Effect of Dietary Amino Acid Balance on the Excretion of Urinary N Compounds and their Ratios* By SHUHACHI KIRIYAMA and HIROYUKIWAO

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1 1 [Agr. Biol. Chem., Vol. 28, No. 5, p , 1964] Effect of Dietary Amino Acid Balance on the Excretion of Urinary N Compounds and their Ratios* By SHUHACHI KIRIYAMA and HIROYUKIWAO The National Institute of Nutrition, Shinjukuku, Tokyo Received November 27, 1963 When weanling male rats were fed amino acid diets which were gradedly varied tryptophan level only (0.18, 0.09 and 0.06%), allantoin and urea excretions were determined, and (allantoin/urea) ~ protein intake values were calculated. (Allantoin/urea) ~protein intake values almost linearly increased with the increment of tryptophan level of the amino acid mixture. From these data, it was assumed that one of the main factors affecting these ratios is in amino acid balance in diet. It was also found that the equivalent weight gains observed between 0.18 and 0.09% Try. groups were mainly due to body fat accumulation of the latter. In previous papers1 `3), it was reported that there existed the close correlation between the quality of ingested protein and the ratio of allantoin to urea excreted. Dietary protein quality and quantity are characteristically reflected in the ratio of al lantoin to urea. If the value of allantoin (A)/urea (U) ratio multiplied by the protein intake (Ip), (A/U) ~Ip, is adopted consider ing the protein consumption at that. time, nitrogen source of weanling rats. EXPERIMENTAL Weanling male rats of Wistar strain were used in this study. They were housed in individual cages and fed the 16% casein diet (N ~6.25=13.6%) ad libitum for 5 days to allow the to adapt to the new environ ment. The rats that showed a progressive weight gain were selected and divided into 4 groups (5 rats/group) TABLE I. COMPOSITIONS OF BASAL AND TEST AMINO ACID DIETS this value falls into a certain constant point, corresponding to the quality of dietary pro tein, independently of varying the protein level or protein intake. So, one of the authors suggested that there existed the possibility that the nutritional quality of dietary protein could be effectively compared by these values. The present experiments showed that one of the main factors influencing the regulari ties was in amino acid balance, using casein simulated amino acid mixture as a sole dietary * This study was supported in part by a grant from Tanahe Amino Acids Research Foundation, Osaka. 1) S. Kiriyama and K. Ashida, J. Japan. Biochem. Soc., 32, 185 (1960). 2) S. Kiriyama and K. Ashida, Japan. J. Nutrition, 20, 121 (1962). 3) S. Kiriyama and K. Ashida, J. Nutrition, 82, 127 (1964). ) Average nitrogen and protein (N ~6.25) content are 1.69 and 10.5%, respectively. 2) See the test and Table II. 3) This mixture contains minerals in following relative ratio: NaCl, 4.58; CaCO3, 6.37; MgSO4, 5.45; 0.15; NH4Fe KI, citrate, 1.18; Na2HPO4 E7H2O, 3.47; KH2PO4, Ten grams this mixture milligrams): 4) of contain (in thiamine hydrochloride, 3.0; riboflavin, 5.0; pyridoxine hydrochloride, 4.0; nicotinic 2.0; Ca pantothenate, 20; p-aminobenzoic acid, acid, 100; inositol, 100; folic acid, menadione, 3.0; choline 2.0; chloride, 1000; and vitamin A, 1250 IU; vitamin D, 125 IU.

2 308 Shuhachi KIRIYAMA and Hiroyuki IWAO on the basis of body weight. All rats were fed the same diet for further 7 days, and the test diets were given for subsequent 12 days. The composition of test diets is shown in Table I. Variations of protein levels in basal and test diets were controlled by changing the proportion of ƒ - starch. Composition of amino acid mixture incorpo rated into test diets is shown in Table II. Of all amino acids only the tryptophan content was varied by three graded levels. The amino acid mixture providing 1.82g of trypto phan per kg of diet was simulated to the amino acid composition of casein4) and conventionally regarded as a "Standard" mixture. The abbreviations of 1/2 Try. and 1/3 Try. repre sent the mixtures providing 0.91 and 0.61g of trypto phan per kg of diet, respectively. Decreased nitrogen content due to decrement of tryptophan was compensated by glycine to make iso nitrogenous. Cystine was entirely replaced by DLmethionine. Since the D-isomers of tryptophan5) and methionine6) have been considered equal to the cor- TABLE II. AMINO ACID COMPOSITION OF "STAND ARD", 1/2, AND 1/3 TRYPTOPHAN DIETS (expressed as g/kg of diet) responding L-forms in promoting rat growth, they were used in the same relative amount as they occur in casein, though Oesterling and Rose7) have reported that D-tryptophan is less effective than L-form. D-Threonine cannot replace the L-isomer, but the former has no toxic action8) Therefore, DL-threonine was used doubly the analytical data of L-threonine in casein. Isoleucinine used in this study is the mixture which contains L-leucine, L-isoleucine, and L-valine in per centage of 67 }2, 28 }2, and below 5, respectively. As shown in Fig. 1, animals were fed 16% casein diet for 12 days and urines were collected for the last 2 days of this period, called the First Period (P-I), by the method as will be described below. Then, animals received the test diets and after the initiation of test period, urines were collected further three times for each 2 days at the intervals of 2 days, called the Second (P-II), Third (P-III) and Fourth Periods (P-IV), respectively. At the end of each urine collection period rats were removed from metabolic cages. The cages, screens, and the funnels which were fitted with filter paper were washed down with hot distilled water. Pooled 2-day urine samples obtained from each rat were filled with distilled water up to 250ml and a portion of this diluted sample was kept with about 0.2ml of chloroform in the deep-freez ready for analysis. The urine samples were analyzed for allantoin and urea by colorimetry using Kotaki electrophotometer in duplicate, the methods employed being as described previously 1,3). Total nitrogen determination of urine and feces was carried out by the Kjeldahl method. 1) See the text. 2) This amount is less than analytical data, since it was difficult to obtain much. 4) R. J. Block and K. W. Weiss, "Amino Acid Handbook", Charles C. Thomas, Springfield, Illinois, ) C. P. Berg, J. Biol. Chem., 104, 373 (1934). 6) R. W. Jackson and R. J. Block, ibid., 122, 423 (1938). FIG. 1. Feeding Procedures and Urine Collectio Periods. 7) M. J. Oesterling and W. C. Rose, ibid., 196, 33 (1952). 8) S. A. Singal, S. J. Hazan, V. P. Sydenstricker and J. M. Littlejohn ibid., 200, 883 (1952).

3 Effect of Dietary Amino Acid Balance on the the Excretion of Urinary N Compounds and their Ratios 309 TABLE III. AVERAGE PROTEIN INTAKES WHEN FED "STANDARD" AND AMINO ACID DIETS TO WEANLING RATS (expressed as g/day/rat) RESULTS AND DISCUSSION As shown in Table III, protein intakes in test periods were less in all groups than in the First Period (16% casein diet). Up to the Fourth Period it seemed to gradually decrease. Especially, the decrement rate of protein intake in 1/3 Try. group was faster than the others. Body weights in both groups of "Standard" FIG. 2. The Growth Rate of Each Group of Rats Fed on Various Diets. The arrow represents the start of test diet Feeding and 1/2 Try. increased linearly, but their rates of weight gain were still less as compared to that of 16% casein period. Judged only from the rate of growth, 1/2 Try. diet seems to be the same as "Standard" diet. In 1/3 Try, group, initial body weight was hardly maintained (Fig. 2). As shown in Table IV, when fed 16% casein diet (in the First Period) all groups excreted about the equal amounts of urea, while after replacing to test diets the differences of urea excretion among 3 groups became significant. Comparison of the protein consumptions given in Table IV reveals that apparently these results were not due to the variation of amounts of ingested protein, but mainly de pendent on the difference of the amino acid composition among test diets. The values of urea excretion (mg) per pro tein intake (mg) are shown as percentage in Table V. From these values, it seems that 1/2 Try. mixture is catabolized into urea in about the same ratio as casein per unit of the ingested protein. "Standard" group was about half of this value, and 1/3 Try. group, about twice. It can be assumed from these data that grossly, "Standard" mixture has higher quality than casein and other test mix tures differing with the conclusion from growth data. As in urea excretion, allantoin was excreted at the same rate in all groups in the First Period. From the Second to the Fourth Period, there was no significant difference between "Standard" and 1/2 Try. group, but

4 310 Shuhachi KIRIYAMA and Hiroyuki IWAO TABLE IV. AVERAGE UREA EXCRETION AT THE VARIOUS PERIODS WHEN FED "STANDARD" AND AMINO ACID DIETS TO WEANLING RATS (expressed as mg/day/rat with standard error) TABLE V. RATIO OF UREA (mg) TO PROTEIN INTAKE (mg) IN VARIOUS PERIODS, WHEN FED ON 16% CASEIN AND TEST DIETS (expressed as percentage) TABLE VI. AVERAGE ALLANTOIN EXCRETION AT THE VARIOUS PERIODS WHEN FED "STANDARD" AND AMINO ACID DIETS TO WEANLING RATS (expressed as mg/day/rat with standard error) TABLE VII. RATIOS OF ALLANTOIN TO UREA MULTIPLIED BY PROTEIN INTAKE WHEN FED "STANDARD" AND AMINO ACID DIETS TO WEANLING RATS (expressed as (A mg/u mg)ipg ~100) 1): Standard error of tire mean. 1/3 Try. group continued to decrease from the Third Period to the final (Table VI). The (allantoin/urea) ~ protein intake values are summarized in Table VIL As can be supposed from the urea and allantoin ex cretion, there is no difference among all groups in this value at the First Period. The difference among them became clear from the Second Period. "Standard" group gave the value of two times casein diet and continued to maintain this level to the Fourth Period. Thus, their own values of "Standard" and 1/2 Try. groups are maintained at the same levels throughout the Periods, but that of

5 Effect of Dietary Amino Acid Balance on the the Excretion of Urinary N Compounds and their Ratios 311 FIG. 3. The Relationship Between Tryptophan Levels in Amino acid Diets and Values of (allantoin/ urea) ~ Protein Intake, (A/U) ~ Ip, at the Second and Third Periods. C, 1/2, and 1/3 represent relative tryptophan levels contained in amino acid diets corresponding to "Standard", 1/2 try., and 1/3 Try. groups, respectively. 1/3 Try. group decreased to about 1/4 of "Stand ard" at the Second Period and further gradually decreased. As seen from the curves of body weight changes in Fig. 2, the methods of nutritive evaluation of dietary protein based on the rate of body weight gain could riot distinguish the quality of "Standard" diet from 1/2 Try. diet in such a case as this experiments, though there existed a distinct difference in (A/U) ~ Ip values of both groups, corresponding to the alteration of quality of amino acid mix tures owing to the degree of amino acid bal ance. Therefore, in order to know the in trinsic composition of apparent weight gain, crude carcass fat content was determined by ether extraction. These results were as fol lows: "Standard" group, 38.8 }3.2; and 1/2 Try. group, 47.5 }1.4 per cent based on dry carcass weight, respectively. Thus, it was re vealed that even when the growth rate was equal in appearance, weight gain could not necessarily reflect the nitrogen retention or retention %, and that the larger parts of gain of 1/2 Try. group was attributable to the ac cumulation of carcass fat as compared to "Standard" group, at least in the present ex periments. So, in growth rate methods, when the composition of weight gain is not known, there is a possibility that do misevaluate the protein quality, while it has been evident that (A/U) ~ Ip value has a parallelism with nitrogen balance method in tendency as ob served in earlier studies3), and furthermore, it linearly responds to the alteration of single essential amino acid content in each amino acid mixture, that is, to the qualities of amino acid mixtures (Table VII and Fig. 3). Thus, even the slight alterations in balance among amino acids due to their delicate com binations can be reflected in (A/U) ~ Ip values which can be simply determined. It might be considered to be a useful procedure to evaluate biologically the quality of dietary

6 312 Shuhachi KIRIYAMA and Hiroyuki IWAO protein of rats. Moreover, the fact that this value linearly corresponds to graded supple mentation of single essential amino acid sug gests that one of the most important factors dominating the value is in amino acid bal ance of dietary protein, which primarily lim its the biological quality.