J Nutr Sci Vitaminol, 1998, 44, 37-45 Dietary Protein as a Factor Affecting Vitamin B6 Requirement Mitsuko OKADA, *Mayumi SHIBUYA, 1 Tomoko AKAZAWA, Hitomi MUYA and Yoko MURAKAMI Faculty of Health and Living Sciences, Naruto University of Education, Naruto 772-8502, Japan1 Junior College, Shikoku University, Tokushima 771-1192, Japan (Received October 8, 1997) Summary Rats were fed 20 or 70% casein diets with varying amounts of vitamin B6 (B6), and the B6 content and B6-dependent enzymatic activity in their tissues were examined to determine the minimum re quirement of B6 for animals subjected to different levels of dietary protein (i.e., 20%: 0, 1.45, 2.90, 5.80mg pyridoxine (PN)/kg diet; 70%: 0, 2.90, 5.80, 8.70mg PN/kg diet). B6 requirements for the rats were almost met in the 1.45mg PN/kg 20% casein diet and the 2.90mg PN/kg 70% casein diet when judged from the hepatic B6 content. However, almost twice the PN was required in both 20 and 70% casein diets when judged from PLP-enzymatic activity. The content of B6 vitamers in plasma appeared to be most sensitive to B6 status, though the satisfactory level is not known. It was confirmed that, in any case tested, a high-protein diet increased the requirement of B6. Key Words vitamin B6 requirement, vitamin B6 content, aspartate aminotransferase, glycogen phosphorylase Vitamin B6 (B6) is found in biological tissues and fluids as pyridoxine (PN), pyridoxal (PL), pyridoxamine (PM) and their phosphorylated derivatives. The vitamers function as coenzymes of glycogen phosphorylase and many amino acid metabolic enzymes. A high-protein diet increases the requirement for B6 as follows: First, a high-protein diet containing a low B6 content increases the urinary excre tion of xanthrenic acid (an abnormal metabolite of tryptophan) and some other tryptophan metabolites such as kynurenine and hydroxykynurenine (1); second, in animals fed a B6-free, high-protein diet, growth is retarded and is accompanied by a severe decrease in the activity of pyridoxal enzymes compared to animals fed a B6-free, low-protein diet (2, 3); and third, an increase in B6 intake increases phosphorylated pyridoxal (PLP) concentration, whereas increased protein intake decreases plasma PLP concentration (4). The accurate determination of B6 in biological samples is essential for assessing nutritional and metabolic requirements. There have been many attempts to analyze * To whom correspondence should be addressed. 37
38 M OKADA et al the vitamin content of biological samples, including microbiological, colorimetric and fluorometric methods combined with ion-exchange column chromatography; however, these methods are not very accurate or sensitive. Recently, a combined method of separation of the vitamin by high-pressure liquid chromatography (HPLC) and post-column derivatization of PLP with sodium bisulfate was found to be more sensitive and accurate, but also more time consuming (5,6). In this study, we fed rats 20 or 70% casein diets with varying amounts of B6, and examined the B6 content and the PLP-enzymatic activity in their tissues to determine the minimum requirement of B6 for animals subjected to different levels of dietary protein. MATERIALS AND METHODS Animals. Two series of experiments were performed. In experiment 1, 25male weanling (3weeks old) rats of the Wistar strain were divided into 4 groups (7 for 1 group and 6 for the other groups). Each group was fed a 20% casein diet containing 0mg/kg (Group 20-0), 1.45mg/kg (Group 20-0.5), 2.9mg/kg (Group 20-1) or 5.8mg/kg (Group 20-2) pyridoxine hydrochloride. In experiment 2,24male weanling (3weeks old) rats of the Wistar strain were divided into 4 groups as well. Each group was fed a 70% casein diet containing 0mg/kg (Group 70-0), 2.9mg/kg (Group 70-1), 5.8mg/kg (70-2) or 8.7mg/kg (70-3) pyridoxine hydrochloride. The animals had free access to the prescribed diet and water for 4 weeks. The complete composition of the diet, other than pyridoxine, has been described previously (7). All dietary materials were obtained from Oriental Yeast Co., Tokyo, Japan. Chemicals. All reagents used were of the highest grade available and were purchased from Wako Pure Chemicals Co. (Osaka, Japan), Boehringer (Mannheim, Germany), Sigma Chemicals Co. (St. Louis, MO, USA) or Oriental Yeast Co. (Osaka, Japan). Preparation. The rats were decapitated and blood was collected into he parinized tubes. The blood was centrifuged at 2,000rpm for 10min. The resultant precipitate was washed twice with an equal volume of saline, suspended in 2 volumes of water and homogenized with a Polytron. The homogenate was centrifuged at 10,000 ~g for 20min, and the supernatant was used to measure aspartate aminotransferase [EC 2.6.1.11 (AST) activity and hemoglobin. The plasma fraction was used for the determination of B6 vitamers as described previously (8). Briefly, plasma (0.5mL) was diluted to 1mL with water, 27ƒÊL of 9N HCl4 was added, the sample was centrifuged at 10,000 ~g for 5min and the resultant supernatant was used for analysis of the B6 content. Rat tissues were immediately removed divided into 2 or 3 pieces depending on the analysis and, except for a piece of liver tissue for the measurement of AST, all tissue samples were frozen in liquid nitrogen, and stored in a freezer until they were used. Pieces of liver (not frozen) were homogenized with 9 volumes of 0.25M sucrose-5mm phosphate (ph 7.4) (v/g) buffer using a Teflon homogenizer, centrifuged at 40,000rpm for 60min and the J Nutr Sci Vitanninol
B6 Requirement and Dietary Protein 39 supernatant used to analyze cytosolic AST activity (9). A piece of gastrocnemius muscle was homogenized with 9 volumes of 0.1M PIPES buffer (ph 6.0) (v/g) (10) and the supernatant, centrifuged at 10,000 ~g for 20min, was used to analyze phosphorylase [EC 2.4.1.1] and AST activity. Another piece of each liver and muscle tissue was homogenized with 9 volumes of 1N HClO4 (v/g) and the supernatant, centrifuged at 10,000 ~g for 10min, was used to analyze B6 content. Methods. B6 vitamers were determined using an HPLC system according to the method originally described by Edwards et al (5) and described previously (6, 8). A Shimadzu RF-535 fluorescence monitor was used to detect the fluorescence of B6 (ex. 325nm, em. 420nm). Glycogen phosphorylase activity was determined by analyzing inorganic phosphate levels during the formation of glycogen in the presence of 1mM AMP (10,11). AST activity was determined in the presence or absence of 10-4M PLP by measuring the rate of NADH oxidation in the presence of malate dehydrogenase as described by Karmen (12). AST activity in the erythrocytes was followed by preincubation with lactate dehydrogenase to remove endogeneous pyruvate prior to starting the AST reaction (13). Hemoglobin was determined using a Wako Hemoglobin B Kit. Statistical analysis. Data were analyzed using Scheffe's multiple test to determine which means were significantly different from each other (i.e.,p<0.05). RESULTS Effects of dietary protein and B6 contents on growth of animals and tissue weights Rats were fed a 20 or 70% casein diet containing various amounts of B6 for 4 weeks. The growth of rats fed a vitamin B6-free diet (20-0, 70-0) was significantly impaired (Table 1). The growth of rats in the 70-0 group was especially affected. The weights of the liver and gastrocnemius muscle from these animals are shown as gram per 100g body weight (Table 2). There were no significant differences in the weights of the tissues between groups, but the weight of liver tissue from the 70% casein groups tended to increase in the 70-3 group. Effect of dietary B6 levels on the B6 content in rat tissues B6 content was measured in rat tissues (i.e., plasma, liver and gastrocnemius muscle) (Table 3). The B6 content in the plasma of rats fed a B6-free diet was below the detection limit (20-0 and 70-0; not shown). PLP and PL, the main components of B6 in the plasma, gradually increased as B6 intake increased. The level of B6 vitamers in the plasma of rats fed a 20% casein diet was higher than that in the 70% casein groups when there was an equal amount of B6 in the diets. In the liver, PMP and PLP were the main form of B6 compounds detected, and the B6 content in both the 20-0 and 70-0 groups was significantly lower than in the other groups. According to the increase in dietary B6 levels, B6 content in the liver gradually increased in both the 20 and 70% groups, then tended to approach to saturated level which was different for the 20 (lower) and 70% (higher) groups. Vol 44, No 1, 1998
40 M OKADA et al Table 1. Growth of rats fed a 20 or 70% casein diet with different amounts of vitamin B6. Groups 20 and 70 were fed at different times so data were analyzed separately. Data were analyzed by Scheffe's multiple test. Within a column, values are not significantly different if and only if superscripts contain at least one common letter (p<0.05). Table 2. Tissue weights of rats fed a 20 or 70% casein diet with different amounts of vitamin B6. Data were analyzed by Scheffe's multiple test. Within a column, values are not significantly different if and only if superscripts contain at least one common letter (p<0.05). In the gastrocnemius muscle, PLP was the main component of the B6 compounds, however, some PMP was also detected. The level of the vitamer in rats fed the B6-deficient diet was approximately 20% of that of the rats given a saturated amount of B6 (20-1, 2 and 70-2, 3). Of the groups with 20% casein and 70% casein diets, 20-1 and 70-2 had saturated levels of B6. J Nutr Sci Vitaminol
B6 Requirement and Dietary Protein 41 Table 3. Vitamin B6 content in the tissues of rats fed a 20 or 70% casein diet with different amounts of vitamin B6. Data were analyzed by Scheffe's multiple test. Within a column, values are not significantly different if and only if superscripts contain at least one common letter (p<0.05). Effect of dietary B6 levels on enzymatic activity AST activity in the erythrocytes, liver and muscle, and glycogen phosphorylase activity in the muscle were measured in these animals (Tables 4 and 5). In erythrocytes, the effect of the addition of PLP to the nast activity in the 20-0 and 70-0 groups was larger than 1.8, a value used for the assessment of the B6 status Vol 44, No 1, 1998
42 M OKADA et al Table 4. Aspartate aminotransferase activity in the tissues of rats fed a 20 or 70% casein diet with different amounts of vitamin B6. Data were analyzed by Scheffe's multiple test. Within a column, values are not significantly different if and only if superscripts contain at least one common letter (p<0.05). Asterisks signify significantly different values between -PLP and +PLP (p<0.05). in humans (14), and those activities were very low as compared to the values obtained from the B6-fed groups, even in the presence of PLP. As dietary B6 increased, AST activity gradually increased both with and without PLP, but the effect of the addition of PLP in each group was not significant. In the liver, cytosolic AST activity in the rats fed the 20% casein diet had normal values in all groups, including the B6-deficient group. On the other hand, the AST activity in the 70-0 group was significantly lower than that in the other 70% groups, and thus, the 70-0 group was judged to be deficient although the effect of the addition of PLP showed AST activity of 1.59-fold. Muscle AST activity was significantly lower in both the J Nutr Sci Vitaminol
B6 Requirement and Dietary Protein 43 Table 5. Muscle phosphorylase activity in rats fed a 20 or 7000 casein diet with different amounts of vitamin B6. Data were analyzed by Scheffe's multiple test. Within a column, values are not significantly different if and only if superscripts contain at least one common letter (p<0.05). 20-0 and 70-0 groups than that in the other groups, and judged to be deficient because the addition of PLP gave AST activity of 2.89-fold for the 20-0 group and 2.52-fold for the 70-0 group. As shown in Table 5, glycogen phosphorylase activity was not affected by the addition of PLP in all groups tested; however, activity in the 20-0 and 70-0 groups was significantly lower than that in the other groups. Moreover, activity in the 20-0.5 and 70-1 groups was also lower than in the other groups fed diets containing much higher amounts of B6. Thus, the 20-0 and 70-0 groups were judged to be severely deficient, and the 20-0.5 and 70-1 groups were mildly deficient as determined by the levels of glycogen phosphorylase. DISCUSSION In this study we examined the B6 content and some PLP-enzyme activities in several tissues of rats fed either a 20 or 70% casein diet containing different levels of B6 (Tables 1-5). The B6 content in the plasma of rats fed a 70% casein diet was approximately half of that in the 20% casein groups (20-1 vs 70-1, 20-2 vs 70-2). B6 vitamers in the liver occurred mainly as PMP and PLP in a ratio of 2:1 in all cases tested except for the groups fed B6-free diets. B6 requirements for the rats were almost met in the 20-0.5 and 70-1 groups when judged from the hepatic B6 contents. For muscle, however, a much higher level of B6 was necessary (i.e., groups 20-1 and 70-2), and the main component of the vitamer was PLP, which accounted for 75% of the total coenzyme. The B6 requirements for rats fed the 20 or 70% casein diets were also determined from the AST and glycogen phosphorylase activity in several tissues. In erythrocytes, although the addition of PLP (10-4M) increased AST activity in both B6-free groups Vol 44, No 1, 1998
44 M OKADA et al (Table 4), activity after PLP was added was still lower than that in the B6-fed groups, suggesting that apo-ast protein may be lower for some reason (i.e., increased degradation or decreased synthesis of the enzyme protein). Taking these data into account, the use of the ratio+plp/-plp for the assessment of B6 status is not satisfactory, but the value of final activity in the presence of PLP may need to be considered. B6 deficiency was not observed for hepatic cytosolic AST activity in rats fed a 20% casein diet. On the other hand, B6 deficiency was observed in the 70-0 group, while B6 levels were evidently sufficient for the liver in the 70-1 group. The muscle tissue of the 20-0 and 70-0 groups appeared to be deficient, but the other groups had sufficient B6 levels when determined by AST activity. Glycogen phosphorylase activity was not increased by the addition of PLP in any case, suggesting that no apo-enzyme is contained in the tissue. B6 deficiency results in a decrease in the enzyme protein of glycogen phosphorylase (10), and in this study, 20-0, 20-0.5, 70-0 and 70-1 groups were estimated as B6 deficient when judged from this enzyme activity. Thus, 2.90mg PN/kg in 20% casein diet and 5.80mg PN/kg in 70% casein diet is necessary to maintain health when judged from both the content of B6 vitamers and B6-enzyme activity. B6 vitamers contained in the diet are known to appear in the plasma mainly as PL (15) and are incorporated into various tissues according to need. Based on the finding of a high content of glycogen phosphorylase in muscle, Krebs and Fischer proposed that muscle phosphorylase acts as a repository of B6 (16). They calculated that 60% of the B6 present in rat muscle was present in the phosphorylase. As phosphorylase constitutes nearly 5% of the soluble protein in muscle (17) and muscle accounts for approximately 40% of the body mass, this enzyme would have a substantial capacity for the storage of B6. In this study, we also found that more than 80% of the muscle B6 exists as PLP, which is the sole coenzyme form of phosphorylase. Black et al reported that muscle phosphorylase and total muscle B6 increased steadily in rats fed high levels of B6 (18). Their results support the hypothesis of Krebs and Fischer that muscle phosphorylase acts as a reservoir for B6 in animals. In this study, we found that when the intake of B6 becomes inadequate, B6 is preferentially incorporated into the liver and is bound to the cytosolic AST, and thus liver appears to resist B6 deficiency. We also found that PL incorporated into the muscle is preferentially bound to the AST molecule rather than to glycogen phosphorylase. Our data indicate that a high protein intake increases the requirement for B6 when the content of B6 vitamers and PLP-enzymatic activity were accounted as the indicators of B6 status, and also that a role of the muscle phosphorylase is to act as a reservoir for B6 in animals. REFERENCES 1) Linkswiler H. 1967. Biochemical and physiological changes in vitamin B6 deficiency. Am J Clin Nutr 20: 547-557. J Nutr Sci Vitaminol
B6 Requirement and Dietary Protein 45 2) Okada M, Ochi A. 1970. The effect of dietary protein level on transaminase activities and fat deposition in vitamin B6-depleted rat liver. J Biochem 70: 581-585. 3) Itoh R, Okada M. 1973. Effect of dietary protein level on pyridoxal content in tissues and excretion of pyridoxic acid into urine in normal or pyridoxine-deficient rat. J Nutr Sci Vitaminol 19: 523-528. 4) Miller LT, Leklem JE, Shultz TD. 1985. The effect of dietary protein on the metabolism of vitamin B-6 in humans. J Nutr 115: 1663-1672. 5) Edwards P, Liu PK, Rose GA. 1989. A simple liquid-chromatographic method for measuring vitamin B6 compounds in plasma. Clin Chem 35: 241-245. 6) Takashi Y, Shibuya M.1994. Response of B6 vitamers in blood, tissues, and 4-pyridoxic acid in urine to three different levels of vitamin in rats. Bull Shikoku Univ (B)2: 57-62. 7) Okada M, Suzuki K. 1974. Amino acid metabolism in rats fed a high protein diet without pyridoxine. J Nutr 104: 287-293. 8) Okada M, Miyamoto P, Nishida T, Tomita T, Shibuya M. 1997. Effect of vitamin B-6 nutrition and diabetes on vitamin B-6 metabolism. J Nutr Biochem 8: 44-48. 9) Okada M, Hirose M. 1979. Regulation of aspartate aminotransferase activity associated with change of pyridoxal phosphate level. Arch Biochem Biophys 193: 294-300. 10) Okada M, Ishikawa K, Watanabe K. 1991. Effect of vitamin B6 deficiency on glycogen metabolism in the skeletal muscle, heart, and liver of rat. J Nutr Sci Vitaminol 37: 349-357. 11) Fiske CH, Subbarow Y. 1925. The colorimetric determination of phosphorus. J Biol Chem 66: 375-400. 12) Karmen A. 1955. A note on the spectrophotometric assay of glutamic oxalacetic transaminase in human blood. J Clin Invest 34: 131-133. 13) Bergmeyer HU, Horder M, Rel R. 1986. Approved recommendation (1995) on IFCC methods for the measurement of catalytic concentration of enzymes. J Clin Chem Clin Biochem 24: 497-510. 14) Leklem JE. 1990. Vitamin B-6: A status report. J Nutr 120: 1503-1507. 15) Sakurai T, Asakura T, Mizuno A, Matsuda M. 1991. Absorption and metabolism of pyridoxamine in mice I. Pyridoxal as the only form of transport in blood. J Nutr Sci Vitaminol 37: 341-348. 16) Krebs EG, Fischer EH. 1964. Phosphorylase and related enzymes of glycogen metabolism. Vitam Horm 22: 399-410. 17) Fischer EH, Heilmeyer LMG Jr, Haschke RH. 1971. Phosphorylase and the control of glycogen degradation. Curr Top Cell Regul 4: 211-251. 18) Black AL, Guirard BM, Snell EE. 1977. Increased muscle phosphorylase in rats fed high levels of vitamin B6. J Nutr 107: 1962-1968. Vol 44, No 1, 1998