u. s, V ExpeY Dep* kg. G A b r r_L culture iron Admin* iion^u Oil' iota of illystin No) 121 LL S. DEPARTMENT QF AGRICULTURE. — / OH'FICE OF EXPERIMENT STATIONS BULLETIN NO. 121. A. C. TRUE, Director. EXTKIJINLTCNTS THE METABOLISM OF NITROGEN, SULPHUR, AND PHOSPHORUS IN THE HUMAN ORGANISM. lEPOSITORY H. C. SHERMAN, Ph. D., Instructor in Analytical Chemistry, Columbia University. CONDUCTED IN COOPERATION WITH COLUMBIA UNIVERSITY. WASHINGTON: GOVERNMENT PRINTING OFFICE, 1902. LIST OF PUBLICATIONS OF THE OFFICE OF EXPERIMENT STATIONS ON THE FOOD AND NUTRITION OF MAN. Note.— For those publications to which a price is affixed application should be made to the Super? fntendent of Documents, Union Building. Wartiingl tie ameer designated bylaw Government publications. Publications marked with an asterisk <*) are not available for distribution. ♦Charts. Food and Diet. By W. O. Atwater. (Four charts, 26 by 4F AGRICULTURE. O.FIGE OF EXPERK, - NO. 121. A. C. TRUE, D EXPERIMENTS THE METABOLISM OF NITROGEN, SULPHUR, AND PHOSPHORUS IN THE HUMAN ORGANISM. BY H. C. SHERMAN, Ph. D.. lor in Analytical Chemistry, Columbia Uni CONDUCTED IX COOPERATION WITH COLUMBIA UNIVERSITY, WASH I NGTON: GOV E R N M E N T PRINTI'NQ OFFIC E . 1 9 2. OFFICE OF EXPERIMENT STATIONS. A. C. True, Ph. D., Director. E. W. Allen, Ph. D., Assistant Director and Editor of Experiment station Record. C. F. Langworthy, Ph. D., Editor and Expert on Foods and Animal Production. NUTRITION INVESTIGATIONS. W. O. Atwater, Ph. D., Chief of Nutrition Investigations, Middletown, Conn. C. D. Woods, B. S., Special Agent at Orono, Mi . F. G. Benedict, Ph. D., Physiological Chemist, Middletown, Conn. R. D. Milner, Ph. B., Editorial Assistant, Middletown, Conn. 2 1 LETTER OF TRANSMITTAL. U. S. Department of Agriculture, Office of Experiment Stations, Washington, D. C., October 1, 1902. Sir: I have the honor to t ransmit herewith a report on experiments on the metabolism of nitrogen, sulphur, and phosphorus in the human organism, carried on by II. C. Sherman, Ph. D., instructor in analyt- ical chemistry at Columbia University, New York, in cooperation with this Department. The investigations were conducted under the imme- diate supervision of Prof. W. O. Atwater, chief of nutrition investi- gations, and form a part of the investigations on the food of man conducted under the auspices of this Office. Doctor Sherman's investigations have for their special object a study of the cleavage of protein, with reference particularly to the waj 7 in which this nutrient serves for building tissue and as a source of energy. The results given herewith constitute a progress report. The report is submitted with" the recommendation that it be pub- lished as Bulletin No. 121 of this Office. Respectfully, A. C. True, Director. Hon. James Wilson, Secretary of AgricuLtwre. 3 CONTENTS. Page. Introduction 7 Metabolism and elimination of sulphur 7 Metabolism and elimination of phosphorus 10 Previous work on the comparative metabolism of nitrogen, sulphur, and phosphorus 13 Purpose and plan of the experiments . _- 15 Analytical methods 16 Composition of food materials 18 Composition of feces 19 Experiments on the digestibility of bread and milk 19 General description of experiments 19 Digestion experiment No. 1 - . . 21 Digestion experiment No. 2 22 Digestion experiment No. 3 23 Digestion experiment No. 4 23 Digestion experiment No. 5 24 Digestion experiment No. 6 25 Digestion experiment No. 7 ■ 25 Digestion experiment No. 8 26 Digestion experiment No. 9 27 Digestion experiment No. 10 . 27 Results of digestion experiments . . 28 Comparison of the metabolism of nitrogen, sulphur, and phosphorus 31 Influence of loss of sleep 34 Lag of elimination after change of diet 36 Comparison of balance of income and outgo 43 Summary 47 5 LLUSTRATIOW Fig. 1. Diagram showing the fluctuations in the daily excretion of nitrogen and phosphorus during the first series of experiments (Nos. 1-3) - Diagram showing fluctuations in the daily excretion of nitrogen and phosphorus during the second series of experiments (Nos. 4 and 5) Diagram showing fluctuations in the daily excretion of nitrogen, sulphur, and phosphorus during the third series of experiments (Nos. 7-10) . 6 2. 3. Pa ire. 39 40 METABOLISM OF NITROGEN, SULPHUR, AND PHOSPHORUS IN THE HUMAN ORGANISM. INTRODUCTION. .Most of fche digesl ion experiments heretofore reported in connection with the nutrition investigations of this Department have included the determination of the balance of income and outgo of nitrogen, while in those carried out in the respiration calorimeter the balance of carbon, hydrogen, and energy arc likewise determined. It is believed that in many cases the determination of income and outgo of sulphur and phosphorus will add considerably to the interest and value of these investigations. The sulphur or phosphorus balance, like the nitrogen balance, may be found by comparing the amounts ingested in the food with those eliminated through the kidneys and intestines. So far as is known no phosphorus and only traces of sulphur escape in the form of vola- tile compounds, and the quantities of sulphates and phosphates in the perspiration are so small that they may probably be neglected, unless in exceptional cases. METABOLISM AND ELIMINATION OF SULPHUR. Small quantities of sulphates occur in foods and in some waters. By far the greater part of the sulphur of the food enters the body in organic combination, in proteids or albuminoids. When proteid ^ rj mat ter is oxidized in the body mos t of the sulphur is burned to su l- phuric acid , the greater part of which appears in the urine as normal 'inorganic sulphates?) A smaller part of the sulphuric acid (in health "usually about one-tenth) is found in the form of e thereal sulphate s, i. e., combined with organic radicles, the latter being usually regarded as derived chiefly from intestinal putrefaction of proteids. Such putrefaction may give rise to the formation of hydrogen sulphid, which may either appear as sulphids, chiefly of iron and the, alkali metals , in the feces, or may be absorbed into the system, or may to some extent escape with the intestinal gases. The total sulphates of the urine may readily be determined by precipitation as barium sulphate after boiling the urine with hydrochloric acid to set free the sulphuric acid in "ethereal" combination. Not all of the urinary sulphur, however, exists in the form of sulphates. About 15 to 20 per cent is 7 8 usually found in less completely oxidized forms, this portion being called " unoxidixccl " or •• neutral" sulphur, to distinguish it from the fully oxidized sulphate-sulphur. The existence of sulphur in other forms than sulphates in the urine was discovered by Ronalds at Giessen in 1846,° but was first bronghl into prominence by Bischoff and Voil in I s . Several compounds have been described as con- tributing to the "neutral" sulphur of the urine. The taurin of the bile is held to be largely reabsorbed from 1 he intestines and eliminated through the kidneys. If taurin be fed directly the amount of neutral sulphur in the urine incr< jcording to Salkowski," and in experi- ments upon a dog with a biliary fistula the neutral sulphur was found to decrease, but did not entirely disappear.'' Among other sulphur compounds which have been found in the urine maybe mentione d sul - phocyanids, originally derived from swallowed saliva.' thiosnlphates. small quantities of cvstin./ of mucin, and occasionally of i iydrogen sulphid^ Abel-' has described a body which yields ethyl sulphid, and it is probable that other compounds remain to be discovered, since the quantities of the above compounds believed to exist in normal urine are not sufficient to account for all of the neutral sulphur found. According to Spiegel 7 ' the appearance of cystin and hyposulphites in the urine points to a condition of diminished oxidation, since these compounds though constantly formed in the bod}' are not normally end products of metabolism. The following quotations from recent text-books (which are given in chronological order) are believed to fairly represent the present general teachings in regard to the significance of the sulphur metabolism and its relation to the metabolism of nitrogen. Halliburton ' says: The sulphuric acid of the urine is in part combined as ordinary sulphates, in part as ethereal sulphates. It is derived to a small extent from the food, but chiefly from the metabolism of proteids, the amounts of sulphuric acid and urea in the urine running parallel. According to Hammarsten:^ The sulphuric acid of the urine originates only to a very small extent from the sulphates of the food. A disproportioiially greater part is formed by the burning ^Falck's Beitrage zur Physiologie, Hygiene, etc., p. 102. &Gesetze der Ernahrung des Fleischfressers. pp. 279-284. 302-303. I c Ci. Lusk. American Text-book of Physiology. Vol. I, p. 507. tfKunkel. Arch. Physiol. [Pfluger]. 14 (1887), p. 353. ^Leared. Proc. Royal Soc. London. 1870. pp. 16. IS: I. Musk, Arch. Path. Anat. u. Physiol. [Virchow]. GO (1877). p. 354. /G-oldniann and Baumann. Ztschr. Physiol. Chem.. 12 (1888). p. 254. <7Ztsckr. Physiol. Chem . '20 (1894). p. 353. *Arch. Path. Anat. u. Physiol. [Virchow]. 166 (1901), pp. 364-371: abs. in Jour. Chem. Soc. [London]. 82 (1902), No. 471. II. p. 93. MSchaffer's Text-book of Physiology. Vol. I. 1898. p. 79. I J Text-book of Physiological Chemistry, trans, by J. A. Mandel. 1898, p. 515. of the proteida containing sulphur within the body, and it is chiefly this forma- tion of snlphuric acid from the proteida which gives rise to the previously men- tioned excess of acids over the bases in the arine. The quantity of snlphnric a id eliminated by the urine amounts to 2.5 grams B 8 SO.nii !„■') • l- A complete parallelism ran hardly be expected, as in tin- first place, a pari »>t' the sulphur is always eliminated as neutral sulphur, and secondly, because the low quantity of sulphur in different protein bodies undergoes greater variation as compared with the high quantity of nitrogen con- tained therein. Generally the relationship between the elimination of nitrogen and sulphuric acid under normal and d seased conditions runs rather parallel. In Novy's" opinion: The proteins of the food and of the tissues constitute almost the sole source of the sulphur containing waste products. A small amount of waste sulphur com- pounds is eliminated as sulphocyauate by the saliva, gastric juice, etc. Another small portion leaves the body as taurin in the taurocholic acid of the bile. With these exceptions almost all the sulphur resulting from protein disintegration appears in the urine. Inasmuch as the sulphates contain most of the waste sul- phur it follows that the total sulphates in the urine furnish an excellent index of proteid disintegration. According to Ogden : b The total quantity of sulphuric acid in the twenty-four hours' amount of urine of an adult taking a mixed diet is from 1.} to 3 grams, or an average of 2 grams. About one-tenth of the total sulphuric acid is in the form of ethereal sulphates. In general it may be stated that the variation in the quantity of ordinary sulphates eliminated in the urine runs parallel to that of urea. Lusk. c states that : Sulphur is built in the proteid molecule of the plant from the sulphates taken from the ground. It is found in albuminoids, especially in keratin. As taurin it occurs in muscle and in bile, as iron and alkaline sulphids in the feces, as sul- phureted hydrogen in the intestinal gas, as sulphate and other unknown com- pounds in the urine. * * * The total amount of sulphur in the urine runs icu^^f* 2 proportionately parallel with the amount of nitrogen; that is to say, the amount 1^^' t is proportional to the amount of proteid destroyed. * * * When an animal eats proteid and neither gains nor loses the same in his body, the amount of sul- phur is equal to the sum of that: found in the urine and feces. Sulphates eaten pass out through the urine. They play no part in the life of the cell. Thus there is general agreement in regarding the sulphur of the urine as essentially derived from katabolism of proteid in the body, so that the quantity eliminated is, like that of nitrogen, an indication of the amount of proteid matter broken down. This agreement in regard to the parallelism of the nitrogen and sulphur excretion is, however, by no means exact, since in some cases the reference is to total sulphur, in others to total sulphates, and in still others to "ordi- nary " sulphates. « Physiological Chemistry, second ed., 1898, pp. 194. 195. & Clinical Examination of the Urine, 1900, p. 111. <• American Text-book of Physiology, second ed.. Vol. I. 1900. pp. 505, 507. 10 Of the authors quoted. Hammarsten is the only one to call atten- t ion to the fact thai this parallelism will be affected by th e variations in the relative proportions of nitrogen and sulphur in different pro- teids . Thai these variations are very large will be seen from a com- parison of the accepted analyses of a few representative proteids. Taking, for Instance, the elementary analyses recently compiled by Osborne in connection with his discussion of the sulphur in proteid bodies," Ave estimate from the percentages given thai the ratio of nitrogen to sulph ur is, in jegumin a s L6.9: 1 ; in f.cin . 26.9: 1 ; in edeatin . 21.2:1; in bynin , 19.4:1; In ffliadi n, L7.2: 1; and in leucosin, 13.1:1. From this it will appear thai the typical proteids oi wheat furnish about three times as much sulphur, with a given amount of nitrogen, as the typical proteid of the legumes. The ratio in casein (19.7:1) is about twice as great as in egg albumin (9.6: J). Among the proteid constituents of the body the differences are even greater than among the food proteids just mentioned. In o xyhemoglobin the ratio is 44.6:1; in myosin , 13.1:1; in serum globuJrtn. 14.3: lj in fibrinogen, 13.3:1; in s erum albumin from human exudation, 7.06:1; in chon- droniucoid/ 5.2:1; in tendon-mucin / 5:1, and in o sseomucoid, 6 5: 1. Thus it would appear that the katabolismof sufficient glucoproteid to yield a gram of nitrogen would result in the elimination of about three times as much sulphur as the katabolism of an equivalent amount of myosin, serum globulin, or gliadin, and nearly ten tines as much as would come from an equivalent amount of oxyhemoglo- bin or of legumin. It is evident, therefore, that the ratio of nitrogen to sulphur in the urine may undergo considerable variation as the result of changes in the kind of proteid given in the food or in the kind of body tissue katabolized in case the protein of the food is insufficient. The interesting investigations of Kolpatcka noted below (p. 13) are based largely on these variations. There is, however, everjr reason to believe that so long as the diet is uniform, and othe] conditions normal, the metabolism and elimination of sulphur wil be nearly parallel with that of nitrogen ; and this seems to be true not only as concerns the twenty-four hours' urine, but usually for shorter periods as well. (See p. 45.) METABOLISM AND ELIMINATION OF PHOSPHORUS. Phosphorus enters the body in organic combination in the form of nucleins, n ucleo-prote ids, l ecithin , p rotagon , and perhaps glycerol - « Connecticut State Sta. Rpt. 1900, p. 464: Jour. Amer. Chem. Soc, 24 (1902), p. 140. b The ratios given for the glucoproteids are from figures given by Hawk and Gies (Amer. Jour. Physiol., 5 (1901), p. 416. In the case of osseomucoid the aver- age of the later and purer preparations is taken. The figures for tendon-mucin (Chittenden and Gies) have recently been confirmed by Cutter and Gies (Amer. Jour. Physiol. . 6 (1902) . p. 155. The figures for chondromucoid are from the work of Morner (Ztschr. Physiol. Chem.. 18 I L893), p. 213, 11 phosphoric acid, but a Larger quanl ity i< taken as mineral phosphates in the food. The proportion of phosphorus eliminated by the intes^ tine depends mainly on the nature of the food and the alkalinity or the blood. Berbivora excrete nearly all of the phosphorus with the feces, and in man 1 he a mount 1 h us excreted is greatest on a vegetable diet or one ri ch in lime salts, a nd may he Largely increased by feed- ing alkaline cit rate and calcium carbonate, the first to furnish the more alkaline reaction to the blood and urine, the second to form with the phosphoric acid the insoluble phosphate of lime (Lusk). a / The phosphorus of the urine is present chiefly as phosphates of I /tin' alkalies, with a much smaller quantity of phosphates of they alkaline earths. A very small proportion is present in organic com- bination^ This has been believed to exist as glycerol-phosphoric acid. Jolly/' however, claims to have found in the urine certain peculiar nitrogenous compounds, which retain some mineral phos- phate in such intimate association that the phosphoric acid is not precipitated by the usual reagents, and he believe^ that it is these phosphates and not. glycerol-phosphoric acid or any incompletely oxi- dized form of phosphorus which escapes precipitation by the ordinary methods. Since t lie phosphorus of the urine comes so largely from the simple passage through the system of the phosphates taken in the food, it follows that variations in the quantity eliminated are more apt to be connected with the diet than with the metabolism of bod} T mate- rial. The idea once held that the quantity of phosphorus eliminated is principally dependent upon the metabolism of nervous tissue was soon abandoned. In this connection Voit* stated that the bones contain about 1.400 grams of phosphorus, the muscles about 130 grams, and the brain and nervous sj'Stem about 12 grams. Moreover, by comparing the loss of weight of different organs in the starving dog. with the changes in the ratio of nitrogen to phosphorus in the urine, he was able to show that the body material katabolized was largely contributed by the bones. Recent work tends to emphasize the importance of the nucleins and related bodies and to confirm the view thai the phosphates found in a ll the organs and tissues of the body a re t o a considerable extent in ch emical combinatio n with the proteid matter. Thus it is stated that, when the body stores proteid a proportionate amount of phosphoric acid is retained for the new protoplasm, while on destruction of pro- teid the phosphoric acid corresponding to it is eliminated. d «See also the recent work of Paton and his associates (Jour. Physiol.. 25 (1900), p. 212), comparing the metabolism and elimination of phosphorus in the dog and in the goat. »Conipt. Rend. Acad. Sci. Paris. 127 i 1898), 118. <" Hermann's Handhnch des Physiologic vol. 6. pt. 1, p. 80. 'Lnsk. American Text-book of Physiology, second ed.. 1900. Vol. I. p. 575. 12 Hie significance of the phosphorus metabolism from the medical standpoint is quite fully discussed by Bergell.* Several investigators* have recently studied the urinary excretion of phosphates as influenced by those conditions which are believed to be especially connected with the met al >ol ism of nudeins. An intimate connection between changes in the phosphorus elimi- nated and in the kaiab >lism of nueleins is evidently assumed by Dunlop, Paton, Stockmann, and Maccadam in interpreting the results of their investigations of the effects of muscular exertion/ In these experiments each subject maintained a uniform diet for seven days. on the fourth of which as much exercise (bicycle riding) was taken as the subject could endure without serious discomfort. In each case the day or days following the exertion showed an increased elimina- tion of nitrogen and sulphur, but only when the subject was in poor training was there a corresponding increase in the elimination of phosphates and of uric acid. From this it was concluded that with the -subject in good training only muscle proteid is broken down, while if the subject be in poor training this consumption of muscle proteid is accompanied b} T the consumption of the material of other tissues which contain nucleo-proteid. In this connection it is inter- esting to note the observation previously made by Preysz, (/ that the increased elimination of phosphoric acid resulting from walking a given distance (2d kilometers) was considerably greater when the distance was walked at a rapid rate, causing a more intense though less pro- longed exertion. As already stated, the greater part of the phosphorus eliminated comes from the phosphates of the food. fwhen, however, the diet is uniform, a variation in the phosphorus elimination must be taken as showing some change either in body metabolism or in the condition of the body with reference to its store of phosphates^ Whether or not the connection between urinary phosphates and the katabolism of nueleins is as intimate as some investigators seem to assume, it is evi- dent that the study of the phosphorus balance may give valuable information which could not otherwise be obtained regarding the nature of the changes taking jjlace in the body. «Fortschr. Med., 1G (1898), p. 1. Bedeutung der Phosphorsaure in mensch- lichen und thierschen Organisuien. Inaug. Diss.. Berlin. 1898. 'Oloraczewski. Arch. Path. Anat. vi. Physiol. [VirchowJ . 151 (1898). p. 88; Milroyand Malcolm. Jour. Physiol.. 23 (1898), p. 817, audio (1899). p. 103: White and Hopkins. Ibid.. 24 (1899). p. 42: Loewi. Ar< h. Exper. Path. u. Pharmakol.. 44 (1900-1901), p. 1: abs. in Jour. Chem. Soc. [London]. 78. 1900. II. p. 417. ejour. Physiol.. 22 (1897-98). p. 68. tfUngar, Arch, Med.. 1 (1892-93), p. 38: reviewed in Arch. Physiol. [Pfltiger]. 54 (1893), p. 81. 18 PREVIOUS WORK ON THE COMPARATIVE METABOLISM OF NITROGEN, SULPHUR, AND PHOSPHORUS. The coursi of tlu elimination during tin day. —Considerable atten- tion lias been given by differenl investigators to tin' course of the elimination of nitrogen and of phosphorus daring the day. The recent work of Rosemann a on nitrogen and of his pupil Roeske* on phosphorus may be especially aoted. (Jnfortunately such studies have usually been made upon only one element at a time. In some recent experiments carried out in the Laboratories of Wesleyan Univer- sity' the course of elimination of nitrogen, sulphur, and phosphorus has been observed simultaneously, the urine being collected in the three- hour periods during the day with one nine-hour period at night. The rates of elimination of nitrogen and sulphur were found to run nearly parallel, rising and falling twice during the day and reaching a minimum during the night. The fluctuations, though quite reg- ular, were not very great, the highest rate of elimination found dur- ing the day being usually about one-fourth greater than the average rate for the nine hours of the night. The elimination of phosphorus, on the other hand, did not run parallel witli that of nitrogen and sulphur, and the fluctuations, though less regular, were considerably larger, the maximum rate of elimination being two to three times as great as the minimum. Moreover, the minimum rate of elimination of phosphorus was reached not during the night, but at some time in the forenoon, usually from one to three hours, but sometimes from four to six hours after rising. Comparativt metabolism during periods of a day or more. — Many metabolism experiments have been made in which nitrogen and phos- phorus were determined and a smaller number in which sulphur was also included. Several of these investigations will be referred to later in connection with the discussion of the results of experiments here report e< I. The investigations of Kolpatcka d are, however, so sugges- tive that they should be mentioned here. The subjects were in all cum^ dogs, and the object of the work was to learn the real source of the nitrogen in the urine — to determine whether it is derived directly from the protein of the food, from protein stored in the body, or from actual proteid tissue — and further, to study the nature'of the stored protein. Arch. Physiol. [Pfluger], 66 (1896), p. 343. •ber den Verlauf der Phosphorsaure Ausscheidung beim Menschen. Inaug. Diss., Greifswald, 1897. c Sherman and Hawk, Amer. Jour. Physiol.. 4 (1900). p 25, and unpublished results by At water and Hawk and by Hawk and Chamberlain.. These experi- ments are more fully described in connection with the discussion of "lag" on p. 36. (l Phiziologicheskii Sbornik. A. I. and V. I. Danilevski. editors. Kharkov, 1888, Vol. I. p. 33; abs. in U. S. Dept. Agr., Office of Experiment Stations Bui. 45, pp. 308, m 14 Kolpatcka endeavored to solve these problems by comparing the ratios of phosphoric acid to nitrogen and of sulphur to nitrogen in the food consumed and in the urine. The ratios found in the foods osed were as follows: In meat, P 8 5 :N:: 1:7.3; S:N:: 1:15.6. In gelatin, which contains no P.>0 5 , the ratio Is as follow-: S:N:: L:22.5. In white- of eggs, P 2 5 :X:: 1:47.6; S:X::1: 9.8. In yolks of eggs, 1' « l : X:: 1: L.8. Knowing the ratios of these elements in the food and in the urine during partial or complete fasting, it was held to be possible to judge whether the nitrogen in the urine for any particular period came from the food consumed, from stored protein, or from actual body tissue. Thus on a meat diet the ratio of P 2 5 :X in the urine was nearly the same as in the food, and it was concluded that the excreted nitrogen came directly from the food. During a period of lasting following the meat diet the relative proportion of phosphorus excreted gradually increased until the fifth day, after which the ratio was nearly constant. P.,0 5 : X:: 1: 4 (about). A similar change in the ratio was found after a change from meat diet to a diet of fat and starch. These results are held to show that when the supply of pro- tein is cut off there follows a katabolism, first of protein simply stored from the previous diet and not yet organized, then of protein from body tissue, this last being the sole source after the fifth day and yielding a relatively large proportion of phosphorus. The increased proportion of "earthy" phosphates led to the belief that some of the "tissue protein" came from the bones, a conclusion reached several years ago by Voit. (See p. 11.) On passing from a meat ration to a ration of white of egg there was a diminution of phosphoric acid and an increase of sulphur. The ratios, however, varied considerably, and a relatively large amount of phosphoric acid in the first Milk ( experiments Nos. 7-9) Milk (experiment No.10).. Per ct. Per ct. Per ct. 9.32 1.610 10.06 86. 51 . 86.74 i . 9.11 ffi . Ms 10.81 1. 7.31 1. 86. 73 86. 50 . 535 3.34 542 3.39 180 1.13 4!C) 3.09 820 11.38 680 10.50 506 3.16 Per ct. 6.21 4.42 4.26 86. 97 4.26 6.18 6. 4!) „: 4.53 Per 72. 4. 4. 4. 7i). 73. 4. ct. 32 Per ct. 2.09 98 . 75 86 . 75 ... •.'. 79 90 .67 39 1.84 38 2. 32 81 .73 01 .70 Cats. 4.221 .767 .778 B.010 .780 4.172 4.301 .795 Per ct. 0.130 Phos- pho- rus. Per ct. 0. 110 .036 . 042 .034 .143 .130 .034 .115 .109 .094 ajonr. Aiuer. Chem. Soc. 24 (1902), p. 1100. 19 COMPOSITION OF FECES. As slated above, the feces were analyzed by the same methods as the food materials. The composition of the feces from the various experiments here reported is show n in Table 2, the results being given on the water-free basis, since the amounl of water in the fresh feces lias no bearing on the questions here studied. Table 2. — Composition of feces. u = z. si p (Mid 661 662 663 r,ti, 665 666 667 668 669 Feces. Experiment No. 1 . Experiment No. 2 . Experiment No. 3 - Experiment No. 4 . Experiment No. 5 . Experiment No. (>. Experiment No. 7 . Experiment No. 8 Experiment No. 9 .. i Experiment No. 10 Total amount. >. f a-d > I - 4 < c ? 1 z Heat of combus t ion ( d 1 e r - mined). 650 Crackers 651 Milk. 653 Butter... Grains. 1,200 8.160 160 Grams. 1.063.0 1,039.5 140.9 Grams. 120. 7 272. 5 1.8 Grains. 74.:> 360.6 139.1 Grams. 867.8 406.4 Grains. 25.1 61.2 4.5 Grams. 19.32 43.66 .29 Calories. 5,066 fi. 256 1,282 Total 2.243.4 395 574.2 1.274.2 90.8 63.27 12.605 661 Feces (water free > 99.1 69.7 2.173.7 96.9 16.8 95.8 16.5 557. 7 97.1 36.4 1.237.8 97.2 29.3 61.5 2.70 60.57 95.8 58.69 564 12,041 95.5 Nitrogen and beat of combustion of urine Energy of food oxi- 438 11,603 Per cent of energy 92.3 28 During this experiment thesubjecl eliminated 3,451 grams of urine, containing 58.69 grams of nil rogen. This makes i be average ail rogen balance per day as follows : Income in food. L5.82 grams; outgo in urine, L4. 67 grams, and in feces, 0.67 gram; indicating a gain to the body of 0.48 gram of nitrogen, corresponding fco 3 grams of protein. DIGESTION EXPERIMENT NO. 3. This experiment began with breakfast July 28, L 900, and continued four days. The weight of the subject (without clothing) at the begin- ning was ill kilograms, at the end 60.9 kilograms. Table 5. — Results of digestion experiment No. S (serial No. a Pi >.= it Kind of food. & a .a '5 Total organic matter. Protein(Nx6.25). -t-> CO 9 o 3 i 525 jr. ■' o = -: -.£■= ; - - H 650 651 653 Crackers Grq,ms. 1,200 lint ins. 1.063.0 Grams. 120.7 872.5 1.8 Grams. 74. 5 360.6 139.1 Grams. 867.8 406.4 Grams. 25.1 61.2 4.5 Grams. 19.32 43.66 .29 Calories. 5,065 Milk.. 8,160 1,039.5 160 140.9 6,258 Butter Total 1,282 2,243.4 395 574.2 1,274.2 90.8 63.27 12,605 662 Feces (water free) ... Amount digested 95 66.7 2,176.7 97 17.4 377.6 95.6 15.2 559 97.4 34.1 1.240.1 97.3 28.3 62.5 68.8 2.78 60.49 95.60 56.65 519 12,086 95.9 Nitrogen and heat of combustion of urine Energy of food oxi- 435 11.651 Per cent of energy utilized 92. 4 During this experiment the subject eliminated 4,071 grams of urine, containing 5(5.65 grams of nitrogen. This makes the average nitro- gen balance per day as follows: Income of food, 15.82 grams; outgo in urine, 14.16 grams, and in feces, 0.70 gram, indicating a gain of 0.96 gram of nitrogen or 6 grams of protein. DIGESTION EXPERIMENT NO. 4. This experiment began with breakfast August 11, 1900, and con- tinued four days. The weight of the subject (without clothing) at the beginning was 60.6 kilograms, at the end 62 kilograms. '24 Table 6. — Results of di serial No. >. t '- - Kind of food. ~- 1 •-- - - 1 Fat > Ajfa \ 11 ro - -. ?- ■- - - - . - - = - mn 4,000 240 Gin 1, is. 500.4 211.4 ms. 163.0 135. 6 170.4 Grams. 1,171.5 194. 4 30 lira ins. ■ 1 Milk .. Butter - water fri Amount diL r '-sTed. — Per cent digested Nitrogen and heat of combustion < >f urine Energy of food oxi- dized in the bodv . . . 3,112 1 . 922 »1, 610.2 226 1,024.4 53 70.6 36.14 S.9IU 66:3 70.4 41.2 B 11.3 96.9 16.1 11.6 41.4 2.21 319 M 4 Per cent of em utilized ■ a Three-fourths of total amount: urine for first day lost. The urine for the first day of this experiment was lost. During the remaining three days the subject eliminated 1,990 grams of urine, containing 31.28 grams of nitrogen. This makes the average niti \ balance per day as follows: Income in food. 12.05 grams; outgo in urine. 10.43 grams, and in feces. 0.74 gram: indicating that the body gained 0.88 gram of nitrogen, or 5.50 grams of protein per day. DIGESTION EXPERIMENT NO 5. This experiment began with breakfast August 15, 1900, and con- tinned four days. The weight of the Bubject (without clothing) at the beginning was 62 kilograms, at the end 60.8 kilograms. Table ?. — Results of digestion experiment No. 5 (serial N 150 Kind of food. Crackers Milk - 12,240 Grams. 425. 8 1,531.2 415 ■ 52L4 Grams. 347.1 594.8 Grams. 10 - 66.34 2,026 9. 523 Total.. 1,966.4 463. 3 551.2 £41.9 101.8 74. 07 11.549 Feces i water free > Amount digested Per cent digested 123. 5 81.9 1,874.5 21.8 441.5 95. 3 12.1 539.1 97.8 48 41.6 60.2 59.1 3.48 95.3 66.01 635 in. 914 94.5 Xitrogen and heat of combustion of urine 4-7 Energy of food oxi- dized in the bodv. . . 10.427 Per cent of energy utilized «... 90.3 1 25 During this experiment the subject eliminated 7,8$9 grams of urine, containing 66.01 grams of nitrogen, making the average nitrogen bal- ance per day as follows: [ncome in food. L8. 52 grams; outgo in urine, L6.50 grams, and in feces, u . v 7 gram; implying a gain of L. 15 grams of nitrogen or 7.19 if protein. DIGESTION EXPERIMENT NO. 6. This experiment began with breakfast July l. L901, and continued fourdays. Theweighl ofthesubject i without clothing) al the begin- ning was 61.45 kilograms, at the end 60.13 kilograms. Tabu: 8. — Results of digestion experiment No. G (serial No. 5 4. t; 1*9.1 Grams. 29. 7 260. 7 Grams. 337. it 299. 8 Grams. 8.8 41 Grams. 30.28 Calories. 4.774 1,171.8 24a : *W 4 637.7 49.8 39.03 ♦5.777 - water free ) . . . Amount digested 37.2 25.5 1,146.3 6.6 237.1 97.3 14.9 11.7 38.1 1.06 37.97 97. 3 43177 205 Per cent digested '.17 Nitrogen and heat of combustion of urine . Energv of food oxi- dized in the body . . . 350 6.222 Per cent of energy utilized- 91.8 • During this experiment the subject eliminated 3,232 grams of urine, containing 43.77 grams of nitrogen. The average nitrogen balance pci- day was therefore: Income in food, 9.76 grams; outgo in urine, 10.94 grams, and in feces, 0.27 gram; indicating a loss of 1.45 grains of nitrogen or 9.0(3 grams of protein. DIGESTION EXPERIMENT NO. 7. This experiment began with breakfast July 14, 1001, and continued five days. The weight of the subject (without clothing) at the begin- ning was 60 kilograms, at the end 59.2 kilograms. 26 T\r.i'.K 9. — Results of digestion experiment No. : {serial No. 330), u a ~ - - So • Kind of food. 1 r s is - "2 boh ~ - oS S Protein I N 6.86). i - I 6 4 < l ■- 1% 3 fl a «— 3 i _ - w Rftfi Crackers... Grams. 750 7,500 Grams. 077. 8 940.4 lira ins. ■>s.± 237 (iriinis. 4*. 7 342. 7 Grams. 550.3 360.7 Grams. 17. 4 54.7 Grams. 12.60 37.96 Calories. 3,226 r,:,; Milk 5.9ii3 Total.. l.tils.:.' 315. 8 381.4 911 72. 1 50.55 9.189 666 Feces (water free) ... Amount digested 77 54. 1 1,554.1 96.7 14.2 301.6 95.5 9.4 388 97.6 30.5 880.5 96.7 22.9 49.2 68.2 2.26 48. 28 95.5 :,; 58 429 8,760 95 3 Nitrogen and heat of combustion of urine Energy of food oxi- 460 8,300 90.3 Per cent of energy utilized During this experiment the subject eliminated 3,027 grams of urine, containing 57.53 grams of nitrogen. This makes the average daily nitrogen balance as follows: Income in food, 10.11 grams; outgo in urine, 11.51 grams, and in feces, 0.45 gram; corresponding to a daily loss of 1.85 grams of nitrogen or 11.56 grams of protein. DIGESTION EXPERIMENT NO. 8. This experiment began with breakfast July 19, 1901, and continued five days. The weight of the subject (without clothing) at the begin- ning was 59.2 kilograms, at the end 60.7 kilograms. Table 10. — Results of digestion experiment No. S (serial No. 331). Is a & .f z Kind of food. -p 1 ■o 8. n °^ si ■*= CO X $ 1 -t-> B g •a >> ■s ■s 8 4 < 1 Heat of combus- tion (deter- mined). 656 Crackers Grams. 1,500 15, 000 Grams. 1.355. 5 1,881 Grams. 157.5 474 Grams. 97.3 685.5 Grams. 1.100.7 721.5 Grams. 34.8 109.5 Grams. 25.20 75.90 Calories. 6,452 657 Milk 11,925 Total 3,236.5 631.5 782.8 1,822.2 144.3 101. 10 18. 377 Feces ( water free) Amount digested 667 188.6 132.9 3,103.6 95.9 32.4 599.1 94.9 31.9 750.9 95.9 68.6 1.753.6 96.2 55.7 88.6 61.4 5.19 95.91 94.9 77.62 1,090 17,287 Per cent digested 94.1 Nitrogen and heat of 606 Energy of food oxi- 16,681 Per cent of energy 90.8 1 27 During this experiment, which followed No. 7 without intermission, the subject eliminated 5,223 grams of urine containing 77.62 grams of nitrogen. This makes the average uitrogeu balance per day as fol- lows: Income in food, 20.22 grams; outgo in urine, L5.52 grams, and in feces, L.04 grams; implying a storage in the body of 3.66 grams of uitrogen, corresponding t<> 22.87 grams of protein. DIGESTION EXPERIMENT NO. 9. This experiment began with breakfast July 24, L901, and continued five days. The weight of the subject (without clothing) at the begin- ning was GO. 7 kilograms, at the end 50.3 kilograms. Table 11. — Results of digestion experiment No. i) {serial No. k Kind of food. 1 a "2 e3 . H CO o Eh «e X g o u 1 08 >, A 1 o 4 < a a> be o Heat of combus- tion (deter- mined). 656 657 668 Grams. 750 7,500 Grams. 677.8 940.4 Grams. 78.8 237.0 Grams. 48.7 342.7 Grams. 550.3 360.7 Grams. 17.4 54.7 drains. 12.60 37.95 Calories. 3.226 Milk 5.963 Total 1,618.2 315.8 391.4 911.0 721 50.55 9,189 Feces (water free) Amount digested 77.7 53.6 1,564.6 96.7 13.5 302.3 95.7 9.0 382.4 97.7 31.1 879.9 96.6 24.1 48.0 66.6 2.15 48.40 95.7 64.33 428 8,761 Per cent digested 95.3 Nitrogen and heat of 479 Energy of food oxi- dized in the body 8,282 Per cent of energy utilized 90.1 During this experiment, which followed No. 8 without intermission and which was a duplicate of No. 7, the subject eliminated 4,310 grams of urine containing 64.33 grams of nitrogen. The average daity nitrogen balance was therefore: Income in food, 10.11 grams: outgo in urine, 12.87 grams, and in feces, 0.43 gram; indicating a loss of 3.19 grams of nitrogen, or 19.94 grams of protein. DIGESTION EXPERIMENT NO. 10. This experiment began with breakfast July 29, 1901, and continued three days. The weight of the subject (without clothing) at the begin- ning was 59.3 kilograms, at the end 60 kilograms. 28 Table 12.- -Results of digestion experiment No. 10 (serial No. U a p. fa 666 658 669 Kind of fo Weight of mate- rial. - - ~- I z u 0* 1 g >> 1 ■a < 2sh 2© 9 © •- - - - ■- - - = s — - W Crackers Milk Crams. 900 9,000 Grams. 813.3 1,152.0 (.rums. 94.5 293. 4 Grams. 58. 1 407.7 C ra ins. 660.4 450.9 Grams. 63.0 Grams. L5. L2 16. 98 Calorics. 3,871 7,020 Total... 387. 9 466.1 1,111.3 83.9 62.10 10.891 Feces I water Prei Amount digested 100.3 68.1 1,897.2 96.5 19.7 94.9 10.5 455. 6 37.9 1,073.4 96. 6 32. 2 51.7 61.6 3. 15 58.95 94.9 -tit. 74 529 10.362 Per cent digested 95.2 Nitrogen and heat of combustion of urine 374 Energy of food oxi- dized in the body. . . 9,988 Per cent of energy utilized _ 91.7 This experiment followed No. 9 without intermission. The diet was nearly the same as in Xo. 8. During the three days of this experi- ment the subject eliminated 4,290 grams of urine containing 49.74 grams of nitrogen. The average nitrogen balance per day was there- fore: Income in food, 20.70 grams; outgo in urine, 1G. 58 grams, and indicating a gain of in feces, 1.05 grams corresponding to 19.19 grams of protein 3.07 grams of nitrogen, RESULTS OF DIGESTION EXPERIMENTS. In Table 13 are summarized the results obtained in the various experiments on the digestibility of the total food eaten. Although the diet was composed in each case of bread (in the form of soda crack- ers) and milk, with butter in some cases, the relative proportions of these two food materials, as well as the quantities taken, varied in the different experiments, as will be seen from the details of the experiments given above. Table 13. — Coefficients of digestibility of n utrients and availability of energy. Ex- peri- ment num- ber. Kind of food. Protein. Fat. Carbo- hy- drates. Ash. Energy. 1 Percent. 95.3 95.8 95. 6 93.9 95.3 97.3 95.5 94.9 95.7 94.9 P< rcent. 97.7 97.1 97.4 96.9 97.8 98.6 97.6 95.9 97.7 1*7.7 Percent. 97.2 97.2 97.3 98.4 94.9 97.7 96.7 96.2 96.6 96.6 Percent. 70. 67.7 78.1 59.1 76.5 68.2 61.4 66.6 61.6 Percent. 92. 3 2 ...do 92.3 3 do - 92. i 4 do... 93. :> 5. Bread and milk 90.3 6... do 91.8 ...do . 90.3 8. .. ...do 90.8 9 do 90.1 10. do... 91.7 29 As explained above, the experiments were varied in nit hers in I he Larger per- centage of the protein digested, the digestibility of the t'.u being also increased bu1 not to such a marked degree. This result is not due to the relative proportions of bread and milk- in the diet, since in this respect the experiment is intermediate between experiments Nos. 5 and 7. The amount of protein taken in the food was somewhal less than in experiment No. 7, and very much less than in experiment No. 5. This fact would of course be favorable to the more complete absorp- tion of the protein, as would also the circumstance that during Hie week previous to the test the subject had eaten less food than usual. These circnmstances may account for the rather unusual figures obtained in this period, and as the feces were collected, dried, and weighed by the subject himself it would seem improbable that any serious loss could have occurred without being detected. Neverthe- less, the amounts of total dry matter, nitrogen, and phosphorus found in the feces for this period are so small that the results are given with some hesitation, and in comparing the determined and calculated fig- ures for digestibility we have averaged the experiments both with and without No. 6. Table 14 shows for each experiment and for the average of all the experiments : (1) The percentage of protein actually digested, as deter- mined; (2) the digestibility as calculated, assuming that 85 per cent of the protein from cereals and 97 per cent of the protein from milk were digested, and (3) the figures calculated on the assumption that 90 per cent of the protein of the bread and 97 per cent of the protein of the milk were digested. Table 14. — Coefficients of digestibility of protein, calculated and determined. Results cal- Results cal- culated, as- culated, as- suming that suming that Results actually found. 85 per cent of bread pro- tein and 97 90 per cent of bread pro- tein and U7 per cent per cent of milk pro- of milk pro- tein were tein were digested. digested. Pera nt. Percent. Per cent . Experiment No. 1 95. :< 93.3 94.9 Experiment No. 2 95. 8 93.3 94.9 Experiment No. 3 95. ii 93.3 94.9 Experiment No. 4 93.9 90.5 98. 2 95 3 95 8 96 3 Experiment No. ti 97.3 93.9 95 3 Experiment No. 7 95.5 94.0 95. 3 Experiment No. 8 94.9 94.0 95.3 Experiment No. 9 95. ; 94.0 95 3 Experiment No. 10 94.9 94.0 95 3 Average of all 95.4 93.6 95.1 Average, omitting experiment No. 6 95.2 93.6 95 30 It will be seen that the digestibility of the protein of the diet, as cal- ciliated on the assumption that 85 per cent represents the digestibility of bread protein and 97 per cent that of milk protein are in 9 out of the 10 cases noticeably lower than the results actually obtained, the aver- age being 1.8 or L.6 percent lower than the average actual value, according as we do or do not include experiment No. 6. If, however, we assume that 90 per cent of the bread protein was digestible, and use the same factor as before (97 per cent) for the milk, we find thai (with the exception of experiment No. 6) the calculated and determined values agree in every instance within 1 per cent, while the averages agree within one-quarter of 1 per cent, a varia- tion which may well be considered as negligible. The factor 90 per cent for the digestibility of the bread protein was suggested !r\ T the fact that this is about the value found for white bread by Woods and Merrill in an extended series of experiments with a number of different subjects, and also in tests with one of the four subjects employed by Snyder.* As might be expected from the fact that fat is supplied in an emulsi- fied and readily available form in milk, its digestibility in these experiments was rather higher than is usually found. A detailed comparison, such as that given for the protein, is, however, impracti- cable, (1) because of the impossibility of distinguishing between ani- mal and vegetable fats in the crackers used, and (2) because those portions of the feces designated "fats" and "carbohydrates" really consist largely of other substances. During experiment No. 2, in which the diet was the same as in experiments Nos. 1 and 3, there was (as will be more fully described beyond, p. 35) a very considerable loss of sleep. This, however, does not seem to have had any appreciable effect upon the proportion of either of the nutrients digested. Experiments Nos. 7, 8, 9, and 10 throw some light upon the digesti- bility of liberal and restricted diets. These were carried out in series, and the relative proportions of milk and bread were uniform through- out. The amount eaten per day was, however, twice as great in experiments Nos. 8 and 10 as in experiments Nos. 7 and 9. On the smaller diet the percentage digested was slightly higher. The differ- ence is quite small, less than 1 per cent, but as all other experimental conditions Avere carefully maintained uniform, and as the agreement between the similar experiments is almost complete, it would seem that the better digestibility shown by experiments Nos. 7 and 9 over experiments Nos. 8 and 10 must be attributed to the fact that less food was taken. Larger but more variable differences have already been observed by Snyder (loc. cit.) in similar experiments. Experiments Nos. 7 to 10 were carried out without intermission, and «U. S. Dept. Agr., Office of Experiment Stations Bui. 85, p. 32. &U. S. Dept. Agr., Office of Experiment Stations Bui. 101, p. 33. 31 covered a period of eighteen days. Experiments Nbs. I bo 3 covered similarly a period of twelve days. The results obtained from these experiments make ii evidenl thai an extremely simple diet maybe continued for a very considerable number of days wi1 hout necessarily diminishing iis digestibility. COMPARISON OF THE METABOLISM OF NITROGEN, SULPHUR, AND PHOSPHORUS. As has been explained, the experiments above discussed as diges- tion tests were also designed to include a study of the comparative metabolism of nitrogen, sulphur, and phosphorus. In each experi- ment the diet was uniform and the urine for each twenty-four hours was collected and examined. Aliquot portions of each day's urine were mixed to give a composite sample representing the entire period. Nitrogen was determined in the urine of each day, and the results were verified by the analysis of the composite sample. Phosphates in the daily urines were determined volu metrically by titration with standardized uranium acetate solution in the usual manner. The total phosphorus of the urine for the whole i>eriod was determined as described in the section on analytical methods, above. It will be seen from the results as tabulated below that the sum of the figures obtained by titration of the daily urines ranges in the different experiments from 95.5 to 98 per cent of the total by the gravimetric method in the composite for the period. These variations are very likely due as much to errors in the volumetric determinations as to differences in the amount of " organic " phosphates present. If the methods and manipulation were free from error the results would indicate from 0.02 to 0.05 gram of phosphorus (or 0.04 to 0.12 gram P. < ,G 5 )per day eliminated in forms not precipitated by uranium. This amount is so small that it appears quite sufficient to use the volumet- ric method when one desires merely to follow the general course of the phosphorus excretion, determining the total phosphorus by the standard gravimetric method in cases where an accurate balance of income and outgo is to be determined. In this connection it may be noted that recent investigations by Ceconi and others a of the so-called organic phosphates of the urine have given quite variable results and have not tended to emphasize the importance of the small amount of phosphorus thus combined. On account of unavoidable interruptions it was impossible in the experiments carried out in 1900 to determine sulphur in the urine of each day. The amount of total sulphur and of sulphate sulphur was, however, determined for each period. The same determinations were "TVerhandl. Cong. Innere Med. Rome. 1896; abs. in Jahrb. Thier.-Chein.. 2 7 (1897). p. 362. Jolly, Compt. Rend. Acad. Sci. Paris. 127 (1898), p. 118. Oertel. Ztschr. Physiol. Chem., 26 (1898), p. 123. Keller, Ibid.. 29 (1900), p. 146. 32 included in experimenl No. 6. In experiments Nos. 7 to LO the sul- phate sulphur was determined for each day and the total sulphur for each period. Comparing fche " sulphate " and "total" sulphur in the differenl experiments, ii would appear thai from 83.1 to 89.6 percent of ili«' sulphur in the urine was in the form of sulphates. The sulphur in forms other than sulphates — so-called "neutral" sulphur — has recently been studied by Reale and Velardi,* Harnack and Kleine,* FivuihI. Pel ry, ' and doubtless others, and will probably repay fur- ther investigation. In the present experiments, however, time did not permit of any study of this question. Neither did tin- analyses include the separate determination of the ethereal sulphates which, as the protein consumed came principally from milk, were probably present in less than the usual proportions.' The final results of the examinations of the urine are brought together in Table 15, which shows the data for each experimental day. a-> well as the total for each of the ten periods. Partial analyses of the urine for the four days immediately following experiment Xo. 5 are also given. Table 15. — Data of examination of urine in experiments Nos. 1—10. Exper- Date. Total amount voided. Specific gravity. Nitro- gen. Sulphur. Assul- T , phates. 10Tal - Phosphorus. Heat of com- bus- tion. nuni- ber. By ti- tration. Total. 1. 1900. Julv 20-21. Gram*. 817 908 998 838 1.0280 L0270 1.0260 1.0280 Grains. 15.38 13.89 14. 28 13. 68 Grams. 1.30 1.34 1.40 1.41 Grams. Cals. Jul v 21-22 .. Jul v 22-23 July 23-21. Total July 24-85 July 25-26. July 26-27 July 27-28 3.550 57.23 3. 37 3. 80 5. 45 .5.63 433.8 2- 852 782 851 966 1.0280 1.0300 1.0310 1.0285 13.96 14. 04 15. 63 15.06 1.28 1.34 1.43 1.66 Total July 28-29.. 3. 451 58.69 3. 46 3. 86 5.71 5.90 438.2 3 1.160 978 952 1.001 1.0250 1.0270 1. 0270 L0255 15. 65 13.67 13. 51 13. 82 1.51 1.34 1.26 1.40 July 29-80 July 30-31 July 31-August 1 . . Total August 12-13 August 13-14 August 14-15 Total August 15-16 August 16-17 August 17-1 s August 18-19 Total 4.(>71 56.65 3. 24 3. 74 5.51 5.66 434.7 4 609 711 670 1.0325 1.0806 1.0305 10.19 10.92 10. 17 .94 1.06 1.02 85.6 38 : 83.4 1.990 31.28 1.85 2.16 3.02 3.10 257.7 2,180 2. 130 1,859 1,720 1.0130 1.0120 1.0145 1.0150 15. 12 16.07 16.98 17.84 1.48 1.72 1.79 1.78 110.6 117.4 127. 3 131.8 7.889 66.01 3. 94 4. 48 6.77 6.96 487.1 aStndii di clinica medica, Xapoli, 1895; abs. in Arch. Yerdauungskrankh.. 2 (1896-97). p. 141. eZtschr. Biol.. 37 (1899). p. 417. cZtschr. Physiol. Chem., 29 (1900), p. '24. dlbid., '1\) (1900), p. 45. e See results by Laquer. Yerhandl. Cong. Innere Med.. 16 (1898). p. 546; abs. in Jahrb. Their. -Chem., 28 (1898), p. 336. 33 i i."». -Data of > xamination of / //. nta Noa. t-10— Continued. Exper Total amount voided. Specific gravity. Nitro- gen. Sulphur. Phosphorus. Heat of com- tion. ber. phates. Total. By ti- tration. Total. 1900-Contd. August 19-20 August 20 21 August 21 22 August 22-28 Total Grants. 1,698 1,730 1 . ■ 15 1,835 L.0150 L.0150 L.0170 L.0150 Grams. L8. L2 17.78 Grams. Grams. L.70 L.71 l.i.'.' 6,808 72.82 l , 6.86 1 L901. July 4-6 . 6 879 641 760 952 L0230 1 275 L.0270 L.0215 ! LO 58 .'.Mi 1.01 1.10 L.21 July 5 6 in. !:.' L1.32 J 1.45 July 6-7 juiv : a Total . . 3. 232 43.77 | 2.57 3.00 4. 2* L. 46 350. 3 July 14-15 __ 7 681 605 620 566 555 1.0320 L.0320 1.0320 1.0350 1.0350 11.65 11.12 11.35 11. studying the "lag" in the elim- ination when the diet was suddenly changed and the new diet maintained for several days. On passing from the did of experiment No. 4 to that of experiment No. 5, there was little change in fuel value, but the amounts of nitro- gen and phosphorus ingested were largely increased. Under these conditions there was a "lag" of some days, i. e., some days were required before the rate of elimination become approximately uniform. Experiment No. 4 lasted three days and although experiment No. 5 properly continued but four days, the diet was maintained and the elimination of nitrogen and phosphates determined for an additional four days. The course of the excretion of nitrogen and phosphorus for the eleven days is shown in fig. 2, in which the curves are plat led in the same manner as in fig. 1 above. It will be seen that while the phosphorus elimination reaches a maximum on the third da}", the maximum elimination of nitrogen is reached only on the sixth day. It must be noted, however, that the increase of phosphorus in the diet was considerably greater in pro- portion than the increase of nitrogen, so that although the curves meet on the fifth da}" it does not follow that equilibrium was then restored. When the elimination of phosphates was at the maximum there was a storage of phosphorus in the body, whereas during the maximum elimination of nitrogen the body was losing that element. The body was in fact nearly in nitrogen equilibrium when the maxi- mum elimination of phosphorus was reached. In experiments Xos. 7 to 10 the lag was studied under different cir- cumstances from those just described. Instead of attempting to keep the fuel value approximately uniform while changing the amount s of certain constituents, the diet was here kept qualitatively the same, so that every change affected each of the constituents to the same extent. The general outline was as follows: For five days the subject took a restricted diet, which it was thought, would be just about sufficient to enable him to do his usual work without becoming uncomfortably hungry. As a matter of fact there was practically no sensation of hunger, but the subject lost during the five days somewjiat over grams of nitrogen and about 2 pounds in weight. During a second period of five days twice the original diet was taken. Then the sub- ject returned to the original diet for another period of five days, the object here being to study the lag after a decrease as well as after an increase in the diet. At the end of the third period the diet was again doubled, and the double diet was this time maintained for three days. The results for the eighteen days covered by this series of experiments 39 are shown in fig. 3, in which t he curves are platted in i he same mannei as in figs, l and i ) . In this case the sulphate sulphur was also deter- mined, and is represented in the figure bya broken line, nitrogen and phosphorus being represented respectively by solid and dotted lines. The rises and falls in the curves in Ihis figure on passing from one experimental period to another are not large as compared with the - 180 ..''' >... 170 .■' "v. ■ "*""--. 160 / / 150 > 140 j .7 130 U if 120 ij II 1 10 100 Ij v^.._ ij / 90 80 70 EXPT. N°-4. EXPERIMENT N°5. DAYS 1 -3. DAYS 4- II. Pig. 2.— Diagram showing fluctuations in the daily excretion of nitrogen and phosphorus during the second seriesof experiments (Nos. 4 and 6). The curves are platted in the same manner as those in fig. 1 . change in the diet. Thus 1 lie diet in experiment No. 8 was twice as greal as in experiment No. 7, but the greatest daily elimination was only about one-half* larger for nitrogen and sulphur and two-thirds larger for phosphorus. This is mainly because, as would bo expected, there was a loss of body material on the small diet and a gain on the Large diets. The daily gains and losses are shown in Table 16, the complete balance for each experiment being tabulated beyond. 40 •J k (A o z 7 a. <2 LU a \ \ \ 1 \ \ IV ^^^v ^v*^**""* ^^^. / > \ 05 Ol z . in Q. X LU .. /■' J "V V. \ j / / / / J "" ,-- - „*» - * -0- »«^* '^^-^ / X 00 Ol z . o £? I? *2 LU CL X N- v. - \ \ / \ / 7\ /I / 1 / 1 / 1 / 1 / J \ / - V / \ **«». ^^ii^ ^^*^?» i \ 1 Ol z LJ CL X LJ \ i \ ' \ l\ •J 1 \ \ \ V / V \ i i ooooooooooo NcOlO^tcQcvI OOC0N .; - C H 41 As has already been stated, the total sulphur and total phosphorus eliminated by the kidneys during each period were determined by analysis of a composite sample of urine. The sulphate Bulphurand the phosphorus precipitable by uranium acetate were determined both in the composite sample and in the urine of each day. From the data thus found it is easy to estimate the total sulphur or total phosphorus for the urine of each day if we assume thai the ratio of "unOxidized" to total material is constanl during the experimental period. While slighl errors mighl result from this -assumption, they would be far too small to affect i he present discussion. The amounts thus estimated are therefore used in the following table: Table 16. -Daily gains and losses of nitrogen, sulphur, and phosphorus in experi- ments Nos. 7-10. Bzperi ment num- ber. 10. Date. July 14-15 July 15-16 July 16-17 July 17-18 July 18-19 July 19-20 July 20-21 July 21-22 July 22-23.. ... Jul'y 23-24 July 24 25- July 25-26 July 25-27 July 27-28 July 28-29 July 29-30 July 30-31 July 31-Aug. 1 Nitrogea. Sulphur. Phosphorus. Gain(+) Gain( + X [n urine. or loss (— ). In urine. or In urine Grams. Grams. Gram. Grams. LI. 65 - L.99 0.82 -0.16 11.12 -1.43 .81 - .15 .92 11.35 -1.69 .87 - .21 1.05 11.66 -2 .82 - .16 1.09 LI. 75 -2.09 .83 - .17 1.05 14.83 1.04 + .27 1.23 15.81 +3.37 1.00 + .31 1.48 15, L9 +3. 99 1.00 + .31 1.47 14.91 +4.27 1.08 + .23 1.65 us. m +2. 30 1.16 + .15 1.62 12.64 -2. 96 .89 - .23 1.31 12.57 -2.89 .86 - .20 1.27 13. 01 -a. 3b .91 - .25 1.43 12. 73 -3.05 .83 - .17 1.31 12.88 -3.20 .90 - .24 1.27 16.23 +3.58 1.19 + .07 1.39 16.71 +2.94 1.23 + .03 1.50 16.80 1.22 + .04 1.57 Gain(+) or I «8 Gram + . + . + . + • + . Id 06 06 11 07 51 26 25 09 .12 23 .19 .35 .19 .50 . 32 It will be seen from this table that the gains and losses were con- siderable, and that equilibrium was not reached in any experiment, even after the continuance of a uniform diet for five days. In view of the large amounts of material gained and lost there is danger that any inferences in regard to lag which could be drawn from these results might be subject to unknown errors arising from the breaking down of body material on the one hand or the transformation of food protein into body protein on the other. It will be shown, however, that, except in cases where the balance was evidently affected by the lag, the proportion of sulphur to nitrogen was nearly the same in the material stored or lost as in the food material actually absorbed. With phosphorus the variations are larger, but a similar relation appears to exist. Hence the question of lag is quite as important here as in the cases where it has been more especially studied, but here it represents not merely the time required for the metabolism of the ingested materials, but to some extent also the time nece> for the body to adapt itself to the increased or decreased diet. On 42 passing from the insufficient diel of experimenl No. 7 to the abundant did of experimenl No. 8 the nitrogen elimination rises on the first day and then remains nearly stationary for three days, during which a Large amounl of nitrogen is stored, as though the body during these d;i\ s was replacing the protein previously Lost. Then on the fifth day the elimination again arises, though not far enough to establish nitrog- enous equilibrium. The elimination of sulphur rises somewhat more sharply on the firsl day than thai of nitrogen, then continues about uniform for two more days, and begins to rise again on the fourth day. continuing to rise on the fifth and reaching a relative rate somewhat higher than that of the nitrogen. The sulphur curve is therefore similar to the nitrogen curve, but the changes arc somewhat more marked, and in one case appeal' to begin earlier. The phosphorus rises sharply during the first and second daysand again on the fourth day. What lias been suggested with reference to the nitrogen and sulphur appears to be true to a lesser extent of the phosphorus. The increased diet immediately increases the excretion, the increase in this case continuing two days, then for a short period — in this case one day — the elimination is nearly constant, while a considerable pro- portion is stored in the body, after which the rate of elimination again rises. While the phosphorus does not reach equilibrium as regards income and outgo, it more nearly approaches this condition than either the nitrogen or the sulphur. When the diet was reduced to one-half, the rate of elimination of each of the three elements studied fell sharply on the first day and showed little if anj r fall thereafter. Thus, in each case the eliminat ion lagged less in falling than in rising. This is the more striking, in view of the fact that the elimination of both nitrogen and sulphur was rising at the time the change in diet was made. In the final period, when the double diet was again resumed, the changes in rate of elimination were similar to those found in the first instance, except that the rise shown by the sulphur was somewhat more pronounced. Pressure of other work prevented the continuance of this experiment after the third day. A somewhat marked but temporary increase in the phosphorus elimination will be noticed on the third day of experiment No. 9, and an examination of the curves shows that simultaneously there occurred a smaller but distinct increase of sulphur, and an increase of nitrogen which relatively is much smaller still and would scarcely have been noticed had only the nitrogen metabolism been studied. Although careful note had been taken of anything which seemed likely to affect metabolism, it is difficult to assign a reason for this increase. During the early parts of the two preceding nights there had been slight rest- lessness, which was attributed to the warm weather, but previous experiments had indicated thai simple loss of sleep, even when very marked, had no great influence upon the metabolism of this subject 43 and increased the relative amounl of phosphorus Little, if any, more than that of nitrogen. Such instances as this would sec in to lend some support to the view, apparently quite generally held, that the nervous condition of the subject has a greater influence upon the metabolism of phosphorus than upon thai of nitrogen. A factorwhich is perhaps Liable to be overlooked in such cases is the influence of the degree of alkalinity of the blood upon the elimination of phosphates through the kidneys. COMPARISON OF BALANCE OF INCOME AND OUTGO. It is now generally recognized thai the daily balance of income and outgo of nitrogen in the human organism may be influenced hya variety of factors, some of which can not be controlled or even satis- factorily defined. The same is doubt less 1 rue of sulphurand probably fed a greater extent of phosphorus. In general, it is believed that in - the present experiments the metabolism was comparatively free from the influence of such obscure factors, but in interpreting the figures obtained for the balance we must take into account (1) actual errors in the determination of income and outgo, and (2) the elimination on a given day of material whose katabolism is to be attributed to the diet or other conditions of some preceding day or days — in other words, the "lag" in the elimination. Errors in determination of income and outgo fall into two groups — analytical errors, and losses of material. The analytical work was carefully performed by the methods already described. In several cases the constituent sought was present in very small amount, which must have increased the relative errors, and it may be stated that in the opinion of the writer the determina- tions of sulphur were less satisfactory than those of nitrogen and of phosphorus. As regards the loss of material, it is believed that no appreciable mechanical loss of either food or excretory products could have occurred in any of the experiments, hut there ma}' have been larger losses through the perspiration. As the experiments were all made in summer and only one-third to one-half of the ingested water appeared in the urine, considerable quantities of water must have passed through the skin, and more or less loss of the elements studied doubtless occurred in this way. The elimination of nitrogen through the skin has been briefly discussed in a previous bulletin," where it is shown that different estimates of the amount which may be thus lost per day vary from 0.2 gram to 1.36 grams. Very little data seems to be available from which to form an idea of the amounts of sulphur and phosphorus which may have been lost through the skin. Favre 5 found in the perspiration only traces of phosphates, but reported one-fourth as much of alkaline sulphates as of urea, corre- «U. S. Dept. Agr., Office of Experiment Stations Bui. 98, p. 51. "Compt. Rend. Acad. Sci. Paris, 35 (1852), p. 721; Schaffer's Text-book of Physiology, Vol. I, 1898 ; p. 671. 44 sponding to an elimination by the skin of about 1 part by weight of sulphur to 8 parts of nil rogen. Little can be said regarding the amounts of nitrogen and sulphur given off as volatile compounds by the intestine or Lost in drying the feces in the air at 100° C. It is known that some nitrogen is thus lost from the feces, probably mainly as ammonia. Loss of ammonia may be avoided by adding acid before drying, but this would result in a loss of sulphur present as sulphids. Hydrogen sulphid is stated to be a normal constituent of the intestinal gases, but the amount of sulphur lost from the body must have been very small in these experi- ments. It follows from what has been said that , aside from any errors of manipulation or analysis, the figures given for nitrogen and sulphur in urine and feces do not quite represent the total outgo from the body. The average daily balance, as actually determined for each experiment, is given in Table 17. Table 17. — Balance of income and outgo of nitrogen, sulphur, and phosphorus — average per day. Fuel value of diet per day. Nitrogen. Sulphur. Phosphorus. Experi- ment number. T3 o o o . •3,2 3 o o M o PI 6 .2 8 • +2 pj m o o Pi 1— 1 i V 9 'u u o . +1 iJ 1. 2 . Calories. 2, 908 2,901 2,913 2,607 1,555 1,660 3,336 1,656 3,329 Gms. 15. 82 15.82 15.82 12.05 18. 52 9. 76 10.11 20.22 10.11 20. 70 Guts. 0.74 . 67 .74 . 87 .27 .45 1.04 .43 1.05 Gms. 14.31 14.67 14.16 10.43 16. 50 10.94 11. 51 15.52 16. 58 +0. 77 + .48 + .'.HI +1.15 -1.45 +3.66 - 3. 19 +3.07 Gms. 1.12 1.12 1.12 . 94 1.44 .69 .70 1.40 .70 1.36 Gm. 0.06 .03 .06 .09 .i). .02 .04 .09 .04 .10 Gms. 0.95 .97 .93 .72 1.12 .75 1.TO .88 1.21 Qm. +0. 1 1 +■ .09 .13 + .13 + .24 - .17 + .25 - .22 + .05 Gms. ?.. 29 Gms. 73 Gms. 1.41 1.48 1.42 1.03 1.74 1.12 1.00 1.49 1.32 1.49 Gm. 40.10 + .08 3 2. 29 . 78 1.40 ■ .44 3.07 1.10 + .09 - .07 + .23 4 5 .. 6. 1.60 1.58 3.16 1.58 3.26 .37 .60 1.42 .50 1.37 + .11 - .02 8 9 10. + .25 - .24 + .40 The first three experiments show apparent daily gains of one-half to 1 gram of nitrogen and about one-tenth gram of sulphur and phos- phorus. These apparent gains may be largely due to the undetermined losses of urea, ammonium salts, sulphates and phosphates through the skin, and of volatile compounds of nitrogen and sulphur through the intestines. In experiment No. 5, in addition to the sources of error just mentioned, we have the effects of "lag" continuing through the experiment, as explained above. Experiment No. G shows a moderate loss of nitrogen and a corresponding loss of sulphur, but a slight apparent gain of phosphorus. In this ease, however, the elimination of phosphorus (as also of nitrogen and sulphur) by the intestine is relatively so small as to mark the experiment as somewhat exceptional. It may be said that in these six experiments the sulphur balance follows that of nitrogen, but in no ease is the gain or loss great enough to justify calculations of the composition of the material stored or broken down. 45 In experiments Nos. 7, 8, and 9 the gains and losses are Larger, and here it is probably safe i<> draw inferences regarding t be composil ion of the materia] stored or lost by the body, though the undetermined errors already discussed will of course affecl the accuracy of such deductions. Neglecting these errors, the figures given for ■• balance" would Indicate thai in the materia] lost in the first of these experi- ments ilic ratio of sulphur to nitrogen was as I :10.9, in thai stored in the second as 1:14.6, and in thai lost in the third as 1 : 14. The ratio in the food consumed was L:14.4, in the food material actually absorbed (food minus feces) it was 1:14.6. It was calculated above that this ratio in serum globulin is L-.14.3, in myosin 1 :13.1, in serum albumin L:7.1, and in the glycoproteids of connective tissue and of bone as about 1:5. Thus the ratio is narrower in the tissue proteids gener- ally than in the food here consumed, but in serum globulin the ratio is about the same, and in myosin not greatly different. A strict inter- pretation of these ratios would thus lead to the conclusion that in the first period the body metabolized the food eaten and some of its own material in which the ratio is narrower; that in the second, a part of the protein of the food is either stored without essential change or con- verted for storage into some form of body protein in which the ratio is practically the same (as in the case with serum globulin), and that the proteid lost in the third period was of the same nature as that stored in the second period. Such a method of interpretation is in line with that followed by Kolpatcka in his studies upon dogs, but for the reasons already given it is believed that such conclusions must be accepted with reserve until more is known of the conditions influencing the "balance" and the "lag." These experiments, however, do at any rate strongly emphasize the close parallelism between the meta- bolism of nitrogen and that of sulphur when the diet is normal and is continued uniform for a period long enough to practically eliminate the effects of the lag. In experiment Xo. 10, which continued but three days, the balance is much influenced by the lag, so that in this case the apparent gains show no relation to the proportions of the two elements in the food. On the other hand the phosphorus metabolism does not show such a close parallelism to the metabolism of nitrogen. In experiment Xo. 7, where the loss of nitrogen was nearly constant throughout, there was at first a slight gain and later a slight loss of phosphorus, the net result being an almost perfect balance. This is probably due to the comparative richness of the diet in phosphorus, so that it supplied sufficient of this element for the needs of the body, while the protein of. the diet was so far insufficient as to result in considerable loss of nitrogen and sulphur. During the live days of abundant diet (experi- ment Xo. 8) there was a storage of 1.25 grams of phosphorus, and almost exactly the same amount was given up during the following five days of restricted diet. The three days of experiment No. 10 4o show a large apparent gain of phosphorus, but this is largely due to the " v Lag" and can not be considered to represent permanently-stored material. Any comparison of the nitrogen and phosphorus metabolism in the human organism is complicated by the varying proportions of phos- phorus eliminated in the feces. Thus in experiments Nos. 8 and 9the diet was qualitatively the same, ye1 in the former 44.9 per cent and in 1 lie latter only 31.6 per eenl of the phosphorus in the food was found in I lie feces. Two explanations suggest themselves, (1) thai only a part of the phosphates from body katabolism may appear in the urine, the remainder being eliminated through the intestine, as in the herbivo- rous animals; (2) that the proportion which the body absorbs may depend not only upon the nature of the ingested phosphates, but also upon the condition and needs of the body. The former is probably true to some extent, but it seems probable that the latter also oper- ates in some cases, as in that just mentioned, where, on doubling the diet, a much smaller proportion of the phosphorus present was assim- ilated. The larger proportion of ingested phosphorus which appears in the feces makes the proper separation of the latter a much more impor- tant matter in experiments in which the balance of phosphorus is to be determined than in those in which onty nitrogen or nitrogen and sulphur are studied. In view of the results which have recently been obtained upon the assimilation of the phosphorus of casein/' the phosphates found in the feces in these experiments should probably be attributed mainly to the calcium phosphate of the food. It should be remembered also that in the present experiments the diet was unusually rich in phos- phates, and the proper interpretation of the results must await the completion of similar experiments upon different diets. The experiments here reported afford no data for a direct compari- son of the nutritive values of different proteids, the food materials used having been similar through the whole series. However, in view of the recent work upon the nutritive value of the proteids of milk, 6 it is interesting to note the tendency shown in experiments Nos. 1 to 4 to store protein on a diet considerably smaller than that usually estimated for a subject of vigorous appetite and doing a considerable amount of work. «Marcuse, Arch. Physiol. [Pfliiger], G7 (1897), p. 373; Knopf elm acher, Wiener Klin. Wchnschr., 12 (1899), p. 1308; Nicko. Ztschr. Biol., 39 (1900), p. 430; Miiller, Ibid., p. 451. b Among the many recent papers may be noted: Marcuse, Arch. Physiol. [Pfliiger] , 64 (1896) , p. 223; Steinitz, Ibid.. 72' (1898) . p. 75; Rohmann, Berl. Klin. Wchnschr., 35 (1898), p. 789; Albn, Fortschr. Med., 17 (1899), p. 505: Poda and Prausnitz, Ztschr. Biol., 39 (1899-1900). p. 279. IT SUMMARY. The digestibility of the protein of the bread and milk diel as found ni nine of the teD experiments agreed closely with the results calcu- lated, assuming 97 per cenl as the coefficienl for milk and 90 per cent as that for bread. The digestibilityjwas nol appreciably influenced by loss of sleep nor by the eont in nance of the diet lor twelve or eighteen days. The proportions of protein digested from a restricted diet were about 0. 7 per ceiit higher than those digested from a liberal diet of the same composition. .Marked loss of sleep for three successive nights resulted in a small increase in the amounts of nitrogen, sulphur, ami phosphorus excreted. The increase of sulphur was proportional to that of nitrogen and the increase of phosphorus was wry slighl ly larger, the relal ive difference being no greater than might be attributed to the usual daily varia- tions. The increased elimination resulting from loss of sleep did not appear until the third day, while changes resulting from alteration of the diet were always perceptible on tin 1 first day. The data collected regarding the relative "lag" of nitrogen, sul- phur, and phosphorus are not j^et sufficient to permit general conclu- sions to be drawn. In general the metabolism and "balance" of sulphur ran approxi- mately parallel with that of nitrogen. The renal elimination and "balance" of phosphorus showed fluctu- ations similar to those of nitrogen, but not so closely parallel as in the case of sulphur. The elimination of phosphorus by the intestine was large and variable, making the accurate separation of the feces an important factor in the determination of the phosphorus balance. The above statements are intended merely to summarize the results of the experiments here reported. As these were all made upon a single subject and with only two orthreefood materials, it would obvi- ously be unsafe to generalize broadly from the results. As already explained, the work was undertaken not so much with a view to obtain- ing results of intrinsic interest as to get data regarding methods of work and possible sources of error, and thus facilitate the stud} r of the sulphur and phosphorus metabolism in connection with certain of the series of nutrition investigations to which the present experiments belong. O LIST OF PUBLICATIONS OF THE OFFICE OF EXPERIMENT STATIONS ON THE FOOD AND NUTRITION OF WAN-Continued. Bui. tw. a Description of Some Chin ble Food Materials and Their Nutritive and Economic Value. By W. C. Blasdale 1| 10 cents. Bui. 69, Experiments on the Metabolism of Matter and Energy in the Hainan Body. ByW.O. Atwater and F. Gh Benedict, with the ooperatioa of .\. W. Smith and A !\ Bryant Pp. ii:.'. Price, 10 cents. Bui. 71. 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