EFFECT OF AN EXCESS INTAKE OF LEUCINE, WITH AND WITHOUT ADDITIONS OF VITAMIN B6 AND/OR NIACIN, ON TRYPTOPHAN AND NIACIN METABOLISM IN RATS

Transcription

1 J. Nutr. Sci. Vitaminol., 23, , 1977 EFFECT OF AN EXCESS INTAKE OF LEUCINE, WITH AND WITHOUT ADDITIONS OF VITAMIN B6 AND/OR NIACIN, ON TRYPTOPHAN AND NIACIN METABOLISM IN RATS Itsiro NAKAGAWA and Atsuko SASAKI1 Department of Nutrition and Biochemistry, Institute of Public Health, Tokyo, Japan (Received May 13, 1977) Summary Three experiments were carried out on a total of 78 female rats to examine the effect of an excess intake of leucine, with and without additions of vitamin B6 and/or niacin, on the excretion of tryptophan and niacin metabolites, and on branched-chain amino acid transaminase activity in some organs and on serum amino acid levels. An excess in take of leucine (10% leucine) retarded the growth of rats, and vitamin B6 and niacin deficiencies did not enhance this effect. Urinary excretion of N-methyl-2-pyridone-5-carboxamide (pyridone) and niacin was scarcely affected by the excess intake of leucine, but the excretion of N L-methylnicotinamide (N-MNA) seemed to decrease. Urinary excre tion of quinolinic acid decreased during the experimental period in all the rats. These facts suggest that the conversion of quinolinic acid to nicotinamide adenine dinucleotide phosphate (NADP) may not be in hibited by an excess intake of leucine. Urinary excretion of 5-hydroxy indoleacetic acid increased in all the rats, and there was no significant difference among them. Branched-chain amino acid transaminase ac tivity decreased in vitamin B6 deficiency, but rather increased in excess leucine. The serum leucine level did not increase in humans in our previous experiment, but in the present experiment in rats, it increased on the 17th and the 21st days of administration of 10% leucine. Serum valine and isoleucine levels decreased. On the contrary, serum tryptophan level increased. An excess intake of leucine, even with a large amount of it, did not seem to be related to the incidence of pellagra, so far as the urinary excretions of tryptophan and niacin metabolites are concerned. GOPALAN et al. (1-3) reported that excess intake of leucine is a cause of pel 535

2 536 I. NAKAGAWA and A. SASAKI lagra in a district in India where jowar (sorgham) is taken as a staple food, and leucine induced an increase of N-MNA and quinolinic acid, and a decrease of pyridone and 5-hydroxyindoleacetic acid. In another experiment, BELAVADY et al. (4) reported that 3 of 5 pups receiving supplements of leucine with a complete diet including nicotinic acid developed signs of nicotinic acid deficiency. In a previous paper (5), we reported the effects of excess leucine and valine deficiency, respectively, on the above metabolites and on plasma amino acid levels in humans. In the present work, the effect of excess leucine, with and without additions of niacin and/or vitamin B6, on tryptophan and niacin metabolism was examined in the rat. EXPERIMENTAL PROCEDURE Three experiments were carried out with a total of 78 female rats. The rats were fed an 18% casein diet (control diet) for 7-9 days after weaning and then were separated into four (in experiment 1 and 3) or five (in experiment 2) groups. Littermates were evenly distributed in different groups. The composition of diets given to each group is shown in Table 1. Each rat was housed in a separate cage with an inverted bottle containing tap water provided freely. The amount of diet intake was made the same in all the rats, as far as possible, by restricting Table 1. Composition of experimental diets. a (mg/100g diet); NaC1, 1,253; KI, 0.025; KH2PO4, 1,715.5; CaHPO4 E2H2O, 21.5; CaCO3, 1,464.5; MgSO4 E7H2O, 499; FeC6H5O7 E6H2O, 31.15; CuSO4 E5H2O, 7.80; ZnCl2, 1.00; MnSO4 E H2O, 6.05; (NH4)6Mo7O2 E4H2O, b (mg/100g diet); thiamin, 0.5; riboflavin, 0.8; pyridoxine, 0.5 (omitted in expt. 1 and 2); nicotinic acid, 4.0 (omitted in expt. 2 and 3); d-ca-pantothenate, 4.0; folic acid, 0.4; cyanocobal amin, 0.003; biotin, 0.04; myoinositol, 10; p-aminobenzoic acid, 10; choline chloride, 200; retinol, 1,000 IU; ergocalciferol, 100 IU; a-tocopherol, 4; menaquinone, 0.5.c In experiment 1, four diets (basal, basal without addition of vitamin B6, excess leucine, and excess leucine without addition of vitamin B6 diets) were used. In experiment 2, five diets (basal, excess leucine, excess leucine without addition of vitamin B6, excess leucine without addition of niacin, and excess leucine without additions of vitamin B6 and niacin diets) were used. In ex periment 3, four diets (basal, excess leucine, basal without addition of niacin, and excess leucine without addition of niacin diets) were used.

3 EXCESS LEUCINE AND NIACIN METABOLITES 537 the amount to be taken by the rats in the basal diet group. Determinations of N-MNA (6), pyridone (7, 8), niacin (9), quinolinic acid (10), 5-hydroxyindoleacetic acid (11), and 4-pyridoxic acid (12) were made on 24-hour urinary samples ob tained during the last three days of each feeding period. All the animals were killed, and branched-chain amino acid transaminase activities (13) in heart, kidney, and liver were determined. A serum aminogram was examined by the use of an automatic amino acid analyzer. As a sufficient amount of blood for amino acid analysis cannot be withdrawn from one rat after overnight fasting, the same amount of blood was collected from each of 3 rats (in experiment 1) or 6 rats (in experiment 3) and then mixed for analysis. Data were analyzed statisti cally with Duncan's multiple comparison procedure (14) and Student's t-test. RESULTS Experiment 1 Rats were divided into 4 groups as shown in Table 2, after feeding them with a control diet for 9 days. The rats were then fed the respective experimental diet Table 2. Urinary excretion of N-MNA, pyridone, nicotinic acid, quinolinic acid, and 4-pyridoxic acid at the intake of excess leucine diet with and without addition of vitamin B6. (17 days) Data were tested with DUNCAN's multiple comparison procedure (14), Values in the same column that do not have the same superscript letter are significantly different (p<0.05). Values of niacin metabolites in the urine in expt. 1 are higher than those in expt. 2, and perhaps it may be due to the difference of feeding method during preexperimental period: Rats of basal group were fed with leucine-excess diet, rats of excess-leucine group fed with basal diet, rats of vitamin B6-deficient group fed with excess-leucine and vitamin B6-deficient diet, and excess-leucine and vitamin B6-deficient group fed with vitamin B6-deficient diet respectively, for 14 days before exchanging diets. for 14 days, and 3 rats of each group were sacrificed to determine the transaminase activity. Another 3 rats of each group were fed further for 17 days, but diets were exchanged (from basal diet to leucine-excess diet, leucine-excess diet to basal diet, vitamin B6-deficient diet to leucine-excess diet without vitamin B6, and leucine excess diet without vitamin B6 to vitamin B6-deficient diet, respectively). Deter minations of N-MNA, pyridone, niacin, quinolinic acid and 4-pyridoxic acid were

4 538 I. NAKAGAWA and A. SASAKI made on 24-hour urinary samples obtained during the last 3 days. A serum amino acid (Table 3) and branched-chain amino acid transaminase activity in organs (Table 4) were examined respectively on the last day of experiment. Table 3. Effect of excess intake of leucine diet with and without addition of vitamin B6 on serum levels of amino acids. (17 days) Table 4. Branched-chain amino acid transaminase activities in heart, kidney and liver of rats fed the excess leucine diet with and without addition of vitamin B6. * Rats of basal group were fed with leucine-excess diet, rats of excess-leucine group fed with basal diet, rats of vitamin B6-deficient group fed with excess-leucine and vitamin B6-deficient diet, and excess-leucine and vitamin B6-deficient group fed with vitamin B6-deficient diet respec tively, for 14 days before exchanging diets. Data were tested with DUNCAN's multiple comparison procedure (14). Values in the same column that do not have the same superscript letter are significantly different (p<0.05). Growth in body weight is shown in Fig. 1. Excess intake of leucine affected body weight, regardless of the presence or abscence of vitamin B6. Table 2 shows concentrations of various metabolites excreted in urine. Urinary excretion of N-MNA tended to decrease both in excess leucine and in vitamin B6 deficiency, but not with significance (p>0.05). Urinary excretion of pyridone decreased in vitamin B6 deficiency, regardless of excess intake of leucine. Excretion of niacin in the urine decreased with excess leucine, regardless of the presence or absence of vitamin B6. Urinary quinolinic acid did not change with excess intake of leucine (p>0.05). Urinary excretion of 4-pyridoxic acid decreased markedly in vitamin B6 deficiency, regardless of excess leucine.

5 EXCESS LEUCINE AND NIACIN METABOLITES 539 Fig. 1 (expt. 1). Growth in body weight. I: Rats were fed basal diet ( ~). II: Rats were fed basal diet with addition of excess leucine ( œ). III: Rats were fed basal diet without addition of vitamin B6 (*). IV: Rats were fed excess leucine diet without addition of vitamin B6 ( ). I>II, etc., indicates that the difference is statistically significant (p < 0.05). Vertical lines show S. D. of the mean. a) Body weight (g), day 7:I>II, IV; III>II, IV; day 14; III>II, IV. b) Body weight (g), day 0:IV>I, II; day 7: III>II III>IV., IV; day 14: III> 11, IV; day 31: Branched-chain amino acid transaminase activities in heart, liver and kidney seemed to decrease with vitamin B6 deficiency and to increase with excess intake of leucine (Table 4). Recently, ICHIHARA (15) reported that the enzyme activity was very high in the stomach. However, we did not determine it at that time. The fasting serum level of leucine increased and those of valine and isoleucine decreased on the 17th day of excess intake of leucine. Tryptophan level tended to be high with excess leucine and to be low with vitamin B6 deficiency. The presence or absence of vitamin B6 did not affect the valine level, but the isoleucine level seemed to decrease with vitamin B6 deficiency (Table 3). The weight of the heart, liver, and kidney tended to decrease with excess leucine, but a definite conclusion could not be drawn from these data (Table 5). Experiment 2 After being fed a control diet for 8 days, rats were divided into 5 groups and fed further for 17 days with basal, excess-leucine, excess-leucine and vitamin B6-deficient, excess-leucine and niacin-deficient, and excess-leucine, vitamin B6 and niacin-deficient diet, respectively. Growth in body weight is shown in Fig. 2. The amount of diet intake was adjusted to be the same in all the rats, as in experiment 1. However, excess intake

6 540 I. NAKAGAWA and A. SASAKI Fig. 2 (expt. 2). Growth in body weight. I: Rats were fed basal diet ( ~). II: Rats were fed basal diet with addition of excess leucine ( œ). III: Rats were fed excess leucine diet without addition of vitamin B6 ( ). IV: Rats were fed excess leucine diet without ad dition of nicotinic acid ( ). V: Rats were fed excess leucine diet without additions of vitamin B6 and nicotinic acid ( ). I>II, etc., indicates that the difference is statistically significant (p <0.05). Vertical lines show S. D. of the mean. Body weight (g), day 7:I>II, III, IV, V; day 13:I>11, III, IV, V; day 17:I>II, III, IV, V. Table 5. Organ weight at the intake of excess leucine diet with and without addition of vitamin B6. weight. Values are means }S. D. Numbers in parentheses indicate organ weight per gram body of leucine affected body weight, and vitamin B6 and niacin deficiencies did not enhance the effect of leucine. Urinary N-MNA, pyridone, niacin, quinolinic acid, and 5-hydroxyindole acetic acid were determined three times during the experimental period and results

7 EXCESS LEUCINE AND NIACIN METABOLITES 541 obtained are shown in Fig. 3 a-e. Urinary excretion of N-MNA in rats fed the basal diet gradually increased during the experiment. However, urinary N-MNA decreased markedly in rats fed the niacin-deficient diet, and tended to decrease in rats fed the excess leucine diet and rats fed the excess leucine diet with vitamin B6. Therefore, the difference between the rats fed the basal diet and those in other groups became marked with progress of the experiment. Urinary pyridone was scarcely affected by excess intake of leucine, but decreased markedly in niacin deficiency. The excretion of niacin in rats fed a basal diet differed from that of rats fed excess leucine, from a cross-sectional observation at the last day of the experiment. However, the difference between those seemed to be insignificant, from a longitudinal observation during the experimental period. Urinary niacin decreased naturally in rats fed niacin-deficient diet. The excretion of quinolinic acid decreased in rats of all the groups during the experiment, and seemed to be remarkable in rats fed niacin-deficient diet. 5-Hydroxyindoleacetic acid in creased in all the rats. The weight of the heart, liver, and kidney of rats in the basal diet group were larger than those of other groups, but their organ weight per gram body weight was lower (Table 6). Table 6. Organ weight at the intake of excess leucine diet with and without additions of vitamin B6 and/or nicotinic acid. (expt. 2) weight. Values are means }S. D. Numbers in parentheses indicate organ weight per gram body Experiment 3 After being fed a basal diet for 7 days, rats were divided into 4 groups and rats of each group were fed further 21 days with basal, excess leucine, niacin deficient or excess-leucine and niacin-deficient diet. Growth in body weight is shown in Fig. 4. Although the amount of diet intake was adjusted to be the same in all the rats, as in the previous experiments, rats used in experiment 3 were older than used in experiment 2, and therefore they were heavier in weight and took more amount of food. Rats fed excess

8 542 I. NAKAGAWA and A. SASAKI Fig. 3.

9 EXCESS LEUCINE AND NIACIN METABOLITES 543 Fig. 4 (expt. 3). Growth in body weight. I: Rats were fed basal diet ( ~). II: Rats were fed basal diet with addition of excess leucine ( œ). III: Rats were fed basal diet without addition of niacin ( ). IV: Rats were fed excess leucine diet without addition of niacin ( ). I, II, etc., indicates that the difference is statistically significant (p<0.05). Vertical lines show S. D. of the mean. Body weight (g), day 7:II<III; day 14:I>II, IV, II<III, III> IV; day 21:I>II, II<III. leucine showed poor growth compared to the rats fed a basal diet. The concentrations of tryptophan and niacin metabolites in the urine were shown in Fig. 5. Excretion of N-MNA in rats used in experiment 3 was greater than that in experiment 2, perhaps due to larger amount of diet intake. However, the tendency of excretion during the experiment was similar to that shown in the previous experiments. With an excess intake of leucine, urinary excretion of N-MNA tended to decrease, but did not differ from that in the basal diet group. The excretion of N-MNA in rats fed niacin-deficient diet decreased remarkably. Urinary excretion of pyridone tended to decrease in the rats fed the excess leucine, but the difference from that of rats fed the basal diet was not significant. Niacin Fig. 3 (expt. 2). Urinary excretions of tryptophan and niacin metabolites. I: Rats were fed basal diet ( ~). II: Rats were fed basal diet with addition of excess leucine ( œ). III: Rats were fed excess leucine diet without addition of vitamin B6 ( ). IV: Rats were fed excess leucine diet without addition of nicotinic acid ( ). V: Rats were fed excess leucine diet without additions of vitamin B6 and nicotinic acid ( ). I>II, etc., indicates that the difference is statistically significant (p<0.05). Vertical lines show S. D. of the mean. a) N-MNA, 10 day: I>IV, I>V, II>IV, II>V, III>IV, III>V. 17 day: I>II, I>III, I>IV, I>V, II>IV, II>V, III>IV, III>V. b) Pyridone, 10 day: I>IV, I>V, II>IV, II>V. 17 day: I>II, I>III, I>IV, I>V, II>IV, II>V, III>IV, III>V. c) Niacin, 10 day: I>III, I>IV, I>V, II>III, II>IV, II>V. 17 day: I>III, I>IV, I>V, II>IV, II>V, III>IV, III>V. d) Quinolinic acid, 17 day: I<II, I<III, II>IV, II>V, III>V. e) 5-Hydroxyindoleacetic acid, 17 day: I<II, I<III, I<V.

10 544 I. NAKAGAWA and A. SASAKI Fig. 5 (expt. 3). Urinary excretions of tryptophan and niacin metabolites. I: Rats were fed basal diet ( ~). II: Rats were fed basal diet with addition of excess leucine ( œ). III: Rats were fed basal diet without addition of nicotinic acid ( ). IV: Rats were fed excess leucine diet without addition of nicotinic acid ( ). I>II, etc., indicates that the difference is statistically significant (p<0.05). Vertical lines show S. D. of the mean. a) N-MNA, 7 day: I>III, I>IV, II>III, II>IV. 21 day: I>III, I>IV, II>III, II>IV. b) Pyridone, 7 day: I>III, I>IV, II>III, II>IV. 21 day: I>II, I>III, I>IV, II>III, II>IV. c) Niacin, 7 day: I>II, I>III, I>IV, II>III, II>IV, 21 day: I>II, I>III, I>IV, II> III, II>IV. d) Quinolinic acid, 21 day: II<III. e) 5-Hydroxyindoleacetic acid, 7 day: I<II, II>III.

11 EXCESS LEUCINE AND NIACIN METABOLITES 545 deficiency markedly affected the excretion of pyridone. Rats in all the groups excreted a larger amount of niacin in the beginning of the experimental period than those in experiment 2. After 1 week, however, the excretion of niacin de creased rapidly in rats of all the groups and thereafter remained at a constant level, having a higher level in basal diet and excess-leucine diet groups than in the niacin-deficient diet group. Urinary quinolinic acid decreased in rats of all the groups just as in experiment 2. It decreased in the first week, and thereafter remained at a constant level. No significant difference was found among dif ferent groups. Excretion of 5-hydroxyindoleacetic acid tended to increase in rats of all the groups. The blood was withdrawn from the rats after overnight fasting, and a serum aminogram was examined on the 21st day of excess intake of leucine (Table 7). Table 7. Effect of excess intake of leucine diet with and without addition of niacin on serum levels of amino acids. Serum leucine level increased and serum valine and isoleucine levels decreased. Deficiency of niacin did not affect serum levels of these amino acids. With excess intake of leucine, serum tryptophan, methionine, and phenylalanine levels in creased and glycine level decreased, but serum glycine level increased in niacin deficiency. The weight of the heart, liver, and kidney tended to decrease with excess leucine, but the weight of these organs per gram body weight did not differ from those of other diet groups (Table 8). DISCUSSION In our experiment in humans (5) and OHGURI'S experiment in rats (5% leucine, unpublished data), effect of excess leucine was not observed on body weight or in urinary excretion of tryptophan and niacin metabolites. In our present experiment, with increased intake level of leucine to 10%, growth in weight was retarded, perhaps due to the toxicity of leucine. Vitamin B6 and

12 546 I. NAKAGAWA and A. SASAKI Table 8. Organ weight at the intake of excess leucine diet with and without addition of nicotinic acid. (expt. 3) weight. Values are means }S. D. Numbers in parentheses indicate organ weight per gram body niacin deficiencies did not seem to give further effect on it. With excess intake of leucine, excretion of pyridone and niacin tended to decrease, compared to that of the basal diet group, but was the same as in the case of vitamin B6 deficiency. Urinary N-MNA tended to decrease in all dietary conditions tested, except that of rats fed a basal diet in which their excretion seemed to increase. TANNOUS et al. (16) also reported that urinary excretion of N-MNA was not affected by the excess intake of leucine. Niacin deficiency naturally decreased the excretion of niacin and its metabolites. Vitamin B6 deficiency did not strengthen the effect of excess leucine on the excretion of N MNA, pyridone, and niacin. HANKES et al. (17) reported that quinolinic acid, which is not converted into NADP because of leucine inhibition, increased. Recently, KRISHNASWAMY et al. (18) reported that vitamin B6 could successfully counteract the effects of leucine on quinolinic acid excretion in urine, and sug gested that vitamin B6 might have a role in the metabolism of quinolinic acid, and administration of leucine resulted in a reduction in the activity of quinolinate phosphoribosyl transferase. Besides, they indicated that vitamin B6 supplements could overcome to a great extent the decrease of urinary 5-hydroxyindoleacetic acid induced by leucine. In our experiment, the excretion of quinolinic acid de creased, and urinary 5-hydroxyindoleacetic acid increased regardless of the presence or absence of vitamin B6. On the other hand, IWAI (19) stated that ad ministration of leucine did not result in any reduction in the activity of quinolinate phosphoribosyl transferase which he isolated from liver and pancreas. According to MURAMATSU et al. (20), addition of 5% leucine alone did not retard growth in body weight, but deprivation of vitamin B6 from the 5% leucine diet caused retarded growth. For this reason, they suggested that the branched chain amino acid transaminase activity in the kidney, heart, and liver decreased in vitamin B6 deficiency, and excess leucine could not be metabolized sufficiently and affected metabolism of other amino acids indirectly, so that the growth was retarded. However, in our case (10% leucine) growth retarded regardless of the presence or absence of vitamin B6. According to TANNOUS et al. (19), the rat fed

13 EXCESS LEUCINE AND NIACIN METABOLITES 547 a high-leucine diet showed a severe growth depression initially, but they became adapted to this diet and started to gain weight slowly. Branched-chain amino acid transaminase activities of organs seemed to de crease in vitamin B6 deficiency and to increase in excess leucine. Therefore, this enzyme activity in rats fed excess leucine diet without vitamin B6 did not differ from that in rats fed the basal diet except that of heart in the second analysis in experiment 1. However, apparent conclusions could not be drawn. In our previous experiment in humans, excess leucine decreased the plasma valine level, but it did not affect the leucine level. However, 10% leucine in creased the serum leucine level on the 17th and 21st days of administration in rats. According to TANNOUS et al. (19), the leucine concentration rose sharply on the first day in plasma from rats fed a high-leucine diet (5% leucine). By the 12th day the plasma concentration of leucine of rats fed a diet containing 5 leucine was not appreciably elevated, including that the ability of the animals to remove excess leucine had improved during this period. By the 12th day the rats were gaining weight and the concentration of isoleucine and valine in plasma increased. These changes may be the result of an adaptation which occurs in response to a high leucine intake. However, in our experiments, serum leucine remained at a high level, and serum valine and isoleucine levels decreased. Neither vitamin B6 nor niacin deficiency affected these amino acid levels. On the other hand, with excess intake of leucine, serum tryptophan level increased, and it remains to be determined whether it can be expressed as an amino acid imbalance. REFERENCES 1) GOPALAN, C.. and SRIKANTIA, S. G. (1960): Leucine and pellagra. Lancet, 1, ) BELAVADY, B., SRIKANTIA, S. G., and GOPALAN, C. (1963): The effect of oral administra tion of leucine on the metabolism of tryptophan. Biochem. J., 87, ) GOPALAN, C. (1969): Possible role for dietary leucine in the pathogenesis of pellagra. Lancet, 1, ) BELAVADY, B., MADHABVAN, T. V., and GoPALAN, C. (1967): Production of nicotinic acid deficiency (blacktongue) in pups fed diets supplemented with leucine. Gastroenterology, 53, ) NAKAGAWA, I., OHGURI, S., SASAKI, A., KAJIMOTO, M., SASAKI, M., and TAKAHASHI, T. (1975): Effect of excess intake of leucine and valine deficiency on tryptophan and niacin metabolites in humans. J. Nutr., 105, ) HUFF, J. W., and PERLZWEIG, W. A. (1947): The fluorescent condensation product of N L-methylnicotinamide and acetone. II. A sensitive method for the determination of N L-methylnicotinamide in urine. J. Biol. Chem., 167, ) PRICE, J. M. (1954): The determination of N-methyl-2-pyridone-5-carboxamide in human urine. J. Biol. Chem., 211, ) WALTERS, C. J., BROWN, R. R., KAIHARA, M., and PRICE, J. M. (1955): The excretion of N-methyl-2 pyridone-5-carboxamide by man following ingestion of several known potential precursors. J. Biol. Chem., 217, ) MIYAZAWA, S. (1952): Determination of vitamin by use of micro-organisms. Annual Rept. of Takamine Lab., 4,

14 548 I. NAKAGAWA and A. SASAKI 10) HENDERSON, L. M. (1949): Quinolinic acid metabolism. II. Replacement of nicotinic acid for the growth of the rat and neurospora. J. Biol. Chem., 181, ) DALGLIESH, C. E. (1958): Determination of 5-hydroxyindoleacetic acid. Advan. Clin. Chem., 1, ) REDDY, S. K., REYNOLDS, M. S., and PRICE, J. M. (1958): The determination of 4-pyri doxic acid in human urine. J, Biol. Chem., 233, ) ICHIHARA, A., and KOYAMA, E. (1966): Transaminase of branched chain amino acids. I. Branched chain amino acids-ƒ -ketoglutarate transaminase. J. of Biochem., 59, ) DUNCAN, D. B. (1955): Multiple range and multiple F tests. Biometrics, 11, ) ICHIHARA, A. (1976): Speciality in amino acid metabolism. Res. Comm. Essen. Amino Acids (in Japanese), No ) TANNOUS, R. I., ROGERS, Q. R., and HARDER, A. E. (1966): Effect of leucine-isoleucine antagonism on the amino acid pattern of plasma and tissues of the rat. Arch. Biochem. Biophys.,113, ) HANKES, L. V., LEKLEM, J. E., BROWN, R. R., and MEKEL, R. C. P. M. (1971): Tryptophan metabolism in patients with pellagra: problem of vitamin B6 enzyme activity and feedback control of tryptophan pyrrolase enzyme. Amer. J. Clin. Nutr., 24, ) KRISHNASWAMY, K., RAO, S. B., RAGHURAM, T. C., and SRIKANTIA, S. G. (1976): Effect of vitamin B6 on leucine-induced changes in human subjects. Amer. J. Clin. Nutr., 29, ) IWAI, K. (1976): Comment during the 234th Vitamin B Committee. Vitamin (in Japanese), 50, ) MURAMATSU, K., and ODAGIRI, H. (1971): Amino acid toxicity in vitamin B6 deficiency. Growth and metabolism of young rats fed diets containing excess leucine. Rep. Res. Comm. Essen. Amino Acids (in Japanese), No. 52, 9-10.