Vitamin B,. Dependence oÃ-Selenomethionine Selenite Utilization for Glutathione Peroxidase in the Rat
and
ABSTRACT The biological availability of selenium from sodium selenite and selenomethionine for glutathione peroxidase activity was studied. Rats were fed ad libitum for 2 weeks a basal diet déficientin both selenium and vitamin B6, and then for the subsequent 2 weeks the same diet supple mented with vitamin BB (2.5 ¿tgas pyridoxine•HCl/g diet) or selenium ( 2 /tg/g diet ) or both. In the presence of vitamin B6, selenite and seleno methionine increased equally the glutathione peroxidase activity in both the liver and erythrocytes above that of selenium-unsupplemented controls. In the absence of vitamin Be, selenomethionine was less effective in the liver and ineffective in the erythrocytes while selenite was equally effective in both tissues and was as effective as in the presence of vitamin B«.These results indicate that selenite selenium is readily available for glutathione peroxidase induction as compared with selenomethionine, and establish that vitamin Bc is involved in the metabolism of selenomethionine to supply selenium for glutathione peroxidase. J. Nutr. 109: 760-766, 1979. INDEXING KEY WORDS vitamin B«•selenite •selenomethionine glutathione peroxidase Selenium has been shown to be an essential trace element for experimental animals (1). The central metabolic function of selenium in animal tissues is believed to be as an integral part of the enzyme glutathione peroxidase ( glutathione-hydrogen peroxide oxidoreductase, EC 1.11.1.9) (2, 3). The enzyme activity increases with increasing dietary levels of selenium (4, 5), and is severely depressed in its deficiency (2). This depletion is expected to account for many of the manifestations of selenium deficiency. The biological availability of dietary selenium seems to depend primarily on its chemical nature rather than on its digestion or absorption characteristics in the intestine (6). Dietary selenium is usually found in association with protein as selenoamino acids in plants and in as yet incompletely understood
forms
in animals.
A large
tion, if not all, of selenium in such animal tissue seems to be prone to alkali treatment to give selenite (7) or selenite and elemental selenium (8). Thomson et al. (9) have presented evidence to suggest that the initial metabolism by rats of selenium compounds depends upon the particular compound, although the selenium ultimately enters into the same metabolic pool, Selenomethionine and selenite are equally effective in preventing selenium deficiency lesions ( 10 ). In animal tissues conversion of selenomethionine into selenite occurs rather readily but the reverse rarely occurs (7, 8). This difference in metabolism gives rise to the conjecture that they may differ with respect to availability for glutathione peroxidase activity. The present study compares the biologi-
por-
Received for publication September 14, 1978. 760
Downloaded from https://academic.oup.com/jn/article-abstract/109/5/760/4770713 by guest on 15 October 2018
KYODEN YASUMOTO, KIMIKAZU IWAMI ANDMUNEHIRO YOSHIDA Department of Food Science and Technology, Faculty of Agriculture, Kyoto University, Sakyo-ku, Kyoto, 606 Japan
PYRIDOXINE-DEPENDENT
UTILIZATION
cal effectiveness of selenite and selenomethionine in raising the glutathione peroxidase activity in vitamin B6-deficient rats. The results show that dietary vitamin Be influences utilization of selenomethionine but not of selenite, suggesting that seleno methionine as such is not incorporated into the prosthetic group of the enzyme. AND METHODS
Male rats of Wistar strain, weighing 50 to 60 g each, were housed individually in hanging wire-meshed cages in a humiditytemperature controlled room with natural light-dark cycle. The rats had free access to distilled water and food during the whole experimental period. The rats were fed for 2 weeks a basal diet which was deficient in vitamin BOand selenium. The basal diet contained: (in %) vitamin-free casein,1 20; corn starch, 50; sucrose, 15; soybean oil,2 6.4; cod liver oil,3 1.6; salt mix,4 4; cellulose powder, 2; and vitamin mix (vitamin B6 excepted)/' 1. The rats were divided into 6 groups and, for an additional 2 weeks, fed the basal diet which was supplemented with vitamin B6 or selenium or both so as to provide six different combinations of the supplements. Selenium was added as either sodium sele nite or DL-selenomethionine at 2 /ig selenium per g diet, and pyridoxine • HC1 at 2.5 /¿g per g diet. After receiving one of these test diets for 2 weeks, rats were stunned with a sharp blow to the head and decapitated with a guillotine. Blood samples were centrifuged at 750 X g for 15 minutes. The cells were washed 3 times with cold saline, and then lysed in 25 rriM phosphate buffer of pH 7.0 for the enzyme assays. The livers were ho mogenized in a Potter-Elvehjem homogenizer in 10 volumes of 50 HIM phosphate buffer containing 0.25 M sucrose at pH 7.0. The homogenates were centrifuged at 6,000 X g for 15 minutes and the supernatant solutions were used for the enzyme assays. Glutathione peroxidase was assayed by a modification of the method of Little et al. (11) with terÃ--butyl hydroperoxide as the peroxidase substrate and coupled to NADPH oxidation via glutathione reduc Ã-ase (EC 1.6.4.2). Aspartate aminotransferase (EC 2.6.1.1) and cystathionase (EC
761
4.2.1.15) were determined by the methods of Furuno et al. (12) and Matsuo et al. (13), respectively. Glutathione S-transfer ase (EC 2.5.1.18) activity was assayed ac cording to Habig et al. ( 14 ) with 1-chloro2,4-dinitrobenzene as substrate. Protein was determined by the method of Lowry et al. as modified by Miller ( 15). Experimental data were tested using analysis or variance; when F-tests were significant ( P < 0.05 ), comparisons were made using the Least Significance Difference test to determine which pairs of means were significantly different (P
and
ABSTRACT The biological availability of selenium from sodium selenite and selenomethionine for glutathione peroxidase activity was studied. Rats were fed ad libitum for 2 weeks a basal diet déficientin both selenium and vitamin B6, and then for the subsequent 2 weeks the same diet supple mented with vitamin BB (2.5 ¿tgas pyridoxine•HCl/g diet) or selenium ( 2 /tg/g diet ) or both. In the presence of vitamin B6, selenite and seleno methionine increased equally the glutathione peroxidase activity in both the liver and erythrocytes above that of selenium-unsupplemented controls. In the absence of vitamin Be, selenomethionine was less effective in the liver and ineffective in the erythrocytes while selenite was equally effective in both tissues and was as effective as in the presence of vitamin B«.These results indicate that selenite selenium is readily available for glutathione peroxidase induction as compared with selenomethionine, and establish that vitamin Bc is involved in the metabolism of selenomethionine to supply selenium for glutathione peroxidase. J. Nutr. 109: 760-766, 1979. INDEXING KEY WORDS vitamin B«•selenite •selenomethionine glutathione peroxidase Selenium has been shown to be an essential trace element for experimental animals (1). The central metabolic function of selenium in animal tissues is believed to be as an integral part of the enzyme glutathione peroxidase ( glutathione-hydrogen peroxide oxidoreductase, EC 1.11.1.9) (2, 3). The enzyme activity increases with increasing dietary levels of selenium (4, 5), and is severely depressed in its deficiency (2). This depletion is expected to account for many of the manifestations of selenium deficiency. The biological availability of dietary selenium seems to depend primarily on its chemical nature rather than on its digestion or absorption characteristics in the intestine (6). Dietary selenium is usually found in association with protein as selenoamino acids in plants and in as yet incompletely understood
forms
in animals.
A large
tion, if not all, of selenium in such animal tissue seems to be prone to alkali treatment to give selenite (7) or selenite and elemental selenium (8). Thomson et al. (9) have presented evidence to suggest that the initial metabolism by rats of selenium compounds depends upon the particular compound, although the selenium ultimately enters into the same metabolic pool, Selenomethionine and selenite are equally effective in preventing selenium deficiency lesions ( 10 ). In animal tissues conversion of selenomethionine into selenite occurs rather readily but the reverse rarely occurs (7, 8). This difference in metabolism gives rise to the conjecture that they may differ with respect to availability for glutathione peroxidase activity. The present study compares the biologi-
por-
Received for publication September 14, 1978. 760
Downloaded from https://academic.oup.com/jn/article-abstract/109/5/760/4770713 by guest on 15 October 2018
KYODEN YASUMOTO, KIMIKAZU IWAMI ANDMUNEHIRO YOSHIDA Department of Food Science and Technology, Faculty of Agriculture, Kyoto University, Sakyo-ku, Kyoto, 606 Japan
PYRIDOXINE-DEPENDENT
UTILIZATION
cal effectiveness of selenite and selenomethionine in raising the glutathione peroxidase activity in vitamin B6-deficient rats. The results show that dietary vitamin Be influences utilization of selenomethionine but not of selenite, suggesting that seleno methionine as such is not incorporated into the prosthetic group of the enzyme. AND METHODS
Male rats of Wistar strain, weighing 50 to 60 g each, were housed individually in hanging wire-meshed cages in a humiditytemperature controlled room with natural light-dark cycle. The rats had free access to distilled water and food during the whole experimental period. The rats were fed for 2 weeks a basal diet which was deficient in vitamin BOand selenium. The basal diet contained: (in %) vitamin-free casein,1 20; corn starch, 50; sucrose, 15; soybean oil,2 6.4; cod liver oil,3 1.6; salt mix,4 4; cellulose powder, 2; and vitamin mix (vitamin B6 excepted)/' 1. The rats were divided into 6 groups and, for an additional 2 weeks, fed the basal diet which was supplemented with vitamin B6 or selenium or both so as to provide six different combinations of the supplements. Selenium was added as either sodium sele nite or DL-selenomethionine at 2 /ig selenium per g diet, and pyridoxine • HC1 at 2.5 /¿g per g diet. After receiving one of these test diets for 2 weeks, rats were stunned with a sharp blow to the head and decapitated with a guillotine. Blood samples were centrifuged at 750 X g for 15 minutes. The cells were washed 3 times with cold saline, and then lysed in 25 rriM phosphate buffer of pH 7.0 for the enzyme assays. The livers were ho mogenized in a Potter-Elvehjem homogenizer in 10 volumes of 50 HIM phosphate buffer containing 0.25 M sucrose at pH 7.0. The homogenates were centrifuged at 6,000 X g for 15 minutes and the supernatant solutions were used for the enzyme assays. Glutathione peroxidase was assayed by a modification of the method of Little et al. (11) with terÃ--butyl hydroperoxide as the peroxidase substrate and coupled to NADPH oxidation via glutathione reduc Ã-ase (EC 1.6.4.2). Aspartate aminotransferase (EC 2.6.1.1) and cystathionase (EC
761
4.2.1.15) were determined by the methods of Furuno et al. (12) and Matsuo et al. (13), respectively. Glutathione S-transfer ase (EC 2.5.1.18) activity was assayed ac cording to Habig et al. ( 14 ) with 1-chloro2,4-dinitrobenzene as substrate. Protein was determined by the method of Lowry et al. as modified by Miller ( 15). Experimental data were tested using analysis or variance; when F-tests were significant ( P < 0.05 ), comparisons were made using the Least Significance Difference test to determine which pairs of means were significantly different (P
