of proteins would improve nitrogen retention then adjust it to fulfill specific objectives. and! tested in adult human subjects.

Transcription

1 TIRE AMERICAN JOURNAL OF CLINICAL NUTRITION Vol. 20, No. 3, March, 1967, pp Printed in U.S..4. Effect of Four Multiples of a Basic Mixture of Essential Amino Acids on Nitrogen Retention of Adult Human Subjects1 2 3 HELEN E. CLARK, PH.D.,4 KAY FUGATE, M.S.,5 AND PATRICIA E. ALLEN, M.S.5 JJ IETARY PROTEINS I)IFFER not only in the quantities and relative proportions among the essential amino acids (1) but also in the percentage of total nitrogen supplied by them (2). Both factors may be important (3). Egg and milk, which have been suggestedi by the FAO/WHO Expert Group as reference proteins (4), contain relatively high concenti-ations of essential amino acids in suitable proportions. Essential amino acids in amounts that represented from g of egg protein caused! a stepwise improvement in nitrogen balances of men (5). Amino acid requirements of many people must be fulfilled, however, by proteins that contain essential amino acids in lower concentrations and! in less satisfactory pro- than those present in the reference Although minimal amino acid requirements of men (6) and women (7) are known, the zone of tolerance (8) for each amino acid has not been established. it is important therefore to ascertain whether ingestion of increasing amounts of a poorly balanced protein or combination From Purdtle University Agricultural Experiment Station, and School of Home Economics, Department of Foods and Nutrition, I.afavettc, Indiana. Journal paper This work was supported in part by Public Health Service Grant AM from the National Institute of Arthritis and Metabolic Diseases. Preliminary reports have been presented before the American Institute of Nutrition and the Seventh International Congress on Nutrition in Professor, Department of Foods and Nutrition. Instructor. of proteins would improve nitrogen retention steadily or depress it by accentuating an existing imbalance. A clear understanding of the quantitative relationships that should exist among essential amino acids and between essential and nonessential amino acids is needed for effective utilization of rapidly accumulating data concerning amino acid composition of dietary proteins. Because administration of many different sources of protein would be time consuming, it seemed expedient in studying the utilization of different quantities and proportions of essential amino acids to devise a mixture of amino acids that would maintain nitrogen equilibrium and then adjust it to fulfill specific objectives. Such a mixture now has been developed and! tested in adult human subjects. PROCEDURE The experimental approach was generally similar to that described in earlier papers from this laboratory (8, 9). Amino acids were provided in part as white wheat flour and in part as purified amino acids. In the first experiment, five combinations of essential amino acids were cornpared to determine which would meet minimal requirements of all subjects without providing an excess of any amino acid; and in two later experiments, four multiples of a modified form of one of these mixtures were administered at differ. cut levels of dietary nitrogen. Essential A in mo A rids A drninistered in Experiment I Modifications of two patterns of amino acids were tested. Mixtures IA and IB, described in 233

2 234 Clark et al. I Essential Amino Acids in Experimental Diets in Experiment I,#{176}milligramns per day Amino Acid Flour I Mixture - IA ID 1IA IIB IIC Isoleucine Leucine Lysine Methionine Phenylalanine Threonine Tryptophan Valine Total EAA, g Nitrogen, g a All diets also contained 350 mg of cystine and 590 mg of tyrosine , ,020 1, , a All diets also contained 290 mg of cystine and 480 mg of tyrosine. The total amounts of essential amino 1,200 1,630 1,150 1,110 1, , acids (EAA) in each mixture include those present in the flour. Table 1, provided approximately 1.25 and 1.50 times the quantities reported as minimal amino acid requirements of men (6). The daily intake of each amino acid was increased above the reported minimal level because unpublished data have indicated that the amounts which were adequate for men when each amino acid was quantitated in the presence of large amounts of other purified amino acids (6) did not consistently maintain nitrogen equilibrium when wheat flour Essential Amino Acids in Experimental Diets in Experiments II and JJJ,a milligrams per day Amino Isoleucine Leucine Lysine Methionine Phenylalanine Threonine Tryptophan Valine Acid Total EAA, g Nitrogen, g II Multiple of Basic Mixture , , ,380 1,950 1,290 1,250 1, , ,780 2,600 1,740 1,880 2,360 1, , ,0 3,260 2,190 2,520 3,080 1,510 2, ,l , , , , , , supplied part of the amino acids whereas twice these quantities always induced strongly positive balances. Mixture ha was developed after consideration of data from earlier experiments in which wheat flour provided significant amounts of amino acids (8-10). It contained smaller quantities of all essential amino acids than did Mixture IA. Quantities of essential amino acids (EAA) in Mixture JIB were the same as in Mixture ha except that tryptophan was increased 50%, and in Mixture hg methionine only was increased 80%. Essential Amino Acids Administered in Experiments II and III The data obtained in experiment I indicated that a combination of essential amino acids based on either Mixture IA or JIB would be useful in studying the effect of different quantities and proportions of essential amino acids. Mixture JIB was selected for modification in experiments II and III because the quantities of all essential amino acids except tryptophan were lower than in Mixture IA and would therefore be more nearly comparable to those present in limited amounts of dietary proteins. The basic mixture shown in Table II was developed from Mixture IIB, but tryptophan was reduced to 310 mg and methionine to 610 mg in the presence of 350 mg of cystine. It was anticipated that sulfur-contain-

3 Essential Amino Acids and Nitrogen Retention 235 ing amino acids would be inadequate for some subjects. Four multiples of this basic mixture were fed in experiments II and III (Table II). Sources of Amino Acids All diets tested in the first experiment contained 130 g of all-purpose white wheat flour. In later experiments this was increased to 141 g, the maximum amount that could be included without raising leucine and phenylalanine considerably above the desired level. Limited amounts of fruits and other low nitrogen foods also were served. Amino acids in the flour and foods were determined by ion exchange chroinatography using the method of Spackman, Stein and Moore (11). The best values obtained after refluxing in 6 N HC1 for 24 hr were used. Flour and a few foods provided significant amounts of all essential amino acids (Table i). In addition, crystalline L-isomers of essential amino acids were administered so that subjects consumed daily from all sources the total amounts of amino acids (EAA) shown in Tables and ii. The following nonessential amino acids also were supplied by 130 g of flour, in milligrams per day: arginine 620, histidine 330, alanine 490, aspartic acid 850, glycine 580, serine 800, proline 1,820, and glutamic acid 5,810. The Basal Diet The wheat flour was prepared and served as biscuits, cookies, and shortbread. Anhydrous milk fat, cornstarch, sugar, and candy were added to regulate the energy value of individual diets as indicated in Table my, last column. A vitamin-mineral supplement#{176} provided daily: vitamin A 5,000 USP units, vitamin D 500 USP units, d-a-tocopheryl acid succinate 5 IU, thiamine mononitrate 3 mg, riboflavin 3 mg, pyrid!oxine-hc1 1 mg, niacinamide 20 mg, d- calcium pantothenate 5 mg, ascorbic acid 50 mg, and vitamin B,, 2 sg. Additional minerals were furnished by a specially prepared mixture and by a phosphate type baking powder. Each subject consumed from all sources, in milligrams per day: calcium 800, phosphorus 1,100, potassium 500, magnesium 200, iron 17, copper 2, manganese 2, iodine 0.15, and zinc 1.5. Total Dietary Nitrogen The flour supplied 2.38 g of nitrogen in experienent I and 2.92 g in the others. Fruits and a Vigran M, Squibb. Sources of Nitrogen in Experiment II,#{176} Source III grams per day Multiple of Basic Mixture Flour, white Fruits, etc EAAb NEAA Total a In Experiment III total nitrogen was reduced to 6.00 g by omitting 3.00 g of nitrogen from the NEAA mixture of glycine, glutamic acid, and diammonium citrate. Essential amino acid mixture. few low nitrogen foods contained 0.25 g of nitrogen. Whereas nitrogen supplied by flour and other foods was constant during each experimerit, nitrogen present in crystalline essential amino acid supplements varied from 0.29 to 0.35 g in experiment I and from 0.22 to 1.50 g in experiments II and III. Glycine, glutamic acid, and diammonium citrate contributed 50, 20, and 30%, respectively, of the nonspecific supplement (NEAA) needed to increase total dietary nitrogets to the predetermined level. In experiments I and II subjects consumed 9.00 g of nitrogen per day, as illustrated for experiment II in Table mm. In experiment III this was reduced to 6.00 g by decreasing the nonspecific supplement; and in one additional period total nitrogen was dropped to 4.5 g for three men. Subjects All participants (Table Iv) were graduate students between 20 and 25 years of age who were approved by a physician. The 14 men and 3 women continued their usual activities. Mean body weights of the men in the successive experiments were 72, 73, and 69 kg, and the corresponding lean body mass values based on measurement of potassium 40 in a whole body scintillation counter (12) were 55, 60, and 49 kg. Plan of Experiment During an initial adjustment period of 12 days subjects consumed twice the minimal require-

4 236 Clark et al. IV RESULTS Characteristics of Subjects Subjecta Weight, kg Height, cm LtnBO1Y CaIoies Experiment GE ,100 DI ,250 DM ,800 JQ ,350 WT ,200 Experiment RA ,150 BW ,200 JK ,300 DL ,050 A? ,250 PW I 2,200 Experiment PA I ,250 WD I ,700 GH ,050 MR ,100 JS ,900 JV ,250 a All were men except GE, DM, and PW. ments reported by Rose (6) or 2.5 times the basic mixture of essential amino acids, and attained nitrogen equilibrium. Thereafter a stratified design was used in assigning treatments to individuals. Each treatment was imposed for 9 days, the first 3 days of which permitted satisfactory adjustment. Nitrogen balance data obtained only in the last 6 days of each interval are reported. A nalyses Nitrogen in foods and excreta was determined by a boric acid modification of the macro-kjeldahi procedure with potassium sulfate and copper sulfate as catalysts. Creatinine in the urine was measured by a modified Folin-Wu procedure. The urine collected in experiment II was prepared by the method of Stein (13) and analyzed by ion exchange chromatography to determine whether differences in the four levels of amino acids consumed would be reflected in the urine. Analysis of variance was used to detect whether nitrogen balances were influenced significantly by treatment, subject, or period within the experiment. The Duncan new multiple range test was applied when appropriate. II III I Body weights of all subjects remained constant or decreased slightly, thus indicating that energy needs were met fully. Mean daily nitrogen balances of men and women who consumed the various combinations of essential amino acids in the three experiments are present in Table v. The values for fecal nitrogen and urinary creatinine also are included. Experiment I. Variable Proportions of Essential A mino Acids Mean balances of the group, and g, were essentially the same whether 1.25 or 1.50 times the minimal requirements reported for men (6) were administered. All subjects retained nitrogen except JQ, a very active man, whose balances were slightly negative. Nitrogen retention of two individuals doubled when the higher level of amino acids was fed, two remained the same, and one decreased. Mixture IIA contained only 80% of the tryptophan present in Mixture IA and from 88 to 95% of the other amino acids. The mean balance, g, that resulted from Mixture ha was significantly lower (P < 0.01) than that obtained with Mixture IA, and three of the four subjects tested retained less nitrogen. Retention improved significantly (P < 0.01) when tryptophan alone was increased from 250 to 375 mg in Mixture JIB, and nitrogen balances of all subjects were positive. The mean balance, g, did not differ significantly from that obtained with Mixture IA or lb. In contrast, retention was only slightly higher, versus g, when methionine in Mixture JIG was increased from 730 to 1,330 mg in the presence of 290 mg of cystine. Only three of five subjects were in positive balance. Mixture ha apparently could be improved by adding tryptophan but not methionine alone. Differences between nitrogen balances of

5 Essential Amino Acids and Nitrogen Retention 237 V Mean Daily Nitrogen Balancesa of Subjects in Experiments I, II, and Ill Subject Treatment Exp. I: Proportions among EAA; 9.00 g of nitrogen IA LB ha JIB hic Mean Fecal N Creatinineb GE DI DM JQ WT Mean RA BW JK DL AP ± ± ± ± ± Mean -0.04±0.20 PA WD GH MR is iv Mean ± ± ± 0.08 Exp. ill. Multiples of basic mixture; 6.00 g of nitrogen ± ± 0.l30.56 ± ± 0.14 a Expressed as grams per day; mean and standard error are given. Urinary creatinine in grams per day. individuals in experiment I were highly significant (P < 0.01) when all treatments were considered. Subject WT retained more nitrogen and JQ less than did GE, DI, and DM who did not differ from each other. These deviations can be attributed in part to variations in fecal nitrogen and urinary creatinine. Subjects tended to retain more nitrogen in the final period regardless of the treatment. Exp. 11. Multiples of basic mixture; 9.00 g of nitrogen ± ± 0.20 t).55 ± ± ± 0.15 Mean 0.34 ± ± ± ± ± 0.19 Mean 0.02 ± ± ± ± ± ± Fecal N Fecal N Experiment II. Four Multiples of a Basic Mixture of Essential Amino Acids and 9.00 g of Total Nitrogen Creatinineb Creatmines The basic mixture of essential amino acids induced a mean nitrogen balance of g in the five men (Table v). It was satisfactory for one man, borderline for two, and inadequate for two. Whether this was due to a deficit in methionine or in lysine is not entirely clear. Rose (6) re-

6 238 Clark et al. ported that methionine requirements ranged from 800 to 1,100 mg/day in the absence of cystine, and that 80-89% of the methionine could be replaced by cystine (14). Clark and Woodward (10) noted that only four of six men maintained equilibrium when they consumed 320 mg of methionine plus 770 mg of cystine in a diet similar to that tested in the present experiment. The total amount of sulfur containing amino acids absorbed in experiment II may have been insufficient for some men. The possibility also exists that lysine was inadequate, since the microbiological assay values for lysine in the baked foods were lower than expected in all experiments and total lysine therefore fell below the requirements established for certain men when a similar (liet was used (9). Mean nitrogen balances of the group were , , , and g, respectively, when they consumed 1.0, 1.5, 2.0, and 2.5 times the basic mixture of essential amino acids. The regression of nitrogen retention on intake of essential amino acids was highly significant (P < 0.01). Differences between treatments also were highly significant (P < 0.01), but differences between individuals or periods were not significant. Provision of 1.5 and 2.5 times the basic mixture of essential amino acids while nitrogen intake remained constant at 9.00 g improved nitrogen retention by 400 and 900 mg, respectively. Mean nitrogen balances of subject PW, a small woman, in response to 1.0, 1.5, 2.0, and 2.5 times the basic mixture were , , , and g, respectively. Fecal nitrogen was 0.39 g/ day and urinary creatinine 1.02 g. Although no explanation can be offered for the negative balance resulting from the basal mixture, the data suggest that small individuals may reach a plateau of retention whereas there was no evidence of this response by the men over the range of amino acid intakes that was tested. Experiment III. Four Multiples of a Basic Mixture of Essential Amino Acids and 6.00 g of Total Nitrogen Because of the current interest in the relative amounts of essential and nonessential amino acids (3, 4), the four multiples of the basic mixture of essential amino acids again were administered in experiment III but total nitrogen was reduced from 9.00 to 6.00 g. The mean nitrogen balance of the men when they consumed the basic mixture was g, almost identical with that observed in experiment II. Five values fell between and g, and one was lower. The regression of nitrogen retention on intake of essential amino acids again was highly significant (P < 0.01). Mean retention increased by 300 and 800 mg, respectively, when 1.5 and 2.5 times the basic mixture were fed. The improvement in retention as quantities of essential amino acids increased was therefore comparable to that observed when 9.00 g of nitrogen were fed instead of 6.00 g. Differences in nitrogen retention of individuals were significant but differences between periods were not. It should be noted that lean body masses of the men averaged 49 kg in experiment III in contrast to 60 kg in the earlier test although body weights differed little (69 vs. 73 kg). Since there was not a significant difference in the regression of nitrogen balance on intake of essential amino acids whether 9.00 or 6.00 g of nitrogen were consumed, data obtained in the two experiments were pooled. The resulting regression equation, shown in Fig. 1, was: V = 550X - 585, when V is nitrogen balance in milligrams per day and X is multiple of the basic essential amino acid mixture. Under these experimental conditions, simultaneous increments in all es-

7 Essential Amino Acids and Nitrogen Retention 239 sential amino acids improved nitrogen retention significantly and steadily, despite the fact that at least one amino acid in the basic mixture was suboptimal for certain stmbjects. There was no advantage in increasing supplementary nitrogen to pro- ide 9.00 instead of 6.00 g of total nitrogen. To test the effectiveness of the basic mixture of essential amino acids when supplementary nitrogen was restricted further, three men consumed only 4.50 g of nitrogen in a terminal period of which 3.39 g were supplied by flour, foods, and crystalline essential amino acids. Mean balances of subjects WD, GH and JS decreased from , , and g when 6.00 g of total nitrogen were provided to , , and g, respectively. Since mean nitrogen retention fell by 0.27 g and all subjects were affected adversely, the earlier report from this laboratory (15) that 6.00 g of total nitrogen permitted mriore efficient utilization of essential amino acids than did 4.50 g was confirmed. LTrinary Excretion of Amino Acids in Experiment II The mean daily amounts of amino acids and other ninhydrin-reacting compounds in the urine of the five men are summarized in Table VI. Quantities of all essential c 0.4, c z 0.4 a 9.0 g N. 6.0 g N.. 550X Multipi. Of EAA Mixture FIG. 1. Regression of nitrogen balance (I ) in milligrams per day on X, multiple of basic mixture of essential amino acids. Ninhydrin-Reacting Urinary Excretion of Ninhydrin-Reacting Substancess in Experiment II Essential amino acids Other Isoleucine Leucine Lysine Methionine Phenylalanine Threonine Valine Alanine Asparagine compounds /3-Aminoisobutyric Cystine Cystathionine Ethanolamine Glycine Serine Histidine 3-Methyl-histidinc Ornithine Taurine Tyrosine Substance acid a In milligrams per day. VI Multiple of Basic Mixture amino acids were small (20 mg or less) and not influenced by intake except for Ii an upward trend in lysine, methionine, and phenylalanine as amino acids increased from 1.0 to 2.5 times the basic mixture. Certain nonessential amino acids and other compounds were influenced more sharply, however. Taurine increased 20% and cystathionine sevenfold, reflecting the alteration in dietary methionine. Glycine and serine fell steadily to 55% of the initial level as the source of supplementary nitrogen, which was high in glycine, was replaced by essential amino acids. Alanine declined slightly. Histidine dropped 20% but 3-methyl-histidine rose 10% although dietary histidine was supplied only by the flour and therefore remained constant. With these few excep

8 240 Clark et al. tions, the quantities of amino acids and related substances in the urine were not altered conspicuously by increasing essential amino acids to 2.5 times the quantities in the basal diet. Considerable variation between individuals at all levels of intake was noted, especially in asparagine, glycine, cystathionine, histidine, lysine, and taurine. These findings are in general agreement with the recent report of Block, Markovs and Steele (16) that doubling dietary protein raised a-aminoadipic acid and cystathionine, and that a very large methionine supplement elevated methionine, cystathionine, and histidine but depressed glycine. COMMENTS That nitrogen retention of men improves steadily as a result of concomitant increases in the quantities of all essential amino acids has been demonstrated clearly at two levels of dietary nitrogen. Although the basic mixture of essential amino acids did not maintain nitrogen equilibrium in all subjects, this limitation was overcome at the higher levels of essential amino acids without causing an imbalance. The procedure followed in these experiments can now be adapted to simulate the proportions of essential amino acids in certain foods that are important in meeting nutritional needs of man even though they are not adequate in all essential amino acids when fed at a low level. The equation reported herein that expresses regression of nitrogen retention on intake of essential amino acids should be useful as a basis of comparison in future experiments. These findings are in general agreement with reports from other laboratories although sources and proportions of essential amino acids differed. Kies and Linkswiler (5) reported that increasing amounts of crystalline essential amino acids patterned as in egg protein induced a marked improvement in nitrogen retention when essential amino acids progressively replaced the nonspecific nitrogen source up to a total nitrogen intake of 5.8 g of nitrogen. Similarly, increases in degerminated cornmeal changed the nitrogen balances of men from negative when 4.00 g of nitrogen were supplied by cornmeal to positive when 8.00 g of nitrogen from the same source were given (17). Interest has been expressed by the FAO/ WHO Joint Expert Group not only in the quantities of essential amino acids present but also in the ratio between essential amino acids and total nitrogen, the E/T ratio (4). The essential amino acids in flour plus purified L-isomers provided 15, 22, 30, and 37% of the 6.00 g of nitrogen in experiment III, comparable to representative dietary proteins (2), but only 10, 15, 20, and 25% of the 9.00 g of nitrogen supplied in experiment II. The E/T ratios of the four mixtures of essential amino acids were, respectively, 1.25, 1.89, 2.52, and 3.15 when 6.00 g of nitrogen were provided; and 0.84, 1.26, 1.68, and 2.10 with 9.00 g of nitrogen. The value of 3.15 corresponds with the E/T ratio of egg and milk and 2.52 with the E/T ratio of certain cereals and legumes (4). However, nitrogen retention was just as satisfactory when the same quantities of essential amino acids were present although the E/T ratio was decreased markedly by increasing total nitrogen to 9.00 g with supplementary nitrogen. Also, subjects maintained equilibrium in experiment I when the E/T ratio of Mixture IA was only 0.86 and of Mixture lb 1.05 in the presence of 9.00 g of total nitrogen. It is clear that caution must be exercised in the use of any ratio to express the relation of essential amino acids to total nitrogen. The effect of supplementary nitrogen on nitrogen retention has been reported in several papers (3, 14, 17, ). Provision of nonessential amino acids or nitrogenous sources from which they can be synthesized permits essential amino acids to per-

9 Essential Amino Acids and Nitrogen Retention 241 form their unique functions (15). On the other hand, the mean balance of those who consumed increasing amounts of essential amino acids in the egg pattern was near equilibrium only when as much as 3.2 g of nitrogen (60% of the total) were supplied as essential amino acids (5). In the first experiment in the present series the subjects attained equilibrium when they received as little as 0.84 g of nitrogen from essential amino acids and 9.00 g of total nitrogen; and the mean balance approached zero with 0.87 g of nitrogen from essential amino acids and either 6.00 or 9.00 g of total nitrogen. There was not a significant difference in the response to 6.00 or 9.00 g of total nitrogen when the basic mixture of essential amino acids was administered in the present experiment or when essential amino acids equivalent to 20 g of egg protein were fed in an earlier experiment (15). On the other hand, addition of supplementary nitrogen to a suboptimal amount of cornmeal steadily improved retention (). It is evident therefore that the adequacy of the mixture of essential amino acids being tested influences the effectiveness of the nitrogenous supplement, and at the same time the presence of supplementary nitrogen spares the essential amino acids. Certain points stand out clearly when the data from these experiments are examined in relation to those already in the literature: 1) relatively small quantities of the eight essential amino acids will maintain nitrogen equilibrium in men and women, and the pattern of essential amino acids may be modified to some extent without affecting nitrogen retention adversely although an intricate balance exists; 2) nitrogen retention improves steadily as the quantities of essential amino acids increase simultaneously, even if the basic assortment is not perfect; and 3) the presence of supplementary nonspecific nitrogen is beneficial but the relationship is not necessarily linear as in the case of the essential amino acids. It may be postulated therefore that the quantities of individual essential amino acids are of primary importance in determining man s ability to utilize a dietary protein effectively and that the relative value of supplementary nitrogen depends on the amounts and proportions of essential amino acids that are present. SUMMARY Two mixtures of essential amino acids were developed that contained limited amounts of all essential amino acids and maintained nitrogen equilibrium in adult human subjects when part of the amino acids was supplied by white wheat flour and part by crystalline amino acids. A modification of one of these mixtures was useful in studying the effect of simultaneous increments of all essential amino acids on nitrogen balance. Mean nitrogen balances were -0.07, +0.22, +0.56, and g, respectively, when 1.0, 1.5, 2.0, and 2.5 times this basic mixture were administered. The regression of nitrogen retention on intake of essential amino acids was linear, and did not differ when 6.00 or 9.00 g of total nitrogen were consumed. Grateful acknowledgment is made to Dr. Wayne V. Kessler, Bionucleonics Dept., for the lean body mass data; to all of the subjects for their excellent cooperation; to Susan MacDonald and Susan Meyers for assistance in the management of the experiment; to Yasuo Yamamura for microbiological assay of lysine; and to Shirley Griebe and Eileen Kidd for technical assistance. REFERENCES 1. ORR, M. L., AND B. K. WATT. Amino Acid Content of Foods. U. S. Dept. Agr. Home Econ. Res. Itept. Pub!. no. 4. Washington, D. C., CLARK, H. E. Utilization of essential amino acids by man. In: Newer Methods of Nutritional Biochemistry, edited by A. A. Albanese. New York: Academic, 1965, vol. 2, p SWENDSEID, M. Protein needs of adult man. In: Proceedings of Symposium on Protein Nutrition and Metabolism, Spec. Pub!. no. 4. Urbana, Ill.: Univ. of Illinois, Coll. of Agr., 1963.

10 242 Clark et al. 4. Protein Requirements. Rept. Joint FAO/WHO Expert Group. FAO Nutr. Meetings Rept. Ser., no. 37. Geneva: World Health Organ., 1965, p Kits, C. V., AND H. M. LINKSWILER. Effect on nitrogen retention of men of altering the intake of essential amino acids with total nitrogen held constant. J. Nutr. 85: 139, RosE, \V. C. The amino acid requirements of adult man. Nutr. Abstr. Rev. 27: 631, LEVERTON, R. M. Amino acid requirements of young adults. In: Protein and Amino Acid Nutrition, edited by A. A. Albanese. New York: Academic, 1959, p CLARK, H. E., P. MYERS, K. GOYAL AND J. RINE- HART. Influence of variable quantities of lysine, tryptophan and isoleucine on nitrogen retention of adult human subjects. Am. I. Clin. Nutr. : 91, CLARK, H. E., S. P. YANG, W. WALTON AND E. T. MERTZ. Amino acid requirements of men and women. II. Relation of lysine requirement to sex, body size, basal caloric expenditure and creatinine excretion. J. Nutr. 71: 229, CLARK, H. E., AND L. WOODWARD. Influence of variable quantities of methionine on nitrogen retention of adult human subjects. Am. J. Clin. Nutr. : 100, SPACKMAN, D. H., \V. H. STEIN AND S. Moot. Automatic recording apparatus for use in the chromatography of amino acids. Anal. Chein. 30: 1190, CHRISTIAN, J. E., L. W. COMBS AND W. V. KESSLER. The body composition of obese subjects. A en. I. Clin. Nutr. 15: 20, STEIN, W. H. A chromatographic investigation of the amino acid constituents of normal urine. J. Biol. Chemn. 201: 45, RosE, W. C., AND R. L. Wixost. The amino acid requirements of man. XIII. The sparing effect of cystine on the methionine requirement. I. Biol. Chem. 216: 763, CLARK, H. E., M. A. KENNEY, A. F. GooDwiN, K. GOYAL AND E. T. MERTZ. Effect of certain factors on nitrogen retention and lysine requirements of adult human subjects. IV. Total nitrogen intake. j. Nutr. 81: 223, BLOCK, W. D., M. E. MARKOVS AND B. F. STEELE. Effect of protein and of free L-methiOnine intake on amino acid excretion by human subjects. J. Nutr. 86: 256, KIE5, C., E. WILLIAMS AND H. M. Fox. Determination of first limiting nitrogenous factor in corn protein for nitrogen retention in human adults. J. Nutr. 86: 350, KIss, C., E. WILLIAMS AND H. M. Fox. Effect of non-specific nitrogen intake on adequacy of cereal proteins for nitrogen retention in human adults. J. Nutr. 86: 357, 1965.