UNIVERSITY OF ILLINOIS I ,r ^RY AT URBANA-CHAMPAIGN BOOKSTACKS Digitized by the Internet Archive in 2014 https://archive.org/details/collectedpapers101yale UNIVERSITY LIBRARY UNIVERSITY OF ILLINOIS AT URBANA-CHAMPAIGN The person charging this material is responsible for its renewal or return to the library on or before the due date. The minimum fee for a lost item is $125.00, $300.00 for bound journals. Theft, mutilation, and underlining of books are reasons for disciplinary action and may result In dismissal from the University. Please note: self-stick notes may result in torn pages and lift some inks. Renew via the Telephone Center at 217-333-8400, 846-262-1510 (toll-free) orcirclib@uiuc.edu. Renew online by choosing the My Account option at: http://www.library.uiuc.edu/catalog/ THE LABORATORY OF PHYSIOLOGICAL CHEMISTRY SHEFFIELD SCIENTIFIC SCHOOL YALE UNIVERSITY Collected papers 191 1-1912 NEW HAVEN, CONNECTICUT U. S. A. THE LABORATORY OF PHYSIOLOGICAL CHEMISTRY RUSSELL H. CHITTENDEN Professor of Physiological Chemistry LAFAYETTE B. MENDEL Professor of Physiological Chemistry FRANK P. UNDERHILL Assistant Professor of Physiological Chemistry CONTENTS The Influence of Urethane in the Production of Glycosuria in Rabbits after the Intravenous Injection of Adrenalin. By Frank P. Undrrhill. (From the Journal of Biological Chemistry, 1911, ix, 13.) Mucic Acid and Intermediary Carbohydrate Metabolism. By William C. Rose. (From the Journal of Biological Chemistry, 1911, x, 123.) Experimental Studies on Creatine and Creatinine. — I. The Role of the Carbohydrates in Creatine — Creatinine Metabolism. By Lafayette B. Mendel and William C. Rose. (From the Journal of Bio- logical Chemistry, 191 1, x, 213.) Experimental Studies on Creatine and Creatinine. — II. Inanition and the Creatine Content of Muscle. By Lafayette B. Mendel and Wil- liam C. Rose. (From the Journal of Biological Chemistry, 1911, x, 255.) Experimental Studies on Creatine and Creatinine. — III. Excretion of Creatine in Infancy and Childhood. By William C. Rose. (From the Journal of Biological Chemistry, 1911, x, 265.) Studies in Nutrition. — I. The Utilization of the Proteins of Wheat. By Lafayette B. Mendel and Morris S. Fine. (From the Journal of Biological Chemistry, 1911, x, 303.) Studies in Nutrition. — II. The Utilization of the Proteins of Barley. By Lafayette B. Mendel and Morris S. Fine. (From the Journal of Biological Chemistry, 191 1, x, 339.) Studies in Nutrition. — III. The Utilization of the Proteins of Corn. By Lafayette B. Mendel and Morris S. Fine. ( From the Journal of Biological Chemistry, 191 1. x, 345.) Studies in Nutrition. — IV. The Utilization of the Proteins of the Legumes. By Lafayette B. Mendel and Morris S. Fine. (From the Journal of Biological Chemistry, 1912. x, 433.) Studies in Nutrition. — V. The Utilization of the Proteins of Cotton Seed. By Lafayette B. Mendel and Morris S. Fine. ( From the Journal of Biological Chemistry, 1912, xi. 1.) Studies in Nutrition. — VI. The Utilization of the Proteins of Extrac- tive-Free Meat Powder; and the Origin of Fecal Nitrogen. By Lafayette B. Mendel and Morris S. Fine. (From the Journal of Biological Chemistry, 1912, xi, 5.) r? 4 ^ I CONTENTS III Studies in Carbohydrate Metabolism. — I. The Influence of Hydrazine upon the Organism, with Special Reference to the Blood Sugar Content. By Frank P. Underbill. (From the Journal of Bio- logical Chemistry, ion, x, 150.) Studies in Carbohydrate Metabolism. — tl. The Prevention and Inhibi- tion of Pancreatic Diabetes. By Frank P. Underbill and Morris S. Fine. (From the Journal of Biological Chemistry, 1911, x, 271.) The Production of Glycosuria as a Result of the Intravenous Injection of Witte's Peptone. By Yandell Henderson and Frank P. Underhill. (From the Proceedings of the Society for Experi- mental Biology and Medicine, 191 1, viii, 80.) The Behavior of Fat-Soluble Dyes in the Organism. By Lafayette B. Mendel and Amy L. Daniels. (From the Proceedings of the Society for Experimental Biology and Medicine, 191 1, viii, 126.) The Production of Glycosuria by Adrenalin in Thyroidectomized Dogs. By Frank P. Underhill. (From the American Journal of Physiology, 1911, xxvii, 331.) The Metabolism of Dogs with Functionally Resected Small Intestine. By Frank P. Underhill (with the collaboration of Chester J. Stedman and Jessamine Chapman). (From the American Journal of Physiology, 1911, xxvii, 366.) Acapnia and Glycosuria. By Yandell Henderson and Frank P. Under- hill. (From the American Journal of Physiology, 191 1, xxviii, Nutrition Investigations on the Carbohydrates of Lichens, Algae, and Related Substances. By Mary Davies Swartz. (From the Trans- actions of the Connecticut Academy of Arts and Sciences, 191 1, xvi, 247.) A Consideration of Some Chemical Transformations of Proteins and their Possible Bearing on Problems in Pathology. By Frank P. Underhill. (From the Archives of Internal Medicine A 191 1, viii, The Role of Different Proteins in Nutrition and Growth. By Thomas B. Osborne and Lafayette B. Mendel. (From Science, 191 1, xxxiv, 722.) The Action of Salts of Choline on Arterial Blood Pressure. By Lafayette B. Mendel, Frank P. Underhill, and R. R. Renshaw. (From the Journal of Pharmacology and Experimental Therapeutics, 1912, iii, 649.) The Influence of Tartrates upon Phlorhizin Diabetes. By Frank P. Underhill. (From the Proceedings of the Society for Experi- mental Biology and Medicine, 1912, ix, 123.) The Haemagglutinating and Precipitating Properties of the Bean (Phaseolus) . By Edward C. Schneider. (From the Journal of Biological Chemistry, 1912, xi, 47.) The Value of Inulin as a Foodstuff. By Howard B. Lewis. (From the Journal of the American Medical Association, 1912, lviii, 1176.) The Influence of Cocaine upon Metabolism with Special Reference to the Elimination of Lactic Acid. By Frank P. Underhill and Clarence L. Black. (From the Journal of Biological Chemistry, 1912, xi, 235.) 275.) 356.) ADVERSITY Of iv CONTENTS The Physiological Action of Some Pyrimidine Compounds of the Barbi- turic Acid Series. By Israel S. Kleiner. (From the Journal of Biological Chemistry, 1912, xi, 443.) The Influence of Sodium Tartrate upon the Elimination of Certain Urinary Constituents during Phlorhizin Diabetes. By Frank P. Underhill. (From the Journal of Biological Chemistry, 1912, xii, 115.) Feeding Experiments with Fat-Free Food Mixtures. By Thomas B. Osborne and Lafayette B. Mendel (with the cooperation of Edna L. Ferry). (From the Journal of Biological Chemistry, 1912, xii, 81.) The Role of Gliadin in Nutrition. By Thomas B. Osborne and Lafay- ette B. Mendel (with the cooperation of Edna L. Ferry). (From the Journal of Biological Chemistry, 1912, xii, 473.) Maintenance Experiments with Isolated Proteins. By Thomas B. Osborne and Lafayette B. Mendel (with the cooperation of Edna L. Ferry). (From the Journal of Biological Chemistry, 1912, xiii, 233.) A Study of the Mechanism of Phlorhizin Diabetes. By Frank P. Underhill. (From the Journal of Biological Chemistry, 1912, xiii, 15.) The Behavior of Fat-Soluble Dyes and Stained Fat in the Animal Organ- ism. By Lafayette B. Mendel and Amy L. Daniels. (From the Journal of Biological Chemistry, 1912, xiii, 71.) Beobachtungen iiber Wachstum bei Fiitterungsversuchen mit isolierten Nahrungssubstanzen. By Thomas B. Osdorne and Lafayette B. Mendel (with the cooperation of Edna L. Ferry). (From the Zeitschrift fur physiologische Chemie, 1912, lxxx, 307.) The Behavior of Some Hydantoin Derivatives in Metabolism. — I. Hydan- toin and Ethyl Hydantoate. By Howard B. Lewis. (From the Journal of Biological Chemistry, 1912, xiii, 347.) The following additional papers from the Laboratory are not included in this volume : Theorien des Eiweissstoffwechsels nebst einigen praktischen Konsequenzen derselben. By Lafayette B. Mendel. Ergebuissc der Physiologie, 1911, xi, 418. Feeding Experiments with Isolated Food-Substances. By Thomas B. Osborne and Lafayette B. Mendel (with the cooperation of Edna L. Ferry). Carnegie Institution of Wasliington, Publication 156, Parts I and II. 1912. pp. 138. Zur Wirkung intravenoser Einspritzungen von Konzentrierten Salz-und Zuckerlosungen. By Frank P. Underhill. Archiv fur experi- mentellc Pathologie unci Pharmakologie , 191 1, lxix, 407. Ein StofTwechselkafig und Fiitterungsvorrichtungen fiir Ratten. By Thomas B. Osborne and Lafayette B. Mendel. Zeitschrift fiir biologische Technik und Methodik, 1912, ii, 313. Biochemical and Bacteriological Studies on the Banana. By E. Monroe Batley. Journal of the American Chemical Society, 1912, xxxiv, 1706. Reprinted from The Journal of Biological Chemistry, Vol. IX, No. 1, 1911. THE INFLUENCE OF URETHANE IN THE PRODUCTION OF GLYCOSURIA IN RABBITS AFTER THE IN- TRAVENOUS INJECTION OF ADRENALIN. By FRANK P. UNDERHILL. {From the Sheffield Laboratory of Physiological Chemistry, Yale University, New Haven, Connecticut.) (Received for publication, December 8, 1910.) The vast accumulation of literature relative to adrenalin gly- cosuria contains very few records of attempts to demonstrate a quantitative relationship between adrenalin administration and sugar elimination. For the most part investigators have been content with the knowledge that a certain quantity of adrenalin injected subcutaneously or intraperitoneally almost invariably causes the appearance in the urine of significant quantities of dextrose. Moreover, it has been generally accepted that adrenalin given by mouth entirely fails to provoke glycosuria and that the intraperitoneal administration gives rise to a greater sugar excre- tion than the introduction of the drug directly into the circulation. What relationship exists between the quantity of adrenalin injected and the sugar eliminated, and how this relation may vary with change in the manner of adrenalin introduction are questions of some importance since adrenalin effects are constantly employed in the study of problems having a far reaching significance. Perhaps the most suggestive investigation in this particular direction is the recent communication of Ritzmann 1 . He has shown that the degree of glycosuria is dependent upon the quantity of adrenalin present in the blood at any given moment. So long as adrenalin is present in the blood sugar in the urine is in order but 1 Ritzmann: Arch. f. exp. Path. u. Pharmakol., lxi, p. 231, 1909. See also Straub: Munch, med. Wochenschr., 1909, No. 10; Pollak: Arch. f. exp. Path. u. Pharmakol, lxi, p. 376, 1909. 13 14 Urethane and Adrenalin Glycosuria glycosuria ceases as soon as adrenalin disappears from the circula- tion and almost immediately reappears when the drug is again introduced. Extremely dilute adrenalin solutions are potent in eliciting a relatively large excretion of sugar. There exists, accord- ing to Ritzmann, a direct relationship between the quantity of adrenalin introduced into the circulation and the amount of sugar eliminated in the urine, and for each rate of adrenalin injection there is a corresponding grade of glycosuria. Furthermore, adrena- lin administered subcutaneously is not capable of inducing as much sugar to appear in the urine as a much smaller quantity of the drug introduced intravenously. In Ritzmann's experiments cats and rabbits were employed and the adrenalin was introduced into the jugular or femoral veins by a modification of the Kretchmer method. The injections were made with the animals under anaes- thesia. In general when rabbits were used narcosis was produced -with urethane given by stomach sound. Urine was collected through a permanent catheter. In the course of an investigation it became desirable to make use of Ritzmann's observations and trials were made in order to deter- mine whether in our hands entirely corroboratory results could be obtained. Our method of introducing the drug directly into the circulation of the experimental animal (the rabbit) was that indicated in a former paper, 1 that is, adrenalin (Parke, Davis and Company), suitably diluted with 0.9 per cent sodium chloride solution, was injected into the ear vein under pressure. This obvi- ated the necessity of narcosis. Urine was obtained at the desired intervals by compression of the bladder through the body wail. Our experimental conditions conformed in every other respect with those of Ritzmann. In spite of the harmony existing between the experimental con- ditions of Ritzmann's investigation and our own all attempts to provoke glycosuria by intravenous injections of dilute solutions of adrenalin into normal rabbits failed. Two experiments are given below in detail which will serve as typical examples of a large number of similar observations. Table 1 shows the results obtained in an attempt to duplicate Experiment 4 (p. 239) of Ritzmann while the data in Table 2 are those yielded in an en- 1 Underhill and Closson: Amer. Journ. of Physiol., xv, p. 321, 1906. Frank P. Underhill deavor to duplicate Ritzmann's Experiment 15 (p. 240). In Ritzmann's Experiment 4, 0.18 gram dextrose was present in the urine after the introduction of 75 cc. of 1 :500000 adrenalin solu- tion in 40 minutes, whereas in the present investigation, Table 1, no sugar appeared after nearly 200 cc. had been injected at the same rate. It is shown in Experiment 15 of Ritzmann's work that TABLE 1. Female rabbit of 2JfiO grams weight. The urine in the bladder contained no reducing substances. Adrenalin solution, 1:500000. TIME. ADRENALIN INJECTED. URINE EXCRETED. REDUCTION TEST.* CC. CC. 11.22 11.42 41 25 negative 12.02 35 10 negative 12.22 40 8 negative 12.42 32 10 negative 1.02 50 20 negative *With Benedict's reagent. Benedict: Journal of Biological Chemistry; 1908, v. p. 485. TABLE 2. Male rabbit of 2200 grams weight. Urine in bladder did not reduce. Adrenalin solution employed, 1:250000. TIME. ADRENALIN INJECTED. URINE EXCRETED. REDUCTION TEST. CC. CC. 3.19 3.39 49 • negative 4.05 34 5 negative 4.27 35 20 negative 4.47 50 17 negative 4.58 15 7 negative 5.18 50 10 negative 5.39 50 18 negative 6.00 50 11 negative 0.28 gram sugar was present in the urine when 49 cc. of 1:250000 adrenalin solution had been injected in 20 minutes. Our introduc- tion of 333 cc. (Table 2) of the same strength and at the same rate of injection did not induce any glycosuria. In an endeavor to account for the discrepancy between Ritz- mann's results and our own control experiments were carried through 1 6 Urethane and Adrenalin Glycosuria without suggesting a probable explanation. Finally observations were made upon animals that had received urethane by mouth. All experiments in which sufficient urethane had been given yielded results in entire accord with those of Ritzmann. Positive results were obtained invariably only when sufficient urethane had been intro- duced. The quantity of urethane necessary appears to be about one gram per kilo of body weight. Smaller quantities will, however, frequently furnish positive results but the smaller quantities cannot be relied upon, whereas with doses of one gram per kilo not a single experiment failed to induce glycosuria. Table 3 presents data obtained upon an animal without ure- thane narcosis and in Table 4 may be found results from the same animal under urethane narcosis. Urethane alone is incapable of TABLE 3. Male rabbit of 2000 grains weight. Bladder empty when tested. 1.5 grams ure- thane in 20 cc. water by stomach tube. Adrenalin solution employed, 1:250000. TIME. ADRENALIN INJECTED. URINE EXCRETED REDUCTION TEST. CC. CC. 3.25 3.45 38 negative 4.05 33 negative 4.25 43 2 negative 4.45 42 3 negative 5.05 45 6 negative 5.25 45 26 negative 5.45 45 23 negative inducing glycosuria and negative results are also yielded when so- dium chloride is introduced into a urethane narcotized rabbit in the quantities and at the rate employed in the adrenalin experiments. From these observations it would therefore appear that urethane renders the rabbit organism unusually sensitive to the glycosuria- inducing action of adrenalin. It has been demonstrated by Froh- lich and LoewiHhat cocaine causes the organism of the cat and dog to become more sensitive to adrenalin with respect to its influence upon blood pressure, salivary secretion and mydriasis. It is possible ^rohlich and Loewi : Arc h. f. exp. Path. u. Pharmakol., lxii, p. 159, 1910. Frank P. Underhill 17 that adrenalin plays a role in adrenalin glycosuria somewhat analo- gous to that of cocaine observed by the above mentioned authors. Moreover it is possible that other narcotics and anaesthetics may exercise an influence similar to that of urethane in this and other forms of experimental glycosuria. In another portion of Ritzmann's paper there appears a compari- son of the influence of adrenalin when administered subcutaneously and intravenously. It is shown that a very small quantity of adrenalin injected intravenously will cause very much more sugar to appear in the urine than a much larger quantity of adrenalin introduced subcutaneously. This comparison seems hardly fair in view of the influence of urethane noted above since in Ritz- mann 's experiments the narcotic was employed only when adrena- TABLE 4. Same animal that was employed in previous experiment — four days later. Urine in bladder gave no reduction, 2.0 grams urethane in 25 cc. water by mouth. Adrenalin employed, 1 -.250000. TIME. ADRENALIN INJECTED URINE EXCRETED. REDUCTION TEST. CC. CC. 10.46 11.05 34 10 negative 11.25 39 5 negative 11.45 38 4 negative 12.05 38 22 Strong 12.25 34 20 very strong lin was given intravenously. From a consideration of the above observations concerning the role played by urethane in the pro- duction of glycosuria after intravenous injections of dilute solu- tions of adrenalin it appeared desirable to determine whether in the non-narcotized animal a definite quantity of adrenalin injected intravenously in dilute solutions would yield more sugar in the urine than the same quantity of adrenalin administered subcutane- ously in the dilution, 1 :1000. Table 5 shows the results of four such experiments. The rabbits were maintained under constant con- ditions of diet throughout so that the divergences in sugar elimina- tion can not be ascribed to such an origin. Neither can they be attributed to lack of glycogen in the body since a lapse of time was allowed between the injections sufficient for the production of a 1 8 Urethane and Adrenalin Glycosuria new store of glycogen. From an inspection of Table 5 it will be at once apparent that the intravenous administration of adrenalin in the dilution employed is far less potent in the non-narcotized rabbit than the usual subcutaneous injection with respect to the appearance of sugar in the urine. These results are in direct opposi- tion to those of Ritzmann. The use of urethane in Ritzmann's experiments is undoubtedly the factor responsible for our failure to corroborate his conclusions. Our experiments also indicate the variation in the quantity of sugar eliminated by the normal rabbit maintained under constant conditions when equal doses of adrenalin are subcutaneously administered at different periods, an observation which has been corroborated for the dog. TABLE 5. RABBIT. SUGAR IN URINE AFTER SUBCUTANEOUS INJECTION OF ONE MILLIGRAM ADRE- NALIN PER KILO IN DILUTION 1:1000 SUGAR IN URINE AFTER INTRAVENOUS INJECTION OF ONE MILLIGRAM ADRENALIN PER KILO IN First injection Second injection dilution 1:125000. 1 3.58 4.17 0.0 2 1.91 3.50 0.0 3 2.78 3.39 0.38 4 3.47 4.93 0.0 CONCLUSIONS. Data are furnished from which it is concluded that adrenalin introduced in very dilute solutions (1:500000 to 1:125000) fails to induce glycosuria in the normal rabbit. On the other hand, when the animal is under the influence of urethane narcosis these dilute adrenalin solutions are a sufficient stimulus for the production of glycosuria. From these observations.it is apparent that urethane renders the rabbit organism unusually sensitive to the glycosuria-inducing action of adrenalin. The subcutaneous administration of adrena- lin in the usual dilution (1:1000) to normal rabbits is far more efficacious in causing glycosuria than the same quantity of adrena- lin introduced intravenously in much greater dilution. The same quantity of adrenalin injected subcutaneously at differ- ent periods into the same animal under constant conditions causes the appearance in the urine of variable quantities of sugar. Reprinted from The Journal of Biological Chemistry, Vol. X, No. 2, 1911 MUCIC ACID AND INTERMEDIARY CARBOHYDRATE METABOLISM. 1 By WILLIAM C. ROSE. (From the Sheffield Laboratory of Physiological Chemistry, Yale University, New Haven, Conn.) (Received for publication, July 16, 1911.) INTRODUCTORY. Various views have been debated regarding the intermediate processes through which the carbohydrates pass in metabolism until they are finally eliminated in the form of their end products. As might be expected those theories fashioned from the experience gained in the study of the fermentation of sugars have received spe- cial attention. 2 Thus the formation of lactic acid from carbohy- drates through the agency of microorganisms finds a counterpart in the occurrence of the same substance in the tissues and secre- tions of the animal body. Metabolism of the carbohydrate molecule by the mechanism here suggested, involves an early splitting of the carbon chain. A distinctly different theory demands a direct oxidation of the carbohydrates without preliminary cleavage of the chain, with the formation of such intermediate products as glycuronic, sac- charic and oxalic acids, as represented in the following formulas: CH 2 OH I (CHOH). COOH I (CHOH) 4 — COOH (CHOH) 4 — COOH I CHO Dextrose CHO Glycuronic Acid COOH Saccharic Acid COOH Oxalic Acid 1 A preliminary report of this investigation was presented at the meet- ing of the American Society of Biological Chemists, December, 1910; see Proceedings, this Journal, ix, p. xii, 1911. 2 The more recent aspects of this subject are reviewed by Harden: Alcoholic Fermentation, 1911. 123 124 Metabolism of Mucic Acid This view was early supported by Schmiedeberg and Meyer, 1 who considered glycuronic acid a metabolic oxidative product of dextrose, on account of the related chemical structures of the two substances, and the ease with which dextrose is converted into glycuronic acid by oxidizing agents outside of the body. This view was also accepted by Emil Fischer and Piloty. 2 From feed- ing experiments with thymotinpiperidid, Hildebrandt 3 concluded that glycuronic acid may be formed from dextrose in the animal organism. He claims that fatal doses of this base, which conju- gates with glycuronic acid, exert no toxic action in rabbits if the animals have previously received dextrose. According to P. Mayer, 4 the elimination of glycuronic acid may be markedly in- creased in severe disturbances of respiration, such as dyspnoea, and in diabetes mellitus. The observations of Mayer are, how- ever, not substantiated by the more recent investigations of Fen- nyvessy, 5 and the statements on these questions are contradictory. That glycuronic acid is not a product of sugar metabolism seems to be indicated by the work of Loewi. 6 This investigator fed dogs with camphor during phlorhizin diabetes, and found that although enormous quantities of campho-glycuronic acid were excreted, the sugar elimination was only very slightly diminished. A much larger reduction in urinary dextrose ought to have occurred if it had been the mother-substance of glycuronic acid. Somewhat conflicting views in regard to this matter are given by Mandel and Jackson 7 and by Jackson. 8 In experiments upon rabbits, Mayer 9 noted an increase in the output of oxalic acid after the introduction of glycuronic acid, either per os or subcutaneously. The livers of animals receiving glycuronic acid were found to be richer in oxalic acid than those Schmiedeberg and Meyer, H.: Zeitschr. f. physiol. XJhem., iii, pp. 422-50, 1879. 2 Fischer and Piloty: Ber. d. deutsch. chem. Gesellsch.,xxiv, pp. 521-28, 1891. 3 Hildebrandt: Arch. f. exp. Path. u. Pharm., xliv, pp. 278-316, 1900. 4 Mayer: Zeitschr. f. klin. Med., xlvii, pp. 68-108, 1902. 5 Fennyvessy: Arch, internat. d. pharmacodyn., xii, pp. 407-20, 1904. 8 Loewi: Arch. f. exp. Path. u. Pharm., xlvii, pp. 56-67, 1902. 7 Mandel and Jackson: Amer. Journ. of Physiol., viii, p. xiii, 1902-03. 8 Jackson: Amer. Journ. of Physiol., viii, p. xxxii, 1902-03. 9 Mayer: loc. cit. William C. Rose 125 of control animals which had been similarly fed without glyc .ironic acid. Autolytic experiments indicated that liver extracts con- tained enzymes capable of producing the change from glycuronic to oxalic acid. Thierfelder 1 has shown that saccharic acid results from the oxi- dation of glycuronic acid in vitro, but there is no direct evidence that this change can occur in the animal organism. Mayer 2 reported an increase in urinary oxalic acid after subcutaneous injections of sodium saccharate. The increase, however, is very slight. Pohl, 3 after giving five grams of sodium saccharate to a small dog, was unable to detect any secondary oxidation products. The urine was reported to contain neither saccharic acid nor an increase in oxalic acid. On the other hand, Schott 4 in experi- ments on rabbits and dogs has recently found that saccharic acid is not oxidized in the body, but is excreted unchanged in the urine. After giving large doses of sugar, an increase in urinary oxalates has been observed in dogs by Baldwin, 5 and in rabbits by P. Mayer 6 and Hildebrandt. 7 Baldwin interprets the oxaluria as due to a special kind of gastric fermentation, while Hildebrandt and Mayer consider it as indicative of an oxidation of sugar through the oxalic acid stage. But, as Magnus-Levy 8 has pointed out, the extremely large doses of sugar (40 grams for a rabbit) may exert a toxic action leading to tissue disintegration which latter might give rise to the oxalic acid. In fact there is considerable evidence indicating the possibility of oxalic acid being an intermediate product in the metabolism of the protein substance, or of the purines. As early as 1875, Furbringer 9 claimed that diabetics excrete excessive amounts of oxalic acid, owing to decreased oxidative thierfelder: Zeitschr. f. physiol. Chem., xi, pp. 388-409, 1887. 2 Mayer: loc. cit. 3 Pohl: Arch. f. exp. Path. u. Pharm., xxxvii, pp. 413-25, 1896. 4 Schott: Ibid., lxv, pp. 35-7, 1911. 5 Baldwin: Journ. of Exp. Med., v, pp. 27^L6, 1900-01. 6 Mayer: loc. cit. 7 Hildebrandt: Zeitschr. f. physiol. Chem., xxxv, pp. 141-52, 1902. 8 Magnus-Levy : Oppenheimer' s Handb. d. Biochem., iv, Part i, p. 331, 1909. 9 Furbringer: Deutsch. Arch. f. klin. Med., xvi, pp. 499-526, 1875; xviii, pp. 143-92, 1876. 126 Metabolism of Mucic Acid functions, but Luzzatto 1 has been unable to observe any rise in the elimination. Indeed, it may be said that there is no. convinc- ing evidence supporting the assumption that glycuronic, saccharic, mucic, and oxalic acids are produced under physiological condi- tions by an incomplete combustion of carbohydrates. Considering, however, the paucity of data in regard to the behav- ior and fate of mucic acid when introduced into the animal organ- ism, and since if the unsplit-chain oxidation theory is correct one would expect mucic acid to result from the combustion of galac- tose, just as saccharic acid would result from the oxidation of dextrose, it seemed desirable to conduct experiments with mucic acid similar to those of P. Mayer with glycuronic and saccharic acids. Baumgarten 2 administered 20 to 50 gram doses of the potas- sium salt of mucic acid per os, to normal and diabetic dogs and men, anol was unable to recover any of the acid in the urine. From these experiments he concluded that mucic acid, in such quanti- ties, is readily and completely oxidized in the body. The urines were not analyzed for possible oxidation products. Baer and Blum 3 found that the feeding of mucic acid is without effect on the keton- uria induced by phlorhizin diabetes. This indicates that if mucic acid is oxidized in the body, it is unable to replace carbohydrate as an acidosis-inhibiting agent. Baer and Blum also observed that mucic acid exerted a toxic action, resulting in the death of their animals. It is probable that this was due to the presence of impurities in their preparation. The crude product always contains toxic nitro-compounds, which are removed only after repeated recrystallization. In the present investigation mucic acid was fed to rabbits and dogs, and the urines examined for mucic and oxalic acids. In the earlier experiments the mucic acid used was made from lactose by oxidation with nitric acid, according to the method of Kent and Tollens 4 and subsequently purified by recrystallization until the luzzatto: Salkowski's Festschrift, pp. 239-52, 1904. 2 Baumgarten: Zeitschr. f. exp. Path. u. Therap., ii, pp. 53-74, 1906. "Baer and Blum: Arch. f. exp. Path. u. Pharm., lxv, pp. 1-32, 1911. 4 Kent and Tollens: Liebig's Annalen, ccxxvii, pp. 221-32, 1885. William C. Rose 127 resulting product was perfectly non-toxic and melted at 213° C. Later Kahlbaum's " Schleimsaure" was used with equally satis- factory results. EXPEKIMENTAL PART. 1 Methods. The oxalic acid was estimated according to the method of Sal- kowski 2 which, from the result of comparative analyses made recently by MacLean, 3 seems to be less subject to error than the more commonly used method of Autenrieth and Barth. 4 It was impossible to determine the mucic acid excretion quanti- tatively, on account of want of an adequate method; but quali- tative tests were made by oxidizing the urines with concentrated nitric acid, as Bauer 5 has proposed in testing for galactose. For this purpose, 100 cc. -portions of the urine were introduced into beakers, each portion treated with 20 cc. of concentrated nitric acid (sp. gr., 1.4), and evaporated on the water-bath to a volume of approximately 20 cc. 6 The contents of the several beakers were then combined, trans- ferred to a small crystallizing dish, further evaporated, and allowed to stand in a cool place over night. The pure white crystals were collected on a small filter paper, washed two or three times with water and alcohol, dried in a dessicator, and identified by the melting point. Preliminary tests showed that 0.2 gram of mucic acid, when added to 100 cc. of urine, could be readily detected by this method. Five-tenths of a gram could be detected with the greatest ease. In one test where the latter quantity was added to 100 cc. of urine, 0.26 gram, or over 50 per cent, was recovered after oxidation. 1 This investigation was undertaken at the suggestion of Professor Lafayette B. Mendel, and the experimental data are taken from the thesis presented by the author for the degree of Doctor of Philosophy, Yale University, 1911. 2 Salkowski: Zeitschr. f. physiol. Chem., xxix, pp. 437-60, 1900. 3 MacLean: Ibid., lx, pp. 20-24, 1909. 4 Autenrieth and Barth: Ibid., xxxv, pp. 327-42, 1902. 6 Bauer: Ibid., li, pp. 158-66, 1907. 6 A flocky, gelatinous, precipitate of silica usually separated when rab- bits' urine was oxidized with nitric acid. This was removed by filtra- tion before crystallizing the mucic acid. 128 Metabolism of Mucic Acid In testing for mucic acid in the feces, the following procedure was em- ployed. The total fecal excretion for the period was rubbed up with water, the mixture made distinctly alkaline with sodium hydroxide, heated below boiling for five to ten minutes, and strained through absorbent gauze. The residue was in this manner extracted three times with water and alkali, the extracts combined, divided into 100 cc. -portions, and oxi- dized with nitric acid, 20-30 cc. of concentrated nitric acid being used for each 100 cc. -portion of the extract. The total volume of the extract was 800-1000 cc. for the excreta of a dog for two-day periods. In testing the delicacy of this method, it was found that half a gram of mucic acid when added to the day's fecal discharge of a dog, could be almost quantita- tively recovered. The urines of the experimental animals were collected in periods of forty-eight hours. In the rabbits, the complete two days' excre- tion was obtained by squeezing out the bladders. With the dogs, no attempt was made to mark off the periods sharply. Usually the animals were kept upon constant diets throughout the experiments, but occasionally they refused to eat as much during the experimental period as they did during the fore period. In such cases a corresponding reduction was made in the diet of the after period, so that in every case the urinary findings during the period of mucic acid feeding are comparable with those result- ing from the same diet without the acid. The attempt to give the acid in the form of the neutral sodium salt almost invariably evoked diarrhoea, so that this method of administration was aban- doned. It is probable that the large amount of sodium chloride formed in the stomach, through the action of the hydrochloric acid upon the sodium mucate, was responsible for the diarrhoea, by inducing a large secretion of water into the intestine. This ex- planation is rendered more probable inasmuch as doses of 5 grams of sodium chloride produce diarrhoea in rabbits. Hence, the rabbits received the free mucic acid suspended in water, by the stomach sound. With the dogs,. the acid was mixed with the finely hashed meat and dog biscuit of the diet, and fed at regular intervals. Rabbits. The results of the nine series of experiments on rabbits are sum- marized in Tables I to III. In animals 1, 2, 3, and 4, the urine was not tested for mucic acid, the oxalic acid excretion alone being William C. Rose 129 TABLE I. RABBIT DURA- TION OF PERIOD VOLUME URINE MUCIC ACID GIVEN OXALIC ACID OUTPUT DIET, NOTES, ETC. days CC. gms. mgms. 2 132 6.2 100 gms. carrots per day. 100 gms. carrots per (1) 1.6 kilos < day. Mucic acid neu- 2 100 10 13.4 ijl clllZiCU. W 1 \jUL lx avll, and given in two doses, 8 hrs. apart. 2 200 7.5 100 gms. carrots per day. 2 420 10.2 250 gms. carrots per day. 250 gms. carrots per (2) 2.1 kilos - 2 480 10 14.1 day. Free Mucic acid given in 5 gm. UUBCO, £rt Ulo. dJJdl 0. 2 320 9.0 250 gms. carrots per day. '200 gms. carrots, 25 2 365 11.7 gms. corn, and 100 cc. water per day. 200 gms. carrots, 25 gms. corn, and 100 (3) 2.0 kilos < 2 360 10 18.6 cc. water per day. Mucic acid given in two equal doses, 24 ^ hrs. apart. 200 gms. carrots, 25 2 375 5.9 gms. corn, and 100 ^cc. water per day. (4) 2.2 kilos | 500 gms. carrots and 2 525 9.1 200 cc. water during period. 130 Metabolism of Mucic Acid TABLE I. — Continued. BABBIT DURA- TION OF PERIOD VOLUME URINE MUCIC ACID GIVEN OXALIC ACID OUTPUT DIET, NOTES, ETC. days. CC. gms. mgms. Ate only 355 gms. car- rots during period. Given 200 cc. water. 2 255 15* 10.2 Mucic acid given in two doses :-lst, 10 (4) 2.2 kilos • gms.; 2nd, 5 gms., 24 L hrs. apart. '355 gms. carrots and 2 350 7.7 1 200 cc. water dur- ing period. *On evaporating the urine for the oxalic acid estimation, 0.1 gm. mucic acid separated. M.P.=213°C. determined. In these experiments as in those on animals 6, 7, 8, and £), there was only a slight increase in the oxalic acid output during the experimental periods, over that of the normal periods. The greatest percentage increase was noted in rabbit 7. Here, the output was 1.9 milligrams for the fore periods, 7.2 milligrams after receiving 10 grams, and 9.8 milligrams after receiving 20 grams of mucic acid. Even here the actual increase is extremely small — far less than one would expect if mucic acid were normally converted into oxalic acid in process of oxidation. Part of the mucic acid was recovered unchanged in the urine in every case after giving doses as large as 15 grams, with the possible exception of rabbit 7. Here after 20 grams given in two doses of 10 grams each, only a trace of an organic precipitate was obtained, having the crystalline form of mucic acid. In the periods when 10 grams of mucic acid were given, the unal- tered acid was recovered only once in amount large enough to identify, and here the total quantity given was introduced in one dose. When the acid was given in two doses of 5 grams each, 24 hours apart, only a very small amount or none could be detected in the urine. From the urine of animal 7, a trace of unaltered acid was recov- ered after a dose of 20 grams, while the urine of rabbit 8, a larger animal, yielded 0.2 gram of acid after a dose of 10 grams. Whether William C. Rose 131 TABLE II. BABBIT DURA- TION OF PERIOD VOLUME URINE MUCIC ACID GIVEN MUCIC ACID RECOVERED OXALIC ACID OUTPUT DIET, NOTES, ETC. days 2 CC. 230 gms. gms. mgms. Constant diet of 150 gms. car- rots npr rlav xyjvkj v^i. viewy throughout the PYnprimpnt. (5) 1.3 kilos I 2 340 15 0.5 (M.P.= 209°) fist day, 5 gms. | mucic acid. 1 2d day, 10 gms. [ free mucic acid. 2 345 2 250 2 225 f400 gms. carrots I and 50 gms. coin [ during period. 2 300 10 Ate only 300 gms. carrots and 50 gms. corn dur- ■ ing period. Mu- cic acid given in 2 equal doses, 24 hrs. apart. (6) 2.0 kilos.... 2 195 f300 gms. carrots < and 50 gms. corn [ during period. 2 200 15 0.15 (M.P.= 209°) 3.9 Same diet as in preceding pe- riod. 1st day, • 10 gms. free mucic acid. 2d day, 5 gms. free mucic acid. 2 250 3.4 fSame diet as in \preceding period. 132 Metabolism of Mucic Acid TABLE II— Continued. BABBIT DURA- TION OF PERIOD VOLUME URINE MUCIC ACID GIVEN MUCIC ACID RECOVERED OXALIC ACID OUTPUT DIET, NOTES, ETC. days CC. gms. gms. mgms. \ Constant diet of 2 260 1.9 1 200 gms. carrots per day through- ouT/ experiment. Mucic acid given (7) 1.6 kilos ... ■ 2 350 10 7.2 ! in two equal doses, 24 hrs. apart. 1st day, 10 gms. 2 450 20 Trace* 9.8 1 free mucic acid. 2d day, 5 gms. free mucic acid. 2 450 5.4 * Very small precipitate was obtained which was identified as organic matter. this was due to individual variations in the ability to oxidize mucic acid, or to differences in rate and degree of absorption, could not be determined. Dogs. Two experiments were made upon dogs. Dog 10 (Table IV) was a small animal, and excreted relatively large amounts of the ingested mucic acid. Throughout the experiment he was kept upon a constant diet, consisting of 100 grams of lean meat, 40 grams of cracker meal, and 25 grams of lard per day. While the urines did not represent exactly 48 hour periods, the oxalic acid excretion agrees very well with the results obtained in rabbits. Here also the actual increase during the mucic acid periods is slight and insignificant. In animal 11, a short experiment was made to determine whether or not any of the mucic acid failed to be absorbed, and was excreted in the feces. The dog was fed 15p grams of meat, 100 grams of dog biscuit, and 30 grams of lard per day. The feces were marked William C. Rose 133 TABLE III. RABBIT DURA- TION OF PERIOD VOLUME URINE MUC1C ACID GIVEN MUCIC ACID RECOVERED OXALIC ACID OUTPUT DIET, NOTES, ETC. days 2 CC. 210 gms. gms. mgms. 5.9 { '400 gms. carrots during period. 2 170 10 0.2 (M.P.= 212°) 6.9 Ate only 350 gms. carrots during period. Mucic acid given in one dose. (8) 1.8 kilos.... • 2 300 7.1 Same diet as in preceding peri- k od. 2 350 20 0.5 (M.P.= 213°) 8.2 Same diet as in preceding peri- od. Mucic acid given in two equal doses, 24 hrs. apart. 450 7.9 r Same diet as in preceding peri- od. • 2 275 5.6 Constant diet of 150 gms. car- rots per day throughout ex- periment. (9) 1.4 kilos 2 350 • 10 Trace* 8.5 < Mucic acid given in two equal doses, 24 hrs . apart. 2 350 6.4 2 400 15 0.5 (M.P.= 212°) 10.0 1st day, 10 gms. mucic acid. 2d day, 5 gms. mucic acid. 2 300 6.7 *Organic matter. Melting point not determined. 134 Metabolism of Mucic Acid TABLE IV. Dog 10; 6.8 kilos. DURATION OF PERIOD VOLUME URINE SPECIFIC GRAV- ITY REAC- TION TO LITMUS MUCIC ACID GIVEN MUCIC ACID RECOVERED OXALIC ACID OUTPUT NOTES days cc. gms. gms. mgms. 2 500 1.020 acid 13.4 z 1 HQ9 acid n u u IRA ID . D 2 300 1.032 acid 10 0.3 (M.P.= 210°) 1.3 25.1 fm*. • Mucic acid was I given in one dose. fMucic acid was 2 410 1.037 acid 20 (M.P.= 210°) 30.5 \ given in two equal [ doses, 24 hrs. apart. 2 275 1.027 acid 7.8 TABLE V. Dog 11; 13.2 kilos. >URATION OF PERIOD VOLUME URINE SPECIFIC GRAV- ITY REACTION TO LITMUS MUCIC ACID GIVEN MUCIC ACID RECOV- EREDIN URINE MUCIC ACID RECOV- ERED IN FECES NOTES days CC. gms. gms. gms. 1 176 1.050 alkaline fMucic acid given 2 300 1.046 acid 20 < in two equal doses, [ 24 hrs. apart. 250 1.027 acid 1st day of period. 170 1.025 alkaline 2nd day of period. off into two-day periods with lamp-black, and analyzed for mucic acid. The results are shown in Table V. After a dose of 20 grams, not a trace of the acid was recovered in the feces. Apparently, mucic acid is readily absorbed even when given in the free state. None was recovered in the urine of this animal after the mucic acid ingestion. The urine, which was alkaline to litmus for some William C. Rose 135 unknown reason during the fore period and last day of the after period, was rendered strongly acid for three days after giving the mucic acid. Whether or not the increased urinary acidity was due to a slight rise in the output of oxalic acid, or to the presence of traces of mucic acid too small to be detected by the method, was not determined. Since rabbits and dogs — at least dog 10 — excrete unaltered mucic acid in the urine after 20-gram doses, it seems scarcely prob- able that mucic acid is an intermediary product in the combustion of galactose and galactose-yielding carbohydrates. To further test this hypothesis, however, a series of experiments were made in which animals received the calculated amount of lactose (or gal- actose) necessary to yield 20 grams of mucic acid in the organism, and the urines were analyzed for mucic acid. The results follow: Experiment 1. A rabbit weighing 2300 grams, received by the stom- ach-tube 35 grams of lactose, in two equal doses, twenty-four hours apart. Thirty-five grams of lactose, if oxidized through the mucic acid stage, should yield 21.4 grams of mucic acid. The animal was well fed on carrots and oats, and the urine collected for sixty hours. The urine contained no sugar or mucic acid. Experiment 2. A rabbit weighing 1800 grams, received 35 grams of lactose in two equal doses, twenty-four hours apart. The urine excreted during the following fifty hours contained no sugar or mucic acid. Experiment 3. A rabbit weighing 2020 grams, received two doses of lactose, 17.5 grams each, twenty-four hours apart. The urine of the following fifty hours contained no sugar or mucic acid. Experiment 4. A rabbit weighing 2200 grams, received 8.6 grams of galactose. After an interval of twenty-four hours, a second dose of the same amount was administered. The total amount given (17.2 grams), if oxidized through the mucic acid stage, should yield 20 grams of mucic acid . The urine for the next sixty hours, contained a minute trace of sugar. No mucic acid was obtained after oxidizing with nitric acid. The amount of sugar present, if galactose, was too small to be detected by the Bauer method. Experiment 5. A dog weighing 5.4 kilos, was given 35 grams of lac- tose in two equal doses mixed in with the food. The urine of the following fifty hours gave a slight reduction of Benedict's solution, no test with Feh- ling's solution, and yielded no mucic acid on oxidation with nitric acid. Experiment 6. The same animal used in Experiment 5, received 35 grams of lactose in one dose. The urine of the following forty-eight hours gave a distinct reduction of Fehling's solution. The sugar was removed by fermenting with ordinary yeast (showing that the sugar was not lac- tose), the yeast filtered off, and the filtrate oxidized with nitric acid. No mucic acid was obtained. 136 Metabolism of Mucic Acid Experiment 7. A dog weighing 6.2 kilos, received 35 grams of lac- tose in one dose. The urine of the following forty-eight hours contained a minute trace of sugar. Direct oxidation with nitric acid yielded no mucic acid. GENERAL DISCUSSION. The results of the mucic acid feeding experiments, particularly those upon dogs, are not in accord with the findings of Baumgarten 1 This investigator was unable to detect mucic acid in the urine of a medium size dog, after giving 20 grams, and in the urines of nor- mal and diabetic men after giving 50 grams of the acid. The failure to detect mucic acid in the urine in any case, was probably due, at least in part, to the inadequate method employed, which consisted in evaporating the urine made alkaline with sodium hydroxide to dryness, extracting the residue with hot absolute alcohol to remove the urea, and preparing the double hydrazine compound of mucic acid, C 4 H 4 (OH)4. (CO . N 2 H2.C 6 H5)2, by boil- ing on the water-bath with sodium acetate and phenylhydrazine- hydrochloride. Billow, 2 who first prepared the compound, heated the mucic acid and phenylhydrazine on an oil-bath, at a tempera- ture of 120° to 140° C. He states that the reaction is not complete until all water has been evaporated. Baumgarten seems to have overlooked these facts, although he cites the paper of Biilow in describing the method. It was found impossible to obtain the compound by Baumgarten's method of heating on a water-bath, even after the addition of 2 grams of mucic acid to the 100 cc. of urine used in the test. It seems scarcely probable that Baum- garten would have used the method without first testing its deli- cacy, but if such tests were made no mention of the fact is to be found in his paper. In both the rabbit and dog experiments, the increase in oxalic acid excretion observed after giving mucic acid compares favor- ably in amount with that found by Mayer 3 after giving doses of 15 to 20 grams of sodium glycuronate or sac char ate, and interpreted by him as indicating an oxidation of these substances to oxalic acid in process of metabolism. A similar interpretation is made 1 Baumgarten: loc. cit. 2 Biilow: Liebig's Annalen, ccxxxvi, pp. 194-97, 1886. 3 Mayer: loc. cit. William C. Rose 137 of the increased oxalic acid excretion observed after giving rabbits 40-gram doses of dextrose, although the largest actual increase obtained was from 1.2 milligrams before, to 4.7 milligrams after the sugar administration. It would seem that the explanation of this small increase in oxalic acid suggested by Magnus-Levy 1 and already alluded to, is much more probable — namely, that the abnormally large doses of foreign substances produce a slight dis- integration of body tissue, sufficient to occasion a rise in oxalate elimination. It is not at all likely that oxalic acid is an intermediary product in the normal metabolism of any of the food-stuffs. Under cer- tain abnormal conditions, at present little understood, it arises in the body, and is immediately excreted. This conception would seem to be indicated from the results of feeding experiments with oxalic acid. The inability of the organism to oxidize it, when intro- duced per os or subcutaneouly, is conclusively shown by the re- searches of Gaglio 2 and Pohl. 3 Recently the latter investigator, 4 has corroborated his former work, having quantitatively recovered in the urine all the oxalic acid introduced into the organism. At any rate, no evidence has been obtained from feeding experiments with mucic, saccharic, and glycuronic acids, that oxalic acid is a normal intermediary product of carbohydrate metabolism. Again, from the results of the lactose feeding experiments, the oxidation of the end carbon atoms of the sugar molecule without preliminary cleavage seems unlikely. In all experiments with the exception of experiment 6, (in which the lactose was given in one dose,) the sugar was completely utilized by the animals. Usually the sugar was given in two doses, 24 hours apart, in order to make the experiments exactly comparable with the mucic acid experiments. Twenty grams of mucic acid given in this manner, are not entirely oxidized by the body, but reappear in part in the urine. Stoichiometrically equivalent amounts of galactose-sugars, given under the same conditions, fail to evoke the appearance of mucic acid in the urine. The conclusion is obvious — mucic acid is not a product of the physiological metabolism Magnus-Levy: loc. cit. 2 Gaglio: Arch. f. exp. Path. u. Pharm., xxii, pp. 235-52, 1887. 3 Pohl: loc. cit. i Pohl: Zeitschr. f. exp. Path. u. Therap., viii, pp. 308-11, 1910. 138 Metabolism of Mucic Acid of galactose. It may be objected, that in the earlier experiments too much acid was introduced into the circulation at one time to allow complete oxidation, while in the latter series the change from lactose to mucic acid was very slow, only small amounts arising each moment. This explanation is, however, scarcely valid, inasmuch as mucic acid is relatively insoluble and its absorp- tion must be very slow. Considerable time would be necessary for the content of the blood in mucic acid to be appreciably increased. Hence, there should be ample time for oxidation to occur, before the circulation becomes flooded with the acid. It is probable, therefore, that sugars are normally oxidized by some method other than that indicated by the unsplit-chain theory. SUMMARY. 1. Mucic acid, in doses of 10 to 20 grams, was not completely oxidized by rabbits, but was in part excreted unchanged in the urine. 2. When given in doses of 20 grams to a medium sized dog, mucic acid was excreted unchanged in the urine in amounts large enough to identify. The greater portion of the acid was not recov- ered. 3. After large doses of mucic acid, only a very small increase in oxalic acid elimination occurs in rabbits and dogs. This increase is by no means as large as would be expected if mucic acid were one of the precursors of oxalic acid. 4. Rabbits and dogs receiving the amounts of galactose and lactose stoichiometrically equivalent to 20 grams of mucic acid, excrete no mucic acid in the urine. Obviously, therefore, mucic acid is not an intermediary product in the metabolism of galactose- yielding sugars. Reprinted from The Journal of Biological Chemistry, Vol. X, No. 3, 1011 EXPERIMENTAL STUDIES ON CREATINE AND CREATININE. 1 I. THE ROLE OF THE CARBOHYDRATES IN CREATINE- CREATININE METABOLISM. By LAFAYETTE B. MENDEL and WILLIAM C. ROSE. {From the Sheffield Laboratory of Physiological Chemistry, Yale University, New Haven, Connecticut.) (Received for publication, August 14, 1911.) INTRODUCTORY. Despite the enormous amount of data that have been accumu- lated in recent years in regard to the occurrence and excretion of creatine and creatinine, our knowledge of the significance of these substances is still far from adequate. The incidence of the excretion of creatinine (cf. Mendel, '09 and Myers, '10), is perhaps better understood than that of creatine. Folin ('05a, '05b, '05c), van Hoogenhuyze and Verploegh ('05), Klercker ('06), Closson ('06), and Shaffer ('08b) have shown that the daily output of creatinine, on a meat-free diet, is remarkably con- stant for the same individual, and is independent of the total nitrogen and volume of the urine. This constancy in the excre- tion of creatinine indicates that it is of endogenous origin, and as Folin ('05b) says, is an " index of a certain kind of protein- metabolism occurring daily in any given individual." Folin ('06) further believes that the creatinine of the urine has no connec- tion with the muscle creatine, as the latter is converted into creat- inine only with great difficulty. 1 A preliminary report of these studies was presented to the Society for Experimental Biology and Medicine, May 17, 1911. 213 2 14 Creatine and Creatinine Metabolism The excretion of creatinine in disease frequently undergoes marked variation. 1 Mellanby ('07) found the creatinine-coef- ficient to be very low in diseases of the liver. This was partic- ularly true of individuals with hepatic carcinoma. The urines of such patients contained large amounts of creatine. Mellanby ('08) believes that the liver is intimately connected with the production and excretion of creatinine. He suggests that the liver is continuously forming it from substances brought from other organs by the blood. In the developing muscle, this creat- inine is transformed into creatine, and stored until the mus- cles have reached a certain saturation point. After this point has been reached, creatinine is continuously excreted. Both creatine and creatinine are excreted in the urine of dogs with Eck fistulas, according to Salaskin and Zaleski ('00), Lon- don and Boljarski ('09), and Foster and Fisher ('11). London and Boljarski found that the administration of creatinine to such animals did not increase urinary creatinine. Results contra- dictory to these were obtained by Foster and Fisher, who report that the ingestion of creatinine increases the creatinine output in the urine. The results of both investigations are in accord in finding that the feeding of creatine caused no increase in creatine excretion, but was followed by a slight rise in eliminated creat- inine. The observations of van Hoogenhuyze and Verploegh ('05) and Shaffer ('08a) as well as of certain earlier investigators, indicate that increased or decreased muscular activity, with adequate food, has per se no effect on the excretion of creatinine. Creatine is never normally present in the urine of adult mammals, unless creatine is taken in with the food. As early, however, as 1868 Meissner ('68) found that creatine almost en- tirely replaces creatinine in the urine of birds. These observa- tions were subsequently verified by Paton ('09-' 10) and Voegtlin and Towles ('11). In mammals, creatine is excreted in the urine during inanition. This was first observed by Benedict ('07), and later verified in 1 For the literature on the excretion of creatinine in various pathological conditions, see Leathes ('06-07), Spriggs ('07a, '07b), Benedict and Myers ('07a), Forschbach ('08), Shaffer ('08b), and Levene and Kristeller ('09). Lafayette B. Mendel and William C. Rose 215 collaboration with Diefendorf (Benedict and Diefendorf, '07) on a fasting woman. At about the same time, but independently, Cathcart ('07a, '07b) noted the excretion of creatine in starvation. Recently this author (Cathcart, '09) has published the results of experiments made upon himself and others in which he reports that creatine is found in the urine, in relatively large amounts, after fasting periods of forty hours. In starvation experiments made upon rabbits, Dorner ('07) noted the elimination of crea- tine. Similar results were obtained in dogs by Underhill and Kleiner ('08), Richards and Wallace ('08), and Howe and Hawk Cii). Pathologically, creatine occurs in a variety of conditions. Benedict and Myers ('07b) found it in the urines of a large num- ber of insane patients, most of whom were in poor nutritive con- dition, which probably accounted for its appearance. In con- valescence after typhoid fever Foster ('10) found an elimination of creatine. Shaffer ('08b) observed creatine to be invariably excreted where there was a rapid loss of muscle protein, such as in acute fevers, in the acute stages of exophthalmic goitre, in tumor cachexia, and in women during the first week post partum, when the resolution of the muscular wall of the uterus is proceeding most rapidly. This has also been observed in dogs by Murlin ('08-'09). According to this author, the creatine first appeared in the urine two days before parturition, and reached a maximum on the fifth day after parturition. He suggests that the latter date probably marks the maximum of the involution process. Levene and Kristeller ('09) noted the excretion of creatine in a variety of diseases. The largest amounts were found in the urine of patients with anterior poliomyelitis and muscular dystrophy. In many of these instances — notably fevers and hepatic car- cinoma — under-nutrition is undoubtedly an important contrib- uting factor in the production of creatine. In this connection the recently published paper of Underhill and Rand ('10) is of particular interest. These authors found large amounts of crea- tine in the urines of women with pernicious vomiting of preg- nancy, and Underhill suggests that the changes observed in the urine are induced by the accompanying inanition. Evidence tending to substantiate this view is furnished by the obser- vation that the perverted creatine metabolism, as well as the 2i6 Creatine and Creatinine Metabolism metabolism of the other nitrogenous constituents of the urine, tends rapidly to resume the normal on the rectal administration of dextrose, without necessarily exerting any influence on the pathological state of the patient. The presence of carbohydrates appears in these experiments to be the all-important factor in preventing the abnormal partition of urinary nitrogen associated with starvation. In the absence of sufficient carbohydrates the energy-yielding substances in the body seem to be utilized with great difficulty. The inability to oxidize carbohydrates may, therefore, explain the observations of Shaffer ('08b) and Dreib- holz ('08); and more recently of Krause ('10), Krause and Cramer ('10), and Taylor ('10, '11), that creatine is a constant product of the metabolism of patients with diabetes mellitus. In consideration, therefore, of such observations as the above, a series of experiments was conducted on starving rabbits, to determine the influence of carbohydrates on the creatine metab- olism during a period of inanition unaccompanied by any other abnormal factor. From two experiments upon geese, Paton ('09-' 10) reached the conclusion that the administration of glucose in fasting has no specific action on the excretion of creatine. But his experiments were entirely too few in number, and extended over too short periods of time, to render his results conclusive. While the present work was in progress, an interesting paper appeared by Cathcart ('09), in which he reports that the creatine excretion induced by fasting is reduced to nil by administering a carbohydrate diet " practically nitrogen and fat free;" whereas with a fat diet, the amount of creatine excreted is increased. He further states that the addition of protein food (carbohydrate-free) during the fat period, does not markedly reduce the creatine excre- tion. The experiments were made upon men, which necessarily limited the duration of the fasting to short periods (usually forty hours). The author reports that this inanition always brought about an output of some hundred and fifty milligrams of creatine, which seems surprisingly large for a fast of so short duration. Usu- ally, several days of starvation are necessary to induce the excre- tion of appreciable amounts of creatine in man (cf. Benedict and Diefendorf, '07, and Underhill and Rand, '10). Lafayette B. Mendel and William C. Rose 217 The chief criticism, however, of Cathcart's experiments involves the nature of the carbohydrate diet used to reduce the creatine output. This consisted of tapioca, sugar, honey, corn-flour, and banana meal, all of which — with the exception of the sugar — con- tain small amounts of nitrogen. In one experimental period table 1. CathcarVs carbohydrate diet. ARTICLE OF FOOD AMOUNT INGESTED PER DAY N N INTAKE grams per cent grams Banana meal 454 0.64 2.80 Honey 230 0.23 0.53 3.33 (Cathcart, '09, p. 316) the diet consisted of banana meal and honey, and contained according to Cathcart's own analyses, 3.33 grams of nitrogen (see Table I). It is true that this is a small nitrogen intake, and that for most purposes the diet might be considered " practically nitrogen free;" but, a priori, we have no way of know- ing whether the nitrogen accompanied by the carbohydrates, or the carbohydrates per se are responsible for the reduction in crea- tine elimination. Chittenden 1 has shown that man can live in perfect health, and remain in nitrogen equilibrium, on an intake of 5 or 6 grams of nitrogen per day, when sufficient carbohydrates and fats are ingested. May it not, therefore, be possible, that an intake of half of the amount of nitrogen necessary to meet the needs of the body is capable, when accompanied by carbohydrates, of exerting some influence on creatine elimination during starva- tion? Certainly we have no answer to this question in the results of Cathcart. Hence, it was determined to test the influence of a carbohydrate diet absolutely nitrogen-free on the creatine elim- ination in animals during inanition. 1 Chittenden : Physiological Economy in Nutrition, New York, 1904. 2lS Creatine and Creatinine Metabolism EXPERIMENTAL PART. 1 Methods. The animals used in the investigation were large rabbits, pre- viously well fed on oats, cracked corn and carrots. In several experiments the urines were analyzed for three or four days before the fasting periods were begun, in order to determine whether or not creatine is normally excreted by rabbits on a mixed diet. The urine was always collected at the end of twenty-four hour periods, unless otherwise stated; the complete excretion for the day's cycle being obtained by squeezing out the bladder. Total nitrogen was estimated by the Kjeldahl-Gunning method, ammonia nitrogen 2 and preformed creatinine 3 by the Folin methods, and "total creatinine," 4 i.e., after conversion of creatine present to creatinine, by the Benedict-Myers modification of the Folin method. In describing the creatinine determination in rabbits' urine, Dorner ('07) alludes to the appearance of a flocky precipitate after treating with picric acid and sodium hydroxide, the precipitate persisting even after dilution of the mixture to 500 cc. He suggests filtering the solution before making the colorimetric readings. It has been the experience of the writer that this procedure is entirely unnecessary. The precipitate which is com- posed largely or entirely of phosphates, is so small in amount that it offers no hindrance to matching the color accurately with the standard bichromate solution. Frequently, in sufficiently dilute urines, no visible precipitate appears at all. Often a difference of three or four minutes in the time the urine is allowed to stand after the addition of the picric acid and sodium hydroxide, pro- duces a variation of a millimeter or more in the colorimetric reading. The maximum depth of color is obtained in about ten minutes. Consequently, in all the determinations of creatine and creatinine recorded below, ten minutes were allowed for the completion of the reaction. The solutions were then diluted and the readings taken immediately. 1 The experimental data are taken from the thesis presented by William C. Rose for the degree of Doctor of Philosophy, Yale University, 1911. 2 Folin: Amer. Journ. Physiol., xiii, pp. 45-65, 1905. 3 Folin: Zeitschr. f. physiol. Chem., xli, pp. 223-42, 1904. 4 Benedict and Myers: Amer. Journ. Physiol., xviii, pp. 397-405,1907. Lafayette B. Mendel and William C. Rose 219 The influence of inanition on the creatine-creatinine excretion in rabbits, with some observations on the elimination of ammonia. Four rabbits were allowed to starve until death resulted, the urines being analyzed for total nitrogen, ammonia nitrogen, total and preformed creatinine. The analytical data are summarized in Tables II to V. Throughout the experiments the animals were given water ad libitum. Creatine usually appeared in the urine on the second day, and progressively increased in actual amount until death. There was generally also an increase in the percentage of the total nitrogen present as creatine, but considerable varia- tion is noted owing to the large increase in total nitrogen. The elimination of nitrogen in the form of creatinine is remarkably constant. Though slight fluctations are noted — especially a tendency to decrease just before death — as in rabbits 1 and 3, the change is by no means commensurate with that observed in the creatine elimination. The percentage of creatinine-nitrogen invariably decreases as inanition progresses, on account of the increased total nitrogen elimination. These results agree with those of Dorner ('07). Ammonia Elimination. Incidentally it is of interest to note the elimination of ammonia nitrogen. So far as the writer is aware, no determinations of the ammonia excretion in rabbits dur- ing starvation have been previously published. It was expected that the percentage of the total nitrogen present in the form of ammonia would be increased, but such is not the case. With the exception of rabbit 1, where the results are very irregular, all animals excreted progressively smaller percentages of their nitro- gen as ammonia. Usually, there is a tendency for the absolute amount to slightly increase before death, but this increase does not keep pace with the increase in the output of total nitrogen. Hence, the percentage steadily decreases. No explanation can be given for the irregular results obtained with rabbit 1. As will be seen in the tabulated data (Table II), ammonia nitrogen was entirely absent, or present in amounts too small to be determined, on the 19th and 22d, but markedly in- creased until the 24th, when it represented 2.3 per cent of the total nitrogen. On the 25th and 26th it was still high, though the percentage had slightly fallen. This rabbit was a small animal, 220 Creatine and Creatinine Metabolism V C3 ft? N N N •biuouitiiv N N N ■Biuouirav 73 73 73 o o o o o o O O O £ £ rH IN Ol O lO 00 H « N CO TjH O 10 00 01 00 CO rH rH rH O rH OOHNM^iOCON 9 J. e N N N ■ejuouiuiv N N N ■Btuouiuiv £ .s I 1 CS O CP g ^fl 73 "C T3 f § O ^ O O O O O 72 O O O O O O "S £ £ £ £ 55 £ < N »0 CD H M O H H H H N lO O 00 CO N lO CN rH rH © © o © © l> *C »C d rtH CO CN OS O g rH CM CO O IN COq Lafayette B. Mendel and William C. Rose 221 < H els N N N Bjaorarav OQ CD >> e3 O O * -(J fori Pi O <+-< © ine Uri Ur "O* T3 T3 OOO O a «£ «2 «2 O <4-4 OOO O £ £ £ § rH GO 00 GO r-i i-H i-H O N auweaiQ N aniuweaiQ N eiaouiray N l B * X uoho'88'h; . 10 rH !» i-t do ■hh 1-1 OS CO ■"O" ^ "O* a o w ^ << C IO O CO CO M lO 8 GO 73 N CO H Oi H H H O rH H ^ (N OS rH ^ CO GO rji CO 1— I rH 1— I CO i-H 3 OOOlOH(MCO^»OtO > 9-1 5 ft? .5 .S B 3 &> O O T3 T3 O O O O O O O i-H GO CO T}H GO CO GO CO o h C8 rHrHrHrHrHOflCQ 222 Creatine and Creatinine Metabolism scarcely full-grown, and was in very poor nutritive condition at the beginning of the experiment. Possibly this may explain the relatively large output of ammonia nitrogen in the last stages of inanition. It is interesting to note that the elimination of crea- tine nitrogen was greater in this animal than that observed in any other experiment, thus emphasizing the importance of the nutritive condition for the nitrogenous metabolism. The fact that starvation does not induce an absolute or per- centage increase in the elimination of ammonia nitrogen in rab- bits, is quite in contrast with the findings of Brugsch, 1 Cathcart, 2 Grafe 3 and others on men. It would seem, therefore, that her- bivora are either not subject to acidosis to the same extent that omnivora and carnivora are, or that they utilize bases other than ammonia for the neutralization of the acid products. In this connection, the suggestion of Burridge 4 — that creatine may serve as a neutralizing agent for lactic acid — is an interesting but unverified assumption. In . subsequent experiments upon rabbits the ammonia deter- minations were omitted. The influence of a carbohydrate diet upon the creatine-creatinine excretion in rabbits during inanition. Diet. Numerous attempts were made to give starving rab- bits dextrose and sucrose by the stomach tube in solutions of varying strengths, but the results were invariably very unsatis- factory. Even when small doses of the sugar solutions were introduced into the stomach at intervals of several hours, diar- rhoea was evoked, with resulting contamination of the urine. A more serious difficulty, however, was the fact that frequently the kidney excretion was practically stopped after giving the sugar for two or three days, and the animals died with symptoms of uremic poisoning. Hildebrandt 5 observed that large doses of dextrose 1 Brugsch: Zeitschr. f. exp. Path. u. Therap., i, pp. 419-30, 1905. 2 Cathcart: Biochem. Zeitschr., vi, pp. 122-23, 1907. 3 Grafe: Zeitschr. f. physiol. Chem., lxv, pp. 21-52, 1910. 4 Burridge: Journ. of Physiol., xli, pp. 303-04, 1910. 5 Hildebrandt: Zeitschr. f. physiol. Chem., xxxv, pp. 141-52, 1902. Lafayette B. Mendel and William C. Rose 223 exert a toxic action in rabbits fed upon a diet of oats. He attrib- uted the toxicity to the production of large amounts of oxalic acid through the incomplete combustion of the sugar, and found that the addition of calcium carbonate to the diet neutralized the oxalic acid and prevented the appearance of abnormal symp- toms. He makes no mention, however, of a decreased kidney excretion. A typical experiment illustrative of this inhibition of kidney function, is summarized in Table VI. The sugar-feeding was begun on December 13. On the 15th, the animal had severe diarrhoea and contaminated most of the urine. On the 16th, the inability to excrete urine was very evident. The complete excre- tion for twenty-four hours was 10 cc, containing only 0.04 gram of total nitrogen. On the 17th, only 4 cc. of urine were excreted. On the 18th, an attempt was made to increase the urine elimina- tion by giving 100 cc. of water by the stomach tube, but only 50 cc. of a very dilute urine were obtained, containing 11 mgms. of nitrogen as total creatinine. The 50 per cent retention of the water could not have been due to a depletion of the tissue mois- ture, for the animal had received water daily throughout the exper- iment. Retention is further indicated by the fact that the ani- mal began to increase in weight on the 15th, and continued to increase until death on the 19th. Before death severe diarrhoea occurred, accompanied by a twitching of the neck and shoulder muscles, and followed by coma. No explanation can at present be given of these observations. Out of some eight or ten experiments in which sugar was adminis- tered, satisfactory results were obtained only once (rabbit 6, Table VII), and this animal differed from the others in readily eating loaf-sugar, thus obviating the necessity of giving sugar solutions by the stomach tube. In consequence of the difficulties associated with the sugar- feeding, this diet was abandoned. In the remaining experiments (Tables VIII to XI), soluble-starch suspended in water was given by the stomach tube with very satisfactory results. In no case did this diet interfere with kidney function. The Composition of the Urine. In the following experiments, as in those previously described, creatine is a constant constit- uent of the urine of starving rabbits. In rabbit 6, creatine did TABLE VI. Rabbit 5 — Starvation; carbohydrate feeding. DATE BODY WEIGHT URINE DIET, NOTES, ETC. Vol- ume Specific Reaction gravity 1 to litmus Total N Creat- inine N Crea- tine N Nov. gms. cc. gms. mgms. mgms. 30 2240 220 1 012 Alkaline 0.67 30 Animal ate 300 gms. carrots and 50 gms. Dec. cracked corn. 1 2260 210 1 014 Alkaline 0.47 27 Animal ate 300 gms. carrots. 2 2240 55 1 020 Alkaline 0.51 32 2 No food. 3 2160 46 1 028 Acid 0.86 28 13 No food. 4 2080 37 1 032 Acid 0.80 28 4 No food. 5 2040 38 1 033 Acid 0.77 29 3 No food. 6 1980 36 1 035 Acid 0.77 27 9 No food. 7 1920 38 1 032 Acid 0.77 30 No food. 8 1860 44 1 025 Acid 0.85 27 1 No food. 9 1800 48 1 022 Acid 0.92 23 2 No food. 10 1740 55 1 018 Acid 0.86 22 7 No food. 11 1700 72 1 018 Acid 0.96 23 19 No food. 12 1640 72 1 020 Acid 1.29 23 29 No food. 13 1580 46 1 025 Acid 0.97 24 35 Animal was given 15 gms. sucrose, (loaf sugar) . 14 1510 21 Acid 0.26 22 16 30 gms. sucrose by stomach - sound in 50 per cent sol. in six equal doses. 15 1550 2* Acid 0.02* 3* 0* 35 gms. ditto in seven equal doses. Part of a day's urine. Ani- mal had diarrhoea. 16 1600 10 Acid 0.04 3 1 25 gms. ditto in five equal doses. Com- plete kidney excre- tion for twenty-four hours. 17 1620 4 Acid 2 trace 4 gms. olive oil given by stomach-sound. Complete kidney ex- cretion for twenty- four hours. 18 1620 50 1 .010 Neutral 7 4 No food. 100 cc. water given by stomach-sound. 19 1660 Animal died in after- noon with severe diarrhoea. *Not a complete day's urine, see notes. Lafayette B. Mendel and William C. Rose 225 not appear in appreciable amounts until after a surprisingly long period of inanition. Small amounts were periodically excreted from February 7 to March 1, but the quantities were too small to be of any significance. For ten days, beginning with February 14th, the animal was given loaf-sugar each day, but the only effect observed was a decrease in the total nitrogen elimination, with a corresponding increase in the percentage of creatinine nitro- gen. On February 25, starvation was resumed, and creatine first appeared in significant amount on March 2. This remarkably long period of starvation was undoubtedly made possible by the excellent nutritive condition of the animal, it having been fed liberally for several weeks before beginning the experiment. The store of glycogen probably prevented the ap- pearance of much urinary creatine, until the liver and muscles had had their glycogen supply nearly depleted. On March 2, sugar-feeding was again begun with the result that the creatine nitrogen fell from 14 mgms. on the 2d, to zero on the 5th. After two days of fasting the creatine nitrogen again increased until it was more than twice as large in amount as the creatinine nitrogen. On resuming the sugar diet, however, it was rapidly reduced to zero. On March 13, an attempt was made to give a protein diet consisting of coagulated egg-white, but diar- rhoea resulted, followed by the death of the animal on the next day. Results similar to these were obtained on rabbits 7, 8 and 9, each of which received the soluble-starch diet instead of the sugar. The creatine elimination was reduced at will by giving an abso- lutely nitrogen- and fat-free carbohydrate diet. Usually the amount of total nitrogen and creatine nitrogen increased the first day of the carbohydrate administration, but rapidly decreased on con- tinuing the feeding. Total nitrogen and total creatinine appear to have a common source, for an increase or decrease in the latter is always accompanied by a similar change in the former. The sig- nificance of this will be discussed later. The experiment upon rabbit 10 is particularly interesting because of the results following the administration of alcohol. The protein-sparing effect of small doses of alcohol is well known. 1 1 For the literature on this subject cf. Rosemann: Oppenheimer' 's Hand- buch der Biochemie, iv, part 1, p. 433, 1909. 226 Creatine and Creatinine Metabolism ^3 O x SoOcOrHC^iNOOO'rJHOCOOOOOOOT-HCN SMCOMCOWMMCOWMCOWMMCOMINMCON CO O N tO N005HNCOOOIMNOCO »Ol>00(»NCONO0N»OM oooooooooooooooooooo '"O ""O 'XS ""O *T3 ^ ""O '"O '"O "^3 ""^ T3* ""O '"O ""C5 ""O ""O S '3 "3 '3 '3 °3 '3 '3 '3 '3 '3 '3 '3 '3 '3 '3 '3 '3 '3 '3 j> _> % CD ? B o . 03 a £ OS ^ o • 43 -o a 2 . o o3 d ^ 00 05 w o -° £ 'd T3 O

¥ «N § B o 03 co £ rB bfi o r-H 03 5 a a ° B » co c3 13 bfi . CM B B o cp co £ J, bfi o 3 a a s B 1/3 <5 CO . a 03 g 1 & S •* 03 co .S B .5 o3 a o ■5 ^ te bfi 1 bfi bfi 03 bfi ^ 03 o B cp bfi to o3 B 8*2 co _ 03 C3 bfi OO < CM 03 c3 03 03 .§5 B B -2 > CO CO O O 6 C3 O O CO o 00 OHO CO CO >o O O) O O O O CM CM CM CM 00 lO O H T)< 10 10 00 (M 00 N GO CO O 73 73 a a 73 73 0) o> o o o3 c3 u u fn OJ fn 0) +s 0> g * g g i i .S 5=1 -2 73 H3 73 0)0)0) 15 ^ 12 a fl d OJ ft ft ft 5 to m S 3 3 03 U o3 ft £ « fl » S £.2 .B 73 o3 0) o o 02 02 00 73 w 73 73oO©Oo3o3c3 O^J OJ © <« o3 O • fH o o3 O O l>- fH a > •-H 0> OJ 02 a "*J ft 03 3 a 02 a -d < o fH 73 02 o> ji a * a 02 fi !•§ c3 fn fn fl) be a MS COMNOllOlOHCO^ O - o> o> d a d d ^3 ^ ""O "73 "73 73 73 73 73 73 73 73 73 <^ OHNM^iocDNoOO)0 Lafayette B. Mendel and William C. Rose 229 t3 N N N N ogjoadg in a a o o g I * * 8 § 8 ogg n d a xn xn m oq +J +3 +3 o3 c3 c3 oS OOOO *d T3 d d d d c3 c3 c3 o3 -t-= § o S d d § 2 2 ft Oh a CO CO 3 3 rd rd o g o OOOO jO *o 'o CD CO CO O CD o M CO CP d 15 ID ft d >> co c3 g W) co 3 O 73 d 73 d o <3 O ^T^TJ-dOOOO© CD CD CD CD <*_ t«_ c_ xxxx 00000 DQ s bD fcJD bC Ol CO O i-H CO O O CO O OOOOOOt^(N(NcNi-HOOcNC0 T)lNlO00CON^HNlOHC5»O' a cn co tjh h tji - .9 .3 .5 .5 _ c3 c3 c3 °3 °o "o O O o O O O O O O <3 0 OOOO 00 00 ^ CO CO CO (N CDNOOClOHlNCOTjiiOcONCiOOlO 230 Creatine and Creatinine Metabolism o N N 0) g & 9< ft d d ^ GO m 02 ^d .d rj (D (B jj ^d j= 'o o T3 TJ 'o 73 0000 0000 8 I O »0 to \-r Tj* ^ Tji M CD h Or-HrHCOr-lO'-lOCO N N ogioadg amnion. OOOtMOOOOt^ N H H COt^OOOOOCNCOt-i rH ->tf CO CO iC O H 10 CO CO Tt< CO CO CO CO i-( o (N ^ CO CO T-H^H(MOO00 CO CO tJH CO 00 O O) ^ O O) lO CO id CO CO CO (N (M t-h i>. OCOCOt^i-HOt^T-H 7 CO CO H ft rH CO CO to CO 8 gN to H (M CO a CM o ^> rH O >o £ O CM 1=3 g .2 .2 S 3 3 1:0 I go O OOOOOO^^HO O O O O O O O • • * O «+-| <+-| %-| O O f-t a a bX) O OOOOOO^ _ O ^ 00 .2 •2 § a a 0) o <~ c3 CD *~ S d 'u o p 00 H IC odd O 00 N O O rH O >— 1 «— 1 O 10 O N d cd Tji N auiuweaiQ Ol OMMN05«00 00N^ CO MMMMIM(NH(NP5d N N aajnueaiQ ogpadg g O i N H Tj< O H H co o o CM rH 3 9 S§ S # * CO N CO CM rH COCOMHMOHNO) OONOONONWOJN lO 00 OJ N CO N OOOOrHr-trHOOO H H O *"CJ ""^^ ^0 F "C3 '"CJ ^5 'o 'o *o 'o 'o 'o 'o *o 'o "o X) T3 ["C5 'o "S '3 > 1 § H CO 73 T3 T3 O O O O *0 "O "O "d O O O O O o o o o o o g ° . 03 H O CO o o "S 2 * =1 03 o o fc 5 d 02 3 o Vl CD o3 £ ^£ o3 d d 03 o 03 'm 03 03 O A • s ® Q & •+= 02 -+3 03 a Is 03 "E 03 03 d ft "d X oo o oooooro ** £ ***** 73 o3 O 14-1 o *♦* o o o d « § *C .2 -S d « £ « g « l! & ^ "S *° 03 ^ 03 03 o 03 a ° a CO N N 8 £ O O CO 00 O CO O O O H i— I CM r-i CO CO »o O H lO CO CO CO CO CO CM CO ogpedg a o o g o i> o OS CM 00 CO CO . CM CD N CM 00 O CM rH CM CO O O OOOOO (MO I>- 83 3 u CO ^ ^ ^ So 2 00 Tt< O O CO d o o . 00* © S3 is § -§ ^ ^ o3 o3 „ 03 03 O bC S bO o3 3 o d -h 03 03 5 Q rd "3d "d n d 03 O 53 03 _ O d g 03 d ^ d M d o o 03 'o3 bfi 03 13 d «~ o o o o So £ £ S d r ® u o a § 03 a 03 5d^ CO "§■3 o3 03 03 03 5h d rd o ^ d O 03 d 03 03 'So So bC to a 03 bC o 03 d d 03 d o 2 8 o ° ^ .3 O » 03 d o rd g 03 OQ CO C3 O 03 73 O 03 ^ 03 h .2 .S d d 03 03 > > "3b 'So -d a O C? o3 o3 -t-=> 0Q 03 03 03 3 3 © .2 -a £ © o 03 DD 03 & 2 r3 © d !n © d ® 03 03 a a o3 o3 bC bO bC -5 >> d © 2 03 03 .s © ^ © O H ax N § (N d Tt* CO O rH CM tH i-i 1-H *C CM CO CO CO N M W ^' N H CO N K CD lO id 00 00 N feW m' h o o o N lO CO H t}( H H H H CO IM tN N * CO rj< rJH CM 3 s N » CO tJ( 00 N CD H | CO CM © CO © iO "H H N CO CO H N W 00 H d 00 00 "cH CO O CO CO i—i i—l i—l i—l O O ■"d ^3 t3 73 'o 'o 'a "5 *8 '5 << -!3 'o '8 "S 'o 'o *o «J 03 r- -1 s; 'C a 03 N O O O r- I rH 1— I r- I rH rH rH r- I N C HNCOiONOONCON £ mnmcomnn'hh p. N N N 1«*<\L g M^cO^iQiOiOcOO b- OS O 5, t-i 1— 1 t-h t-i r-i i-H cq 5 CDCOHHOOiOtOO-* i 3 a 3 g00 00 C0 00 00OiOiT-(T^ O rH ^OOOOOOOi-irH rH rH rH uonoua'jj *r3 *"d ""O r, o *"0 r d r o ""O ""O 'o '3 '3 "3 '3 '3 '3 '3 "3 '3 '0 '3 ^^^^^^^^^ o lO g g 8 3 8 »o o o o 10 03 O CO -P CO o a o T-l TH P f3 1— I 88 O O Q O O (M cM iO iO 88 00 i-hcNCjiOlOOt^ CO CO O 00 CD O "O N t-H H H Ol N CO i-H « 00 S 10 co co ^ §3 >0 r)H CO CN CN CO N 00 >o O CO N GO H CO H 10 (N O h 00 CO O Ol N ^ O O M CO N H N O O) N ffi ffi >0 CO O JO T3 T3 °3 'a '3 '3 ~©~oo lo ©~ «1 0 10 00 10 o g o o >o 00 iO CM CM O to TfH O OiOQOOiOQ 1^ OJ N O ^ N N O CM H (M N (N (N ^ CO o o iO o CO N uo CO CN CM 05 O O O / — — - CM O .2 • • • -IOCM ■ CO (M O O Oi CO N • • ? CD CD CD iO • ■ IO iO COCO HHHHlOlOHHTfTjl^^COMMHH CM •H © i0tDN00©OHNC0Tj(iOC0N00O)OHCH(NC0^ hhhhhiN(NCNN^CNCN(NCNCNCOC03 246 Creatine and Creatinine Metabolism siderable amounts is of particular interest in view of the recently published paper of Frank and Isaac. 1 These investigators found that during phosphorus poisoning, the amount of sugar in the blood practically disappears. If carbohydrates are necessary for normal cellular metabolism, or for the conversion of creatine into creatinine, as has been suggested, then creatine would be expected under such conditions. Lusk ('07) was unable to detect any significant change in creatinine output after phosphorus poisoning, in one experiment on a fasting dog. No creatine estimations were reported. Lefmann ('08) concluded that the creatinine elimination was increased during the poisoning not accompanied by fasting, while the creatine output remained unal- tered. His data, however, are so irregular that definite conclu- sions are impossible. The parallelism between total creatinine and total nitrogen elim- ination is very striking in the phosphorus-intoxicated dog as well as in the experiments previously described. An increase in total creatinine is, without exception, accompanied by an increase in total nitrogen. There is, however, one difference in the results obtained on dogs as contrasted with those obtained on rabbits. In dogs the excretion of creatine is usually accompanied by a decrease in creatinine, though not commensurate with the increase in creatine; while in rabbits the creatinine is comparatively con- stant in amount, or shows only a very slight tendency to decrease during the last days of the fasting period. The behavior of dogs would seem to indicate that creatine and creatinine have the same origin in the organism, and that while the production of creatine increases as starvation progresses, less and less conversion to the anhydride is accomplished. The protocols of Cathcart ('09) show a decrease in creatinine excretion in man, coincident with the increase in creatine, but contrary to the present findings no parallelism exists between the elimination of total creatinine and total nitrogen in his experiments. Moreover, he makes no reference to the possible importance of carbohydrates in bringing about the conversion of creatine to cre- atinine. 1 Frank and Isaac: Arch. f. exp. Path. u. Pharm., lxiv, pp. 274-92, 1911. Lafayette B. Mendel and William C. Rose 247 GENERAL DISCUSSION. Two fundamental facts are emphasized by the experiments recorded: (1) An increase in the elimination of total creatinine (i.e., creatine plus creatinine) is always accompanied by an increase in the output of total nitrogen; and (2) Carbohydrates, in contrast to the other food-stuffs, are capable of preventing the excretion of crea- tine, and are therefore indispensable for normal creatine-creatinine metabolism. Although an increased output of nitrogen in the form of creatine and creatinine is associated with a rise in total nitrogen elimination, it by no means follows that the reverse is true. It is necessary to remember that under ordinary conditions we have in the animal body three sources of urinary nitrogen, namely: food nitrogen, reserve nitrogen, and tissue nitrogen. During fasting the food nitrogen does not enter into the problem. The urinary nitrogen in inanition, therefore, has its origin in (a), reserve nitrogen (variously termed circulating protein, Vorratsei- weiss, Reserveeiweiss, Zelleneinschluss, labiles Eiweiss, by differ- ent writers); and (6) disintegration of organized body tissue — so-called endogenous nitrogenous metabolism. If the former were its source, no accompanying increase in total creatinine should occur, for it is probable that this form of nitrogen is metabolized just as the (exogenous) food nitrogen. When, on the other hand, it becomes necessary for the tissues to disintegrate to furnish energy for the organism, an increase in creatine or creatinine neces- sarily occurs. The most abundant tissue, and one which suffers great loss in weight, namely, muscle tissue, contains considerable creatine. This will be liberated and might be expected to appear in the urine either unaltered or as creatinine. But this is not the only factor involved. Considerable evidence has been accumulated in recent years, indicating that creatine is a product of endogenous metabolism, and that an increased for- mation of creatine occurs when the tissue catabolic processes are accelerated. There is evidence, also, that creatine can in part disappear in the body, and in part be converted into creatinine. Of importance in this connection, is the work of Gottlieb and his collaborators. Gottlieb and Stangassinger ('07, '08), and Stan- gassinger ('08), found that during the autolysis of muscle and other 248 Creatine and Creatinine Metabolism organs, a formation of creatine occurs. The creatine so arising, as well as creatine added to the autolytic mixture is, by the action of an enzyme, partially converted into creatinine. Both creatine and creatinine are by long continued autolysis, destroyed through the action of specific enzymes (creatase and creatinase). The very complex curves representing the amounts of creatine and creatinine in an autolytic mixture depend, therefore, upon the balance between formation, conversion, and destruction of these substances. The results of Gottlieb's experiments have been severely criti- cised by Mellanby ('08), who concluded that when the autolytic experiments were kept rigorously free from bacteria, and when precautions were taken to prevent the conversion of creatine into creatinine by heating, no change occurred during autolysis. The experiments have, however, been repeated with improved technic by Rothmann ('08) and van Hoogenhuyze and Verploegh ('08), and results obtained similar to those of Gottlieb. Whether one accepts Gottlieb's or Mellanby's experiments, the fact remains that in muscle tissue an increased production of crea- tine may actually occur under appropriate conditions. Weber ('07) and Howell and Duke ('08) found that the beating heart liberates creatine into the perfusion fluid. Weber also observed that the creatinine excretion is considerably higher in dogs poi- soned with cinchonine, indicating that increased tonus may lead to creatine or creatinine formation. Likewise Graham-Brown and Cathcart ('08) found that stimulation of isolated frog muscles brought about an increase of from 7 to 13 per cent in the crea- tine content. Van Hoogenhuyze and Verploegh ('08) found the excretion of creatinine to be greater during the day, when the mus- cle wear and tear is increased, than during the night when the mus- cle tonus is reduced. More recently Pekelharing and van Hoogenhuyze ('10a) have demonstrated an increased formation of creatine in the muscle during rigor caloris, rigor mortis, and heightened tonus. These authors believe that during tonus muscle protein is decomposed to furnish energy, and that creatine is a product of this endogenous catabolism. The sources of the energy utilized for the maintenance of tonus and for the performance of ordinary muscular work are, according to this view, entirely different. If this theory is correct, an increased formation of creatine should occur during fasting Lafayette B. Mendel and William C. Rose 249 after the supply of non-nitrogenous energy-yielding food-stuffs and of nitrogenous reserve material has been depleted, and should not occur until such a depletion has been effected. The theory and the facts obtained in the experiments already described are, therefore, entirely in accord. The numerous other observations of creatine excretion in condi- tions associated with wasting of muscle tissue, as in fevers (Shaffer, '08b; and van Hoogenhuyze and Verploegh, '08), during the post partum resolution of the uterus (Shaffer, '08b; and Murlin, '08-'09), and in muscular disease (Levene and Kristeller, '09), all point to the same conclusion, namely, that whenever muscle protein is decomposed, creatine is a product of the disintegration. Further evidence of this will be found in a marked increase of the creatine content of muscle during starvation, in hens and rabbits. 1 At the present time the most probable explanation of the pro- duction of creatine and creatinine may be sought in the catabolism of the tissues (i.e., endogenous metabolism). Under appropriate nutritive conditions, the small amount of creatine arising from muscle wear and tear, is converted into creatinine and excreted. When, however, an undue creatine production occurs, the conver- sion to creatinine may become inadequate, and creatine as such appear in the urine. Possibly some may be oxidized and not appear at all. In this case creatine and creatinine would be anal- ogous to uric acid and represent a balance between formation and destruction. They would then be intermediary rather than end products. These views are represented in the accompanying scheme. Tissue N Food N Reserve N Creatine No Creatine No Creatine (Oxidized) ? Urinary Creatinine Urinary Creatine ir rhe experimental proof of this will be furnished in the next paper. 250 Creatine and Creatinine Metabolism It may be objected that if this theory were true creatine intro- duced per os or parenterally should ordinarily be converted into creatinine, and that this is contrary to the observations of most investigators (Folin, '06, Klercker, '06 and '07, Lefmann, '08). But this does not follow any more than that creatine arising dur- ing starvation should be converted into creatinine. It is possible that the organism is capable of converting only a definite amount of creatine into the anhydride, and that when this amount is exceeded, unaltered creatine appears in the urine. On the other hand, a slight conversion was observed by van Hoogenhuyze and Verploegh ('08). In the most recent paper along this line, Pekel- haring and van Hoogenhuyze ('10b) found that creatine parenter- ally introduced into rabbits and dogs, was partly transformed into creatinine, partly oxidized, and partly excreted unaltered. It must be remembered also that in injection experiments the cir- culation may be so flooded with creatine, that there is not suffi- cient time for conversion before elimination occurs. In many of the earlier experiments, where creatine was given by mouth, it is not improbable that it was largely decomposed by bacteria in the alimentary canal (cf. Czernecki, '05, and Nawiasky, '08). Plimmer, Dick and Lieb ('09-' 10), in experiments in man, found that 2.5 grams of creatine had to be given by mouth before any could be recovered in the urine. This may serve to explain the entire disappearance of creatine in many feeding experiments, and its failure to increase the creatinine output. With our present knowledge, it is impossible to formulate any- thing definite as to the chemical processes by which creatine arises in tissue catabolism. The striking similarity between its structure and the structures of many other substances occurring in muscle tissue, or derived from proteins, indicates that its origin in tissue catabolism is by no means inconceivable. Attempts, however, to associate these compounds with creatine and creatinine experimentally, have thus far been unsuccessful or doubtful (cf. Burian, '05, Jaffe, '06, Achelis, '06, Dorner, '07, and Lefmann, '08). It is difficult to form any chemical picture of the influence car- bohydrates may have in preventing the excretion of creatine. As already suggested, they may be necessary for the conversion of creatine into creatinine, or in their presence creatine may be more readily oxidized and excreted as urea. Again, the tissue cells Lafayette B. Mendel and William C. Rose 251 may not functionate properly when the normal amount of carbo- hydrate food is wanting, and in this case the elimination of crea- tine would be analogous to the production of the acetone bodies, which is also inhibited by the administration of carbohydrates. More work will be necessary to elucidate these problems. With- out question the metabolism of creatine is intimately associated with carbohydrate metabolism. SUMMARY. 1. The excretion of creatine induced by starvation, is inhib- ited in rabbits by feeding a diet of carbohydrates absolutely free from proteins and fats. When the carbohydrates are given in liberal amounts, creatine entirely disappears from the urine. 2. The creatine elimination is not reduced by feeding a diet of fat alone, or by a diet of fat and protein. 3. Experimental interference with carbohydrate metabolism leads to the elimination of creatine. After phlorhizin diabetes which depletes the store of carbohydrates, and during phosphorus poisoning, which disturbs the glycogenic functions, the output of creatine in dogs is decidedly increased. 4. An increase in the output of creatine plus creatinine (total creatinine), is always accompanied by an increase in total nitrogen elimination. This parallelism of total creatinine and total nitro- gen outputs in inanition and with nitrogen-free diets is ascribed to a common source, namely, true tissue or endogenous metabolism. The metabolism of exogenous or reserve proteins is not accom- panied by the production of creatine or creatinine. 5. The intimate relation of creatine excretion (or the failure of conversion into creatinine) to carbohydrate metabolism, is discussed in detail. BIBLIOGRAPHY. Achelis: Zeitschr. f. physiol. Chem., 1, pp. 10-20, 1906. Benedict: Carnegie Inst., Washington, Publication No. 77, pp. 386- 95; 458-9, 1907. Benedict and Diefendorf: Amer. Journ. of Physiol., xviii, pp. 362- 76, 1907. Benedict and Myers: ibid., xviii, pp. 377-96, 1907a. Benedict and Myers: ibid., xviii, pp. 406-12, 1907b. 252 Creatine and Creatinine Metabolism Burian: Zeitschr. f. physiol. Chem., xliii, pp. 545-6, 1905. Cathcart: Journ. of Physiol., xxxv, pp. 500-10, 1907a. Cathcart: Biochem. Zeitschr., vi, pp. 109-48, 1907b. Cathcart: Journ. of Physiol., xxxix, pp. 311-30, 1909. Cathcart and Taylor: ibid, xli, pp. 276-84, 1910. Closson: Amer. Journ. of Physiol., xvi, pp. 252-67, 1906. Czernecki: Zeitschr. f. physiol. Chem., xliv, pp. 294-308, 1905. Dorner: ibid., lii, pp. 225-78, 1907. Dreibholz: Inaug. Dissertation, Greifswald, 1908. Folin: Amer. Journ. of Physiol., xiii, pp. 45-65, 1905a. Folin: ibid., xiii, pp. 66-115, 1905b. Folin: ibid., xiii, pp. 117-38, 1905c. Folin: Hammarsten's Festschr., pp. 1-20, 1906. Forschbach: Arch. f. exp. Path. u. Pharm., lviii, pp. 113^10, 1908. Foster: Proc. Soc. Exp. Biol, and Med., viii, pp. 30-32, 1910. Foster and Fisher: Journ. Biol. Chem., ix, pp. 359-62, 1911. Gottlieb and Stangassinger: Zeitschr. f. physiol. Chem., lii, pp. 1- 41, 1907. Gottlieb and Stangassinger: ibid., lv, pp. 322-37, 1908. Graham-Brown and Cathcart: Journ. of Physiol., xxxvii, p. xiv, 1908. van Hoogenhuyze and ten Doeschate : Annal. d. gynecol. et d'obstet. Jan. et Fev., 1911. van Hoogenhuyze and Verploegh: Zeitschr. f. physiol. Chem., xlvi, pp. 415-71, 1905. van Hoogenhuyze and Verploegh: ibid., lvii, pp. 161 266, 1908. Howe and Hawk: Journ. Amer. Chem. Soc, xxxiii, pp. 215-54, 1911. Howell and Duke: Amer. Journ. of Physiol., xxiii, pp. 174-79, 1908. Howland and Richards: Journ. of Exp. Med., xi, pp. 344-72, 1909. Jaffe: Zei'schr. f. physiol. Chem., xlviii, pp. 430-68, 1906. Klercker: Hofmeister's Beitrdge, viii, pp. 59-61, 1906. Klercker: Biochem. Zeitschr., iii, pp. 45-87, 1907. Krause: Quart. Journ. of Exp. Physiol., iii, pp. 289-96, 1910. Krause and Cramer: Journ. of Physiol., xl, p. lxi, 1910. Leathes: ibid., xxxv, pp. 205-14, 1906-07. Lefmann: Zeitschr. f. physiol. Chem., lvii, pp. 476-514, 1908. Levene and Kristeller: Amer. Journ. of Physiol., xxiv, pp. 45-65, 1909. Lindsay: Biochem. Journ., v, pp. 407-26, 1911. London and Boljarski: Zeitschr. f. physiol. Chem., lxii, pp. 465-67, 1909. Lusk: Amer. Journ. of Physiol., xix, pp. 461-67, 1907. Meissner: Zeitschr. f. rat. Med., xxxi, p. 234, 1868. Mellanby: Journ. of Physiol., xxxvi, p. xxiii, 1907. Mellanby: ibid, xxxvi, pp. 447-87, 1908. Mendel: Science, xxix, pp. 584-91, 1909. Mendel and Hilditch: Amer. Journ. of Physiol., xxvii, pp. 1-23, 1910. Lafayette B. Mendel and William C. Rose 253 Murlin: Amer. Journ. of Physiol., xxiii, p. xxxi, 1908 09. Myers: Amer. Journ. Med. Sci., cxxxix, pp. 256-64, 1910. Nawiasky: Arch. f. Hyg., lxvi, pp. 209-43, 1908. Osterberq and Wolf: Journ. of Biol. Chem., iv, p. xxiii, 1908. Paton: Journ. Physiol., xxxix, pp. 485-504, 1909-10. Pekelharing and van Hoogenhuyze: Zeitschr. f. physiol. Chem., lxiv, pp. 262-93, 1910a. Pekelharing and van Hoogenhuyze: ibid., lxix, pp. 39.5-407, 1910b. Plimmer, Dick, and Lieb: Journ. of Physiol., xxxix, pp. 98-117, 1909- 10. Richards and Wallace: Journ. Biol. Chem., iv, pp. 179-95, 1908. Rothmann: Zeitschr. f. physiol. Chem., lvii, pp. 131-42, 1908. Salaskin and Zaleski: ibid., xxix, p. 545, 1900. Shaffer: Amer. Journ. of Physiol., xxii, pp. 445-55, 1908a. Shaffer: ibid., xxiii, pp. 1-22, 1908b. Spriggs: Quart. Journ. Med., i, pp. 63-87, 1907a. Spriggs: Biochem. Journ., ii, pp. 206-16, 1907b. Stangassinger: Zeitschr. f. physiol. Chem., Iv, pp. 295-321, 1908. Taylor: Brit. Med. Journ., p. 1343, (October 29), 1910. Taylor: Biochem. Journ., v, pp. 362-77, 1911. Underhill and Kleiner: Journ. Biol. Chem., iv, pp. 165-78, 1908. Underhill and Rand: Arch. Intern, Med., v, pp. 61-91, 1910. Voegtlin and Towles: Journ. Biol. Chem., ix, p. xi, 1911. Weber: Arch. f. exp. Path. u. Pharm., lviii, pp. 93-112, 1907. Reprinted from The Journal of Bioloqical Chemistry, Vor,. X, No. 3, 1911 EXPERIMENTAL STUDIES ON CREATINE AND CREATININE. n. INANITION AND THE CREATINE CONTENT OF MUSCLE.* By LAFAYETTE B. MENDEL and WILLIAM C. ROSE. (From the Sheffield Laboratory of Physiological Chemistry, Yale University, New Haven, Connecticut.) (Received for publication, August 14, 1911.) Comparatively few investigations have been made in regard to the creatine content of muscle in normal and abnormal condi- tions. Such data as we have were, for the most part, obtained with the older and inadequate analytical method of Neubauer and are consequently usually unreliable. Many of these researches were undertaken with a view of determining the influence of muscular work on the content of creatine, but because of the errors in technic the results have been widely different. Liebig ('47) was the first to begin systematic studies on the effect of activity on tissue composition. In 1847 he made his classi- cal investigation of the creatine content of muscle in fatigue and found that the muscle of a fox killed in the chase contained ten times the amount of creatine present in an equal weight of muscle from a resting animal. Sarokow ('63) also found an in- crease of creatine after work. He believed that coincident with the formation of creatine, the muscular activity brought about a partial conversion into creatinine. A similar increase in creatine was reported by Sczelkow ('66) as the result of tetanus, while rest was supposed to produce a decrease. On the other hand Naw- rocki ('66) in experiments on dogs, found no difference in the crea- tine content of tetanized and resting muscle. The results of Voit ('68) were again different. In experiments on frogs, he decided that tetanus produced a decrease in the 1 The experimental data are taken from the thesis presented by William C. Rose for the degree of Doctor of Philosophy, Yale University, 1911. 255 256 Creatine and Creatinine Metabolism creatine content of muscle. According to this investigator the creatine is transformed during work to a substance not identical with creatinine. The most consistent of the earlier results on the effect of work were obtained by Monari ('87) who found that an increase in creatine invariably occurred in fatigue, and that coincident with the increase a much larger amount of creatinine was detectable. The protocols of Monari appear very conclusive, and were accepted and frequently quoted in subsequent papers. It seems prob- able now from the investigations of Mellanby ('07- 08), that much of the precipitate which Monari weighed as pure creatine, was composed of other substances. The creatinine obtained by him was undoubtedly derived from creatine in the evaporation of the acid tissue extracts. That preformed creatinine does not exist in freshly killed muscle is definitely proven by the researches of Grindley and Woods ('06-'07), Mellanby ('07-'08), and Paton C09-'10). Since the introduction of the Folin colorimetric method for the estimation of creatinine, several papers have appeared on the influ- ence of activity on muscle creatine. Weber ('07) found that work caused a slight decrease in the creatine content, and that the beating heart gave off creatine or creatinine into the perfusion fluid. According to this author, less creatine was present in degen- erated than in normal muscle. Mellanby ('07-'08), on the other hand, found that the performance of muscular work, as well as the survival of isolated muscle, leaves creatine unaffected. No change in the creatine content was found by von Fiirth and Schwarz ('11) after tetanizing the hind-leg muscles of a dog for more than an hour. Graham-Brown and Cathcart ('08) report that stimulation pro- duces an increase in the amount of total creatinine in isolated frog muscles, whereas it leads to a slight decrease when the circu- lation is left intact. In a later communication, these authors (Graham-Brown and Cathcart, '09) report the results of studies made on rabbits, in which they find that with the circulation in- tact, stimulation induces a constant though small decrease in the amount of total creatinine (i.e., creatine plus creatinine). Practically no data have been published on the effect of factors other than activity on the creatine content of muscles. By the Lafayette B. Mendel and William C. Rose 257 use of the Neubauer method Demant (79) observed an increase in the percentage of creatine in the breast muscles of pigeons dur- ing starvation. All figures given by this author are much lower than those obtained by the use of the Folin method. From the analysis of the muscle of a single starving rabbit Dorner ('07, p. 261), using the Folin method, concluded that there was a decrease in creatine during inanition. Recently, in a preliminary report, Howe and Hawk ('11) claim that a very marked reduction in the creatine of dog's muscles is brought about by fasting. Such studies are of prime importance in determining the origin of urinary creatine and creatinine. If it is universally true that a decrease in the creatine content of muscle occurs during inani- tion, the creatine found in the urine during starvation must have its origin in a " washing-out" of muscle creatine. If, on the other hand, the accelerated endogenous metabolism during star- vation occasions an increased formation of creatine, as has been suggested in a previous paper (cf. Mendel and Rose '11), then urinary creatine and creatinine would represent local metabolic end-products, rather than substances merely washed out of the tissues. The excess of creatine produced during such a process might be entirely excreted or oxidized, or partly retained in the muscle. The muscle analyses would then show either no change or an increase in the creatine content, but never a decrease. To determine this question a series of analyses was made of muscle tissue removed from normal and starving rabbits and hens, the results of which are summarized in Tables II and III. The animals were allowed to starve for varying lengths of time and killed by bleeding. To avoid errors which might arise from differ- ences in the amount of creatine in different muscles, similar mus- cles were always selected in the control and experimental animals. In the rabbits, the muscle tissue from the hind legs and back was completely removed, freed as much as possible from connective tissue, and thoroughly ground in a hashing machine. From the uniform mixture samples were rapidly weighed for the analyses. In the fowl, only the pectoral muscles were used. The total creatinine was estimated according to the procedure of Mellanby. 1 l Mellanby: Journ. of Physiol., xxvi, pp. 453-4, 1907-8. ft, 258 Creatine and Creatinine Metabolism For this purpose, the finely ground muscle was killed by covering with 95 per cent alcohol. The alcohol was poured off through silk gauze, the muscle pressed out, and repeatedly extracted by shaking with five portions of water, about half an hour being allowed for the extraction with each por- tion. The alcoholic and watery extracts were then combined and evapor- ated to dryness on the water-bath. The residue was extracted five times with 75 per cent alcohol, which removed the creatine and creatinine, but left most of the protein behind. The alcohol was removed by evaporation, the solution made up to a known volume with water — usually 15G cc. — fil- tered, and the total creatinine determined on 10 cc. portions by the Folin- Benedict-Myers method. Preliminary analyses showed that very good duplicates could be obtained by this procedure. In the rabbits, simultaneous determinations were made of the water, ether-extract, and ash of the muscles. Since only slight variations in the percentages of ether-extract and ash occurred, these estimations were omitted in the fowl. In the rabbits the creatine content is calculated on the moist muscle, and on the ether-extract- and fat-free dry material; while in the fowl the per- centages are calculated on the bases of moist and dry tissue. The amount of water in the normal tissues is reasonably con- stant. The higher figure obtained with rabbit 4 is due to the fact that the animal had received an intravenous injection of 150 cc. of dilute adrenalin solution, in connection with other investigations. Only a small amount of the injected water had been excreted when death occurred. In the starving animals, the water content progressively increased as starvation was pro- longed. Similar results were obtained by v. Boethlingk 1 on mice. This increase in water may have caused the apparent decrease in the amount of creatine observed by Dorner, and calculated by him on the moist material. The ether-extract is also constant in the normal animals, and shows a decrease during fasting. The figures agree with those found in starving rabbits by Rubner. 2 This author reports that in starvation the muscles of rabbits contain 2 to 3 per cent of fat calculated on the dry material. Assuming that the muscles of his animals contained approximately 80 per cent of water, his figures agree with those reported in Table II. The percentage of fat in the well-fed rabbits is somewhat lower than that reported *v. Boethlingk: Arch. d. scienc. biol., v, p. 395, 1897. 2 Rubner: Zeitschr. f. Biol., xvii, p. 229, 1881. Lafayette B. Mendel and William C. Rose 259 for the wild hare by Konig and Farwick, 1 who found 1.07 per cent of ether-extract in the muscles of the extremities. Moreover, the fat content of other animals is usually higher than that of the rabbit. According to the analyses of Almen, 2 the muscles of lean oxen contain 1.5 per cent of fat and 76.7 per cent of water, while the muscles of pigeons contain 1.0 per cent of fat. 3 This is in accord with the well known fact that rabbits usually have relatively little subcutaneous adipose tissue. They seem to store fat only with difficulty. table 1. The creatine content of muscle. AUTHOR REFERENCE ANIMAL CONDITION OP MUSCLE CREATINE IN MOIS1 TISSUE Mellanby Journ. Physiol., xxxvi, p. per cent 472, 1907-8 Frog Normal 0.302 Graham- Brown and » Biochem. Journ., iv, p. Frog Normal 0.377 Cathcart A 01 IOAO Frog Isolated and 0.413 stim. Mellanby Journ. Physiol, xxxvi, p. 472, 1907-8 Fowl Normal 0.360 Mellanby Journ. Physiol., xxxvi, p. 460, 1907-8 Rabbit Normal (leg) 0.520 Mellanby Journ. Physiol., xxxiv, p. 460, 1907-8 Rabbit Normal (back) 0.505 Mellanby Journ. Physiol., xxxiv, p. 460, 1907-8 Rabbit Stimulated 0.506 Dorner Zeitschr. f. physiol. Ch., lii, p. 265, 1907 Rabbit Normal 0.529 Dorner Zeitschr. f. physiol. Ch., lii, p. 265, 1907 Rabbit Normal 0.496 Dorner Zeitschr. f. physiol. Ch., lii, p. 265, 1907 Rabbit Normal 0.496 Dorner Zeitschr. f. physiol. Ch., lii, p. 265, 1907 Rabbit Normal 0.505 Dorner Zeitschr. f. physiol. Ch., iii, p. 265, 1907 Rabbit Starving 0.414 1 Konig and Farwick: Zeilsch'. f. Biol., xii, p. 497, 1876. 2 Alm6n: Nova Act. Reg. Soc. Scient. Upsal., vol. extr. ord., 1877. 3 Cf. Konig: Chem. d. menschl. Nahrungs- u. Genussmittel, i, p. 42, 1903 Creatine and Creatinine Metabolism K ss Kfl g O 2 OhimioioooOhCOOOIN^O d d MMMOOWOOOiOOcDN ooooooooooo C©C0OC0^t-i00I>-00tHC0O»O --f h (N M (M QOO<£> o B gg « d si REMARKS SAMPLE CHILD React! litm — . d o— < d Tota fcl Pref( Crea crea Crea per tlni years mgms. mgms. mgms. 20 n 1.010 Acid ID 14. o 19 1^ . Perfectly nor- mal child. 21 ii 1.005 Acid 17 1 4 7 10 Oo . o Perfectly nor- mal child. 22 3 1.030 Neu- tral DO 38 uo 1 ^ 9R 3 Perfectly nor- mal child. 23 3 1.018 Acid 68 40 28 41.2 Perfectly nor- mal child. 24 5 1.030 Alka- line 72 60 12 16.7 Mumps. 25 7 1.019 Acid 61 27 34 55.7 Perfectly nor- mal child. -26 7 1.020 Acid 66 50 16 24.2 Sample from same child as No. 25. 27 7 1.024 AlL-o AiKa- line fiO oo 7 11 7 Perfectly nor- mal child. 28 8 1.021 Acid 4.1 7 17 1 1/ . 1 Perfectly nor- mal child. 29 8 1.030 Acid on yu Oo Q9 OO . D Sample from same child as No. 28. 30 11 1.913 Acid 64 52 12 18.8 Perfectly nor- mal child. 31 11 1.020 Acid 7Q /y oy in 1U 19 7 Perfectly nor- mal child. 32 12 1.020 Acid 91 62 29 31.9 Minute trace of protein. 33 13 1.025 Acid 91 91 Perfectly nor- mal child. 34 13 1.016 Acid 60 49 11 18.3 Perfectly nor- m q 1 pVnlH 35 13 1.022 Acid 117 86 31 26.5 Perfectly nor- mal child. 36 15 1.023 Acid 83 61 22 26.5 Trace of pro- tein present. 37 15 1.016 Acid 72 60 12 16.7 Same as 36. Protein present. 38 20 1.014 Acid 69 69 Normal. 39 21 1.016 Acid 81 81 Normal. William C. Rose 269 The urines were preserved with toluene, and analyzed within twenty-four hours after excretion. Except in the cases indicated (Nos. 9, 32, 36 and 37), protein and sugar were entirely absent. Contrary to the findings of Schwarz, children of five years and over excrete considerable creatine. Indeed, with the exception of two cases, creatine was present in all specimens from children under fifteen years of age. A boy of ten and a girl of thirteen failed to have creatine in their urines. No progressive decrease in the percentage of the total creatinine in the form of creatine coincident with increase in age is apparent; nor is the percentage of creatine constant for the same individual. For instance, one specimen (No. 3) obtained from a child of five years contained 24.2 per cent of the total creatinine in the form of creatine, while a second sample (No. 4) from the same child a few days later, contained 79.2 per cent of the total creatinine as creatine. It was impossible to obtain information as to the amount and kind of food eaten by the children. Most of the specimens were obtained from the city orphans home or from private families, and the subjects of the experiments were probably ingesting more or less meat. It is possible, therefore, that the oxidation or con- version of creatine into creatinine may be difficult for young indi- viduals to accomplish, and in this case the creatine of the urine may, in part, represent ingested creatine; or the glycogenic func- tions may be imperfectly developed, and the store of carbohy- drates be insufficient to exert its regulatory influence over me- tabolism during childhood. Frank 1 has shown that the percentage of sugar in the blood of infants is greater than that of adults. It is conceivable that the demand for carbohydrates for the histo- genetic processes may be so great that the cells are left in partial carbohydrate hunger, and are unable to perform the "endo-cat- abolic ,, activities as perfectly as in later life. At any rate it is of some interest statistically to find that creatine is usually pres- ent in the urine until or after the age of puberty. 1 Frank: Zeitschr. f. physiol. Chem., lxx, pp. 129-42, 1910. 270 Creatine and Creatinine Metabolism BIBLIOGRAPHY. Amberg and Morrill: Journ. Biol. Chem., iii, pp. 311-20, 1907. Amberg and Rowntree: Johns Hopkins Hosp. Bull., xxi, pp. 40-44, 1910. Closson: Amer. Journ. of Physiol., xvi, pp. 252-67, 1906. Funaro: Biochem. Zeitschr., x, pp. 467-71, 1908. van Hoogenhuyze and ten Doeschate : Annal. d. gynecol. et d'obstet., Jan. et Fev., 1911. van Hoogenhuyze and Verploegh: Zeitschr. f. physiol. Chem., xlvi, pp. 415-71, 1905. Mellanby: Journ. of Physiol., xxxvi, pp. 447-87, 1908. Mendel and Leavenworth: Amer. Journ. of Physiol., xxi, pp. 99-104, 1908. Rietschel: Jahrb. f. Kinderheilkunde, lxi, p. 615, 1905. Schwarz: Ibid., lxxii, pp. 549-74; 712-35, 1910. Sedgwick: Journ. Amer. Med. Assoc., lv, pp. 1178-80, 1910. Reprinted from The Journal of Biological Chemistry, Vol. X. No. 4, 1911 STUDIES IN NUTRITION. I. THE UTILIZATION OF THE PROTEINS OF WHEAT. By LAFAYETTE B. MENDEL and MORRIS S. FINE. (From the Sheffield Laboratory of Physiological Chemistry. Yale University, New Haven, Connecticut.) (Received for publication, September 2, 1911.) CONTENTS. Introduction 303 Factors involved 305 Earlier studies with bread 306 Earlier studies with isolated proteins 307 Methods 308 Experimental part 310 Products employed 310 Metabolism experiments 311 "Glidin" 311 Gluten 313 Glutenin 317 Gliadin 321 Nitrogen balances 324 Summary 324 INTRODUCTION. For many years unlike values in nutrition have been ascribed to the proteins of animal and vegetable origin. Now that the chemical individuality and physiological specificity of the so- called proximate principles are asserting their importance, 1 fur- ther study of the availability of the foodstuffs seems especially 1 Reference may be made to the recent researches on the amino-acid requirements of the animal organism by Abderhalden, Henriques, Michaud, (see bibliography), and others. Cf. also the studies of Hunt (Hygienic Laboratory, Public Health and Marine Hospital Service of the United States, Bull. 69, 1910), which indicate that the resistance of animals to certain poisons may Vary with the character of their diet. 303 304 Utilization of Wheat Proteins desirable. The opinion is freely expressed that the animal pro- teins are far better utilized than those of plant origin, and the following statistics compiled by Atwater and Bryant 1 may be quoted on this point: CHARACTER OF DIET Animal foods. . . Cereals Legumes, dried. Vegetables Fruits Vegetable foods Total food PROTEIN UTILIZED per cent 97 85 78 83 85 84 92 It can scarcely be said that we are yet in a position to explain adequately the poorer utilization of the nitrogen components of certain vegetable foods. Voit 2 and his followers 3 were early aware that structural peculiarities of plant products, such as cellulose walls, etc., render the proteins comparatively inaccessible to the digestive juices, thus in part explaining the possibility of poorer utilization. The question of the relative availability and nutri- tive value of the vegetable proteins per se has received little atten- tion, owing in large part to the technical difficulties in securing suitable isolated products for study. We have undertaken a detailed investigation of some of the factors involved in the digestibility and utilization of the proteins of vegetable food materials. An attempt has been made to elim- inate many of the unfavorable conditions or factors which attend the use of these plant products, and above all to study the nutri- tive value of their proteins as such. Incidentally it has become necessary to devote some attention to various features of the ali- mentary functions, such as the origin of the feces, which have an important bearing on the interpretation of experimental results. 4 1 Atwater and Bryant : Report of the Storrs Agricultural Experiment Station, 1899, p. 86. 2 Voit: Sitzungsberichte der Bayerischen Akademie, ii (4), 1869. s Cf. Rubner: Zeitschrift fur Biologie, xix, p. 45, 1883. 4 The data in this and succeeding papers of this series are taken from the dissertation of M. S. Fine, Yale University, 1911. Lafayette B. Mendel and Morris S. Fine 305 FACTORS INVOLVED. The low nitrogen content of most vegetable foods necessitates the ingestion of a relatively large volume. This generally in- creased bulk of vegetable food may of itself lead to more rapid evac- uation and lessen the possibilities of digestion and absorption. Again, in comparison with products of animal origin the vege- table foods may present an unfavorable texture. In older plants the cell walls may be quite tough and even supplemented with lignin. There is evidence that cellulose is not digested to any considerable extent by the higher animals, 1 and the vegetable membranes are not always easily permeable to the digestive juices. 2 It is of primary importance for digestion that the plant cells should be thoroughly disintegrated. That ordinary cooking is not suffi- ciently rigorous treatment to bring about complete rupture, is brought out in a subsequent paper of this series. Observations by Wicke indicate that the nitrogen utilization of diets containing much bread becomes more and more unfavorable with increase in the cellulose content. Thus, with an almost constant nitrogen intake, the nitrogen utilization was 79, 75, 63, 69, 53 per cent respectively with a cellulose concentration in the food of 0.2, 0.3, 1.1, 1.3, 1.6 per cent. This condition is probably due in great part to the incomplete rupture of the cells. Whether the cellu- lose per se exerts an unfavorable influence, that is when it cannot be accused of rendering the nutrients inaccessible to the digestive agents, is a question which has received comparatively little atten- tion. The matter will be discussed fully in a later paper. Mechanical factors may influence the rate of passage through the alimentary canal. This applies to coarse particles such as are derived from seed coats in bran. Wheat bran and similar products contain phytin to which a laxative action has been attributed in the case of cattle. 3 The fermentative development of acid and gas with the consequent stimulation of peristalsis has likewise 1 Cf . Scheimert and Lotsch: Biochemische Zeitschrift, xx, p. 10, 1909. 2 Cf. Rubner, loc. cit. 3 Cf. Jordan, Hart, and Patten: American Journal of Physiology, xvi, p. 268, 1906; Hart, McCollum and Humphrey: Wisconsin Agricultural Experiment Station, Research Bull. No. 5, 1909; also Mendel and Underhill: American Journal of Physiology, xvii, p. 75, 1906. 306 Utilization of Wheat Proteins been pointed out in connection with utilization. For example, Menicanti and Prausnitz noted that poor utilization of bread nitro- gen is accompanied by high acidity of the feces. In addition to the preceding considerations we must ask our- selves whether the vegetable proteins by themselves exhibit any inherent resistance to the digestive enzymes of man. 1 To this ques- tion we have devoted special attention. EARLIEK STUDIES WITH BREAD. The actual nutritive value of bread was early investigated by Bischoff and Voit on dogs. They found the nitrogenous constit- uents of bread to be 80 to 84 per cent available. E. Bischoff reported a more extended study, in which the nitrogen of bread was shown to be 82 to 85 per cent utilized. When the nitrogen and starch of bread were replaced by the nitrogen of meat and pure starch, the utilization was 92 per cent, which result would lead one to believe that the low digestibility of the bread nitrogen might be attributed to the unfavorable texture. Meyer also found that the texture plays an important role in the utilization. "Semmel" — white bread made of the finest flour — was 80 per cent available, while " Pumpernickel' 1 had a digestibility of but 58 per cent. Rubner obtained results essen- tially the same as those reported by Meyer. Wicke found decorticated wheat bread to be more thoroughly utilized than undecorticated, thus being in accord with Rubner's experiments, in which it was shown that the nutritive value of bread becomes lower as the bran content increases. From the studies of Menicanti and Prausnitz it appears that the nitrogen of rye bread is less digestible (70 per cent) than that of wheat (87 per cent), while bread made of equal parts rye and wheat had a nutritive value between the two (80 to 82 per cent). From the data 2 thus briefly cited, it is apparent that the nitrog- enous constitutents of products made of decorticated finely ground 1 Cf . Moore, in Schdfer's Textbook of Physiology, p. 441, 1898, and Hammar- sten: Lehrbuch der physiologischen Chemie, 1909. 2 For other experiments, in which bread formed a larger or smaller part of the diet, see the digest by Atwater and Langworthy: Office of Exper- iment Stations, Bull. 45, 1897. Lafayette B. Mendel and Morris S. Fine 307 wheat are the most thoroughly digested, 1 although evidently not as completely as meat, whereas the coarse breads, made of undecorticated flours, are very poorly utilized. Between these there are all gradations, depending upon the texture. It is wor- thy of note that under apparently the same conditions of texture, etc., rye bread is less well utilized than wheat bread. In the studies above reviewed the nitrogen of bread has in no case been shown to be as available to the organism as that of meat. However, it is difficult to deduce satisfactory conclusions from these experiments as there has usually been some complicating influence — bran, cellulose, acidity, etc. In the properly conducted exper- iment one would employ the pure protein, free from bran, starch, and cellulose, or the latter at least thoroughly disintegrated. The starch can very easily be washed out of flour, and thus a fairly pure protein preparation obtained. The resulting material — gluten — is a common commercial article. A similar product — "aleuronat" — is slightly changed gluten. EARLIER STUDIES WITH ISOLATED PROTEINS. Rubner found the utilization of macaroni noodles with and with- out gluten to be 89 and 83 per cent respectively. However, the nitrogen intake in the former diet was twice as great as that in the latter, and there is thus the possibility that with equal nitro- gen intakes the coefficients of digestibility would have been more nearly alike. In an experiment on a man Constantinidi found gluten to be 94 per cent digested, and the utilization in two exper- iments on a dog was 97 per cent. Potthast showed this material to be 92 per cent available, and Lusk obtained the somewhat less favorable result of 87 per cent. Kornauth found the utiliza- tion of gluten to be 91 per cent against 78 to 82 per cent for dried meat protein. The thorough digestibility of " aleuronat" has been demonstrated by many workers, notably Bornstein, Laves, Wintgen, and Sal- kowski. In recent years gliadin, among other proteins, has been the object of study, with particular reference to its ability to main- 1 Cf. Woods and Merrill: Office of Experiment Stations, Bull. 85, 1900. These authors give the utilization of the protein of white bread as 86 per cent, being the average of thirteen experiments. 3 o8 Utilization of Wheat Proteins tain nitrogen equilibrium. Incidentally we may glean some data bearing upon the subject of protein utilization. Abderhalden found gliadin to be 94 to 98 per cent digested, this result being even better than the utilization of 90 per cent for horse meat. Michaud obtained coefficients of digestibility for "glidin" of 86 to 96 per cent. Buslik and Goldhaber also worked with " glidin" and found its utilization to be as good or better than that of meat nitrogen. METHODS. The ideal method for the elucidation of the question as to the relative degree of digestibility of animal and vegetable proteins would seem to be the feeding of such mixtures of the pure foodstuffs protein, sugar and fat — free from starch and cellulose. 1 By this procedure one would avoid the complicating factors of excessive volume, characteristic of plant food, and the inaccessibility of the food materials due to the inclusion of these substances within the impenetrable cells. In some instances this ideal has been strictly followed; in others the cellulose was not removed, but the plant cells were thoroughly broken by heating or grinding to an impalpable powder. In general, for the present experiments, periods of meat feeding were interposed between the experimental periods. The animals were thus kept in good condition; any disturbing influence of one diet would probably be overcome before the feeding of the next food under investigation; and finally all experimental foods were adequately controlled by the thoroughly digested meat diets. The fat content 2 of the meat was not determined; hence it cannot be stated to exactly what extent the calorific intakes in the differ- ent periods were comparable. One or more of the proteins of wheat supplied all the nitrogen of the diet. The nitrogen intakes were practically the same over long periods of time; when for any reason the nitrogen intake was changed, a preliminary period of two to three days always preceded the period of actual observa- tion on the new nitrogen level, thus giving an opportunity for readjustment. 1 For criticism of this viewpoint cf. Bryant and Milner : American Journal of Physiology, x, p. 84, 1903. 2 Arbitrarily assumed to be 10 per cent. Lafayette B. Mendel and Morris S. Fine 309 The feces accruing from the various diets were identified by giving a capsule of lampblack or carmine 1 with the first meal of each period. Unless dry when collected, the feces were preserved in acidified alcohol until all the feces of the period had been assem- bled, whereupon they were dried on the water bath and finally ground and analyzed. 2 Current discussion 3 would seem to indicate that this process of drying on the water bath occasions a loss of nitrogen. The error incident to this procedure, however, did not appear to us to warrant serious attention, at least not until certain details of metabolism operations, such, e. g., as the accurate division of feces belonging to successive periods, reach a higher stage of perfection. The feces have only occasionally been diarrhoeal and the ani- mals have never been observed to dispose of their excrement. In many instances agar agar or bone ash or both of these indigestible materials were added to the diet. This served to offset the diffi- culty in obtaining satisfactory separations of successive periods due to infrequent defecations. On the other hand the objection may very properly be made that the addition of these materials unfavorably influences the protein utilization. Indeed, the experi- ments of Lothrop, 4 and also some of our own studies, amply confirm the validity of these objections. Lothrop's work shows, for exam- ple, that the addition to a meat diet of 1 gram of bone ash per kilo of body weight may almost double the nitrogen loss through the feces. However, it seems reasonable to suppose that the utilization of all diets would be similarly influenced; and thus the results for various periods would still be comparable. The actual specific influence of these indigestible materials will be discussed in a subsequent paper. 1 Each capsule contained an average of 0.35 gram carmine =0.02 gram nitrogen— a negligible quantity. 2 The weights of the feces and the percentage nitrogen composition are based upon the air-dried specimens. The comparisons of these values which will be frequently made throughout this work are, nevertheless, considered permissible since variations due to this cause would undoubt- edly be smaller than incidental variations from other causes. 3 Cf. Howe, Rutherford, and Hawk: Journal of the American Chemi- cal Society, xxxii, p. 1683, 1910. For the literature see Emmet and Grindley : ibid., xxxi, p. 569, 1909. 4 Lothrop: American Journal of Physiology, xxiv, p. 297, 1909. Utilization of Wheat Proteins EXPERIMENTAL PART. Products Employed. I. "Glidin" 1 a commercial preparation. This material is a slightly yellowish and tasteless white powder, which, according to Bergell, 2 and Thiemer 3 is prepared from wheat flour by a process of washing and centrifuging. II. Gluten, 4 a commercial preparation manufactured by the Kel- logg Food Company, of Battle Creek, Mich. It consisted of thin, flat, yellow scales, approximately yq inch in diameter. III. Glutenin. 5 The specimen used in these trials was prepared as follows: Wheat flour was washed thoroughly with water to remove the starch. The resulting gluten was then extracted four times with 70 per cent alcohol, i. e., until the extracts were color- less, and practically all gliadin was removed. The crude glutenin thus obtained was dried, finely ground, extracted once with ether, and finally ground to an impalpable powder. It was not dissolved in alkali and reprecipitated with acid, and hence still contained practically all the cellulose of the original wheat flour. On the other hand, the possibility of the protein being changed by solu- tion in alkali was avoided. IV. Gliadin. 6 The 70 per cent alcoholic extract obtained in preparing glutenin, as described above, was concentrated to a thick syrup, and precipitated by pouring into water containing a little salt. This glue-like material was dissolved in alcohol, whose final strength was 70 per cent, and this time precipitated by pouring into 95 per cent alcohol. The material thus obtained was dried with alcohol and ether and ground to an impalpable powder. Dr. T. B. Osborne very kindly supplied us with both glutenin and gliadin in sufficient quantities. 1 Obtained from Menley and James, New York City. The material contained 14.5 per cent nitrogen, and did not give a starch reaction. Con- siderable of this preparation dissolved in warm 70 per cent alcohol, repre- cipitating on pouring into cold water — a characteristic behavior of gliadin. 2 Bergell: Medizinische Klinik, No. 41, p. 1042, 1905. 3 Thiemer: Wiener medizinische Presse, No. 47, p. 2431, 1906. 4 This material was very kindly furnished by Dr. Kellogg. It contained 14 per cent nitrogen. 5 For description of glutenin and gliadin, see T. B. Osborne: "Die Pflanzenproteine," Ergebnisse der Physiologie, x, p. 47, 1910. 6 Cf. preceding footno'e. Lafayette B. Mendel and Morris S. Fine 311 Metabolism Experiments. "Glidin" — Tables 1 to 3. Detailed information on the nature of the food ingredients may be obtained from the tables. In the periods of meat feeding, the mixture of cane sugar, lard, agar and bone ash was heated on the water bath, till the lard had melted, TABLE 1. 'Glidin" with Agar and Bone Ash. SUBJECT, DOG 5 Weight at beginning, 5.4 Kg. Weight at end, 5.2 Kg. PERIOD III (4 days) Meat Feeding PERIOD IV (4 days) "Glidin" Feeding PERIOD v (5 days) "Glidin" Feeding (4 days) Meat Feeding Composition of daily diet Nitrogen output. Urine nitrogen, gm.... Total nitrogen, gm Nitrogen in food, gm.. Nitrogen balance, gm. Feces. Weight air dry, gm. . . . Nitrogen, gm Nitrogen, per cent Nitrogen utilization, per cent grams Meat 150 Sugar 20 Lard 20 Agar 3 Bone Ash 7 Water 100 Estimated calories 520 grams "Glidin" 34 Sugar 20 Lard 30 Agar 3 Bone Ash 7 Water 200 Estimated calories 470 grams "Glidin" 34 Sugar 20 Lard 30 Agar 3 Bone Ash 7 Water 200 Estimated calories 470 grama Meat 150 Sugar 20 Lard 20 Agar 3 Bone Ash 7 Water 100 Estimated calories 520 Daily Averages Daily Averages Daily Averages Daily Averages 3.80 4.16 4.90 +0.74 14.5 0.36 2.48 92.7 4.63 4.95 4.93 -0.02 15.5 0.32 2.03 93.6 4.85 5.20 4.93 -0.27 15.4 0.35 2.25 93.0 3.71 4.11 4.93 +0.82 15.5 0.40 2.60 91.8 whereupon the meat 1 and water were added, 2 the whole being thoroughly mixed. In the "glidin" periods, the food mixture was warmed on the water bath the day before feeding, the water thor- I 1 Preserved frozen, according to the method of Gies. 1 Just before feeding. Utilization of Wheat Proteins oughly incorporated, and the whole allowed to stand over night, thus giving ample time for " hydration" 1 of the material. Such food mixtures were fed for periods of 3 to 5 days to three small bitches. The food was disposed of in one meal at 9 :00 to 9:45 each morning. TABLE 2. "Glidin" with Agar and Bone Ash. PERIOD IV (5 days) Meat Feeding PERIOD V (5 days) "Glidin" Feeding PERIOD VI (4 days) Meat Feeding grams Meat 150 Sugar 20 Lard 20 Agar 3 Bone Ash. 7 Water 100 Estimated calories 520 arams 11 Glidin" 34 Sugar 20 Lard 30 Agar 3 Bone Ash 7 Water 200 Estimated calories 470 grams Meat 150 Sugar 20 Lard 20 Agar 3 Bone Ash 7 Water 100 Estimated calories 520 Daily Averages Daily Averages Daily Averages 3.70 4.06 4.93 +0.87 4.83 5.14 4.93 -0.21 4.13 4.48 4.93 +0.45 13.6 0.36 2.64 13.6 0.31 2.31 14.5 0.35 2.42 92.7 93.6 92.9 SUBJECT, DOG 6 Weight at beginning, 5.0 Kg. Weight at end, 4.9 Kg. Composition of daily diet. Nitrogen output. Urine nitrogen, gm Total nitrogen, gm Nitrogen in food, gm Nitrogen balance, gm Feces. Weight air dry, gm Nitrogen, gm Nitrogen, per cent Nitrogen, utilization, per cent From an examination of the tables, it will be observed that the data without exception point to the fact that 11 glidin 17 is as thoroughly utilized as meat under identical conditions. 1 When first added to the food mixture there appeared to be little tend- ency for the water to be absorbed. On standing a few hours, however, and especially the next morning, a thick thoroughly hydrated mush resulted. Lafayette B. Mendel and Morris S. Fine 313 TABLE 3. "Glidin" with Agar and Bone Ash. SUBJECT, DOQ 7 Weight, 4.9 Kg. PERIOD III (S days) Meat Feeding PERIOD IV (5 days) "Glidin" Feeding PERIOD V (3 days) Meat Feeding Composition of]daily diet. < Nitrogen output. Urine nitrogen, gm Nitrogen in food, gm Nitrogen balance, gm Feces. Nitrogen, gm Nitrogen, per cent Nitrogen utilization, per cent grams Meat 100 Sugar 20 Lard 25 Agar 3 Bone Ash 7 Water 100 Estimated calories 480 grams "Glidin" 23 Sugar 20 Lard 30 Agar 3 Bone Ash 7 Water 175 Estimated calories 430 grams Meat 100 Sugar 20 Lard 20 Agar 3 Bone Ash 7 Water 100 Estimated calories 430 Daily Averages Daily Averages Daily Averages 2.58 2.81 3.29 +0.48 12.8 0.23 1.81 93.0 3.21 3.48 3.29 -0.19 11.0 0.27 2.43 91.9 2.63 2.89 3.29 +0.40 12.7 0.26 2.04 92.1 Commercial Gluten — Tables 4 to 9. In Tables 4 to 6 are recorded experiments wherein the utilization of gluten, fed with agar and bone ash, is compared with meat diets in which identical additions of these indigestible materials were made. Data on the comparative utilization of gluten and meat, where no agar or bone ash was employed, are reported in Tables 7 to 9. An examination of these tables will readily convince one that the present sample of commercial gluten is as thoroughly utilized as meat. One might take exception to this generalization, observing that the persistently high nitrogen content of the gluten-feces, as com- pared with that of the corresponding meat-feces, indicates that a portion of the gluten had been lost through the feces. Moreover, 3U Utilization of Wheat Proteins careful scrutiny will disclose the fact that the utilization of the gluten is consistently — if only slightly — lower than that of meat. This criticism is indeed valid. However, had the gluten been finely divided, objections of the above nature, which in any case are concerned with small differences, would without doubt be untenable. TABLE 4. Gluten with Agar and Bone Ash. SUBJECT, DOG 5 Weight at beginning, 5.2 Kg. Weight at end, 5.2 Kg. PERIOD VIII (5 days) Meat Feeding PERIOD IX* (6 days) Gluten Feeding Nitrogen output. Urine nitrogen, gm Total nitrogen, gm Nitrogen in food, gm Nitrogen balance, *gm Feces. Weight air dry, gm Nitrogen, gm grams Meat 150 Sugar 20 Lard 20 Agar 3 Bone Ash 7 Water 100 Estimated calories 520 grams Gluten 36 Sugar 20 Lard 30 Agar 3 Bone Ash 7 Water 200 Estimated calories 480 Daily Averages Daily Averages 4.09 4.44 4.80 +0.36 15.0 0.35 2.32 92.8 4.78 5.22 4.90 -0.32 14.5 0.44 3.05 91.0 * On last two days of period, about half the food was forced. Lafayette B. Mendel and Morris S. Fine 315 TABLE 5. Glu ten with A g ar and Bone Ash. SUBJECT, DOG 6 Weight at beginning, 4.7 Kg. Weight at end, 4.5 Kg. PERIOD VIII (5 days) Meat Feeding PERIOD IX (5 days) Gluten Feeding Nitrogen output. Total nitrogen, gm Nitrogen in food, gm Nitrogen balance, gm Feces. grams A/Too + 1 KCi Sugar 20 Lard 20 Agar 3 Bone Ash 7 Water 100 Estimated calories 520 grams ^jriuien oo Sugar 20 Lard 30 Agar 3 Bone Ash 7 Water 200 Estimated calories 480 Daily Averages Daily Averages 4.23 4.62 4.80 +0.18 15.0 0.39 2.59 91.9 4.81 5.24 4.90 -0.34 14.4 0.43 3.00 91.2 Nitrogen, per cent Nitrogen utilization, per cent TABLE 6. Gluten with Agar and Bone Ash. SUBJECT, DOG 7 Weight at beginning, 4.6 Kg. Weight at end, 4.6 Kg. PERIOD VII (5 days) Meat Feeding PERIOD VIII (5 days) Gluten Feeding Nitrogen output. Urine nitrogen, gm Feces. Nitrogen, gm grams Meat 100 Sugar 20 Lard 20 Agar 3 Bone Ash 7 Water 100 Estimated calories 430 grams Gluten 24 Sugar 20 Lard 30 Agar 3 Bone Ash 7 Water 175 Estimated calories 430 Daily Averages Daily Averages 2.55 2.79 3.20 +0.41 12.8 0.24 1.86 92.6 3.24 3.52 3.27 -0.25 12.2 0.28 2.31 91.4 Nitrogen, per cent Nitrogen utilization, per cent 3i 6 Utilization of Wheat Proteins TABLE 7. Gluten without Agar or Bone Ash. SUBJECT, DOG 5 Weight at beginning, 5.9 Kg. Weight at end, 5.8 Kg. PERIOD XX (4 days) Meat f eeding PERIOD XXII* (5 days) Gluten Feeding Composition of daily diet < Nitrogen output. Feces. grams Meat 150 Sugar 25 Lard 20 Water 100 Estimated calories 530 grams Gluten 34 Sugar 25 Lard 25 Water 225 Estimated calories 440 Daily Averages Daily Averages 4.08 4.30 4.59 +0.29 4.5 0.22 4.95 95.2 4.83 5.05 4.77 -0.28 2.8 0.22 7.68 95.5 Nitrogen, per cent * About half of the food forced each day. TABLE 8. Gluten, without Agar or Bone Ash. SUBJECT, DOG 6 Weight at beginning, 6.1 Kg. Weight at end, 6.2 Kg. PERIOD XXI (4 days) Meat Feeding PERIOD XXIII (5 days) Gluten Feeding Composition of daily diet < Nitrogen output. Total nitrogen, gm Nitrogen in food, gm Nitrogen balance, gm Feces. Weight air dry, gm Nitrogen, gm grams Meat, 150 Sugar 25 Lard 20 Water 100 Estimated calories 530 grams Gluten 34 Sugar 25 Lard 25 Water 225 Estimated calories 440 Daily Averages Daily Averages 3.55 3.77 4.59 +0.82 3.5 0.22 6.34 95.2 4.92 5.17 4.77 -0.40 3.6 0.25 6.93 94.8 Nitrogen, per cent Nitrogen utilization, per cent Lafayette B. Mendel and Morris S. Fine 317 TABLE 9. Gluten without Agar or Bone Ash. SUBJECT, DOG 7 Weight at beginning, 5.9 Kg. Weight at end, 6.3 Kg. PERIOD XX (4 days) Meat Feeding PERIOD XXII (5 days) Gluten Feeding Composition of daily diet I Nitrogen output. Total nitrogen, gm Feces. Weight air dry, gm grams Meat 150 Sugar 25 Lard 20 Water 100 Estimated calories 530 grams Gluten 34 Sugar 25 Lard 25 Water 225 Estimated calories 440 Daily Averages Daily Averages 3.55 3.74 4.59 +0.85 3.2 0.19 5.93 95.8 4.68 4.81 4.77 -0.04 1.8 0.13 7.40 97.2 Glutenin — Tables 10, 11. Man, Table 10: The subject of this experiment, M. S. F., was 23 years of age, about 57.5 kilos in weight. Throughout the experiment he was engaged in the routine con- nected with such work. The general plan of the experiment was as follows : A preliminary period of three days was obtained, dur- ing which the body was enabled to attain nitrogen equilibrium and to adjust itself to the experimental conditions. As will be noted from the accompanying table, during the preliminary, fore, and after periods, a varied mixed diet was consumed; and during the experimental period, the meat, nut butter and part of the egg were replaced by glutenin. 3i8 Utilization of Wheat Proteins Character of the Diet. PRELIMINARY, FORE AND AFTER PERIODS EXPERIMENTAL PERIOD Daily Averages Daily Averages Gm. Gm. Cereal 20 20 60 60 Egg 110 50 Pine nut butter 50 Meat 110 62 Potato 110 110 Banana 140 140 270 250 160 180 Milk 40 40 Sugar 110 180 Butter 40 80 Cereal coffee, tea 600 600 TABLE 10. Glutenin. SUBJECT, MAN Weight at beginning, 57.8 Kg. Weight at end, 57.6 Kg. Composition of daily diet. Nitrogen output. Urine nitrogen, gm Total nitrogen, gm Nitrogen in food, gm — Nitrogen balance, gm.... Feces. Weight air dry, gm Nitrogen, gm Nitrogen, per cent Nitrogen utilization, per cent PERIOD I (6 days) Mixed Diet Meat nut butter, cereal,potato fruit, etc. Estimated calories 2400 Daily Averages 9.85 11.10 11.30 +0.25 23.0 1.25 5.45 89.0 PERIOD II (4 days) Glutenin Glutenin cereal, potato, fruit, etc. 67.2 per cent of the total nitrogen fur- nished by glutenin. Estimated calories 2500 Daily Averages 10.80 12.11 11.12 -0.99 22.1 1.31 5.92 88.2 PERIOD III (4 days) Mixed Diet Meat, eggs, nut butter, cereal, potato, fruit, etc. Estimated calories 2600 Daily Averages 9.98 11.36 11.15 -0.21 24.7 1.38 5.63 87.6 Lafayette B. Mendel and Morris S. Fine 319 These diets furnished about 11.2 grams nitrogen and 2,500 calories per day; during the experimental period 67 per cent of the nitrogen was supplied by the glutenin. During the evening of the third day of the experimental period, slight indigestion was manifest, which continued to the following morning, when 100 cc. of 0.2 per cent HC1 were taken. 1 The glu- tenin had a peculiar ether-alcohol odor which could not be removed by drying at 130° C. for eight consecutive hours. This odor was very disagreeable and nauseating and could not be covered by mixing with other foods. It was found that mixing the glutenin with egg and potato, and frying this mixture, was practically the only way in which the glutenin could be consumed in any quantity. Under such circumstances it is probable that the psychic secretion was a negligible quantity and this may account for the symptoms of indigestion noted. With the exception of the feeling of nausea attending the meals of the four days of glutenin feeding, the subject felt perfectly well. Throughout the experiment defecation was accomplished regu- larly every morning, the volume of feces being apparently alike from day to day. The feces were of semi-solid consistency, never well formed. There was, however, nothing approaching diarrhoea, throughout the experiment, except on the last day of glutenin feeding. In spite of the nausea with its possible secretory consequences, the glutenin appeared to be as well digested as the materials which it replaced in the control diets. It should be observed, however, that about 25 per cent of the nitrogen of the control diets was furnished by nut butter, and, according to Jaffa, 2 the nitrogen of nuts is in general only about 75 per cent utilized. Hence, in order to show the readily digestible nature of glutenin, the coefficient of diges- tibility in the experimental period should have been somewhat greater than those for the control periods. Nevertheless, it is significant to note that in two experiments on the same subject, where mixed diets were employed, which contained no nut prep- arations but were otherwise similar, the utilization was 88 and 86 per cent, respectively. 1 This affects one day out of four; and since the HC1 gave no noticeable relief, the influence of the acid even in this day was probably very small. * Jaffa: Office of Experiment Stations, Bull. 132, p. 69, 1903. 320 Utilization of Wheat Proteins TABLE 11. Glutenin with Agar and Nutrient "Salts." SUBJECT, DOG 4 Weight at beginning, 5.1 Kg. Weight at end, 5.0 Kg. Composition of daily diet . Nitrogen output. Urine nitrogen, gm Total nitrogen, gm Nitrogen in food, gm Nitrogen balance, gm Feces. Weight air dry, gm Nitrogen, gm Nitrogen, per cent Nitrogen utilization, per cent PERIOD I (5 days) Meat Feeding PERIOD II* (5 days) Glutenin Feeding PERIOD III (3 days) Meat Feeding grams Meat 150 grams Glutenin 43 grams Meat 150 Sugar 15 Starch 5 Sugar 25 Starch 5 Sugar 25 Starch 5 Lard 20 Lard 30 Lard 20 Agar 8 Salts 2 Agar 8 Salts 4 Agar 8 Salts 4 Water 200 tit j _ nr\r\ Water 300 Water 200 Estimated Estimated Estimated calories 520 calories 520 calories 560 Daily Averages Daily Averages Daily Averages 4.71 4.71 4.18 5.04 5.01 4.48 5.18 5.18 5.18 +0.14 +0.17 +0.70 13.6 15.0 12.7 0.33 0.30 0.31 2.40 1.99 2.42 93.7 94.3 94.1 * About half of the food forced each day. For mixed diets on man 93 per cent is given as the average coef- ficient of digestibility by At water and Bryant, 1 being considerably higher than the coefficients obtained in our experiments. The difference may be accounted for by the presence in the diets of relatively large amounts of fruits and vegetables, which contain considerable indigestible material. 2 Dog, Table 11: The plan of the experiment did not differ essen- tially from those already described. The character of the indi- 1 See footnote, 1 p. 304. 2 Cf. Bryant and Milner : American Journal of Physiology, x, p. 96, 1903. Lafayette B. Mendel and Morris S. Fine 321 gestible materials was somewhat different, larger amounts of agar being used, without bone ash, the latter being replaced by a salt mixture. 1 Despite the necessity of forced feeding, the glutenin was quite as thoroughly utilized as meat fed under similar experi- mental conditions. Gliadin— Tables 12, 13. Man, Table 12: The plan of the experiment was essentially that outlined under glutenin, except that slightly more meat was eaten in the control periods; and in the gliadin period the egg, meat and nut butter were completely replaced by gliadin, which supplied 85 per cent of the total nitro- gen. The gliadin when mixed with water forms a veritable glue, which cannot possibly be eaten. This glue-like consistency was avoided by mixing with cornstarch, salt, sugar, and baking powder, which mixture was finally baked. Even in this condition, the material soon evoked nausea which, on the last day of the period, made a discontinuance necessary. Aside from the nausea attend- ing the meals of the gliadin period, the subject felt in excellent con- dition throughout the experiment, diarrhoea and symptoms of indigestion being entirely absent. From Table 12, it is evident that gliadin is as thoroughly utilized as the materials of the control periods, which it replaced. The salts of the baking powder seemed to have no appreciable influence. The apparently low digestibility of the mixed diets has already been discussed under glutenin. Dog, Table IS: The plan of this experiment was exactly the same as that described on page 320 under the glutenin experiment on a dog. The food mixture was like so much glue, but the animal 1 With one or two modifications this is the salt mixture proposed by Rohmann (Allgemeine medizinische Central-Zeitung, No. 9, 1908). Itcon. sisted of the following ingredients: Gram Calcium phosphate 10 Acid potassium phosphate 37 Sodium chloride 20 Sodium citrate 15 Magnesium citrate 8 Calcium lactate 8 Ferric citrate 2 322 Utilization of Wheat Proteins TABLE 12. It/? nni Yl SUBJECT, MAN PERIOD I PERIOD II PERIOD III Weight at beginning, 57.0 Kg. (5 days) (4 days) (4 days) Weight at end, 57.6 Kg. Mixed Diet Gliadin Mixed Diet ivleat, eggs, tjriiauin, Meat, eggs, nut butter, potatoes, nut butter, potatoes, fruit, etc. potatoes, fruit, etc. 84.8 percent of fruit, etc. Composition of daily diet. < the total ni- trogen sup- plied by glia- din. Estimated Estimated Estimated calories 2600 calories 2800 calories 2900 Daily Averages Daily Averages Daily Averages iV itrogen output. in 79 1U . i z 11 . OO 10 in 1U. 1U 19 1Q LZ . lo 1Z .00 1 1 QQ 11 .OO 19 79 LZ . i Z LZ . \JZ 19 ftfi 1Z . Do Nitrogen balance, gm +0.60 -0.80 +1.30 Feces. Weight air dry, gm 23.6 22.5 26.8 Nitrogen, gm 1.41 1.27 1.53 Nitrogen, per cent 5.96 5.53 5.74 Nitrogen utilization, per cent 89.0 89.4 87.9 ate it with apparent relish. From Table 13 the glutenin appears to have been quite thoroughly utilized. Abderhalden obtained plus balances with gliadin, but notes that his preparations contained 0.35 per cent lysine — -an amino- acid not found in pure gliadin. He infers further that it is not known whether really pure gliadin can maintain nitrogen equilib- rium. We are unable to state whether or not the gliadin employed in our experiments was free from lysine, but the negative nitrogen balances, even with large intakes, are significant. Henriques ob- tained plus balances with gliadin in rats, but Abderhalden ques- tions the purity of his preparations. 1 1 Osborne and Mendel (Feeding Experiments with Isolated Food-Sub- stances, Carnegie Institution of Washington, Publication No. 156. p. 21, Lafayette B. Mendel and Morris S. Fine 323 TABLE 13. Gliadin with Agar and Nutrient "Salts." SUBJECT, DOG 4 PERIOD III PERIOD IV PERIOD V Weight at beginning, 4.9 Kg. (3 days) (5 days) (4 days) Weight at end, 4.9 Kg. Meat Feeding Gliadin Feeding Meat Feeding gram gram gram Meat 150 Gliadin 32 Meat 150 ougar 60 ougar ZiO ougar ao Starch 5 Starch 5 Starch 5 Lard 20 Lard 30 Lard 20 Oomnosition of dailv dipt < Agar 8 Agar 8 Bone Ash 10 Salts 4 Salts 4- Water 200 Water 200 Water 250 TT!g f" 1 TY\ Q "f ori TTlcf"! m {jfon TT.stim n tpd cfiloriGS 520 calories 560 Daily Averages Daily Averages Daily Averages Nitrogen output. Urine nitrogen, gm 4.18 5.09 4.24 Total nitrogen, gm 4.48 5.37 4.50 Nitrogen in food, gm 5.18 5.10 5.40 Nitrogen balance, gm +0.70 -0.27 +0.90 Feces . 12.7 13.4 15.0 Nitrogen, gm 0.31 0.28 0.27 Nitrogen, per cent 2.42 2.07 1.79 Nitrogen utilization, per cent 94.1 94.5 95.0 It is difficult at present to account for the persistent negative balances with the other protein preparations. Michaud did indeed obtain minus balances with " gliding ' but his nitrogen intakes were very small, while those in our experiments were relatively large. 1911) state that in rat experiments at least 10 per cent of the excreted nitro- gen may be lost in connection with the difficult manipulation attending metabolism experiments with small animals. This condition may in part account for the positive balances obtained by Henriques. 324 Utilization of Wheat Proteins Nitrogen Balances. TABLE 14. Average Daily Nitrogen Balances for the Wheat Proteins and Correspond- ing Values for Meat. SUBJECT TABLE "glidin" "gluten" GLUTENIN GLIADIN MEAT Dog 5.. i -0.02,-0.27 +0.74,-1-0.82 Doer £i — 91 in 07 in &K Dog 7.. 3 -0.19 +0.48, +0.40 Dog 5.. 4 -0.32 +0.36 Dog 6. . 5 -0.34 +0.18 Dog 7.. 6 -0.25 +0.41 Dog 5.. 7 -0.28 +0.29 Dog 6.. 8 -0.40 +0.82 Dog 7.. 9 -0.04 +0.85 Man . . . 10 -0.99 +0.25,-0.21 Dog 4. . 11 +0.17 +0.14, +0.70 Man . . . 12 -0.80 +0.60,+1.30 Dog 4. . 13 -0.27 +0.70, +0.90 SUMMARY. The problems associated with the utilization of food products of plant origin have been reviewed as an introduction to a series of experimental studies on the nutritive value of vegetable pro- teins. It is pointed out that two distinct questions must be con- sidered, namely: (1) the availability of the products existing more or less in their native condition, with accompanying structural elements, as in bread; (2) the specific utilization of the proteins themselves. The latter aspect is the one which primarily calls for further investigation. In our feeding experiments an attempt has been made to con- trol the extraneous factors as far as possible, by improving the texture and mechanical condition of the crude products, or puri- fying the individual proteins. The present paper deals with wheat. The experimental trials on man and dogs indicate that "glidin," gluten, and the two characteristic proteins of wheat, gliadin and glu- tenin, are as thoroughly utilized as the nitrogenous components of fresh meat. Lafayette B. Mendel and Morris S. Fine 325 BIBLIOGRAPHY. Abderhalden: 1909, Zeitschrift fiir physiologische Chemie, Ix, p. 418. Bischoff, E. : 1869, Zeitschrift fur Biologie, v, p. 454. Bischoff, T. and Voit: "Die Erndhrung des Fleischfressers." Bornstein: 1897, Berliner klinische Wochenscrhift, No. 8, p. 162. Buslik and Goldhaber: 1911, Zeitschrift fiir physikalische und didtetische Therapie, xv, p. 93. Constantinidi: 1887, Zeitschrift fiir Biologie, xxiii, p. 433. Erismann: 1901, Zeitschrift fiir Biologie, xlii, p. 672. Fauvel: 1907, Maly's Jahresbericht iiber die Fortschritte der Tierchemie, xxxvii, p. 605. Henriques: 1909, Zeitschrift fiir physiologische Chemie, lx, p. 105. Kornauth: 1892, Oesterreichisches landwirtschaftliches Centralblatt. Laves: 1900, Miinchener medizinische Wochenschrift, p. 1339. Lehmann, K. B. : 1894, Archiv fiir Hygiene, xx, p. 1; 1902, ibid., xlv, p. 177. Lusk: 1890, Zeitschrift fiir Biologie, xxvii, p. 459. Menicanti and Prausnitz : 1894, Zeitschrift fur Biologie, xxx, p. 328. Meyer, G.: 1871, Zeitschrift fiir Biologie, vii, p. 1. Michaud : 1909, Zeitschrift fiir physiologische Chemie, lix, p. 405. Potthast: 1887, Inaugural Dissertation, Leipzig Prausnitz : 1893, Archiv fiir Hygiene, xvii, p. 626. Romberg: 1897, Archiv fiir Hygiene, xxviii, p. 274. Rubner: 1879, Zeitschrift fiir Biologie, xv, p. 115; 1883, ibid., xix, p. 45. Salkowski: 1909, Bioche.vAsche Zeitschrift, xix, p. 83. Wicke: 1890. Archiv fiir Hygiene, xi, p. 349 (see the corrections made by Rubner, ibid., xiii, p. 123, 1891). Wintgen: 1902, Zeitschrift fiir Untersuchung der Nahrungs- und Ge- nussmittel, v, p. 289. Woods and Merrill: 1900, Office of Experiment Stations, Bull. 85. Reprinted from The Journal of Biological Chemistry, Vol. X, No. 4. 1911 STUDIES IN NUTRITION. H. THE UTILIZATION OF THE PROTEINS OF BARLEY. By LAFAYETTE B. MENDEL and MORRIS S. FINE. (From the Sheffield Laboratory of Physiological Chemistry, Yale University, New Haven, Connecticut.) (Received for publication, September 25, 1911.) Data regarding the utilization of the proteins of barley are con- fined almost entirely to the Japanese literature, 1 according to which they are but 40 to 76 per cent available. However, the experiments from which these data are taken were not exempt from those unfavorable conditions which render it difficult to draw conclusions. 2 METHODS. The routine incident to the metabolism experiments did not differ essentially from that described in the previous paper of this series. The concentration of hemicelluloses in the barley preparation and in the resulting feces was determined. For this purpose 2.5 to 3.5 grams of the dried material were boiled for four hours with 220 cc. of 2 per cent hydrochloric acid. 3 Proteoses and peptones, which disturb the subsequent precipitation of copper oxide, were removed by the addition of a solution of phosphotung- 1 Cf . Oshima: U. S. Department of Agriculture, Office of Experiment Stations, Bull. 159, 1905. 2 Cf. Mendel and Fine: This Journal, x, p. 303, 1911. 3 From the studies of Swartz (Transactions of the Connecticut Academy of Arts and Sciences, xvi, p. 323, 1911) it appears that maximum reduction after hydrolysis is obtained in three to four hours. The crude barley pro- tein preparation of these experiments yielded but 0.6 per cent more hemi- celluloses when boiled four hours than when the interval was reduced to two and a half hours as called for in the official method for the estimation of starch (U. S. Dept. of Agriculture, Bureau of Chemistry, Bull. 107, p. 53, 1910). 339 340 Utilization of Barley Proteins stic acid. 1 The resulting precipitate was washed, the united fil- trates neutralized and made up to 500 cc, of which 25 cc. were used for the sugar estimation according to the Allihn gravimetric method. The hemicellulose was computed by multiplying the dextrose thus obtained by O.9. 2 EXPERIMENTAL PART. Product Employed. The crude barley protein employed in this study was prepared as follows: About seven pounds of barley flour, 3 in one pound lots, were made into a thin homogeneous mush with water and heated in an autoclave at about 120° C. for ten to twenty minutes. After the material had cooled to a tem- perature of about 60° C. an amylolytic preparation was added. It was neces- sary to repeat this process at least three times before the cells were com- pletely disintegrated and no starch reaction was obtained in a test tube trial. The insoluble material thus obtained settled very readily and was washed with ease four to six times with water by decantation. It was finally filtered, dried on a water bath and ground to an impalpable powder. Analysis gave the following values: per cent Protein (N X 5.7 4 ) 51.0 Carbohydrate by hydrolysis (hemicelluloses) 21.7 Ether extract 8.3 Ash 1.0 Moisture 3.0 Crude fiber (by difference). 15.0 When treated with iodine and examined under the microscope, starch particles were so infrequent as to be considered absent; yet 1 Cf. Abderhalden' s Handbuch der Biochemischen Arbeitsmethoden, iii, 1, p. 271, 1910. With this method the barley preparation yielded 21.7 per cent hemicelluloses; when the phosphotungstic precipitation was omitted, the yield was reduced to 18.5 per cent. Swartz (loc. cit., p. 343) adopted the expedient of clarifying with charcoal with satisfactory results. 2 This is believed to be justifiable in view of the similarity in ultimate analysis of starch and hemicelluloses. 3 Mr. M. F. Deming of the Cereo Company, Tappan, N. Y., kindly con- tributed this material. 4 Factor proposed by Atwater and Bryant : Report of the Storrs Agri- cultural Experiment Station, p. 79, 1899. Lafayette B. Mendel and Morris S. Fine 34 j on hydrolyzing with dilute acid, 22 per cent of carbohydrate was obtained. This condition would suggest that the values for starch, as ordinarily found by acid hydrolysis, cannot be relied upon, since a body other than starch may be present, which on hydroly- sis yields reducing substances. Indeed Schulze 1 has shown the wide-spread occurrence of hemicelluloses, a group of substances readily attacked by dilute acids, yielding reducing sugars, and only very slowly affected by enzymes. 2 Apparently the untreated barley contains at least 5 per cent of hemicelluloses. Metabolism Experiments. Crude barley protein was fed to two bitches as detailed in the first two tables. Casual inspection of these data would indicate that barley protein is relatively poorly utilized, having a coeffi- cient of digestibility of 85 per cent against 97 per cent for meat fed, as it might at first sight appear, under identical conditions. However, each day's supply of barley protein preparation contained about 8 grams of crude fiber — practically indigestible 3 — and 11 grams of hemicelluloses, of which 9 grams reappeared in the feces in both experiments. 4 The barley-protein feces thus contained about 17 grams of undigested non-nitrogenous material, and these experiments should therefore not be held comparable to trials with meat where such unfavorable conditions were not in evi- dence. 1 Cf. Schulze and Godet: Zeitschr.f. physiol. Chem., lxi, p. 281, 1909. 2 Cf. Swartz: hoc. cit. (contains the literature). 3 Cf. Swartz: hoc. cit., p. 268 ff. (contains the literature). 4 The barley-protein feces from both animals yielded 10 grams of dextrose (= 9 grams of hemicellulose) on hydrolysis. The criticism might be offered that a portion of the dextrose thus obtained is due to the cane sugar of the food mixture, its digestion and absorption having been diminished by the excessive fecal discharges (22 grams dry daily). Against this objection are the following facts : (1) meat diets containing 10 grams of bone ash but otherwise identical to those in this paper yielded 14 to 15 grams of dry carbo- hydrate-free feces daily; (2) cotton seed diets, containing 25 grams of cane sugar as in the above trials, produced 23 to 24 grams of dry feces, which yielded only 4 to 6 grams of reducing carbohydrate on hydrolysis — quantities attributable to undigested hemicelluloses of the food. 342 Utilization of Barley Proteins The importance of comparing experiments in which the indiges- tible non-nitrogenous material, 1 in addition to the nitrogen intake and accessory articles of diet, are similar, was adequately appre- ciated only after the major portion of the studies of this series was completed. Trials in which these principles were consistently applied not being at hand, we must be content with grouping data which will as far as possible enable one to interpret properly the results on protein utilization. Table 3 contains such an arrange- ment. Meat diets containing 6 grams of fiber and 13 grams of indigestible materials were 91 and 89 per cent utilized, respectively. Thus the utilization of 85 per cent for the barley preparation with its 8 grams of fiber and 11 grams of hemicelluloses (of which 9 grams reappeared in the feces) leads one to believe that under favorable conditions, barley protein, like that of the closely related cereal wheat, would be almost perfectly digested. TABLE 1. Crude Barley Protein. SUBJECT, DOG 5 PERIOD XXVIII PERIOD XXIX* Weight at beginning, 6.2 Kg. Weight at (5 days) (4 days) end, 6.3 Kg. Meat Feeding Barley Protein Feeding grams grams Meat 150 Barley protein 52 Sugar 25 Sugar 25 Composition of daily diet ( Lard 20 Lard 30 Water 100 Water 200 Estimated Estimated calories 530 calories 490 Daily Averages Daily Averages Nitrogen output Urine nitrogen, gm 3.68 3.99 Total nitrogen, gm 3.84 4.67 Nitrogen in food, gm 4.64 4.64 Nitrogen balance, gm +0.80 -0.03 Feces Weight air dry, gm 3.4 22.0 Nitrogen, gm 0.16 0.68 Nitrogen, per cent 4.62 3.10 Nitrogen utilization, per cent 96.6 85.3 •About half the food forced. 1 The influence of such substances upon nitrogen utilization will be dis- cussed more fully in a subsequent paper. Lafayette B. Mendel and Morris S. Fine 343 TABLE 2. Crude Barley Protein.. SUBJECT, DOG 7 PERIOD XXVIII PKRIOD XXtX Weight at beginning, 6.6 Kg. Weight at (5 days) (5 days) end, 6.7 Kg. Meat Feeding Barley Protein Feeding grams qtcliyis Meat 150 Barley protein 52 Sugar 25 Sugar 25 Composition of daily diet < Lard 20 Lard 30 Water 100 Water 200 Estimated Estimated calories 530 calories 490 Nitrogen output Daily Averages Daily Averages 3.60 3.75 Total nitrogen, gm 3.75 4.45 Nitrogen in food, gm 4.64 4.64 Nitrogen balance, gm +0.89 +0.19 Feces 3.8 22.4 Nitrogen, gm 0.15 0.70 Nitrogen, per cent 3.82 3.12 Nitrogen utilization, per cent 96.9 85.0 TABLE 3. Utilization with Reference to Indigestible Materials in the Diet. Daily Averages. DOG PERIOD DAYS NATURE OF INGESTA FIBER IN OR ADDED TO FOOD TOTAL INDIGES- TIBLE MATERIAL IN FOOD N INTAKE N UTILIZA- TION AVER- AGE N UTILIZA- TION grams grams grams per cent per cent 5 xxix 4 1 Crude barley f 8 19t 4.6 85.3 | 85.2 7 xxix 5 / protein \ 8 19t 4.6 85.0 5 xviii* 4 6 6 3.3 90.5 6 xix* 4 J Meat | 6 6 3.3 89.2 J 91.0 7 xviii* 4 6 6 3.3 93.3 5 XV* 4 1 Meat 6 13 3.3 91.6 6 xvi* 4 \ AgarJ 2 gm. j 6 13 3.3 87.7 J 89.2 7 XV* 4 J Bone ash§ 5 gm. [ 6 13 3.3 88.3 * The details of these experiments will be published in a subsequent paper of this series, t This includes 11 grams of hem icelluloses, which were 18 per cent utilized (see page 341, this paper). JSaiki (this Journal, ii, p. 251, 1906) recovered all but 17 per cent of the agar fed. The amount of agar thus failing to reappear in the feces of these experiments is too small to be considered. § We are unable to state exactly how completely the bone ash reappears in the feces. Steel and Gies (Amer. Journ. of Physiol., xx, p. 350, 1907) found no change in urinaiy calcium or phosphorus when large amounts of bone ash were added to the diet. Reprinted from The Journal of Biological Chemistry, Vol. X, No. 5, 1911 STUDIES IN NUTRITION. III. THE UTILIZATION OF THE PROTEINS OF CORN. By LAFAYETTE B. MENDEL and MORRIS S. FINE. (From the Sheffield Laboratory of Physiological Chemistry, Yale University, New Haven, Connecticut.) (Received for publication, September 25, 1911.) Corn has been produced extensively as a food for man in America for many hundreds of years 1 It is scarcely a century since corn has ceased to be the most important of our food cereals, this high place in our dietary having since been assumed by wheat because of its superior bread-making qualities. Corn has been, and is, an important article of diet in other parts of the world also. A very popular dish in Italy is maize cooked with water to a stiff mush, to which a little cheese is added. This preparation is called polenta, and. according to Ranke, 2 it is the main article of diet in certain parts of Italy. Rubner reported a study on the availability of polenta seasoned with meat extracts The digestibility of the whole was 85 per cent, but assuming the meat extracts to be completely absorbed, the apparent utilization would fall to 80 per cent. In experiments on himself Malfatti found the digestibility of maize to be 82 per cent. The utilization of this material was lowered to 68 per cent by the addition of much butter and raised to 93 per cent when cheese was added to the diet. The work of Grandeau, on the availability of maize in the horse, is of interest in that it shows that even in an animal with a long intestine, where the food may remain for a greater length of time, and the cellulose be dissolved to a considerable extent — even here the digestibility was an average of only 69 per cent. The gener- ally low utilization of polenta is further shown by Albertoni and Novi, and by Erismann. 1 Cf . Merrill (1906) : see bibliography. 2 Ranke: Zeitschrift fur Biologie, xiii, p. 130, 1877. 345 34^ Utilization of Corn Proteins The utilization experiments of Merrill, in which the protein of the corn of various corn preparations was found to vary in diges- tibility from 73 to 86 per cent, lend further support to these data, as does the later work of the same author, in which the protein of corn was calculated to be only 61 per cent utilizable. The above data on the digestibility of maize are quite contrary to those obtained for "roborat," 1 an albumose-like preparation. "Roborat" has been the subject of considerable investigation, notably by Laves, Loewy and Pickart, Wintgen, Hoppe, and Som- merfeld, the consensus of opinion being that this commercial mate- rial is quite as well utilized as meat. Pertinent objections may be raised to all the foregoing data on the digestibility of the proteins of corn. The maize employed contained considerable starch, and it is an open question as to what extent the cells were ruptured. The unfavorable influence of these conditions has been discussed at length in a previous paper. 2 While these objections do not apply to the thoroughly digested "roborat," still here the protein has been considerably changed, being present in part as proteoses. Investigations on the pure unchanged protein would of course be free from these criticisms. Maize contains several proteins, 3 one of which is zein, an alcohol-soluble protein, which makes up about half the total protein. The experiments on zein are prac- tically limited to those of Rockwood on dogs. In two experi- ments this observer found the utilization of zein to be 78 and 90 per cent respectively. It should be noted that the zein employed by Rockwood was hard and not very finely divided, and it is pos- sible that this may have been a factor contributing to the poor utilization. Henriques has studied zein with reference to its ability to main- tain nitrogenous equilibrium in rats. Incidentally one notes from his tables that the zein varied in its apparent utilization be- tween 50 and 90 per cent. However, zein, which had been pre- viously subjected to tryptic digestion was only 74 per cent utilized; 1 Vor an account of the properties of this material compare Loewy and Pickart (see bibliography). 2 Cf. Mendel and Fine: This Journal, x, p. 303. 1911. » See T. B. Osborne: "Die Pflanzenproteine," Ergebnisse der Physiologie, x, p. 47, 1910. Lafayette B. Mendel and Morris S. Fine 347 and zein obtained by precipitation from its solution in alkali with acid was but 64 per cent digested. Szumowski's 1 observa- tions may be of interest in this connection. A 4 kg. dog was fed with a mixture of 100 grams of finely ground zein, sugar, lard and water. Five hours later the dog was bled to death, and there were recovered 61 grams of zein from the stomach, 18 grams from the small intestine and 6 grams from the large intestine. In an- other instance, a solution of alkali-zein was fed, which resulted in irritation of the stomach and diarrhoea. It may be that the poor utilization obtained by Henriques for zein subjected to tryptic digestion, and for the zein previously dissolved in alkali, is due to the production of diarrhoea. Indeed it is well known that proteose preparations from meat and other sources may show apparently poor utilization for this very reason. The literature thus assembled would seem to favor the view that the unchanged protein of corn is poorly utilized; but it should be borne in mind that the conditions attending these experiments have in practically no instance been free from objection. EXPERIMENTAL PART. Product Employed. The present studies were confined to corn gluten, 2 which con- tained practically no starch. A large amount of this substance was obtained by Dr. T. B. Osborne, who very kindly supplied all of this material necessary for our experiments. Metabolism Experiments. The gluten, ground to an impalpable powder, was fed to three bitches, the usual method of procedure prevailing. 3 Dogs 6 and 7 (Tables 2 and 3) in the experimental periods received no other nitrogenous substance except the corn gluten; while dog 5 (Table 1) received two-thirds of the total nitrogen as corn gluten, and the remainder in the form of meat. 1 Szumowski: Zeiischrift fur physiologische Chemie, xxxvi, p. 198, 1902. 2 This material contained 7.6 per cent nitrogen. 3 See Mendel and Fine: This Journal, x, p. 303, 1911. 348 Utilization of Corn Proteins TABLE 1. Corn Gluten with Agar and Bone Ash. SUBJECT, DOG 5 Weight at beginning, 5.7 Kg. Weight at end, 5.4 Kg. PERIOD I (5 days) Meat Feeding PERIOD II (4 days) Corn Gluten and Meat Feeding PERIOD III (4 days) Meat Feeding Composition of daily diet Meat Sugar Lard Agar Bone ash Water grams 160 20 20 3 7 100 grams Estimated calories 530 Nitrogen output. Urine nitrogen, gm Total nitrogen, gm Nitrogen in food, gm Nitrogen balance, gm Feces. Weight air dry, gm Nitrogen, gm Nitrogen, per cent Nitrogen utilization, per cent Corn gluten Meat Sugar Lard Agar Bone ash Water Estimated calories 470 Corn gluten furnished 66.6 per cent of the total nitrogen 43 50 20 25 3 7 175 Meat Sugar Lard Agar Bone ash W ater grams 150 20 20 3 7 100 Estimated calories 520 Daily Averages Daily Averages Daily Averages 4.44 4.73 4.89 +0.16 13.2 0.29 2.22 94.0 4.21 4.72 4.91 +0.19 23.5 0.51 2.16 89.7 3.80 4.15 4.90 +0.75 14.5 0.36 2.48 92.7 TABLE 2. Corn Gluten with Agar and Bone Ash. SUBJECT, DOG 6 Weight at beginning, 5.3 Kg. Weight at end, 5.1 Kg. PERIOD I (4 days) Meat Feeding PERIOD II (5 days) Corn Gluten Feeding PERIOD III (5 days) Corn Gluten Feeding PERIOD IV (5 days) Meat Feeding Composition of daily diet 1 Nitrogen output. Urine nitrogen, gm. . . . Total nitrogen, gm Nitrogen in food, gm . . . Nitrogen balance, gm. . Feces. Weight, air dry, gm. . . Nitrogen, gm Nitrogen, per cent.... Nitrogen utilization, per cent grams Meat 150 Sugar 20 Lard 20 Agar 3 Bone ash 7 Water 100 Estimated calories 520 grams Corn gluten 65 Sugar 20 Lard 25 Agar 3 Bone ash 7 Water 175 Estimated calories 420 grams Corn gluten 65 Sugar 20 Lard 25 Agar 3 Bone ash 7 Water 200 Estimated calories 420 grams Meat 150 Sugar 20 Lard 20 Agar 3 Bone ash 7 Water 100 Estimated calories 520 Daily Averages Daily Averages Daily Averages Daily Averages 3.89 4.16 4.93 +0.77 12.2 0.28 2.28 94.3 4.79 5.29 4.95 -0.34 26.0 0.50 1.92 89.9 4.71 5.17 4.95 -0.22 21.0 0.46 2.17 90.7* 3.70 4.06 4.93 +0.87 13.6 0.36 2.64 92.7 "By an accident, about half the feces of this period were charred, giving rise to possible in analj^sis. Lafayette B. Mendel and Morris S. Fine 349 TABLE 3. Corn Gluten with Agar and Bone Ash. SUBJECT, DOQ 7 Weight at beginning, 5.6 Kg. Weight at end, 4.9 Kg. Composition of daily diet Nitrogen output. Urine nitrogen, gm Total nitrogen, gm Nitrogen in food, gm. . . . Nitrogen balance, gm . . Feces. Weight air dry, gm Nitrogen, gm Nitrogen, per cent Nitrogen, utilization, per cent PERIOD I (4 days) Meat Feeding Daily Averages 2.98 3.26 3.29 +0.03 12.5 0.28 2.26 91.4 PERIOD II (4 days) Corn Gluten Feeding grams grams grams Meat 100 Corn Meat 100 Sugar 20 gluten 43 Sugar 20 Lard 20 Sugar 20 Lard 25 Agar 3 Lard 25 Agar 3 Bone ash 7 Agar 3 Bone ash 7 Water 100 Bone ash 7 Water 100 Water 160 Estimated Estimated Estimated calories 430 calories 390 calories 470 Daily Averages 3.30 3.66 3.27 -0.39 19.2 0.36 1.86 89.0* PERIOD III (5 days) Meat Feeding Daily Averages 2.58 2.81 3.29 +0.48 12.8 0.23 1.81 93.0 * By an accident, about half the feces of this period were charred, giving rise to possible errors In analysis. From the accompanying brief summary it is apparent that the protein of the corn gluten is only slightly less well utilized than meat similarly fed. Summary of the Data on Nitrogen Utilization (see Tables 1-3). DOG CORN GLUTEN MEAT (AVERAGE) per cent per cent 5 89.7 93.3 6 89.9 93.5 6 90.7 93.5 7 89.0 92.2 35° Utilization of Corn Proteins Certain considerations lead us to believe that under ideal con- ditions the utilization of corn would compare even more favorably with that of meat. The proportion of protein to crude fiber in corn flour 1 is approximately 8:1. It is probable therefore that the corn gluten of our experiments had a crude fiber concentra- tion approximating 6 per cent. In the light of experiments to be reported in detail in a subsequent paper, it is conceivable that the cellulose, etc., thus included in the diet would in part account for the difference in the coefficients of digestibility of the proteins of corn and meat. TABLE 4. Utilization with Reference to Indigestible Materials in the Diet. Daily Averages. DOG PERIOD DAYS NATURE OF INGESTA FIBER IN OR ADDED TO FOOD TOTAL INDIGES- TIBLE MATERIAL IN FOOD N INTAKE N UTILIZA- TION AVER- AGE N UTILIZA- TION grams grams grams grams per cent per cent 5 XV* 4 1 Meat f 6 13 3.3 91.6 6 xvi* 4 > Bone ash 5 < 6 13 3.3 87.7 J 89.2 7 XV* 4 J Agar 2 [ 6 13 3.3 88.3 6 ii 5 1 Corn gluten 4 14t 4.9 89.9 6 iii 5 > Bone ash 7 I 4 14t 4.9 90.7 J 89.9 7 ii 4 J Agar 3 [ 3 I— « CO 3.3 89.0 * The details of these experiments appear in a subsequent paper in this series, t These values are low if anything, a certain amount of indigestible hemicelluloses probably being present. From Table 4 it is apparent that meat diets containing amounts of indigestible materials comparable to those in the corn gluten diets were no more thoroughly utilized than the corn gluten. From this point of view, the proteins of corn are evidently quite thoroughly utilized. There may be some doubt as to the validity of certain of the data in Tables 2 and 3 as is pointed out in foot-notes to the tables; but the data are in such close accord with others, about which there are no such uncertainties, that we are inclined to accept them as giving true pictures of the actual state of affairs. 1 See Atwater and Bryant: U. S. Department of Agriculture, Office of Experiment Stations, Bull. 28 (revised), p. 56, 1906. Lafayette B. Mendel and Morris S. Fine 351 A rather unsuccessful attempt was made to determine directly the influence of indigestible materials upon the utilization of corn proteins. Agar and bone ash were omitted from the food mixtures. (See Table 5.) A new sample of gluten was employed, toward which the animals acted very differently than they did toward the first sample. Dogs 5 and 6 vomited practically all their food, and dog 7 yielded results which cannot be accepted entirely without question, since, as pointed out in the foot-note to the table, a por- tion of the food here also was ejected. Here the utilization of the corn protein is decidedly inferior to that of meat, and even thus, the apparent utilization recorded is better than is actually the case, since not all the food noted in the table was submitted to diges- tion. Why the animals acted so differently toward these two samples of corn gluten, we are unable to explain. Although quite finely ground, the second sample was not an impalpable powder, and this may account for its low digestibility This explanation is not especially satisfactory to us TABLE 5. Corn Gluten without Agar and Bone Ash. SUBJECT, DOG 7 Weight at beginning, 6.6 kg. Weight at end, 6.6 kg. Composition of daily diet Nitrogen output. Urine nitrogen, gm Total nitrogen, gm Nitrogen in food, gm Nitrogen balance, gm Feces. Weight air dry, gm Nitrogen, gm Nitrogen per cent Nitrogen utilization, per cent PERIOD XXVII* (3 days) Corn Gluten Feeding grams grams Corn gluten 61 Meat 150 Sugar 25 Sugar 25 Lard 25 Lard 20 Water 225 Water 100 Estimated Estimated calories 450 calories 530 PERIOD XXVIII (5 days) Meat Feeding Daily Averages Daily Averages 4.38 3.60 4.78 3.75 4.76 4.64 -0.02 +0.89 10.7 3.8 0.41 0.15 3.82 3.82 91.4 96.9 | * For the first two days, the food was greedily eaten; on the last day a small part of the food had to be forced. About one-tenth of the food of the last day was vomited. 352 Utilization of Corn Proteins From Table 6 it is apparent that with one exception, negative balance resulted in the corn gluten periods while positive balances obtained throughout the meat periods. The one exception is that of Dog 5, and in this case it will be recalled that one-third of the daily nitrogen was supplied by meat. Henriques failed to estab- lish nitrogenous equilibrium with zein in rats TABLE 6. Average Daily Balances. — Nitrogen, in grams. SUBJECT TABLE GLUTEN PERIODS CORRESPONDING MEAT PERIODS Dog 5 1 +0.19 +0.16, +0.75 Dog 6 2 -0.34, -0.22 +0.77, +0.87 Dog 7 3 -0.39 +0.03, +0.48 Dog 7 5 -0.02 +0.89 SUMMARY Corn proteins, partially purified, were somewhat less thoroughly utilized than meat. Evidence was presented to indicate that this small difference may in great part be attributed to the cell resi- dues remaining in the corn preparation employed BIBLIOGRAPHY. Albertoni and Novi: Archiv fur die gesammte Physiologie, lvi, p. 213> 1894. Erismann: Zeitschrift fur Biologie, xlii, p. 672, 1901. Grandeau, LeClerc, and Ballacey: Quoted from Szumowski: Zeit- schrift fur physiologische Chemie, xxxvi, p. 198, 1902. Henriques: Zeitschrift filr physiologische Chemie, lx, p. 105, 1909. Hoppe: Milnchener medizinische Wochenschrift, xlix, p. 479, 1902. Laves: Milnchener medizinische Wochenschrift, p. 1339, 1900. Loewy und Pickart: Deutsche medizinische Wochenschrift, p. 821, 1900. Malfatti: Sitzungs-Berichte der Wiener Academie, xc, (3), p. 323, 1884. Merrill: Maine Agricultural Experiment Station, Bull. 131, 1906; Ibid., Bull. 158, 1908. Rockwood: American Journal of Physiology, xi, p. 355, 1904. Rubner: Zeitschrift fur Biologie, xv, p. 115, 1879. Sommerfeld: Archiv fiir Kinder heilkunde, xxxvi, p. 341, 1903. WintgExNt: Zeitschrift fiir Untersuchung der Nahrungs-und Genussmittel, v, p. 289, 1902. Reprinted from The Journal or Biological Chemistry, Vol. X, No. 6, 1912 STUDIES IN NUTRITION. IV. THE UTILIZATION OF THE PROTEINS OF THE LEGUMES. By LAFAYETTE B. MENDEL and MORRIS S. FINE. {From the Sheffield Laboratory of Physiological Chemistry, Yale University, New Haven, Connecticut.) (Received for publication, September 25, 1911.) CONTENTS. Earlier studies 433 Experimental part 435 Products employed 435 Metabolism experiments 437 Soy bean 437 White bean 446 Crude bean protein , 448 Phaseolin 454 Pea globulin 454 Nitrogen balances 456 Summary 457 EARLIER STUDIES. The literature on this subject has been so adequately reviewed by Wait, that only the most cursory consideration of the earlier work need find place here. In experiments on a man, Hoffmann found the nitrogen of a diet of lentils, bread and potatoes to be 58 per cent available, against a utilization of 82 per cent for the nitrogen of meat. Woroschiloff compared the utilization of the protein of peas with that of meat protein. In three cases the meat protein was 90, 92, and 96 per cent utilized against 83, 88, and 90 per cent for the digestibility of the protein of the peas. Striimpell found the nitrogenous constituents of "leguminose" — a finely ground commercial preparation, consisting of a mixture of lentils, peas and rye — to be 90 per cent available, against a utilization of 433 434 Utilization of Legume Proteins but 60 per cent in an experiment with unground lentils. Rubner has pointed out that in the experiments of both Woroschiloff and Strumpell, materials other than legumes were eaten, and these accessories may have exerted a favorable influence. Accordingly, Rubner conducted two experiments with thoroughly cooked hulled peas which were the only food consumed. The utilization was 72 to 83 per cent. In Malfatti's experiments, peas were 86 per cent utilized and Potthast found lentils to be 74 per cent digested. In an experiment by Prausnitz, white beans, soaked for several hours and then cooked till soft, yielded 70 per cent available nitrogen. Erismann found the nitrogen of peas to be 80 per cent digested. Richter obtained a utilization of 90 per cent for the nitrogen of peas cooked in distilled water, against 83 per cent when hard water was employed in the process of cooking. Under the latter condition particles of apparently unchanged peas were ob- served in the feces. The poor digestibility of the peas cooked in hard water is attributed in part to the formation of difficultly digestible alkali earth albuminates, and in part to the digestive disturbance due to the magnesium salts in the water. Snyder reported a utilization of 80 per cent for the protein of peas, and obtained a similar result with beans. In their experiments with the Maine lumbermen, Woods and Mansfield estimated the pro- tein of beans to be at least 78 per cent utilizable, and an average digestibility of 65 per cent is reported in Oshima's compilation of Japanese investigations. In a very thorough study, Wintgen found the average coefficients of digestibility of lentils, beans, and peas to be 78, 80 and 86 per cent respectively. Wintgen's results are in accord with those obtained in an extensive investigation by Wait, in which a utilization of 77 to 78 per cent was obtained for bean protein, and 70 to 83 per cent for cow pea protein. In commenting upon this literature one can but reiterate the statements made in a previous paper 1 of this series, and point out the necessity for studying the utilization of the isolated protein, or material in which the protein is more readily accessible to the digestive juices. 1 Mendel and Fine: This Journal, x, p. 303 ; 1911. Lafayette B. Mendel and Morris S. Fine 435 EXPERIMENTAL PART. Products Employed. 1 . Soy Bean. 1 This material was an impalpable yellow pow- der, which betrayed no cellular structure under the microscope. In respect to consistency, it would thus appear to be ideal for digestion experiments. As may be observed from the accompany- ing analysis, 2 the soy bean offers several points of interest : Its content of protein and fat far exceeds that of any other legume, which condition seems to have been appreciated in Japan ; for, according to Oshima, it is next to rice in importance in the Japanese dietary. 3 In addition to cane sugar, the presence of galactans and of pentosans has been detected by Schulze and his collaborators. 4 The soy bean does not give the ordinary iodine test for starch. 5 2. White Bean. This was the ordinary white bean of com- merce. 3. Crude Bean Protein. Experiments with the ordinary white bean are subject to the same criticism as has been offered in connection with the work of previous investigators. The attempt was here made to thoroughly rupture the cells and dissolve and wash away the starch. The method in brief was as follows: about 1 Mr. M. F. Deming of the Cereo Company, Tappan, N. Y., very kindly contributed this material. 2 Reported by Ruhrah: Journal of the American Medical Association, liv, p. 1664, 1910. 3 Oshima, (see bibliography) gives an interesting account of the various soy bean preparations, which are common articles of diet in Japan. 4 For the literature, see Schulze and Godet: Zeitschrift fur physiologischc Chemie, lxi, p. 279, 1909. 5 Cf. Oshima: loc. cit. p. 26. per cent Protein Fat. 44.6 19.4 9.3 4.2 2.3 5.3 14.8 Cane sugar Mineral matter Crude fiber Moisture Non-nitrogenous extract 436 Utilization of Legume Proteins 5 pounds of finely ground hulled beans 1 were mixed with water and heated in a glycerol bath. After the mixture had been held near 100° C. for about an hour, the thin mush which had formed was cooled below 75° C. and a glycerol extract of malt diastase added, as a result of which, after a few minutes, starch could no longer be detected with iodine in a test-tube trial. The material thus obtained was washed by decantation and the water driven off by heat until about 20 per cent was made up of solid matter. The resulting preparation was a thick mush, which could be conven- iently pressed into cakes and preserved frozen. Although no iodine test for starch was obtained in a test-tube trial, nevertheless, when a sample treated with this reagent was examined under the microscope, not infrequently starch grains were observed within cells, which had apparently not in any way been affected by the treatment to which they had been subjected. This insufficient rupture undoubtedly accounts for the incomplete conversion of the starch. ' Analysis of Crude Bean Protein (calculated for anhydrous material). per cent Protein (N X 6.25) 51 . 1 Sugar from insoluble carbohydrate (by hydrolysis) 28.9 Sugar from soluble carbohydrate (by hydrolysis) 2.4 Ash 2.6 Ether extract* 4.0 Crude fiber (by difference) 11 .0 ♦Estimated from Atwater and Bryant: U. S. Department of Agriculture, Bull. 28 (Revised), p. 65, 1906. Material was not available for analysis. Attention is called to the fact that a barley protein preparation 2 with approximately the same concentration of protein contained practically no cellular structure or starch, yet yielded 20 per cent of carbohydrate by hydrolysis. The latter was believed to be hemicelluloses. It is thus probable that a not inconsiderable portion of the " carbohydrate by hydrolysis" of the above analysis was in reality also made up of hemicelluloses. 3 1 Furnished by Mr. Deming who also prepared a considerable portion of the crude bean protein for us according to the method outlined. 2 Mendel and Fine: This Journal, x, p. 340, 1911. 3 At the time of proof reading we learn through a private communication from Prof. E. Schulze that hulled beans, phaseolus vulgaris, contain 12.9 per cent hemicellulose. We estimate the hemicellulose concentration of our preparation at approximately 25 per cent. Lafayette B. Mendel and Morris S. Fine 437 4. Phaseolin. This material was very kindly furnished by Dr. T. B. Osborne. It was dried, ground to an impalpable powder, and found by analysis to contain 13 per cent of nitrogen. 5. Pea Globulin. This material was prepared as follows: dried peas were finely ground and repeatedly extracted with 10 per cent NaCl solution. The perfectly clear extract thus obtained was saturated with ammonium sulphate, the resulting precipi- tate being collected on a filter paper, suspended in a small amount of water to which toluene had been added and dialyzed for about two weeks, that is, until free from sulphates. Part of the prepar- ation was obtained by dialyzing the saline extract, thus avoiding the necessity of the precipitation with ammonium sulphate. The resulting precipitate was dried at 40° to 50° C, ground to an im- palpable powder and found by analysis to contain 16 per cent of nitrogen. Metabolism Experiments. Soy Bean. Man, 1 Table 1: The ordinary routine was followed : a fore period (preceded by a three day adjustment period), during which a mixed diet was consumed; experimental period, in which over 90 per cent of the nitrogen ingested was furnished by soy bean; and an after period essentially like the fore period. The character of the diet is outlined below: Character of Diet. PRELIMINARY AND FORE PERIODS EXPERIMENTAL PERIOD AFTER PERIOD Daily Averages Daily Averages Daily Averages grams grams grams Cracker 70 70 Egg 100 200 Peanut butter 75 Meat 140 190 Soy bean 165 Potato 100 120 Tomato 250 375 200 Apple 200 200 200 Orange 180 180 180 Milk 60 60 60 Sugar 130 140 130 Butter 50 100 90 Cereal coffee, tea 600 600 1 The subject was one of us (M. S. F.) twenty-four years of age, leading the usual active life of the laboratory. 438 Utilization of Legume Proteins As will be observed, during the experimental period the cracker, egg, meat, and nut butter were completely replaced by soy bean, which furnished 91 per cent of the total nitrogen intake of this period. The daily nitrogen and calorific intakes in these periods were fairly constant, averaging about 12.6 grams and 2500 calories, respectively. The soy bean was boiled in water for one-half hour, salted to taste, and the tomatoes thoroughly incorporated into the resulting mush. The palatability of the mixture was still further increased by the addition of a very small amount of pap- rika. On the whole it may be said that this fare proved quice agreeable, no unpleasant symptoms appearing throughout the period of six days. TABLE 1. Soy Bean. SUBJECT, MAN PERIOD I PERIOD II PERIOD III ' Weight at beginning, 56.8 Kg. (5 clays) (6 days) (5 days) Weight at end, 56.6 Kg. Mixed Diet Soy Bean Mixed Diet Meat, egg, Soy bean, Meat, egg, po- nut butter, fruit, etc., tato, fruit, potato, 90.5 per cent etc. (No nut Composition of daily fruit, etc. total nitro- butter). gen supplied by soy bean. Estimated Estmated Estimated calories 2400 calories 2400 calories 2600 Nitrogen output. Daily Averages Daily Averages Daily Averages Urine nitrogen, gm 9.56 10.81 9.69 Total nitrogen, gm 11.11 12.72 11.16 Nitrogen in food, gm 12.78 12.93 12.22 Nitrogen balance, gm. . . . +1.67 +0.21 +1.06 Feces. Weight air dry, gm 25.0 26.6 25.2 Nitrogen, gm 1.55 1.91 1.47 Nitrogen, per cent 6.18 7.15 5.88 Nitrogen utilization, per cent 87.9 85.3 88.0 The subject felt in excellent condition throughout the entire experiment. Defecation took place regularly every morning and no diarrhoea occurred. Lafayette B. Mendel and Morris S. Fine 439 It will be observed from Table 1, that the soy bean nitrogen is distinctly (if only slightly) less well utilized than that of the preced- ing and succeeding mixed diets. The nitrogen concentration of the feces of the soy bean period is higher than in any other experi- ment on this subject which indicates that some soy bean protein escaped absorption. Soy Bean. Dogs (with agar and bone ash) — Tables 2 to 4 : Dog 1, Table 2, was fed with a mixture of soy bean, lard, agar, bone ash and water. It was heated on the water bath for four to six hours, the purpose being to thoroughly " hydrate" the material, which, as fed to the animal, was a thick mush. Dogs 5 and 7, Tables 3 and 4, were fed with similar ingredients and sugar in addition. The mixture, including the water, was not heated, but allowed to stand over night, after which the material appeared to be thoroughly "hydrated." The plan of experimentation differed in no particular from those previously followed, and we may therefore proceed directly to an examination of the tables, which contain all the essential details. In the dog, the soy bean was in every case strikingly less well util- ized than the meat fed under similar experimental conditions, and one also notes the persistently higher nitrogen concentration of the feces of the soy bean periods as compared with that of the meat- feces. This is an indication, as noted above, that some soy bean protein has probably escaped digestion. 44° Utilization of Legume Proteins TABLE 2. Soy Bean with Agar and Bone Ash. SUBJECT, DOG 1 Weight at beginning, 15.0 Kg. Weight at end, 14.6 Kg. PERIOD III (5 days) Meat Feeding PERIOD ivf (5 days) Soy Bean Feeding PERIOD V (4 days) Meat Feeding Composition of daily diet ' iv til uyvii viAiiputi, Urine nitrogen, gm Total nitrogen, gm Nitrogen in food, gm Nitrogen balance, gm. . . . Feces. Weight air dry, gm Nitrogen, gm Nitrogen, per cent Nitrogen utilization, per cent grams Meat 300 Lard 60 Agar* 5 Bone ash 15 Water 300 Estimated calories 1070 grams Soy bean 147 Lard 60 Agar 5 Bone ash 15 Water 500 Estimated calories 1110 grams Meat 300 Lard 60 Agar 5 Bone ash 15 Water 300 Estimated calories 1070 Daily Averages Daily Averages Daily Averages 10.07 10.60 10.38 -0.22 29.4 0.53 1.79 95.0 9.25 10.94 10.44 -0.50 55.6 1.69 3.05 83.8 8.81 9.38 10.44 +1.06 29.5 0.57 1.95 94.5 * On the first two days of the period, the "indigestible" was represented by 20 grams bone ash. This produced brittle feces, hence in the remaining three days agar and bone ash were em- ployed as noted in the table. t Forced feeding necessary throughout the period— no vomiting. TABLE 3. . Soy Bean with Agar and Bone Ash. SUBJECT, DOG 5 Weight at beginning, 5.2 Kg. Weight at end, 5.2 Kg. PERIOD VI (4 days) Meat Feeding PERIOD VII* (4 days) Soy Bean Feeding PERIOD VIII (5 days) Meat Feeding Composition of daily ( diet | Nitrogen output. Urine nitrogen, gm Total nitrogen, gm Nitrogen in food, gm Nitrogen balance, gm Feces. Weight air dry, gm Nitrogen, gm Nitrogen, per cent Nitrogen utilization, per grams Meat 150 Sugar 20 Lard 20 Agar 3 Bone ash 7 Water 100 Estimated calories 510 grams Soy bean 69 Sugar 20 Lard 25 Agar 3 Bone ash 7 Water 200 Estimated calories 570 grams Meat 150 Sugar 20 Lard 20 Agar 3 Bone ash 7 Water 100 Estimated calories 510 Daily Averages Daily Averages Daily Averages 3.71 4.11 4.93 +0.82 15.5 0.40 2.60 91.8 3.01 4.26 4.90 +0.64 34.0 1.25 3.67 74.5 4.09 4.44 4.80 +0.36 15.0 0.35 2.32 92.7 Cystitis developed but was cured in the course of two days by means of AgNOj solution. Lafayette B. Mendel and Morris S. Fine 441 TABLE 4. Soy Bean with Agar and Bone Ash. SUBJECT, DOG 7 PERIOD V PERIOD VI PERIOD VII Weight at beginning, 4.9 Kg. (3 days) (6 days) (5 days) Weight at end, 4.6 Kg. Meat Feeding Soy Bean Feeding Meat Feeding QTdTilS QTC177XS QTQ7718 Meat 100 Soy bean 47 Meat 100 Sugar 20 Sugar 20 Sugar 20 Lard 20 Lard 25 Lard 20 Composition of daily Agar 3 Agar 3 Agar 3 diet 1 Bone ash 7 Bone ash 7 Bone ash 7 Water 100 Water 175 Water 100 Estimated Estimated Estimated calories 430 calories 490 calories 430 Nitrogen output. Daily Averages Daily Averages Daily Averages Urine nitrogen, gm O AO Z.bo o oo 2.00 9 «Q z . oy 6 . IHt 9 7Q z . /y Nitrogen in food, gm 3.29 3.34 3.20 Nitrogen balance, gm. . . . +0.40 +0.30 +0.41 Feces. Weight air dry, gm 12.7 20.5 12.8 Nitrogen, gm 0.26 0.66 0.24 Nitrogen, per cent 2.04 3.22 1.86 Nitrogen utilization, per cent 92.1 80.2 92.6 Soy Bean. Dogs (without agar and bone ash) — Tables 5 to 18: These experiments were conducted in essentially the same manner as those just reported, except that the indigestible adjuvants — agar and bone ash — were omitted. Tables 8 to 10 contain the results of trials instituted after the intestinal tract had been sub- jected to a thorough treatment with indigestible non-nitrogenous materials, the purpose being to remove as far as possible the accu- mulated intestinal debris. Proceeding directly to a study of the tables, we again note the poor utilization of the soy bean nitrogen. A fuller discussion of these data with a consideration of the attending conditions will be offered below 1 in connection with the discussion of the results obtained with the crude bean protein. In Oshima's compilation one notes that certain soy bean products {e.g., tofu) are as much as 96 per cent utilizable. Tofu, however, is probably of an albumose nature and such favorable results should be correspondingly interpreted. 1 P. 452. 44 2 Utilization of Legume Proteins TABLE 5. Soy Bean without Agar or Bone Ash. SUBJECT, DOG 5 Weight at beginning, 5.9 Kg. Weight at end, 6.0 Kg. PERIOD XX (4 days) Meat Feeding r Hi IXIKJU AAl (5 days) Soy Bean Feeding Composition of daily diet < Nitrogen output. Urine nitrogen, gm Total nitrogen, gm Nitrogen in food, gm Feces. Weight air dry, gm Nitrogen, gm Nitrogen, per cent Nitrogen utilization, per cent grams Meat 150 Sugar 25 Lard 20 Water . 100 Estimated calories 530 grams Soy bean 64 Sugar 25 Lard 20 Water 225 Estimated calories 530 Daily Averages Daily Averages 4.08 4.30 4.59 +0.29 4.5 0.22 4.95 95.2 3.96 4.66 4.61 -0.05 17.8 0.70 3.90 85.0 One-quarter to one-half of the food forced each day. TABLE 6. Soy Bean without Agar or Bone Ash. SUBJECT, DOG 6 Weight at beginning, 6.1 Kg. Weight at end, 6.3 Kg. PERIOD XXI (4 days) Meat Feeding PERIOD XXII (5 days) Soy Bean Feeding Composition of daily diet < Nitrogen output. Total nitrogen, gm Nitrogen balance, gm Feces. Weight air dry, gm Nitrogen, gm Nitrogen, per cent Nitrogen utilization, per cent grams Meat 150 Sugar 25 Lard 20 Water 100 Estimated calories 530 grams Soy bean 64 Sugar 25 Lard 20 Water 225 Estimated calories 530 Daily Averages Daily Averages 3.55 3.77 4.59 +0.82 3.5 0.22 6.34 95.2 3.42 4.16 4.61 +0.45 18.2 0.74 4.04 84.0 Lafayette B. Mendel and Morris S. Fine 443 TABLE 7. Soy Bean without Agar or Bone Ash. SUBJECT, DOG 7 Weight at beginning, 5.9 Kg. Weight at end. 6.3 Kg. PERIOD XX (4 days) Meat Feeding PERIOD XXI (5 days) Soy Bean Feeding Nitrogen output. Urine nitrogen, gm Feces. Weight air dry, gm Nitrogen, gm Nitrogen, per cent grams Meat 150 Sugar 25 Lard 20 Water 100 Estimated calories 530 grams Soy bean 64 Sugar 25 Lard 20 Water 225 Estimated calories 530 Daily Averages Daily Averages 3.55 3.74 4.59 +0.85 3.2 0.19 5.93 95.8 3.41 4.21 4.61 +0.40 16.2 0.80 4.91 82.8 TABLE 8. Soy Bean without Agar or Bone Ash. SUBJECT, DOG 5 Weight at beginning, 6.2 Kg. Weight at end, 6.2 Kg. PERIOD XXVI* (3 days) Soy Bean Feeding PERIOD XXVIII (5 days) Meat Feeding Composition of daily diet < Nitrogen output Total nitrogen, gm Nitrogen in food, gm Nitrogen balance, gm Feces. Weight air dry, gm.* Nitrogen, gm Nitrogen, per cent grams Soy bean 64 Sugar 25 Lard 20 Water 225 Estimated calories 530 grams Meat 150 Sugar 25 Lard 20 Water 100 Estimated calories 530 Daily Averages Daily Averages 3.66 4.31 4.61 +0.30 18.5 0.65 3.52 85.9 3.68 3.84 4.64 +0.80 3.4 0.16 4.62 96.6 * About half of the food forced each day. 444 Utilization of Legume Proteins TABLE 9. Soy Bean without Agar or Bone Ash. SUBJECT, DOG 6 Weight at beginning 6.6 Kg. Weight at end, 6.6 Kg. PERIOD XXVII (3 days) Soy Bean Feeding PTRTflTl YTTY jrSU±\l.\JU AAiA (5 days) Meat Feeding Composition of daily diet < Nitrogen output. Urine nitrogen, gm Total nitrogen, gm Nitrogen in food, gm Nitrogen balance, gm Feces. Nitrogen, gm Nitrogen, per cent Nitrogen utilization, per cent Soy bean 64 Sugar 25 Lard 20 Water 225 Estimated calories 530 Meat 150 Sugar 25 Lard 20 Water 100 Estimated calories 539 Daily Averages Daily Averages 3.45 4.06 4.61 +0.55 16.7 0.61 3.64 86.8 3.35 3.59 4.64 +1.05 4.4 0.24 5.42 94.9 TABLE 10. Soy Bean without Agar or Bone Ash. SUBJECT, DOG 7 Weight at beginning, 6.6 Kg. Weight at end, 6.6 Kg. PERIOD XXVI (4 days) Soy Bean Feeding PERIOD XXVIII (5 days) Meat Feeding Composition of daily diet < Nitrogen output. Feces. Weight air dry, gm Nitrogen, gm Nitrogen, per cent Nitrogen utilization, per cent grams Soy bean 64 Sugar 25 Lard 20 Water 225 Estimated calories 530 grams Meat 150 Sugar 25 Lard 20 Water 100 Estimated calories 530 Daily Averages Daily Averages 3.78 4.35 4.61 +0.26 15.9 0.57 3.60 87.6 3.60 3.75 4.64 +0.89 3.8 0.15 3.82 96.9 Lafayette B. Mendel and Morris S. Fine 445 TABLE 11. Soy Bean without Agar or Bone Ash. SUBJECT, DOG 5 Weight at beginning, 6.0 Kg. Weight at end, 5.9 Kg. PERIOD XVI (4 days) Meat Feeding PERIOD XVII (4 days) Soy Bean Feeding Composition of daily diet < Nitrogen output. Urine nitrogen, gm Total nitrogen, gm Nitrogen in food, gm Nitrogen balance, gm Feces. Weight air dry, gm Nitrogen, per cent Nitrogen utilization, per cent grams Meat 100 Sugar 25 Lard 20 Water 150 Estimated calories 450 grams Soy bean 46 Sugar 25 Lard 20 Water 225 Estimated calories 460 Daily Averages Daily Averages 2.77 2.79 3.28 +0.49 0.4 0.02 5.32 99.4* 2.83 3.48 3.31 -0.17 14.0 0.65 4.67 80.2 * Feces evidently separated imperfectly. TABLE 12. Soy Bean without Agar or Bone Ash. SUBJECT, DOG 6 Weight at beginning, 6.2 Kg. Weight at end, 6.1 Kg. PERIOD XVII (4 days) Meat Feeding PERIOD XVIII (4 days) Soy Bean Feeding Composition of daily diet < Nitrogen output. Urine nitrogen, gm Total nitrogen, gm Nitrogen in food, gm Feces. Weight air dry. gm Nitrogen, gm Nitrogen, per cent Nitrogen utilization, per cent grams Meat 100 Sugar 25 Lard 20 Water 150 Estimated calories 450 grams Soy bean 46 Sugar 25 Lard 20 Water 225 Estimated calories 460 Daily Averages Daily Averages 2.41 2.54 3.28 +0.74 1.9 0.13 7.03 96.0 2.49 3.21 3.31 +0.10 14.0 0.72 5.15 79.3 446 Utilization of Legume Proteins TABLE 13. Soy Bean without Agar or Bone Ash. SUBJECT, DOG 7 Weight at beginning, 6.0 Kg. Weight at end, 5.9 Kg. PERIOD XVI (4 days) Meat Feeding PERIOD XVII (4 days) Soy Bean Feeding Composition of daily diet < Nitrogen output. Total nitrogen, gm Nitrogen in food, gm Feces. Weight air dry, gm Nitrogen, gm Nitrogen, per cent Nitrogen utilization, per cent grams Meat 100 Sugar 25 Lard 20 Water 150 Estimated calories 450 grams Soy bean 46 Sugar 25 Lard 20 Water 225 Estimated calories 460 Daily Averages Daily Averages 2.85 2.95 3.28 +0.33 1.5 0.1 j 7.06 96.8 2.83 3.37 3.31 -0.06 11.0 0.54 4.77 83.8 White Bean. Man, Table H: The original intention was to investigate the digestibility of hulled beans which had been finely ground and thoroughly cooked, and in which the starch had in great part been dissolved by an amylolytic preparation. This mixture, however, produced such violent nausea that a successful experiment with it was entirely out of the question. Indeed other workers, notably Strumpell, have reported similar difficulties with bean experiments. However, the subject was unwilling to have the fore period stand for naught, so the experiment was carried through using ordinary unhulled beans, which were cooked or baked in the usual way. In this form the beans were far from unpalatable. The character of the dietary employed in this experiment is given below: Lafayette B. Mendel and Morris S. Fine 447 PRELIMINARY AND PORE PERIODS EXPERIMENTAL PERIOD Dally Averages Dally Averages grams grams 70 35 Egg 200 90 Meat 200 Beans 230 100 250 300 Apple 200 200 Orange 180 180 120 140 Milk 60 60 Sugar 120 130 Butter 75 80 Cereal coffee, tea 600 600 TABLE 14. White Bean. SUBJECT, MAN Weight at beginning, 56.4 Kg. Weight at end, 56.0 PERIOD I (4 days) Mixed Diet PERIOD II (4 days) White Bean Composition of daily diet < Nitrogen output. Urine nitrogen, gm Total nitrogen, gm Nitrogen in food, gm Nitrogen balance, gm Feces. Weight, air dry, gm Nitrogen, gm Nitrogen, per cent Nitrogen utilization, per cent Meat, egg, pota- to, fruit, etc. Estimated calories 2600 Beans, egg, fruit, etc. 68.2 per cent of total nitrogen supplied by the beans. Estimated calories 2700 Daily Averages Dally Averages 9.19 10.90 12.25 +1.35 26.2 1.71 6.54 86.0 8.00 10.70 12.20 +1.50 44.2 2.70 6.11 77.9 The plan of the experiment differed in no essential from those on man already reported. The preliminary period of adjustment was only one day in duration, and the after period was entirely 443 Utilization of Legume Proteins omitted. The results are in general accord with those obtained by previous observers. The factors which probably contribute to the unfavorable util- ization of the protein of beans have already been discussed. 1 The plan was conceived of avoiding these unfavorable influences as far as possible, and to that end, as already described, 2 hulled and powdered beans were thoroughly cooked, and the greater part of the starch removed. Experiments with this material follow. Crude Bean Protein 3 — Dogs, Tables 15 to 21: The material was not dried; but when in the stage of evaporation the proportion of solid matter became about 20 per cent, it was pressed into pack- ages containing the daily supply of protein, and preserved frozen until ready for use. In some cases, no additional water was given, as the moisture of the product sufficed. Such details may be most readily learned from the tables. In the experiments reported in Tables 15 to 17, the food mixture was of the consistency of putty and made a rather large volume of material. The animals experienced some difficulty in chewing because of the unusual consistency, but in spite of this Dogs 6 and 7 appeared to relish the fare; and even Dog 5, upon whom forced feeding had to be practiced, did not seem to find the food especially repellent. It was thought that possibly this large mass of food overburdened the digestive tract thus accounting for the rather poor utilization, and hence experiments reported in Tables 18 to 20 were instituted, where the food nitrogen was only two-thirds as great as in the pre- ceding three experiments. In all the above experiments, agar and bone ash were included in the diet, and it was therefore desired to eliminate the influence of these "indigestibles" for comparison. Hence the experiment recorded in Table 21. 1 Cf. Mendel and Fine: This Journal, x, p. 305, 1911. 2 Pp. 435-436. 3 As far as we are aware the only experiments with similar material were conducted by Edsall and Miller (see bibliography) on infants and on man. The infants digested 90 per cent or over of the nitrogen in the bean periods and the man utilized 94 per cent. However, the bean protein fur- nished but 25 per cent of that of the infant's food and only about 12 per cent of the protein in the man's dietary, whereas in our experiments all the pro tein was supplied by the bean preparation. Lafayette B. Mendel and Morris S. Fine 449 TABLE 15. Crude Bean Protein with Agar and Bone Ash. SUBJECT, DOG 5 Weight at beginning, 6.5 Kg. Weight at end, 6.2 Kg. PERIOD X (4 days) Meat Feeding PERIOD XI* (3 days) Bean Protein Feeding Composition of daily diet < Nitrogen output. Urine nitrogen, gm Total nitrogen, gm Nitrogen in food, gm Nitrogen balance, gm Feces. Weight air dry, gm Nitrogen, gm Nitrogen, per cent Nitrogen utilization, per cent grams Meat 150 Sugar 20 Lard 20 Agar 2 Bone ash 5 Water 100 Estimated calories 520 grams Bean protein 300 Sugar 20 Lard 25 Agar 2 Bone ash 5 Water (contain- ed in the bean protein) 240 Estimated calories 560 D&ily .A. vcrsi^cs 4.65 4.97 5.23 +0.26 11.0 0.32 2.95 93.8 4.24 5.08 5.10 +0.02 39.3 0.84 2.15 83.4 * Forced feeding necessary throughout the period. TABLE 16. Crude Bean Protein with Agar and Bone Ash. SUBJECT, DOG 6 Weight at beginning, 7.1 Kg. Weight at end, 6.8 Kg. PERIOD XI (4 days) Meat Feeding PERIOD XII (3 days) Bean Protein Feeding Composition of daily diet < Nitrogen output. Urine nitrogen, gm Total nitrogen, gm Nitrogen in food, gm Nitrogen balance, gm Feces. Weight air dry, gm Nitrogen, gm Nitrogen, per cent Nitrogen utilization, per cent grams Meat 150 Sugar 20 Lard 20 Agar 2 Bone ash 5 Water 100 Estimated calories 520 grams Bean protein 300 Sugar 20 Lard 25 Agar 2 Bone ash 5 Water (contain- ed in the bean protein) 240 Estimated calories 560 Daily Averages Daily Averages 3.84 4.20 5.23 +1.03 9.2 0.36 3.91 93.1 1 3.62 4.59 5.10 +0.51 35.3 0.97 2.75 81.0 450 Utilization of Legume Proteins TABLE 17. Crude Bean Protein with Agar and Bone Ash. SUBJECT, DOG 7 Weight at beginning, 6.7 Kg. Weight at end, 6.4 Kg. PERIOD X (4 days) Meat Feeding PERIOD XI (3 days) Bean Protein Feeding Composition of daily diet < Nitrogen output. Urine nitrogen, gm Total nitrogen, gm Nitrogen in food, gm Nitrogen balance, gm Feces. Weight air dry, gm Nitrogen, gm Nitrogen, per cent Nitrogen utilization, per cent grams Meat 150 C? mm**** -mm OA sugar ZD Lard 20 Agar 2 Bone ash 5 Water 100 Estimated calories 520 grams Bean protein 300 ougar zU Lard 25 Agar 2 Bone ash 5 Water (contain- ed in the bean protein) 240 Estimated calories 560 Daily Averages Daily Averages 3.84 4.16 5.23 +1.07 10.0 0.32 3.20 93.9 3.51 4.37 5.10 +0.73 36.3 0.86 2.38 83.1 TABLE 18. Crude Bean Protein with Agar and Bone As) i. SUBJECT, DOG 5 Weight at beginning, 6.2 Kg. Weight at end, 5.9 Kg. PERIOD XII (4 days) Meat Feeding PERIOD XIII* (4 days) Bean Protein Feeding PERIOD XIV (4 days) Meat Feeding Compostion of daily diet ' Nitrogen output. Urine nitrogen, gm Total nitrogen, gm Nitrogen in food, gm Nitrogen balance, gm Feces. Weight air dry, gm Nitrogen, gm Nitrogen, per cent Nitrogen utilization, pei grams Meat 100 Sugar 25 Lard 20 Agar 2 Bone ash 5 Water 150 Estimated calories 450 grams Bean pro- tein 200 Sugar 25 Lard 25 Agar 2 Bone ash 5 Water (160 gm. in the bean protein) 225 Estimated calories 480 grams Meat 100 Sugar 25 Lard 25 Agar 2 Bone ash 5 Water 150 Estimated calories 450 Daily Averages Daily Averages Dally Averages 3.15 3.43 3.49 +0.06 11 0.28 2.59 91.8 3.00 3.54 3.34 -0.20 26.5 0.54 2.05 83.8 2.64 2.90 3.28 +0.38 11.5 0.26 2.27 92.1 * Forced feeding necessary throughout the period. TABLE 19. Crude Bean Protein with Agar and Bone Ash. SUBJECT, DOG 6 Weight at beginning, 6.6 Kg. Weight at end, 6.3 Kg. PERIOD XIII (4 days) Meat Feeding PERIOD XIV (4 days) Bean Protein Feeding PERIOD XV (4 days) Meat Feeding Composition of daily diet I Nitrogen output. Urine nitrogen, gm Nitrogen in food, gm Nitrogen balance, gm. . . . Feces. Weight air dry, gm Nitrogen, gm Nitrogen, per cent Nitrogen utilization, pei cent grams Meat 100 Sugar 25 T.nrH 90 1 Jill < l ZA) Agar 2 Bone ash 5 Water 150 Estimated calories 450 grams Bean pro- tein 200 Sugar 25 UnrH 9 Bone ash, 5 grams I 6 13 3.3 87.7 89.2 7 XV 4 J Agar, 2 grams [ 6 13 3.3 88.3 * This problem will be treated in detail in a subsequent paper of this series. t Including respectively about 5 grams and 3 grams hemicelluloses which escaped digestion. t Including approximately 4 grams Indigestible hemicelluloses (exclusive of agar), i.e., the aver- age of 5 grams and 3 grams. An actual determination of the hemicelluloses of the feces of this experiment was not made. 8 Cf. Crawford: Journ. of Pharm. and Exper. Ther., i, No. 5, p. 519, 1910. Reprinted from The Journal ok Biological Chrmistry, Vol. XI, No. I, 1912 STUDIES IN NUTRITION. VI. THE UTILIZATION OF THE PROTEINS OF EXTRACTIVE-FREE MEAT POWDER; AND THE ORIGIN OF FECAL NITROGEN. By LAFAYETTE B. MENDEL and MORRIS S. FINE. {From the Sheffield Laboratory of Physiological Chemistry, Yale University, New Haven, Connecticut.) (Received for publication, September 25, 1911.) CONTENTS. The utilization of the proteins of extractive-free meat powder 5 Earlier studies with this and related materials 5 Experimental part 6 Product employed 6 Metabolism experiments 6 On the origin of fecal nitrogen 10 Earlier studies 11 Experimental part 15 Influence of indigestible non-nitrogenous materials upon the nitrogen statistics of the feces •. 15 Influence of poorly utilized highly nitrogenous matter upon the nitrogen statistics of the feces 18 Simultaneous influence of both types of materials upon the nitrogen statistics of the feces 19 Effect of previous thorough evacuation upon utilization 21 Estimation of "metabolic" products in the feces 21 The utilization of the vegetable proteins 23 The Utilization of Extractive-Free Meat Powder. earlier studies. Forster was the first to conduct an investigation with this mate- rial. His immediate problem was the question of salt metabolism, but incidentally we note that the nitrogen was 91 to 96 per cent available. During the past twenty years, considerable attention has been paid to the comparative utilization of fresh meat and S 6 The Utilization of Proteins dried meat preparations, for example, "soson," "somatose," "tropon," and the meat residues from meat extract factories. Passing over the literature previous to 1901, we may dwell briefly upon the results obtained by Prausnitz, which are in general accord with those of the earlier workers. The average coefficient of digestibility of dried meat was 90 per cent against a coefficient of 93 per cent for fresh meat. Moreover the nitrogen concentra- tion of the dried-meat-feces was 1.35 to 1.76 per cent higher than the fresh meat feces. These facts make it probable that a portion of the dried meat had escaped absorption. Prausnitz also showed that dried meat was less readily digested in artificial gastric juice than fresh meat. He accounted for these phenomena on the assumption that a not inappreciable length of time elapses before the dried meat particles are sufficiently "hydrated" to permit the digestive enzymes to operate. Max Voit found similar although less striking differences. Considerable work has also been accomplished with dried blood preparations, but a consideration of these investigations would lead us too far afield. EXPERIMENTAL PART. Product Employed. The meat residue 1 employed in the present studies was a light brown impalpable powder, containing 13.2 per cent of nitrogen, 8.9 per cent of ether extract, 2.5 per cent of ash, and 7.0 per cent of moisture. Metabolism Experiments. Tables 1-3. During these experiments, the methods described in a previous paper 2 were followed. The utilization of the nitrogen of meat powder is distinctly, although slightly lower than that of fresh meat. The relatively high nitrogen concentration of the meat powder feces is indicative of a loss of this material through the excrement. These points are concisely presented in the accompanying brief tabular summary. 1 Obtained from Armour and Company. 2 Mendel and Fine: This Journal, x, p. 303, 1911. Lafayette B. Mendel and Morris S. Fine 7 Summary of the Data on Nitrogen Utilization (see Tables 1-8) MEAT POWDER FRESH MEAT (AVERAGES) DOG Nitrogen utilization Nitrogen in feces Nitrogen utilization Nitrogen in feces 1 4 4 per cent 91.3 89.3 91.0 per cent 2.98 3.81 3.87 per cent 94.0 94.5 93.7 per cent 1.94 2.04 2.36 TABLE 1. Extract-free Meat Powder. SUBJECT, DOG 1 Weight at beginning, 14.6 kg. Weight at end, 14.6 kg. period v (4 days) Meet Feeding PERIOD VI* (5 days) Meat Powder Feeding PERIOD VII (4 days) Meat Feeding Composition of daily diet I Nitrogen output. Urine nitrogen, gm Total nitrogen, gm Nitrogen in food, gm Nitrogen balance, gm Feces. Weight air dry Nitrogen, gm Nitrogen, per cent Nitrogen utilization, per cent grams Meat 300 Lard 60 Agar 5 Bone ash 15 Water 300 Estimated calories 1070 grams Meat Pow der Lard Agar Bone Ash Water 80 60 5 15 500 Daily Averages 8.81 9.38 10.44 +1.06 29.5 0.57 1.95 94.5 Estimated calories 860 Daily Averages 8.76 9.68 10.53 +0.85 31.0 0.92 2.98 91.3 Meat grams 300 60 Lard Agar 5 Bone Ash 15 Water 300 Estimated calories 1070 Daily Averages 8.47 9.15 10.46 +1.31 35.2 0.68 1.92 93.5 * Food almost entirely forced. 8 The Utilization of Proteins TABLE 2. Extract-free Meat Powder. SUBJECT, DOG 4 Weight at beginning 4.9 kg. Weight at end, 5.1 kg. PERIOD V (4 days) Meat Feeding PERIOD VI (5 days) Meat Powder Feeding PERIOD VII (4 days) Meat Feeding Composition of daily diet. < Nitrogen output. Total nitrogen, gm Nitrogen in food, gm Nitrogen balance, gm Feces. Weight air dry, gm Nitrogen, gm Nitrogen, per cent Nitrogen utilization, per cent grams Meat loU Sugar 25 Starch 5 Lard 20 Bone Ash 10 Water 200 Estimated calories 570 grams Meat Powder 39 Sugar 25 Starch 5 Lard 25 Agar 8 "Salts" 4 Water 260 Estimated calories 510 grams Meat 150 Sugar 25 Starch 5 Lard 20 Agar 8 "Salts" 4 Water 200 Estimated calories 570 Daily Averages Daily Averages Daily Averages 4.50 5.40 +0.90 15.0 0.27 1.79 95.0 o . yo 4.50 5.13 +0.63 14.4 0.55 3.81 89.3 4.70 5.40 +0.70 14.0 0.32 2.29 94.1 Lafayette B. Mendel and Morris S. Fine TABLE 3. Extract-free Meat Powder. SUBJECT, DOG 4 Weight at beginning, 5.1 kg. Weight at end, 5.2 kg. Composition of daily diet. Nitrogen output. Urine nitrogen, gm Total nitrogen, gm Nitrogen in food, gm. . . Nitrogen balance, gm. . Feces. Weight air dry, gm Nitrogen, gm Nitrogen, per cent Nitrogen utilization, cent per PERIOD XI (5 days) Meat Feeding 4.28 4.62 5.20 +0.58 12.4 0.34 2.77 93.4 PERIOD XII (5 days) Meat Powder Feeding grams grams grams Meat 150 Meat Powder 40 Meat 150 Sugar 25 Sugar 25 Sugar 25 Starch 5 Starch 5 Starch 5 Lard 20 Lard 25 Lard 20 Agar 8 Agar 8 Agar 4 "Salts" 4 "Salts" 4 Bone Ash 8 Water 200 Water 300 Water 200 Estimated Estimated Estimated calories 570 calories 510 calories 570 Daily Averages Daily Averages Daily Averages 4.51 4.98 5.26 +0.28 12.3 0.47 3.87 91.0 PERIOD XIII (4 days) Meat Feeding 4.46 4.77 5.22 +0.45 16.0 0.31 1.96 94.0 IO The Utilization of Proteins On the Origin of Fecal Nitrogen. In previous papers 3 of this series we have followed the current custom of basing the data for nitrogen utilization upon the rela- tion of the nitrogen appearing in the excrement to that of the ingesta. This procedure would be strictly correct only in case the fecal nitrogen consisted entirely of food residues. As a matter of fact, there is abundance of evidence in the literature to demon- strate that fecal nitrogen in great part emanates from " metabolic products." 4 Obviously an adequate understanding of the source of fecal nitrogen and the conditions influencing its excretion is essential for the proper interpretation of experiments on nitrogen utilization. In the earlier papers referred to we have at times pointed out that an apparently poor utilization was probably in- duced by the indigestible matter — cellulose, hemicellulose — inher- ent in the experimental material. The influence of such materials upon utilization has not always been fully appreciated. Rubner, and later Wicke, did indeed call attention to the unfavorable effect of cellulose upon the utilization of bread nitrogen; but in these cases it is difficult to decide in what measure the insufficiently ruptured cells are responsible for the low coefficients of digesti- bility, and to what extent the latter is to be attributed to the cel- lulose per se. This question is not satisfactorily answered by the poor utilization of meat obtained by Hoffmann when coarsely cut straw was added to the diet. Such coarse particles probably unduly irritated the digestive tract, resulting in increased secre- tion and peristalsis. Lothrop demonstrated an increased elimi- nation of fecal nitrogen when bone ash was added to the diet. In the present paper the nitrogen of the excrement under a variety of conditions is discussed briefly from the historical aspect; 5 data purporting to show to what extent indigestible non-nitroge- nous substances may influence the amount and character of the feces are presented; and a plan of experimentation is proposed, 3 See footnotes 20-24, pp. 23 and 24. 4 By this term is understood intestinal secretions, cast off cells, bacteria, etc. For a consideration of the important role of bacteria in this respect and the literature related thereto, see MacNeal, Latzer and Kerr: Journ. of Infect. Dis., vi, p. 123, 1909. 5 For a more detailed review reference is made to Tsuboi (see bibli- ography). Lafayette B. Mendel and Morris S. Fine n with which it seems possible to approximately determine to what degree the nitrogen excreted in the feces is derived from undigested or indigestible nitrogenous constituents of the ingesta. Were this known, the term " utilization" would be eminently appropriate. EARLIER STUDIES. Feces in Starvation. Man. The accompanying table presents oft quoted data 6 obtained from the professional fasters, Cetti and Breithaupt, and from certain patients. Daily Nitrogen Excreted through the Feces in Starvation. gram Cetti . 0.32 Breithaupt 0.12 Patient (stenosis of oesophagus) 0.45 Neurasthenic 0.22 Neurasthenic 0.17 Average 0.26 Dogs. Bidder and Schmidt, and Voit early observed that during starvation black pitch-like feces were obtained from dogs. The latter obtained daily 2 grams of feces (= 0.15 gram of nitrogen) from a dog of 30 kilos. The studies of Miiller offer further illus- trative data. Daily Feces Obtained from Starving Dogs (Miiller, 1884). BODY WEIGHT FECES WEIGHT DRY FECAL NITROGEN FECAL NITROGEN PER KILO BODY WEIGHT kilo grams per cent grams gram 43 4.8 5.0 0.24 0.005G 30 2.4 8.0 0.19 0.0063 30 1.4 8.0 0.11 0.0037 23 2.8 5.3 0.15 0.0065 7 0.7 7.5 0.05 0.0071 0.0058 6 Taken from Schmidt and Strasburger (see bibliography), p. 115. The Utilization of Proteins Benedict has pointed out that the amount of feces formed during starvation is probably much smaller than is indicated by earlier studies. Fasting feces are in great part derived from retained fecal matter, resulting from the food immediately preceding the period of inanition. This is owing to diminished peristalsis con- sequent upon the withdrawal of food. With Nitrogen-Free Diets. The accompanying table embodies results obtained by Rieder. Nitrogen Eliminated through Feces on Nitrogen-free Diet (Rieder). SUBJECT FECES WEIGHT DRY FECAL NITROGEN FOOD grams per cent gram Man 13.4 4.08 0.54 485 grams cakes of starch, sugar and fat. Man 15.4 5.69 0.87 159 grams cakes of starch, sugar and fat. Man 13.4 5.85 0.78 147 grams cakes of starch, sugar and fat. Dog 3.0 3.67 0.11 70 grams starch. Dog 6.0 3.85 0.22 140 grams starch. Rubner (1879) reported similar results. Tsuboi fed dogs for periods of six to nine days on cakes made of starch, fat and sugar, and obtained data, which are in accord with the above. Nitrogen Eliminated through Feces on Nitrogen-free Diet (Tsuboi). FECES WEIGHT DRY FECAL NITROGEN FOOD Starch Sugar Fat grams per cent gram grams grams grams 2.6 5.1 0.14 5.8 4.1 0.24 70 12 50 12.9 4.4 0.57 200 25 80 There can of course be no question as to the source of fecal nitrogen in the above experiments. Lafayette B. Mendel and Morris S. Fine 13 With Meat Diets. The most interesting work bearing upon the nitrogen of the feces obtained with meat diets and the relation of the amount of meat ingested to the nitrogen thus eliminated was contributed by Muller. Influence of Meat Diet on Fecal Nitrogen in Dogs (30-35 kilos) (Muller). MEAT FECES WEIGHT DRY FECAL NITROGEN NITROGEN UTILIZED grams grams per cent gram per cent 2.0 7.96 0.15 500 5.1 6.50 0.30 98.2 1000 9.2 6.50 0.55 9.8.4 1500 10.2 6.50 0.67 98.7 1800 10.3 6.50 0.70 98.9 2000 11.1 6.50 0.80 98.8 2500 15.4 6.50 1.00 98.8 It is clear from this summary that the nitrogen of the feces does not increase in proportion to the amount of meat eaten. That the fecal nitrogen incident to a meat diet is essentially of metabolic origin, 7 is very convincingly brought out by Fritz Voit. After a loop of the intestine had been isolated, a dog was fed with meat. It was found that the contents of the loop resem- bled the feces in appearance and nitrogen content. Moreover, when calculated to unit surface the absolute amount of dry sub- stance in the loop compared favorably with that of the feces. Equally significant is the recent study of Mosenthal, who also worked with isolated intestinal loops. This author estimated that the succus entericus contained nitrogen equivalent to 35 per cent of the nitrogen ingested, and 300 to 400 per cent of the nitrogen of the feces. Nitrogen equivalent to at least 25 per cent of that of the intake must therefore have been reabsorbed. From the foregoing there can be no doubt that the feces resulting from a thoroughly digestible food such as meat are almost solely of "metabolic origin." Prausnitz has attempted to give this more widespread application. 7 By an ingenious microscopical method, Kermauner (see bibliography) showed that in man but one per cent or less of the ingested meat reappeared in the feces. j 4 The Utilization of Proteins Composition of Feces on Various Diets (Prausnitz). FECES — DRY NUMBER PERSON MAIN FOOD Nitrogen Ether Extract Ash per cent per cent per ccTit 1 H. Rice 8.83 12 A 15.4 2 H. Meat 8.75 16.0 14.7 3 M. Rice 8. 37 18.2 11.0 4 M. Meat 9. 16 16.0 12.2 5 W.P. Rice 8.59 15.9 12.6 6 W.P. Meat 8.48 17.5 13.1 7 J.Pa. Rice 8.25 14.5 8 J.Pa. Meat 8.16 15.2 9 F.Pi. Rice 8.70 16.1 10 F.Pi. Meat 9.05 15.1 11 d.Cl. (vegetarian) Rice 8.78 18.6 12.0 Average 8.65 16.4 13.8 12 M. Mixed diet 6.76 25.3 12.0 13 H. Mixed diet 6.63 25.8 14.9 14 H. Mixed diet 6.07 30.1 15.0 The excreta from the above diets (Nos. 1 to 11) contained no starch, and the composition of the feces did not alter materially as the character of the food changed. Such feces Prausnitz con- sidered "normal feces. " When, however, the food contains mate- rial of a less digestible nature, the composition may change. Where this indigestible material is cellulose the nitrogen content of the feces is lowered (Nos. 12 to 14); if a nitrogenous substance, the nitrogen content might be expected to be raised. Schierbeck recognizes three types of individuals: (1) those that consistently have feces with low nitrogen concentration (about 4 per cent) whatever the nature of the diet may be; (2) those that under these conditions have feces of high nitrogen percentage (6-7 per cent) ; and (3) those in whom coarse food yields feces of low nitrogen percentage, and readily absorbed material produces feces with nitrogen concentration as high as 8 per cent. We are inclined to agree with Benedict that during starvation the formation of feces is reduced to a practically negligible quan- tity. When a material such as meat is eaten whose protein utilization, estimated according to the usual custom, is at least 95 Lafayette B. Mendel and Morris S. Fine '5 per cent, the resulting feces are for the most part of metabolic origin. It has been shown that the feces from such a diet represent a very small portion of the originally secreted intestinal juice, the latter having been absorbed in great part before reaching therectum. Ob- viously the degree to which this secretion is reabsorbed will depend upon the rate of peristalsis, which in turn is influenced by the mass and character of material in the intestine. Hence, if to a meat diet an indigestible or less digestible material is added, thus stimulating peristalsis, more metabolic products* must escape reabsorption If we deal with a non-nitrogenous material, e.g., agar, bone ash or crude fiber, the percentage nitrogen of the feces will of course be lower. If the comparatively indigestible material is highly nitrogenous like protein, the nitrogen concentration will be higher; and if both types of indigestible materials are present, the percentage of nitrogen may be indistinguishable from that found in meat-feces. Illustrative data follow. EXPERIMENTAL PART. The conduct of these experiments did not differ essentially from that of trials described in previous papers. The quantities of meat and indigestible non-nitrogenous materials can be learned from the tables; the amounts of water, sugar and lard approxi- mated those employed in previous experiments. The influence of indigestible non-nitrogenous materials upon the nitrogen statistics of the feces is illustrated in Tables 4 and 5. In Table 4 the contrast is made between feces resulting from meat and feces accruing from an identical diet to which 3 grams of agar plus 7 grams of bone ash had been added daily. In Table 5 a similar contrast is drawn between meat- and meat-crude-fiber feces. The data are briefly summarized in Table 6. The increase in absolute fecal nitrogen due to the addition of indigestible materials to the diet is manifest, although the nitrogen intake did not vary. Thus the fecal nitrogen of (1) is increased 60 per cent by the addition of 10 grams of indigestible non-nitrogenous sub- stances, and that of (3) is augmented 133, 133, and 192 per cent 9 8 Possibly also food residues and products of digestion. 9 Too great a quantitative significance should not be placed upon these figures, as an accurate isolation of pure meat-feces is almost impossible even when special precautions are taken. i6 The Utilization of Proteins TABLE 4. Influence of Agar + Bone Ash upon the Feces Resulting from a Meat Diet. Daily Averages. NATURE OF INGEST A +3 >> XX xxviii i iii iv viii / Meat, sugar, lard = 4.6 \ \ to 4.9 gm. nitrogen j As above + Agar 3 gm. Bone Ash 7 gm Average of 1 and 2. Average of 3 to 6. . . grams 4.5 3.4 13.2 14.5 15.5 15.0 4.0 14.5 gram 0.22 0.16 0.29 0.36 0.40 0.35 0.19 0.37 per cent 4.95 4.62 xxi xxix iv vi viii Meat, etc., as for Dog 5 Meat, etc., with indiges- tible materials, as for Dog 5 Average of 7 and 8. Average of 9 to 12. . 3.5 4.4 12.2 13.6 14.5 15.0 4-0 13.8 0.22 0.24 0.28 0.36 0.35 0.39 0.23 0.34 xx xxviii i iii v vii Meat, etc., as for Dog 5 Meat, etc., with indiges- tible materials, as for Dog 5 Average of 13 and 14- Average of 15 to 18. . . 3.2 3.8 12.5 12.8 12.7 12.8 3.5 12.7 0.19 0.15 0.28 0.23 0.26 0.24 0.17 0.25 Lafayette B. Mendel and Morris S. Fine 17 TABLE 5. Influence of Crude Fiber upon the Feces Resulting from a Meat Diet. Daily Averages. xvn xix xvi xviii Xll xiv xv xv xvi xiv xv NATURE OF INGESTA Meat, etc., = 3.3 gm. nitrogen As above + 6 gm. crude fiber* Meat, etc., as for Dog 5 Meat, etc., + 6 gm. crude fiber as for Dog 5 Meat, etc. , as for Dog 5 Meat, etc., + 6 gm. crude fiber as for Dog 5 Average of 1,3,5 , Average of 2, 4,6 Meat, etc., + 2 gm. agar + 5 gm. bone ash The same The same + 6 gm. filter paper Meat, etc., + 2 gm. agar + 5 gm. bone ash The same The same + 6 gm. filter paper Meat, etc., + 2 gm. agar +5 gm. bone ash The same The same + 6 gm. filter paper Average of 7, 8, 10, 11, 13, 14 Average of 9, 12, 15 0.4f 10.1 1.9 10.0 1.5 8.5 1.7 9.5 11.0 11.5 17.7 10.5 10.0 18.0 8.5 9.5 0.02f 0.30 0.13 0.34 0.10 0.21 0.12 0.28 0.28 0.26 0.27 0.35 0.31 0.40 0.23 0.26 18.0 38 2 14 88.3 10.2 28 2 79 91.6 17.9 35 1 97 89.2 per cent 5.32 2.97 7.03 3.52 7.06 2.51 6.4' 3.00 2.59 2.27 1.55 3.37 3.07 2.23 2.74 2.70 w « u S3 £ 3 per cent 99.4J 90.5 96.0 89.2 96.8 93.3 96.4 91.0 91.8 92.1 91.6 89.8 90.6 87.7 93.3 92.2 * Newspaper (0.1 per cent nitrogen) was thoroughly disintegrated under water, t These values are abnormally low owing to poor separation of feces of successive periods. They are not included in the averages, t Omitted from the averages. THE JOURNAL OF BIOLOGICAL CHEMISTRY, VOL. XI NO. 1. i8 The Utilization of Proteins TABLE 6. The Influence of Indigestible Non-Nitrogenous Materials upon the Nitrogen Statistics of Meat-Feces. (Summary of Tables 4 and 5) . Daily Averages. DUMBER NUMBER OF EXPERI- MENTS AVERAGED TAKE UME INDI- ION-NITRO- ATERI AL fHE MEAT FECES 'ILIZATION REFERENCE J NITROGEN IN TOTAL V O L 1 GESTIBLE I GENOUS M ADDED TO 1 Weight (Air Dry) Nitrogen Nitrogen NITROGEN Ul grams grams grams gram per cent per cent 1 6* 4.6 3.8 0.20 5.2 95.7 2 12** 4.6 10 13.7 0.32 2.3 92.7 3 3t 3.3 1.7 0.12 6.5 96.4 4 3| 3.3 6 9.5 0.28 3.0 91.0 5 6§ 3.3 7 10.2 0.28 2.8 91.6 6 3|| 3.3 13 17.9 0.35 2.0 89.2 • Cf. Table 4, Nos. 1, 2, 7, 8, 13, 14. ** Cf. Table 4, Nos. 3 to 6, 9 to 12, 15 to 18. t Cf. Table 5, Nos. 1,3,5. k | Cf. Table 5, Nos. 2, 4, 6. § Cf. Table 5, Nos. 7, 8, 10, 11, 13, 14. || Cf. Table 5, Nos. 9, 12, 15. by the addition to the diet of 6, 7, and 13 grams respectively of such materials. The low nitrogen concentration of the feces of (2), (4), (5), and (6) is characteristic of diets of thoroughly util- ized materials including much indigestible non-nitrogenous matter. The nitrogen concentration, however, is not sufficiently diminished to compensate for the increased volume of feces — hence the above increment in absolute fecal nitrogen and the correspondingly low- ered coefficients of digestion. Illustrations of the influence of poorly utilized highly nitrogenous matter upon the nitrogen statistics of the feces are especially con- spicuous in certain data already published 10 and which are repro- duced in Table 7. The nitrogen concentration of the phaseolin- feces is 6.1 per cent against 2.3 per cent for that of feces resulting from a meat diet fed under conditions identical with those attend- ing the phaseolin feeding. A similar though less striking example 10 Mendel and Fine: This Journal, x, p. 433, 1912: Table 24 (phaseolin); Table 25 (pea globulin); Tables 10-11 (soy bean). Lafayette B. Mendel and Morris S. Fine 19 is offered in the case of the pea globulin experiment. Nos. 5 to 8 of this table disclose how closely the nitrogen concentration of feces accruing from diets containing both poorly utilized highly nitrogenous materials and indigestible non-nitrogenous materials may simulate the corresponding value for meat feces. TABLE 7. The Influence of Poorly Utilized Highly Nitrogenous Materials upon the Nitrogen Statistics of the Feces. Daily Averages. REFERENCE NUMBER NATURE OF INGESTA NITROGEN INTAKE Weight (Air Dry) FECES Nitrogen Nitrogen NITROGEN UTILIZA- TION grams grams grams per cent per cent 1 Phaseolin 5.2 20.0 1.21 6.1 76.9 2 Meat* 5.2 10.9 0.26 2.3 95.0 3 Pea Globulin 4.8 1,5.6 0.56 3.6 88.3 4 Meatf 4.8 14.7 0.37 2.5 92.4 5 Soy Bean 4.6 15.9 0.57 3.6 87.6 6 Meat 4.6 3.8 0.15 3.8 96.9 7 Soy Bean 3.3 14.0 0.65 4.7 80.2 8 Moot, 3.3 0.4| 0.02| 5.3 99.4 * Average of fore and after periods, t Average of fore and after periods. X See second note to Table 5, this paper. Obviously the fecal nitrogen concentration by itself is not a safe criterion 11 by which to judge the digestibility of a material. The nitrogen of the voluminous meat-cellulose-feces may be almost entirely of metabolic origin and yet be present in relatively low concentration; whereas a soy bean diet may yield feces composed in great part of highly nitrogenous undigested food residues, the nitrogen concentration, 12 however, being comparable to that of meat-feces. 11 Tsuboi (see bibliography), p. 80, likewise believes that one should be conservative in drawing conclusions from this one factor. 12 Tsuboi (loc. cit., p. 81), has made a similar statement. He points out that in Rubner's studies, peas were poorly utilized (72 per cent) and yet the nitrogen concentration of the feces was 7.3 per cent, thus according closely with that of 6.9 per cent for the nitrogen concentration of feces from meat which was 97 per cent utilized. 20 The Utilization of Proteins Benedict has called attention to the difficulty encountered in satisfactorily isolating feces accruing from a particular diet, owing to the lagging behind of fecal material from the preceding diet. Our own experience testifies to this difficulty. It was especially pronounced where the experimental, preceding and succeeding TABLE 8. Influence of Thorough Evacuation upon Nitrogen Statistics of Feces. Daily Averages. FECES b5 NUMBE o o a DAYS PERIOD NATURE OF INGE8TA Weight Air Dry Nitrogen Nitrogen NITROGE UTILIZATI grams gram per cent per cent 1 5 4 xxiii Meat, etc., + 10 gm. Agar ( = 4.6 gm. Nitrogen 15.7 0.52 3.33 88.6 2 5 3 xxiv Meat, etc., + 10 gm. Bone Ash 13.8 0.26 1.90 94.3 3 5 4 XXV Meat, etc., + 10 gm. Agar 13.5 0.40 2.95 91.4 4 6 4 xxiv Meat, etc., + 10 gm. Agar 15.5 0.53 3.42 88.5 5 6 3 XXV Meat, etc., + 10 gm. Bone Ash 15.2 0.32 2.12 93:1 6 6 4 xxvi Meat, etc., + 10 gm. Agar 12.8 0.37 2.92 92.0 7 7 4 xxiii Meat, etc., + 10 gm. Agar 14.8 0.45 3.02 90.3 8 7 3 xxiv Meat, etc., + 10 gm. Bone Ash 14.0 0.28 1.99 94.0 9 7 4 XXV Meat, etc., + 10 gm. Agar 11.9 0.32 2.70 93.1 Average of 1, 4,7 15.3 0.50 3.26 89.1 A verage of 2,5, 8 14.3 12.7 0.29 2.00 93.8 Average of 3,6,9 0.36 2.86 92.2 Lafayette B. Mendel and Morris S. Fine 21 diets were all composed of thoroughly digested materials and the resulting feces were not adequate stimuli to peristalsis. This difficulty was obviated in a measure when the experimental period was preceded and succeeded by a 2-3 day period of a meat diet including 10 grams of bone ash daily. This lag and the effect of previous thorough evacuation upon util- ization is illustrated in Table 8. The first period for each dog (Nos. 1, 4, 7) was preceded by a period of wheat gluten, which is very well utilized. 13 After thorough evacuation, it is clear (Nos. 3, 6, 9) that the apparent utilization is considerably improved. Estimation of " Metabolic 11 Products in the Feces. Investigators have sought a method whereby the prominent part taken by alimentary waste products in the formation of feces could be determined with some degree of accuracy. This would enable one to estimate what proportion of the feces is due to undi- gested food residues. Processes have been proposed which involve treating the feces with pepsin-HCl or dilute alkali. Data thus obtained are of doubtful value. Equally unsatisfactory are those procedures which involve subtracting from the experimental feces the equivalent of fecal material obtained during starvation or on a thoroughly digested non-nitrogenous diet. The plan generally followed in the present work, namely the comparison of experimen- tal feces with feces obtained from a control meat diet is likewise not always free from objection. None of the above methods take into account the influence of undigested masses upon the degree of reabsorption of the intestinal juice. We propose the following plan 14 which seems to avoid most of the above shortcomings: 1. Determine the volume and nitrogen of feces resulting from the material under investigation. 2. Determine the fecal nitrogen resulting from a nitrogen-free diet to which has been added an amount of indigestible non- 13 Cf. Mendel and Fine: This Journal, x, p. 324, 1911. 14 Tsuboi has applied a similar principle to certain results reported by Rubner. The nitrogen eliminated on a starch diet was subtracted from that excreted in feces of comparable volume resulting from diets of wheat bread and maccaroni. The food nitrogen actually escaping utilization could thus be computed. 22 The Utilization of Proteins nitrogenous matter 15 that will yield approximately the same vol- ume of feces as was obtained in (l). 3. Subtract the fecal nitrogen of (2) from that of (1). This excess of nitrogen is presumably due to undigested or unabsorbed nitrogenous matter of the food material. An experiment with a nitrogen-free diet including indigestible non-nitrogenous matter follows: A 6 kilo bitch was fed for four days on a mixture of 35 grams of sugar, 45 grams of lard, 200 grams of water and 10 grams of agar. On this diet 13.2 grams of feces with a nitrogen concentration of 2.44 per cent were obtained daily. There were thus eliminated through the feces 0.32 gram of nitrogen daily, which was obviously of metabolic origin. This result makes it prob- able that the feces from meat diets, containing similar amounts of indiges- tible non-nitrogenous matter, (see Table 6) are likewise made up entirely of alimentary waste — proof in itself that meat nitrogen is 100 per cent utilized. From the single experiment above reported and from Table 6, No. 6, we may conclude that a thoroughly digested material may yield 13.2 to 17.9 grams of feces and yet the nitrogen (0.32-0.35 grams) thus eliminated will be of "metabolic" origin. Hence in feces of comparable volumes 16 all nitrogen in excess of 0.32-0.35 gram may be attributed to the nitrogen of the food. This prin- ciple is applied in Table 9. "Utilization, "as the term is employed in the last column of this table, exactly expresses our meaning. The actual utilization of soy bean nitrogen 17 is 90.3-92.8 per cent and that for the crude bean protein is 91.8 per cent. If anything the latter value is low, as 24.6 grams of meat-feces would probably contain more than 0.35 gram of nitrogen. 15 The choice of indigestible adjuvant is a matter of some moment, as these materials may vary in their ability to stimulate peristalsis. 16 This of course applies only for dogs of approximately the same weight (5 to 7 kilos.) as those in these experiments. 17 Soy bean is reported (Wolff -Lehmann : Landw. Fiitterungslehre, cited by Schulze und Castoro: Zeitschr. f. physiol. Chem., xli, p. 455, 1904) as having 10 per cent of its nitrogen present as non-protein. The latter may be more thoroughly utilized than the protein constituents, and thus the utili- zation calculated for the total nitrogen intake would be greater than is actually the case for the soy bean protein. Excepting the soy bean and cotton-seed flours, the preparation of the materials employed in this series of studies renders contamination with nitrogenous non-protein matter unlikely. Lafayette B. Mendel and Morris S. Fine 23 The Utilization of the Vegetable Proteins. About the thorough utilization of the proteins of wheat 18 there is no question. The probability that those of barley 19 and corn 20 are equally available was pointed out in previous papers of this series. With regard to the legume proteins 21 we must for the present conclude that the presence of indigestible non-nitrogenous materials camiot entirely account for their low coefficients of diges- TABLE 9. Utilization as Estimated from the Portion of Fecal Nitrogen Derived from Food Residues. Daily Averages. NUMBER OP EX- PERIMENTS AVERAGED NATURE OF INGESTA NITROGEN INTAKE FECES NITROGEN FROM UNDIGESTED FOOD NITROGEN UTILIZATION Weight (Air Dry) Nitrogen As Ordinarily Estimated Actual Utilization gram grams gram gram per cent per cent 1 Nitrogen-free diet including 10 gm. agar 0.0 13.2 0.32 0.00 3 Meat diet* 3.3 17.9 0.35 0.00 89.2 100.0 6 Soy beanf 4.6 17.2 0.68 0.33 85.3 92.8 3 Soy bean| 3.3 13.0 0.64 0.32 81.1 90.3 1 Crude bean protein§ 3.4 24.6 0.63 0.28 81.5 91.8 * Cf . Table 6, No. 6 this paper. t Cf. Mendel and Fine: This Journal, x, p. 433, 1912. Tables 5-10. t Cf. Mendel and Fine: loc. cit. Tables 11 to 13. § Cf. Mendel and Fine: loc. cit. Table 21. tibility. These proteins appear to be less readily affected by the digestive processes than those of barley or corn. This resistance is even more pronounced in the case of the cotton-seed protein. 22 Nevertheless, future research with the isolated proteins may modify our opinion with regard to these two last classes of materials. The lack of animal extractives in vegetable materials has at times been thought to be the cause of the apparently poor utili- zation of plant foods in comparison with those of animal origin. 18 Cf. Mendel and Fine: This Journal, x, p. 303, 1911. 19 Cf. Mendel and Fine: Ibid, x, p. 339, 1911. 20 Cf. Mendel and Fine: Ibid., x, p. 345, 1911. 21 Cf. Mendel and Fine: Ibid, x, p. 433, 1912. 22 Cf. Mendel and Fine: Ibid, xi, p. 1, 1912. 24 The Utilization of Proteins The evidence bearing upon this seems to be inconclusive. Bischoff showed that meat extracts did not appreciably influence the diges- tibility of bread. Thompson came to the opposite conclusion. Effront noted that meat extracts exert a favorable influence upon the availability of vetegable diets, but this could not be confirmed by Wintgen. The fact that the proteins of wheat, and probably those of barley and corn also, are thoroughly utilized lends support to the view that the secretory influences of the extractive materials play a minor role in the ultimate utilization. It was pointed out in an earlier paper that certain wheat preparations evoked intense nausea in man, and necessitated forced feeding in the dog experi- ments, but were, nevertheless, thoroughly digested. This would suggest that psychic secretion does not influence the ultimate utilization to any great extent. 23 It would be interesting to study the relation of gastric secretion to ultimate utilization by means of a sequestered stomach. Studies on the digestibility of vegetable proteins in vitro are not lacking. Rothe 24 found that in 24 out of 26 of his experiments, the coefficients of digestibility were above 90 per cent. The coefficients for the legumes averaged about 95 per cent. In these studies, 2 grams of material were acted upon by 250 cc. of concentrated gas- tric juice for forty-eight hours. As yet, it is uncertain to what extent in vitro experiments of this kind can be held comparable to studies on the utilization of proteins from the alimentary tract. It may be noted that artificial digests are not contaminated with " metabolic" products, and this may explain the high coefficients of digestibility obtained in such trials compared with those result- ing from experiments in vivo. Studies on animals where this factor has been taken into account yield results not very unlike those obtained in artificial digestion experiments (see Table 9) . A study of the literature on the availability of vegetable materials reveals an interesting situation. In instances where the protein has been very poorly utilized, the carbohydrate, on the contrary, has rarely been less than 95 per cent digested. This is illustrated in the accompanying table, containing data gathered at random. 23 Cf. Schmidt und Strasburger: "Die Fazes des Menschen," p. 17, Berlin 1910. Also Osborne and Mendel: Carnegie Institution of Washington, Publication No. 156, p. 5, 1911. 24 Rothe: Zeitschr.f. physiol. Chem., li, p. 185, 1907, (contains the literature). Lafayette B. Mendel and Morris S. Fine 25 Table Illustrating the Simultaneous Poor Utilization of Protein and Good Utilization of Carbohydrate. NATURE OF FOOD Kidney beans White beans Cow peas Rice, barley, and vegetables Rice and barley Cooked rice Barley COEFFICIENTS OF DIGESTIBILITY Protein Carbohydrate per cent per cent 77 94 78 96 70 87 76 97 67 99 76 99 60 97 AUTHOR OR COMPILER Wait 25 Wait Wait Oshima 26 Oshima Oshima Oshima It is possible that the starch has been completely utilized and the carbohydrate of the feces is in reality hemicellulose. BIBLIOGRAPHY. THE UTILIZATION OF MEAT POWDERS AND ALLIED MATERIALS. Abderhalden und Ruehl: Zeitschrift fur physiologische Chemie, lxix, p. 301, 1910. Ellinger: Zeitschrift fur Biologie, xxxiii, p. 190, 1896. Forster: Ibid., ix, 1873. (cited by Atwater and Langworthy: U. S. Dept. of Agriculture, Office of Experiment Stations, Bull. 45, 1897. Hildebrand: Zeitschrift fur physiologische Chemie, xviii, p. 180, 1894. Imabuchi: Ibid., lxiv, p. 1, 1910. Maas : Medizinische Klinik, No. 8, 1906. Muller: Miinchener medizinische Wochenschrift, xlvii (2), p. 1769, 1900. Neumann: Ibid., xlv (1), p. 72, 1898 and xlvi (l), p. 42, 1899. Neumeister: Deutsche medizinische Wochenschrift, xix, pp. 866 and 1169, 1893. Plauth : Zeitschrift fur diatetische und physikalische Therapie, i, p. 62, 1898. Prausnitz: Zeitschrift fur Biologie, xlii, p. 377, 1901. Salkowski: Biochemische Zeitschrift, xix, p. 83, 1909. Schmilinsky und Kleine: Miinchener medizinische Wochenschrift, xlv (2), p. 995, 1898. Strauss: Therapeutische Monatshefte, xii, p. 241, 1898. Voit: Zeitschrift filr Biologie, xlv, p. 79, 1904. 26 Wait: U. S. Dept. of Agriculture, Office of Experiment Stations, Bull. 187, p. 53, 1907. 26 Oshima: Ibid., Bull. 159, 1905. 26 The Utilization of Proteins ORIGIN OF FECAL NITROGEN AND CONDITIONS INFLUENCING ITS EXCRETION. Benedict: " The Influence of Inanition on Metabolism," p. 347, Carnegie Institution of Washington, 1907. Bidder und Schmidt: "Die Verdauungssdfte und der Stoffwechsel," 1852. Bischoff: Zeitschrift fur Biologie, v, p. 454, 1869. Effront: Fiinfter internationeller Kongress fiir angewandte Chemie, p. 97, 1904. (Cited by Wintgen.) Hoffmann: (Cited by Voit: Sitzungsberichte der bayerischen Akademie, ii, (4), 1869). Kermauner: Zeitschrift fur Biologie, xxxv, p. 316, 1897. Lothrop: American Journal of Physiology, xxiv, p. 297, 1909. Mosenthal: Journal of Experimental Medicine, xiii, p. 319, 1911. Muller: Zeitschrift fur Biologie, xx, p. 326, 1884. (Cited by Tsuboi.) Prausnitz: Ibid., xxxv, p. 335, 1897. Rieder: Ibid., xx, p. 378, 1884. (Cited by Tsuboi.) Rubner: Ibid., xv, p. 115, 1879; also Ibid., xix, p. 45, 1883. Schmidt und Strasburger: "Die Faeces des Menschen," p. 115, Berlin, 1903. Schierbeck: Archiv fur Hygiene, li, p. 62, 1904. Thompson: Zentralblatt fur Physiologie, No. 17, p. 814, 1910. Tsuboi: Zeitschrift fur Biologie, xxxv, p. 68, 1897. Voit (C): Ibid., ii, p. 308, 1866. (Cited by Tsuboi.) Voit (P.): Ibid., xxix, p. 325, 1893. (Cited by Tsuboi.) Wicke: Archiv fiir Hygiene, xl, p. 349, 1890. Wintgen: Veroffentlichungen aus d. Gebiete des Militdrsanitdtswesens, xxix, p. 56, 1906. Reprinted from Tiik Journal of Biological Chemistry, Vol. X, No. 2, 1911 STUDIES IN CARBOHYDRATE METABOLISM. I. THE INFLUENCE OF HYDRAZINE UPON THE ORGANISM, WITH SPECIAL REFERENCE TO THE BLOOD SUGAR CONTENT. 1 PLATE III. By FRANK P. UNDERHILL. {From the Sheffield Laboratory of Physiological Chemistry, Yale University, New Haven, Connecticut.) (Received for publication, July 28, 1911.) The constancy of the sugar content of the blood under normal conditions constitutes one of fundamental axioms of physiology. It has been universally assumed that a certain supply of sugar in the blood is essential to normal metabolic rhythm and that even under distorted physiological conditions, as in inanition, the organ- ism is capable of furnistiing the requisite materials from its own economy for the maintenance of the blood sugar constant. f , Attempts to upset this nice adjustment have resulted in the main in the temporary establishment of an excessive quantity of sugar in the blood. Thus hyperglycaemia may follow the introduction of various drugs into the body or may be produced by the induc- tion of different pathological states. On the other hand, with the exception of phloridzin and uranium glycosurias, little has been known concerning the conditions necessary to diminish the content of blood sugar. ; The practical significance of the investigations upon changes in blood sugar content obviously lies in the importance which the results obtained from such research may bear to the interpreta- tion and treatment of human diabetes. It has been assumed that if the abnormal state which exists in diabetes could be experimen- tally reproduced, some hope for the prevention or alleviation of the condition might be realized. Such, however, has not been X I am much indebted to Dr. M. S. Fine for aid in carrying out some of these experiments. 1 5Q 160 Hydrazine and Carbohydrate Metabolism the case for, with the possible exception of pancreatic diabetes, an exact experimental duplication of the conditions existing in human diabetes has not yet been made. Recent investigations in the production of hypoglycaemia, notably those of Frank and Isaac, 1 with phosphorus, suggest a possible control of the blood sugar content which may lead to a distinct advance in our knowledge concerning certain phases of carbohydrate metabolism. The present paper is the first of a series in which it is planned to present various aspects of carbohydrate metabolism under conditions in which the sugar of the blood is experimentally dimin- ished. The method employed for the production of this condi- tion was the administration of the diamine, hydrazine. THE INFLUENCE OF HYDRAZINE UPON THE ORGANISM IN GENERAL. In their paper on "The Influence of Hydrazine upon Inter- mediary Metabolism in the Dog," Underhill and Kleiner 2 have described the general effects of hydrazine upon the organism in the following words: "The researches of Borissow, 3 of Pohl 4 and of Poduschka 6 have demonstrat- ed the relatively great toxicity of this compound and have defined the series of manifestations following its introduction into the body. With doses of 0.1 gram hydrazine sulphate per kilo of body weight subcutaneously injected vomiting is observed which is succeeded by extreme restlessness. There is augmentation of the heart beat which later falls below the normal and respiratory difficulty is accompanied by general paralysis. At this stage a short period of coma usually ensues which terminates in death. The entire cycle of events is completed within a very few days. Coincident with the symptoms noted above is the appearance in the urine of varying quan- tities of protein and bile pigments " From his histological study of the action of hydrazine, Wells 6 concluded that "hydrazine seems to be a poison with an almost specific effect upon the cytoplasm of the parenchymatous cells of the liver, for when the poison is 1 Frank and Isaac: Arch. f. exp. Path. u. Pharmakol., lxiv, p. 274, 1911. * Underhill and Kleiner: This Journal, iv, p. 165, 1908. 'Borissow: Zeitschr. f. physiol. Chem., xix, p. 499, 1894. 4 Pohl: Arch. f. exp. Path. u. Pharmakol, xlviii, p. 367, 1902. 6 Poduschka: Ibid., xliv, p. 59, 1900. •Wells: Journ. of Exp. Med., x, p. 457, 1908. Frank P. Underhill 161 given subcutaneously this tissue alone shows evident structural alterations, although equal or greater amounts must reach other organs or tissues. It seems to have remarkably little effect upon other than hepatic cells, and does not cause any appreciable destruction of red corpuscles; slight hemor- rhages are occasionally produced, but much less than by other poisons with a similar effect upon the liver. It attacks only the cytoplasm of the liver cells, never affecting the nucleus primarily, and causes a profound fatty metamorphosis of the type commonly referred to as "fatty degeneration." In this respect it resembles phosphorus, from which it differs in two impor- tant particulars. Hydrazine attacks first the cells in the center of the lobules, while phosphorus shows its first and most marked effects upon the peripheral cells; and secondly, phosphorus usually causes marked fatty changes in the myocardium, the kidneys, and indeed throughout the body, whereas the effects of hydrazine seem to be limited almost absolutely to the liver. The unknown poisons of acute yellow atrophy and eclampsia, and most of the bacterial poisons, attack first and chiefly the nuclei of the liver cells, in contrast to the strictly cytoplasmic effects of hydrazine. Phos- phorus also effects the nuclei more than does hydrazine. On this account the recovery of the liver to normal after hydrazine poisoning is remark- ably rapid and complete, there being no permanent anatomical alterations after recovery from a most severe non-fatal poisoning." Despite the grave injury to the liver it has been demonstrated that "the most striking feature of the action of hydrazine upon the animal body is the absence of abnormal relationships in the principal urinary constituents" 1 and the presence of abnormal substances, such as lactic, oxybutyric and diacetic acids, acetone or reducing bodies could not be detected in the urine, although in one instance there was a separation of a small quantity of cystine. 2 Subsequent experience with hydrazine has afforded additional facts concerning the general influence of this poison upon the body of the dog which are of considerable importance for the conduct of future investigation. In the first place the subcutaneous admin- istration of hydrazine sulphate in the dosage of 50 mgms. per kilo presents an entirely different series of symptoms than a simi- lar introduction of 100 mgms. per kilo body weight. In both instances there is the same initial picture, i.e., vomiting and ex- treme restlessness. With the smaller dosage, however, the ani- mal appears merely drowsy and stupid during the first day. In general upon the second day the dog seems practically normal with the noteworthy exception that there may be evidence of 1 Underhill and Kleiner: loc. ext. 2 Underhill and Kleiner: loc. ext. 1 62 Hydrazine and Carbohydrate Metabolism extreme weakness especially noticeable in the hind limbs. Food is refused. The animals may show a considerable loss of body weight, much more than can be accounted for by the few days starvation. Upon the fourth or fifth day food may be greedily eaten. After this stage is reached, the ultimate recovery of the animal is assured. In general nearly all dogs receiving the smaller dosage make complete recovery in spite of the fact that during the second and third day after the administration of hydrazine the liver presents, a typical, light colored appearance of " fatty degeneration." Bile may or may not appear in the urine and in general the urine contains no protein. Methods. In the investigations here recorded, hydrazine (Kahlbaum) was always introduced subcutaneously as the sulphate in a 2.5 per cent solu- tion which is a practically saturated solution at room temperature. Esti- mations of the blood sugar content were made according to the method indicated in a former paper, 1 the copper finally being determined gravimet- rically. Glycogen in the liver was determined by Pfluger's method. 2 Blood pressure was recorded by a mercury manometer connected with a femoral artery. In blood pressure experiments narcosis was induced by a mixture of morphine and atropine (10 mgms. morphine sulphate and 1 mgm. atropine sulphate per kilo of body weight). Ether was not necessary after the preliminary operative procedures. Injections were made into the femoral vein. THE ACTION OF HYDRAZINE UPON THE BLOOD SUGAR CONTENT. It has been previously intimated that there is a paucity of liter- ature relating to the artificial production of hypoglycaemia. Aside from the consideration of the well known examples in connection with phloridzin and uranium salts, the experiments of Frank and Isaac 3 with phosphorus have shown for the first time the com- plete disappearance of sugar from the blood of rabbits. A decrease in blood sugar content has also been observed by Porges 4 after extirpation of the adrenals in dogs and in cases of adrenal insuffi- ciency caused by Addison's disease. In all these instances the decreased sugar content of the blood is accompanied by a rapid decrease or complete disappearance of the liver glycogen in spite of the ingestion of relatively large quantities of carbohydrate. 1 Underhill: This Journal, i, p. 113, 1905-06. 2 Pfluger: Arch. f. d.jes. Physiol, cxi, p. 307, 1906. 3 Frank and Isaac: loc. cit. 4 Porges: Zeitschr. f. klin. Med., lxix, p. 341, 1909; lxx, p. 243, 1910. Frank P. Underhill What becomes of the glycogen of the liver and the sugar of the blood is at present largely a matter of conjecture. Whether the carbohydrate is merely transformed into some incompletely metab- olized product and the latter eliminated, or whether more rapid metabolism is induced resulting in increased carbohydrate combus- tion remain problems for future determination. Experiments with Dogs. Observations have been made to ascertain the influence of hydrazine upon the blood sugar content of dogs. From the data presented in Table 1 it may be seen that an appreciable hypoglycaemia follows the introduction of hydra- zine in doses of 50 mgms.per kilo of body weight, and that twice this quantity does not necessarily exert a more marked influence. In a large number of experiments hardly a single individual ani- mal has failed to exhibit this phenomenon although variations in the degree of diminution may occur. The symptoms of extreme weakness in dogs after hydrazine treatment may be directly cor- related with the diminished store of carbohydrate as indicated by lowered blood sugar percentage. TABLE 1. The Influence of Hydrazine upon Blood Sugar Content and Liver Glycogen of the Dog. NUMBER OF ANIMAL SUBCUTANEOUS INJEC- TION OF HYDRAZINE SULPHATE SUGAR CONTENT OF BLOOD GLYCOGEN CONTENT OF LIVER EXPRESSED AS GRAMS OF DEX- TROSE REMARKS mgms. per kilo per cent 1 50 0.02 0.10 Three days after hydra- zine injection. 2 100 0.05 0.22 One day after hydra- zine injection. 13 50 0.03 Two days after hydra- zine injection. 15 50 0.04 Two days after hydra- zine injection. A 0.14 Fasted six days. B 0.15 Fasted six days. C 0.13 Emaciated from lack of food. 164 Hydrazine and Carbohydrate Metabolism That starvation per se is not responsible for the lowered blood sugar content may be concluded from experiments A, B, and C, in which dogs were allowed to fast greater number of days than those in hydrazine experiments. Experiments with Rabbits. When hydrazine is adminis- tered to rabbits in doses of 50 mgms. per kilo the animals refuse food for a period of at least two days. No other noteworthy symptoms are conspicuous. Since starvation plays a role in hydra- zine experiments of this type it is imperative that the influence of this factor shall be determined. Accordingly determinations have been made of the sugar content of the blood and the glyco- gen of the liver in normal rabbits fed and kept under the usual laboratory conditions as a comparison for similar determinations made upon animals in every way comparable except that all food was withheld for a period of two days. This period was chosen for the reason that after hydrazine introduction a like period of time was allowed to elapse previous to killing the animals. The results of these trials (Table 2) indicate what is gener- ally accepted, namely, that the blood sugar content of normal rabbits is approximately 0.10 per cent, and that usually the liver contains a considerable quantity of glycogen although the quantity in individual rabbits may show a noteworthy varia- tion. It is also evident that a period of two days inanition is sufficient to practically eliminate the liver's store of glycogen. On the other hand, the percentage of the blood sugar undergoes no appreciable change. When hydrazine is administered in the doses indicated it is clear that in the majority of cases the drug is capable of decreasing the sugar content of the blood, in some instances to a remarkable degree, in others only slightly, while a practically normal blood sugar content is maintained by a third group of individuals. From this diversity of results it is obvious that the rabbit can not be relied upon to invari- ably exhibit hypoglycaemia after hydrazine introduction. Hence results of experiments planned to supply data of carbohydrate metabolism from the standpoint of hyperglycaema may be of questionable value. The problem as to whether hydrazinized rabbits are capable of maintaining blood sugar content and glycogen store unchanged was subjected to experiment by subcutaneously introducing dex- Frank P. Underhill 165 TABLE 2. The Behavior of Blood Sugar Content and Liver Glycogen in Rabbits Treated with Hydrazine. NUMBER OF ANIMAL SUBCUTANEOUS INJEC- TION OF HYDRAZINE SULPHATE SUGAR CONTENT OF BLOOD GLYCOGEN CONTENT OF LIVER EXPRESSED AS GRAMS OF DEX- TROSE REMARKS mgms. per kilo per cent 10 0.10 6.53 Well-fed rabbit. 11 0.09 11.07 Well-fed rabbit. 8 0.12 0.01 Two days fast. 9 0.11 0.02 Two days fast. 1 50 0.003 0.06 No food after hydrazine injection. 2 50 0.016 0.00 No food after hydra- zine injection. 3 50 0.009 0.02 No food after hydra- zine injection. 12 50 0.08 0.00 No food after hydra- zine injection. 18 50 0.06 0.00 No food after hydra- zine injection. 19 50 0.09 0.00 No food after hydra- zine injection. 20 100 Died within five hours. 16 100 Died within twenty- four hours. 15 • 50 0.11 Subcutaneous injection 4 grams dextrose per kilo twice on day after hydrazine administra- tion. trose into animals on the day following hydrazine injection. On the next day the animals were killed and the blood sugar content and glycogen of the liver were determined. The results of one such experiment (Rabbit 15, Table 2) are given. It is obvious from what has been said that the exact interpretation of these results is not easy since from the data furnished one is unable to know whether hydrazine exerted any appreciable influ- ence upon this animal's carbohydrate store. It may be of inter- 1 66 Hydrazine and Carbohydrate Metabolism est, however, to point out that in the two experiments, one of which is detailed in Table 2, Rabbit 15, carried out with this object in view both animals emitted sharp cries and exhibited a notice- able hyperpnoea immediately following the second injection of dex- trose. This respiratory disturbance lasted about one minute, after which the animals appeared normal. To determine the influence of small doses of hydrazine continued over a considerable period of time, two well-fed rabbits of 2100 and 2200 grams body weight respectively were selected and for twelve days each received 25 mgms. of hydrazine sulphate daily without obvious detrimental influence of any kind. The only effect noted was an apparent ravenous appetite. After this period the dosage was increased to 75 mgms. daily for eighteen days without any appreciable influence. Body weight had changed too little to be of significance. The animals were then killed. The blood sugar content of the two animals was 0.07 and 0.12 per cent respectively. The glycogen in the liver amounted to 1.56 grams and 5.1 grams expressed as dextrose. In none of the hydrazinized rabbits was there any evidence of the characteristic light colored liver seen with dogs. THE ACTION OF SUBCUTANEOUS INJECTIONS OF DEXTROSE UPON HYDRAZINIZED DOGS. Since hydrazine invariably causes a diminution in the per- centage of blood sugar it was assumed that some light might be thrown upon the fate of the blood carbohydrate by a determination of the assimilation limits for dextrose injected subcutaneously into hydrazinized dogs. If dextrose of the blood undergoes a more rapid combustion in the body subsequent to hydrazine administration, one might conjecture that the organism would be capable of utilizing larger quantities of dextrose. It has been shown that normal dogs completely utilize dextrose injected hypodermically in doses of 5 grams per kilo. 1 As hydra- zinized animals refuse food it is essential to determine whether animals without food for a period of two days — the time selected 1 Scott: Journ. of Physiol., xxviii, p. 107, 1902; Underhill and Closson: This Journal, ii, p. 117, 1906. Frank P. Underhill 167 for sugar introduction subsequent to hydrazine injections — show as high assimilation limits as the normal animal. Three well-fed animals were selected for this purpose. Dog 21 was a bitch of 5.1 kilos. Dog 22 was a dog of 7.6 kilos. Dog 24 was a hound of 15.7 kilos. To these animals, after a prelim- inary inanition period of two days, subcutaneous injections of dextrose were given in doses of 5 grams per kilo. The sugar was a 30 per cent solution. For two days subsequent to the sugar injection, urine was collected and tested for dextrose. In no case was there the least trace of reducing substance. No food was given on the two days following the administration of dextrose. After a two days period during which the animals were well fed on a mixed diet, hydrazine was subcutaneously injected in doses of 50 mgms. per kilo body weight. On the second day following the hydrazine introduction quantities of dextrose were injected sub- cutaneously exactly equal to the amounts previously administered. The sugar injections were all made in the late afternoon and when left for the night the animals were apparently in normal condition. The next morning all animals were found dead. The bladder of Dog 21 contained 13 cc. of urine; 37 cc. of urine were obtained from Dog 22, while the bladder of Dog 24 held 70 cc. of urine. In no case was there evidence of reducing substances. Autopsy revealed nothing abnormal except the exhibition of the typical light colored liver. Three experiments in every respect similar except that the preliminary period of starvation and sugar injection was omitted yielded results exactly comparable. All animals died within a period of twelve hours after the sugar injection. The dosage of hydrazine alone was not sufficient to cause the death of the animals in this length of time, i.e., within three days. In fact, it has been our experience that with this dose, 50 mgms. per kilo, practically all animals recover. It is apparent that the combination of the two factors, hydrazine and dextrose, was nec- essary for the unexpected result. In some of the animals a noteworthy anuria was observed which may be of significance in the interpretation of the results obtained. Why sugar solutions thus introduced should exert such a prompt toxic influence upon dogs treated with hydrazine remains to be ascertained. This problem is being subjected to further study. 1 68 Hydrazine and Carbohydrate Metabolism THE INFLUENCE OF HYDRAZINE UPON BLOOD PRESSURE. During the course of this investigation it became desirable to know the action of hydrazine upon the arterial blood pressure. That hydrazine exerts some influence in this respect might be concluded from the observation of Borissow 1 that hydrazine at first greatly increases the cardiac beat but subsequently causes a very significant slowing of the heart. Experiments to test this point have been made. A tracing (Plate III) is appended showing that even in relatively strong solutions hydrazine sulphate when introduced directly into the circulation fails to exhibit any im- mediate significant influence upon arterial blood pressure. In this experiment a 10 kilo dog was employed. 15 cc. of 0.1 per cent, 0.5 per cent, 1.0 per cent, and 2.5 per cent hydrazine sulphate solution were injected successively. The injections lasted about one minute. In the tracing time is recorded in half seconds. CONCLUSIONS. Lethal Dose. Hydrazine sulphate subcutaneously injected into dogs and rabbits in doses of 100 mgms. per kilo invariably cul- minates in death, while all animals usually recover from admin- istration of 50 mgms. hydrazine per kilo. Hypoglycaemia. Doses of 50 mgms. per kilo hydrazine sul- phate subcutaneously injected into dogs leads to a distinct lowering of the percentage of sugar in the blood; with rabbits this effect is not constant. During a short period of inanition, dextrose assimilation after hypodermic administration is as good as in normal well-fed dogs. Dextrose in doses of 5 grams per kilo promptly causes death when subcutaneously injected into dogs previously treated with non-fatal doses of hydrazine. Hydrazine introduced directly into the blood stream shows no appreciable immediate influence upon arterial blood pressure. 1 Borissow: loc. ext. rHE JO THE JOURNAL OF BIOLOGICAL CHEMISTRY, VOL. X. PLATE III. Reprinted from The Journal, of Biological Chemistry, Vol. X, No. 3, 1911 STUDIES IN CARBOHYDRATE METABOLISM. II. THE PREVENTION AND INHIBITION OF PANCREATIC DIABETES. By FRANK P. UNDERHILL and MORRIS S. FINE. (From the Sheffield Laboratory of Physiological Chemistry, Yale University, New Haven, Connecticut.) (Received for publication, August 16, 1911.) The literature 1 relative to the production of pancreatic diabetes in dogs is so well-known among physiologists that a detailed recital of the conditions attending the induction of the above men- tioned pathological state is unnecessary. Nevertheless, for the full appreciation of the import of the present communication it may not be out of place to call attention briefly to a few of the most salient features connected with the production of pancreatic diabetes. For temperamental and anatomical reasons the dog is best suited for this type of experimentation. With rabbits the production of pancreatic diabetes may not be constantly successful, owing to the diffuse distribution of the pancreas through the mesentery of this animal. Accepting then as typical the results obtained with the dog, one may reasonably assume certain facts as fundamental for the pur- pose of further investigation. Thus, it is a matter of universal experience that glycosuria is always provoked by the practically complete removal of the pancreas in dogs whether the animals are maintained in a well-fed or fasting condition. The time of the first appearance of sugar in the urine subsequent to the opera- tion in question is not usually specifically stated in the literature incident to such experimentation, hence such data have been sup- plied in the present paper under conditions strictly comparable to those found to be essential for the purposes aimed at. Another feature which may be assumed to be a constant factor in pancre- 1 cf. Biedl: Innere Secretionen, Berlin, 1910. 271 272 Hydrazine and Pancreatic Diabetes atic diabetes is the accompanying hyperglycaemia. Finally, the assumption may be made, without meriting such universal acceptance, that pancreatic diabetes is either identical with or closely akin to the pathological conditions existing in the diabetes of man. Leaving out of consideration the decision as to the identity of experimental pancreatic diabetes with the human abnormal state one may reasonably assume an exceedingly close relationship between the two conditions. Any procedures, therefore, which will aid in the better understanding of the experimental diabetes, or in the prevention, inhibition or alleviation of the symptoms, are likely to have an important bearing upon our conception and treat- ment of the human type of pathological carbohydrate metabolism. In a previous paper 1 it has been pointed out that the diamine, hydrazine, is capable of inducing an appreciable hypoglycaemia in dogs. The relation of the action of hydrazine to pancreatic diabetes forms the basis of the present communication. EXPERIMENTAL. Methods. Unless specifically stated otherwise all operations were carried out under ether anaesthesia only. Aseptic precau- tions were taken throughout the operative technique. After operation the animals were carefully cared for, attention being paid to assure proper temperature of the animal room. After death of the animals operated upon for removal of the pancreas, autopsy revealed the practically entire absence of pancreas in every instance. Analysis of the blood sugar was made according to the method usually employed in this laboratory. 2 Sugar in the urine was estimated with a Schmidt and Haensch triple shadow saccharimeter. Experiments to determine the first appearance of sugar in the urine of dogs subsequent to removal of the pancreas. In our previous paper intimation has been made that hydrazine is fatally toxic in large doses but that relatively small quantities iUnderhill: This Journal, x, p. 159, 1911. 2 Underhill: loc. ext. Frank P. Underhill and Morris S. Fine 273 of the drug exert only a temporary detrimental influence. When the dog is subjected to a combination of hydrazine poisoning and pancreas removal death follows within a comparatively few hours. Therefore in order to determine correctly the action of hydrazine upon the course of events following pancreas removal it is impera- tive to know the behavior of the criterion adopted, i.e., presence of sugar in the urine, in animals deprived of the pancreas but with- out the administration of hydrazine. Two such observations have been made upon animals kept under conditions in every way identical with those depancreatized dogs subjected to the influence of hydrazine to be reported below. Experiment 1. From a full-grown bitch of 5 kilos the pancreas was removed. The operation was completed at 4:15 o'clock on the afternoon of May 2. Upon catheterization at 10:00 p. m. of the same day 10 cc. of urine were obtained which contained approximately 1 gram of dextrose. The 150 cc. of urine obtained by catheterization the following morning at 10:30 yielded 5.4 grams dextrose. Experiment 2. A bull bitch was depancreatized. At 12:30 p. m., May 8, the operation had been completed. In the 100 cc. of urine obtained by catheterization two hours subsequent to the operation 7.33 grams of dextrose were eliminated. These observations indicate that dextrose may appear in signi- ficant quantities in the urine within a very few hours after removal of the pancreas, in some instances after an interval of two hours. It would seem therefore that extirpation of the pancreas has an almost immediate effect in evoking glycosuria, that there is little or no significant latent period, which would conform to the blood sugar content values obtained under practically comparable con- ditions. 1 These experiments also indicate that the pancreas removal was sufficiently complete to insure the speedy initiation of pancreatic diabetes. The prevention of pancreatic diabetes by the subcutaneous injection of hydrazine. The observation that hydrazine will almost invariably establish a condition of hypoglycaemia in the dog at once suggested the possibility that by this method pancreatic diabetes could be pre- 1 Underhill: This Journal, i, p. 113, 1905-06. 274 Hydrazine and Pancreatic Diabetes vented. It is well known that during this disturbance of carbo- hydrate metabolism the sugar content of the blood is above the normal. The assumption has been made that in this form of dia- betes sugar appears in the urine only after the dextrose content of the blood has risen to a certain unknown point beyond which the kidneys become permeable to the carbohydrate. Theoretically at least if the sugar content of the blood could be kept below this point no dextrose should appear in the urine. In other words the appearance of sugar in the urine would depend upon which of the two factors at issue, namely, hydrazine with its hypoglycaemia producing action or the unknown force set free by removal of the pancreas exerting an influence toward blood sugar accumulation, would display the greater activity. With the combination of pancreas removal and hydrazine injec- tion it is therefore possible to imagine at least three conditions in which theoretically glycosuria may not appear. In the first place, the hydrazine effect may be very much stronger than the influence of pancreas removal, in which event sugar would not be elimin- ated in the urine. Secondly, the two effects may exactly neutral- ize each other causing a normal blood sugar content and thus pre- vent glycosuria. Finally, the effect induced by removal of the pancreas may be greater than the hydrazine influence, but the action of the latter may be sufficient to prevent accumulation of sugar in the blood to the point at which sugar elimination begins. These theoretical considerations have been put to the test. The details of these experiments may be found in the following proto- cols. Experiment 3. Dog 9. On May 9 at 4:00 p. m. a full-grown terrier bitch of 8 kilos received a subcutaneous injection of 16 cc. of a 2.5 per cent hydrazine sulphate solution (50 mgms. per kilo). At 11:15 a. m., May 11, the pancreas had been entirely removed; the operation being performed under ether anaesthesia. The animal was wrapped in absorbent cotton and placed in a cage in a warm room. The urine (60 cc.) obtained by catheter- ization at 3:30 yielded no trace of reducing substance with Benedict's solu- tion. 1 A similar negative result was given with 40 cc. urine obtained by catheterization at 5:15 p. m. Upon catheterization at 11:30 p. m. 65 cc. of urine were drawn which contained no dextrose. When left the dog was in good condition and responded to petting. The following day at 8:00 1 Benedict: This Journal, v, p. 485, 1908. Frank P. Underhill and Morris S. Fine 275 a. m. the animal was found dead. 40 cc. of urine taken from the bladder gave negative tests with Benedict's solution. Autopsy revealed entire absence of pancreas; the liver presented the typical pale appearance of hydrazinized dogs. Experiment 4. Dog 10. At 1 :00 p. m., May 13, 20 cc. of a 2.5 per cent solution of hydrazine sulphate were administered hypodermically to a well nourished bitch of 10 kilos. On May 15 the operation for pancreas removal was completed at 11:30 a. m. Immediately after the operation the animal was given a subcutaneous injection of 150 cc. 0.9 per cent sodium chloride solution to supply fluid to the tissues. The urine (50 cc.) obtained by cath- eterization at 2:30 p. m. did not reduce Benedict's solution. At 6:00 p. m. catheterization yielded 45 cc. urine which contained no reducing substance. Benedict's test was also negative with the 110 cc. of urine obtained by cath- eterization at 11:30 p. m. The following morning 150 cc. of urine obtained at 9:00 a. m. by use of the catheter contained 3.65 grams dextrose. The animal was immediately given 10 cc. hydrazine hypodermically. At 12:30 p.m. the dog voided urine which gave a faint test for sugar. The quantity present was too small, however, to be estimated with the polariscope. Only 5 cc. of urine were obtained by catheter at 3:00 p. m. and this gave a nega- tive Benedict's test. At 6:00 p. m. the few cubic centimeters of urine obtained did not reduce Benedict's solution. The animal at this time appeared to be failing rapidly. It was apparent that the kidneys were not functionating as well as normally, therefore, the dog was given a subcuta- neous injection of 100 cc. 0.9 per cent sodium chloride solution. The 10 cc. of urine procured at 11:15 p. m. failed to give any evidence of reducing com- pounds. The following morning the dog was found dead. The bladder was empty. The liver was very light colored. No evidence of pancreatic rests could be seen. Experiment 5. Dog 6. A well-fed bitch of 11.5 kilos received a sub- cutaneous injection of 23 cc. of a 2.5 per cent hydrazine sulphate solution on the afternoon of April 24. The pancreas was removed April 28, the operation being completed at 4:30 p. m. A few hours previous to the oper- ation 10 cc. hydrazine (2.5 per cent solution) were given. The urine ob- tained at 12:30 a. m. April 29 did not reduce Benedict's solution. At 9:00 a. m. of the same day the dog was found dead. No reducing substance was present in the 100 cc. of urine found in the cage bottle. The liver presented the typical light colored appearance. No trace of pancreas was in evidence. The results of these observations warrant the conclusion that in the quantities employed the subcutaneous administration of hydra- zine is capable of preventing the appearance of sugar in the urine of depancreatized dogs. The non-appearance of sugar in the urine under the experimental conditions can not be ascribed to a latent period attendant upon pancreas removal for in the control experi- ments given above glycosuria promptly follows extirpation of the pancreas. 276 Hydrazine and Pancreatic Diabetes The sugar content of the blood of hydrazinized dogs after pancreas extirpation. For the correct interpretation of the above mentioned results obtained with hydrazinized dogs after pancreas removal some knowledge of the behavior of the blood sugar is imperative. Such data would afford a much better idea of the changes which occur in sugar metabolism than can be gained by a study of the appear- ance of sugar in the urine alone. It is true that preliminary exper- iments previously reported have demonstrated the action of hydra- zine in causing hypoglycaemia but it does not necessarily follow that the same activity prevails after pancreas removal. It can be conceived for instance that hydrazine may have at least a two- fold activity — one causing a temporary hypoglycaemia, the other an action rendering the kidney less permeable to sugar poured into the blood after pancreas removal, in a manner opposite to that which has been ascribed to phloridzin. Improbable though this seems, it was necessary to test the matter experimentally. table 1. The sugar content of blood of hydrazinized dogs after pancreas removal. NUMBER OP DOG AND BODY WEIGHT SUBCUTANEOUS INJECTION OP HYDRAZINE SULPHATE SUGAR CONTENT OF BLOOD IM- MEDIATELY BE- FORE PANCREAS REMOVAL SUGAR CONTENT OF BLOOD AFTER PANCREAS REMOVAL Arter half hour After three hours After six hours 13.* 12 kilos mgms. per kilo 50 50 50 per cent 0.029 0.09 0.04 per cent 0.031 0.15 per cent 0.016 0.13 0.03 per cent 0.12 0.01 14.* 16.4 kilos 15.* 8.5 kilos 'No sugar appeared in the urine throughout the experiment. In the experiments reported in Table 1 the animals were subjected to the action of hydrazine for a period of two days after which the pancreas was removed. The blood sugar was determined in arterial blood drawn from a femoral artery. The observations reveal at least two indications: (a) Hydrazine maintains its typi- Frank P. Underhill and Morris S. Fine 277 cal effect upon the blood sugar by keeping it far below normal in spite of the opposite accumulative tendency characteristic of pancreas extirpation, and (6) in animals in which hydrazine does not appear to greatly reduce the blood sugar, its influence is still sufficient to keep the content of blood sugar below the point at which the kidney becomes permeable to it. In presenting these data the realization is borne in upon us that the duration of these experiments is not very great. They repre- sent, however, the extreme limit of time that our animals could withstand the combined action of hydrazine poisoning, pancreas removal and extensive hemorrhage. The latter alone is quite detrimental since for exact determination of small quantities of sugar in the blood fairly large amounts of blood should be employed. We have used between twenty and thirty grams of blood for each estimation. The inhibition of pancreatic diabetes in dogs by subcutaneous injec- tions of hydrazine. The non-appearance of sugar in the urine of depancreatized dogs previously subjected to the influence of hydrazine is possibly open to the criticism that the pancreas extirpation was not sufficiently complete to have invariably induced glycosuria. Although we consider this criticism hardly valid inasmuch as we have never failed to produce glycosuria after pancreas removal in normal dogs it is nevertheless true that the inhibition of pancreatic dia- betes by hydrazine would be of far more significance than the prevention only. Details of the experiments planned to demonstrate this feature of the investigation are to be found in the following protocols. Experiment 6. Dog 7. The pancreas was removed from a full-grown bitch of 5 kilos on May 2. The operation was completed at 4:15 p. m. Urine collected by catheter at 10:00 p. m. contained about 1 gram of dex- trose. The urine contained in cage bottle and that obtained up to 10:30 a. m., May 3, was found to contain 5.4 grams dextrose. At this time 5 cc. 2.5 per cent solution hydrazine sulphate were injected subcutaneously. At 3:30 p. m. 50 cc. of urine obtained by catheterization showed a content of 2.83 grams dextrose. A second injection of hydrazine (10 cc. of the above solution) was administered. The urine (250 cc.) yielded from this time up to 10:30 a. m., May 4, showed the presence of 1 gram dextrose. Cath- eterization at 3:30 p. m. furnished 45 cc. of urine which gave a very faint 278 Hydrazine and Pancreatic Diabetes test with Benedict's solution. The quantity of dextrose was too small to be indicated by the polariscope. The animal was catheterized at 6:00 p. m. and at 9:00 p. m. and the quantity of urine obtained each time consisted of only a few drops which gave a negative test with Benedict's solution. The animal was found dead the next morning. From this experiment it is evident that hydrazine had a very decided influence upon the pancreatic diabetes, at first by greatly diminishing the quantity of dextrose eliminated and finally by completely inhibiting its appearance in the urine. This result may perhaps be seen better from the following consideration. The sugar eliminated from the time of operation on May 2 to 3:30 p. m. on May 3 amounted to 9.23 grams, that excreted from May 3 at 3:30 p. m. to May 4 at 3:30 p. m. was only 1 gram. After 3 :30 p. m. on May 4 the urine was sugar-free. The decrease from 9.34 grams of dextrose in approximately twenty-four hours to 1 gram during the succeeding day shows the remarkable rapidity with which hydrazine must accomplish its action. From the rapid falling off in urine secretion it is also evident that hydrazine must exert a distinct influence upon the kidney secretion. Experiment 7. Dog 11. On May 16 the pancreas was removed from a bitch of 7 kilos the operation being completed at 11 :00 a. m. As soon as the animal was under the influence of ether preparatory to the operation a subcutaneous injection of 50 mgms. hydrazine sulphate per kilo was given. Catheterization at 12:30 p. m. yielded 40 cc. of urine which was sugar-free. The 30 cc. of urine obtained at 3:30 p. m. yielded a small quantity of dex- trose. At 6:00 p. m. the dog was again catheterized and the 20 cc. of urine yielded no reducing body. At this time 100 cc. 0.9 per cent sodium chloride were injected subcutaneously. From the catheterization at 11:20 p. m. 30 cc. of urine obtained gave no evidence of the presence of dextrose. On the following day 100 cc. of urine were obtained at 9:00 a. m. which showed the presence of the merest trace of dextrose. At this time 10 cc. of hydra- zine sulphate (2.5 per cent solution) were given. The urine furnished at 3 :30 p. m. and 5 :30 p. m. gave evidence of a mere trace of reducing substance. At 11:45 p. m. the urine (10 cc.) obtained by catheterization furnished evi- dence of the presence of dextrose in the slightest degree only; by Benedict's test the merest precipitate formed on cooling. The animal was still living at 10:00 a. m., May 18, but was in a somewhat comatose condition. About 5 cc. of urine obtained from the bladder contained no reducing substance. The animal died during the morning. The results of this experiment demonstrate that the influence of hydrazine was almost sufficient to completely inhibit the elim- Frank P. Underhill and Morris S. Fine 279 ination of sugar in the urine. At one time the inhibition was complete, but during the night of May 16 it is evident that the action of hydrazine was insufficient to overcome the sugar accumu- lative power of the depancreatized dog and sugar was found in the urine, its appearance finally being prevented by a second dose of hydrazine. Experiment 8. Dog 25. From a bitch of 12 kilos the pancreas was removed resulting in the elimination of 11.20 grams of dextrose for the suc- ceeding twenty-four hours. Then a subcutaneous injection of 20 cc. hydra- zine sulphate in a 2.5 per cent solution was administered. Two hours later the urine excreted held 0.69 gram dextrose. An hour later 11 cc. of urine obtained by catheterization yielded the merest trace of dextrose, too small to be indicated by the polariscope. Six hours after the hydrazine adminis- tration the urine was sugar-free. The data submitted justify the conclusion that hydrazine admin- istered to dogs during pancreatic diabetes is capable of completely inhibiting the elimination of sugar by the kidney. How much hydrazine is necessary to prevent pancreatic diabetes and how long does its action persist? The importance of the determination of the quantity of hydra- zine necessary to prevent pancreatic diabetes is obvious. How long the peculiar action of the drug endures is a question also partic- ularly worthy of investigation. These topics have as yet been considered in the crudest manner only and hence the results thus far obtained do not allow us to make more than the most general statements. To find out the limit of time during which a single subcutaneous injection of 50 mgms. hydrazine sulphate per kilo is still capable of exerting its preventive action toward pancreatic diabetes, the following experiment was carried through. Experiment 9. On June 24 a 12 kilo bitch received the usual injection of hydrazine. Four days later the pancreas was removed. Within a few hours after the operation the appearance of sugar in the urine in fairly large quantities (10 to 12 grams per day) was noted. The animal lived two days after the operation. From this result it is apparent that a single subcutaneous injec- tion of 50 mgms. hydrazine sulphate per kilo body weight is not 28o Hydrazine and Pancreatic Diabetes capable of preventing the appearance of sugar in the urine of dogs deprived of the pancreas four days after the hydrazine adminis- tration. The time during which this influence prevails must lie, therefore, somewhere between two and four days. A single experiment to test the effect of smaller doses of hydra- zine is detailed in Experiment 10. Dog 26. On July 8 a bitch of 17.4 kilos received a subcutaneous injection of 25 mgms. hydrazine sulphate per kilo. Two days later the pancreas was removed. In this experiment it was necessary to employ a small quantity of morphine in order to anaesthetize the animal. The pancreas had been removed at 11:30 a. m. and 4.3 grams dextrose were found in the urine at 2 :30 p. m. At 5 :30 p. m. the urine held 3.2 grams dex- trose. A second injection of the same dose of hydrazine was now given. The dog was found dead at 9:00 a. m. the following morning. From the bladder 30 cc. of urine were obtained which reduced Benedict's solution. This observation indicated that a single injection of 25 mgms. hydrazine sulphate per kilo is insufficient to prevent the elimina- tion of dextrose in the urine of dogs deprived of the pancreas two days after the initial dose of hydrazine. A tentative hypothesis to account for the action of hydrazine upon sugar metabolism. Porges 1 has demonstrated that adrenal extirpation as well as adrenal insufficiency exemplified in Addison's disease leads to a significant hypoglycaemia and a disappearance of glycogen from the liver. Phosphorus 2 produces the same effects and Neubauer and Porges 3 have ascribed to phosphorus an influence upon the adrenals leading to an insufficiency of adrenal secretion, presum- ably adrenalin, thereby causing the liver glycogen to disappear and the blood sugar content to decrease. This theory was based upon experiments in which Neubauer and Porges failed to obtain evidence of adrenalin in extracts of the adrenals after phosphorus administration. 1 Porges: Zeitschrift fur klinische Medizin, lxix, p. 341, 1910; lxx, p. 143. 2 Frank and Isaac: Archiv fur experimentelle Pathologie und Pharmakol- ogie, lxiv, p. 274, 1911. 3 Neubauer and Porges: Biochemische Zeitschrift, xxxii, p. 290, 1911. Frank P. Underhill and Morris S. Fine 281 Since phosphorus and hydrazine are now observed to be alike in their effect upon blood sugar content and liver glycogen disappear- ance, it is reasonable to assume that the influence of the two might possibly be directed upon the same object, namely, upon the adrenal. The explanation as to the manner in which adrenal secretion influences carbohydrate metabolism is lacking. As a working hypothesis we have made use of the following scheme of inter- action. DIAGRAM INDICATING INFLUENCE OF THE ORGANS ON THE METABOLISM OF CARBOHYDRATE. (Sugar Content) Blood Liver (Glycogen Store) (Sugar Content) Blood (Sugar Content) Blood V Pnncreas Secretion/ s > < /Adrenal Secretion \ Liver (Glycogen Store) PANCREAS REMOVAL (indicated by broken lines) leads to Hyperglycaemia Anuria Diuresis No Glycosuria Glycosuria 282 Hydrazine and Pancreatic Diabetes In the first place we have assumed that there is an interrelation between the pancreas, the adrenals and the liver by the equilibrium of which the sugar content of the blood is kept practically constant. To accomplish this the pancreas is assumed to pour an internal secretion into the blood which tends to increase carbohydrate catabolism, hence to diminish the blood sugar. This influence we have called the pancreas secretion factor. On the other hand the adrenal is assumed to give an internal secretion to the blood which has the opposite effect, namely, it has a tendency to check carbohydrate catabolism, hence leads to an accumulation of sugar in the blood. This action we have designated the adrenal secretion factor. According to this idea of what occurs to keep blood sugar content normal, the quantity of glycogen which will be taken from the liver's store to accomplish this purpose is entirely dependent upon which of the two factors is predominant at any given moment. If we apply these ideas to pancreas extirpation or adrenal re- moval, the effects which should theoretically follow according to our scheme agree perfectly with recorded observations which for the most part have universal acceptance. When the pancreas has been removed from an animal our hypoth- esis assumes that the pancreas factor is taken away, in other words what we have called the influence that " facilitates carbohydrate catabolism" is lost. Consequently the blood sugar equilibrium is upset because the adrenal factor, which "inhibits carbohydrate catabolism" is no longer counterbalanced by the pancreas factor, the influence normally furnished by the pancreas. Without the check of the pancreas upon it the adrenal factor now has full play, there is less carbohydrate catabolism than normally, sugar, poured out of the liver's store of carbohydrate, accumulates in the blood (hyperglycaemia), diuresis is induced in the effort to eliminate the sugar through the kidneys, and then glycosuria is in evidence. If this condition of affairs is maintained sufficiently long the liver will be more or less depleted of its glycogen owing to the body's vain effort to furnish sufficient energy for its needs. On the other hand, removal of the adrenals from a normal ani- mal results in a series of events of the opposite order. The power which has a tendency to " inhibit carbohydrate catabolism," the adrenal factor, has been lost, hence the pancreas factor holds Frank P. Underhill and Morris S. Fine 283 full sway, resulting in the rapid catabolism of all available carbo- hydrate material even to the depletion of the liver glycogen, leading to hypoglycaemia, fall of blood pressure, hence anuria, and no glycosuria. Extirpation of both pancreas and adrenals should yield results in correspondence with which of the organs was first removed. If the adrenals were extirpated first hypoglycaemia should be in evidence; if the pancreas were removed before the adrenal, hyper- glycaemia should prevail. The extirpation of both would ulti- mately mean a complete upset of the sugar regulation of the organ- ism. If we assume that hydrazine acts in a manner similar to the action ascribed to phorphorus it is evident that here hydrazine activity upon the blood sugar content would be equivalent to partial or complete extirpation of the adrenals. Hence according to our tentative hypothesis hydrazine may prevent pancreatic diabetes by its inhibition or at least suppression of adrenal func- tion. Viewed from another standpoint it is possible that hydra- zine has an action upon sugar metabolism entirely similar to that exerted by the internal secretion of the pancreas. According to this idea injections of hydrazine cause hypoglycaemia by in- creasing the efficiency of the pancreatic secretion or by augment- ing its output. All the results thus far obtained could be inter- preted upon this basis. Assuming that hydrazine does exert some inhibitory influence upon adrenal secretion one might expect perhaps to obtain some indication of this by testing adrenal extracts furnished by hydrazinized dogs for the generally accepted active principle of adrenal secretion, namely, adrenalin. Is the secretion of adrenalin inhibited by hydrazine administration? The communication of Neubauer and Porges 1 demonstrating the absence of adrenalin in the adrenals as a sequel to phosphorus administration would lead one to infer that possibly hydrazine has a similar influence, more especially as both these agents have the same action upon the blood sugar causing it to be significantly decreased. The exhibition 2 of adrenalin in the adrenals is usu- 1 Neubauer and Porges: loc. cit. *cf. Biedl: Innere Secretionen, for literature to color reactions. 284 Hydrazine and Pancreatic Diabetes ally made by color reactions, among which may be mentioned the development of a red color on the addition of mercuric chloride, or green coloration caused by adding a neutral solution of ferric chloride to aqueous extracts of these organs. In our first attempts to gain evidence of the presence of adre- nalin in watery extracts of the adrenals by the above mentioned color reactions we were unsuccessful. It soon developed, how- ever, that the accompanying turbidity, presumably of a protein nature, was the cause for our failures, for if the filtrates could be obtained water clear the reactions proved to be very delicate. Our final method for demonstrating the presence of adrenalin con- sisted in grinding the adrenals to a pulp in a mortar with fine sand and a very little water or physiological salt solution. This mix- ture was filtered and the turbid filtrate clarified by the addition of just enough mercuric chloride to precipitate the protein (usu- ally two or three drops of a 10 per cent solution). The clear fil- trate, on the addition of a little more mercuric chloride, soon yielded the pink or red color characteristic of adrenalin. The development of the coloration was much more rapid and intense if the solution was warmed somewhat. The green color reaction with ferric chloride was obtained in a similar manner, the pre- liminary addition of mercuric chloride for the purpose of clarify- ing the turbid filtrate being without any apparent detriment to the reaction. In fact, so little mercuric chloride was added that it is probable that practically all of it was removed in combina- tion with the precipitate. With this method the extracts of the adrenals taken from seven normal dogs showed the presence of significant quantities of adre- nalin. Dogs poisoned with hydrazine, the dose of which was in some instances large enough to kill the animals within twenty-four hours, yielded adrenals giving apparently just as strong adrenalin reactions as normal animals. This result makes it evident, therefore, that even if hydrazine does have an inhibitory influence upon adrenal produc- tion the inhibition is not complete; it is a quantitative influence. Frank P. Underhill and Morris S. Fine 285 CONCLUSIONS. Removal of the pancreas from normal dogs may be followed by the appearance of sugar in the urine within a period of two hours. Glycosuria fails to manifest itself after pancreas extirpation in dogs that have received previous injections of hydrazine. In general this effect is produced by a single subcutaneous injection of 50 mgms. per kilo hydrazine sulphate. The influence of the hydrazine administration lasts between two and four days. A single injection of 25 mgms. per kilo hydrazine sulphate does not prevent sugar elimination in the urine, under the experimental conditions, after pancreas removal. The blood sugar content of animals treated with hydrazine and then depancreatized remains below the normal, or at least hyper- glycaemia is not in evidence. Hydrazine introduced into dogs during pancreatic diabetes is capable of completely inhibiting the excretion of sugar by the kidney. After hydrazine administration the presence of adrenalin in the adrenals may still be demonstrated. In this respect hydra- zine differs from phosphorus. A tentative hypothesis to account for the phenomena is presented. We are greatly indebted to Professor Lafayette B. Mendel for aid in some of the operations for pancreas extirpation and for criticism of the manuscript. Reprinted from the American Journal of Physiology. Vol. XXVII. — January 2, 1911. — No. III. THE PRODUCTION OF GLYCOSURIA BY ADRENALIN IN THYROIDECTOMIZED DOGS. By FRANK P. UNDERHILL, [From the Sheffield Laboratory of Physiological Chemistry, Yale University, New Haven, Connecticut.] THE failure of adrenalin to induce glycosuria after removal of the thyroids (the parathyroids being intact) reported by Ep- pinger, Falta, and Rudinger 1 was not corroborated by Underhill and Hilditch. 2 In the experiments of the latter adrenalin called forth the appearance of sugar in the urine of all the thyroidectomized animals tested. In a reply by Falta and Rudinger 3 the following criticisms con- cerning the work of Underhill and Hilditch are offered: (i) with a single exception the latter authors injected larger quantities of adren- alin than were employed by Eppinger, Falta, and Rudinger. The positive outcome of the single exception is given the doubtful ex- planation that some thyroid secretion might still be in circulation such a short time (two days) after thyroidectomy. On the other hand, when in the same animal adrenalin in the proper dosage called forth glycosuria eighty-one days after removal of the thyroids, Falta and Rudinger point out that "Es darf aber nicht vergessen werden, dass bei Hunden sich gar nicht so selten akzessorische Schilddriisen langs des Oesophagus nach abwarts finden, welche bis zum Pericard herabsteigen konnen." 4 (2) Falta and Rudinger claim that comparison can be made with their experiments only when the irritability of the thyroidectomized animal to an electrical current has been determined 1 Eppinger, Falta, and Rudinger: Wiener klinische Wochenschrift, 1908, p. 241; Zeitschrift fur klinische Medizin, 1908, lxvi, p. 1, and 1909, lxvii, p. 1. 2 Underhill and Hilditch: This journal, 1909, xxv, p. 66. 3 Falta and Rudinger: Zentralblatt fur die gesamte Physiologie und Pathologie des StorTwechsels, 1910, xi, p. 81. 4 Falta and Rudinger: Loc. cit., p. 83. 33i 332 Frank P. Underhill. in an endeavor to discover latent tetany. Underhill and Hilditch made no such observations. (3) Finally, according to Falta and Rudinger, Underhill and Hilditch made no autopsies to demonstrate whether perchance thyroids or accessory thyroids were still present in these animals. In reply to the criticisms outlined above it should be stated, first of all, that apparently Falta and Rudinger read only a review 5 of the work by Underhill and Hilditch. In the original article 6 there are given at least two instances, out of a possible four, in which it may be seen that the same or a smaller quantity of adrenalin than that employed by Eppinger, Falta, and Rudinger induced glycosuria in dogs after thyroidectomy. Falta and Rudinger explain sugar ex- cretion in the single exception noted by them (Dog A 7 or Dog I) by assuming that the adrenalin was administered too soon after removal of the thyroids (two days). This explanation will not suffice, how- ever, for the appearance of dextrose in the urine when adrenalin was injected according to the Eppinger, Falta, and Rudinger standard of dosage in the case of Dog D. 8 This injection occurred six days after thyroidectomy, and the statement is made by the above-mentioned authors that at least three days after thyroidectomy adrenalin failed to produce glycosuria. Falta and Rudinger admit in the case of Dog A (Dog I), in which glycosuria was caused eighty-one days after removal of the thyroids, that there can be no question here of a latent tetany, but they inti- mate that accessory thyroids may be present. At the time of the publication of our former investigation it was considered undesirable to kill Dog A (Dog I) in order to determine the presence of accessory thyroids. Since then, however, an autopsy has been performed upon this animal with this end in view. Every piece of tissue in any way bearing a resemblance to thyroid tissues was carefully preserved and sent for identification to Professor H. Gideon Wells of the Uni- versity of Chicago. In his report concerning these tissues Professor 5 Underhill: Zentralblatt fur die gesamte Physiologie und Pathologie des Stoffwechsels, 1909, x, p. 641. 6 Underhill and Hilditch: Loc. cit. 7 In the article by Underhill and Hilditch the animals were called A, B, C, etc. In the review by Underhill Dog A was called Dog I, Dog B was designated Dog II, etc. 8 Underhill and Hilditch: Loc. cit. The Production of Glycosuria by Adrenalin. 333 Wells makes the statement that he finds no evidence of thyroid tissue or accessory thyroids; nothing except parathyroid tissue was in evi- dence. During the present summer an autopsy was also performed upon Dog C (Dog III). The result demonstrated the presence of two somewhat hypertrophied parathyroids but no trace of thyroid TABLE I. Dog. Time after thyroidectomy. Adrenalin chloride per kilo injected. Dextrose in urine. mgm. gm. March 3 1.0 0.0 A 1 16 months j March 15 1.0 4.79 21 2 21 days 1.0 0.76 22 3 4 days 1.0 3.67 23 4 3 days 1.0 14.38 1 This animal was Dog A employed in experiments by Underhill and Hilditch. The dog never showed any signs of abnormality. The body weight was 12.1 kilos. 2 A dog of 8 kilos in splendid nutritive condition. Both thyroids were removed, leaving two parathyroids intact. Fed a mixed diet. Autopsy revealed absence of thyroid tissues. 3 Well-fed dog of 13.2 kilos. No evidences of thyroid tissues on autopsy. 4 Dog of 12 kilos, in good condition. No thyroid tissues on autopsy. None of these animals showed any abnormality. tissue, nor could any thyroid tissue be found at the autopsy of Dog D. The results of the autopsy of Dog B (Dog II) were noted in our former communication. The criticism of Falta and Rudinger concerning the points just discussed are not extremely potent in view of the autopsy findings now reported, together with the observation that all dogs gave glyco- suria after removal of the thyroids on treatment with adrenalin, and that two of the four reacted positively with the same dosage employed by Eppinger, Falta, and Rudinger. Nevertheless, in order to decide the question even more definitely further experiments have been un- dertaken the results of which may be seen in Table I. The methods employed were identical with those outlined in our former commu- 334 Frank P. Underhill. nication, except that in the observations here reported all adrenalin injections were made subcutaneously. It may be seen from these data that adrenalin chloride administered subcutaneously to dogs in the dosage of i mgm. per kilo body weight is capable of provoking the appearance in the urine of significant quantities of dextrose, three, four, and twenty-one days, and thirteen months after thy- roidectomy. In no case were there any abnormal manifestations, nor could any thyroid tissues be found on autopsy. In the present investigation, as in the previous one, we have deemed it unnecessary to determine the response of the thyroidectomized animal to electrical stimulation in order to discover latent tetany. The observation that none of the animals selected behaved in an abnormal manner, together with the fact that Dog A (Dog I) and Dog C (Dog III) of our previous experiments lived more than six- teen months without tetany, that like conditions obtained for Dog D, killed ten days after thyroidectomy because of an abscess at site of injection, and that Dog 21 of the present investigation was allowed to live more than twenty-one days after removal of the thyroids — these facts all speak against the idea that latent tetany was present. The criticism that adrenalin injection was made too soon after operation in the case of Dog A (Dog I) of our former investigation will not hold for our present experiments, since in no case was adrenalin introduced under three days after removal of the thyroids. A survey of the work of Eppinger, Falta, and Rudinger reveals that in several instances their own injections were made three and four days after thyroidectomy. In the paper by Falta and Rudinger two new experiments 9 are detailed designed to corroborate former statements. Concerning the first experiment the question may well be asked, "Why was the dose of adrenalin, 10.5 mgm. (less than 1 mgm. per kilo for a 13-kilo dog) divided into two portions (6 mgm. and 4.5 mgm.) and these injected on two separate days?" Such a procedure does not add weight to the statement of Eppinger, Falta, and Rudinger that 1 mgm. adrenalin per kilo body weight administered subcutaneously or in- traperitoneally into thyroidectomized dogs is incapable of causing glycosuria. The failure of these quantities of adrenalin to provoke the appearance of sugar in the urine can be duplicated at times in 9 Falta and Rudinger: Loc ciL, p. 82. The Production of Glycosuria by Adrenalin. 335 normal dogs. In fact, even 1 mgm. per kilo often fails to induce glycosuria (see Dog 22, Table II), and in Dog A (Dog I) without thyroids (see Table I) it may be seen that this dose failed on March 3, whereas on March 15 it caused the excretion of 4.79 gm. dextrose. From our experience with normal dogs we have been led to the con- clusion that, although in general 1 mgm. adrenalin per kilo body weight administered subcutaneously or intraperitoneally is capable of causing glycosuria, animals are frequently encountered in which this dose provokes no glycosuria. On the other hand, these same animals at other times behave in the usual way and react to doses of 1 mgm. adrenalin per kilo. The second experiment of Falta and Rudinger shows that 5 mgm. adrenalin injected intraperitoneally before removal of the thyroids caused a slight glycosuria. After thyroidectomy the same quantity injected intraperitoneally failed. Later 10 mgm. introduced sub- cutaneously into two places also failed to provoke glycosuria. In several experiments we have noted the absence of dextrose in the urine following the intraperitoneal introduction of 5 mgm. adrenalin into normal dogs smaller than the one employed by Falta and Rudinger. The fact that 5 mgm. in this instance failed to produce glycosuria does not necessarily mean that this has been due to thy- roid removal. When the subcutaneous injection was given, the animal weighed 16.2 kilos, and yet only 10 mgm. adrenalin were ad- ministered. If Falta and Rudinger desired to offer evidence in sup- port of their former statement that 1 mgm. adrenalin per kilo will not cause glycosuria in thyroidectomized dogs, why did they not inject enough adrenalin to comply with their own conditions? Again in a footnote the following is given concerning the dog of the second experiment: "Der Hund bekam vor dem 2. Versuch auch Pituitri- num infundibulare. Dieses hat nach unseren Untersuchungen keinen EinfTuss auf den Kohlehydratstoffwechsel." 10 Nevertheless, in an experiment the results of which may be so significant it would have been much better to have eliminated this last unnecessary conflict- ing factor. The experiments of Falta and Rudinger do not support the original statement of Eppinger, Falta, and Rudinger that 1 mgm. adrenalin per kilo administered subcutaneously or intraperitoneally fails to 10 Falta and Rudinger: Loc. cit. 336 Frank P. Under hill. provoke glycosuria in thyroidectomized dogs, since in neither of the protocols reported is there any indication that these authors intro- duced i mgm. adrenalin per kilo body weight. In an article by Grey and de Sautelle 11 the conclusion is drawn that after thyroidectomy in dogs glycosuria evoked by adrenalin is much smaller than in the normal animal. The method of procedure adopted by these investigators was as follows : Dogs were kept upon a fixed meat diet for several days. They were then injected with adrenalin, after which thyroidectomy was performed. During re- covery from the operation a mixed diet was fed. Then meat was again given and adrenalin administered a second time. In the two experiments recorded less sugar in the urine was obtained after thy- roidectomy, as a result of adrenalin injection, than was excreted by the normal dog. The results obtained by these authors, namely, the appearance of sugar in the urine of thyroidless dogs after administration of less than i mgm. adrenalin per kilo, stand in direct opposition to those reported by Eppinger, Falta, and Rudinger, but they are in perfect harmony with the observations of Underhill and Hilditch. Grey and de Sautelle were also unable to find any thyroid tissue at autopsy. On the other hand, the experiments cited hardly warrant the con- clusion drawn by the authors, namely, that after thyroidectomy the glycosuria, produced by adrenalin in the normal animal, is greatly reduced. The investigation was evidently carefully planned and executed, but was based upon an assumption the correctness of which is questionable. Consequently the conclusion drawn is not firmly established. From the data presented it is apparent that these authors assumed that if the same normal dog is kept under constant conditions and given equal doses of adrenalin at two different times the quantity of sugar excreted after these injections should be approxi- mately the same. If that assumption was not made, then the ex- periments are purposeless. All experimental evidence, however, points against such an assumption, for the same normal dog under constant conditions does not necessarily excrete the same quantity of sugar with the same dosage of adrenalin given at two different times. If a normal dog will not invariably respond to adrenalin 11 Grey and de Sautelle: Journal of experimental medicine, 1909, xi, p. 659 # The Production of Glycosuria by Adrenalin, 337 twice alike, it is fallacious to attribute a lessened elimination of sugar to the loss of the thyroids without at least the support of a great number of experiments all showing the same marked tendency. In an experiment having as its object the study of the influence of the thyroids upon carbohydrate metabolism, it is, therefore, apparent that quantitative changes in sugar excretion should be given little weight. As corroboratory to the views just expressed the data in Table II are submitted. In these experiments the plan followed was very similar to that outlined by Grey and de Sautelle, and although the details differ somewhat the end aimed at, to keep conditions of diet constant, was attained. The animals had been fed upon meat for several days before the experiments began. They were then fed upon a constant mixed diet for a period of five days. Then adrenalin was given subcutaneously, — 1 mgm. per kilo body weight. On the day of the injection the usual quantity of water was given but no food. As a rule sugar elimination ceased within twenty-four hours after adrenalin administration. The dogs were then fed upon meat until five days before the second adrenalin injection. During these five days the mixed diet was again given. As before, no food was offered on the injection day. After the second adrenalin administra- tion the thyroids were completely removed, but at least two para- thyroids, one on each side, were left intact. This was confirmed at autopsy. During the period of recovery from the operation the dogs received the meat diet. Five days before the third adrenalin injec- tion the mixed diet was fed, and as previously no food was given on the day of injection. By such a regime the animals remained prac- tically constant in weight. In every instance 1 mgm. adrenalin per kilo was administered subcutaneously in the region of the lower ribs. The subcutaneous injection possesses the following advantages: animals do not die so frequently as with the intraperitoneal injection, and are not so likely to vomit or have diarrhoea or bloody urine. In this particular point we differ from Grey and de Sautelle, since their injections were made intraperitoneally. The principle, however, is identical in the two cases, since the mechanism involved in each case is the same. Furthermore, there is no basis for assuming that adrenalin given intraperitoneally will show a different behavior concerning the point under discussion than adrenalin administered subcutaneously. 338 Frank P. Underbill. The data presented in Table II demonstrate conclusively that adrenalin administered twice to the same normal animal under like conditions does not necessarily provoke the same degree of glycosuria in the two instances. Moreover, in every case reported adrenalin induced glycosuria after thyroidectomy, and the quantity of sugar TABLE II. Sugar in Urine in Grams. Dog. Before thyroidectomy. After First injection. Second injection. thyroidectomy. A Xi 1.27 ... | 0.16 | March 3 0.0 March 15 4.79 Died during operation 21 9.70 1.20 0.76 22 0.0 3.40 3.67 23 1.22 4.64 14.38 1 This animal was a dog of 7.0 kilos. The details concerning the other dogs are given in the footnotes of Table I, p. 333. In each ex- periment 1 mgm. adrenalin per kilo was injected subcutaneously. eliminated after the operation was not uniformly decreased. In fact, in two of three experiments detailed more sugar was excreted by the thyroidectomized dog than appeared in the urine of the normal dog. Conclusions. Renewed investigation concerning the efficiency of adrenalin in provoking glycosuria in thyroidectomized dogs leads to a reiteration of our former conclusion that adrenalin chloride administered sub- cutaneously in doses of i mgm. per kilo body weight causes a signifi- cant glycosuria in dogs deprived of both thyroids but retaining at least two parathyroids. The criticisms of Falta and Rudinger with respect to our former experiments have in no way invalidated this conclusion. The Production of Glycosuria by Adrenalin, 339 In the investigcation by Falta and Rudinger, put forth in support of the conclusions deduced by Eppinger, Falta, and Rudinger, they have failed to comply with the conditions laid down by the latter. Consequently the results offered by Falta and Rudinger cannot be accepted as proof that adrenalin administered to thyroidectomized dogs in doses of 1 mgm. per kilo is incapable of causing glycosuria. The observations of Grey and de Sau telle are in harmony with our own results and stand in direct opposition to the position taken by Eppinger, Falta, and Rudinger. The validity of the conclusions drawn by Grey and de Sautelle, however, may be questioned, since the investigation was based upon an assumption the correctness of which has not yet been established. Experiments are reported demonstrating that adrenalin admin- istered subcutaneously to normal dogs in doses of 1 mgm. per kilo causes a widely varying degree of glycosuria. The same quantity of adrenalin introduced into thyroidectomized dogs under like condi- tions is capable of inducing as great or even a greater glycosuria than occurs with the normal animal. Reprinted from the Proceedings of the Society for Experimental Biology and Medicine, 1911, viii, pp. 126-127. 76 (60l) The behavior of fat-soluble dyes in the organism. By LAFAYETTE B. MENDEL and AMY L. DANIELS. [From the Laboratory of Physiological Chemistry, Sheffield Scientific School, Yale University, New Haven, Conn.] It is well known that the fat-soluble dye, Sudan III., is readily deposited in the adipose tissue of animals. An attempt was made by the authors to study the movements of the dye under conditions where fat transport takes place (e. g., in starvation, phlorhizin- and phosphorus poisoning). The dye readily migrates into the blood with the fat under these conditions, but is rarely found in the liver tissue into which large quantities of fat enter (fatty infiltra- tion). This is explained by the observation that the Sudan III. is abundantly excreted with the bile into the intestine from which it may be reabsorbed. Sudan III., which is insoluble in water, is not excreted through the kidneys except where alimentary lipuria is induced (in rabbits and rats). The elimination from the liver is not accomplished through the solvent medium of fat excreted in the bile (lipocholia) ; but the dye is soluble in bile as well as in solution of the isolated bile salts. We have thus estab- lished a path of elimination for fat-soluble (or bile-soluble) substances through the biliary secretion. An investigation of a considerable number of water-insoluble, fat-soluble compounds — mostly non- toxic aniline dyes and food colors — showed comparable conditions justifying the above general conclusion. It has further been established that these water-insoluble compounds do not experi- ence absorption from the intestine in the absence of bile. Dis- solved in fat-emulsion and introduced into the organism by ali- mentary, subcutaneous, or intravenous paths, these dyes are al- ways eliminated with the bile into the intestine. When there is a paucity of fat in the diet the fat-soluble dyes may be absorbed through the agency of reabsorbed bile, but they are speedily Scientific Proceedings (44). 2 eliminated again by the liver channels; with an abundance of fat to act as carrier, they travel with it through the lymphatics into the circulation. The distribution of fat-soluble dyes within the organism depends on the presence of fat and its migrations. Thus they may be carried to or from adipose tissues, be deposited in the egg-yolk, or be secreted in company with fat in the milk of animals; they apparently do not traverse the placenta. The dyes have not been detected in the lipoids of the nervous tissue. We have failed to note any inability on the part of animals to utilize fats in which Sudan III. has been deposited. Reprinted from the Proceedings of the Society for Experimental Biology and Medicine, 191 1, viii, p. 80. 47 (572) The production of glycosuria as a result of the intravenous injection of Witte's peptone. By YANDELL HENDERSON and FRANK P. UNDERHILL. [From the Physiological Laboratory, Yale Medical School and the Sheffield Laboratory of Physiological Chemistry, Yale University.] Renewed investigation concerning the phenomena provoked by intravenous administration of Witte's peptone has demon- strated the production of a marked glycosuria, following such in- jections. The appearance of sugar in the urine is accompanied by hyperglycemia. The experiments were carried out for the most part upon dogs. When Witte's peptone is injected into the rabbit glycosuria is not in evidence, an observation which is in entire accord with the failure of this substance to induce certain other phenomena in this animal which are brought about in the dog. The tentative hypothesis is advanced that the presence of sugar in the urine is induced as a result of the respiratory disturbances set up by the "peptone" injection. The phenomena connected with the injection of "peptone" mixtures are being subjected to further investigation. Reprinted from the American Journal of Physiology. Vol. XXVII. — February 1, 1911. —No. IV. THE METABOLISM OF DOGS WITH FUNCTIONALLY RESECTED SMALL INTESTINE. By FRANK P. UNDERHILL. (With the Collaboration of CHESTER J. STEDMAN and JESSAMINE CHAPMAN.) [From the Sheffield Laboratory of Physiological Chemistry, Yale University, New Haven, Connecticut.] r I A HE necessity of removing varying lengths of intestine from man has made imperative extensive investigations concerning the influence of such surgical procedures upon nutritional processes. This is particularly true with respect to the mechanisms involved in digestion and absorption. For obvious reasons the experimental demonstration of the effects incident to resection of portions of the enteric tract has been made for the most part upon the dog and cat. Of particular importance in this connection have been the observa- tions recorded by Harlay, 1 Senn, 2 Trzebicky, 3 Monari, 4 De Filippi, 5 Erlanger and Hewlett, 6 Flint, 7 and others. 8 From these investiga- tions it may be stated that extirpation of portions of the small intes- tine generally entails a decreased absorption of the nitrogenous and fatty constituents of the food. The extent of lessened absorption depends upon the relative length of intestine removed, and also upon the period which has elapsed after the operation, that is, whether compensation has been established. The composition of the food 1 Harlay: Proceedings of the Royal Society, London, (B), 1899, lxiv, p. 255. 2 Senn: Experimentelle Beitrage zur Darmchirurgie, Basle, 1892, quoted. 3 Trzebicky: Archiv fur klinische Chirurgie, 1894, xlviii, p. 54. 4 Monari: Beitrage zur klinischen Chirurgie, 1896, xvi, p. 479. 5 De Filippi: Archives italiennes de biologie, 1894, xxi, p. 445. 6 Erlanger and Hewlett: This journal, 1901, vi, p. 1. 7 Flint: Transactions Connecticut State Medical Society, 1910, p. 283. A complete discussion of the earlier literature upon this subject both in connection with the human subject and with the lower animals may be found in this article. 8 London and Dmitriew: Zeitschrift fur physiologische Chemie, 1910, lxv, p. 213; Carrel, Meyer, and Levene: This journal, 1910, xxv, p. 439. 366 The Metabolism of Dogs. 3fy may play a significant role, since it has been established that large quantities of fat bring about a much poorer utilization of food nitro- gen than occurs when smaller quantities of fat are ingested. Dimin- ished utilization of fat is particularly noticeable in these experimental animals. With respect to carbohydrate absorption the observations are somewhat at variance, since in some instances it has been reported that the faeces held reducing substances, and in other experiments none were found. In the investigation to be reported study of three problems was held in view, (1) the absorption of foodstuffs after functional removal of varying lengths of small intestine, (2) a study of food absorption at different intervals after the operation, and (3) the determination of carbohydrate utilization under varied conditions. Experimental. Description of the animals employed. — The dogs used in these ex- periments were placed at our disposal through the kindness of Pro- fessor Joseph Marshall Flint, and for convenience will be designated Dog A, Dog B, and Dog No. 12. In these three instances a portion of the small intestine was short-circuited. Dog A was a water spaniel of 8.3 kilos in splendid nutritive condi- tion. Two weeks after the operation, when she came into our posses- sion, January 26, 1910, the wound was well healed. The stools of this animal were fairly well formed, and throughout the entire period of observation diarrhoea was not once noticed. On autopsy about nine months later it was found that the entire intestine was 412.5 cm. long and that 162.5 cm. or 39 per cent had been short-circuited. Dog B was a mongrel bitch weighing 13.5 kilos when she came into our possession, January 26, 1910. The animal was in fair nutritive condition, but suffered from persistent diarrhoea, discharging copious liquid stools of exceedingly foul odor. On her entrance to the labora- tory two weeks after the operation the wound was well healed. On autopsy about nine months later measurement showed the entire length of intestine to be 525 cm., of which 350 cm. had been short- circuited, — 66 per cent. Dog No. 12 was the animal called Dog No. 12 in Professor Flint's report. She was received into the laboratory January 8, 1909, al- 368 Frank P. Under hill. most six months after the operation. At that time she weighed 7.4 kilos; a distinct loss of weight which was never regained. Diarrhoea was persistent and the appetite was ravenous. On autopsy it was found that of the 324 cm. of small intestine 235 cm. or 73 per cent had been short-circuited. Methods. — In general the methods followed were those usually employed in metabolism experiments in this laboratory. Urine was collected in twenty-four-hour periods by catheterization. The water content of the faeces was determined by drying them upon the water bath under acid alcohol and preserving them air-dry. Carbohydrates in the faeces were estimated according to the method of Tsuboi. 9 Food was given in two portions daily. One meal was at nine o'clock in the morning, the other at four o'clock in the afternoon. Description of Experiment I. In this experiment Dog A and Dog B were employed. For several days previous to the investigation the animals had received an ade- quate mixed diet. The experiment was planned to determine the ability of these dogs to maintain nitrogenous equilibrium upon suffi- cient mixed diets the composition of which was to be radically altered at intervals with respect to the content of fat and carbohydrate. As originally planned, each period of the experiment was to extend over five days. Owing to faecal contamination of the urine of Dog B, it was practically impossible to obtain urine and faeces unmixed for five consecutive days, with the exception of the first period. Accordingly in the other periods balances have been made covering the consecu- tive days on which the urine was uncontaminated. Although it was out of the question at times to include the excreta in the balance, food was given as usual and all other conditions remained constant, so that when a balance of less than five days is reported the results are probably fairly representative of the animal's condition through- out the entire period. The periods of this experiment have been designated Periods 1, 2, 3. Diets. Dog A. — The diet for Period 1 consisted of 100 gm. meat, 50 gm. cracker meal, 20 gm. lard, 10 gm. bone ash, and 150 c.c. 9 Tsuboi: Zeitschrift fur Biologie, 1897, xxxv, p. 68. The Metabolism of Dogs. 369 water, the total nitrogen content of which amounted to 4.39 gm., or 56 gm. nitrogen per kilo. The estimated fuel value was 505 calories, or 64 calories per kilo. In Period 2 the diet was made up of 100 gm. meat, 75 gm. cracker meal, 36 gm. lard, 10 gm. bone ash, and 230 c.c. water, containing 4.81 gm. nitrogen and 750 calories, or 0.62 gm. nitrogen and 96 calories per kilo. The composition of the food in Period 3 was as follows: 100 gm. meat, 100 gm. cracker meal, 10 gm. bone ash, and 300 c.c. water. The diet contained 5.23 gm. nitrogen, and a calculated fuel value of 520 calories, or 0.69 gm. nitrogen and 67 calories per kilo body weight. Further details concerning Experiment I, Dog A, may be found in Tables I and III, pages 370 and 372 respectively. Diets. Dog B. — In the first period this dog was placed upon a diet consisting of 200 gm. meat, 80 gm. cracker meal, 30 gm. lard, 20 gm. bone ash, and 300 c.c. water. This diet contained 8.44 gm. nitrogen and had an estimated fuel value of 837 calories, or 0.63 gm. nitrogen and 63 calories per kilo body weight. In Period 2 the diet was made up of 200 gm. meat, 120 gm. cracker meal, 50 gm. lard, 30 gm. bone ash, and 400 c.c. water, and contained 9. 1 1 gm. nitrogen and n 80 calories (calculated), or 0.71 gm. nitrogen and 93 calories per kilo. The diet in Period 3 had the following composition: 200 gm. meat, 160 gm. cracker meal, 30 gm. bone ash, and 450 c.c. water. The food contained 9.78 gm. nitrogen with an estimated fuel value of 875 calories, or 0.79 gm. nitrogen and 70 calories per kilo body weight. For further details of Experiment 1, Dog B, see Tables II and III, pages 371 and 372 respectively. Discussion of Results of Experiment I. Throughout the entire first experiment, Dog A maintained body weight and furnished large positive nitrogen balances (see Tables I and III). Nitrogen utilization was practically the same as that of a normal animal upon a mixed diet. The effect of increasing carbohy- drate and fat intake had little influence upon nitrogen equilibrium, although fat utilization on this diet was somewhat diminished. A further increase is to be noted when lard was entirely removed from 370 Frank P. Underbill. TABLE I Experiment I. — Dog A, 39 per Cent Intestine Resected. 1 Period 1. — Food: 100 gm. Meat; 50 gm. Cracker Meal; 10 gm. Lard; 10 gm. Bone Ash; 150 c.c. Water. Nitro- gen in food. Urine. Faeces. Date. Body weight. Vol- ume. Total Weight. Water Nitro- gen. Ether extract. nitro- gen. Moist Air- dry. con- tent. 1910. Jan. 29 kilos. 7.8 gm. 4.39 c.c. 160 gm. 1.95 gm. 3.3 gm. 2.7 per cent. 18 gm. gm. " 30 7.7 4.39 160 3.06 " 31 7.7 4.39 180 3.15 29.0 > 2.66 11.96 Feb. 1 7.7 4.39 100 2.70 36.5 22.5 38 " 2 7.7 4.39 100 3.21 59.6 38.0 36 Period 2. — Food: 100 gm. Meat; 75 gm. Cracker Meal; 36 gm. Lard; 10 gm. Bone Ash; 230 c.c. Water. Feb. 3 7.7 4.81 200 3.21 " 4 7.7 4.81 200 2.93 77.2 46.7 39 " 5 7.7 4.81 200 4.08 26.8 > 3.68 27.35 " 6 7.7 4.81 200 3.03 40.8 21.0 48 " 7 7.7 4.81 200 3.43 53.8 28.7 46 Period 3. — Food: 100 gm. Meat; 100 gm. Cracker Meal; 10 gm. Bone Ash; 300 c.c. Water. Feb. 8 7.7 5.23 230 4.26 36.8 17.9 51 - " 9 7.6 5.23 240 4.02 55.6 26.0 53 " 10 7.6 5.23 240 3.90 > 3.67 9.80 " 11 7.7 5.23 280 3.84 32.6 20.4 37 " 12 7.7 5.23 310 4.26 97.5 43.7 55 1 In all experiments the urine showed an acid reaction to litmus. The Metabolism of Dogs. 371 table n. Experiment I. — Dog B, 66 per Cent Intestine Resected. Period 1. — Food: 200 gm. Meat; 80 gm. Cracker Meal; 30 gm. Lard; 20 GM. Bone Ash; 300 c.c. Water. Urine. Faeces. Body weight. Nitro- gen in food. Date. Vol- ume. Total Weight. Water Nitro- gen. Ether extract. nitro- gen. Moist. Air- dry. con- tent. 1910. Jan. 29 kilos 13.3 gm. 8.44 c.c. 550 gm. 9.24 gm. 23.9 gm. 9.7 per cent. 59 gm. gm. " 30 13.0 8.44 350 6.90 93.8 52.7 44 " 31 13.0 8.44 420 5.40 104.7 50.2 52 > 5.33 29.26 Feb. 1 12.9 8.44 320 8.16 119.8 45.8 62 " 2 12.7 8.44 310 8.16 73.2 36.8 50 Period 2. — Food: 200 gm. Meat; 120 gm. Cracker Meal; 50 gm. Lard; 30 GM. Bone Ash; 400 c.c. Water. Feb. 5 12.5 9.11 350 7.86 " 6 12.6 9.11 410 8.24 153.5 81.5 47 ►3.41 15.66 " 7 12.6 9.11 450 7.89 114.2 64.5 43 Period 3. — Food: 200 gm. Meat; 160 gm. Cracker Meal; 30 gm. Bone Ash; 450 c.c. Water. Feb. 10 " 11 12.4 12.3 9.78 9.78 430 360 7.80 7.86 155.7 129.7 59.3 50.2 62 61 1 3.37 5.48 the diet and carbohydrate intake again augmented. Carbohydrate utilization was perfect upon all diets of this experiment. With Dog B there was a gradual but steady loss of body weight upon diets which would have been entirely adequate for a normal animal of the same weight. In the first period (five days) a negative balance of 0.99 gm. nitrogen or minus 0.19 gm. nitrogen per day was 372 Frank P. Underhill. obtained. During this period nitrogen of the food was utilized to the extent of 87 per cent. The fat utilization was 85 per cent, while that of the carbohydrate was perfect — that is, no trace of carbohydrate could be demonstrated in the faeces. In the second period (three TABLE III. Summary. — Experiment I. Dog A, 39 per Cent Nitrogen. Periods. Food. Excreta. Balance. Urine. Faeces. Total. Per period. Per day. 1 gm. 21.95 gm. 14.07 gm. 2.66 gm. 16.73 gm. +5.22 gm. + 1.04 2 - 24.05 16.68 3.68 20.36 +3.69 +0.74 3 26.15 20.28 3.67 23.95 +2.20 +0.44 Dog B, 66 per Cent 1 42.20 37.86 5.33 43.19 -0.99 -0.19 2 27.33 23.99 3.41 27.40 -0.07 -0.02 3 19.56 15.66 3.37 19.03 +0.53 +0.26 days) the dog was in almost perfect nitrogenous equilibrium, a total negative balance of only 0.07 gm. or 0.02 gm. nitrogen per day being obtained. During the period both carbohydrate and fat intake had been markedly increased, nitrogen increase being slight. In spite of these increases the utilization of the three components of the diet re- mained practically unchanged. The third period (two days) reveals a positive nitrogen balance of 0.53 gm. nitrogen, or plus 0.26 gm. nitrogen per day. No lard was fed during this period, but carbohy- drate was much increased. The utilization of nitrogen was not quite so good as in previous periods. Fat utilization was greatly dimin- ished. A portion of this apparent diminution may probably be ex- The Metabolism of Dogs. 373 plained by the presence in the faeces of ether soluble intestinal ex- cretory products which in the absence of truly unutilized food fat causes a distortion of the percentage utilization. Although the car- bohydrate intake was twice that of the first period, no trace of sugar- . TABLE III. Summary Experiment I. Intestine Resected. Fat (ether extract). Carbohydrate. Utiliza- tion. Food. Faeces. Utilization. Food (calcu- lated). Faeces. Utilization. per cent. 87 gm. 125.50 gm. 11.96 per cent. 90 gm. 172 gm. per cent. 100 84 207.55 27.35 86 273 100 86 29.55 9.80 66 364 100 Intestine Resected. 87 199.45 29.26 85 292 100 87 181.59 15.66 91 262 100 82 22.36 5.48 75 233 100 yielding substances could be detected in the faeces. A point of interest in connection with the faeces is the variable water content. From these experiments upon animals with different lengths of intestine put out of function shortly after the operation only small differences can be detected in the ability of the two dogs to utilize their food, although in one case, Dog A, only 39 per cent of the en- tire small intestine was not functionating, whereas with Dog B 66 per cent was non-functional. Furthermore, notable increases in fat intake appeared to cause little or no change in fat, nitrogen, or car- bohydrate utilization. Large increases in carbohydrate did not result in impaired utilization. The carbohydrate utilization was 374 Frank P. Underhill. very much better than that of normal dogs upon practically the same diets. For instance, unpublished experiments of Dr. Mary D. Swartz make it evident that only 90 to 95 per cent of ingested carbohydrate is utilized in the normal dog. Only in the case of fat can the utiliza- tion be called poor. TABLE IV. Experiment II. — Dog A, 39 per Cent Intestine Resected. Period 1. — Food: 100 gm. Meat; 50 gm. Cracker Meal; 10 gm. Lard; 10 gm. Bone Ash; 150 c.c. Water. Nitro- gen in food. Urine. Faeces. Date. Body weight. Vol- Total Weight Water Nitro- Ether ume. nitro- gen. Moist. Air- dry. con- tent. gen. extract. 1910. May 25 kilos. 10.5 gm. 4.43 c.c. 75 gm. 3.77 gm. 23 gm. 13 per cent. 43 • gm. gm. " 26 10.4 4.43 80 3.70 32 17 47 j> 1.60 5.88 " 27 10.4 4.43 65 3.28 38 25 34 Period 2. — Food: 100 gm. Meat; 75 gm. Cracker Meal; 36 gm. Lard; 10 gm. Bone Ash; 230 c.c. Water. May 28 10.5 4.88 85 3.44 16 7 56 " 29 10.5 4.88 110 3.51 49 27 45 " 30 10.6 4.88 180 3.42 41 22 46 > 3.44 13.22 " 31 10.5 4.88 160 3.28 43 21 51 June 1 10.5 4.88 220 3.33 68 35 48 Description of Experiment II. At the completion of Experiment I the dogs were allowed to run in large airy cages and were fed upon adequate mixed diets. As time progressed, it became noticeable that Dog B developed an almost in- satiable thirst. Diarrhoea was persistent, the stools discharged being notably clay-colored. A constant but gradual loss of weight also The Metabolism of Dogs. 375 occurred. On the other hand, Dog A appeared to be perfectly normal and steadily gained in weight. The rest period for these animals extended from February 12 to May 23. From this time until June 2, TABLE V. Experiment II. — Dog B, 66 per Cent Intestine Resected. Period 1. — Food: 200 gm. Meat; 80 gm. Cracker Meal; 30 gm. Lard; 20 gm. Bone Ash; 300 c.c. Water. Nitro- gen in food. Urine. Faeces. Date. Body weight. Vol- ume. Total Weight. Water Nitro- gen. Ether extract. nitro- gen. Moist. Air- dry. con- tent. 1910. May 23 kilos. 10.2 gm. 8.50 c.c. 205 gm. 7.83 gm. 137 gm. 50 per cent. 64 gm. gm. " 24 10.4 8.50 150 6.39 223 67 70 " 25 10.4 8.50 175 7.78 140 59 58 > 6.93 66.96 " 26 10.2 8.50 190 7.37 166 60 64 " 27 10.2 8.50 150 7.05 165 64 64 Period 2. — Food: 200 gm. Meat; 120 gm. Cracker Meal; 50 gm. Lard; 30 gm. Bone Ash; 400 c.c. Water. May 28 10.2 9.22 170 6.99 118 32 73 " 29 10.2 9.22 175 6.96 315 106 65 " 30 10.0 9.22 165 6.96 262 110 59 > 8.63 102.37 " 31 10.0 9.22 155 7.26 372 150 60 June 1 10.0 9.22 185 7.56 63 25 60 two periods of five days each were carried out upon diets practically identical with those of Experiment 1. The food fed in these two periods corresponded with that given in the first two periods of Ex- periment 1. Owing to slight differences of nitrogen content of the meat, the total nitrogen intake varied slightly from that in the previ- ous experiment. See Tables IV, V, and VI. 376 Frank P. Under hill. Discussion of Results of Experiment II. During the second period of observation (Table VI) Dog A furnished only positive nitrogen balances. This animal had 39 per cent of its small intestine short-circuited. Fat utilization may be fairly com- TABLE VI. Summary. — Experiment II. Dog A, 39 per Cent Nitrogen. Periods. Excreta. Balance. Food. Urine. Faeces. Total. Per period. Per day. 1 gm. 13.29 gm. 10.75 gm. 1.60 gm. 12.35 gm. +0.94 gm. +0.31 2 24.40 16.98 3.44 20.42 +3.98 +0.79 Dog B, 66 per Cent 1 42.5 36.42 6.93 43.35 -0.85 -0.17 2 46.1 35.75 8.63 44.38 + 1.72 +0.34 pared with that of a normal dog on a mixed diet and was better than in Experiment I several months earlier. Increase of fat intake had little if any influence upon utilization of any of the foodstuffs. • Car- bohydrate utilization remained perfect. The body weight of Dog A was 7.8 kilos at the beginning of Experiment I, and at the end of Ex- periment 2 had increased to 10.5 kilos — a gain of 2.7 kilos. It is at once apparent from an inspection of Table VI, Dog B, that at a period three months after functional resection of two thirds of the intestine fat utilization was much lower than shortly after the opera- tion. Increasing fat intake within somewhat narrow limits did not markedly impair utilization of any of the foodstuffs. Carbohydrate utilization was still perfect. Nitrogen utilization, however, was some- what lowered and appeared to undergo a still further slight diminution The Metabolism of Dogs. 377 by increase in fat intake. At this later period of observation the dog furnished a slight negative nitrogen balance for the first five days and a positive nitrogen balance for the second period. The body weight at the beginning of Experiment I was 13.3 kilos and at the end of Experiment II was 10.0 kilos — a loss of 3.3 kilos. TABLE VI. Summary. — Experiment II. Intestine Resected. Fat (ether extract). Carbohydrate. Utiliza- tion. Food. Faeces. Utilization. Food (calcu- lated). Faeces. Utilization. per cent. 87 gm. 75.48 gm. 5.88 per cent. 92 gm. 109 gm. per cent. 100 86 206.20 13.22 94 273 100 Intestine Resected. 83 201.28 66.96 66 292 100 81 301.90 102.37 66 437 100 Description of Experiment III. The food received by Dog No. 12 was the usual mixture of raw meat, cracker meal and lard which was fed several days previous to the actual period of observation and in sufficient quantities to main- tain a normal dog in nitrogenous equilibrium. Water was given ad libitum. Preliminary trials demonstrated the separate collection of urine and faeces to be almost impracticable owing to the persistent diarrhoea. To overcome this obstacle the animal was fed small quan- tities of finely ground agar-agar with the food — a procedure which resulted in the passage of stools which while not formed were also not fluid. The number of defalcations was just as great as without 378 Frank P. Underhill. the agar, but the character of the stools was so entirely altered that contamination of the urine was prevented. In all probability the insoluble agar produced this change in the texture of the faeces by imbibing the water from the intestinal contents. Utilization of nitrogen, fat, and carbohydrate. — In the first observa- tion with this dog it was planned to bring about nitrogenous equi- TABLE VII. Experiment III, Period 1. — Dog No. 12, 73 per Cent Intestine Resected. Date. 1909. Body weight. Nitrogen in food. Urine. Faeces. Volume. Total nitrogen. Ammonia nitrogen. Indican fehlings sol. = 100. We .22 'o 3 ight. •i £ Water content. Nitrogen. Ether extract. Jan. kilos. gm. c.c. gm. gm, gm. gm. per cent. gm. gm. 14 7.0 8.92 130 6.48 0.34 10 210 36 83 15 7.0 8.24 125 6.00 0.55 10 232 37 84 ^8.70 18.70 16 7.0 8.24 220 8.40 0.70 12 67 79 14 82 17 6.8 8.24 210 6.86 12 7 librium as nearly as possible and then to determine the utilization of the different foodstuffs. To accomplish this purpose a few days previous to the real observation the dog received a diet consisting of 200 gm. meat, 120 gm. cracker meal and 10 gm. lard containing 8.92 gm. nitrogen and furnishing approximately 775 calories, or 1.27 gm. nitrogen and no calories per kilo body weight, amounts which would be far in excess of the requirements for a normal dog of this size. This diet was continued through the first day (January 14) of obser- vation and was then reduced, since the animal appeared to have difficulty for the first time in devouring these large amounts of food. The new diet contained 200 gm. meat, 80 gm. cracker meal, 10 gm. lard, and 10 gm. agar. The nitrogen content amounted to 8.24 gm. This diet was eaten readily. During the four days of observation, the results of which may be seen in Tables VII and IX, it is apparent that the dog was not in a condition of nitrogenous equilibrium in spite of the previous ingestion of large quantities of food, the nitrogenous balance for the four days The Metabolism of Dogs. 379 being minus 2.80 gm., or minus 0.7 gm. nitrogen per day. Turning to the utilization of nitrogen and fat, it may be observed that both were poor, nitrogen being utilized to the extent of only 74 per cent fat TABLE VIII. Experiment III, Periods 2 and 3. — Dog No. 12, 73 per Cent Intestine Resected. Period 2. — Food per Day: 100 gm. Meat; 25 gm. Gelatin; 80 gm. Cracker Meal; 10 gm. Lard; 10 gm. Agar- agar. Total Nitrogen = 8.24 gm. Urine. Faeces. Date. Vol- Total nitro- gen. Indican fehl- Weight. Water Nitro- Ether ume. ings solution = 100. Moist. Air-dry. con- tent. gen. extract. 1909. Jan. 22 c.c. 120 gm. 6.38 7 gm. 112 gm. 20 per cent. 82 gm. gm. " 23 160 6.57 9 48 " 24 175 7.49 8 201 51 80 > 7.19 15.80 " 25 250 10.10 8 86 18 80 " 26 160 6.72 10 159 24 84 Period 3. — Food per Day: 50 gm. Gelatin; 80 gm. Cracker Meal; 10 gm. Lard; 10 gm. Agar-agar. Total Nitrogen = 8.96 gm. Jan. 27 450 8.88 11 1 146 26 82 " 28 " 29 430 170 7.44 7.58 8 8 161 35 29 82 > 8.91 24.90 " 30 160 7.51 9 32 to the extent of 72 per cent. These figures agree well with those of Erlanger and Hewlett. In spite of the extremely foul odor of the faeces the quantity of indican eliminated through the urine was not excessive. The figures for ammonia nitrogen are high when com- pared to the output of the normal dog. Another point of interest was the rather large percentage of water contained in the faeces of this animal. Normally the water content of the air-dry faeces rarely 3 8o Frank P. Under hill. exceeds 75 per cent, whereas those passed by this animal had a water content of 80 to 85 per cent (see also Table VIII). It is not unlikely that the water content of the faeces may have been increased by the presence of agar, which would imbibe a certain quantity of water and prevent its absorption. It is hardly probable, however, that this is the sole explanation since the fluidity of the faeces passed when TABLE IX. Summary. — Experiment III, Dog No. 12, 73 per cent Intestine Resected. Peri- ods. Nitrogen. Fat (ether extract). Food. Excreta. Balance. Utili- zation. Food. Faeces. Utili- za- tion. Urine. Faeces. Total. Per period. Per day. gm. gm. gm. gm. gm. gm. per cent. mg. gm. per cent. 1 33.64 27.74 8.70 36.44 -2.80 -0.70 74 67.60 18.70 72 2 41.20 37 26 7.19 44.45 -3.25 -0.64 82 96.50 15.80 84 3 35.84 31.41 8.91 40.32 -4.48 -1.12 75 59.20 24.90 58 no agar was given was such that a large water content was obvious. By varying the amount of carbohydrate even up to four times the requirement for a normal dog of the same weight, no change in the utilization of this foodstuff could be observed. It has been demon- strated repeatedly in this laboratory that a diet sufficient for a normal dog of this weight may consist of 200 gm. meat, 30 gm. cracker meal, and 25 gm. lard. Dog No. 12 received approximately this diet, then the cracker meal was increased to 80 gm. and the lard reduced to 10 gm. Finally, the cracker meal was still further increased to 120 gm. In all cases carbohydrate utilization was complete. 10 In these experiments designed to test carbohydrate utilization agar was not given with the food for obvious reasons. The Influence of Gelatin Feeding upon the Elimination of Urinary Indican. In a previous communication 11 it was demonstrated that the re- placement of meat by gelatin in a mixed diet results in a diminution 10 Cf. Flint: hoc. ciL 11 Underhill: This journal, 1904-1905, xii, p. 176. The Metabolism of Dogs. 381 in the excretion of indican in the urine of the dog. This observation is in accord with the now well-established origin of indican. In Table VIII (Periods 2 and 3) are given the results of observations made with a view of determining whether a dog with a short-circuited in- testine would behave in a manner similar to a normal animal when a portion or all the meat of the diet was replaced by gelatin. It is ob- vious from these figures that the substitution of gelatin for meat was without significant influence upon urinary indican elimination. With this animal only negative nitrogen balances were obtained. In the second gelatin period nitrogen utilization amounted to 75 per cent, which is comparable to the utilization obtaining when meat was fed (see Table IX, Period 1). The fat utilization in the first gelatin period (see Table IX, Period 2) when some meat was fed was much better than when the meat was entirely replaced by gelatin. In the first instance utilization amounted to 84 per cent; in the second gelatin period (see Table IX, Period 3) only 58 per cent of the fat was utilized. It would appear that meat in the diet of this animal had a tendency to aid fat utilization. Summary. From the foregoing observations it is apparent that as much as 39 per cent of the small intestine of a dog may be short-circuited without causing significant detrimental changes in the utilization of the various foodstuffs, and the animal may gain in weight. This statement is equally true when observations are made either at a period shortly after operation or at a period several months later. When as much as 66 per cent of the small intestine has been func- tionally resected, the nutritive condition of the animal presents an entirely different aspect. Under these conditions fat utilization is particularly decreased and the dog displays a decided tendency to furnish negative nitrogen balances. A small though steady loss of weight is especially noticeable. Food utilization is in general ap- parently much better immediately after the operation than at a later period. In neither animal did a material increase in fat intake cause significant change in the utilization of this or other foodstuff. When about three quarters of the small intestine of the dog has been short-circuited, food utilization for the most part is seriously 3 82 Frank P. Underbill. iir paired, at least at a period several months after the operation. This is particularly true for fat utilization. Indican elimination through the urine is not materially altered under these conditions by replace- ment of meat in the diet with gelatin, an observation directly opposed to that obtained with the normal dog. The animal with a short-circuited intestine displays a greater ability to utilize carbohydrate than does the normal dog. Even though the carbohydrate intake may be much, in one case several times, greater than the normal animal requires, carbohydrate utiliza- tion is complete whether the test is made shortly after the operation or months later. This observation may prove of practical importance in the dietary treatment of the human subject who has undergone extensive intestinal resection. Reprinted from the American Journal of Physiology. Vol. XXVIII. — August 1, 1911. — No. V. ACAPNIA AND GLYCOSURIA. By YANDELL HENDERSON and FRANK P. UNDERHILL. [From the Physiological Laboratory, Yale Medical School, and the Sheffield Laboratory of Physiological Chemistry, Yale University, New Haven, Connecticut.] I. The Point of View. GLYCOSURIA is induced by an extraordinarily large number of widely differing conditions. This fact indicates that it is the result of a disturbance of a complex balance involving many factors. With the exception of phloridzin diabetes, practically all forms of glycosuria, whether experimental or clinical, are the result of a hyper- glycemia. The excess of sugar in the blood is in turn, not a funda- mental phenomenon, but the expression of diminished ability on the part of the tissues to utilize dextrose. Thus the seat of the complex equilibrium is in the cells, not the fluids, of the body. The problem of the normal glycogenic function and of experimental diabetes consists in determining the various factors in this equilibrium and denning their relations. The problem of any clinical form of glycosuria lies in discovering which particular factor or set of factors is altered from its normal strength, and how. One of the most important of the sets of factors involved in the normal balance is the internal or tissue respiration. In his investiga- tion^ on various forms of experimental diabetes, Underhill 1 noted the casual connection of these conditions with disturbances of breathing. Araki 2 has shown that an insufficient supply of oxygen to the cells (e. g. y in CO poisoning) results in a notable glycosuria. Macleod 3 reached the conclusion that in asphyxia it is the excess of CO2 and not the deficiency of oxygen, which stimulates hepatic glycogenolysis. The 1 Underhill, F. P.: Journal of biological chemistry, 1905, i, p. 113. 2 Araki: Zeitschrift fur physiologische Chemie, 1891, xv, p. 351. 3 Macleod, J. J. R.: This journal, 1909, xxiii, p. 302. 275 276 Yandell Henderson and Frank P. Under hill. work of Edie, 4 and of Edie, Moore, and Roaf 5 seems to demonstrate that an excess of CO2 in the air breathed apart from oxygen deficiency may result in glycosuria. We proposed to ourselves the question: Will acapnia also upset the capacity of the tissues to hold sugar? The observations to be here reported indicate that such is the case. One may bring a see-saw to the ground as well by lessening as by increasing the weight on one end. Our observations indicate also that acapnia is a concomitant of some forms of experimental glycosuria in which Edie, Moore, and Roaf have held that hypercapnia must occur. Glycosuria is known to follow violent emotion in human subjects and to occur in cats which rage at being tied. 6 Henderson 7 has pointed out that the stormy breathing .of anger involves excessive pulmonary ventilation. He has likewise shown that ether anaesthesia in dogs without morphin nearly always involves a greater or less degree of acapnia, never hypercapnia. It is noteworthy that a tem- porary glycosuria is a frequent sequel of ether anaesthesia. Prolonged ether excitement may reduce the C0 2 content of the blood to half the normal amount, and result fatally. 8 These effects, including the glyco- suria, are solely due to the excessive breathing induced by ether excite- ment. 9 We have never found sugar in the urine of dogs which had been brought into deep ether anaesthesia quietly. We believe that both emotional glycosuria and polyuria are frequently the result of acapnia. Acetone has an influence even more powerful than ether in excit- ing respiration to excessive activity. When administered to normal animals it has been shown to produce glycosuria. 10 The close asso- ciation of this substance with the most important clinical form of 4 Edie: Biochemical journal, 1906, i, p. 455. 5 Edie, Moore, and Roaf: Biochemical journal, 1911, v, p. 325. 6 Bohm and Hoffmann: Archiv fur experimentelle Pathologie und Pharma- kologie, 1878, viii, p. 271. 7 Henderson, Y.: This journal, 1910, xxv, p. 311. 8 Henderson, Y.: This journal, 1910, xxvi, p. 280. 9 Underhill: hoc. cit. 10 Albertoni: Archiv fur experimentelle Pathologie und Pharmakologie, 1884, xviii, p. 218; v. Jaksch: Zeitschrift fiir klinische Medizin, 1886, x, p. 362; Ruschhaupt: Archiv fiir experimentelle Pathologie und Pharmakologie, 1900 xliv, p. 127; Muller: Ibid., 1901, xlvi, p. 67. Acapnia and Glycosuria. 277 glycosuria raises the question to what extent acapnia may occur, and what its significance may be, in diabetes mellitus. Acetone is prob- ably a factor in that " nervousness " of diabetics which is in part the subjective expression of their proneness to hyperpncea. In diabetic coma a degree of acapnia has been observed more in- tense than in any other known condition. Magnus Levy 11 found only 3.3 per cent of CO2 in the blood just before the death of a patient, — less than one tenth of the normal. It is generally assumed that this is not a true acapnia such as results from excessive breathing, but that it is merely a pseudo-acapnia due to the expulsion of CO2 from the bicarbonates of the blood by organic acids. This view involves, how- ever, a large measure of assumption. To prove it would require simul- taneous determinations of the CO2 tension, C0 2 content, and hydro- + gen ion concentration (H) of the blood. No one has yet performed this difficult task. 12 The line of reasoning usually adopted starts from the fact that (1) Acids are formed in the tissues and pass into the blood in acidosis. (2) Acids liberate CO2 from bicarbonates. And (3) during acidosis the CO2 content of the blood is greatly diminished. From these three facts the conclusions are drawn that: (1) In acidosis the acids are produced in an amount sufficient to explain the low content of CO2 wholly by expulsion. (2) The capacity of the blood to carry CO2 is destroyed or greatly diminished. And (3) the acidity or (H) is in- creased. The facts scarcely warrant any of these conclusions. Beddard, Pembrey, and Spriggs 13 found that diabetic blood is quite capable of taking up large quantities of CO2 when exposed to the gas in vitro. Evidently the alkalies of the blood even in acute diabetic acapnia are far from being completely neutralized by oxybu- tyric and other organic acids. These investigators have also recorded the exceedingly important observation that during diabetic coma the CO2 tension of the alveolar air of the lungs is far below normal. Thus they have demonstrated that the acapnia incident to acidosis is mainly 11 Magnus Levy: Archiv fur experimentelle Pathologie und Pharmakologie, 1901, xlv, p. 389. 12 For methods see Krogh, Skandinavisches Archiv fur Physiologie, 1908, xx, p. 279; Hasselbalch: Biochemische Zeitschrift, 1910, xxx, p. 317. Michaelis and Rona: Ibid., 1909, xviii, p. 317. 13 Beddard, Pembrey and Spriggs: Journal of physiology, 1904, xxxi, p. xliv. 278 Yandell Henderson and Frank P. Underbill. a true low tension acapnia due to hyperpncea. From this fact does + it not follow that the (H) of diabetic blood is below normal, and that the blood during acidosis is less "acid " than normally, in spite of the neutralization of a part of its alkali bicarbonates by strong organic acids? The pulmonary ventilation of this condition is so excessive that it exhales not only the CO2 liberated from combination with alkalies, but also a part of that amount which is normally held in the blood in simple solution. It is preeminently that portion of carbonic acid which is merely dissolved, uncombined with alkalies, which according to the formulae + of L. J. Henderson 14 determines the (H) of the blood. This portion obeys the law of Henry. At equal partial pressures of CO2 in the lungs the carbonic acid dissolved as such will be the same in normal and in acidosis blood. Whichever is exposed to the lower gas pres- sure will have the less CO2 in simple solution, and to this extent the + lower (H). As L. J. Henderson has pointed out in his discussion of the neutrality equilibrium of the body, lactic, oxybutyric, and the other diabetic acids when thrown into the blood in moderate amount are almost completely neutralized by alkalies from bicarbonates and phosphates. Short of so intense an acidosis as to absorb practically + all of the alkalies of the bicarbonates, the (H) continues to depend principally just as under normal conditions upon the pressure of CO2 in the alveolar air of the lungs. In acute acidosis this pressure is much below normal. Furthermore the body aids itself in neutralizing the diabetic acids by an abnormally large amount of nitrogen in the form of ammonia in the blood and urine. The process of urea formation is, like glycoge- nolysis, an equilibrium of many factors, of which carbonic acid is one. 15 From the foregoing considerations it is evident that the excessive respiration associated with many forms of glycosuria cannot be due to an unusually high pressure of CO2 in the blood. Neither can it be due to a high acidity. It is almost certainly due to the acidosis sub- stances, of which a certain quantity is present in the blood even in health; but in our opinion it is probably the ethereal qualities of 14 Henderson, L. J.: Ergebnisse der Physiologie, 1909, viii, p. 254. 15 Macleod and Haskins: Journal of biological chemistry, 1906, i, p. 319. Acapnia and Glycosuria. 279 these bodies and not their acidic influence which, when added to the C0 2 remaining in the blood, excites the respiratory centre to maintain + the tension of this gas, and with it the (H), below normal. That such a conception is a possibility is proven by Henderson's observations that a fatal acapnia may be induced by means of ethyl ether, and by the fact that acetone is an even more powerful respiratory excitant. A number of investigators have noted that the intravenous infusion of acids exerts a very brief influence upon breathing. The acids are immediately neutralized into their salts; the CO2 thus displaced is + exhaled; and the (H) depends as before upon the carbonic acid in solution. The effect of the administration of alkalies in diminishing the dyspnoea during diabetic coma can in like manner be reconciled with our view. 16 If the activity of the respiratory centre is due to the sum- + mated influence of the ethereal acidosis bodies and the C0 2 or (H) in the blood, then diminishing the second term in this sum must reduce their combined stimulating influence in precisely the same manner as that by which alkalies lessen the breathing of a normal subject. It matters not whether the influence of carbonic acid upon the respiratory + centre be regarded as depending upon (H) (Winterstein 17 ) or whether, as appears to us more probable, this centre is specifically irritable to CO2 apart from its acidity. With such tentative conceptions as these in mind we undertook the experiments here to be reported. They show that acapnia is in fact a constant accompaniment of all the forms of experimental glycosuria studied by us. Upon the more important point, whether or not prevent- ing the development of acapnia will prevent glycosuria, the evidence which we have to report is incomplete. Apparently in some of these conditions such is the case, in others not. We have found it far more difficult than we had expected to administer an atmosphere so rich in CO2 as to prevent acapnia. As a suggestion to others who may in future work on this or related topics, and as a criticism upon much of the work previously published we would point out that absolutely the only way to find out whether an animal under any experimental con- ditions is acapnic or hypercapnic is by means of analyses either of the 16 Cf. Labbe and Violle: Presse medicale, Paris, 1911, xix, p. 292. 17 Winterstein, H. : Archiv fur die gesammte Physiologie, 191 1, cxxxviii, p. 167. 280 Yandell Henderson and Frank P. Underbill. blood or the alveolar air. In many of our own experiments we should have supposed the C0 2 content to be above normal when analyses actually showed it to be much below. Furthermore, as a commentary upon the theorizing of previous workers, it must always be remem- bered that the only certain criterion of whether the tissues of the body are adequately supplied with oxygen is the demonstration of a considerable quantity of this gas in the venous blood. The arterial blood may be saturated and arterial pressure high, yet if the blood stream is small the supply of oxygen will be insufficient for internal respiratory needs and the resulting hyperglycemia may be mistakenly assigned to other than its true cause. Most of the forms of acapnia thus far described by Henderson 18 have been the result of excessive breathing induced by external influ- ences upon afferent nerves. We would point out that perversions of metabolism (e. g., in diabetes and in fever) may produce substances capable of exciting the respiratory centre to abnormal activity. Thus acapnia may be a factor in the pathologic physiology of the diseases of internal medicine no less than in those of surgery. II. Peptone Glycosuria. The functional disturbances induced by intravenous injection of proteoses have been studied by a long series of investigators. In addi- tion to the effects of such injections upon the coagulability of the blood and upon arterial pressure, one of the most obvious results of such injections is that upon respiration. The animal exhibits great excitement with struggles, cries, and labored breathing. It was found by Lahousse 19 in Ludwig's laboratory that in dogs in which the arterial blood contained 33.4 to 39.5 volumes per cent of CO2 prior to peptone injection, a sample of blood in four minutes after the injection (i. e., when the condition of shock had fully developed) contained only 16.3 to 22.4 per cent C0 2 . The oxygen content of the blood was slightly increased. The indications were against the view that the tissue oxidations were diminished. Lahousse observed that the acapnia lasted as long as the condition of shock, and that the CO2 content of the blood rose again toward normal as recovery progressed. 18 Henderson, Y. : This journal, 1910, xxvi, p. 280. 19 Lahousse: Archiv fur Physiologie, 1889, p. 77. Acapnia and Glycosuria. 281 Glycosuria has never, so far as we can find, been observed as a re- sult of peptone injection. The consideration advanced in the previous section led us, however, to expect that it must occur. Our observations confirm those of Lahousse and in addition verify our expectation that glycosuria or at least hyperglycemia is a sequel of peptone shock, and that it is due to acapnia. 20 They demonstrate that the excessive respiration results in acapnia and is succeeded by a period of subnormal breathing, during which the CO2 content of the blood rapidly returns to normal. Glycosuria fol- lows. If, however, during the period of excitement excessive pul- monary ventilation is prevented by compelling the subject to breathe through a long tube attached to the trachea, neither glycosuria nor hypercglyaemia occur. Our experiments further indicate that the lowering of arterial pressure following peptone injection is not a factor in the disturbance of sugar control. Dog of 4 kilos under ether. Urine free from sugar. At 11.07 m ~ jected 2.5 gm. Witte peptone in 35 c.c. water into jugular vein. Vio- lent hyperpncea for a few minutes. At 1 2 .00 the animal had practically recovered. Urine was full of sugar. The NH 4 fraction of its nitrogen was 13.5 per cent. Cat of 2.1 kilos under ether. At 11.30 injected 1.0 gm. peptone in 20 c.c. water into jugular vein. Hyperpncea resulted. -At 12.21 urine was full of sugar. Experiment of Oct. 29, igoy. — Dog of 10 kilos. Ether. Injected into femoral vein 3 gm. Witte peptone in 40 c.c. water. Vigorous hyperpncea. Time. Arterial gases. Blood sugar. Urinary. Notes. o 3 co 2 per cent. NH : N. 9.50 18.0 30.0 0.16 6.9 10.08 Peptone injected. 10.12 20.5 21.0 0.12 10.55 9.0 Urine full of sugar. 11.30 20.0 26.0 0.26 20 Henderson and Underhill: Proceedings of the Society for Experimental Biology and Medicine, 191 1, viii, p. 80. 282 Yandell Henderson and Frank P. Underbill. Experiment of Dec. 5, igoy. — Dog, 23 kilos. Ether. Injected 12 gm. pep- tone in 100 c.c. water. Hyperpncea resulted. Time. Arterial gases. Venous gases. Arterial pressure. T>1 _ _ J Jdiooq sugar. Notes. o 2 C0 2 o 2 C0 2 MM.Hg. % 12.40 24.0 40.3 16.6 45.6 145 0.15 Urine free from sugar. 12.44 55 Peptone injected. Hy- perpncea. 1.00 18.7 30.6 6.6 39.0 45 1.40 23.0 16.0 16.4 33.4 3.12 25.0 12.6 38.8 95 0.27 Urine free from sugar. Experiment of Dec. 14, igoy. — Dog, 11.5 kilos. At first chloroformed; then ether. Injected 6.0 gm. peptone. Hyperpncea resulted. Tube at- tached to trachea 2 m. long with 13 mm. above. Time. Arterial gases. Venous gases. Arterial pressure. mm.Hg % Blood sugar. Notes. o 2 C0 2 o 2 C0 2 11.45 150 11.46 50 Peptone injected. 11.55 24.1 32.7 13.6 45.8 Blood does not clot. 12.00 Tube attached to trachea. 12.44 75 Tongue pink; breathing deeply. 1.12 23.9 38.5 14.3 51.3 85 Blood does not clot. 2.15 0.17 Urine free from sugar. III. Piqure Diabetes. The Bernard puncture of the floor of the fourth ventricle in a rabbit is usually followed immediately by hyperpncea. This excessive breath- ing continues for five or ten minutes, and is succeeded by a compen- Acapnia and Glycosuria. 283 satory period of subnormal breathing. From half an hour to an hour after the operation polyuria and glycosuria set in. These conditions usually last for four or five hours and then pass off, leaving the animal in apparently normal condition. The experiments detailed below show that a marked acapnia results from this hyperpncea. Lahousse 21 has observed that during the time which we have denominated above as the compensatory period the respiratory quotient is low. He regards this fact as indicating an al- teration in the nature of the substances undergoing combustion in the tissues. A simpler explanation appears to us to be that, owing to the acapnia, breathing in this period is subnormal. It is sufficient to supply all the oxygen the tissues need, but diminishes the elimination and thus allows reaccumulation of CO2. During any period when an animal is diminishing its store of CO2 by hyperpncea or reaccumulat- ing its normal stock by subnormal breathing the respiratory quotient would be correspondingly increased or diminished. If at the same time the quantity of oxygen absorbed by the lungs continues at an approxi- mately normal rate, the respiratory quotient may afford an entirely misleading indication of the nature of the substances undergoing combustion. Another object of our experiments was to determine whether the pre- vention of acapnia would likewise prevent glycosuria after piqure. For this purpose the rabbits were placed immediately after the opera- tion in a small chamber supplied with an ample stream of air to which known quantities of C0 2 had been added. To our surprise we found it by no means easy to prevent acapnia. With small percentages of C0 2 (5 per cent or less) the hyperpncea induced by piqure is sufficient to result in a marked reduction of the body's store of C0 2 . Owing to the loss of time involved in learning this, we were obliged, by pres- sure of other work, to leave many points undecided. Three years have passed without our finding an opportunity to resume the in- vestigation. In the recent paper of Edie, Moore, and Roaf 22 it is argued that hypercapnia is a factor in many forms of experimental diabetes. Our data demonstrate that such is not the case in any of the forms of experimental glycosuria studied by us. 21 Lahousse: Archives mternationales de physiologie, 1907, v, p. 105. See also La Franca: Zeitschrift fur experimentelle Pathologie und Therapie, 1910, vi, p. 1. 22 Edie, Moore, and Roaf: hoc. cit. 284 Yandell Henderson and Frank P. Underhill. The data here tabulated show that the prevention of acapnia obviates polyuria and probably to some extent delays and diminishes, but does not entirely prevent, the excretion of sugar after piqure. It is note- worthy that in one of our control experiments no apparent disturb- ance of respiration resulted from the piqure. In this case the appear- ance of sugar in the urine was delayed and no diuresis was observed. In all of our experiments the procedure was as follows. The rabbits were anaesthetized with ether. All urine was so far as possible pressed out of the bladder, and tested for sugar. No sugar was found in any case. A sample of blood was drawn from the femoral artery for the gas analyses. 23 Piqure was then performed. The animal was immedi- ately placed in the ventilated chamber and supplied either with air alone or with air and carbon dioxide of the percentage shown in the table. The chamber was so arranged that samples of blood could be drawn and urine pressed out of the bladder without removing the ani- mal's head and forequarters from the chamber. An attempt to obtain urine in this way was made every fifteen minutes. The data of ten typical experiments are summar'zed in the table on page 285. IV. Pancreatic Diabetes. From the single experiment outlined below it is apparent that when glycosuria is induced by removal of the pancreas at first a notable diminution of the CO2 content of the arterial blood occurs. The animal breathes excessively and later subnormally. We have no reason to suppose that preventing the development of acapnia would prevent glycosuria or delay the fatal result materially. Even this single ex- periment shows, however, that pancreatic diabetes does not necessarily involve hypercapnia (cf. Edie, Moore, and Roaf on page 283 of this paper). The circulation fails rapidly in all such experiments, as Un- derhill has frequently had occasion to observe, and the venous blood soon shows by its color an almost entire lack of oxygen. Thus, although the arterial blood may be rich in oxygen, the tissues may be insuffi- ciently supplied because of failure of the circulation. We are not yet 23 Barcroft and Haldane: Journal of physiology, 1902, xxviii, p. 234. The flasks used by us were three times as large as those of Barcroft and Haldane, and the blood samples were 3.0 c.c instead of only 1.0 c.c. Acapnia and Glycosuria. 285 prepared to follow the lead of those who hold that deficiency of oxygen is not capable of disturbing sugar metabolism. ARTERIAL BLOOD GASES. Exp. no. Before piqure. 1 hr. after piqure. 2 hrs. after piqure. 7 hrs. after piqure. Appear- ance of glyco- suria in min. Polyuria. o 2 C0 2 2 C0 2 c 2 C0 2 2 C0 2 1 1 13.5 51.1 13.3 28.7 13.5 45.7 2 1 14.5 58.0 14.8 37.9 14.5 42.0 30 3 1 12.0 40.0 12.0 26.3 8.5 34.3 12.8 30.0 45 4 1 12.0 38.9 16.4 17,1 60 5 2 13.0 36.0 75 6 3 210 7 4 12.1 50.0 14.5 47.2 135 8 B 14.5 45.8 7.3 61.5 9.2 69.5 75 96 16.2 25.7 15.2 44.5 60 10 7 1 Breathed air. Hypernoea. 2 No hypernoea. 3 Breathed 10 per cent C0 2 . 4 Breathed 15 per cent C0 2 . 5 Breathed 25 per cent C0 2 . 6 Breathed 25 per cent C0 2 . 7 Normal rabbit not anaesthetized; no piqure, breathed.25 per cent C0 2 for one hour. No trace of sugar appeared in the urine. 8 . . indicates marked polyuria, and zero indicates no apparent diuresis. Experiment of February 14, igo8. — 9.45. Dog under chloroform. 10.00. Arterial gases, 02:19.3; CO2 42.3 per cent. 1 1 . 1 5 . Pancreas completely removed. 11.30. Respiration 30 per minute, and full. 1. 10. Urine full of sugar. 1. 15. Arterial gases, O2 119.4 per cent; CO2 128.6 per cent. 2.00. Tube attached to trachea 2 cm. in diameter and 130 cm. long. Respiration very weak. 2.55. 0.12 gm. morphin sulphate subcutaneously. 286 Yandell Henderson and Frank P. Under hill. 3.50. Arterial gases, 02:23.2 per cent; 002:31.0 per cent. 4.45. Urine full of sugar. Respiration almost failing. 5.15. Saline saturated with CO2 placed in abdomen. Attached tube to trachea. Deep respiration follows. 7.30. Animal stopped breathing. V. Piperidin Diabetes. Underhill has observed that when piperidin is painted upon the pancreas a notable hyperpncea followed ^by subnormal breathing occurs. Herter 24 had previously demonstrated that painting piperidin upon the pancreas results in a marked glycosuria which persists for several hours. Although we have performed but a single experiment, it appears sufficient to show that hypercapnia is not a factor in this form of glycosuria, but that, on the contrary, the animal develops a notable acapnia. During the period of subnormal breathing which follows the tissues may be insufficiently supplied with oxygen, because of the low content of oxygen in the venous blood. In the earlier experiments of Underhill it was found that this form of diabetes was prevented when oxygen was given. It is possible that the principal factor in this result was the prevention of acapnia, as the process of breathing into a mask necessarily involves more or less re-breathing. This prevention of acapnia would later obviate also that tendency to failure of respiration and consequent anoxhaemia which were observed by Underhill. February 25, igo8. — Dog, 15 kilos. 11.30. Chloroform — then ether. 12.00. Arterial gases, 02:19.9; 002:33.7. Venous gases, 02:15.2; CO2: 40.9. 12.10. Urine free from sugar; blood pressure, 75 mm. Hg. Piperidin solution — 10 per cent — painted on pancreas. Less than 1 c.c. used. Respiration immediately accelerated. 12.15. Arterial pressure, 95 mm.; hyperpncea. 12.25. Arterial pressure, no mm.; coma. Urine free of sugar. 1. 10. Arterial gases, 2 : 18.9 per cent; C0 2 :27.o per cent. Venous gases, 02:11.1 per cent; CO2: 28.0 per cent. 1. 1 5. Arterial pressure, 130 mm. 24 Herter: Medical news, 1902, xxx, p. 865. Acapnia and Glycosuria. 287 1.45. No sugar in urine. 1 c.c. piperidin painted on pancreas. 2.15. 1 c.c. piperidin 10 per cent painted on spleen. 2.45. Urine contains sugar. 3.20. Arterial gases 02:9.9 per cent; 002:46.9 per cent. Venous gases, 02:3.8 per cent; 002:57.2 per cent. 3.00 to 3.20. Very feeble respiration. Animal barely alive. 3.25. Slow heart beat; 65 per minute; respiration poor. Arterial pressure varying from 50 to 120 mm. Urine contains sugar. 3.50. Animal died of failure of respiration. VI. Glycosuria after Laparotomy and after Excessive Artificial Respiration. It has been shown by Henderson 25 that when the intestines are handled in a current of warm moist air acapnia develops. The first of the two experiments given below indicates that hyperglycemia and glycosuria may follow the acapnia. The most direct procedure for the experimental production of acap- nia is excessive artificial respiration. Henderson 26 has found that artificial respiration administered with a hand bellows while the thorax is intact, usually produces little or no acapnia. The elastic recoil of the thorax in expiration under these conditions is so slow that an exces- sive ventilation is difficult to obtain. It is necessary that the apparatus employed for a dog with intact thorax should not only inject fresh air, but also that it should quickly and forcibly suck out again the other- wise slowly expired air. Excessive ventilation can, how T ever, be ob- tained merely with a hand bellows after the thorax has been opened. Under these conditions the lungs collapse and expel their air rapidly in the intervals between the strokes of the bellows. This method of inducing acapnia was employed in the second experiment reproduced below. No urine was obtained after acapnia had developed, but the blood sugar exhibited a notable increase. Aeration of Intestine. November ig, igo8. — Vigorous bulldog; weight, 7.5 kilos. 10.30. Etherized until 10.45. 25 Henderson, Y.: This journal, 1909, xxiv, p. 66. 26 Henderson, V.: This journal, 1910, xxv, p. 322. 288 Yandell Henderson and Frank P. Underbill. n.oo. Arterial gases, 02:23.3 per cent; C0 2 :3Q.6 per cent. Blood sugar, 0.18 per cent; respiration, 42. Pulse, 160; urine free from sugar. NH 4 — N fraction : 5.0 per cent. 11. 10. Tracheotomized; abdomen opened; intestines aerated; shallow respiration. Very little ether necessary. 12.25. Gases in blood from mesenteric vein; oxygen, 10.3 per cent; 002:31.3 per cent. 12.28. Comatose; arterial gases, 02:17.5 per cent; C0 2 :25.8 per cent. I. 00. Respiration, 18; pulse, 170. Good arterial pressure; urine full of sugar; blood sugar, 0.30 per cent; NH 4 — N fraction of urine, 8.3 per cent. December 3, 1907. — Young dog, 10 kilos. First chloroform; then ether; resists anaesthesia. 10.50. Arterial, 02:17.0 per cent; 002:36.1 percent. Venous, 2 : 14.1 per cent; 002:38.4 per cent. Arterial pressure, 150 mm.; pulse, 150. Rapid respiration. II. 15. Thorax opened; rapid and full artificial respiration ad- ministered. 11.30. Urine free from sugar. 12.30. No anaesthetic necessary; animal in profound shock. Arte- rial gases, O2: 17.2; CO2: 14.5 per cent. Venous, O2: 6.9 per cent; CO2: 33.9 per cent. Intestines relaxed; not irritable; bladder empty. ^. 10.50. Blood sugar, 0.16 per cent. ' (12.30. Blood sugar, 0.22 per cent. VII. Conclusions. Acapnia is a frequent concomitant of glycosuria or at least of hyper- glycaemia both under clinical and experimental conditions. In some artificial forms of diabetes prevention of acapnia obviates disturbance of the sugar-regulating function. We believe that glycosuria after etherization is due to acapnia, and that traumatic and emotional glycosurias also are usually due to this cause. The work of previous investigators is here quoted to show (a) that in diabetic coma an acute acapnia occurs; (b) that this is a true acapnia resulting from hyperpncea and is not merely due to the expulsion of CO2 from the bicarbonates of the blood by acids; (c) that in acidosis + the acidity, i. e. (H), of the blood is probably below normal, instead of Acapnia and Glycosuria. 289 above, as usually assumed; (d) that the hyperpncea of diabetic coma is induced by the ethereal, not the acid, acidosis bodies, e. g., acetone. In conclusion we would point out that it is often impossible to infer the interior conditions of tissue respiration from the external condi- tions to which an animal may be exposed. The only certain cri- terion of acapnia or hypercapnia is analysis of alveolar air or blood gases. The only certain criterion of insufficient oxygen supply to the tissues is the demonstration that the venous blood contains no oxygen or only a minimal amount. TRANSACTIONS OF THE CONNEGTIGUT ACADEMY OF ARTS AND SGIENGES Incorporated A.D. 1799 VOLUME I 6, PAGES 247-382 APRIL, 1911 Nutrition Investigations on the Carbohydrates I of Lichens, Algae, and Related Substances BY MARY DAVIES SWARTZ FROM THE LABORATORY OF PHYSIOLOGICAL CHEMISTRY SHEFFIELD SCIENTIFIC SCHOOL YALE UNIVERSITY NEW HAVEN, CONNECTICUT, U. S. A. YALE UNIVERSITY PRESS NEW HAVEN, CONN. 1911 COMPOSED AND PRINTED AT THE WAVERLY PRESS The Williams & Wilkins Company Baltimore, U. S. A. CONTENTS. I. INTRODUCTION. PAGE Lichens, Algae, Tree Bark and Certain Tubers and Foodstuffs 253 II. HISTORICAL PART. Introduction 259 Cellulose 262 (a) Occurrence and Nature 262 (b) Occurrence of Cytases (Cellulases) 264 (1) In the Vegetable Kingdom 264 (2) In Lower Animals 266 (3) In Higher Animals , 267 (c) Digestion and Utilization 268 (1) By Animals 268 (2) By Man 269 The Pentosans 272 (a) Occurrence and Nature 272 (b) Role in Plant Physiology 274 (c) Occurrence of Pentosanases 275 (1) In the Vegetable Kingdom 275 (2) In Lower Animals 276 (3) In Higher Animals 276 (d) Digestion and Utilization 278 (1) By Animals 278 (2) By Man 278 The Galactans 282 (a) Occurrence and Nature 282 (b) Occurrence of Galactanases 284 (1) In the Vegetable Kingdom 284 (2) In the Animal Kingdom 285 (c) Digestion and Utilization by Animals and Man 285 The Mannans 289 (a) Occurrence and Nature 289 (b) Occurrence of Mannanases 291 (1) In the Vegetable Kingdom 291 (2) In the Animal Kingdom 292 (c) Digestion and Utilization by Animals and Man 293 The Levulans 295 (a) Occurrence and Nature 295 (b) Occurrence of Levulanases 296 (1) In the Vegetable Kingdom 296 (2) In the Animal Kingdom 297 (c) Digestion and Utilization by Animals 297 249 250 Contents The Dextrans 300 (a) Occurrence and Nature 300 (b) Occurrence of Dextranases 301 (1) In the Vegetable Kingdom 301 (2) In the Animal Kingdom 302 (c) Digestion and Utilization by Animals and Man 302 III. EXPERIMENTAL PART. Introduction 306 Chemical Investigations. General Methods 307 Pentosan Preparations 309 (a) Dulse (Rhodymenia palmata) 309 (b) Hawaiian Seaweeds 313 (1) Limu Lipoa (Haliseris pardalis) 313 (2) Limu Eleele (Enteromorpha intestinalis) 313 (3) Limu Pahapaha {Ulza lactuca, etc) 314 Galactan Preparations 314 (a) Irish Moss (Chrondus crispus) 314 (b) Hawaiian Seaweeds 316 (1) Limu Manauea (Gracilaria coronopifolia) 316 (2) Limu Huna (Hypnea nidijica) 316 (3) Limu Akiaki (Ahnfeldtia concinna) 316 (4) Limu Uaualoli (Gymnogongrus vermicularis Americana, etc) 316 (5) Limu Kohu (Asparagopsis sanfordiana) 316 (c) Slippery Elm (Ulmus fulva) 317 A Mannan Preparation — Salep (Orchis) 318 A Levulan Preparation — Sinistrin (from Scilla Maritima) 321 Summary 322 BACTERIOLOGICAL INVESTIGATIONS. Introduction 323 Trials with pure cultures of aerobes 324 Trials- with mixtures of aerobes 325 Trials with anaerobes 327 Discussion and summary 328 PHYSIOLOGICAL INVESTIGATIONS. Introduction 331 Experiments with Enzymes 332 Parental Injections 332 (a) Methods and Technique 332 Contents 251 (b) Subcutaneous and Intraperitoneal Injections 335 (1) Dulse 335 (2) Irish Moss 336 (3) Salep 338 (4) Sinistrin 340 Feeding Experiments 342 (a) Methods and Technique 342 (b) Digestibility of Pentosans 344 (1) Dulse 345 (2) Limu Eleele 346 (3) Limu Pahapaha 347 (4) LimuLipoa 347 (c) Digestability of Galactans 348 (1) Irish Moss 349 (2) Limu Ma'nauea 350 (3) Limu Huna 351 (4) Limu Akiaki 351 (d) Digestibility of Mannan 1 ... 353 (1) Salep 354 Discussion and Summary 356 IV. CONCLUSIONS. V. BIBLIOGRAPHY. Lichens and Algae — Composition and uses 365 Cellulose 366 Pentosans 369 Galactans 373 Mannans 376 Levulans 379 Dextrans 381 This paper has been prepared from the author's dissertation submitted for the degree of doctor of philosophy, Yale University, 1909. I. INTRODUCTION. Lichens, Algae, Tree Bark and Certain Tubers as Foodstuffs. From the earliest times, the food of man has included lichens and algae, and even the tender branches and inner bark of certain trees and shrubs, such as elm, birch, pine, and the staff-tree or bitter-sweet {Celastrus scandens). When the bark of trees is so used, it is freed from cork and the hard outer rind; is cleaned, dried, mixed with more or less meal, and made into "bark bread." Such substitutes for bread are commonly resorted to only in northern lands where there is scarcity of cereal crops, or in other regions during periods of famine. Johnson (7) records that elm bark is so employed in some continental countries, and Dillingham (4) relates that certain tribes of North American Indians, 'in times of extreme dearth, were accustomed to keep body and soul together by boiling and eating the bark of the staff-tree.' Poulsson (17) states that in Finland and northern Russia, sphagnum mosses are similarly employed; and Schneider (21) agrees with these other writers, saying that in general lichens are used as articles of diet only in cases of special need, principally because all lichens contain a bitter principle, which not only gives an unpleasant flavor and is difficult to remove, but also exerts an irritating effect upon the digestive tract, causing inflammation. Nevertheless, in the northern parts of the Scandinavian Peninsula, where cereal crops are always scanty or uncertain, great interest attaches to two species of lichen widely distributed through Europe, and through Arctic and Antarctic regions: namely, Celraria islandica and Cetraria nivalis, which, as Poulsson (17) observes, 'have been considered nutritive and easily digestible since olden times. ' Cetraria islandica, whitened and freed from its bitter principle by washing with dilute alkali, is a rather appetizing substance; it has sometimes been used as a foodstuff by Polar navigators, and Dr. Hansteen, chief lecturer in the Agricul- tural school at Aas, Norway, has gone so far as to prophesy that moss is destined to become the great popular food for the masses, because of its cheapness and nutritive properties. Of marine algae, many tons are gathered and eaten annually in various parts of the world, the largest quantities being consumed 253 254 Mary Davies Swartz, by the Japanese, Chinese, and Hawaiians. These algae are found in great variety and widely distributed. In Japan, the general name applied to them is "Nori," which is also given to several prepared products. According to H. M. Smith (23), the most important Japan- ese seaweed preparations are: "Kanten," or seaweed isinglass, made from various species of Gelidium, the principal one being Gelidium comeum, often adulterated with similar seaweeds; "Kombu" made from Kelps, especially numerous species of Laminaria, Arthothamnus , and Alaria; "Amanori," from species of Porphyra; and "Wakame," from Undaria pinnatifida. Kanten is used largely for food, in the form of jellies, and as an adju- vant of soups and sauces. According to H. M. Smith (23), it is also employed in foreign countries 'in jellies, candies, pastries, and many desserts, in all of which it is superior to animal isinglass.' It has recently also attained popularity as a therapeutic agent in chronic constipation, being sold under various trade names, either plain or impregnated with laxative drugs, as cascara or phenolphthalein. 1 Kombu enters into the dietary of every Japanese family, being cooked with meat, soups, etc., and also served as a vegetable, or made into a relish with Soy-bean sauce. Amanori is eaten fresh or else is chopped and sun-dried in thin sheets, which are toastsd over a fire before eating. The crisp amanori is crushed between the hands and dropped into sauces or soups to impart flavor; or broken into pieces, dipped in sauce and eaten alone. Sheets of amanori, spread with boiled rice and covered with strips of meat or fish, are rolled and cut into trans- verse slices, and take the place of the American sandwich. Wakame is eaten as a salad, or cooked like amanori. In Hawaii, edible algae are called "limu. " Of these there are over seventy distinct species used for food, more than forty being in general use (18). Tons of limu are gathered for eating in Hawaii annually, and large quantities are also imported from the Orient and San Fran- cisco. Some idea of the extent of their use may be gained from the following statement by Miss Reed (18): " Ancient Hawaiians prob- ably seldom ate a meal without some kind of limu, and even today no Hawaiian feast is considered quite complete without several varieties served as a relish with meats or poi." 2 Since, with the exception of a few experiments reported by Oshima (15) and Saiki (20), there are no l Cf. Galactans, p. 283. 2 Poi is a thick paste made from the root of the taro plant, and takes the place of rice or bread in the native diet. Nutrition 1 nvestigations. data upon the digestibility of marine algae, an investigation of some of these Hawaiian limu seemed highly desirable; and through the kindness of Miss Reed, a number have been obtained for this purpose. Their occurrence and uses will therefore be described in some detail. 1 These limu are washed carefully after gathering, salted, and usu- ally broken, pounded, or chopped into small pieces. They may then be eaten uncooked, as a relish with poi, meats or fish; boiled with meats; put into soups for thickening or flavoring; or roasted with pig in a pit. Served raw and crisp, they take much the same place in the diet as our salads. Among the most popular varieties are Limu Eleele (Enter o- morpha of various species), Limu Kohu (Asparagopsis sanfordiana) and Limu Lipoa (Haliseris partialis) . Next in favor come Limu Ma- nama (Gracilaria cor onopif olio), Limu Huna (Hypnea nidifica) and Limu Akiaki (Ahnfeldtia concinna). Limu Pahapaha (Ulva fasciata and Ulva lactuca) is widely distributed but not very popular. Limu Uaualoli (Gymnogongrus vermicularis americana and Gymnogongrus disciplinalis) is limited to certain islands, and hence not in such gen- eral use and favor as some of the others. Limu eleele is a great favorite, forming a part of every native feast. It is generally eaten uncooked, sometimes being dropped into hot gravy, broth or meat stews just before serving. Limu kohu is always pounded in cleaning to free it from bits of coral and soaked 24 hours in fresh water to remove the bitter iodine flavor. It becomes slightly fermented and acquires a somewhat sour taste. Limu lipoa is popular on account of its penetrating spicy flavor, and is frequently used as a condiment, taking the place of sage and pepper in Hawaiian foods. Limu huna is especially prized for boiling with squid or octo- pus, though limu manauea and limu akiaki are often used as substi- tutes. These limus, as well as limu kohu, yield large amounts of mucilaginous extract on boiling, limu manauea being considered es- pecially fine for thickening chicken broth. Many of the seaweeds used in Hawaii and Japan occur also along the coasts of the United States and Europe, and are to some extent used as food in both regions. The very species of Gelidium from which the Japanese prepare their Kanten grow in abundance on our Pacific coast. Irish moss (Chondrus crispus), the "Tsunomata" of Japan, has long had considerable commercial value as a foodstuff in Ireland. In this country it is found from North Carolina to Maine, being especially abundant north of Cape Cod. After cleansing, cur- sor fuller description see Reed (18). 256 Mary Dames Swartz, ing, and bleaching it is to some extent used for making blanc mange or a demulcent for coughs. Through the kindness of Dr. C. F. Lang- worthy, Nutrition Expert, United States Department of Agriculture, I have obtained the following interesting data concerning the use of Irish moss, from the Journal of the South-Eastern Agricultural Col- lege, Wye, Kent (1): " Professor D. Houston, of the Royal College of Science, Dublin, has favored us with the following notes on this sub- ject: Chondrus crispus (carrageen, or Irish moss) is a seaweed plentifully distributed along our northern, western and southern coasts. It is gathered and sold to local chemists, who retail it, in some parts at all events, at 6d. per pound. It is used by many people as an article of food in the west, and generally for colds, for which pur- pose it is boiled in milk. Several of my students tell me that it is used for feeding weak calves and with striking results, bringing about an alteration of condition within four days. One student tells me that in one case at his own farm a batch of twelve calves took a kind of wasting disease, and nine died; the other three on the verge of death were given this plant, and all three recovered. It is prepared by putting one pound of the "weed" in a net bag and boiling in a gallon of water. The water on cooling sets to a jelly. The calves are given one glass of jelly in their milk each meal and wonderful results are said to be obtained." The high proportion of mineral matter is noteworthy; 1 but without making a fuller investigation, it is impossible to say precisely wherein lies the value of this seaweed. Purple laver (Porphyra laciniata), a source of Japanese amanori, is found in abundance on the rocky shores of America and Europe generally ; but it is not used in this country save sparingly by the Chi- nese, who usually import it directly from China, and by some of the Indians of our northwest coast. In Ireland it is known as 'sloak,' and is boiled and served with butter, pepper, and vinegar as an ac- companiment of cold meats, or is served with leeks and onions. Dulse (Rhodymenia palmata) is found abundantly on rocky shores both in this country and in Ireland. It is very abundant in New England, where it is rough-dried in the sun and eaten as a relish. In Philadelphia it is called sea-kale and eaten as a vegetable. In Scot- land it has long been used both in the fresh state and dried. In the Scotch Highlands, "a dish of dulse boiled in milk is," it is said, "the best of all vegetables." In Ireland, it is eaten with fish or boiled in milk with rye flour. Purple dulse (Iridea edulis), which occurs on the Pacific coast, is often eaten like Rhodymenia palmata. l Cf. Analysis of Chondrus crispus, p. 254. Nutrition Investigations. 257 Besides such lichens and algae, and the bark of trees, various tubers are used as food for man. In Japan, the tubers of Hydrosme rivieri (Conophallus Konjaku) are extracted with lime water, and the result- ing gelatinous mass is cut into small cakes. These, cooked with "shoyu" or Soy-bean sauce form a common article of diet. The tubers of many species of Orchis and Eulophia, native to Turkey, the Caucasus, Asia Minor and the greater part of Central and Southern Europe, furnish a food material known as Salep. The small ovoid, oblong or palmate tubers are decorticated, washed, heated till horny and semi-transparent, and finally dried. An abundant mucilaginous extract is obtained by macerating the bulbs in water. Frequently the tubers are ground to powder, and the powder used like sago or tapioca. Royal salep, said to be used as food in Afghanistan, is pre- pared from Allium Macleanii. A former instructor in the American College for Girls, in Constantinople, reports that salep is a very com- mon article of diet in Turkey. It is sold in the markets in powdered form, and is made into a sort of sweetened gruel with milk. Not only is it used as a warm drink in the household, much as we use cocoa or chocolate, but it is also sold in the streets by venders, who either stand in booths along the way, or go about carrying huge brass urns strapped to their shoulders, clinking their cups and calling " Taze- Sahlep!" 1 It is especially popular in districts of the city where peo- ple work late at night. In the month of Ramazon, the time of all-day fasting, hot salep finds a ready sale at night. It is no uncommon thing to see the workman standing with his salep cup in hand, waiting for the firing of the sunset cannon. In spite of the fact that there have been almost no scientific inves- tigations as to the digestibility of such mucilaginous plant substances there seems to be a special virtue attached to mucilages in the popular mind. The prevailing impression is shown in some of the following remarkable statements. The United States Dispensatory, 1908, not only says that the mucilaginous extract of slippery elm bark (Ulmus fulva, Michaux) is nutritious, but adds, " We are told that it has proved sufficient for the support of life in the absence of other food." Of salep Smith (25) says in his dictionary of economic plants: "It con- tains a chemical substance called bassorin, which is said to contain more nutritious matter than any other vegetable product, one ounce per diem being sufficient to sustain a man"! The United States Dis- pensatory also assures us that salep is "highly nutritious." Johnson ^resh salep. 258 Mary Davies Swartz. (7) particularly recommends Iceland moss (Cetraria islandica) as a diet for consumptives, as "it seems to be both extremely nutritious and very easy of digestion, though of course, only capable of use as a substitute for starchy matters." In regard to Irish moss (Chondrus crispus), he is a little more uncertain. "It is much used for invalids, especially in cases of consumption, but with doubtful advantage when substituted for more nutritious food." Schneider (21) says of Ice- land moss: "Inhabitants of Iceland, Norway, and Sweden mixed this lichen with various cereals and mashed potatoes, from which an un- commonly healthful bread was prepared." Until the matter has been thoroughly investigated, we must suspend our judgment as to the ac- curacy of such statements. After a few metabolism experiments, Oshima (15) far more conservatively remarks concerning the algae of Japan: "Their actual value doubtless depends in considerable measure upon the mineral salts they contain." In view of the scarcity of any scientific investigations as to the be- havior of all these substances in the body, further experiments upon their nature and digestibility seem highly desirable, since they are not only widely distributed, and already form a considerable portion of the diet of many persons; but because, if they possess any real nutri- tive value, a wider use of such comparatively cheap materials would be an economic advantage; and because, under the prevailing notions as to their food value, they are sometimes relied upon as a source of nutriment in diseases (as diabetes) where the character of the diet is particularly important. The present work has been undertaken to throw some light on this interesting subject. A survey of the litera- ture shows that even the chemical nature of many of these algae has scarcely been investigated; and if this were known, we should still be under the necessity of studying their behavior in the animal body, for it is impossible to tell from chemical analysis alone whether a given substance will or will not prove digestible, as Rubner has long since warned us. II. HISTORICAL PART. Introduction. According to the current practice of agricultural analysts, the car- bohydrates of plants are reported as crude fiber and nitrogen-free extract. Crude fiber is the term applied to the resistant mixture form- ing the mature cell wall, shown as long ago as 1864 by Henneberg and Stohman (41) to have no definite chemical composition. It is there- fore not identical with cellulose, but consists of a mixture of cellulose with incrusting substances, lignin and cutin, the relative proportions of which have recently been exhaustively studied by Konig (51), Fiirstenberg (39), and Murdfield (63). Cellulose is the chief consti- tuent; the other two are usually present in varying proportions. Schulze (74) to whom much of our knowledge of the composition of the plant cell wall is due, has classified the carbohydrates of the nitro- gen-free extract as follows: Water-soluble carbohydrates. To this class belong the mono-, di-, and tri-saccharides, and some soluble polysaccharides. Carbohydrates insoluble in water, but yielding sugar under the action of diastase. The chief member of this group is starch. Carbohydrates insoluble in water and resistant to the action of diastase, never being changed by it into sugar. This group is called the Hemicelluloses. The term hemicellulose, as used by recent writers 1 seems to be inter- preted to include some polysaccharides of the first group. It is there- fore used here as a group name for those carbohydrates which are dis- tinguished from cellulose by being capable of hydrolysis on boiling with dilute mineral acids, and from the other polysaccharide carbohy- drates by not being readily digested by diastase. According to the kind of sugar yielded on hydrolysis, the hemicelluloses are designated as Pentosans or Hexosans, the latter including Galactans, Mannans, Dextrans, Levulans, etc. After a general review of the chemical ii. III. ^.g., Lohrisch. 259 260 Mary Davies Swartz, nature of lichens and algae, each of these classes will be discussed separately in detail. The percentage composition of some common species of algae is shown in the following table: FOOD MATERIAL. WATER. PROTEIN. FAT. CARBOHYDRATES. ASH. Nitrogen- free Extract. Crude I.* Cystophyllum fusiform, 15 74 11 .37 .49 54.84 17.56 Ecklonia bicyclis, dried. . 18 75 9.58 .46 51.63 9.79 9.79 Enteromorpha linza, dried (Limu eleele)... 13 53 19.35 1 .73 46 .18 19.21 Laminaria sp., dried.. . . 23 08 7. 11 .87 47 .70 21.24 Porphyra laciniata, 13 98 33.75 1.30 41 .22 t 9.75 Ulopteryx pinnatinda, dried 18 92 11.61 .31 37.81 31.35 H.t Ahnfeldtia concinna, fresh (Limu akiaki).. . 80 00 1.4 0.0 14.4 4.2 Ulva fasciata and U. Jactuca, fresh (Limu pahapaha) 80 00 3.7 0.0 12.5 3.8 Gracilaria coronopifolia, fresh (Limu manauea) 80 00 1.8 0.0 14 .1 4.1 IILJ Chondrus Crispus, dried. 13 40 13.06 2.59 54.16 2.57 14.22 IV. § 0.32 1.2 43.3 5.3 2.2 *Oshima (15). t Reed (18), (calculated on uniform water basis). tAnnet (1). § Schmidt (24), first studied the ash and reported a notable amount of calcium and potassium phosphates. He found no nitrogen. Blondeau (3) reported 21.36 per cent nitrogen. || Brown (334). Until 1905 the chemical nature of the constituents of algae had received little attention. Analyses of many species of algae from Japan and China were reported recently by Konig and Bethels (8), the results of which are given in the following table on page 255. According to Oshima and Tollens (16) the carbohydrates of Por- phyra laciniata consist largely of anhydrides of d-mannose and i-ga- lactose. Miither and Tollens (13) studying various species of Fucus (F. vesiculosus, F. nododus, F. serratus), Laminaria, and Chondrus crispus, found a methyl-pentosan (fucosan), in Fucus and Laminaria; and glucose, fructose, galactose and pentose groups in Chondrus Krefting reports a reserve carbohydrate in Laminaria digitata in win- Nutrition Investigations . si si rsi si 'Si si si s si s. (U PU|fL|P-|pl H pL H pL|pL|P_|P H PUpL|P-| SIS1S1S)S1U)S1S1S1S1 oooooooooo oouoooocjuo MJ-lS-ll-iS-wSHlHt-lJ-IJ-l t/2 Si o o o o OJ > >> r3 I-l ^ o o 5 3 3 o oj V3 a ^ 3 i2 "3 1 jfii a, a o 3 *c o o » ^ ">> ^ •3 -5 3 3 0) CD CD +-> d d ^d d d d cd > >> fa fa 4) c/3 m m m o o o o d d c d d d a d d cd cJ ^ ^ S ^ ^ ^ *T3 ^ CD CD t/3 t/3 t/3 m O O O O o o o cj d rf 9 9 9 9 ^3 t3 ^ cd cd cd in m m O O O CJ CJ CJ 3 3 3 o o o 3 ^ *h d cu •£ 6 £ o IS •3b O O c3 U U U is o £ | 8 £ J 5 o 2 u 4h ft O CU cn C/2 Ph C/3 Ph P ,±5 d HNn^mcoNoocJOHN 264 Mary Davies Swartz, CYTASES IN THE VEGETABLE KINGDOM. By the early investigators, Haubner (40), Henneberg and Stohman (41), Kiihn, Aronstein, and Schulze (54), it was accepted without much question that, since cellulose disappeared from the alimentary tract of herbivora, it is digested like starch, and equally valuable as a nu- trient. But after Tappeiner (78), in 1884, showed that cellulose could be decomposed by micro-organisms, and promulgated his theory that this was the only way to account for the disappearance of cellulose from the alimentary canal of ruminants, the matter fell into great dis- pute, 1 and the question is not yet definitely settled as to how cellulose is digested and what are the products of its digestion. A diligent search has been made for enzymes capable of attacking it (cytases), but so far, such cytases have been proved to exist only in plants and lower animals. Many of these so-called cytases act upon hemicel- lulose rather than true cellulose, and will be discussed in connection with the hemicelluloses, though it is not always possible to make a sharp distinction between the two. A careful review of the subject of cytases in plant physiology up to 1898, has been made by Bieder- mann and Moritz (34), from which it appears that the penetration of wood by the mycelia of moulds is due to such cytases, and that a powerful cellulose-dissolving enzyme has been derived from Peziza sclerotium by de Bary (37) and from another botrytis (presumably a Peziza) by Ward (84), while Brown and Morris (36) have described cytases existent in germinating grasses which dissolve their cell walls. That this is anything more than a diastatic enzyme is denied by Rei- nitzer (67) ; but Newcombe (64) considers the assumption of the iden- tity of all cell- wall dissolving enzymes with diastase as far from jus- tifiable. Bergmann (32) reports such cytases in hay and straw. Scheunert and Grimmer (71), on the contrary, find none in oats, corn, horse-beans, lupine seeds, buckwheat or vetch. Thus we see that even in the case of plants, these enzymes need to be isolated and identified before we can arrive at any satisfactory conclusions. That cellulose can be dissolved by bacteria has been demonstrated for such forms as Amylobacter butyricus, Vibrio regula and Clostridium polymyxa (34). Omelianski (65) has described two organisms which ferment cellulose, and Ankersmit (31) finding Omelianski's bacteria on hay, has studied their behavior when introduced into the alimen- tary canal of the cow on its food. He finds that they do not increase : For a review of this discussion cf. Lohrisch (56). Nutrition Investigations. 265 in number during their passage through the digestive tract, and there- fore concludes that they play a very inconsiderable role in the decom- position of cellulose. According to Van Iterson (81), certain aerobic bacteria, attacking cellulose, form from it products which nourish other forms (spirilla); certain anaerobes are also shown to attack it. Eberlein (38), rinding in the first stomach of herbivora Infusoria which utilize cellulose for food, suggests that these protozoa, digested farther along in the alimentary tract, serve as means of transforma- tion of cellulose into products which the animal can digest; but there is nothing to indicate that such forms occur in sufficient numbers to be worthy of much consideration. Since 1906 three investigators have given the problem careful at- tention. Scheunert (68) has concluded from experiments in vitro that bacteria play an exclusive role in the solution of crude fiber in the coe- cal contents of horses, swine, and rabbits. He found that filtered coecal fluid acted on cellulose much less than unfiltered or simply strained coecal contents. This is contrary to the opinion of Hof- meister (45) and Holdefleiss (48), who attribute the phenomenon to the action of enzymes, and explain the loss of power occasioned by filtering as due to the effect of exposure to the air upon the enzymes. Lohrisch (57) has reported that fresh coecal fluid is effective in destroy- ing cellulose while heated fluid is not. On the other hand, implanting the sterilized fluid with coecal bacteria and protozoa would not restore its activity. Coecal fluid kept at 38° C. any length of time gradually lost its cellulose-dissolving power, while that kept on ice remained active, v. Hoesslin and Lesser (47) have attempted to explain these apparent contradictions, and conclude from their own experiments that anaerobic bacteria are the most effective agents in cellulose de- composition in the intestine. Equal volumes of non-sterilized and sterilized coecal fluid of the horse, to which weighed amounts of cel- lulose had been added, were suspended in sterile physiological salt solu- tion under practically anaerobic conditions and digested for periods of from 9 to 35 days. The disappearance of cellulose with the non-steril- ized coecal fluid amounted to from 55.7 per cent to 71.2 per cent; with sterilized fluid, to from 6.2 per cent to 42.4 per cent. It was also found that the addition of 1-5 grams of dextrose would effectively protect the cellulose from digestion by the non-sterilized fluid, the bacteria preferring the more easily attacked carbohydrate. The gases evolved in these fermentations were characteristic of bacterial action, being chiefly methane, carbon dioxide, and hydrogen. The retarding effect of exposure to the air is explained by the theory that anaerobes are 266 Mary Davies Swartz, the effective agents. So, also, the fact that Lohrisch was unable to get cellulose digestion in sterilized fluid again inoculated with unsteril- ized fluid is attributed to the medium's being an unfavorable one for the development of these organisms, inasmuch as the addition of pep- tones to similar preparations caused in several cases an increased de- composition. It seems fairly well established, therefore, that the action of the coecal fluid of the horse is due to enzymes of bacterial origin. CYTASES IN LOWER ANIMALS. There is no doubt that cytases occur in some of the lower forms of animal life. Biedermann and Moritz (34) found a powerful cellulase in the secretion of the liver of the common snail (Helix pomatia), and their observation was verified by E. Muller (61), also by Lohrisch (57) who reports two series of experiments in which snails fed tender let- tuce leaves digested from 40.1 per cent to 81.6 per cent of the cellulose present. On the other hand, Muller (61) could not verify Knauthe's report of a cellulase in the hepato-pancreas of the carp (50) ; Pacault found none in the saliva of Helix pomatia (66) ; and Biedermann none in the digestive juice of the meal worm (Tenebrio molitor) or of the cabbage worm (Pieris brassica) (34). Biedermann also examined the faeces of the cabbage worm microscopically and found unaltered par- ticles of leaves, from which he concluded that much of the plant food eaten is excreted unchanged. Lohrisch (56) has obtained similar re- sults with caterpillars of sphinx moths (Sphinx euphorbiae), not only in experiments with intestinal juice in vitro, but also in feeding expe- riments in which the cellulose was quantitively excreted. Selliere (75-76) has recently added some interesting contributions to this subject, showing that cotton treated in various ways; namely, that recovered after solution in Schweitzer's reagent, that treated with concentrated zinc chloride, or with 25 per cent caustic alkali hot or cold until the fibers are swollen, and subsequently washed with 1 per cent acetic acid and water, is attacked by Helix pomatia much more readily than the untreated substance. Subsequent drying of the treated cotton diminished its digestibility somewhat, suggesting that the physical condition of the cellulose is a definite factor in its utilization. Selliere believes that only the more tender portions of plant cellulose are attacked by the digestive juice of this snail. It would seem that the previous treatment of th 3 cellulose is a factor to be kept in mind in the interpretation of the results of feeding experiments. 1 *Cf. the experiments on cellulose utilization in the dog, p. 263. N utrition Investigations. 267 CYTASES IN HIGHER ANIMALS. There is at present no proof of the existence of cytases in any of the higher animals. The literature on the subject has been exhaustively reviewed by Bergmann (32), and Lohrisch (55, 56, 57) and it appears that there is no cellulase in the saliva or pancreatic juice of swine, horses, cattle, or sheep. The old observation by MacGillawry 1 (cited by Biedermann and Moritz(34) that a cytase can be extracted from the vermiform appendix of the rabbit has been denied by Zuntz and DegtiarefT (88). Schmulewitsch's 2 statements (also cited by Bieder- mann and Moritz) are worthless because he employed no antiseptics. E. Miiller (61) found no sugar formed from the decomposition of cel- lulose in the stomach of the goat, and Lusk (59) observed no increase in sugar elimination after feeding a phlorhizinized dog 20 grams of cauliflower, or a phlorhizinized goat 10 grams of paper. Lohrisch (57) fed pure cellulose (5-20 grams) to a phlorhizinized rabbit and found that it had no marked influence on the sugar output, and no nitrogen- sparing effect. Scheunert (70) has made further investigation on the action of the saliva and salivary glands in sheep, and confirms the earlier experiments with the saliva of this animal. On the other hand, Selliere (77) reports that the specially treated cellulose mentioned above is converted into dextrose by the intestinal secretions of the guinea pig in some instances. Practically nothing is known concerning the way in which cellulose disappears from the alimentary tract of man. Schmidt and Loh- risch (73) fed pure cellulose to diabetics and observed a disappearance averaging 77.7 per cent, and no increase in the elimination of sugar. They believe that most of it is absorbed in soluble form and not de- stroyed by fermentation in the intestines. Lohrisch, having fed cel- lulose in various diseases of the alimentary tract, 3 calls attention to the fact that in constipation, where there is the least bacterial action, the utilization of cellulose is highest, while in fermentation dyspepsia, in which one might expect a marked disappearance, the utilization is lowest. He therefore considers the digestion of cellulose as due at least in part to enzymes. x Archiv Neerland, Vol. XI. 2 tjber das Verhalten der Verdauungssafte zur Rohfaser der Nahrungsmittel. Bulletin de l'Academie Imperial de St. Petersburg, 1879. 3 See results, p. 264. 268 Mary Davies Swartz, Digestion and Utilization of Cellulose by Animals. The literature on the digestion of cellulose up to 1909 has been so exhaustively reviewed by Lohrisch that it is unnecessary to enter into a detailed discussion of it. From tables (55) showing the results of all previous experiments on the utilization of crude fiber in herbivora, carnivora, and birds, it appears that in the case of herbivora, especi- ally ruminants, 20-28 per cent of the crude fiber ingested with food disappears from the alimentary canal; that in case of carnivora 1 and birds 2 there is no utilization whatever. Lohrisch (56) himself reported three experiments in which dogs were fed pure cellulose and digested 31.1 per cent, 37.45 per cent and 5.4 per cent respectively, but Scheu- nert and Lotsch (72) repeating Lohrisch's work with a somewhat dif- ferent method of determining cellulose found that the administration of 40 grams of prepared white cabbage, containing 7.37 grams of pure cellulose, resulted in the recovery of the total amount ingested. Cook- ing the cabbage in bouillon did not increase its digestibility. They attribute the apparent utilization in the preceding experiment to des- truction of cellulose by the reagents used for its purification. Since the publication of their paper, Lohrisch has repeated his work with the dog (57), and reports complete recovery of the cellulose fed. He explains the error in the earlier investigation as due to the fact that the ingested cellulose was twice subjected to purification (before feed- ing and in faeces) with consequent increase in percentage of loss, which was not taken into account. He points out the inevitable loss of some cellulose by any method at present in use for its determina- tion, and defends his own as sufficiently accurate for all practical pur- poses if conditions are carefully observed. 3 ^he only experiments on record are by Voit and Hoffmann on the dog and by von Knieriem on the hen. Experiments by Weiske on the goose, and by von Knieriem on the hen. 3 Lohrisch used the method of Simon and Lohrisch, in which the cellulose is dis- solved by heating for an hour on a water bath with 50 per cent potassium hydroxide, then adding f cc. of 30 per cent hydrogen peroxide, and digesting from |tof hour longer if necessary. The cellulose is then precipitated by adding to the solution one half its volume of 96 per cent alcohol and 6-7 cc. of concentrated acetic acid; filtered off, washed with water, dilute acetic acid, alcohol and ether, dried and weighed. Scheunert and Lotsch mix the substance to be analyzed with 100 cc. of cold water, add 100 grams of potassium hydroxide and heat for an hour on a water bath, then filter through a hard filter paper, wash the residue on the paper with boiling water till only a trace of alkali remains, transfer it to a beaker and thence to a weighed ' Nutrition Investigations. 269 Cellulose digestion in the dog has been almost simultaneously stud- ied by v. Hoesslin (46). Two dogs on a meat-fat diet to which was added daily 2 grams of specially prepared white cabbage (containing 63.25 per cent of pure cellulose), for five periods of five days each, excreted on the average 99.7 per cent and 94.5 per cent respectively. This long experiment is significant as showing no adaptation of the digestive glands to the type of food. By these independent workers it seems now well established that the dog is unable to utilize cellulose. Hoffmann (42) has just published the results of some investigations on the influence of cellulose on the nitrogen balance and on phlo- rhizin-diabetes in the rabbit, from which it appears that after inges- tion there is no increase of sugar excretion, and no glycogen formation, yet he thinks that cellulose and hemicelluloses have a favorable influ- ence in phlorhizin-diabetes. 1 It seems to follow from this, that even in case of herbivora cellulose is not utilized in the manner customary for starch and sugar. DIGESTION AND UTILIZATION OF CELLULOSE BY MAN. A similar tabulation of results of feeding experiments on man, shows that cellulose is not so well utilized as by herbivora, but does disap- pear in appreciable amounts. With one exception, the cellulose in all these experiments was administered as crude fiber. Hofmeister (43) fed pure cellulose and reported 75.7 per cent soluble cellulose and 5.6 per cent insoluble cellulose digested. Konig and Reinhardt (53) added to a diet rich in protein and fat, but free from cellulose, in sev- eral experiments, green peas and ripe shelled peas, red cabbage, w T hite filter, on which it is washed successively with hot water, dilute acetic acid, hot water, alcohol and ether, and finally weighed. Scheunert and Lotsch claim that by Lohrisch's method the cellulose is altered in character, and as much as 40 per cent lost in the process; and that subsequent treat- ment of the recovered material causes an even greater per cent of loss, while by their method the loss in the first case is not over 6.8 per cent, and that in the second case even less. For the details of this controversy over method see the following : Simon and Loh- risch; Zeitschrift fur physiologische Chemie, Vol. 42, p. 55, (1904). Scheunert; Berliner tierarztliche Wochenschrif t, No. 47, p. 826, (1909) . Scheunert and Lotsch; Ibid., p. 867, (1909); also Biochemische Zeitschrift, Vol. 20, p. 10, (1909); and Zeitschrift fur physiologische Chemie, Vol. 65, p. 219, (1910). Scheunert and Grimmer; Berliner tierarztliche Wochenschrif t, No. 48, p. 152, (1910). Lohrisch; Zeitschrift fur physiologische Chemie, Vol. 69, p. 143, (1910). Unfortunately the original paper was not accessible. 270 Mary Davies Swartz, beans, graham and soldiers' bread and found 30.27 per cent to 76.79 per cent of the added cellulose digested. Lohrisch (55) finds that the cellulose of a common vegetable diet disappears from the alimentary tract in large amounts, the actual quantity varying with the age, source and tenderness of the cellulose. Thus he finds that for normal individuals, of cellulose from lentils, 45 per cent is digestible; from kohlrabi, 79.1 per cent; from white cabbage, 100 per cent. Under abnormal conditions in the digestive tract, he has obtained the fol- lowing results: CONDITION. CELLULOSE UTILIZATION IN PER CENT. Normal 57.9 Chronic Constipation 81.4 Fermentation Dyspepsia 37.8 Gastrogenic Diarrhea 29.5 Fatty Faeces in Icterus 27.8 Fatty Faeces in Disease of Pancreas 20.9 According to Lohrisch, two diabetics on a cellulose-free diet, to which white cabbage was added in quantities to yield about 6 per cent of cellulose per day, digested 68.6 per cent and 84.5 per cent respectively, without increased output of sugar in the urine. Since the only way to determine definitely the energy value to the organism of such amounts of cellulose as are absorbed, is by means of respiration experiments, Lohrisch (57) has performed such an expe- riment on man, using the Zuntz-Geppert apparatus. In fasting, the respiratory quotient averages about 0.76. After ingestion of carbo- hydrates such as starch, it rises gradually in two to three hours, to 0.9-1.0, and when the carbohydrate has been consumed, sinks again to a lower level. Since the respiratory quotient for fat is 0.7 and for protein about 0.8, it is possible to determine in this way to what extent the carbohydrate replaces protein and fat in metabolism. Hence if cellulose is absorbed and oxidized as a carbohydrate, the res- piratory quotient should rise. If it is decomposed by bacteria, the respiratory quotient should not rise, since the theoretical respiratory quotient for fatty acids, such as butyric and acetic, is, according to Munk (62) and Mallevre (60), 0.6 and 0.5 respectively. Now Loh- risch, feeding a man moist cellulose equivalent to 73.6 grams of dry substance, of which 25 per cent was digested (18.5 grams) obtained the following results : Nutrition Investigations . •NOIX -saoNi asonii'iao aO 0NINNI03S aaXiV 1 •NOixonaoHd zoo § H|N H|lN H|N iH|N b 1 1 1 1 1 + 1 + •NOIXdHJlSNOD «0 »i He* S him h|c* CD O H(NlOCO(OHH^ + 1 + + + + + + •axnNiH aaa NOixonaoaa zoo ccm. 156.34 146.50 151.42 142 . 79 143.82 150.16 147.81 141.36 157.78 146.26 159.23 •axaNm H3d NOIXdHXlSNOO &0 ccm. 194.37 189.89 192.13 194.47 187.38 202.69 203.49 203.26 223.82 211.37 219.53 "0 'H oot>t^i>^t^t^r^t^©t^ ooooooooooo •Noixonaoaa zoo Per cent. 3.33 3.68 3.37 3.40 3.23 3.10 3.22 3.10 3.01 3.01 *NOIXJPin.SNOO ^00 Per cent. 4.14 4.77 4.59 4.43 4.36 4.27 4.63 4.44 4.35 4.15 'axnNrre Had aaavKNi aimoA • lOi-h NO05 00OHOO §0300 MW^tOOJ^iOOl S O OS (NCNCONCOOOOIN U CO T^TjH^-H^TiHlOTtltO •saxn •"IxiJ'V Ix± JLiN. killU 1 a. cl Cl -xa ao Noixvana •XNararaad -xa ao ONifjNioaa v." M iO rHt^cOOOOOCMCO tq CO N OH^HN^CDN •xNara -raaaxa ao naaiinN ,-H M^»OCDNOOOJO 272 Mary Davies Swartz, The respiratory quotient attains its highest value in the fourth hour, instead of the second or third, showing that cellulose is absorbed more slowly than starch. The rise is too slight to indicate that cellu- lose exercises any considerable protein- or fat-sparing effect. It is unfortunate that the amount of cellulose absorbed was so small. It is striking that the 02-consumption decreases at the very time that the respiratory quotient rises, and the C0 2 -production scarcely in- creases. Lohrisch interprets this as indicating that the increased 02- consumption required for oxidation of the cellulose is compensated by a sparing of protein and fat. The differences seem too small to draw any satisfactory conclusions as to the energy value of cellulose. The low respiratory quotient in the later hours of the experiment, together with the increased Oo-consumption, indicates the utilization of some of the cellulose in the form of fatty acids. We must bear in mind that no formation of sugar or glycogen from cellulose, in men or ani- mals, has been demonstrated. Further investigations would seem to be necessary before we can agree with Lohrisch in saying, " Wir wissen, dass Cellulose und Hemi-cellulosen votn Menschen reichlich verdaut werden, wir haben alien Grund anzunehmen, dass ihre Verdauung nach Analogie der Starke ablduj I . . . Die resorbirten Mengen werden im menschlichen Organismus vollstdndig verbrannt. Dabei wird Eiweiss und Fett von der Verbrennung geschiitzt." In any event, the quanti- ties of cellulose which the alimentary tract of man is capable of ab- sorbing are, apparently, too small for it to play a role of any impor- tance in the diet of a normal individual. Occurrence and Nature of Pentosans. The anhydrides of the 5-carbon sugars are collectively designated as pentosans. These are not reported to occur in the animal kingdom, but the pentose sugars are found forming a part of the nucleic acid radical of the nucleo-protein molecule. In the vegetable kingdom, pentosans are very widely distributed, as has been shown by many investigators, especially Tollens and his pupils. 1 They occur in all kinds of plants, from the lowest to the highest, and are limited to no 1 Tollens, Landw. Vers., V. 39, p. 401, (1891); Tollens, Jour. f. Landw., Vol. 44, p. 171 (1896). For an exhaustive review of the literature on the occurrence of the pentosans see v. Lippmann, Chemie der Zuckerarten, 3rd Edition, Vol. I, pp. 44-60; 116-123; and Czapek, Biochemie der Pflanzen, Vol. I, pp. 537-545 (1905). Nutrition Investigations . 273 particular organ or tissue, being found abundantly in roots, stems, leaves or seeds. In regard to solubility in water, pentosans show all possible varia- tions. De Chalmot (108) found them present in the watery extract of the leaves of many plants; Winterstein (167) in the somewhat mucila- ginous hot water extract of the seeds of Tropaeolum majus; Schulze (146) , in both soluble and insoluble form in the cotyledons and endosperms of the seeds of Lupinus luteus and other legumes, where they are doubt- less stored as reserve material for the growing plant; and in the cell walls of the mature plants, where in most cases they approach true cel- lulose in character. It is difficult to differentiate these highly resis- tant pentosans of the cell wall, which are commonly included in the term crude fiber, from the ligno-celluloses and oxycelluloses also found there, which as Cross, Bevan and Beadle (104) have shown, 1 are like true pentosans in yielding furfurol on distillation with dilute hydrochloric acid. Besides hemicelluloses yielding pentoses (xylose and arabinose) exclusively, occur many yielding also methyl-pentoses (fucose, rhamnose) . These yield on distillation with dilute hydrochloric acid, methyl-furfurol, which is precipitated by phloroglucin, and hence included in quantitative estimations of pentosans by the method of Tollens andKrober (121). The distribution of methyl-pentosans has been studied especially by Tollens and his pupils. Japanese "Nori" (Porphyra laciniata, Laminaria, and other seaweeds) (129), tragacanth and many other gums (163) contain fucosan. Rhamnose occurs also widely distributed in the plant kingdom, but more frequently in the form of a glucoside. Rohmann (134) reports a rhamnosan in Ulva lactuca. It is a very common thing to find pentosans and hexosans occurring together. In fact, it is absolutely impossible, in treating of hemicellu- loses, to draw any sharp dividing lines, for they are not only intimately associated, but frequently chemically combined. Schulze (146) has given the name paragalactan to the carbohydrate yielding arabinose and galactose, which occurs in the seeds of many legumes. Winter- stein (167) finds galacto-xylan in the water extract of Tropaeolum majus, and numerous other examples of such combinations might be cited. A class of substances to which has been given a distinctive name because of their peculiar gelatinizing property, is the Pectins. As Czapek 2 remarks, "It is uncertain whether they form a definite : For further details see v. Lippmann; Chemie der Zuckerarten, Vol. I, pp. 160-169^ 2 Die Pektin-Substanzen; Czapek, Biochemie der Pflanzen, Vol. I, p. 545. 274 Mary Dames Swartz, class of cell wall substances, or whether they should be classified as 'hemicelluloses' or 'pentosans.' " In 1868, Scheibler (141) found a sugar which he called pectinose, but which was later shown to be ara- binose (142). In 1875, Reichardt (132) obtained a pectin body from carrots and beets, which he called 'pararabin,' expressing the view that pectins should hardly be considered as a special class of carbo- hydrates. Tromp de Haas and Tollens (160) have found from numer- ous analyses, that the pectins do not differ from other carbohydrates in their relative proportions of hydrogen and oxygen so much as earlier workers supposed, and hence they may be classified with other hemi- celluloses according to the products of their hydrolysis (pentoses; galactose and other hexoses). Cross (106) believes them to be allied to the ligno-celluloses. The whole matter is still in a state of uncer- tainty. Herzfeld (116) has shown that arabinose can be obtained from most pectins, and consequently they have been included among the pentosans, though from the frequency with which they yield ga- lactose, they might equally well be discussed with the galactans. Ac- cording to Czapek while pectins occur frequently in phanerogams, ferns and mosses, their presence in algae is doubtful, although it is possible that soluble carbohydrates of algae yielding arabinose or ga- lactose are closely related to the pectins of other plants. 1 Role of the Pentosans in Plant Physiology. Comparatively little is known of the role of pentosans in plant phys- iology. De Chalmot's (108) observation that they decrease in quan- tity in seeds — peas and corn — during germination, and reappear in the stems and roots of the growing plant, would seem to indicate that they form a part of the reserve material in the seed; but Schone and Tollens (145), finding no diminution in the amount of pentosans in grains during germination, but rather a slight increase, declare that they do not belong to the reserve-stuff of the seed; so the question may be regarded as still unsettled. Changes in the relative amounts of pentosan in plants at different stages of growth, studied by Cross, Bevan and Smith (105), Gotze and Pfeiffer (113), Calabresi (98), and others, show that the increase of pentosans runs parallel to the forma- tion of the skeletal substance; and have led to the idea that they arise through the transformation of a part of the cellulose, and along with lignin and cutin, take part in wood formation. Ravenna and Cereser J Cf. also Bigelow, Gore, and Howard (92). Nutrition I nvestigations. 275 (131) find in the case of dwarf beans that when the food is wholly dex- trose administered to the leaves, pentosans increase greatly, especially in the light, and that when the functioning of chlorophyll is prevented for long periods the amount of pentosans decreases. They conclude that the simple sugars exert a preponderating influence in pentosan formation, and that these serve as a reserve material when the plant has exhausted its more readily available food materials. PENTOSANASES IN THE VEGETABLE KINGDOM. Our knowledge of enzymes inverting pentosans is meager, and rather indefinite. The action of such forms as Hymenomycetes upon wood seems to be of chemical nature. At any rate it is evident (107-146) that they are able to utilize xylan. Bourquelot and Herissey (95) have isolated an enzyme from malt diastase which produces reducing sugar from pectins, and call it pectinase. This is not to be confused with the so-called pectase which causes the coagulation of pectin bodies. Bigelow, Gore and Howard (92) also find that the enzymes of Asper- gillus partially hydrolyze the pectin of gentian root. According to Harrison (114), Bacillus oleraceoe produces a cytase capable of dissolv- ing the cell walls of potatoes, turnips, cauliflower and allied plants, which acts particularly on the middle lamella, the supposed seat of pectin. 1 The latter is not an inverting enzyme. In Persian Berries (Rhamnus) (162), in Penicillium glaucum, and Botrytis cinerea (90), an enzyme (rhamnase) has been found which splits off rhamnose from some of its glucosides (rhamnetin and rhamnazin). An early observa- tion of the presence of rhamnase in the rutin of garden rue was made by Borntrager (94). That some of the so-called cytases described under cellulose 2 may act on pentosans seems possible, but there is no direct evidence that such is the case. On the contrary, Cross and Bevan (105) believe that pentosans once formed in the plant, remain thenceforth unaltered. Tollens and Glaubitz (159) assert that the pentosans do not undergo lactic or butyric acid fermentation, and are otherwise unaffected by yeast, as has also been shown by Lintner and Dull (125) . The pento- sans are very resistant toward the action of bacteria. Slowtzoff (154) found that a small amount of pure xylan in a putrefying mixture, x Ci. Czapek, Biochemie der Pflanzen. 2 Cf. Biedermann and Moritz (34), Brown (35), Brown and Morris (36), Berg- mann (32), Griiss (184), Newcombe (64). 276 Mary Davies Swartz, kept at a temperature of 40° C, did not entirely disappear from the solution before the ninth or tenth day. Two widely distributed fer- menting agents acting on hemicellulose {Bacillus aster os poms Arth. Meyer, and Bacillus clostridieforme, Burri and Ankersmit), studied b}' Ankersmit (89), are said by him to occur in insufficient numbers to make their activity of any significance in the alimentary canal of the cow. PENTOSANASES IN LOWER ANIMALS. Extensive investigations regarding the occurrence of pentosan- splitting enzymes in lower animals, have been made by Selliere since 1905. The secretion of the hepato-pancreas of the common snail (Helix pomatia) not only digests cellulose in vitro, 1 but also xylan, ac- cording to this writer (148). In feeding experiments, analyses of the food (oak wood) and excreta of these xylophages showed a higher per- centage of xylan in the former than in the latter (149). Hence xylan must have been digested. In 1907, he showed that pentoses were actually liberated and absorbed, by testing the blood of these snails, which gave the phloroglucin reaction (151). That sugar can be found in their blood is denied by Couvreur and Bellion (99), but this Selliere attributes to the fact that the sugar content is much less than in higher animals, and hence has been entirely overlooked. - Xylanase also occurs in other species of snail (150) such as Helix aspera Miill., Helix nemoralis L., Limax arborum Bouch., Limax variegatus Drap., Arion rufus L., Patella vulgata L., Littorina lit'.orea L., Littorina littoralis L., and in a representative of the Coleoptera, Phy- tnatodes variabilis L. The presence of a xylanase in Patella vulgata and the Littorinae is especially significant, as their food consists in pentosan-rich algae. Selliere (150) and Pacault (130) have independ- ently discovered a xylanase in the salivary glands of Helix pomatia. According to Rohmann(134), Aplysia, which subsist largely upon Viva lactuca, do not, digest the soluble methyl-pen tosan (rhamnosan) present in this alga. He finds this carbohydrate present in the glands of the midgut, but regards it as a food residue. PENTOSANASES IN HIGHER ANIMALS. There have been only a few investigations as to the presence in higher animals of enzymes hydrolyzing pentosans. Slowtzoff (154) l Ci. Biedermann and Moritz (34). Nutrition Investigations. 277 found that pure xylan was not digested by saliva, gastric or pancre- atic juice, but could be gradually hydrolyzed (in two or three days) by 0.2 per cent hydrochloric acid. Bergmann (91) digested pure xylan with extracts of the intestines of many animals (hen, goose, guinea- pig, sheep, ox, horse), and of the vermiform appendix of rabbits, but in no case found a xylanase. These experiments were performed with suitable antiseptics and controls in all cases. An old experiment by Fudakowski (112), attributing an inverting action upon gum arabic to pepsin, and another by Schmulewitsch (144), attributing such an action upon crude fiber to pancreatin,must be disregarded, as no anti- septics whatever seem to have been used. According to Selliere (152), neither the pancreatic juice of rabbits, nor a mixture pancreatic and in- testinal juices, will hydrolyze xylan. Negative results were also ob- tained by him with macerated intestines of these animals. On the other hand, chloroform extracts of the intestinal contents of rabbits and guinea-pigs fed fresh hay and bread, produced pentoses in a 5 per cent xylan solution after 48 hours digestion at 37 degrees C, while negative results were obtained with boiled controls. This indicates that the enzymes causing hydrolysis were of bacterial origin, a conclusion sub- stantiated by later work of the same author (153). No xylanase was detected in the excreta of carnivora such as the lion, panther, and wolf. From a centrifugalized extract of human faeces and soluble xylan, di- gested under aseptic conditions, xylose was obtained after 15-20 hours; but in meconium of calves and human beings in which bacteria were absent no xylanase could be found, although the intestinal glands were functioning. McCollum and Brannon (126) have shown that in the case of the cow intestinal bacteria destroy pentosans under anaerobic conditions, the degree of destruction varying with the kind of plant. Corn, wheat and oat feeds were incubated with fecal bacteria of this animal, and digestions continued 14 days in atmospheres both of car- bon dioxide and hydrogen, with the following average results: MATERIAL Com Fodder Corn Fodder Wheat Straw. . . . Wheat Straw Oat Straw Oat Straw ATMOSPHERE. PER CENT OF PENTOSANS DISAPPEARING. co 2 H C0 2 H C0 2 H 51.78 76.13 28.09 37.99 30.66 54.00 From this review it is evident that the presence of pentosanases in the higher animals has not yet been demonstrated. 278 Mary Davies Swartz, DIGESTION AND UTILIZATION OF PENTOSANS BY ANIMALS. In the case of men and animals subsisting on a mixed diet, the hex- oses and their derivatives so overbalance the pentosans, under normal conditions, that the utilization of the latter is a question of theo- retical rather than of practical importance. But in the case of herbi- vora, limited to a diet in which pentosans occur in considerable amounts, the extent of pentosan utilization becomes a question of economic importance. It is not surprising to find, therefore, that since the development of satisfactory methods of quantitative deter- mination, a considerable number of investigations have been made upon such utilization by animals. The results of these experiments are shown in tables on pages 274 and 275. The results in these experiments were obtained by analysis of food and faeces. Lindsey (123) Gotze and Pfeiffer (113) and Tollens (157) found no measurable amount of pentoses or pentosans excreted in the urine of sheep, but Neuberg and Wohlgemuth (128) state that pento- sans always occur in the urine of rabbits, only disappearing when the vegetable diet is compensated by pentose-free material. They report that 9 per cent of soluble araban (cherry gum) fed to rabbits was ex- creted in the urine. Slowtzoff (154) found 1.4-4.5 per cent of xylan in the urine of rabbits, but no reducing sugar. He also found that if the animal were killed shortly after xylan feeding, xylan could be de- tected in blood, liver and muscles. Hence xylan must have been ab- sorbed from the digestive tract. The feeding experiments show that herbivora digest, on the aver- age, 55-60 per cent of the pentosans in their diet, but since no animal enzymes hydrolyzing pentosans have been demonstrated, and there is always the possibility of bacterial decomposition in the intestines, the most conclusive experiments as to the actual nutritive value are those of Kellner (118) with the respiration calorimeter. From the slight difference in loss of potential energy, when the furfurol-yielding rye straw preparation was substituted for starch, he concludes that furfurol-yielding substances participate in the formation of fat in the animal body. DIGESTION AND UTILIZATION OF PENTOSANS BY MAN. We have seen that pentosans can be digested by herbivora to a considerable extent. Can they be digested by man? The only feeding experiments on record are by Konig and Reinhardt (120). Nutrition Investigations . 279 In 1902, they conducted researches on two men whose main diet con- sisted of meat and butter or other fat, and beer; to this, in the various experiments, were added respectively (along with sugar, butter, beef extract, etc., used in preparing them) the following substances: Experiment I. Green Peas. Experiment II. Ripe Shelled Peas. Experiment III. Red Cabbage. Experiment IV. Canned White Beans. Experiment V. Soldiers' Bread. Experiment VI. Graham Bread. From analyses of food and faeces the following results were obtained: TOTAL PENTOSANS IN GRAMS. EXP. I. ii. in. IV. V. VI. 15.55 23.15 14.01 12.80 52.64 41.26 0.79 0.59 0.70 1.12 8.66 4.06 Per cent not utilized, estimating Pen- tosans in Beer as unutilized 5.08 2.55 5.0 8.75 16.45 9.84 7.47 3.24 7.75 14.32 20.24 12.97 Hence we see that of the total pentosans in the diet 3.24-20.24 per cent were excreted. Only a little furfurol-yielding substance was found in the urine. From the small percentage recovered in these experiments, Konig and Reinhardt (120) conclude that the pentosans are to a high degree utilized by man, but they take no account of pos- sible destruction by bacteria. 1 Since pentosans do disappear from the alimentary tract of men and animals, it behooves us to consider whether, on the assumption that they are hydrolyzed like starch, the pentose sugars so produced are as well utilized as dextrose. Konig and Reinhardt (120) found some furfurol-yielding substance in the urine, and Blumenthal (93) observes that after eating huckleberries, cherries and prunes, pentosans are excreted, but no reducing sugar. Cominotti (100) finds pentoses ab- sent from the urine of man on a meat diet, but always present on a mixed diet. He agrees with Konig and Reinhardt that the output in the urine is small compared with the amount of pentosans in the food, and proposes to investigate the possibility of glycogen formation from pentosans. The behavior of pentoses in the body has been exhaustively reviewed by Neuberg(127). 2 It appears from the work of Cremer (102, 103), 1 Cramer (101) has shown (according to a recent review, the original paper was not accessible) that bacteria are essential to hemicellulose transformation. 2 For a recent discussion of the absorption and utilization of pentoses see A. Mag- nus-Levy, Oppenheimer's Handbuch der Biochemie der Menschen und der Tiere, 1909, Vol. IV, pp. 395-407. 280 Mary Dames Swartz, CO O (M © O N bO be d d 4) o O a a "3 ^ o co o 2 h-1 • M lO N lO M iO tO ^ ^ ^ M en f£ 13 fl .2 CO O ctf aj U > O co *■ £ 13 13 ^ '§ ^ CJ d ™ ^ §0 CO O J h s 1 s> < S * 2 13 bb£ (~-^ ^ ,_> • — < r be to ji S-H d Oh O PQ S ^ . W to CO CO WcocoMMgococoggco O CO 3 G £ c &-I M O W w m h N M lO OS OS OS 00 OO GO iO CO OS OS CO 00 o OS o 00 OS r-l CO o o OS OS en cy d _ iO 13 T— I Ch v — ' d aj CD +j b> C/3 C/3 l> 13 N 0h S O d CO »o „ w CO •§ > CO t> Nutrition I nvestigations. 281 (MOO GO 00 (N CO O © ^ CO N in H N N ^ (i Ol N O iO 00 N C3 • ' in (M CO Ol CD (N Ol 00 LO CM CM CO ^ CO o CM LO CO © LO in -v 1- t> l> CO 00 OS CO CM CI A 00 CO CO LO OS -CH d LO »o CO 43 cj u d 2 _1_ « ~r i 5 cj is c ^ cj w C + 1S 2+1 « a 2 g >-> is 43 +3 P< co H ^ < M U b0 cu 2 £ -a o ■o i w + ^ c3 cj <3 PQ CD £ O ^ to 'bb o o C/5 _3 cu (2 M I 5 3° n & cu pq "3 o 282 Mary Davies Swartz, Ebstein (109), Frantze (111), Neuberg and Wohlgemuth (128), Sal- kowski (137), v. Jacksch (117),Lindemann and May (122), Brasch (96) and others, that the pentoses and methyl-pentoses (rhamnose) are ex- creted more readily than the hexoses; that they exert an unfavorable effect in diabetes; and that there is no evidence of their acting as gly- cogen-formers in man. Consequently, even if further experiments justify Konig and Reinhardt's conclusions, the pentosans must appar- ently still play a very small part in the nutrition of man. Occurrence and Nature of Galactans. Next to the pentosans, no hemicelluloses seem to be so widely dis- tributed as the galactans; both occur together in the plant cell, and often in a more or less intimate chemical combination. The pure galactans, i.e., those yielding exclusively galactose upon hydrolysis, have been differentiated into several classes, chiefly by differences in s jlubility or specific rotation, namely: 1. a-galactan, so named by Miintz (199), the first to identify galactan as an anhydride of galactose; it composes 42 per cent of luzerne seeds and occurs also in beans, barley, and malt. 2. jS-galactan, isolated from the lime residues in the sugar beet industry by Lippmann (192). 3. 7-galactan, first isolated from Chinese moss (Sphaerococcus lichenoides) by Payen (262), in 1859, and by him called "gelose." He also identified it in agar-agar 1 (Gelidium corneum) and other algae. The carbohydrates of agar-agar were again studied by Reichardt in 1876, who obtained a substance of the formula C12H22O11 and con- sidered it identical with the "pararabin" which he found in carrots and beets. 2 In 1881 and 1882, Greenish (180, 181) investigated the carbohydrates of Fucus amylaceus (Ceylon agar-agar) and obtained on hydrolysis a sugar-yielding mucic acid (galactose). From Sphaero- coccus lichenoides he also obtained a substance resembling Payen's " gelose." In 1884, Bauer (169) showed that agar-agar yields galac- tose; and in 1905, Konig and Bettels (190) gave the following per- centage composition of Japanese agar-agar from Gelidium: Per cent. Per cent- Galactans 33 Ash 3.5 Water 20 Pentosans 3. 1 Protein 2.6 Crude fiber 0.4 x The term agar-agar is applied to the hot water extract of various red algae, mainly species of Gelidium. 2 See Pentosans, p. 268. Nutrition Investigations. 283 Another species of marine algae in which galactan has been fully identified, is Chondrus crispus (Irish moss). This is also a red alga. C. Schmidt (210) first examined it, in 1844; he demonstrated that the gelatinizing substance was a carbohydrate and yielded sugar on hydrolysis. Fliickinger and Mayer (178), in 1868, discovered that the water extract of this alga yielded consieterable mucic acid. In 1875, Bente (171) obtained levulinic acid from the products of its hydrolysis, and in 1876, reported that it yielded a non-crystallizing syrup (172). The first quantitative analysis was made by Hadike, Bauer and Tollens (185), who showed that the water extract yielded mucic acid corresponding to about 25 per cent of galactan. Sebor (220), in 1900, found in the products of hydrolysis, glucose, fructose and a small quantity of pentose. These observations were verified by Miither (200) in 1903, who further identified the galactose as a (/-galactose. From the large yield of mucic acid, the water extract of Chondrus may therefore be regarded as chiefly galactan, together with some dextran and levulan, and a very little pentosan; groups which, according to Hadike, Bauer and Tollens (185), may be partly or entirely bound into ester-like compounds. Examples of galactans occurring in combination, or close associa- tion with other hemicelluloses are numerous. Lupeose, from luzerne seeds, originally called /3-galactan, yields 50 per cent galactose and 50 per cent fructose (214). The tuberous roots of Stachys tuberifera contain a soluble crystallizable carbohydrate yielding 37 per cent mucic acid, along with an unidentified sugar (225). Para-galactan (galacto-araban) forms a large proportion of the reserve material of many seeds. 1 Rothenfusser (204) finds that the mucilaginous extract of flaxseed yields equal parts of pentosans and hexosans, the latter being mainly galactose. Galactans and pentosans, as already indicated, 2 occur together in many lichens and algae, and also in the pectins. 3 Herissey (187) has shown that the "galactine" of Muntz (199) yields equal parts of galactose and mannose. Galacto-mannans also fre- quently occur in the reserve material of seeds, as in those of the date and other species of palm, and in coffee beans; in the American honey x Cf. Schulze (215), Schulze, Steiger and Maxwell (217), Schulze and Castoro (218), Castoro (176), and Goret (179). Also Schulze and Godet, Zeitschrift fur physiologische Chemie, V. 61, p. 279, for a very complete review of the work of Schulze and his pupils. 2 See Chemical Nature of Lichens and Algae:- Konig and Bettels (8), Escombe (6), K. Miiller (11), Ulander (26). 3 Cf. Pentosans, p. 268. 284 Mary Davies Swartz, locust (Gleditschia triacanthus) , Goret (179) found the albumen to yield 66-70 per cent galactose and 22-23 per cent mannose; he has shown, in fact, that the carbohydrate reserve of almost all seeds with horny albumen consists largely of a mixture of mannans and galac- tans. 1 GALACTANASES IN THE VEGETABLE KINGDOM. The hydrolysis of the paragalactan of lupine seeds during germina- tion was first observed by Schulze and his co-workers. That ordi- nary diastatic enzymes do not form sugar from the para-galactan of Lupinus hirsutus was demonstrated by Schulze and Castoro (218). Ptyalin, pancreatin, malt diastase and"taka" diastase, will, however, in the course of 5 or 6 days' digestion at 35-40° C. render this carbo- hydrate soluble in water to the following extent: Per cent. Per cent. Malt diastase 38 Ptyalin 40 Taka diastase 35 Pancreatin 15 , Griiss (184) has made exhaustive microchemical investigations upon the germinating date endosperm, in which he has been able to observe the solution of the galactans by enzymes developed during germina- tion. Bourquelot and Herissey (174) find a soluble enzyme hydrolyz- ing galactan, 2 produced by the germinating embryos of the seeds of the carob, Nux vomica, fenugrec and luzerne. Shellenberg (208), studying the action of moulds on hemicelluloses, found at least four different ferments showing considerable specificity in their action; seeds of Lupinus hirsutus (containing paragalactan) were attacked by most of these moulds (Mucor neglectus, Mucor piriforme, Rhizopus nigricans, Thamnidium elegans, Penicillium glaucum). Similarly, Herissey (187) found galactose produced from manno-galactans by Aspergillus niger and Aspergillus fuscus; Saiki (205) obtained sugar from Irish moss by digesting it with inulase prepared from Aspergillus niger and Penicillium glaucum; and with "taka" diastase prepared from another mould, Eurolium oryzae. Little is known of the action of bacteria upon galactans. Gran (182) found sugar produced from agar-agar by Bacillus gelaticus, through the action of an enzyme which he calls " gelase." Saiki (105), ^f. Mannans, p. 283; for a further discussion of the occurrence of Galactans see v. Lippmann, Chemie der Zuckerarten, Vol. I, pp. 686-697. 2 Cf. Mannans, p. 284. Nutrition Investigations. 285 in experiments with B. coli communis, on culture media containing different kinds of comminuted seaweed, found a slight gas production in one culture, in media with agar-agar and Irish moss. GALACTANASES IN THE ANIMAL KINGDOM. The only discovered instance of a galactanase in lower animals is cited by Bierry and Giaja (173), who found that the hepato-pan- creatic juice of Helix pomatia produced galactose from extracts of carob seeds (Ceratonia siliqua) ; later experiments upon agar-agar, with extracts from a number of crustaceans (Astacus fluviatilis Rondel., Homarus vulgaris Bel., Maja squinado Rondel., Carcinus moenas L., and Platycarcinus pagarus L.) were entirely negative; the galactans of luzerne and fenugrec were attacked with difficulty by the extract from Astacus. Strauss (221) could find no enzyme attack- ing agar-agar, in the larvae and puppae of various species of Lepidop- tera and Diptera. No galactanases have been found in higher animals. Bierry and Giaja (173), using extracts of luzerne seeds, got negative results with digestive juices of dogs and rabbits , and Sawamura (207) ob- tained similar results with extracts of different sections of the alimen- tary canal of swine and horses. Saiki (205) found saliva, pancreatic, and intestinal juices unable to hydrolyze Irish moss. DIGESTION AND UTILIZATION OF GALACTAN BY ANIMALS AND MAN. The first study of the digestibility of galactans in higher animals was made in 1903, by Lindsey (191). Alsike clover-seed, containing 8 per cent galactan, was fed in connection with hay, the digestibility of which had been previously determined; from analyses of food and faeces, the galactan in the hay (1.72 per cent) was found to be 75 per cent digestible, and that in the clover 95.78 per cent digestible. Saiki (205) fed agar-agar and Irish moss to dogs and recovered a large part in the faeces, as shown by the increased amount of carbohydrate excreted. Lohrisch (194) fed dogs and rabbits agar-agar in its usual form, and also " soluble-agar " prepared from ordinary agar by Dr. Karl Dieterich of Dresden, Director of the Helfenberg Chemical Fac- tory. This product seems to be partially hydrolyzed in its prepara- tion, since it is not only readily soluble in water, but has slight reduc- ing action; it yields on boiling with Fehling's solution, 3.5^.1 per cent sugar, and if a watery solution is allowed to stand 18 hours at 286 Mary Davies Swartz, 37° C, it is further hydrolyzed and yields then 16.9-20.4 per cent sugar. The results of Lohrisch's experiments appear in the folowing table: ANIMAL. FOOD. HEMICELLULOSE EQUIVALENT OF AGAR FED. HEMICELL- ULOSE EXCRETED. HEMICELL- ULOSE DIGESTED. Per cent. Rabbit I Ordinary agar 18.77 = 14.48 7.1 50.9 Rabbit II Ordinary agar 11.8 = 9.11 4.71 48.3 Rabbit III Soluble agar 95.9 = 65.02 14.2 78.1 (given in 9 days) Dog Same as III 53.0 = 35.9 11.7 67.3 Lohrisch (194) has also studied the utilization of agar-agar in starv- ing herbi'vora. In two experiments, rabbits starved for two days were fed ordinary agar as long as they would eat it, other animals of the same weight being kept in starvation as controls; in a third expe- riment, " soluble agar" was fed. Urine and faeces were collected and analyzed. Of the ordinary agar, about 50 per cent was excreted in the faeces; of " soluble agar," about 25 per cent. No positive evidence of any change in nitrogen excretion attributable to the agar fed, can be drawn from the protocols. One animal died through accident, another survived its control but one day, and the third, in spite of its apparently good digestion of the "soluble agar," died a week before its control. In the case of rabbits made diabetic with phlorhizin and then fed 20-40 grams of both ordinary and soluble agar, Lohrisch (194) found that the D : N ratio remained fairly constant throughout each experi- ment, showing no marked increase in sugar excretion. We see, there- fore, no grounds for assuming that agar-agar (galactan) forms glycogen in rabbits. The first studies on the utilization of galactan by man were made by Saiki (205) (1906). In feeding experiments in which various car- bohydrates were at different times added to a uniform diet, consisting of 513 grams beefsteak, 500-600 grams bread, 40 grams sugar, 31 grams butter, 2 eggs and 2 apples — a diet on which over 98 per cent of the carbohydrates were digested, he obtained the following results: Nutrition Investigations . 287 NO. SUBSTANCE ADDED TO DIET. EQUIVALENT OF SUBSTANCE IN DEXTROSE. CARBOIIYDRATES IN FAECES CAL- CULATED AS DEXTROSE. HEMICELLULOSE DIGESTED. Grams. Grams. Per cent. 1 10 9.2 8 2 24 grams agar 12 8.8 27 3 40 grams wakame 4.7 3.4 28 4 11.4 2.5 78 Lohrisch has also studied the digestibility of "soluble agar" in man. Sometimes it is not well borne, especially if given in quanti- ties over 50-60 grams per day and causes gas formation, diarrhoea, and other intestinal disturbances; in other cases, large amounts (100 grams per day) cause no unpleasant symptoms whatever. The agar was dissolved in some beverage, and the diet was otherwise carbohy- drate-free. Some of the results are shown in the following table (194) : NO. DURATION OF EXPERIMENT. AMOUNT DIGESTED. HEMICELL- ULOSE DIGESTED. HEMICELL- ULOSE DIGESTED. As Air Dry Soluble Agar. As Hemicel- lulose. HEMICELLULOSE EXCRETED. Grams. Grams. Grams. Grams. Per cent. 1 1 day 100 61.9 46.06 15.84 25.6 2 1 day 100 61.9 39.1 22.8 36.8 3 3 days 235 145.4 90.5 54.9 37.7 4 3 days 240 148.5 40.8 107.7 72.5 5 1 day 100 61.9 25.4 36.5 58.9 6 1 day 110 67.8 23.4 44.4 65.5 No. 4 was a case of chronic constipation; the high percentage of hemi- cellulose digested is in accordance with the observations of Lohrisch (193) and Pletnew (203), on the extraordinarily good utilization of all foodstuffs in chronic constipation. Two of these experiments were on diabetics, and showed that the 18.36 grams of "soluble agar" ab- sorbed per day caused no increase of sugar in the urine, and had no noticeable effect on nitrogen metabolism. From these experiments, we see that ordinary agar is digestible to a very small extent, and that even when changed to an easily hydro- lyzed form, it is only digested to about 50 per cent. Is the part digested absorbed and utilized as galactose? The recent exhaustive 288 Mary Davies Swartz, discussion of the behavior of galactose in the animal body by Brasch (175) renders any details on the utilization of this sugar unnecessary. Hofmeister (188) showed that of all sugars it is most readily excreted. That galactose can form glycogen in dogs and rabbits, has been shown by Weinland (226), Kausch and Socin (189), Cremer (177), Voit (223), Brasch (175), and others. 1 Brasch (175) has shown that the assimila- tion limits for galactose lie, for normal man, between 30 and 40 grams, while for dextrose they lie between 100 and 150 grams. Voit (224), Sandmeyer (206), Bauer (170), and others have shown that galactose, even in small amounts increases the sugar excretion in diabetes. It would seem, therefore, that if soluble agar were absorbed as sugar, it would increase the sugar output in the urine. To throw some light on this problem Lohrisch (194) has conducted three respiration ex- periments on men after ingestion of 100-110 grams of soluble agar, of which, on the average, about 63 per cent was absorbed. The changes in the respiratory quotient are shown in the following table: Respiratory Quotient. NO. IN FASTING. NUMBER OF HOURS AFTER INGESTION OF SOLUBLE AGAR. 1 2 3 4 5 6 7 I II III 0.768 0.786 0.739 0.768 0.766 0.835 0.794 0.815 0.860 0.825 0,800 0.770 0.767 0.774 0.735 0.724 0.714 NO. IN FASTING. NUMBER OF HOURS AFTER INGESTION OF SOLUBLE AGAR. 8 9 10 n 12 13 I II III 0.768 0.786 0.739 0.693 0.730 0.703 0.618 0.669 The distinct rise in the respiratory quotient in the fourth hour (beginning in the third hour in Experiment I) would indicate that car- bohydrate was being oxidized, which in this case must come from the agar. The low value in the later hours seems due to the oxidation of fatty acids; 2 that such acids may be formed from soluble agar by bacteria, appears probable also from the intestinal fermentation pro- *Cf. Magnus-Levy, Verwerthbarkeit der Galactose in normalen Organismus : Op- penheimer's Handbuch der Biochemie der Menschen und der Tiere, Vol. IV, p. 379. 2 Cf . respiration experiments described under Cellulose. Nutrition Investigations. 289 duced when large amounts of this preparation are taken. A slight increase in acetone output, shown in the metabolism experiments with diabetics, points to the same conclusion. Perhaps, as Lohrisch suggests, the very slow digestion of the carbohydrate, may enable the organism to utilize the galactose formed, and account for its non-ex- cretion, but this requires further demonstration. According to these experiments by Lohrisch, cellulose and the solu- ble galactan show little difference in their physiological behavior. Both can be digested to about 50 per cent. Ordinary agar, as Saiki's experiments show, is largely recovered in the faeces; in fact, a thera- peutic practice which has been recently established is based upon the recognized indigestibility of agar, namely, its employment as a remedy in cases of chronic constipation. It is especially valuable, as Mendel (196) points out, in those cases where the difficulty is due to an ex- tremely complete digestion and absorption of all foodstuffs from the alimentary tract, which causes the formation of dry, hard faecal masses (scyballa) difficult to evacuate. The agar, remaining undigested and retaining a high percentage of water, gives bulk and softness to the faeces, and facilitates their daily elimination. Being resistant towards bacterial action, it causes neither gas formation nor produc- tion of harmful decomposition products. According to A. Schmidt (209), it can be advantageously taken in quantities up to 25 grams per day, part with the breakfast cereal, and part with sauce or cream, at another meal. In view of such facts as these, we are hardly prepared to agree with Lohrisch, that ' Cellulose and Hemicelluloses are readily digested. ' Occurrence and Nature of Mannans. As widely diversified in origin and character as the galactans, and very intimately associated with them are the Mannans. They show all possible degrees of solubility, from the readily soluble mucilage found in certain legumes, to the completely insoluble "reserve-cellu- lose," which forms the horny albumen in such seeds as the date, and which was long confused with true cellulose. A few examples will serve to show the diverse places in which man- nans may be found. They occur in yeast: 1 (258) in algae, as Por- phyra laciniata; (278) in moulds, as Penicillium glaucum; (285) in the leaves and roots of the Japanese plant, Conophallus konjaku (280) ; in the bark and wood of many American trees (272). *For further discussion see v.Lippmann, Chemie der Zuckerarten, Vol.1, pp. 641- 649, and Czapek, Biochemie der Pflanzen, pp. 325-329. 290 Mary Barnes Swartz, The most extensive study has been given to the mannans of various seeds, in which, as already shown, 1 mannans and galactans seem al- most invariably to occur together. The seeds of the carob tree (Ce- ratonia siliqua) contain a hemicellulose originally called "caruban" by Effront (241) (1897), but shown by van Ekenstein (282) to yield mannose, and by Bourquelot and Herissey (232) (1899), ^-galactose. The first elaborate studies of "reserve-cellulose" were made by Reiss (264), who showed that the horny albumen of the seeds of Phytelepas macrocarpa, Phoenix dactylifera and other species of palm, Allium cepa, Asparagus officinalis, Iris pseudacorus, Strychnos nux vomica and Caffea arabica, differed chemically from true cellulose in their color reactions, in the ease with which they can be hydrolyzed, and in yielding, instead of dextrose, a sugar which he called "seminose," but which proved to be identical with Fischer and Hirschberger's (242) previously described mannose. Mannan also occurs richly in the tubers of the many species of Or- chis and Eulophia which are the source of commercial salep. On ex- traction with water, they yield a mucilaginous extract which was first studied by C. Schmidt (270) in 1844, and called by him "salep- bassorin"; on hydrolysis with dilute sulphuric acid he obtained, be- s'des some gummy substance and cellulose, a fermentable sugar which he thought to be dextrose. Mulder (259) considered the salep mucilage a mixture of starch and gum or pectin acids, while Franck (243) thought it a modification of cellulose, and Girand (248) a trans- formation of a starchy substance into a variety of dextrin swelling in water. Pohl (263) by precipitation with neutral salts, distinguished an "a-Schleim" and a "jS-Schleim. " According to Thamm (276), who has made the most recent investigations, "a-Schleim" does not occur in German salep. Tollens and Gans (277) showed that on hydrolysis, besides dextrose, mannose or, as they called it, " isomanitose " was formed, but this was shown by Fischer and Hirschberger (242) to be identical with d-mannose. Thamm (276) and Hilger (254) have shown conclusively, that the starch-free water extract contains an anhydride of mannose only. A very resistant type of mannan occurring in some plants, has been designated as manno-cellulose by Schulze (273). Bertrand (227) finds it taking the place of xylan in the woody tissues of gymnosperms. x Cf . Schulze and his coworkers, and Goret, under Galactans. Also Schulze and Godet, Zeitschrift fur physiologische Chemie, V. 61, p. 279, for a very complete review. Nutrition I n vest i gat ions . 291 MANNANASES IN THE VEGETABLE KINGDOM. There is very little literature concerning the action of bacteria upon mannans. Sawamura (267) observed that extracts of Hydrangea pa- niculata, used in the manufacture of Japanese paper, which contain mannan (along with galactan and araban) , became liquefied on stand- ing. In bacteriological studies with extracts of this plant, and of roots of Cono phallus konjaku, he found that only B. mes enter icus ml- gatus dissolved these mannans. The action was greatly facilitated, and sugar formation increased if a certain wild yeast, in itself inactive, were present. Traces of a similar enzyme seem to occur in B. prodi- giosus. In his studies of the action of moulds on hemicelluloses, Schellen- berg (269) found that the seeds of Ruscus aculeata, which yield almost exclusively mannose (237-240), were attacked only by Penicillium glaucum. Herissey (253), using pure cultures and water extracts of cultures of Aspergillus niger (grown on media rich in mannose and ga- lactose to incite the development of mannanase and galactanase), with suitable antiseptics and controls, obtained mannose — and galactose — from seeds of Ceratonia siliqua and Gleditschia triacanthus, and an abundant yield of mannose from salep; similar results were obtained with Aspergillus fuscus. As early as 1862, Sachs (266) observed the change of the thickened cell-walls of the date endosperm into sugar during germination. The cytases producing this change in 'reserve-cellulose' were later care- fully investigated by Reiss (264), Brown and Morris (230), Newcombe (261), Gniss (251), and others. Still more recently, Bourquelot and Herissey have made many studies on the specific characteristics of these plant enzymes. An exhaustive review of the literature on man- nans and the action of enzymes upon them has been published by Herissey (253), consequently this subject will only be reviewed very briefly here. Griiss (251) has demonstrated that the solution of the date embryo (Phoenix dactylifera) is due to a ferment, the product of whose activ- ity is galactan and mannose. Effront (241) (in 1897) attributed the solution of the albumen of carob seeds (called by him caruban) to a "caroubinase," but thought that the product of its activity was not identical with the products of hydrolysis; in 1899, however, Bourque- lot and Herissey (233) showed the possibility of obtaining mannose by the action of a soluble ferment derived from these seeds, which they called "seminase." Shortly afterwards, a similar enzyme was 292 Mary Davies Swartz, isolated by them from the seeds of Phoenix canariensis. Herissey (253) has been able to show that seeds of such legumes as luzerne, fenugrec, and common genet have, at least at the time of germination, ferments capable of transforming mannans — and galactans — into their corresponding sugars. Experiments in vitro show that they are not limited to action upon the seeds by whose embryos they are pro- duced, but act on the reserve-cellulose of seeds from very distinct groups of plants. However, the luzerne ferment does not digest all mannans and galactans; it will hydrolyze the mannans of the tubers of the Orchis family (and commercial salep prepared from them), but not those of the albumen of palm seeds. Griiss (251) has also shown that the enzyme of the date endosperm hydrolyzes starch, although this does not occur in the date seed, and that malt diastase works on a-mannan (the soluble mannan of date seeds, according to Griiss) which does not occur in the barley endo- sperm. Griiss considers diastatic enzymes a group working not only on starch, but also on hemicelluloses. Herissey thinks that diastase and seminase are found together in varying proportions in barley, legumes, carob seeds, etc., and that neither is a simple ferment, but a " superposition de ferments," and defines "seminase" as a "ferment or group of soluble ferments, causing the transformation of the car- bohydrates of horny albumens of the seeds of Leguminosae into as- similable sugars." Gatin (247) has made further researches upon the nature of seminase, and states that during the germination of certain seeds whose reserve is in the form of mannan, the presence of mannose is exceptional, but dextrose occurs in abundance. This phenomenon he attributes to a "manno-isomerase," which transforms the mannose, as fast as formed by the seminase, into dextrose. Experiments in vitro seem to indicate that this is a soluble ferment. MANNANASES IN THE ANIMAL KINGDOM. There are only a few instances on record of mansases occurring in lower animals. Bierry and Giaja (228, 229) found that the hepato- pancreatic juice of Helix pomatia was capable of producing mannose from extracts of carob seeds and salep; that of Astacus fluviatilis, Ho- marus vulgaris, and Maja squinado, from the ivory nut (Phytelepas ma- crocarpa) , the two latter hydrolyzing it at ordinary room temperature. On the other hand, the mannans of fenugrec and luzerne were hydro- lyzed with difficulty, or not at all, by very pure gastro-intestinal juice. No mannanase was found by Strauss (275) in the larvae and Nutrit ion Investigations 293 puppae of Lepidoptera and Diptera. Similar negative results have been obtained with the digestive enzymes of higher animals. Kino- shita (257) found that emulsin and invertin did not hydrolyze the man- nans of Conophallus konjaku and Gatin (245, 246) tried the blood of rabbits, chicken serum, the pancreatic juice of dogs, the macerated intestines and pancreas of chickens and cattle, upon salep and carob seeds with negative results; on the other hand, Sawamura (268) re- ports a mannanase in the extracts from different sections of the ali- mentary tract of swine and horses. DIGESTION AND UTILIZATION BY ANIMALS AND MAN. There are also very few records in the literature of feeding experi- ments with mannans. In a paper in the Zeitschrift fur Biologie, Voit (283) in 1874 1 described one by Hauber, who fed a medium sized dog 390 grams of dry salep powder in the course of eight days. The faeces of the feeding period were roughly marked off, and Hauber reported no unchanged salep present in them, because there was no swelling in water as with the original powder. Calculations based on the yield of sugar from the faeces on hydrolysis showed that at least 50 per cent of the salep was absorbed. This seems to have been a very crude ex- periment, and cannot be considered of convincing value. In 1879, Weiske (284) fed carob-beans {Ceralonia siliqua) to sheep, along with meadow hay, and compared the nutritive value of this ra- tion with one in which the carob-beans (210 grams) were replaced by an equivalent weight of starch, sugar and protein (from crushed peas). The coefficients of digestibility and nitrogen balance were so nearly the same on the two rations, that Weiske pronounced "Johannis- brod" (carob beans) an acceptable and digestible feed for sheep. In 1890, Schuster and Liebscher (274) tried feeding the sawdust of ivory nut (Phytelepas macrocarpa) to sheep, having previously found that it had a favorable effect on cattle. Merino sheep gained consider- able fat when fed oat straw and vetch fodder, plus ivory nut sawdust furnishing 50 per cent of the digestible carbohydrates. The ration, exclusive of the ivory nut, did not yield enough energy for such a re- sult to be possible, hence the latter must have been utilized. The coefficient of digestibility, both for the nitrogen-free extract and crude fiber of this material, was at the same time shown by Niebling (262) to be 82 per cent for sheep. x This paper reviews the early literature on gums. 294 Mary Davies Swartz, From these experiments, mannan would seem to be well utilized by herbivora. The only experimental data regarding the nutritive value of mannans to man, are cited by Oshima (15) from work by Kano and Iishima (255), who found the coefficient of digestibility of konjaku 82 per cent (prepared from Conophallus konjaku). Further investigations seem highly desirable, in view of the fact that in certain regions food stuffs like salep and konjaku, consisting of almost pure mannan, are among the chief articles of the poor man's diet. It is also a question whether the nutritive value of bark, especially of coniferous trees, is due to mannan present. According to Dillingham (239) the quantity of mannan present does not justify such an assumption, aside from the question of its digestibility. We have finally to inquire whether mannan can be hydrolyzed within the organism, and if so, whether the mannose produced can be retained and form glycogen. From the literature on the subject, it appears that mannose is well utilized by rabbits, dogs and men. Ac- cording to Neuberg and Mayer (260), the d-form is better utilized than the /- or i-form. Mannose is readily converted to dextrose in the organism; thus Neuberg and Mayer found that a rabbit, receiv- ing 10 grams of /-mannose per os, excreted 1 gram /-mannose and 4-5 grams /-glucose; 10 grams of ^-mannose given rabbits per os, or sub- cutaneously, were almost completely oxidized. Rabbits fed 30 grams d-mannose by Cremer (238) excreted 3-4 grams in the urine, and dogs given 20 grams by Rosenfeld (265), excreted over 4 grams. This is somewhat more than would be excreted on giving equally large quan- tities of dextrose or levulose. Cremer (238) found no sugar in the urine of a man after feeding 3-12 grams of mannose. That mannose can act as a glycogen former in rabbits, has been demonstrated by Cremer (238) and also by Rosenfeld (265). Neu- berg and Mayer (260) found only a small amount of glycogen in the livers of starving rabbits after feeding /-mannose, but even this form is utilized to some extent. There is good reason for assuming, there- fore, that if mannans can be converted into mannose in the process of digestion, they may be considered as true nutrients for the organ- ism, the mannose being to a high degree capable of absorption and con- version into glycogen. Nutrition Invest iga Hons. 295 Occurrence and Nature of Levulans. A number of polysaccharide carbohydrates yielding levulose on inversion have been described. They are all levo-rotatory, more or less soluble in cold water and insoluble in alcohol, and easily hydro- lyzed by dilute acid, but have not been investigated sufficiently to permit any conclusion to be drawn respecting their relation to one another. The most important of these substances and their sources are shown in the following table:* NAME. SOURCE. INVESTIGATOR. Inulin Tubers of dahlia, artichoke, Jerusalem artichoke, elecampane; bulbs of onion, garlic, narcissus, hyacinth, and tube- rose; flowers, seed, etc., of various compositae Tanret (321) Chevastelon (291) Pseudo-inulin Inulenin Helianthin Synanthrin Tubers of dahlia, artichoke, Jerusalem artichoke, elecampane; bulbs of onion, garlic, narcissus, hyacinth, and tube- rose; flowers, seed, etc., of various compositae Tanret (321, 322) Levulin Tubers of Helianthus tuberosus (Jeru- salem artichoke) Reidemeister (314) and others Phlein Rootstalks of Phleum praetense (Tim- othy) Ekstrand and Jo- hanson (296) Cerosin Unripe grains Tanret (320) Graminin Rootstalks of various grasses, e.g., Trisetum alpestre Ekstrand and Johan- son (296) Harlay (301) Triticin Dracaena australis and rubra, Triti- cum repens (couch grass) Reidemeister (314) Sinistrin Bulbs of Scilla Maritima (Sea onions or squills) Schmiedeberg (318) Reidemeister (314) Levulan Molasses in beet-sugar industry v. Lippmann (309) * Cf. v. Lippmann, Chemie der Zuckerarten, Vol. I, pp. 795-807. 296 Mary Davies Swartz, The best known member of this group is inulin, 1 closely associated with which are the four levulans described by Tanret; these seem to be intermediate products between inulin and levuiose, all having greater solubility than inulin, but less levo-rotatory power. The other carbohydrates mentioned are also more soluble than inulin, but have higher specific rotation. LEVULANASES IN THE VEGETABLE KINGDOM. Comparatively few studies have been made upon the action of enzymes on the levulans, and these have been for the most part lim- ited to inulin. Certain micro-organisms as B. Coli communis (295), Clostridium pastorianum (328), and several Schizomycetes, decom- pose inulin, but without any production of sugar. Yeast, according to Tanret (321) does not ordinarily ferment it, but Lindner (308) asserts that certain forms of top yeast change it readily. Levulin is fermented by yeast, according to Levy (307), and triticin, in the course of four or five days, according to Reidmeister (314); but it seems probable that the first changes are due to gradual hydrolysis on standing in water, or to other organisms. The effect of vegetable enzymes on these carbohydrates, as far as they have been studied, is shown in the following table: NAME OF LEVULAN. Inulin Levulin . . Graminin . Triticin. . Sinistrin . INVERTIN OF YEAST. - (8) + (D (2) MALT DIASTASE. -(3) -(4) + (5) -(6) TAKA DIASTASE. (3) INULASE OF ASPERGILLUS. + (7) + very slowly (4) (1) Levy (307) (2) Reidemeister (314) (3) Chittenden (292) (4) Harlay (301) (5) Reidemeister (314) (6) Schmiedeberg (318) (7) Dean (293) and others (8) Komanos (303) Discovery of the best known ferment for any levulan is due to Green (300) who, in 1888, extracted such an enzyme from the tubers of the Jerusalem artichoke (Helianthus tuberosus), and named it "in- x For description and early literature see Kiliani (302) and Dean (294). Nutrition I nvestigations. 297 ulase. " Subsequently, Bourquelot (289) found inulase in Asper- gillus niger and Penicillium glaucum; and Chevastelon (291) showed that this enzyme would hydrolyze the inulin of the monoctyledons. Dean (293) has studied the properties of inulase exhaustively, and shown that in Aspergillus and Penicillium it exists only as an endo- enzyme. Went (327) has found inulase also in Monilia sitophila and other Amylomyces. LEVULANASES IN ANIMALS. The first instance of an inulase in an animal organism has been cited by Strauss (319). In 1908, he reported studies on the enzymes of seven species of Lepidoptera and Diptera, during their various stages of development (Euproctis chrysorrhea, Ocneria disparata, Bom- byneustria, Bombyx mori, Galleria melonella, Hyponomenta, Calliophera vomitoria), but found inulase present only in the eating larvae of Bombyx mori and Hyponomenta. No inulase was present in the larvae of these species after they had ceased eating, nor in the pupae and imagines. The results of Kobert (304) in 1903, with extracts of May beetles, cross spiders, scorpions, cockroaches, ascarides, pupae of pine spiders, and house flies, were entirely negative; so also have been the experi- ments in vitro with digestive juices of higher animals, as shown by table on following page. DIGESTION AND UTILIZATION BY ANIMALS. Inulin is hydrolyzed by very dilute acid (0.05-0.2 per cent at 40° C. according to Chittenden), so that its more or less complete inversion by the gastric juice is possible, and has led many to believe that in spite of the negative results obtained with amylolytic enzymes shown above, it might be converted into levulose, and as such be read- ily utilized by the animal organism. It has therefore frequently been recommended for the diet of diabetics, who show a special tol- erance for levulose; in fact, simply because inulin did not reappear in the urine as sugar, when fed to diabetics, its utilization has been as- sumed by many, no account being taken of its possible reappearance in the faeces. This reappearance is well demonstrated in an experi- ment of Sandmeyer (317) in which, after feeding 80 grams of inulin to a diabetic dog, over 46 grams were recovered in the faeces. 298 Mary Dames Swartz, AUTHORITY. DATE. SOURCE OF ENZYME. KIND OF LEVULAN. RESULT. Komanos (303) 1875 Saliva Inulin — Pancreatic juice Inulin — Schmiedeberg (318; .... 1879 Saliva Sinistrin Chittenden (292) 1898 Saliva Inulin — Pancreatic juice Inulin — Bierry and Portier(288) 1900 Macerated pancreas and intestines of dog, rabbit and seal Inulin • Bierry andPortier (288) . 1 nnn 1900 Macerated pancreas and in- testines of dogs, rabbits; fed three months on arti- chokes to induce formation oi an inulase* Inulin TT„ Jo,, /9Al\ 1 nm 19U1 Saliva Lrraminin Bierry (28bj 1905 Pancreatic juice of dog Inulin Pancreatic juice of dog + macerated intestines of dogs and rabbits t i; Inulin 1910 Pancreatic juice of dog from pancreatic fistula after in- jection of secretin Inulin Same pancreatic juice added to macerated intestines of dog and rabbit, in slightly acid, slightly alkaline and neutral solutions Inulin Hepato-pancreatic juice of Helix pomatia Inulin Levulose Enzyme prepared from he- pato-pancreatic juice of Helix pomatia Inulin Levulose Weinland (326) 1905 Extract of small intestine of dog Inulin *Cf. Richaud, (326). Attempts to induce glycogen formation in rabbits have not justi- fied the hopes of the dieto-therapists in regard to inulin as a food for diabetics. The earlier experiments were either negative or open to criticism on account of faulty technique. The more discriminating work of recent investigators (Miura [313]; and Mendel and Naka- seko [312]), has shown that little glycogen is formed from inulin, even under the most favorable circumstances. A brief survey of the expe- riments in this field is given in the following table: «_ be o an d a ure less ding inu ho > en en a a .5 ° d d 5 g I 1 io a, > LO tn O S CO o LO lo io O rt< OTf( © © © © O O a a u J* 3 u u V '.s Js LO CO co d d d gms. ions gms. ions gms. ions o LO o to o LO T-H 2 2 a - 5 «> cfl O LO d 'C (M o 300 Mary Dames Swartz, Excluding the experiment of Luchsinger (310) which was estimated on a very low specific rotation for glycogen, only four out of the 17 experiments before Miura's (313) are positive, and in these the gly- cogen was estimated without purification, so that the figures are prob- ably high. In more reliable experiments of Miura (313), and Mendel and Nakaseko (312), the glycogen content of the rabbits' livers was as low or lower than the starvation maximum for the rabbit, as esti- mated by Kulz (309), so that glycogen formation from inulin must be regarded as doubtful, or very slight. When inulin is introduced parenterally into the organism, there is no inversion or utilization, as shown by the experiments of Mendel and Mitchell (311). They injected warm solutions into the peritoneal cavity, and determining the output of inulin in the urine (which was sugar-free) by calculations from the specific rotation, recovered 2.2 grams of 2.8 grams injected. In an experiment in which the sugar- free urine was hydrolyzed, and the output of inulin calculated from the amount of reducing sugar obtained, 1.43 grams were recovered out of 2.2 grams injected. Weinland (326) after subcutaneous injec- tions of inulin into dogs, continued for a month, found no inulase produced thereby. On the other hand, Saiki (316) succeeded in pro- ducing a definite anti-inulase in rabbit's serum. We see, therefore, that inulin is not attacked by animal enzymes, as far as investigated, with the possible exception of two species of inver- tebrates; and by a very few vegetable enzymes. It appears to a con- siderable extent in the faeces after being fed per os in spite of the abil- ity of the gastric juice to hydrolyze it. In spite of the accepted fact that levulose is capable of being directly utilized by the animal body there is no conclusive evidence of glycogen formation from inulin. Whether other levulans resemble this hemicellulose in these respects has not been investigated. Occurrence and Nature of Dextrans. In the higher plants, starch, dextrin, and cellulose occur almost to the exclusion of other anhydrides of dextrose. A few hemicelluloses yielding dextrose have been described, however, such as "a-amylam" (soluble in hot water) and "/3-amylam" (soluble in cold water), dis- covered by O 'Sullivan (343) in wheat, rye and barley; those in the mucilaginous extracts of flax-seed and rleabane, described by Bauer (329) and Rothenfusser (345); and that in Colocasia antiquorum, described by Yoshimure (352). Nutrition Investigations. 301 Even in the lower plants, dextrans do not occur to any great extent. They have been observed in bacteria (338), yeast (339), fungi (350), and liverworts (337), but occur most abundantly in lichens and algae 1 the lichens, as already stated, yielding dextrans to which the names lichenin, isolichenin, usnin, everniin, etc., have been given. Especial interest is attached to the dextrans of Cetraria islandica (lichenin and isolichenin) which together form 80-90 per cent of the total carbohy- drates of this lichen, because of its abundance in northern lands and its use there as a foodstuff; hence these carbohydrates have received more attention from chemical investigators than any other dextrans. Ever since Berzelius (333), in 1808, studied the hot water extract of Cetraria islandica, and called the carbohydrate mixture so extracted "moss-starch," on account of its giving a blue color with iodine, the idea that it is, like starch, a valuable nutrient, has prevailed. That this hot water extract contained two carbohydrates, one soluble in cold water (isolichenin) and the other in hot, was demonstrated by Berg (332) in 1873, who also showed that the blue coloration with iodine was a property of isolichenin, but not of lichenin. Lichenin was first found to yield dextrose by Klason, in 1886 (337). The next year the two carbohydrates were more fully investigated by Honig and St. Schubert (336), who have carefully reviewed the earlier literature on this subject. That lichenin and isolichenin yield dextrose on hydroly- sis, has been verified by Karl Miiller (341), Brown (334), and Ulander (348), who have also shown the hemicelluloses of the water-insoluble part to consist of dextran, mannan, and galactan, with a small amount of pentosan. Escombe's (335) observation that lichenin yields gal- actose has proved to be incorrect. DEXTRANASES IN THE VEGETABLE KINGDOM. Honig and St. Schubert (336) subjected isolichenin to the action of malt diastase, and observed a rapid disappearance of the iodine color reaction, and the formation of a dextrin-like substance precipitable by alcohol — a result verified by Brown (334) in 1898. Berg (332) treated lichenin with malt diastase but was unable to observe any change produced in it; his results also have been verified by Brown (334). The only experiments in which sugar has been obtained from lichenin by the action of vegetable enzymes have been carried out by Saiki (346) with "Taka" diastase from Eurotium oryzae and inulase from Aspergillus niger. *Cf. p. 255, also v. Lippmann, Chemie der Zuckerarten, Vol. I, pp. 215-220. 302 Mary Davies Swartz, DEXTRANASES IN THE ANIMAL KINGDOM. Attempts to hydrolzye lichenin by animal enzymes have been uni- formly unsuccessful. The most exhaustive researches were made by Nilson (342), in 1893, partly with pure lichenin and partly with the powdered lichen itself. Digestions were made with human gastric juice for 24 hours, in neutral, acid, and alkaline solutions; with pancreatic extracts; with gastric juice followed by pancreatic extract; and with these same extracts, using preparations treated with J per cent sodium hydroxide solution for 24 hours before the digestion. Nilson signifi- cantly remarks that this resistance to sugar-forming enzymes is worthy of note, inasmuch as certain lichens have been considered valuable food for man, and that it is hard to understand how reindeer utilize the car- bohydrates of lichens. His negative results with animal enzymes have been substantiated by Brown (334) — who found digestion with 0.2 per cent to 0.4 per cent hydrochloric acid equally ineffective — and by Saiki (346). Torup (347) reports that the dextran isolated from La- minaria digitata by Krefting is not hydrolyzed by ptyalin, amylopsin or diastase. DIGESTION AND UTILIZATION IN ANIMALS AND MAN. Interest in the digestibility of lichenin arises, not only from its use in the diet of normal individuals, but in the possibility of its furnish- ing a substitute for other carbohydrates in the diet of diabetics. After this idea was set forth by Kiilz (305), in 1874, it is not surprising to find, in 1879, the Italian physician Cantani, 1 and the Norwegian physician Bugge 2 reporting experiments in the use of Cetraria bread for diabetics. Without any further observations than that the sugar in the urine was not increased, the idea prevailed which Voit expres- sed in his monograph on Nutrition in 1881 (348) and Poulsson repeated in 1906 (344), that in some way or other, the "moss-starch," or lichenin, was changed into sugar in the alimentary tract, and served as a true nutrient. Poulsson undertook to verify this by feeding experi- ments with two diabetics, but as Mendel (340) has taken pains to point out, the results obtained, namely that 45-49 per cent of the car- bohydrates of the Cetraria bread eaten were utilized, are unreliable, since the carbohydrates of the faeces were calculated by difference, instead of being determined directly by analysis. iCited by Poulsson (344). 2 Bugge, Forhandlingar i det medicinske selskap, Kristiania, 1879, p. 179 (cited by Poulsson). Nutrition Investiga lions. 303 The few feeding experiments made with animals do not sustain the claims made for the value of Cetraria as a foodstuff. Brown (334) found only 1.25-0.7 per cent glycogen in the livers of rabbits after Cetraria feeding, but these results are not very satisfactory, since the rabbits would not eat it very well. An old experiment by von Mering (351), in which 16 grams lichenin were fed to each of two rabbits, shows 0.56-0.63 grams of glycogen in the liver, but Miura (313) has pointed out that his glycogen estimates were probably too high. Saiki (346) fed Cetraria extract, containing 2 per cent dry matter, in por- tions of 292 cc. and 300 cc. on two successive days, to a meat-fed dog. The faeces of the feeding period were marked off at the beginning of the Cetraria diet by fine quartz, and at the end by cork. Their com- position is shown in the following table: DIET WEIGHT AIR DRY, GRAMS. CARBOHYDRATE. AS DEXTROSE. Meat 2 days 10 5.8 0.68 Meat + Cetraria extract . . 2 days 15* 25.8 3.90 Meat 2 days 5* 24.5 1.20 Meat 2 days 6 3.2 0.19 * Faeces of Cetraria Period. The Cetraria extract contained 6.3 grams carbohydrate estimated as dextrose, the faeces 5.1 grams. Feeding experiments on man, in which the intake and output of carbohydrate have been carefully determined by direct analysis of the carbohydrate as dextrose, have recently been conducted in Pro- fessor Mendel's laboratory. The data have not yet been published in detail, but from a preliminary description given by Mendel (340) is taken the following report of one experiment* : FAECES. Weight Air Dry. CARBOHYDRATE. As Dectrose. CETRARIA FED. Grams. Grams. Per cent. I. Fore period = 3 days 35 2.1 1 146 38.0 56 80 g. = 56g. as dextrose II. Fore period = 2 days 68 6 4 Fore period = daily 34 6 2 Cetraria period = 1 day 53 24 13 20g. = 14|g as dextrose After period = 2 days 29 6 2 * From unpublished experiments by Dr. V. C. Meyers, Sheffield Laboratory of Physlologi- ca 1 Chemistry. 304 Mary Davits Swartz, In this experiment, the Cetraria islandica was carefully washed, extracted with a dilute solution of potassium carbonate, to remove the bitter principle; again thoroughly washed, dried and ground to a powder. This preparation contained 72.5 per cent carbohydrate as dextrose. The carbohydrates of the diet, throughout the experiment, were limited to fine white bread and zwieback, forms in which they are utilized in man to 98 per cent. The faeces were hydrolized with dilute acid, and the carbohydrates determined as dextrose by Allihn's gravi- metric method. It is evident that nearly all of the Cetraria carbo- hydrate escaped digestion and was recovered in the faeces. Through the kindness of Professor Mendel, the protocol of a similar experiment, by Mr. S. W. MacArthur, is also reproduced, in which the technique was practically the same as described for Dr. Myers's experiment. PERIODS. DIET. COMPOSITION OF THE FAECES. Cellulose-Free. Weight Weight Moist. Air Dried. Dextrose. Dextrose. Fore = 3 days .... Mid = 3 days .... After = 3 days .... Meat, etc. Meat + Cetraria* Meat, etc. Grams. Grams. 281 1 90.0 542 ! 149.0 284 | 87.5 Per cent. 4.4 27.6 4.9 Grams. 3.96 34.5* 4.2 * Amount Cetraria eaten = 47 grams, which would be equivalent to 34.1 grams of dextrose in faeces. It is evident that the results of this experiment simply confirm those of Dr. Myers, and demonstrate that uncooked Cetraria, although taken in a form as favorable as possible for its digestion, is scarcely affected by its passage through the alimentary canal, and must be classed among the indigestible carbohydrates. Very desirable expe- riments on the digestibility of the peculiar carbohydrate of Cetraria — lichenin — are also being conducted, which may throw new light on the digestibility of the dextrans, but at present we certainly have no grounds for assuming that this group of hemicelluloses deserves to be classed with the true nutrients; all experiments show that they are not attacked by animal enzymes, and are recovered unchanged in the faeces after feeding. In conclusion, attention may be called to certain data from Japan- ese dietary studies, given by Oshima (15), as to the digestibility of Nutrition Investigations. 305 some dried marine algae, which have not been mentioned in connec- tion with the different classes of hemicelluloses. The coefficient of digestibility for each species studied is given in the following table: ALGAE DRIED. OTHER SUBSTANCES IN DIET. COEFFICIENT OF DIGESTIBILITY (Carbohydrates includ- ing crude fiber). Ecklonia bicyclis Shoyu* and sugar 36.2 Laminaria sp Shoyu 75.2 Laminaria sp Shoyu and cleaned rice 55.0 Ulopteryx pinnatifida Cleaned rice, shoyu, sugar 72.3 Average 67 . 7 * Soy-bean sauce. III. EXPERIMENTAL PART. Introduction. The foregoing review has emphasized the limits of our knowledge, both in regard to the chemical composition of marine algae, and their fate in the alimentary tract of men and animals, as determined by actual measurement of intake and output, and as explained by the ac- tion of bacteria and enzymes in vitro. Ten species of marine algae have, therefore, been made the basis of the present investigations. Eight of them were Hawaiian Limu, obtained, as already stated, through the kindness of Miss Minnie Reed, Science teacher in the Ka- mehameha Boys' School, Honolulu. They were dried in the sun, with the salt water adhering to them, before shipping to America. The other two (dulse and Irish moss) were easily obtained in our Eastern markets. That the carbohydrates of algae are chiefly hemicelluloses, is indi- cated by the analyses which have already been made; that in many species, these are to a great extent water-soluble, is also well known. In as much as such soluble forms are thus particularly well adapted for nutrition investigation on account of their freedom from all in- crusting substances, which end to interfere with digestion, the present studies have been confined as far as possible to them. Since it was desirable to study the different groups of hemicelluloses, and man- nans and levulans were not found in the seaweeds in sufficient quanti- ties for metabolism experiments, these were obtained from other sources; a mannan from salep, and a levulan (sinistrin) from squills (Scilla maritima). Other investigators in this laboratory are working on a dextran which would naturally be included here, namely lichenin from Cetra- ria islandica; consequently no experimental studies on this group of hemicelluloses have been made. In considering any classifications of these materials, it must be borne in mind that most of these carbo- hydrates are more or less complex in nature, and can be grouped only with reference to what appears to be the chief constituent in any given case. The following list comprises al the species examined, arranged upon this plan: 306 Nutrition Invest igations . 307 I. The Pentosans: Dulse (Rhydomenia palmata), Limu Lipoa (Haliseris pardalis) , Limu Eleele (Enteromorpha intestinalis) , Limu Pahapaha (Ulva lactuca laciniata and Ulva fas data). II. The Galactans: Irish Moss (Chondrus crispus), Limu Manauea (Gracilaria coronopifolia), Limu Huna (Hypnea nidifica), Limu Akiaki (Ahnfeldtia concinna), Limu Uaualoli (Gymnogongrus vermicularis americana and Gymnogongrus disciplinalis) , Limu Kohu (Asparagospis sanfordiana), Slippery Elm (Ulmus fulva). III. The Mannans: Salep (Species of Orchis and Euiophia). IV. The Levulans: Sinistrin (Urginea or S cilia maritima). The primary object of these investigations has been to determine the fate of these substances in the alimentary canal of man, since they are all used as foodstuffs except sinistrin, and are all representative of a large class of materials so employed. The experiments con- ducted have been Chemical, Bacteriological and Physiological in character, and each of these phases will be taken up separately in turn in the following pages. Chemical Investigations. The aim of the experiments was to isolate, identify, and pre- pare for bacteriological and physiological experiments, any water- soluble carbohydrates present in sufficient amount in the materials under consideration; and to determine such of their properties as would facilitate their detection, isolation, and quantitative estimation in these experiments. GENERAL METHODS. All the seaweeds, with the exception of Irish moss, were washed re- peatedly in cold tap water, to remove salt, sand, and other foreign substances, and for convenience, dried by spreading in thin layers 308 Mary Davies Swartz, over steam radiators. The Irish moss, being comparatively free from salt, etc., and largely soluble in pure water, was quickly washed once, and extracted immediately. All hydrolyses of carbohydrates were made with 2 per cent hydro- chloric acid, by boiling with a reflux condenser over a free flame. After cooling, the acid was neutralized with potassium hydroxide, using phenolphthalein as an indicator, when the solutions were suf- ficiently light in color; in other cases, litmus paper was employed. When the products of hydrolysis served to determine the nature of the carbohydrates, they were evaporated on a water bath nearly to dryness, the residues extracted with hot 95 per cent alcohol the alcohol removed from the filtered solution by evaporation, the residues fre- quently taken up in a little water and decolorized with charcoal, con- centrated, and again extracted with absolute alcohol. All qualitative tests for reducing sugar were made with Fehling's solution; all quantitative tests by Allihn's gravimetric method for dextrose, the results being calculated as dextrose in view of the com- plex nature of most of the products, and the advantage of uniformity. On all preparations used for feeding experiments, the length of time in which the maximum yield of sugar could be obtained has been de- termined, as a criterion in analyses of faeces. Five grams of dry air material were hydrolyzed in 500 cc. of 2 per cent hydrochloric acid, 50 cc. being removed at intervals of one or more hours, cooled, neu- tralized, made up to 100 cc. and reducing power determined as dex- trose by Allihn's gravimetric method. Tests for the presence of fermenting sugars have been made in fermentation tubes with fresh compressed yeast, using as controls solutions of the substance to be tested, without yeast, and dextrose solutions with yeast. All carbohydrate solutions for polariscopic examination have been clarified by addition of an equal volume of alumina cream. Qualitative tests for pentosans have been made by boiling the sub- stance to be tested in a small Erlenmyer flask with 12 per cent hydro- chloric acid and testing for furfurol with anilin-acetate paper. Quantitative tests for pentosans have been made by the furfurol- phloroglucin method. 1 Tests for galactans or galactose have been made by oxidation with nitric acid to mucic acid, and the mucic acid identified by its melting point (212° C.-215° C). l Described in "Official and Provisional Methods of Analysis," United States Department of Agriculture, Bureau of Chemistry, Bull. No. 107, 1907. Nutrition Investigations . 309 Qualitative tests for mannose have been made by Storer's (271) method. The products of hydrolysis, freed from the greater part of the salts, gums, etc., in the manner already described, were taken up in a little water, and portions of 1 cc. or 2 cc. placed in test tubes. The reagent for testing was freshly prepared by shaking together 1 cc. of phenylhydrazin, 2 cc. of glacial acetic acid, and 10 cc. of distilled water. 3-16 drops of this reagent were added to each of the test tubes, and after standing several hours at room tem- perature, they were examined for precipitates of mannose-hydrazone. These precipitates were examined under the microscope, because they usually contained considerable amorphous matter. The mannose- hydrazone itself does not come down as colorless rhombic plates at first, but as globules of greenish-yellow or brownish-yellow color, sometimes smooth and resembling large yeast cells in the way they c uster together, and at other times covered with blunt points or spines. When these globules were observed, the precipitate was care- fully washed with water, sometimes without removing from the test- tube, the last drops being taken up with filter paper, and then dissolved in warm diluted alcohol (3 parts of 95 per cent to 1 part water), which was not filtered, but decanted from the amorphous insoluble portion, and allowed to evaporate slowly to facilitate the formation of crys- tals. Unless these crystals could be obtained, the tests were consid- ered negative, although Storer has pointed out that they are sometimes difficult to obtain, even when true mannose-hydrazone balls are present. All quantitative determinations have been made in duplicate un- less otherwise stated. PENTOSAN PREPARATIONS. Dulse. A pure, water-soluble pentosan-preparation has been obtained from dulse (Rhodymenia palmata). After boiling in water, in an open vessel, with occasional stirring, for several hours, this dark, reddish- brown seaweed yielded a carbohydrate, non-mucilaginous in character, which could be precipitated from its solutions by alcohol. About 12 hours' boiling proved to be necessary for complete extractions. The hot, brown, watery extract was first filtered through gauze, and then through cotton, as it clogged up filter paper very quickly. This filtrate, concentrated to a syrup on a water bath, was poured while 310 Mary Dames Swartz, still warm into about three times its volume of acetone, which expe" rience showed to be a more satisfactory precipitant than alcohol* Most of the carbohydrate came down very soon, in large, flocculent, yellowish-white masses, but a portion remained in suspension as a fine white powder, which made filtration difficult. The bulk of the precipitate was therefore removed by filtering through three or four thicknesses of fine gauze, and the rest obtained by distilling off the ace- tone, concentrating the residue, and reprecipitating the carbohydrate in solution with acetone. This precipitate was very hydroscopic, and was therefore transferred immediately to 95 per cent alcohol. This was replaced by fresh alcohol after a few hours, and the whole boiled on a reflex condenser for half an hour. A yellowish, granular powder was thus obtained, which was filtered, washed with ether, and the ad- herent ether allowed to evaporate. It was then redissolved in a small volume of water, filtered hot through paper, on a jacketed funnel, reprecipitated with acetone, again put into 95 per cent alcohol, and finally into absolute alcohol, in which it was allowed to stand for several weeks. It was then filtered off, washed with ether, and dried in vacuo over sulphuric acid. The product was a cream-white powder, and apparently not at all hydroscopic. From about two kilograms of crude commercial dulse, approximately 75 grams of this material were obtained, and used subsequently for feeding experiments. An attempt made to remove the dark red coloring matter by extrac- tion with 1 per cent sodium carbonate, led to the discovery that this carbohydrate is readily extracted by dilute alkaline solutions. For preparations on a large scale, it was therefore found more satisfactory to use the following method, based on Salkowski's method (139, 140) of obtaining xylan and araban by precipitation with Fehling's solu- tion. This method could be applied exactly as described, but there was an evident tendency for the carbohydrate to dissolve in the Feh- ling's solution. The dulse was accordingly extracted with 1 per cent potassium hydroxide solution for 48 hours, with occasional stirring, the extract removed by a hand press, and the extraction with fresh alkali repeated for 24 hours. 1 These extracts were filtered through several thicknesses of gauze, and to this filtrate a solution of copper sulphate was added till the reaction was just neutral. A flocculent, bluish-green precipi- tate formed. Into this solution was stirred carefully the alkaline Rochelle salt-potassium hydroxide solution used for Fehling's solu- tion, until the precipitate clumped together in heavy granular masses. *A third extraction contained so little of the material that it was discarded. Nutrition Investigations. 311 This was easily filtered off through gauze, as much liquid as possible removed by pressure, and the precipitate washed quickly with a little water to remove the excess of alkali. The carbohydrate was freed from its copper compound just as described by Salkowski (140). The precipitate was placed in a mortar and rubbed to a cream with diluted hydrochloric acid (1 volume of water to 1 volume of concen- trated acid) the acid being added until all blue particles had disap- peared. It was then poured into 90 per cent alcohol, the precipitate filtered off upon plaited paper and washed with 50 per cent alcohol, replaced in 90 per cent alcohol acidified with hydrochloric acid, and allowed to stand several hours to dissolve out the copper. It was then filtered, dissolved in dilute potassium hydroxide, and the dark brown, muddy solution filtered through paper on a hot funnel, the carbohy- drate reprecipitated with acid alcohol, and redissolved and reprecipi- tated until free from copper. When it no longer came down readily in alcohol, acetone was substituted, in which it formed white fibrous masses resembling paper pulp. Washed with absolute alcohol and ether, and dried in vacuo over sulphuric acid, it became a cream- white powder. Both of these methods yielded a product readily soluble in cold water, forming a clear, limpid, amber-colored solution. It gave no color reaction with iodine, and contained no reducing substance. In Fehling's solution it formed a very flocculent white precipitate, was not precipitable by lead acetate, neutral or basic, in neutral solution, but formed a precipitate in alkaline solutions. A test for mucic acid gave negative results, but a strong furfurol reaction was obtained on boiling with hydrochloric acid, indicating the presence of pentosans. A 1-gram sample of material, prepared by the method first described, was tested quantitatively for pentosans. It contained 26.8 per cent moisture, and 2.48 per cent ash, and yielded 0.076 grams of phloro- glucid, from which the yield of pentosans, according to Krober's tables, 1 is calculated as 72 per cent. The phoroglucid precipitates were afterwards extracted with 95 per cent alcohol, according to Ellett and Tollen's 2 method for quantitative determination of methyl-fur- furol. The Gooch crucibles containing the precipitates were warmed 10 minutes to 60° C. with 15-20 cc. of alcohol, the extract filtered off, and the extraction repeated till the alcohol was colorless. The pre- cipitates were then dried at 100° C. and weighed. The loss of weight was 0.0047 grams or 6 per cent of the original precipitate. The dulse preparation therefore contained a small amount of methyl-pentosan ^eitschrift fur physiologische Chemie, XXXVI, appendix. 2 Berichte der deutschen chemischen Gesellschaft, Vol. 38, p. 492 (1905). 312 Mary Dames Swartz, The products of hydrolysis were tested for fermenting sugar, with negative results, but after heating with phenyl-hydrazin-hydrochloride and sodium acetate, an abundant yield of osazones was obtained. These crystallized out only on cooling, were pale yellow, soluble in hot water only with great difficulty, but very soluble in alcohol, acetone, or pyridin. After four or five recrystallizations from alcohol, they melted at 152° C. and this melting point remained constant after ten or twelve recrystallizations. However, there were very minute points at which melting seemed to occur about 140° C. Under the microscope, clusters of long needles were seen, each with a tuft of small tine needles springing from its very tip. Dissolved in glacial acetic acid, and examined in a 100 mm. tube, these osazones showed no rotation of polarized light. A very white sample of the dulse carbohydrate was used to deter- mine its specific rotation. It contained 7.1 per cent moisture and 1.68 per cent ash. Two determinations were made, one on a 0.6 per cent solution and the other on a 1.0 per cent solution for which the polariscope readings in a 200 mm. tube were respectively —0.90° and —1.52°. The specific rotation, calculated from these readings was therefore [a] D = —75.2° and —76.2°, or corrected for moisture and ash, [o] D = -82.4° and -83.6°, average, -83°. The rate of hydrolysis and maximum reducing power were deter- mined as follows: 5 grams of the material dissolved in 500 cc. of 2 per cent hydrochloric acid were boiled in the usual way. At the end of two hours, and at intervals of one hour thereafter, 50 cc. portions were removed, neutralized and made up to 100 cc, and the amount of reducing sugar present determined as dextrose. The following results were obtained: That the results vary greatly with the concentration, is shown by the fact that a 0.3 per cent solution boiled 5 hours yielded 67.1 per cent of sugar as dextrose. Having established the fact that this dulse preparation consists of pentosans, with the properties described, further investigations into the exact chemical nature of the carbohydrates composing it were not considered within the province of this work. TIME OF BOILING. SUGAR AS DEXTROSE. Hours. 2 3 4 5 Per cent. 87.2 87.2 89.4 89.5 Nutrition Investigations. 313 Hawaiian Seaweeds. Beside the dulse preparation, three seaweeds have been included in this group which yielded little or no soluble carbohydrates, namely, Limu Lipoa (Haliseris pardalis), Limu Eleele (Enter vmorpha intesti- nalis) and Limu Pahapaha (Ulva lactuca, etc.). Limu Lipoa. Limu Lipoa contained a small amount of non-muci- laginous carbohydrate, soluble in cold water as well as hot. It was precipitated by alcohol, in which it came down as a white fibrous mass. On hydrolysis, it yielded a dextro-rotatory fermenting sugar; a test with phenylhydrazin acetate for mannose was negative, as were tests for pentosans. The total amount of this carbohydrate was so small as to be almost negligible, as far as feeding experiments were concerned, hence the original washed material was used, after grinding to a powder in a coffee mill. It contained a very high percentage of inorganic matter because the thalli were so encrusted with calcareous substances, that it was impossible to remove them entirely by washing. This preparation gave a strong f urf urol test, and a single quantitative test for pentosans gave the following results: The sample, weighing 1 gram, contained 10.5 per cent moisture and 18.5 per cent ash. It yielded 0.161 grams of phloroglucid, which according to Krober's tables 1 is equivalent to 0.147 grams pentosans, or 14.7 per cent of the crude substance. Tests for starch and reducing sugar were negative. Only a minute quantity of mucic acid was obtained; a quantity too small to purify and determine the melting point. The products of hydrolysis showed slight fermentation, which was doubtless due to the mannan of the water-extract. A determination of the reducing power made in the same manner as already described, gave the results: Limu Eleele. Limu Eleele yielded no appreciable amount of water- soluble carbohydrate, even after boiling 3 or 4 hours. The dried TIME OF BOILING. SUGAR AS DEXTROSE. Hours. Per cent. Very little H 3 4 6 S 14.3 14.7 12.9 12.8 1 Zeitschrift fiir physiologische Chemie, XXXVI, appendix. 314 Mary Davies Swartz, seaweed was therefore simply finely ground for use in feeding experi- ments. It gave a strong furfurol test, but yielded a mere trace of mucic acid. Tests for starch and reducing sugar were negative. The products of hydrolysis contained no fermenting sugar. From this it was evident that the hemicelluloses were chiefly pentosans. Determination of the reducing power gave the following results: TIME OF BOILING. SUGAR AS DEXTROSE. Hours. Per cent. 2 16.8 3 16.9 4 18.1 5 16.8 Limu Pahapaha. Ulva lactuca is said by Rohmann (134) to con- tain a water-soluble methyl -pentosan, rhamnosan; but if this occurs in Limu Pahapaha, it must be in very small amount, as an extract of 50 grams of the dried seaweed, made by boiling 3 or 4 hours, gave very little residue on evaporation to dryness. For feeding experi- ments, the dry crude substance was simply ground to a powder. Like Limu Eleele, it gave a strong furfurol test, but yielded no mucic acid. Starch was present, but no reducing sugar. Fermentation with yeast was marked in 12 hours, probably due chiefly to the hy- drolysis of the starch. Determination of reducing power gave the following results: TIME OF BOILING. SUGAR AS DEXTROSE. Hours. Per cent. 2 28.8 4 31.8 GALACTAN PREPARATIONS. Irish Moss. The carbohydrates of Irish moss are, as already noted, readily soluble in cold water, after the salt has been removed from the sea- weed. By allowing the moss to stand for 24 hours in cold water (about 10 liters to 250 grams of dry substance), an almost colorless, semi- transparent, mucilaginous extract was obtained. By straining this off through gauze, and allowing it to stand over night, for minute particles of cellulose held in suspension to settle, a solution almost entirely free from insoluble material was obtained by decantation. N utrition Investigations. 315 This was considered sufficiently pure for feeding experiments, and was quickly dried by pouring into broad shallow dishes and placing over a steam radiator. It formed yellowish, translucent scales, which were easily removed, and finely ground. Subsequent extractions were made in a steam sterilizer, heating several hours at a time. Tests showed that the carbohydrate was not hydrolyzed by this repeated subjection to high temperature. The several extracts were first strained off through gauze and then filtered hot through cotton, to remove the cellulose particles. As these clogged even cotton filters very rapidly, it was found most satisfactory to let the extracts stand over night, decant off the supernatant fluid as far as possible, and filter in a water-jacketed funnel. Solutions containing over 1 per cent dry substance could not be filtered through paper. For experi- ments where a perfectly clear fluid was desired, a \ per cent solution was filtered hot through plaited paper, and then concentrated on a water bath to the desired strength. One per cent solutions formed a soft jelly on cooling; 2 per cent solutions, a firm jelly. Even when evaporated to a thick syrup, the carbohydrates of the Irish moss extract are not readily precipitated by comparatively large volumes of 95 per cent alcohol, but form a voluminous, transparent, gelatinous mass. This was found to be more or less characteristic of all the galactans examined. They could be brought down most satisfactorily by addition of sodium chloride to the extract before pouring it into the alcohol. In this way a white precipitate of fine fibers was obtained from the moss. The carbohydrate could also be precipitated by saturation with potassium acetate, and freed from inorganic salts by dialysis, according to the method described by Pohl (263). It could not be precipitated by Fehling's solution, nor by lead acetate in neutral solution. Owing to the opacity of its solutions, and to the fact that its gelat- inizing property made the use of very dilute solutions necessary, no satisfactory determination of its specific rotation could be obtained. A 0.5 per cent solution, clarified with alumina cream, and examined in a 200 mm. tube, showed a rotation of +0.34°, and other trials gave positive evidence that it was dextro-rotatory. The products ot hydro- lysis were also dextro-rotatory, and yielded osazones, which after one recrystallization from alcohol, had a melting point of 184°-185° C. The carbohydrate gave a red- violet color with iodine, and con- tained no reducing sugar. A faint furfurol test was obtained. Oxi- dation with nitric acid gave a rich yield of mucic acid. Since Hadike, 316 Mary Dames Swartz, Bauer and Tollens (185), and Miither (200) have already shown that Irish moss contains galactan, levulan, dextran and pentosan groups, these tests were simply verifications of some of their observations. Determination of the reducing power gave the following results: TIME OF BOILING. SUGAR AS DEXTROSE. Hours. Per cent. 2 45.6 3 48.6 4 45.8 I Hawaiian Seaweeds. Limu Manauea (Gracilaria cor onopif alia), LimuHuna (Hypnea nidifica), Limu Akiaki (Ahnfeldtia concinna), Limu Kohu (Asparagopsis sanfordiana), Limu Uaualoli (Gymnogongrus) . k These five seaweeds all contained soluble carbohydrates, which were extracted by boiling in water in an open vessel over a free flame for two hours or longer. Limu Manauea, Limu Huna, and Limu Akiaki, which consist largely of soluble gelatinizing hemicelluloses, yielded most of these on boiling two or three hours. The extracts were strained off through gauze, filtered hot through cotton, and dried in thin sheets as described for Irish moss. While the preparations were dark colored, and had a decided "sea" flavor, they were not unpleasant, and were used in feeding experiments without further purification. As already stated, the carbohydrates were not easily precipitated with alcohol unless a neutral salt (as sodium chloride) was present. Limu Kohu and Limu Uaualoli contained only a small proportion of soluble hemicelluloses, and this was obtained only after boiling 8 to 24 hours. The extracts were also much less gelatinous in charac- ter. The thalli of Limu Kohu are almost like wire when dry, and remain tough and hard even after many hours' boiling. The extracts of these two species were more readily precipitated by alcohol than the others, but the precipitation was greatly facilitated by adding sodium chloride. The carbohydrate of Limu Kohu was precipitated as a white cheese-like cake, floating on the surface, while that of Uaua- loli came down as a mass of coarse white fibers. These precipitates were transferred to absolute alcohol, and after standing several days, were filtered off, washed with ether and dried at 40°-50° C. The Nutrition Investigations. 317 Kohu preparation should have been dried in vacuo, for it proved to be slightly hydroscopic, and instead of remaining a fine white powder, became somewhat brownish. The Uaualoli preparation dried easily to a grayish white, light, fibrous mass. Tests for starch and reducing sugar were negative on all these substances. Tests for galactans and pentosans were positive in every case. Three-gram samples of the air-dry preparations of Limu Akiaki, Limu Uaualoli and Limu Kohu respectively yielded 0.53 grams, 0.92 grams and 0.64 grams of mucic acid, recrystallized once from ammonium carbonate. 1 The products of hydrolysis in no case contained fermenting sugars. It is evident therefore, that these five preparations from the foregoing Hawaiian seaweeds consisted chiefly of galactans^ accompanied by some pentosan-groups. From the frequency with which methyl-pentosans have been shown to occur in all seaweeds previously investigated, it is very likely that they occur in all these varieties and it would be desirable to make tests for methyl-pentosans. Determinations of the reducing power were made, as shown in the following table: SPECIES OE SEAWEED. SUGAR AS DEXTROSE. 1 Hour. 2 Hours. 3 Hours. 4 Hours. Per cent. Per cent. Per cent. Per cent. Limu Manauea 41.9 44.6 39.8 Limu Huna 43.6 58.4 55.6 30.8 Limu Akiaki 36.0 36.0 34.0 Slippery Elm. For the preparation of the carbohydrate which forms the mucilaginous extract of slippery elm bark, pieces of the latter were torn into narrow strips and allowed to stand over night in cold water, 2 and then the mucilage expressed by squeezing through gauze. This process was repeated until the bark became a mass of separate fibers. The mucilaginous principle swells in cold water to a transparent jelly, but is soluble only to a very limited extent. It was found impossible to filter it, even through gauze, and therefore, although it contained small particles from the disintegrated bark *For method cf. Bull. No. 107, p. 55, Bureau of Chemistry, United States Dept. of Agriculture. 2 It was found impossible to extract the mucilaginous principle in hot water. 318 Mary Dames Swartz, fibers, the carbohydrate was precipitated by pouring the thick slimy mass into about six times its volume of 95 per cent alcohol. After standing some hours, a transparent, gelatinous precipitate settled to the bottom, and was filtered off through several thicknesses of gauze. Dehydrated by means of absolute alcohol and ether, it formed a gray- ish-brown powder. This was found to be soluble in dilute alkali, and was subsequently purified by dissolving in 1 per cent potassium hy- droxide, filtering through cotton and reprecipitating with 95 per cent alcohol. The product was somewhat lighter in color than at first, but still far from white. It was soluble in hot Fehling's solution, but precipitable with lead acetate. It gave no color with iodine, although a small amount of starch was present in the original bark. Furfurol tests were faint showing only traces of pentosans, but the yield of mucic acid was large, 0.15 grams of mucic acid being obtained from ] gram of the air dry powder. The products of hydrolysis were dextro-rotatory and contained no fermenting sugars. Hence this preparation consisted chiefly of galactan. A MANNAN PREPARATION. Since none of the algae which form the basis of these studies yielded mannan, save Limu Lipoa, and that in amounts inadequate for the experiments proposed, this hemicellulose was obtained in soluble form from salep. Both the small, horny dried tubers and the grayish- white powder made from them, werj purchased from Schieffelein& Co., New York. A preparation of pure mannan was made in the following way: The tubers were soaked in cold water 24 hours, washed thoroughly and ground in a meat chopper. To this mass, cold water was added in large volume, and the whole allowed to stand over night, then the dissolved mannan filtered off through gauze. According to Hilger (254), the extract made in this way should contain no starch. But when the tubers are heated before drying, the starch is made soluble, and in this instance the cold water extract gave a blue color with iodine. 1 Hence subsequent extractions were made with hot water on a water bath, for several hours. The salep swells very much in water so that a very large portion was required to get the mannan all into 1 Salep tubers purchased since this work was done yielded only a trace of starch ;n the cold water extract. Nutrition I nvestigations. 319 solution. 1 The extracts, strained through cheese cloth, were digested 24 hours with malt diastase to free from starch, then concentrated to a thick syrup on a water bath, and poured into three times their volume of 95 per cent alcohol. A voluminous, flocculent, and some- what fibrous, snow-white precipitate formed, which was filtered off, pressed free from alcohol, redissolved in hot water, and reprecipitated. (This was done largely to free it from sugar produced by the diges- tion of the starch.) It was then transferred to absolute alcohol and allowed to stand three or four, days, after which it was washed with ether, and dried in a vacuum desiccator. A somewhat coarse white powder resulted, containing 6.94 per cent moisture and 0.74 per cent ash. 2 It swelled up very readily in water, but dissolved exceedingly slowly to a colorless, semi-transparent mucilaginous solution, which did not reduce Fehling's solution, and examined in the polariscope, after clarification with alumina cream, appeared optically inactive. However, on reprecipitating the carbohydrate with alcohol, and examining the alcoholic filtrate, sugar was found to be present in small amount. A solution absolutely sugar-free became optically active. A sample in which the sugar had been removed by fermen- tation with yeast, was used to determine the specific rotation. The following results were obtained: (1) A 2 per cent solution in a 200 mm. tube read —1.59°; applying corrections for moisture and ash, [a] D = —43.1°. (2) A sample containing in 100 cc. 0.5868 grams mannan dried to constant weight at 105° C. read —0.48°; corrected for 0.4 per cent ash, [a] D = —43.8°. According to Thamm (276), salep extract is inactive. In the above experiments, the levo-rotatory nature of the mannan was at first obscured by the presence of traces of reducing sugar formed by the hydrolysis of the starch, which could not be detected by testing directly by Fehling's solution. Thamm, however, in several ways carefully tested salep hydrolysis products for dextrose with negative results, so that the only way to account for these conflicting results seems to be to attribute it to difference in the specimens of Orchis which furnished the mannan. Salep-extract is readily precipitated by Fehling's solution in floccu- lent white masses. It is not precipitated by lead acetate in neutral solution (nor, according to Thamm [276], in solutions of other neutral salts), but is precipitated by basic lead acetate. A furfurol test was faintly positive, verifying the report of traces of pentosans by Tollens and Widtsoe (163), and also by Thamm (276). *15 liters of water to 100 grams salep powder, according to Thamm (276). 2 Thamm found 0.483 per cent. 320 Mary Dames Swartz, The products of hydrolysis were dextro-rotatory and contained sugar fermentable with yeast. A rich yield of mannose-hydrazone was obtained with phenyl-hydrazine acetate, melting on recrystalliza- tion at 188° C. According to Thamm (276), salep extract yields ex- clusively mannose on complete hydrolysis. Hydrolyzed for three hours, the reducing power of this mannan was 91.6 per cent. Determinations of ash, moisture, starch, and mannan were made on the salep obtained in the form of a powder. Starch and mannan were determined as follows: 1 gram of air dry powder was boiled in 250 cc. water, and after cooling to 37.5° C, the starch hydrolyzed with malt diastase, dialyzed sugar-free. The solution was then filtered, con- centrated to small volume, and the mannan precipitated with absolute alcohol. The precipitate was filtered off, dissolved in a little water and reprecipitated, to obtain any sugar retained in the first precipi- tation. The mannan was then dried at 100° C. and weighed. The filtrates were combined, freed from alcohol, hydrolyzed with 2 per cent hydrochloric acid 45 minutes to convert all the maltose to dextrose, and sugar determined by Allihn's method. The results of these analy- ses are shown in the following table: Per cent Per cent Moisture 0.77 Starch 26.4 Ash 8.9 Mannan 19.5 According to Dragendorf 1 the composition of Orchis tubers is as follows : Per cent Per cent Starch 27.3 Protein 4.9 Mucilage 48.1 Cellulose 2.4 Sugar 1.2 Thamm also reports a yield of 40-45 per cent mucilage from the salep powder used in his investigations. Hence the powder used in this the present experiment was for some reason very deficient in mannan. Its reducing power was as follows: TIME OF BOILING. SUGAR AS DEXTROSE. Hours. Per cent 2 74.2 3 75.8 5 75.8 iCited in the National Dispensatory (1884), also by Thamm (276). NiUr it ion InVi 'Stig a I ions. 321 A LEVULAN PREPARATION. Commercial Squills, consisting of the dried and broken leaves of the bulbs of Scilla maritima (or Urginea Scilla Stenh.) yield, as dis- covered by Schmiedeberg (318), the levulan sinistrin. They were finely ground in a coffee mill, and the sinistrin prepared according to Schmiedeberg's directions. To the dry powder sufficient water was added to make a thin cream, and then a saturated lead acetate solu- tion until further addition produced no precipitate. To the clear, straw-colored filtrate, freed from lead with hydrogen sulphide, was added freshly prepared milk of lime, with constant stirring, until a somewhat creamy consistency was produced. To facilitate the for- mation of sinistrin-calcium carbonate, this mixture was concentrated on the water bath for some time (as suggested by Reidemeister) [314]. The precipitate was then sucked dry on a Biichner funnel, washed thoroughly with cold water (being rubbed up in a mortar for the pur- pose), again sucked dry, rubbed to a cream with water, and treated with carbon dioxide until the fluid was no longer alkaline to litmus. After heating to facilitate the complete separation of the calcium carbonate, the sinistrin in solution was filtered off, a little oxalic acid carefully added to remove the last traces of lime, and the solution then decolorized with charcoal, and evaporated to a syrup at a temperature of about 40° C. From this solution the sinistrin was precipitated with 95 per cent alcohol, as a white gummy mass. Transferred to abso- lute alcohol, and allowed to stand 24-36 hours it became very tenacious, but on longer standing, with occasional stirring, it grew brittle, and finally crumbled to a coarse white powder, which was dried in a vacuum desiccator. This material was readily soluble in cold water. (According, to Schmiedeberg [318], even solutions of 20-30 per cent are not syrup-like.) It gave no color with iodine, did not reduce Fehling's solution, and was not precipitated by it. This preparation, at first, contained 13 per cent moisture and 0.76 per cent ash. De- termination of the specific rotation then gave the following results: A 2 per cent solution in a 200 mm. tube, read —1.32°; corrected for moisture and ash, [a] D = —38.2°. After longer standing (three months) over sulphuric acid, the moisture content was 4.8 per cent, and determination of specific rotation gave the following results: A 1 per cent solution in a 200 mm. tube, read —0.55°; corrected for moisture and ash, [a] D = —29.1°. Schmiedeberg (318) found the average for [a] D = —41.4°, and Reidemeister (314), [a] D = —34.6°. It is impossible to account for these differences. Reidemeister claims 322 Mary Dames Swartz, that the rotation increases on standing, but in these solutions there was no change in 48 hours, at room temperature. On hydrolysis, sinistrin yields a levo-rotatory, reducing sugar, fer- menting with yeast, Schmiedeberg (318) reports this as a mixture of levulose and an inactive sugar, but Reidemeister (314) declares that it is neither a mixture of levulose and an inactive sugar, nor of levu- lose and dextrose, in spite of the fact that he found for it |a] D = —88°, while for levulose, [a] D = —106°, a difference for which he is unable to account. SUMMARY. The composition of the preparations which have been described is best shown in the following table: NATURE OF CARBOHYDRATES PRESENT. SOURCE OF MATERIAL. Pentosans. Galactan. Mannan. Levulan. Dextran. Dulse (Rhodymenia Palmata) + Limu Lipoa {Haliseris Par- + Limu Eleele {Enter omorpha intestinalis) + Limu Pahapaha {Ulva lac- tuca, etc.) + (Starch) Irish Moss {Chondrus crispus) Trace + + + Limu Manauea {Gracilaria + + Limu Huna (Hypnea nidifica) + + Limu Akiaki {Ahnjeldtia con- cinna) + + Limu Uaualoli {Gymnogon- grus) + + Limu Kohu {Asparagopsis + + Slippery Elm (Ulmus) + Trace + Squills (JJrginea s cilia) [Sinis- trin] + The foregoing observations correspond with those of Konig and Bettels (8), in that the marine algae all yield pentosans, and fre- quently galactans. The gelatinizing principle in every case appears to be due to the galactan groups. No specific tests have been applied Nutrition Investigations. 323 for fructose, the polysaccharide of which also appears to be common in algae, but the absence of fermenting sugar in all the algae except Limu Lipoa, indicates that if present, it is in too small amount to be detected in the hydrolysis products of 5-10 grams of crude material. The reducing power has been determined on each substance used in feeding experiments; the results of all determinations are summarized in the following table: SUBSTANCE. SUGAR AS DEXTROSE AFTER BOILING. 1 Hour 2 Hours. 3 Hours 4 Hours. 5 Hours 6 Hours, 8 Hours. Per cent. Per cent. Percent. Dulse Limu Lipoa Limu Eleele Limu Pahapaha Irish Moss Limu Manauea Limu Huna Limu Akiaki Salep (Powder) Salep (Pure mannan) 87 2 87 3 14 3 16 8 16 9 28 8 45 6 48 G 41 9 44 6 58 4 55 6 36 34 74.2 75.8 91.6 Per cent, 89.4 14.7 18.1 31.8 45.8 39.8 30.6 Per cent. 89.5. 16.8 75.8 Per cent. 12.9 Per cent. 12.8 Bacteriological Investigations, introduction. It is an accepted fact that even cellulose, with its high powers of resistance, is to some extent decomposed in the alimentary tract by bacteria. It is therefore reasonable to expect that the less resistant hemicelluloses will also be attacked and decomposed by bacteria. The object of these experiments has been to throw some light on the problem as to what organisms are most likely to effect such a decom- position, and whether there is an appreciable production of sugar as a result of bacterial activity. The four classes of hemicelluloses under special investigation have been represented by the following sub- stances: Pentosans Dulse. Mannans Salep. ~ , J Irish Moss. Levulans Sinistrin. Galactans < _ . [ Limu Manauea. 324 Mary Davies Swartz, Both aerobic and anaerobic cultures have been made, in neutral, faintly alkaline, and faintly acid reaction, with solutions made from the carbohydrates alone, and with the addition of small amounts of such nutrients as beef extract or peptone to facilitate the growth of the organisms. Anaerobic cultures in test tubes have been made by the Wright method ; anaerobic cultures in Erlenmeyer flasks, by passing a stream of hydrogen through for half an hour, and then sealing hermetically. The aerobes which have been employed all occur in the human digestive tract. Both aerobic and anaerobic cultures from the faeces of human subjects have also been used, in conjunction with soil bacteria from street sweepings. Tests for the presence of reducing sugar have been made by pre- cipitating the carbohydrates in solution with absolute alcohol, evapor- ating the alcoholic extract to dryness, taking up the residue in 2 or 3 cc. of water, and boiling two minutes with Fehling's solution. Suitable controls have been used in all cases. TRIALS WITH PURE CULTURES OF AEROBES. One per cent solutions of the preparations from dulse, Irish moss and salep, neutral, acid, and alkaline in reaction, and consisting of, (1) pure carbohydrate: (2) carbohydrate plus J per cent beef extract and \ per cent sodium chloride; (3) carbohydrate plus 1 per cent peptone and i per cent sodium chloride, have been used as culture media. Five cc. portions of each of these solutions were placed in test-tubes with a pipette, and inoculated with the following organisms: B. Coli communis, B. Pyocyaneus, B. Prodigiosus, B. Proteus vulgaris, B. Pyogenes foetidus. To approximate the conditions in ordinary digestion of these car- bohydrates, they were incubated for three days at a temperature of 37.5° C. At the end of this time, nearly all gave evidence of some bacterial growth. Salep-peptone cultures of B. Pyocyaneus showed a brilliant green; salep solutions containing B. Pyogenes foetidus, and B. Coli in alkaline-beef extract media, had changed from trans- parent colorless solutions to an opaque white jelly insoluble in water. The carbohydrates were then precipitated with alcohol, and after standing several days were compared with controls similarly prepared, to see whether any change could be observed in the nature or amount of carbohydrate. The results were in all cases negative. These pre- cipitates were then transferred to small folded filter papers of uniform Nutrition Investigations. 325 weight, previously prepared. The alcoholic filtrates were tested for sugar; the precipitates were dried, and their weight compared with that of the control. It was thought that this rather crude method would show whether any considerable amount of the carbohydrate had disappeared. The results were so largely negative that weighings of every precipitate were not made. There seemed to be a slight loss of dulse, in some of the cultures of B. Proteus vulgaris, B. Pyogenes foetidus, and B. Coli communis, but repetition of these experiments allowing the organisms in question to grow two weeks, not only in dulse but also in salep media, did not justify any conclusion that an appreciable amount of carbohydrate had disappeared. All tests for reducing sugar were negative. Four per cent solutions of Irish moss, and two per cent solutions of limu manauea were then prepared, with reactions and additions of nutrient material as described in the first series of experiments. These formed firm jellies, which were used to study the possibility of lique- faction or gas formation. Stab cultures were made, and grown at a temperature of 25°-30° C. for one to three weeks. No liquefaction or gas formation was observed in any case. TRIALS WITH MIXTURES OF AEROBES. Mixtures of B. Pyocyaneus, B. Prodigiosus, B. Proteus vulgaris, and B. Pyogenes foetidus, were used, also mixtures of faecal and soil bacteria. These were first inoculated into nutrient bouillon, the former from pure cultures, the latter from human faeces and street sweepings, and incubated 24 hours. Five cc. portions of these cultures were then introduced into 50 cc. of neutral solutions of each of the different carbohydrates, in small Erlenmeyer flasks, and these cul- tures allowed to grow for four weeks at 37.5° C. At the end of this time, no marked change had taken place save in the salep culture of B. Pyocyaneus, B. Proteus vulgaris, B. Pyogenes foetidus and B. Prodigiosus. This had changed from a colorless, semi-transparent, slightly mucilaginous fluid, to a firm, white opaque jelly, insoluble in water, but readily soluble in dilute alkali; a phenomenon already observed with this carbohydrate in cultures of B. Coli communis and B. Pyogenes foetidus. No liquefaction had taken place with Irish moss nor limu manauea. The carbohydrates were then precipitated with alcohol, the alco- holic extracts tested for sugar, and the precipitates hydrolyzed by boiling with 2 per cent hydrochloric acid, neutralized, made up to a 326 Mary Davies Swartz, definite volume, and examined in a polariscope. The results of these experiments are shown in the following table. Mixtures of B. Pyo- cyaneus, B. Prodigiosus, B. Proteus vulgaris and B. Pyogenes foetidus are designated A, and mixtures of faecal and soil bacteria, B. REDUCTION ROTATION AFTER HYDROLYSIS. SUBSTANCE BACTERIAL OF CULTURE fehling's solution. Experiment. Control. Dulse B + 0.13° +0.20° A +0.20° B +0.27° +0.20° Limu Manauea .... A Not determined. Salep A Not determined. Salep B +0.17° +0.20° Sinistrin A -0.97° —0.97° The action of putrefactive organisms upon the dulse preparation was also studied, according to the method used by Slowtzoff (154) in the case of xylan. One hundred grams of chopped lean beef and 10 grams of sodium carbonate were added to 1 liter of water, and the mixture allowed to stand in a warm place for three days. Two hundred and fifty cc. were then removed for a control, and to the remainder 0.5 gram of dulse was added. This solution gave a strong pentosan reaction; the control was pentosan-free. The two solutions were put in a warm place, and tested daily for pentosans. After five days' digestion, the reaction of the dulse solution was very much fainter than at first, but it did not entirely disappear till the twelfth or thirteenth day. Slowtzoff found that xylan disappeared in nine or ten days, but his solution was kept at a temperature of 40° C, while these mixtures remained at a temperature of from 30° to 35° C, a condition less favorable for rapid decomposition. Solutions of Irish moss were digested with faecal mixtures in the following manner: Human faeces were rubbed to a mud with water. Ten cc. portions of this material were added to flasks containing 50 cc. of a 1 per cent "moss" solution, and allowed to digest in a warm place for 24 hours. A portion of water inoculated in the same way was used as a control. Small portions of these solutions were then evap- orated nearly to dryness, extracted with alcohol, and tested for reduc- ing sugar. The results were wholly negative. That limu manauea is not entirely resistant to the action of putre- fying organisms is shown by the following: A solution was made Nutrition Investigations. 327 up to contain 2 per cent of the air dry extract, 1 per cent peptone, \ per cent beef extract and \ per cent sodium chloride. This could be filtered through paper only on a hot, water-jacketed funnel, from which it dropped as a clear, amber-colored jelly. After standing unsterilized over night in a warm room, this was found to be entirely broken up by the formation of gas throughout the whole mass. The reaction, which had been neutral, was now acid to litmus. This material was placed in a flask and allowed to stand for two months, at the end of which time, the greater portion was liquefied, the former lumps of jelly being reduced to small particles distributed throughout the liquefied portion. Alcoholic extracts did not reduce Fehling's solution. A sterile preparation of the plain manauea extract in test tubes was inoculated with some of this material, but without produc- ing the same striking results. There were evidences of growth, but none of liquefaction or gas formation, in the course of two weeks. TRIALS WITH ANAEROBES. The action upon Irish moss of pure cultures of the powerful putre- factive organisms B. Putrificus, Bienstock, B. Maligni cedematis, and B. Anthracis symptomatici, was tried in the following way. A 4 per cent solution of the moss was prepared, which would not become liquefied at a temperature of 30°-35° C. From this material culture media were prepared, neutral, alkaline, and acid in reaction, using the solution plain, and with the addition of \ per cent beef extract and \ per cent salt, or 1 per cent peptone and \ per cent salt. Test tubes were inoculated from fresh, active cultures, and the organisms allowed to grow for one to three weeks, being examined at first daily, and later every three or four days, for liquefaction and gas formation. The results were negative in all cases, save that in the peptone media an occasional small bubble was seen, with cultures of the bacilli of malignant cedema and symptomatic anthrax. However, the same phenomena were observed in peptone-agar tubes used as controls. Mixtures of B. Anthracis symptomatici and B. Maligni cedematis were tried upon solutions of dulse, Irish moss, salep and sinistrin, in the following way: Small Erlenmeyer flasks containing 50 cc. of 1 per cent solutions of each of these carbohydrates, and 5 cc. of ordi- nary nutrient bouillon, were inoculated with fresh cultures of these organisms, rendered anaerobic, and incubated for four weeks at 37.5° C. On inspection, no change was apparent. The carbohydrates were removed, the alcoholic extracts examined for reducing sugar, and 328 Mary Davies Swartz, the carbohydrate residues hydrolyzed and examined in the polari- scope, as in similar trials with aerobes. The results are shown in the following table: REDUCTION OF ROTATION AFTER HYDROLYSIS. NAME OF SUBSTANCE. fehling's SOLUTION. Experiment. Control. Dulse Lost by accident Irish Moss + 0.24° + 0.20° Salep + + 0.13° + 0.20° Sinistrin + - 0.27° - 0.97° Mixtures of soil and faecal bacteria were also tried, the experiments being carried out just as described for mixtures of the bacilli of symp- tomatic anthrax and malignant cedema. The results are shown in the following table: NAME OF SUBSTANCE. reduction of febxing's solution. ROTATION AFTER HYDROLYSIS. Experiment. Control. Dulse Irish Moss Salep + + 0.13° + 0.20° + 0.03° + 0.20° + 0.20° + 0.20° DISCUSSION AND SUMMARY. It seems reasonable to expect, that if the hemicelluloses used in these trials were readily attacked by micro-organisms, there would have been some evidence of change in three days, if conditions for growth were favorable as regards reaction and temperature; but although the concentration of the solutions was moderate, the reaction varied, and temperature 37.5° C, results were negative, even in the cases where nutrients were added to facilitate bacterial growth. Apparently all of the material was recovered in unaltered condition, save in certain instances where salep underwent an insoluble modification. In trials where the cultures were allowed to grow from one to three weeks, no difference in the results could be detected, by the methods employed. In solid media there was no liquefaction and practically no gas formation, except in the case of the peptone-beef extract preparation of limu manauea, on exposure to the air. Nutr it ion Investigations . 329 Marked evidences of change were observed in one trial with a putre- factive mixture (on dulse), and in some of the four-week cultures. Irish moss was the most thoroughly investigated and proved the most resistant. In the long experiments (4 weeks) where the other carbohydrates suffered more or less change this one remained appar- ently unaltered. The results of this series are summarized in the following table: Irish Moss. CULTURES USED. Mixture of Pure Aerobes reduction of fehling's solution. ROTATION OF UNALTERED CAR- BOHYDRATE AFTER HYDROLYSIS. Irish Moss. Control. + 0.20° + 0.20° + 0.27° + 0.20° + 0.24° + 0.20° + 0.20° + 0.20° Mixture of Faecal and Soil Bacteria (aerobic) Mixture of Bacilli of Malignant Oedema and Symptomatic Anthrax . . Mixture of Faecal and Soil Bacteria (anaerobic) The single experiment with the galactan, limu manauea, under the same conditions, with the mixture of pure aerobes, gave similar results, but the fact that liquefaction occurred in the peptone-beef extract culture medium after exposure to the air, shows that general conclusions as to the behavior of galactans cannot be drawn from study of a single representation of the class. We have, however, further proof that the galactans are not easily decomposed by bacteria, in the fact that aqueous solutions of all the galactans included in the present series, could be left several days in the warm atmosphere of the laboratory without any apparent change taking place; and in the fact that agar-agar, so widely used in bacteriological laboratories on account of its indifference to bacterial action, is a member of the galactan group. It has been suggested 1 that extracts of other sea- weeds might prove good substitutes for agar-agar as culture media, if fully investigated. So far, the greatest objection to use of Irish moss in this way is that it tends to liquefy at body temperature; strong solutions (4 per cent) can, however, be kept fairly firm at a x Cf. Reed (18). 330 Mary Davies Swartz, temperature of 30° C. The extract of limu manauea is free from these objections, but extensive experiment is still necessary to demon- strate its powers of resistance. The soluble dulse pentosan is certainly decomposed not only by putrefactive organisms under the most favorable conditions (e.g., in meat mixtures), but by aerobes and anaerobes in solutions where the carbohydrate is the chief source of nutriment. The results of the four weeks' digestions are summarized in the following table: Dulse. CULTURES USED REDUCTION OP fehling's solution. ROTATION OF UNALTERED CARBONYDRATE AFTER HYDROLYSIS. Dulse. Control. Mixture of Faecal and Soil Bacteria (aerobic) +0.13° (Lost by Accident) +0.13° +0.20° +0.20° +0.20° Mixture of the Bacilli of Malignant Oedema and Symptomatic Anthrax. . Mixture of faecal and Soil Bacteria In the present studies, this pentosan stands second to the galactans in degree of resistance. Sawamura (267) thought that he observed a slight hydrolysis o: mannan by B. Prodigiosus, an observation which has not been verifiec in these experiments. No reducing substance was detected in the three-day cultures nor the four-weeks cultures, in which this organism was present. The opaque jelly, insoluble in water, formed from salep by the action of B. Coli communis, B. Prodigiosus, and mixed cultures containing these organisms, resembles an intermediary product o: the acid hydrolysis of salep-mannan described by Thamm (276) He isolated and examined two such products, one forming an opales- cent solution in water, the other insoluble, but passing over into the soluble form by treatment with dilute alkali; both were anhydrides of mannose. It seems reasonable to inquire whether this insoluble material produced by bacterial action may not be regarded as an early stage in the hydrolysis of the carbohydrate under consideration especially in view of the fact that in all the other four-week trials a very definite reduction of Fehling's solution was noted, corresponding Nutrition Investigal it ms. 331 in strength with the loss of unaltered carbohydrate, as shown in the following summary: Salep. CULTURES USED. ROTATION OF UNALTERED CARBO- REDUCTION OF j HYDRATE AFTER HYDROLYSIS. FEHLING 'S SOLUTION. Salep. Control. Mixture of Pure Aerobes . Mixture of Faecal and Soil Bacteria (aerobic) (Insoluble jelly) + Mixture of the Bacilli of Malignant Oedema and Symptomatic Anthrax. . Mixture of Faecal and Soil Bacteria (anaerobic) Not det ermined + 0.17° +0.20° + 0.13° +0.20° + 0.03° +0.20° These experiments give some grounds for expecting the hydrolysis of salep in the alimentary tract, through the action of bacteria. Two experiments with sinistrin gave the following results: Sinistrin. ROTATION OF UNALTERED CAR- REDUCTION OF BOHYDRATE AFTER HYDROLYSIS. CULTURES USED. FEHLING 'S SOLUTION. Sinistrin. Control. Mixture of Pure Aerobes - 0.97° -0.97° Mixture of Bacilli of Malignant Oedema and Symptomatic Anthrax + - 0.27° - 0.97° Sinistrin is therefore hydrolyzed by the anaerobic putrefactive organisms, but further experiments are necessary to determine how readily this change takes place. Physiological Investigations. In the physiological experiments, attempts have been made to answer the following questions: (1) To what extent are hemicelluloses digested by animal and vegetable enzymes? (2) Can they be ab- sorbed and utilized without intervention of the alimentary tract? 332 Mary Dames Swartz, (3) Do they reappear in the faeces after administration per os? The various experiments will accordingly be discussed in these three groups: (1) Trials with Enzymes; (2) Parenteral Trials; (3) Feeding Experiments. TRIALS WITH ENZYMES. Approximately 1 per cent solutions of the various hemicelluloses (with the exception of Limu Lipoa, which was finely ground and sus- pended in water), have been digested for 24 hours at 37.5° C. in the presence of toluene, with the following enzymes: (1) Filtered human saliva. (2) Malt diastase, dialyzed sugar-free. (3) "Taka" dias- tase (Eurotium oryzae). (4) Chloroform extract of pig's pancreas. (5) Fresh pancreatic juice of dogs. (6) Chloroform water extract of dog's intestines. (7) Glycerol extract of pig's stomach. Digestions have also been made with 0.2 per cent hydrochloric acid, to determine whether any of the action of the artificial gastric juice might be due to the acid present. The activity of the amylolytic enzymes has always been tested first with starch paste, and that of the gastric extract with fibrin. Boiled controls have been employed in every instance, and all trials have been made in duplicate. Tests for reducing sugar have been conducted in the following manner: At the end of 24 hours the solutions were evaporated to thick syrups on the water bath, to free from toluene and to concen- trate so that the undigested hemicelluloses could be readily precipi- tated by absolute alcohol. The alcoholic extracts were filtered off and evaporated to dryness; the residues were taken up in a few drops of water and tested for sugar with Fehling's solution. The results of all digestion trials are shown in the table on opposite page. PARENTERAL INJECTIONS. Methods and Technique. Small dogs were used for all injections, after a confinement in cages long enough to obtain samples of normal urine. The carbohydrates employed in these experiments were preparations of dulse, 1 Irish moss, 2 salep, 3 and sinistrin. 4 They were introduced subcutaneously , by means iCf. p. 303. 2 Cf. p. 308. 3 Cf. p. 312. C >o © © O0 to CM © i-i i-i CM OS i-h d CD O £ 00 O 00 CO OS i-H i—l M N CM CM CM CM CM H o <2 N CO Ol OO Tt< O CM CM a 3 bo 52 "c3 550 a g s 8 g + S 2 s 1-1 U 73 tn J2 s ^ So O t/i CO g 00 w S S p o M 5 CM S J U co .2 ^> to g I) a; bo O «h O "V ^ 8 + o co O co to g to 0) O >i O O *V ^ c to % | + . § 3 CJ 3 1> «3 4> S ^ I S I O0 U CO CO en en CO CM U 0> «5 £ tn >»>>>, ■d t3 i3 CM CM rfl 3£s cn cn tn >» >» >> T) T) CM CM ^ II II II < £ ^ LIMU RE- COVERED. Per cent. o to o 00 H CO LIMU FED (As Dextrose). Grams. CD CO tO tO O X ^ C5 CO © ▼■H CO 00 t>! r-i ,-H CO o CO t>- C5 CO CO i-l O CO 00 00 OS 00 >0 TH i-H O (N (M lO n >^ cj cd o3 T) T3 U CI bO o p 53 §■ Oh LIMU RECOVERED. Per cent. 66 LIMU FED (As Dex- trose). Grams. 1.0 of Faeces. pentosan (As Dextrose) . Grams. 1.0 3.8 2.1 Composition Per cent. 4.8 8.1 8.2 WEIGHT AIR DRY. Grams. 22.5 46.4 26.8 WEIGHT MOIST. Grams. 65.7 95.4 193.4 diet. Cellulose-free Same + 30 gms. Limu Paha- paha, boiled | hr. Same as Fore Period PERIOD. f Fore = 2 days I Mid =4 days* [ After = 2 days WEIGHT. SUBJECT. Z (Woman) d 00 SE- RIES. < e s- e (X, .°° ?! y. ' — i a SE o »o OS »o © tO CO lO 00 CO OS 00 O CM co ^" TfH 00 CM T— 1 O a as -g O . 4) 60 S u o o O U) g CO h|n ^ « S J +^ 3 tf -s | | | 8 days days days (M II II CM II f Fore Mid k After CM T— 1 Dog OS 348 Mary Dames Swartz, The coefficients of digestibility of the pentosan preparations, as determined in the usual way from the preceding experiments, are set forth in the following table: SERIES A. PENTOSAN. COEFFICIENT OF DIGESTIBILITY. EXPERIMENT NO. For the Dog. For Man. 1 Dulse 80 2 Dulse 66 3 Dulse 100 4 Dulse 100 5 Limu Eleele 50 6 Limu Eleele 20 7 Limu Eleele 69 8 Limu Pahapaha 34 9 Limu Lipoa 16 It is evident from these figures, that pentosans in soluble form dis- appear from the alimentary tract of dogs to a very considerable extent (average 73 percent), and that small quantities, ingested by man, do not reappear in the faeces. The insoluble limu preparations appear much more indigestible, an average of 28 per cent being digested by dogs, and 51 per cent by man. It must be borne in mind, in interpreting the results of these metabo- lism experiments, that they are at best only approximate. The dif- ficulty of strict separation of the faeces, the fact that the human sub- jects were not kept on a uniform weighed diet, and the errors unavoid- ably introduced by determining many different kinds of sugar as dex- trose, make all of the figures given as "coefficients of digestibility," in this and succeeding sections, comparative rather than absolute. The Digestibility of Galactans. In these experiments, preparations of the water extracts of Irish moss, Limu Manauea, Limu Huna and Limu Akiaki have been fed, without any disagreeable symptoms. The results are given in the tables which follow: 7 2 2 o o 2«! 00 OS © CO CO CO CO r-H 11 bO I— I O C/3 . s i uo bo g c/3 co © W> g 4 V- ' © §5 £ e I 3 P CD CD o o ^ r-i CM CO ^ 00 O) ri H CO o o 00 00 O iO ^ CM i—l tO CD i— I CO CO 1-1 CO TjH CD o o C2 to iO (M i-l i-H CM O CM © bo g ^ J u S "2 o o ri in a f B " & £ bC g a o &o 3* 8 tfT3 c3 9 6 13 O en CO g _T bfi ri 3 ^ £ + h o3 »3 g S H Ctf U. w < hi U co o .2 1 e ri # 3 * £ o §3 §1 c3 bC cc! 2 ai g =3 ri r «K oo U + ri + a § s 3 c 3 00 00 M 2 w m ft CO cj cci CM CM 0) 0) £ o co co ^ b b cj Co Co XI ^ XI CM CM tO < £ S £ £ £ Tl x) CM CM to < S CO T3 CM c A pq be o pq pq CD CD 00 h N (K CO © © 00 O ^ h N J>- CO ' LQ lO tO CO tJH CO H IN H S3 K a .§ o CO m w a © bC g ^ « 3 a + ^ ■si! s (U tJ ?i c ^ i-5 U co D-4 03 a £ CO a a bO g O Ml 3 -g 1 § ^ i_q CJ co 3 K a a a + a M £ © J + .2 3 co O co a3 CO » a id "d CO pq 352 Mary Dames Swartz, The coefficients of digestibility of the galactan preparations are given in the following table: SERIES B. EXPERIMENT NO. GALACTAN. COEFFICIENT OI For the Dog. DIGESTIBILITY. For Man. Per cent. Per cent. 1 Irish Moss 46 2 XI loll itlUjo 3 Irish Mioss H 4 Alloil 5 Limu Manauea 55 6 Limu Manauea 12 7 Limu Manauea 30 8 Limu Manauea 30 (av.) 9 Limu Huna 30 (20 gms. fed) 10 Limu Huna 83 (7 gms. fed) 11 Limu Huna 10 12 Limu Akiaki 60 Although these preparations were administered in small quanti- ties, under the most favorable conditions for digestion in, the only instance where the utilization in any degree approaches that of starch (Limu Huna) , the quantity fed (7 grams) was so small that this exper- iment can hardly be taken as a criterion of digestibility. Exclusive of this experiment, the average of five trials with dogs is 32 per cent, while that of six trials with human subjects is 23 per cent. In both cases, the averages are lower than that of Lohrisch (194) for " soluble agar," 50 per cent. Where the quantity of galactan fed was 10 or more grams, the in- fluence on the character of the faeces was usually noticeable. The increase in bulk, after ingestion of 45 grams of Irish moss, is well illus- trated in a photograph of the dried and ground faeces of the dogs used in experiments 1 and 2: 1 A represents the fore-period (3 days), B the mid-period, during which 15 grams of moss were ingested daily (3 days), and C the after-period (3 days). The separation of the faeces at the beginning of experi- ment 1 (on the right) was not very satisfactory. The dog had pre- viously been fed bone-ash, and the marked faeces were undoubtedly contaminated with this, so that they appear unusually bulky. Exper- iment 2 is typical of the results obtained in most of the experiments 1 Cf. p. 343. Nutrition Investigations. 353 with human subjects. In these, the undigested hemicelluloses gave frequently a peculiar, wax-like consistency, especially noticeable with imu Huna in the experiment recorded, 1 and in another not reported, ecause the faeces for part of the time were lost. In the experiment with Limu Akiaki (No. 12), 1 the galactan was excreted after the first day's feeding, in a tough mass almost impossible to break up with a EXP. 2 The Influence of Irish Moss upon the Mass of the Faeces. A. Fore Period: 3 Days on a Cellulose-free Diet. B. Mid Period: 3 Days on a Cellulose-free Diet to Which 15 grams of Irish Moss were Added Daily. C. After Period: 3 Days of a Cellulose-free Diet. spatula. That of the second day was not excreted till the third day after feeding, the subject being inclined to constipation. It seems likely that the high coefficient of digestibility is due to this fact, or else to the method of determination, which is not altogether satisfac- tory, in view of the complexity of the products of hydrolysis, the dan- ger of decomposing a part of the sugar from the easily inverted polysaccharides by the long boiling necessary for the more resistant, and the great difference in reducing power of the sugars so produced. The Digestibility of Mannan. In four experiments, the commercial salep powder (containing 19 per cent mannan and 26 per cent starch) was administered; in the others, pure mannan prepared from the Orchis tubers. The results of seven trials are tabulated on the following pages. 1 Cf. p. 345. o © l> 1> oq CO CO 00 t~- vO C5 O i-i (N CO iO Tti , & d cc? XJ xi XJ X) X) CI CM II M 0) £ b H < S 12 £ he o Q U 356 Mary Davies Swartz, The coefficients of digestibility of the salep preparations are shown in the following table : SERIES C. EXPERIMENT NO. COEFFICIENT OF DIGESTIBILITY. For the Dog. Per cent Per cent 1 Salep Powder 70 2 Salep Powder 100 3 Salep Powder 100 4 Salep Powder 94 5 Salep Mannan 10 6 100 7 100 For Man. Thus we see that in every case, except that in which a dog received, in one day, 10 grams of pure mannan, the greater portion of the salep fed was digested, the coefficient of salep powder for dogs averaging 85 per cent, and for man, 97 per cent; while that of pure mannan for man is 100 per cent, in spite of the fact that it is not attacked by diges- tive enzymes! The contrast between the volume of faeces produced when a galac- tan such as Irish moss was fed, and that when a more digestible hemi- cellulose was given, is shown in the photograph of the faeces from experiments Nos. 1 and 2 of Series C, 1 on the next page, in which A represents the fore-period, B the mid-period, and C the after-period, each period being three days in duration. The group on the right represents experiment No. 1, in which 70 per cent of the hemicellulose and starch of the salep powder was digested, and that on the left, experiment No. 2 in which apparently all of these were digested. DISCUSSION AND SUMMARY. A glance at the table on page 327 clearly shows that none of the hemicelluloses under consideration are readily attacked by the ordi- nary animal or vegetable enzymes. The results are for the most part entirely negative. Even where there has been hydrolysis with 0.2 per cent hydrochloric acid, the amount of sugar produced in 24 hours was relatively small. The hydrolyzing action of the gastric juice is probably largely due to the presence of acid, although no compari- son of the relative amounts of sugar produced by gastric juice or by l Cf. p. 348. Nutrition Investigations. 357 0.2 per cent acid alone has been made. It is noticeable that even the very soluble hemicellulose, sinistrin, which is so speedily hydrolyzed by acid (in § hour at 37° C. with 0.2% hydrochloric acid) is not attacked by ordinary diastatic enzymes within 24 hours. The parenteral introduction of these carbohydrates has resulted in their speedy and apparently complete elimination through the kid- neys without any change in character. The carbohydrates prepared from Dulse, Irish Moss, Salep and Sinistrin have all been isolated and identified in the urine, after subcutaneous and intraperitoneal injec- tions. These results are not surprising, in view of the commonly ac- The Influence of Salep upon the Mass of the Faeces. A. Fore Period: 3 Days on a Cellulose-free Diet. B. Mid Period: 3 Days on a Cellulose-free Diet to Which 15 grams of Salep Powder were Added Daily. C. After Period: 3 Days on a Cellulose-free Diet. cepted fact that carbohydrates must be converted into monosacchar- ides before they can enter into the processes of intermediary meta- bolism. Experimental evidence in support of this fact is given by such investigators as F. Voit 1 and Blumenthal, 2 who found that even di- saccharides, as lactose and saccharose, were eliminated almost quanti- ^iinchener medicinische Wochenschrift, 1896, p. 717; Deutsches Archiv fur klinische Medicin, v. 58, p.521 (1897). 2 Beitage zur chemischen Physiologie, v. 6, p. 329 (1905). 358 Mary Dames Swartz, tatively after subcutaneous injection in man and the rabbit; or as Mendel and Mitchell, 1 who have shown that polysaccharides like dextrin, soluble starch, glycogen, inulin, and isolichenin are recovered to a considerable extent in the urine, whether introduced subcuta- neously, intraperitoneally, or intravenously. In the present experiments, the dulse pentosan was the most slowly eliminated, being found in the urine four or five days after injection; Irish moss and salep were not detected after the third day; while si- nistrin seemed to be all excreted within the first 24 hours. The average coefficients of digestibility for the ten preparations which have formed the basis of the feeding experiments, are summar- ized in the following table : Coefficients of Digestibility of Hemicelluloses. HEMICELLULOSE. Class. Source. Dog. Man. Per cent. Per cent. Pentosan Dulse 73 (2 exp.) 100 (2 exp.) Pentosan Limu Eleele 35 (2 exp.) 9 (2 exp.) Pentosan Limu Pahapaha 34 (1 exp.) Pentosan Limu Lipoa 16 (1 exp.) Galactan Irish Moss 33 (2 exp.) 6 (2 exp.) Galactan Limu Manauea 33 (2 exp.) 30 (3 exp.) Galactan . Limu Huna 56 (2 exp.) 10 (1 exp.) Galactan Limu Akiaki 60 (1 exp.)* Mannan Salep Powder 85 (2 exp.) 97 (2 exp.) Salep Mannan 10 (1 exp.) 100 (2 exp.) SUBJECT OF EXPERIMENT. Subject with chronic constipation. That the low coefficients enumerated above are not due to inabil- ity of the various subjects to utilize carbohydrates, is shown by the following figures. The coefficient of digestibility for cracker meal in the experiments on dogs, determined by taking the average of all the fore-periods of the feeding trials, in which five different dogs were used, was 99.0 per cent. This is much higher than London and Polowzowa's 2 coef- ficient for carbohydrate digestibility in dogs on a bread diet, 96 per cent. 1 American Journal of Physiology, v. 14, p. 239 (1905). 2 Zeitschrift fur physiologische Chemie, 56, 513 (1908). Nutrition Investigations. 359 For the four women who were subjects of feeding experiments, the average daily amount of carbohydrate excreted in the faeces, on a cellulose-free diet, estimated as dextrose, by averaging the fore-pe- riods of all trials, was 0.8 gram. The utilization of carbohydrates was therefore unusually good, since Atwater and Bryant's 1 coefficient of digestibility for such a diet is 98 per cent, and undoubtedly every one of these individuals consumed over 50 grams of carbohydrate per day. With the exception of the subject of a single experiment who had chronic constipation, these were all normal, healthy individuals, free from disturbances of the alimentary tract. The three seaweeds fed in toto, Limu Eleele, Limu Pahapaha, and Limu Lipoa, show an average digestibility of 51 per cent. This is higher than that obtained in Professor Mendel's laboratory for un- cooked Cetraria islandica 2 (average of three experiments, 15 per cent) and much lower than that reported by Oshima for dried marine algae 3 (average 77 per cent). In man, with the exception of dulse and salep, which almost entirely disappeared in the alimentary tract, the average digestibility of all preparations is only 34 per cent, a figure in contrast to those of Loh- risch (194), who finds cellulose and hemicellulose 50 per cent digestible. In dogs, the average of all preparations is 42 per cent. Considering that the pentosan of dulse was in a form most favorable for digestion, the results with this hemicellulose are in harmony with those of Konig and Reinhardt (120) who reported 75 per cent of the pentosans as disappearing from the alimentary tract in man; and with the averages obtained by the various investigators on ani- mals, which show these carbohydrates 40-70 per cent diges- tible in herbivora. 4 It would be desirable to repeat the experiments with larger quantities, although the process of preparing the material is rather laborious. It must be borne in mind, that the dulse pento- san is not attacked by ordinary diastatic enzymes, but can be decom- posed by soil and faecal bacteria; and although this decomposition did not occur readily in pure solutions of the carbohydrate, or even in a putrefying mixture, it still remains to be demonstrated whether the complete disappearance from the alimentary tract is not largely due to the more favorable conditions for bacterial activity within the 1 Report Storr's Agricultural Experiment Station, 1899, p. 86. 2 Cf . pp. 297-298. 3 Cf. p. 299. 4 Cf. pp. 274-275. 360 Mary Davies Swartz, organism. While we have, in the case of herbivora, some convincing evidence that the pentosans are a true source of energy, 1 we have as yet no real grounds for this assumption in the case of man. The insoluble pentosans of the Hawaiian algae are manifestly less digestible than the soluble forms. The coefficient of digestibility is approximately the same as Slowtzoff's (154) average for pure xylan in rabbits, 55 per cent. While it would be perhaps desirable to de- termine the pentosans directly by the furfurol-phloroglucin method, rather than by estimation of sugar after acid hydrolysis, a trial with dulse by both methods gave practically identical results: hence, con- sidering that the hemicelluloses of these algae are chiefly pentosans, it seems safe to assume that the results reported represent the amount of pentosan excreted, within the limits of error for all of the feeding experiments. The galactans were all soluble, and were ingested in quantities not exceeding 15 grams per day, yet the coefficient of digestibility is lower than for any other hemicellulose group (26 per cent) . The resistance of Irish moss is particularly striking, but is not surprising in view of its utter indifference to attacks of digestive enzymes or bacteria. Its influence on the character of the faeces was not so marked as that of Limu Huna, owing probably to a greater tendency to liquefy at body temperature. The latter would seem to be a very effective agent in constipation; a comparison of its efficiency with that of agar-agar would be extremely interesting. Saiki (205) found the coefficient of digestibility for agar (average of two experiments) 17 per cent. In view of the negative results of digestions in vitro and of trials with bacteria, we can scarcely be surprised at the results of these met- abolism experiments, especially as we recall that Lohrisch (57) found that his "soluble agar," already partially hydrolyzed, was only diges- tible to 50 per cent (average). The mannans stand in striking contrast to the galactans. In the present studies, 99 per cent of the salep administered has been uti- lized, a result in accordance with Kano and Iishima's (255) coefficient of digestibility for the Japanese mannan, Konjaku, 82 per cent. Pure mannan fed to a dog, was excreted the succeeding day, seem- ingly unaltered, since it formed a semi-transparent gelatinous mass in the faeces, from which, later, a rich yield of mannose-hydrazone was obtained. The very different result with salep powder, of which 85 per cent was digested by dogs, may perhaps be accounted for by the 3 Cf. Kellner, p. 274. Nutrition Investigations. 361 fact that it contained a high percentage of starch (26 per cent). The amount of undigested carbohydrate excreted in the faeces is in close agreement with the quantity of pure mannan ingested. However, as tests for mannose-hydrazone were negative in these cases, further experiments are necessary before an authoritative statement can be made in regard to this question. It is manifestly possible for faecal and soil bacteria to produce sugar from mannan; 1 hence it is not unlikely that hemicelluloses of this group are inverted in the intestines through the activity of micro- organisms, and that the sugar so produced is absorbed and becomes a true source of energy for man, in spite of the resistance of mannans to the action of digestion enzymes. Further investigations to determine its exact nutritive value seem highly desirable. In considering the proper place in the dietary for marine algae, lichens and similar substances, we must not disregard the possibility of their having a valuable function entirely aside from the question of energy production. As Oshima (15) points out, they may be val- uable for their inorganic salts. The non-irritating, laxative proper- ties of many species make them desirable adjuncts to the diet of per- sons with a tendency to constipation; 2 and even if they disappear, in marked degree, from the alimentary tract during the process of diges- tion, they may perhaps still play an important role as stimulants to intestinal activity, being in fact what Prausnitz 3 calls "faeces-forming foods." An illustration of this effect is afforded by the experiments in which salep powder was fed to dogs. 4 The periods were equal in length, and in one case (No. 2 in photograph) the utilization of carbo- hydrates was equally good for all three; yet in the mid-period there is a decided increase in the bulk and weight 5 of the faeces, not more than 1 gram of which is by any possibility attributable to the cellu- lose of the salep powder, and in the other experiment, the increased amount of faeces cannot be wholly accounted for by the amount of undigested carbohydrate present. Mendel (196) has already sounded a warning against the hasty assumption that every carbohydrate, by virtue of its ultimate chem- ical composition, stands in the category of true nutrients for the human organism. The results of the present investigations emphasize the 1 Cf . Sawamura (267) . 2 Cf. p. 283. 3 Zeitschrift fur Biologie, v. 35, p. 335 U897). 4 Cf. pp. 348-349. 6 Cf. Table, p. 348, Series C, Experiments Nos. 1 and 2. 362 Mary Dames Swartz, necessity of drawing our final conclusions only from exact metabolism experiments. The soluble hemicelluloses show great diversity of behavior in the alimentary tract, although equally resistant to diges- tive enzymes in vitro; some disappear entirely, others reappear in the faeces in varying degree, up to 100 per cent. It is evident that the latter do not constitute a source of energy for the organism: how far the former actually do so, remains to be demonstrated. IV. CONCLUSIONS. 1. The hemicelluloses of the ten species of marine algae included in these investigations are chiefly pentosans and galactans. The pen- tosans are largely insoluble in water, but a soluble form in consider- able quantity has been isolated from Rhodymenia palmata. The ga- lactans are soluble in hot water, and are characterized by their gela- tinous nature. Small quantities of soluble pentosans have been found associated with them in every case. 2. In order of resistance to the action of bacteria, the hemicellu- lose groups studied stand as follows, — galactans, pentosans, levu- lans, mannans, the galactan of Chondrus crispus being entirely unaf- fected by common micro-organisms. 3. Aerobic and anaerobic cultures of soil and faecal bacteria, and cultures of B. anthracis symptomatici and B. maligni oedematis, caused inversion of salep mannan, with actual production of reducing sugar. The latter cultures also hydrolyzed the pentosan of Rhodymenia pal- mata, and the levulan, sinistrin. In a mixture of aerobes, salep ap- peared to be partially hydrolyzed, forming an insoluble transition product. 4. Digestion experiments in vitro, continued for 24 hours at body temperature under antiseptic conditions, have been almost entirely negative in result. The only exceptions are the hydrolysis of the pento- san of dulse, the galactan of limu kohu, and the levulan, sinistrin, by "Taka" diastase; and of sinistrin, and the galactans of limu kohu, limu akiaki, and slippery elm bark, by artificial gastric juice or 0.2 per cent hydrochloric acid, the action of the gastric juice being in all probability due to its acidity. 5. After parenteral injection, whether subcutaneous or intra- peritoneal, the hemicelluloses are excreted through the kidneys, and can be recovered unaltered in the urine. The pentosan of dulse is completely eliminated in four to five days, and the carbohydrates of Irish moss, salep and sinistrin, in one to three days. 6. Feeding experiments show that those hemicelluloses most readily attacked by bacteria disappear most completely from the alimentary tract. The average coefficient of digestibility for man is, in the case of the pentosan of dulse and the mannan of salep, 99 per 363 364 Mary Davies Swartz, cent notwithstanding their apparent resistance to amylolytic enzymes and the hydrolyzing influence of the gastric juice; their disappear- ance seems therefore directly attributable to bacterial activity, and the possibility of sugar formation by this agency having been demon- strated, it remains to be shown by means of respiration experiments to what extent materials so hydrolyzed can serve as true nutrients for the organism. Dogs can also utilize the dulse pentosan to a consider- able degree, but their power to digest mannan is still an open question. In striking contrast to the above hemicelluloses stand the galac- tans, with their high degree of resistance to bacterial decomposition; they show in man, an average digestibility of approximately 25 per cent, in dogs of 45 per cent. It is manifestly impossible to treat of the digestibility of hemicelluloses as a class, in view of such diversity in the groups. Not only must each type receive special considera- tion, but distinction must be drawn between soluble and insoluble forms, as is illustrated by the pentosans, the ratio of the digestibility coeffi- cient of the former to the latter being approximately 100 to 50 in man, and 75 to 25 in dogs. We may, however, say in general, that they disap- pear from the alimentary tract of men and animals to an extent seem- ingly proportional to their susceptibility to attacks of micro-organ- isms, and give little justification for any high claims made for them as sources of energy in nutrition. They may, however, have a valuable function as adjuvants in the dietary, as therapeutic agents in consti- pation, or as sources of inorganic salts. The author gratefully acknowledges the helpful suggestions and criticism freely given by Professor Lafayette Mendel throughout the progress of this work and the kindly interest and assistance of Pro- fessor Rettger in the bacteriological problems. V. BIBLIOGRAPHY. LICHENS AND ALGAE. — COMPOSITION AND USES. (1) Annett, Darbishire and Russell: The Journal of the South-Eastern Agricultural College, Wye, Kent, No. 16, pp. 204, 205, (1907). (Extract obtained from Dr. C. F. Langworthy, U. S. Dept. of Agriculture.) (2) Berzelius: Recherches sur la nature du lichen dTslande et sur son emploi comme aliment. Annales de Chimie, V. 90, p. 277, (1814). (3) Blondeau: De la Goemine, substance neutre extraite du Goemon (Fucus crispus). Comptes Rendus, V. 60, p. 860, (1865). (4) Dillingham: The Staff Tree as a Former Food Supply of Starving Indians. The American Naturalist, June, 1907. (5) Dillingham: A Contribution to the History of Bark Bread. Bulletin of the Bussey Institution, Vol. Ill, Part V, p. 120, (1906). (6) Escombe: Chemie der Membranen der Flechten und Pilze. Zeitschrift fur physiologische Chemie, V. 22, p. 288, (1896). (7) Johnson, C. Pierpont: The Useful Plants of Great Britain, (1862). (8) Konig and Bettels: Die Kohlenhydiate der Meeresalgen und daraus hergestellter Erzeugnisse. Zeitschrift fur Untersuchung der Nahrungs- und Genuss- mittel, V. 10, p. 487, (1905). (9) Krefting: A New Carbohydrate from Laminaria Digitata. Biochemisches Centralblatt, V. 8, p. 770 (1909). (From Pharmacia, V. 6, p. 151.) (10) Mendel: Das Verhalten einiger unverdaulicher Kohlenhydrate im Ver- dauungstrakt. Zentralblatt fur die gesammte Physiologie und Pathologie des Stoffwechsels, No. 17, p. 1, (1908). (11) Muller, Karl: Die chemische Zusammensetzung der Zellmembranen bei verschiedenen Kryptogamen. Zeitschrift fur physiologische Chemie, V. 45, p. 264, (1905). (12) Muther: Untersuchungen iiber Fucusarten, Laminaria und Carragheen- moos, sowie die hydrolytisch daraus entstehenden Substanzen und iiber Derivate derselben, besonders Fucose und Fuconsaure. Inaugural Dissertation, Gottingen, (1903). Berichte der deutschen chemischen Gesellschaft, V. 37, p. 298, (1904). (13) Muther and Tollens: Uber die Producte der Hydrolyze von Seetang (Fucus), Laminaria, und Carragheenmoos. Zeitschrift des Vereins der Deutschen Zucker Industrie. Band 54, Heft 576, s. 59, (1904). (14) Nilson: Zur Kenntniss der Kohlenhydrate in den Flechten. Upsala Lakareforenings Forhandlingar, Vol. 38, also Jahresbericht fur Thierchemie, V. 23, p. 53, (1893). (15) Oshima: A Digest of Japanese Investigations on the Nutrition of Man. Office of Experiment Stations, United States Department of Agriculture, Bulletin 159, p. 34, (1905). (16) Oshima and Tollens : Uber das Nori aus Japan. Berichte der deutschen chemischen Gesellschaft, V. 34, p. 1422, (1901). 365 366 Mary Davies Swartz, (17) Poulsson: Untersuchungen iiber das Verhalten einiger Flechten-Kohlen- hydrate im menschlichen Organismus und iiber die Anwendung derselben bei Diabetes mellitus. Festschrift fiir Olof Hammersten, XIV, pp. 1-25. Upsala Lakareforenings Forhandlingar, (1906), supplement. (18) Reed, Minnie: The Economic Seaweeds of Hawaii and Their Food Value. Report of Hawaiian Agricultural Experiment Station, (1906). (19) Richards: Some Edible Seaweeds. Science, N. S., V. 21, p. 895, (1905). (20) Saiki: The Digestibility and Utilization of some Polysaccharide Carbo- hydrates Derived from Lichens and Marine Algae. Journal of Biological Chemistry, V. 2, p. 251, (1906-7). (21) Schneider: A Text-book of Lichenology, p. 23, (1897). (22) Setchell: Limu. University of California Publications, Botany, Vol.2, No. 3, p. 91, (1905). (23) Smith, H. M.: The Seaweed Industries of Japan; and the Utilization of Seaweed in the United States. Bulletin of Bureau of Fisheries, V. 24, p. 133, (1904). (24) Schmidt, C: Uber Pflanzenschleim und Bassorin. Liebig's Annalen, V. 51, p. 56, (1844). (25) Smith: Dictionary of Popular Names of Economic Plants, p. 418, (1882). (26) Ulander: Untersuchungen iiber die Kohlenhydrate der Flechten. Dis- sertation, Gottingen, (1905). (27) Ulander and Tollens: Untersuchungen iiber Kohlenhydrate der Flechten. Berichte der deutschen chemischen Gesellschaft, V. 39, p. 401, (1906). (28) Winterstein : Zur Kenntniss der in den Membranen der Pilze enthaltenen Bestandtheile. Zeitschrift fiir physiologische Chemie, V. 19, p. 521, (1895). (29) Wisselingh: Microchemische Untersuchungen iiber die Zellwande der Fungi. Jahrbucher fiir wissenschaftliche Botanik, V. 31, p. 619, (1897). (30) Yendo: Uses of Marine Algae in Japan. Postelsia, 1901. II. CELLULOSE. (31) Ankersmit: Untersuchungen iiber die Bakterien im Verdaungskanal des Rindes. Centralblatt fiir Bakteriologie, Parasitenkunde und Infektionskrank- heiten. Abt. I, V. 40, p. 100, (1905). (32) Bergmann: Studien iiber die Digestion der Pflanzenfresser. Skandi- navisches Archiv fiir Physiologie, V. 18, p. 119, (1906). (33) Biedermann: Beitrage zur vergleichenden Physiologie der Verdauung. Pfluger's Archiv fur Physiologie, V. 72, p. 105, (1898); V. 75, p. 1, (1899). (34) Biedermann and Moritz: tiber ein Celluloselosendes Enzym im Leber- secret der Schnecke, (Helix Pomatia). Pfluger's Archiv fiir Physiologie, V. 73, p. 236, (1898). (35) Brown: On the Search for a Cellulose-dissolving (Cyto-hydrolytic) En- zyme in the Digestive Tract of Certain Grain-feeding Animals. Journal of the Chemical Society, London, V. 61, p. 352, (1892). (36) Brown and Morris: On the Existence of a Cellulose-dissolving Enzyme (Cyto-hydrolyst) in the Germinating Seeds of the Grasses. Journal of the Chemical Society, London, V. 57, p. 497, (1890). (37) De Bary: tiber einige Sclerotinien und Sclerotienkrankheiten. Bota- nische Zeitschrift, V. 44, p. 377, (1886). N utrition Investigations. 367 (38) Eberlein: Ueber die im Wiederkauermagen vorkommenden ciliaten Infusorien. Zeitschrift ftir wissenschaftlichen Zoologie, V. 49, p. 233, (1895). (Cited by Scheunert, Berliner tierarztliche Wochenschrift, No. 47, p. 826, (1909). (39) Furstenberg: Das Verhalten der pflanzlichen Zellmembran wahrend der Entwicklung in chemischer und physiologischer Hinsicht. Zentralblatt fur Physiologie, V. 22, p. 567, (1908). (40) Haubner: Journal fiir Landwirtschaft, 1855, p. 177. (Cited byLoh- risch.) (41) Henneberg and Stohman: Beitrage zur Begriindung einer rationellen Fiitterung der Wiederkauer. Heft. 2, 1864. Cited in Zeitschrift fiir Biologie, V. 21, p. 613, (1885). (42) Hoffmann: Uber den Einfluss von Hemizellulosen und von Zellulose auf die Stickstoffbilanz und den Phlorhizindiabetes von Kaninchen. Dissertation, Halle, 1910. (Cited in Zentralblatt fur Biochemie und Biophysik, V. 10, p. 794, (1910). (43) Hofmeister: Die Rohfaser und einige Formen der Cellulose. Land- wirtschaftliche Jahrbiicher, V. 17, p. 239, (1888). (44) Hofmeister: Die Cellulose und ihre Formen. Ibid., V. 18, p. 767, (1889). (45) Hofmeister: Uber Celluloseverdauung beim Pferde. Archiv fiir wissen- schaftliche und practische Thierheilkunde, V. 11, p. 46, (1885). (46) v. Hoesslin: Zur Kenntnis der Zelluloseverdauung. 2. Mitteilung. Die Ausniitzung der Zellulose beim Hunde. Zeitschrift fiir Biologie, V. 54, p. 395, (1910). (47) v. Hoesslin and Lesser: Ueber die Zersetzung der Zellulose durchden Inhalt des Coecums des Pferdes. Zeitschrift fiir Biologie, V. 54, p. 47, (1910). (48) Holdefleiss: Die Bedeutung des verdauten Antheils der Rohfaser fiir die tierische Ernahrung. Berichte aus dem physiologischen Laboratorium und der Versuchsanstalt des Landwirtschaftlichen Instituts der Universitat Halle. Heft. 2, (1895). (Cited by Lohrisch.) (49) Kellner: Untersuchungen iiber den Stoff- und Energieumsatz der erwach- senen Rinder bei Erhaltungs und Productionsfutter. Landwirtschaftliche Versuchs- Stationen, V. 53, p. 457, (1900). (50) Knauthe: Uber die Verdauung und den Stoffwechsel die Fische. Archiv fiir Anatomie und Physiologie, Physiologische Abtheilung, V. 22, p. 149, (1898). (51) Konig: Zur Bestimmung der Rohfaser und zur Trennung von Cellulose, Lignin und Cutin in derselben. Berichte der deutschen chemischen Gesellschaft, V. 41, p. 46, (1908). (52) Konig: Die Zellmembran und ihre Bestandtheile in chemischer und physiologischer Hinsicht. Landwirtschaftliche Versuchs-Stationen, V. 65, p. 55, (1907). (53) Konig and Reinhardt: Uber die Ausnutzung der Pentosane beim Men- chen. Zeitschrift fur Nahrungs- und Genussmittel, V. 5, p. 110, (1902). (54) Kuhn, Bronstein and Schulze: Bericht iiber die auf der landwirt- schaftlichen Versuchs-Station Weende ausgefiihrten Versuche. Journal fiir Land- wirtschaft, V. 14, p. 269, (1866). (55) Lohrisch: Uber die Bedeutung der Cellulose im Haushalte des Menschen. Zeitschrift fur physiologische Chemie, V. 47, p. 200, (1907). (56) Lohrisch: Uber die Verdauung und Verwertung der Rohfaser und Zellu- lose im tierischen und menschlichen Organismus. Zentralblatt fiir die gesammte Physiologie und Pathologie des Stoffwechsel s, V. 21, (1907). 368 Mary Davies Swartz, (57) Lohrisch: Der Vorgang der Cellulose und Hemicellulosen Verdauung beim Menschen, und der Nahrwerth dieser Substanzen fur den menschlichen Organismus. Zeitschrift fur experimentelle Pathologie und Therapie, V. 5, p. 478, (1908). (58) Lohrisch: Bemerkungen zur Frage der Cellulose verdauung beim Hund und iiber die Methoden der quantitativen Cellulosebestimmung. Zeitschrift fur physiologische Chemie, V. 69, p. 143, (1910). (59) Lusk: On the Question Whether Dextrose Arises from Cellulose in Diges- tion. American Journal of Physiology, V. 6, p. xiii, (1901-2). (60) Mallevre: Der Einfluss der als Gahrungsprodukt der Cellulose gebildeten Essigsaure auf den Gaswechsel. Pfliigers Archiv fur Physiologie, V. 49, p. 460, (1891). (61) Muller, E.: Ein Beitrag zur Frage der Cellulose verdauung im Darm- canale. Pfliigers Archiv fur Physiologie, V. 83, p. 619, (1901). (62) Munk: Der Einfluss des Glycerins, der fluchtigen und festen Fettsauren auf den Gaswechsel. Pfiuger's Archiv fur Physiologie, V. 46, p. 303, (1890). (63) Murdfield: Das Lignin und Kutin in chemischer und physiologischer Hinsicht. Inaugural Dissertation, Minister, 1906. (Cited by Konig, Fursten- berg and Murdfield.) (64) Newcombe: Cellulose Enzymes. Annals of Botany, V. 13, p. 49, (1899). (65) Omelianski: Sur la Fermenentation Formique de la Cellulose. Cen- tralblatt fur Physiologie, V. 16, p. 522, (1902). (From Archives des Sciences Biologiques, St. Petersbourg, V. IX, No. 3, p. 251.) (66) Pacault: Sur deux proprietes diastatiques de la salive de l'escargot. Comptes Rendus de la Societe de Biologie, V. 59, p. 29, (1909). . (67) Reinitzer: Uber das Zellwandlosende Enzym der Gerste. Zeitschrift fur physiologische Chemie, V. 23, p. 175, (1897). (68) Scheunert: Beitrage zur Kenntniss der Zelluloseverdauung im Blinddarm und des Enzymgehaltes des Caecalsekretes. Zeitschrift fur physiologische Chemie, V. 48, p. 9, (1906). (69) Scheunert: Uber die Celluloseverdauung bei den Haustieren. 2. Mitteil- ung. Vermag der Hund Cellulose zu verdauen? Berliner tierarztliche Wochen- schrift, No. 47, p. 867, (1909). (70) Scheunert: Vermag Schafspeichel Zellulose zu losen? Berliner tierarzt- liche Wochenschrift, No. 48, p. 113, (1910). (71) Scheunert and Grimmer: Zur Kenntniss der in den Nahrungsmitteln enthaltenen Enzyme und ihrer Mitwerkung bei der Verdauung. Zeitschrift fur physiologische Chemie, V. 48, p. 27, (1906). (72) Scheunert and Lotsch: Vermag der Hund Cellulose oder Rohfaser zu verdauen? Biochemische Zeitschrift, V. 20, p. 10, (1909). (73) Schmidt and Lohrisch: Uber die Bedeutung der Cellulose fur den Kraft- wechsel der Diabetiker. Deutsche medicinische Wochenschrift, No. 47, p. 1938, (1907). (74) Schulze: Uber die zur Gruppe der stickstofffreien Extractstoffe gehoren- den Pflanzenbestandtheile. Die Landwirtschaftliche Versuchs-Stationen, V. 52, (1904). (75) Schulze, Steiger and Maxwell: Zur Chemie der Pflanzenzellmem- branen. Zeitschrift fur physiologische Chemie, V. 14, p. 227, (1890). Nutr ition Investigations . 369 (76) SELLliSRE: Rcmarques sur l'hydrolyse diastatique de la Cellulose du Coton et de quelques autres Polysaccharides. Comptes Rendus de la Societe de Biologie, V. 63, No. 34, (1907); Centralblatt fur Biochemie, V. 7, p. 88, (1908). (77) Selliere : Sur la digestion de la cellulose. Comptes Rendus de la Societe de Biologie, V. 68, p. 107, (1910). (78) Tappeiner: Untersuchung iiber die Garung der Cellulose, insbesondere iiber deren Losung im Darmkanale. Zeitschrift fur Biologie, V. 20, p. 52, (1884). (79) Tollens: Untersuchungen iiber Kohlenhydrate. Die Landwirtschaft- liche Versuchs-Stationen, V. 39, p. 401, (1891). (80) Ustjanzew: Zur Frage iiber die Rolle der Rohfaser in dem Stickstoffum- satz des tierischen Organismus. Die Landwirtschaftliche Versuchs-Stationen, V. 56, p. 463, (1902). (81) Van Iterson. Die Losung der Cellulose durch aerobe Microorganis- men. Biochemisches Zentralblatt, V. I, p. 678, (1903). (82) Voit and Hoffman: Sitzungsberichte der Bayerischen Akademie, 1869. (Cited by Tappeiner.) (83) von Knieriem : Uber die Verwertung der Cellulose im tierischen Organis- mus. Zeitschrift fur Biologie, V. 21, p. 67, (1885). (84) Ward: On a Lily Disease. Annals of Botany, V. 2, p. 319, (1889). (85) Weiske and Mehlis : Uber das Verhalten der Rohfaser im Verdauungs- apparate der Ganse. Landwirtschaftliche Versuchs-Stationen, V. 21, p. 411, (1878) . (86) Weiske: Beeinflussen die in Vegetabilien vorkommenden Fermente der Ausnutzung der Nahrung im Organismus? Zeitschrift fur physiologische Chemie, V. 19, p. 282, (1894). (87) Wieler: Analysen der Jungholzregion von Pinus Silvestris und Salix Petendra nebst einem Beitrag zur Methodik der Pflanzenanalyse. Landwirt- schaftliche Versuchs-Stationen, V. 32, p. 307, (1886). (88) Zuntz: Gesichtspunkte zum Kritischen Studium der neueren Arbeiten auf dem Gebiete der Ernahrung. Landwirtschaftliche Jahrbiicher, V. 8, p. 65, (1879) . in. pentosans. (89) Ankersmit: Untersuchungen iiber die Bakterien im Verdauungskanal des Rindes. Centralblatt fur Bakteriologie, Parasitenkunde und Infectionskrank- heiten, V. 40, Abt. I, p. 100, (1905). (90) Behrens: Beitrage zur Kenntnis der Obstfaulnisse. Chemisches Cen- tralblatt, V. 98b, p. 1027, (1898). (91) Bergmann: Studien iiber die Digestion der Pflanzenfresser. Skandi- navisches Archiv fur Physiologie, V. 18, p. 119, (1906). (92) Bigelow, Gore and Howard: Studies on Apples. United States De- partment of Agriculture, Bulletin of the Bureau of Chemistry, No. 94, (1905). (93) Blumenthal: Die Pentosurie, Deutsche Klinik, Vol. 3, (1903). (Cited by Lohrisch.) (94) Borntrager: Uber die Rutinsaure. Liebig's Annalen, V. 53, p. 385, (1845). (95) Bourquelot and Herissey: Sur l'existence dans Forge germee d'un ferment soluble agissant sur la pectine. Comptes Rendus, V. 127, p. 191, (1898). 370 Mary Dames Swartz, (96) Brasch: Uber das Verhalten nicht-gahrungsfahiger Kohlenhydrate im tierischen Organismus mit Beriicksichtigung des Diabetes. Zeitschrift fiir Biolo- gie, V. 50, p. 113, (1907). (97) Brown: On the Search for a Cellulose-dissolving (Cytohydrolytic) En- zyme in the Digestive Tract of Certain Grain-feeding Animals. Journal of the Chemical Society, London, V. 61, p. 352, (1892). (98) Calabresi: Uber die Bildung und die physiologische Aufgabe der Pento- sane in den Pnanzen. Centralblatt fur Agriculturchemie, V. 37, p. 93, (1908). (99) Couvreur and Bellion: Sur le sucre dans le sang d'escargot. Comptes Rendus de la Societe de Biologie, V. 63, p. 339, (1907). (100) Cominotti. Ueber das Vorhandensein der Pentosen im Harne des Men- schen und der Tiere. Biochemische Zeitschrift, V. 22, p. 106, (1909). (101) Cramer: Untersuchungen uber Hemicellulosevergarung durch mensch- lichen Kot. Dissertation, Halle, 1910. (Cited in Zentralblatt fiir Biochemie und Biophysik, V. 10, p. 800, (1910). (102) Cremer: Uber das Verhalten einiger Zuckerarten im thierischen Organis- mus. Zeitschrift fur Biologie, V. 29, p. 484, (1892). (103) Cremer: Uber die Verwertung der Rhamnose im tierischen Organismus, und einige damit zusammenhangende Fragen der Physiologie der Kohlehydrate. Zeitschrift fur Biologie, V. 42, p. 428, (1901). (104) Cross, Bevan and Beadle: Die naturlichen Oxycellulosen. Berichte der deutschen chemischen Gesellschaft, V. 27, p. 1061, (1894). (105) Cross, Bevan, and Smith: Uber einige Vorgange in der Gerstenpflanze. Berichte der deutschen chemischen Gesellschaft, V. 28, p. 2604, (1895). (106) Cross: liber die Constitution der Pektinstoffe. Ibid., V. 28, p. 2609, (1895). (107) Czapek: Zur Biologie der holzbewohnenden Pilze. Berichte der deut- schen botanischen Gesellschaft, V. 17, p. 166, (1899). (108) De Chalmot: Pentosans in Plants. American Chemical Journal, V. 15, p. 276, (1893). (109) Ebstein: Einige Bemerkungen zum Verhalten d i Pentosen im mensch- lichen Organismus. Virchow's Archiv, V, 129, p. 401, (lSt2). (110) Fraps: The Digestibility of some Non-nitrogenous Constituents of Certain Feeding Stuffs. North Carolina Experiment Station, Bulletin No. 172, (1900). (111) Frentzel: Uber Glycogenbildung im Thierkorper nach Fiitterung mit Holzzucker. Pfliiger's Archiv fur Physiologie, V. 56, p. 273, (1894). (112) Fudakowski: Spaltung der Arabinsaure durch Pepsin. Berichte der deutschen chemischen Gesellschaft, V. 11, p. 1069, (1878). (113) Gotze and Pfeiffer: Beitrage zur Frage iiber die Bildung resp. das Verhalten der Pentaglykosen in Pflanzen- und Thierkorper. Die Landwirtschaft- liche Versuchs-Stationen, V. 47, p. 59, (1896). (114) Harrison: A Bacterial Disease of Cauliflower (Brassica and Oleraceae), and Allied Plants. Centalblatt fur Bakteriologie, Abtheilung II, V. 13, p. 185, (1904). (115) Hofmeister: Die quantitative Trennung von Hemicellulose, Cellulose, und Lignin, und das Vorkommen der Pentosane in diesen. Die Landwirtschaft- liche Versuchs-Stationen, V. 50, p. 347, (1898). (116) Herzfeld: Die Pektinsubstanzen. Chemisches Centralblatt, V. II, p. 618, (1891). Nutrition Investigations . 371 (117) V. Jacksch: Uber Alimentare Pentosurie. Zeitschrift fur Heilkunde- V. 20, p. 195, (1899). (118) Kellner and Kohler: Untersuchungen iiber den Stoff- und Energie- umsatz des erwachsenen Rindes bei Erhaltungs- und Productionsfutter. Die Landwirtschaftliche Versuchs-Stationen, V. 53, p. 457, (1900). (119) Konig: Die Zellmembran und ihre Bestandteile in chemischer und physiologischer Hinsicht. Die Landwirtschaftlichen Versuchs-Stationen, V. 65, p. 55, (1907). (120) Konig and Reinhardt: Ausnutzung der Pentosane beim Menschen. Zeitschrift fur Untersuchung der Nahrungs- und Genussmittel, V. 5, p. 110, (1902) ; V. 7, p. 729, (1904). (121) Krober: Untersuchungen iiber die Pentosanbestimmungen mittelst der Salzsaure-Phloroglucin Methode, nebst einiger Anwendungen. Journal fur Landwirthschaft, V. 48, p. 357, (1900). (122) Lindemann and May: Die Verwerthung der Rhamnose vom normalen und vom diabetischen menschlichen Organismus. Deutsches Archiv fur klinische Medicin, V. 56, p. 283, (1895-6). (123) Lindsey: The Pentosans. Fifteenth Massachusetts State Report, p. 69, (1903). (124) Lindsey and Holland: Twelfth Massachusetts State Report, p. 175, (1900). (Cited by Lindsey.) (125) Lintner and Dull: Uber die chemische Natur des Gerstengummi. Chemiker Zeitung, V. 15, Repertorium p. 266, (1891). (126) McCollum and Brannon: The Disappearance of Pentosans from the Digestive Tract of the Cow. Journal of the American Chemical Society, V. 31, p. 1252, (1909). (127) Neuberg: Die Physiologie der Pentosen und der Glukuronsauren. Ergebnisse der Physiologie, V. 3, p. 421, (1904). (128) Neuberg and Wohlgemuth: Uber das Verhalten stereo-isomer Sub- stanzen im Tierkorper. Zeitschrift fur physiologische Chemie, V. 35, p. 41, (1902). (129) Oshima and Tollens: Uber das Nori aus Japan. Berichte der deutschen chemischen Gesellschaft, V. 34, p. 1422, (1901). (130) Pacault: Sur deux proprietes diastatiques de la salive de l'escargot. Comptes Rendus de la Societe de Biologie, V. 59, p. 29, (1905). (131) Ravenna and Cereser: Origin and Physiological Function of Pen- tosans in Plants, Journal of the London Chemical Society, V. 96, p. 1946, (1909). (From Atti R. Accad. Lincei, 1909 [V] 18, ii, p. 177.) (132) Reichardt: Pararabin, ein neues Kohlehydrat. Berichte der deut- schen chemischen Gesellschaft, V. 8, p. 807, (1875). (133) Reinhardt: Die Bestimmung der Cellulose und ihr Verhalten, sowie das der Pentosane im Darmcanal des Menschen. Dissertation, Munchen, (1903) . (134) Rohmann: Einige Beobachtungen iiber die Verdauung der Kohlehydrate bei Aplysien. Centralblatt fur Physiologie, V. 13, p. 455, (1899). (135) Rubner: tlber den Werth der Weizenkleie fur die Ernahrung des Menschen. Zeitschrift fur Biologie, V. 19, p. 45, (1883). (136) Rudzinski: Uber die Bedeutung der Pentosane als Bestandtheile der Futtermittel, insbesondere des Roggenstrohes. Zeitschrift fur physiologische Che- mie, V. 40, p. 317, (1904). (137) Salkowski: tlber die Gahrung der Pentosen. Zeitschrift ftir physiolo- gische Chemie, V. 30, p. 478, (1900). 372 Mary Dames Swartz, (138) Salkowski: Uber das Verhalten der Pentosen im Thierkorper. Cen- tralblatt fur die medicinische Wissenschaften, No. 11, p. 193. (1893). (139) Salkowski: Uber das Verhalten des Arabans zu Fehling'scher Losung. Zeitschrift fur physiologische Chemie, V. 35, p. 240, (1902). (140) Salkowski: Ueber die Darstellung des Xylans. Ibid., V. 34, p. 162, (1901-2). (141) Scheibler: Uber den Pectinzucker (Pectinose), eine neue durch Spal- tung der Metapectinsaure entstehende Zuckerart. Berichte der deutschen chemi- schen Gesellschaft, V. 1, p. 108, (1868). (142) Scheibler: Uber das Vorkommen der Arabinsaure (Gummi) in den Zuckerriiben und liber den Arabinzucker (Gummizucker). Berichte der deut- schen chemischen Gesellschaft, V. 6, p. 612, (1873). (413) Sherman: The Insoluble Carbohydrates of Wheat. Journal of the American Chemical Society, V. 19, p. 308, (1897). (144) Schmulewitsch: Bulletin de l'Academie de St. Petersbourg, V. 25, p. 549, (1879). (Cited by Bergmann.) (145) Schone and Tollens : Untersuchungen uber Kohlehydrate. Die Landwirtschaftlichen Versuchs-Stationen, V. 40, p. 377, (1892). (146) Schorstein: Zur Biochemie der Holzpilze. Centralblatt fiir Bakterio- logie, Abtheilung II, V, 9. p. 446, (1902). (147) Schulze, Steiger and Maxwell: Zur Chemie der Pflanzenzellmem- branen. Zeitschrift fiir physiologische Chemie, V. 14, p. 227, (1892). - (148) Selliere : Sur la presence d'une diastase hydrolysant dans le sue gastro- intestinal d'escargot. Comptes Rendus de la Societe de Biologie, V. 58, p. 409, (1905). (149) Selliere: Sur une diastase hydolysant la xylane dans le tube digestif de certaines larves de Coleopteres. Ibid., V. 58, p. 940, (1905). (150) Selliere: Sur la presence de la xylanase chez differents mollusques gasteropodes. Ibid., V. 59, p. 120, (1906). (151) Selliere: Sur Pabsorption et la presence dans le sang chez l'escargot des produits de l'hydrolyse digestion de la xylane. Comptes Rendus de la Societe de Biologie, V. 63, No. 36, (1907). (152) Selliere: Sur la digestion de la xylane chez quelques mammiferes herbivores. Ibid., V. 64, p. 941, (1908). (153) Selliere: Sur la digestion de la xylane chez les mammiferes. Comptes Rendus de la Societe de Biologie, V. 66, p. 691, (1909). (154) Slowtzoff: Uber das Verhalten des Xylans im Thierkorper. Zeit- schrift fiir physiologische Chemie, V. 34, p. 181, (1901). (155) Stone: Die Verdaulichkeit der Pentosane. Berichte der deutschen chemischen Gesellschaft, V. 25, p. 563, (1892). (156) Stone and Jones: Verdaulichkeit der Pentosane. Centralblatt fiir Agriculturchemie, V. 22, p. 677, (1893). (157) Tollens: "Uber die in den Pflanzenstoffen und besonders den Futter- mitteln enthaltenen Pentosane, ihre Bestimmungsmethoden und Eigenschaften. Journal fiir Landwirthschaft, V. 44, p. 171, (1896). (158) Tollens: Untersuchungen liber Kohlenhydrate. Die Landwirtschaft- lichen Versuchs-Stationen, V. 39, p. 401, (1891). (159) Tollens and Glaubitz: Uber den Pentosan-gehalt verschiedener Materialien, welche zur Ernahrung dienen, und in den Garungs-Industrieen ange. Nutrition Investigations. 373 wendet werden, und tiber den Verbleib des Pentosans bei den Operationen, welcher die obigen Materialien unterworfen werden. Journal fiir Landwirthschaft, V. 45, p. 97, (1897). (160) Tromp De Haas and Tollens: Untersuchungen iiber die Pectionstoffe. Zeitschrift des Vereins fiir die Riibenzucker-Industrie der Deutschen Reiches, V. 45, p. 473, (1895). Liebig's Annalen, V. 286, p. 278, (1895). (161) Utzjanzew: Zur Physiologie des Blinddarmes bei den Pflanzenfressern. Biochemische Zeitschrift, V. 4, p. 154, (1907). (162) Ward and Dunlop: On Some Points in the Histology and Physiology of the Fruits and Seeds of Rhamnus. Annals of Botany, V. I, p. 1, (1887). (163) Widtsoe and Tollens: Uber Arabinose, Xylose und Fucose aus Traganth. Berichte der deutschen chemischen Gesellschaft, V. 33, p. 132, (1900). (164) Weiser: tjber die Verdaulichkeit der Pentosane. Die Landwirt- schaftlichen Versuchs-Stationen, V. 58, p. 238, (1903). (165) Weiser and Zaitschek: Beitrage zur Methodik der Starkebestimmung und zur Kenntnis der Verdaulichkeit der Kohlenhydrate. Pfliiger's Archiv fiir Physiologie, V. 93, p. 98, (1903). (166) Weiske tjber die Verdaulichkeit der in den vegetabilischen Futter- mitteln enthaltenen Pentosane. Zeitschrift fiir physiologische Chemie, V. 20, p. 489, (1895). (167) Winterstein: Uber das pflanzliche Amyloid. Zeitschrift fiir physiolo- gische Chemie, V. 17, p. 353, (1893). (168) Zuntz and Utzjanzew: Zur Bedeutung des Blinddarmes fiir die Ver- dauung beim Kaninchen. Archiv fiir Anatomie und Physiologie, physiologische Abtheilung, 1905, p. 403. IV. GALACTANS. (169) Bauer: Ueber den aus Agar-agar entstehenden Zucker, iiber eine neue Saure aus der Arabinose nebst dem Versuch einer Classification der Gallertbilden- den Kohlehydrate nach den aus ihnen enstehenden Zuckerarten. Journal fiir praktische Chemie, V. 30, p. 36, 7, (1884). (170) Bauer: Weitere Untersuchungen uber Alimentare Galaktosurie. Zen- tralblatt fiir innere Medizin, 1906, p. 1176. (171) Bente: Ueber anderweitige Darstellung von Levulinsaure. Berichte der deutschen chemischen Gesellschaft, V. 8, p. 417, (1875). (172) Bente: Zur Darstellung der Levulinsaure und iiber Carragheenzucker. Berichte der deutschen chemischen Gesellschaft, V. 9, p. 1157, (1876). (173) Bierry and Giaja : Digestion des mannanes et des galactanes. Comp- tes Rendus, V. 148, No. 8, p. 507, (1909). (174) Boureuelot and Herissey: Germination de la graine de Caroubier. Comptes Rendus, V. 129, p. 614, (1899). (175 J Brasch: Ueber das Verhalten nicht-garungsfahiger Kohlehydrate im tierischen Organismus mit Beriicksichtigung des Diabetes. Zeitschrift fiir Biolo- gie, V. 50, p. 113, (1907). (176) Castoro: Beitrage zur Kenntnis der Hemicellulosen. Zeitschrift fiir physiologische Chemie, V. 48, p. 96, (1905); V. 49, p. 96, (1906). (177) Cremer: Ueber das Verhalten einiger Zuckerarten im tierischen Organis- mus. Zeitschrift fur Biologie, V. 29, p. 484, (1892). 374 Mary Dames Swartz, (178) Fluckinger and Mayer: Neues Repertorium fur Pharmacie, 1868, p. 350. (Cited by Haedike, Bauer and Tollens.) (179) Goret: Etude chimique et physiologique de quelques albumens comes de graines de Legumineuses. These, Paris, 1901. (Cited by Herissey.) (180) Greenish: Untersuchung von Fucus Amylaceous. Berichte der deut- schen chemischen Gesellschaft, V. 14, p. 2253, (1881). (181) Greenish: Die Kohlehydrate des Fucus Amylaceous. Ibid., V. 5, p. 2243, (1882). (182) Gran: Die Hydrolyse des Agars durch ein Enzym. Centralblatt fur Bacteriologie, Abtheilung II, V. 9, p. 562, (1902). (183) Gruss: Studien iiber Reservecellulose. Botanisches Centralblatt, V. 70, p. 242, (1897). (184) Gruss: Ueber den Umsatz bei der Keimung der Dattel. Berichte der botanischen Gesellschaft, V. 20, p. 36, (1902). (185) Hadike, Bauer and Tollens: Ueber Galactose aus Carragheenmoos. Liebig's Annalen, V. 238, p. 302, (1887). (186) Herissey: Isolement du galactose crystallise dans les produits de digestion par la seminase, des galactans, des albumens cornes. Comptes Rendus de la Societe de Biologie, V. 54, p. 1174, (1902). (187) Herissey: Recherches chimiques et physiologiques sur la digestion des mannanes et des galactanes, par la seminase, chez les vegetaux. Revue Generate de Botanique, 1903, p. 345. > (188) Hofmeister: Ueber Resorption und Assimilation der Nahrstoffe. Archiv fur experimentelle Pathologie und Pharmakologie, V. 25, p. 240, (1888). (189) Kausch and Socin: Sind Milchzucker und Galactose directe Glycogen- bildner? Archiv fur experimentelle Pathologie und Pharmakologie, V. 31, (1893). (Cited by Lohrisch.) (190) Konig and Bettels: Die Kohlenhydrate der Meeresalgen und daraus hergestellter Erzeugnisse. Zeitschrift fur Untersuchung der Nahrungs- und Genuss- mittel, V. 10, p. 487, (1905). (191) Lindsey: The Digestibility of Galactan. 17th Annual Report, Massa- chusetts State Experiment Station, p. 78, (1905). (192) Lippmann: Ueber ein neues Galactan und einige Eigenschaften der Galactose. Berichte der deutschen chemischen Gesellschaft, V. 20, p. 1001, (1890). (193) Lohrisch: Die Ursachen der chronischen habituellen Obstipation im Lichte systematischer Ausnutzungsversuche. Deutsche Archiv fur klinische Medizin, V. 79, p. 383, (1904). (194) Lohrisch: Der Vorgang der Cellulose- und Hemicellulosenveirdauung beim Menschen und der Nahrwerth dieser Substanzen fur den menschlichen Or- ganismus. Zeitschrift fur experimentelle Pathologie und Pharmakologie, V. 5, p. 478, (1908). (195) Mallevre: Der Einfluss der als Gahrungsprodukt der Cellulose gebil- deten Essigsaure auf dem Gaswechsel. Pfliiger's Archiv fur Physiologie, V. 49, p. 460, (1891). (196) Mendel: Das Verhalten einiger unverdaulicher Kohlehydrate im Ver- dauungstrakt. Zentralblatt fur die gesammte Physiologie und Pathologie des Stoffwechsels, No. 17, p. 1, (1908). (197) Muller, Karl: Die chemische Zusammensetzung der Zellmembranen bei verschiedenen Kryptogamen. Zeitschrift fur physiologische Chemie, V. 45, p. 264, (1905). Nutrition Investigations . 375 (198) Munk: Der Einfluss des Glycerins, der fliichtigen und festen Fettsauren anf den Gaswechsel. Pfluger's Archiv, V. 46, p. 303, (1890). (199) Muntz : Sur la Galactine. Comptes Rendus, V. 94, p. 453, (1882). (200) Muther: Untersuchungen iiber Fucusarten. Laminaria und Carraghe- enmoos, sowie die hydrolytisch daraus entsehenden Substanzen und iiber Derivate derselben, besonders Fucose und Fuconsaure. Inaugural Dissertation, Gottingen, 1903. (201) Muther and Tollens : Ueber die Producte der Hydrolyse von Seetang (Fucus) Laminaria, und Carragheen-Moos. Berichte der deutschen chemischen Gesellschaft, V. 37, p. 298, (1904). (202) Payen: Sur la g61ose et les nids de salagane. Comptes Rendus, V. 49, p. 521, (1859). (203) Pletnew: Vergleichende Ausnutzungsversuche an normalen und habi- tuell obstipirten Menschen. Zeitschrift fur experimentelle Pathologie und Thera- pie, V. 5, p. 186, (1908). (204) Roihenfusser : Der Schleimkorper des Leinsamens. Jahresbericht fur Thierchemie, V. 34, p. 78, (1904). (205) Saiki: The Digestibility and Utilization of some Polysaccharide Carbo- hydrates derived from Lichens and Marine Algae. The Journal of Biological Chemistry, V. 2, p. 251, (1906). (206) Sandmeyer: Ueber die Folgen der partiellen Pankreasextirpation beim Hund. Zeitschrift fur Biologie, V. 31, p. 32, (1895). (207) Sawamtjra: Uber Enzyme im Verdauungskanal. Bulletin of the Col- lege of Agriculture, Tokyo Imperial University, V. No. 2, (1902); Jahresbericht fur Thierchemie, V. 32, p. 419, (1902). (208) Schellenberg : Untersuchungen iiber das Verhal ten einiger Pilze gegen Hemicellulose. Flora, V. 98, p. 257, (1908). Biochemisches Centralblatt, V. 7, p. 767, (1907). (209) Schmidt, A.: Neue Beobachtungen zur Erklarung und rationellen Behandlung der chronischen habituellen Obstipation. Miinchener medizinische Wochenschrift, V. 52, p. 1970, (1905). (210) Schmddt, C. : Ueber Pflanzenschleim und Bassorin. Liebig's Annalen, V. 51, p. 56, (1844). (211) Schmddt and Lohrisch: Weitere Beobachtungen iiber die Bedeutung der Zellulose (Hemicellulose) fur die Ernahrung der Diabetiker. Deutsche medizi- ische Wochenschrift, No. 47, p. 1, (1908). (212) Schulze: Ueber die Zellwandbestandtheile der Cotyledonen von Lupi- nus luteus und Lupinus angustifolius, und iiber ihr Verhalten wahrend des Keim- ungsvorganges. Zeitschrift fur physiologische Chemie, V. 21, p. 392, (1896). (213) Schulze : liber die zur Gruppe der stickstoff freien Extractstoffe gehoren- den Pflanzenbestandtheile. Journal fur Landwirtschaft, V. 52, (1904). (214) Schulze: Zur Kenntnis des /3-Galactans. Berichte der deutschen chemischen Gesellschaft, V. 25, p. 2213, (1893). (215) Schulze: Ueber das Vorkommen eines unloslichen Schleimsaure gebenden Kohlehydrats in den Rothklee- und Luzerne-pfianzen. Die Landwirt- schaftliche Versuchs-Stationen, V. 42, p. 9, (1893). (216) Schulze: Zur Kenntnis der in den Leguminosensamen enthaltenen Kohlehydrate. Ibid., V. 41, p. 207, (1892). (217) Schulze, Steiger and Maxwell: Zur Chemie der Pflanzenzellmem- branen. Zeitschrift fur physiologische Chemie, V. 14, p. 227, (1890). 376 Mary Davies Swartz, (218) Schulze and Castoro: Beitrage zur Kenntnis der Hemicellulosen. Ibid., V. 37, p. 41, (1902); V. 39, p. 318, (1904). (219) Schulze and Steiger: Untersuchungen uber die stickstofffreien Reser- vestoffe der Samen von Lupinus luteus und iiber die Umwandlungen derselben wahrend des Keimungsprozesses. Die Landwirtschaftliche Versuchs-Stationen, V. 36, p. 391, (1889). (220) Sebor: Ueber die Kohlenhydrate des Carragheen-Moos. Botanisches Centralblatt, V. 86, p. 70, (1901). (221) Strauss: Ueber das Vorkommen eini ger Kohlehydratefermente bei Lepidopteren und Dipteren in verschiedenen Entwicklungsstadien. Zeitschrift fur Biologie, V. 52, p. 95, (1908). (222) Ulander: Untersuchungen iiber die Kohlenhydrate der Flechten. Dissertation, Gottingen, 1905. (223) Volt: Ueber die Glykogenbildung nach Aufnahme verschiedener Zuckerarten. Zeitschrift fur Biologie, V. 28, p. 245, (1891). (224) Volt: Ueber das Verhalten der Galactose beim Diabetiker. Zeit- schrift fur Biologie, V. 29, p. 147, (1892). (225) Von Planta and Schulze: Ueber ein neues krystallisirbares Kohle- hydrat. Berichte der deutschen chemischen Gesellschaft, V. 23, p. 1692, (1890). (226) Weinland: Ueber die Bildung von Glykogen nach Galactose-fiitterung. Zeitschrift fur Biologie, V. 40, p. 374, (1900). V. MANNANS. (227) Bertrand: Sur la presence de la mannocellulose dans le tissu ligneux des plantes gymnospermes. Comptes Rendus, V. 129, p. 1025, (1899). (228) Bierry and Giaja: Sur la digestion des mannanes et des galactanes. Comptes Rendus de la Societe de Biologie, V. 60, p. 945, (1906). (229) Bierry and Giaja: Digestion des mannanes et des galactanes. Comptes Rendus, V. 148, pp. 735 and 507, (1909). (230) Brown and Morris: On the Existence of a Cellulose-dissolving Enzyme (Cytohydrolyst) in the Germinating Seeds of the Grasses. Journal of the Chemical Society, London, V. 57, p. 497, (1890). (231) Bourquelot and Herissey: Sur la composition de l'albumen de la graine du Phoenix canariensis et sur les phenomenes chimiques qui accompagnent la germination de cette graine. Comptes Rendus, V. 133, p. 302, (1901). (232) Bourquelot and Herissey: Sur la composition de l'albumen de la graine de caroubier: production de galactose et de mannose par hydrolyse. Ibid., V. 129, p. 228, (1899). Sur la composition de la graine de caroubier. Ibid., V. 129, p. 391, (1899). (233) Bourquelot and Herissey: Germination de la graine de caroubier: production de mannose par un ferment soluble. Ibid., V. 129, p. 614, (1899). (234) Bourquelot and Herissey: Sur les ferments solubles produits pendant la germination par les graines a albumen corne. Comptes Rendus, V. 130, p. 42, (1900). (235) Bourquelot and Herissey: Sur l'individualite de laseminase, ferment soluble secrete par les graines de legumineuses a albumen corne pendant la germina- tion. Ibid., V. 130, p. 340, (1900). Nutrition Investigations . 377 (236) Bourquelot and Herissey: Sur la mecanisme de la saccharification dcs mannanes du corrozo par la seminase de la luzerne. Ibid., V. 136, p. 404, (1903). (237) Castoro: Beitrage zur Kenntnis der Hcmicellulosen. Zeitschrift fiir physiologische Chemie, V. 49, p. 96, (1906). (238) Cremer: Verhalten einiger Zuckerarten im tierischen Organismus. Zeitschrift fur Biologie, V. 29, p. 484, (1892). (239) Dillingham: A Contribution to the History of the Use of Bark Bread. Bulletin of the Bussey Institution, Vol. Ill, Part V. 120, (1906). (240) Dubat: Composition des hydrates de carbone de reserve de I'albumen des graines de quelques Liliacees et en particulier du Petit Haux. Comptes Rendus, V. 133, p. 942 (1901). (241) Effront: Sur une nouvelle enzyme hydrolytique, "la caroubinase." Ibid., V. 125, pp. 38 and 116, (1897). Sur la caroubinase. Ibid., V. 125, p. 309, (1897). (242) Fischer and Hirschberger: Ueber Mannose. Berichte der deutschen chemischen Gesellschaft, V. 21, p. 1805, (1888); V. 22, pp. 365 and 1155, (1889). (243) Franck: Ueber die anatomische Bedeutung und die Enstehung der vegetabilischen Schleime. Jahrbucher fiir wissenschaftliche Botanik, V. 5, p. 161, (1866). (244) Gans and Tollens: Ueber die Bildung von Zuckersaure aus Dextrose haltenden Stoffen, besonders aus Rafhnose, und iiber die Untersuchung einiger Pflanzenschleimarten. Liebig's Annalen, V. 249, p. 215, (1888). (245) Gatin: Action de quelques diastases animales sur certaines mannanes. Comptes Rendus de la Societe de Biologie, V. 58, p. 847, (1905). (246) Gatin and Gatin: tiber die Verdaulichkeit der Mannanen durch Dias- tasen der hoheren Tiere. Chemisches Centralblatt, 1907 (2), p. 1181. (247) Gatin: Isomerisation de mannose en glycose sous Taction d'un fermenl soluble. Comptes Rendus de la Societe de Biologie, V. 64, p. 903, (1908). (248) Girand: Etude comparative des gommes et des mucilages. Comptes Rendus, V. 80, p. 477, (1875). (249) Goret: Sur la composition de I'albumen de la graine de fevier d'Ameri- que (Gleditschia triacanthos L., Legumineuses) . Comptes Rendus, V. 131, p. 60, (1900). (250) Gruss: Studien iiber Reserve-Cellulose. Botanisches Centralblatt, V. 70, p. 242, (1897). (251) Gruss: Ueber den Umsatz bei der Keimung der Dattel. Berichte der deutschen botanischen Gesellschaft, V. 20, p. 36, (1902). (252) Herissey: Sur la digestion de la mannane des tubercules d'Orchidees. Comptes Rendus, V. 134, p. 721, (1902). (253) Herissey: Recherches chimiques et physiologiques sur la digestion des mannanes et galactanes par la seminase chez les vegetaux. Revue Generale de Botanique, 1903, p. 345. (254) Hilger: Zur Kenntnis der Pflanzenschleime. Berichte der deutschen chemischen Gesellschaft, V. 36, p. 3198, (1903). (255) Kano and Iishima: Bulletin of the College of Agriculture. Tokyo Im- perial University, II, No. 2, 1894. (Cited by Day, Bulletin 202. Office of Ex- periment Stations, United States Department of Agriculture.) 378 Mary Davies Swartz, (256) Kinoshita: On the Occurrence of two kinds of Mannan in the Root of Conophallus Konjaku. Bulletin of the College of Agriculture, Tokyo Imperial University, No. V, p. 205, (1902). (257) Kinoshita: On the Occurrence of Mannan. Ibid., No. V, p. 253, (1902). (258) Meigen and Spreng: Ueber die Kohlehydrate der Hefe. Zeitschrift fur physiologische Chemie, V. 55, p. 48, (1908). (259) Mulder: Ueber Pflanzenschleim. Journal fur praktische Chemie, V. 37, p. 334, (1846). (260) Neuberg and Mayer: Schicksal der drei Mannosen im Kaninchenleib. Zeitschrift fur physiologische Chemie, V. 37, p. 530, (1902). (261) Newcombe: Cellulose Enzymes. Annals of Botany, V. 13, p. 49, (1899). (262) Niebling: Untersuchungen iiber die kiinstliche Verdauung landwirth- schaftlicher Futtermittel nach Stutzer, und Pepsinwirkungen im allgemeinen. Landwirtschaftliche Jahrbucher, V. 19, p. 149, (1890). (263) Pohl: Ueber die Fallbarkeit colloider Kohlenhydrate durch Salze. Zeitschrift fur physiologische Chemie, V. 14, p. 159, (1890). (264) Reiss: Ueber die Natur der Reserve-Cellulose. Berichte der deutschen botanischen Gesellschaft, V. 7, p. 322, (1889). (265) Rosenfeld: Untersuchungen iiber Kohlehydrate. Centralblatt fur innere Medizin, V. 21, p. 177, (1900). (266) Sachs: Zur Keimungsgeschichte der Dattel. Botanische Zeitung, 1362, p. 241. (267) Sawamura: On the Liquefaction of Mannan by Microbes. Central- blatt fur Bakteriologie, Abtheilung II., V. 11, p. 21, (1903-4). (268) Sawamura: On the Digestive Power of the Intestinal Canal. Bulletin of the College of Agriculture, Tokyo Imperial University, No. V, p. 155, (1902). (269) Schellenberg : Untersuchungen iiber das Verhalten einiger Pilze gegen Hemicellulosen. Flora, V. 98, p. 257, (1908). (270) Schmidt, C: Ueber Pflanzenschleim und Bassorin. Liebig's Annalen V. 51, p. 29, (1844). (271) Storer: Testing for Mannose. Bulletin of the Bussey Institution, Vol. Ill, Part II, p. 13, (1902). (272) Storer: Notes on the Occurrence of Mannan in the Wood of Some Kinds of Trees, and in Various Roots and Fruits. Ibid., V. Ill, Part III, p. 47, (1903). (273) Schulze: Zur Chemie der pflanzlichen Zellmembranen. Zeitschrift fur physiologische Chemie, V. 16, p. 387, (1892). (274) Schuster and Liebscher: Der Nahrwerth der Steinnussspahne. Landwirthschaftliche Jahrbucher, V. 19, p. 143, (1890). (275) Strauss: Ueber das Vorkommen einiger Kohlehydratefermente bei Lepidopteren und Dipteren in verschiedenen Entwicklungsstadien. Zeitschrift fur Biologie, V. 52, p. 95, (1908). (276) Thamm: Ein Beitrag zur Kenntnis der Pflanzenschleime. Dissertation, Miinchen, 1903. (277) Tollens and Gans: Mannose oder Isomanitose aus Salepschleim. Berichte der deutschen chemischen Gesellschaft, V. 21, p.2150, (1888). (278) Tollens and Oshima: Uber das Nori aus Japan. Berichte der deut- schen chemischen Gesellschaft, V. 34, p. 1422, (1901). (279) Tollens and Widstoe : Uber die Reactionen des Methvl-Furfurols und der Methyl-Pentosane. Ibid., V. 33, p. 132, (1900). Nutrition Investigations. 379 (280) Tsuji: Mannan as an Article of Human Food. Bulletin of the College of Agriculture, Tokyo Imperial University, No. II, p. 103, (1894). (281) Tsukamato: Ueber die Bildung von Mannan in Amorphophallus Konjak. Chemisches Centralblatt, V. 97a, p. 930, (1897). (282) Van Ekenstein: Sur la caroubinose et sur la d-mannose. Comptes Rendus, V. 125, p. 719, (1897). (283) Voit: Ueber die Aufnahme des Pflanzenschleims und des Gummis aus dem Darme in die Safte. Zeitschrift fur Biologie, V. 10, p. 59, (1874). (284) Weiske: Versuche iiber die Verdaulichkeit und den Nahreffect des Jobannisbrodes. Journal fur Landwirthschaft, V. 27, p. 321, (1879). (285) Zanotti: Untersuchungen iiber einige zusammengesetzte Kolhehy- drate. Chemisches Centralblatt, V. 99a, p. 1209, (1899). VI. LEVULANS. (286) Bierry: Recherches sur la digestion de Pinuline. Comptes Rendus de la Societe de Biologie, V. 59, p. 256, (1905). (287) Bierry: Recherches sur la digestion de l'inuline. Comptes Rendus, V. 150, p. 116, (1910). (288) Bierri and Porter: Recherches sur la digestion de l'inuline. Ibid., V. 52, p. 423, (1900). (289) Bourquelot: Inulase et fermentation alcoholique indirecte de l'inuline. Comptes Rendus, V. 116, p. 1143, (1893). (290) Bourquelot: Remarques sur les ferments solubles secretes par l'As- pergillus et le Penicillium. Comptes Rendus de las Societe de Biologie, V. 9, p. 653, (1893). (291) Chevastelon: Sur l'inuline d'ail, de la jacinthe, de l'asphodele et de la tub£reuse. Journal de Pharmacie, V. 4, p. 2, (1895). (Cited by Dean.) (292) Chittenden: The Behavior of Inulin in the Gastro-intestinal Tract. American Journal of Physiology, V. 2, p. XVII, (1898). (293) Dean: Experimental Studies on Inulase. Botanical Gazette, V. 35, p. 24, (1903). (294) Dean: On Inulin. American Chemical Journal, V. 32, p. 69, (1904). (295) Ducamp: Beitrag zum Studium der Unterscheidung des Colibazillus, Wirkung der Bazillen der Colityphusruhrgruppe auf die Kohlehydrate. Jahres- bericht fur Thierchemie, V. 37, p. 952, (1907). (296) Ekstrand and Joh anson : Zur Kenntnis der Kohlehydrate, I. Berichte der deutschen chemischen Gessellschaft, V. 20, p. 3310, (1887). Zur Kenntnis der Kohlehydrate, II. Ibid., V. 21, p. 594, (1888). (297) Finn: Experimentelle Beitrage zur Glycogen- und Zuckerbildung in der Leber. Arbeiten aus dem physiologischen Laboratorium, Wiirzburg, 1877. (Cited by Miura.) (298) Fitz: Ueber Schizomycetengahrungen. Berichte der deutschen chemi- schen Gesellschaft, V. 11, p. 42, (1878). (299) Frerichs: Zur Glykogenbildung in der Leber. Dissertation, Wiirz- burg, 1876. (Cited by Miura.) (300) Green: On the Germination of the Jerusalem Artichoke (Helianthus tuberosus). Annals of Botany, V. I, p. 223, (1888). (301) Harlay: De l'hydrate de carbone de reserve dans les tubercules de 'avoine a chapelets. Comptes Rendus, V. 132, p. 423, (1901). 380 Mary Dames Swartz, (302) Kiliani: Ueber Inulin. Liebig's Annalen, V. 205, p. 145, (1880). (303) Komanos: Ueber die Verdauung des Inulins und seine Verwendung bei Diabetes Mellitus. Dissertation, Strassburg, 1875. Jahresbericht fur Thier- chemie, V.6, p. 180, (1876). (304) Kobert: Ueber einige Enzyme wirbelloser Tiere. Pfliiger's Archiv fur Physiologie, V. 99, p. 116, (1903). (305) Kulz: Beitrage zur Pathologie und Therapie des Diabetes Mellitus. Jahresbericht fur Thierchemie, V. 4, p. 448, (1874). (306) Kulz: Beitrage zur Kenntnis des Glycogens. Centralblatt fur Physio- logie, 1890, p. 789. (307) Levy: De la fermentation alcoholique des topinambours, sous l'influence des levures. Comptes Rendus, V. 116, p. 1381, (1893). (308) Lindner: Garversuche mit verschiedenen Hefen und Zuckerarten. Chemisches Centralblatt, 1901, p. 56. (309) v. Lippmann: Ueber das Lavulan, eine neue, in der Melasse der Riiben- zucker-fabriken vorkommende Gummiart. Berichte der deutschen chemischen Gesellschaft, V. 11, p. 57. (1881). (310) Luchsinger: Zur Glykogenbildung in der Leber. Pfluger's Archiv fur Physiologie, V. 8, p. 289, (1874). (311) Mendel and Mitchell: On the Utilization of Various Carbohydrates without Intervention of the Alimentary Digestive Processes. American Journal of Physiology, V. 14, p. 239, (1905). ' (312) Mendel and Nakaseko: Glycogen Formation after Inulin Feeding. Ibid., V. 4, p. 246, (1900). (313) Miura: Wird durch Zufuhr von Inulin die Glykogenbildung gesteigert? Zeitschrift fur Biologie, V. 32, p. 255, (1895). (314) Reidemeister: Sinistrin, Levulin, und Triticin. Chemisches Central- blatt, 1880, p. 808; Jahresbericht fur Thierchemie, V. 11, p. 68, (1881). (315) Richaud: Sur quelques points relatifs a l'histoire physiologique de l'inuline chez les animaux. Comptes Rendus de la Societe de Biologie, V. 52, p. 416, (1900). (316) Saiki: Anti-inulase. Journal of Biological Chemistry, V. 3, p. 395, (1907). (317) Sandmeyer: Ueber die Folgen der partiellen Pancreasextirpation beim Hund. Zeitschrift fur Biologie, V. 31, p. 32, (1895). (318) Schmiedeberg: Ueber ein neues Kohlehydrat. Zeitschrift fur physio- logische Chemie, V. 3, p. 112, (1879). (319) Strauss: Ueber das Vorkommen einiger Kohlehydrate fermente bei Lepidopteren und Dipteren. Zeitschrift fur Biologie, V. 52, p. 95 (1908) . (320) Tanret: Sur la levulosane, nouveau principe immediat des cereales Comptes Rendus, V.: 112, p. 293, (1891). (321) Tanret: Sur l'inuline et deux principes immediats nouveaux. Ibid., V. 116, p. 514, (1893). (322) Tanret: Sur les hydrates de carbone du topinambour. Ibid., V. 117, p. 50, (1893). (323) Tanret: Sur une nouvelle glucosane, la levoglucosane. Ibid., V. 119, p. 158, (1894). (324) Von Mering: Zur Glykogenbildung in der Leber. Pfluger's Archiv fur Physiologie V. 14, p. 274, (1877). Nutrition Investigations . 381 (325) Wallach: Zur Kcnntnis der Kohlchydrate. Liebig's Annalen, V. 234, p. 364, (1886). (326) Weinland: Ueber das Auftreten von Invertin im Blut. Zeitschrift fur Biologie, V. 47, p. 280, (1906). (327) Went: Monilia sitophila (Mont.) Sacc, ein technischer Pilz. Che- misches Centralblatt, V. (1901) b, p. 650. (328) Winogradsky: Clostridium pastorianum, seine Morphologie und seine Eigenschaften als Buttersaureferment. Chemisches Centralblatt, V. 1902, b, p. 709. VII. DEXTRANS. (329) Bauer: Ueber eine aus Leinsamenschleim entstehende Zuckerart. Die Landwirtschaftlichen Versuchs-Stationen, V. 40, p. 480. (330) Bauer: Ueber eine aus Laminariaschleim entstehende Zuckerart. Berichte der deutschen chemischen Gesellschaft, V. 22, p. 618, (1899). (331) Bauer: Ueber die Arabonsaure und die aus Lichenin entstehende Zuckerart. Journal fur praktische Chemie, V. 34, p. 46, (1886). (332) Berg: Jahresbericht fur Chemie, 1873, p. 848. (From Russ. Zeitschr. Pharm., 1873, pp. 129 and 161.) (333) Berzelius: Recherches sur la nature du lichen d'Islande, et sur son emploi comme aliment. Annales de Chimie, V. 90, p. 277, (1814). (334) Brown: Notes on Cetraria Islandica. American Journal of Physiology, V. I, p. 455, (1898). (335) Escombe: Chemie der Membranen der Flechten und Pilze. Zeitschrift fur physiologische Chemie, V. 22, p. 288, (1896). (336) Honig and St. Schubert: Ueber Lichenin. Monatshefte fur Chemie, V. 8, p. 452, (1887). (337) Klason: tiber die durch Inversion von Lichenin entstehende Zucker. Berichte der deutschen chemischen Gesellschaft, V. 19, p. 2541, (1886). (338) Meyer: Ueber Reservestoffe, Kerne und Sporenbildung der Bakterien. Chemisches Centralblatt, V. 1900, b. p. 56, (1900). (339) Meigen and Spreng: Ueber die Kohlehydrate der Hefe. Zeitschrift fur physiologische Chemie, V. 55, p. 49, (1908). (340) Mendel: Das Verhalten einiger unverdaulicher Kohlehydrate im Ver- dauungstrakt. Zentralblatt fur die gesammte Physiologie und Pathologie des Stoffwechsels, No. 17, p. 1, (1908). (341) Muller, Karl: Die Chemische Zusammensetzung der Zellmembranen bei verschiedenen Kryptogamen. Zeitschrift fur physiologische Chemise, V. 45, p. 264, (1905). (342) Nilson: Kenntnis der Kohlenhydrate in den Flechten. Upsala Lakareforenings Forhandlingar, V. 28. (Jahresbericht fiir Thierchemie, V. 23, p. 53, (1893). (343) O'Sullivan: Amylam in Wheat, Rye and Barley. Chemical News, V. 44, p. 258, (1881). (344) Poulsson: Untersuchungen iiber das Verhalten einiger Flechten- kohlehydrate im menschlichen Organismus und iiber die Anwendung derselben bei Diabetes-Mellitus. Festschrift fiir Olof Hammarsten XIV, Upsala Lakarefore- nings Forhandlingar, (1906). 382 Mary Dames Swarlz. (345) Rothentusser : Der Schleimkorper des Leinsamens. Dissertation, Miinchen, 1903. (Jahresbericht fur Thierchemie, V. 34, p. 78, 1904. (346) Saikj: The Digestibility and Utilization of Some Polysaccharide Carbo- hydrates derived from Lichens and Marine Algae. Journal of Biological Chem- istry, V. 2, p. 251, (1906). (347) Torup: A New Carbohydrate from Laminaria Digitata. Biochemisches Centralblatt, V. 8, p. 770, (1909). (From Pharmacia, V. 6, p. 153.) (348) Ulander: Untersuchungen iiber die Kohlenhydrate der Flechten. Dissertation, Gottingen, (1905). (349) Voit: Hermann's Handbuch der Physiologie, V. 6, p. 413, (1881). (350) Winterstein: Zur Kenntnis der in den Membranen der Pilze enthal- tenen Bestandtheile. Zeitschrift fur physiologische Chemie, V. 19, p. 521, (1895). V. 21, p. 152, (1897). (351) Von Mering: Zur Glykogenbildung in der Leber. Pfliiger's Archiv fiir. Physiologie, V. 14, p. 274, (1877). (352) Yos,hihura: Ueber einige Pflanzenschleime. Jahresbericht fiir Thier- chemie, V. 25, p. 51, (1895). (Bulletin II, No. 4, College of Agriculture, Tokyo Imperial University.) A CONSIDERATION OF SOME CHEMICAL TRANSFORMA- TIONS OF PROTEINS AND THEIR POSSIBLE BEAR- ING ON PROBLEMS IN PATHOLOGY* FRANK P. UNDERHILL NEW HAVEN, CONN. Recent investigations concerning the structure of proteins have led to a readjustment of our ideas with respect to the manner in which these substances may become an integral part of the organism; and the study of the changes which occur from the time when protein has been built up into cell structure until its exit from the body in the form of waste products is at present only in its infancy. According to the newer conception, the protein molecule is a huge complex consisting of the union of a large number of simple amino-acids. This conception is due largely to the researches of Emil Fischer, who has succeeded in fastening together various combinations of amino-acids in such a manner that the resulting compound behaves in some respects like certain of the proteins. Some of these substances, called polypeptids, have indeed been isolated from the decomposition of native protein. According to Fischer, proteins are merely mixtures of complex polypeptids and cannot be considered as chemical individuals. This view, however, is not shared by other protein chemists. In its passage through the alimentary canal the large protein complex undergoes a degradation, — a transformation into simpler molecules which are still regarded as simple proteins, together with a long chain of amino- acids belonging to the aliphatic, aromatic and heterocyclic series. Our modern view of the nature of protein forbids the acceptance of the older idea that the necessity for alimentary treatment of protein is merely to transform protein into a condition suitable for absorption. Obviously, solubility and diffusibility are only a small portion of the process ; other- wise it would be unnecessary to entail so much labor on the gastro-enteric tract by the apparently needless formation of amino-acids. The need for nitrogenous substances is to replace cellular structures worn out through metabolic activities. Just how this replacement occurs is problematical. Certain it is that the only detectable nitrogenous food supply for such structures is to be found in the proteins of the blood. No amino-acids or other protein decomposition products have been isolated from the * The Middleton Goldsmith Lecture for 1911; read before the New York Pathological Society at the Academy of Medicine, March 18, 1911. 2 blood. 71 ' 3 An explanation for the necessity for the extensive disintegra- tion of the protein molecule has been offered as follows : "Every species of animal — in fact, every individual — has its specifically con- stituted tissues and cells. If the diet were always the same, the formation of the tissues might bear some close relation to the components of the food. The diet varies, however, and, especially in the case of human beings and the omnivora, is exceedingly diverse in nature and to make its organism independent of the outer world in the matter of food taken, it disintegrates the nutrient it receives, and utilizes those components which may be of service to it in building up new complexes." 1 Many important functions have been attributed to the intestinal wall, but perhaps none of more significance than a selective power for the synthesis of amino-acids into serum albumins. According to this view, the intestine receives a series of more or less simple amino-acids and by uniting them in varying proportions forms definite compounds, probably serum proteins, to meet the organism's requirements. The serum proteins are then drawn on to furnish nitrogen requirements for the specific organ or group of cells. This probably entails a further transforma- tion. Such a theory accounts for the necessity of the complete degrada- tion of protein in the gastro-enteric canal and accords with the entire absence of cleavage products in the blood. On the other hand, this extensive degradation, synthesis and further disintegration appears quite uneconomical physiologically. In the first place, only a comparatively small amount of nitrogen is needed to rebuild tissue, and the excess must be transformed into some form easy of elimination, all of which entails loss of energy and tissue waste. It is a matter of common observation that when protein is fed, practically all the nitrogen is rapidly excreted, and one is inclined strongly to believe that this portion has never been re-synthesized into protein. If amino-acids or other decomposition products could be found in the blood, the solution of the problem would be at hand. The failure to discover them does not disprove their presence, however, since the quantity in the portal vein, at any one moment, may be so small as to escape detection with our present methods. Whichever way one views this problem, the fact remains that sooner or later intermediary processes must be concerned, primarily with a series of amino-acids. These are the substances that must be metabolized in either case, and it is to some of the transformations that these com- pounds may undergo within the organism that I particularly desire to call attention. It is my purpose to indicate the possible bearing of certain types of intermediary processes upon problems in pathology, for it is my belief that the hope for a complete understanding of some of the phases of disease will be realized in proportion as our knowledge of intermediary processes increases. 3 DEAMINATION AND THE SIGNIFICANCE OF AMMONIA IN THE BODY Modem investigations teach that when amino-acids obtain entrance to the tissues, a process of deamination rapidly occurs; the nitrogen is split off in the form of ammonia. In other words, the amino-acid is converted into a nitrogenous and a non-nitrogenous portion. It is probable that this process of deamination takes place for the most part in the liver, although the liver is by no means the only organ capable of performing this reaction, as has been pointed out by Jacoby 51 and Lang. 57 The older observation of Nenki, that blood flowing from the intestine is richer in ammonia than the blood of any other vessel, may also be regarded as probably our first indication that the intestinal wall may carry out the process of deamination. Ordinarily, the ammonia split off reappears in the urine as urea, being synthesized into this closely related compound in the liver. That deamination occurs also with certain protein complexes, has been conclusively demonstrated by Cohnheim. 24 The further transformation of the non-nitrogenous part will be consid- ered later. Under normal conditions the quantity of ammonia excreted in the urine varies within certain well defined limits, and in general is directly proportional to the intake and total output of nitrogen. The elimination of ammonia in disease may vary enormously, and usually when a changed excretion is observed it is in the direction of a greatly increased output. In fact, ammonia output in the urine is probably more easily affected than the elimination of any other single urinary constituent, except the excretion of urea which bears a reciprocal relation to ammonia. If the literature relating to ammonia output in disease is reviewed, it is found that increased ammonia excretion 77 is characteristic of pathological con- ditions apparently widely diverse in nature. For instance, an augmented ammonia output has been observed in cholera, intestinal hepatitis, carcinoma of the liver, cirrhosis of the liver, pneumonia, polyarthritis, typhoid, various other fevers, acute uremia, phosphorus poisoning, gastro-enteritis, starvation, diabetes, pernicious vomiting of pregnancy, eclampsia, etc. Ammonia in the normal urine is looked on to-day as an index to the quantity of acids present. Acids are toxic to the organism, as has been demonstrated repeatedly, and ammonia is diverted from its trans- formation into urea to neutralize these acid radicles. 42 ' 44 » 25 > 31 > 52 The same idea prevails with respect to diseased conditions in which increased ammonia in the blood and urine is regarded as evidence of increased production of acid radicles. 77 It is a well-known observation that this excessive elimination may be greatly diminished by feeding other alkalies, as, for instance, in diabetes. If ammonia is employed in the tissues merely to neutralize acid radicles, it would seem fair to assume that if 4 sufficient alkali were introduced, no ammonia should appear in the urine. Experimentally, such a condition has never been brought about in spite of very large doses of alkali. From this fact it would appear that a certain quantity of ammonia is continually present in the blood. This holds true also for the herbivora, even though these animals are assumed to neutralize acid radicles by alkalies other than ammonia. Throughout the literature relating to acidosis and ammonia excretion in the urine, emphasis is laid on acid radicles as toxic agents. Little or no attention has been devoted to the role which may be played by ammonia itself when viewed from the same standpoint. As a matter of fact, ammonia is exceedingly poisonous and few salts exceed those of the ammonium series in comparative toxicity. On the other hand, of the acid radicles which are supposed to exert such deleterious effects, as, for example, in diabetes, not one has been shown to have any strikingly toxic properties, at least in the normal organism. From these statements the possibility is offered that increased ammonia output in the urine may really mean increased ammonia production resulting from decreased urea formation. Experimentally, there is no evidence that such may not be the case, and according to this view ammonia must be neutralized, which would account for the presence of organic acids in the urine, as in diabetes, starvation, liver disorders, etc., precisely as the introduction of camphor, menthol, thymol, etc., accounts for the appearance of glycuronic acid in the urine. In this connection another interesting problem presents itself. In diabetes, beta-oxybutric acid is the acid formed in large quantity, whereas in other conditions, as in cirrhosis of the liver, lactic acid is found. As a rule, the two do not occur together except in starvation and in pathological conditions in which inanition is an accompaniment. The presence of these two compounds in the urine would indicate two different types of mechanism slightly diverted from the normal. 93 In entire accord with the theory that ammonia may be the toxic agent are the observations of Carlson 21 and Jacobson. 49 The former has shown an increased quantity of ammonia in the blood after complete thyroidectomy and parathyroidectomy. The latter has demon- strated that the concentration of ammonia in the blood necessary to produce experimental ammonia tetany is practically equal to that found during parathyroid tetany. "This supports the view that the increased ammonia in the blood of parathyroidectomized animals is directly respon- sible for the tetany and the depressive symptoms." 49 If, on the assumption that a common law underlies all the various pathological conditions mentioned, one attempts to account for the pres- ence of ammonia in the urine, the suggestion presents itself that it is in some way connected with the metabolism of the carbohydrates. In practi- cally every condition in which ammonia in the urine is characteristic, 5 there is either a lack of carbohydrates, as in prolonged starvation, or a faulty utilization of carbohydrate, as in diabetes, cirrhosis of the liver, or after complete removal of the thyroids and parathyroids, etc. 92 It would appear, therefore that carbohydrate is the factor controlling the regulation of ammonia production. 93 Thus in prolonged starvation, ammonia in the urine may be greatly diminished by feeding carbo- hydrate. 77 ' 93 In the solution of the problem as to the part played by carbohydrate, at least two possibilities are presented: 1. Carbohydrate influences the output of ammonia indirectly by its effect on the combus- tion of fat, for it is well known that fat is burned much more readily when carbohydrate is present than when the store of this substance in the body is greatly depleted. 2. It may be possible that the liver cell, for instance, is incapable of effecting urea synthesis in the presence of insuf- ficient carbohydrate in that organ. The latter hypothesis certainly pre- sents interesting problems, both for the physiologist and for the patholo- gist. Attempts have been made in our laboratory to follow this hypothesis to its logical conclusion. The results thus far obtained, however, have not been far-reaching for the reason that it is exceedingly difficult to obtain the proper experimental conditions. The investigation has been carried out on animals entirely. In the first place, an endeavor has been made to remove the carbohydrate store from the animal's body by various means with the hope of inducing increased ammonia in the urine. Prolonged starvation, phosphorus poisoning, a combination of the two, phloridzin intoxication alone and together with prolonged inanition, and cocain poisoning have proved unsuccessful in inducing increased output of ammonia. This result is in harmony with those obtained by other inves- tigators (Jackson and Pearce, 48 Eichards and Wallace, 83 Underhill and Kleiner 94 ). These facts by no means invalidate the hypothesis, for it is well known from the researches of Pniiger and his pupils that it is exceedingly difficult to get dogs glycogen-free. Since it is well known that dogs are refractory in this respect, inanition experiments were car- ried out also with the pig and rat, omnivora, and hence, presumably, allied to man in metabolism. The results were in entire accord with those with the dog. Ammonia in the urine was not increased. From this point of view it seems most probable that the decision of this question can be obtained most readily with the human subject. If ammonia is to be regarded merely as an alkali for neutralization purposes, rather than as a toxic agent per se, it may be cited as a splendid illustration of the "factor of safety" principle enunciated by Meltzer 67 for other mechanisms. Ordinarily, ammonia is transformed to urea, but if acid radicles are floating in the body fluids they are rendered inert by union with ammonia, hence less urea is formed. Therefore, increased ammonia in the urine, with a corresponding diminution in the urea may 6 be regarded as an indication that the body is endeavoring to maintain normal conditions and not that there is necessarily any inability to form nrea on the part of the organism, or that lesions in one or another organ are necessarily related to increased ammonia output. Indeed, even when acute yellow atrophy of the liver is at its height, the quantity of ammonia in the urine may be normal. 77 Closely associated with problems concerning the significance of ammonia, are those having to do with the relation of this substance to the amino-acids and the mechanism of deamination. Within the last few years discussion of the relation of so-called defective or insufficient deami- nation in a series of pathological conditions has come into vogue. It has been assumed that the liver was incapable of performing its function and that the condition was indicated by high ammonia and a high unde- termined nitrogen in the urine. It does not seem to have occurred to those who advocate this point of view that high ammonia and the pres- ence of amino-acids in the urine present incongruous conceptions if the sum of urea and ammonia nitrogen is normal. In other words, if deami- nation were defective, one would expect low ammonia in the urine when amino-acids are present. Much stress has been laid latterly on the occur- rence of amino-acids in the urine. In health a little glycocoll has been isolated in the urine, which is not strange when one considers the role of glycocoll as a compound intended to render toxic substances innocuous as in the case of benzoic acid, and the comparative difficulty with which glycocoll is burned. Leucin and tyrosin have been isolated from the urine of patients with acute yellow atrophy, and with phosphorus poisoning. 75 According to Ignatowski, 47 > 4 > 33 > 34 > 104 > 88 > 41 - 65 > 78 glycocoll, leucin, tyrosin and aspartic acid are found in pneumonia and leukemia. Similar finds are obtained in scarlatina and typhoid. 50 ' 39 Insults to the pan- creas 17 may lead to the excretion of a polypeptid which on hydrolysis yields tyrosin, and according to several observers 70 ' 2 a similar compound may be obtained from diabetic urines. As a result of disturbed meta- bolism induced by lack of oxygen, 61 ' 62 amino-acids may appear in the urine as well as after ether-chloroform narcosis. 5 These bodies may also be present in exudates and in the fluids formed in edema. 76 ' 75 To account for the presence of amino-acids in the blood and hence also their excretion by the kidney, it is unnecessary to assume that defec- tive deamination is responsible. They may be produced, as for example, by increased autolysis of a tissue or organ in a part of the body far remote from the organ or organs possessing the power of deamination and may therefore be eliminated through the urine before the organs of deamina- tion have had an opportunity of performing their function. In other words, these compounds may be formed in the muscles, for instance, and be eliminated in a large part by the kidney before the liver, which may be 7 cited as an organ of examination, has had an opportunity of acting on them. In harmony with this idea may be cited the conditions which obtain in this respect in cystinuria. 75 In certain of these patients, the amino-acid, cystin, is eliminated through the urine either alone or in company with other amino-acids, for example, leucin and tyrosin. Appar- ently, the cystinuric patient is incapable of deaminating and burning the cystin. Nevertheless, when cystin 11 ' 102 is fed to such individuals they experience no difficulty in completely deaminating and burning this amino-acid, and it has also been demonstrated that certain, at least of the cystinurics, can handle other amino-acids, as tyrosin 90 and aspartic acid, 103 when fed. The reason for the presence of amino-acids in the urine under the pathological conditions previously cited, may be similar to that fre- quently given for cystinuria, namely, that the cystin eliminated arises as a result of processes within the organism, not necessarily from the food. And in neither case is it necesary to assume defective deamination. Until recently our knowledge concerning the exact mode of decompo- sition of the cleavage products of protein has been extremely limited. It has been assumed for years that the non-nitrogenous portion of the amino- acid is oxidized and hence may be looked on either as a direct source of energy, or as potential energy residing in carbohydrates or fats synthesized from such material. Eecent observations, however, have tended to give us a more enlightened view as to the exact way in which these amino- acids are handled. Thus, from the investigations of Emden, Salomon and Schmidt, 35 it may be seen that leucin added to the blood of a dog and made to pass through the surviving liver causes a considerable increase in the acetone contained in the circulating blood. Under normal conditions some acetone is a product of liver activity. 53 In order to understand the steps necessary for the production of acetone from leucin, certain other facts must be taken into consideration. Knoop 53 has conclusively demonstrated that the aromatic fatty acids are decomposed in the body in such way that there is an oxidation at the beta carbon atom followed by a cleavage in the side chain between the alpha and beta carbon atoms. Thus, phenylbutyric acid is decomposed into acetic acid and phenylacetic acid. The latter appears in the urine, and the acetic acid is presumably decomposed to C0 2 and H 2 0. THE FURTHER FATE OF AMINO-ACIDS COOH phenyl butyric ucd 8 It is perfectly possible that the breaking down of the aliphatic fatty acids takes place in somewhat the same fashion. Eeasoning from this viewpoint it is probable that the transformations which occur in the production of acetone from leucin are as follows : CH 3 CH, CH, CH 3 CH Z CH 3 CH 3 CH, \/ 7 v a r 1 v.C0\/ \/ anamination ^ c»eava 5 e r> r- /■» -f\ CH CH C ' eaW5e . PCH k C=0 CH 2 CH Z CH NH 2 C=0 COOH COOH [too] " acid with ammo acid ketone acid / €SS cai-bon one (Leucn) (Isovaleric add) (Ac*ton%) Leucin first undergoes oxidative deamination by which a ketone acid is formed. Then by oxidative C0 2 cleavage isovaleric acid is produced. Next cleavage takes place between the alpha and beta carbon atoms. On the other hand, Baer and Blum 12 have found an increased output of beta oxybutyric acid in the urine after giving leucin to a diabetic and the following transformations have been attributed to it. CH CH CH CHOH II I I CH 2 > CH, > CHo * CH J I J \ CH.NH 2 CHOH COOH COOH L CH 3 CH, CH 3 CH 3 CH 3 . OXypnenyl <*C«fi'c acid. According to these reactions, a deamination of the aromatic amino- acid must occur in the large intestine. The further reaction for the pro- duction of p. oxyphenylacetic acid involves the cleavage of carbon dioxid from the carboxylic group and subsequent oxidation of the carbon atom. If phenol is produced from cresol, then demethylation must occur. These reactions if correct, must involve the processes of deamination, cleavage of carbon dioxid, oxidation and demethylation. From trypto- phan the following products are formed. C.CHXH 2 .C00H CCH 2 .CH 2 .C00H CXH 2 .COOH C.CH, /? amino tic acid Indol pt-opionic. Acid Xtidol acetic acieC Sfcatol From these reactions it is obvious that the same chemical changes have occurred as in the transformations for tyrosin, namely, deamination, carbon dioxid cleavage, oxidation and finally demethylation. On the other hand, it has been suggested recently that indol may arise in part as a result of intermediary processes quite distinct from those involved in putrefaction. 16 Ordinarily, when intestinal putrefaction is mentioned one invariably thinks of indol and skatol as being responsible for the series of disturb- ances which may be associated with this condition. It has been assumed that a long list of pathological conditions may be closely related to increased intestinal putrefaction. Thus, this condition has been held responsible in part for sciatica, tetany, epilepsy, eclampsia, many forms of dermatitis, cirrhosis, arteriosclerosis, various types of nervous diseases, chlorosis, myxedema, cretinism, pernicious anemia and nephritis. 98 ' 73 12 Although there is abundant clinical evidence that excessive intestinal putrefaction may be associated with or responsible for marked disturb- ances, the substances thus far isolated from intestinal contents can not be said to possess very profound toxicity. It is true that indol when administered in quantities up to 2 gm. per day causes frontal headache, irritability, insomnia and confusion, 45 and it has been shown further that indol and skatol cause muscle to react to stimuli like fatigued muscles. 60 But the comparatively slight toxicity can hardly be responsible for some of the symptoms observed. Various explanations have been offered to account for the discrepancy noted between clinical evidence of intestinal intoxication and the fact that the substances formed thus far isolated are only slightly toxic. The most plausible explanation for the discrepancy mentioned is that the list of compounds which may be formed in putre- faction has not yet been exhausted, and it is possible, and indeed probable, that in time other compounds of putrefactive origin will be found that will adequately account for the clinical symptoms observed. On the other hand, it be possible that indol and skatol may exert quite different effects on the normal organism from those which it exerts on a body whose resist- ant powers have been lowered as a result of other pathological processes. In other words, the receptive condition of the body under the two condi- tions mentioned may be entirely different, producing in turn quite rad- ically differing symptoms. Amines and Their Formation. 13 — That we have by no means isolated all the active principles from putrefactive mixtures may be well illus- trated by the investigations recorded during the last three years. In 1907 Dixon and Taylor 29 aroused considerable interest by the publication of their observation that alcoholic extracts of the human placenta when injected intravenously caused a marked rise in blood-pressure and con- tractions of the pregnant uterus. On repetition of this work, Kosenheim 87 failed to corroborate the findings of Dixon and Ta}4or when extracts of perfectly fresh placentas were employed. When, however, extracts of placentas in various stages of putrefaction were intravenously admin- istered, results were obtained identical with those of Dixon and Taylor. A substance responsible for these effects has been separated and identified oy Barger and Walpole, 15 according to whom the active principle is p. oxyphenylethylamin and may be derived from tyrosin as a result of the following reaction: 0H 0H CH. NK Icool H 13 Moreover, iso-amylamin has been isolated from putrefactive mixtures probably being derived from leucin in accordance with the following reaction : CH, CH- CH CH, V V CH CH. NH. I COO H CH CH Z i CH 2 I (Leucin) I s ©any 1 ami n These results have led the authors to remark that they are induced "to emphasize the probability that the amins which we have isolated are nor- mally formed by putrefaction in the intestine and are absorbed from it." 15 The compound, p. oxyphenylethylamin, is of peculiar interest for sev- eral reasons. In the first place, it was originally isolated by Emerson 36 from an autolysis of pancreas and its mode of formation from tyrosin has always been considered unique. It is obvious at present that it was prob- ably produced by putrefaction in this case also. Much more interest attaches to this substance from its great resemblance, both structurally and pharmacologically, to epinephrin. OH /\ OH p.oxypAenyierAyJam/',, CH. OH ( CH a I ad, "P. oxyphenylethylamin has an action very similar to that of adrenalin [epinephrin], reproducing both the motor and inhibitory effects of nerves of the true sympathetic system. It produces the motor more powerfully than the inhibitory effects. Its action differs from that of adrenalin in being weaker, and slower in onset, and in being less strictly though mainly peripheral. It is absorbed from the subcutaneous tissues and the alimentary canal and produces its effects when so administered." 27 Isoamylamin has a similar action. Finally, it is of exceeding great interest to note that p. oxyphenyl- ethylamin is one of the substances which give to ergot 14 its characteristic 14 action on the uterus. It is also probable that it is identical with the urohypertensin of Abelous and Bardier. 6 These observations also indicate the necessity for controlling all possible sources of bacterial contamination when extracts of animal tissues are employed in demonstration of specific action. Again, "Many observations have been published recently con- cerning the presence in the blood-serum and urine in various pathological conditions, of substances which cause dilatation of the pupil of the enucleated eye of the frog. The fact that both these amins have this action casts some doubt at least on the validity of the assumption, made by certain observers, that the substance in serum, responsible for this effect, is adrenalin." 27 On introduction into the body, the base is elimi- nated as p. oxyphenylacetic acid, 40 another example of deamination. OH OH f>. oty phenyl ethyl- or"' Oxy phenyl • acid amin When histidin is subjected to the action of putrefactive bacteria, a compound 7 is produced which holds promise of being responsible in a measure for certain reactions which have long been unexplained. CH hn /"\r c CH 2 CH^ /3 imina^olylethylam'tn This substance, beta-iminazolylethylamin, resembles in some respects p. oxyphenylethylamin in that both compounds are contained in ergot extracts, and both substances exert similar influences on the muscle of the uterus. In addition to the reactions possessed in common with p. oxyphenylethylamin, beta-iminazolylethylamin is capable of calling forth symptoms practically identical with those induced by injections of peptone solutions, or by serum or other protein in the sensitized guinea- pig, that is, producing anaphylactic shock. 27 The base has also a mild, direct, stimulant effect on the activity of the salivary glands and the pancreas. This secretory action, being paralyzed by atropin, may be regarded as a weak action of the pilocarpin type ; the association has some interest in that pilocarpin also contains an iminazole ring. More recently this base 28 has been isolated from extracts of the intestines, thus lending support to the suggestion that under normal conditions it may exert a more or less definite function in the maintenance of nutritional rhythm. Closely associated with p. oxyphenylethylamin in extracts of placentas is another compound which may also be considered as an amin and which has an action antagonistic to that of p. oxyphenylethylamin. This com- pound, cholin, has been the subject of a great deal of discussion during ^■CH a .CH 2 .0H the last few years. It undoubtedly has its origin in a lipoid compound, lecithin, a constituent of practically all cells. It is, therefore, apparent that in putrefactive processes in the tissues, cholin may arise in relatively large quantity and, although not highly toxic when given by mouth, it may be exceedingly poisonous when allowed to come in contact with nervous tissue. Thus, Donath 30 observed severe tonic and clonic con- vulsions after cholin had been injected directly into the cortex or under the dura. This investigator offers the opinion that cholin may be respon- sible for epileptic convulsions — an opinion founded on the fact that he with other investigators 43 ' 86 has been able to demonstrate the presence of cholin in large quantities in the cerebrospinal fluid of epileptics and in other conditions associated with destruction of nervous tissue. In accord- ance with this idea, unsuccessful attempts 95 have been made to demon- strate the presence in the blood of cholin in animals during the tetanic convulsions caused by complete removal of thyroids and parathyroids. The removal of the glands has been shown to result in marked changes in certain areas of the nervous system, 97 and it was assumed that these histo- logical changes were due to chemical reactions whereby cholin might be liberated and produce a secondary effect. Chemically related to cholin is neurin, a substance easily formed from cholin by oxidation, which is nearly twenty times as toxic as the latter. The possibility presents itself that neurin may be formed from cholin within the organism under certain pathological conditions and may be responsible for some of the symptoms characteristic of certain abnormal conditions as salivation, vomiting, diarrhea and a specific action in caus- ing arrest of respiration. This formation of neurin within the organism and any relationship which it may bear to deranged metabolism has never 16 been conclusively demonstrated. It has been shown, however, that neurin may be excreted, at times at least, through the urine. 56 These compounds, together with muscarin and betain, constitute one of the groups of the ptomains, so-called, and hence heretofore have been interesting chiefly because of their advent into the organism through the introduction of decomposing tissue taken as food. Nevertheless, inas- much as they arise outside the body as a result of putrefactive process, there remains open the possibility that they may be formed in the alimen- tary canal or in tissues either in a process of degeneration or putrefaction. The old idea, that there is a hard-and-fast line to be drawn between plant alkaloids and animal poisons is rapidly disappearing, and the fact that one toxic compound, as for instance muscarin, occurs as a rule in plant tissue does not exclude the possibility of the presence of the same body as a result of chemical reactions within the animal organism. The best recognition of this disappearing demarcation line is to be found in the new publication by Winterstein and Trier, 101 where all basic substances, whether of animal or plant origin, are considered as alkaloids. From this review of the more recently discovered compounds which may arise within the body by putrefactive processes, one fact stands forth with striking clearness, namely, the possible functions which some of these substances may exert in physiological processes and their significance in problems concerned with disease. We have seen that one compound resem- bles epinephrin, both structurally and in physiological activity, while another stimulates the activity called forth by pilocarpin. Epinephrin and pilocarpin are in daily use as drugs, the degree of whose activities may be regulated at will. The protein derivatives mentioned have practically similar actions but, as they arise within the organism, can not be subjected to voluntary control. It is likely that under normal conditions, only small quantities of these compounds may be thrown into the blood-stream and that there is some nice adjustment of mechanism which may have an influence on the further beneficial disposition of these bodies. It is con- ceivable, however, that at times an undue quantity of such material may overwhelm the regulating mechanism to such a degree that substances which perhaps normally aid in the maintenance of physiological rhythm may indeed become responsible for the advent of abnormal reactions. The suggestion is therefore offered that some of the disturbances asso- ciated with excessive intestinal putrefaction may have such an origin. A hypothesis of this sort readily furnishes an explanation for the vaguely cholin neurin. 17 defined headache and general malaise characteristic of the previously men- tioned pathological states. DIAMINES Cystinuria has been given a great deal of consideration by investiga- tors for the excellent opportunity it afforded of elucidating some of the complex processes underlying the principles of intermediary metabolism. It has been and still is a matter of great uncertainty as to the origin and significance of cystin and the diamins, putrecin and cadaverin, which appear in the excretia of certain individuals. The appearance of cystin has been the subject of such widespread interest, and the results of study of cystinuria have been so extensive and are so well known that further discussion of this subject in this place appears superfluous. 75 On the other hand, the diamins have not received so much attention. The structure of these bases is as follows : CH 2 . CH 2 . CH 2 . CH 2 CH 2 . CH 2 . CH 2 . CH 2 . CH 2 NH 2 NH 2 NH 2 NH 2 Tetramethylendiamin Pentamethylendiamin Putrecin Cadaverin A third diamin, neuridin or saprin, is isomeric with cadaverin. The diamins are eliminated, not only in the urine but also in the feces. As has been stated previously, they are found usually associated with cystin, although the condition responsible for the appearance of cystin in the urine does not seem necessary for the condition of diaminuria, for these substances have also been eliminated under certain other pathological conditions ; principally, intestinal disturbances, as for instance, in various infections, in cholera, 85 dysentery, gastro-enteritis, 84 and from one case of pernicious anemia tetramethylendiamin was isolated. 46 The origin of these bodies appears to be in two diamino-acids that are products of normal digestion. From lysin, which has the following formula: CH 2 . CH 2 . CH 2 . CH 2 . CH . COOH NH 2 C0 2 is split off, forming cadaverin, with the following formula : CH 2 .CH. 2 CH 2 .CH 2 .CH 2 NH a NH 2 Putrecin is derived ultimately from arginin, guanidin and amino- valerianic acid. Kossel and Dakin 55 have demonstrated the existence of an enzyme, arginase, in certain tissues of the body which is capable of splitting arginin into urea and ornithin. Thus: CH 2 . CH 2 . CH 2 . CH . COOH NH NH 2 CH 2 .CH 2 .CH 2 .CH.COOH I I I * C-NH 2 NH 2 NH 2 | Ornithin NH 2 Arginin 18 From ornithin, the formula for which is as follows : CH 2 . CH 2 . CH 2 . CH . COOH NHa NH 2 C0 2 is split off, forming putrecin, the formula below : CH 2 . CH 2 . CH 2 . CH 2 NH 2 l!fH 2 In intestinal disturbances, it is probable that these compounds are the result of the bacterial activity — indeed they may be the metabolic prod- ucts eliminated by microorganisms. In cystinuria, however, it is possible that a different 63 explanation for diaminuria is pertinent. It may be assumed, for instance, that in the beginning cystinuria and diaminuria are brought about through a similar, or indeed the same cause, or causes, for example, a gradually changing type of metabolism induced by some unknown agency, resulting in an anomaly of metabolism. If the anomaly is slight in character, cystin alone is eliminated as a result, whereas if the change in metabolism is sufficiently pronounced diamins are also excreted. If this assumption is accepted, it is easy to explain why in some cases of cystinuria the diamins are absent, and that gradually one or both of these compounds disappear, that cystinuria persists, but that cystinuria does not cease and leave diaminuria. Thus far all attempts to produce diaminuria experimentally have been unsuccessful. These bodies are possessed of a certain interest, aside from their chemical significance in that, like nearly all the amins, they are more or less toxic. It has been demonstrated experimentally that the diamins may also exert an influ- ence on certain intermediary processes. Thus, according to Pohl, 81 feed- ing diamins inhibits certain well-known protective reactions which the organism is capable of putting forth, as for instance, the formation of glycuronates and the synthesis of hippuric acid. The toxicity of these compounds calls to mind the poisonous action NH 2 of another diamin, not found in the organism, namely, hydrazin, 94 ^ This substance is, however, very much more toxic than the diamins just mentioned. Its introduction into the organism is followed by marked histological changes" in various tissues, especially in the liver. In fact, the action of this substance is directed almost specifically on the liver, provoking fatty degeneration of that organ. Only the cytoplasm of the cell is attacked. Although the liver is almost completely transformed under the influence of hydrazin, no noticeable change in nitrogenous intermediary metabolism can be demonstrated through a study of the urinary constituents. 11) APORRHEGMAS In a recent communication Ackermann and Kutscher 8 have proposed a new designation for the transformation products of amino-acids which are formed by life processes, whether in the animal or the vegetable kingdom. The term employed for these bodies is "aporrhegma." It is interesting to note the number of such compounds which may arise from putrefaction alone. The above-mentioned authors 8 ' 9 have given a list of these substances, together with the amino-acids from which they are derived. AMINO-ACID APORRHEGMA Histidin Iminazolylethylamin Iminazolpropionic acid Arginin Ornithin Tetramethylendiamin Aminovalerianic acid Lysin Pentamethylendiamin Glutaminic acid Aminobutyric acid Asparaginic acid Alanin Succinic acid Glycocoll Methylamin ( ? ) Leucin Isoamylamin Isovalerianic acid Prolin Pyrrolidin Phenylalanin Phenylethylamin Phenylacetic acid Phenylpropionic acid Tyrosin Oxyphenylethylamin Oxyphenylacetic acid Oxyphenylpropionic acid Tryptophan Indol Skatol Indolpropionic acid Indolacetic acid METHYLATION IN THE ORGANISM Until the last year or two, methylation within the organism was looked on as a reaction which occurred only rarely. With the exception of the well-known examples of the methylation of tellurium 66 and selenium, our knowledge of this type of physiological activity was exceed- ingly limited. Renewed interest in the process of methylation has been aroused by the recent investigations of Engeland, 37 who has demonstrated that complete methylation of most of the amino-acids derived from the protein molecule is far from a difficult reaction. The betains comprise all that group of bodies of the aliphatic series which are basic in character, thus, such compounds as the various amins, cholin, muscarin, urea, creatin, etc. It is further probable that the wide-spread distribution of the betains, both in the animal and vegetable kingdoms, is to be explained solely by decomposition of protein. Moreover, it has been suggested that all the betains which arise from amino-acids formed by decomposition of protein bear a relation to the so-called "alkaloids." 20 A methylated amino-acid was unknown in the animal kingdom until completely methylated glycocoll was isolated in considerable quantity from crab meat. 8 Accompanying methyl glycocoll was found trimethyl- amin oxid. 91 CH a . COOH J Methyl glycocoll Trimethylamin oxid Under normal conditions methylation of amino-acids does not occur to any considerable extent in the body of the warm-blooded animals, owing probably to the fact that if such compounds are formed, they are at once oxidized and serve as energy-producing material. When abnormal conditions are induced, however, as in phosphorus poisoning, where, perhaps, oxidative processes are less active the appearance of methylated compounds may be observed. Thus, the methylated gamma amino- butyric acid has been isolated recently from the urine of a dog poisoned with phosphorus. 38 This substance probably arises from glutaminic acid, being transformed into gamma amino-butyric acid, then completely methylated and eliminated. coo *— - CH.NH, CH, / 2 « ' CH a >► CH, CH a .NH 2 I CHj, COOH COOH J amino-butyric methylated y ami no- Glutafliinic acid acid butyric acid In plants and the lower animals methylated glycocoll is an amino-acid which is very widely distributed. Glycocoll is exceedingly resistant to decomposition by putrefaction, as was long ago demonstrated by Nencki. It is the only amino-acid which appears regularly in the urine, in the form of hippuric acid. It is found in its mono-methylated form as sarcosin combined with the guanidin residue, or modified urea rest, forming creatin of the muscle or creatinin of the urine of the higher animals. NH 2 NH CO NH 2 NH.CH 3 I I I | | C-NH C-NH CH 2 C-NH C-NH CH 2 | I I | H 3 C-N H 3 C-N CH 2 COOH Creatinin Glycocoll Methyl CH 2 glycocoll COOH Creatin Creatin and Creatinin. 68 ' 72 — At present, perhaps the most interesting example of methylation may be found in the origin of the creatin of muscle and the creatinin of the urine. The exact significance of these 21 compounds is exceedingly obscure. It is known, for example, that under physiological conditions creatin is absent from the urine and that creatinin elimination is practically constant for a given individual. This elimina- tion, however, is different for different individuals, but bears no relation to the volume of the urine or the total nitrogen excreted. From a long series of investigations, it has been concluded that creatinin is an index to some special form of normal metabolism, as yet unknown, but undoubtedly connected with processes concerned with muscle tissue. There is apparently a somewhat close relationship between muscle efficiency and creatinin elimination. Creatinin excretion varies greatly under abnormal conditions, being increased in fevers and diminished in a large number of other pathological states. Particularly striking is the diminution in excretion in abnormal metabolism of muscle tissue and of the liver. Ordinarily, creatin is absent from the urine, but may be present in large quantities under certain pathological conditions, especially those associated with inanition, depression of liver function, or an abnormal state of muscle. The appearance of creatin in the urine under these circumstances might lead one to infer, by analogy with the presence of the betain of amino-butyric acid noted previously, that when abnormal processes are in order, certain chemical reactions are held in abeyance resulting in the elimination of creatin as an incompletely disintegrated intermediary product. From the most recent investigations 22 concerning creatin and creatinin, it appears likely that there is an intimate relation- ship between these substances and carbohydrate metabolism, although, at present, this relationship is not more than a mere indication. For example, creatin is present in the urine during fasting both in man and animals. Administration of carbohydrate under these circumstances causes the rapid disappearance of urinary creatin. It has also been suggested that one function of creatin being a base, is to serve as an alkali in the muscle to neutralize lactic acid which arises as a result of muscle activity. 18 Future investigations will, undoubtedly, reveal the significance of the interrelation of creatin, creatinin, muscle and carbo- hydrate and if the prophecy of Folin, uttered a few years ago before the Harvey society, comes true, the unraveling of this mystery will mean much in the domain of pathology. THE PROTEOSES Since the classic experiments of Schmidt-Muhlheim, 89 the behavior of the proteoses when injected into the blood stream has occupied the attention of a long series of investigators, the aim of whom has been to explain the significance of the reactions induced. It had been assumed for years previous to recent times that when proteoses were formed in digestion, they were absorbed into the portal vein, carried to the liver and there were detoxicated. As a matter of fact, it was also discovered 22 that proteoses introduced into the portal vein were incapable of provoking the typical effects induced by injection into the systemic circulation, an observation in entire accord with the then current views regarding the processes of digestion and absorption. Eenewed activity directed toward the solution of the problem con- cerning the normal presence of proteoses in the blood was awakened by the more recent investigations having to do with the degradation of the protein molecule in the enteric tract. According to one school, proteoses are absorbed into the blood, thus casting doubt on the prevalent idea that proteoses are broken into their simplest cleavage products, which are absorbed, and then further worked over into the needful compounds. Opposed to this theory is the school that is unable to find any evidence of proteoses in the blood normally. Amid these conflicting views and observations, the apparent consensus of opinion is that proteoses are absent from the blood under normal conditions. 19 Hence, when these substances are introduced into the blood-stream, they act as poisons calling forth certain characteristic reactions, fever, fall of pressure, changes in respiration, increased flow of lymph, saliva, and other secre- tions, but causing a somewhat prolonged anuria. There has been con- siderable controversy as to whether pure proteoses are really toxic. All the older observations were subjected to severe criticisms by Pick and Spiro, 80 who maintained that proteoses as such are non-toxic and that the toxic principle is merely an adhering contamination, derived from the animal enzymes employed in the preparation of the proteoses. According to these authors, this substance, peptozyme, can be rendered non-toxic by subjecting the proteoses to a certain chemical treatment. Later observations, 96 however, have demonstrated that this conclusion is erroneous, since proteoses prepared from the action of vegetable enzymes on vegetable proteins and also naturally occurring vegetable proteoses induce the same train of symptoms as proteoses made from animal proteins and enzymes. Moreover, cleavage of proteoses beyond the biuret-yielding stage does not produce any symptoms. From the obser- vations of Popielski, it is concluded that the substance responsible for the so-called proteose action may be removed in large measure by treat- ment with alcohol. This compound has been designated vasodilatin and, according to Popielski, 82 is not protein in nature. The fact, however, that this investigator has not succeeded in separating the vasodilatin from proteoses, together with the well-known fact that a portion of the proteoses are soluble in alcohol, militate against the correctness of the view that the proteoses action is separable from these bodies. On the other hand, it has been suggested 28 that a portion of the reactions provoked by "peptone" may be induced by the presence in the "peptone" of beta iminazolylethylamin which is capable of calling forth symptoms in nearly every respect similar to those produced by the "peptone." The single 23 exception noted is that beta iminazolylethylamin does not render the blood non-coagulable, which is distinctly characteristic for "peptone." In our laboratory the observation has been made recently that the intravenous injection of proteoses produces a significant glycosuria. Coincident with the appearance of sugar in the urine, there is a marked hyperglycemia. The cause and possible significance of this reaction is being investigated. It appears a little strange that, of the long series of investigations carried out with the proteose injections, in not a single instance is there a recorded observation indicating glycosuria. When proteoses are injected into the blood-stream, the major portion of these compounds promptly reappears in the urine, although there is some evidence that they may be partially transformed into smaller molecules. 23 Albumosuria, so-called, is indicative of the formation of proteoses within the organism and is considered of importance in diagnosis. Thus, albumosuria may be observed in suppuration of all kinds, resolution of pneumonia, involution of the uterus, carcinoma, atrophy, eclampsia, leukemia, absorption of simple and inflammatory exudates, febrile conditions with destruction of tissue, and ulcerating pulmonary tuberculosis. 100 It is possible, and indeed may be probable, that the proteoses formed within the body and thrown into the blood-stream may be responsible for some of the symptoms which are characteristic of some of the abnormal conditions cited. The fact that these substances are fairly toxic is almost evidence for the possibility just indicated. The significance of these compounds, both in physiology and pathology, warrants further investi- gation of the cause of the symptoms induced. It is hardly probable that any one specific group in these complex polypeptids is responsible for all the reactions noted and future study will undoubtedly demonstrate the presence of several distinct entities which specifically call forth certain symptoms. The modern conception of the living cell makes enzymes responsible for the numerous types of known activities; thus we speak of enzymes facilitating reduction, oxidation, deamination, cleavage, etc. One organ is found to contain enzymes active to a high degree in one direction, another organ in another direction, and still a third organ which may furnish an almost unlimited number of enzymes capable of performing varied types of reactions. Ordinarily when enzymes are mentioned, one thinks involuntarily of the best-known agents, the digestive ferments. Our ideas concerning the exact mode of action of the enzymes that undoubtedly play an important role in intermediary metabolism are not well defined. It is probably, however, not an unwarranted position to assume that the several types of reactions discussed in previous portions of this paper have enzyme activity as their basis. With the acceptance of enzyme activity as the foundation of cellular chemical activity, hence, intermediary processes, it is easy to conceive 24 how the induction of abnormal environment for these agents may lead to the production of distinctly pathological phases of metabolism with consequent injury to the whole physiological economy. In our endeavor to discover the reason, or the cause, for abnormal metabolism we have been led into the error of looking for changes of too great magnitude. Enzymes are exceedingly sensitive to all sorts of changes in environmental conditions. Too much acid, or too little, lack of the requisite inorganic salts, perhaps absence of carbohydrate — that group of substances vaguely designated as co-enzymes — may retard or hold in abeyance certain types of reaction the effects of which, over a considerable period of time, work injury to other types of enzymes until finally a large number of processes may be carried out only imperfectly. 105 Perhaps one of the best examples of the point under discussion may be taken from the recent work of By waters, 20 who found that the inverting power of an aqueous extract of yeast is increased ten- to fifteen-fold by the addition of acetic acid. On the other hand, this activity given by acid addition is susceptible of removal by alkalies, which subsequent addition of acid is capable of counteracting. Under one form of environment the power disappears and when changed to another the power is restored. With data of this type at hand, is one unwarranted in suggesting the probability that pathological metabolism of various types may be the direct result of changed environmental conditions of intracellular enzymes ? I believe that such a viewpoint will do much toward a solution of many problems concerning metabolism both from the standpoint of physiology and pathology, the dividing line of which is exceedingly narrow. The advances of the future are to be made, I believe, by a careful study of small changes, details which at first thought perhaps appear insignificant but if followed will lead to far-reaching results. One of the best examples that can be cited in this connection may be taken from a recent paper on "The Influence of Alcohol on Nitrogenous Metabolism" by Mendel and Hilditch. 69 As a result of their study, the authors conclude that "the most significant impression, perhaps, which the analytical data afford, is the absence of pronounced alterations indicative of markedly disturbed protein metabolism." Emphasis on the words "absence of 'pronounced alteration" shows an entire appreciation of the presence of small differences. In this particular case, the organism was capable of using certain doses of alcohol to its distinct advantage; beyond this limit changes in purin output were observed. The cellular mechanism was altered in such a manner that certain types of processes were changed in a measure, the extent of the change being indicated by the altered purin output. The changed output was not large, but was it not just as good an indication of deranged metabolism as if the change had been twice as great? If the view suggested is correct, then the hope of future advances along the line of intermediary metabolism lies in an 25 appreciation of the significance of small differences and changes in the environmental conditions of the cell. 445 Orange Street. BIBLIOGRAPHY 1. Abderhalden : Text-Book of Physiological Chemistry, transl. by Hall, 1908. 2. Abderhalden: Ztschr. f. physiol. Chem., 1905, xliv, 17. 3. Abderhalden and Oppenheimer : Ztschr. f. physiol. Chem., 1904, xlii, 155. 4. Abderhalden and Schittenhelm : Ztschr. f. physiol. Chem., 1906, xlvii, 339. 5. Abderhalden and Schittenhelm: Ztschr. f. physiol. Chem., xlv, 468. 6. Abelous and Bardier: Jour, de physiol., 1909, xi, p. 34. 7. Ackermann: Ztschr. f. physiol. Chem., 1910, lxv, 504. 8. Ackermann and Kutscher: Ztschr. f. physiol. Chem., 1910, lxx, 265. 9. Ackermann: Ztschr. f. physiol. Chem., 1910, lxx, 273. 10. Albrecht and Zdarek: Ztschr. f. Heilk., 1902, xxiii, 366, 379. 11. Alsberg and Folin: Am. Jour, of Physiol., 1905, xiv, 54. 12. Baer and Blum: Arch. f. exper. Path. u. Pharmakol., 1906, lvi, 92. 13. Barger and Dale: Jour. Physiol., 1910, xli, 19. 14. Barger and Dale: Arch. f. exper. Path. u. Pharmakol., 1908, lviii, 366. 15. Barger and Walpole: Jour. Physiol., 1909, xxxviii, 343. 16. Blumenthal: Biochem. Ztschr., 1910, xxix, 472. 17. Blumenthal and Bergell: Arch. f. d. ges. Physiol., 1904, ciii, 627. 18. Burridge: Jour. Physiol., 1910, xli, 285. 19. Bywaters: Biochem. Ztschr., 1909, xv, 344. 20. Bywaters: Jour. Physiol., 1910, xli, p. 168. 21. Carlson: Am. Jour. Physiol., 1910, xxv, 403. 22. Cathcart: Jour. Physiol., 1909, xxxix, 311. 23. Chittenden, Mendel and Henderson: Am. Jour. Physiol., 1899, ii, 142. 24. Cohnheim: Ztschr. f. physiol. Chem., 1909, lix, 239. 25. Coranda: Arch. f. exper. Path. u. Pharmakol., 1880, xii, 76. 26. Dakin: Jour. Biol. Chem., 1908, iv, 419; 1908-09, v, 173, 303; 1909, vi, 203, 221, 235; 1910, viii, 11, 25. 27. Dale and Dixon: Jour. Physiol., 1909, xxxix, 25. 28. Dale and Laidlaw: Jour. Physiol., 1910, xli, 318. 29. Dixon and Taylor: Brit. Med. Jour., 1907, ii. 1150. 30. Donath: Ztschr. f. physiol. Chem., 1903, xxxix, 526. 31. Dunlop: Jour. Physiol., 1896, xx, 82. 32. Embden: Ztschr. f. physiol. Chem., 1893, xvii, 182 and 1894, xviii, 304. 33. Embden and Marks: Beitr. z. chem. Phys. u. Path. (Hofmeister) , 1908, ii, 308. 34. Embden and Reese: Beitr. z. chem. Phys. u. Path. (Hofmeister), 1905, i, 411. 35. Embden. Salomon and Schmidt: Beitr. z. chem. Phys. u. Path. (Hofmei- ster), 1906, vii, 129. 36. Emerson: Beitr. z. chem. Phys. u. Path. (Hofmeister), 1902, i, 501. 37. Engeland: Sitzungsberichte zur Beforderung der gesamten Naturwissen- schaften zu Marburg, 1909, Feb. 10. 38. Engeland and Kutscher: Ztschr. f. physiol. Chem., 1910, lxx, 282. 39. Erben: Ztschr. f. physiol. Chem., 1905, xliii, 320 and Ztschr. f. Heilk., 1904, xxv, 33. 40. Ewins and Laidlaw: Jour. Physiol., 1910, xli, 78. 41. Forssner: Ztschr. f. physiol. Chem., 1906, xlvii, 15. 42. Gaethgens: Centralbl. f. d. med. Wissensch., 1872, 833. 43. Halliburton: Ergebnisse der Physiologie, 1904, iv. 23. 44. Hallervorden: Arch. f. exper. Path. u. Pharmakol., 1878, x, 125. 45. Herter: New York Med. Jour., 1898, lxiii, 89. 46. Hunter: Tr. Med. Soc, London, 1890, xiii, 386. 47. Ignatowski: Ztschr. f. physiol. Chem., 1904, xlii, 388. 48. Jackson and Pearce: Jour. Exper. Med., 1907, ix, 552. 26 49. Jacobson: Am. Jour. Physiol., 1910, xxvi, 407. 50. v. Jaksch: Ztschr. f. klin. Med., 1902, xlvii, 1 and 1903, 1, 167. 51. Jacoby: Ergebnisse der Physiologie, 1902. 52. Klein and Moritz: Arch. f. klin. Med., 1910, xcix, 162. 53. Knoop: Beitr. z. chem. Phys. u. Path. ( Hofmeister ) , 1905, vi, 150. 54. Knoop: Centralbl. f. Physiol., 1910, xxiv, 815. 55. Kossell and Dakin: Ztschr. f. physiol. Chem., 1904, xli, 181 and 321. 56. Kutscher and Lohmann: Ztschr. f. physiol. Chem., 1906, xlviii, 1. 57. Lang: Beitr. z. chem. Phys. u. Path. (Hofmeister), 1904, v, 321. 58. Langstein and Meyer: Arch. f. klin. Med., 1903, lxxviii, 161. 59. Langstein: Beitr. z. chem. Phys. u. Path. (Hofmeister), 1904, iv, 145. 60. Lee: Jour. Am. Med. Assn., 1906, xlvi, 1499. 61. Loewy: Deutsch. med. Wchnschr., 1905, No. 44. 62. Loewy: Biochem. Ztschr., 1907, iii, 439. 63. Loewy and Neuberg: Biochem. Ztschr., 1907, ii, 438. 64. Lusk: Jour. Am. Chem. Soc, 1910, xxxii, 671. 65. Malfatti: Ztschr. f. physiol. Chem., 1909, lxi, 499. 66. Mead and Gies: Am. Jour. Physiol., 1901, v, 105. 67. Meltzer: Jour. Am. Med. Assn., 1907, xlviii, 655. 68. Mendel: Science, 1909, xxix, 584. 69. Mendel and Hilditch: Am. Jour. Physiol., 1910, xxvii, 1. 70. Mies: Miinehen. med. Wchnschr., 1894, 34. 71. Morawitz and Dietschy: Arch. f. exper. Path. u. Pharmakol., 1905, liv, 88. 72. Myers: Am. Jour. Med. Sc., Feb., 1910. 73. Myers and Fischer: Zentralbl. f. d. ges. Physiol. u. Path. d. Stoffwechs., 1908, new series iii, p. 849. 74. Neubauer: Deutsch. Arch. f. klin. Med., 1909, xlv, 211. 75. Neuberg in Oppenheimer's Handbuch der Biochemie des Menschen und die Tiere, 1910, iv, 338. Literature. 76. Neuberg and Strauss: Berl. klin. Wchnschr., 1906, No. 9. 77. v. Noorden: Handbuch der Pathologie des Stoffwechsels, i, 1907. Literature. 78. Oehler: Biochem. Ztschr., 1909, xxi, 485. 79. Osier: Lancet, London, 1904, i, p. 10. 80. Pick and Spiro: Ztschr. f. physiol. Chem., 1900-01, xxxi, 237. 81. Pohl: Arch. f. exper. Path. u. Pharmakol., 1898, xli, 97. 82. Popielski: Arch. f. d. ges. Physiol., 1909, cxxvi, 483. 83. Richards and Wallace: Jour. Biol. Chem., 1908, iv, 179. 84. Roos: Ztschr. f. physiol. Chem., 1891, xvi, 192. 85. Roos: Berl. klin. Wchnschr., 1893, No. 15. 86. Rosenheim: Jour. Physiol., 1906-07, xxxv, 465. 87. Rosenheim: Jour. Physiol., 1909, xxxviii, 343. 88. Samuely: Ztschr. f. physiol. Chem., 1906, xlvii, 376. 89. Schmidt-Miihlheim: Arch. f. Physiol., 1880, p. 30. 90. Simon: Ztschr. f. physiol. Chem., 1905, xlv, 357. 91. Suwa: Arch. f. d. ges. Physiol., 1909, cxxviii, 421. 92. Underhill and Hilditch: Am. Jour. Physiol., 1909, xxv, 66. 93. Underhill and Rand: The Archives Int. Med., 1910, v, 61. 94. Underhill and Kleiner: Jour. Biol. Chem., 1908, iv, 165. 95. Underhill and Saiki: Jour. Biol. Chem., 1908, v, 225. 96. Underhill: Am. Jour. Physiol., 1903, ix, 345. Literature. 97. Vassale and Donaggio: Arch. ital. de biol., 1896, xxvii, 129. 98. Weintraud: Ergeb. d. allg. Path., 1897, iv, 17. 99. Wells: Jour. Exper. Med., 1908, x, 457. 100. Wells: Chemical Pathology, 1907. Literature. 101. Winterstein and Trier: Die Alkaloide, Berlin, 1910. 102. Wolf and Shaffer: Jour. Biol. Chem., 1908, iv, 439. 103. Wolf and Williams: Jour. Biol. Chem., 1909, vi, 337. 104. Wohlgemuth and Neuberg: Med. Klin., 1906, No. 9. 105. Wohlgemuth: Berl. klin. Wchnschr., 1910, Nos. 48 and 49. Literature. [Reprinted from Science, N,&, Vol. XXXIV., No. 882, Pages 722-732, November 24, 1911] THE ROLE OF DIFFERENT PROTEINS IN NUTRITION AND GROWTH Interest in the study of the problems of nutrition has largely been coincident with the development of the chemical aspects of physi- ology, in distinction from the physical and mechanical phenomena which earlier attracted the attention of investigators. The subject of nutrition has, in large measure, been con- sidered in the past from what might be desig- nated as a statistical standpoint. The balance of income and outgo of energy and matter, nutritive needs and dietary standards, and the effect of external factors on these, are illustra- tions of the type of questions which has called for discussion. With the progress in the study of physiological chemistry have come impor- tant additions to our knowledge of the make- up of the foodstuffs and of the real signifi- cance of the processes which take place in the alimentary tract. The conception of digestion as a simple act of solution has evolved into that of an intricate and carefully regulated chemical transformation. The intermediary changes which characterize the metabolism of food materials after absorption and incident to the real nutritive reactions of the body within its tissue cells have at length become the subject of experimental inquiry. With this development has come about an appreciation of the specific role of foodstuffs. Various incidents have favored this trend of physiology. The study of enzymes and their striking specificity has served to emphasize the necessity of digestion before the nutrients can satisfy their purposes. Observations on the 2 unique responses of various parts of the ali- mentary tract to different kinds of chemical compounds have brought to light the remark- able interrelations of the secretory and motoi functions of the digestive tract and their de- pendence on special (chemical) stimulants. But more important than all this, perhaps, have been the disclosures of the past decade in respect to the chemical structure of the so- called proximate principles, and the proteins in particular. The development of this field of study has been little short of epoch making, so that it seems timely to begin to apply some of the newer knowledge to the investigation of problems in nutrition. The idea that proteins of different origin may possess an unlike physiological value is not entirely new. Gelatin, for example, has long been pointed out as an illustration of an inadequate protein. It has been impossible experimentally to sustain life with a diet in which gelatin formed the sole source of nitro- genous intake. To-day one can cite other illustrations of proteins, e. g., zein, gliadin, hordein, casein, which lack some of the char- acteristic amino-acid complexes readily ob- tainable from other albuminous materials which are vaguely regarded as " complete." In still other cases, e. g., edestin and glutenin, the relative proportions of these constituent complexes are so markedly different from the average as to raise the question of compara- tive nutrient values. Overabundance of glut- aminic acid groups must necessarily be at- tended by relative deficiency in other so-called " building stones " of the protein fundament. If, then, a minimum of some of these is an indispensable requirement of tissue mainte- nance or growth or repair, problems of relative values at once suggest themselves. 1 To this 1 These and related questions are discussed in detail by Mendel, "Ergebnisse der Physiologie, ' ' 1912, XL 8 may be added the question of protein synthesis in animals which has been so vigorously de- bated in recent years. Here we touch upon problems quite independent of the energy needs of the organism, yet equally important. No sooner has the idea of the isodynamic re- placement of nutrients found acceptance, than the practical limitations of this law are sub- jected to critical examination. The foremost reason why so little is known in the directions noted lies in the fact that the individual foodstuffs have, with very few ex- ceptions, rarely been examined heretofore in respect to their actual nutrient role. Meat and cereals have, it is true, been crudely an- alyzed in terms of protein (N X 6.25), fat, carbohydrate and ash, and fed as assumed mixtures of the composition indicated. Physi- ologists are, however, just beginning to recog- nize the extreme chemical complexity of such animal and plant tissues. How much of the nutritive failures or successes shall be ascribed to either presence or paucity of some inci- dental component, as lime or iron, as lipoid or nitrogenous " extractive " of specific physi- ological import, such as is attributed to the " hormones " % It is, indeed, only in very recent years that the perfection of biochemical technique has permitted the preparation of isolated proteins in what may be called comparative purity. We believe, from the experience which one of us has gained during many years of experi- ment in this field, that the vegetable proteins to-day are in general easier of access for chemical investigation and isolation than the related compounds of animal origin. And it is this fact which encouraged us to undertake what Carl Voit long ago proclaimed as the ideal method, viz., the feeding of isolated food- stuffs under controllable conditions. The la- borious and costly investigations which are under way have been made possible by the 4 cooperation of the Carnegie Institution of Washington. A detailed report of the first two years' work and the literature pertaining thereto is available in Publication 156, Parts I. and II., of the Carnegie Institution. 2 The following pages are intended to call attention very briefly to some aspects of these studies. We have undertaken to investigate certain features of nutrition by feeding isolated food substances to albino rats. The selection of this animal has been determined by several in part obvious considerations. The white rat is easily reared and cared for. Its small size reduces the food requirement to a magnitude which falls within the range of experimental possibility where the preparation of special dietaries by laborious processes is a funda- mental prerequisite. Furthermore, the longev- ity of this animal is, according to Donaldson, about three years ; so that the first year of life corresponds to a long span in terms of human years. Not insignificant is the additional fact that the white rat has in recent years been made the subject of exceptionally extensive measurements in respect to growth and vari- ous features of development at the Wistar Institute in Philadelphia. In this way phys- ical standards, so to speak, have been estab- lished for this animal. At the outset numerous problems of experi- mentation have arisen quite apart from the main question itself. Can rats be kept in health indefinitely under cage conditions which permit the control of the food intake and col- lection of the excreta ? For the description of the cages and experimental technique we must refer to our detailed publication (Part I.). How successful this has been is best answered 2 "Feeding Experiments with Isolated Food- substances," by Thomas B. Osborne and Lafay- ette B. Mendel, with the cooperation of Edna L. Ferry, Carnegie Institution of Washington, Publi- cation 156, Parts I. and II., 1911. 5 by the statement that rats have been main- tained for many months at all ages with ap- parent success. Far more important than the ability to withstand confinement in a re- stricted space has been the demonstration of the possibility of maintaining rats on an " artificial " food paste of unaltered uniform composition during a large span of their life. Herein we have apparently been far more suc- cessful than any of our predecessors; for the supposed monotony of diet has been the stumbling block leading to failure in the records of various investigators. 3 Their ani- mals have failed to eat and have declined as an obvious result of insufficient food intake. We are inclined to lend emphasis to the result of the excellent hygienic environment and care of our animals. And whereas nutritive de- cline has commonly been attributed to the anorexia consequent upon the monotony of diet, we are more than ever inclined to shift the explanation in many such cases to mal- nutrition as a primary cause. From this point of view improper diet and malnutrition may be the occasion rather than the outcome of the failure to eat — a distinction perhaps not suffi- ciently recognized heretofore. As the criterion of the nutritive status of the rats their body weight has been adopted, and this has proved to be an advantageous index. It soon became apparent that one must distinguish sharply between maintenance and growth in any such study of nutrition. The white rat shows a very characteristic curve of growth (plotted from the body weight) which becomes practically stationary within 300 days. According to Donaldson the body weight changes from 5 grams at birth to 270 grams in the case of the male, or 225 grams in the female at the age of 300 days. To judge of 8 These earlier studies are reviewed in Publica- tion 156, Part I., Carnegie Institution of Wash- ington, 1911. 6 the effect of a dietary regime by noting the subsequent duration of life, as is still fre- quently done by investigators, is misleading; for the incidence of death may depend on the previous nutritive condition — the store of fat and glycogen — where food is insufficient. An error less readily appreciated consists in de- scribing the nutritive status as necessarily satisfactory because an animal maintains an undiminished body weight over long periods under the conditions imposed. A man who maintains his weight may be in excellent nu- tritive condition ; but a child which does like- wise is failing to grow. Childhood demands of a perfect ration the possibility of normal growth, not simply maintenance. This can not be emphasized too strongly. Furthermore, growth in the sense of an increase in the size of some structural part of the body or some organ may proceed independently of the cor- related development of the body as a whole. Even with the existence of unquestionable malnutrition, skeletal growth may manifest itself in a conspicuous degree; so that the length or height of an individual may mark- edly increase while the total body weight re- mains stationary or even declines. One part of an organism may thrive at the expense of other tissues. The complexity of these rela- tionships of absolute and relative (or propor- tionate) growth have likewise commanded at- tention in our experiments. A study of physiological literature will make it evident that no convincing reply has been given to the question: can life be main- tained and is growth possible with a single protein in the dietary. "Protein" has been used in this connection in a generic sense; and one of the (chemically) simplest foods, milk, contains at least two proteins of marked individuality. Casein and lactalbumin are chemically unlike. How widely two exten- 7 sively used food proteins may differ in their chemical make-up is indicated below. The individuality of proteins of different biological origin is further indicated by their Casein 4 Ze:ii r - Ammo-acids Per Cent. Per Cent. Glycocoll 0.00 0.00 Alanine 1.50 4 9.79 Valine 7.20 4 1.88 Leucine 9.35 6 1 9.55 Proline 6.70 7 9.04 Asparticacid 1.39 4 1.71 Glutaminic acid 15.55 4 26.17 Phenylalanine 3.20 8 6.55 Tyrosine 4.50 9 3.55 Serine 0.50 10 1.02 Oxyproline 0.23 10 Histidine 2.50 u 0.82 Arginine 3.81 u 1.55 Lysine 5.95 u 0.00 Tryptophane 1.50 8 0.00 Diaminotrioxydodecanic acid 0.75 12 Ammonia 1.61^ 3.64 Sulphur 0.76 14 0.60 Phosphorus 0.85 14 0.00 4 Osborne and Guest, Journal of Biological Chemistry, 1911, IX., p. 333. 'Osborne and Liddle, American Journal of Physiology, 1910, XXVI., p. 304. 'Levene and Van Slyke, Journal of Biological Chemistry, 1909, VI., p. 419. 7 Van Slyke, Berichte der deutschen chemischen Gesellschaft, 1910, XLIV., p. 3170. 8 Abderhalden, Zeitschrift fur physiologische Chemie, 1905, XLIV., p. 23. •Beach, Virchow's Archiv, 1899, CLVIIL, p. 288. 10 Fischer, Zeitschrift fur physiologische Chemie, 1903, XXXIX., p. 155. n Osborne, Leavenworth and Brautlecht, Amer- ican Journal of Physiology, 1908, XXIIL, p. 180. "Fischer and Abderhalden, Zeitschrift fur physiologische Chemie, 1904, XLII., p. 540. 13 Osborne and Harris, Journal American Chem- ical Society, 1903, XXV., p. 323. 14 Hammarsten, Zeitschrift fur physiologische Chemie, 1883, VII., p. 227. 8 specific immunity reactions. The published feeding experiments in which a single purified protein has been administered to animals are all limited in their duration to periods of days or weeks which are too brief to furnish con- vincing data. Indeed, one will scan the lit- erature in vain for properly controlled experi- ments in which isolated and purified proteins have been fed successfully. 15 Without citing here the numerous failures and the successive changes instituted in our earlier trials, we may briefly call attention to some of the purely " nutritive " factors which have had to be taken into consideration. The energy requirement must obviously be satisfied in an available form. A minimum protein requirement must likewise be provided in any event. Experiments which are to continue over more than very few days must include a suitable quota of inorganic salts — so-called mineral nutrients. This is in itself a problem of fundamental importance, the study of which has barely been begun in any synthetic way. One may, it is true, imitate the " ash " of milk or blood; but the elements occur here in combinations quite different from those prevailing in the tissue fluids themselves or in the native foods. The balance of acid and basic groups, the changing need for individual elements like phosphorus, calcium, chlorine and iron, furnish a series of complex variables which are probably as indispensable to certain aspects of nutrition as they are unappreciated. If to all this is added the uncertain signifi- cance of the as yet largely unidentified com- pounds such as cholesterol and phosphatides which occur in all natural food mixtures, the experimental difficulties begin to appear in their true light. At the outset it is only fair to remark that 16 Cf. Osborne and Mendel, Publication 156, Part L, Carnegie Institution of Washington, 1911, for the literature on these topics. 9 a successful feeding experiment with isolated food mixtures is of greater import than a V Food e iten 50- X -A 100 1 dead Nitrofre 1 balance jJtr- ^4 ] Days Fig. 1. (Taken from Carnegie Publication No. 156, page 26.) Showing the continued decline of a rat on a dogbiscuit-lard diet for 103 days. failure may be; for ill health may be occa- sioned by incidents quite apart from those already outlined. Accident and acquired dis- ease, unrecognized or uncontrollable, enter into the life of every individual and serve to upset an otherwise normal nutritive equilibrium. Turning to the present experiments, our earlier attempts were largely based on those of our predecessors. Comparative trials with food mixtures precisely alike except for both content and character of the inorganic in- gredients soon showed the great importance of this feature. A fairly suitable salt mixture was thus empirically selected. The table be- low may serve to illustrate the character of the earlier food mixtures which experience showed to be most suitable. In such a mixture the protein can be varied without serious change in the fuel value. With one protein, viz., zein from maize, nu- tritive decline was apparent from the outset. 10 The failure, as actual investigation showed, can not be attributed solely to poor utilization. With all the other proteins, such as casein, legumin, edestin, glutenin or gliadin, in the mixtures indicated, grown rats have been maintained in body weight for much longer \ Bod ^ — \ r f V | fooi HM \ d Tl- t— ri i 1 Hi'.rcje A balance [rn -rf ♦ 0.05 o £ e I 1 v \ > 1 ( f ) 0) o * c , •2 \ ) 1 1 j5 c r ■I— 1 1 i — j_ ■A h -t--- _ n c± r 1 L 1 P o cS £ S from the outset, the second and third as ex- amples of a relatively successful attempt over a period of 169 days and 259 days, respectively. 12 In every case — and we might cite very many such experiments under varied conditions — a decline ultimately ensued leading to death unless a dietary change was instituted. It early seemed unlikely that the protein was responsible for failures of this character; for this foodstuff forms so large an essential A Body weigh! ^ 2 Food eaten \ / y — i. _ ... . A \\ v v r ~ ! \ / \ \ / \ / V \ — ' — x \ \ ! ' — .r u-r Nitrogor balance -J" rfl- ! ' 20 40 £0 20 100 120 1^0 ISO :20 200 220 240 Fig. 4. (Taken from Carnegie Publication No. 156, page 45.) This rat was fed 210 da on a diet containing casein and excelsin as the only proteins. component in the mixture that a defect ought soon to manifest itself — as indeed it did with zein. Nor did the animals fare better when more than one protein was present. Here, too, the ultimate decline in the grown rats in- evitably showed up, as will be seen in the illustrative charts below. Remarkable in this connection were the ob- servations made on small white rats during the period of active growth. Lacking food, at this stage, the animals speedily die, since the reserve stores are small or wanting. With an appropriate mixed diet growth is vigorous, and the rate of gain is strikingly similar in healthy 13 animals of related origin. When young rats are fed on diets containing a single protein in the mixtures described above they fail to grow, although they can be maintained at uniform body weight and size for long periods. Here then is an evident distinction between main- tenance and growth in respect to the function of the ration. An illustration of the stunting of animals in this manner is graphically af- forded by the appended curves in which a dwarfed animal is compared with a suitably fed one from the same litter. 2 Body weight dead — V Food eaten 1 rl -n-r Nitrcger balance 1 O 20 40 60 80 100 120 1*0 160 Doys Fig. 5. (Taken from Carnegie Publication No. 156, page 46.) This rat was fed 160 days on a diet containing casein and pea legumin as the only proteins. What is the factor or what are the causes connected with the ultimate failure of the older rats to thrive on the dietaries outlined, or of young rats to grow? Evidence which need not be reviewed here pointed to some- thing other than the character of the protein, fat or carbohydrate. Animals will thrive and grow on a "mixed" diet of corn, vegetables, 14 etc.; but we have, furthermore, noted that their nutritive needs can be met with an " arti- ficial " food mixture in which dried milk and fat form the sole ingredients. 17 That the Fig. 6. Showing the body weight and food in- take of a small rat grown normally on a diet of dried milk and lard in the upper curves. The lower curves are charted from a rat of the same litter maintained without growth on a diet con- taining glutenin as the sole protein in a mixture unsuitable for growth. 17 The dried milk used is the commercial "Tru- milk, " furnished by the Merrell-Soule Co., of Syracuse, N. Y. 16 stunted or malnourished rats in these earlier experiments have not lost their capacity to groiv or, in the case of the adults, have not become permanently disorganized from a nu- tritive standpoint can be readily demon- strated ; for they will resume growth or become realiirented, as the case may be, as soon as 14© 4X i 2*0 'A lie no foot So- t5 S -J( •-- -K k . BE 3'* tt , - rn Fig. 7. Showing the real i mentation of a rat practically moribund, by the addition of 'otein-free milk to the diet containing a single protein (casein). mixed food is furnished. The milk mixtures are as efficient as mixed food in promoting growth and restoring nutritive equilibrium. Rats have been carried through two genera- tions on a food mixture of the following com- position : 1(3 Per Cent Milk powder 60.0 Starch 15.7 Salt mixture 1.0 Lard 23.3 100.0 Obviously the milk contains the nutrient elements essential to success which had previ- ously not been satisfactorily imitated in the artificial food mixtures. It occurred to us to attempt to locate these as yet unknown com- ponents by removal of the proteins from milk and concentration of the protein-free (and fat- free) residues. The product thus obtained (and which may conveniently be termed " pro- tein-free milk " 1S ) has fulfilled our expecta- tion and enabled us at length to study the relative value of added proteins in the dietary. The protein-free milk contains the milk sugar in addition to inorganic salts and other as yet unknown components of the milk. Whether it is the peculiar combinations of the latter, or some ideal " balancing " of the inorganic ions therein, or the presence of traces of essen- tial organic compounds, or all of these, which guarantee the successful outcome, remains to be ascertained. What has been accomplished thus far with the new possibilities of investigation at hand may be mentioned in brief. Rats which have developed marked symptoms of decline on mixtures of isolated food substances contain- ing a single protein have been revived in a way little short of marvelous by the substitu- tion of the protein-free milk in place of part of the previous (non-protein) food. Instances have occurred where successful realimentation has thus followed in animals practically mori- bund. The chart below furnishes a graphic illustration. 18 A detailed description of the preparation and composition of protein-free milk is given in the detailed papers, Part II. SI 4- .g £ s Pi 1=1 3LC .9 .9 § & ^4 43 fl be 2 O M ►•a "8 & ft 5K •V 111 S O Pi 00 ■§ ? ad * I . pi © 2 *h H O bo -3 O C3 -M pi 18 Even more interesting is the role of this protein-free milk in facilitating growth. By the use of protein-free milk to furnish the " accessory " portions of the diet the relative deportment of different proteins in growth has been investigated. Thus adequate growth has been noted where the sole protein was either the casein of milk, the lactalbumin of milk, crystallized egg albumin, crystallized edestin from hempseed, the glutenin of wheat, or gly- cinin from the soy bean. But not all proteins suffice to promote growth under otherwise favorable conditions. The gliadin of wheat (notably lacking in glycocoll and lysine) and the hordein of barley (closely resembling gliadin in its chemical constitution) suffice for maintenance without growth. Zein, the tryptophane-, lysine- and glycocoll-free protein of maize, is alone insufficient for the main- tenance requirement. How well growth can proceed under these somewhat artificial condi- tions of diet is shown in a few charts in which the curve of growth on mixed food is simul- taneously plotted. How entirely different the results are when an " inadequate " protein is alone furnished, despite an abundant ingestion of food, is strikingly shown by the drawings. The ani- mals A and B were of one age and differed simply in having been sustained on different proteins. It will be noted that the older but stunted animals do not vary materially in size from properly nourished younger animals which have attained the same body weight. Herein they differ essentially from young animals which, maintained at constant body weight by underfeeding, continue to grow in size. Such conditions have been described in cattle, 1 ' "Waters, Proceedings Society for the Promo- tion of Agricultural Science, 1908, XXIX., p. 3; also Ibid., XXX., p. 71. 19 dogs 20 and children ; 21 and they lead to dispro- portioned forms. Our (malnourished rather than undernourished) rats have merely main- tained themselves, if we except the possibility of a continued development of the nervous c This drawing shows the influence of different proteins on growth. A and B are rats of the same brood which were fed from the time of weaning on foods of the same composition except that the diet given to A contained pure casein while that given to B contained pure gliadin as its only pro- tein. The appearance of B at the age of 140 days closely resembles that of C, a normally nourished rat, which at the age of 36 days had the same weight as B. (Sketch from photographs in Publi- cation 156, Part II., Carnegie Institution of Wash- ington, 1911.) system of which we have furnished some evi- dence elsewhere. 22 Aside from the nutritive inequalities of dif- ferent proteins, as well as the apparent com- parable suitability of chemically and biolog- ically unlike proteins — all of which remains to be subjected to more refined experimental in- "Aron, Biochemische Zeitschrift, 1910, XXX., p. 207 ; Philippine Journal of Science, Sec. B., 1911, VI., p. 1. 21 Fleischner, Archives of Pediatrics, October, 1906. 22 Osborne and Mendel, Carnegie Institution of Washington, Publication 156, Part II. 20 vestigation — it is worth while to point out numerous other incidental findings. Animals which have grown from small size, e. g., 40 grams, to adult form, e. g., 160 grams, and have thus quadrupled their weight on a diet furnishing its nitrogen in the form of a simple protein like edestin, have by some process per- fected the synthesis of purines and nucleo- proteins, perchance of phosphoproteins and nitrogenous phosphatides, and of ferruginous proteins (like hemoglobin) from iron-free pro- tein precursors and " inorganic iron." With what powers of synthesis in such directions is the body provided by nature? What modifications, if any, can be introduced into the organism in respect to structure, function or inheritance by the possibility of a successfully regulated control of the character of the most important foodstuff, the protein? Such physiological and broader biological questions appear to lend themselves to experi- mental study by the methods which we have initiated. There are, further, pathological aspects involving abnormal growth, dwarfism, recuperation and senescence which similarly suggest themselves. The program for the future is limited only by the success and effi- ciency of the methods adopted. To the biological chemist, no feature of these problems appeals more strongly, perhaps, than the question of how an organism can build such diverse nitrogenous tissues from a single dietary protein. It is true that the newer conceptions of the extensive role of hydrolysis in digestion prior to absorption have extended the inquiry a step further, so that we may ask what is the minimum of this or that amino-acid or simple polypeptide re- quired. But we have seen rats grow for months with casein — thoroughly purified and glycocoll-free — as the sole source of these amino-acids. During this time, one animal even brought forth two broods of young and 21 secreted milk in sufficient quantity to bring her young to the age when they were able to care for themselves. Another pair of rats maintained 178 days on gliadin as the sole protein of the diet produced healthy young and successfully reared them. It is most unlikely from all that is otherwise known, that the tissues of our experimental animals are chem- ically imperfect or essentially unlike those of normally fed rats which presumably do contain glycocoll and lysine groups. Have we hereto- fore underrated the ultimate synthetic capac- ities of animal cells % 28 The observation that animals long main- tained on diets of the character used in our feeding trials voraciously eat the feces of normally fed rats led us to experiment in another direction. It has been noted as a result of this that in a not inconsiderable number of instances the feeding of small por- tions of " normal " rat feces tended to check the decline of rats kept on pastes of isolated food substances containing the earlier salt mixture. The possibility of altering the bacterial flora of the alimentary tract by dietetic conditions at once suggests itself in this connection, and reference may be made to the significant studies of Herter and Ken- dall, 24 among others, which elucidate this ques- tion. To what extent is the cooperation ©f bacteria either essential or useful in the ali- mentary functions? This is, indeed, still a debated question. 25 But one can not dispel the 23 One is reminded of the recent studies of Knoop, Zeitschrift fur physiologische Chemie, 1910, LXVIL, p. 489, and Embden and Schmitz, Biochemische Zeitschrift, 1910, XXIX, p. 423, bearing on such possibilities. Cf. Mendel, Ergeb- nisse der Physiologie, 1912, XI. 24 Herter, ' ' The Common Bacterial Infections of the Digestive Tract," The Macmillan Co.; Herter and Kendall, Journal of Biological Chemistry, 1910, VII., p. 203; Kendall, Journal of the Amer- ican Medical Association, April 15, 1911. 22 idea that bacteria might, after all, enter into reconstructive reactions which may furnish new nitrogenous complexes from amino-acids. Viewed in this light, the immediate hydrolysis products of our foodstuffs may become avail- able only after they have in greater or less part been reconstructed by the preeminently synthetic symbiotic bacteria into products of more uniform character, possibly widely dif- ferent from the original intake. Nucleopro- tein synthesis, for example, may thus become referable to bacterial intervention; and the subtle influence of the indeterminable non- protein factors may lie in some measure in the regulation which they exert upon the micro- organisms of the gastro-intestinal tract. 2 ' In any event such suggestions need to be dealt with. It is hoped to continue these nutrition studies, the possible scope of which has barely been indicated in what has gone before. They seem to us to justify the effort which has been involved. Indeed only by unremitting re- gard for details, such as the careful purifica- tion and preparation of the materials fed and attention to the animals, can the uncertain factors be limited, comparable results obtained and definite conclusions safely drawn. We realize that only a beginning has been made, and believe that further progress is possible. Thomas B. Osborne, Connecticut Agricultural Experiment Station Lafayette B. Mendel, Sheffield Laboratory of Physiological Chemistry, Yale University New Haven, Connecticut 25 Nuttall and Thierf elder, Zeitschrift fur physi- ologische Chemie, 1895, XXI., p. 109; Sehottelius, Archiv fiir Hygiene, 1908, 67, pp. 177-208. 26 Cf. Armsby, "The Nutritive Value of the Non-protein of Feeding Stuffs," Bureau of Ani- mal Industry, Bulletin 139, 1911. Reprinted from The Journal of Pharmacology and Experimental Therapeutics Vol. Ill, No. fi. July, 1912 THE ACTION OF SALTS OF CHOLINE ON ARTERIAL BLOOD PRESSURE LAFAYETTE B. MENDEL, FRANK P. UNDERHILL, and R. R. RENSHAW From the Sheffield Laboratory of Physiological Chemistry, Yale University, New Haven, Connecticut, and the Chemical Laboratory of Wesleyan University, Middletown, Connecticut The discovery of the presence of choline in many of the animal tissues either preformed or as a derivative of larger complexes, and the possible relationship cf this base to some of the so-called " hormone" effects in the organism has lent a new interest to the precise determination of its physiological role. The contention that absolutely pure choline salts fail to induce the characteristic fall in blood pressure commonly described as a typical effect of the injection of this compound was supported several years ago by Popielski and his pupil Modrakowski. 1 They believed that pure choline causes only a rise of pressure; and they maintain that those investigators who report a fall of pressure have worked with impure or deteriorated preparations. This contradiction of the customary teaching regarding the physiological action of choline naturally elicited a speedy reinvestigation of the question, to which we, among others, contributed a preliminary reply in 1910. 2 One might assume that our experience, together with the experi- mental studies of Abderhalden and Muller, 3 Lohmann, 4 Berlin, 5 1 Modrakowski : Pfliiger's Archiv fur die gesammte Physiologie, 1908, cxxiv, p. 601. 2 Mendel and Underhill: Zentralblatt fur Physiologie, 1910, xxiv, p. 251 (June 25). 3 Abderhalden and Muller: Zeitschrift fur physiologische Chemie, 1910, lxv. p. 420; 1911, lxxiv, p. 253. * Lohmann: Zeitschrift fur Biologie, 1911, lvi, p. 1. 6 Berlin: Zeitschrift fur Biologie, 1911, lvii, p. 1. 649 650 L. B. MENDEL, F. P. UNDERHILL AND R. R. RENSHAW Hunt and Taveau 6 — to mention only a part of those which have arisen — had adequately controverted the newer claims and re- stored confidence in the earlier view point. However, the con- tinued reiteration of Popielski's allegations in recent papers 7 induces us to present a few further details of our trials. 8 They were carried out entirely with synthetic preparations made by Dr. Renshaw at Middletown according to the improved method devised by him. 9 Since the controversy centers largely in the purity of the salts used, our attention has been devoted primarily to this point. We have been unable to confirm Popielski and Modrakowski's claims respecting the absence of depressor effects when the purity of the choline products is sufficiently assured. PREPARATIONS OF CHOLINE USED — METHODS Eight different preparations of choline chloride and sulfate were tested in numerous trials. The animals (usually cats) were maintained in deep ether anaesthesia. The importance of this deserves the emphasis which Abderhalden and Mliller have given to it; for when the narcosis is not adequate it is easy to obtain transitory rise of pressure reflexly by even slight manipulation of the animal prior to the act of intravenous injection. Unless specifically stated otherwise, the choline salts were dissolved in 0.9 per cent NaCl solution just prior to the injection into an exposed vein. Popielski remarks : "Die Schwierigkeit der physiol- ogischen Untersuchung des Cholins ist auf die grosse Schwierigkeit der Gewinnung von chemisch reinen Praeparaten zuriickzufuh- 6 Hunt and Taveau: Bull. No. 73. Hygienic Laboratory, U. S. Pub. Health and Mar. Hosp. Service, Washington, 1911, p. 12. Details of the literature will be found in this paper and in the contributions of Abderhalden and Miiller, so that they need not be repeated here. Cf. also Gautrelet: Journal de physiologie, 1909, xi, p. 227; v. Fiirth: Probleme der physiologischen und pathologischen Chemie, 1912, p. 186, ff. 7 Cf . Studinski: Archiv fur experimented Pathologie und Pharmakologie, 1911, lxv, p. 155 (Popielski's laboratory); also Samelson: ibid., 1912, lxvi, p. 347. 8 A report was presented to the American Society for Pharmacology and Experi- mental Therapeutics at the meeting in December, 1911. Cf . Journal of Experimen- tal Pharmacology and Therapeutics, 1912, iii, p. 457. 9 Renshaw: Journal of the American Chemical Society, 1910, xxxii, p. 128; Abcfer- halden's Biochemisches Handlexikon, 1911, iv, p. 829. CHOLINE AND BLOOD PRESSURE 651 ren." 10 Accordingly we shall describe some of our products in sufficient detail to permit the reader to form a more critical judg- ment as to their probable purity. Choline chloride: Preparations 1, 2 and 3 Preparation 1 was the product of the sixth reprecipitation of an alcoholic solution of our synthesized material with ether. Not more than one-third of the dissolved product originally used was removed from the solution, thus creating ideal conditions for the exclusion of soluble impurities. An analysis gave: Cl found 25.45 per cent, 25.49 per cent; calculated, 25.41 per cent Preparation 2 was obtained after five additional reprecipitations of 1. Preparation 3, our purest specimen, was the product of the fifteenth reprecipitation. The second of three partial precipitations on both the first and the second solutions of the choline chloride was taken as the sample for purification. From the third on the procedure was as indi- cated. The reason was, of course, to eliminate the possibility of any amount of a somewhat more insoluble product always pre- cipitating with the chloride. Had such a substance been present even in fair amount this procedure would have eliminated all but such quantities as the solvent could have taken care of. Such a possibility was very remote; but on account of the con- troversy it seemed worth while to eliminate every point that might be criticised. None of these preparations had any odor of trimethylamine, nor did they develop it rapidly when exposed to sunlight. This is important in relation to the alleged speedy deterioration of choline salts discussed later. In view of the differ- ences in the extent of purification attempted one would expect wide variations in the degree of purity and a consequent differ- ence in physiological action dependent upon the amount of con- taminating impurity; i.e., preparation 3 ought (according to the 10 Popielski: Zeitschrift fur physiologische Chemie, 1910, lxx, p. 250. 652 CHOLINE AND BLOQJ) PRESSURE 653 explanation of depressor effects on the impurity hypothesis) to be less active physiologically than 1 or 2. These preparations were delivered in sealed tubes and injected the same day. Twenty-one months later the experiments with these products were duplicated, the preparations having been kept meanwhile in glass-stoppered bottles in a dark desiccator. There is no evidence for any lack of, or quantitative differences in, the depressor effects of measured doses of choline chloride 1, 2 or 3; nor has the long time interval noticeably altered the behavior of the purest product, 3, despite Modrakowski's contentions of the extreme instability of choline salts. The illustrative blood pressure tracings should be read from left to right. A few of the numerous data are summarized in tabular form in the Appendix. It is unnecessary to report the observations with larger doses, since they were not essentially different in character. Furthermore the fact that a fall was always produced even with the very small doses is significant of itself. Comparative effects of choline chloride 1 (recrystallized six times) and 3 (recrystallized fifteen times) on blood pressure. The prep- arations were not more than twenty-four hours old Further evidence of the failure of this pure product to develop any extreme toxicity on standing was shown by experiments made with choline chloride 3. The solution was prepared one month after the preparation of the salt and then allowed to stand thirty- two days in the laboratory before being tested. Surely here was abundant opportunity for the development of the extreme de- pressor effects assumed to be characteristic of old preparations and depicted in the curves of other investigators for " crude" choline. The tracing shows merely the typical transitory fall regularly found when the same product was used immediately after its delivery. 654 L. B. MENDEL, F. P. UNDERHILL AND R. R. RENSHAW Choline chloride 3 — old solution This curve may also be compared with that obtained in 1910 with the fresh preparation. (Cf. Zentralblatt fur Physiologie, 1910, xxiv, p. 252.) Cat. 2.8 kgm. Injection of 2.4 mgm. (0.8 cc.) The solution stood one month in the laboratory before being used. Similar depressor results were obtained on dogs with the fresh purest choline chloride 3, even with doses of 0.1 mgm. per kilo- gram. The salt was used within thirty hours after it was made. CHOLINE AND BLOOD PRESSURE 655 Choline sulfate A pure preparation of this salt 11 was likewise examined. The depressor action was never missed even in small doses. It was not found increased when the same preparation, carefully pre- served, was tested three months later; i.e., there was no evi- dence of a production of the hypothetical depressor derivative on standing. Since Modrakowski has especially emphasized the method of Gulewitsch 12 as essential to obtain pure choline we have also followed this plan of purification. A solution of choline chloride was evaporated until no amine odor was detectable. It was converted into the platinic salt, recrystallized five times, decomposed with hydrogen sulfide, and finally obtained as a pure crystalline chloride = choline chloride 4. This salt, injected within a day after its preparation gave the usual characteristic fall of pressure, as reported likewise by Abder- halden and Mtiller and by Lohmann for products similarly purified. Inasmuch as the hypothetical "impurities" which are alleged to account for the fall in pressure are assumed to be eliminated by this process of purification we searched for them in' the wash solutions or mother liquors of choline chloride 4. They were precipitated with ether. The new product, choline chloride 5 in which the decomposition product might be concentrated, might be expected to show the depressor effects in marked degree. This was, however, not the case. The result was merely the typi- cal transitory fall of arterial pressure characteristic of similar doses of the purest choline salts. It occurred to us that possibly the manipulations to which Modrakowski subjected his solutions — the evaporations to remove the amine odor, the treatment with hydrogen sulfide, a possible failure to remove the last traces of platinic sulfide which separate with difficulty — might have developed some antagonistic sub- stance and thus " masked" the typical depressor effect despite the 11 Cf. Renshaw: Journal of the American Chemical Society, 1910, xxxii, p. 129. 12 Gulewitsch : Zeitschrift fur physiologische Chemie, 1898, xxiv, p. 513; Cf. Modrakowski: Pfliiger's Archiv fur die gesammte Physilogie, 1908, cxxiv, p. 619. 656 L. B. MENDEL, F. P. UNDERHILL AND R. R. RENSHAW chemical purification of the choline salts. Solutions of our prep- arations were accordingly evaporated and treated with hydrogen sulfide alone without altering the physiological effects obtained. One solution was evaporated to remove the amine odor com- pletely, recrystallized, redissolved in alcohol, and reprecipitated with ether five times. A blood pressure tracing of this choline chloride 6 made within twenty-four hours is reproduced here. Cat. 2.8 kgm. Injection of 0.5 mgm. (0.5 cc.) Choline chloride 6 — repeatedly purified That traces of platinic salts do not alter the results and thus account for the alleged rise of pressure was plainly shown by numerous trials with varying proportions of the heavy metal added. Pi w « W CO pi} H-3 H ee P 3 03 ne o t> W so <■ — s TE o o «! P g H g fc CM OP 05 o .2 O Q3 o rt Eh p ►J O a o 03 a Oh 6 u o w > w o w ti 0) o .2 <5 W 'o ^3 § d a ; o '5" 057 658 L. B. MENDEL, F. P. UNDERHILL AND R. R. RENSHAW OTHER FACTORS Modrakowski has maintained (p. 620) that the characteristic blood pressure effect of pure choline is precisely comparable with that obtained from " impure" preparations after atropine is administered. We have found that atropine always abolishes the depressor effects of choline salts; but according to our experience a rise in pressure under these conditions is only obtained when the doses of choline salts administered are relatively large. This is shown in the appended tracings. Choline sulfate following atropine Section of the vagi does not alter the effect of pure choline salts. It is only fair to point out that our preparations do not exhibit any of the extreme depressor effects, with attendant symptoms, which have sometimes been described by those who have used crude commercial products. Modrakowski has pointed out, for example, that the latter in doses of 1 to 3 mgm. per kilogram lead to prolonged inhibition of the heart beat and that repeated injec- tions may impair the peripheral endings of the vagi and be followed by rise in pressure. "Bei rasch hintereinander erfolgenden In- jektionen vermag die blutdrucksteigernde Cholinwirkung doch durchzudringen" (p. 620). We have never obtained more than a transitory fall of pressure even with larger doses of our choline preparations than are reported in this paper. On the other hand a rise of pressure was never observed (except after the use of atropine) even when many repeated injections were undertaken. The deterioration of choline salts on standing Our experience throws some light upon this question which is obviously of moment in the controversy at hand. In pointing out the extraordinary instability of even pure salts of choline Modra- kowski wrote (p. 622): "Das Kahlbaum'sche Praparat war, wie bereits erwahnt, bei seinem Eintreffen vollkommen frei von Tri- methylamingeruch. Gelegentlich einer kurzen Unterhaltung im Laboratorium hielt ich dasselbe einige Augenblicke in der Sonne; da erregte der Umstehenden und meine Aufmerksamkeit ein deutlicher Trimethylamingeruch, welcher der halboff enen Flasche, CHOLINE AND BLOOD PRESSURE 659 die die Cholinchloridkrystalle enthielt, entstromte. Es zeigte sich also, dass unter geeigneten Bedingungen die Zersetzung dieses Praparates fast momentan erfolgen kann. Die Moglich- keit erschien daher ausserst wahrscheinlich dass kiirzere oder lan- gere Aufbewahrung die Wirkung des Cholins verandern konnte." Lohmann, on the other hand, insists that choline chloride does not decompose even when exposed to the direct sunlight for months. 13 This is in accord with experience of Dr. Renshaw in similar trials. Abderhalden and Muller are uncertain as to whether Kahlbaum's preparations are contaminated or undergo secondary decomposition. Their own synthetic chloride gave no odor of trimethylamine after being preserved sealed in the dark for six months. "Wir mochten uns aus diesen Griinden vorlaufig nicht dariiber aussern, ob ganz reines Cholinchlorhydrat, unter geeigneten Bedingungen (verschlossen, unbelichtet) , auf- bewahrt, unbegrenzt haltbar ist." 14 We have failed to note a development of odor after two years in preparations thus pre- served. In any event no deterioration is detectable, as already pointed out, from the standpoint of quantitative alterations in blood pressure effects which, after all, Modrakowski used as his chief criterion. CONCLUSIONS The experiments recorded above, along with numerous further records, afford added evidence that the views promulgated by Popielski and his pupils about the physiological behavior of salts of choline are not tenable. Even with exceptionally pure syn- thetic salts we have never failed to observe the characteristic transitory fall of arterial pressure — a fall not profound or pro- longed, but never absent even when fractions of a milligram of purest products are injected. The " contamination" theory is rendered improbable by the fact that choline salts showed no quantitative differences in the physiological effect when different specimens of presumably unequal purity were investigated. Fur- thermore, it seems extremely doubtful if properly prepared and preserved choline salts readily decompose. 13 Lohmann: Zeitschrift fur Biologie, 1911, lvi, p. 16. 14 Abderhalden and Muller: Zeitschrift fur physiologische Chemie, 1911, lxxiv, p. 264. 660 L. B. MENDEL, F. P. UNDERBILL AND R. R. RENSHAW APPENDIX Selected protocols of blood pressure experiments with salts of choline (only trials with small dosage are reported here) PREPARATION USED DOSE PER KILOGRAM PALL OF PRESSURE AFTER THE REMARKS mgm. mm. Hg. { 0.1 4 This salt was recrystallized six times. Choline chloride 1 0.3 26 See tracing I 0.5 36 0.1 5 This salt was recrystallized fifteen 0.3 22 times. See tracing I 0.5 30 1.0 24 See tracing in Zentbl. Physiol., 1910, xxiv, 252. Choline chloride 3 • 0.23 26 After being preserved twenty-one 0.45 36 months without deterioration 0.9 26 The solution stood one month in the laboratory before being used. See tracing II 0.1 6 Dog 0.2 28 Doe Choline chloride 4 0.05 15 Purified by method of Gulewitsch Choline chloride 5 0.35 16 Fraction which ought to contain the hypothetical depressor product in marked concentration Choline chloride 6 0.18 24 Repeatedly purified. See tracing III 3.0 30 ' 0.05 11 Choline sulfate < 0.1 17 0.5 32 Reprinted from the Proceedings of the Society for Experimental Biology and Medicine, 1912, ix, pp. 123-124. 87 (696) The influence of tartrates upon phlorhizin diabetes. By FRANK P. UNDERBILL. [From the Sheffield Laboratory of Physiological Chemistry, Yale University, New Haven, Conn.] A recent communication of Baer and Blum (Archiv fur Exper- imented Pathologie und Pharmakologie, 191 1, 65, p. 1) shows that the subcutaneous administration of a number of organic com- pounds, containing two carboxyl groups, exercises a remarkable inhibitory influence upon the elimination of urinary nitrogen and dextrose in dogs with phlorhizin diabetes. Among the substances possessing this property may be mentioned glutaric and tartaric acids. In an endeavor to explain the mechanism of the unique influ- ence exerted by these compounds investigations have been carried out with tartrates upon both dogs and rabbits under conditions similar to those established by Baer and Blum. We have been able to corroborate the findings of Baer and Blum with respect to the action of tartrates although Ringer (Proc. Soc. Exp. Biol, and Med., 1912, 9, p. 54) failed to obtain the reported results with glutaric acid. Our interpretation of the diminution of the urinary constituents is, however, entirely different from that offered by Baer and Blum. Tartrates subcutaneously injected cause a prompt disintegration of the cellular elements of the kidney tubules, leading to partial or complete loss of secretory activity, and in many cases to anuria. Hence, in phlorhizin diabetes urinary nitrogen and sugar are not eliminated to an appreciable extent. Reprinted from The Journal of Biological Chemistry, Vol. XI, No. 1, 1912 THE HAEMAGGLUTINATING AND PRECIPITATING PROPERTIES OF THE BEAN {Phaseolus). By EDWARD C. SCHNEIDER. (From the Department of Biology of Colorado College, Colorado Springs, Colorado. 1 ) (Received for publication December 4, 1911.) The extracts of a number of kinds of seeds are capable of pro- ducing in vitro an agglutination and sedimentation of the red blood corpuscles of various animals. This peculiar property is largely confined to species of the Leguminosae and to a few Sol- anaceae, although an occasional member of other families may possess it. The property was first noted among certain toxic seeds; the several species of Ricinus, Abrus pecatorius, and Croton tiglium. 2 In recent years the list has been enlarged by a careful search for haemagglutinin bearing seeds. Landsteiner and Raubit- schek 3 found this property in extracts of beans, Phaseolus, peas, Pisum, vetches, Vicia, and lentils, Ervum; and v. Eisler and v. Portheim 4 report its presence in five species of Datura. Mendel 5 added the following: sweet pea, Lathyrus odoratus; lentil, Lens esculenta; yellow locust, Robinea pseudacacia; five species of Vicia, Wistaria Chinensis, Caragana arborescens; senna, Cassia Mari- landica; and sweet rocket, Hesperis malronalis. He also found among beans that the haemagglutinins are absent in the Lima 1 Most of the work here reported was done in the Sheffield Laboratory of Physiological Chemistry of Yale University. The writer wishes to ex- press his hearty thanks to Professor Lafayette B. Mendel for the sugges- tion of the problem and for his kindly interest. 2 For the early literature on these see Jacoby: Biochemische Centralblatt. i, p. 289, 1903. 3 Landsteiner and Raubitschek: Centralblatt fur Bakteriologie, 1 Abtei- lung, xlv, pp. 660-67, 1907. 4 V. Eisler and v. Portheim: Zeitschrift fur Immunitdtsforschung und experimentelle Therapie, i, p. 151, 1908. 5 Mendel: Archivio di fisiologia, vii, pp. 168-177, 1909. 47 4 8 Haemagglutinin of the Bean bean. Wienhaus 6 reports that this property occurs in the soy bean, Glycine or Soja hispida; and Assmann 7 found it is the seeds of Canavalia ensiformis, Datura stramonium, and three species of Lathyrus. The agglutinative property is not necessarily coincident with the toxic activity of seeds. It varies greatly in the seeds known to contain haemagglutinin and does not manifest itself equally well with the blood of different kinds of animals. Among laboratory animals Mendel 8 reports the blood of the rabbit to be most sus- ceptible, and those of the pig and the sheep the most refractory. The extract of a number of the seeds noted above reacts well with rabbit's blood but gives negative results with all other bloods tested. The reaction is strongest with suspensions of serum-free corpuscles. Landsteiner 9 found the normal blood serum of many kinds of blood capable of checking the process but that agglu- tination occurred readily when washed corpuscles were used. Several workers have suggested methods for obtaining purified preparations of the agglutinins from the crude extracts. Land- steiner and Raubitschek 10 found that (1) the addition of a little acid produced a precipitate which contained only a trace of the agglutinin, the chief portion remaining in the filtrate. (2) When alcohol was added an agglutinative precipitate was obtained. It was also observed that when this precipitate was redissolved there was no loss of power. (3) The agglutinin was also salted out with the proteins on saturation with ammonium sulphate. From the extract of beans Wienhaus 11 separated a mixture of proteins to which he has applied the name of Phasin. Ten grams of bean meal were extracted with 500 grams of 0.9 per cent sodium chloride solution for twenty-four hours and then filtered. To the filtrate an equal volume of alcohol was added. A voluminous precipitate of albumin and globulin was secured in which the agglu- tinin is held quantitatively. On drying this precipitate in a 6 Wienhaus: Biochemische Zeitschrift, xviii, pp. 228-60, 1909. 7 Assmann: Pfluger's Archiv, cxxxvii, pp. 489-510, 1911. 8 Mendel : Loc. cit. 9 See Raubitschek: Hamagglutinine pflanzlicher.Provenienz und ihre Anti- korper; Kraus and Levadite's Handbuch der Technik und Methodik der Immunitatsforschung, p. 625, 1911. 10 Landsteiner and Raubitschek: Loc. cit. 11 Wienhaus: Loc. cit. Edward C. Schneider 49 vacuum he secured a white powder which yielded to physiological salt solution all of the agglutinin and some inactive proteins. He suggests that he hopes later to free the "Phasin" from proteins by digestion. Landsteiner 12 employed the characteristic of erythrocytes that causes them to give up to the suspension fluid, when gently heated, the agglutinins with which they are combined. To this end he agglutinated, in an ice chest, sensitive serum-free corpus- cles with purified bean extract for several hours. The corpuscles were then washed with cold isotonic salt solution in a centrifuge until no trace of agglutinin was found in the washing solution. The agglutinated corpuscles were next suspended in a small amount of salt solution and stirred for an hour at 45° C. With precautions to avoid cooling they were then centrifugalized. By this means he obtained a clear but often red colored solution containing the agglutinin. This he found he could further purify by dialysis or with ammonium sulphate. Thus far the nature of these vegetable haemagglutinins has not been satisfactorily determined. Landsteiner and Raubitschek conjecture it to be a protein by analogy with the very pure ricin isolated by Osborne, Mendel, and Harris. 13 The latter investi- gators separated the proteins of the castor bean, Ricinus Zanzi- bar ensis, by dialysis and fractional precipitation with neutral salts and found the physiological properties, toxic and haemagglutina- tive, to be associated with the coagulable albumin. The agglu- tinative action was absent in the globulin and proteose fractions, and very active in the albumin fractions. SEPARATION OF THE PROTEIN CONSTITUENTS OF THE BEAN. In view of the experience of Osborne, Mendel, and Harris an attempt has been made to separate the haemagglutinin of the Scarlet Runner bean, Phaseolus multiflorus, Willd. A preliminary examination of a number of varieties of beans was made for the purpose of determining which is richest in haemagglutinins. 12 See Raubitschek in Kraus and Levadite's Handbuch der Technik und Methodik der Immunitdtsforschung, p. 625, 1911. 13 Osborne, Mendel and Harris: American Journal of Physiology, xiv, pp. 259-86, 1905. THE JOURNAL OP BIOLOGICAL CHEMISTRY, XI, NO. 1. 5o Haemagglutinin of the Bean Among these were the dwarf wax-podded varieties Burpee's Kid- ney, Wardell's Kidney Wax, Red Kidney, Dwarf Champion, and Early Six Weeks; and the climbing wax-podded variety Golden Champion of P. vulgaris, L. ; also the Scarlet Runner, P. multi- florus, Willd. The extracts prepared from equal weights of bean meal were almost equally active. The Scarlet Runner seed is much larger than the seeds of the other varieties which favored the removal of the seed coat. The Scarlet Runner beans were first passed through a very coarse grinder. Much of the seed coat was thus broken away from the substance of the coty- ledons and was blown out with an air blast. Afterward these cracked beans were ground to a coarse meal and treated with benzine to remove the oil. Following this the coarse meal was ground to a powder and 1 kilo of it was extracted with 5 liters of a 2 per cent sodium chloride solution that had been previously heated to 60° C. After frequent stirrings for two hours it was placed in a cold room over night and then filtered perfectly clear. The extract was dialyzed in running water for thirty-six hours. The precipitate I which separated was filtered from the solution B and dried. Unfortunately precipitate 1 was dried so slowly that more than two-thirds of it was changed into an insoluble protean. Solution B was further dialyzed three days and yielded a heavy precipitate II which, when dried, was more than 85 per cent soluble. The solution while dialyzing tended to become acid in reaction and required frequent neutrali- zation. It was protected against decomposition with toluene. Solution Bl, which remained after filtering off precipitate 11, was again dialyzed four more days and yielded a small amount of precipitate 111. Precipitates 1 and 11 were the globulin phaseolin; and 111 was probably the other globulin, phaselin, separated by Osborne 14 from the kidney bean. The proteins remaining in solution B2 (obtained from Bl on filtering off precipitate 111) were salted out by saturating with ammonium sulphate. This procedure yielded precipitate IV and solution B3. Solution B3 was then dialyzed in running water until free from salts when it was found it did not contain a trace of the haemagglutinin. Precipitate IV was dissolved in a small volume of water and the clear solu- tion C was then saturated with magnesium sulphate and weakly acidulated with acetic acid. The small amount of precipitate V, which will be called albumin, was then filtered from solution CI. The albumin precipitate, V, was redissolved and precipitated with mag- nesium sulphate and then dissolved in a very small volume of water. From this solution the salt was removed by dialysis. The solution was next evap- orated at a low temperature and yielded 0.3 gram of albumin. 14 Obsorne: Journal of the American Chemical Society, xvi, p. 635 and p. 707, 1894. Edward C. Schneider 5i Solution CI was dialyzed for several weeks until free from salt, then was evaporated in low dishes at 48° C. Almost a gram of proteoses was secured from this solution. THE ACTION OF THE BEAN PROTEIN PREPARATIONS ON BLOOD. Tests were always made with defibrinated rabbit's blood diluted (1:5) with 0.9 per cent sodium chloride solution. One cubic centimeter of this blood mixture was placed in a small and very narrow test tube; and 2 cc. of the protein preparation, dissolved in the salt solution, were added. The time of the visible beginning of agglutination and the condition at the end of two, four, and twelve hours were noted. Preliminary tests with the phaseolin and phaselin (preparations I, II, and III) revealed the presence of haemagglutinin. Prepara- tion II was most active but none of the globulin preparations exhibited the property in a striking degree. Believing that these proteins adsorbed the haemagglutinin, an attempt was made to purify precipitate II. About half of preparation II was dissolved in 0.9 per cent sodium chloride solution. One-half of this solution was dialyzed until it yielded its phaseolin, preparation Ha, and the other half of the solution was saturated with magnesium sulphate. The resulting precipitate was redissolved in water and on dialysis yielded preparation lib. Preparations Ha and lib were less active than preparation II. This weakening in activity by purification indicates that the haemagglutinin in these preparations is held there by adsorption. The albumin, purified from precipitate V, was also active but less so than the globulins. The degree of activity of the albumin and globulins is given in Table I. The proteose preparation was found to be rich in haemagglu- tinin. It produced strong agglutination when present in blood dilutions of one part to 100,000 and more. Wienhaus 15 found his crude product "Phasin" completely agglutinated rabbit's blood in the ratio of 1:7000 in fifteen hours; and with cat's blood, which reacted still better, in dilutions of 1:11,000 in eighteen hours and 1 :60,000 in twenty-three hours. Assmann 16 also working with a 15 Wienhaus: Biochemische Zeitschrift, xviii, p. 232-33, 1909. 16 Assmann: Pfliiger's Archiv, cxxxvii, pp. 489-510, 1911. 52 Haemagglutinin of the Bean TABLE I. Agglutination Trials with Protein Preparations. PREPARATION USED MILLIGRAMS ADDED TO 1 CC. OF BLOOD MIXTURE FIRST INDICATION OF AGGLUTINATION REMARKS No. I. Phaseolin .. , 1912 THE INFLUENCE OF COCAINE UPON METABOLISM WITH SPECIAL REFERENCE TO THE ELIM- INATION OF LACTIC ACID. By FRANK P. UNDERHILL and CLARENCE L. BLACK. {From the Sheffield Laboratory of Physiological Chemistry, Yale University, New Haven, Connecticut.) (Received for publication, February 27, 1912. ) The introduction of cocaine into the organism is followed by such well defined symptoms that an almost specific influence upon the nervous system is indicated. In the main, it is to this aspect of its action upon the body that the very extensive literature 1 regard- ing this drug relates. Definite knowledge of the effect of cocaine upon general metabolism is meagre although the picture presented by the cocaine habitue* is sufficiently characteristic to lead one to infer that ultimately at least the nutritional rhythm must be altered. The widespread employment of cocaine as an ingredient of various types of proprietary remedies and the large number of cases of cocainism makes pertinent at this time an inquiry into the in- fluence upon metabolism of the drug under discussion. The observation of Araki 2 that lactic acid appears in the urine in unusually large quantities after cocaine injections considered in connection with the.findings of Wallace and Diamond 3 that cocaine causes vacuolization of the liver cells of rabbits suggested the pos- sibility of a disturbance in intermediary metabolism. In the present paper the relation of cocaine poisoning to lactic acid out- put is shown and the influence of the nutritive condition of the animal upon this type of acidosis is discussed. It is also demon- strated that in spite of the marked symptoms characteristic of 1 Cf. Richet: Dictionnaire de physiologie, iv, p. 1, 1900. 2 Araki: Zeitschr. f. phyriol. Chem., xv, p. 335, 1891. 3 Reported at the 19th Annual Meeting of the American Physiological Society, New York, 1907. 255 THE JOURNAL OF BIOLOGICAL CHEMISTRY, VOL. XI, NO. 3 236 Influence of Cocaine upon Metabolism chronic cocaine poisoning general metabolism is only slightly changed from the normal even though the quantity of drug admin- istered is sufficient to finally cause death. These observations serve as a further illustration of the tenacity with which the organ- ism adheres to the fundamental laws underlying its metabolic processes; in other words, another example of the "factor of safety" principle is encountered in cocaine poisorfing. THE INFLUENCE OF COCAINE UPON METABOLISM, AS INDICATED BY ITS EFFECT UPON NITROGENOUS EQUILIBRIUM AND PROTEIN AND FAT UTILIZATION. Methods. The experiments were planned so that the animals (dog and rabbit) employed were kept upon a fixed diet and cocaine administered subcutaneously at a time sufficiently long after a meal to avoid the danger of food being vomited. During the first period of the experiments the drug was given once daily, later the animal was kept under the influence of cocaine the greater portion of each day by repetition of the injection. Lactic acid was estimated by the Ryffel 4 procedure. The Folin method as modified by Steel 5 was employed in the deter- mination of ammonia in the urine of rabbits. The other deter- minations were carried out according to the well known meth- ods usually employed in this laboratory. Urine was collected in twenty-four hour periods by catheterization (dogs) or by pressure on the bladder through the body wall (rabbits) . Unless otherwise noted all urines of dogs were acid in reaction to litmus. The rab- bits' urines were alkaline throughout. Description of experiments. Experiments 1 and 2. In these observations dogs 50 and 51 were kept for several days previous to the investigation upon the diet arranged for the experimental trials in order to bring them as nearly as possible into a condition of nitrogenous equilibrium. A fore-period was followed by an interval during which the animals received daily subcutaneous injections of cocaine hydrochloride (Kahlbaum's crystalline pro- duct) dissolved in water. In addition to a constant diet through- out the experiment the animals received, also, a fixed water intake. 4 Ryffel: Journ. of Physiol., xxxix, p. v. 1909-10. 5 Steel: This Journal, viii, p. 365, 1910-11. Frank P. Underhill and Clarence L. Black 237 Protocol of Experiment 1. Dog 50, weighing 12.8 kilos, was normal in every respect except that she was extremely deaf. The diet consisted of 200 grams meat, 80 grams cracker meal, 40 grams lard, 10 grams bone ash, and 300 cc. water. The total nitro- gen intake amounted to 7.40 grams nitrogen daily with sufficient fat and carbohydrate to yield approximately 70 calories fuel value per kilo of body weight. Each day food was given at 9 :30 a.m. and the first cocaine injection at 3:30 p.m. On October 20 the cocaine period was begun. Just before the cocaine injection the rectal temperature was 38.6° C. and two hours later had risen to 39.0° C. The pupils showed extreme dilatation. October 21. In the morning the dog seemed normal and ate food with evident relish. Temperature before cocaine administration was 38.6° C. and had risen to 40.0° C. two hours later. About 45 minutes after the injection the animal exhibited peculiar movements of the head which were constant. The dog was extremely restless. The pupils were greatly dilated. October 22. The dog was apparently normal at meal time. Symptoms after cocaine injection similar to those of previous days. October 22. Symptoms unchanged. October 24. Rectal temperature at 9:30 a.m. = 38.8° C, just before injection at 3:30 p.m. = 38.6° C. ; at 4:30 p.m. = 40.9° C. At 4:30 p.m. the heart action was very slow but strong. Arhythmic beating was in evidence. There was extreme dilatation of pupil. The animal was very much excited and the head was constantly moved up and down. Usually the animal was too deaf to pay attention to any sound, but at this time it would respond to a call. October"25. In the morning the dog appeared normal and devoured food as usual. Temperature at 9:30 a.m. = 38.8° C, just before injection; at 3:30 p.m. = 38.8° C; at 5:00 p.m. = 41.1° C. The movements of animal were more pronounced and there was much more excitation after cocaine administration than had been observed at any previous time. The peculiar irregularity of the heart was again in evidence at 5:00 p.m. although previous to the injection, the beat was normal. October 26. The appetite of animal was ravenous. Temperature at 9:30 a.m. = 38.6° C, just before injection; at 3:30 p.m. = 38.6° C, at 4:00 p.m. = 41.6° C at 5:00 p.m. = 40.9° C. It was apparent that the animal had become much more sensitive to the cocaine since the usual daily injection was followed by greatly augmented symptoms of excitation. These lasted for a period of two hours. October 27. The dog devoured food with apparent relish. Temperature at 3:15, just before injection = 38.6° C; at 3:45 = 41.2° C. ; at 4:15 = 41.6° C; at 4:45 = 409° C. ; at 5:15 = 39.8° C. The symptoms of excitation and pupil dilatation appeared within fif- teen minutes after cocaine administration. Apparently the peculiar head movements were caused by an attempt to push the head out of the cage 238 Influence of Cocaine upon Metabolism TABLE 1. Experiment I — Dog 50. Fore Period. (Daily Nitrogen Intake = 7.40 grams.) H URINE FECES O fa DATE Z H M vity c » u ater otal Ni ther E: 05 > w H < Q ' Eh H 1910 mgms. kilos cc. gms. gms. mgms. gms. gms. per cent gms. gms. October 15 12.8 300 1.020 6.15 0.28 49 26.8 11.6 56 *(4.5) 16 12.8 300 1.020 6.18 0.30 49 32.6 14.0 57 (4.8) 17 12.8 310 1.021 6.03 0.27 48 19.0 12.5 34 3.30 6.09 (4.4) -18 J2.8 300 1.020 6.24 0.29 49 29.0 15.0 49 (4.6) 19 12.8 300 1.020 6.18 0.26 48 96.0 46.0 51 (4.2) Average per day.. . . 12.8 302 1.020 6.15 0.28 48 40.7 19.8 49 0.66 1.22 (4.5) J^irsi Cocaine Period. 20 128 12 8 410 1 .020 6 45 0.26 (4.3) 54 51 26 49 21 128 12 7 275 1 .025 5 88 0.24 (4.0) 56 60 28 53 . 22 128 12 6 260 1 020 6 15 0.34 60 29 15 0. 49 (5.5) 23 128 12 6 270 1 021 5 64 .28 64 35 17 51 (4.9) 1 24 128 12 6 210 1 026 5 73 0.35 61 47 24 49 (6.1) 25 128 12 5 240 1 030 6 75 0.34 (5.0) 54 35 20 42 8.0717.83 26 128 12 4 170 1 040 6. 66 0.30 (4.5) 53 51 30 41 * Figures in brackets indicate percentages of total nitrogen. Frank P. Underhill and Clarence L. Black 239 TABLE 1— Continued First Cocaine Period — Continued tngms 128 128 128 128 128 kilos 12.3 12.3 12.2 12.2 165 I 1.045 160 I 1.046 170 j 1.040 165 1.041 12.5" 227 i 1.032 gms. gnts. 12 0.27 (4.4) 6.54| 0.30 ■ (4.5) 6.12 0.33 ' (5.3) 6.06! 0.30 4.9) 6.19| 0.30 I (4.8) Weight 70 76 71 78 63 cent sms - 40.0 24 .0 40 50.0 24.0 52 47.0 25.0 47 44.0,24.0 45 41.0 23.0 47 0.73 1.62 Second Cocaine Period. November 1 256 12.2 170 1.040 8.25 0.36 (4.3) 84 46.0 21.0 54 2 256 11.4 140 1.050 6.84 0.35 (5.1) 83 52.0 32.0 39 3 256 11.3 120 1.052 5.94 0.31 (5.2) 79 53.0 33.0 38 3.55 13.95 4 256 11.2 125 1.050 6.18 0.31 (5.0) 80 50.0 23.0 54 Average per day. . . 256 11.5 138 1.048 6.80 0.33 (4.9) 81 50.0 27.0 46 0.88 3.48 240 Influence of Cocaine upon Metabolism Balances Fore Period grams Nitrogen in food 37.00 Nitrogen in excreta: Urine 30.78 Feces 3.30 34.08 Nitrogen balance +2.92 Per day +0.58 Nitrogen Utilization = 91 percent. Ether extract in food . Ether extract in feces . grams 323.20 . 6.09 Fat utilized. . . Fat utilization . .. 317.11 per cent. First Cocaine Period. Nitrogen in food 81.40 Nitrogen in excreta : Urine 68.10 Feces 8.07 76.17 Ether extract in food 711.04 Ether extract in feces 17.83 Nitrogen balance +5.23 Per day +0.47 Nitrogen utilization = 90 per cent. Fat utilized 693.21 Fat utilization = 98 per cent. Second Cocaine Period. grams Nitrogen in food 29 .60 Nitrogen in excreta: Urine 27.21 Feces 3.55 30.76 Ether extract in food 258 . 56 Ether extract in feces 13 .95 Nitrogen balance — 1.16 Per day - 0.29 Nitrogen utilization = 88 per cent. Fat utilized 244 .61 Fat utilization = 94 per cent. toward the light. During the remainder of this period which was concluded on October 31 no new features developed. It was planned to begin the second cocaine period on October 31 by giving two injections of the drug, at 12:00 m. and 4 :00 p.m. respectively. The first injection caused vomiting which contaminated the urine. This period was therefore, commenced on the next day, November 1. On this date cocaine in doses of 128 mgrms. each was administered at 3:00 p.m. and 5:00 p.m. Just previous to the first injection the temperature was 38.5° C, at 5:p.m. ; 40.0° C, at 6:00, p.m., 40.9° C. The dog was in a state of extreme activity during this time. November 2. Cocaine was injected as on November 1. The conditions of the animal had, however, undergone a marked change since all movements were executed in a weak and uncertain manner. Frank P. Under hi 11 and Clarence L. Black 241 TABLE 2. Dog 51. Fore Period. (Daily Intake of Nitrogen = 4.72 grams) w URINE FECES < a 4> U) Weight O DATE DAILY DOSE OF BODY WEIGHT B "0 > Specific Gravit; Total Nitrogen Ammonia Nitr Lactic Acid >> u Q u 1 Total Nitrogen Ether Extract 1910 mgms. kilos cc. firms. gms. mgms. per cent gms. gms. November 30 8.3 120 1.040 4.56 0.21 *(4.6) 45 December 1 8.2 175 1.035 4.25 0.23 (5.1) 46 52.0 23.0 56 2 8.2 165 1.036 4.23 0.23 (5.1) 51 17.0 10.0 42 3 8.2 165 1.036 4.24 0.17 (4.0) 51 29.0 18.0 38 2.10 5.46 4 8.2 125 1.040 4.21 0.16 (3.7) 45 20.0 11.0 45 5 8.2 175 1.030 4.20 0.18 (4.2) 48 15.0 10.0 33 Average per day. 8.2 154 1.036 4.28 0.19 (4.4) 47 22.0 12.0 42 0.35 0.91 Cocaine Period. 6 123 8.2 7 123 j 7.6 8 123 j 7.6 9 123 7.6 10 123 j 7.6 220 200 155 150 1.026 1.030 1.035 1.034 4.44 0.18| 58 11.0 5.0 55 (4.0); 3.84 0.14' 73 23.0 11.0 51 (3.6) 4.35 0.16 74 I (3.6)1 4.29 0.17 70 (3.9), 155 1.035 3.80 0.16| 79 42.0 22.0 46 (4.2) * Figures in brackets indicate percentages of total nitrogen. 242 Influence of Cocaine upon Metabolism TABLE 2— Continued Cocaine Period — Continued 1910 December 11 12 13 14 15 mgms.\ kilos 123 123 123 123 123 Average per day. 123 7.6 7.6 250 130 7.5 140 7.5 7.5 7.6 135 145 o s i 2 m , EH I gms. gms. 1.030 4.21 0.18 (4.2) 1.040 4.50 0.18 (4.0) 0.16 (5-5) 1.035 2.88 1.037 3.15 j 1.033 4.56 Weight gms. 168 0.16 (5.0) 0.19 (4.1) 1.033 4.00 0.17 (4.2) 85 90 j 50.0 79 1 11.0 90 j 44.0 j 20 .0 79 49.0 per cent 78 :25.0 25.0, 50 6.0 H j W gms. j gms. 3.89 (32.54 45 31.0! 30 12.0 40 25.0 49 13.7 45 0.39! 3.25 Balances Fore Period. grams grams Nitrogen in food 28.32 Ether extract in food 243.60 Nitrogen in excreta: Urine 25.69 Feces 2.10 Ether extract in feces 5.46 Fat utilized 238.14 Fat utilization = 97 pei cent. Nitrogen balance +0.53 Per day +0.08 Nitrogen utilization = 92 per cent. Cocaine Period. grams grams Nitrogen in food 47.20 Ether extract in food 406.00 Nitrogen in excreta : Urine 40.02 Feces 3.89 43.91 Nitrogen balance +3.29 Per day +0.33 Nitrogen utilization = 91 per cent. Ether extract in feces . 32.54 Fat utilized 373.46 Fat utilization = 91 per cent. Frank P. Underhill and Clarence L. Black 243 November 8. The dog showed signs of diminished appetite. Conditions remained unchanged. November 4- Conditions about as usual. Animal appears weak. November 5. The dog died twenty-five minutes after the first cocaine injection. Just before death the dog was in a state of extreme activity. This was rapidly followed by a period of partial paralysis culminating in respiratory failure. Further data concerning this experiment may be found in Table 1, pp. 238-240. Protocol of Experiment 2. Dog 51 . A fox terrier bitch of 8.3 kilos was placed upon a fixed diet composed of 125 grams meat, 60 grams cracker meal, 20 grams lard, 10 grams bone ash and 150 cc. water for a period of 10 days previous to the actual fore period of the experiment. The nitrogen content of this diet amounted to 4.72 grams; the fuel value was approximately 69 calories per kilo body weight. November 30. On this date the fore period of six days was begun. December 6. The cocaine period was commenced by the injection of 123 mgms. cocaine at 3:00 p.m. No rise in temperature could be observed. The only symptoms noticeable were salivation and pupil dilatation. December 7. About one-half hour after the administration of cocaine the dog became markedly excited, the bodily movements not being under perfect control. Pupil dilatation was extreme and the arhythmic heart beat was evident. Each day up to December 12 the symptoms of excitement etc. were noticeable but unchanged in character. December 12. Shortly after the cocaine injection the animal became completely paralyzed in the hind-quarters. The j aws and tongue were kept constantly in motion as though the animal was tasting something unpleas- ant. The dog remained in this condition for several hours during which she appeared deaf and blind. December 13. The animal seemed normal although somewhat Weak. The weakness became more and more noticeable and on December 15 the experiment was terminated. For other data associated with this animal see Table 2, pp. 241-242. DISCUSSION OF RESULTS. From the details of the protocols and tables submitted it is apparent that the most obvious symptoms arising from cocaine injections in the doses given are distinctly of nervous origin. A significant influence is also exerted upon the heat regulating mech- anism whereby the temperature is quite markedly increased for a short period after which there is a gradual return to the nor- mal. 6 With daily doses of 10 mgms. of cocaine hydrochloride •Reichert: Centralbl. f. d. med. Wissenschaften, 1889, p. 444. 244 Influence of Cocaine upon Metabolism per kilo of body weight no appreciable influence can be detected upon the course of nitrogenous metabolism nor upon the utiliza- tion of protein and fat although body weight shows an appreciable decline. When injections of 15 mgms. cocaine per kilo are daily admin- istered fat utilization is very slightly impaired and is accompanied by a decreased body weight. Doses of 20 mgms. per kilo per day divided into two injections show a fairly distinct detrimental in- fluence upon both protein and fat utilization and for the first time a slight negative balance was in order. Body weight was markedly diminished under this dosage. The water excretion of Dog. 50 was quite distinctly diminished under cocaine when compared with that of the fore-period. This finding does not hold true for Dog 51. The difference may be explained perhaps by the fact that Dog 50 was apparently much more sensitive in its reaction to cocaine with respect to the tem- perature raising influence than was Dog 51. Assuming this to be true more water was probably eliminated by the lungs in the first case than in the second which would account for lessened water elimination by the kidney. THE INFLUENCE OF COCAINE UPON THE ELIMINATION OF LACTIC ACID IN THE URINE. The presence of lactic acid in the urine in appreciable quanti- ties has been a subject of much investigation and discussion result- ing in a multiplicity of conflicting theories with respect to its sig- nificance. Out of the enormous literature 7 relative to lactic acid only a few references that have a bearing upon the present paper may be cited. Thus, Araki 8 has demonstrated that lactic acid appears in the urine in the absence of a sufficient supply of oxygen induced by various types of toxic compounds and epileptic seizures. The older work of Spiro 9 indicating that increased muscular activity leads to lactic acid excretion finds confirmation in the recent investiga- 7 Ryffel: Quarterly Journ. of Med., in, p. 413, 1909-10. 8 Araki: loc. cit. 9 Spiro: Zeitschr. f. physiol. Chem., i, p. Ill, 1877. Frank P. Underhill and Clarence L. Black 245 tions of Ryffcl 10 and Feldman and Hill. 11 According to the latter authors the appearance of lactic acid in the urine may be greatly diminished by breathing oxygen before and after exertion. They conclude that the increased production of lactic acid by the muscles is due to oxygen want, a view that was earlier denied by Ryffel. 12 Viewed from the standpoint of ultimate origin, it is possible that lactic acid is intimately associated with the carbohydrate store of the body; for Araki found, under the experimental conditions, less lactic acid in the urine of starving animals than could be dem- onstrated in the urine of those well fed. On the other hand, phosphorus, which leads to a disappearance of the carbohydrate store, causes a large output of lactic acid which may be accompan- ied by an increased elimination of ammonia. 13 It is presumed that the increase of the latter urinary constituent is for the purpose of neutralizing the lactic acid produced. In the experiments to be recorded the rabbits were kept upon a diet consisting of 300 grams of carrots and 20 grams oats which experience had demonstrated would usually be entirely eaten each day. Experiment 3. Rabbit B. During each day of the fore period this animal left small portions of the carrots uneaten. After the subcutaneous cocaine injections no food was ever left. For the first two days of the cocaine period no evidences of ab- normal symptoms were observed. On the third day, however, there was considerable dilatation of the pupil. Beginning with November 9, the tenth day of administration, irritability and restlessness were noticeable. The appetite remained good, all food being eaten shortly after the daily cocaine administration. About 10 minutes after cocaine injection on November 11 the animal was seized with convulsions and respiration almost ceased, but recovery was complete three-quarters of an hour later. On the succeeding two days convulsions were in evidence shortly after cocaine administration, but in each instance recovery was complete. The animal died in a convul- sion on November 14. The liver which was immediately excised contained 8 per cent of glycogen. From the data in Table 3 it will be observed that the injections of cocaine were progressively increased from approximately 15 mgms. per kilo to 20 10 Ryffel: Journ. of Physiol., xxxix, p. xxix, 1909. 11 Feldman and Hill: Journ. of Physiol., xlii. p. 439. 1911. 12 Ryffel: Journ. of Physiol., xxxix, p. xxix, 1909. 13 Mandel and Lusk: Amer. Journ. of Physiol., xvi, p. 129, 1906. 246 Influence of Cocaine upon Metabolism TABLE 3. Rabbit B. Fore Period. DATE DAILY DOSE OF COCAINE BODY WEIGHT URINE Volume Specific 1 Giavity Total Nitrogen Ammonia Nitrogen Lactic Acid 1910 October 26 27 28 zy 30 mgms. kilos 2.38 2.38 2.34 2.32 2.32 cc. 200 110 120 105 125 1.016 1.025 1.024 1.025 1.025 grams 1.00 0.76 0.75 0.93 0.96 mgms. 2.5 (0.25)* 1.8 (0.23) 1.8 (0.23) 1 1 . 8 (CI 1 q\ (u. iy; 1 A 1 .4 (r\ 1 (0.15) mgms. 9 8 10 1 A 1 O J z Average v per day . . 2.34 132 1.023 0.88 1.8 (0.21) 10 Cocaine Period. 31 33 2.32 125 1.025 0.90 1.0 11 (0.11) November 1 34.5 2.32 160 1.021 0.97 1.08 11 (0.19) 2 34.5 2.32 190 1.020 0.93 2.7 13 (0.29) 3 34.5 2.32 215 1.018 0.80 2.7 10 (0.33) 4 34.5 2.32 215 1.019 0.74 2.3 13 (0.31) 5 34.5 2.32 250 1.015 0.71 2.7 12 (0.18) 6 34.5 2.32 210 1.016 0.73 1.8 14 (0.24) 7 46 2.30 185 1.018 0.67 1.8 12 (0.27) 8 57.6 2.30 210 1.016 0.59 1.8 15 (0.30) Frank P. Underhill and Clarence L. Black 247 TABLE 3 Continued ( loca inc. Period — Continued DATE DAILY DOSE OF COCAINE BODY WEIGHT 1910 7TXQ TTl S November 9 69 2.26 10 89 2.22 11 101 2.24 12 101 2.28 13 101 2.30 Average per day . . 2.29 Volume 235 195 180 200 185 196 pGCtnc Total A m mon \n JU9;Ct'lC Gravity N ltrogen Nitrogen Acid grams mgms. mgms. 1.015 0.61 1.8 26 (0.30) 1.020 0.61 6.3 25 (1.0) 1.020 0.63 1.8 33 (0.28) 1.024 0.82 1.1 39 (0.13) 1.024 0.88 1.1 51 (0.12) 1.018 0.75 2.2 20 (0.30) * Figures in brackets indicate percentages of total nitrogen. mgms. on November 7, to 25 mgms. per kilo on November 8, to 30 mgms. on November 9, to 40 mgms. on November 10, and finally to 45 mgms. per kilo on November 11. Frequent tests throughout the cocaine period failed to demonstrate an appreciable rise in rectal temperature. Experiment 4- Rabbit C. This animal behaved in a manner very similar to Rabbit B. A rise in rectal temperature of about 0.5° C. was the maximum increa e shown dur- ing the period of observation. The daily dose of cocaine given varied from approximately 10 mgms. per kilo on November 29 and 30, to 20 mgms. on December 1 to 6 inclusive, and from this time to the end of the experiment the animal received approximately 34 mgms. cocaine per kilo body weight. From the data in Tables 3 and 4 with rabbits and those in Tables 1 and 2 with dogs, it is evident that cocaine causes an appreciable increase in the elimination of lactic acid in the urine. In a general way the quantity of lactic acid thus excreted is in direct proportion to the amount of cocaine injected. The output of ammonia, however, does not appear to be significantly increased by the augmented elimination of lactic acid, an indication that in 248 Influence of Cocaine upon Metabolism TABLE 4. Rabbit C. Fore Period. DATE DAILY BODY URINE DOSE OF COCAINE WEIGHT Volume Specific Gravity Total Nitrogen Ammonia Nitrogen Lactic Acid mgms. kilos . c.c. grams mgms. mgms. 1910 November 21 2.26 290 1.014 0.80 1.0 24 22 2.24 295 1.014 0.85 1.0 CO 12) 20 23 2.20 245 1.015 0.83 3.6 (0.43) 23 24 2.18 235 1.016 0.83 4.5 (0.54) 22 25 2.20 230 1.016 0.80 3.6 (0.45) 22 26 2.22 190 1.018 0.82 3.6 (0.44) 20 27 2.24 200 1.019 0.81 4.5 (0.55) 21 . 28 2.26 230 1.018 0.83 4.5 (0.54) 23 Average per day . . 2.22 239 1.016 0.82 3.2 (0.39) 22 Cocaine Period. 29 20 2.26 270 1.017 1.18 4.5 (0.38) 23 30 20 2.24 245 1.020 1.40 4.5 (0.32) 23 December 1 45 2.26 240 1.021 1.30 3.6 (0.27) 24 2 45 2.24 230 1.021 1.26 3.6 (0.27) 24 3 45 2.26 220 1.022 0.97 3.6 (0.37) 25 4 45 2.30 230 1.022 0.86 4.5 (0.52) 26 Frank P. Underhill and Clarence L. Black 249 TABLE 4— Continued. DATE DAILY BODY UKINK DOSE OP COCAINE WEIGHT Specific Total Ammonia Lactic Volume ( tT*M Vi t V V_X J V H/J Nitrogen Nitrogen Acid mgms. kilos. c.c. grams mgms. mgms. December O QA Z . oU 91 ^ 1 099 1 . \}ZL ^0 OU /lo^ a O Qfl Z . oU 215 1 099 SO 00 (0.67) 7 75 O OA 205 1.022 0.74 6.3 33 (0.85) 8 75 2.24 225 1.020 0.72 8.1 36 • (1.12) 9 75 2.26 190 1.024 0.83 9.0 40 (1.08) 10 75 2.30 230 1.020 0.95 9.0 42 • (0.94) Average 2.26 226 1.022 0.99 5.5 30 per day . (0.55) * Figures in brackets indicate percentages of total nitiogen. this connection lactic acid may be neutralized by some base other than ammonia. This is particularly true for dogs, but does not hold quite so well with rabbits, for with Rabbit C. the output of ammonia paralleled closely the elimination of lactic acid. The influence of diet upon lactic acid elimination under the experimental conditions may be indirectly inferred from the data of . Table 5 obtained from Dog 52 during a period of inanition. Here it will be observed that in spite of largely increased doses of cocaine lactic acid output fell considerably. The larger quantities of lactic acid excreted during the first few days of the experiment may perhaps be explained on the assumption that the carbohy- drate store of the body during this interval had not been depleted. As soon as this condition had been reached a diminution in lactic acid output took place. These results are in harmony with the theories outlined by Araki, but are in opposition to the observa- tions reported for pernicious vomiting of pregnancy where lactic acid is eliminated 14 in the urine probably as a result of the inanition "Underhill: This Journal, ii, p. 485, 1906-07; see also Underhill and Rand: Arch, of Int. Med., v, p. 61, 1911. 250 Influence of Cocaine upon Metabolism TABLE 5. Dog 52 — Inanition. DAILY URINE DATE DOSE OF . COCAINE BODY WEIGHT Volume Specific Total Ammonia Lactic Gravity Nitrogen Nitrogen Acid rngms. kilos cc. grams gram. mgms. 1910 ■ November 10 120 10.2 160 1.050 6.57 0.31 (4.7)* 41 11 120 10.2 120 1.058 4.23 0.39 (9.2) 38 12 120 9.9 180 1.025 3.06 0.34 (11.1) 39 13 120 9.6 140 1.035 2.73 0.26 (9.5) 36 14 120 9.3 | 200 15 150 120 9.0 1.040 4.92 0.31 (6.3) 32 16 2x 150 8.9 70 1.030 1.80 0.13 (7.3) 13 17 2x150 8.8 160 1.030 3.60 0.25 (6.9) 21 18 2 x 150 8.6 100 1.018 0.48 0.03 (6.2) 5 19 2x150 8.5 100 1.020 3.25 0.12 (3.6) 25 * Figures in brackets indicate percentages of total nitrogen. which accompanied this pathological state. The observations noted above are also opposed to the results obtained in phosphorous poisoning 15 a condition in which carbohydrate is almost missing from the liver and blood. On the other hand, hydrazine 16 which behaves in a manner similar to phosphorus 'with respect to its influence upon the carbohydrate of the organism does not lead to the appearance of appreciable quantities of lactic acid in the urine. From these contradictory results it is apparent that lactic acid must have a diverse origin under the different conditions mentioned. The ammonia content of the urine voided by the dog in a state of inanition was not greatly influenced by the cocaine injections and did not bear a direct relationship to the elimination of lactic acid. 16 Frank and Isaac: Arch. f. exp. Path. u. Pharm., lxiv, p. 374, 1911. 1G Underhill: This Journal, x, p. 159, 1911. Frank P. Underbill and Clarence L. Black 251 From the observations here recorded the conclusion may be drawn that the appearance of lactic acid in increased quantity during cocaine poisoning is probably associated with the attendant increased muscular activity induced by the action of the drug upon the nervous system. What relation augmented lactic acid out- put bears to lack of oxygen as claimed by Araki is a problem dif- ficult of decision unless one accepts the view put forth by Feldman and Hill 17 that increased muscular work results in a decreased amount of oxygen in the muscles, which in turn causes an increased production and subsequent excretion of lactic acid. It is also apparent that in cocaine poisoning greater quantities of lactic acid are eliminated by given doses of cocaine to well-fed animals than occurs under the same conditions during an interval of starvation. The average elimination of lactic acid during co- caine poisoning in a state of inanition was less than that of other animals maintained in a well-fed condition, but without cocaine administration. It seems probable, therefore, that during cocaine poisoning, carbohydrate material may be intimately associated with the production of lactic acid. conclusions. In confirmation of previous investigation, it is found that co- caine introduced subcutaneously into dogs causes a temporary but significant increase in body temperature. With daily doses of 10 mgms. of cocaine hydrochloride per kilo of body weight for short periods of time no influence can be de- tected upon nitrogenous metabolism nor upon fat utilization. Fat utilization is slightly impaired and body weight is consider- ably decreased when daily injections of 15 mgms. cocaine are administered. When the dose of cocaine is increased to 20 mgms. per kilo body weight per day a distinct lowering of both nitrogen and fat utilization is noted. This may be accompanied by a slight nega- tive nitrogen balance. , Lactic acid excretion in the urine is markedly increased in well- fed dogs and rabbits as a result of cocaine injection. In a starving 17 Feldman and Hill: loc. cit. TH E JOURNAL OF BIOLOGICAL CHEMISTRY, VOL. XI, NO. 3. 252 Influence of Cocaine upon Metabolism condition the dog eliminates less lactic acid after cocaine injections than is excreted by the normal well-fed animal. It is not unlikely that the increased lactic acid elimination after cocaine injections is associated with increased muscular activity induced by the drug. The ammonia output apparently bears little relation to lactic acid elimination under the experimental conditions. Lactic acid and carbohydrate metabolism are presumably inti- mately associated although there are indications that lactic acid may at times arise from more than a single antecedent. Reprinted from The Journal of Biological Chemistry, Vol. XI. No. 5, 1912 THE PHYSIOLOGICAL ACTION OF SOME PYRIMIDINE COMPOUNDS OF THE BARBITURIC ACID SERIES. By ISRAEL S. KLEINER. {From the Sheffield Laboratory of Physiological Chemistry, Yale University, New Haven, Connecticut.) (Received for publication, March 27, 1912.) Aside from the fact that certain pyrimidine compounds are con- stituents of the nucleic acid molecule, their possible biochemical importance is attested by a structural relation to the purines, creatine, creatinine, allantoi'n and other compounds of physiologi- cal interest. Moreover a few pyrimidines are known to have a marked pharmacological action. The first substance of this type used in physiological experiments was alloxantine, which is formed by the reduction of alloxan. HN— CO HN— CO OC— NH I I +H2 - II 'I 2 OC CO ^ 2 OC CH— O C— OH CO II > I I I I HN— CO HN— CO OC NH Wohler and Frerichs 1 fed 5 to 6 grams of this to men but could not recover any in the urine; nor was alloxan found. The urine was rich in urea, and a breaking down of alloxantine to urea and other products was believed to be probable. No mention is made of any toxic effects, although if any had been experienced they would undoubtedly have been described because the subjects were human beings. Koehne 2 fed alloxan and alloxantine in 8-gram doses to dogs. Each caused a mild diarrhea without other symptoms. No alloxan or alloxantine was excreted in the urine; but small amounts of oxalic and parabanic acids were found. Working independently of Koehne with the same compounds Lusini 3 obtained results different in some respects at least. In his experi- 1 Wohler and Frerichs: Ann. d. Chem. u. Pharm., lxv, pp. 335-349, 1848. 2 Koehne: Inaugural Dissertation, Rostock, 1894, 39 pp. 3 Lusini: Ann. di chim. e di farmacol., xxi, pp. 145-160, 1895; pp. . 41-257; and xxii, pp. 341-351, 1895; pp. 385-394: from Chem. Centralbl., 1895, i, p. 1074; ii, p. 838. 443 THE JOURNAL OF BIOLOGICAL CHEMISTRY, XI, NO. 5 444 Action of Certain Pyrimidines ments he found that both of these substances attacked the skin of frogs, dogs and rabbits. Both acted upon the cerebro-spinal centers, this action being divided into two periods, (a) hyper-reflex-excitability followed by rigidity, and (b) hypo-reflex-excitability and paralysis. This second stage had previously been noted by Curci, 4 who because of the use of a larger dosage had overlooked the first stage. These and other minor effects varied slightly with the compound used, alloxan being in general more toxic than alloxantine. Alloxantine strongly reduced the hemoglobin of the blood both in vitro 5 and in vivo. Among other phenomena produced in frogs by alloxan, mydriasis is noteworthy. Alloxan also had a powerful influence on the heart; the contractions were diminished in vigor, diastolic pauses lengthened, and finally the heart stopped in diastole. According to Lusini, alloxan was non-toxic when given per os, 0.5 gram being easily borne. It did not reappear in the urine but parabanic acid and alloxantine were found. When Lusini fed alloxantine he recovered only slight traces in the urine. A small amount of dialuric acid was found together with parabanic acid and murexide in larger quantities. Lusini HN— I reached the conclusion that the group, OC is able to stimulate and then I . ' HN — HN — CO inhibit the nerve centers, and that the grouping, q^L, has no such power. I It is, according to Lusini, the ketone-like CO which seems to have the stimu- lating property, and the abundance of these groups increases the toxicity of alloxan. More recently Steudel 6 has attempted to ascertain whether pyrimidines may be built up to purines in the animal body. The compounds used in- cluded those which Behrend and Roosen 7 had described as intermediate products in the synthesis of uric acid in the chemical laboratory. At the outset it may be stated that a purine synthesis in vivo was not established. Steudel fed the substances to a bitch weighing 6.2 kg. in doses of 1 gram per day with meat and attempted to isolate them or their purine deriva- tives in the urine. 4-Methyluracil and 5-nitrouracil were found unchanged in the urine. 5-Nitrouracil-4-carboxylic acid, however, did not reappear in the urine. Steudel believes that it underwent a complete decomposition in the organism, although he does not consider the possibility of the non- absorpti n from the alimentary tract and does not report any analyses of 4 Curci: Cited by Lusini: Ann. di chim. e di farmacol., xxi, pp. 145-160, 1895; from Chem. Centralbl., 1895, i, p. 1074. 6 This property was described by Kowalewsky: Centralbl. f. d. med. Wis- sensch., xxv, pp. 1-3, 17-18, 1887. 6 Steudel: Zeitschr. f. physiol. Chem., xxxii, pp. 285-290, 1901. 7 Behrend and Roosen: Ann. d. Chem., ccli, pp. 235-256, 1889. Israel S. Kleiner 445 the feces. Of the following pyrimidines none was recovered in the urine after feeding, nor was any difficultly soluble condensation product detected : isobarbituric acid, isodialuric acid, thymine and uracil. This author points out the striking difference in behavior between thymine (5-methyl-2,6- dioxypyrimidine) and 4-methyluracil. Structurally they differ only in the position of the methyl group but the former is broken down in the body while the latter is not. If, however, a nitro group is substituted for the methyl in thymine the physiological character of the pyrimidine is reversed; for it now passes unchanged through the kidney. Although a purine synthesis could not be demonstrated, Steudel deter- mined to extend his experiments with other pyrimidines, namely, 2,4-di- HN— CO I I amino-6-oxypyrimidine, H2N — C CH and 2,4,5-triamino-6-oxypy- II II N— C— NH 2 HN— CO I 1 rimidine, H2NC C — NH2 which Traube 8 had obtained as intermediate II II N— C— NH 2 products in his synthesis of guanine. Both were administered as the sul- phates in 1-gram dose in the manner above described. Both were reported to be toxic, which was surprising inasmuch as none of the other compounds had been accompanied by any untoward symptoms. Feeding of the 2, 4-diaminopyrimidine was followed by vomiting and the triamino compound provoked equally serious disturbances. About an hour after the substance was taken, there occurred attempts at vomiting without any vomitus being ejected. The animal had no appetite and lay on one side almost all day. The urine contained protein, hyaline cylindroids and the unchanged tri- amino compound. The last was recovered as the sulphate and identified by the violet color produced by saturating it with ammonia. By subcu- taneous injections the lethal dose for rats was determined as 0.2 gram for 2,4-diamino-6-oxypyrimidine sulphate and 0.1 gram for 2,4,5-triamino-6- oxypyrimidine sulphate. Autopsy of the rats poisoned with the diamino substance revealed nothing characteristic; but the kidneys of the animals which had received the triamino compound contained numerous concre- tions and resembled microscopically the kidneys of dogs poisoned with adenine. 9 From these results, Steudel concluded that the attachment of amino groups to the pyrimidine ring transforms harmless, indifferent substances into poisonous ones. The toxicity of adenine, 6-aminopurine, he regards as an analogous phenomenon in the purine series. He believes that an examina- tion of other amino derivatives of the pyrimidine and purine compounds will prove the universality of this law. No analyses of the two amino- 8 Traube: Ber. d. deutsch. chem. Gesellsch., xxxiii, pp. 1371-1383, 1900. 9 Minkowski: Arch f. exp. Path. u. Pharm., xli, pp. 375-420, 1898. 446 Action of Certain Pyrimidines pyrimidines fed are presented by Steudel, nor are any data as to their solu- bility given. In a later contribution Steudel 10 reported the investigation of other mem- bers of this series. Pseudo-uric acid and isouric acid did not result in a purine synthesis although both have been transformed into uric acid in vitro. A similar result was obtained when hydrouracil was fed. 2-Thio-4-methyl- uracil, like 4-methyluracil described above, was quickly excreted in the urine. 2-Amino-4-methyluracil, which differs from thiomethyluracil only in the substitution of an amino group for sulphur in the 2-position did not appear in the urine, nor was any other characteristic product found. Steu- del concludes that none of the pyrimidines used by him are adapted to a synthesis of purine compounds in the dog. The pharmacological action of some pyrimidines was studied by Fischer and von Mering 11 with interesting results. They discovered that certain alkyl derivatives possess an action similar to that of sulphonal. The latter, diethylsulphone-dimethylmethane, is rich in alkyl groups; and it was the idea of these authors to experiment with other alkyl organic compounds, many of which Fischer had synthesized, in the hope of ascertaining the essential or most effective groupings for hypnotic action. Of especial inter- est are the cyclic compounds employed, which are derivatives of barbituric acid and of malonyl guanidine. It was found that the 5-monoalkyl deriva- tives of barbituric acid have no hypnotic action, nor has the 5, 5-dimethyl derivative; but when both hydrogen atoms in the 5 position are replaced by alkyl groups, at least one being higher than methyl, the compound acquires sleep-producing powers. This reaches its maximum in 5, 5-dipro- pylbarbituric acid. Some of the compounds studied proved toxic; for example, substitution of the H in the 1 position by CH 3 or of the O in the 2 position by S transformed 5,5-diethylbarbituric acid into a toxic com- pound. However, 5,5-dipropylmalonyl guanidine, HN — CO as well as diethylmalonuric acid, H2N COOH had no pharmacological I I /C3H7 HN = C C< I I X C 3 H 7 HN— CO 5, 5-Diethylbarbituric acid, HN — CO I I /C 2 H 5 oc c< I I X C 2 H 5 HN— CO has been used widely in 10 Steudel: Zeitschr. f. physiol Chem., xxxix, pp. 136-142, 1903. 11 Fischer and von Mering: Therapie der Gegenwart, N. F., v, pp. 97-101, 1903. Israel S. Kleiner 447 medicine as an hypnotic under the name "veronal," its sodium salt as "medinal," and the dipropyl compound to a less extent as "proponal." Fischer and von Mering 12 have found that most of the, veronal is excreted from the body unchanged. Recently P. Fischer and Hoppe, 13 Bachem, 14 Grober, 15 and Jacobj 16 have added many new facts to the literature of veronal. Wolf 17 injected uracil, thymine, and cytosine in 10 to 50 mgs. doses into the circulation of cats, but observed no effect upon arterial pressure, intesti- nal volume, respiration, or rate of blood-clotting. Sweet and Levene 18 fed thymine to a dog with an Eck's fistula (on the basis of Steudel's contention that thymine is destroyed by the normal dog). A marked diuresis resulted and thymine was found in the urine in considerable amount. This is inter- esting in view of the close relationship between this methylated pyrimidine and the methyl substituted xanthines: theophylline, theobromine, and caffeine which are also diuretics. Mendel and Myers 19 have however recently shown that thymine is not completely destroyed normally by the dog, nor is uracil nor cytosine. Diure- sis was not observed by them after the administration of thymine. The output of purines, creatinine and urea + ammonia was not influenced by administering any of these to rabbits, dogs or men. None of the compounds had any marked pharmacological effects. This is especially interesting because cytosine is an amino-pyrimidine, closely related to the compound alleged by Steudel to be toxic. EXPERIMENTAL PART. 20 Dogs, rabbits, and guinea pigs were used in the physiological studies. The dogs were not catheterized, as the time relations were not of interest; but in the case of rabbits the urine was removed by artificial means in some experiments. The dogs' food always was mixed with bone so that the feces became firm and did not contaminate the urine. For the same reason the rabbits and guinea pigs were given some grain in addition to carrots. The compounds were administered subcutaneously, intraperitoneally, or by mouth. 12 Fischer and von Mering: Therapie der Gegenwart, April, 1904. 13 P. Fischer and Hoppe: Miinahener med. Wochenschr., 1909, p. 1429. 14 Bachem: Arch. f. exp. Path. u. Pharm., lxiii, p. 228, 1910. 15 Grober: Bio'chem. Zeitschr., xxxi, p. 1, 1911. 16 Jacobj: Arch. f. exp. Path. u. Pharm., lxvi, p. 241, 1911. 17 Wolf: Journ. of Physiol., xxxii, pp. 171-174, 1905. 18 Sweet and Levene: Journ. of Exp. Med., ix, pp. 229-239. 1907. 19 Mendel and Myers: Amer. Journ. of Physiol., xxvi, pp. 77-105, 1910. 20 The experimental data in this paper are taken from the writer's dis- sertation for the degree of Doctor of Philosophy, Yale University, 1909. 44 8 Action of Certain Pyrimidines The substances used were barbituric acid and its amino-deriv- atives. HN— CO I I OC CH 2 I I HN— CO Barbituric acid (Malonyl urea) HN— CO I I HN = C CH 2 I I HN— CO Malonyl guanidine HN— CO HN— CO HN = C CHNH 2 I I HN— CO 5-Aminomalonyl guanidine HN— CO H 2 NC CH II II N— CNH 2 2, 4-Diamino-6-oxy- pyrimidine H 2 NC CNH 2 N — CNH 2 2, 4, 5-Triamino-6-oxy- pyrimidine Barbituric Acid. Barbituric acid was made essentially according to Michael's 21 method, the principle of which consists in condensing urea with diethylmalonate in the presence of sodium ethylate. The yield represented 71 per cent of the theoretical and the acid obtained gave the following results on analysis (Kjeldahl-Gunning method). N Calculated for C4H4N2O3: 21.87 Found: 21.87 21. 65 22 21. 53 22 Barbituric acid crystallizes in two forms, the anhydrous as needles, and the hydrated as rhombic prisms. It is slightly soluble in water. A rough solubility determination showed that a 2.68 per cent solution can be prepared at 40 to 43°. Efforts to obtain a method for estimating barbituric acid quanti- tatively in urine were unsuccessful, although a qualitative color test afforded a means of getting rough values colorimetrically. The difficulty lies in the fact that many of the properties of bar- bituric acid are possessed also by hippuric acid. As the latter 21 Michael: Journ. f. prakt. Chem., (2), xxxv, pp. 449-459, 1887. 22 These two analyses were made several months later. Israel S. Kleiner 449 occurs constantly in all ordinary urines, and in considerable amount in the urine of herbivora, it proved an effective bar. Both com- pounds are precipitated by mercuric sulphate and by silver nitrate; both are soluble in ethyl acetate and amyl alcohol, and insoluble in ligroin, petroleum ether and benzene. The color reaction referred to is based on Baeyer's 23 observation that nitroso-barbituric acid, in the presence of ferrous acetate, yields a deep prussian blue color. The directions for this test are as follows: 3 cc. of urine are treated with three drops of 2 per cent sodium nitrite solution; about 0.5 cc. of 10 per cent sulphuric acid is added and the solution is now made alkaline with sodium carbonate solution; on addition of one or two drops of strong ferrous sulphate solution a beautiful blue appears in the presence of barbituric acid. When the expression ' 'NaN0 2 -FeS0 4 reaction" is employed hereafter it will be understood that this test is meant. Other members of this series give this reaction but thy- mine, cytosine and uracil do not. 24 Since urine frequently assumes a deeper color when subjected to this treatment, a direct colori- metric estimation was not attempted but a crude method was worked out, in which the greatest dilution allowing a positive test was considered the standard. It was thus found that a 0.0023 per cent solution of barbituric acid in water is the limit for this test, and hence the standard for comparison. Barbituric acid is precipitated by mercuric sulphate solution. It gives Jaffe's reaction as applied to creatinine. A red color results when ferric chloride solution is added to barbituric acid. The sodium salt was made by dissolving the acid in the amount of NaOH calculated to form the disodium salt, concentrating and allowing the salt to crystallize. Needle crystals were obtained; but that they were probably a mixture of the mono- and disodium salts is evident from the nitrogen determination. 23 Baeyer: Ann. d. Chem. u. Pharm., cxxvii, pp. 199-236, 1863. 24 None of the compounds of the barbituric acid series give the character- istic reactions of uracil, thymine or cytosine. For example, if thymine in substance be treated with diazobenzol-sulphonic acid a reddish purple color results ; tested in the same way barbituric acid gives a red, malonyl guani- dine and cyanacetylguanidine a deep orange and the others a yellow or green color. When uracil or cytosine is dissolved in about 5 cc. of water, bromine water added in slight excess and the solution boiled, a deep purple precip- itate results on the addition of baryta water. None of the barbituric acid series studied gives this test. 450 Action of Certain Pyrimidines N Calculated for Calculated for C^NzOsNa*: C^s^OsNa: . .. 16.28 18.66 Found: 17.58 As illustrations of the general method employed in the animal experiments two typical protocols will first be given. Experiment 1. A rabbit weighing 2 kg. was given 0.519 gram barbituric acid in about 25 cc. of water at 40°, hypodermically. The urines of the next two days were precipitated with mercuric sulphate, the precipitate decom- posed with hydrogen sulphide and, after removing the mercuric sulphide, the colorimetric determination made. The amount excreted was estimated at 0.026 gram. No hypnotic or toxic action was exerted by the compound. Its acidic character, however, made it harmful to the tissues at the point of injection; this caused an opening in the body wall which led to the death of the animal seven days after the injection. Experiment 17. 0.64 gram of sodium barbiturate in 45 cc. water contain- ing 0.1 cc. & NaOH at 38° were injected intraperitoneal!}^ into a rabbit weighing 1.6 kg. Diarrhea resulted in about two hours and this condition persisted for five days. The urines of the first two days gave positive NaNCVFeSCX tests and these corresponded to 0.04 gram of barbituric acid. These as well as other experiments with barbituric acid are tabulated on the opposite page. From this table it is seen that the fatal termination of 'Experi- ments 1 and 3 must be ascribed to the acidic properties of barbi- turic acid; for when larger amounts of the sodium salt were given as in Experiments 12 and 17 no toxic effects resulted. The only physiological effect, which may be ascribed to its structure, is its diarrheal action ; but a greater number of experiments need to be done to settle this point. In this connection it is interesting to recall the fact, noted above, that Koehne 25 observed a mild diar- rhea after feeding alloxan and alloxantine. Again, the fact that barbituric acid has no hypnotic action harmonizes with Fischer and von Mering's 26 experiments on substituted barbituric acids, in which, as detailed above, they found that the lower the substi- tuted alkyl groups, the less hypnotic the influence possessed by the complex. In barbituric acid, the lowest degree is reached and no hypnotic action is observed. 25 Koehne: Inaugural Dissertation, Rostock, 1894. 26 Fischer and von Mering: Therapie der Gegenwart, v, pp. 97-101, 1903. Israel S. Kleiner 45 1 Animal Experiments. TABLE I. Barbituric Acid (Malonyl urea). NUMBER AND ANIMAL (1) Rabbit (2) Rabbit. (3) Rabbit. (4) Rabbit. (6) Rabbit, (12) Guinea pig.. (17) Rabbit. AMOUNT GIVEN Total kg. \ gram 2.0 ! 0.52 2.1 i 0.32 2.2 [ 0.53 1.9 0.2 1.9 0.6 0.5 1.6 0.3 0.64 Per kilo- gram MODE OF ADMINISTRATION gram 0.26 Subcutaneously 0.15 Subcutaneously 0.24 Intraperitoneally 0.10 Intraperitoneally 0.3 Per os.. 0.6 Subcutaneously 0.4 Intraperitoneally REMARKS AND RESULTS Not toxic except for necrosis at point of injection, which caused death seven days later. Some excreted. Not toxic. Excret- ed about one-third (?) Death in three days. . Diarrhea at first. Diminished flow of urine (28 cc. in two days) containing 0.09 gram (?). Au- topsy revealed fib- rinous adhesions in peritoneal cavity. Recrystallized prep- aration used. Not toxic. Under ob- servation fifty-six days. Marked diarrhea. Excreted about -jV in urine. Na salt used. Not toxic. Excreted 0.01 gram. (?) Na salt used. Ex- creted about 0.04 gram (?) Diar- rhea for five days, otherwise not tox- ic. Under obser- vation thirty-one days. 452 Action of Certain Pyrimidines Malonyl Guanidine. In synthesizing malonyl guanidine Michael's 27 procedure was essentially followed. The pyrimidine was obtained in the form of its sodium salt which was dissolved in water and dilute NaOH,and the free pyrimidine precipitated with acetic acid. Malonyl guani- dine crystallized in fine white needles which, after drying in a desiccator, were analyzed for nitrogen. Calculated for C4H 6 N,0 2 +H 2 0: C4H6N3O2: Found: N 28.96 33.07 32.05 31.91 The low nitrogen values are probably due to incomplete removal of the water of crystallization by simple desiccation. Inasmuch as the analysis was fairly close and the preparation was pure white no further purification was attempted. It was only slightly solu- ble in water. At 40 to 43° a 0.049 per cent solution was the strong- est obtainable. This, of course, renders malonyl guanidine itself unsuitable for injection experiments and the sodium salt was accordingly used. In preparing this, the pyrimidine was dissolved in NaOH, as little in excess of the calculated amount as would bring about solution being used. On concentration, fine pale pink needles crystallized out. From the analyses, which follow, this salt must contain four molecules of water of crystallization which are lost in the desiccator very slowly. Calculated for Found : C4H4NaN 3 02+4H 2 0: (air-dry) N 19.00 18.77 Calculated for Found: C4H4NaN 8 2 : (desiccated) N 28.19 25.31 A method for recovering malonyl guanidine from urine is at once suggested by the slight solubility of 'the free substance. However, if urine is acidified and allowed to stand, uric acid and, if concentrated sufficiently, hippuric acid will also crystallize out. The NaN0 2 -FeS0 4 reaction described above for barbituric acid is also applicable to malonyl guanidine. The limit for this test in urine is 0.004 per cent. Another mode of estimation by means of this color reaction was tried as follows: 0.002 gram in 27 Michael: Journ. f. prakt. Chem., xlix, pp. 26-43, 1894. Israel S. Kleiner 453 3 cc. water was converted to the prussian blue compound and dilu- tions made until the blue was no longer distinctly discernible in a 100 cc. cylinder. The concentration just above this was con- sidered the standard. By such a rough method it was found that a distinct blue can be seen when there is an amount corresponding to 0.0004 per cent present. Sodium malonyl guanidine is not precipitated by ammoniacal silver nitrate solution, but is precipitated quantitatively by mer- curic sulphate solution. With picric acid and alkali a red color is formed as in Jaffe's test for creatinine. From the animal experiments (see Table II) it is seen that mal- onyl guanidine is non-toxic, at least in the doses for the animals used. The failure to detect the substance or a related compound in the urine of Experiment 5 may be due to the small amount injected. TABLE II. Animal Experiments. Malonyl Guanidine. NUMBER AND ANIMAL (5) Rabbit . . . (9) Rabbit. . . (26) Rabbit . . (24) Dog. kg. 2.2 1.9 2.1 10. AMOUNT GIVEN Total gram 0.09 0.41 0.22 2.1 Per kilo- gram gram 0.04 0.21 0.10 0.21 MODE OF ADMINISTRATION REMARKS AND RESULTS Subcutaneously Subcutaneously Subcutaneously Per os Sodium salt used. No effects. Not detected in urine. Sodium salt used. No effects. De- tected in urine. Sodium salt used. Mild diarrhea; no other effects. All (?) excreted in urine. Free malonyl guani- dine used. Some absorbed and ex- creted, in urine. No toxic effects. 454 Action of Certain Pyrimidines 5-Aminomalonyl Guanidine. This compound is quite difficult to obtain in good yield as it decomposes very easily. The most advantageous method was found to be a modification of one described by Traube 28 in which the sulphate of this compound can be prepared directly from mal- onyl guanidine. The directions of Traube were followed as far as the formation of 5-aminomalonyl guanidine sulphate by reduc- tion with H 2 S, but instead of extracting this salt with hot water, the sulphur was removed by means of CS 2 and the sulphate con- verted into the hydrochloride by treatment with BaCl 2 . Traube's suggestion of adding alcohol to induce crystallization was not found to be advantageous since the crystals, when finally obtained, had a pink tinge. Consequently, the fluid was concentrated under diminished pressure and allowed to crystallize. Light yellow ros- ettes of needles formed very slowly. Analysis of this preparation (A) by the Kjeldahl-Gunning method showed that, in spite of the tinge of yellow color, the salt was quite pure. Another preparation (B) made by the same method gave a higher nitrogen percentage. Its solution, which is acid to litmus, very quickly turns red, owing undoubtedly to a slight oxidation. It stains the tissues red and has a faint disagreeable odor. Boiling with NH 4 OH yields a solu- tion colored like potassium permanganate and this changes to dark blue on addition of KOH. It will give the NaN0 2 -FeS0 4 reaction, but not readily or brilliantly. The Jaffe color reaction for creatin- ine is not given by this salt. It is precipitated both by ammoni- acal silver nitrate and mercuric sulphate, but as very little can be injected into an animal and as it was found to be toxic no attempt was made to isolate it from urine. The toxicity of this compound is shown in the following illus- trative protocols and the accompanying table (Table III) which summarizes all the experiments. The hydrochloride was used in each case. Calculated for (C4H 6 N402)HC1+H 2 0: Found: 28.55 28.08 29.41 N 28.57 28 Traube: Ber. d. deutsch. chem. Gesellsch., xxvi, pp. 2551-2558, 1893. Israel S. Kleiner 455 Experiment 7. November 30. 3:10 p.m. A rabbit weighing 1.4 kg. was given subcutaneously 0.37 gram in 35 cc. water. 3:20 p.m. Has defecated very soft stools. Moves around restlessly. 4:15 p.m., 4:50 p.m. Apparently well. December 1. 8:45 a.m. Rabbit found dead. Autopsy: kidneys are very light colored; intestines intensely reddened; liver, light brown; large amount of bloody fluid in peritoneal cavity. Experiment 23. March 11. 11 :00 a.m. A guinea pig weighing 450 grams was given 0.036 gram of the salt in about 10 cc. water subcutaneously. March 12. 2:15 p.m. Has eaten 60 grams carrots and 9 grams oats. Urine, 64 cc, alkaline; specific gravity, 1.017; albumin present, but no casts. March 13. 2:40 p.m. Has eaten 70 grams carrots and 3 grams oats. Urine, 43 cc; alkaline; specific gravity, 1.024; large amount of albumin; granular, granular partly hyaline, and cellular casts found; NaNCVFeSC^ test negative. March 14. 2 :35 p.m. Has eaten 85 grams carrots and 4 grams oats. Urine 30 cc; alkaline; albumin present; casts. March 15. 2:50 p.m. Has eaten 93 grams carrots and 3 grams oats. Urine 57 cc; alkaline; specific gravity, 1.021; albumin; casts. March 16. 8:40 a.m. Animal appears well. Weight 390 grams. The animal daily ate more food until March 20, when the usual amount (150 grams carrots and 15 grams oats) was entirely consumed. On March 18, its weight had dropped to 360 gram but then rose to 440 grams on March 24. The urine still contained a trace of albumin. On April 3 — twenty-three days after the injection — the animal was still living and apparently well. Experiment 22. March 11. 4:00 p.m. A female rabbit weighing 2.44 kg. was given a subcutaneous injection of 0.19 gram in about 40 cc. water. March 12. 9:00 a.m. Stools partly diarrheal. 2:15 p.m. No urine. Has eaten 145 grams carrots but no oats. March 13. 2:40 p.m. No urine. Has eaten 130 grams carrots but no oats. March 14. 11:00 a.m. No urine, no feces. Has eaten 85 grams carrots and 8 grams oats. March 15. 2:50 p.m. Has eaten 46 grams carrots but no oats. Urine, 163 cc; alkaline; specific gravity, 1.013; NaN0 2 -FeS0 4 test negative; albu- min and granular casts present; slight reduction of alkaline copper solu- tion (after removing albumin). March 16. 8:40 a.m. Has eaten 35 grams carrots. Animal is very weak, breathes slowly and can not hold its head up. 10:05 a.m. Breathes more quickly but head is on floor of the cage. 11:50 a.m. Still breathing; extremely weak. 2:10 p.m. Found dead. Urine, 52 cc; alkaline; specific gravity, 1.010; albumin and casts present; reduction positive. Autopsy. Weight 2.26 kg. All viscera hyperemic; blood of liver does not clot readily; kidneys edematous; bladder empty; animal is quite fat. Sections of tissues preserved. 456 Action of Certain Pyrimidines table nr. Animal Experiments. 5-Aminomalonyl guanidine. NUMBER AND ANIMAL (7) Rabbit.. (22) Rabbit. (8) Rabbit.. (10) Guinea pig.. (27) Guinea pig-- (25) Guinea pig... (23) Guinea pig... AMOUNT GIVEN kg. 1.4 2.4 2.6 0.41 0.54 0.54 0.45 Total gram 0.37 0.19 0.11 0.05 0.061 0.048 Per kilo- gram gram 0.26 0.08 0.04 0.12 0.11 0.09 0.036 0.08 MODE OF ADMINISTRATION Subcutaneously Subcutaneously Subcutaneously Subcutaneously Subcutaneously Subcutaneously Subcutaneously REMARKS AND RESULTS Fatal in less than eighteen hours. Albuminiuria; casts; glycosuria. Death in five days. Fifty-three cubic centimeters urine in first forty-eight hours. Albumi- nuria until fourth day. No glyco- suria. Recovery. Albuminuria. Death in four days. Au- topsy: organs ap- pear normal. Blood does not clot read- iiy. Not fed on day of injection. Albu- minuria: mucus cylindroid seen. Fatal in less than two days. Autop- sy: one fetus pres- ent; large amount of bloody subcu- taneous effusion. Kidneys seem con- tracted. Albuminuria for at least seven days; casts and leucocy- tes in urine; recov- ery; under obser- vation twelve days . Albuminuria; casts; recovery. Israel S. Kleiner 457 TABLE III— Continued. NUMBER AND ANIMAL AMOUNT GIVEN MODE OF ADMINISTR \TION WEIGHT Total Per kilo- gram REMARKS AND RESULTS (21) Guinea pig... kg. 1.7 0.52 gram 0.18 0.04 gram 0.10 0.08 Per os Per os NTn svmntoms' no albuminuria. Un- der observation eighteen days. No symptoms ; n albuminuria. Un- der observation thirty days. From these results it appears that a lethal subcutaneous dose for rabbits is 0.08 gram per kg. and for guinea pigs 0.11 gram per kg. It is also evident that when the compound is fed it is not toxic. In Experiment 20, the urine was repeatedly examined for substances giving the NaNCVFeSC^ test but with negative results. The feces, however, in both Experiments 20 and 21 were tinged with pink at times. Probably not enough of the compound is absorbed from the alimentary tract at one time to prove toxic; it may be mentioned, however, that the hydrochloride is fairly solu- ble. That the compound acts mainly on the kidneys is evident from the protocols and the table, but substantiating evidence is given by the histological examination, made by Professor H. Gideon Wells to whom I am greatly indebted for the following report. Experiment 22 — Rabbit. Kidney. Shows extensive necrosis of the con- voluted tubules, perhaps one-fourth of the tubules seen in section showing total necrosis of the epithelium. The necrotic epithelium desquamates into the lumen of the tubule which it fills up, and all stages of transition from masses of necrotic epithelium to granular and hyaline casts which pack the collecting tubules can readily be made out. These casts, being very abundant and staining intensely with eosin, give the sections a striking appearance. The tubular epithelium where not necrotic is strikingly little altered, some tendency to vacuolization of the cytoplasm being the chief abnormality noted. Glomerules congested, swollen, and in some a little granular material and occasional red corpuscles free in the space outside the tuft; in general the glomerules show relatively little change. There is an occasional small area of interstitial hemorrhage. To summarize, the poison has caused a marked necrosis of the epithelium of the convoluted 458 Action of Certain Pyrimidines tubules, but without affecting other renal structures to any considerable degree. Liver. No definite changes except the accumulation of masses of yellow- ish brown pigment in many of the stellate cells. Spleen. Some of the endothelial cells of the splenic sinuses contain brownish pigment, otherwise no change. The pigmentation of the liver and spleen suggests a hemolytic action by the poison. Experiment 27 — Guinea Pig. Kidney. Shows the same necrosis of the secretory epithelium of the tubules and the same formation of casts as described in Rabbit 22, but very much less marked, only occasional tubules showing the lesion. Liver. No pigmentation or other distinct changes. Spleen. Much more pigment than in Rabbit 22. No other changes. Adrenal. No changes. Experiment 10 — Guinea Pig. Kidney. Granular and hyaline casts are very abundant and conspicuous, although there are fewer tubules showing necrosis than in either of the other specimens. When found it is typical, exactly the same in appearance as in 22 and 27. The casts much more often show desquamated epithelial cells within them. Marked congestion, but no other changes. The constancy of the finding of necrotic tubular epithe- lium in all three kidneys is conclusive evidence that this is a specific effect of the poison given. Liver. No distinct alterations. 2,4-Diamino-6-oxypyrimidine. Both 2,4-diamino-6-oxypyrimidine and its precursor, cyana- cetylguanidine, were used in the experiments on animals. They were made by Traube's 29 method with some modifications. Guani- dine hydrochloride, according to this procedure, is condensed with cyanethylacetate forming, in part, the pyrimidine; but mainly cyanacetylguanidine, which is easily converted into the pyrimi- dine by alkali. H 2 N COOC2H5 I I HN = C + CH 2 I I H 2 N CN The yield of cyanacetylguanidine was 35.8 per cent of the theo- retical, if this were the sole end-product. The mother-liquor was of a dark red color and on concentration yielded a large amount HN— CO HN— CO -> HNC CH 2 > H 2 NC CH 2 II II I H 2 N CN N— CNH 29 Traube: Ber. d. deutsch. chem. Gesellsch., xxxiii, pp. 1371-1383, 1900. Israel S. Kleiner 459 of material which was used in the preparation Of the pyrimidine. The first crop was recrystallized from hot water, pulverized and desiccated. To determine whether the substance obtained was cyanacetylguanidine or the pyrimidine, advantage was taken of the fact that the latter crystallizes with one molecule of water of crystallization while the former is water-free. Calculated for (C4H 6 N40)+H 2 0: C 4 H 6 N40: Found: H 2 12.5 0.0 1.2 This preparation was consequently cyanacetylguanidine with very little, if any, pyrimidine admixture. Nitrogen determina- tions by the Kjeldahl-Gunning method gave low figures, perhaps because some HCN may have been formed and lost or because of a very slight admixture of the pyrimidine. Calculated for C4H6N4O: Found: N 44.44 41.99 41.96 A much better yield is obtained by using guanidine sulphocy- anide in place of the hydrochloride, as the mother liquor in this case is not as dark colored and may be evaporated to dryness without much loss of material. In this modification, when used as a step in the preparation of the pyrimidine, it is not necessary to remove the NaSCN formed until the 2,4-diamino-6-oxypy- rimidine is precipitated as the sulphate, since the latter can be washed free from inorganic salts with water. Cyanacetylguanidine is quite soluble in water — a 2.5 per cent solution being easily maintained at 40° — and is suitable for injec- tion. Cyanacetylguanidine forms a rose red isonitroso compound (or is converted into the isonitroso derivative of 2,4-diamino-6- oxypyrimidine) on adding NaN0 2 and H 2 S0 4 to its solution; as this is quite insoluble it may be isolated from the urine. Accord- ing to Traube the isonitroso compound has an intense yellow or yellowish green color; however, with our preparation the brilliant red compound formed first and did not become yellow until addi- tional acid was used. The color test with NaN0 2 and FeS0 4 as described above is also positive for cyanacetylguanidine. For the transformation of cyanacetylguanidine into its isomer, 2,4-diamino-6-oxypyrimidine, it was put into boiling 2-5 per cent THE JOURNAL OF BIOLOGICAL CHEMISTRY, VOL. XI, NO. 5. 460 Action of Certain Pyrimidines NaOH, animal charcoal added, boiled a few minutes, and filtered into a beaker placed in an ice-bath. As some NH 3 is split off by- boiling with alkali in this way, the operation must necessarily be performed quickly. The solution was now made weakly acid with H2SO4 and white or yellowish needle-like crystals of the sulphate of the pyrimidine appeared. When recrystallized from hot water large silky, grayish needles were obtained and these were again recrystallized from water in the presence of dilute H 2 S0 4 and some charcoal; the crystals resulting were of a light yellow, almost white color. According to Traube the sulphate, when recrystallized from water, contains one molecule of water of crystallization which is not driven off at 100°. Its composition is (C 4 H 6 N 4 02). H 2 S0 4 -|- H 2 0. Our preparation agreed in its nitrogen content with this formula, as the following analyses indicate. Calculated for (C 4 H6N40)2-H 2 S04+H20: Found: N 30.43 30.42 30.27 This salt is sparingly soluble in water. A rough solubility de- termination showed that at 43° a greater concentration than 0.49 per cent could not be maintained and at a slightly lower tempera- ture much of the substance instantly crystallized out. An aqueous solution gives a positive NaN0 2 -FeS0 4 test. Because of the poor solubility no injection experiments were performed. However, Steudel's 30 experiment, in which he reports this compound toxic when fed to a dog, was repeated in exactly the same manner and with the same relative dosage. Expeeiment 15. February 15. 10:20 a.m. Bitch weighing 9.6 kg. fed 180 grams chopped meat with bone meal, to which was added 1.55 grams of the sulphate of the pyrimidine. 10:20 to 11:40 a.m. Under observation almost continually. The animal, which has always been playful, shows no unusual behavior, but is appar- ently normal. 2 :00 p.m. Animal still lively. 2:15 p.m. Ate some meat and drank water. No nausea observed. 5 :10 to 5 :20 p.m. Animal well. February 16. 9:00 a.m. Fed meat, cracker and bone meal. Urine, 170 cc. ; specific gravity, 1.055; acid; no albumin. On adding NaNC>2 and H 2 S0 4 30 Steudel: Zeitschr. f. physiol. Chem., xxxii, pp. 285-290, 1901. Israel S. Kleiner 4 6i a rose-colored precipitate appeared which was filtered off and washed with hot water, alcohol and ether. For the total volume of urine this would have amounted to 1.17 grams. It was dissolved in KOH, reprecipitated by HC1, filtered etc. and analyzed by the Kjeldahl-Gunning method (for nitrates). Calculated for isonltroso derivative of 2,4-diamino-6-oxy- monamino-dioxy- pyrimidlne pyrimidine ( = C4H B N 5 02): ( = dH4N408): Found: N 45.16 35.89 39.25 39.56 Feeding suspensions of the salt to a guinea pig and to a rabbit gave simi- lar non-toxic results (see Table IV). The sulphate, as in Steudel's investi- gation, was used in every case. TABLE IV. Animal Experiments: 2, 4-Diamino-6-oxy pyrimidine. NUMBER AND ANIMAL AMOUNT GIVEN MANNER OF ADMINISTRATION WEIGHT Total Per kilo- gram REMARKS AND RESULTS kg. gram gram (15) Dog 9.6 1.55 0.16 Per os No toxic effect. Large (18) Guinea proportion excret- ed; deaminized (?) pig.... 0.16 0.14 0.87 Per os Fed in saccharose suspension from pipette. No symp- toms. Under ob- servation seven days. (19) Rabbit.. 1.96 0.51 0.26 Per os Given in suspen- sion in water. No albuminuria. Na- N0 2 - Fe S0 4 test positive. Unable to obtain an isonitro- so compound as in Experiment 15. It is therefore evident that his pyrimidine is not toxic when given per os as the sulphate. Doses larger than those reported toxic by Steudel were without effect upon the rabbit and guinea pig as Experiments 18 and 19 indicate. 462 Action of Certain Pyrimidines 2 ,4,5 -Triamino-6-oxy pyrimidine. This pyrimidine was prepared according to Traube's 31 directions and was isolated as the sulphate. When crystallized quickly the salt appears as small rods or rectangular prisms but if allowed to crystallize slowly large needles are formed. After desiccation, an analysis gave the following results. The solubility of this salt is about the same as that of the diamino compound, i.e., it was found to be possible to obtain a 0.49 per cent solution at 43°. In this case, however, the fluid became dark dur- ing the manipulation and, after drying, the residue was dark brown in color. It is thus evident that some chemical change — a decom- position or oxidation — occurred and hence the determination can only be regarded as an evidence of the very slight solubility of the substance at low temperatures and of its instability, when in solution, at a high temperature. According to Traube, if an ammoniacal solution of the sulphate be shaken so as to afford contact with the air the fluid assumes an intense violet color resembling permanganate solution. This re- action, according to our experience, is better performed and with more uniform success, if a few milligrams of the substance are placed on a porcelain surface together with one or two drops of NH4OH and evaporated to dryness on a water-bath; the violet tinge is here seen against the white surface. In trying to dissolve some of the salt in 50 per cent alcohol it was discovered that although very little went into solution the latter became colored with this same violet tint. This pyrimidine also resembles uric acid in two reactions, namely, the murexide and SchifFs tests; the murexide test is given very brilliantly indeed. Addition of bro- mine water to an aqueous solution was found to produce a deep reddish-purple color which vanished, leaving a yellow solution, when the bromine was in excess. The NaN0 2 -FeS0 4 reaction is positive if the triamino pyrimidine be first dissolved in boiling water; this is probably due to a trace of the diamino being formed by the action of the water as, theoretically, if the 5 position is occupied by an amino group no isonitroso derivative can be formed. N Calculated for (C4H7N £ 0)H 2 S04+H 2 0: 27.24 Found: 27.87 31 Traube: Ber. d. deutsch. chem. Gesellsch., xxxiii, pp. 1371-1383, 1900. Israel S. Kleiner 463 In using this salt in physiological experiments we again obtained results quite different from those reported by Steudel. Experiment 14. February 11. 2:45 p.m. Bitch weighing 9.8 kg. (same animal as in Experiment 15) was given 1.58 grams of the sulphate of this pyrimidine mixed with about 180 grams of chopped meat and some bone meal. No unusual symptoms were noticed by 4:00 p.m. 4:30; 5:00; 5:30; 7:40; 9:15 p.m.; animal observed and was well and play- ful. February 12. 8:50 a.m. Animal well. Urine, 134 cc; dark orange-red in color; specific gravity, 1.049; acid; no albumin. Some of the urine was made acid with H 2 S0 4 and was concentrated to small volume. HgS0 4 solution was added and the precipitate filtered off; a few crystals were found and, as they gave a violet color on treatment with NH 4 OH and evaporation, were probably some triamino-sulphate which had crystallized before adding the HgS0 4 . The mercury precipitate, was unfortunately lost through an accident. 4:00 p.m. Fed meat and bone. February 13. a.m. Urine light yellow in color. Relatively larger doses were fed in suspension to a rabbit and a guinea pig with similarly negative results; these are summed up in the following table (Table V). table v. Animal Experiments: 2, 4, 5-triamino-6-oxypyrimidine. NUMBER AND ANIMAL (14) Dog. (10) Guinea Pig (young). kg. 9.8 (13) Rabbit.. 2.5 0.13 AMOUNT GIVEN Total gram 1.58 0.2 0.5 0.13 Per kilo- gram MANNER OF ADMINISTRATION REMARKS AND RESULTS gram 0.16 Per os 0.08 Per os 0.20 1.0 ! Per os No toxic effect. Some excreted (?). Urine red. No toxic effect. Sec- ond dose four days after first. Urine red after second dose. Fed, suspended in saccharose, solu- tion, from a pip- ette. No toxic effects. Urine col- ored dark red. 464 Action of Certain Pyrimidines It is accordingly evident that even the triamino compound, which Steudel claims is the more toxic of the two, has no harmful influence upon the organism when administered by way of the mouth. Cyanacetylguanidine. HN— CO I I HN = C CH 2 I I H 2 N CN The preparation and properties of cyanacetylguanidine are described above in the description of the process of making 2,4- diamino-6-oxypyrimidine. This compound was used because it is a precursor of the diamino and triamino pyrimidines just described and might readily be pres- ent as an impurity if these compounds were carelessly prepared. In- asmuch as from the following experiments it is seen to be toxic after injection, a reason for the difference between our results and SteudeFs is thus suggested. Experiment 28. March 30. 12:30 m. Injected subcutaneously, into guinea pig weighing 680 grams, 0.38 gram cyanacetylguanidine in 15 cc. water. 4:00 p.m. Animal shows hyperexcitability. 6:20 p.m. Still very excitable. March 31. 1:15 p.m. Apparently well except for continued hyperexcit- able state, which is not as great as on the previous day. 4:00 p.m. Has eaten 15 grams oats and 95 grams carrots during twenty- four hours. Weight 668 grams. Urine, 43 cc; alkaline; specific gravity, 1.031; no albumin present; strong NaN0 2 -FeS0 4 test; upon addition of NaN0 2 in substance, and H 2 S0 4 a pink isonitroso derivative was obtained which amounted to 0.161 gram, if computed to total volume. This was analyzed with the following results. Calculated for C4H5N5O2 ( = isonitroso derivative of cyanacetylguanidine) : Found : N. 45.16 36.47 5:10 p.m. Apparently well. April 1. 4:00 p.m. Has eaten 15 grams oats and 105 grams carrots. Weight, 664 grams. Urine, 46 cc. ; alkaline; specific gravity, 1.030; no albu- min; NaN0 2 -FeS04 test positive. Israel S. Kleiner 465 April 2. 4:00 p.m. Weight, 667 grams, Urine, 38 cc; NaN0 2 -FeS0 4 test negative. Experiment 31. April 2. 11:45 a.m. Young guinea pig, weighing 191 grams, given 0.4 gram cyanacetylguanidine in 17 cc. water by subcutan- eous injection. 12:15 m. Apparently well. 2:10 p.m. Animal found in violent spasms, especially the posterior parts of the body. There is hyperexcitability. 2:20 p.m. Head raised a little more and pig runs around some, pawing at its chin at intervals. Twitchings continue. 4 :13 p.m. Violent convulsion; lies on its side and moves its limbs rapidly. 4:18 p.m. Animal gradually rights itself and grips the side of the wire cage with its teeth. Waves of convulsions, starting at the posterior part and running forward, occur. 4:22 p.m. Dies in the same position; body quickly in rigor. The urine excreted, 2 cc, was found to contain no albumin but on addition of NaN0 2 and H2SO4 a pink precipitate appeared which after dissolving in Na2CC>3 and adding FeS0 4 produced the deep prussian blue color. Experiment 30. March SI. 2:55 p.m. A dog weighing 5.8 kg. was fed 100 grams chopped meat containing 0.94 gram cyanacetylguanidine. 3:10 to 3:20; 4:25 to 4:30 p.m. Apparently no effects. 4:50 p.m. Drank water; no nausea. April 1. 9:15 a.m. Dog apparently well. 3:00 p.m. Fed meat, lard, bone and cracker meal. Urine, 226 cc; acid; specific gravity, 1.025; no albumin; strong NaN02-FeS04 test. To an aliquot portion was added solid NaN0 2 and H2SO4 and the reddish brown precipitate which amounted to 0.389 gram analyzed. April 2. 3:00 p.m. Dog well. Urine, 138 cc; specific gravity, 1.032; acid; no albumin; strong NaNCVFeSC^ test. No loss of appelite or other unfavorable symptoms. April 3. 9:30 a.m. Dog well. Weight, 5.6 kg. Urine gives uncertain NaN0 2 -FeS0 4 test. These and one other experiment are summarized in Table VI. The low nitrogen values found in Experiments 28 and 30 suggest the possibility of a deaminization of cyanacetylguanidine in the body. The substitution of O for NH in its isonitroso derivative would result in a compound containing 35.90 per cent of nitrogen; the figures found, 36.47 per cent and 34.43 per cent, correspond with this percentage. Calculated for C4H5N5O2 ( = isonitroso derivative of cyanacetylguanidine) : 45.16 N Found: 34.43 466 Action of Certain Pyrimidines TABLE VI. Animal Experiments: Cyanacetylguanidine. NUMBER AND AMOUNT GIVEN MANNER OP WEIGHT ANIMAL Total Per kilo- gram ADMINISTRATION REMARKS AND RESULTS kg. gram gram (28) Guinea Hyperexcitability . pig.... 0.68 0.38 0.56 Subcutaneously Excreted consider- able as a deamin- ized (?) substance. (31) Guinea Hyperexcitability. pig.... 0.19 0.40 2.1 Subcutaneously Violent convul- sions. Fatal in four and three quarters hours. (29) Dog 8.4 0.70 0.08 Per os No symptoms. Urine gave positive Na- N0 2 -FeS0 4 test. No albuminuria. (30) Dog 5.8 0.94 0.16 Per os No harmful effect. Considerable ex- creted asadeamin- ized(?) substance. This toxic action agrees with the results' of some unpublished trials by Mr. J. J. Costello, who observed similar effects in Pro- fessor Mendel's laboratory when the sulphate of this compound was subcutaneouly injected. A few of his figures follow. Dose per kilogram of guinea pig. Results . 92 Hyperexcitability-recovery. 0.96 Hyperexcitability for two days-recovery. 0.96 Death in sixteen hours. 1.02 Death in fourteen hours. 2.26 Death in three and one-half hours. DISCUSSION. In considering the physiological and pharmacological behavior of the members of this series the most striking fact is the toxicity of 5-aminomalonyl guanidine with its chief effect upon the epithe- lium of the convoluted tubules. Its harmlessness when adminis- Israel S. Kleiner 467 tered per os may be due either to an absorption so slow as to allow of elimination before a toxic concentration is reached, or to a trans- formation — perhaps by deaminization — into a non-toxic compound in the intestinal wall. The toxicity after subcutaneous adminis- tration may possibly be attributable to some hydrolytic or oxida- tion product formed during solution inasmuch as the solution quickly assumes a red color. The absence of hypnotic powers in barbituric acid and malonyl- guanidine is in harmony with the ineffectiveness of the lower alkyl barbituric acid derivatives and of 5,5-dipropylmalonylguanidine. 32 The diarrheal action of barbituric acid is noteworthy because of a similar action ascribed to alloxan. 33 Steudel's 34 claim that 2,4-diamino-6-oxypyrimidine and 2,4,5- triamino-6-oxypyrimidine are toxic, cannot be substantiated. In duplicating his experiments in which he fed these compounds to a dog, no similar results could be obtained; the animal used was a very playful one as was SteudeFs but it did not become less lively after ingesting these substances, nor was vomiting or albuminuria observed or any other of the effects noted by that author. The lethal doses for rats he gives as 0.2 gram and 0.1 gram for the sul- phates of the diamino and triamino compounds, respectively, when injected subcutaneoulsy. The smallest volumes which can possi- bly contain these amounts at 43° are 40 cc. and 20 cc. respectively. Moreover, it was shown above that such concentrations are not suitable for injection and this leads us to believe that Steudel used products which were more soluble than these aminopyrimidines. Moreover he published no analyses of his compounds. Cyanace- tylguanidine, however, is a precursor of both pyrimidines; it is quite soluble as is also its sulphate; and finally, when injected sub- cut aneously it is toxic. These properties would indicate that this compound was an admixture of Steudel's preparations and would account for their toxic action. However, when fed to dogs, cyana- cetylguanidine is not toxic although his preparations were; and the only apparent explanation for this is that still another com- taminating substance was responsible in this case. That cyana- cetylguanidine is toxic is not surprising since, from its structure, 32 Fischer and von Mering: Therapie der Gegenwart, v, pp. 97-101, 1903. 33 Koehne: Inaugural Dissertation, Rostock, 1894, 40 pp. "Steudel: Zeitschr. f. physiol. Chem., xxxii, pp. 285-290, 1901. 4 68 Action of Certain Pyrimidines HN— CO I I HNC CH 2 I I H 2 N CN it might possess the properties of guanidine or of nit riles. Guani- dine, the toxicity of which' has long been known, causes 35 peculiar shaking movements of the head and ears, paralysis of the hind limbs, clonic muscular contractions and muscular twitchings of the entire body. Different nitriles have different effects but the typi- cal phenomena are described 36 as vomiting, dyspnoea, tetanic con- vulsions and opisthotonus. Hence, probably cyanacetylguanidine embraces some of the toxic effects of both of these poisons (see Experiments 28 and 31). The behavior of 2,4-diamino-6-oxypyrimidine and cyanacetyl- guanidine in the body affords suggestions for further work upon the intermediary metabolism of these substances, as the few experi- ments indicate that a deaminization may occur in vivo. The dif- ferences between the theoretical percentage of N for the compounds administered and those recovered from the urine are too great (6 to 11 per cent) to be ascribed to the method of analysis or to faulty technique. Moreover, an analysis of the pure isonitroso derivative of the diamino pyrimidine by the same method gave a satisfactory nitrogen value. The possibilities for the transforma- tion of this pyrimidine are shown by the following scheme. HN— CO HN— CO HN— CO II .11 II H 2 NC CH + H 2 = NH 3 + OC CH 2 or HNC CH 2 II II II II N— CNH 2 HN— CNH HN— CO With cyanacetylguanidine a somewhat similar problem is pre- sented as deaminization can result in one of three compounds: HN— CO HN— CO HN— CO HN— CO II lilt II HNC CH 2 + H 2 = NH 3 + OC CH 2 or OC CH 2 or HN— C CH 2 II I I I I II H 2 N CN H 2 N CN HN— CNH HN— CO 35 Gergens and Baumann: Arch. f. d. ges. Physiol., xii, pp. 205-214, 1876; Pommerenig : Beitr. z. chem. Physiol, u. Path., i, pp. 561-566, 1901. 86 Kobert : Lehrbuch der Intoxikationen, Stuttgart, ii, p. 862, 1906. Israel S. Kleiner 469 If either of the last two complexes result it is of great interest as no precisely similar transformation of an acyclic into a cyclic compound is known in physiology. HN— I Lusini's conclusion that the grouping OC has first a stimulat- I HN— ing and then an inhibiting action on the nerve centers and that HN— CO the grouping 1 has no such power can not be substan- I tiated inasmuch as barbituric acid, which is non-toxic, contains this urea grouping and differs very little in structure from alloxan which Lusini found to be toxic. SUMMARY. The administration of barbituric acid per os is followed by no marked physiological effects except diarrhea; when given subcu- taneously the free pyrimidine has a local action on the tissues due to its acid properties. The sodium salt has no local action. Malonyl guanidine when fed, or when injected subcutaneously as the sodium salt, provokes no noteworthy symptoms. 5-Amino- malonylguanidine hydrochloride, 2, 4-diamino-6-oxypyrimidine sul- phate and 2,4,5-triamino-6-oxypyrimidine sulphate, when fed, are also without marked action. Subcutaneous injection of 5-aminomalonylguanidine hydro- chloride leads to grave changes in the tubular epithelium of the kidney; casts and albumin abound in the urine; and death fre- quently results. 2,4-Diamino-6-oxypyrimidine sulphate and 2,4,5 1 triamino-6-oxy- pyrimidine sulphate, which Steudel reported as toxic, are too insol- uble to inject in appreciable quantity. Inasmuch as cyanacetyl- guanidine, a precursor of both of these, is quite soluble, and was found to be toxic when injected subcutaneously, doubt is expressed as to the purity of the diamino and triamino pyrimidines used by Steudel, especially as this author also observed nausea, etc., after feeding them to dogs, whereas no symptoms whatever occurred in the present investigation under similar conditions. 47o Action of Certain Pyrimidines A color reaction is described which is common to all of this series, although 2,4,5-triamino-6-oxypyrimidine and 5-aminomalonylgua- nidine do not react well. By aid of this reaction and in other ways, evidence was gained that, after administration of a compound of this series there was excreted in the urine the compound used (or a derivative) in every case except with 5-aminomalonylguanidine, and perhaps 2,4,5-triamino-6-oxypyrimidine. Evidence is presented to indicate that 2,4-diamino-6-oxypy- rimidine and cyanacetylguanidine may be deaminized in the body. ' My thanks are due Prof. Lafayette B. Mendel who directed the physiological investigations and Prof. Treat B. Johnson, who aided and advised in the syntheses of the compounds employed as well as in the questions of organic chemistry involved. Reprinted from The Journal of Biological Chemistry, Vol. XII, No. 1, 1912. THE INFLUENCE OF SODIUM TARTRATE UPON THE ELIMINATION OF CERTAIN URINARY CONSTITU- ENTS DURING PHLORHIZIN DIABETES. By FRANK P. UNDERHILL. {From the Sheffield Laboratory of Physiological Chemistry, Yale University, . New Haven, Connecticut.) (Received for publication, May 25, 1912.) In recent communications by Baer and Blum 1 it is pointed out that the subcutaneous administration of a series of organic com- pounds containing two carboxyl groups exercises a remarkable inhibitory influence upon the elimination of urinary nitrogen and dextrose in dogs with phlorhizin diabetes. Among the substances possessing this property may be mentioned glutaric and tartaric acids and their salts. The results obtained by these authors are so striking and of such fundamental importance in the interpretation of the mechanism of phlorhizin diabetes that a reinvestigation of the problem seemed desirable. Accordingly experiments have been planned similar to those of Baer and Blum the details of which are appended. 2 The investigation has corroborated the reported results but has yielded an explanation for the phenomena observed which is differ- ent from that put forth by Baer and Blum. Our work has been confined to the study of the action of a single compound used by Baer and Blum, namely, sodium tartrate, prepared by neutrali- zation of the racemic crystalline tartaric acid (Kahlbaum and other preparations) with sodium carbonate. Both rabbits and dogs were employed as experimental animals. *Baer and Blum: Hofmeister's Beitrdge, x, p. 80, 1907; xi, p. 102, 1908; Arch. f. exp. Path. u. Pharm., lxv, p. 1, 1911. 2 A notice of this investigation was communicated to the Society for Ex- perimental Biology and Medicine, May 15, 1912. ii5 1 1 6 Sodium Tartrate and Phlorhizin Diabetes After the completion of our experiments a preliminary account of the influence of glutaric acid on phlorhizin diabetes was reported by A. I. Ringer. 3 In this communication Ringer has entirely failed to confirm the reported results of Baer and Blum with respect to glutaric acid. Methods. The general plan of experimentation was similar to that of Baer and Blum. Phlorhizin diabetes was established for a preliminary period (usually three days) in the fasting animal, the drug being given subcutaneously once daily in sodium carbonate solution. Water was allowed ad libitum. Urine was collected in twenty-four-hour periods either by compression of the bladder in rabbits or by catheterization in the case of dogs. Tartrate admin- istration occurred immediately after the phlorhizin injection and the quantities of tartaric acid specified in the tables were subcutane- ously injected subsequent to neutralization with sodium carbonate. In our preliminary trials we repeated the work of Baer and Blum employing rabbits instead of dogs. As may be seen from tables 1, 2, 3, and 4, sodium tartrate administered subcutaneously to rabbits with phlorhizin diabetes promptly causes a very decided diminution in the output of total nitrogen and dextrose. It will also be observed, however, that urine secretion is greatly diminished and in some of the experiments, of which the appended are a few examples only, was completely inhibited. From the data in the first four tables it is evident that suppression of urine is sufficient to account for the very great decrease in the output of the urinary con- stituents under consideration. When the urine secretion was not entirely inhibited we have at times obtained water-clear twenty- four-hour specimens of fair volume in which no trace of nitrogen or dextrose could be detected. Experiments with dogs yielded results in accord with those obtained with rabbits as may be seen from the examples cited in tables 5 and 6. In experiment 7, dog 1, table 5, the change in the output of urine and of the urinary constituents under discussion shows a striking similarity to that observed in rabbits. Experi- ment 8, dog 3, table 6, is inserted to show that at times one dog may perhaps be less susceptible to the action of tartrate than other indi- viduals, and that water may be eliminated by the kidney even 3 Ringer: Proceedings of the Society for Experimental Biology and Medicine, ix, p. 54, 1912. Frank P. Underhill 117 TABLE 1. EXPERIMENT 1, RABBIT A. Male rabbit of 2200 grams received daily subcutaneous injection of 0.25 gram phtorhizin. DATE 1911 Volume UR Specific gravity INE Total Nitrogen Dextrose REMARKS November cc. grams grams 14 90 1 .040 1 .85 2.72 15 100 1.026 1.60 1.80 16 100 1.030 1.99 1.43 17 30 OK) o 13 u . uu OUUCU IHIltJO Uo 1I1J cl LlOIl . of 3 crvnmc; f.nrf.Qrir* clUlLl, lie U tl d>l±£i*5\JL WJ.LI1 Na 2 C0 3 , in 40 cc. water. 18 8 0.00 0.00 Animal lies in deep coma. Does not re- spond to stimulation. 19 Heart beat and respi- ration very slow. 20 Animal found dead. Bladder empty. No urine secreted for 24 hours. when the latter is no longer in a condition to normally secrete the organic constituents of the urine. With rabbits exactly analogous conditions may obtain. In other words, a dose of sodium tartrate which in the majority of rabbits or dogs causes suppression of urine may exert only a slight influence in this direction in a small number of individuals. An inspection of the data presented by Baer and Blum points to the same conclusion, and it is possible that the .nega- tive results reported by Ringer may be due to the same fact. This seems hardly likely, however, and as a possible explanation of Ringer's failure to corroborate the findings of Baer and Blum we would call attention to the fact that Ringer administered his glutaric acid solutions in three equal doses during the course of the day and in this way failed, perhaps, to overwhelm the capacity of the animal to transform the compound into a harmless derivative. 1 1 8 Sodium Tartrate and Phlorhizin Diabetes TABLE 2. EXPERIMENT 2, RABBIT B. Female rabbit of 2300 grams received daily subcutaneous injection of 0.25 gram phlorhizin. DATE 1911 URINE REMARKS Volume Specific gravity Total nitrogen Dextrose November cc. grams grams 14 115 1.030 1.66 3.58 15 75 1.036 1.58 1.39 16 100 1.030 1.82 1.28 17 55 0.06 0.17 Ssi l r*pii t €k n pah c iniopf ir\M OUUtliliallCUUS 1I1J CO UlUIl r»f 3 D"Tfimcj tnrf nrir npifl npiitrn 1 1 7Pfl wif.Vi cliV-'lVl, 11C Li Ul CLlHiCU. Willi Na 2 C0 3 , in 40 cc. water. 18 10 0.05 0.00 19 Animal lies in cage, can- not stand. Has no muscular control. Heart beat and respi- ration are greatly ac- celerated. 20 Found dead. No urine secreted for twenty- four hours. From the data in tables 1, 2, 3, and 4, it is evident that sodium tartrate produces its unique influence upon the elimination of urinary nitrogen and dextrose in phlorhizinized animals by causing a partial or complete suppression of urine. In order to determine further the correctness of this conclusion, sections of the kidneys were preserved and sent to Prof. H. Gideon Wells of the University of Chicago to whom I am greatly indebted for the examination of the tissues. In his report Professor Wells says in part, "The greater part of the epithelium of the convoluted tubules is entirely necrotic, and most of the tubules, almost all, in fact, are occluded by large hyaline and granular casts, frequently containing more or less hemoglobin. It is easy to understand that such a kidney could not secrete. It is quite as severe a change as I have ever seen in experimental nephritis. There is very little difference between any Frank P. Underhill 119 TABLE 3. EXPERIMENT 3, RABBIT C. Female rabbit of 2200 grams received daily subcutaneous injection of 0.25 gram phlorhizin. URINE DATE 1911 Volume Specific gravity Total nitrogen Dextrose REMARKS November cc. grams grams 21 85 1.026 0.95 1.88 Drank 80 cc. water. 22 75 1.030 1.30 1.16 Drank 45 cc. water. 23 75 1.030 1.18 0.87 Drank 50 cc. water. 24 30 1.012 0.00 0.00 Subcutaneous injection of 1.75 grams tartaric acid, neutralized with Na 2 C0 3 , in 30 cc. water. Drank 140 cc. water. 25 40 1.015 0.07 0.00 Would not drink. Par- tial prolapse of uterus. Animal killed. On autopsy all organs appeared normal ex- cept the kidneys • which seemed very pale and soft. of the specimens, and that only in degree. The glomerules show almost no change beyond an occasional small hemorrhage." Concerning the histology of the dog kidneys Professor Wells reported that vacuolization was much more prominent than necrosis. The histological picture therefore coincides with the other data. In their investigation Baer and Blum apparently made no histolo- gical study of the kidneys, hence their failure to fully recognize the cause of the diminished excretion of the urinary constituents. It is only fair, however, to add that the suspicion of a kidney factor must have entered into their ideas since they have recorded some experiments with this point in mind. 4 But they tested the secretory power of the kidney by means of inorganic salts only and 4 Baer and Blum: Arch. f. exp. Path. u. Pharm., lxv, p. 1, 1911. i2o Sodium Tartrate and Phlorhizin Diabetes TABLE 4. EXPERIMENT 4, RABBIT D. Female rabbit of 2200 grams received daily subcutaneous injection of 0.25 gram phlorhizin. DATE 1911 Volume URINE Specific Total gravity nitrogen Dextrose REMARKS November cc. grams grams . 21 125 1.024 1.12 2.75 Drank 70 cc. water. 22 GO 1.036 1.17 1.28 Drank 25 cc. water. 23 50 1.040 0.92 1.19 Drank 45 cc. water. 24 18 0.05 0.00 Subcutaneous injection of 2.0 gram tartaric acid, neutralized with Na 2 C0 3 , in 40 cc. water. Animal drank 125 cc. water. 25 Ml 2 drops n on Tivitip /*Mr\"fc in i ckl 1 \i liL-o Millie tlUlo 111 Jell J -Hive mass. Would not drink. 26 3 0.00 Would not drink. 27 At 5 p.m. animal was seized with convul- sions and died id few moments. At autop- sy all organs appeared normal except the kid- neys which were pale and soft. report no change in the elimination of these compounds and there- fore conclude that kidney secretory factors are not primarily account- able for their results. However, it does not necessarily follow- that in a given form of nephritis inorganic salts alone may not be eliminated, nor is it fair to assume that, because one substance may be excreted, a second compound of an entirely different chemi- cal nature will behave in the same manner. If these results of Baer and Blum with inorganic salts are accepted our results indicate the correctness of our contention. It is not our intention, however, at this time to enter more deeply into the conditions attendant upon tartrate nephritis but rather to indicate that a nephritic # Frank P. Underhill I 2 I TABLE 5. EXPERIMENT 7, DOG 1. Full-grown bitch of 9 kilos received daily subcutaneous injection of 1.5 grams phlorhizin. tJRINE DATE 1912 REMARKS Volume Specific gravity Total nitrogen Dextrose February cc. grama grams 6 220 1.070 6.42 32.82 D : N ratio = 5.11. Animal drank 120 cc. water. 7 415 1.074 11.70 43.33 D : N ratio = 3.70. Animal drank 250 cc. water. 8 600 1.053 11.98 40.06 D : N ratio = 3.34. Animal drank 70 cc. water. 9 125 1.008 0.16 0.00 8.0 grams tartaric acid, neutralized with Na 2 - C0 3 , were subcutane- ously injected, dis- solved in 50 cc. water. 10 10 _ 0.075 0.25 Animal has lost control • of muscles and lies in cage. Vomits any water given. At the close of this day it was apparent that dog would not survive the night. Animal was killed by chloro- form. All organs ap- peared normal. condicion must be taken into consideration in the discussion of the results reported by Baer and Blum . Some of the factors of tartrate nephritis will be detailed in a subsequent communication shortly to appear, the work of which has already been completed. The present problem has also been attacked from another stand- point. It is quite conceivable that phlorhizin, acting, presumably, specifically upon the kidney structure, may render the latter unus- ually sensitive to tartrate action and therefore that the combina- 122 Sodium Tartrate and Phlorhizin Diabetes TABLE 6. EXPERIMENT 8, DOG 3. Full-grown bitch of 11.0 kilos received daily subcutaneous injection of 1.5 grams phlorhizin. URINE DATE 1912 Volume Specific gravity Total Nitrogen Dextrose REMARKS March cc. grams grams 5 240 1.070+ 4.44 31.05 D : N ratio = 6.90. Animal drank 370 cc. water. 6 240 1.070+ 8.11 29.44 D : N ratio = 3.63. Animal drank 250 cc. water. t 1.060 8.79 zy . / o D : JN ratio = 3.30. A * 11 1 AAA Animal drank 290 cc. water. 8 442 1.035 3.05 12.88 Subcutaneous injection of 10.0 grams tartaric acid, dissolved in 50 cc. fluid and neu- tralized with Na 2 C03. 9 335 1 . uou 7 fiS 15.12 After injection ani- mal vomited repeat- edly. Could not drink because of vomiting. 10 209 1.045 1.27 4.06 11 240 1.016 1.08 1.02 Animal developed ab- cess at site of injec- tion. In a weak con- dition. Vomits con- tinually. Killed with chloroform. At au- topsy all organs ap- peared normal. tion of the two drugs may bring about changes in the kidney that neither alone could accomplish. If, however, sodium tartrate has a specific action upon kidney secretion this should be manifested by exclusion of the phlorhizin effect, all other conditions remaining unchanged. We have endeavored to compass this result and in order to measure the extent of kidney secretion (in the absence of Frank P. Underhill 123 TABLE 7. EXPERIMENT 5, RABBIT E. (Control). Male rabbit of 2%00 grams. No phlorhizin was given throughout experiment. URINE DATE 1911 Total nitrogen Creat- inine Creatine REMARKS November cc. grams milli- grams milli- grams 22 105 1.012 0.85 • 84 9 Animal drank 150 cc. water. 23 65 1.022 n si 01 24 70 1.020 1.22 78 63 Animal drank 25 cc. water. 25 30 1.015 O 19 Trace, too small to es- timate Trace, too small to es- timate QUUO ULctilCUUb JUJLJ cl; L1UH of 3.0 grams tartaric acid, neutralized with Na 2 C0 3 , in 40 cc. water. Animal drank 30 cc. water. No symp- toms followed injec- tion . 26 less than 1 cc. - Animal drank 110 cc. water. 27 — — Animal drank 60 cc. water. Animal ap- pears normal except that head is rotated to to the right. During morning had one con- vulsion. Recovered. • Found dead. At autop- sy peritoneal cavity contained 30 cc. of clear fluid that readily clotted. The kidneys presented an injected appearance. All other organs seemed normal. Bladder contained no urine. 124 Sodium Tartrate and Phlorhizin Diabetes TABLE 8. experiment 6, rabbit f. (Control). Female rabbit of 2200 grams. No phlorhizin was given throughout experiment. URINE DATE REMARKS 1911 Volume Specific gravity Total nitrogen Creat- inine Creatine November cc. grams milli- grams milli- grams 22 115 1.011 0.74 90 21 Animal drank 90 cc. water. 23 70 1.020 0.97 111 39 Animal drank no water. 24 60 1.025 0.90 84 48 Animal drank 50 cc. water. 25 32 1.015 0.15 Trace, but too small to es- timate Trace, but too small to es- timate Subcutaneous injection of 3.0 grams tartaric acid, neutralized with Na 2 C0 3 , in 40 cc. water. No symptoms followed injection. Animal drank 60 cc. water. less Animal drank 25 cc. 26 than 5 cc. water. Peculiar posi- tion of head similar to that of Rabbit E. 27 Animal in light coma. Killed with chloro- form. Kidneys were very pale and soft. All other organs were apparently normal. Bladder was empty. glycosuria), have noted the excretion of urine and total nitrogen as usual and, in addition, the elimination of creatinine and creatine, the latter compound being constantly present in the urine of our fasting animals. In the data presented in tables 7 and 8 the water intake was observed in order to discover whether diminished volume of urine could be accounted for by lack of water consumption. It will be seen that there is no strict correlation between the intake and the output of water in the tables mentioned, a fact which also applies to the data contained in all the other tables. From the Frank P. Underhill experiments with fasting rabbits, given subcutaneous injections of tartrate only, it is evident that the result presented is one induced specifically by the tartrate and apparently bears little or no relation to the application of phlorhizin. The histological examination revealed no recognizable differ- ences in the kidney changes of specimens taken from animals receiving both phlorhizin and tartrate and from those to whom only tartrate had been administered. In his partial report above, Pro- fessor Wells says, ' 4 There is very little difference between any of the specimens, and that only in degree. " The kidneys taken from animals represented in tables 7 and 8 were sent to Professor Wells mixed in the lot excised from animals having had an injection of both phlorhizin and tartrate. From these observations it is appar- ent that sodium tartrate alone is capable of inducing a particularly severe form of nephritis when subcutaneously introduced into rab- bits and dogs. Our conception of the mechanism responsible for the diminution in urinary constituents as reported by Baer and Blum also furnishes a reasonable explanation for the toxicity of tartaric acid observed by these investigators. In their last paper 5 upon the subject tar- taric acid action is discussed as follows, "Weiterhin besass die Saure eine erhebliche Giftigkeit. Ohne bemerkenswerte Symtome starb die Mehrzahl unserer Hunde kurz nach Beendigung, einzelne Tiere sogar vor Beendigung des Versuchs. Immerhin glauben wir diese giftige Wirkung als etwas Akzidentelles auffassen zu diirfen, v nicht als die Ursache des Einflusses auf Zucker-, Stickstoff- und Acidosekorperausscheidung. " SUMMARY. The observation of Baer and Blum that sodium tartrate subcu- taneously injected may greatly diminish the output of nitrogen and dextrose in the urine of phlorhizinized dogs has been substantiated by the results of our investigation on the subject, but we differ from these authors in the interpretation of the phenomena provoked. Our experience shows that sodium tartrate subcutaneously admin- istered to phlorhizinized rabbits and dogs induces distintegrative changes 5 Baer and Blum: Arch. f. exp. Path. u. Pharrn., lxv, p. 16, 1911. 126 Sodium Tartrate and Phlorhizin Diabetes in the kidney tubuli sufficient to account for the lessened elimination of urinary nitrogen and dextrose, observed by Baer and Blum. Under strictly comparable experimental conditions similar results may be obtained in animals that have not received phlorhizin, thus demonstrating that sodium tartrate acts specifically in this direc- tion and that phlorhizin probably contributes little or nothing to the detrimental influence under discussion. ****** (TOm T« J(n ,„„ A , M ^ xjii ^ i9[2 FEEDING EXPERIMENTS WITH FAT-FREE FOOD MIXTURES. 1 Bv THOMAS B. OSBORNE and LAFAY ETTE B . MENDEL With the Cooperatzon oe Edna L . Ferby (Received for publication, May 20, 1912.) Ca ^ates ents of the diet. I * her them T V^ 8 ™ 1 ^ com P°- food intake nutrit on so lt Z f '^^^ however liberal the energy valu s " th eC ° meSnotabl y defective, be. The ketonuria ano T u I remamm g nutriment may Phenomena when carbohvdt ^ aPP6al " a '° ng with ot her familiar; and thTneSfofSS at '° n ^^'^y fails are tal postulates of phystlogy ° n — / -4 b l - / — / r / - f f r *• 7 / / j J. 1 ; / - y / / 0- 1 '/ \ 1 * 1 / y f / c a, Si d •n / f fit - f r f 1 i t V ■~ Days Chart 5, Rat 529, cf . The fat-free diet had the following percentage com- position: Casein Edestin Sucrose Starch "Artificial" protein-free milk Period 2 22.0 0.0 . 20.0 . 28.5 . 29.5 100.0 Period S 0.0 22.0 20.0 . 28.5 29.5 100.0 In so far as one can judge by appearance and body weight these experiments with fat-free diets show growth quite as successful as 88 Growth on Fat-free Food that attained with natural or artificial mixtures of all the types of food stuffs. Although we cannot claim a complete freedom from " lipoids" for the foods prepared as described above, it is scarcely likely that products so carefully isolated can include any signifi- cant quantities of cerebrosides or phosphatides. This is peculiarly true of experiment 6 in which the sole possibilities of contamination are associated with the recrystal- lized phosphorus-free protein edestin and refined starch. McCollum 14 has demonstrated that the phosphorus needed by an animal for phosphatide forma- tion can be drawn from inorganic phosphates, and that phospha- tides can be synthesized anew in the animal body. Rohmann 15 asserts the possibility of lecithin synthesis in mice which were maintained into the second gen- eration on lecithin-free food. Our own experiments point in the same direction with regard to the lipoids in general; and they give positive evidence of the dis- pensableness of true fats for growth. 16 14 McCollum: Amer. Journ. of Physiol, xxv, p. 120, 1909; McCollum and Halpin: This Journal, xi, 1912, Proc. Soc. Biol. Chem., p. xiii; also Fingerling: Biochem. Zeitschr., xxxviii, p. 438, 1912. 15 Rohmann : Biochemie, 1908, p. 109. 16 In agreement with Stepp, we have not yet succeeded similarly in induc- ing adequate growth in mice with similar diets. Stepp, who used crude food substances, is, however, cautious in his statements. He says : " Wenn nach den mitgeteiltenVersuchen und den anschliessenden Erorterungen der Schluss sich aufdrangt, dass gewisse alkohol-atherldsliche Substanzen fur die Erndh- rung von Mausen ' unentbehrlich sind, so mochte ich diesen Schluss nicht ohne eine Einschninkung aufrechterhalten. Die Untersucher, die sich mit dem Chart 6, Rat 640, & . The fat- free food had the following per- centage composition: Edestin 22.0 Sucrose 20.0 Starch 28.5 "Artificial" protein free-milk . 29.5 100.0 Thomas B. Osborne and Lafayette B. Mendel 89 The possibilities of the method of study introduced by us are manifest. The problems of the origin of fats in animals and their genesis from various carbohydrates or proteins are thus made ap- proachable by experiment. 17 We hope to return to these ques- tions later. Studium der Lipoide beschaf tigten, haben, wie schon kurz erwahnt, an diesen Korpen Eigenschaften gefunden, die man in der Chemie bisher kaum kannte. Die Lipoide haben eine ganz ausserordentliche Fahigkeit, auf die Loslichkeit anderer Stoffe einzuwirken und ihnen Loslichkeit in den spezi- fischen Lipoidlosungsmitteln zu verleihen, in denen die Stoffe sonst ganzlich unloslich sind. So ware es nicht undenkbar, dass gemeinschaftlich mit den Lipoiden irgendwelche unbekannte lebenswichtige Stoffe in Losung gehen und dass so die Lipoide gewissermassen zu Tragern f ur diese Stoffe wi'irden, dass mit anderen Worten bei der Entfernung von Lipoiden die unbekannten Korper mit entfernt und bei Zusatz von Lipoiden mit diesen zugesetzt wer- den. Ein Hinweis auf eine derartige Moglichkeit erscheint notwendig, so- lange es nicht gelingt, die Versuche mit chemisch reinen Korpern durchzu- fiihren." l7 Lummert (Pfliiger's Archiv, lxxi, p. 176,1898) has made attempts in the same direction. Reprinted from Tun Journai, or Biological Chemistry Vol. XII, No. 3, 1912 THE ROLE OF GLIADIN IN NUTRITION. 1 By THOMAS B. OSBORNE and LAFAYETTE B. MENDEL, With the Cooperation of Edna L. Ferry. (From the Laboratories of the Connecticut Agricultural Experiment Station and the Sheffield Laboratory of Yale University, New Haven, Connecticut.) (Received for publication, July 31, 1912.) Our notions regarding the relation of the food proteins to tissue proteins, and the role of proteins in nutrition have experienced radical changes in recent years. Side by side with the increasing evidence of distinct structural differences between the albuminous compounds of different origin and the chemical dissimilarity which may even characterize two proteins derived from a common source, such as some particular seed, has arisen the well founded conviction that it is impossible to develop marked changes in the character of the tissues of animals correlated with the character of the food ingested. Whatever may be the source, or chemical make-up, of the latter previous to its involvement in the nutritive processes, the resulting tissue cells and fluids remain characteristic and specific for the species. "Der Artcharakter wird durch die Art der Ernahrung nicht beeinnusst" (Abderhalden). How this possibility of the fixity of the tissues in the midst of diversity of food types results is made apparent by the newer knowledge respecting the role of digestion in nutrition. The structural pesuliarities which determine the individuality of the proteins are lost by the digestive process; hence we have ultimately to deal with the fragments of the original complexes in the problems pertaining to nutrition. Our food stuffs are currently assumed to leave the alimentary tract largely, if not entirely, in the form of 1 The expenses of this investigation were shared by the Connecticut Agricultural Experiment Station and the Carnegie Institution of Washing- ton. 473 THE JOURNAL OF BIOLOGICAL CHEMISTRY, VOL. XII, NO. 3. 474 Gliadin in Nutrition the so-called ammo-acid "Bausteine." It is these which become our immediate concern in the intermediary problems of metabo- lism that result in the construction or renewal of the specific body protein. Quoting Abderhalden: "Unsere Korperzellen erfahren niemals, welcher Art die aufgenommene Nahrung war." In the organism proper the proteins, as such, may be responsible for various physiological functions. "At present we cannot fully comprehend the role of the proteins, but we must assume that many of the enigmatical properties of living matter depend on this activity of intact protein molecules. We can obtain some idea of the possible variety in the combinations of the protein Bausteine by recalling the fact that they are as numerous as the letters in the alphabet which are capable of expressing an infinite number of thoughts. Every peculiarity of species and every occurrence affecting the individual may be indicated by special combinations of protein Bausteine, that is to say by specific pro- teins. Consequently we may readily understand how peculiarity of species may find expression in the chemical nature of the pro- teins constituting living matter, and how they may be transmitted through the material contained in the generative cells." 2 As one of us has written earlier: "The results of my work have shown that no two seeds are alike in their protein constituents, and that those proteins which appear to be alike are found only in seeds that are botanically closely related. As I have elsewhere pointed out, it would seem that these differences in the reserve food sub- stances of the endosperm must have an important bearing on the character of the developing embryo which derives its first food from them. This food substance, and the embryo as well, are the final products of the series of chemical changes which led to their formation. When the embryo begins its development it finds at hand a definite food, which for each individual of the same species is the same, but for the individuals of different species is different. Each member of a species begins its independent life under similar chemical conditions, but under chemical conditions which are different from those of every other species. When, therefore, each individual plant reaches that stage of development at which its organs of assimilation are able to furnish it with nutri- 2 Kossel: Lectures on the Herter Foundation. The Proteins. Johns Hopkins Hospital Bulletin, xxiii, p. 76, 1912. T. B. Osborne and L. B. Mendel 475 ment from its external surroundings, it is highly probable that its chemical processes have already been established along definite lines which it must follow throughout the rest of its life." 3 In the preliminary processes of metabolism, however, the char- acter of the amino-acid fragments apparently assumes a dominating- importance. The modern chemistry of the proteins has dis- closed the fact that the variations between the different albumi- nous compounds in respect to their Bausteine may be both quanti- tative and qualitative in character. This has raised the question of the relative physiological value of unlike proteins. "The fact that so many of the vegetable proteins, which serve extensively as food, have been shown, by our present investigation, to yield such different proportions of the various nitrogenous decomposi- tion products, as compared with the animal proteins, makes it a matter of the greatest interest and importance to know some- thing more of the processes involved in this synthesis." 4 Whether protein can be suitably utilized when administered in its completely digested or abiuret form as well as in its natural condition need not concern us here; since the possibility of main- taining individuals in satisfactory nutritive balance, at least for a not inconsiderable time, on an intake made up exclusively of Bausteine has been demonstrated. It would seem, therefore, as if the problem of replacing the larger protein complexes by their elementary constituent fragments had been to a certain extent solved. 5 If we assume, in harmony with some of the prevailing views of metabolism, and notably that supported by Abderhalden, that the animal must construct its tissue proteins from the amino- acid fragments which are furnished by protein hydrolysis, it is obvious that deficiencies in quantity in the Bausteine or a lack of one or more of them must lead to serious nutritive disturbances. The chemical fixity of the tissues under widely differing nutrient environment points in the same direction. Abderhalden has maintained that, so long as there is no evidence that amino-acids can readily experience a transformation into one another in the organism, the extent of protein construction in the body must be 3 Osborne: Proc. Soc. Exp. Biol, and Med., v, p. 105, 1908. 'Osborne and Harris: Jour. Amer. Chem. Soc, xxv, p. 323, 1903. 5 Cf . Abderhalden : Synthese der Zellbausteine in Pflanze und Tier, Berlin, 1912. 476 Gliadin in Nutrition limited by the amino-acid which is present in the smallest relative amount in our intake. The fact that certain proteins, such as gelatin and zein, which are notably defective in respect to the number of the amino-acids which they yield, are unable by them- selves to promote nutritive equilibrium and supply the nitrogenous needs of the diet might be quoted in support of the views mentioned above. If Abderhalden's hypothesis regarding the nature of protein metabolism is correct it follows that those food proteins which approach most nearly to the tissue proteins in their amino-acid make-up should most easily supply the protein needs of the animal. Michaud 6 has undertaken to demonstrate, in accord with this, that the protein minimum of dogs can be maintained at a lower level when the intake is in the form of dog tissue than in the form of proteins differing widely therefrom in their chemical make-up; yet the investigations heretofore recorded with these proteins lead to the belief that they are, at least to some degree, utilized as food by the animal ; even when they are fed as the sole source of nitrogen. Some of these proteins lacking one or more of the cleavage prod- ucts known to be necessary for the formation of the proteins of the animal body are of relatively high efficiency in preventing loss of body nitrogen due to endogenous metabolism, although they are insufficient for growth. 7 It is evident that "the processes of replacing nitrogen degraded in cellular metabolism are not of the same character as the processes of growth. It seems also to be a necessary conclusion that the processes of cellular cata- bolism and repair do not represent a series of chemical changes involving the destruction and reconstruction of an entire protein molecule." 8 Regarding the necessity of distinguishing carefully between maintenance, repair and growth in nutrition we shall have more to say later. Undoubtedly the failure to bear these distinctions in mind has led to much confusion in the past. Fur- thermore, investigators have heretofore been so largely concerned with the functions of proteins as a whole in important biological processes that the possibility of their individual participation 6 Michaud: Zeitschr. f. physiol. Chem., lix, p. 405, 1909; cf. also Frank and Schittenhelm : ibid., lxx, p. 99, 1910; lxxiii, p. 157, 1911. 7 Cf . Osborne and Mendel : Carnegie Institution of Washington, Publi- cation 156, pt. ii, 1911; also Zeitschr. f. physiol. Chem., 1912 (in press). 8 McCollum: Amer. Journ. of Physiol., xxix, p. 215, 1911. T. B. Osborne and L. B. Mendel 477 and use has been generally overlooked. As Kossel lias lately said: "Hitherto the appearance of protein Bausteine in the living organism has always been ascribed to protein decomposition. But this supposition is unjustified. We must rather assume that these Bausteine may appear and disappear in the body without at any time forming part of a protein molecule. And further we may suppose that only under certain circumstances, for definite physiological purposes, are these independent groups stored in a collected form — the protein substances." 9 Attempts have been made at various times in the past to perfect the so-called abnormal or incomplete proteins by adding to them in the diet one or more ammo-acids which are known to be lacking from the complex. This is true of studies made with gelatin— which yields no tyrosine, tryptophane or cystine — and with zein, — a protein which yields no tryptophane, and from which no lysine or glycocoll can be obtained. These trials have, all in all, not been very satisfactory. Other experiments in which an amino- acid, such as tryptophane, has been intentionally eliminated from the food mixture have speedily exhibited a nutritive defect in the dietary. In any event it seems clear, from such evidence as is available at the present time, that the cyclic compounds, tyro- sine, phenylalanine, histidine and tryptophane, are indispensable for the welfare of the organism. Indeed W. A. Osborne has expressed the view that the essential difference between the animal and the plant organism lies in their respective ability or inability to synthesize substances of the cyclic type. Cyclopoiesis, accord- ing to him, is a property exhibited solely by the vegetable organism. Are the other amino-acids equally indispensable? At the present moment it is impossible to give any definite answer to the question as to whether an amino-acid like leucine, for example, can be replaced by alanine, or any other closely related form. In one case, in any event, the possibility of a synthesis of an amino- acid de novo in the animal organism has been admitted. Prolonged feeding experiments with casein from which glycocoll has not been obtained, as well as the enormous production of glycocoll for the hippuric acid synthesis after the administration of benzoic •Kossel: Lectures on the Herter Foundation. The Proteins. Johns Hopkins Hospital Bulletin, xxiii, p. 76, 1912. 47 8 Gliadin in Nutrition acid 10 — a production out of all proportion to the assumed content of preformed glycocoll in the food intake, or the body tissues themselves — leave little doubt of the capacity of the animal cell to synthesize at least one amino-acid. For evidence of the formation of amino-acids more complex than glycocoll, the recorded experiments with gliadin must be taken into consideration. This substance, an alcohol-soluble pro- tein of the prolamine type, possesses a special interest in that it yields so little of the diamino-acid, lysine, as well as of glycocoll, that these have not heretofore been obtained from it by the usual analytical methods. It furthermore contains a relatively small proportion of both arginine and histidine, and extremely large proportions of glutaminic acid and ammonia yielding groups. Its ready digestibility has been demonstrated repeatedly 11 in contrast to the greater resistance of the "abnormal" protein zein. Henriques 12 reported that he kept rats in nitrogenous equilibrium on a diet in which gliadin constituted the sole form of nitrogenous intake, although he failed when the tryptophane-free zein was used. Abderhalden and Funk 13 were similarly successful with gliadin fed to dogs. They state, however, that in one case the preparation of gliadin fed by them contained 0.35 per cent of lysine; and they intimate that the nutritive equilibrium secured by Henriques on a diet containing gliadin as the sole protein was due to an impure preparation. The question of lysine synthesis in the body is expressed by Rona 14 as follows: "Das Problem is also durch die Versuche von Henriques noch nicht gelost, hingegen sprechen alle unsere Erfahrungen dafur dass die Aminosauren, Glykokoll ausgenommen, im Organismus nicht neugebildet werden." In other experiments Henriques and Hansen 15 have stated that they were able to get rats into a state of nitrogenous equilibrium on a diet containing the nitrogen solely in the form of the mono- amir.o fraction of a digest. Here too, as in the experiments with 10 Cf. Magnus-Levy: Biochem. Zeitschr., vi, p. 523, 1907; Ringer: this Journal, x, p. 327, 1911; Epstein and Bookman: ibid., x, p. 353, 1911. 11 Cf. Mendel and Fine: this Journal, x, p. 303, 1911. 12 Henriques: Zeitschr. f. physiol. Chem., lx, p. 105, 1909. 13 Abderhalden and Funk: ibid., lx, p. 418, 1909. 14 Rona: Oppenheimer's Handbuch der Biochemie, iv, pt. i, p. 550. 15 Henriques and Hansen: Zeitschr. f. physiol. Chem., xliii, p. 417, 1904; and xlix, p. 113. 1906. T. B. Osborne and L. B. Mendel 479 gliadin, a synthesis of nitrogenous compounds of the type 4 , precipi- table by phosphotungstic acid must be assumed if the nutritive equilibrium of the experimental animals was at all adequate. It will be seen, therefore, that the problem of di-amino synthesis has heretofore largely hinged upon the validity of the work of Henriques. The situation has been summed up by Rona in these words: "Vorlaufig mussen wir also daran festhalten, daes eine Ueber- fuhrung einer Aminosaure in eine andere, bezw. eine Neubildung einer Aminosaure (Glykokoll ausgenommen) im tierischen Organ- ismus nicht stattfindet." 16 EXPERIMENTAL PART. Employing the methods which ' we have developed in recent years in connection with our feeding experiments with isolated food substances 17 we have accumulated a large number of data which refer directly to the nutrient r61e of gliadin in the animal organism. Inasmuch as we have succeeded, by the application of care in the management of the rats, by furnishing suitable hygienic environment and appropriately selected diet, in main- taining these animals in good nutritive condition on mixtures of isolated food stuffs over periods of more than 500 days, we believe that some of the criticisms which have been aimed at experiments carried out on rats are thereby met. The guiding considerations which have led to the special proportions of nutrients, etc., in the food mixtures reported below have been discussed in some detail in our previous publications. 18 The upshot of our trials has been the demonstration that the gliadins of wheat and rye, as well as the closely related alcohol-soluble hordein of barley — all of which are similar in the proportion of their Bausteine — suffice for the maintenance of rats without growth. 16 Rona: Oppenheimer's Handbuch der Biochemie, iv, pt. i, p. 550. 17 Osborne and Mendel: Carnegie Institution of Washington, Publi- cation 156, pts. i and ii, 1911; Zeitschr. f. biol. Technik u. Methodik, ii, p. 313, 1912; and Zeitschr. f. physiol. Chem., 1912 (in press). 18 Osborne and Mendel: Carnegie Institution of Washington, Publi- cation 156, pt. ii, 1911; Science, N. S., xxxiv, p. 722, 1911; Zeitschr. f. physiol. Chem., 1912 (in press). Gliadin in Nutrition Preparation and composition of gliadin. The gliadin used for these feeding experiments was made from very thoroughly washed wheat gluten from which all proteoses and other water-soluble proteins had been removed as completely as possible. The alcoholic extract of this gluten was filtered water- clear, thereby separating any suspended glutenin or other proteins insoluble in 70 per cent alcohol. After concentrating the alcoholic extract the residual gliadin was dissolved in alcohol, and its solution poured in a thin stream into a very large volume of cold water, thereby removing any water-soluble substance which might possi- bly be set free when the gluten was dissolved. The precipitated gliadin was again dissolved in alcohol, and its syrupy solution poured into a very large quantity of absolute alcohol, and thus precipitated as a coherent mass. This was then digested with fresh quanti- ties of absolute alcohol, and finally with ether and was easily reduced to a powder. After drying in the air, the gliadin thus ob- tained formed a snow white powder which was completely soluble in 70 per cent alcohol. It is difficult to see how gliadin, thus prepared, can contain any other proteins than those soluble in alcohol, or how any purer preparation could be made. As it was of the greatest importance to know whether or not this gliadin was entirely free from lysine, we made a very careful examination of two portions of 100 grams each, according to the method of Kossel and Kutscher, with which we have had extensive experience. The result was in each case entirely negative, corre- sponding with our earlier experience, as well as with that of Kossel and Kutscher and of Abderhalden. However, in view of the rela- ti ely considerable precipitate produced by phosphotungstic acid, it seemed possible that some lysine might be contained therein, under conditions which rendered its separation as the picrate difficult. This seemed the more probable in view of our previous experience in attempting to isolate lysine as the picrate directly from the products of the hydrolysis of casein. In this attempt we obtained less than one-half as much as by Kossel and Kutscher's method, thus showing the effect of the presence of other amino-acids. We accordingly made renewed efforts to obtain lysine picrate from our solutions*. In one case fractional precipitation with phosphotungstic acid was employed without success. In the other T. B. Osborne and L. B. Mendel 481 case the alcoholic solut ion to which picric acid had boon added was divided into two parts, one of which was allowed to evaporate slowly until nearly dry. The semicrystalline residue was extracted with alcohol. The insoluble residue when recrystallized gave 0.2 gram of lysine picrate. The other half of the solution was neutral- ized with acetic acid and allowed to evaporate slowly until a considerable quantity of free amino-acids separated. These were filtered out and washed with alcohol. The alcoholic filtrate was neutralized with sodium hydroxide and treated with spdium picrate, whereupon a small precipitate of lysine" picrate formed, which when recrystallized weighed 0.23 gram. We thus obtained 0.43 gram of lysine picrate from 100 grams of gliadin correspond- ing to 0.15 per cent of lysine in this preparation. Whether or not this represents all of the lysine in this gliadin cannot, of course, be determined; but our previous experience with casein has convinced us that it is very difficult to separate all of the lysine picrate from solutions containing other amino- acids. Whether the presence of this lysine is to be ascribed to contamination of our preparation of gliadin with other proteins, or to the presence of a small amount of lysine in gliadin, is likewise difficult to determine. All we can say is that we do not know how any purer preparation of the substance heretofore known as gliadin can be made, and our present opinion is that future in- vestigations will show that gliadin does in fact yield a little lysine. The question is, therefore, raised: can the absence of any amino- acid from any protein be assumed solely because it cannot be separated by direct crystallization? In our opinion it cannot be so assumed. The known difficulty encountered in trying to thus separate all of any of the amino-acids from mixtures of them sup- ports this view, as does also the experience of Osborne and Jones and the more recent experience of Abderhalden. Both of these investigations showed that less than one-half the glycocoll, alanine or aspartic acid could be recovered from mixtures containing known quantities of pure amino-acid§. Whether or not gliadin is actually deficient in glycocoll or lysine, we do know from incontrovertible evidence that it yields relatively very little glycocoll, arginine, histidine or lysine and ex- tremely large quantities of glutaminic acid, proline and ammonia. Gliadin, therefore, has a unique constitution, very different from 482 Gliadin in Nutrition the tissue proteins of animals, as well as from most of the other proteins which are commonly present in the foods of men and animals. We should consequently expect to find the value of gliadin in nutrition to be different from that of other proteins which yield amino-acids in proportions corresponding more closely with those obtained from proteins of animal origin. Accordingly we have made a large number of prolonged feeding trials on both mature and growing rats, with the results described in the following pages. Maintenance experiments with grown rats. The illustrative protocols which are presented in graphic form are largely self-explanatory. The abscissae of the curves represent days and the ordinates actual body weight (solid line) or food- intake (dotted line) in grams. In the charts for ungrown animals the average (normal) curve of growth, plotted from body weight data available for normally growing animals of the same sex, is represented by a broken line for comparison. The food-intake curve is plotted from the weights of food eaten per week. Where numbers are marked on body weight curves they indicate the time at which changes in the character of the feeding were insti- tuted. In Charts 1, 2 and 3 are represented the results of prolonged maintenance trials with full' grown rats in which gliadin formed the sole nitrogenous intake. 19 These experiments far exceed the longest records of trials in any way comparable with our own which have been reported in the literature. Henriques' records, for example, extend at best over only 23 days. 20 A study of the dietaries quoted in connection with these charts will show that, for long periods, in several of our experiments, there was no possi- bility of the inclusion of any other protein than the gliadin itself in the make-up of the food, except in the very small quantity of feces supplied during period 2. 21 Thus rat 130, Chart 1, was 19 Some of the earlier portions of the charts in this paper have already been published elsewhere. 20 Henriques: Zeitschr. f. physiol. Chem., lx, p. 105, 1909. 21 See Osborne and Mendel: Carnegie Institution of Washington, Publi- cation 156, pt. ii, p. 60 for discussion of the effect of feces thus fed. T. B. Osborne and L. B. Mendel 483 fed for 290 days on a food entirely free from any other protein than gliadin, before his condition became such as to render a change in his diet necessary. That the failure to be longer maintained in a satisfactory condition was not due to deficiencies in the gliadin is proved by the rapid recovery of health and weight when the non-protein constituents of the food were changed by replacing the inorganic constituents and a part of the carbohydrate with " protein-free milk." 22 A similar condition is shown by rat 134, Chart 2, but in this experiment the decline in weight occurred much earlier and the change in the non-protein constituents of the food had to be made after only 72 days. Rat 147, Chart 3, was kept on the original gliadin food for 256 days, but at that time its loss of weight and physical condition was such that it could only be restored by changing the protein to casein. Later (see period 6), the failure to thrive on the original gliadin food was completely remedied by the addition of "protein-free milk" to the diet. A possible criticism of these experiments concerns the residual content of milk protein in the " protein-free milk." Such analyses as we have made have indicated that the extent of this contami- nation cannot exceed 0.6 per cent of the entire food mixture — a quantity of "normal protein" far too small, as we have convinced ourselves by other studies directed to this point, to meet the nutrient deficiency of gliadin in respect to growth. However, the experi- ments which have been conducted without the use of the protein- free milk bear direct testimony in favor of the conclusion that possible traces of contaminating milk protein cannot in any way explain the satisfactory maintenance of our animals, but that some other substance than the protein is the cause of rapid recovery induced by the addition of the protein-free milk. In the light of such long continued experiments, extending as they do over a very considerable portion of the natural life of an animal whose longevity has been estimated at about three years, one must accept these observations as evidence that, so far as main- tenance is concerned, the protein of the food can differ very widely in its amino-acid make-up from the tissue proteins of the animal without affecting the well being of the latter. 22 See ibid., p. 80. 4 8 4 Gliadin in Nutrition Maintenance experiments with growing rats — failure to grow. The following charts illustrate the inability of wheat gliadin and other prolamines to promote growth under dietary conditions in which other single proteins have been eminently satisfactory. In Charts 4, 5 and 6 the curves of growth with casein, edestin, and glutenin will be found to correspond closely, during the first 100 days, to those observed on animals receiving mixed food. The contrast of the trials with gliadin'are striking in the extreme ; and the results are the same if the alcohol : soluble hordein from barley or the gliadin from rye are used. We have tested the gliadin of wheat, 23 Charts 7, 8 and 9; of rye, 23 Charts 10 and 11, and the similarly constituted hordein of barley, 23 Charts 12 and 13. The results are the same, whether the trials be made at a very early age (compare Chart 7) or somewhat later (compare Chart 12). In corroboration of the statement that the results described repre- sent true maintenance without growth, we present two experi- ments, one with gelatin, Chart 14, and one with zein, Chart 15, which show by contrast the failure of maintenance when an abso- lutely inadequate protein, like those mentioned earlier, forms the nitrogenous constituent of the food intake. The youthful appearance of animals thus maintained without growth corresponds in every respect, so far as external characters go, with the size rather than the age of the animal. 24 That the failure to grow is in nowise attributable to any toxicity or inhibi- tory property of the specific proteins used is shown by experiments (see Charts 16 and 17) in which the addition of a small proportion of an ''adequate" protein has sufficed to induce noteworthy growth. To determine whether growth could in any way be induced by largely increasing the content of the " inadequate' ' protein in the food mixture, special experiments were undertaken (see Charts 18, 19 and 20). The relative variations in body weight in relation to the larger protein intake in the two series are too small to be of marked significance. 23 The preparation and properties of these proteins are given by Osborne : Abderhalden's Handbuch der biochemischen Arbeitsmethoden, ii, 1909. 24 Photographs of some of our ungrown animals maintained on gliadin will be found in Osborne and Mendel: Carnegie Institution of Washington, Publication 156, pt. ii. 1911. T. B. Osborne and L. B. Mendel 485 Aside from their interest in furnishing a physiological differ- entiation between various proteins, as exemplified in the capacity or failure of maintenance, and the capacity or failure of growth, these experiments have a large field of interest in presenting a method whereby the effective stunting of animals can be induced at any stage in the normal period of growth. We have as yet not determined the possible alterations in the histological make-up of the organs and tissues which may be correlated with the suppres- sion of growth. There is much in the recent literature on infan- tilism which suggests that the dwarfing may be secondary to defects or alterations in organs, such as the ductless glands. One point alone may be emphasized here, namely, that the capacity to grow is by no means lost even after very prolonged periods of stunting with the gliadin diet. This is shown in Chart 7 which exhibits satisfactory growth on a suitable dietary after a continuous suppression of growth lasting 277 days, when the animal was 314 days old — an age at which normally little or no growth takes place. 25 Gliadin and gestation. Long continued feeding with gliadin as the sole source of nitro- gen by no means impairs the capacity of the animal to produce healthy young and suitably nourish them. The two animals whose records are presented in Charts 21 and 22 were 'paired and the female (Rat 129) gave birth to a litter of four at the end of 178 days on the gliadin food mixture. The young rats whose growth records are reproduced in Charts 23, 24, 25 and 26 were nourished satisfactorily by the mother during the first month of their existence, in so far as one can judge by their increase in weight, in comparison with that of normally reared rats. At the end of 30 days, three rats were removed from the mother and put upon diets of casein food, edestin food and milk food respectively. The fourth animal was allowed to remain in the cage with the mother whose sole source of nutriment was the original gliadin food mixture. It will be noted from the records that whereas the three removed animals manifested a normal growth on their new dietaries, which had likewise proved adequate 25 See also charts cxx, cxxi, cxxii and cxxiii, Publication 156, pt. ii, Car- negie Institution of Washington. 486 Gliadin in Nutrition for growth in many other instances, the rat kept with the mother began to evince a failure to grow at about the period (30 days) when young rats are wont to depend upon extraneous food for nourishment. In the present case this means that the young animal, forced to depend upon the gliadin food mixture in place of the milk of its mother, showed the typical failure to grow on the "inade- quate" diet upon which the mother had not only been maintained but had actually produced young and secreted milk sufficient in quantity and quality to induce normal growth in her offspring. No doubt can remain, we believe, that in this experiment, in which there has unquestionably been a renewal, or new formation, of body tissue, very large in proportion to the original weight of the mother animal, there must have occurred a synthesis not only of the "Bau- steine" deficient in the protein intake, but likewise of tissue and milk components like the nucleic acids (with their content of purines, pyrimidines and organically combined phosphorus), and phospho-proteins, like casein, etc., which were completely missing in the special food intake that had formed the sole food of the mother during several months. Unless one were prepared to maintain a profound alteration in the chemical make-up of this " gliadin family" it must be admitted that synthesis in animal nutrition has here been demonstrated in a striking manner. We have elsewhere 26 taken cognizance of the possible role of alimentary bacteria in furnishing some of the components which may be deficient in the dietary. These synthetic organisms may well be able to build new amino-acids out of a variety of substrates; and the possibility is thereby suggested of the production, through bacterial intervention, of complexes missing or deficient in the original food intake. We can hardly regard this possibility as an explanation of the ability of animals to be maintained on the abnormal proteins, gliadin and hordein; otherwise there is no apparent reason why they should not likewise be maintained by zein or gelatin, nor why growth should not also be possible with every digestible protein through the intervention of the bacterial protein complexes manufactured. It is more likely that growth 26 Cf. Osborne and Mendel: Carnegie Institution of Washington, Publi- cation 156, pt. ii, p. 61, 1911. T. B. Osborne and L. B. Mendel 487 hinges on the intervention of some protein complex not essential for the endogenous metabolism of the individual. The unique features of growth and maintenance on the special proteins here considered serve to emphasize the fact that mainte- nance experiments alone cannot suffice to solve the problem of the full biochemical value of dietaries. Nutrition involves an ensemble of processes which are determined or modified by factors whose real significance is only beginning to reveal itself. No method of study involving well controlled conditions need be cast aside; but hasty judgment formed as the result of brief feeding trials on larger animals containing an abundant reserve supply must henceforth be accepted with extreme caution. 488 Gliadin in Nutrition Chart 1, Rat 130 9 , shows long continued maintenance on a diet containing feeding by diseased lungs and a large parasite, over 40 cm. long, encysted in I The diet during the different periods is shown below. During period 2 effect of this is discussed in Publication 156, pt. ii, p. 61, Carnegie Institution of PERIODS 1, 2, AND 3. Gliadin (wheat) . Starch Sucrose Agar.... Salt mixture I.. Lard per cent. .... 18.0 29.5 17.0 5.0 2.5 J28J) 100.0 Gliadin (wheat).. Protein-free milk. Starch Agar Lard T. B. Osborne and L. B. Mendel 489 JB o « 00 « 20 ^0 460 480 500 520 J40" |le protein. The animal's life was terminated after 546 days of experimental ur-dry feces from rats on a mixed diet was given each week. The possible PERIOD 5. per cent. •...18.0 Milk powder C ™\ .... 28.2 Starch .... 20.8 Lard " Efc« 5.0 .... 28.0 60.0 12.0 28.0 100.0 100.0 49Q Gliadin in Nutrition Chart 2, Rat 134 9 , shows long continued maintenance on a diet containing gl by an ulcer of the pylorus. The diet during periods 1 and 2 was : PERIOD 1. Gliadin (wheat). Starch Sucrose Agar Salt mixture I . . Lard T. B. Osborne and L. B. Mendel 491 500 520 ,320 340 3fo0 380 400 420 4 40 4fo0 480 otein. The animal's life was terminated after 511 days of experimental feeding milk. per cent. .... 18.0 .... 28.2 .... 20.8 .... 5.0 .... 28.0 100.0 492 Gliadin in Nutrition 1 Chart 3, Rat 147 9 , shows long continued maintenance on lung disease after 445 days of experimental feeding The diet during the different periods is shown below. Du legend on Chart 1. PERIODS 1, 2, 3 AND 6. per cent. Gliadin (wheat) 18 Starch - 29 • 5 Sucrose 15 Agar 5.0 Salt mixture 1 25 Lard ■ • 300 100.0 Gliadin (wheat) Protein-free mil Starch Lard. T. B. Osborne and L. B. Mendel 493 280 300 320 340 3&0 380 400 4-20 440 460 |adin as the sole protein. The animal's life was terminated by I quantity of feces from rats on a mixed diet was supplied. See per cent. .... 18.0 .... 28.2 . ... 20.8 ... 5.0 . .. . 28.0 100.0 PERIODS 5 AND 8 per cent. Casein (cow's milk) 18 Protein-free milk 28 2 Starch 23 8 Agar Lard 5.0 25.0 100.0 494 Gliadin in Nutrition _£< • ^OOOOOio f j \\ is i s \ \N — - B 4;- c \ \\ ^ -f- c s — N '\ ^ > •v. ^ 3 ! O if §"3 „ o ^ O CM 00 O O S OO X CO o * Eh M o CD III 3 2 J S 5 H T5 T. B. Osborne and L. B. Mendel 495 l3o 120 80 / / s s / / / / f / / // / / / / / 1 1 1 / 1 1 1 / 1 f / * G'lwte nm t P rotei"-"f ree «rv k > * O Dat^s 20 * 4-0 60 80 100 Chart 6, Rat 284 cf , shows normal growth on a diet containing glutenin which, together with an approximately equal quantity of gliadin. forms about 80 per cent of the proteins of the wheat kernel. The diet was: per cent. Glutenin (wheat) 18.0 Protein-free milk 28.2 Starch 23.8 Agar 5.0 Lard 25 100.0 49 6 Gliadin in Nutrition DaifS 20 Chart 7 Ra» 240 9 , shows failure to make more than slight growth o rate after 276 days of stunting. At this time the rat was 314 days old, The diet during periods 1 and 2 was : PERIOD 1. Gliadin (wheat).. . ■ Protein-free milk. Starch T. B. Osborne and L. B, Mendel 497 300 320 340 3fc0 38 o 4o o 420 440 4feO 480 SoT jg gliadin as the sole protein, and capacity to resume growth at a normal ps normally grow very little more. PERIOD 2. 'der. per cent. .... 60.0 .... 16.0 ... . 24.0 100.0 49 8 Gliadin in Nutrition T. B. Osborne and L. B. Mendel 499 7 / \ \ \ ..43 \ V v_ V. *+■ c \ c \ \ \ \ T C c s ) \ \ X— \ s \ \ ). ^ » •a & CD CO h CM §^ CD +3 CD . 1 "% % CD I * o » ■+3 -+■= fl ^ CD o3 s *3 ^2 SH CO tein-f»-ee din n,lk> - - R^e Dai^s 20 40 fcO 80 100 120 |40 160 180 2C0 Chart 11, Rat 549 d% shows failure to make more than slight growth on diets containing gliadin from rye and later gliadin from wheat. This experiment was terminated by the death of the rat after 172 days of experi- mental feeding. The only abnormal condition revealed by the autopsy was a collection of hair balls in the stomach. The die during periods 1 and 2 was: PERIOD 1. PERIOD 2. per cent per cent. Gliadin (rye) 18.0 0.0 Gliadin (wheat) 0.0 18.0 Protein-free milk 28.0 28 Starch 28.0 26.0 Lard 26.0 28.0 100.0 100.0 500 zoo Chart 12 ; Rat 255 9 , shows failure to make more than slight growth on a diet con- ming hordein from barley as the sole protein. Hordein is very much like gliadin in phys- il properties and amino-acid make-up and appears to have a similar value in nutrition, us rat died suddenly after 249 days of experimental feeding but no cause for death was- own by the autopsy. The diet during periods 1 and 2 was: per cent. Hordein lg q Protein-free milk 28.2 Starch jg g Agar Lard 5.0 0.0 100.0 per cent. Casein jg q Protein-free milk 28 2 Starch 18 8 A e ar 5.0 Lard 30.0 100.0 Chart 13, Rat 256 9, shows .fail- ure to make more than slight growth on a diet contain- ing hordein from barley as the sole protein. Hordein is very much like gliadin in physical properties and amino-acid make- up and appears to have a similar I ln nutritl °n. The animal died suddenly after 220 days of experimental feeding but itopsy failed to show anything abnormal, ^he diet during periods 1 and 2 was: 'ordeln tein-free milk. ^gar. iard. per cent. 18.0 28.2 l itar CA 18 ,g . 5.0 . 30.0 100.0 PERIOD 2. per cent. Casein jg q Pcotein-free milk 28 2 Starch 18 8 A sar '['" 5.'o Lard 30.0 100.0 SOI 502 Gliadin in Nutrition \2 ^ «._G e la Prote Ir tm 1- -free mil Gelol -- -Kfre« free m t Prote ; milk. 5 \ Do^S 20 Chart 14, Rat 615 cf, shows failure to grow or even be maintained during period 1 on a diet containing gelatin as its sole protein, and recovery when one- half of the gelatin was replaced by glia- din. The final fall in weight was due to diseased lungs which caused death. The diet during periods 1 and 2 was: PERIOD 1. per cent. Gelatin 18.0 Protein-free milk 28.0 Starch 27.0 Lard 27 100.0 PERIOD 2. Equal parts of gela- tin food (as in period 1) and gliadin food. per cent. Gliadin 18.0 Protein-free milk 28.0 Starch 26.0 Lard 28.0 100. 4 / i/ \ ! \ I' ^ \ \ w %— — 4 *■ k _- C | a. CL -> p— N —i i Chart 15, Rat 634 cf, shows a rapid decline in weight, despite a food intake quite sufficient for maintenance, when the diet contained zein as its sole pro- tein. Note the rapid repair and growth when the zein was replaced by lactal- bumin and sudden decline when the rat was again placed on the zein food. The d et in the different periods was: PERIODS 1 AND 3. PERIOD 2. grams. per cent. Zein 18.0 Lactaibumin 18.0 Protein-free milk 28 Protein-free milk 28.0 Starch 24.0 Starch 29.0 30.0 Lard 25.0 100.0 100.0 Water 15 cc. T. B. Osborne and L. B. Mendel 503 120 s J7 / / // / /I / 1 e- Cosei 1 f opd 2 / 1 / / Gl.o< UtProte n free m Ik 7S%> / / / T 1/ if Daijs 20 fcO 80 100 120 Chart 16, Rat 287 cf , shows nor- mal growth on a diet in which the protein consisted of 1 part casein and 3 parts of gliadin. Note the effect on the rate of growth induced by this small proportion of casein. Cf. Charts 7, 8, 10, 13, 18 and 19. The diet consisted of a mixture of one part of the casein food with three parts of the gliadin food. ^GLIADIN POOD. CASEIN FOOD. 'percent. percent. Gliadin 18.0 Casein 18.0 Protein-free milk 28 . 2 Starch 32.5 Starch 28.8 Sucrose 17.0 Agar 5.0 Agar 5.0 Lard ^28 .0 Salt m ixture I... , 2.5 100.0 Lard • 25.0 100.0 Chart 17, Rat 280 cf , shows nor- mal growth on a diet in which the protein consisted of 1 part casein and 3 parts of gliadin. Note the ef- fect on the rate of growth induced by this small proportion of casein. Cf. Charts 7, 8, 10. 13, 18 and 19. The diet consisted of a mixture of one part of the casein food with three parts of the gliadin food. GLIADIN FOOD. per cent. Gliadin 18.0 Protein-free milk 28.2 Starch 28.8 Agar 5.0 Lard 28.0 100.0 CASEIN FOOD. per cent. Casein 18.0 Starch 32.5 Sucrose 17.0 Agar 5.0 Salt mixture I... 2.5 Lard 25.0 100.0 Datjs ZO — Gh P.col P-otem--f Chart 18, Rat 588 9 , shows fail- ure to grow on a diet containing gliadin as the sole protein and an artificial imitation of the natural pro- tein-free milk. After 114 days the artificial protein-free milk was re- placed by natural, but the decline in weight which had begun was not stopped by this change. The autopsy showed no adequate cause for death. The diet in periods 1 and 2 was: ODotjS 20 44 60 80 '00 I PERIOD 1. per cent. Gliadin 18.0 Artificial protein-free milk. 30.0 Starch 22.0 Lard 30. 100.0 PERIOD 2. per cent. Gliadin 18.0 Protein-free milk 28 Starch 26.0 Lard ■ 28.0 100.0 T 50 ortrflc ■f r ol Prote \ bO 80 100 Chart 19, Rat 594 c?, shows failure to make more than slight growth on a diet containing gliadin as its sole pro- tein and an artificial imitation of the natural protein-free milk. The animal died after 92 days of experimental feed- ing. Calculi were found in the bladder and left kidney. The diet was: per cent. Gliadin 25.0 Artificial protein-free milk 30.0 Starch 15.0 Lard ■ 30.0 100.0 / / / / / / / / 1— Y / .-Glyodu 1 —f 35%ror1 f.c.al ?t ot^Kfre e m> Ik / . /^-""^ Bo Chart 20, Rat 603 cf, shows failure to grow at normal rate on a diet containing gliadin as the sole protein. In this ex- periment an artificial imitation of the natural protein-free milk was used. This animal died after 105 days of experi- mental feeding with diseased lungs. The diet was: per cent. Gliadin 35.0 Artificial protein-free milk 30.0 Starch 5.0 Lard • 30 100.0 504 T. B. Osborne and L. B. Mendel 505 506 Gliadin in Nutrition sr 1 J* -Gl.od m + Pre fein-Fn :i \ y — \ c +- *> T3 — td 1 1 i \ / Chart 22, Rat 168 cf, shows maintenance and fertility on a diet containin gliadin as its sole protein. After 154 days this rat was paired with Rat 129, fou young being the result of the mating. The animal died with diseased lungs afte 230 days of experimental feeding. The diet was: PERIOD 1. per cent. Gliadin 18.0 Protein-free milk 28.2 Starch 20.8 Agar 5.0 Lard >. 28.0 100 PERIOD 2. per ce Edestin 18. Protein-free milk Starch 20. Agar •• •■• 9 Lard T. B. Osborne and L. B. Mendel 20 40 60 100 120 140 160 180 200 220 240 260 280 300 Iflb w , ^ ' ° WS n ° rmal gr ° Wth during 284 days on a diet obtaining all of the ingredi- fclk Note the vigorous growth of this animal which was produced and suckled period ther whose food during the previous 178 days contained gliadin as its sole protein ' period 2 Milk powder. Starch Lard per cent. 60.0 16.0 24.0 100.0 508 Gliadin in Nutrition / / o /' / 1 — 1 o // -V A // // o // // / / / / \-- V 5 ■ / / / / j— 3 1 1 / ; / -rf ? — // //« 5 / / / 2/ / — V c »sein t Prote, i-free r -> i ■ , p. 61, Carnegie Institution of Washington. The animal's life was ter- PERIOD 6. per cent. per cent. ... 18.0 Edestin (hempseed) 18.0 . .. 28.2 Protein-free milk 28.0 Starch 26.0 28.0 23. 5.0 Lard. 25.0 100.0 100.0 250 Maintenance with Isolated Proteins 100 120 140 160 180 200 Chart 2, Rat 133 9 , shows maintenance during 465 days on a diel time the animal's life was terminated by a tumor of the spleen. No" r ganic salts and a part of the carbohydrate of the original food mixture, The diet during the different periods is shown below. PERIODS 1 AND 3. per cent. Edestin (hemp seed) 18.0 Starch 29.5 Sucrose 17.0 Agar «>.0 2.5 28.0 Salt mixture I. Lard Edestin (hempseed). Protein-free milk — Starch Agar Laid 100.0 T. B. Osborne and L. B. Mendel I \ od - in t prof Jin-free niilk-- | 2 O *■ D c v \ \ |€e- f* V N i 280 300 320 34-0 360 38o 400 420 44-0 4-60 4-80 n from hempseed formed the sole protein. At the end of that rery in periods 2 and 4 when protein-free milk replaced the inor- PERIOD 5. per cent. per cent. ... 18. Edestin (hempseed) 22.0 . .. 28.2 Artificial protein-free milk 29.5 Starch 28.5 20.0 20. 5.0 Sucrose. 28.0 100.0 100.0 Maintenance with Isolated Proteins Chart 3, Rat 147 9 , shows long continued maintenance on a die"] lungs after 445 days of experimental feeding. The diet during the different periods is shown below. During p on Chart 1. PERIODS 1, 2, 3 AND 6. per cent. Gliadin (wheat) 18.0 Gliadin (wheat)..' Starch '. . . v 29.5 Protein-free milk- Sucrose 15.0 Starch JL Agar 5.0 Agar .£ Salt mixture 1 2.5 Lard Lard 30.0 100.0 T. B. Osborne and L. B. Mendel 253 \ V V. 7 I- 00/ \ -4) \ i / c /Prot< osei n : 1 n-f ree + Milk GI * > 1 j is (j •/> U 280 300 320 340 360 380 400 420 n as the sole protein. The animal's life was terminated by diseased antity of feces from rats on a mixed diet was supplied. See legend PERIODS 5 AND 8. per cent. per cent. ... 18.0 Casein (cow's milk) 18. ... 28.2 Protein-free milk 28.2 ... 20.8 Starch 23.8 ... 6.0 Agar 5.0 ... 28.0 Lard 25.0 100.0 100.0 254 Maintenance with Isolated Proteins o o - ■ ■ ■ o — — o o— o o 1 la&'in + Prote. i-f ^ee milk- J G Onus 20 40 60 80 100 120 140 160 ,a ° 200 220 Chart 4, Rat 240 9 , shows failure to make more than slight growt normal rate after 276 days of stunting. At this time the rat was 314 d The diet during periods 1 and 2 was: PERIOD 1. ' Gliadin (wheat) { Protein-free milk Starch Agar Lard T. B. Osborne and L. B. Mendel 300 320 34-0 3fo0 380 420 -440 460 460 500 lining gliadin as the sole protein, and capacity to resume growth at a which rats normally grow very little more. PERIOD a. der. per cent. ... 60.0 ... 16.0 ... 24.0 100.0 256 Maintenance with Isolated Proteins u ■ — — , 2 — <; \ \ y > L. J 320 340 3fo0 380 400 420 440 4b0 480 500 520 54S ie sole protein. The animal's life was te minated after 511 days of experi- PERIOD 2. per cent. heat) 18.0 emilk 28.2 20.8 B 50 28.0 *100.0 Maintenance with Isolated Proteins « Cq sem t Gl utenin — G lutemn / 2 7 A- ^- — \ 4 — — — v' V 1 40 Go do ioo 140 ifeo 180 2oo 2Zi Chart 6, Rat 71 d% shows long-continued feeding of isolated foodstuffs mal's life was terminated after 531 days of experimental feeding by an abscess; The diet during the different periods was as follows : PERIOD 1. per cent. Glutenin (wheat) 6.0 Casein (cow's milk) 12.0 Starch , 24.5 Sucrose 15.0 Agar 5.0 Salt mixture 1 2.5 Lard •. 35.0 PERIODS 2 AND 5. Glutenin (wheat). Starch Sucrose Agar Salt mixture I Lard 100.0 T. B. Osborne and L. B. Mendel 300 320 34o 36o 38o 400 420 440 4£0 480 >00 S20 54o j^nued maintenance on glutenin from wheat as the only protein. The nade eating impossible. PERIOD 3. per cent. ain (wheat) 9 in (hemp seed) . ixture I. 9.0 33.5 18.5 5.0 2.5 22 5 100.0 PERIODS 4 AND Mixed per cent. Glutenin (wheat) 18.0 Protein-free miik 28.2 Starch 23 8 Agar Lard 5.0 25.0 100.0 260 Maintenance with Isolated Proteins 60 80 100 120 140 2C0 220 I Chart 7, Rat 150 9 , shows long-continued maintenance on a diet conta days of experimental feeding. m The diet during the different periods is shown below. During period 2 a PERIODS 1, 2 AND 3. Casein (cow's milk) S tarch Sucrose Salt mixture 1 2 6 Lard Lard per cent. .. 18.0 Casein (cow's mlllj' .. 32.5 Protein-free milk.' .. 21.9 Starch 25.0 100.0 T. B. Osborne and L. B. Mendel 300 22.0 340 360 380 400 420 440 460 480 500 520 540 5 sole protein. The animal's life was terminated by diseased lungs after 538 feces from rats on a mixed diet was supplied. See legend on Chart 1. PERIOD 5. Percent. percent. 18.0 Milk powder 60 28.0 Starch WW'.'. 12 27.0 Lard ...WW. 28 27.0 100.0 100.0 262 Maintenance with Isolated Proteins Chart 8, Rat 141 9 , shows long-continued maintenance on a diet containing diseased lungs. The diet during the different periods is shown below. PERIODS 1 AImD 4. per cent. Casein (cow's milk) 18-0 Casein (cow's milk) Starch 32 5 f arch Sucrose 219 ^crose Salt mixture I 2 6 Salt mixture I Lard 25 Lard PEBIOD 2. 100.0 T. B. Osborne and L. B. Mendel. 263 ein. After 587 days of experimental feeding the animal's life was terminated by AND 5, PERIOD 0. per cent - per cent pmllk) 18.0 Milk powder 60 ; Jmilk 28.0 Starch 120 .... 27.0 Lard \\" t \\\ 2 8 27.0 100.0 100.0 Maintenance with Isolated Proteins — _ — < Casern f pod Co setn i- p cotem-fr; A / \ / i Kr£ J 3 y V zo 40 60 SO 100 \Z0 140 160 180 200 Chart 9, Rat 139 d" , shows long-continued maintenance on a die mental feeding, but no cause for death was shown by the autopsy. The diet during the different periods is given below. PERIODS 1 AND 4. per cent. Casein (cow's milk) 18.0 Casein (cow's milk, Starch 32.5 Starch Sucrose 21.9 Sucrose Salt mixture 1 2.6 Salt mixture I . . Lard 25.0 Lard S 100.0 T. B. Osborne and L. B. Mendel 265 280 300 320 340 3fe0 3&0 400 420 44<5 460 480 gin as its so'e protein. The animal died after 468 days of experi- PERIODS 3 AND 5. per cent. ■ ■■ 36.0 Casein (cow's milk), ■ • 22 .5 Protein-free milk . . . ... 13.9 Starch .. 2.6 Lard .. 25 per cent. ... 18.0 ... 28.0 ... 27.0 . . 27.0 100.0 100.0 266 Maintenance with Isolated Proteins Chart in Rat 124 9 , shows long-continued maintenance on a diet containing edestj 609 days of' experimental feeding (which is, so far as we are aware the longest reco weight Dnring period 2 the rat received small quanttt.es of feces from nor, PERIODS 1, 2 AND 3. per cent. . 18 Edestin (hem] Edestin (hemp seed) lou , „ , ' , 29 5 Artificial proteii Starch , .... 17.0 Starch Sucrose , 5 . Lard A e ar 2 5 Salt mixture I Lard 100.0 T. B. Osborne and L. B. Mendel 267 340 360 38o 400 420 440 460 4&0 Soo 520 540 560 560 600 t oh^ J? f > t w f d u atUral P rotein - free milk for " days out of the entire W T rt ? ? *5£ at 6nd ° f tMs Peri0d the animal is ^mewhat above its e legend on Chart 1. This rat is still at its original weight after 637 days per cent. ■ ■■ 18.0 Edestin (hempseed) • ■ • 29. 5 Natural protein-free milk • •• 24.5 Starch • •■ 28.0 Lard PERIOD 5. per cent .. 18.0 .. 28.0 .. 26.0 .. 28.0 100.0 100.0 68 Maintenance with Isolated Proteins WW foO L 3 i- _ Glia dm foo jfiodm food -t- feces j^. _G l a d D<4ms 20 4-0 60 60 100 120 i40 'CO 1 80 200 ZZ0 Z40 Chart 11, Rat 130 9, shows long continued maintenance on a diet cont mental feeding by diseased lungs and a large parasite, over 40 cm. long, enc The diet during the different periods is shown below. During period 2 tl PERIODS 1, 2 AND 3. per cent. Gliadin (wheat) 18.0 Gliadin (wheat) . . . Starch 29.5 Protein-free milk..' Sucrose 17.0 Starch Agar 5.0 Agar > Salt mixture 1 2.5 Lard Lard 28.0 100.0 T. B. Osborne and L. B. Mendel 2 \5 '' \ ' \ St ^ \ \ Gl.odm ♦ Prote m-free trti I k Mi «* )0 32.0 340 3b0 380 400 420 440 460 480 500 SZO .540 I the sole protein. The animal's life was terminated after 546 days of experi- r. nail quantities of feces from normally fed rats. See legend for Chart 1. PERIOD 5. per cent. per cent. ... 18.0 Milk powder 60.0 ... 28.2 Starch 12.0 ... 20.8 Lard 28.0 ... 5.0 ... 28.0 100.0 100.0 270 Maintenance with Isolated Proteins 200 220 2.40 260 28< Chart 12 Rat 271 9, shows long-continued maintenance on a purely artificial diet containing casein as its sole protein. The animal died, after 277 days of experimental feeding, with diseased kidneys. The diet during the different periods is shown below. PERIOD 1. PERIOD 2. Casein (cow's milk) . Starch Lactose. .. i Agar Salt mixture I Lard per cent. 18.0 Casein (cow's milk) 24 . 5 " Artificial' ' protein-free milk . ... 24.0 Starch 5.0 Lard ... 2.5 per cent. ... 18.0 ... 29.6 ... 20.4 ... 26.0 26.0 100.0 100 Chart 13, Rat 588 9 , shows maintenance on a diet containing gliadin as the sole protein and an artificial imitation of the natural pro- tein-free milk. After 114 days the artificial protein-free milk was replaced by the natural, but the decline in weight which had begun was not stopped' by this change. The autopsy showed no adequate cause for death. The diet in periods 1 and 2 was : PERIOD 1. Gliadin Artificial protein-free milk Starch Lard per cent. .. 18.0 30.0 22.0 30.0 PERIOD 2. per cent. Gliadin 18.0 Protein-free milk 28.0 Starch 26.0 Lard 28.0 100.0 De»w|S 20 °rO 100.0 T. B. Osborne and L. B. Mendel 271 180 20 I \2 J— — u 1 - -Gli id in -foe d -- - ■§~ J M -Gliadi ifbod * n ormal f< -£ -a O v 20 40 60 60 100 IZ0 140 160 l8o 200 220 Chart 14, Rat 142 9 , shows maintenance on a diet containing gliadin as its sole protein. During periods 2 and 3 the rat received small quantities of feces from normally fed rats. For a discussion of the effect of the sterilized and normal feces see Publication 156, p. 62, Carnegie Institution of Washington. The animal died suddenly after 219 days of experimental feeding, but unfortunately no autopsy was made. The diet was as follows. PERIODS 1, 2 AND 3. per cent. Gliadin (wheat) 18.0 Starch 29.5 Sucrose 17.0 Agar 5.0 Salt mixture 1 2.5 Lard 28.0 100.0 272 Maintenance with Isolated Proteins y — - — — — - \ 1 f \ V c C £ Q_ V S? 4- * £ t 5- c c "5 }!" u c - s — -* U / \ _ / 60 Dai PEEIOD 2. per cent. Gelatin food (as in period 1) 50.0 Casein food (as in period 4) 50 .0 Chart 15, Rat 477 d" , shows rapid de- cline on a diet containing gelatin as its sole protein, and subsequent mainte- nance and repair when the gelatin was either partially or entirely replaced by casein. After 158 days of experimental feeding the animal's life was terminated by diseased lungs. The diet during the different periods is shown below. PERIOD 1. Gelatin food. per cent. Gelatin (horn).... 18.0 Protein-free milk . 28.0 Starch 27.0 Lard 27.0 100.0 PERIOD 3. per cent. Gelatin food (as in period 1) 25.0 Casein food (as in period 4) 75.0 100.0 100.00 PERIOD 4. Casein food. per cent. Casein (cow's milk) 18.0 Protein-free milk. 28.0 Starch 27.0 Lard 27.0 J. X 8$ i ' if- -St--*- Up 3 !■> 1 '/ 3 — *i c f~~ °~ / + -r C C % 8 i 2 J £ s I 7 , / 80 100.0 Chart 16, Rat 592 cf , shows rapid decline on a diet containing gelatin as its sole protein, followed by normal growth when the gelatin is replaced by casein. The animal's life was terminated after 119 days of experimental feeding by calculi in the bladder. The diet during the different periods is shown be- low. PERIOD 1. PERIOD 2 Gelatin food. Casein food. per cent. per cent. Gelatin (horn). ... 18.0 Casein (cow's Protein-free milk 28.0 milk) 18.0 Starch 27.0 Protein-free milk. 28.0 Lard 27.0 Starch 27.0 Lard 27.0 100.0 100.0 PERIOD 3. per cent. Gelatin food (as in period 1). . . . 50.0 Casein food (as in period 2 50.0 100.0 T. B. Osborne and L. B. Mendel 273 4 i 4 ■4 i V Ufa h ' 1 -4 k V _o ./ a 2 Chart 17, Rat 598 9, shows rapid decline on a diet containing gelatin as its sole protein, and re- covery when one-half of the gela- tin was replaced by gliadin, a pro- tein incapable of inducing more than very slight growth when it forms the sole protein constituent of the dietary. The animal's life was terminated by diseased kid- neys after 120 days of experimen- tal feeding. The diet during the different periods is shown below: PERIOD l. PERIOD 2. per cent. Gelatin (horn) 18.0 Protein-free milk 28.0 Starch 27.0 Lard 27.0 100.0 per cent. Gelatin food (as in period 1) 50.0 Gliadin food 50.0 100.0 Gliadin food. Gliadin (wheat) . . Protein-free milk. Starch Lard gram. . 18.0 28.0 26.0 28.0 100.0 E ' f £ r !■ "o ; 0--t- . Ze. *pro-tein-f liodm+prot :in-fr*emil V — NO-* Chart 18, Rat 659 9 , shows rapid de- cline on a diet containing zein as its sole protein, followed by recovery when the zein was entirely or partially replaced by gliadin or casein. The diet during the different periods is shown below. 60 PERIOD 1. grams. Zein (maize) 18.0 Protein-free milk. 28.0 Starch 24.0 Lard 30.0 100.0 Water 15 cc. PERIOD 2. per cent. Gliadin (wheat). 18.0 Protein-free milk 28.0 Starch 24.0 Lard 30.0 PERIOD 3. 100.0 per cent. Zein food (as in period 1) 50.0 Casein food 50.0 100.0 Casein food. grams. Casein 18.0 Protein-free milk 28.0 'Starch 29.0 Lard 25.0 PERIOD 4. per cent. Zein food (as in period 1) 25.0 Gliadin food (as in period 2).... 75.0 100.0 100.0 274 Maintenance with Isolated Proteins fv 3 iH- £ ' E V c 2? I «r \ \ £ / c 6 c o a. 6-Zemt v- : milk-. ZeirY> pi-oteiin.fr se milk ^ free milk E -S -- -X / -/- - -o - - / ^ K- £ h 2 1 \ \ 60 80 140 160 Day 200 Chart 19, Rat 146 a 71 , shows rapid decline on a diet containing zein as its sole protein, followed by speedy recovery when the zein was partially or entirely replaced by casein or edestin. The experiment was terminated after 266 days of experimental feeding. The diet during the different periods is shown below. PERIODS 1 AND 3. PERIOD 2. PERIOD 4. grams. per cent. per cent. Zein (maize) 18.0 Zein food (as in period 1) 50.0 Zein food (as in period 1) . 50. Protein-free milk 28.2 Casein food ({ is in period Edestin food (as in period 23.8 6) 50.0 5) 50.0 Agar 5.0 25.0 100.0 100.0 100.0 Water PERIOD 5. per cent. per cent. Edestin (hempseed) . . , 18.0 Casein (cow's milk) . . 18.0 28.2 Protein-free milk . 28.2 Starch 20.8 Starch 23.8 Agar 5.0 5.0 Lard 28.0 25.0 100.0 100.0 ♦ T. B. Osborne and L. B. Mendel 275 / 3 E u Vi- >i « iff «h\ E olin ■ £ i 1 E 1' JC n. 11 4- ■*- 03 3 3 03 a ? o 2 g ct % r3 m W o3 ~ ■A Si 03 03 c3 ^ 5 ft 02 02 3 3 o o 03 03 c c c3 c3 -4-3 o o -Q Si 03 3 ^ O O r y y & bfi 03 b£ 03 02 qj 02 ° Z ° b£ b£ 03 5s o o a 8 o 3 a S3 £ Q w o oil 'o o z o3 ~ 03 03 03 03 03 03 .a 3 P '« 'Si -4-3 -4-3 +3 02 02 02 02 02 02 02 bfi bD bD bC bC b£ b£) 3 3 # a 3 3 3 3 ^3 o *9 c 03 03 u 03 o c 03 03 03 03 03 03 03 Em -< « ^ 3 - .ft H H I— I u_i g |— | 33 c 3 rt 3 d c3 o3 c3 c» c3 "73 "d ""3 m t$ 3 3 1 3 3 ~ 3 CC 00 CO CO < CO 02 5 J c3 3 C2 O «3 .~ 3 IT O ,o 03 o 7 6 Behavior of Fat-Soluble Dyes stained. The blood of rabbits taken from two to six hours after intravenous injections of indophenol-blue dissolved in oil emulsion yielded pink residues on extraction. These results point to the reduction of the indophenol-blue to indophenol by the tissues. The presence of active reductases in the various tissues of the animal body has been observed by Ehr- lich, 7 Herter, 8 Harris 9 and others. Heffter 10 reports that the liver is particularly rich in this enzyme, a fact which was further demonstrated by us as follows: Ground liver tissue, to which oil stained with indophenol-blue had been added, was allowed to autolyze in the presence of toluene at a tem- perature of 30°C. After twenty-four hours, the mixture had lost its blue color and had become pink; the addition of hydrogen peroxide brought back the blue color. No change in color took place in a control experi- ment, carried out with boiled liver tissue under identical conditions. i The localization of fat-soluble dyes in the tissues. Analysis of the various tissues of the animal body shows that the largest quantity of fat (ether extract) is found in the subcuta- neous tissue, the fatty tissue of the abdominal cavity and the bone marrow; however, the muscular, glandular and nervous tissues contain estimable amounts. It is reasonable to suppose, therefore, that animals containing Sudan-stained adipose tissue would like- wise have stained fat in the other fat-bearing tissues, especially since this dye readily reveals the presence of fat in histological sections of these tissues. The only investigators who even suggest that the fat of other than the adipose tissues may not be colored are Mann 11 and S. P. and S. H. Gage. 12 The basis for Mann's statement that " animals fed on oil colored with Sudan III show only the adipose tissue stained" is not clear. S. P. and S. H. Gage failed to find the stain in the nerve fibres of the chicks developed from the Sudan-stained eggs, although the adipose tissues of these were distinctly colored. 7 Das Sauerstoffbediirfniss des Organismus, Berlin, 1885. 8 Amer. Journ. of Physiol., xii, pp. 207, 457, 1904-5. 9 Bio-chem. Journ., v, p. 143, 1911. 10 Medizinisch naturwissenschaftliches Archiv, i, p. 81, 1907-8. 11 Physiological Histology, 1902, pp. 36-7. 12 Science, xxviii, p. 494, 1908. Lafayette B. Mendel and Amy L. Daniels 77 Bondi and Neumann 18 found that the bone marrow and livers of rabbits were distinctly blue after the injection of an emulsion of fat, stained with indophenol, and that the Kupfer cells of the •livers of rabbits became distinctly pink after the injection into the circulation of an oil emulsion stained with Scharlach Rot. The animals were killed a few hours after the injection; the adipose tissue had not become stained in this short time, and the fact that the liver cells contained the color of the dye injected cannot be taken as proof that these cells store fat. The results of subsequent experiments in this investigation pertaining to the mode of elimi- nation of fat-soluble dyes, to which reference will be made later, have thrown some light upon this point, and make it evident that these observations of Bondi and . Neumann may be otherwise interpreted. Expekimental. In order to ascertain whether stained fat, other than that in the distinctly adipose tissue, is present in the bodies of animals into which fat-soluble dyes have been introduced, 2-gram portions of the tissues to be examined were freed, as far as possible, from extraneous fat and connective tissue, finely divided, dried and extracted with ether in accordance with the method already described. The dyes were administered dissolved in olive oil or in lecithin emulsion of peanut oil. The results are summarized in the table on pages 78 and 79. Discussion. Negative results were always obtained from nervous and renal tissues; from muscle when it was freed from con- nective tissue or extraneous fat as in starvation: and in general from liver tissue. Livers however from which blood had not been removed by perfusion or bleeding sometimes showed traces of the dye. In two cases the livers from rats which had been fed on a diet containing 75 per cent of deeply stained lard, yielded con- siderable quantities of the dye. These livers were distinctly pink, owing undoubtedly to the storage of the absorbed fat in the liver cells. Microscopic examinations of frozen sections, however, failed to disclose the dyes, even when chemical isolation demon- strated their presence. . The explanation of these results is not clear. It may be that the form of the fat in the nervous, muscular and glandular tissues 13 Zentralbl. f. Biochem. u. Biophysik, x, p. 1453, 1910. 78 Behavior of Fat-Soluble Dyes CO 5s> c ^ 3 f- O -3 m O « 3 M £ >■ r a; f-i S-i t-< iZ, +^ "t- 5 02 oQ t» j£ g a a ^ O o O o3 £ £ £ =3 QQflfi 2 3 + + + + 11+ + + + + + + I I I I I I + I I I I I I I I I I- I I ' I I I I + + + I + + + + + I I I I I I I I I I + o 5 w s « 9 o o 73 g CD 43 ® 3 3 c3 O 5 ° O CD bfi bfi bfi bfi b£) bfi c g g 3 g a bfi bfi bfi bfi G G (3 G bfi bfi g g £ +3 — XXX^'Z'Z o +J ^ 4J ^ X! -Q g c3 c3 c3 o3 c3 o3 ,2j p4 pL, Lafayette B. Mendel and Amy L. Daniels i 1 1 8 S § 9 1 1 e 1JT3 1 + 1 + + + + ■ + 1 1 1 + + + + 1 r- 1 1 + + + 1 1 11111 1 1 1 p=< f=< f=< p=, ^ Ill ' * 10 2 p3 P3 p3 p4 Ph p3 pd 8o Behavior of Fat-Soluble Dyes of the body is quite different from that in the adipose tissue — that it is held in some loose chemical combination which is no longer capable of taking up the stain. The present methods of fat extrac- tion and staining may result in a disintegration of this complex molecule. MacLean and Williams 14 have advanced the theory that the fat removed by extraction from animal tissues does not represent the form in which the fat exists in these tissues, and that the fat is made evident as the result of certain post-mortem changes by which the compound is broken up and the fat liberated. Leathes 15 and Abderhalden and Brahm 16 have suggested that the fat of the active tissues differs from that of the storage tissue. In the present investigation it was found that the isolated ether-soluble substances of the brain can take up the stain. This observation, together with the fact that the nervous tissue of Sudan-stained animals is always free from the dye, even when the embryonic fat contained an abundance, as was demonstrated by S. P. and S. H. Gage 17 in chicks developed from the stained eggs, adds weight to the theory outlined above. An explanation of the fact that in a large number of the experi- ments the liver tissue was found to be free from the dye, is afforded by the observation that the fat-soluble dyes are more soluble in bile than in fat; and when these dyes are introduced into the body in solu- tion in the fat they are eliminated in the bile. Added evidence in favor of this explanation is found in the fact that the fat complex in the liver is not incapable of holding the dye in combination. This is shown by the following experiment: A solution of Sudan III in bile was injected, under pressure, into the common bile duct of a rabbit. After 20 cc. had been forced in, the liver was removed, perfused with physiological saline solution, comminuted, washed in cold running water for twenty-four hours and filtered; the resi- due, which was distinctly pink, was washed until the filtrate gave no test for bile salts with Pettenkofer's reaction; ether extracts of the dried residue were distinctly pink. There could be no doubt that the fat had absorbed the stain. Conclusions. Stained fats, introduced into the animal body intraperitoneally, intravenously, subcutaneously or by absorption 14 Bio-chem. Journ., iv, p. 455, 1909. 16 Problems in Animal Metabolism, 1906, p. 72. 16 Zeitschr. f. physiol. chem., lxv, p. 330, 1910. 17 Science, xxviii, p. 494, 1908. Lafayette B. Mendel and Amy L. Daniels 81 from the alimentary tract, are laid down in the adipose tissue and marrow. The renal and nervous tissues are free from the stain, even when the fatty tissue is deeply colored; muscle tissue, when freed from fat, as in extreme starvation, contains no stained fat; the dye is found in the liver only when the blood contains an abundance, as in starvation, or when the animal has been fed food containing a large amount of stained fat a few hours before the examination. Liver fat, in situ, is capable of taking up the stain. Indophenol-blue is reduced in the body; this reduction takes place, in part, in the liver; hence adipose tissue is not stained with this dye. AVAILABILITY OF STAINED FAT IN METABOLISM. Riddle 18 has suggested that adipose tissue stained with Sudan III is less available to the organism than unstained adipose tissue. Inasmuch as the dye enters into no chemical union with the fat, but is merely dissolved therein, 19 it does not seem probable that the Sudan III can so change the nature of the fat that it cannot be used as effectively by the organism as unstained fat. An indif- ferent material, like Sudan III, might be toxic, or might form toxic combinations in the body, and thus affect organs dealing with fat combustion; but that the fat itself is rendered unavailable scarcely seems tenable. The non-toxicity of Sudan III has been shown by feeding animals over long periods of time without apparent deleterious results. Experimental. In order to determine if Sudan-stained fat is less available to the organism, starvation experiments were carried out with Sudan-stained rats and pigeons; comparable experi- ments were conducted with normal animals. 1. Pigeon B. Fed with pulverized dog biscuit, lard deeply stained with Sudan III, and cracked corn for three weeks before the beginning of the fasting period. Subcutaneous tissue became pink. Weight of pigeon at beginning of fast was 297 grams. Death in ten days. It had lost 116.5 grams, 39 per cent of its initial weight; all visible fat had disappeared. No pink color was to be seen. A slight trace of Sudan III was found in 18 Journ. of Exp. Zobl., viii, p. 163, 1910. 19 Michaelis: Virchoiv's Archiv., clx, p. 263, 1901. THE JOURNAL OF BIOLOGICAL CHEMISTRY, VOL. XIII, NO. 1. 82 Behavior of Fat-Soluble Dyes ether extract of the tail gland, bone marrow and liver; the muscle, kidney and brain contained no trace of the dye. 2. Pigeon C. Preliminary feeding same as B. Subcutaneous tissue became noticeably pink. Weight at beginning of fast 294 grams. Death in eleven days. Loss of weight 165 grams, 56 per cent. No visible fat remained; tissues showed no pink color; ether extract of liver and bone marrow slightly pink; of muscle, kidney and brain, colorless. 3. Pigeon D. Fed Sudan-stained food as in B and C. After sixteen days of fasting this animal died. It had lost 41 per cent of its initial weight. All visible fat and stain had disappeared from the body. The ether extract of the bone marrow was slightly pink; that from the liver and muscle showed no pink color. 4. Pigeon E. A normal well-fed bird, was starved for sixteen days, dur- ing which it lost 229 grams or 54 per cent of its initial weight. All visible fat had disappeared from the body. 5. Pigeon F. A well-fed normal bird which died fifteen days after the fasting period began. The loss in weight was 144 grams or 45 per cent of its initial weight. All visible fat had disappeared. It should be noted that Pigeons B and C were kept during November in an unheated room with the windows open. This doubtless explains their earlier death as compared with pigeons D, E and F which were kept at about 20°C. In every case, however, the fatty tissue had entirely disap- peared from the body. Experiments with rats gave similar results. 6. Rat A. Fed with ground dog biscuit mixed with lard deeply stained with Sudan III for seven days. Fasting period, three days. Loss in weight, 42 grams, 42 per cent of initial weight. The body was free from all traces of visible fat and stain. 7. Rat C. Preliminary feeding period, same as A. Fasting period ap- proximately sixty hours. All visible fat disappeared from the body. The ether extract of the brain, liver, muscle, kidney and subcutaneous tissue left no pink residue. 8. Rat B. A normal well-fed rat, which died after a 60 hours' fast. The loss in weight was 40 grams or 22 per cent of its initial weight. The body was free from all visible fat. 9. Control experiment. Rat D. Was fed on Sudan-stained food as in the previous experiments, for seven days. The subcutaneous tissue, omen- tum and fatty tissue about the kidneys were deeply pink. The result of the control experiment affords evidence tljat the adipose tissue of the experimental animals was similarly stained at the beginning of the fasting periods. The further observation was made that rats, and in some cases rabbits, stained as the result of feeding with deeply stained, fat-rich food, excreted urines which Lafayette B. Mendel and Amy L. Daniels 83 were distinctly pink; such urines were found to contain both fat and Sudan III. In two experiments Sudan-stained pigeons were fed with un- stained foods after long fasting periods — other pigeons, fasting the same length of time and under similar conditions, had died. At autopsy, the fatty tissue of these was found to be unstained. 10. Pigeon G. Fed with Sudan-stained fat, described in protocols 1-3, starved thirteen days; loss of weight, 104 grams or 29 per cent. It was re-fed and examined some months later. All trace of Sudan had disap- peared; the ether extract of the tissues left no pink residue. 11. Pigeon A. Previously fed with Sudan-stained food; starved eleven days; loss of weight, 104 grams or 32 per cent. It was re-fed until it had gained 16 grams. Upon examination, no stained tissue was found. Ether extract of subcutaneous fat, tail gland and omentum showed no pink color. Discussion. The results of these trials are not in agreement with those reported by Riddle. Sudan-stained pigeons and rats died in no less time than the unstained control animals. In both cases the visible fat had entirely disappeared, and, in the stained animals, the dye as well. Those animals which were fed after long fasting periods until there was a marked increase in body weight, contained no trace of the former Sudan-stained fatty tissue. One must conclude from these results that stained adipose tissue is no less available to the organism than the non-Sudan-stained fat and that it is used quite as readily and completely. The disparitybetween our results and those of Riddle is difficult to explain. His observations that chicks fed on stained food developed more slowly than normal chicks and that hens ceased to lay after considerable quantities of the dye had been ingested may have resulted from other causes than the ingestion of the dye. It is conceivable that the apparent failure of starving stained animals in his experiments to utilize their fatty tissues as do normal animals was the result of impurities in the dye fed. Mann 20 has observed that Scharlach Rot given to half grown kittens in large doses causes vomiting. We gave large doses of Sudan III, put up by an American manufacturer, to two cats. These died within a comparatively short time apparently from the effect of some im- purity in the dye. Other cats, given equally large doses of the Kahlbaum dye, experienced no ill effects. Riddle's deductions from 20 Personal communication. 8 4 Behavior of Fat-Soluble Dyes his second series of fasting experiments that stained animals under- went a greater percentage loss of weight during starvation than do unstained are unconvincing by reason of the fact that an important part of his weighing records was lost. THE FATE OF FAT-SOLUBLE DYES IN THE ORGANISM. The observations cited above have shown that Sudan III, depos- ited in the tissues as the result of adding the dye to the food, dis- appears completely during starvation. Experiments upon cats and rats gave no reasons for thinking that this disappearance is due to elimination of the dye by the kidneys. The fact that the excreta of starving Sudan-stained pigeons contained the dye and the observation that the dye was present in the gall bladders of Sudan-stained animals subjected to starvation or poisoning with phosphorus or phlorhizin turned our attention first to the elimina- tion of fat-soluble dyes by way of the bile. It is well-known that the bile is the normal path of elimination of many substances. From the work of Abel and Rountree 21 on phenoltetrachlor- phthalein the assumption seems justifiable that substances which leave the body exclusively by way of the bile must be insoluble in water and soluble in bile or substances contained therein. Two preliminary experiments were made upon cats, previously fed with Sudan III and starved for four days preceding the experi- ment. The bile, collected as it was secreted by the liver, and the blood yielded pink ether extracts, while those obtained from the liver tissue, washed free of blood and bile, were colorless. These results pointed to a transport of Sudan-stained fat to the liver with subsequent storage of the fat in the liver and elimination of Sudan III in the bile. The elimination of fat-soluble dyes under normal conditions. The dyes, dissolved in lecithin-oil-emulsion, were introduced into the circulations of cats, dogs and rabbits by injections into the femoral veins. In each case the urine contained in the bladders, as well as the liver tissue and bile, was examined for the injected stain. The approximate time of the appearance of the dye in the 21 Journ. of Pharmacol, and Exp. Therapeutics, i, p. 231, 1909. Lafayette B. Mendel and Amy L. Daniels 85 bile after its introduction into the blood stream was incidentally noted. The results of the experiments are summarized in the following table : The excretion of fat-soluble dyes introduced dissolved in fat. ANIMAL DYE COLOR OP DYE RESIDUE FROM ETHER EXTRACT OF BILE TIME OF AP- PEARANCE OF DYE IN BILE DYE IN URINE DYE IN LIVER TTtitllliCS Cat II, 8 Indophenol Blue Blue 30f None Dog II, 14. . . . Sudan Red Pink 30 None Present Dog II, 16. . . . Sudan III Red Pink 20 None None Cat II, 21. . . . Indophenol Blue Til Blue 60t None Present* Dog 11,21.... Sudan III Red Pink Cat II, 22. . . . Oil Green Green Green 30 None Nonef Cat II, 23.... Oil Green Green Green 90 None None Rabbit II, 28. Biebrich Scar- let Red Pink 60 None Present Dog III, 21 . . Sudan III Red Pink 60 None None Cat III, 24. . . Sudan III Red Pink 75 None None Cat IV, 21.... Butter Color Yellow Pink 55 None None Cat V, 2 Oil Orange Orange Yellow- red 90 None None Cat V, 6 Blue Base Blue Pink 50 None Present^ Cat V, 15 Butter Color Yellow Pink None * The animal died fifteen minutes after the second injection of dye. t The animal died one-half hour after the injection. t The addition of dilute hydrochloric acid to the liver tissue resulted in a blue color. The animal died four hours after the injection. In a number of the experiments the residues from the ether extracts of the excreted bile were examined for fat. The ethereal filtrates were allowed to evaporate from watch glasses; the residues were heated gently; no melting of the material took place and no grease spot was formed on soft tissue paper by this residue. The dyes were excreted dissolved in bile and not in combination with fat. In some cases, the color of the residues from the ether extracts of the bile was not precisely like that of the dyes injected. This change in color is the result of the passage of the dyes through the body where they are brought in contact with hydroxylions. The action of dilute alkalies on the dyes outside the body causes a sim- ilar change. 86 Behavior of Fat-Soluble Dyes The residues from the ether extracts of the liver tissue were not always colored; when the animals were killed some time after the injection of the stain, or when only a small amount had been intro- duced, the liver was found to be free from the dye. The urines were consistently free from stain. It is obvious from these results that fat-soluble dyes, when intro- duced into the circulation in solution in fat, become separated from the fat and are eliminated in the bile. Absorption of fat-soluble dyes into the portal circulation. The next experiments were designed to determine the roles played by the bile and by the fat in the absorption of fat-soluble dyes from the intestine. 22. Dog: 25 kgm. Fed at 7.20 a.m. Two hours later the animal was anaesthetized, and a cannula was inserted in the thoracic duct. Twenty cubic centimeters of Sudan-stained oil were injected into the duodenum at 11.15 a.m., followed by 1 gram of desiccated ox bile in solution. At 12.15 the lymph was intensely pink. At 2.00 p.m., a cannula was inserted in the bile duct. The animal was killed by bleeding at 5.30 p.m. Two-gram por- tions of the liver tissue, 10 cc. samples of the blood, 50 cc. of the lymph and from 2 to 4 cc. of the bile were examined. The ether residues from the dried lymph and bile were distinctly pink, while those from the blood and liver showed no trace of the dye. 23. Cat, full-grown. Fed at 8.15 a.m., was anaesthetized at 9.30 a.m. A cannula was inserted in the thoracic duct at 10.45 a.m.; after 10 cc. of Sudan-stained emulsified oil had been injected into the duodenum, a cannula was placed in the common bile duct and the bile collected therefrom. The lymph flowed freely, and at 2.30 p.m. it was distinctly pink in color. The ether residues from the lymph and bile were distinctly pink. The blood (10 cc.) taken at 5.00 p.m. yielded no pink residue. The liver was also free from the dye. These experiments show clearly that although Sudan III, intro- duced with fat into the intestine, is absorbed by the lacteals and appears in the thoracic lymph, it is still absorbed and eliminated in the bile, under conditions which preclude the entrance of the lymph into the blood. In the latter case, neither the blood of the general circulation nor the liver tissue is stained. This behavior is explained in part by the observation that the dyes studied are more soluble in bile than in fat and by the results of the following experiments which show that the dyes may be absorbed from the intestine into the portal circulation in solution in bile. Lafayette B. Mendel and Amy L. Daniels 87 Pasting animals were anaesthetized, a cannula inserted into the bile duct and bile solutions of the various dyes used in the earlier experiments were injected into the small intestine. The results are summarized below. The excretion of fat-soluble dyes absorbed from alimentary tract, dissolved in bile. ANIMAL DYE COLOR OF DYE RESIDUE FROM ETHER EXTRACT OF BILE DYE IN LIVER DYE IN BLOOD DYE IN URINE Cat II, 8 Sudan III Red Pink None None None Cat II, 8 Blue Base Blue Blue None None Cat III, 10 Indophenol Blue Blue None None None Cat III, 10 Oil Green Green Brown pink None None None Rabbit III, 20. . . Biebrich-Scar- let Red Pink None None Cat V, 29 Oil Yellow Orange Yellow None pink The presence of the dye in the bile in these experiments and its absence from the blood of the general circulation show clearly that it is absorbed with the bile by the portal circulation and eliminated with the bile by the liver. That none of the dye entered into the general circulation is evidenced by the fact that the blood of the animals examined — five out of six— gave no indication of even traces of the dye when tested by a method capable of detecting 0.00001 gram of Sudan III in 10 cc. The two following experiments show that when bile is not present with the dye in the intestine, no absorption of the dye in the portal circulation occurs. Stained fat was introduced into a loop of the upper intestine after this had been washed out with physiological saline solution to remove all traces of the adherent bile. The bile excreted under these conditions was free from the stain, although in one case (cf. protocol 24) the thoracic lymph showed that a slight amount of fat absorption had taken place; in the other experiment (cf. proto- col 25) both bile and lymph contained no dye until after the intro- duction of a bile solution into the intestinal loop, when the excreted bile was found to contain Sudan III, although the lymph was still colorless. 88 Behavior of Fat-Soluble Dyes 24. Dog: 20 kgm. Narcotized with morphine and ether. A temporary lymph cannula was inserted at 10.00 a.m., and a bile cannula at 10.45 a.m. A 12-inch loop of the intestine was tied off just below the pylorus and washed out with physiological saline solution, at body temperature, until the washings were clear. Sudan-stained oil, together with a solution of 0.1 per cent HC1, introduced to increase the pancreatic secretion, were injected into the intestinal loop at 11.00 a.m. Bile, collected at 12.00 m., 2.00 p.m. and 3.30 p.m., when dried and extracted, left no pink residues. Seventy cc. of lymph, collected between 2.00 and 3.30 p.m., contained a small amount of Sudan III; the ether extract of dried blood was unstained. 25. Dog: 8 kgm. Anaesthetized with morphine and ether at 9.45 a.m. The insertion of the temporary lymph cannula immediately preceded that of the bile cannula. The intestine was ligated just below the pylorus and 14 inches below it. This loop was washed out with physiological saline solution until the washings were clear. Approximately 10 cc. of Sudan-stained emulsified oil, together with 10 cc. of 0.1 per cent HC1 were introduced into this loop. The bile collected at 3.30 p.m. left no pink resi- due; the lymph also was free from dye. At 3.30 p.m., 10 cc. of a solution of desiccated ox bile were injected into the intestinal loop. The bile col- lected at 7.30 p.m., 3.5 cc, showed the presence of the dye, while the lymph taken at this time, 25 cc, left no pink color when extracted. The elimination of the dye in the bile during fat absorption, under conditions where the stained fat was prevented from entering the general circulation, was undoubtedly due to the migration of the dye from the fat to the bile in the intestine and its subsequent absorption. There is no reason to believe that the dye in the excreted bile was the result of absorption of stained fat into the portal circulation. Had such been the case, the dye would have been present in the excreted bile in experiment 24, as well as in the lymph. The time required for the absorption and deposition of fat, studied by means of fat-soluble dyes. The fact that fat-soluble dyes are eliminated in the bile explains some hitherto inexplicable phenomena observed in the work with Sudan III. Earlier in this investigation an attempt was made to determine the length of time required to lay down the fat absorbed from the alimentary tract. Stained fat was fed to rabbits and cats; and samples of blood, taken from the ear veins of the rabbits and the jugular veins of the cats, were examined for the circulating dye. The stain was still found to be present in the blood of rabbits Lafayette B. Mendel and Amy L. Daniels 89 one week after the last stained feeding; and the blood of the cats; tested from four to five weeks after the last Sudan-feeding, left distinctly pink residues. Oil emulsions stained with Sudan III and Biebrich Scarlet gave similar results when injected into the cir- culation of rabbits. The blood of these was found to contain the dye three weeks after the last injection. These observations find their explanation in the fact that dye, absorbed from the intestine into the lymph with the fat and into . the portal blood with the bile, again enters into the intestine with the bile. Thus a closed circulation of the dye is established and it is possible that the blood of a once Sudan-stained animal may become quite free from the stained fat only after long periods under normal conditions of feeding. Animals examined months after the Sudan-stained feeding had ceased contained deeply stained fatty tissue. The time required for the elimination of circulating fat-soluble dyes. An attempt was made to ascertain (in cats and dogs) the length of time necessary for the separation of the dye from the circulating fat and its elimination through the bile. Emulsions of stained fat, in amounts varying from 1 to 10 cc, wei*3 injected directly into the circulation; cannulae were placed in the common bile ducts and samples of blood and bile were taken every two or three hours. In order to facilitate the flow of bile, solutions of desiccated ox bile were injected into the upper intestine. The bile, blood, and liver tissue after it had been washed free from blood, so far as possible, were examined for the stain. Nine and one-half hours was the longest period during which observations were made in any experiment; and although in that instance only 1 cc. of the stained emulsion was injected, both blood and bile, collected at the end of this time, showed that a consider- able quantity of stained fat was still in circulation. In those cases in which the experiments continued over a comparatively long time, or when a small amount of the stained fat had been intro- duced, the liver was free from the stain. The liver evidently does not store up stained fat; the dye becomes separated from the fat as the stained fat comes in contact with the bile in the liver cells. Discussion. Fat-soluble dyes introduced into the body in solution in fat are secreted in the bile. These dyes may enter the 90 Behavior of Fat-Soluble Dyes body from the alimentary tract in two ways: (1) in the lymph, in solution in fat; (2) through the portal circulation, dissolved in reabsorbed bile. When the dyes are absorbed dissolved in bile, they apparently do not pass beyond the liver, but are speedily reexcreted into the gut, and do not enter the general circulation unless fat is present in the intestine. The blood of Sudan-stained animals, under normal conditions of feeding, is never free from the fat stain. The dye put out in the biliary secretion is reabsorbed in the digesting fat, and a continuous circulation from gut to blood, and return is established. The elimination of the stain from the circulation, when all possibility of reabsorption is removed, takes place slowly. The stained fat was found in the blood of a cat nine and one-half hours after it had been injected into the femoral vein. FAT TRANSPORT IN STARVATION AND PATHOLOGICAL CONDITIONS: PHOSPHORUS AND PHLORHIZIN POISONING. We have attempted to follow the migrations of Sudan-stained fats under conditions in which a transport of fat is well-known to occur, namely, in starvation and after poisoning with phosphorus or phlorhizin. The experimental animals were fed in advance for a period of three to five weeks on Sudan-stained food. Phos- phorus was administered subcutaneously, dissolved in oil; phlor- hizin similarly in solution in sodium carbonate. Other details of selected protocols are summarized in tabular form: Sudan III in pathological fat transport and starvation. ANIMAL DURATION OF EXPERI- MENT FAT CONTENT OF LIVER STAIN IN Bile Blood days per cent Cat I, 13 5 56.0 Present Present Starvation j Cat I, 18 5 33.5 Present Present Guinea pig 1 7.9 Present Phosphorus J Cat XI, 23... . 9 64.3 Present Present Hen X, 20 4 59.0 Present poisoning 1 Hen XI, 29 . . . 19 40.5 Present Phlorhizin j Cat XII, 6. . . . 12 15.8 Present Present poisoning \ Cat XI, 19. . . . 7 . 11.1 Present Present Lafayette B. Mendel and Amy L. Daniels 91 Neither in the foregoing nor in numerous other comparable experiments in which a transport of fat (fatty infiltration) was induced, was any evidence obtainable of dye in the extracts of the liver tissue or in frozen sections thereof. The constant finding of the Sudan III in both the blood and bile makes it evident that the dye migrates from the stained adipose tissue and is brought to the liver where it is eliminated in the bile. The observations give an additional indication that the fatty livers in these patho- logical conditions are produced by infiltration of fat; for it is diffi- cult to believe that, if the high content of liver fat had been ob- tained by a degeneration process in the hepatic tissue, such an accumulation of dye in the bile would have taken place. FAT TRANSPORT TO THE EMBRYO. The question of the origin of foetal fat has been much debated; 22 It involves the broader problem of the passage of substances through the placental barrier. S. H. and S. P. Gage ('08) failed to find the adipose tissues of the young stained, when stained fats were fed to pregnant mothers. Hofbauer ('05) believed that he found particles of dye in the foetal blood and assumed that they had become separated from fat metabolized by the embryo. His method — microscopic examination — is scarcely adapted to deter- mine this point, however. Numerous experiments in which we have fed rats and cats with Sudan-stained food or Biebrich Scarlet throughout the period of gestation have uniformly shown an absence of the dye in the foetus or the newly-born young. Two illustrative protocols, selected from many similar ones, will suffice to show our method of inves- tigation. Rat C. Sudan-feeding was begun sixteen days before the young were born. The alimentary tract was removed from one of them soon after birth. Its contents were distinctly pink (from mothers' milk). The ether extract of the entire residual body was uncolored. Subsequent examina- tion of the mother showed deeply stained adipose tissue. 22 Cf. Ahlfeld: Centralbl. f. Gynaekol, i, p. 265, 1877; Thiemich: Cen- tralbl. f. Physiol, xii, p. 850, 1898; Jahrb. f. Kinderheilk., lxi, p. 174, 1905; Hofbauer, J.: Arch. f. Gynaekol., lxxvii, p. 139, 1906; Oshima: Zentralbl. f. Physiol., xxi, p. 297, 1907; Bondi: Arch. f. Gynaekol.,' xciii, p. 189, 1911. 92 Behavior of Fat-Soluble Dyes Cat B. Was fed 80 ragms. of Sudan III every second day for eighteen days prior to birth of kittens. Aside from the stomach contents there was no pink in the ether extract of tissues of the young examined soon after birth. The adipose tissue of the mother was deeply stained. Although it is unlikely from such findings that stained fat can pass through the placenta, this is not necessarily conclusive evidence that the foetal fat has its origin in substances other than fat. The findings in the case of the alimentary epithelial tissues and glandu- lar structures however add little likelihood to the transport or deposition of the fat in a non-stainable combination. FAT TRANSPORT INTO MILK. The precise relation of milk fat to food fat and the extent to which the latter can pass directly into the mammary secretion without first becoming a part of the body stores is not easily deter- mined. S. H. and S. P. Gage ('09) found Sudan III in the milk of rats after prolonged feeding with the dye; this, however, is no proof of the immediate origin of the milk fat from the food, since the fat depots of the rats were also stained. In explanation of the observations that foreign food fats have more frequently been found secreted in the milk of smaller animals (goats, sheep, dogs, rats) than of cows, it has been suggested that the milk secretion is more directly dependent upon the food supply in the smaller species. 2 " However, the marked differences in the time required to stain the adipose tissue of guinea pigs and rabbits with Sudan III in comparison with cats and rats, suggests that the discrep- ancies noted above may bear some relation to the readiness with which the different animals absorb and store fat. Experimental. We have investigated the appearance of Sudan III in the milk after feeding the dye both before and during the period of lactation. When animals, notably cats, have refused to eat stained fat, the dye has been administered in capsules either directly before or after a meal rich in fat. This fact is important for successful results. In the case of cats and rats the character of the milk was determined by examining the stomach contents of suckling young. Needless to say great care must be taken to 23 Cf. Lusk: Science of Nutrition, 1909, p. 237. Lafayette B. Mendel and Amy L. Daniels 93 have the cages scrupulously free from stained food which might lead to erroneous conclusions. It is scarcely necessary to repeat here the details of the many trials, since the methods are fairly obvious. Both Sudan III and Biebrich Scarlet were found to be secreted into the milk by rats; Sudan excretion was likewise observed in cats, guinea pigs and a goat. In the case of the goat, one gram of Sudan III dissolved in oil was added to the feed twice daily during six successive days. 24 The milk drawn nine hours after the first dose showed the presence of the dye, the tint increasing with the subsequent milkings.' The guinea pigs received 2 cc. of stained olive oil every other day. An important fact in this connection is the observation that the color disappears from the milk when the Sudan-feeding is discontinued, despite the persistence of the stain in the adipose tissues of the secreting animals. This was likewise true in exper- iments with Biebrich Scarlet. The following protocol illustrates the transport of storage stained fat during starvation and the passage of the dye into the milk: Rat 11. Was fed Sudan-stained food during the period of gestation. Soon after the birth of the young, April 30, the cage was cleaned and un- stained food thenceforth employed. On May 14 the ether extract of milk found in the stomach of a suckling rat was uncolored. The mother was now starved two days. At the end of this time the milk in the stomach of another one of the young gave a faintly pink ether extract. The adipose tissue of the adult was found to be stained still. This experiment was duplicated with another mother. Like S. H. and S. P. Gage, we have failed to induce the secretion of Sudan III in the milk of cows. A Holstein cow was given 7.5 grams, twice daily, dissolved in olive oil and added to the mash feed on three successive days, without positive results. In con- sidering this we recall that the milk of the goat and guinea pig — animals in whose diet fat likewise plays a comparatively small role — was decidedly faint in color in comparison with the milk of cats and rats. Bearing in mind the necessity of fat for the transport of the dye an explanation at once suggests itself for the inequalities here observed. 24 This experiment was conducted at the New York Agricultural Ex- periment Station in Geneva, through the kindness of Director W. H. Jor- dan. 94 Behavior of Fat-Soluble Dyes SUMMARY. Some of the fat-soluble dyes, introduced into the organism by various paths, are deposited in the adipose tissues and bone marrow. The renal and nervous tissues are free from the stain, even when the fatty tissues are deeply colored. Muscle probably does not take up the dye. It is seldom found in the liver, because the fat- soluble dyes, which are insoluble in water, dissolve readily in the bile and are excreted thereby into the intestine from which they can be reabsorbed. The fat-soluble dyes may enter the organism from the alimentary tract through the lymphatics, in solution in fat; or by the portal circulation, dissolved in reabsorbed bile. They do not pass beyond the liver unless fat is present to transport them. Then they may be found in the blood, which is rarely free from the dye in a nor- mally fed animal that has once been stained. A cycle between intestine, bile and blood becomes established. No elimination of the dyes occurs through the kidneys, except when an alimentary lipuria arises (in rabbits and rats). Contrary to the assertion of others, the stained fat is no less available to the organism than the unstained. In cases conducive to fat transport — in starvation, phosphorus - and phlorhizin-poisoning — stained fat migrates from the stained depots to the blood and the liver cells. Here the dye is separated and secreted into the bile; so that the liver, though having a high content of fat, may be free from the dye. Stained fat does not traverse the placenta. The blood of the foetus and the fat of young born of Sudan-stained mothers is free from dye. The excretion of Sudan III and Biebrich Scarlet in milk, when they are given with food fat, suggests that the latter may pass directly into the mammary secretion. With cats and rats the results are striking, but the dye excretion in milk ceases when the stained food is no longer fed. In guinea pigs and goats the secre- tion of dye in the milk is positive; in the cow it has not yet been demonstrated. The variation in the outcome in the different species may be due to variations in the relative abundance In the dietaries of fat necessary for the absorption and transport of the dye. This explanation is emphasized by the observation that those Lafayette B. Mendel and Amy L. Daniels 95 animals (cats, rats, hens, pigeons) for which fat enters more largely into the diet, become stained more easily or speedily than animals which are accustomed to ingest relatively smaller amounts of fat. BIBLIOGRAPHY OF EXPERIMENTS WITH SUDAN III AND OTHER FAT-SOLUBLE DYES. Biedermann: Arch. f. d. ges. Physiol., lxxii, p. 105, 1898. Bondi and Neumann: Zentralbl. f. Biochem. u. Biophysik, x, 1453, 1910. Daddi: Arch. ital. de biol., xxvi, p. 142, 1896. Fischer: Centralbl. f. allgemeine Pathol, u. pathol. Anat., xiii, p. 943, 1902. Franz and Von Stejskal: Zeitschr. f. Heilkunde, xxiii, p. 441, 1902. Gage, S. H. and S. P.: Science, xxviii, p. 494, 1908. Gage, S. H. and S. P.: Anat. Rec, iii, 1909. Hofbauer L. : Arch. f. d. ges. Physiol., lxxxi, p. 263, 1900. Hofbauer I.: Grundziige einer Biologic der menschlichen Placenta mitbe- sonderer Beriiksichtigung der Fragen der fdtalen Erndhrung, Wien und Leip- zig, 1905. Jacobsthal: Verhandl. d. deutsch. pathol. Gesellsch, xiii, p. 380, 1909. Mann: Physiological Histology, p. 306-07, 1902. Mendel: Amer. Journ. of Physiol., xxiv, p. 493, 1909. Michaelis: Deutsch. med., Wochenschr., xxvii, p. 183, 1901. Neisser and Braeuning: Zeitschr. f. exper. Pathol, u. Therap., iv, p. 747, 1907. Pfluger: Arch. f. d. ges. Physiol., lxxxi, p. 375, 1900. Riddle: Science, xxvii, p. 945, 1908. Riddle: Journ. of Exper. Zobl., viii, p. 163, 1910. Sitowski: Anz. d. Akad. d. Wissensch. in Krakau, p. 542, 1905. Staniewicz: Zentralbl. f. Biochem. u. Biophysik., x, 1435, 1910. Whitehead: Arrier. Journ. of Physiol., xxiv, p. 294, 1909; xxv, p. xxviii, 1909-10. HOPPE-SEYLEK'S ZFJTSCHKIFT fiir PHYSIOLOGISCHE CHEMIE unter Mitwirkung von E. ABDERHALDEN-Halle, SVANTE ARRHEN I US-Stockholm, G. v. BUNGE-Basel, 0. COHNHEIM-Heidelberg, P. EHRLICH-Frank- furt a. M., A. ELLINGER-Konigsberg, H. EULER-Stockholm, EMIL FISCHER- Berlin, W. v. GULEWITSCH-Moskau, 0. HAMMARSTEN- Upsala, S. G.HEDIN-Upsala, V. HENRIQUES-Kopenhagen, G.HOPPE- SEYLER-Kiel, Wm. KUSTER- Stuttgart, FR. KUTSCHER-Marburg, E. LUDWIG-Wien, CARL TH. MORNER- Upsala, K. A. H. MORNER- Stockholm, W. OST WALD-GroBbothen, I P. PAWLOW-St. Petersburg, C. A. PEKELHARING-Utrecht, E. SAI.KOWSKI-Berlin, M. SIEG- FRIED-Leipzig, S. P.L. SORENSEN-Kopenhagen, H. STEUDEL-Berlin, H. THIERFELDER-Tubingen, R. WILLSTATTER-Zurich, R. v. ZEYNEK-Prag herausgegeben von A. KOSSEL, Professor der Physiologie in Heidelberg. Beobachtungen iiber Wachstum bei Futterungsversuchen mit isolierten Nahrungssubstanzen. Von Thomas B. Osborne und Lafayette B. Mendel, unter Mitwirkung von Edna L. Ferry. Mit 65 Kurvenzeichnungen im Text. Separat-Abdruck aus Band 80, Heft 5, STRASSBURG VERLAG von KARL J. TRUBNER. 1912. ACHTZIGSTER BAND, FUNFTES HEFT. In halt. Seite Osborne, Thomas B., Lafayette B. Mendel und Edna L. Ferry. Beobachtungen uber Wachstum bei Futterungsversuchen mit isolierten Nahrungssubstanzen. Mit 65 Kurvenzeich- nungen im Text . . 307 Rogozinski, F. Zur Methylierung des Glupeins 371 Pringsheim, Hans. Uber den fermentativen Abbau der Hemi- cellulosen. I. Mitteilung. Ein Trisaccharid als Zwischen- produkt der Hydrolyse eines Mannans 376 Fiir die nachsten Hefte sind Arbeiten eingegangen von: L. Wacker, G. Trier, E. Abderhalden und A. Eodor, e. Letsche, G. Th. Morner, A. DorneiyP. Rohland. Hoppe-Seyler's Zeitschrift fur phy siologische Ghemie erscheint in Banden von 6 oder mehr Heften, im Gesamtumfang von 30 bis Si Bogen. Preis des Bandes 12 Mark. Die in dieser Zeitschrift zu publizierenden Arbeiten werden, wenn es nicht aus technischen Griinden unmoglich ist, in der Reihenfolge, in welcher sie der Redaktion zugehen, aufgenommen. — Kurze Notizen oder Bemerkungen zu anderen Arbeiten werden in der Regel am Schlufi des Heftes und aufterhalb der Reihenfolge des Eingangsdatums mitgeteilt. — Bereits in anderen Zeitschriften veroffentlichte Arbeiten, sowie Referate uber bereits publizierte Arbeiten werden nicht aufgenommen. Das Honorar betragt fiir den Druckbogen 25 Mark. Von jeder Arbeit werden dem Verfasser 75 Separat-Abdriicke gratis geliefert. In bezug auf die Rechtschreibung der Fachausdriicke sind bis auf weiteres die Publikationen der Deutschen chemischen Gesellschaft mafi- gebend. In zweifelhaften Fallen wird der etymologische und internationale Standpunkt vor dem phonetischen bevorzugt. Beobachtungen uber Wachstum bei Futterungsversuchen mit isolierten Nahrungssubstanzen. 1 ) Von Thomas B. Osborne und Lafayette B. Mendel, unter Mitwirkung von Edna L. Ferry. Mit 65 Kurvenzeichnungen im Text. (Aus dem Laboratorium der Connecticut Agricultural Experiment Station und dem Sheffield-Laboratorium fur physiologische Chemie der Yale Universitat, New Haven, Connecticut, Vereinigte Staaten von Amerika.) (Der Redaktion zugegangen am 29. Mai 1912.) Inhalt. Einleitung. Veranderungen des Wachstums, die durch aufterhalb der Ernahrung liegende Faktoren oder durch ungeniigende Nahrungszufuhr verursacht werden. Anderungen des Wachstums durch qualitativ (che- misch) ungeeignete Nahrungszufuhr. Eiweifi und Wachstum. Warum wachsen die Tiere bei gewissen Ernahrungsformen nicht? Eiweifrkorper der Leguminosen und Wachstum. Quantitative Gesichtspunkte uber Wachstums- hemmung. Kunstliche Salzmischungen und Wachstum. Wachstum bei einer von atherloslichen Substanzen freien Diat. Die Unterdriickung des Wachstums und die Fahigkeit, das Wachstum wieder aufzunehmen. Einige Bemerkungen und SchluMolgerungen. Einleitung. Die vorliegenden Beobachtungen setzen voraus, daB die Wachstumszunahme der jungen weiBen Ratte durch eine charakteristische Kurve ausgedriickt werden kann. Die Auf- merksamkeit war deshalb besonders darauf gerichtet, die Fak- toren, welche das normale Wachstum hemmen oder vollstandig behindern, zu bestimmen. Welche Ernahrungskomponenten sind fur eine ange- messene Entwicklung unentbehrlich ? In welchen Quantitaten mussen sie an die Tiere verfuttert werden? Kann nach ge- hemmtem oder unterdriicktem Wachstum eine Wiederherstel- l ) Die Ausgaben fur diese Untersuchung trug die Connecticut Agricultural Experiment Station und die Carnegie Institution of Wash- ington, D. C. Hoppe-Seyler's Zeitschrift f. physiol. Chemie. LXXX. 21 308 Thomas B. Osborne und Lafayette B. Mendel, lung erwartet werden und wenn so, wie? Gibt es feststehende qualitative Unterschiede zwischen der zur Erhaltung und der zum Wachstum notigen Ernahrung? Dies sind einige Probleme, die der Losung harren. Und es wird sich zeigen, daB das Studium der feststellbaren Wachstumshemmung, einige wenige Falle ausgenommen, die Formulierung positiver Ergebnisse moglich gemacht hat. Die vorliegende Arbeit beriehtet in ausgedehnter Weise iiber schon friiher an weiBen Ratten vorgenommene Fiitterungs- versuche, in welchen die Feststellung einer geeigneten Er- nahrung mit Mischungen einzelner Nahrungsstoffe angestrebt wurde. Unsere friiheren Experimente haben wir gleichzeitig mit der angegebenen Literatur sowie den Methoden der Ge- fangenhaltung, Futterung und Pflege der Tiere anderweitig bereits veroffentlicht. 1 ) Aus den bereits veroffentlichten Fest- stellungen wollen wir nur wiederholen, daB, verschiedentlich debattierte Schwierigkeiten des Versuchsplans anlangend, die Einformigkeit der Ernahrung als kein ernstliches Hindernis sich erwiesen hat. Aufzeichnungen iiber in Perioden vonl— 2Jahren mit unveranderter Kost ernahrten Ratten lassen iiber diese Tatsache keinen Zweifel aufkommen. Als Bestatigung dienen die Ver such e von Hart, Mc Gollum, Steenbock und Hum- phrey, welche durch 3 Jahre eine im wesentlichen unveran- derte Kost an ihre Tiere verfiittert haben und zu dem Schlusse kamen, daB die Einformigkeit der Ernahrung kein storender Faktor und bei den Futterungsversuchen keineswegs von solcher Tragweite ist, wie gewohnlich behauptet wird. 2 ) Ferner er- reichen weiBe Ratten trotz unserer Gefangenhaltung und der begrenzten Freiheit in Bewegung und Leben ein verhaltnis- *) T. B. Osborne und L. B. Mendel, Futterungsexperimente mit isolierten Nahrungssubstanzen, Carnegie Institution of Washington, Publi- cation 156, Parts I and II, 1911; Science N. S., 1911, Bd. 34, S. 722—732, und Zeitschrift fur biologische Technik und Methodik, 1912, Bd. 2, S. 313—318. 8 ) E.B.Hart, E. V. Mc. Collum, H. Steenbock u. G. C.Humphrey, Physiological Effect on Growth and Reproduction of Rations Balanced from Restricted Sources. Univ. of Wisconsin Agri. Expt. Station, Research Bulletin Nr. 17, June 1911, p. 131—205; cf. also Journal of Biological Chemistry, 1912, Bd. 11, S. XII. Uber FiUterungsversuchen mit isolicrten Nahrungssubstanzen. 309 maBig hohes Alter im Vergleich zu Tieren, die in dieser Be- ziehung weniger Einschriinkung erleiden mussen, eine Beobach- tung, in der wir uns mit der jiingst veroffentlichten Ansicht von Slonaker 1 ) in Einklang befinden. Die GroBe des Wachs- tums, gemessen durch den Wechsel des Korpergewichts und graphisch aufgezeichnet, ist noch immer ein durchaus ge- niigender Index fur die Schwankungen, mit welchen wir uns beschaftigen. Die gut bekannten Kurven von Donaldson 2 ) uber das wechselnde Korpergewicht der Albinoratte sind durch Slonakers Messungen bestatigt worden; 3 ) diesen konnen wir nunmehr eine betrachtlich groBere Anzahl von eigenen hinzu- fugen. Die Resultate werden in Kurve 1 gezeigt. Kurve 1 zeigt die durchschnittlichen Schwankungen im Korpergewicht der mannlichen und weiblichen Albinoratte, wie sie durch Bestimmungen von Donaldson, Slonaker und Osborne und Mendel festgestellt worden sind. Es wird sich zeigen, daB unsere Ratten aus Griinden, die wir noch nicht genugend haben bestimmen konnen, die Neigung zeigen, etwas kleiner als die von Donaldson zu bleiben. Dies muB beim Studium unserer Wachstumsdaten im Auge be- halten werden. Veranderungen des Wachstums, die durch aufierhalb der Ernahrung liegende Faktoren oder durch ungemigende Nahrungszufuhr verursacht werden. Gewisse Faktoren bedingen so ersichtlich eine Hemmung des Wachstums, daB sie keiner ausfuhrlichen Besprechung be- diirfen. Bekannt ist dies von MiBhandlungen, angeborenen Defekten und Krankheiten, mogen sie nun familienweise z. B. *) J. R. Slonaker, Die normale Lebensfahigkeit der Albinoratte von ihrer Geburt bis zu ihrem normalen Tod, ihre Wachstumszunahme und Lebensdauer. Journal of Animal Behavior, 1912, Bd. 2, S. 20—42. 2 ) H. H. Donaldson, Ein Vergleich der weifien Ratte mit dem Menschen in bezug auf Wachstum des ganzen Korpers. Boas Memorial Volume, New York, 1906. Cf. also Osborne and Mendel, Carnegie Institution of Washington, Publication 156, Part I, S. 14, Part II, S. 87. s ) J. R. Slonaker, Journal of Animal Behavior, 1912, Bd.2,S.20-42. 21* 310 Thomas B. Osborne und Lafayette B. Mendel, Kurve 1. Uber Fiitterungsversuche mit isolierten Nahrungssubstanzcn. 311 als Infektion usw. oder auch in mehr kryptogener Form auftreten. *) Eine andere wichtige Hemmung des normalen Wachstums bildet die Unzulanglichkeit im Energieersatz, 2 ) obwohl die Wachstumshemmung keineswegs immer mit Gewichtsstillstand zusammenfallt. 3 ) Dies sind Erscheinungen, welche in den vor- liegenden Untersuchungen nicht Gegenstand spezieller Studien sein konnen. Die Moglichkeiten, welche sie darstellen, miissen, so besonders der Eintritt von Krankheiten, als Gelegenheiten zur Gewichtsabnahme betrachtet werden, die keineswegs der Diat allein zuzuschreiben sind. So muB also ein Unterschied gemacht werden zwischen aktueller Herabsetzung des Er- nahrungszustandes (Sinken des Korpergewiehts) und Unter- druckung des Wachstums bei gleichbleibendem Korpergewicht in Zeiten, in welchen ein normales Wachstum erwartet werden konnte. Anderungen des Wachstums durch qualitativ (chemisch) ungeeignete Nahrungszufuhr. In unseren friiheren Studien haben wir bereits beobachtet, daB Tiere in ersichtlich geniigendem Ernahrungszustande und gleichem Korpergewicht und -maB verbleiben konnen, wenn sie sich in einem Alter befinden, in dem ein kraftiges Wachstum ohnehin zu erwarten ist. Obgleich bei diesen Tieren wahrend langer Perioden keine sichtbare Abnahme oder sonstige Anzeichen von Gesundheitsstorungen bestanden, wuchsen sie aber nicht in einem MaBe, das dem normalen entsprochen hatte. Solche Falle zeigen, was wir Gleichgewicht ohne Wachstum nennen. *) Manche Debatten uber diese Fragen finden sich in der Literatur uber Kinderheilkunde. Gf. E. Schloss, Die Pathologie des Wachstums im Sauglingsalter, Berlin 1911. 8 ) J. Rosens tern, Uber Inanition im Sauglingsalter. Ergebnisse der inneren Medizin und Kinderheilkunde, 1911, Bd. 7, S. 332. 3 ) H. Aron, Biochemische Zeitschrift, 1910, Bd. 30, S. 207; Phi- lippine Journal of Science (B), 1911, Bd. 6, Nr. 1, S. 1—50; H. J. Waters, Das Bestreben der Tiere, unter verschiedenen Bedingungen zu wachsen. Proceedings Society for the Promotion of Agricultural Science, 1908, Bd. 29, S. 3. 312 Thomas B. Osborne und Lafayette B. Mendel, Entweder die Nahrung ermangelte einiger spezifischer che- mischer Substanzen, die zur Bildung neuer Gewebe oder zum Wachstum notig waren, oder das beziigliche Verhaltnis der einzelnen Bestandteile der Nahrung zueinander war nicht das richtige. Unsere Nahrungsgemische bestanden zu dieser Zeit aus einzelnen EiweiBkorpern, x ) Fett, Starke und Zucker, und einer Salzmischung nach Angaben von Rohmann 2 ) welch letztere folgende Zusammensetzung hatte: Salzmischung I. Ca 3 (P0 4 ) 2 10,0 g K 2 HP0 4 37,0 » NaCl 20,0 » Na-Gitrat 15,0 » Mg-Citrat 8,0 » Ca-Lactat 8,0 » Fe-Citrat 2,0 » 100,0 g. Seit der spater folgende Versuch gezeigt hat, daB ein normales Wachstum erfolgt, wenn der Zucker durch Lactose, die hier angewandten Salze durch eine veranderte Mischung ersetzt werden, sofern die iibrigen Nahrungsbestandteile in bezug auf Quantitat und Qualitat unverandert bleiben (S. 49), miissen wir die folgende erste Versuchsreihe als ein Beispiel von Wachstumsunterdruckung ohne merklichen Verfall betrachten *) Es mufi bemerkt werden, daft auf die Darstellung der bei diesen Untersuchungen angewandten Eiweiftkorper die groftte Sorgfalt angewandt wurde. Die Produkte waren so rein, als man sie fur den Zweck einer genauen Eiweifianalyse nur erwarten konnte. Die Notwendigkeit einer Genauigkeit in dieser Richtung kann nicht streng genug betont werden, seit, wie wir gezeigt haben, kleine Beimischungen entschieden die Re- sultate eines Fiitterungsversuchs andern. Cf. Carnegie Institution of Wash- ington, Publication 156, Part II, 1911, S. 84. 2 ) Rohmann, Allgemeine med. Zentralzeitung, 1903, Nr. 1; 1908, Nr. 9. Cf. Malys Jahresbericht, 1903, Bd. 33, S. 823; 1908, Bd. 38, S. 659. Die Griinde fur den Gebrauch dieser Mischung sind in unseren ersten Berichten angegeben, Publication 156, Carnegie Institution of Washington, Part I, 1911, S. 32. Uber Ftttterungsversuche mil isolierten Nahrungssubstanzen. 313 und sie den Nicht-EiweiB- und Nicht-Fettbestandteilen der Nahrung zuschreiben. Die folgenden Kurven 2 und 3 iilu- strieren dies. 1 ) 7 1 / / s 1 | i f / / / / / -> 4^ ^ /<5tf s / s / / / / — /« — / / / / / — ^ ?.5 ~ (r ¥0 Kurve 9. 60 80 Kurve 9 (Ratte 59) und Kurve 10 (Ratte 193 ?) zeigen eine Un- terdriickung des Wachstums in verschiedenen Altern bei einer in der beigegebenen Tabelle ge- zeigten Ernahrung; Kurve 11 (Ratte 380?) zeigt das Wachstum, wenn die Nahrung «eiweififreie Milch* enthalt. 160 1*0 Edetfw ohn E/wvss-free Mt/ch ^^7 Edestin (Hanfsamen) «Eiweififreie Milch* Starke Zucker Agar Salzmischung I Fett 7 tOO f20 fVO Tape Kurve 10. Nahrung : Ratte 59 und 193 «) °/o 18,0 0,0 29,5 15,0 5,0 2,5 30,0 220 2^0 Ratte 380, Periode •/. 18,0 28,2—28,0 20,8—26,0 0,0 5,0—0,0 0,0 28,0 *) In Periode 2 wurden kleine Retrage von «normalen» Faeces gelegentlich verfuttert, siehe Publication 156, Part II, Carnegie Institution of Washington, p. 61. 320 Thomas B. Osborne und Lafayette B. Mendel, Kurve 12. Kurve 13. Kurve 16. 322 Thomas B. Osborne und Lafayette B. Mendel, Kurve 12 (Ratte 56) zeigt ungeniigendes Wachstum; Kurve 13 (Ratte 565 ?) und Kurve 14 (Ratte 531 zeigt geniigendes Wachstum, wenn die Nahrung « eiweiBfreie Milch » enthalt. Nahrung : Ratte 56 Ratte 565, Periode 2 Ratte 531, Periode 2 o/o o/o °/o Casein (Kuh) 12,0 18,0 0,0 Excelsin (Brasilnufi) 6,0 0,0 18,0 «Eiweififreie Milch» 0,0 28,0 28,0 Starke 29,5 28,0 28,0 Zucker 15,0 0,0 0,0 Agar 5,0 0,0 0,0 Salzmischung I 2,5 0,0 0,0 Fett 30,0 26,0 26,0 Kurve 15 (Ratte 101 d") zeigt vollige Unterdruckung des Wachstums, und Kurve 16 (Ratte 284 ✓ / / _ t / / / / y / / / / f / / 1 4. 1 1 1 F -> I 80 60 20 W 60 SO fOO 120 «H) \ 20 W 60 s / / / / / / / t 1 1 1 /— / -> 7 100 120 f+O 160 180 200 220 2V0 Tape Kurve 26. m 120 100 60 20 20 44 60 €0 Kurve 27. / / / / / / / / L. Ze/n. -Hr I" 2/ 20 W 60 Kurve 28. 400 120 Kurve 27 (Ratte 549 #). Nahrung : Roggen-Gliadin «Eiweififreie Milch » Starke Fett > 18,0 28,0 28,0 26,0 330 Thomas B. Osborne und Lafayette B. Mendel, Periode 2. Zein (Mais) «Eiweiftfreie Miich» Starke Agar Fett Wasser Kurve 28 (Ratte 503 Nahrung: g 18,0 28,2—28,0 23,8—24,0 5,0— 0,0 25,0—30,0 Periode 3. > Zein Futter (wie in Periode 2) 50,0 Edestin Futter 50,0 Edestin (Hanfsamen) 18,0 «Eiweiftfreie Milch » 28,0 Starke 26,0 Fett 28,0 In Periode 3 veranlaftte der Zusatz von Edestin zur Nahrung auf einmal eine Wiederaufnahme des Wachstums. Das zeigt an, daft Zein an sich nicht giftig ist. 1/77 _^ — + 20 Tage Kurve 29. Kurve 29 (Ratte 554 so l ) L. B. Mendel and M. S. Fine, Journal of Biological Chemistry, 1911, Bd. 10, S. 303, 339, 345, 433; 1912, Bd. 11, S. 1 und 5. 334 Thomas B. Osborne und Lafayette B. Mendel, Ratten iiber Perioden von 500 Tagen bei einer Nahrung, in der als alleiniges Protein das Gliadin gereicht wurde, erfolg- reich futtern konnten, ist ein guter Beweis fur die glanzende Ver- wertbarkeit des Gliadins. Beschrankte Quantitaten von anderen entsprechenden EiweiBkorpern wie Casein oder Edestin fiihren bald zu einer Herabsetzung des Korpergewichts. Wir haben also keinen Grund zu der Annahme, daB Gliadin nicht ebenso ausnutzbar wie andere EiweiBkorper ist. EiweiBkorper der Leguminosen und Wachstum. Wir wollen nun nach vorhergegangenen Gesichtspunkten die EiweiBkorper der Leguminosen betrachten. Trotz der Tat- sache, daB die modernen Methoden der Proteinhydrolyse und die Bestimmung der Aminosaure diese EiweiBkorper als vollstandig bezeichnen, gelang es uns doch nicht, mit einer Ernahrung, in welcher die Leguminosenproteine die einzige N-Quelle waren, Wachstum zu erzielen, ausgenommen mit den EiweiBkorpern der Sojabohne. Dies erhellt deutlich aus den beigegebenen Kurven (32 und 33), in welchen die Verwendung des Phaseolins *) der Schminkbohne, Phaseolus vulgaris, als einzig verfutterter EiweiB- korper sofort von Wachstumstillstand und Gewichtsabfall be- gleitet ist, wahrend schnell wieder ein Ansatz und eine Gewichts- zunahme erfolgt, wenn dieses Phaseolin durch Milchcasein oder Edestin des Hanfsamens ersetzt wird. Diese Wachstumshemmung ist nicht nur ein Gharakteristikum der ersten Entwicklungs- periode, sie erfolgt vielmehr in verschiedenen Jugendstadien. Und sie wird in ahnlicher Weise illustriert in den Versuchs- tabellen mit Konglutin aus den gelben Lupinen, Lupinus luteus, Kurve 34; Erbsenlegumin aus der Gartenerbse, Pisum sativum, Kurve 35; Vignin aus der Kuherbse, Vigna catjang, Kurve 36; Legumelin aus der Sojabohne, Soja hispida, Kurve 37. Die nachfolgenden Kurven zeigen den EinfluB der Legu- minoseneiweiBkorper auf das Wachstum. ') In bezug auf Herstellung dieses Praparats vgl. T. B. Osborne, Abderhaldens Handbuch der biochemischen Arbeitsmethoden, 1909, Bd. 2, S. 311. Uber Futterungsversuche mit isolierlen Nahrungssubgtanzen. 335 Kurve 33. Kurve 34. Thomas B. Osborne und Lafayette B. Mendel, 120 y / / ✓ / A- / / / / / / / f <— - ^ «tf 9,0 28,0 22,0 9,0 5,0 27,0 / / A — r / / / / / t 1 1 i J Casern -> 20 40 60 60 Tape Kurve 41. f20 60 rape Kurve 42. Kurve 41 (Ratte 323 Edestin (Hanfsamen) 9,0 «Eiweiftfreie Milch » 28,0 Starke 20,0 Lactose 9,0 Agar 5,0 Fett 29,0 160 1¥0 120 100 80 60 s * s C- s — y y / / / / <• / _/_ / y 12% ? _ 3* /\ f V. — 20 ¥0 60 fOO 120 1*0 160 *80 Tape Kurve 47. 346 Thomas B. Osborne und Lafayette B. Mendel, Kurve 47 (Ratte 369 ?) zeigt Wachstum bei einer Diat, welche 12°/o Edestin enthalt. Nahrung: Edestin (Hanfsamen) 12,0 «EiweiMreie Milch * 28,0 Starke 20,0-25,0 Lactose 6,0 Agar 5,0-0,0 Fett 29,0 Kurve 48 (Ratte 372 'ss // fe/f £c/es> y/7 20 W 60 80 Kurve 55. Kurve 54 (Ratte 373 / -It / s AS > i/ ij — h i // 2/ 1 / CO o ts O O iO vo o .2 o" of of go" o~ i* CM CM CM > 03 1 § _, 03 03 t> ^ is ■-3 ^ is W 03 ?-» ' 1 hfl c © Z; Qj 03 g ® I *0 <5tf 80 & -B « 2 o 03 CO C J* cs t ^ 2 s -23 03 S3 03 ]S ^ •s .£? § £ 5s Q 03 Q . r 2 20 uo 60 Tage Kurve 59. 80 too *) Rubner, Archiv fiir Hygiene, 1908, Bd. 66, S. 82. 360 Thomas B. Osborne und Lafayette B. Mendel, Nahrung: Periode 1 Periode o/o O/o Edestin (Hanfsamen) 18,0 0,0 Milchpulver 0,0 60,0 Starke 29,5 15,7 Zucker 15,0 0,0 Salzmischung I 2,5 1,0 Agar 5,0 0,0 Fett 30,0 23,3 120 60 4 f / / / t // / y / / Weizen- Viadin - // _ _ a - -> 7 IS 20 to 60 120 160 180 Kurve 60. Kurve 60 (Ratte 381 ?) zeigt die Wiederaufnahme des Wachstums nach einer lange fortgesetzten, durch nicht ent- sprechendes EiweiB verursachten Unterdriickung desselben. Beweis, daB das wieder aufgenommene Wachstum durch Ver- futterung einer Mischung von isolierten Nahrungssubstanzen verursaeht wurde. Nahrung: Periode 2 Periode 3 °/o 7° Gliadin (Weizen) 18,0 0,0 Casein (Kuh) 0,0 18,0 «EiweiMreie Milch* 28,2 28,2—28,0 Starke 20,8 23,8—27,0 Agar 5,0 5,0—0,0 Fett 28,0 25,0-27,0 Uber FiUterungsversuche mit isolierten Nahrungssubstanzen. 301 Die Wiederaufnahme des unterdruckten Wachstums infolge quantitativer Unzulanglichkeit der Ernahrung wird in ahnlicher Weise ira folgenden gezeigt. f s y *ase/n f8% / / / £ Casein / / / /"" / / 1 J / 2 20 W 60 80 iOO 120 I'M 160 180 Kurve 61. Kurve 61 (Ratte 340 ?) zeigt die Wiederaufnahme des Wachstums nach einer Unterdnickung desselben infolge quan- titativer Unzulanglichkeit der EiweiBkorper in der Diat. Der wesentliche Unterschied in den Ernahrungsformen der zwei Perioden besteht im Gehalt an Casein. Ahnliche Resultate zeigt die Kurve 45, in welcher das Wachstum nach einer uber 100 Tage dauernden wachstumslosen Periode wieder aufge- nommen wurde. Nahrung: Periode 1 Periode 2 °/o °/o Casein (Kuh) 12,0 18,0 «EiweiMreie Milch» 28,0 28,0 Starke 22,0 22,0—27,0 Lactose 6,0 0,0 Agar 5,0 5,0-0,0 Felt 27,0 27,0 362 Thomas B. Osborne und Lafayette B. Mendel, Die Perioden, nach welchen eine Wiederaufnahme des unter- brochenen Wachstums erfolgt, sind hier verhaltnismaBig kurz. GroBeres Interesse verdient es, wenn der Wachstumsstillstand bis zu einem Alter ausgedehnt wird, in dem gewohnlich kurze Zeit spater das Wachstum der Tiere vollendet ist. DaB der Wachstumsimpuls in diesem spateren Alter nicht erloschen isl, zeigen die Kurven 62 und 63, in welchen in einem Alter von 237 und 304 Tagen ein erneutes normales Wachstum erfolgte. Kurve 62 (Ratte 196 ?) zeigt die Wachstumskapazitat nach einer 177 Tage dauernden Wachstumsunterbrechung. Die einzigen Unterschiede in der Diat bestanden in dem Ersatz der anorganischen Salzmischung und des Zuckers der Periode 1 bis 4 durch die eiweiBfreie Milch der Periode 4. Nahrung: Periode 1,2,3 Periode 4 °/o •jo Edestin (Hanfsamen) 18,0 18,0 «Eiweiftfreie Milch* 0,0 28,2 Starke 29,5 25,8—20,8 Zucker 15,0 0,0 Agar 5,0 0,0—5,0 Salzmischung I 2,5 0,0 Fett 30,0 28,0 Uber andere Versuche, welche die Wachstumskapazitat nach bedeutend kurzeren Perioden von Wachstumsunterbrechung demonstrieren, siehe «Futterungsexperimente mit isolierten Nah- rungssubstanzen» Carnegie Institution of Washington, Publi- cation 156, Part II, p. 92, 93, 98, 103, 109, 112, 113, 122, 123, 124, 133, 134; Charts XXVIII, XXIX, XXXVII, XL VI, XLVII, LXV, LXXI, LXXII, LXXVIII, XCVI, XCVII, XCIX, C, CXX, CXXI, CXXII, CXXIII. Kurve 63 (Ratte 240 ? ) zeigt die Wachstumskapazitat nach einem fur 265 Tage durch die Verfiitterung von Gliadin, als alleinigem EiweiBkorper der Nahrung, unterbrochenen Wachs- tum. tiber Futterungsversuche mil isolierten Nahrungssubslanzen. 363 Thomas B. Osborne und Lafayette B. Mendel. Nahrung: Periode 1 Periode 2 Gliadin (Weizen) °/0 18.0 % 0,0 Milchpulver 0,0 60,0 «EiweiGfreie Milch* 28,2 0,0 Starke 20,8 16,0 Agar 5,0 0,0 Fett 28,0 24,0 Solche Erfolge miis- sen scharf von derWie- derernahrung solcher Tiere unterschieden werden, die zunachst Gewichtsverluste erlit- ten haben. Die Wieder- herstellung desGewichts i. e. Wiederernahrung. mag unter Bedingungen erfolgen, unter welchen ein Wachstum unmog- lich ist. Kurve 64 (Ratte 147 ?)zeigtdie Wieder- herstellung desGewichts in Periode 2 nach einem Gewichtsabfall in Perio- de 1. Dieser Typus der Wiederherstellung muB unterschieden werden von der Gewichts- zunahme, die fur ein wirkliches Wachstum charakteristisch ist und durch Gliadin nicht veranlaBt werden kann. Nahrung: Li «J .UJ ■ i _ We/ze 7 -G/iad s-fre/e n ' s \ -\ Z -v \ \ V. V > 20 60 rage Kurve 64. too J2o Periode 1 Periode 2 0/0 7° Gliadin (Weizen) 18,0 18,0 «EiweiBfreie Milch > 0,0 28,2 Starke 29,5 20,8—25,8 Zucker 17,0 0,0 Agar 5,0 5,0-0.0 Salzmischung I 2.5 0,0 Fett 28.0 28.0 Uber Futterungsversuche mit isolierten Nahrungssubstanzen. 365 Einige Bemerkungen und SchluBfolgerungen. Der Erfolg, welcher die im Vorhergehenden wiederge- gebenen Versuche begleitet hat und der darin besteht, daB es gelang, bei Tieren ein charakteristisches Wachstum durch iso- lierte Nahrungssubstanzen zu erzielen, ofTnet den Weg fur wert- vollere Forschungen liber die zahlreichen individuellen Faktoren im Wechsel der Entwicklung. Es hat sich gezeigt, daB es mog- lich ist, ein junges Tier durch den groBten Teil seiner selbst- tatigen Wachstumsperiode zu Ziehen, wahrend welcher Zeit sein Korpergewicht sich unter Darreichung einer Mischung von sorg- faltig gereinigten EiweiBkorpern, Starke. Zucker, Fett und an- organischen Salzen mehrfach vervielfacht. Wenn, wie es mog- lich erscheint, diese Mischung insofern weiter vereinfacht werden kann, daB die Fette und alle anderen atherloslichen Substanzen (in bezug auf Reinheit die unsichersten Komponenten) aus- geschaltet werden. sind die chemischen Ernahrungsprobleme einer erfolgreichen experimentellen Losung um so naher geriickt. Rosenstern hat kurzlich geschrieben: «Wenn man bis- lang noch nicht imstande ist, einen Organismus mit einem kiinst- lichen Nahrgemisch am Leben zu erhalten, so spielt dabei u. a. wohl ein Mangel an Reizstoffen eine Rolle, auf deren Bedeutung die Pawlowschen Untersuchungen ein Licht geworfen haben.* x ) Abgesehen von irgend einer Wirkung, w r elche die Nahrstoffe an sich und die anorganischen Salze haben, kann ein beson- derer «Reiz»-Faktor jedoch kaum als wesentlich fiir den nutri- tiven Erfolg in Betracht kommen. Vage Ansichten iiber solche hypothetische Komponenten sollten uns aber nicht langer ver- wirren. Die experimentellen Daten dieser Arbeit haben gezeigt, was zu erwarten war, daB namlich ein gewdsses Minimum an EiweiBzufuhr fur das Wachstum notig ist. Betrage von EiweiBzu- fuhr, die unterhalb des Wachstumsbedarfs liegen, sind — gleichen Energieersatz vorausgesetzt — natiirlich keineswegs mit einer Erhaltung des Lebens, sogar ohne Gewichtsverlust, unverein- *) J. Rosenstern, Ergebnisse der inneren Medizin und Kinder- heilkunde, 1911, Bd. 7, S. 393. 366 Thomas B. Osborne und Lafayette B. Mendel. bar. Es besteht Erhaltung statt Wachstum. Diese Unterscheidung wurde von anderer Seite als «Betriebsstoffwechsel» und «Baustoff- wechsel» ausgedriickt. Es ist wichtig, daB ein verhaltnismaBig kleiner Betrag an EiweiB geniigt, um ein ausreichendes Wachs- tum zu unterhalten ; ein starkeres Wachstum kann durch uber- maBige EiweiBzufuhr nicht hervorgerufen werden. Ahnliche Resultate ergaben sich in unseren Versuchen auch im Hinblick auf das Optimum und Minimum der anorganischen Bestandteile der Nahrung. Weiterhin interessierte der EinfluB, welchen die qualita- tiven Eigenschaften der EiweiBkorper und Salze auf das Wachs- tum ausiiben. Die Zahl der EiweiBkorper, welche sich als von giinstigem EinfluB auf das Wachstum erwiesen haben, ist nicht gering. Sie umschlieBt EiweiBkorper verschiedenster Herkunft und zweifelsohne verschiedenster Zusammensetzung. 1 ) Tafel I zeigt die Gewichtszunahme (oberhalb des Null- punkts) und die Gewichtsabnahme (unterhalb des Nullpunkts) wahrend der ersten 30 Tage, in welcher Zeit mit verschiedenen EiweiBkorpern sowohl tierischen wie pflanzlichen Ursprungs gefuttert wurde. Die unter Phaseolin- und Leimernahrung verzeichneten Gewichtsverluste sind nur geschatzt, da die Versuche mit diesen Proteinen nicht durch 30 Tage hin- durch gefuhrt werden konnten wegen der groBen Gewichts- verluste der jungen Tiere. Die groBten Gewichtsverluste, welche unter Phaseolinfutterung gezeigt werden, erfolgten bei zwei Ratten von ca. 50 g Anfangsgewicht, die kleineren bei Ratten von ca. 35 g Anfangsgewicht. Bei Betrachtung der Unterschiede zwischen diesen ein- zelnen Versuchsreihen muB in Betracht gezogen werden, daB diese Versuche in ihrer Anordnung neu sind und an groBeren Zahlen von Tieren wiederholt werden miissen. Worin die Unzulanglichkeit mancher dieser EiweiBkorper liegt, sind wir nicht imstande zu sagen. Es muB hervorge- l ) Fiir Angaben iiber die verschiedenen Typen von N-Kombinationen in den meisten dieser Eiweiftkorper siehe T. B. Osborne, C. S. Leaven- worth and C. A. Brautlecht, American Journal of Physiology, 1909, Bd. 23, S. 180. Uber Futtcrungsversuche mit isoliertcn Nahrungssubstanzen. 3 hoben werden, daB durch irgend einen EiweiBkorper , dem die zyklische Gruppe, wie sie im Tyrosin und Tryptophan ge- fundenwurde, 1 ) fehlt, ein Wachstum nicht vollendet werden kann. W. A. Os- borne hat in der Tat die Hypothese aufgestellt, daB die «Cyclopoiese» in diesem Sinne eine Eigentiimlichkeit der pflanzlichen Zelle ist, und daB der tierische Organismus « acyclo- poietisch» und fiir gewisse Typen seiner Nahrung vom pflanz- lichen Leben ab- hangig ist. Diese un- wirksamen EiweiB- korper sind aber nicht urspriinglich toxisch, das erhellt aus der Tatsache, daB manche unter ihnen sich als durch- aus ausreichend fiir die Erhaltung des Ge- wichtsstillstandes bei *) Gf.auchE. Abder- h al d e n , Diese Zeitschr., 1912, Bd. 76, S. 22. Hon *e/n Z_ ] £n 'sen Phas^o/rn I lew \Leim \ Konghjrm \Rogg 'n-g/t'aitn ■g f/aaf/n \u 6 . Safe. ///? Ma/s y/u/e///? £xce/s//7 /(urd/ssa/nen- g /o fa ///'/?■ Ova t6u/r?/n odse/n \Uvo- 368 Thomas B. Osborne und Lafayette B. Mendel, wachsenden und erwachsenen Tieren erwiesen haben, wahrend, wie bekannt, andere, so Zein und Leim, zu diesem Behufe nicht genugen. Ferner geniigen Beimengungen von kleinen Mengen eines wirksamen EiweiBkorpers zur Anregung des Wachstums bezw. Erhaltung des gleichen Gewichts. 1 ) Trotz des normalen Charakters, der aus manchen unserer erfolgreicheu Versuche entnommenen Wachstumskurven sind wir noch nicht imstande, unter den kiinstlich geschaffenen Be- dingungen eine vollstandig normale Entwicklung hervorzurufen. Wenn wir an die Ernahrung des Kindes denken, so fallt uns auf, daB manches Kind eine ausreichende Gewichtszunahme zeigt, dabei aber offenbar anamiseh ist und eine schlecht ent- wickelte Muskulatur hat. Wenn ein solches Kind Anstrengungen, die tiber das physiologische MaB hinausgehen, ausgesetzt wird, dann kann es in einer Weise, die bei dem wirklich normalen Kind nicht moglich ist, versagen. Ein Beispiel dafur ist die klinische Beschreibung eines kiinstlich ernahrten Kindes: «Der Zustand, in dem sich die Kinder dabei finden, ist aber zweifellos nicht als ein normaler zu bezeichnen, das zeigt sich, wenn aus therapeutischen Griinden ein Hunger eingeleitet wird oder wenn eine Ernahrungsstorung das Gedeihen unterbricht. Es treten dann in kurzer Zeit bis- weilen ganz rapide Gewichtsverluste ein, begleitet von schweren Allgemeinerscheinungen. » 2 ) Chemische und histologische Untersuchungen der bei den Experimentaltieren vorhandenen Gewebe, Ziichtungs- versuche, Immunitatsversuche und andere Untersuchungen sind notig, um Licht in die Materie zu bringen. Der Ein- fluB von spezifischer Ernahrung auf die Hauptdrusen, welche bekanntlich eine wichtige Bolle innehaben, ist zugleich zu er- forschen. Es ist z. B. moglich, daB der durch manche unserer dargereichten Nahrungsstoffe verursachte Gewichtsstillstand *) Zur Veranschaulichung dieser Tatsache siehe Karte CXX— CXXIII in unsere Publication 156, Carnegie Institution of Washington, 1911, Part II, S. 133 u. 134. 8 ) Rosenstern, Ergebnisse der inneren Medizin und Kinderheil- kunde, 1911, Bd. 7, S. 393. Uber Fiilterungsversuche mit isoliertcn Nahrungssubstanzen. 369 — mag er durch qualitativ oder quantitativ schlechte Ernahrung veranlaBt sein — die indirekte Veranlassung zu einer Unter- entwicklung von Thymus, Thyreoidea, der Sexual- oder anderer Driisen ist. Es ware eine interessante Betrachtung, die Be- ziehungen unserer «Zwerge» zum menschlichen Infantilismus 1 ) zu untersuchen. In dieser Beziehung muB hervorgehoben werden, daB die Versuche iiber Gewichtsstillstand, wie er durch Gliadin als EiweiB veranlaBt wurde — wobei eine Unterdruckung des Wachstums iiber eine verhaltnismaBig lange Periode der ab- geschatzten Lebensdauer des Tieres ausgedehnt wird — , eine giinstige Gelegenheit darbieten, um die WachstumsgroBe in einer vollstandig anderen Weise zu untersuchen, als dies die Re- generation und Wiederherstellung ermoglicht. Mc Gollum 2 ) hat kiirzlich klar gezeigt, daB die Wiederher- stellungsprozesse sich von den Wachstumsprozessen in ihrem Charakter unterscheiden. Nach ihm verursachen die Prozesse der Zellerneuerung keine Zerstorung und Neusynthese eines ganzen Proteinmolekiils. Hierin liegt vielleicht das Geheimnis, daB die sogenannten «unvollstandigen EiweiBk6rper» imstande sind, einen Gewichtsstillstand zu erhalten, wahrend sie zur Bildung neuer Gewebe, d. h. zum Wachstum nicht ausreichen. Es ist zu bedenken, daB der Wachstumsimpuls nicht leicht er- lischt, wie angenommen wird. DaB diese Kraft lange Zeit zu- ruckgehalten werden kann, ist in anderer Hinsicht durch die Erscheinung der verschiedenen Formen von abnormem Wachs- tum gezeigt worden, wie Tumoren, Garcinom und Sarcom, welche allgemein als auf Wachstum bedachte Zellen, die der hindernden Kontrolle entbehren, aufgefaBt werden. Da die mit Ratten an- gestellten Versuche iiber Gewichtsstillstand Bedingungen zeigen, unter welchen die Zellen in mehr oder weniger jungem Zu- stande gehalten werden, konnen wir verstehen, warum der 1 ) Gf. G. A. Herter, Infantilism, New York 1908; G. Peritz, Der Infantilismus, Ergebnisse der inneren Medizin und Kinderheilkunde, 1911, Bd. 7, S. 405. 2 ) E. V. McGollum, American Journal of Physiology, 1911, Bd. 29, S. 215. 370 Th. B. Osborne u. L. B. Mendel, Uber Fiitterungsversuche usw. Wachstumsimpuls nicht erlischt. Deshalb miissen in dem Ge- webe bei sorgfaltiger Untersuchung mehr jugendliche als senile Eigenschaften erwartet werden. Zum Schlusse darf die Richtung der hier angefiihrten Ver- suche, die von der Frage der Synthese im tierischen Korper handelt, nicht iibersehen werden. Die Physiologen waren nicht geneigt, die Moglichkeit einer Synthese von Aminosauren dem Korper de novo zuzuschreiben. Die GewiBheit, die mit Riick- sicht auf das Glykokoll erbracht wurde, schien dann auBerhalb jeden Zweifels. J ) Andere weitere Daten weisen in die Richtung der Syn- thesefahigkeit der tierischen Zelle. 2 ) Die synthetische Bildung von Glykokoll bei unseren Ratten, welche mit dem glykokollfreien Casein gefuttert wurden, muB als feststehend angenommen werden. Entweder erfolgte die Synthese in ihren Korperzellen oder unter der Wirkung der Darmbakterien. Und wenn wir uns erinnern, daB alle unsere Versuchsfiitterungen purinfrei und einige vollig frei von organisch gebundenem Phosphor sind, wird die Tatsache der Synthese uns auf einmal klar. Phosphor- proteine und Nucleoproteine miissen in solchen Organismen ohne Zweifel durch komplizierte synthetische Umwandlungen ent- stehen. 3 ) Bis zu welcher Ausdehnung und in welchen Richtungen diese letzteren moglich sind, muB die Zukunft erschlieBen. 1 ) Cf. Ringer, Journal of Biological Chemistry, 1911, Bd. 10, S. 327, und Epstein and Bookman, ibid., S. 353. Tiber die friihere Literatur wird in diesen neuen Arbeiten berichtet. 2 ) Cf. Knoop, Diese Zeitschrift, 1910, Bd. 67, S. 489; Zentralblatt fur Physiologie, 1910, Bd. 24, S. 815; Embden und Schmitz, Biochem. Zeitschrift, 1910, Bd. 29, S. 423; 1912, Bd. 38, S. 392; Kondo, ibid., S. 407; Fellner, ibid, S. 414. 3 ) Cf. E. V. McCollum, American Journal of Physiology, 1909, Bd. 25, S. 120; Abderhalden, Diese Zeitschrift, 1912, Bd. 76, S. 22; Fingerling, Biochemische Zeitschrift, 1912, Bd. 38, S. 448; McCollum and Halpin, Journal of Biological Chemistry, 1912, Bd. 11, S. 13. Verlag von J. V. Bergmanp in Wiesbaden. Lehrbuch der Physiologischen Chemie von Olof Hammarsten, ehcm. o. o. Professor der med. und physiol. Chemie an der Universit'at Upsala. Siebente vbllig umgearbeitete Auflage. Preis Mk. 23.—, geb. Mk. 25 AO. Das Hammarstensche Lehrbuch wird von jedem als ein sehr lieber, unentbehrlicher Freund und Ralgeber begriifit, so oft es in neuer Gestalt erscheint. Es ist ja in der Tat das Lehrbuch der physiol. Chemie, und wird es wohl noch lange bleiben. Wenigstens in seiner Art als absolut zuverlassiges Hand- und Nachschlagewerk und zur kritischen Ein- fuhrung in bestimmte Fragen. An der Anordnung ist nichts geandert, nur ein sehr willkommenes Namenregister angefugt. Dafi die Literatur soweit als irgend moglich bis auf die letzte Zeit beriicksichtigt ist, braucht nicht erwahnt zu werden. Biochemisches Zentralblatt. Analyse des Harns. Zum Gebrauch fur Mediziner, Checker und Pharmazeuten zugleich Elfte Auflage von Neubauer-Hupperts Lehrbuch. Bearbeitet von A. Ellinger-Konigsberg, F. Falk-Wien, L. Henderson-Boston, j F. N. Schultz-Jena, K. Spiro-Strafiburg und W. Wiechowski-Wien. J /. Hdlfte. — Preis Mk. 15.—. .... Neu ist an der Bearbeitung besonders auch, daft jetzt die qualitativen und quantitativen Bestimmungen sich der Besprechung der einzelnen Stoffe unmittelbaj anschliefien, was fur den, der nach dem Buche arbeiten will, entschieden eine grofie Eiieichterung darstellt. Der Inhalt des Buches ist zu reichhaltig, um auf Einzelheiten einzugehen, lafit aber nirgends Vollstandigkeit und Uebersichtlichkeit vermissen. Die Autoren durfen ihr Werk der Oeffentlichkeit ubergeben in dem BewuBt- sein, einem dringenden Bedurfnis entsprochen und Mustergiiltiges geleistet zu haben. Zentralblatt f. Innere Medizin. Dynamisehe Bioehemie Chemie der Lebensvorgange. Von Professor Dr. Sigmund Frankel, Wien. Preis Mk. 18.60, gebunden Mk. 20.20. Gewissermaften als zweiter Band zu des gleichen Autors «Deskrip- tiver Bioehemie » folgt diese dynamisehe Bioehemie, in welcher das Haupt- gewicht auf das chemische Geschehen im Organismus gelegt wird. In sehr geschickter Weise wird das weitschichtige Gebiet, welches ja den grofieren und, abgesehen vom Kreislauf, praktisch wichtigsten Teil der Lebensvorgange umfaftt, dargestellt. Fiir die Lesbarkeit des Werkes ist es wohl ein Vorzug, dafi der Autor in der Auswahl des zu besprechenden Stoffes eine gewisse Beschrankung sich auferlegt hat. Deutsche mediz. Wochenschrift. Verlag von KARL J. TRUBNER in Strafibnrg. Im September gelangt zur Ausgabe: Chemie der Fette vom physiologisch-chemischen Standpunkte. Von Prof. Dr. Adolf Jolles, in Wien. Zweite vermehrte und verbesserte Auflage. 8°. VII, 148 Seiten. Geheftet M 4.—, in Leinwand geb. Ji 4.50. Urteile der Presse iiber die erste Auflage: «Das Werkchen ist eine sehr fleiftige Sammlung der neueren For- schungen auf dem Gebiete der Fettchemie, insoweit sie physiologisch bedeutsam sind, im Zusammenhang mit den in Betracht kommenden allgemeinen Erfahrungen der organischen und physikalischen Chemie. Die Anzahl der die wissenschaftlichen Arbeiten veroffentlichenden Zeitungen und Zeitschriften ist zu einer erstaunlicben Hohe angewachsen und macht es dem Einzelnen schon schwer, selbst auf kleinerem Gebiete, alle Ab- handlungen und Leistungen zu verfolgen. Wer nun weifi, welches Mafi von Miihe und Zeitaufwand c-s kostet, bei diesen Mengen von Publikationen eine so vielfach zerstreute einschiagige Literatur zu bearbeiten, wird dieser gewissenhaften Zusammenfassung Dank wissen.> Osterreichische Chemiker-Zeitung 1907, Nr. 16. «. . . Die Arbeit stellt sich als ein mit. grofiem Fleifi zusammen- getragenes Sammelreferat dar, welches mit seinen zahlreichen Literatur- angaben jedem willkommen sein mufi, der sich theoretisch oder praktisch mit der physiologischen Fettchemie befafit oder sich iiber den heutigen Stand dieser Wissenschaft schnell zu orientieren wunscht.» Apotheker-Zeitung 1907, Nr. 95. <. . . Das Btichlein wird Chemikern und Physiologen iiber alle auf die Fette bezuglichen Daten, Fragestellungen und Ergebnisse eine rasche Orientierung ermoglichen.* Deutsche Literaturzettung 1908, Nr. 14. U. Dn Mont-Schauberg, Strassburg. — 365. Reprinted fiom The Journal of Biological Chemistry, Vol. XIII, No. 3, 1912. THE BEHAVIOR OF SOME HYDANTOIN DERIVATIVES IN METABOLISM. I. HYDANTOIN AND ETHYL HYDANTOATE. By HOWARD B. LEWIS. {From the Sheffield Laboratory of Physiological Chemistry, Yale University, New Haven, Connecticut.) (Received for publication, October 16, 1912.) The demonstration of the occurrence of pyrimidine derivatives as constituents of the nucleic acid molecule has awakened a wide interest in the physiological behavior of the pyrimidine ring. The possible biochemical significance of this group is attested by its structural relationship to the purines, creatinine, allantoin and other physiologically important compounds. Although hydantoin and its derivatives have not yet been found present as constitu- ents of any tissues of the body, the behavior of the hydantoin nucleus, a structure similar to the pyrimidine grouping but contain- ing one less carbon atom, deserves consideration in connection with intermediary metabolism. N— C C C N— C Pyrimidine nucleus. N— C N— C Hydantoin nucleus. The close relationship between hydantoin, allantoin, creatinine, purine and imidazole may be seen by a comparison of their struc- tural formulae. NH— CO NH— CO NH 5 NH— CO NH— CH N; C = C = C = C = NH CH II II II II NH— CH 2 NH— CH— NH (CH 3 )N— CH 2 N— CH N C N<^ Hvdantoin. Allantoin. Creatinine. Imidazole. Purine. CH C— NH- II II 347 348 Behavior of Hydantoin Derivatives None of these substances with the possible exception of the imidazole nucleus have been demonstrated to be destroyed when introduced into the organism. Dakin and Wakeman 1 have shown by perfusion experiments with the liver that some slight decom- position of histidine, which contains the imidazole nucleus, may take place with the formation of acetoacetic acid, but they con- clude that the effect is too slight to formulate any promising hypoth- esis for the catabolism of histidine. Feeding experiments with histidine 2 leave the fate of this substance in the organism in doubt. The failure of these related compounds to experience disintegration in metabolism renders the behavior of hydantoin, a compound simpler than any of the others, of particular interest. From another viewpoint, the behavior of hydantoin seems worthy of study. Lusini 3 working with alloxan and alloxantin NH— I reached the conclusion that the grouping C = functions to stim- t NH— ulate and then inhibit nerve centers. It is, according to Lusini, the ketone-like group ^)CO which has the stimulating property and an abundance of these groups increases the toxicity. More recently Kleiner 4 was unable to confirm Lusini's conclusions since barbituric acid, which Kleiner studied and which is non-toxic, contains the ketone 'group and differs little from the toxic sub- stance alloxan. Inasmuch as hydantoin also contains the alleged NH— I toxic group, CO , the question of its toxicity is of interest. I NH— The present paper -deals with the behavior of hydantoin and 1 Dakin and Wakeman: this Journal, x, p. 499, 1912. 2 Abderhalden and Einbeck: Zeitschr. f. physiol. Chem., Ixii, pp. 322-32, 1909; Abderhalden, Einbeck and Schmid: ibid., lxviii, pp. 395-99, 1910; Kow- alevsky: Biochem. Zeitschr., xxiii, pp. 1-4, 1910. 3 Lusini: Ann. di chim. e di farmacol., xxi, pp. 145-60, 241-57, xxii, pp. 341-51, 385-94, 1895; Chem. Centralbl, i, p. 1074; ii, p. 838, 1895. 4 Kleiner: this Journal, xi, pp. 443-70, 1912. Howard B. Lewis 349 the ethyl ester of hydantoic acid, introduced in different ways into the organism of various species. The hydantoic acid ester was prepared from glycocoll ester hydrochloride and potassium cyanate, according to the method of Harries and Weiss. 5 On evaporating the ester to dryness with concentrated hydrochloric acid, it is converted to the hydantoin. The latter is then purified by recrystallization from absolute alcohol. NH 2 • CO • NH • CH 2 • COOC 2 H 5 Ethyl ester of hydantoic acid Analysis (Kjeldahl nitrogen determination) of the hydantoin prepared gave the following result: Calculated for C»H4N 2 2 : Found: N 28.00 per cent. 27.88 per cent. For the identification of the hydantoin in the urine, use was made of the insoluble benzalhydantoin. The urine was acidified and evaporated to small volume on a water bath, decolorized with animal charcoal and evaporated to dryness. The product was then condensed with benzaldehyde in the presence of glacial acetic acid, acetic anhydride and dried sodium acetate, as de- scribed by Wheeler and Hoffman. 6 For the identification of the ester, the urine was evaporated to dryness with concentrated hydrochloric acid to convert the ester into hydantoin, and the benzal derivative was prepared as before. The analytical procedures included the Kjeldahl-Gunning meth- od for nitrogen and Folin's methods for urea and creatinine. Blank experiments with hydantoin showed that this substance is not attacked by Folin's urea method. In the experiments with rabbits the bladder was emptied by pressure at the same hour daily. The substances, when fed, were dissolved in water and introduced through a gastric sound. In the experiments with dogs the animals were catheterized at reg- 5 Harries and Weiss: Ber. d. deutsch. chem. Gesellsch., xxxiii, p. 3418, 1900. 8 Wheeler and Hoffman: Amer. Chem. Journ., xlv, p. 368, 1911. NH— CO I C = I NH— CH 2 Hydantoin 350 Behavior of Hydantoin Derivatives ular twenty-four-hour intervals. Here the substances fed were mixed with the food. EXPERIMENTS WITH HYDANTOIN. Rabbit. I. Diet, 300 grams of carrots daily. This was com- pletely consumed except on the day of the hydantoin administra- tion when the animal ate only 270 grams. No toxic effects of any sort were noted. The protocol follows: Rabbit; weight, 1.8 kgms. a VOLUME SPECIFIC GRAVITY TOTAL N « p UREA + NHs-N TOTAL N N NOT UREA + NHj N REMARKS CC. grams gram per cent gram 1 280 1.012 0.876 0.768 87.6 0.108 2 295 1.012 0.918 0.750 80.1 0.168 3 245 1.015 0.900 0.756 84.0 0.144 fl.5 gm. hydantoin 4 145 1.026 1.308 0.738 56.4 570 \ = 0.42 gram N, in - 5 220 1.016 0.696 0.600 8( 1.4 0.096 [ traperitoneally. 6 205 1.024 0.690 0.630 90.1 0.060 About 0.3 gram of benzalhydantoin, after purification by re- crystallization from alcohol, was obtained from the urine of the fourth day. The benzalhydantoin isolated melted at 217° and when mixed with a pure synthetic sample did not alter the melting point of the latter. No increase in the urea + ammonia nitrogen on the day of the injection was observed, although the increase in the elimination of total nitrogen excreted accounted for all the nitrogen admin- istered as hydantoin. This fact, together with the identification of hydantoin in the urine, indicates that hydantoin is unaltered in its passage through the body. Another experiment with the same animal a few days later, in which the same amount of hydantoin was administered per os, gave similar results. Rabbit. II. Diet, 300 grams of carrots. On the day on which Howard 13. Lewis 35i the hydantoin was fed, the animal consumed the full daily ration. No toxic symptoms of any sort were noted. The protocol follows: Rabbit; weight, 1.64 kgms. < Q VOLUME SPECIFIC GRAVITY TOTAL N < « P 5 n + H K P >j H O H P REMARKS CC. gram gram per ceni gram 1 275 1.015 0.900 0.786 87.3 0.114 2 240 1.018 0.708 0.642 90.6 0.066 3 150 1.016 0.624 0.558 89.4 0.066 fl.5 gm. hydantoin 4 205 1.021 0.918 0.576 1.7 342 \ = 0.42 gram N, 5 280 1.019 0.726 0.570 79.1 156 { per os. 6 160 1.020 0.600 0.516 86.0 0.084 7 160 1.025 0.570 0.474 83.1 0.096 From the urine of the experimental day a small amount of ben- zalhydantoin melting at 218° was obtained. Dog. A female was fed on a constant daily diet of 200 grams of lean meat, 50 grams of lard, 30 grams of sugar, 5 grams of bone ash, 2 grams of salt and 250 cc. of water with a total nitrogen content of 6.84 grams. The hydantoin was dissolved in the water of the diet. The animal ate eagerly on the experimental day as at other times. Dog A; weight, 12.4 kgms. < H a p o > « O £ o w H M B K « pi i+* « H g g -J sa REMARKS o > Cm to O H p PS o cc. grams grams per cent grams gram 1 160 1.055 8.45 7.63 89.8 0.82 0.279 2 3 4 180 300 300 1.042 1.025 1.030 6.69 6.38 6.87 6.24 5.80 5.67 93.3 90.9 82 6 0.45 0.58 1 20 0.294 0.294 0.298 2.5 gm. hy- dantoin = 0.7 gm. N, with food. 5 320 1.024 5.99 5.39 90.0 0.60 0.306 6 350 1.023 6.12 5.44 88.8 0.68 0.284 7 300 1.028 6.37 5.76 90.4 0.61 0.297 352 Behavior of Hydantoin Derivatives From the urine of the experimental day 3.3 grams of benzal- hydantoin were obtained corresponding to 1.7 grams of hydantoin ( = 0.476 gram N) in the day's urine. This was purified by solu- tion in potassium hydroxide and reprecipitation with acid. Cat. A cat weighing approximately 4 kgms. received 3 grams of hydantoin mixed with raw meat. The urine of the next twenty- four hours was collected and examined for the presence of hydan- toin as above. The benzalhydantoin obtained melted at 214° and after purification weighed 1.7 grams. A nitrogen determination (Kjeldahl) on the mixed products obtained in this and the three preceding experiments gave the following results: Calculated for C10H8N2O2: Found: N 14.92 per cent. 14.59 per cent. In all our experiments the recovery of the administered hydan- toin from the urine leaves no doubt as to its absorption. The increase in the total nitrogen of the urine on the experimental day also points to the same conclusion. In no case was the urea + ammonia nitrogen output increased, the difference between the total and urea + ammonia nitrogen nearly always approach- ing the value of the nitrogen administered as hydantoin. In the rabbit II, there was observed a slight lag, part of the hydantoin probably being eliminated on the day after the administration. No effect on the creatinine elimination was observed in the dog. Hydantoin appears to be without influence on nitrogenous me- tabolism and is not destroyed or changed by the organism. No NH — I toxicity attributable to the group C = O as alleged by I^usini was NH — observed. EXPERIMENTS WITH ETHYL HYDANTOATE. In order to ascertain whether the inability of the organism to break down hydantoin was due to the cyclic structure, experiments were conducted with the ethyl ester of hydantoic acid. This acid is converted to hydantoin with the loss of a molecule of water Howard 13. Lewis 353 and bears the same relation to hydantoin that creatine hears to creatinine. NH— CO NH 2 CONH-CH,COOH Hydantoic acid c=o + H 2 NH— CH 2 Hydantoin Hydantoic acid may also be considered as a uramino acid, uraminoacetic acid. Koehne, 7 working with the ethyl ester of the homologous uraminoformic or allophanic acid, found that it dis- appeared in the body. Rabbit 1. Diet, 300 grams of carrots daily. On the exper- imental day the animal showed no unusual symptoms and resumed eating immediately after the feeding of the ester. The protocol follows: Rabbit; weight, 1.7 kgms. >< a a o ECIFIC GRAVITY lEATININE ITAL N < a 351 Aba ■fJS el ' A a REMARKS 6 > 5 o EH 8 cc. gram gram gram per cent gram 1 220 1.011 0.055 0.810 0.729 90.0 0.091 2 220 1.012 0.056 0.443 0.347 78.3 0.096 r 2 gm. hydan- 3 200 1.013 0.047 0.443 0.396 89.8 0.047 toic acid es- 4 295 1.017 0.071 845 0.420 49 6 425 ter = 0.38 5 220 1.016 0.063 0.458 0.347 73.5 0.111 gram N, per 6 270 1.014 0.061 0.414 0.369 89.1 0.045 OS. From the urine of the experimental day, benzalhydantoin was prepared in the usual manner. An amount equivalent to 0.67 gram of the ester (= 0.13 gram N.) was obtained. The ben- zalhydantoin melted at 218° and did not change the melting point of the pure synthetic substance when mixed with it. A nitrogen (Kjeldahl) determination gave the following 'results: Calculated for C10H8N2O2: Found: N 14.92 per cent. 14.67 per cent. 7 Koehne: Inaugural Dissertation, Rostock, 1894, p. 17. 354 Behavior of Hydantoin Derivatives Rabbit 2. Diet, 300 grams of carrots daily. The animal ap- peared normal on the day of the injection and ate as usual. The protocol follows: Rabbit; weight, 1.5 kgms. DAY VOLUME SPECIFIC GRAVITY TOTAL N CREATININE REMARKS CC. grams gram 1 95 1.026 1.172 0.103 2 180 1.020 0.702 0.070 3 220 1.013 0.611 0.078 fl.5 grams hydantoic acides- 4 210 1.013 0.878 0.081 \ ter = 0.286 gram N, sub- 5 240 1.012 0.513 0.084 [ cutaneously. 6 240 1.012 0.374 0.076 7 230 1.016 0.444 0.073 From the urine of the experimental day a small amount of benzalhydantoin was prepared which melted at 217° and did not affect the melting point of the pure synthetic substance. Rabbit 3. Diet, 300 grams of carrots and 30 grams of oats daily. On the day of the injection only 270 grams of carrots were eaten. No abnormal symptoms were noted. Rabbit; weight, 2.34 kgms. < a a (3 O ECIFIC GRAVITY < H < « A + B « TOTAL N tEATININE REMARKS Q > 00 O H S3 P D m U CC. grams grams per ce«< gram gram 1 2 265 220 1.010 1.013 1.20 0.855 1.020 0.716 85.0 82.9 0.180 0.139 0.122 0.092 2 gm. hydan- toic acid es- ter = 0.38 grams N. intr aperi- toneally. 3 4 220 260 1.012 1.015 1.018 1.476 0.840 1.035 83.3 70.3 0.180 0.441 0.110 0.145 5 6 185 160 1.015 1.015 0.996 0.990 0.852 0.900 85.5 90.9 0.144 0.090 0.123 0.135 7 125 1.018 0.945 0.810 85.7 0.135 0.081 From the urine of the experimental day benzalhydantoin was prepared and purified by solution in potassium hydroxide and reprecipitation with acid. It weighed 0.75 gram = 0.58 gram ester Howard B. Lewis 355 in the day's urine. A nitrogen (Kjeldahl) determination gave the following results. N Calculated for C10II8N2O2: 14.92 per cent. Found: 14.88 per cent. Dog. A female received a standard daily diet (see experiments on hydantoin). The food was eaten as usual on the experimental day. Dog A ; weight, 12.6 kgms. < s p J > SPECIFIC GRAVITY TOTAL N