ACID, ALKALI AND SALT ON BLOOD CATALASE 371 each was introduced into bottles containing equal quantities of neutral hydrogen peroxide. One cubic centimeter of blood taken from the same sample was added to each of these bottles and the amount of oxygen liberated from each was determined. It was found that the bottle containing the ash of the carbonate blood liberated practically the same amount of oxj^gen as that containing the ash of the normal blood. If the increase in catalase after the introduction of alkalies into the intestine were due to the neutralizing effect on the acid of the hydro- gen peroxide, then the introduction of acids into the alimentary tract should decrease the catalytic activity of the blood. In figure 2 it may be seen that the introduction of acids as well as acid salts increased the catalase of the blood instead of decreasing it. Furthermore, if alkalies when introduced into the alimentary tract of an animal increase catalytic activity by neutralizing the acid of the hydrogen peroxide, then the catalytic activity of the blood of the portal should be increased parallel with that of the liver. In figure 2 it may be seen under ammonium carbonate, sodium carbonate and sodium phosphate that such is not the case, but that the catalase of the liver blood is increased much more rapidly than that of the portal. This was interpreted to mean that these substances produced an increase in catalase by stimulating the liver to an increased output of this enzyme. Determinations similar to the preceding were carried out using sodium phosphate instead of sodium carbonate with practically the same results. From these experiments the conclusion may be drawn that while a very small percentage of the increase in the power of the blood to decompose hydrogen peroxide after the introduction of alkalies and alkaline salts into the alimentary tract of animals is due to an increase in the catalytic activity when acid hydrogen peroxide is used, by far the larger part of the increase is due to an actual increase in catalase brought about by the stimulation of the alimentary glands to an increased output of this enzyme. It might be well to mention again in this connection that neutral or slightly alkaline hydrogen peroxide was used in the experi- ments reported in this paper on the effect of acids, alkalies and salts on the blood catalase, hence none of the oxygen liberated in these experiments can be attributed to the neutralizing effect of the alkalies. Chvostek (9) showed that when hydrochloric acid was administered to a rabbit it decreased oxidation, while Lehmann (10) showed that an alkali, such as sodium carbonate, increased oxidation. In figure 1 it may be seen that the administration of hydrochloric acid to rabbits 372 W. E. BURGE produced a decrease in catalase, which is offered in explanation for the decreased oxidation as observed by Chvostek. The following experi- ment will show that the decrease in the blood catalase after the adminis- tration of hydrochloric acid to the rabbit is due in part to the inhibiting effect of the acid on the catalase, and in part to the direct destruction of the enzyme. Ten cubic centimeters of blood were drawn from the jugular of a large rabbit and defibrinated. Two grams per kilo of hydrochloric acid dissolved in 75 cc. of water per kilo were introduced into the stomach of the animal. Ninety minutes later another 10 cc. of blood were drawn and defibrinated. The blood taken before the introduction of the acid will be referred to hereafter as normal blood and that after the introduction of the acid as acid blood. Catalase determinations were made of these two samples of blood and it was found that the acid blood liberated about 15 per cent less oxygen than the normal blood. One cubic centimeter of each of the samples was added to tubes contain- ing 10 cc. of 0.9 per cent sodium chloride and heated at 80°C. until the catalase was destroyed. The heated normal blood as well as the heated acid blood was added to neutral or slightly acid hydrogen peroxide and catalase determinations were made using the normal blood. It was found that the bottle containing the heated acid blood liberated 10 per cent less oxygen than that liberated by the bottle containing the heated normal blood. This is interpreted to mean that of the 15 per cent decrease produced by the introduction of hydrochloric acid into the stomach of the rabbit, 10 per cent was due to the inhibiting action of the acid while the remaining 5 per cent was due to the direct destruc- tion of the enzyme. In figure 2 it may be seen that the introduction of hydrochloric acid into the intestine of a dog increased the blood catalase instead of de- creasing it, as was found to be the case with the rabbit in figure 1. It may be seen further in figure 2 that ammonium chloride is a very effective stimulant to catalase production, 0.9 per cent per kilo being sufficient to bring about a great increase in this enzyme. Since the dog is a carnivorous animal, a relatively large amount of ammonium chloride is formed, when hydrochloric acid is administered, in the neutralization of this acid by ammonia, whereas the rabbit being a herbivorous animal, a relatively small amount is formed. Hence the increase in catalase after the introduction of hj^drochloric acid into the intestine of a dog is attributed to the stimulating effect of the ammonium chloride on the liver giving an increased output of this enzyme. EFFECT OF ACID, ALKALI AND SALT ON BLOOD CATALASE 373 Grafe (11) found that ammonium chloride increased oxidation in the rabbit. Lusk (12) showed that the ingestion of an amino acid, such as glyeocoll, increased oxidation in the dog. The increase in catalase with resulting increase in oxidation after the ingestion of glyeocoll is attributed to the stimulation of the liver by the glyeocoll as well as by the ammonium carbonate and acetic acid resulting from the deamina- tion of the glyeocoll. Further evidence that catalase is probably involved in the oxidative processes is found in the following observations. Miiller-Thurgau (13) found that the respiratory metabolism is greatly decreased in potatoes kept for several days at 0°C., that it is greater at the beginning than at the end of the rest period, and also that it is greater in large mature potatoes than in small immature potatoes. Appleman (14) found that there was an increase in catalase in potatoes under the same conditions that Miiller-Thurgau had found an increase in the respiratory me- tabolism and a decrease in catalase where he had found a decrease in metabolism. Appleman also found that there occurred an increase in catalase in the greening as well as the sprouting of potatoes parallel with the increase in the respiratory metabolism, while the oxidases were not increased and, in fact, were shghtly decreased. Hasselbalch (15) found that oxidation or metabolism is very low dur- ing the first month of life, and Magnus-Le\'y^ and Falk (16) that it is high during childhood. As a result of the work of these three observers and of Bailey and Murlin (17), Murlin and Hoobler (18), Rowland (19), Benedict and Talbot (20), Benedict, Emmes, Roth and Smith (21), Palmer, Means and Gamble (22) and others, it is now considered that oxidation or metaboUsm is low during the first month of life, high during childhood and low after the onset of old age. Battelli and Stearn (23) found that the catalase content of most of the tissues and particularly of the liver of newly-born pigs is lower than the corresponding tissues of the mother, but that the catalase activity rapidly increased until at the end of the seventh or eighth day it was as high as that of the adult. We repeated and confirmed these observations, using the dog and newly-born puppies. We also determined the catalase content of the tissues of puppies that were about ten weeks old and found that the tissues generally were richer in catalase than those of the mother. The catalytic activity of the liver, for example, of the ten-week-old puppies was about 30 per cent greater than that of the liver of the mother. The catalase was determined by adding 1 gram of the hashed tissue, that had been washed free of blood to hydrogen peroxide and THE AMEBICAN JOURNAL OF PHYSIOLOGY, VOL. 52, NO. 2 374 W. E. BURGE the amount of oxygen liberated in 10 minutes was taken as a measure of the amount of catalase. Warburg (24) found that during the process of fertilization, oxidation was greatly increased in the sea-urchin egg. Winternitz and Rogers (25) showed that the unfertilized hen's egg showed no catalytic activity even after prolonged incubation, whereas the incubated fertilized egg rapidly acquired the power of decomposing hydrogen peroxide. Doubt- less if Winternitz and Rogers had determined the intensity of oxidation in the fertilized hen's egg, they would have found that it increased parallel with the increase they observed in catalase, and if Warburg had determined the catalase content of the fertilized sea-urchin egg, he would have found that this enzyme increased parallel with the increase he observed in oxidation. J. Loeb (26) attributes the development of the fertilized egg to the increase in oxidation, and the increase in oxidation to a change in the cortex of the egg which makes the entrance of ox^^gen, and hence oxida- tion, possible, while R. Lillie (27) holds that the cortical layer of the unfertilized egg prevents the diffusion of carbon dioxide from the egg and that this carbon dioxide prevents oxidation and hence development. A more plausible explanation for the increased oxidation or metabolism in the fertilized egg, and hence for the development of the egg, would seem to be that the spermatazoon furnishes a substance which stimulates the egg to an increased formation of catalase. Further evidence in support of this view is afforded by the fact that the very same chemicals (amines, alkalies, acetates, butyric acid, etc.) which Loeb found would bring about increased oxidation and artificial parthenogenetic develop- ment of the egg, we found, when introduced into the alimentary tract of animals, stimulated the alimentar}^ glands, particularly the liver, to an increased output of catalase with resulting increase in oxidation. SUMMARY 1. The increase in oxidation produced by the introduction of an alkali such as sodium carbonate or phosphate into the alimentary tract of an animal is attributed to the increase in catalase brought about by the stimulation of the alimentary glands, particularly the liver, to an increased output of this enzyme. 2. The decrease in oxidation after the administration of an inorganic acid, such as hydrochloric, to rabbits is attributed to a decrease in catalase brought about by the inhibiting effect of the acid and bj^ the EFFECT OF ACID, ALKALI AND SALT ON BLOOD CATALASE 375 direct destruction of the enzyme. The increase in oxidation following the ingestion of organic acids, such as acetic, proprionic and butyric, is attributed to an increase in catalase. 3. The increase in oxidation after the ingestion of the amino acids is attributed to the stimulation of the liver to an increased output of catalase by the ammonium carbonate and the organic acids resulting from the deamination of the amino acids, as well as by the amino acids themselves. The increase in catalase after the introduction of hydro- chloric acid into the intestine of the dog is attributed to the stimulation of the liver to an increased output of this enzyme by the ammonium chloride formed in the neutralization of this acid by anunonia. 4. The increased oxidation in the sprouting of potatoes and the ger- mination of grain with resulting increase in metabohsm and develop- ment is attributed to an increase in catalase. 5. It is suggested that the low respiratory metabohsm of the ovum before fertilization may be attributed to the low catalase content of the egg, while the increase in the respiratory metabohsm after fertiHzation with resulting development may be due to an increase in catalase brought about by the stimulation of the egg by the spermatazoon to an increased production of catalase. Similarly the relatively low metab- ohsm of the newly-born may be attributed to the poorness of the tissues in catalase, due to the small output of this enzyme from the liver, while the high metabohsm characteristic of childhood and youth is a result of the richness of the tissues in catalase brought about by a large output of this enzyme from the liver. BIBLIOGRAPHY (1) Burge: This Journal, 1919, xlviii, 2, 133; 1918, xlv, 4, 388. (2) Jacobson: Zeitschr. Physiol. Chem., 1892, xvi, 340. (3) Loevenhart: This Journal, 1905, xiii, 171. (4) IssAjEw: Zeitschr. Physiol. Chem., 1905, xlv, 331; 1905, xliv, 546. (5) Senter: Zeitschr. Phys. Chem., 1903, xliv, 257. (6) Winternitz and Rogers: Journ. Exper. Med., 1910, xii, 755. (7) Mendel and Leavonworth : This Journal, 190-8, xxi, 85. (8) Bodansky: Journ. Biol. Chem., 1919, xl, 127. (9) Chvostek: Maly's Jahresber., 1893, xxiii, 410. (10) Lehmann: Maly's Jahresber., 1885, xv, 384. (11) Grafe: Deutsch. Arch. f. klin. Med., 1915, cxviii, 1. (12) Lttsk: Journ. Biol. Chem., 1912, xiii, 155. (13) Ml'Ller-Thurgau : Landwirtsch. Jahrb., 1885, xiv, 859. (14) Appleman: Maryland Agric. Exper. Sta. Bull., 191, 1915. (15) Hasselbalch: Bibliotek for Laeger, Copenhagen, 1904, viii, 219. 376 W, E. BURGE (16) MAGxrs-LEVY AND Falk : Arch. f. Anat. u. Physiol., 1899, Suppl. 315. (17) Bailey andMtjrlin: Amer. Journ. Obstet., 1915, Ixxi, 1. (18) MuRLix and Hoobler: Amer. Journ. Dis. Child., 1915, ix, 81. (19) Howlaxd: Zeitschr. f. Physiol. Chem., 1911, Ixxiv, 1. (20) Benedict axd Talbot: Carnegie Inst, of Washington, Pub. 201, 1914; Amer. Journ. Dis. Child., 1914, viii, 1. (21) Bexedic't, Emmes, Roth axd Smith: Journ. Biol. Chem., 1914, xviii, 139. (22) Palmer, Meaxs axd Gamble: Journ. Biol. Chem., 1914, xix, 239. (23) Battelli axd Stearx: Arch. d. Fisiol., 1905, ii, 471. (24) Warburg: Zeitschr. f. physiol. Chem., 1908, Ivii, 6. (25) Wixterxitz and Rogers: Journ. Exper. Med., 1910, xii, 12. (26) Loeb: Artificial parthenogenesis and fertilization, Chicago, 1913. (27) Lillie: This Journal. 1910, xxvii, 289. ON THE PERMEABILITY OF THE PLACENTA FOR ADRE- NALIN IN THE PREGNANT RABBIT AND ALBINO RAT ^ YOSHITAICl SHIMIDZU From the Wistar Institute of Anatomy and Biology, Philadelphia Received for publication March 30, 1920 INTRODUCTION At the present time the hormones are of great interest to many in- vestigators. Not only the specific character of each hormone, but the interrelations between them give rise to ver}^ interesting problems and are the subject of many discussions. Under normal conditions. the interrelation of the several hormones is self-regulated so as to maintain the physiological balance and keep the animal normal. Whenever this equilibrium is disturbed, there occurs a compensatory formation of hormones in several endocrine glands which tends to bring about a readjustment. Sometimes in consequence of this forced activity his- tological changes in these glands are more or less demonstrable. During pregnancy the corpus luteum elaborates its hormone activeh' and consequently calls into responsive action, it is said, some of the other endocrine glands, as the thyroid, hypophysis, suprarenals, etc. Meanwhile, the ductless glands of the fetus develop to a certain degree and begin their function as endocrine organs and their products, in- cluding the hormones of the sex glands, are present in the fetal cir- culation. In multiparous mammals there may be several fetuses of both sexes in one uterus. Thus one pregnant annual incloses several hormone systems in her body, but in deciduate mammals these hormone systems are separated in a measure from each other by the placenta. From such considerations the important question inevitabl}^ arises whether the placenta is permeable for the hormones or not. If the placenta is permeable, there will be a constant exchange of hormones ^ The observations on the rabbits were made at the Zoological Laboratory of the University of Chicago. I wish to acknowledge my obligation to Prof. F. R. Lillie who gave me the best of laboratory facilities and gave me also many helpful suggestions. 377 378 YOSHITAKA SHIMIDZU not only between mother and fetus, but also between the fetuses them- selves, though indirectly, by the intermediation of the mother's circu- lation, so that the male hormone, for example, will be delivered to the mother and the female fetus, and the hormone of the mother and the female fetus to the male fetus. Thus the interrelation of these hor- mones or hormone systems becomes much more complicated in preg- nancy than in the non-pregnant state. From this point of view, the problem of the permeability of the placenta is very important, not only for the development of the fetus but also for the differentiation of sex. For the solution of this question it seems to be important as a first step to learn whether a hormone administered to a pregnant animal can be transferred to the fetus through the placenta. As is well known, most of the hormones have not j^et been isolated in a pure state ; con- sequently their chemical character and constitution are still obscure, except in the case of adrenalin. Of course adrenalin may not represent the .entire suprarenal hormone, but there is no doubt that adrenalin is an essential component of the suprarenal hormone, and we are jus- tified therefore in taking adrenalin as a representative of the sup- rarenal hormone. If, in the first instance, we could get an experi- mental result by using such a well-characterized substance, we might apply the result to the next experiment and so gradually approach the solution of the problem. With this idea in mind I have examined the effect on the fetus of the injection of adrenalin into a pregnant animal, using for this purpose rabbits and albino rats. GENERAL PLAN OF EXPERIMENTS The aim of my present experiment is simply to learn whether adre- nalin administered to a pregnant animal is able to increase the adrenalin in the contained fetus. At the end of pregnancy the suprarenal glands of the fetus elaborate their internal secretion and the fetal blood contains a certain amount of adrenalin. Therefore the proof of the presence of adrenalin in the blood of the fetus does not give a conclusive answer to the question. In order to say that the adrenalin administered to the mother has passed to the fetus, there must be a significant increase of adrenalin in the fetal blood as compared with the amount normally there. There are several chemical and biological methods used to test the presence of adrenalin. In testing for adrenalin in blood the biological PERMEABILITY OF PLACENTA FOR ADRENALIN 379 method is considered in general to be better than the chemical. Among the biological methods that which depends on changes in blood pressure seems to be the most sensitive. But, because of its small size, it is almost impossible to measure the blood pressure of a fetus or a new- born individual of the ordinary laboratory animals. A considerable amount of blood is also required in order to examine the effect of adre- nalin on the response of smooth muscle. Trendelenburg's method (1) also needs too much blood for my case. The frog-iris reaction requires only a few drops of blood and the manipulation is very simple, but the result is not always reliable. It is a well-known fact that the parenteral administration of adrenalin causes hyperglycemia, and the animal is generally pretty sensitive to this test. If any increase of adrenalin occurs, it will be followed by hyperglycemia, without fail. Thus, we can diagnose readily anj^ increase of adrenalin by a quan- titative determination of the sugar in the blood, and if we employ a micro-method, the blood sugar can be determined with a little blood, not exceeding the quantity that can be obtained easily from a fetus or several fetuses. Relying on this reaction, I injected adrenalin into pregnant animals . and determined the blood sugar in the contained fetuses. Of course, the increase in sugar is not exactly proportional to the increase in the quantity of adrenalin, but I do not require that in my case because in my experiment it is necessary only to determine whether or not the adrenalin in the fetal blood has increased after the injection of adrenalin into the mother. Furthermore, it should be mentioned here that glucose, as is well known, very easily passes through the placenta and thus sugar in the mother's blood, increased by adre- nalin, may increase the sugar in the blood of the fetus and thus obscure the real adrenalin effect on the fetus. To avoid such a result special precautions were taken, and these will be discussed later on. For experimental material, as already mentioned, I used rabbits and albino rats, and made parallel experiments on both these species. In my experiments the adrenalin used was that prepared by Parke, Davis & Co. The adrenalin was diluted with physiological salt solution before injection and the hypodermic injection was always made between the scapulae. /. Experiments on rabbits As the rabbit is the most convenient and the most thoroughly studied animal in respect of adrenalin hyperglycemia, I took this animal first for my tests. 380 YOSHITAKA SHIMIDZU For the estimation of blood sugar in the rabbit I employed the Lewis- Benedict method (2) following the modification of Pearce (3). This method originally requires 2 cc. of blood for one determination, but it is almost impossible to obtain such an amount of blood from one rabbit fetus. Therefore I cut down the quantity of blood for one determi- nation to one-fourth this amount, keeping all the other conditions — picric acid concentration, sodium carbonate concentration, etc., exactly as in the original formula. The sugar determination was made as follows : To 2 cc. of water 0.5 cc. of blood was added. After the lakingof the blood had taken place, 3.75 cc. of a saturated picric acid solution were added, and the solu- tion agitated. To 3 cc. of the filtrate 1 cc. of saturated picric acid solution and 0.5 cc. of 10 per cent sodium carbonate solution were added. The mixture was heated in the autoclave for thirty minutes under a pressure of 20 pounds per square centimeter. Then the solution was made up to 5 cc. with distilled water and put into the colorimeter tube. The Duboscq colorimeter is too big for my purpose and the Duboscq micro- colorimeter was not at hand, so I constructed a simple micro-colorimeter, using the parts of a burette and some other ordinary laboratory apparatus, and thus imitated the Duboscq ijistrument. This homemade micro-colorimeter gave me satisfactory results. Before proceeding to the experiment proper, I made some necessary preliminary tests. Adrenalin hyperglycemia in normal rabbits. Concerning the increase in the blood sugar in rabbits after the adrenalin injection, there are many reports in the literature. I wished to determine this relation using the adrenalin which had been selected and the modified pro- cedure of the Lewis-Benedict-Pearce method arranged for my own experiments. Three rabbits were used for this purpose. During the night previous to the experiment the animals were kept without food. The next morn- ing each animal received one subcutaneous injection of 5 cc. of 1 :10,000 adrenalin. Just before the injection and after it at intervals of one- half, one, two, three and four hours the blood was taken from the ear vein of each animal. The quantity of sugar found in each specimen of blood is given in table 1. On the whole this result agrees with those previously reported. Adrenalin hyperglycemia in newborn rabbits. There is no doubt that adrenalin causes hyperglycemia in adult rabbits. Now what influence will the adrenalin injection have on the blood sugar in newborn rabbits? Though the cause of the adrenalin hyperglycemia is not yet clearly explained, it is generally considered as due to the mobilization of gly- PERMEABILITY OF PLACEXTA FOR ADRENALIN 381 cogen deposited in the body tissues. Therefore if the newborn rabbit has any glycogen deposit in its bod}', hyperghxemia should be caused by adrenalin. Chipman (4) examined the glycogen of the fetal liver of the rabbit embryo and found that the glycogen appeared at the twenty-second day of gestation and increased rapidly and steadily in amount till the end of pregnane}'. Lochhead and Cramer (5) made a quantitative determination of the gtycogen in the fetal liver and the remainder of the fetal bod}' in an age series of pregnant rabbits from the fourteenth da}' to the end of pregnancy. According to them the fetal liver contains a trace of glycogen at the eighteenth day, though none can be demonstrated histologically till the twenty-second day. At the twenty-fifth day the percentage of glycogen in the liver rises for the first time above the glycogen percentage in the rest of the fetal body, and then there is a rapid increase till on the TABLE 1 Adienalin hypeigly emia in rabbits {0.5 mgm. of adrenalin administered subcutaneously) NUMBER BODY WEIGHT BEFORE INJECTION PERCENTAGE OF BLOOD SUGAR HOURS AFTER INJECTION 1 1 2 3 4 1 2 3 grams 2360 2082 2187 O.OS 0.07 0.10 0.12 0.18 0.20 0.26 0.28 0.31 0.26 0.26 0.28 0.23 0.23 0.25 0.16 0.20 0.21 Average 0.08 0.17 0.28 0.27 0.24 0.19 twenty-ninth day half of the fetal glycogen is stored in the liver. From these facts we can expect the sugar increase in the blood of newborn rabbits after adrenalin injection just as it occurs in the case of adult rabbits. With- this expectation I tried the following expermients: Rabbit 1. At full term of pregnane}' ;2 body weight 2925 grams. Four fetuses were excised by Caesarean section. The body weight of the young varied from 34 to 38 grams. Immediately after removal one fetus was killed by bleeding from 2 In my experiment the copulation of the rabbits was not watched, because it was not so necessary in my case to know the exact age of the fetus. My experi- ment required only rabbits in advanced pregnancy whose fetuses would have plenty of glycogen in their bodies and, at the same time, blood enough for a sugar determination. In rabbits the full term of pregnancy can be diagnosed without any difficulty. Several hours before the parturition they always begin to make a nest. This manoeuver is very evident and no one can miss it. 382 YOSHITAKA SHIMIDZU the neck while each of the others received 0.1 cc. of 1:10,000 adrenalin. At intervals varying from thirty minutes to two hours after the injection these were killed one after the other for blood specimens. The results of the sugar deter- minations are given in table 2. Rabbit 2. At full term of pregnancy; body weight 2737 grams. Five fetuses were obtained by Caesarean section. The body weight of- the young varied from 35 to 40 grams. The young were treated in the same way as those of rabbit 1. The percentages of blood sugar at different times are entered parallel with those for no. 1 in table 2. This result shows that after the adrenalin injection the blood sugar increases in newborn rabbits as in the adults. TABLE 2 Blood sugar of newborn rabbits before and after the adrenalin injection {0.01 mgm. adrenalin subcutaneously) PERCENTAGE OF BLOOD SUGAR NUMBER Before injection Hours after injection i 1 li 2 1 2 0.06 0.08 0.14 0.12 0.22 0.20 0.21 0.16 0.18 The influence of adrenalin injected into a pregnant rabbit on the blood sugar in the contained fetus. From the results of the preliminary experi- ments described above, we can expect an increase of sugar in the fetal blood after the injection of adrenalin into the mother, if the injected adrenalin is transferred to the fetus. As already mentioned, however, the increased sugar in the mother's blood is easily diffusible into the fetus. The increase of sugar in the fetal blood produced in this way is not only meaningless from the standpoint of my experiment, but tends to confuse any possible effect due to the passing of adrenalin into the fetus. Fortunately there is a certain space of time between the adrenalin injection and the distinct appearance of hyperglycemia. Therefore, if the fetus is removed from the mother before it is influenced in a marked way by the sugar in the mother's blood, such confusion will be practically avoided. With these relations in mind, I made the following experiments. Nine pregnant rabbits at full term were used. Their body weights varied from 2532 to 3112 grams. After a short period of inanition, from four to six hours, the animals each received one hj^podermic in- jection of 5 cc. of 1 : 10,000 adrenalin. Then at varying intervals of PERMEABILITY OF PLACENTA FOR ADRENALIN 383 ten, twenty and thirty minutes after the injection the fetuses were removed by Caesarean section under ether. Immediately after removal from the mother, one fetus from each litter was killed bj^ bleeding from the neck, while the others were kept warm till they were examined one after another for blood sugar at periods varying from twenty minutes TABLE 3 Blood sugar in newborn rabbits whose mother received 0.5 mgm. adrenalin before operation for delivenj PERCENTAGE OF BLOOD SUGAR NUMBER Mother New born Before injection At operation Just after delivery 20 minutes after delivery 40 minutes after delivery 1 hour after delivery 1. Fetuses removed from mother ten minutes after the adrenalin injection 1 2 3 0.10 0.06 0.09 0.10 0.07 0.11 0.10 0.06 0.08 0.09 0.07 0.08 0.06 0.06 0.08 0.05 Average 0.08 0.09 0.08 0.08 0.06 0.07 II. Fetuses removed from mother 20 minutes after the adrenalin injection 1 2 3 0.07 0.09 0.06 0.10 0.13 0.15 0.08 0.10 0.12 0.08 0.09 0.10 0.07 0.07 0.08 0.06 Average 0.07 0.13 0.10 0.09 0.07 0.07 III. Fetuses removed from mother 30 minutes after adrenalin injection 1 2 3 0.09 0.09 0.06 0.15 0.14 0.18 0.12 0.12 0.15 0.12 0.10 0.12 0.06* 0.09t Average 0.08 0.16 0.13 0.12 0.11 0.08 IV. Control, without adrenalin injection 1 2 0.09 0.08 0.08 0.08 0.08 0.08 0.07 0.07 0.07 0.07 Average 0.09 0.08 0.08 0.07 0.07 * Two hours after the delivery the blood sugar = 0.07 per cent t Two hours after the delivery the blood sugar = 0.07 per cent; three hours after the delivery = 0.05 per cent 384 YOSHITAKA SHIMIDZU to one or more hours after their dehvery. Every blood specimen was used for a sugar determination. Besides these, two pregnant rabbits at full term were used as control animals. Their body weights were 2435 and 2618 grams respectively. They were treated just as the test animals, but without any injection. In all cases, in the tests as well as in controls, every placenta was carefully sliced and thoroughly examined for hemorrhages or other abnormalities. Where any pathological change appeared, the animal was omitted from the record. The results of the experiments are given in table 3—1, II, III and IV. The data in table 3 (I to III) show a difference in the quantity of sugar in the fetal blood immediately after delivery which increases as the interval between the injection and the operation for deliver}^ in- creases. Nevertheless, the blood sugar of newborn rabbits shows a tendency to decrease in the course of the first hours of extra-uterine life. The interpretation of these facts will be made later. II. Experiments on albino 7rds As the next step in my work I repeated this series of experiments on albino rats. I was fortunately allowed to use pregnant and non- pregnant albino rats from the colony of these animals maintained by the Wistar Institute at Philadelphia. In consequence of the smallness of this animal I employed the Epstein (6) micro-method for the blood sugar determination. This method requires only 0.1 to 0.2 cc. of blood for one determination and is based on the same principle as the Lewis-Benedict method, which is recognized as an admirable method and was emploj'ed by me for rabbits in the present study. The Epstein method does not give very accurate results. However, if it is carefully carried out, it is still fairly available for the usual biological tests, such as this present one, in which accuracy above a certain limit is not necessarily required. The matters to be attended to in connection with this method are the accuracy of the standard color solution and the purity of reagents, especially of the picric acid. Keeping these matters in mind, I got satisfactory results with this method. In my experiments the rats were brought from the colony house in the morning and kept in the laboratory till the evening, without food, to avoid if possible the alimentary hyperglycemia. Then they received the experimental treatment. PERMEABILITY OF PLACENTA FOR ADRENALIN 385 In adult rats the blood was taken from the tail. The fetal blood was obtained from the neck, using two or three fetuses at one time. For one sugar determination 0.1 cc. of blood was always used. The colori- metric work was done almost always under artificial daylight, recom- mended by Gage (7).^ The influence of fear and of ether narcoses on the hloocl sugar. The rat, even when tamed, is not so gentle as the rabbit and is easily excited by unskilful treatment. Even a hypodermic injection or a sharp cut in the tail sometimes excites the animal greatly. Such an emotional disturbance affects the equilibrium of the blood sugar. Also the ether narcosis, when severe, has the same influence on the blood sugar. In working with such an excitable animal it is absolutely necessary to know first, how much influence these factors have on the blood sugar, because either agitation or narcosis was unavoidable in making the injection and obtaining the blood. To solve this question, I made the following tests with two rats: rat 1, male, body weight 182.4 grams, and rat 2, female, body weight 165.8 grams. Both of these were roughly treated with the forceps, making them very angry. Each was then wrapped up in a towel and the blood taken by cutting the tail. To compare with these results samples of blood were taken from rat 3, male, body weight 174.6 grams; rat 4, male, body weight 126.9 grams; and rat 5, female, body weight 132.4 grams. These animals had been treated very gently and carefullyj wrapped in a towel, placed on the table softly, without causing any marked excitement, and the blood obtained by cutting the tail. Finally rat 6, female, body weight 135.3 grams; rat 7, female, body weight 143.6 grams; rat 8, male, body weight 164.7 grams; rat 9, male, body weight 147.8 grams, were lightlj^ etherized and the blood was taken in the same manner from the tail. The blood sugar determination for each animal gave the results entered in table 4. ' The growth of the fetus of the albino rat becomes rapid toward the end of pregnancy. Parturition occurs usually at night. I made my tests therefore almost always at night in order to get the fully-grown fetus, and to obtain as much blood as possible. Thus I was obliged to make the color tests under arti- ficial daylight, because the glucose in the solution diminishes in its reducing power with time and the color of the produced picramate fades pretty rapidly. In my experience the artificial daylight is rather better than natural daylight, if the background is not homogeneous, as is usually the case in cities or towns, where buildings are crowded around the laboratory. 386 YOSHITAKA SHIMIDZU This result shows that the effect of agitation is distinct, while the ether narcosis has no marked influence on the blood sugar, when, as in my case, the narcosis is neither deep nor long. TABLE 4 Blood sugar in excited, not excited and lightly etherized rats BODY MENTAL ETHER BLOOD SUGAR AVERAGE WEIGHT CONDITION NARCOSIS PERCENT PER CENT 1 c? 182.4 Agitated — 0.22 \ 0.21 2 9 165.8 Agitated — 0.20 3 cf 174.6 Quiet — 0.16 \ 4 cf 126.9 Quiet - 0.18 0.16 5 9 132.4 Quiet — 0.15 J 6 9 135.3 Quiet + 0.15 7 9 143.6 Quiet + 0.15 • 0.16 8 d" 164.7 Quiet + 0.17 9 & 147.8 Quiet + 0.16 Rats 6 to 9 were narcotized once more at intervals varying from thirty minutes to one hour after the first narcosis. The blood of each animal at that period gave a percentage of sugar as shown in table 5. TABLE 5 Blood sitgar of twice narcotized rats R.\.T NUMBER PERCENTAGE OF BLOOD SUGAR INTERVAL BETWEEN First blood Second blood minutes 6 0.15 0.15 30 7 0.15 0.14 60 8 0.17 0.16 30 9 0.16 0.16 40 As this table shows, the repeated ether narcosis has no apparent effect on the blood sugar of albino rats, provided the narcosis is neither deep nor long. So we can use ether narcosis without fear of complicat- ing our results. Adrenalin hyperglycemia in normal albino rats. In the following tests each animal received 0.5 cc. of 1 : 10,000 adrenahn subcutaneously. Just before the injection and at intervals varying from thirty minutes to two hours after the injection, the blood was taken from the tail PERMEABILITY OF PLACEXTA FOR ADREXALIX 387 under light ether narcosis. In the control animals 0.5 cc. of physi- ological salt solution was injected in the place of the adrenalin. The result of the sugar determinations is given in table 6. TABLE 6 Adrenalin hyperglycemia in albiiio rats {0.05 ingm. adrenalin injected suhcutaneously) BAT NUMBER BODT WEIGHT PERCENTAGE OF BLOOD SUGAR Before injection Hours after injection Controls grams 1 c? 148.7 0.15 0.16 2 cf 151.6 0.16 0.16 3 ? 159.1 0.17 0.17 4 9 141.8 0.16 0.16 5 6" 123.6 0.16 0.18 6 d" 170.1 0.17 0.15 7 & 177.3 0.16 0.16 8 9 132.2 0.18 0.16 Average 0.17 0.16 0.17 0.17 0.16 Tests 1 d' 130.4 0.13 0.18 2 & 165.5 0.16 0.19 3 cf 136.8 0.14 0.18 4 9 143.2 0.16 0.19 5 cf 155.6 0.15 0.18 6 9 120.8 0.15 0.20 7 9 139.3 0.15 0.22 8 & 148.7 0.19 0.21 9 & 177.1 0.19 0.31 10 & 158.1 0.19 0.31 11 9 114.3 0.15 0.23 12 & 139.2 0.14 0.20 13 & 121.8 0.15 0.24 14 o 146.3 0.15 0.21 15 & 167.2 0.17 0.21 16 & 186.1 0.15 0.19 17 9 131.5 0.16 0.20 Average 0.16 0.18 0.24 0.25 0.20 388 YOSHITAKA SHIMIDZU Table 6 shows that in albino rats the blood sugar rises slowly after the adrenalin injection, reaching its maximum after one or one and a half hours. Though the percentage of the blood sugar at the highest point of the hyperglycemia is not much less in rats than in rabbits, the increase of the blood sugar is not so apparent as in rabbits, owing to the high percentage of normal blood sugar in the rats. In spite of that difference the albino rat is still a fairly available animal for my purpose. Adrenalin hyperglycemia in newborn albino rats. The fetuses of three pregnant rats were removed by Caesarean section at full term.'* Immediately after delivery by operation two or three fetuses from each litter were used for the first blood specimen, while each of the others received a hypodermic injection of 0.05 cc. of 1 : 50,000 adrenalin, and then were kept in an incubator till they were killed at varying intervals of from thirty minutes to one and a half hours after the injection. The blood sugar determination gave the results shown in table 7. TABLE 7 Blood sugar of newborn albino rats before and after the adrenalin injection (0.001 mgm. adrenalin administered subcutaneously) PERCENTAGE OF BLOOD SUGAR RAT NUMBER Before injection Hours after injection 1 1 n 1 2 3 0.14 0.14 0.15 0.18 0.17 0.20 0.20 0.21 0.20 Average 0.14 0.18 0.20 0.21 These results indicate that the newborn albino rats also show a dis- tinct reaction to adrenahn b}^ an increase of the blood sugar. Therefore we can reasonably expect an increase of blood sugar in a fetus after the injection of adrenalin into the mother, if the injected adrenalin is trans- ferred to the fetus, and this sugar increase should continue for one or more hours after the injection, regardless of the removal of the fetus from the mother. * The full term of pregnancy of the rat can be diagnosed by palpation. The growth of the rat fetus in the last days of intra-uterine life is very rapid. The difference between the size of the fetus on the last day and on the day before is quite distinct. Therefore diagnosis of the last day of pregnancy is not very difficult after a little practice. PERMEABILITY OF PLACENTA FOR ADRENALIN 389 The influence of the adrenalin injected into 'pregnant albino rats on the blood sugar of the fetuses. For this series of experiments twelve pregnant rats at full term were used, nine as tests and three controls. Their TABLE 8 Blood sugar of newborn rats whose mother received 0.05 mgm. adrenalin before operative delivery PERCENTAGE OF BLOOD SUGAR NUMBER Mother New born Before injection At operation Just after delivery 20 minutes after delivery 40 minutes after delivery 1 hour after delivery I. Fetuses removed from mother 10 minutes after injection 1 2 3 0.13 0.14 0.14 0.13 0.14 0.14 0.13 0.15 0.14 0.12 0.13 0.12 0.11 0.11 0.12 Average 0.14 0.14 0.14 0.13 0.12 0.12 II. Fetuses removed from mother 20 minutes after injection 1 2 3 0.16 0.15 0.15 0.20 0.18 0.17 0.16 0.16 0.16 0.15 0.15 0.13 0.12 0.13 0.13 Average 0.15 0.18 0.16 0.15 0.13 0.13 injection 1 2 3 0.17 0.14 0.15 0.20 0.19 0.21 0.18 0.17 0.17 0.16 0.15 0.14 0.13 0.15 0.12 Average 0.15 0.20 0.17 0.16 0.14 0.14 IV. Controls: without adrenalin injection 1 2 3 0.12 0.14 0.15 0.12 0.14 0.14 0.11 0.14 0.13 0.13 0.09 0.13 Average 0.14 0.13 0.13 0.13 0.11 body weights varied from 153.6 to 215 grams. Each test animal re- ceived one hypodermic injection of 0.5 cc. of 1: 10,000 adrenalin. At an interval of ten, twenty or thirty minutes after the injection, all the THE AMERICAN JOURN.^ required for any similar task actually performed in ordinary life where the rate of work may be either faster or slower. The series includes experiments with the subject at rest and performing various household tasks. The tasks performed during these preliminary experiments were chosen partly because they represent some of the more common and typical household activities and partly because they seemed likely to indicate how experimental methods should be developed for this type of investigation, especially in the matter of reproducing actual working conditions within the calorimeter. In the experiments with washing no water was used because of the complicating effect it would have on the water vapor and heat measurements, but it is hoped to devise means of overcoming this difficulty in later experiments. The results of fifty-three tests (briefly noted in the report of the work of the Office of Home Economics, presented at the 11th annual meeting of the Ameri- can Home Economics Association, Science Section, Chicago, June, 1918) (3), are summarized in table 1. Rest experiments. The first measurements of energy expenditure by this subject were made in four rest experiments in which the external muscular activity was reduced to a minimum. For this purpose the subject rested easily in a comfortable position in a large swivel chair, and remained very quiet, hardly moving during the two hours compris- ing each rest experiment. The number of calories measured during each of the four experiments was very uniform, indicating that the expenditure of energy by this subject while sitting quietly was practically ENERGY EXPENDITURE IN HOUSEHOLD TASKS 403 constant. The average of the values for these four experiments is used as a basis of comparison with those for experiments in which work was performed, showing the increase in heat output resulting from the muscular activity involved in the performance of the work. Experiments with knitting, crocheting and hand sewing. The measure- ments of the energy expended while knitting, crocheting and hand TABLE 1 Average energtj (heat) output during experiments with a woman engaged in different household tasks CHARACTER OP EXPERIMENT Resting Knitting Crocheting Sewing Scalloping Running Hemming Hemming and running .... Darning Average for sewing Dressing infant (model) Sweeping floor Washing floor Washing towels Ironing towels Dishwashing Table 65 cm. high Table 100 cm. high Table 85 cm. high Average for dishwashing NUMBER OF EXPERI- MENTS 11 4 4 3 4 6 2 4 2 HEAT ELIMINATION Total per 2-hour period calories 121.4 141.2 138.0 140.8 136.4 139.6 142.4 137.4 139.6 168.8 201.4 177.6 220.8 171.6 181.6 190.2 162.2 181.1 Total per hour calories 60.7 70.6 69.0 70.4 68.2 69.8 71.2 68.7 69.8 84.4 100.7 88.8 110.4 85.8 90.8 85.1 81.1 90.5 Per hour per kilogram of body weight calories 1.22 1.42 1.39 1.43 1.39 1.45 1.39 1.40 1.40 1.72 2.06 1.82 2.22 1.75 1.85 1.74 1.65 1.75 Per hour for work alone calories 10.1 8.3 10.2 7.5 10.3 9.8 8.0 9.4 23.6 40.1 29.0 49.6 24.3 30.0 24.4 20.3 24.8 sewing, were all conducted with the subject comfortably seated in an ordinary bentwoocl chair, and all motions except those necessary for the work were reduced to a minimum. The knitting was done on a sweater about half completed. The rate of work was 23 stitches per minute taken to the beat of a metronome. 404 C. F. LANGWORTHY AND H. G. BAROTT In the crocheting experiments, fine cotton thread was used and a simple pattern chosen. The rate of the work was 32 stitches per minute. The experiments with hand sewing included several types of work: a, making a plain unpadded scallop (blanket stitch) on the edge of a small piece of fine linen, at the rate of 18 stitches per minute; h, simple running on light cotton goods, 6 stitches being taken on the needle, one to one beat of the metronome, then the thread pulled through to four beats, with a total of 30 stitches per minute; c, hemming on light cotton goods, at a rate of 30 stitches to the minute; the thread being pulled all the way through after each stitch; d, darning light-weight cotton hose with a thread about 24 inches long. As might be expected these tasks all entail a relatively small expendi- ture of energy, varying from 7 calories per hour for sewing with the running stitch to 10 calories per hour in hemming. The expenditure during knitting (10 to 11 calories per hour) is slightly higher than that during crocheting (8 to 9 calories) , doubtless because the wool sweater which was handled in knitting weighed more than the cotton lace which was being crocheted, and because greater play of the hands and arms was required by knitting with fairly long needles than by the crocheting. Since in the sewing experiments the materials used were of approxi- mately the same weight and ease of handling, the differences there are probabl}^ to be attributed to the extent of movement required ; thus with the running stitch (7 calories per hour) the movement of drawing the thread to its full length was made only every 6 stitches or 5 times per minute, whereas with the hemming (10 to 11 calories per hour) it was made after each stitch or 30 times per minute. This is in line with the practical experience of needleworkers, who for most forms of plain sewing avoid an unnecessarily long thread as causing too much work of the arms. In later experiments it is planned to introduce sewing on articles of different types in order to learn how such factors as weight, shape and texture influence the energy expenditure. Experwients on dressing model of infant. It was desirable that some observation be made on the energy output involved in caring for children and the task of dressing and undressing an infant was chosen for this preliminary test. Because of the difficulties in conducting the experi- ment with a young child, a full-sized model was substituted. The model was about the size of a j-ear-old infant, but it weighed, dressed, only 2 kgm., whereas a child of this size would weigh about 8 to 10 kgm. The clothing of the model consisted of a band (no sleeves), diaper, shirt with sleeves, two underskirts (no sleeves), dress, knitted ENERGY EXPEXDITUEE IX HOUSEHOLD TASKS 405 sack with sleeves, socks, booties and bonnet. The model was dressed and undressed seven times during each 2-hour experiment . The energy output in this experiment, 23.6 calories per hour for work alone, is undoubtedly lower than wouldbereciuiredinthe care of a li\'ing child, as a child of this size would be four or five times as hea\y to lift and its movements would still further increase the energy,- output. These figures, therefore, represent only the labor involved in the act of removing and replacing the clothes plus the necessaiy lifting about and supporting of a 2-kilogram model. Even the unduly low figure thus obtained (24 calories in round numbers per hour) was fulh^ twice as great as that for such light tasks as sewing, similar to that required for dishwashing and ironing and about one-half that for washing clothes. This indicates that such work should be classed as a moderately hea^"^' household task. A part of the program for experiments now being conducted is the measurement of energy expended in the care of li\-ing children and it is beUeved that the value of the results thus obtained will be sufficient to offset the difficulties involved. Experiments with sweeping and icashing floors. These tasks were included as representing what are usually considered rather hard forms of housework. The sweeping was done on the bare floor of the calorim- eter. A long-handled broom was pushed forward on the floor, lifted and moved back. The rate was 38 complete strokes per minute. In the floor-washing experiment , the motions of actual washing were simulated with a dry cloth and empty pail. The subject worked on her knees, giving 85 short rubs of the cloth on the floor in 50 seconds and wringing the cloth in the pail for 10 seconds. When compared with such tasks as knitting and sewing, sweeping the floor would be classified as hard labor, the energy expenditure, 40.1 calories per hour for work alone, being four or five times as great. Plans have been made for experiments with various types of floor coverings. This work should give interesting results as to the relative resistance offered to the broom by other surfaces as compared with the smooth floor of the calorimeter, and the consequent variations in the energy required for sweeping. The average of 29 calories per hour for energy expenditure during floor washing is beHeved to be considerably too low for usual work of this kind. Not only was the task slightly lightened by omitting water, which would add to the work of lifting and wringing the cloth, but the subject, who was unused to such labor, was not able to continue exerting 406 C. F. LANGWORTHY AND H. G. BAROTT as much force as a professional charwoman would expend and her movements were those of light rubbing rather than hard scrubbing. It is planned to repeat the experiment with a stronger subject accus- tomed to such tasks. Experiments with laundry work. In the experiments with laundry work, both washing and ironing were included. The work was done on towels 16 inches square. In washing, the equipment consisted of a small galvanized iron tub and an ordinary scrubbing board. A towel was rubbed on the board 40 times in 30 seconds, wrung by hand for 15 seconds, then exchanged for another towel, 15 seconds being allowed for the exchange. This routine was repeated throughout the experiment. The energ}^ output in washing was 49.6 calories per hour for work alone; this is similar to that found for sweeping floors and like it must be classified as hard labor. It seems probable that the weight of the water in actual washing might increase the labor of moving the towel and also that wet articles might offer slightly more resistance on the scrubbing board and in wringing. Moreover in ordinary family wash- ing the average weight and size of the articles handled are greater than those of the towels used in these tests, and would tend to increase rather than decrease the energy expenditure. The figures here given, therefore, represent less rather than more energy than would be expended for comparable work in actual laundering. It is hoped to devise some way to overcome the difficulties of using water in experiments with such types of work and to approximate more closely the conditions actually found in the household. In the ironing experiment, the work was done with a 5-pound iron on a table of comfortable working height. The rate of work was 70 strokes of the iron in 50 seconds, and 10 seconds to change towels. A cold iron was used because of the complications which heat would cause in the measurements, but it is not probable that this omission would noticeably affect the amount of energj^ expended by the worker. The energy expenditure in the ironing experiments, which in six experiments averaged 24 calories per hour, lies between that of the tasks classed as light work and those classed as heavy work and might be classed as moderate work. Experiments with dishwashing at tables of different heights. In the dishwashing experiments a table of adjustable height was used so that studies might be made of the comparative amounts of energ}- expended when working surfaces of different heights were used. In one set of ENERGY EXPENDITETRE IN HOUSEHOLD TASKS 407 observations the table waS: too low for comfort, in another, too high, while in a third it was set at what was regarded as the correct height for the worker. The dishes, comprising 4 plates, 2 bow:ls, 2 teacups and 2 saucers, were placed in a pan, rubbed with a cloth, plac;ed in a draining pan and then wiped. The motions w^re the same as in. actual washing. Each dish was given 10 rubs, turned, and given 10 more, in time with a metro- nome, beating 130 times per minute, then 10 beats were allowed to change pieces. The work simulating drying was then performed at the same rate. The whole process was repeated twelve times per hour. In the series of tests in which the table top was set too low for the comfort of this subject, the height from the floor to the top of the table was 65 cm. and to the top of the pan 78 cm. In the series in which it was too high, the top of the table was 100 cm. and the top of the dishpan 113 cm. from the floor. In the series in which the height was adjusted to the comfort of the subject the table top was 85 cm. and the top of the pan 98 cm-, from the floor. As regards the general qitcstion of energy expenditure the average figure of 24.8 calories per hour shows that washing table dishes is to be classed as moderate work, about midway between sewing and sweep- ing and washing. Somewhat more heat would no doubt be eliminated with heavier dishes (large platters, kettles, etc.) or ones which require harder ruljbing than was given in these tests. As regards the differences in the energy expended at tables of different heights,the figures show 21 calories per hour when the subject worked in a comfortable position, 25 calories when the pan was set so that her arms were raised during work, and 30 calories when she was obliged to bend over. It is hoped in the future to develop such experiments so that the oncome and effect of fatigue may be noted when similar tasks are performed under vaiying conditions. SUMMARY Fifty-three experiments on energy elimination during the performance of various household tasks were made, using a specially designed respi- ration calorimeter and a young woman subject. The results for such light tasks as sewing, crocheting, knitting, darning and embroidering, gave an average expenditure of 9 calories per hour more than that of the same subject sitting quietly in a chair; other tasks regarded as "harder work" than sewing, such as washing, sweeping and scrubbing 408 C. F. LANGWORTHY AND H. G. BAROTT floors, caused an increased energy expenditure over the expenditure when at rest with the same subject, of about 50 calories per hour. Several other tasks studied gave results between these two figures: thus, ironing, dressing a child (life-size model) and dishwashing each requiring about 24 calories per hour. During the experiment with dishwashing the height of the table used was varied, and a corresponding variation in energy expenditure was noted, a variation of 15 per cent in height of table causing an increase in energy expenditure of 20 per cent to 40 per cent. The observed increase of heat elimination well illustrates the importance of choosing equipment to "fit" the worker. BIBLIOGRAPHY (1) Report of Director, Office of Experiment Stations, 1914, 14-15. Report of Director, Office of Experiment Stations, 1915, 18. Report of Director, Office of Experiment Stations, 1916, 31. (2) Langworthy and Milner: Journ. Agric. Research, 1915, v, 299. (3) Langworthy: Journ. Home Economics, 1919, xi. 13 ^1 THE AMERICAN Journal of Physiology VOL. 52 JULY 1, 1920 No. 3 AMPLIFICATION OF ACTION CURRENTS WITH THE ELEC- TRON TUBE IN RECORDING WITH THE STRING GALVANOMETER! ALEXANDER FORBES and CATHARINE THACHER From the Laboratory of Physiology in the Harvard Medical School Received for publication April 6, 1920 INTRODUCTION In the spring of 1916 Dr. H. B. Williams suggested the use of the electron tube to amphfy those action currents in the nerv'ous system which are too small to record with the string galvanometer alone. In the course of several researches in this laboratory we have encoun- tered difficulties due to the extremely small excursions obtainable with the string galvanometer from the action currents in nervous tissue, notably in the acti^'ity of the spinal cord and in the motor nerve involved in the crossed extension reflex. The suggestion ap- peared therefore to offer possibihties of attacking physiological prob- lems hitherto inaccessible. ^Nlihtary duties prevented the development of this suggestion until the spring of 1919, when conditions in this laboratory became favor- able for the work. Doctor Wilhams was still absent from his lab- orator j^ on duty; but the prospect of utiUty of the method made us feel justified in proceeding to develop it without waiting for his return and the collaboration we should have preferred. We wish to express our appreciation of his generosity in urging us to publish the work as our own, and to thank him for certain helpful suggestions in the later phases of the work since he returned to his laboratory. There is nothing new in the idea of amphfpng electric cm-rents with the electron tube. Especially in the field of radio communica- ^ A preliminary report has appeared; This Journal, 1920, li, 177 (proceedings). 409 THE AMERICAN JOCRNAL OF PHYSIOLOGY, VOL. 52, NO. 3 410 ALEXAKDER FORBES AND CATHARINE THACHER tion, this has been standard practice for years. The usual method of observing the currents ampUfied b}^ this de\'ice is with the telephone receiver. This method is not apphcable to those problems in phj'si- ology which have led us to this work. In using the string galvano- meter to record the amplified currents we are confronted with several new problems which do not enter into the telephone method. These problems are of practical importance to the physiologist who w^ould use the method, and of some theoretical interest to the phj^sicist. It has seemed worth while to carr}^ out a good mam^ experiments and to attempt to correlate them with theoretical expectation, in order to formulate, if possible, the correct method of obtaining the maximum amplification, or at least to outhne the principles on which such a method must depend. We have therefore taken up some of the mathe- matical considerations involved although these may be of little interest to the phj^siologist who wishes merely to know how to use the method. The mathematical discussion maj' readily be skipped, and simple in- structions still be found for obtaining ver}- nearly the maximum possible amplification. THE ELECTRON TUBE The electron tube or thermionic ampUfier (1), commonly known in this country as the audion, consists essentially of a vacuum tube con- taining a cathode in the form of a filament, heated to incandescense b}' the passage of a current, an anode ^--»'**-.,_ in the form of a metal plate, and an auxiliary electrode in the form of a grid (2) placed between the anode and the cathode. A conventional I ^^ ^,^ y^ \ and schematic representation of ^ » f \ y A/ • I these elements is shown in figure 1; ^^ ^^ ■ this is convenient for identification of the parts in circuital diagrams to follow. When the filament (cathode) Fig. 1. Conventional diagram of ig incandescent it emits electrons electron tube elements. F, filament; -^^^^ ^j^g gp^^^ ^^.^^^l^^ ^l^g ^^^^^ jf ^, gri , , p a e. ^^^ means of a battery the plate (anode) is made positive with respect to the filament, these electrons under the influence of the electric field thus set up, travel to the plate and thus enable a current to flow from plate to filament within the AMPLIFICATION OF ACTIOX CURRENT WITH ELECTRON TUBE 411 tube (according to the conventional designation of the direction of current flow). Under ordinary conditions of operation, more elec- trons are emitted by the filament than can be drawn to the plate by the voltage appUed to it; a negative "space charge" results which repels electrons subsequently emitted and causes them to return to the filament. This space charge exerts a limiting effect on the current that passes from anode to cathode. If now a positive potential, with respect to filament, be appHed to the grid, or auxiliary electrode, it will oppose the space charge and enable more current to flow in the tube; if a negative charge be applied to the grid it will still further limit the current. In this way the current in the tube may be regulated by varia- tions in the potential of the grid. In a properlj" designed tube small variations in the potential of the grid may be made to produce large variations in the plate-to-filament current, and in this way the device acts as an amplifying relay, the energy of amplification being supplied by the battery which maintains the high potential of the plate. METHODS IN GENERAL The salient feature of our problem as compared with the ordinary uses of the electron tube was the necessity of protecting the delicate string of the galvanometer from the direct current used to maintain the liigh potential of the plate. Telephone receivers are ordinarily placed directh' in series with the plate and the high voltage battery, as is shown in figure 2. This would be impossible with a sensitive string galvanometer. With the AVestern Electric "D-tube," for instance, the optimum plate poten- tial is 200 volts; under the most favorable working conditions this tube has a plate-to-filament resistance of about 100,000 ohms. Neither string nor telephones placed in series with this would add greatly to the total resistance of the circuit. Therefore, the current that would pass through the string would amount to about 2 milli- amperes. One hundredth of this current is as much as or more than it is safe to pass through a string of 2 or 3 micra diameter, the size best suited to nerve phj^siologj'. Even were it possible to pass the full plate current through the string without destroying it, to work under this condition would be impossible, for such a current would move the string well out of the field accessible to the beam of light required for projection. 412 ALEXANDER FORBES AND CATHARINE THACHER Three methods have presented themselves for utihzing the amph- fying energy of the plate battery without placmg the string directly in the path of a damaging current. The first is to place the primary coil of a transformer in the plate circuit, and connect the galvanometer across the terminals of the secondary coil of the transformer (see PH ONES INPUT H H HH B + Fig. 2. Typical arrangement of electron tube and circuits commonly used for amplifying currents and detecting them with telephone receivers. A, filament- heating battery; B, plate battery. N v^ o B -+- Fig. 3. Transformer method (see text). N, nerve or other tissue giving rise to action current. T, transformer, G, string galvanometer. fig. 3). The second method was proposed by Mr. S. W. Dean, and is based on the principle of the Wheatstone bridge (fig. 4). It may be designated the bridge method. The third method was proposed by ]Mr. Sewall Cabot. It consists in protecting the string galvanometer from the direct current bj^ placing it in series with a condenser, and AMPLIFICATION OF ACTION CURRENT WITH ELECTRON TUBE 413 shunting the plate current by the string and condenser through a resistance of the same order of magnitude as that of the tube (fig. 5).^ Whittemore (3) has described a scheme for measuring with a string galvanometer radio signals rectified by an electron tube. This procedure, though intended for a wholly different purpose from ours, has much in ^mi'ww B Fig. 4. Bridge method (see text). + i^ 1^1^ 1^1- -r\y^ B r^ Nl u <^ R, I = C I Fig. 5. Condenser method. C, condenser. R2, by-pass resistance (see text) . 2 This method was first tried with the plate battery between the filament and the galvanometer, etc. At the suggestion of Prof. G. W. Pierce, the battery was shifted to the location shown, next to the plate. It makes no difference in the amplification, but it is better practice to have, the string on the low potential side|of the circuit, and makes it possible to keep the magnet core of the galvanom- eter grounded. 414 ALEXANDER FORBES AND CATHARINE THACHER common with both the bridge method and the condenser method here described. The transformer method offers the objection that what is recorded is the rate of change of the difference of potential to be ampHfied; a sustained e. m. f . would give no evidence of its presence by this method, except at its beginning and ending. However, as the action current of a nerve-trunk following a single stimulus is of very rapid onset and brief duration, — so brief that no string has time to attain its full excur- sion before. the maximum e. m. f. is over (4, p. 145) — there might be some use in obtaining records that would show a rate of change. It might serve for the comparison of the magnitudes of successive re- sponses in nerve under conditions insuring constancy of time rela- tions. The method seemed at least worth a trial. The bridge method consists in causing the current from the high voltage (plate) battery to pass through a split circuit, one branch con- sisting of 7?i and the tube in series, the other consisting of i?2 and Rs in series (fig. 4). The string connects the two circuits at their middle points. The best results would be obtained with Ri, Rn and Rs all approximately equal to the resistance of the tube, R;, (plate-to-fila- 7? 7? ment), and in any case the proportion — " = — must hold. ^3 Rb This method depends on the balance in the bridge, i.e., the equaliz- ing of potential at the two ends of the string. Starting with such a balance, a change in the input difference of potential (between fila- ment and grid) would, by causing a change in the resistance of the tube (plate-to-filament), unbalance the system and create a difference of potential between the ends of the string. The chief advantage of this method is that changes in the input difference of potential are recorded without distortion; that is, a con- stant e. m. f. is shown as such in the record. The chief drawback lies in the difficulty of obtaining a perfect bal- ance, and of maintaining it through an experiment. Another objec- tion is that the resistance of an}- string commonly in use is so low, compared with those in the bridge, that it approximates a short-circuit and tends to reduce the difference of potential set up between the two points it connects. This tendency will reduce the resulting amplifi- cation. The condenser method, suggested bj^ Cabot, obviates both of these difficulties. There being no current permitted to pass as long as the tube resistance is stead}-, on account of the interposed condenser, the AMPLIFICATIOX OF ACTION CURRENT WITH ELECTRON TUBE 415 problem of balancing does not arise. There is also no such reducing tendency- as was mentioned above. The principle of the method is in the main the same. It depends on the fact that if a constant volt- age drop occurs through two resistances in series, when one of these changes (the other remaining constant), the voltage drop through it must change; consequently, a change of potential occurs at the end of the string connected to the junctional point where the two resistances are connected. At first sight it would appear that the condenser would lay this method open to the same objection that appHes to the transformer method, that it would distort the record, showing only a rate of change in the input, and failing to record a sustained e. m. f. as such. This objection can readih^ be overcome for practical purposes by making the condenser veiy large, so that it provides a virtually constant reser- voir of energy throughout the time that an action current lasts. We are chiefly concerned with the action currents of nerves, and these are of brief duration, most of the effect being over in less than 0.01 second; action currents of cardiac or skeletal muscle rarely require ampUfica- tion. The resistance through wliich the condenser has to discharge it- self with this arrangement under optimum conditions amounts to about 80,000 ohms. Through such a resistance a condenser of 25 mf. requires considerably over 2 seconds for approximately complete discharge; in 0.01 second it would lose 0.5 per cent of its charge. In short, for recording an electrical disturbance lasting only 0.01 second^ the method would not introduce any appreciable distortion with a condenser of this order of magnitude. In the case of action currents of skeletal muscle, although their duration is much greater than those of neiwe, the distortion is still insignificant. When currents of longer duration are studied, it is usually possible to obtain all the amplifica- tion that is needed by slacking the tension on the string. Electron tube amplification, therefore, has its chief use in studying brief and rapidly changing electrical disturbances which a slack string will not follow satisfactorily. APPARATUS The electron tubes used in this research were generously loaned us by the Western Electric Company. On the recommendation of Dr. H. B. Arnold '"D-tubes" and "L-tubes" were tried, two of each being furnished, with a socket to fit them. 416 ALEXAXDER FORBES AND CATHARINE THACHER A transformer was also loaned by the Western Electric Company to enable us to trj- the first-mentioned method. It was an autotrans- former with a closed iron core. The windings were in four sections, two of 12.5 ohms each and two of 62.5 ohms each, making a total re- sistance of 150 ohms. The condenser used in the third method was made of the common Western Electric Company paper condensers of 1 microfarad each, connected in parallel. In nearly all of the experi- ments 15 were used, giving thils a capacitj' of 15 mf. The current for heating the filament was supplied by an Edison storage battery of five tj^pe B-2 cells, having a capacity of 40 ampere- hours. This is conventionally known as the "A-battery." The cur- rent was regulated by a small circular rheostat on a porcelain base, such as is commonly used for this purpose. The battery which sup- plied the plate current (conventionally known as the B-battery) con- sisted during the experimenal stage of the work chiefly of the small dry cells used for flash-lights, connected in series. This was supple- mented by a 24-cell lead storage battery loaned by the Cruft Labora- tory of the Harvard Physics Department. When the apparatus was finally arranged as a permanent installation a B-battery of lead storage cells was made in the following way : A lead strip 1 inch wide by ye inch thick was passed through a press at the Jefferson Physical Laboratory, thus impressing on it a pattern which increased the surface area; then the strip was cut into suit- able lengths. A disc-shaped mould was prepared with two slits thi'ough which the lead strips fitted, and with a wire nail protruding upward through the bottom. Two lead strips were passed through the slits to the proper distance and a string tied round them within the mould, then a short piece of glass tubing was put over the wire nail. Then a mixture of paraffin, beeswax and rosin was poured into the mould and allowed to harden, the string serving to strengthen the wax, and the glass tube providing a vent. Small glass jars about 2 inches by 6 were filled with sulphuric acid, and the wax discs with the lead strips protruding downward were fitted into the necks and the openings sealed with hot wax, all but the vents. A wooden separator was placed between the plates of each cell. One hundred and thirty-five of these cells were made and set up in three trays. Each tray had 45 cells connected in series. In order to form the plates the three trays were' connected in parallel and charged from the 110-volt mains first in one direction and then in the other. The charging current proved to be so small that to hasten the process storage batteries were con- I AMPLIFICATION OF ACTIOX CURRENT WITH ELECTRON TUBE 417 nected in series with the 110-volt mains, giving a total charging volt- age of 130. AMien the plates were formed the batteries were con- nected with a group of three switches so wired that by throwing them all one way the three groups of cells were arranged in series and con- nected with the system, and by throwing them all the other way the three groups were arranged in parallel and connected with wires whereby they could be charged from the 110-volt power mains. This switching arrangement is included in the general wiring plan shown in figure 17. The galvanometer used was an Einthoven String Galvanometer fur- nished by the Cambridge Scientific Instrument Company of Cam- bridge, England ; the same that has been used in this laboratory for several years. In most of the experiments a comparatively coarse platinum string was used, having a diameter of 5 micra and a resist- ance of 910 ohms (designated string B) ; in some experiments we used a gilded quartz string, furnished by Hindle, of 2.5 micra diameter and 20,000 ohms resistance (string G); in the last experiments we used a similar Hindle string of 12,000 ohms. In order to protect the string dming the establishment of the high voltage circuit, and in preliminary tests, a shunt was connected across the terminals of the double-pole switch used to connect the string with all other cu'cuits. For this purpose we used a rheostat of the same type as that used to control the filament current. For projection we used an arc lamp furnished by A. T. Thompson. As usual, a cylindrical lens was used to focus the beam of Hght in a horizontal line across the film. For recording we at first attempted to use a camera in which the film was moved by means of a Sandstrom electric kymograph. This was abandoned for a reason which will be discussed later, and all ex- periments were performed with a newly constructed camera, used for the first time in this research. Since this camera has proved exceedingly useful and convenient for a wide range of physiological work, and has not yet been described, it is thought worth while to include here a brief description, especially since one feature of it proved essential to success under the conditions met with in these experi- ments. The camera was designed to make records on standard per- forated moving picture film, the motion of which is regulated by a sprocket such as is used in moving picture cameras and projectors. The sprocket is so placed behind the illuminated sht that it presents the sm'f ace of the film at the focal plane of a cyhndrical lens set in an adjustable holder in the path of the beam of hght. It is mounted 418 ALEXANDER FORBES AND CATHARINE THACHER on a shaft provided with a conical clutch whereby it may be rapidly started and stopped, the clutch connecting it with a friction drive which is run by an electric motor. The motor is a 12-volt D. C. ma- cine intended for an automobile self-starter, and is completely encased in metal; it runs at 460 r. p. m. This motor turns the disc of the fric- tion drive, which is of cast iron and has a diameter of 32 cm. A spring on the shaft holds this disc firmly in contact with a wheel with a leather rim 18.5 cm. in diameter mounted on the horizontal shaft which / \ M Fig. 6. Horizontal section of photographic recording apparatus at level j of optical axis. M, motor. FD, friction drive. CL, clutch. E, handle for oper- ating clutch, showing spring, lever pivoted at P, and catch. Cam, camera. carries the clutch, and is perpendicular to the motor shaft. The wheel may be moved along the shaft from the center to the rim of the disc, and secured in any desired position with a set-screw, thus providing a wide range of gear ratios (fig. 6). In this way any desired speed of film is obtained, from a minimum of about 7 cm. per second to a maxi- mum of 48 cm. per second. Still slower speeds could be had by putting a resistance in series with the armature of the motor. The film, which comes in 200-foot rolls, is placed in a light-proof magazine of a tj'-pe used in some moving picture cameras: in this magazine it may be in- AMPLIFICATION' OF ACTION CURRENT WITH ELECTRON TUBE 419 serted in the camera in full daylight. The end of the film is led around the sprocket and into a receiving magazine so arranged that the spool on which it is received is coupled to the shaft of a spring clock-work device which serves to wind up the film as fast as it leaves the sprocket (fig. 7). The lever which operates the clutch also moves a screen which cuts off the beam of light when the film is stopped. This pre- vents fogging of the film during short intervals between observations. This fogging spoils the film for about an inch on each side of the point behind the slit; on a series of brief observations at a slow speed of film the percentage of waste would be considerable without this screen. The current which drives the motor is f«rnished by a batter}- of CJould storage cells of large capacity, which maintains practically constant speed. This camera has several advantages over one previous^ described (4, p. 127) and used hitherto in recording with this galvanometer. It carries 200 feet of film at one loading, as compared with 50 feet, which was practically the maximum ca- pacity of the old camera; and it can be loaded by transferring the magazine to the dark room without moving the camera, which is large and heavy. The old camera had only certain fixed speeds depending on the gear ratios of the Sand- strom kymograph. The friction drive makes it possible to select any speed within the limits mentioned. The most important advantage is the un- failing uniformity of speed imparted to the film by the sprocket engaging in the perforations. It was the great difficulty in obtaining vmiform motion with a film pressed against a revolving cylinder bj- rubber rollers, that led to the abandonment of the old camera. In this par- ticular research an unexpected and indispensable advantage was found in the new camera; this has already been alluded to, and will be dis- cussed later. The chief limitation of the new camera lies in the nar- rowness of the film (35 mm.), this being the maximum width made with perforations. This difficulty can be overcome by moving the camera nearer to the galvanometer and thus decreasing the magnification. Sharper definition of the image results, and greater contrast in the film Fig. 7. Vertical section of camera. F, feeding spool in magazine. R, receiving maga- zine. S, sprocket. L, cylin- drical lens. 420 ALEXANDER FORBES AND CATHARINE THACHER at high speeds. If it is desired to reproduce the record on a larger scale it may easily be enlarged, and the increased definition and con- trast ■will facilitate the process. ELEMENTARY EXPERIMENTS For comparison of the results obtained with and without the elec- tron tube, we used in general an artificial source of current to be am- plified, instead of the action current of a nerve or muscle. This had the great advantage of saving time, for anj- desired electromotive force could be drawn at a moment's notice without the labor of dis- secting a nerve, placing it in a moist chamber and connecting it with Fig. 8. Standard wiring diagram (simplified). Ri, resistance in primary cir- cuit. A', kej'' in primary circuit. Rn, resistance representing resistance of tissue in series with galvanometer, G. E, connection to earth. non-polarizable electrodes. Even greater advantages were the de- pendable uniformit}^ of the source of current and the ease with which its quantity and duration could be regulated, conditions impossible of fulfillment with living tissues. The source of current was an Edison cell connected with a resistance of 640 ohms, Ri, in series with a slide- wire 1 meter long and having a resistance of 4.8 ohms. This est abhshed a potential drop of 0.0001 volt per centimeter along the shde-wu-e, and made it convenient to select readily the desired e. m. f . From one end of the slide-wire and from the sliding contact, wires were led either to the string galvanometer or to the filament and grid of the electron AMPLIFICATION OF ACTIOX CURRENT WITH ELECTRON TUBE 421 tube; this may be termed the input circuit. In order to simulate as far as possible the electrical conditions which obtain in recording the action currents of Hving tisues, a resistance representing the resistance of the tissue was introduced in the input circuit in the path from the shding contact to the galvanometer or the grid of the electron tube, as the case might be. In the great majority of experiments this re- sistance, termed Rn, was 10,000 ohms, this value being selected as a convenient one which is about the lower hmit of resistances found in experiments on nerve, and about the upper hmit in the case of muscle preparations. The key which was used to make and break the current was placed in series mth the Edison cell and the resistance Ri, the /v Ma .L.W.

o oT at m bC m fe J < 424 ALEXANDEB FORBES AND CATHARINE THACHER THE CONDENSER METHOD The first method tried was the condenser method suggested by Cabot (fig. 5). This method showed promising results at the out- set. Both D-tube and L-tube were tried, and the D-tube showed in the prehminary test so much greater amphfication that it was there- after adopted and- used regularly in all subsequent experiments. It is possible that more careful study and selection of the values of the various constants would have resulted in some improvement of the performance of the L-tube, but it seemed to us better to concentrate on the analysis of the results with the tube which appeared most promising. The board with the tube and meters, the A- and B-batteries and the condenser were all placed on tables near that on which the cell and resistances sho^uTi in figure 8 were permanentlj^ installed. The wiring of these resistances was then so modified and supplemented as to make the entire arrangement conform to the basic scheme in figure 5. The detail shown in figure 9 applies to all experiments, but most of it is omitted in the other diagrams for the sake of simplicity. The protective shunt across the string, already referred to, was connected with those terminals of the switch leading to the string, which, when the switch was open, were not connected with the string. In this way the condenser obtained its initial charge through the shunt before this switch was closed. This is an important safeguard to the string. Figure 11 shows contrasted the standard arrangement and the con- denser arrangement of the electron tube circuits intended to simulate those of physiological work. The same e. m. f . was tapped from the resistance sUde-wire, representing that arising in the tissue, and the same resistance, Rn, was introduced in each case at that point in the cir- cuit where the resistance of the tissue would be. In some experiments the initial potential of the grid was allowed to remain the same as that of the filament; in that case no cell was introduced at Ec. In others the grid was given an initial "bias," i.e., a positive or negative poten- tial, by inserting one or more dry cells in series between the slide-wire and grid. After a few prehminary tests with the artificial source of current some practical tests were made with action currents in nerve and muscle. The description of these will be reserved for a later section. After adjusting the tension of the string a photographic record was made with the standard arrangement, showing the excursion due to AMPLIFICATION OF ACTIOX CURRENT WITH ELECTRON TUBE 425 the passage of a current through the galvanometer, the cu'cuit being opened and closed by hand with a kej- (A" in fig. 11). Sometimes a metal spring key was used, sometimes a key in which a sharp amalga- mated copper point made contact with a cup of mercury. After mak- ing this control record we rewired the apparatus for the electron tube test without disturbing the adjustment of the string tension. With the string circuit still open and the shunt closed, we turned on the filament current and established the plate current through Rz. When all adjustments were made we closed the switch leading to the string, and finalh^ opened the protective shunt. The string showed a marked displacement, in some instances disappearing from the field. This indicated a slight leakage of current through the condenser. IMeas- urements showed that when 15 microfarads were used the resistance of Fig. 11. Comparison of arrangements for experiments on amplification. A, standard arrangement. B, electron tube arrangement, condenser method. Notation as in other figures. this leak was 32 megohms. It could be avoided by using carefully made mica condensers. A much cheaper way out of the difficulty will be described later. But in most of the experiments we adopted the crude but simple expedient of moving the fibre case of the galvanom- eter laterally till the string, though deflected by the current, was again in the middle of the field. The input e. m. f. was then applied bj^ making and breaking the circuit at X and the resulting excursions observed and recorded photographically. It was at this point that we encountered the difficulty already men- tioned in recording with the Sandstrom kymograph camera. As soon as the kj^mograph motor was started the string began to oscillate vio- lently. The same occurred when we turned on the electromagnetic THE .\MERIC.4N JOURN.U. OF PHYSIOLOGY, VOL 52, NO. 3 426 ALEXANDER FORBES AND CATHARINE THACHER tuning fork which shows hundredths of a second by its shadow on the fihn. The Sandstrom kymograph is run by a 110-volt D. C. motor which works on the regular power Hne; it has a centrifugal governor which maintains a constant speed by breaking the circuit through a part of the armature windings when the speed exceeds this constant value. The action of this governor is attended by visible sparking at the centrifugal contact. The electromagnetic tuning fork was operated on a split circuit from the power mains with a lamp in series and a resistance in parallel. The tuning fork is kept vibrating by means of an electromagnet, on the principle of an ordinary buzzer. Fig. 12. Oscillations induced by 110-volt kymograph motor in string galvano- meter connected with electron tube as in figure 11 B. 7^, 1.10; E, 280; Ec. —1.5; R, 60,000. A, when motor is first started. B, after centrifugal governor has begun to operate (see text). String B. Tension. 1 cm. excursion = 2.75 X 10~^ amp. Speed of film, 15.6 cm. per second. In this and all other photographic records the magnification is 300 diameters. In all records made with electron tubes the following symbols are used: — /a, filament current; E, plate battery voltage; Eh, plate potential; E^, initial grid potential; R, by-pass resistance (hitherto designated R^). In all records made with condenser method condenser capacity was 15 mf. Both the k^-mograph and the tuning fork had two features in common, connection with the 110-volt power circuit, and a circuit-breaking contact at which sparking occurred. In the case of the kymograph motor, some oscillations were visible as soon as the motor was turned on, but they enormously increased in amplitude as soon as the centrifugal governor began to operate with its attendant sparking. (See fig. 12.) It was clearly impossible to AMPLIFICATION OF ACTION CURRENT WITH ELECTRON TUBE 427 record anj^ experiment with a camera depending on this kymograph motor, at least without elaborate shielding. Such shielding as was mechanically feasible failed to stop the oscillations. We therefore substituted for it the new camera described above, which had just been completed for general use with the string galvanometer. It was then found that the friction of the leather rim of the wheel with the iron disc in the friction drive produced static electricity to such an extent that sparks jumped occasionally from the metal part of the wheel to the disc. The running of this motor caused no oscillation of the string when connected with the electron tube arrangement, except when one of these sparks was seen to jump; then the string made a quick excursion. This difficulty was remedied by grounding both the disc and the wheel of the friction drive, using a copper brush to make continuous contact with the revolving disc. After this change it was found possible to obtain perfectly smooth records with this camera, there being no oscillations in the string induced by the running of the motor. It should be noted that this was a 12-volt motor, run from a storage batterj^ in the room, having no connection with the power mains, and that the motor was completely encased in metal. The oscillations induced by the tuning fork were found to be con- siderably reduced in amplitude when, later on, the electron tube and accessories were permanently installed on a lower shelf on the same table with the resistance and switches connected with the galvanometer. The oscillations were still further reduced somewhat when the tuning fork was operated by a 6-volt battery instead of being connected with the power mains (fig. 13). The persistence of oscillations even under these conditions showed the powerful effect of sparking in inducing such disturbances. The cause of the disturbance was evidently some form of electro- magnetic induction, probably due to high frequency currents result- ing from sparking, which by their action on the grid circuit, caused disturbances in the plate circuit, by virtue of the rectifying action of the tube. It was feared that a similar disturbance would result from the use of break shocks of an ordinary induction coil for stimu- lating the tissues to be studied, for the break of the primarj^ current is necessarily attended by a spark. An inductorium was set up in the position commonly used for stimulating, and as large a primary cur- rent as is ever used with this coil was broken while the galvanometer was connected with the electron tube as indicated. No excursion of the string resulted unless the hand of the experimenter touched the 428 ALEXANDER FORBES AND CATHARINE THACHER primary circuit. With such contact estabhshed, the making and break- ing of the primary circuit caused excursions of the string. Evidently such sparking as occurred in the induct orium as used in the laboratory, did not suffice to produce any excursions of the string which might con- fuse an experiment. But it was necessary to insure insulation of the primarj^ circuit from the experimenter, to avoid confusing excursions of the string. In subsequent experiments the tuning fork was not used, and the constancy of the speed of the motor was relied upon for measurements iiiAAaaAAAAAaM i^*^t^A^**^*iAti*^^*iA*^*^^^^^i*^^i^^^^^^^^i^^^^^i^ iAiiAAAAiHililiiiiiiaiiAiiiiAAiiiiiUUaiiiiiiiiiAlilAAAIiliAliiAi Fig. 13. Oscillations induced by electromagnetic tuning fork. A, string H ; tension, 1 cm. = 5.9 X 10"^ 7a, 1.10; E, 300; E^, - 1.5; R, 50,000. B and C string B; tension, 1 cm. - 2.75 X 10-" amp. (as in fig. 12). 7a, 1.10; E, 280; Ec, — 1.5; R, 60,000. In A and B the tuning fork was operated on the 110-volt power circuit; in C it was operated by a 6-volt battery. Time indicated by shadow of tuning fork; each complete vibration = 0.01 second. of time. This constancy was found experimentally to be quite ade- quate for our purposes. A short strip of film with the tuning fork operating was exposed before the experiment, and another one after- wards, by way of speed control. Referring to figures 5 and 11 B, it will l^e noted that part of the voltage of the B-battery is used up in the resistance 7?2, leaving so much less for the tube. In other words, the plate-to-filament resistance of the tube being in series with the resistance /?2, part of the potential AMPLIFICATION OF ACTION CURRENT WITH ELECTRON TUBE 429 drop in the circuit occurs in the latter, and only the remainder within the tube. On this account it is necessary to provide a battery of higher voltage than that which would be required to operate the tube without such a resistance in series. It is also necessary to determine, among other things, the value of Ro which will give the best results. The question of values of the various resistances and voltages in- volved will be discussed later in detail. For the present it will suffice to say that the condenser method was tried with various values of plate battery voltage from 113 to 400, and with values of R2 varying from 10,000 ohms to 120,000 ohms. , , ^ ^ _^ The results of fairh^ typical i^ " * "' ^ HJi experiments are shown in figure ^^^* - ^H 14. A shows the amplification with the low resistance string; B shows the amplification under comparable conditions with the high resistance string. Marked amplification in the excursions of the string is obvious in both cases. It is also notice- able that with the electron tulje arrangement the decline of the current through the string due to the gradual discharge of the condenser is very small in as brief a time as that shown in the figure; in the case of figure 10 B, amounting to about 0.08 second. From this it follows that for disturbances as brief as the action currents of nerve and skel- etal muscle, the distortion from this cause is negligible, as was predicted. It is also noticeable that the amplification is consider- ably greater in the case of the high resistance string than in that of the low resistance string. This is due to the fact that a low resistance string is relatively Vig. 14. Amplification by electron tube, condenser method. Upper row, electron tube arrangement; lower row, standard arrangement. In all records of test cur- rent with electron tube ^Ec is change in grid potential used to make record. Other notation as explained in legend to figure 12. A, string 5; tension, 1 cm. = 2.75 X 10"'^ amp. Upper row:— 7a, 1.25; .& 240; £'b, 170; Ec, - 3.0; R, 90,000; AE^, 0.0006; 7?n. 10,000. Lower row (standard) 0.0006 . ... (i.e., 10,000 ohms in series S + 10,000 with string). Tuning fork shows time in 0.01 sec. intervals. B, string G; tension, 1 cm. = 5.0 X 10~". Upper row:— 7a, 1.25; E, 238; Eh, 170 (appro.K.); Ec, -3.0; R, 90,000; AE^, 0.0020 0.0020; Rn, 10,000. Lower row, ^S-h 10,000 Speed of film, 20 cm. per second. 430 ALEXANDER FORBES AND CATHARINE THACHER placed at a disadvantage as compared with a high resistance string when connected in series with the resistances involved in the electron tube circuit, which are very much greater than the resistance of either string. A large external resistance makes less difference to the result- ing current if the string resistance is large than if it is small. We found that when the initial grid potential was zero, — that is, when no cell was introduced to give it a negative bias, the grid circuit absorbed a certain amount of current. This was shown by the fact that the excursions were made considerably smaller when a 10,000 ohm resistance, R„, was introduced into the grid circuit, than when this resistance was left out. In one experiment this reduction amounted to 32 per cent. When one or more dry cells were inserted in the grid circuit, thus making the grid potential negative to the extent of 1.5 volts or more,^ the grid circuit took no current. This was shown by the fact that the insertion of as much as 130,000 ohms made no difference whatever in the magnitude of excursion. In this sense the apparatus assumed the function of an electrometer. The consequence is that the electron tube will amplify the electrical disturbance of a tissue of high resistance, veiy much more than it will that of a tissue of low resist- ance and the relative gain is greater for a low resistance string than it is for a high resistance string. For instance, in the case of our low resistance string, B, with only 910 ohms resistance, there is no amplification at all in measuring an electrical disturbance set up in a circuit of negligibly small resistance; there is on the contrary a reduc- tion. In other words, when a given difference of potential is applied to the terminals, this low resistance string will give a larger excursion than it will if connected with the electron tube when the same difference of potential is applied to the grid circuit. The conclusion to be drawn from this is that in studying a tissue of low resistance like skeletal muscle, with a low resistance string little or nothing would be gained by use of the electron tube, but that in studying a high resistance tissue such as a nerve trunk very great amplification is to be obtained. This point will be taken up quantitatively later on. THE TRANSFORMER METHOD In testing the transformer method it was still necessary to protect the string by placing a condenser in series with it, since the trans- former at our disposal was an autotransformer, that is, the windings ^ A third D-tube used later in some of the final experiments required 3 volts negative grid potential to bring about this result. AMPLinCATIOX OF ACTION CURRENT WITH ELECTRON TUBE 431 were all connected in series, with a number of taps available for connecting the circuits. Thus it was impossible to insulate the sec- ondary circuit from the primary, and conse- quently without a condenser the string would have been subjected to a large direct current from the B-batteiy. On the advice of Mr. Colpitts, the coil was connected as a step-down trans- former; that is, all four windings were included in the primary, or plate circuit, and only one of the two larger windings was included in the sec- ondary or galvanometer circuit. The reverse arrangement was also tried. We used values of filament current and plate potential which had given good amplification with the condenser method. It was evident at once that the transformer method was inferior in every respect. Figure 15 shows a comparison of the galvanometric excursions obtained with the standard arrangement, with the condenser method and with the transformer method. In this experi- ment the essential electron tube values were the same with the transformer method as with the condenser. It should be noted in this connection that the entire plate battery voltage is applied to the plate, none of it being expended in the trans- former, whose resistance is negligible compared with that of the tube. The record shows that not only does the transformer method record only the rate of change of the electrical disturbance, but that the amplification of the initial excursion is very much less than that obtained with the condenser method. Reversing the transformer produced a record practically identical with that shown in the figure, but a trifle smaller still. No further experiments were tried with this method. Fig. 15. Compari- son of transformer and condenser methods. String G. A, control observa- tion with standard arrangement. Ten- sion, 1cm. = 6.0X10-^ 0.0020 amp. THE BRIDGE METHOD In one experiment we tried the bridge method suggested by Dean (see fig. 4). It proved a difficult matter to balance the four resistances S + 10,000" B, condenser method. Tension, 1 cm. = 6.0 X 10-^ amp. 7.^, 1.25; £",306; Eh, 163; Ec, 0; R, 50,000; AEc, 0.0020; i?n, 10,000. Tuning fork struck by hand just before observa- tion. C, transformer method. Tension, 1 cm. = 5.0 X 10-^ amp. 7^^, 1.25; E, (same as Eb) 163; £"0,0; A^c, 0.0020 ;7?n, 10,000. 432 ALEXANDER FORBES AND CATHARINE THACHER with sufficient accuracy to keep the string of the galvanometer in the field. And after a balance had been found with difficulty, it was also difficult to maintain it, for the string kept travelling slowly sidewise as a result either of the running down of the A-battery or the B-battery, or of the change of temperature in the tube. A few records were taken with this method, and as was expected, they showed no great difference from those taken with the condenser method. The chief difference is that there is no falling off during the record of a continued current, such as occurs in the condenser method. On the other hand the gradual shift of base line due to the disturbance of balance mentioned above, though not as rapid as that due to the discharge of the condenser in the other method, is more troublesome in practice. The amplification was also appreciably less with this method than with the condenser. This is probably due to the fact that the experiment was made with the low resistance string whose resist- ance was so low that it approxi- mated a short-circuit of the bridge. Figure 16 shows records made with the condenser method and the bridge method under approximately com- parable conditions, excepting speed of film. These experiments led us to the conclusion that the condenser method was by far the most satis- factory. We proceeded to install a Fig. 1(3. Comparison of bridge and condenser methods, showing in bridge method absence of decline resulting in condenser method from discharge. String B. A, condenser method. Tension. 1 cm. = 5.50 X 10-' amp. 7.^, 1.25; E, 250; Ec, 0; R, 50,000; XE^, 0.0010; i?n. 10,000. Speed of film, 19.5 cm. per second. B, bridge method. Tension, 1 cm. = 2.75 X 10-' amp. 7.^, 1.18; E, 223; 7^1, 40,000; R., 30,000; i?.,, 70,700; J5;c,0;A£'c, 0.0010; i?n. 10,000. Speed of film 10.7 cm. per second. permanent wiring plan whereby we could rapidly shift from the standard arrangement to the electron tube arrangement, wired in accordance with the condenser method, by merely throwing a few switches. This served a twofold purpose. It enabled us to make rapid comparisons between the excursions obtained with the standard arrangement and those obtained with the electron tube, thereby reducing to a minimum the error introduced by change AMPLIFICATION OF ACTION CURRENT WITH ELECTRON TUBE 433 of tension of the string. It also rendered the method permanently available at a moment's notice for the amplification of any action cur- rents found, in the course of a physiological experiment, to be too small to record with the string galvanometer unaided. This wiring plan is shown in figure 17. In the compactness obtained by installing tlTj^.^^ >i-B -B •cztKl:^ -B A\ PC Ez:;:::xy |^^vv^^^/vwv^^vA/^ Fig. 17. Complete wiring diagram showing switches for rapid shift from con- nection of galvanometer directly with tissue or artificial source of current to connection with electron tube. G, galvanometer (core grounded). E, connection to earth. PS, protective shunt. DS, switch for connecting galvanometer with tissue or substitution resistance in standard arrangement; in electron tube ar- rangement this switch is always thrown to right. R2, in standard arrangement, substitution resistance; in electron tube arrangement, by-pass resistance. N_ non-polarizable electrodes for connection with tissue. C, condenser in series with string. A, filament-heating battery. B, plate battery in three sections connected with switches for charging them in parallel and discharging them in series. M, wires for connection with power mains. Ec, grid bias battery. PC, pole-changing switch in source of current used for test or compensation. D, single-pole double-throw switch for use in electron tube arrangement. When artificial current is used for test or calibration this switch is thrown to left to short-circuit tissue leads; when recording action currents it is thrown to right and switch 2 is open, thereby disconnecting test current circuit from grid circuit. In standard arrangement switches 1 and S are open, switch 2 is closed upwards. In electron tube arrangement / and 3 are closed, DS is thrown to right, 2 is closed downward (or open if D is to right). Cf. reference 4, fig. 1. THE AMERICAN JOtRXAL OF PHYSIOLOGY, VOL. 52, NO. 3 434 ALEXANDER FORBES AND CATHARINE THACHER all the apparatus on shelves of the same table which supported the resistances and switchboard of the old equipment we found the double advantage of convenience and of minimizing the disturbances due to induction in the grid circuit. THE SEARCH FOR MAXIMUM AMPLIFICATION General considerations. Having chosen the condenser method as in- dicated in figure 5 as that which was most worth developing, it re- mained to determine if possible how to find those values of the four vari- ables, filament current, grid potential, plate potential, and by-pass + + R. R Fig. 18. Simplified schema for analysis of condenser method (see text). E, plate battery; Rh. plate-to-filament resistance of tube; R, by-pass resistance (designated Ri in figs. 5 and 11); Es, battery susbtituted for condenser; S, string of galvanometer; O, junctional point assumed for convenience to be at zero po- tential in analysis; Z, junctional point actually put to earth, here assumed to be point of variable potential. resistance, Ro, which would yield the maximum possible ampHfication. Our aim was to find the maximum current in the string which could be made to result from a given change in the difference of potential between grid and filament. For purposes of analysis we may look on the sj'stem as simplified in accordance with the schema shown in figure 18. In this the electron tube and the string are shown simply as resistances, and the condenser is replaced by a battery. In reality a difference of potential is main- tained between the plates of the condenser, equal during the resting state, to the voltage drop in R (representing i?2 in fig. 11), and the AMPLIFICATION OF ACTION CURRENT WITH ELECTRON TUBE 435 capacity of the condenser is so large that for such brief times as we are dealing with, this difference of potential remains practically constant; for purposes of argument we may look on the capacity as infinite. It acts like a battery whose voltage exactly equals the fall of potential in R in the resting state, and it is therefore so represented in the analysis. In practice the zero point which is grounded, and therefore at zero potential, is that point in the system which is connected to the string, the positive end of R, the filament and one side of the input circuit. But in the following discussion it will simplify matters to assume that the point of zero potential is that which is connected with the nega- tive terminal of the B-battery, the negative end of R and the negative plate of the condenser {0 in fig. 18). The potentials of all other points in the system are positive with respect to this point, and we may assign to them absolute instead of relative values if we consider the potential of this point fixed at zero. When the difference of potential between grid and filament is raised the resistance of the tube is de- creased, and the portion of the total plate battery voltage used up in the tube is decreased. Consequently a rise in potential occurs at that point in the system at which the filament, the positive end of R, and the string are connected together (Z in fig. 18). Since the negative plate of the condenser is taken to be fixed at zero potential, the positive plate, (in the present schema, the positive terminal of the battery Es) may be taken to be at a fixed potential Es. When the potential of the point Z is raised, a current will flow through the string from this point to the positive condenser plate. If the potential difference in the grid circuit is lowered the reverse process occurs, everything happening in like manner but in the opposite sense; but to avoid confusion of terms it will be simplest to consider in every case the current resulting from raising the grid potential. The quantities we must deal with may be designated as follows : E = total plate battery voltage. Ef, = difference of potential between plate and filament. Ec = difference of potential between grid and filament, grid potential. Es = difference of potential between plates of condenser. Rf, = plate-to-filament resistance of tube. R = by-pass resistance. (Designated as i?2 in fig. 11.) S = resistance of string in galvanometer. lb = plate-to-filament current within tube. ir = current flowing in R. is = current in string galvanometer. 436 ALEXANDER FORBES AND CATHARINE THACHER In the resting state a constant current flows through the tube and the resistance, R, in series, while no current flows in the string; in other words 4 = ir, and is = 0. It is our aim to make the current in the string, resulting from a given change in grid potential as great as possible; in other words, to make -r^ a maximum/ The immediate cause of the string current, is, is the rise of potential at the point Z, or the difference of potential between this point and the positive plate of the condenser; this results directly from the di- minution of voltage drop within the tube, AEf,. This in turn results directly from the change in resistance of the tube. It is therefore our aim to make the change in tube resistance as great as possible without introducing factors which will offset the advantage thus gained. With these considerations in mind we may attempt to find in the tube char- acteristics a clew to the best method of fulfilling the desired conditions. The problem resolves itself into two phases; (a) how to make a given change of grid potential produce the greatest proportional change in tube resistance; (b) how to make a given change of tube resistance pro- duce the greatest current in the string. We shall consider (b) first, since the result of this consideration will influence our mode of attack upon (a). Reduced to its simplest terms the fall of potential at the point Z is that which occurs at the junctional point of two resistances con- nected in series with a battery of fixed voltage, when one resistance changes and the other does not. This may be illustrated simply in figure 19 {Rb and R being transposed for convenience). Let the nega- tive pole of the battery be kept at zero potential, and the positive pole at the potential E. Then if R remains fixed, the potential of the junctional point, E^, will change when the resistance Ri, changes. It can be shown that for any given ratio bet\yeen R and the initial value of Rb, the change in E^,- {dE^,) depends directly on the proportional or percentage change of R^; i.e., on -—-^ or d log R^. It can also be shown Rb that for a fixed value of E the change in E}, for a given proportional change in R^ is greatest when R is made equal to /?;,. In other words ^ For those unfamiliar with this notation it may be exphiined that A signifies a definite increment (or decrement if negative) of the quantity designated; dy d signifies an infinitesimal increment; — signifies the rate at which a variable dx y is changing per unit change of a related variable x at any given time. AMPLIFICATIOX OF ACTION CURRENT WITH ELECTRON TUBE 437 j-p' -p -T-. — ^-— attains its maximum value - when R a log Rh 4 Rh. When this con- ^ Eh dition obtains, since £"5 is half of E, the value — will equal -^. E 4 " 2 If we are free to make the battery voltage, E, as large as we like, and to make R correspondingly large in order that the actual plate voltage in the tube {E^) may be kept within proper Hmits, we can dE, increase the value of (change of potential at Z for a given pro- d log Rb portional change in R^ still more than is possible if E is fixed and R is made equal to the initial value of R^, as was assumed above. + R, Fig. 19. Schema to illustrate change of potential at junction of resistance in series (see text). flE When E and R are thus increased, , . ^^ approaches the value Ei d log Rb as E becomes infinitelj^ large. In other words if we operate the tube at its proper plate voltage we can make the desired change of potential almost twice as great by making the battery voltage, E, very much larger than the plate voltage Eb, as by making it only twice as large as Eb. On this account it would appear desirable to make the battery voltage as large as possible and to increase R correspondingly. In practice the difficulty and danger of ha\ang in the laboratory a bat- tery of much more than twice the 200 volts at which the plate of one of these tubes should operate, would be so objectionable as to offset any such small increase in potential as could be had in this way. 438 ALEXANDER FORBES AND CATHARINE THACHER From the above we have seen that for our purpose we should seek the maximum proportional change of resistance in the tube for a given change of grid potential. This leads us to study the tube char- acteristics. Van der Bijl (1, p. 180) gives us the general equation correlating plate-to-filament current with plate and grid potentials, 7^ = a{yEi, -\- Ec -{■ e)-, where a, y and e are constants depending on the structure and properties of the tube, and the other symbols have the meanings already assigned to them. Langmuir (5) has given a similar formula chiefly differing from van der Bijl's in that the expo- nent is 3/2 instead of 2. It is generally held that some portions of the characteristic curve obey more nearly the formula of van der Bijl, and other portions the formula of Langmuir. The relations embodied in these formulae axe expressed graphically in figure 10. Referring to figure 10 B, showing the relation between plate current and grid potential, and bearing in mind that at a given plate voltage plate current varies inversely with the resistance of thte tube, we see that the porportional change of resistance for a given change of grid po- tential becomes greater and greater as the grid potential becomes more and more negative. It can be shown mathematically that if either van der Bijl's or Langmuir's equation holds, the maximum value of — tI— -^ (i- e., proportional change of tube resistance for af given dEc change of grid potential) is obtained at the point in the curve where the current ?6 becomes zero: i.e., where the resistance becomes infi- nite. If we go too far toward this point on the curve we shall defeat our ultimate aim, for the current in the string is is always less than 4, and if this is made excessively small a corresponding limit is placed on is. We must seek that point in the characteristic curve of the tube as determined by cathode temperature and by Ei, and Ec which, every- thing considered, will give us the greatest current in the string for a given change in Ec. Two ways are open to seek this end, complete mathematical analysis based on the formulae expressing the tube characteristics, and actual experiment with a large variety of systematically chosen values of the independent variables, — filament current, battery voltage, grid poten- tial, and by-pass resistance. Both methods were tried, and the results will be described in turn. Before leaving the general features of the problem it should be noted that with a given grid potential, Ec, the current increases more rapidly as the plate voltage, E^, is increased than it would if the tube AMPLIFICATION OF ACTION CURRENT WITH EtECTEON TUBE 439 resistance were fixed. That is, the increase in plate voltage actually lowers the resistance of the tube. The effect of this is to reduce the amplification which would otherwise be obtained. The reason is as follows: Taking our standard case in which the grid potential is raised by a small amount, this change, A£'c, causes a diminution of the tube resistance, Rj,. But since the plate voltage is not fixed, but depends on the ratio between Rf, and R in accordance with the relation ■pi D -=^ = - — f—Yii the diminution of Ri, serves to lower E;,. This in turn, ij Rb -T R because of the above noted characteristic of the tube, increases the tube resistance. Thus the change in resistance which a change in Ec tends to produce is in part wiped out by the resultant change in the opposite sense caused by the resulting change in Ei,. Mathematical analysis. In this section we are indebted to Dr. E. L. Chaffee for developing a method of analysis and for many helpful criticisms and suggestions. We shall continue to assume the condenser replaced by a battery and the point (fig. 18) to be maintained at zero potential. The obvious method of searching for the law of maximum amplification dis would be to find an expression for j=^ in terms of all the variables in- aEc volved (except filament current which we may regard in the mathe- matical treatment as fixed), and then by differentiating for each vari- able in turn to find at last the value for each which will give the absolute maximum. Following this method we may begin with the formulae , . Eg -\- Sis Is = % ~ h ^Qd If = R derived from Kirchhoff's laws. Combining these with van der Bijl's law for plate current, 4 = a{yEi, + EcY, (neglecting the quantity e which is insignificant), we obtain the formula is = cc(yEb + E,y - ?1±^% R from which by a simple process of algebra we obtain the formula . RociyEb + E,y - Es I, = — • R + S 440 ALEXANDER FORBES AND CATHARINE THACHER This expresses the current in the string in terms of the controllable quantities. But when we attempt to differentiate this expression with respect to Ec, making allowance for the fact that E^, is a function of Ec, we encounter insoluble expressions, and the method proves im- possible. An ingenious method has been proposed by Doctor Chaffee whereby a verv close approximation to the true value of j=^ can be expressed in comparatively simple terms. The method is as follows: — Referring to figure 18 and applying Kirchhbff's laws we have the following fundamental equations : Es + isS - irR = 0, (1) 4 = ir + is, (2) irR = E - Eb. (3) Combining (1) and (2) and multiplying through by R, we have E,R + ibSR - irR (S + R) = 0; substituting (3) and rearranging, we have {E - Et) (R + S) - E,R lb RS (4) Differentiating, T^ "" ~ ~^ — ^^^ aEf) Kb Changing (2) to read ir = 4 — h, and combining it with (l), we have, E, + i,S - ibR + isR = 0; from which 4 = — + — h (6) R R Differentiating, — * = (7) dig R Thus far the equations are rigorous. At this point we make an assumption which introduces a slight inaccuracj', but greatly simpli- fies the treatment. We assume that the tube is operated under con- ditions such that the curve of ij, plotted against E^ is a straight line. AMPLIFICATIOX OF ACTIOX CURRENT WITH ELECTROX TUBE 441 and that the slope of this Hne is not altered when it is displaced to right or left by a change of Ec. For the small changes of £"{, and Eg involved in this work the assumption is a close approximation to the truth. The operation of the system on this assumption is shown graphicalh' in figure 20. Two curves of 4 plotted against Ei, for slightly different values of Ec, are shown side by side. The slope of di 1 these curves in the region used is such that ^^ = -', r, may thus be a Eh r . Fig. 20. Graphic representation of simplified mathematical analysis by Doctor Chaflfee (see text). regarded as the virtual resistance of the tube. The expression for the curve now becomes h = -{Eh + ii{E,-E;)). r In this expression r, fx, and E^ are constants. Then (^) =1 andf^) = ^\ We mav now seek an expression for -p^, the desired quantitj^; dEc dig _ dig dih dEc dib dEc , ^, . / dib \ dib , n ■ , *The expression I 1 means — — when Ec is kept constant. ^ KdEb/Ec dEb (8) 442 ALEXANDER FORBES AND CATHARINE THACHER Substituting (7) in the above we have dis ^ R X — . dE, R + S dE^ dih The next step is to find -j=- under the conditions in the system. dij, = ^ •dEt,+^' dEc. dE, ' bE, dih dEr, KbEjE, dE, KbEjE,' Substituting the partial derivatives above this becomes: ^ = 1 . ^^ 4- ^. (Q-) dEc r dEc r Now in this system anj- change in Ec causes a change in £"{,. From the analysis of the system in accordance with Kirchhoff's laws as indicated in equations (1) to (5) any change in E^^ must be correlated with % in accordance with the formula dib ^ _ R -h S , dEb RS Therefore any change in E{j produced by a change in Ec must cause a corresponding change in 4 which when plotted as in figure 20 will fall on a line whose slope is indicated by -p^, i.e., the cotangent of R -\- S whose angle with the horizontal is . Let dEb be the differ- Ko ential of Eb when 4 remains constant, i.e., the change of E^, re- quired to restore % to its original value when it has been altered by a change in Ec', dEb will then represent the horizontal distance between the two curves. We shall use dEi express the actual differential of Eb in the system as resulting from a change of Ec. Bj^ definition ~dEb ^ /bEb\ dEc \bEjib bib M bEc r bib 1 bEb r AMPLIFICATION OF ACTION CURKENT WITH ELECTEON TUBE 443 Therefore — ' = - ix. (10) By inspection of figure 20 it is evident that lEl^JMi = - r. (11) dih Combining this with (5) we get dE^- dEj ^ dib ^ (R + S)r dii dEb RS SimpKfying this equation and adding 1 to each side, dEb ^ {R + S)r + RS dEb RS Combining this with (10) we get dEb uRS dE, (R + S)r + RS Substituting this expression in (9) and reducing we get dn ^ ^(R + S) dE, ~ {R + S)r + RS Finally by combining this with (7) we obtain the desired formula dig _ nR dE, ~ (R + S)r + RS (12) (13) (14) (15) di This equation enables us to calculate the quantity ~ , upon which diLc amplification depends, in terms of two known resistances and two constants which may be obtained from the characteristic curves of- the tube, and these curves may readily be obtained by direct experi- ment. Evidently to make this expression large we should choose large values of /x and R, and small values of r and S. But whereas the value of the expression is directly proportional to ju, its increase with increase of R is relatively slight. 444 ALEXANDER FORBES AXD CATHARINE THACHER Experimental. j\Iost of the experiments made in order to determine the method of obtaining the maximum amphfication were performed before the mathematical analysis by Doctor Chaffee had been devel- oped. The end result being a function of four independent variables, it was difficult to isolate the effect of varjdng each quantity bj^ itseff. A large number of experiments was necessary. In order to make the basis of comparison uniform we kept the resistance i?„, representing the nerve, constant at 10,000 ohms. It has alreadj^ been mentioned that with the tubes first used, when an initial negative grid potential amounting to 1.5 volts or more was applied, the resulting excursion of the string became independent of the value of this resistance. There- fore the insertion of J?„ under this condition became unnecessary. Early in the experiments we found that considerably greater amplifi- cation was obtained with a negative grid potential than without. Thereafter all experiments bearing on maximum amplification were made with at least 1.5 volts negative grid potential. For the same reason of uniformity of comparison it was necessary to use the same string throughout a series of experiments, or at least to keep the string resistance constant. ]\Iost of the earlier quantitative comparisons were made with string G, having a resistance of 20,000 ohms. Later this string lost its conductivity, and we were left to complete the experi- ments with only string B, having a resistance of 910 ohms. Therefore in order to make the experiments quantitatively valid we introduced a resistance of 19,000 ohms in series with string B to make the resist- ance of that part of the circuit the same as it had been with string G. In a few experiments we placed 20,000 ohms instead of 19,000 ohms in series with string B for convenience. In comparing results, amph- fication was defined as the ratio between the excursion of the string when connected with the electron tube, caused by placing a given change of potential (one or two millivolts), on the grid, and the excur- sion obtained by placing the same difference of potential in series with the string and the resistance i?„ arbitrarily chosen to represent the resistance of a nerve. Clearly the amplification with a given arrange- ment depends on the magnitude of resistance selected to represent the nerve, since as already stated this magnitude has no effect on the excursion obtained with the electron tube under the best conditions of operation, whereas it makes a great difference in the excursion obtained without the tube. In giving figures for amplification the assumed value of Rn must be stated. AMPLIFICATION OF ACTION CURRENT WITH ELECTRON TUBE 445 In order to simplify the problem as far as possible we decided to select a standard value of filament current and to use it in all quan- titative experiments in order that this should not enter as a variable into the result. As already stated we found that the plate current in- creased rapidly with increase of filament current, until the latter reached a value of about 1.1 amperes, and after this, much less rapidly. Van der Bijl has shown (1, p. 175) that a tube should operate in the latter part of its curve; that is, under the condition that the plate current varies little with change of filament current. It is also easier to maintain uniform working conditions with the conductivity of the tube as insensitive as possible to changes of cathode temperature. On the other hand it is desirable not to operate the tube with too high a cathode temperature, since to do so shortens the life of the tube. For these reasons 1.20 amperes was selected as the standard filament cur- rent and used in a great majority of the experiments. In a few cases this was varied in order to determine the effect of doing so. The values E, R and Ec then remained to be studied with a view to choosing the optimum value of each. In our earlier experiments we varied Ec, keeping E and R constant, and then varied R, keeping E and Ec constant. The results were difficult to interpret, since by changing either one of these variables we changed indirectly the value of Ef,, the actual voltage drop within the tube, thereby changing the tube resistance and producing a complex instead of a simple change of conditions. For instance, if Ec was changed, keeping E and R con- stant, the tube resistance was changed, and that portion of the total battery voltage, E, used up in the tube was correspondingly changed. The consequence was that we were not only studying a different part of the 4, Ec curve but a different part of the 4, £"(, curve as well. In like fashion a change of R was complicated by a similar change of Ei,. We finally decided to csn'ry out some experiments keeping Ei, constant at approximatel}^ 200 volts, the value of the plate potential recom- mended bj' Doctor Arnold as giving the best results with this type of tube. In this way we sought to determine under comparable condi- tions the best values of grid potential and total battery voltage, and as a corollary of the latter the value of the by-pass resistance, R. As nearly as we could judge from an effort to determine the influence of plate potential as distinguished from that of other factors in the earlier experiments, the best amplification was obtained in the neighborhood of 200 volts, although this value was by no means critical: amplification almost as great was obtained with" values higher and lower than this. 446 ALEXANDER FORBES AND CATHARINE THACHER Adherence to approximate^ 200 volts as a standard therefore ap- peared advisable. If the plate potential, Ef,, is to be kept constant the problem of choosing the total battery voltage, E, becomes second- ary to that of choosing the value of the by-pass resistance, R. That is, the choice of E is the means to the end of obtaining the desired ratio between by-pass resistance, R, and tube resistance, R^. Preliminary experiments had shown the greatest amplification to be obtained with values of Ec between — 1.5 and —4.0, and with values ■p of the ratio — lying between 0.4 and 0.9. In order to carry out a Rb complete experiment to show the effect of varying the ratio at dif- Rb ferent values of grid potential, and of varying the grid potential at different values of the resistance ratio, we made a careful series of measurements of the tube resistance with the plate potential kept at 200 volts and the following values of grid potential: —2.0, —2.5, — 3.0, —3.4 and —3.9. We selected the following values of the bat- tery voltage, E: 278, 302, 322, 342, 362, as giving with the desired plate potential a series of resistance ratios ranging from 0.4 to 0.9. Each series of observations was to begin with a battery voltage of 278. We therefore calculated from the above measurements of tube resistance the value of by-pass resistance, R, which for each value of grid potential would make the plate potential 200 volts with a battery voltage of 278. In this way each series of observations would start with the same value of E, the same value of Ef,, and consequently the same resistance ratio — , namely 0.4. With these calculations made beforehand the experiment was begun, and on the first observation of each series, the plate current was read with the milliammeter. There- after each time the battery voltage was increased the resistance, R, was correspondingly increased just enough to maintain the same value of the plate current. In this way we could be certain that we were maintaining the same value of the plate potential, E^, so long as the grid potential and filament current were not altered, and these were carefully controlled. Having completed such a series with a grid potential of —2.0, we began the second series with a grid potential of — 2,5, using the same steps of battery voltage and beginning with the previously calculated value of R, noting the resulting value of the plate current and again keeping this constant throughout the series. In this way five series were carried out, but the last three were incom- AMPLIFICATION OF ACTION CURRENT WITH ELECTRON TUBE 447 plete, since we had not resistances enough to maintain the higher ratios when the tube resistance became very high as a result of using a large negative grid potential. At frequent intervals during the experi- ment the apparatus was rearranged according to the standard wiring plan, and control observations were made with the current sent directly- through the string. From these observations we plotted a curve showing the gradual change of string tension due to heating of the galvanometer, which enabled us to apply the necessary corrections to make the comparison valid. Owing to slight inaccuracies in the previous measurements of tube resistance and possibly to slight TABLE 1 0.40 R/Rb 0.52 0.62 0.75 0.88 -2.0 10.9 11.4 11.2 10.8 10.7 -2.5 10.8 11.0 11.1 10.9 10.5 -3.0 10.9 11.0 11.0 10.6 -3.4 10.6 10.5 10.5 -3.9 9.6 10.1 B (corrected) -2.0 11.7 12.4 12.8 13.0 13.4 -2.5 11.6 12.0 12.7 13.1 13.1 -3.0 11.7 12.0 12.5 12.7 -3.4 11.4 11.5 12.0 -3.9 10.3 11.0 changes in the behavior of the tube, we were unable to keep the resistance ratios precisely the same in each series of observations. But by means of the milliammeter readings of the plate current we were able to be sure that the plate potential remained constant dur- ing each series, and we were able to maintain it at nearly the same value through all of the series. The results are tabulated in table 1 A, the figure for amplification being given on the basis of 10,000 ohms as the value of i?„. From this experiment it would appear that the greatest amplifi- cation occurred with a grid potential of about —2.0 volts and with a value of the bj'-pass resistance about half as great as that of the 448 ALEXANDER FORBES AND CATHARINE THACHER tube. On the other hand it is evident from this and from the prehmi- nary experiments that these values are not at all critical; in fact ver}^ nearly as great amplification is obtained with values of all the quantities varied over a wide range; in the case of grid potential all the way from —1.5 to —4.2; in the case of by-pass resistance all the way from equality with the tube resistance down to less than half of this value. After the completion of this experiment the mathematical analysis given above was developed, and the results of experiments were there- upon compared with the calculated values of amplification based on the formula deduced by Doctor Chaffee, — ^ = . In clE, {R + S)r + RS making these calculations the values of /j. and r were obtained by making careful measurements of the tube characteristics, plotting curves of these on coordinate paper and measuring the slopes of the curves at the proper points. The comparison of observed and calculated values showed in general a striking agreement. In those experiments in which no initial nega- tive grid potential was applied and in which therefore the grid cir- cuit absorbed current, there was a wide discrepancy between the calcu- lated and observed values. But inasmuch as the mathematical analy- sis takes no account of such action in the grid circuit this particular discrepancy is to be expected. It is furthermore unimportant, since the amplification is much less under this condition than with a nega- tive grid potential. Therefore only those results in which there was a negative grid potential, and the system operated as an electrometer, need be considered. In all such cases the greatest discrepancy found amounted to only about 15 per cent; in a majority of observations it was less than 5 per cent; in some observations there was practically none. On the other hand in the experiments just described in which conditions were most carefully controlled with reference to constancy of plate potential, we found an inverse correlation which, though slight, was constant, and therefore led us at first to think that the formula failed to deal adequately with all the factors. According to the formula the 66 per cent increase in by-pass resistance between the second and fifth observations in the top row of table 1 A, in which grid potential was kept at —2.0 volts, should have caused an increase in amplification amounting to 8 per cent. In reality it caused a decrease in amplifica- tion amounting to 7 per cent. Similarly in the second series when the grid potential was kept constant at —2.5 volts a 60 per cent increase in AMPLIFICATION OF ACTION CURRENT WITH ELECTRON TUBE 449 by-pass resistance, instead of causing the calculated 6 per cent in- crease in amplification, did cause a 4 per cent decrease in amplification. As stated above, in each of these comparisons grid potential remained constant, and plate potential was kept constant with the aid of the milliammeter in the plate circuit. Therefore, since we were working at precisely the same point in the characteristic curve of the tube, the greatest source of inaccuracy, viz., the determination of r and n depending on the slopes of the characteristic curves, was completely eliminated. An explanation of the apparent discrepancy was found in the leak- age of the condense'r. It has already' been noted that leakage occurred and that the resistance of fifteen one-microfarad condensers connected in parallel, (the arrangement regularly used) amounted to 32 megohms. When the voltage drop in R is increased, as in this experiment, by in- creasing the resistance ratio and with it the total battery voltage, the difference of potential between the condenser plates is increased and with it the ciu'rent in the string due to leakage. This current is great enough to place the string under appreciably increased tension on account of displacement. This increased tension reduces the excur- sion resulting from a given current, and the greater the displacement the greater will be the reduction. In this waj'^ it seemed probable that the increase in amplification which should have resulted from increas- ing the ratio — , was more than nulHfied bv the concomitant increase in string tension. An experiment was made to test this point. The string was adjusted to the same initial tension that was used in the experiment described, and then deflected by a current of approximately the same magnitude as that resulting from the condenser leakage in the case of the lesser voltage drop. A small difference of potential was then applied and the excursion measured. Then the string was further deflected by a current as great as that leaking through the condenser under the greater voltage, and the excursion resulting from the same difference of potential was measured. The decrease of sensitiveness caused by the smaller displacement amounted to 17 per cent; the de- crease caused by the larger displacement, 26 per cent. A correction for this difference applied to the amplification as observed in the ex- periment under consideration, sufficed to change the apparent loss of amplification to a gain of almost exactly the right amount to correspond with the calculated value. The agreement between observed and cal- culated values was thus brought well within the hmits of observational THE AMERICAN JOURNAL OF PHYSIOLOGY, VOL. 52, NO. 3 450 ALEXANDER FORBES AND CATHARINE THACKER error. The experimental verification of Chaffee's formula thus became as complete and satisfactory as the experimental conditions rendered possible. In table 1 B is shown the result of the experiment as modified by the corrections for change of string tension due to displacement. This shows the amplification which presumably would have occurred in this experiment had there been no leakage in the condenser. In table 2 are shown a number of calculated and observed values of dE. from experiments with different values of ix, r, R and S. The TABLE 2 observed values are corrected for string displacement. They illus- trate the agreement between prediction and result; and considering the difficulty in obtaining accurate values of /i and r, ihcj are quite satisfactorv. PRACTICAL DEDUCTIONS Since the formula for amplification — ^ = dEc {R + S)r + RS IS ex- perimentally proved valid, it should be a simple matter to lay down practical rules for obtaining maximmii amplification. Four distinct quantities are involved and ma}^ be dealt with in turn. If r could be considered by itself we should want to make it as small as possible since it appears onlj' in the denominator; that is, we AMPLIFICATION OF ACTION CURRENT WITH ELECTRON TUBE 451 should want to choose the steepest part of the curve correlating /j, with El, (in fig. 10 A). But since jj. is determined in part by r we must consider the two together. Clearly /x should be made as large y JE> J El, as possil)le. But n is defined as equal to -^^ — ; that is, it is the (-) KdEjE^ ratio between the slope of the 4, E^ curve and the slope of the ?<,, Ec curve, or the relative sensitiveness of plate current to changes in grid potential as compared with its sensitiveness to plate potential. In other words it depends not only on the steepness of the 4, Ec curve but also on the lack of steepness of the 4, £"5 curve. Therefore making r small, while it serves to reduce the denominator of our expression, also reduces the value of m and therefore at the same time reduces the numerator. As already stated, the experimental evidence shows that the value of plate potential, Ei„ is not critical as regards amplification. Very nearly the same results are obtained with values all the way from 150 to 250 volts. Considering both convenience and efficiency, a value between 160 and 200 volts is to be recommended. With the value of r thus approximately established, m should be made as large as possible. This means we should seek the steepest part of the 4, E^ curve. But this leads us into the region where Ee is positive, and it has already been shown that unless Ec is made negative the grid circuit absorbs current, and in that way detracts from the amplification. We should then seek the steepest part of the curve where this will not occur. The results shown in the table appear to substantiate this view, in that the greatest amplification here is obtained with the smallest negative value of grid potential, — 2.0 volts. On the other hand, in other experiments with the same tube under otherwise closely similar conditions we have obtained just as great amplification with a grid potential of —3.0 volts; and in some cases almost as great amplification with —4.0 volts grid potential. It is therefore evident that with this tube the variations in M due to varying grid potential from —1.5 to —4.0 are not large, and do not make much difference in the resulting amplification. These figures must not be rehed on for all tubes of this type. A new D-tube, already referred to in footnote 4, was found to absorb cur- rent with a grid potential of —1.5 volts; it was necessary to use as much as 3 volts negative })ias to render the excursions independent of 452 ALEXANDER FORBES AND CATHARINE THACHER input resistance, /?„, and to bring the tube to its point of maximum amplification. Each tube should be tested in this respect before it is used. In practice the most convenient arrangement is the insertion of either one or two dry cells (according to the reciuirements of the individual tube) in the input circuit with the negative terminal con- nected to the grid. ^Yith regard to l^j-pass resistance, R, it is seen that when allow- ance is made for the effect of string displacement due to condenser leakage, an increase in amplification is oljtained by increasing its value, provided the total battery voltage is increased at the same time enough to keep the actual plate potential the same. However, tlie increase in amplification after the by-pass resistance has attained a value of 60 per cent of the tube resistance is very small, and a prac- tical limit is placed on its further increase by the undesirability of installing a battery of much more than 300 volts for so slight a gain as is to l)e had in this way. The correct value may be readil}^ determined after the ]:)attery voltage, E, is established and the tube resistance, i?^, has been measured at a plate voltage of a1)0ut 200 with the negative grid potential foiuid necessary to prevent the grid circuit from taking curi'ciit. The formula is R = — —. Assuming that the tube E, is to be operated at approximately '200 volts, T?^, the necessar}^ data are provided. The value of S is of some importance in determining the amplifica- tion; larger excursions are ol)tained for a given impressed e. m, f. with a low resistance string tlian with a high resistance string. On the other hand, on account (jf large resistances necessarily involved in this system tliis advantage is less than it is in the case of direct re- cording, except when the tissue is one of very high resistance. Further- more, in the choice of a string the main consideration — lightness and correspondingly small inertia, which enable a string to respond rapidly to currents of brief duration — is just as important when the string is used with the electron tube as without. The string will therefore be selected with reference to its availa])ility for nerve work in general, and may be regarded as a constant in this system, and not as a variable to be selected. In some experiments the effect of varying the cathode temperature was tested. Although most of the records were made with a filament current of 1.20 amperes, some were made with 1.25 and some with 1.10. Those made with 1.25 amperes showed little difference in amplification AMPLIFICATION OF ACTION CURRENT WITH ELECTRON TUBE 453 from those with 1.20. Reducing the filament current to 1.10 amperes in the tube first used caused a slight but appreciable reduction in amplification. For example, in one experiment with the following values: filament current, 1.10; Ec, —1.5; E, 270; R, 50,000 ohms; E^, 185 volts; and a resistance ratio of 0.46, the amplification on the basis of 10,000 ohms as the value of R„, was 10.3; — the greatest amplification on this basis obtained with any combination (not correcting for string displacement) was 11.5. But with the last tube used reduction of fila- ment current below 1.15 amperes caused a marked loss in amplification. In general it is advisable for the sake of the resulting increase in the length of life of the bulb, to use it at the lowest filament temperature that will give satisf actor}' amplification. In choosing the battery voltage, E. to be used when the installation was made permanent, we decided on 135 cells as being exceedingly- convenient and at the same time efficient. Forty-five lead storage cells is the maximum number which, connected in series, can be charged efficiently from 110-volt power mains; 45 cells can be conveniently arranged in trays by placing them in nine rows of five cells to a row. Three such trays can be charged in parallel, and when connected in series will give at least 275 volts. As indicated above this voltage is sufficient to operate the tube at very near its maximum efficiency. In practice we have found that our battery when freshly charged has 300 volts on open circuit. In an earlier section it was pointed out that inasmuch as the elec- tron tube under proper conditions operates as an electrometer, meas- uring the difference of potential arising in the tissue irrespective of the resistance, the amplification of galvanometric excursion obtained by means of it, depends on the resistance of the tissue, since the excur- sion obtained with the galvanometer alone depends on tissue resistance as well as on electromotive force. In other words, since a large tissue resistance will reduce the response of the unaided galvanometer to a given e. m. f. but will not reduce the response of the galvanometer when working with an electron tube, a comparison between the two arrangements will show a greater gain in the case of a large resistance than in that of a small one. This fact is shown quantitatively in figure 21. Here the amplifi- cation is plotted for each of the three strings against the value of R„ expressed in thousands of ohms. The curve for string' G (resistance 20,000 ohms) is plotted on the basis of an amplification of 11.4 (cor- rected) at 10,000 ohms tissue resistance, this being a value obtained 454 ALEXANDER FORBES AND CATHARINE THACHER in a number of experiments and very nearly the greatest value obtained at all, — in one experiment Chaffee's formula agreed exactly with obser- vation in giving this value. The curves for strings B and H, with resistances of 910 and 12,000 ohms, are based on the calculated ampli- fication under those experimental conditions in which this precise agreement was found; that is, the proper quantity was substituted for 20,000 as the value of S in the formula. It is notable that as mentioned earlier, amplification is replaced by reduction \\\\\\ the low resistance string when the tissue resistance 1 60 Ami 'I ! 50 y^ y : ^ i^ ' ^ 40 ^^ ^ 1^ 0,-^ ^ ^^ -f. ■^ ^ ^ 30 y^ ^^ ^ ^ JO ^ ^ --?^ ^ ^ 10 , ^ ^ ^^ >" ^ ^ /^ ^ y^ R. 10 ao 30 40 50 60 70 80 10 100 Fig. 21. Amplification (ordinates) plotted against tissue or input resistance, fln, (abscissae) in thousands of ohms, for the three strings used in experiments, and for the particular case in which m = 40, r = 71,000 and R = 100,000. becomes very small. With string B amphfication ceases when /?„ is reduced to about 1000 ohms. On the other hand with a tissue resist- ance of 42,000 ohms the amplification is identical with all three strings, and with greater tissue resistance the string of lowest resistance ampli- fies the most. With a tissue resistance of 100,000 ohms we obtain with string B a 5o-fold amplification, whereas with the 20,000-ohm- string we obtain 45-fold amplification. These curves are only vahd for the particular values of ju, ?' and R selected in this case. We have found that with a different choice of these values different curves are AMPLIFICATION OF ACTION CURRENT WITH ELECTRON TUBE 455 obtained, similar in character and again all crossing at a point, but at a different point denoting a different value of R„. Below this point the amphfication is always greatest with the string of highest resist- ance; above it the inverse relation always holds. The curves are of necessity straight lines. It should be understood in estimating from these curves the relative merits of low and high resistance strings that the gain of amplification is not an absolute quantity, but only rela- tive; it is merely the gain in excursion over what would be obtained in recording under similar conditions without the electron tube. COMPENSATION FOR CONDENSER LEAKAGE In an earlier section it was stated that in most of our experiments we met the problem of string displacement due to condenser leakage, by simply moving the fiber case laterally and thus bringing the string Fig. 22. Method of compensating for condenser leakage, proposed by Doctor Williams. back into the center of the field. At the suggestion of Dr. H. B, Wil- liams, an electrical method of compensation was tried and found satis- factory. A dry cell was connected with a resistance of 700 ohms. The negative pole of the diy cell was connected with the grounded end of the string. From a point in the resistance separated from this terminal by 200 ohms, connection was made with the other end of the string through the resistance of a graphite line drawn on ground glass, having a resistance of about 150,000 ohms. The arrangement is shown in figure 22. In this way enough current is tapped off from the dr}' cell circuit to neutralize the current in the string due to condenser leakage, and yet the resistance of the shunt circuit around the string is so much 456 ALEXANDER FORBES AND CATHARIXE THACHER higher than that of the string itself that it does not act to an}- consider- able extent as a short-circiiit. The combination described approxi- mately balanced the condenser leakage, so that when the protective shunt was opened very little displacement occurred. The amplifica- tion with this arrangement was measured and compared inmiediately afterward with the amplification obtained with exactly the same values of the usual constants and with the ordinar^^ method of bringing the string into the field bj^ moving the fibre case. The excursion ob- tained with electrical compensation was sHghtly the larger of the two. This shows that if there is any loss of efficiency in this method it is less than the apparent loss of efficiency caused by the increased ten- sion due to the usual displacement. If a standard value of each of the variables is adopted as a matter of permanent installation it is a simple matter also to keep permanently installed a drj' cell with the necessary resistances, and to effect electrical compensation by the simple throwing of a single switch. By tapping from the compensating cir- cuit through a still larger resistance, say a megohm, there would be practically no loss of efficiency. PROPORTIOXALITY OF EXCURSIOX TO IXPUT VOLTAGE For certain purposes it is desirable to know whether our system in amplifying gives a faithful record of the relative values of the disturb- ances amplified. That is, we should ascertain whether an e. m. f. of 2 millivolts applied to the grid vnll produce just twice as great an ex- cursion of the string as 1 millivolt. In some experiments this point was put to test. In some cases the excursions resulting from 1 milli- volt ami 1.0 millivolts were compared in quick succession; in others comparisons were made between 1 and 2 millivolts. The propor- tionality regularly was good; in most observations it was accurate to within 2 per cent, and in no case was the value out of proportion by more than 4 per cent. This is within the limits of experimental and observational error. APPLICATIOX TO PHYSIOLOGY In order to put the electron tube method to the practical test of recording action currents, experiments were made with the sciatic nerves of both cats and frogs, and with the muscles of the human forearm led off through the skin by the usual method employed in making electromyograms (,9j. AMPLIFICATION OF ACTION CURRENT WITH ELECTRON TUBE 457 The experiments on nerves will be described first. The resistance of a given length of a frog's sciatic nerve is very much greater than the resistance of the same length of a cat's sciatic nerve. Therefore the action current in the frog's nerve should be susceptible of far greater amplification than that in the cat's nerve, since the resistance in the tissue serves to reduce the current obtainable in the galvano- meter when directl}^ connected with the nerve, whereas we have seen that this resistance causes no reduction in the excursion of the galvano- meter when used with the electron tube. A preliminary experiment was made with a cat's nerve, using the heavj^ string (string B). This Avas done before the best values of the variables for amplification had been found. The result showed amplification, but not as great as was obtained under the more favor- able conditions developed later. After the experiments on maximum amplification had been completed and the permanent installation had been arranged as described above, an experiment was made to illus- trate the amplification of the monophasic action current in a frog's nerve; in this experiment the new string, H, with a resistance of 12,000 ohms, was used. The sciatic nerve of a good-sized frog was placed in a moist chamber with stimulating electrodes on the central end, and the distal end laid across non-polarizable boot electrodes 15 or 20 mm. apart. The nerve was crushed at a point midway between the boot electrodes to render the response monophasic. The resistance of the section of nerve and the electrodes in circuit with the string was measured by substitution, and found to be 85,000 ohms at the be- ginning of the experiment, rising as a result of drying to over 100,000 ohms at the end. Such a high resistance as this should prove favor- able to great amplification, as indicated above. Specificalty, with the combination of tube constants employed, which had previously shown 10.4-fold amplification with 10,000 ohms for i?„ and a string resistance of 20,000 ohms, there should be with a nerve of 85,000 ohms resistance and a string of 12,000 ohms, a 40-fold amplification. In practice it was found that a maximal stimulus sent the shadow of the string off the film in every observation with the electron tube. The lack of constancy in the condition of the nerve made impossible any accurate quantitative comparisons in the case of submaximal stimuli, but as nearly as such comparison could be made it indicated about 45-fold amplification. Records made in this experiment with and without the electron tube are shown in figure 23. In the first pair, (submaximal responses THE AMERICAN JOURNAL OF PHYSIOLOGY, VOL. 52, NO. 3 458 ALEXANDER FORBES AND CATHARINE THACHER produced by make shocks) the stimuli were of the same strength. In the second pair, since the maximal stimuli with the electron tube caused the excursion to exceed the width of the film, the response to a slightly submaximal break shock with the tube is compared with a maximal response with the unaided galvanometer. The speed of film is the same in all, but since the tuning fork causes interference with the electron tube arrangement, time is only shown on the records taken without it. It is noteworth}^ that the duration of the electrical dis- turbance in the nerve is much greater than one would suppose from the records made with the unaided galvanometer, amounting to as Fig. 23. Action currents in frog sciatic nerve (see text). String H. Tension, 1 cm. = 4.16 X 10~' amp. A, electron tube arrangement; B, standard arrange- ment. Speed of film the same in all. Al:— /a, 1.10; E. 300; Eh, 189; R, 60,000; Ec, - l.o. Stimulus submaximal make shock. A2:— /a, 1.10; E, 300; Eh, 207; R, 50.000; ^c - 1.5. Stimulus, break shock, 8.0 Z units (Martin scale). Bl, Stimulus, make shock of same strength as Al. B2, Break shock, 34.5 Z units. much as a tenth of a second. The observations were made at ordi- nary room temperature, i.e., about 20°C. In this experiment and in another under similar conditions, every action current recorded, whether produced bj^ a weak or strong stimulus, showed this surpris- ingly long duration. These experiments were performed in December and January with one of the original D-tubes mounted rigidly in the AMPLIFICATION OF ACTION CURRENT WITH ELECTRON TUBE 459 open air. Three experiments performed on frog nerves in late Feb- riiar}^ and early ]\Iarch failed to show any such prolonged duration (see fig. 26 A). The experimental conditions were identical except that the new D-tiibe alread}^ mentioned, having slightly different char- acteristics, had been substituted for that used in the earlier experi- ments (this being broken), and the tube was now mounted in the sound- proof box. Tests with an artificial source of current fail to reveal an^'thing in the new tube or its mounting which could account for this great change in the duration of the recorded action current. We are therefore led to the view that the difference in duration is phj^sio- logical, possibly due to some seasonal change in the condition of the nerve. It should also be noted in figure 23 that the direct action of the induction shock ("escape of current'') appears in each record, pre- ceding the action current (8, pp. 186-198). This was the case in all experiments with frog nerves even when the stimuli were subminimal. From its absence in the case of a nerve of low resistance (see fig. 26 B) we ma}' infer that the prominence of this effect depends on a- high resistance in the grid circuit. In figure 24 are shown several electromyograms with and without the electron tul)e, some made with string B, a string of small resist- ance and large inertia, and some with string H having large resist- ance and small inertia. In the later of the two experiments when the high resistance string was used, care was taken to see that the con- tractions in each corresponding pair of observations were of the same strength, as indicated by the readings on the dynamometer which the subject gripped. EXTRANEOUS OSCILLATIONS In figures 23 and 24 it will be seen that those records which were made with the electron tube and string H manifest fine oscillations at a frequency' of about 305 a second. These are superimposed upon the coarser oscillations due to physiological activity. Similar oscillations were found in some of the earher experiments with string G (also one of small inertia) as well as with string H, when used mth the electron tube and an artificial source of current, but they were largest in the experiments with the frog's nerve. Thej^ were absent m all experi- ments made with the heavy string, B. On the other hand it is evi- dent that the}^ are not mere mechanical \abrations of the lighter strings, for they are only evident when the galvanometer is used in connection with the electron tube. • ••iisij^ffttiieiei i'a.i • • • • * ♦ • i i 9 i B i a if' i J %aM a t i a a a a 9^ a a i c D E V aaaaaaisaaaaaaaaaa.aaaai UkUikitikkki&kikknkkkLikkkkUkkUAiJkikkiiiUkkkkk^LiMUkkkkkkkkkkkkUkUkkkk i Mkk s A & ^ iAkkkkikk n Ukt h k kU t M k kkkk k k kkkkUMkkkkkkkkkkkkkkkkkkkkkkkkkkkkkkt AiiiAilUlAiiiiAAiliUAAiUAAAilAiAiliAiiiiAiiiiiiAAAAiiaiAiAAiliiAliiiAilAiii ^-----.v /V-NV "/V-V^- vV . S + 100,000 ' S + 10,000 Amplification in 2 is" somewhat reduced by low value of /a- AMPLIFICATION OF ACTION CURRENT WITH ELECTRON TUBE 465 tion of current, is introduced in this side of the grid circuit, and if the primary resistance, Ri, is arranged in two parts with the key between them as was the case in most of our experiments. The oscillations persisting in the records of frog nerves are probably in part due to the induction from the generator just mentioned. Their irregularity may indicate some further disturbance of phj'siological origin, or may be due to some extraneous cause not yet identified. At all events, the oscillations persisting in spite of the sound-proof box are insignificant as compared with a maximal action current in a nerve trunk; and with a resistance as low as is common in mammalian experi- ments they are imperceptible. PRECAUTIONS Several precautions in the use of the electron tube should be enum- erated and emphasized. The most important of these concerns the avoidance of charging or discharging the condenser through the string. The rule is not to open or close any switch in the filament, grid or plate circuit, in short, anywhere in the electron-tube system, nor to make any adjustment in any part of the system while it is connected with the string galvanometer. All adjustments of current and voltage connected with the tube, and of by-pass resistance, should be made while the switch leading to the string is open and while the protective shunt (or equivalent short-circuit) is closed to enable the condenser to be charged from the B-battery. After all adjustments are made the switch leading to the string may be closed, and finally the protective shunt opened; but it should not be opened until at least 5 seconds after the closure of the switch which permits the charging of the condenser, for on account of the large resistance through which the charging cur- rent must pass, this time is required for the current to subside to a value which is safe to pass through the string. The string should be disconnected again before any switches in the rest of the system are opened. The least neglect of this rule will probably result in the loss of the string. There is one exception; the mere closing of the protec- tive shunt, without disconnecting the string, is sufficient protection during minor adjustments of the filament current if made with suffi- cient caution. If there is any possibility that one of the electrodes may be dislodged from its contact with the tissue under observation during an experi- ment, a shunt should be provided to prevent the grid circuit from THE AMEBICAN JOUBNAI, OF PHYBIOLOQT, VOL. 52, NO. 3 466 ALEXANDER FORBES AND CATHARINE THACHER being broken. If a grid "bias" is used, the change in tube resistance which would result from breaking the grid circuit, would cause a current in the string so great as probably to destroy it. As a safeguard a graphite pencil line of about a megohm resistance, drawn on paper, has been connected across the leads to the non-polarizable electrodes, and has been found by experiment not to reduce measurably the ex- cursion resulting from the action current of a nerve. The filament current should be shut off when the tube is not in use. With the tube out of sight in a sound-proof box, one is apt to overlook this at the close of an experiment. The life of a tube is usually long, but not unlimited. In one case a tube was permanently impaired b\' accident ly leaving this current on until the battery was discharged. Other precautions have been touched on in connection with the electricall}' induced oscillations caused by the Sandstrom kj-mograph motor and by the electrically driven tuning fork, and in connection with the oscillations traceable to vibration of the tube. Two dis- tinct classes of disturbance are met with, those electrically induced and those of a mechanical origin. Two ways of dealing with the electrical disturbances are open, — prevention of the cause, and inter- vention between the cause and effect by shielding. It is desirable to avoid the use of electric motors in the vicinity of the system, and to minimize sparking from any source. To drive the film a low volt- age motor completely encased in grounded metal is satisfactory. To record time a device which is free from the sparking of an electro- magnetic tuning fork, should be used. It has been found that rapid motion of the body close to the grid circuit, or shuffling of the feet produces marked excursions of the string. These are to be explained by the electrostatic induction in the grid circuit by the more or less charged body. On this account the experimenter should stand as still as possible during an observation. This disturbance is somewhat reduced by grounding the bod}' of the experimenter. This has been effected by keeping the hand on the clutch which sets the film in motion, and which is grounded. If, when a stimulating inductoriimi is set up near the electron tube "system, the primary circuit of the inductorium is touched with the hand, an excursion of the string will result when the circuit is made or broken, even if there is no connection between the inductoriimi and the recording sj'stem (see p. 428). It is essential, therefore that if the stimulating key be operated by hand it should be done through an insulated handle. AMPLIFICATION OF ACTION CURRENT WITH ELECTRON TUBE 467 It is important to see that all connections are good; they should be soldered as far as possible. A loose connection will introduce serious disturbance and may result in the loss of a string. It is also very important that all batteries, especially the B-battery, should be in good condition. Dry cells which are nearly exhausted or storage cells in bad condition or bubbling as a result of too recent charging, may cause troublesome excursions of the string. The danger of mistaking static effects or other extraneous disturb- ances for physiological effects is always present in the study of action currents with the string galvanometer. Amplification with the elec- tron tube enormously increases this danger; one cannot be too careful to guard against it both by eliminating all possible sources of dis- turbance and by scrutinizing all results obtained with reference to latent period, dependence on vitality in the tissue studied, and any other possible criteria which may serve to verify the physiological origin of the current observed. SUMMARY 1. The electron tube is a device wherein a stream of electrons emitted from a hot cathode in a vacuum and drawn to an anode (plate) by a high voltage battery, is modified by small changes of potential applied to a third electrode known as the grid, placed in the intervening space. In this way it serves as an amplifying relay and is commonly used as such in radio telegraphy. 2. This device offers great possibilities of amplifying such action currents in the nervous system as are too small to be recorded satis- factorily with the string galvanometer alone. When the string gal- vanometer is used to record the current amplified by this device it cannot be placed directly in the path of the high voltage current, as is done with the telephone, without destruction of the string. This must be protected from the direct current, and yet so connected with the system as to record changes in the electron stream. 3. Three methods of connecting the string with the tube in such a way as to afford the needed protection, were tried. These are desig- nated the transformer method, the bridge method and the condenser method. They are illustrated diagrammatically in figures 3, 4 and 5. The condenser method was the only one which proved wholly satis- factory. It consists in placing a very large condenser in series with the string, and shunting the plate current by both through a resistance 468 ALEXANDER FORBES AND CATHARINE THACHER of the same order of magnitude as the plate-to-filament resistance of the tube. By using a condenser with a capacity of as much as 15 microfarads the distortion due to gradual discharge of the condenser when the system is unbalanced becomes negligibly small in the case of action currents of brief duration. A permanent wiring arrangement was installed by means of which the electron tube system can be made available for the amplification of action currents in a tissue under observation, by merely throwing a few switches and making two or three adjustments, a process requiring less than two minutes. "D-tubes" loaned bj^ the Western Electric Company, have been used in these experiments. 4. An effort was made to determine those values of filament-heating current, plate-battery voltage, grid potential, and by-pass resistance which would afford the greatest possible amplification. In general, amplification depends on a large proportional change in tube resistance for a given change in grid potential, together with other factors whose interaction is rather complex. 5. Complete mathematical analj'sis of the problem, using the em- pirically established formulae for tube characteristics, leads to insol- uble expressions. Dr. E. L. Chaffee devised a method of obtaining an approximate formula for expressing in terms of two easil}^ measur- able quantities in the tube characteristics and of the resistance of the string and the b3^-pass resistance, amplification in anj" given case. The formula is — ^ = . In this is is the cur- dE, (R + S)r-\-RS rent in the string; E^ is grid potential; R is the bj^-pass resistance; S is the resistance of the string; r is the virtual resistance of the tube as determined by the reciprocal of the slope of the curve correlating plate current with plate potential; n expresses the sensitiveness of plate current to grid potential as compared with its sensitiveness to plate potential, in other words, the ratio between the slopes of the curves correlating plate current with grid potential and with plate potential respectively. 6. A large number of experiments in which the amplification was measured with a large variety of combinations of the quantities above enumerated, verified Doctor Chaffee's formula. Unless the grid is given an initial negative potential the grid circuit will absorb current and thereby detract considerably from the amplification. It was found that best results are obtained with a filament current of 1.1 to 1.2 am- peres, with a negative grid potential of 1.5 to 3.0 volts (the larger value AMPLIFICATION OF ACTION CURRENT WITH ELECTRON TUBE 469 being necessary with some tubes), with a plate voltage of approximately 200, and with a plate battery voltage as large as is convenient to install in the laboratory, the difference between plate and plate battery voltage being consumed in the by-pass resistance. This resistance should be at least half as large as the resistance of the tube, and preferably more than that. In practice the combination should depend on preliminary tests of the individual tube. Convenient and efficient arrangements with the D-tubes used have been within the following limits — fila- ment current 1.1 to 1.2; grid potential —1.5 or —3.0 (one or two dry cells); plate battery voltage 300; by-pass resistance 50,000 to 80,000 ohms. 7. The use of paper condensers, which are most convenient when a very large capacit}^ is desired, introduces sufficient leakage of cur- rent to cause a considerable displacement of the string. With the Cambridge string galvanometer the string may be simply brought back into the field by lateral adjustment of the fiber case. A somewhat better method is to tap off from a dry cell through a high resistance an equal and opposite difference of potential. 8. As nearly as we could measure it, the amphfied current in the string galvanometer was strictly proportional to the e. m. f. applied to the grid circuit. 9. If the grid potential is kept negative with respect to the filament by a sufficient amount to prevent the flow of current in the grid cir- cuit, the excursions of the string depend wholly on the e. m. f. im- pressed on the grid, and are independent of the resistance of the input circuit, i.e., with values of resistance at least up to 150,000 ohms. In this way the system operates as an electrometer rather than a galvanom- eter. The amphfication, or ratio between excursions of the string with and without the tube depends both on the resistance of the input or tissue circuit and on the resistance of the string. The electron tube necessarily introduces high resistance into this system; therefore if the current to be recorded arises in a tissue of small resistance, and is recorded with a string of small resistance, the use of the electron tube will afford no amplification. But if either string or tissue has a large resistance there will be amplification. Given the resistance of the string and the amplification obtained with any given tissue resistance, the amplification obtainable with any other tissue resistance may be simply calculated. In the case of a frog's sciatic nerve having a re- sistance of 100,000 ohms between the leading-off electrodes, the ampli- fication may be more than 50-fold. 470 ALEXANDER FORBES AND CATHARINE THACHER 10. Records have been made with the electron tube of the action currents in frog's nerve, and in human muscles during voluntary con- traction, both with a high resistance string and with a low resistance string. These have been compared with action currents recorded di- rectly with the unaided galvanometer under otherwise similar condi- tions. In the case of the frog sciatic nerve an amplification of more than 40-fold has been found. 11. When a motor operated on the 110-volt power mains was used to drive the film for photographic recording, oscillations were induced in the galvanometer through the electron tube system, which we were unable to prevent by screening. It proved necessary to record with a camera designed to work with a 12-volt motor completely encased in metal and run from a storage battery within the room. This camera has certain advantages over those previously used, and a brief descrip- tion is included. Oscillations were also induced by an electrically driven tuning fork. These oscillations were probably traceable to high frequency currents arising in the electric spark and rectified by the tube. It was necessary to dispense with this method of recording time. 12. Other oscillations occurred in the galvanometer when connected with the electron tube system, which were traceable to vibrations in the elements in the tube, causing fluctuations in the electron stream. This difficulty was eliminated by installing the electron tube in a sound-proof box, suspended by the method of Julius. Smaller oscilla- tions persisted in experiments with nerves of very high resistance, traceable probably to induction from the power mains. They are so small as to be insignificant, and were absent in experiments with tissues of medium resistance. 13. In addition to the precautions indicated in the last two sections, it is most important that the string be protected from the high volt- ages used in this sj^stem by careful avoidance of adjustments in any part of the system w^hile the string is connected with it, and by allow- ing the condenser to become charged through a protective shunt or short-circuit before any connection with the string is made. Other confusing effects due to static electricity or bad connections or batteries in poor condition, must be carefully guarded against, if satisfactory results are to be obtained. We wish to express our thanks to Dr. H. B. Williams, Dr. H. B. Arnold, INIr. E. H. Colpitts, Mr. S. W. Dean, i\Ir. Sewall Cabot and AMPLIFICATION OF ACTION CURRENT WITH ELECTRON TIJBE 471 Prof. G. W, Pierce for suggestions and advice, and especially to Dr. E. L. Chaffee for the mathematical analysis, and to Miss M. D. Ring for help in preparing the work for publication. BIBLIOGRAPHY (1) VAN DER Bijl: Phys. Rev., N. S., 1918, xii, 171. (2) Deforest: U. S. Patents no. 841, 387, 1907; no. 879, 532, 1908. (3) Whittemore: Phys. Rev., N. S., 1917, ix, 434. (4) Forbes and Gregg: This Journal, 1915, xxxvii, 118. (5) Langmtiir: Proc. Inst. Radio Engineers, 1915, ill, 261. (6) Einthoven: Annal. d. Physik, 4th Folge, 1905, xvi, 20. (7) JuLiTJs: Annal. d. Physik, N. S., 1895. Ivi, 151. (8) Forbes and Gregg: This Journal, 1915, xxxix. 172. <9) Forbes and Rappleye: This Journal, 1917, xlii, 228. VARIATIONS IN THE RESPIRATORY DEAD AIR SPACE DUE TO CHANGES IN THE DEPTH OF BREATHING R. G. PEARCE AXD D. H. HOOVER From the Cardio-Respiratory Laboratory, Medical Service, Lakeside Hospital Received for publication April 8, 1920 The variation which occurs in the capacity of the dead air space of the lungs because of changes in the tone of the bronchial muscles or in the amount of air inspired at each breath, is unsettled. In a previous paper of this series (1) it was shown that the dead air space has a fairly constant volume at ordinary depths of inspiration, and the great variation in the capacity of the dead air space found hj Haldane (2) and by Henderson (3) and co-workers was not sustained, while the findings of Krogh and Lindhard (4) were in the main substantiated. These latter observers found evidence of a small increase in the capacitj' of the dead air space when maximal breaths were taken. While we were unable to obtain absolute evidence of even this small increase, we believe that our early results suggested such a possibihty. Our experiments were free from many errors present in earlier work, but owing to the manner of combining our figures we obtained averages of the dead air space for all depths of inspiration. Later work bj- us indicated that, at moderate volumes of inspiration, the dead air space was a relatively fixed volume. Unfortunate!}', in the method employed we did not and could not use extreme volumes of inspiration. Moreover the spirometers we used had a dead air space of their own which, although we tried to elmiinate it b}' filling with expired air before the observation, must have contributed toward making our results too high. In order to put this subject to further test, we have devised a method which we beheve will, if properly carried out, give a fairh' accurate measure of the capacity of the air passages at all depths of inspiration. In this method, as in the former method, we used the mathematical formula given in our first paper (5) . By it the carbon dioxide contents of a large and a small expiration following a measured inspiration are compared with the volume of air in a large and a small expiration in 472 VARIATIONS IN RESPIRATORY DEAD SPACE 473 a manner so that the air from the dead space may be determined. In the appHcation of this formula to estimating the capacity of the dead air space at different lung volumes, we assimie that the amount of carbon dioxide which is picked up by the definite volume of air inspired and expired quickly, following a forced expiration to residual air, is fairly constant in amount for a given state of bodily activity. That this is the case we have satisfied ourselves. We also assume that the carbon dioxide in the alveolar air collected for analysis has a uniform concentration. We admit that such is not the case actually, but for practical purposes of the experiment, providing the expirations com- pared do not differ by more than 400 cc, the assumption may be allowed. [ UJ^LLJ^ /Q^^^^- J 5aK Fig. 1 It is also assumed that, given the same volume of inspiration, the capacity- of the air passages will be the same in the different observations necessary to collect the required data. By progressively increasing the amount of air inspired, any variation of the dead air space due to stretching of the bronchial tree can be detected if such variations be greater than the experimental error of the method. The essential feature of this method is that a sharp, quick expiration to residual air be followed by a quick inspiration of a measured volume of air, and this followed by a quick expiration, the first portion of which is collected for measurement and anah'sis. The amount of air collected must be varied between 400 and 800 to 1000 cc. for best results. Briefly the experimental procedure is as follows. Reference is to figure 1. 474 R. G. PEARCE AND D. H. HOOVER Bag v4. is a flat rubber bag capable of holding 5 liters of air. It connects with a three-way stopcock, 6', which in turn connects with stopcock H, which has a mouthpiece, I. Bag S is a flat rubber bag of 1200 cc. capacity attached to cock G. C is an automobile grease gun, delivering 340 cc. of air at a stroke, and D a three-Avay cock for taking air into the gun and delivering it into bag A. E is a 100 cc. Luer syringe, and F a three-way cock attached to bag B for measuring the volume of air in bag B. K is a tube with cock for obtaining samples for analysis. The bags are flat and do not hold an appreciable amount of air when evacuated. Bag A is filled with a measured amount of air by C. The cocks are then as shown in position 1. The subject is seated, and after five minutes of rest makes a quick forced maximal expiration. This must have followed a normal inspiration. Cock H is then turned to position 2, and a quick inspiration empties bag A. The breath is held while cock G is turned to position 3 and a forced expiration is made into bag B. In this case, however, cock G is quickly turned to position 4 during the expiration. By this means it is possible to limit the amount of air collected in bag B. The air in bag B is then analyzed accurately for its carbon dioxide content, and its volume measured by means of syringe E. For each inspiratory volume it is necessary to make at least four observations, in which two small volumes and two somewhat larger volumes of the first portion of the expired air are collected in bag B. The amounts of air collected in bag B and their carbon dioxide con- tent are combined in the formula for the estimation of the dead air space as given in our previous papers. The formula reads: Volume of large multiphed by the volume of the small portions of expired air, multiplied by the difference in the percentage of carbon dioxide in the large and small portions of expired air, divided by the difference in the total volume of carbon dioxide found in the large and small portions of expired air, equals the volume of the dead air space. The vahdity of using this method for obtaining the volume of the dead air space depends on certain factors: a, For each individual series of observations in which a given volume of air is inspired, the amount of carbon dioxide produced per unit of time must be constant during the experiment. For this reason it is absolutely necessary that the subject rest 3 to 5 minutes before each observation, b, The inspiration of the measured amount of air in the large bag must follow the deepest possible expiration, which in turn must have followed a normal inspira- VARIATIONS IN RESPIRATORY DEAD SPACE 475 tion. This must obtain, since otherwise the amount of carbon dioxide in the lung air and the amount which is dehvered by the blood to the lung air during the moment of observation would vary, c, The small bag, which collects the first part of the inspiration, must not contain any air except that which has been received from the first part of the forced expiration, d, The analysis of the carbon dioxide content of the air in the small bag must be very accurate, and the volume of the air collected in the small bag must be measured very accurately, e, The periods of time taken for the observations must be about equal, in order that the carbon dioxide eliminated by the blood be the same for each observation. The results obtained on the subjects are given in tables 1, 2, 3 and 4, and are plotted as curves on cross-section paper, figure 2, the abscissae of which represent the volumes of air inspired and the ordinates the volumes of dead air space found with the respective inspiratory volume. It will be noted that when ordinary volumes of inspiration — 1500 to 2500 cc. of air — are taken after a forced expiration (which amount is practically that which is ordinarily present in the lungs at the end of the normal inspiration), the variations of dead air space are very little but progressively increase; while with larger volumes of inspiration the dead air space increases from 100 to 115 per cent maximum. In other words, the increase in dead air space with the increasing volumes of inspiration is not a linear function to the depth of inspiration but a powered function. The explanation of this phenomenon no doubt rests in the fact that the small bronchioles contribute much to the volume of dead air space and are expanded somewhat during the in- spiratory effort. The greater the inspiratory effort, the greater must be the negative pressure of the intra-thoracic pressure. Since the bronchi and bronchioles are made up of more or less elastic tissue, it is reasonable to suppose that their walls are dilated more or less pro- portionately to the degree of intra-thoracic pressure. Since the area of a circle varies as the square of its radius, it is apparent that a slight progressive increase in the diameter of the bronchioles will cause a relatively disproportionate and larger increase in the area of the cross section of the bronchioles. The curves obtained very strongly suggest that this condition obtains to a greater or less degree during deep breathing. It is interesting to note that the maximum variation of dead air space, obtained when the deepest possible inspiration was made, is about 115 cc, correspond- ing closely to the figures obtained by Krogh and Lindhart (4). It 476 R. G. PEARCE AND D, H. HOOVER TABLE 1 Subject R. G. Pearce, height 5 feet 9 inches, weight 176 pounds. Vital capacity about 4000 cc. EXPERIMENT NUMBER AIR INSPIRED FROM BAG A AIR EXPIRED INTO BAG B CO2 IN BAG B volume of co2 in bagB CC. CC. per cent 1 1700 660 3.50 23.00 2 1700 690 3.55 24.50 3 1700 390 3.10 12.10 4 1700 350 3.00 10.50 1 2380 685 2.90 19.80 2 2380 590 2.80 16.50 3 2380 303 2.25 6.80 1 3060 585 2.60 15.25 2 3060 383 2.15 8.20 1 3740 720 2.40 21.70 2 3740 655 2.85 18.60 3 3740 665 2.80 18.50 4 3740 395 2.10 7.80 5 3740 365 2.10 7.30 When 1700 cc. of air are inspired: By combining 1 and 3 we have 94.5 cc. dead air .space By combining 1 and 4 we have 92.0 cc. dead air space By combining 2 and 3 we have 97.5 cc. dead air space Bj' combining 2 and 4 we have 95.0 cc. dead air space Average dead air space when 1700 cc. are inspired is 94.8 cc. When 2380 cc. of air are inspired: By combining 1 and 3 we find 104 cc. of dead air space By combining 1 and 4 we find 101 cc. of dead air space Average dead air space when 2380 cc. are inspired is 102.5 cc. When 3060 cc. of air are inspired: By combining 1 and 2 we have 143 cc. of dead air space When 3740 cc. of air are inspired: By combining 2 and 3 we find 201 cc. dead air space B}' combining 3 and 5 we find 182 cc. dead air space B3- combining 1 and 4 we find 184 cc. dead air space B3' combining 1 and 5 we find 165 cc. dead air space By combining 3 and 4 we find 196 cc. dead air space By combining 3 and 5 we find 172 cc. dead air space Average dead air space when 3740 cc. are inspired is 183 cc. VARIATIONS IN RESPIRATORY DEAD SPACE 477 Summary 1700 cc. inspired gives dead space of 94.5 cc. 2380 cc. inspired gives dead space of 102.5 cc. 3060 cc. inspired gives dead space of 143.0 cc. 3740 cc. inspired gives dead space of 183.0 cc. TABLE 2 The following results were obtained on D. H. H. between 4 '-00 and 6 :00 p.m., June 3, 1918, the subject being seated in a chair at rest. The experiment ivas the same as above, save that the expired air was collected in a small spirometer. The dead space of the apparatus when these data were collected was probably 20 cc. greater than with the improved method using the rubber bag B. His vital capacity was about 4000 cc. EXPERIMENT NUMBER INSPIRED EXPIRED SPIROMETER B CO2 SPIROMETER B CO2 SPIROMETER B CC. cc. per cent CC. 1 1500 560 2.75 15.40 2 1500 525 2.70 14.20 3 1500 930 3.05 28.40 4 1500 1035 3.10 32.10 1 2100 735 2.70 19.80 2 2100 670 2.60 17.40 3 2100 1070 3.05 32.60 4 2100 1085 3.10 33.60 1 2900 1130 2.50 28.20 2 2900 1060 2.47 26.20 3 2900 500 1.80 9.00 4 2900 525 2.00 10.50 5 2900 525 1.85 9.70 1 3600 1035 2.20 23.20 2 3600 1125 2.32 26.10 3 3600 1105 2.33 26.80 4 3600 620 1.75 10.85 5 3600 630 1.75 11.00 1 4300 680 1.85 12.60 2 4300 675 1.75 11.80 3 4300 1125 2.25 25.30 4 4300 1085 2.15 23.30 478 R. G. PEARCE AND D. H. HOOVER When 1300 cc. of air are inspired: By combining 1 and 4 we find 120 cc. dead air space By combining 2 and 4 we find 121 cc. dead air space By combining 1 and 5 we find 130 cc. dead air space By combining 2 and 4 we find 121 cc. dead air space Average dead air space when 1500 cc. are inspired is 123 cc. When 2100 cc. of air are inspired: By combining 1 and 3 we find 120 cc. dead air space By combining 2 and 3 we find 129 cc. dead air space By combining 1 and 4 we find 140 cc. dead air space By combining 2 and 4 we find 139 cc. dead air space Average dead air space when 2100 cc. are inspired is 132 cc. When 2900 cc. of air are inspired: B\' combining 1 and 3 we find 208 cc. dead air space By combining 2 and 3 we find 206 cc. dead air space By combining 1 and 4 we find 156 cc. dead air space Bj- combining 1 and 5 we find 208 cc. dead air space By combining 2 and 5 we find 209 cc. dead air space Bj' combining 2 and 4 we find 166 cc. dead air space Average dead air space when 2900 cc. are inspired is 192 cc. When 3600 cc. of air are inspired: By combining 1 and 4 we find 238 cc. dead air space Bv combining 1 and 5 we find 245 cc. dead air space B}' combining 2 and 4 we find 261 cc. dead air space By combining 3 and 4 we find 245 cc. dead air space Average dead air space when 3600 cc. of air are inspired is 247 cc. When 4300 cc. of air are inspired: By combining 1 and 3 we have 241 cc. dead air space By combining 2 and 3 we have 280 cc. dead air space By combining 1 and 4 we have 201 cc. dead air space By combining 2 and 4 we have 253 cc. dead air space Average dead air space when 4300 cc. of air are inspired is 243 cc. In this case .3800 cc. was about the vital capacity. Summarjj 1500 cc. inspired gives dead space of 123 cc. 2100 cc. inspired gives dead space of 132 cc. 2900 cc. inspired gives dead space of 192 cc. 3600 cc. inspired gives dead space of 247 cc. 3800 cc. (maximum) gives dead space of 243 cc. This is given in form of curve in which 20 cc. are deducted from each figure because of the dead space present in the spirometer. This figure is an approxi- mate one only for the actual dead space of the Krogh spirometer is difficult to measure accurately. VARIATIONS IN RESPIRATORY DEAD SPACE 479 TABLE 3 Subject R. G. Pearce EXPERIMENT AIR INSPIRED FROM AIR EXPIRED INTO CO2 IN BAG B VOLUME OF CO: IN NUMBER BAG A BAG B EXPIRED AIR CC. CC. per cent 1 1700 830 3.05 25.40 2 1700 925 3.00 27.75 3 1700 560 2.80 15.70 4 1700 455 2.70 12.25 1 2680 720 2.30 16.55 2 2680 520 2.10 10.90 3 2680 470 2.00 9.40 4 2680 340 1.75 5.95 1 3740 750 2.30 17.40 2 3740 663 2.25 14.90 3 3740 430 1.75 7.55 4 3740 425 1.75 7.45 When 1700 cc. of air are inspired: By combining 1 and 3 we find 120 cc. dead air space By combining 1 and 4 we find 101 cc. dead air space By combining 2 and 3 we find 85 cc. dead air space By combining 2 and 4 we find 82 cc. dead air space Average dead air space when 1700 cc. are inspired is 97 cc. When 2680 cc. of air are inspired: By combining 1 and 3 we find 142 cc. dead air space B3- combining 1 and 4 we find 128 cc. dead air space By combining 2 and 4 we find 125 cc. dead air space Average dead air space when 2680 cc. of air are inspired is 132 cc. When 3740 cc. of air are inspired: By combining 1 and 3 we find 176 cc. of dead air space By combining 2 and 3 we find 176 cc. of dead air space By combining 1 and 4 we find 191 cc. of dead air space By combining 2 and 4 we find 189 cc. of dead air space Average dead air space when 3740 cc. of air are inspired is 183 cc. Summary 1700 cc. inspired gives a dead air space of 97 cc. 2680 cc. inspired gives a dead air space of 132 cc. 3740 cc. inspired gives a dead air space of 183 cc. 480 R. G. PEARCE AND D. H. HOOVER TABLE 4 Table 4 gives the data collected in the case of Dr. M. D., who is 5 feet 9\ inches in height and weighs 130 pounds. His tidal air amounts to about 500 cc, and his complemental air to 1200 cc. When breathing in 2200 cc. following a forced expi- ration, his lungs would contain about 500 cc. more than at the end of a normal inspiration, or when breathing 3600 cc. as above, 1900 cc. more than is present at the end of a normal inspiration. His vital capacity was about 8600 cc. EXPERIMENT AIR INSPIRED FROM AIR EXPIRED INTO COj IN SPIROMETER AMOUNT CO2 IN NUMBER BAG .4 SPIROMETER B B SPIROMETER B CC. CC. per cent 1 2200 1000 3.55 35.5 2 2200 1000 3.50 35.0 3 2200 790 3.40 26.8 4 2200 700 3.30 23.1 5 2200 650 3.25 21.2 6 2200 400 2.75 11.0 1 3600 1500 3.00 40.5 2 3600 1050 2.90 30.4 3 3600 800 2.75 22.0 4 3600 575 2.40 13.8 When 2200 cc. of air are inspired: By combining 1 and 6 we find 130 cc. dead air space By combining 1 and 5 we find 132 cc. dead air space By combining 1 and 4 we find 140 cc. dead air space By combining 6 and 3 we find 121 cc. dead air space By combining 6 and 4 we find 128 cc. dead air space Average dead air space when 2200 cc. are inspired is 131 cc. When 3600 cc. of air are inspired: By combining 1 and 4 we find 185 cc. dead air space By combining 1 and 3 we find 182 cc. dead air space By combining 1 and 2 we find 195 cc. dead air space By combining 2 and 4 we find 182 cc. dead air space By combining 3 and 4 we find 196 cc. dead air space Average dead air space when 3600 cc. are inspired is 184 cc. Sximmary 2200 cc. inspired gives a dead air space of 131 cc. 3600 cc. inspired gives a dead air space of 184 cc. VARIATIONS IX RESPIRATORY DEAD SPACE 481 is most important to note that variations in the dead air space in the ordinary volumes of respiration are shght, and this point makes it possible to determine the percentage composition of the alveolar air with reasonable accuracy by the examination of the gaseous content of the expired air, correction being made for the dead air space. It is also interesting to note that the volume of the respiratory dead air space thus determined is somewhat smaller than has hitherto been thought. tC IMSPIRCD Fig. 2 It was hoped that evidence might be obtained of normal variations in the respiratory dead air space at different times of the day and under varying conditions. Such variation has been noted previously by us when the dead air space was determined by the method described in previous articles of this series. The experimental error of the method is probably too great to allow this to be done. THE AMERICAN JOURNAL OF PHYSIOLOGY, VOL. 52, NO. 3 482 R. G. PEARCE AND D. H, HOOVER SUMMARY 1. A new method of determining the capacity of the dead air space is described. 2. The volume of the respiratory dead air space is relatively constant at all volumes of respiration. 3. The changes which occur in the volume of the dead air space when the variations in depth of breathing are such as ordinarily occur during moderate effort, are not of as great magnitude as when forced respiratory efforts are made. 4. The percentage composition of the alveolar air can be determined with approximate accuracy from analysis of the expired air and an assumed volume of the dead air space, if the depth of breathing is not excessive. This method is as reliable as the best direct methods now used for the determination of the percentage composition of alveolar air, providing the respiratory volume is large enough to completely wash out the dead air space. BIBLIOGRAPHY (1) Pearce and Hoover: This Journal, 1917, xliv, 391. (2) Haldane: This Journal, 1915, xxxviii, 20. (3) Henderson, Chillingworth and Whitney: This Journal, 1915, xxxviii, 1. (4) Krogh and Lindhard: Journ. Physiol., 1917, li, 59. (5) Pearce: This Journal, 1917, xliii, 73. THE SUB-ARACHNOID AND INTRA-ARTERIAL ADMINIS- TRATION OF SODIUM BICARBONATE AND OTHER ELECTROLYTES J. B. COLLIP From the Department of Bio-chemistry and Physiology of the University of Alberta, Edmonton, Canada Received for publication April 8, 1920 INTRODUCTION It has been shown that extensive voluntary hyperpnoea may produce mild tetany in the normal human subject (1). The causation of this effect was interpreted as ^ mild alkalosis due to the "washing out" of CO2 from the blood at the lung surface. A certain amount of evi- dence in support of this view is given by the fact that on two occasions mild tetany was produced in dogs by large injections of sodium bi- carbonate. It would therefore appear that such tetany is due either to a decrease in the Ch of the blood or else to a disturbance in the sodium, potassium, calcium equihbrium within the nervous tissue. In order to throw further light upon this subject a number of experiments were carried out on dogs, rabbits, frogs, and turtles, in which the ef- fects of sub-arachnoid and intra-arterial injections of various electro- lytes were studied. It has been shown by Weed and Wegeforth (2) that irrigation of the spinal and cerebral sub-arachnoid spaces with bicarbonate-free Ringer's solution can be carried out without serious effect, whereas irrigation with isotonic solutions of sodium chloride results in the manifestation of severe toxic effects. They found also, when the irrigation fluid was free from KCl that no noticeable toxic symptoms developed, while irrigation with an isotonic Ca-free solution resulted in the same toxic manifestations as when 0.7 per cent to 1 per cent NaCl in distilled water was used. The importance of the Ca ion in relation to physiological activity has frequently been emphasized (3). Locke (4) showed that the Ca ion is necessary for the transference of the excitory process from nerve to muscle and Overton (5) demon- strated that it is equally necessary for the transmission of the excitory 483 484 J. B. COLLIP state through the synapse. It is very probable that a variety of effects which may be elicited in various ways, such as will be subsequently de- scribed, are in a large measure due to a disturbance in the Ca balance within the nerve cell. METHODS The effects of sub-arachnoid injections of various electrolytes were studied in both normal and anesthetised animals. Dogs were used for the most part but experiments were also carried out on rabbits, frogs, and turtles. The animal, in practically all experiments where graphic records were taken, was first placed under morphine-ether an- esthesia, a cannula was then inserted in the left carotid artery and con- nected to the mercury manometer, tracheotomy was performed and anesthesia maintained by the ether bottle method described by Jack- son (6). The respiratory movements were recorded by means of a Marey tambour which was connected by rubber tubing to a glass can- nula which was inserted into the rubber tubing connecting the tracheal cannula with the valved top of the ether bottle. The valves in the ether bottle were so adjusted that uniform anesthesia was maintained, the tracing of the Marey tambour lever then being a relative index of the rate and depth of the respiratory movements. Two needles were, as a rule, placed in the spinal canal, one in the lumbar, the other in the subcerebellar region. Injections into the sub-arachnoid space were made very slowly and cautiously and the volume of fluid injected at any one time was small as the records indicate. As a rule 2 or 3 cc. of spinal fluid (in large dogs) was drawn off before an injection was made or else the obturator was removed from the needle for two or three minutes prior to making the injection, and this latter was not replaced until a few minutes after the injection had been made. These precautions were taken in order to exclude the possibihty of any effects elicited by spinal injection being due to increased pressure of the cerebro-spinal fluid. Intra-cerebral injections were made through a needle placed in the third ventricle, a small opening having first been made in the parietal bone just lateral to the median plane. Intra-venous and intra-arterial injections were also made. The ex- ternal jugular vein was chosen for the former while the vertebral or carotid artery was selected for the latter. Intra-spinal injections were made on rabbits after lumbar puncture had been performed under local anesthesia, while intra-spinal injections SUB-ARACHNOID INJECTION OF SODIUM BICARBONATE 485 were made in frogs and turtles by means of a small hypodermic needle which was placed through the occipito-atlantoid ligament. When dogs were not placed under morphine-ether anesthesia, a small amount of morphine was given b}^ subcutaneous injection and then lumbar punc- ture was performed under local anesthesia. RESULTS Sub-arachnoid and intra-arterial injections of NaHCOs. It was found that the injection of small amounts of sodium bicarbonate solution into the lumbar sub-arachnoid space resulted almost immediately in the manifestation of marked tetany of the musculature of practically the whole body, the muscles of the posterior half of the body being, how- ever, more affected than those of the anterior region (protocols 1, 2 and 3). Experiments have been carried out on twenty dogs and six rabbits and in every instance where NaHCOs has been injected into the spinal canal in appreciable amounts, tetany has resulted. Tetanic convulsions were also elicited in frogs and turtles by intra-spinal in- jections of mere traces of NaHCOg. The effect can be produced in both anesthetised and unanesthetised animals. The strength of the bicarbonate solutions used varied from 1 per cent to 10 per cent. Intra- spinal injections of NaHCOs in addition to producing tetany resulted in intensive and sometimes very much prolonged stimulation of the chief medullary centers which was manifested by a great increase in lung ventilation, rise in blood pressure and various degrees of cardiac vagus activity (fig. 1). If the NaHCOs were injected in sufficient amount to cause most intensive tetanic spasms, respirations were in- hibited and artificial respiration had to be resorted to until the spasm had modulated to a sufficient degree to make spontaneous respiration possible. It was found that tetany could be antagonized to a con- siderable extent by the intra-spinal injection of CaCl2 (fig. 1), while subcutaneous injection antagonized only to a slight degree. The medul- lary stimulation was, however, not counteracted to the same degree by the intra-spinal injection of CaClo. When injections of NaHCOs were made into the sub-cerebellar cistern after occipital-atlantoid puncture, the medullary stimulation was much more marked, while tetany was less readily obtained. If but a small quantity of NaHCOs was injected in this region there was no tetany but only violent hyperpnoea, increased blood pressure and evidence of cardiac inhibition (fig. 2). If the dose was slightly increased, in addition to the above symptoms mild 486 J. B, COLLIP tetany of the muscles of the face, neck, shoulders, and fore limbs was produced, while a still larger injection caused the tetany to become gen- eral. Intra-cerebral injection of NaHCOs through a needle placed in the third ventricle produced somewhat similar effects as injection into the subcerebellar cistern (fig. 3, e). While intra-venous injections of comparativelj' large quantities of NaHCOs were without marked effect it could be shown that the medullary stimulation following the sub- arachnoid injection of NaHCOs was due in part at least to the direct t~Mw\, Jm^unt< Gsnnfvdu.i %ilUluJta/tyj^ \m/'^^^^^ gc.c//L.a^ ^cc/Zcce^ /C.Q. /O/JTLHCO Fig. 1. Tetanic convulsions following intra-spinal injection of XaHCOs. Ef- fect antagonized by CaCU. Time, 3-second intervals. action of the latter upon the nerve cells of the medulla. This was dem- onstrated in the following manner. A cannula was placed in the left carotid artery and connected with a mercury manometer. The right vertebral and carotid arteries were then dissected free and NaHCOs was injected at different periods into both of these vessels. In other instances injections were made into the central end of the ligated carot- id artery. It was found that intra-arterial injection of this type resulted at once in stimulation of the medullar}' centers. SUB-ARACHXOID IXJECTIOX OF SODIUM BICARBONATE 487 The immediate effect of simultaneous stimulation of the respiratory, vasomotor and cardio-inhibitory centers bj- XaHCOs varied in differ- ent animals. The first effect was in some cases complete cardiac in- hibition marked by a great fall in blood pressure (fig. 2). This however passed off in a few seconds, and the blood pressure rose to a higher point than it was formerly. That the cardiac inhibition just mentioned was due to strong stimulation of the cardio-inhibitory center was shown Fig. 2. a. Vagus inhibition following injection of XaHCOs into cisterna magna. b. Same after 4 minutes. Effect of (NH4)2C03 and of XaHCOs. Vagi cut immedi- ately after injection of XaHCOs. Time, 3-second intervals. by cutting both vagi. When the vagi were cut sub-arachnoid injec- tion of XaHCOs resulted in an immediate and extensive rise in blood pressure. As has been stated previoush% slight tetany was obtained in dogs on two occasions after large intra-venous injections of XaHCOs; this observation was the exception, however, and not the rule. It has also been demonstrated that occasionally one may obtain increased lung ventilation in dogs under ether anesthesia following intra-venous in- 488 J. B. COLLIP jection of sodium bicarbonate. Under morphine-ether, however, the usual effect of NaHCOs given by intra-venous injection is a decrease in respiratory activity. Sub-arachnoid injections of Locke's solution. It was found that sub- arachnoid and intra-cerebral injection of normal Locke's solution was without marked effect, while injection of concentrated Locke's solution caused definite stimulation of the medullary centers. Suh-arachnoid injections of distilled water. Sub-arachnoid injection of 2 cc. of distilled water in the lumbar region of an unanesthetised rabbit caused after a latent period of one and one-half minute a tetanic con- vulsion which lasted for a period of seven minutes. Artificial respira- tion was performed during this period. Spontaneous respirations were resumed with the relaxation of the muscular spasm and an increase both in the amplitude and the rate of the respiratory movements was noted (protocol 4). During the next twenty minutes the tetany grad- ually subsided, the animal gaining control of the muscles of the an- terior half of the body first, while at the end of half an hour it was able to run about. Sub-arachnoid injection of distilled water into the lumbar region of anesthetised dogs was followed by a rise in blood pressure, greatly increased lung ventilation and mild tetany of the muscles of the posterior half of the body. Injection of distilled water into the sub-cerebellar cistern caused stimulation of the medullary centers. Rapid injection of distilled water into the vertebral or carot- id artery caused slight increase in blood pressure and very mild stim- ulation of the respiratory center (fig. 4). Sub-arachnoid injections of sodium chloride. Hj'pertonic solutions of NaCl injected into the sub-arachnoid space of the lumbar region of rabbits produced practically the same type of effect as NaHCOs or distilled water (protocol 5). Injection of hypertonic NaCl solution into the lumbar region of the spinal canal of anesthetised dogs caused a mild degree of tetany, increased respiratory movements and a rise in blood pressure. The tetany was antagonized by CaCL; Weed and Wegeforth (2) have previously observed toxic effects during irrigation -of the spinal canal with isotonic NaCl. Injections of hypertonic NaCl solution into the sub-cerebellar cistern resulted in strong stimulation of the medullary centers. If too great a quantity were injected these centers were paralyzed but could be again activated if CaClo were in- jected at once (fig. 3). The injection of hypertonic NaCl into the ver- tebral or carotid artery caused slight stimulation of the medullary centers. SUB-ARACHNOID INJECTION OF SODIUM BICARBONATE 489 _2 ffi ■So o c .5 ^ o _. CI . o o .2 7^ o c .5 2H 5 « Q cj- o "^ t^ bC ■"" 4J c c3 o a S "B -^j o3 W -ti o o3' £3 -fi ,o C 0) a bD « c3 ^^ >. £ , , o c« o o 5s, 3 O) C !s c ^ o s C3 _C -IJ aj U «^ 0) -s o ^ K c3 C bC O W >j " Z H c3 t»— 1 e o , ;-< « a CO rt o rs ^ bC '-U "3 o £ O o e3 e3 c o 490 J. B. COLLIP Sub-arachnoid injections of KCl. Hypertonic solutions of KCl in- jected into the sub-arachnoid space or into the carotid or vertebral artery were found to have the same effect as hj^pertonic solutions of NaCl, but to a much greater degree (fig. 3, g). CaCla antagonized the action of KCl in much the same manner as it did NaCl. S>uh-arachnoid injections of (NH4)2C03, MgS04, Na2HP04 and NaH2P04. (NH4)2C03 was found to have a mild stimulating effect on Fig. 4. Effect of injection into carotid artery of ^j NaOH, yl- neutral phos- phate, distilled water and yi acid phosphate. Time, 10-second intervals. the medulla (fig. 2), while MgS04, as has been well estabhshed, had a depressant action. Both basic and acid phosphates injected into the subcerebellar cistern caused increased lung ventilation while neutral phosphate had a slight stimulating action. Intra-arterial injection of both acid, basic and neutral phosphate solutions caused increased re- spiratory activity (figs. 3 a, and 4). Intra-spinal injection of basic phosphate produced tetany in the rabbit (protocol 6). SUB-ARACHNOID INJECTION OF SODIUM BICARBONATE 491 Suh-arachnoid injections of NaOH. One cubic centimeter of -^-^ NaOH injected into the lumbar sub-arachnoid space of a rabbit caused mild tetany in all muscles of the body which was followed bj- a tempo- rary paralysis of the muscles of the lower extremities (protocol 7). Three cubic centimeters N. sodium hydroxide injected into the lumbar region of the spinal canal of an 8-kilo dog under the influence of mor- phine caused increased tonus of all muscles. Two cubic centimeters of 10 per cent NaHCOs injected into the same region after an interval of twenty minutes caused intensive tetanic spasms of the whole mus- culature. Three cubic centimeters of -3^ NaOH injected into the lumbar region of a dog under morphine-ether anesthesia produced very little effect while 1 cc. of f^ NaOH injected into the sub-cerebellar re- gion of another animal caused a decrease in respiratory movement and a slight fall in blood pressure. One-half cubic centimeter of -2^5 NaOH placed in the sub-cerebellar cistern caused in another instance decreased lung ventilation and stimulation of the cardio-inhibitory and vaso- motor centers. Injection of 10 cc. of f^ NaOH into the carotid artery of one animal caused definite stimulation of the respiratorv center (fig. 4). Irrigation of the spinal canal. Irrigation of the spinal canal with isotonic Ringer-Locke's produced little effect, while irrigation of the sub-arachnoid spaces with Ringer-Locke's containing 0.3 per cent NaHCOs produced intense hyperpnoea. It was found when the cere- brospinal fluid was washed out from the sub-arachnoid spaces and re- placed by isotonic saline that, after a period of half an hour had elapsed, it was practicall}^ impossible to secure fluid from the sub-cerebellar cistern. This was attributed to the inabihty of the practically protein- free fluid to resist absorption from the sub-arachnoid area. The protocols which follow indicate in greater detail the manner in which some of the experiments referred to above have been carried out. PROTOCOLS 1. Rabbit cT, weight 2 kilos 3:55 p.m. Cocaine hydrochloride 0.5 grain subcutaneous and intra-muscular over the fourth lumbar vertebra. 4:00 p.m. Injection of 2 cc. 10 per cent NaHCOs in distilled water into the sub- arachnoid space of the lumbar region. 4:01 p.m. Tetanic convulsion onset of which caused the animal to turn a com- plete somersault. 492 J. B. COLLIP 4:01 to 4:30 p.m. Gradual diminution of the tetany with hyperpnoea through- out. 4:45 p.m. Animal able to run around. Animal died the following day. 2. Dog 9 6.5 kilos 3:45 p.m. 1 gr. morphine subcutaneous. 4:10 p.m. 0.5 gr. of cocaine subcutaneous and intra-muscular over fourth lumbar vertebra. 4:17 p.m. 4 cc. of 5 per cent NaHCOs injected into the lumbar sub-arachnoid space. 4:17J p.m. Tetanic spasm of the entire body but most marked in the posterior half. 4:30 p.m. Tetanj^ practically gone. 4:50 p.m. 2 cc. of 1 per cent CaCla injected into the lumbar sub-arachnoid space. 5:00 p.m. 5 cc. of 5 per cent NaHCOs injected without effect. 5. Dog cf , 40 kilos 10:25 a.m. 4 gr. morphine subcutaneous. 11:00 a.m. Ether anesthesia. 11:15 a.m. Tracheotomy and cannula in left carotid connected for recording blood pressure. 11:40 a.m. Lumbar puncture. Clear fluid from needle. 11:45 a.m. 1 cc. of 10 per cent NaHCOs injected through needle in lumbar sub- arachnoid space. Tetany confined to posterior half of body developed at once, rate and depth of respirations increased, and increase in blood pressure from 120 mm. to 200 mm. Hg. 11:55 a.m. 1 cc. of CaCh by lumbar needle. 11 :58 a.m. 1 cc. of 10 per cent NaHCOs by lumbar needle. No tetany. Hyperp- noea and rise in blood pressure from 130 mm. to 165 mm. Hg. 4. Rabbit cf , 2 kilos 12:00 noon. 0.5 gr. cocaine hydrochloride sub-cutaneous and intra-muscular over fourth lumbar vertebra. 12:05 p.m. Lumbar puncture. 12:06 p.m. 2 cc. distilled water slowly injected into spinal canal. 12:07? p.m. Intense tetanic spasm of all muscles. 12:10 to 12:13 p.m. Artificial respiration. 12:13 to 12:45 p.m. Tetany diminished gradually. 12:45 p.m. Animal has control of fore-limbs, head and shoulders. 12:55 p.m. Animal able to run about. Next day. Completely recovered. SUB-AILICHNOID IXJECTIOX OF SODIUM BICARBONATE 493 5. Rabbit <3^ , 1 .5 kilo 4:00 p.m. 0.5 gr. cocaine hydrochloride sub-cutaneous and intra-muscular over fourth lumbar vertebra. 4:05 p.m. Lumbar puncture. 4:07 p.m. 2 cc. of 10 per cent XaCl intra-spinal. 4:08 p.m. Tetanj- of whole musculature. 4:13 p.m. Tetany considerably diminished. 4:30 p.m. Animal running about. Next daj'. Animal normal. 6. Rabbit 0^,2 kilos 4:00 p.m. 0.5 gr. cocaine hydrochloride sub-cutaneous and intra-muscular over fourth lumbar vertebra. M 4: 05 p.m. 0.5 cc. rz Na2HP04 intra-thecal. lo 4:05| p.m. Tetanic spasm most marked in posterior extremities. 4:10 p.m. Tetany practically gone. 4:15 p.m. Animal running about. 4:20 p.m. 0.5 cc. of XaH2P04 intra-thecal; no effect. 7. Rabbit d", 2 kilos 4:00 p.m. 0.5 gr. cocaine hj'drochloride sub-cutaneous and intra -muscular over the fourth lumbar vertebra. 4:02 p.m. Lumbar puncture. 4:05 p.m. 0.5 cc. of ~ XaOH intraspinal. 4:05? p.m. Mild tetany of whole musculature. 4:15 p.m. Tetany practicalh* gone but animal has no control of lower extremities. 5:00 p.m. Only partial control of hind legs. Next day: Slight paralj'sis of posterior extremities. Five days later: Complete recover}'. DISCUSSION It was thought at first that the tetany following intra-spinal injection of XaHCOs was due in part to the change in the C\ of the nerve cells as a result of increased concentration of bicarbonate and in part to the specific action of the HCO3 ion. When, however, further work disclosed the fact that smiilar effects could be elicited by intra-spinal injections of such substances as distilled water, XaCl, KCl and XaOH, it became apparent that the factors of H and HCO3 ion concentration were prob- ably of secondary importance. The change in concentration of Ca ion within the nerve cell either relatively or absolutelv in relation to the 494 J. B. COLLIP concentration of Na and K ions would seem to be the direct cause of the symptoms manifested in these experiments. As the effect of NaHCOs is relatively greater than that of the other electrolytes studied, especially of NaOH, it is possible that the bicarbonate ion as such may have a specific action. This would concur with the opinion of Macleod (7) that the respiratory center is sensitive to the HCO? ion. It is as- sumed, therefore, that change in the relative as well as absolute concen- trations of the various kations is the chief underlying cause for the va- rious phenomena noted in the experiments which have been quoted. Disturbance of the concentration of the Ca ion is, however, the chief factor. As physiologicalh^ balanced saline solutions are practically without effect while distilled water and solutions of Ca-free electrolj^tes in various concentrations produce such marked symptoms, it is evident that in all these latter instances the concentration of the Ca within the nerve cell must have been reduced as a result of the diffusion of the same out of the cell due to the difference in concentration established as a result of the injection. The administration of weak alkahne solu- tions such as NaOH, NaHCOa and Na.HPO^ would cause as well an actual fixation of a certain amount of Ca within the tissue, the concen- tration of Ca ion thereby being appreciably diminished. The fact that sub-arachnoid injection of sodium bicarbonate caused effects which in magnitude are out of all proportion to the somewhat similar effects produced by NaOH would tend to show that the HCO3 ion or the NaHCOs molecule has a specific stimulatory action. Large amounts of NaHCOs intra-spinal are definitely toxic, and result in par- alysis of the motor nerve cells. It is also true that paralysis of the respiratory center results after large injections of distilled water or almost any neutral electrolyte. It is possible that the phosphate ion may also have a specific stimulatory action on the medulla as both the acid and basic salts produce somewhat similar effects on the medullary centers. Intra-spinal injections into the lumbar region, however, dis- closed the fact that tetany is readily produced by the basic but not by the acid salt. This, taken in conjunction with the medullary stimu- lation by acid, basic and neutral phosphate solutions suggests a certain amount of specific action of the phosphate ion on the medulla. The tetany which is produced by lumbar sub-arachnoid injection of NaHCOs, distilled water, hypertonic solutions of NaCl and other elec- trolytes, is due in the main to direct stimulation of the motor cells in the anterior horn. One cannot exclude, however, the possibility of the stimulation of the anterior horn cells as a result of irritation of the SUB-ARACHNOID INJECTIOX OF SODIUM BICARBONATE 495 afferent fibers in the posterior roots or the intercalated neurones in the spinal reflex arcs. The stimulation of the respiratory and vasomotor center must under the above circumstances be largely reflex as the effect is manifested before the injected fluid can possibly reach the medullary centers. "When, however, one makes the injection into the sub-cere- bellar cistern there is, no doubt, direct stimulation of the nerve cells in the medulla. The fact that cardiac inhibition through the vagi is produced much more readily by sub-cerebellar injection than by lum- bar injection is very significant in this respect. The eardio-inhibitorj' center is not stimulated reflexly to the same degree as are the vaso- motor and respiratory centers, while direct stimulation of the medullary centers results in a ver}' definite cardiac inhibition, which is manifested by a marked fall in blood pressure. This effect then gives way and the blood pressure rises due to general vasoconstriction. The experiments which are quoted in this paper demonstrate very clearly that substances in solution administered by the sub-arachnoid channel act in a very short space of time directly on the nerve cells of the brain and cord. While the effects following sub-arachnoid injec- tions are, for obvious reasons, much more pronounced than those follow- ing injections into the carotid or vertebral artery, yet the similarity of the S3'mptoms manifested after arterial and sub-cerebellar injec- tion of NaHCOs, for example, are very significant. The very definite stimulation of the respiratory and vasomotor centers following intra- arterial injection of a potentially strong alkali like 10 per cent XaHCOs is positive proof of the sensitivity of these centers to the HCO3 ion irrespective of blood Ch- The decrease in lung ventilation which fol- lows, as a rule, slow and continuous intra-venous injections of XaHCOs, is probably due to the lowering of the hydrogen ion concentration of the blood, without there being a concomitant increase of sufficient magni- tude in the bicarbonate ion to neutralize or to overcome the effect of the latter. Definite stimulation of the respiratory center can, however, be occasionally obtained during intra-venous injection of XaHCOs. It is of interest in this respect that such an effect is more likely to be manifested by an animal under ether rather than under morphine-ether anesthesia. When massive doses of XaHCOs are given comparatively rapidly by intra-venous injection, it is possible to obtain sudden collapse of the heart without paralysis of the respiratory center. In one instance spontaneous respiratory movements continued for two minutes after the heart had failed completely. 496 J. B. COLLIP While it is possible to obtain direct action of a substance on the nerve cells by sub-arachnoid injection, the passage of such substance into the general circulation may take place very slowly or in some instances it may not be removed from the spinal canal at all (8). The demonstration of Wilson (9) that parathyroid tetany is asso- ciated with an alkalosis of the blood, and the findings of MacCallum, Kellogg and Voegtlin (10) that symptoms like those of tetany can be induced by deficiency of calcium and symptoms following parathyroidectomy relieved by calcium administration would be amplified by the experiments herein recorded. CONCLUSIONS It would seem logical to conclude that the tonus of effector nerve cells and probably of all nerve cells is regulated not only by the Ch of the blood and spinal fluid but also by the maintenance in them of a definite equilibrium between the various ions. Of these latter the Ca ion is preeminent. Decrease in Ca ion leads to increased tonus, and increase in the Ca ion to decreased tonus. Slight increase in the con- centration of either Na or K is marked by definite stimulation of the medullary centers while the stimulatory.' effect of the bicarbonate ion is very definite. The importance of Na, K and Ca ions as regards reg- ulation of the respirator}^ center has been suggested by Howell (11). It would seem probable that one of the underlying causes of tetany, as observed clinically, is a disturbance in the kation equilibrium within the nerve cells of the brain and cord. Results, such as are herein reported, serve to emphasize the impor- tance of definite ions of electrolytes in definite concentration within the living cell, a principle which has never been lost sight of since the pio- neer experiments of Sidney Ringer (12) were published. In conclusion I wish to acknowledge the assistance of Dr. P. L. Backus in the experimental work reported in this paper. SUMMARY 1. Sub-arachnoid injection on the lumbar region of NaHCOs, NaCl and distilled water is followed by violent tetany and definite stimula- tion of the medullary centers. 2. CaCl2 administered by intra-spinal injection antagonizes tetany so produced. SUB-ARACHNOID INJECTION OF SODIUM BICARBONATE 497 3. Injections of NaHCOs, NaCl and KCl into the sub-cerebellar cis- tern cause marked stimulation of the respiratory, vasomotor and cardio- inhibitory centers. CaCU antagonizes this effect to a hmited extent. 4. Injection into the carotid or vertebral artery of NaHCOa, NaCl or KCl caused definite stimulation of the respiratory and vasomotor centers. 5. Intra-venous injection of NaHCOs may cause increased lung ventilation. 6. It is held that the bicarbonate ion has a specific stimulatory effect upon the nerve cells of the medulla and the motor cells of the anterior horn. 7. Disturbance in the concentration of the various kations particu- larly of the Ca ion within the nervous tissue produces most marked effects. BIBLIOGRAPHY (1) CoLLip AND Backus: This Journal, 1920, H, 568. (2) Weed and Wegeforth: Journ. Pharm. Exper. Therap., 1919, xiii, 317. (3) Howell: This Journal, 1898, ii, 47. (4) Locke: Zentralbl. Physiol., 1894, viii, 166. (5) Overton: Pfliiger's Arch., 1904, cv, 346. (6) Jackson: Experimental Pharmacology, St. Louis, 1917, 38. (7) Macleod and Knapp: This Journal, 1918, xlvii, 189. (8) Becht: This Journal, 1920, li, 1. (9) Wilson, Stearns, Thompson and Thurlow: Journ. Biol. Chem., 1915, xxiii, 89. (10) MacCallum: Journ. Exper. Med., 1909, xi, 118; Ibid., 1913, xviii, 646; Journ. Pharm. Exper. Therap., 1911, ii, 421. (11) Howell: Textbook of Physiology, Philadelphia, 1918, 705. (12) Ringer: Journ. Physiol., 1880, iii, 195; Journ. Physiol., 1884, iv, 29. THE AMERICAN JOURNAL OF PHYSIOLOGV, VOL. 52. NO. 3 THE INFLUENCE OF INTERNAL SECRETIONS ON BLOOD PRESSURE AND THE FORMATION OF BILE ARDREY W. DOWNS From the Physiological Laboratory of McGill University, Montreal, Canada Received for publication April 16, 1920 In connection with work previously reported (1) on the influence of internal secretions on the formation of bile, a study of the blood pres- sure was made. The object of this was twofold: to observe the effect on blood pressure of the particular gland substance being studied, and to determine what relation, if any, existed between blood pressure changes and the amount of bile secreted after a gland substance had been administered intravenously. The following gland substances were employed: mammary, orchic, ovarian, pancreatic, splenic, thj^mic and thyroid. These were all obtained from Armour & Companj-. To this list was added solution of adrenalin chloride prepared by Parke, Davis & Company, and secretin prepared by a method described in connection with other experiments (2). These were all given by intra- venous injection. Nearh' all of the experiments were performed on dogs but occasionally cats were used. A description of the technique will be found in the article to which reference has been made (l) and the numbers of experiments mentioned in this contribution correspond to the experiment numbers there given. All observations were made for arbitrary periods of twent}' minutes each. The usual procedure was to record blood pressure and the number of drops of bile secreted during three twenty-minute periods, one immediateh' preceding the administration of the gland substance, a second commencing with the beginning of the injection, and a third immediately following the second. In a few cases the third record was not secured. The dose in every experiment, except those with adrenalin and secretin, was 10 mgm. of the gland substance per kilogram of body weight of the animal, dissolved in 100 cc. of phj^siological sahne solution. This was warmed to 37°C. on a water bath and injected into the jugular vein by means of a burette. The dose of adrenalin was 0.1 cc. of a 1:1,000 solution of adrenalin chloride per kilogram of body weight, and the dose of secretin was 10 mgm. of a dried acid extract per kilogram. 498 EFFECT OF GLAXD EXTRACTS OX BILE FORMATIOX 499 Adrenalin. Five determinations were made with adrenalin. There was invariably an immediate rise of pressure which averaged 81.25 mm. of mercurJ^ In the case of adrenalin the injections were made much more slowlj' than with the other substances studied and the maximum to which the pressure was allowed to rise was 220 mm. This high blood pressure, with slight fluctuations, was maintained for 5 minutes as an average and at the end of the 20-minute period the pressure had always fallen to or slightly below the original level. The amount of bile secreted after the injection of adrenalin was always less than during the preceding 20 minutes and the striking feature was the marked decrease during the period of high blood pres- sure. For example, in experiment 13 the initial count was 140 drops distributed fairh' uniformly throughout the period; during the first 5 minutes of the injection period 1 drop was secreted, in the next 5 minutes 3 drops, in the third 5 minutes 3 drops, and during the last 5 minutes 42 drops — a total of 49 drops for this 20-minute period. The diminution produced by adrenahn in the amoimt of bile secreted may be explained by a decrease of both the arterial and venous inflow. As has been shown by Burton-Opitz (3), (4) adrenalin injected into the hepatic arter.y or portal vein exerts a local constricting action and diminishes the inflow and it is not improbable that even when the adrenalin is introduced through a vessel more distant from the liver the same action takes place. Mammary. Mammary substance was used eight times and in all but one there was an initial rise in blood pressure followed by a fall and a slow return to normal. The average figures for these experi- ments are as follows: blood pressure at beginning of injection 109.2 mm.; maximum pressure 117.6 mm., reached 26.4 seconds after injec- tion started; minimum pressure 95.4 mm., 71.4 seconds from beginning of injection; pressure 103.8 mm. at end of first 5 minutes, and 106.8 mm. at end of period. Experiment 46, which failed to show an initial rise in blood pressure, was similar in other respects to the experiments of the group. Ott and Scott (5) report a slight fall in blood pressure for a few seconds after intravenous injection of mammary substance but there is no record of the preliminary rise which occurred so uni- formly in our experiments. So far as bile production was concerned the effect of mammary gland substance was not uniform but it would seem to be a temporary decrease. In experiment 11 the falling off in the rate of secretion was most marked immediate^ after the injection. Twenty-three drops of 500 ARDREY W. DOWNS bile were secreted during the twenty-minute period following the injection as compared with an initial count of 46; 14 of the 23 drops came during the last half of the period whereas the original 46 drops had been quite uniformly distributed. Experiment 8 was similar; the initial count was 3 drops, but after the injection there was no secretion until the last 4 minutes of the period when 2 drops were recorded. On the other hand, experiments 16, 46 and 57 showed a progressive decrease in the formation of bile. The other experiments of the group showed no change or an increased production. From these records it appears that not only is mammary substance inconstant in its effect on the secretion of bile but that there is no rela- tion between changes in blood pressure and bile formation under its influence. Orchic. Seven experiments were made with orchic substance. Four showed an initial rise of pressure followed by a fall slightly below nor- mal, a gradual return to the normal level and the pressure at the end of the period approximately the same as at the beginning; two did not show an initial rise, but a slight fall and then a gradual rise above the original level persisting to the end of the period. In one, experiment 4, no blood pressure record was made. The average figures for the first group of experiments, viz., 18, 41, 42 and 55, are as follows: blood pressure at beginning of injection, 128 mm. of mercury; maximum pressure 147.2 mm., reached 62.5 seconds from beginning of injection; minimum pressure 124 mm. at 110.7 seconds after injection com- menced; pressure at end of first 5 minutes, 134.7 mm., and at end of period, 126.5 mm. For the second group of orchic experiments, num- bers 19 and 62, the initial pressure is 113 mm., followed by a minimum of 99 mm. in 20 seconds, a rise to 127 mm. in 125 seconds, 123 mm. at end of first 5 minutes and 132 mm. at the end of the period. The reported observations of the effect of the intravenous injection of testicular extract on blood pressure vary somewhat but agree in general that the pressure is lowered. In 1901 Dixon (6) described an immediate and considerable transient fall of blood pressure accom- panied by cardiac slowing following the injection of orchitic extract. Vincent and Sheen (7) and Miller and Miller (8) note the production of a fall in pressure after testicular extract. Vincent (9) and Ott and Scott (5) state that the fall in pressure is slight and lasts for a few seconds only. Wheelon (10) records a slight fall in blood pressure after castration. Bingel and Strauss (11) were unable to produce a change in pressure by the administration of extract of testis. EFFECT OF GLAND EXTRACTS ON BILE FORMATION 501 Orchic gland substance caused a decrease in the amount of bile formed in five of the seven experiments and in four of these the effect was progressive, a further decrease occurring in the second 20-minute period after the injection. One interesting feature presents itself in an analysis of these records: there was a primary decrease during the first 15 minutes following the injection, a rise during the next 15 min- utes, and a second decline beginning about 30 minutes after the gland substance was administered. This second depression of secretory activity was more marked than the first. A comparison of blood pressure changes with variations in the amount of bile secreted fails to show any uniformity. Of the four experiments showing a rise in blood pressure, then a fall and a gradual return to normal, three gave a decrease in bile formation and one, number 55, an increase of 108.33 per cent. In the two experiments where there was a fall of pressure followed by a rise to a point 18 mm. above the original height at the end of the period, the changes in pres- sure were accompanied by decreased production of bile in one, experi- ment 19, and increased production in the other, experiment 62. Ovary. Ovarian substance was used five times, in four of which blood pressure records were made. Two cases, experiments 20 and 59, showed a rise of pressure shortly after the injection began, then a fall below normal, a second rise above the original level, and a gradual return to normal. Averages: initial pressure, 130 mm. of mercury; first maximum, 148.5 mm., 19.5 seconds after injection began ; minimum 119.5 mm., 44 seconds from beginning; second maximum 137.5 mm. at end of first 3 minutes, and final pressure 132.5 mm. Two other exper- iments, 43 and 56, with ovarian substance failed to show changes similar to the above, the pressure remaining almost constant. In experiment 5 no blood pressure record was secured. The effect of ovarian gland substance or extract of ovary on blood pressure has been reported by Ott and Scott (5), Vincent and Sheen (7), Miller and Miller (8), Vincent (9) and Gonalons (12). These investigators agree that the effect is a lowering of blood pressure which is usually slight and transient. Ovarian substance invariably produced a decrease in the amount of bile formed. The average decrease was 44.59 per cent in the first period and 59.36 per cent in the second period after the injection. Pancreas. This substance was employed in nine experiments and for six of these complete blood pressure records are available. The effects on blood pressure fall into two groups, one showing a prompt 502 ARDREY W. DOWNS and very marked fall in blood pressure, the other showing a prelim- inary slight rise followed by a fall. In all of these records there is a rather characteristic lowering of blood pressure which is still in evidence at the end of the period, but from which there is gradual recovery during the second 20-minute interval. Forty minutes after injection the blood pressure averaged 121 mm. as compared with 123.6 mm. immediately preceding the injection. The first group consists of four experiments, 22, 38, 48, 50, and shows the following: initial pressure 123.2 mm.; fall began in 5.5 seconds and the minimum pressure, 60 mm., was reached 26.7 seconds from the beginning of the injection. At the end of 5 minutes the pressure was 86.5 mm., and at the end of the period, 95.7 mm. The second group, experiments 14 and 52, gives these averages: initial pressure, 124 mm.; 17 seconds later pressure began to rise and reached a maximum of 131.5 mm. 19 seconds after the injection was started; then fell to 77.5 mm., 40 seconds from beginning; at end of first 5 minutes was 118.5 mm., and at end of period 107 mm. With the exception of Popielski (13), who reports a marked and pro- longed rise in blood pressure as the result of the injection of an acidu- lated watery extract of pancreas, investigators agree that injection of the substance of the pancreas or of saline extracts of pancreas causes a fall in blood pressure. Ott and Scott (5) state that the lowering of pressure is more marked than that obtained with ovary, testis, mam- marj^, spleen and thjTnus. Biliary secretion was decreased in every case in which pancreatic substance was administered. The reduction averaged 45.45 per cent in the first period and 44.28 per cent in the second period following the injection. The striking feature in this connection was the great reduc- tion in secretory" activity immediately after the injection. The counts made during the periods preceding the injections show the drops of bile falling at a fairly uniform rate. In the first 10 minutes subsequent to the introduction of the gland substance only 19 per cent of the total secretion for the period was obtained, the other 81 per cent occurring during the second half of the period. In the next 20-minute period, the second after the injection, the rate of secretion was more uniform but still only a little more than one-half the original. Secrcti?i. Seven experiments were carried out with secretin. The blood -pressure records show an average pressure preceding the injec- tion of 114.1 mm. of mercury; 20.2 seconds after the injection was begun pressure commenced to fall and reached a low point of 56.8 mm. EFFECT OF GLAXD EXTRACTS ON BILE FORMATION 503 40 seconds from the starting point; at the end of 5 minutes the pres- sure was 110.5 mm., and at the end of the period, 118.4 mm. In two experiments, 26 and 54 of this series, there was a slight rise of pressure immediately following the injection. This increase above the original pressure averaged 15 mm. of mercury and in experiment 26 was suc- ceeded by a drop similar to that which took place in the other experi- ments of the group; in no. 54 there was no abrupt fall in pressure but a gradual decline with the pressure at the end of the 20-minute period nearly the same as at the beginning. The amount of bile produced was greatly increased in every case in which secretin was employed. This increase averaged 241.52 per cent for the first 20 minutes after the injection and 413.78 per cent for the second 20 minutes. Bayliss and Starhng (14) state, "Acid extracts of the mucous mem- brane ( of the duodenum and jejunum) normally contain a body which causes a fall of blood pressure. This bodj^ is not secretin, and the latter may be prepared free from the depressor substance by acting on desquamated epithelial cells with acid." This has been confirmed by v. Fiirth and Schwarz (15). Matsuo (16) also concludes that the depressor substance is separate from secretin, especialh' as acid injected into the duodenum, while producing copious pancreatic secretion, was followed by no change in blood pressure. He was, however, not able to obtain a secretin preparation which did not produce some fall in blood pressure, but the degree of the fall and the activity of the various preparations were not at all proportionate. In our experiments it will be observed that while there was, as a rule, a fall in pressure immediately following the injection this had been recovered from in 5 minutes and from then on the pressure re- mained within a few millimeters of the original. At the same time the amount of bile was tremendously increased during the 40 minutes the experiments lasted. Spleen. Injection of substance of the spleen was practically without effect on blood pressure. Seven trials were made and the average pressure prior to the injection was 104.4 mm.; 20 minutes after the injection it was 107.4 mm. Only two experiments showed any fluctua- tions that could be attributed to the injection. In experiment 21 the pressure rose from 128 mm. to 135 mm. in 20 seconds, then fell to 124 mm. 22 seconds later, rose to 136 mm. 63 seconds later, and declined gradually to 129 mm. at the end of the period. The initial pressure in experiment 61 was 130 mm. With the beginning of the injection 504 ARDREY W. DOWNS blood pressure commenced to fall and in 40 seconds had reached 124 mm.; from there it rose gradually to 140 mm. at the end of the period. Ott and Scott (5) report a slight fall in blood pressure for a few seconds after the intravenous administration of splenic substance. Vincent and Sheen (7) and Vincent (9) noted the production of a tran- sient fall in pressure by splenic extract. Oliver and Schafer (17) state that spleen produces a prehminary fall of pressure followed by a gradual rise above normal and then a gradual return to normal. Bingel and Strauss (11) found inconstantly a temporary rise of pressure followed by a sharp fall. Miller and Miller (8) state that in their hands saline extracts of spleen invariably caused a rise in blood pressure, which was usuall}' but not always followed by a slight fall below normal. So far as the formation of bile after the introduction of splenic sub- stance by vein is concerned we found no constant effect. In the first period after the injection four experiments gave a decrease, two an increase and one no change. Alterations in the rate of secretion during the second period after injection were also without harmony. Thymus. Five experiments were carried out with thymic substance and no consistent effect on blood pressure noted. Experiment 39 showed a slight rise in pressure followed by a sharp fall, and a return to normal 6 minutes after the injection began. Where any effect on blood pressure was produced by the injection intravenously of thymic gland substance, watery extracts or saline extracts of the thymus, observers are almost unanimous in reporting it as a decrease. Reference is made to Ott and Scott (5), Miller and Miller (8), Schafer (18), Popper (19), Basch (20) and Lucien and Parisoot (21). Popielski (13) states that an acidulated watery extract of thymus caused a rise of blood pressure. Reference to our previous paper (1) on this subject will show that we found the amount of bile secreted after the administration of thymic substance to be decreased. This effect was still in evidence in three out of four experiments in which a count was made for a second period of 20 minutes after the injection. • Thyroid. Substance of the thyroid gland was injected into nine ■dogs. In two of the experiments, 6 and 33, no record of blood pressure was obtained. The other seven all show an initial rise in pressure, a sharp fall, a second gradual rise and a fall to normal. Pressure during the second 20-minute period following the injection was practically the same as in the period preceding the injection. Average figures for these experiments follow: initial pressure 105.8 mm.; first maximum EFFECT OF GLAXD EXTRACTS ON BILE FORMATION 505 119.4 mm., 19 seconds from beginning; minimum 86.5 mm., 43.4 sec- onds from beginning; second maximimi 117.7 mm., 191.4 seconds from beginning, and pressm'e at end of period, 110.2 mm. Other observers have usually found a fall in blood pressure to result from the administration of thyroid extract or thyroid gland substance. Oliver and Schafer (17), Haskovec (22), Georgiewsky (23), Guinard and Martin (24), Fenyvessy (25), v. Cj^on and Oswald (26), v. Fiirth and Schwarz (27), all report a fall in pressure. Ott and Scott (5) used iodothyrin and obtained a marked fall with subsequent gradual rise above normal. Schafer (18) observed a considerable fall when using thyroid extract. Vincent (9) usuallj^ noted a fall but occasionally a rise of pressure. Levy (30) records no appreciable alteration in blood pressure after intravenous injection of Kendall's crystalline thyroid iodine compound. A rise in pressure is reported bj^ Popielski (13), Heinatz (28) and Livon (29). So far as bile formation is concerned thyroid substance was without constant effect. Experiments 6, 28, 40 and 51 show an increase aver- aging 82.29 per cent in the first period after the injection. Experi- ments 12, 17, 33, 37 and 58 show a decrease averaging 33.85 per cent for the same period. Nevertheless, in spite of the wide variations in amount of bile secreted, the blood pressure ran a very similar course in all of these experiments. DISCUSSION While the foregoing experiments are too few in number to permit definite conclusions to be drawn, one thing seems certain, viz., that there is no constant relation between blood pressure and the amount of bile secreted. Adrenalin, it is true, consistently raised blood pres- sure and lowered bile formation; secretin, on the other hand, w'here it caused a change in blood pressure, produced a lower pressure and a great increase in the flow of bile. It might be urged that thyroid gland substance owes any action it exerts upon blood pressure and bile for- mation to the intervention of the adrenals and this cannot be entirely controverted by our experiments. That thyroid substance increases the output of adrenahn has been shown by Biickner (31), Rudinger, Falta and Eppinger (32) and Glej* and Quinquaud (33) . In our experi- ments, however, there was no constant relationship between blood pressure and bile production after the administration of thyroid gland substance. We did not find bile production regularly decreased when blood pressure rose or vice versa. With others of the gland substances 506 ARDREY W. DOWTVS employed the blood pressure might be lowered and bile production decreased at the same time. The most striking example of this is in the series with pancreatic substance. Intravenous administration of substance of the pancreas caused lowering of the blood pressure and lessening of the output of bile. To a lesser extent thjTnic substance acted in the same manner. If we place in contrast with this the effect of secretin it would seem that we are not justified in concluding that the effect on bile formation is due to the alteration in blood pressure. With the other gland substances employed, viz., mammary, orchic, ovarian and splenic, the results were inconstant. Orchic substance, for instance, caused a rise of blood pressure in some of our experiments and a drop in others, while the production of l)ile was definitely lowered. After the administration of substance of the ovary the blood pressure showed oscillations, or waves of higher and lower pressure. The pressure at first rose above the original level, then fell below the initial, rose again and returned to the original. Biliary secretion was lowered in every case and an examination of the individual records of these experiments does not show any synchronism between changes in blood pressure and the rate at which the bile was secreted. CONCLUSIONS 1. As a result of the experiments set forth in this paper we feel inclined to iDelieve that some at least of the endocrine organs exert a specific influence on the secretory activity of the hepatic cells leading to the production of bile. 2. The output of bile in the dog is increased by the administration of secretin. 3. The output of bile in the dog is decreased bj' the administration of adrenalin, and by mammary, orchic, ovarian, pancreatic and thymic gland substances. 4. The amount of bile secreted is not affected in a constant or defi- nite manner by the substance of the spleen and thyroid gland. 5. Blood pressure is raised by adrenalin. 6. Blood pressure is lowei'ed by pancreatic substance and the secretin preparation employed. 7. A fall of blood pressure, ordinarih' preceded I33' a slight rise, is caused by orchic and mammary gland substances. 8. Oscillations of blood pressure are caused by ovarian and thyroid gland substances. 9. Blood pressure is not usually affected b}' splenic and thymic gland substances. (1 (2 (3 (4 (5 (6 (7 (8 (9 (10 (11 (12 (13 (14 (15 (16 (17 (18 (19 (20 (21 (22 (23 (24 (25 (26 (27 (28 (29 (30 (31 (32 (33 EFFECT OF GLAND EXTRACTS ON BILE FORMATION 507 BIBLIOGRAPHY Downs and Eddy: This Journal, 1919, xlviii, 192. Downs and Eddy: This Journal, 1917, xliii, 416. Burton-Opitz : Quart. Journ. Exper. Physiol., 1912, v, 309. Burton-Opitz: Quart. Journ. Exper. Physiol., 1912, v, 329. Ott and Scott: Amer. Med., 1914, xx, 249. Dixon: Journ. Physiol., 1901, xxvi, 244. Vincent and Sheen: Journ. Physiol., 1903, xxix, 242. Miller and Miller: Journ. Physiol., 1911, xliii, 242. Vincent: Internal secretions and the ductless glands, 1912, 29. Wheelon: This Journal, 1914, xxxv, 283. 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