x^ vfe-'^ **L/ Dptiz Art CORNELL UNIVERSITY. THE ilosiucll |J. Slower Eibrarg THE GIFT OF ROSWELL P. FLOWER FOR THE USE OF THE N. Y. STATE VETERINARY COLLEGE. 1897 Digitized by Microsoft® Cornell University Library QP 981.N9M12 '%ese^RCH Byffifcf^ Nq/a iiiiii'iiiH iili) iTmi/ii iiiu'iliniiiiiiiifiiii laiViiiiTili iif™ v i 3 1924 003 311 663 THE UNIVERSITY OF^WISCONiSiN i AGRIGULTIMVL ^ER^NT STATION Nu^leinSyi AUG h- 1810 ■:■ 'i. trie BY .*** y'f.^vCk&QujuM^.. >} if\'\ <*"" v*. "% 5 DigkiM'btf MMom- Introduction.. — «,_. „-.,.; ..:,.. J '.y,.,,.-. ...... Purpose of these experiments. .. .-. . . : s Preparation of .the fooa\ materials ., .. . the.^^|te|ntf ^|i^ft|i;>-.T.,; . ". .. . . . . . , - ExB^riifiljBnts With mature animals . . . .. . . ." ik^rifi^&tj^th -yciiiBger animals, fi. t '^iA^ol expertBiefilw ,$ith casein..I;, ,». '5"iJ!fp«iDje4ts with normal ration. ..... 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McCOLLUM Modem investigations have led physiologists to the belief that in the normal processes of metabolism, the proteins of the tissues of the animal are the products of a regeneration of these bodies from comparatively simple cleavage products of those proteins taken as food. 1 Our knowledge of the fate of nueleins in the body, and of the origin of the body nucleo-proteins, is less clear than of the simple proteins. The nucleo-proteilis are complexes, consisting of sim- ple proteins in union with nucleic acid, the latter containing a high content of phosphorus in combination with purine and pyrimidine bases and a carbohydrate group. The earlier in- vestigators established the fact that the proteolytic digestive enzymes, pepsin and trypsin, do not attack nucleic acids in such' a manner that purine bases are liberated/ More recently Abder- halden and Schittenhelm 3 have studied the behavior of thymus nucleic acid with pancreatic juice of the dog, and found that this substance is changed in some manner so that the character- istics of nucleic acid are lost, but without the liberation of purine bases. They likewise found that thymus nucleic acid, when di- gested with extracts of the pancreas and intestinal mucosa of the cow, was speedily liquefied and purine bases set free. This the authors attribute to the presence of intracellular enzymes in such extracts. The behavior of nucleo-proteins and their cleavage products, the nueleins and purine bases, with individual organs and tissue i Hugo Liithje, Ergebnisse der Physiologie, 1908, 7: 795. A resume of the researches hearing on this subject. 2 Iwanoff, Ztschr. Ehysiol. jChem... 1903. .39.: 31. References to the older literature. U^Wze(TbfN\\CrOSbft® s Abderhalden and Schittenhelm, Ztschr. Physiol. Chem., 1906, 47: 452. S\ 74 Wisconsin Experiment Station. ■extracts, has received much attention during the last few years.* 'The existence of four distinct classes of enzymes concerned with the transf ormations of purines in the body seems to be well estab- lished : 5 1. Nucleases ; which liberate purine bases from the nucleic acid molecule. 2. Deamidizing enzymes ; which liberate ammonia from ade- nine and guanine, forming oxy-purines, hypoxanthine and xan- thine. 3. Oxidizing enzymes; which oxidize hypoxanthine and xan- thine to uric acid. 4. Uricolytic enzymes ; which destroy uric acid. Almost every organ and tissue of the animal body seems to be endowed with the power to bring about one or more changes in the nucleic acids or their products, all of, which lead to the final destruction of the component parts of the molecule. These facts lead to the question: What kind of phosphorus compounds can the animal utilize for the elaboration of the phosphorus con- taining complexes of its cell nuclei? and, Is an exogenous supply of purine bases essential to nuclein formation? The work of the investigators cited above all points to a ■destruction of the nucleic acids taken with the food, rather than a direct transposition of nucleic acid complexes of exogenous origin, and a substitution of these for the portions of the nuclei of the living cells broken down during metabolic activity. Steinitz 6 attempted to throw light on the question as to whether the formation of nucleo-proteins is a chain of syntheses involving inorganic , phosphoric acid, by studying the nitrogen and phos- phorous retention in a dog fed: (a) a phosphorus free protein (myosin) combined with carbohydrate fat and inorganic salts, the phosphorus being supplied in inorganic form, and (b) a phosphorized protein (vitellin) combined with the same sub- stances but without the phosphates. He found a better retention of phosphorus, but poorer retention of nitrogen when vitellin was given and a better retention of nitrogen, but insignificant storage of phosphorus when myosin supplied the protein of the ration. His experiments were conducted only five to eight days. Leip- iBloch, Biochem. Centbl., 1906, 5: 521, 561, 817, 873, gives an ex- tensive resume of the literature. s Mendel and Mitcfi^/fkft^ifcjJiMfcrBBSfff®., 1907, 20: 97. e Steinitz, Arch. Physiol., [Pfliiger], 1898,72: 75. Nuclein Synthesis in the Animal Body. 75 ziger 7 using the same dog, repeated Steinitz' experiment using edestin as the phosphorus free protein and confirmed his observa- tions in a 7-day trial. Zadik 8 and Ehrlich 9 employing edestin and casein reached the same conclusion as a result of similar ex- periments. The above experiments were all of short duration and can hardly be looked upon as positive proof of the point in question. It is well known that the elimination of phosphorus is not neces- sarily constant during a brief period and is influenced by numer- ous factors, especially by the relative amounts of the alkaline earth and alkali salts in the food. 10 In an experiment by Hart, McCollum and Fuller 11 it has been shown that when pigs were fed on a ration containing a very low phosphorus content, and which proved inadequate for the mainte- nance of the animals, the addition of phosphorus in the form of calcium phosphate, corrected all of the pathological disturbances and led to normal growth and development. Their experiment did not furnish proof of a nuclein or phosphatide synthesis from inorganic phosphates, since their ration still contained small amounts of phosphorus in unknown forms. It was not found possible to secure a basal ration entirely free from phosphorus, in sufficient quantity for work with large animals. It was the purpose of the writer in undertaking the present series of experiments, to demonstrate whether an animal can rely wholly upon inorganic phosphorus forms for its supply of this element. Such a series of experiments involves the maintenance of ani- mals during the growing period upon a ration of artificially pre- pared foodstuffs, rendered phosphorus free by appropriate meth- ods of purification, inorganic forms of phosphorus being added. This is necessary since none of our naturally occurring protein containing foodstuffs are free from organic forms of phosphorus. Several attempts by other investigators to maintain animals on a ration made up of relatively pure proteins, carbohydrates, fats and inorganic salts, have been wholly or partially unsuccessful. 7 Leipziger, Arch. Physiol., rPfluger], 1899, 78: 402. s Zadik, Arch. Physiol., [Pfluger], 1899, 77:1. » Ehrlich, Stoffwechselversuche mit P-haltigen und P-freien Eiweiss- korpern. Inoug-Diss. Breslau. 1900. loEhrstrom, Skand. Arch. Physiol. 1903, 14: 82-111. "Hart, McCollum rand.. Fuller, Amer. Jmir, Physiol. 1909, 23:246. wis. Agr. Expt. staMmMmmrosvn® 76 Wisconsin Experiment Station. Socin 12 and Hall 13 attempted to maintain mice on a ration con- sisting of casein, fat and sugar or starch and inorganic salts. Socin gave in one experiment also hemaglobin to furnish a supply of organic iron. His mice lived no longer than 33 days in any case. Hall added cellulose to his ration to serve as an, irritant to the digestive tract and carniferrin to supply organic iron. In no case did he succeed in keeping the animals alive on such a ration more than 40 days. Falta and Noggerath" fed white rats on rations in which the protein was supplied by relatively pure proteins of different sources. Carbohydrates, fats and inorganic salts were given in addition. With a ration in which the nitrogen was given as serum albumin and casein their animals died after 51 to 53 days, having lost weight from the beginning of the experiment. When the nitrogen was given as ovalbumin the rats lived 83 to 94 days. In a trial in which serum albumin, ovalbumin and casein were given together in addition to carbohydrates, fats and inorganic salts the rats lost weight as in the preceding experiment and died in from 71 to 94 days. The addition of sodium nucleinate, cholesterin and lecithin gave no better results. The authors be- lieved that the steady decline of their animals was due to either insufficient intake or to lack of utilization of the food consumed rather than an actual insufficiency in the ration. L. Jacob 15 has contributed some very instructive experiments in this field. Doves were fed a ration consisting of casein, starch and sugar, fat and inorganic salts in the form of ash of milk. The ration was calculated to supply protein, carbohydrate, fat and ash in the same proportions found in the wheat grain. The mixture was pressed into pellets of uniform size. The doves in two experiments were dead or in a dying condition at the end of 37 days. When meat powder was substituted for casein the results were the same. On sectioning, the crops were seen to be filled with a compact dough-like mass. The writer attributed the outcome of the experiment to this difficulty. The birds could not expel the food from the crop, and thereafter vomited everything they attempted to eat. Death was therefore due to inanition. 12 Socin, Ztschr. Physiol. Chem., 1891, 15: 93. is Hall, Arch. Anat. u. Physiol. 1896, p. 49. it Falta and Noggerath. Beitr. .Chem. Physiol. Path., 1905, 7:313. 15 jacoh, n, ztsch£%Kfee$)W memoir® sIuclein Synthesis in the Animal Body. 77 In one experiment in which cellulose was added better results were obtained. Jacob calculated the food value of the pellets fed to the dove and observed that during the four weeks of the experiment the total intake was equivalent to only 70 per cent of the energy requirement of the bird. The loss in weight was pro- portional to the energy deficit of the food taken. The same investigator experimented with rats, feeding a mix- ture of casein, carbohydrate and fat, to which inorganic salts were added. The ration was mixed with cellulose. The rats lost weight steadily from the beginning and died after 42, 73, and 324 days respectively, after the loss of about 40 per cent of their body weight. The difference in the ability of individuals to withstand such a diet is strikingly apparent. Zadik 16 prepared four different rations as follows : 1. Casein, nutrose, bacon, starch, meat extracts and meat salts. 2. Nutrose, bacon, starch and salts. 3. Casein, bacon, starch and salts. 4. Vitellin, bacon, starch and salts. Alternating these rations he was able to mainain a dog 36 days during which time the animal gained 200 grams in weight. At this point the dog became ill and diarrhoea and vomiting neces- sitated the cessation of the experiment. The dog was unable to eat meat without vomiting. Zadik believed the interruption of the experiment resulted from catarrh of the bladder brought on by the continued use of a catheter. G. Marcuse 17 fed a dog casein, rice starch, bacon and salts during 11 days and observed a gain of 730 grams in the ani- mal's weight, and a retention of 11.25 per cent, of the nitrogen ingested. The dog likewise retained phosphorous. Henriques and Hansen 18 fed white rats with a mixture of casein, fat, sugar, cellulose and salts during 13-17 days and ob- served that they gained in weight and retained nitrogen. "Willcock and Hopkins 19 have reported that a diet containing only zein as a nitrogenous constituent is unable to maintain growth in young mice. . The addition of tryptophane, an amino which acid is absent from zein, while not making it capable of maintaining growth, promotes the wellbeing of the animals and greatly prolongs the survival period. ie Zadik, Arch. Ph;wipL. IPfliteerJ-.ISflfl K^l. "Marcuse, G, ArcPmm&FJ^%9f, ( ml>, 64: 223. is Henriques and Hansen, Ztschr. Physiol. Chem., 1904-5, 43: 417. is Willcock and Hopkins, Jour, of Physiol., 1907, 35: 88-102. 78 Wisconsin Experiment Station. Another class of experiments should be mentioned in this con- nection. Abderhalden and Rona- were unable to keep a dog in nitrogen equilibrium with a pancreatic digest of casein. Lesser 21 could not obtain nitrogen equilibrium in a dog fed with a pan- creatic digest of fibrin. Plosz 22 attempted to substitute peptone for protein. He fed a young pup for .18 days on a mixture of peptone, sugar and fat and a salt mixture. The dog gained 501 grams or 37.5 per cent pf its body weight. Loewi 23 fed a dog with a mixture of ammoaeids and other simple products resulting from the auto-digestion of pancreas. The mixture did not give the biuret reaction. During 11 days the dog gained 0.72 grams of nitrogen and 0.649 gram phos- phorus. Loewi concluded that with these simple substances the animal can replace body tissue. In the experiments just described the constituents of the ra- tions used were relatively pure chemical substances except in Zadik's employment of meat extracts and bacon. The former contains unknown nitrogen and phosphorus compounds and the Jatter connective tissue and some cellular materials. We have now to mention several experiments in which a cer- tain amount of naturally occurring mixtures were added to ra- tions composed mainly of simple chemical substances of known composition. Salkowski 24 fed a ration of eucasein (NH., casemate), bacon, rice and meat extract. A dog ate this for 10 days and increased 285 grams in weight. At the end of the period it refused to take food. In another experiment with the same mixture in different proportions a second dog ate the ration for a period of 24 days, gaining 285 grams in weight. It retained nitrogen and was in a normal condition. R6hmann 2,r ' fed mice on a mixture of casein, egg albumen, vi- Tellin. nucleoprotein from liver, wheat starch, potato starch, margarine and ether extract of egg yolk. To these he added 20 Abderhalden and Rona, Ztschr. Physiol. Chem., T905, 44: 198. =i Lesser, Ztschr. Biol.. 1904. 45: 497. 22 Plosz, Arch. Phvsiol. [Pfliigerl, 1874. 9:323, cited from Maly's Jahresbericht der Tier-Chemie, 1875, 4: 21. 23 Loewi. Arch. Expt. Path. u. Pharmakol.. 1902, 48: 303-330. Cited from Maly's Jahresber. Tier-Chemie, 1903, 32: 684. 24 Salkowski. T)eut.lJft]JtJz@&>&y : Wl}W033W® No. 15, p. 225. 2." Rohmann, Klin. Ther. Wohnschr., 1902, 40: 1. Cited from Maly's Jahresber. 1904, 33: 823. Nuclein Synthesis in th^ Animal Body. 79 sodium and magnesium citrates, calcium lactate, calcium phos- phate, dipotassium phosphate and sodium chloride. \ The mice lived 96 days on this ration, produced young and the young continued 94 days on the same ration to which was added about 4 per cent malt. They in turn produced young. Weiske 20 studying the effect of a calcium — poor ration on the composition of the bones, fed a goat (6-7 years old) on wheat straw which had been thoroughly extracted with hydrochloric acid. To this he added casein, sugar, starch and sodium chloride. A second animal received extracted straw, casein, sugar, starch, sodium chloride and sodium phosphate. A third received the same ration as did the second except that calcium carbonate was substituted for sodium phosphate. The second goat would not eat the ration. The third ate the ration readily during 42 days when it began to leave some of its feed. There were no signs of illness except that it grew languid toward the end of the experiment. In another experiment in which sodium phospnate was used instead of calcium carbonate the animal showed no signs of ill- ness but grew feebler day by day and died on the 50th day. In a third series of experiments Weiske and "Wildt 27 fed 2^4 months old lambs with the same mixture to which calcium car- bonate and sodium phosphate were added. The experiment was continued 55 days. No. 1 decreased in weight from 46 to 32 pounds. No. 2 decreased in weight from 47 to 34 pounds. E. Voit calculated from the data furnished by Weiske and Wildt that the intake of food by the animals in these experiments was too small for maintenance, and attributed their loss of weight to inanition. When we consider the causes leading to the unsatisfactory re- sults in the experiments above described, several possibilities are suggested : 1. The animals may have failed because of a lack, wholly or in part, of certain organic complexes in the food given, which the body was not able to supply through its synthetic power from the materials at hand. 2. Certain of the ash constituents essential to the life of the animal can be utilized only when presented in certain organic Digitized by Microsoft® 20 Weiske, Ztschr. Biol., 1872, 7: 1 Abhg. p. 179; 2 Abhg. p. 333. 2r Ibid. 1873, 8:239. Wildt, ibid. 8:266. 80 "Wisconsin Experiment Station. combinations, whereas in several of the experiments described they were given as inorganic salts. Iron and phosphorus might be especially mentioned in this connection. 3. The physical character of the food, especially in respect to lack of bulk and irritating power in the digestive tract. 4. The sameness of the ration. 5. The psychical factor of palatability as influencing the intake and utilization of food. Concerning the two first-mentioned factors, it would seem that they have been excluded by the experiments described. Falta and Noggerath met this objection by feeding sodium nuclinate, lecithins and a variety of proteins with no better results than have been obtained by others with much simpler mixtures in which the iron and phosphorus were given in inorganic forms. That the physical character of the food might be responsible for the observed inadequacy of the rations in certain cases must be admitted. Lunin, 28 however, found that mice lived indefi- nitely, without any ill effects, on evaporated milk alone. Socin 20 fed mice on egg yolk, starch and cellulose during 99 days and they remained in excellent condition. The writer in a preliminary experiment designed to show whether the cellulose was an important factor in Socin's experi- ment, fed five half-grown mice on boiled egg yolk alone from May 19 to October 7. The five mice together at the beginning weighed 37 grams. At the end of the experiment they weighed 16, 17, 20, 14, and 14 grams respectively, their collective weight being 81 grams. This ration contained no indigestible matter but was quite sufficient for normal growth and development of the mice. In another experiment two young white rats were fed on egg yolk alone. The eggs were hard boiled and the whites completely separated. No. 13 (male) gained weight from 71 grams at the beginning to 167 grams at the end of 18 weeks. No. 14 ( female 1 gained weight from 40 grams to 160 grams during the same period. Just before the close of the experiment she gave birth to eight young which she had made entirely from egg yolk. The ration in addition to being entirely free from indigestible matter contained no carbohydrate. The idea that the sameness of the 28 Lunin. Ztschr. Pli^^zeef^ MsrpSOfi® 29Socin, Ibid., 1891, 15: 93. Nuclein Synthesis in the Animal Body. 81 ration necessarily leads to a failure of the appetite is not com- patible with these experiences. The work of Pawlow 30 and his students seems to furnish a more satisfactory explanation of the question. The psychic in- fluence of palatability is one of the most important factors in nutrition, either human or animal. Pawlow and his students have shown that the character of the secretions of the digestive glands is profoundly influenced, both in quantity and quality, by the mental sensations accompanying the taking of food. With food possessing little taste, and giving little or no pleasurable sen- sations on eating, the digestive secretions are scanty and of low digesting power, while the mere enjoyment of eating very pala- table foods, even when these never actually enter the stomach, as was the case in his system of "sham feeding," the gastric and pancreatic juices produced by dogs are very abundant and of high digesting power. Lusk 31 says ' ' Not only the quantity of the food makes for the wellbeing, but the quality as well. No amount of actual fuel value could compel the American soldiers of the Spanish war to eat the "embalmed beef" furnished by the government. The flavor is to the man what oil is to the battleship. Without flavor in the food the digestive apparatus does not run smoothly. ' ' Purpose of These Experiments In the experiments to be described, the hope of success was based mainly on the belief that a ration composed of pure pro- teins, carbohydrates, fats and the necessary salts, could be made sufficiently palatable to insure a satisfactory intake and utiliza- tion of food. The primary object was to limit the phosphorus supply wholly to organic forms. All conceivable devices com- patible with this end were resorted to in order to change the taste and relieve the monotony of food supplied from day to day. The first experiment was carried out with two lots of three rats each. Instead of full grown animals, white rats about half grown or somewhat under half the adult weight were employed since it was desirable to obtain an increase in body weight. It was believed also that at this actively growing period there is a better ability to utilize food than when growth has ceased. 30 Pawlow, Work oPia^&A^iSl88BR®02, p. 76. si Lusk, Science of Nutrition, 1906, p. 189. 82 Wi^coni-in Experiment Station. Preparation of the Food Materials The proteins of the food of Lot I consisted of edestin and zein. It was found impossible to prepare any other proteins in the necessary quantity in a phosphorus free condition. Even with these easily obtainable proteins the necessary degree of purity was attained only with much persistence, the proteins retaining a trace of phosphorus with surprising tenacity. Zein was purified by pouring its alcoholic solution into water, and repeating this a large number of times. It was then dried, ground as finely as possible and soaked in 0.5 per cent HC1. This treatment was very effectual in removing phosphorus. "When the extraction was nearly complete the protein was dissolved in alcohol, repre- cipitated by pouring into water and the process of drying and extracting with HC1 repeated. By presenting new surface for extraction the phosphorus removed by soaking with HC1 was greatly increased. Edestin was repeatedly crystallized by cooling its dilute salt solutions. The content of phosphorus steadily but slowly de- creases during this process of purification. "N. The standard of purity for all foods was the failure of the qual- itative test for phosphorus by the Neumann method on 5 grams of material. Glucose was not at hand in this degree of freedom frojn phosphorus and was prepared from wheat starch. Com- "ymeycial starch contains traces of phosphorus. This can be re- .riibved readily by grinding to a fine condition and agitating with ^-a large volume of 0.2 per cent HC1, allowing the starch to settle and decanting the solution. The cane sugar used was free from phosphorus. Butter fat obtained by melting butter and filtering the clear fat- through paper contains small amounts of phosphorus which can be almost entirely removed by thoroughly agitating the warm fat with slightly acidulated warm water. The fat used gave only a yellowish ti,nt with ammonium molyb- date in nitric acid solution. There was no precipitate from 5 grams after warming the test solution at 60 degrees for an hour. In one instance where bacon fat was given to Lot I near the end of the experiment, this contained a small amount of phosphorus in unknown form. The proteins th ^jffi^$f$i} c f§y$ft{$ an appreciable taste, the edestin much more than the zein, probably due to the great Nuclein Synthesis in the Animal Body. 83 insolubility of the latter in water. Further constituents of the ration were corn starch, wheat starch, butter fat freed from phos- phorus as described, cane sugar, milk sugar, pure glucose, choles- terine, and ash of milk. Calcium phosphate and sodium chloride were always added, and at intervals ferric chloride. At the beginning of the experiment a ration containing 12 per cent of protein, 75 per cent carbohydrate (starch and cane sugar), 5 per cent ash of milk, 5 per cent butter fat, 2 per cent calcium phosphate, and 1 per cent sodium chloride, was made up, mixed with a small amount of finely divided cellulose from filter paper, and enough water added to make a dough. This was dried in an oven at 100 degrees C, cut into pieces and preserved in a Mason jar. The rats ate this with apparent relish for about a week, #fter which there was evidence of a waning appetite. The sugar content of the food was changed and they again ate more readily. At this time the food was baked thoroughly and a portion fed in this form. At one time slightly caramelized sugar was used to give a new flavor to the food. At another the food was moistened with water distilled from a strong cheese which was finely ground. This water . possessed in some degree the cheese flavor and caused the rats to eat with more relish. Good results were frequently obtained by leaving fat out of the food entirely for a few days, changing it as much as possible by the methods mentioned above, then relieving the rats of these flavors by feeding the simple food mixed with fresh butter fat. This in- variably induced a good consumption for a day or two. On some days the ration was presented flavored with a trace of bananna, celery, cinnamon, lemon or vanilla flavors obtained from the com- mercial articles. The rats generally ate the ration on such oc- casions but it can not be determined to what extent the consump- tion was induced by these substances. As time went on it was found that when the mixed foods were not eaten readily, pure edestin would be consumed with avidity, but only for one feeding. Glucose was frequently given sep- arately and considerable quantities were eaten. In one instance toward the end of the experiment, bacon fat, freshly ' rendered and filtered through paper, induced a hearty consumption when every other means failed. Cellulose, ground charcoal and bone ash were given at different times to regulate the condition of the feces. Care was algflfg^k-g^ jjjfy f)Nffl@§($l® content in the ration of sodium chloride at intervals in order to secure the change in taste which it afforded. 84 Wisconsin Experiment Station. This ration contained no purines. Even during starvation there is a regular elimination of uric acid arising from the break- ing down of cell nuclei which necessarily accompanies the func- tioning of the cells. The rats used in this experiment must therefore have lost nuclear material daily or they synthesized these bodies from the food supplied. Table I presents the records kept during this experiment. Table I Records of Weights op Rats in Lot I which Received the Inorganic Phosphorus Ration and No Purines. Date. Eat IV. EatV. Eat VI. May 26 Grams. 170 171 172 165 173 159 150 151 154 150 145 135 (77 days) Grams. 108 104 110 100 104 101 97 99 104 100 97 95 93 86 96 90 (104 days) Grams. 124 123 9 125 " 16 118 " 23 118 " 30 105 July 7 105 " 14 104 " 21 111 " 28 105 100 •• 11 95 •' 18 94 " 25 83 " 31 88 Sept. 7 Rat IV was killed Aug. 11. During 77 days it lost 35 grams in weight or 20.59 per cent of its body weight. Rat V was killed September 7. During 104 days it lost 18 grams in weight, or 16.66 per cent of its body weight. Rat VI was killed on August 31. During 97 days it lost 36 grams, or 29.03 per cent of its body weight. Rat IV was in a feeble condition when killed. The other two were still apparently in good condition. It is interesting to compare the behavior of the rats in this ex- periment with those of Falta and Noggerath 32 and Jacob 33 which were fed with similar rations but with no care to induce a good consumption of food by constant change in the flavor of the ration. Their rats lost weight regularly from the beginning if the experiment. In the experiment of Jacob a rat weighing 193 grams lost on an average 11.6 grams per week and died at the end of the sixth week. Another weighing 190 lost on an average 7.7 grams per week, dying after 11 weeks. The third weighing 32 Falta and NoggvC;... 33 Jacob, K, Ztschr. Biol., 1906, 48: 19 u. Path., 1905, 7: 313. Nuclein Synthesis in the Animal Body. 85 162 grams lost only 3.6 grams per week and died only after 17 ■weeks. In Falta and Noggerath's experiment in one instance a rat of 170 grams, or about two-thirds grown, was 10 grams heavier at the end of four weeks than at the beginning of the experiment. Thereafter it lost weight steadily. All of their other rats were nearly full grown animals. It is very suggestive that in the experience of these authors "those animals which were young and had not attained their growth, withstood the artificial and unpalatable ration much hetter than did adults. In the case of my own experiment Rat Y having the smallest initial weight did better in maintaining its body weight than did the other two. In this case no decided loss of weight began until August 1, after the animal had been •on the artificial ration 66 days. In all of these rats the increased palatability of the ration deferred for some time the decrease in their body weight. Lot II of this experiment, were fed the same ration as Lot I, and the same precautions were taken in both experiments to secure the consumption of food. In addition, however, they received purine bases prepared from liver, and also lean beef which was hydrolyzed with 15 per cent H,S0 4 until the biuret reaction dis- appeared. It is fair to assume that the phosphorus in this product was reduced to inorganic forms. It was thought that this mixture of amino acids might be efficient in rendering the taste of the food more pleasant. It was given only in small amounts and at irregular intervals to aid in relieving the mo- notony of the ration. The record of these animals is shown in Table II. Eat VIII lost during the experiment covering 106 days, 27 grams, or 20.76 per cent of its body weight. Rat IX lost 28 grams during the same period, or 26.41 per •cent of its body weight. It is again apparent that by adding to the palatability of the food, the time at which the steady loss of weight began was de- ferred to about the 50th day in the case of rat IX and to about the 60th day in the case of rat VIII. Rats VIII and IX were still in a fairly good condition at the end of the experiment, but it was evident here as in the case of Lot I that if kept cEtigliizdbidfyMicmwS® would die after a few weeks more. They were therefore killed for analysis. 86 Wisconsin Experiment Station. Table II Weights of Rats in Lot II Which Received the Inor- ganic Phosphorus Ration Together with Purine Bases and Amino Acid Mixture from the Hydrolysis of Beef Muscle. Date. Kat VIII. Eat IX. Date. Eat VII. May 23 Grams. 130 122 124 130 124 135 124 119 128 129 123 119 117 111 103 100 103 (106 days). Grams. 106 99 104 90 104 92 93 96 99 97 95 90 88 85 87 80 78 June 12 Grams. 153 Tune 23 im June 30 168 July 7... 164 July 14... 175 July 21 180 July 28.... 170 July 7 176 July 14 Aut'. 11. (53 days) 169* July 21 July 28 Aug. 18... Aug. 25. . . . 158 102 Aug. 18 (32 days). Aug. 2? Sept. 2 Sept. (i * All phosphorus left out of the ration from this time on. Rat VII was a remarkable individual. She ate the rations offered her with unusual persistence, and as her record shows made an actual gain in body weight of 23 grams in 53 days. It was thought desirable to find how much influence the complete removal of phosphorus in any form from the ration would have on her subsequent behavior. As will be seen from the record, her decline was steady and rapid after this change. From the experiment with Lot II it is evident that in two cases; the addition of purines and of the cleavage products of meat caused no appreciable improvement in the condition of the animals. In the case of rat VII, while it did much better than any other rat in the experiment described, it is questionable whether the purines and meat cleavage products were responsible for her unusual behavior, as will appear later. Experiments with Younger Animals The experience thus gained led to the belief that further ex- periments with still younger animals might give yet more satis- factory results. Three young white rats weighing from 35-46 grams were used in this experiment. The ration was the same as was used with Lot I and the methods of feeding were the same. Table ITT shows therffWji^Mi.th&^anintfik on this ration. Nuclein Synthesis in the Animal Body. 87 Table III. — Weights of Yeky Young Rats on a Kation Supplying Only Inorganic Phosphorus and no Pujunes. Date. Eat XVIII. Rat XIX. Date. Rat XX. Aug. 3 Grams. 37 34 42 39 40 42 48 49 51 48 50 54 56 62 60 66 73 76 (127 days) Grams. 85 31 37 33 35 39 40 4t (56 day ) Nov. 17 Grams. 46 " 11 ■■ 24 46 " 18 Dec. 1 49 * " 25 8 50 Sept. 2 • 15 59 " 10 " 21 54 •' 17 58 " 28 " 12 60 Oct. 6 -.. " 13 (56 days) " 20 •' 27 Nov. 3 " 10 " 17 " 24 Dec. 1 8 Rat XVIII gained in 127 days 39 grams or 105 per cent. Rat XIX gained 6 grams in 56 days or 17.1 per cent. This rat was killed by accident, tHe lid of the cage falling upon it. Rat XX gained 14 grams or 30.4 per cent of its body weight in 56 days. Control Experiment with Casein For the purpose of comparison a control experiment was made in which a phosphorized protefn was contained in the ration. Two young rats were fed tts same ration at Lot I, except that in place of calcium phosphate, casein was given. The casein was prepared fresh before each feeding by precipitating separator skim milk with acetic acid, straining and washing, then dissolv- ing the curd in ammonia and reprecipitating with acid. The casein was always mixed with the other food and was given in a perfectly fresh condition. Ash of milk was given with this ra- tion. Casein was not given every day but usually at intervals of two or three days, the same methods being used as in former ex- periments to give variety to the ration. No purines, hydrolyzed meat nor commercial flavors were given since it was not desired to introduce the two first mentioned substances and a satisfac- tory intake of food was secured without the latter. The results are shown in Table IV. The experiment was broken off at this date, the rats bein^fl^f&Att^^effiflial condition. ■88 Wisconsin Experiment Station. Table IV.— Weights of Rats Fed on Inorganic Phosphorus Ration Used with Lot I and Casein. Date. Eat XV. Rat XVI. Sept. 28 Grams. 87 86 90 87 95 93 103 108 108' 111 107 106 (78 days) Grams. 83 Oct. 6 77 '■ 13 80 " 20 78 •' 27 87 Nov. 3 85 '• 10 95 " 17 103 " 24... 103 Dec. 1 102 8 101 " 15 100 Experiment with Normal Ration For purposes of comparison the rate of growth of normally fed rats, receiving a ration of corn, wheat and rolled oats, Table V is included. Table V. -Rate of Growth of Rats Fed Corn, Wheat and Rolled Oats. Date. Rat I. Rat II. Rat III. May 26 Grams. 172 200 207 223 231 233 240 241 245 248 Grams. 135 158 167 187 196 200 203 212 222 228 Grams. 103 125 9 156 16 138 " 23 30 153 176 July 7 14 21 28 11 18 255 250 Experiment with Mature Rat In order to determine whether the period in the life of the ani- mal is an important factor in relation to maintenance with a ration such as was used in these experiments, a single full-grown rat was placed in a separate cage and given the same ration and care that was given Lot I. His attitude toward the ration is shown by the record of his weight during a period of 33 days : Date Weight op rat Oct. 7 233 grams Oct 25 \\\\\VWP^V.V^MW?'^ f f®\'.'.'.'.'.\ 222 "" Nov. 3 204 Nov. 10 195 Nuclein Synthesis in the Animal Body. 8!> The experiment was discontinued. "When the animal was placed on a corn, wheat and rolled oat ration it speedily recovered its weight. It would not eat a sufficient quantity of the arti- ficially prepared ration to supply its energy requirement. Experiment on Phosphorus Excretion In order to throw additional light on the subject of nuclein metabolism it was desired to obtain data showing the actual amount of phosphorus excreted per day when a phosphorus free ration was taken. That this is not necessarily the same as the- phosphorus excreted during starvation is the opinion of Gev- aerts 34 who found that in rats when fed a ration containing no phosphorus the ratio of phosphorus to nitrogen excreted fell to about one-tenth of the value during starvation. In order to obtain more data on this important question, Rat VII, whose record is shown in Table II, was placed in an inverted bell jar having a hole in the center of the dome. A screen formed a floor on which the rat could stand and move about. Asbestos, thor- oughly extracted with hot nitric acid, was spread over this screen to serve as a bed. Each day the rat was removed and the bottom of the bell jar washed with a small sponge, the washings being collected in a large dish placed below. The screen floor was washed after removing the asbestos and the latter placed in the dish with the washings. Fresh dry asbestos was placed on the screen and the rat replaced in the jar. The washings and asbes- tos bed were then acidified strongly with nitric acid, then boiled and the whole transferred to a suction filter and the asbestoi washed free from phosphorus. The washings were then concen- trated, sulphuric acid added and the solution transferred to a flask and all organic matter destroyed by oxidizing with nitric- acid according to the Neumann method. Phosphoric aeid was determined by precipitating with ammonium molybdate in nitric aeid solution and was weighed as magnesium pyrophosphate. The phosphorus excreted is shown in Table VI. This experiment was begun on August 25th and continued eight days. The ration given during this period consisted of zein, edestin, starch, sugar and butter fat. The ash constituents given were- magnesium oxide, calcium sulphate, potassium chloride, sodium- carbonate, sodium chloride, and ferric chloride. s* Gevaerts, La cM% e A b &W crosom 90 Wisconsin Experiment Station. Table VI Phosphorus Excreted by Rat VII. Day. Phosphorus excreted. Day. Phosphorus excreted. 1 Grams. 0.0085 .0084 .0076 .0114 .0089 7 .. Grams. .0012 2 '8 .. .0051 3 Total 4&5 0.0511 6 0.0063 It is not possible to say how much the rat was eating during the days of this experiment, but it is known that some food was taken each day. Gevaerts 35 found for starving rats an excretion of phosphorus in an animal weighing 210 gram, amounting to 0.007 to 0.0114 gram per day. In another experiment the amounts were 0.0086 to 0.0184 gram per day. When phosphorus free edestin and cane sugar were fed Gevaerts observed an excretion of only 0.001 to 0.004 gram of phosphorus per day for rats weighing 180 to 200 gram. In a second trial with a rat weighing 217 grams, the writer starved it for four days, then fed edestin and cane sugar for a period of four days. The excreta were collected together for the entire period and the phosphorus amounted to 0.017 gram, or 0.0043 gram per day. Analyses op the Carcasses The phosphorus was determined in the entire bodies of nine rats (exclusive of the skeletons), by boiling the rats after opening the stomach and intestines and discarding their contents and removing the skeleton, great care being taken to remove the small bones of the feet. The tissues and water in which the rat was boiled were evaporated to dryness, dried in an oven, ground and sampled. Analysis of the dry tissues of nine individuals, includ- ing all rats in Tables I and II, and three normally fed ones, cal- culated on the basis of the live weight less the weight of the skeleton, showed an average phosphorus content of the body of the skeleton-free living rat to be 0.19 per cent (P). This fur- nishes interesting data for calculation. If a rat weighing 200 grams excrete 0.0Q5.gram^phosp.horus^i£r day, since his body 3= Gevaerts, La, Cellule, 1901, 18: 7. Nuclein Synthesis in the Animal Body. 91 would contain 0.38 gram of this element, in the course of 76 days the entire content of phosphorus in his body would change. Since of the three rats in Tables I and II which were continued beyond 100 days, the loss of body weight was 16.66, 20.76 and 26.41 per cent respectively (average 21.27 per cent), even in these experi- ments it would seem incontrovertible that the animals were utilizing the inorganic phosphorus supplied for nuclein and phos- phatide formation. This is all. the more convincing when the weights of the skeletons of these animals are taken into account. The abnormally large skeletons found in those animals receiving a large supply of inorganic phosphates is strikingly evident even though the body weight did not increase. They were not, there- fore, drawing phosphorus from this source. This is in harmony with the observations of Hart, McCollum and Fuller with pigs. Even admitting Gavaert's lowest figure: 0.001 gram per day, a 100 gram i'at, containing 0.19 gram of phosphorus in its soft tissue 1 -', would metabolize 53 per cent of this during 100 days. This is not in harmony with a loss of 16 to 20 per cent of body weight in the same time. As a possible explanation of the results here presented, it might be urged that the gains in weight observed were due to an exces- sive deposition of fat or an undue accumulation of fluids in the body as sometimes occurs as a pathological condition, the animals used were accordingly in most cases subjected to the following analyses : The weight of the animal at the time of death was as- certained. It was then boiled with water until thoroughly cooked, when the skeleton was completely removed from the tis- sues. The tissues and water in which the rat was boiled were united and the whole evaporated on the water bath to dryness. The dry tissues (less the skeleton) were then dried in an oven at 100 degrees C. for several days, carefully scraped from the dish, ground, weighed and placed in a bottle. The skeleton was dried in an oven at 100 degrees C. and weighed and afterward ignited to a nearly white ash in a muffle. The weight of the ash was obtained. The fat in the dry tissues was extracted with ether for 24 hours, then the tissues were ground a second time and again extracted 12 hours. While this does not give the total fat of the tissues, it is believed to represent nearly all of the fat deposited as such and not the invisible f^yrff/^^/^S^^^J^j^giues. The tissues thus extracted were weighed and the weight taken as fat free tissue. 92 Wisconsin Experiment Station. The data thus obtained for all the rats analyzed is given in Table VII. Table VII. — Composition of Rats in Experiments Rations. with Vakious Eation. Normal Inorganic Phosphorus. [Later P free] Inorganic P Num- ber of rat. I II X VII IV v VI VIII IX XVIII Weight. Grams. 147 157 34 102 135 96 ?t: Skele- ton. Grams. 6.67 6.50 1.33 7.14 9.00 5.78 7.50 6.03 4.58 4.07 Dry tissues less skele- ton. Grams. 38.0 45.0 9.5 25 5 34.8 22.0 20.5 21.5 14.5 17.5 Skele- Ether Ash of ton. ex- skele- ton. Weight of rat. Grams. Grams. Grams. 8.89 3.79 4.45 10.80 3.85 4.14 3.25 .68 3.91 3.42 4.49 7.00 7.00 4.28 6.66 4.01 3.02 . 6.02 3.63 3.18 8.5i 2.67 3.52 5.85 .8d 2.77 5.87 3.40 2.14 . .36 Fat free tissues. Weight of rat. Grams. 19.90- 21.79- 18.39- 21.76- 20.59 18.74 19.17 17.79- 17.50 18.58 The data presented in Table VII shows that while the ratio of fat free tissues to body weight varies considerably in individuals- as would be expected, this variation does not point to an ab- normal composition of the body of any individual examined. The fat content and muscle and organ content of the experi- mental animals must be considered normal. Hence the gains in weight in the case of rats VII and XVIII are to be taken as proof of an actual increase of muscle tissue and of organ tissue. This is true also for rats XIX and XX in which a fair gain in body weight was observed. That the composition of the organs and tissues with respect to phosphorus, calcium and moisture is always maintained normal is shown by the data furnished by Hart, McCollum and Fuller in their analysis of pigs' tissues. That the composition of the tis- sues with respect to other constituents is also normal can not be doubted. The per cent of phosphorus in the ash of the bones o!i all rats described in Table VII was found to be the same. Conclusions The data furnished by these experiments seem to warrant the following conclusions: 1. The palatability of the ration is a most important factor in animal nutrition. Without palatability the ration may possess all the necessary ^M^kiMm^fet fail to nourish an animal properly. Nuclein Synthesis in the Animal Body. 93 2. The failure of previous efforts to maintain animals on a mixture of relatively pure proximate constituents of our food stuffs was due to the lack of palatability of such mixtures. 3. When sufficient care is given to changing the character and flavor of the food supplied in such simple mixtures, it is possible to induce an appreciable amount of growth. 4. Very young animals adapt themselves to a ration possessing a low degree of palatability, much better than do adults. 5. Other things being satisfactory, all the phosphorus needed "by an animal, for skeleton, nuclein or phosphatide formation, can be drawn from inorganic phosphates. fi. The animal has the power to synthesize the purine bases necessary for its nuclein formation from some complexes con- tained in the protein molecule, and does not necessarily use purine bases of exogenous origin for this purpose. Digitized by Microsoft® Digitized by Microsoft® Digitized by Microsoft® Digitized by Microsoft® THE UNIVERSITY OF WISCONSIN Agricultural Experiment Station STATION STAFF The President of the University H. L. Russell, Director i S. M. Babgook, Assistant Director I Iba Herfurth, Executive Olerk ' W. A. Henry, Emeritus ProfessqJ,of Agriculture . A. 8. Alexander, Veterinary Science, In icharge of Stallion Licensing S. M. Babcock, In charge of Agricul- tural Cnemistry - -: L..J. Cole, In charge of Experimental Breeding. E. J. "Dblwighe, Supt. Northern Sub-Sta- tions, -'£?»