mixes containing 20, 60, or 120 mg of Se as sodium selenite/g of salt mix; the fourth treatment was 60 mg of Se as selenized yeast/g of salt mix. Cows given salt.
Effect of Selenium Supplements on the Distribution of Selenium Among Serum Proteins in Cattle F. T. AWADEH,*,1 M. M. ABDELRAHMAN,†,2 R. L. KINCAID,*,1,3 and J. W. FINLEY‡,4 *Washington State University, Pullman 99164-6310 †Jerash University, Jerash, Jordan ‡USDA, ARS, Grand Forks, ND 58202-9034
ABSTRACT The objective of this study was to determine the effects of the amount and chemical form of dietary Se on the distribution of Se among serum proteins. Six growing calves were assigned in a completely randomized design to receive diets containing either adequate (0.41 mg/g) or excess (0.73 mg/g) dietary Se. Proteins in serum collected from the calves were separated into albumin, glutathione peroxidase, and selenoprotein P fractions, and the concentration of Se in each was determined. The concentration of Se within serum was elevated by dietary Se supplementation. The selenoprotein P fraction within serum contained the largest percentage of Se among the serum proteins. In a second study, 12 mature cows were assigned to receive one of four experimental salt mixes containing 20, 60, or 120 mg of Se as sodium selenite/g of salt mix; the fourth treatment was 60 mg of Se as selenized yeast/g of salt mix. Cows given salt with 120 mg of Se as selenite or 60 mg of Se as selenized yeast had the highest concentrations of Se in whole blood; however, concentrations of Se in serum did not differ among treatments. Concentrations of Se in the protein fractions within serum were not affected by treatment. Within serum, the highest concentration of Se was in the selenoprotein P fraction (31.6 ng/ml), the smallest concentration was in the glutathione peroxidase fraction (4.7 ng/ml), and an intermediate amount of Se was obtained from the albumin fraction (8.5 ng/ml). In conclusion, selenized yeast and selenite as sources of Se for supplementation of cattle resulted in similar patterns of Se distribution among proteins in serum. The greatest concentration of Se was found in the selenoprotein P
Received May 2, 1997. Accepted October 23, 1997. 1Department of Animal Sciences. 2Department of Animal Production. 3To whom correspondence should be addressed. 4Grand Forks Human Nutrition Research Center. 1998 J Dairy Sci 81:1089–1094
fraction, which may contribute to Se transportation or function as an antioxidant. ( Key words: selenium, cows, calves, selenoproteins) Abbreviation key: GSHpx = glutathione peroxidase, SeY = selenized yeast. INTRODUCTION Since Rotruck et al. ( 1 5 ) discovered that Se is an integral part of glutathione peroxidase ( GSHpx) , most research on Se has emphasized its role as a cellular antioxidant, and much less attention has been given to other biochemical roles of Se. The functions of GSHpx do not explain all of the biological effects associated with nutritional deficiencies of Se; therefore, other biochemical functions of Se need to be investigated. For example, Beckett et al. ( 3 ) found that Se plays an important role in thyroid hormone metabolism in rats via the Se-dependent enzyme, Type I deiodinase. Recently, Wichtel et al. ( 1 7 ) reported that effects of Se deficiency in calves were at least partially mediated by changes in the metabolism of thyroid hormones. Most Se in animal tissues is bound to protein, and research has shown the presence of numerous selenoproteins other than GSHpx. One such selenoprotein, selenoprotein P, is synthesized mainly in the liver and is secreted into plasma. Burk and Hill ( 4 ) found that, in rats, selenoprotein P contains about 65% of the Se in plasma. Although the function of selenoprotein P is not completely understood, it probably plays a role in Se transport or is an antioxidant. Recently, Chittum et al. ( 6 ) separated five glycosylated forms of selenoprotein P in the plasma of rats. Most of the protein (92 to 93%) was found in the 57-kDa forms ( P 57A, P57B, and P57C) , but smaller amounts ( 7 to 8%) existed in the 45-kDa forms ( P 45A and P45B) . Results of the study by Chittum et al. ( 6 ) also support the hypothesis that selenoprotein P functions as an antioxidant in the extracellular spaces. Little work has been done to identify selenoproteins in ruminants. Thus, the objective of this study was to determine the effect of amount and
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chemical form of Se on the distribution of Se among proteins (albumin, GSHpx, and selenoprotein P ) in the serum of calves and cows. MATERIALS AND METHODS The experimental protocols used in this study were approved by the Washington State University Institutional Animal Care and Use Committee. In the first experiment, six growing Holstein calves (101 ± 15 kg of BW and 92 ± 7 d of age) were randomly assigned to two dietary treatments of adequate (0.41 mg/g) or excess (0.73 mg/g) Se. The calves were housed in groups of three at the Washington State University Dairy Center. Diets were composed of about 36% alfalfa hay and 64% concentrate and were formulated to meet or to exceed the nutrient requirements of the calves (11). A Se premix with sodium selenite was added to the concentrate to provide the supplemental Se to the calves (Table 1). Monthly blood samples TABLE 1. Ingredient and chemical composition of the concentrate fed to calves (Experiment 1). Dietary Se Composition
Adequate
Excessive (%)
Ingredient Barley Soybean meal Molasses Dicalcium phosphate Trace-mineralized salt Trace-mineralized salt with Se1 Vitamin A premix2 Vitamin D premix3 Lasalocid premix4 Se Premix5
83.05 10.00 5.00 1.00 0.50 ... 0.025 0.025 0.40 ...
Chemical CP NDF ADF P Ca Mg Na K
14.60 23.90 5.60 0.59 0.40 0.24 0.44 0.16
Se Zn Cu Mn
82.95 10.00 5.00 1.00 ... 0.50 0.025 0.025 0.40 0.10 ( % of DM)
15.30 24.60 5.60 0.61 0.38 0.23 0.39 0.14 (mg/kg of DM) 0.56 1.06 76.20 69.10 13.30 12.50 37.30 41.30
1Contained 96 to 98.5% NaCl, 0.009% Se, 0.006% Co, 0.01% I, 0.035% Cu, 0.20% Fe, 0.18% Mn, 0.037% Mg, and 0.35% Zn. 2Contained 7,500,000 IU/kg of vitamin A. 3Contained 8,800,000 IU/kg of vitamin D . 3 4Contained 12.2 g/kg of lasalocid sodium. 5Contained 0.02% Se (sodium selenite).
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TABLE 2. Chemical composition of ingredients1 fed to cows (Experiment 2). Haylage CP ADF NDF Ash Ca P Se
20.24 33.42 42.1 11.67 1.02 0.31 0.15
Ryegrass
Concentrate2
( % of DM) 13.69 16.22 31.32 8.5 49.8 15.46 11.36 16.23 2.43 1.23 0.22 1.072 ( mg/g of DM) 0.12 0.30
1The
diet contained approximately 55% haylage, 42% ryegrass, and 3% concentrate. 2Contained 50.00% peas, 32.31% barley, 1.00% animal fat, 0.32% vitamin A (30,000 IU/g as retinyl acetate), 6.37% Bovatec (12.4 mg/g of lasalocid; Hoffmann-LaRoche Inc., Nutley, NJ), 5.00% magnesium oxide, and 5.00% dicalcium phosphate.
were collected using heparinized and nonheparinized vacutainer tubes (Becton, Dickinson and Co., Rutherford, NJ) for whole blood and serum, respectively. The experimental diets were fed for 20 wk. In the second experiment, 12 crossbred beef cows were assigned to one of four experimental treatments of salt mixes containing 20, 60, or 120 mg of Se as sodium selenite/g of salt mix or 60 mg of Se as selenized yeast ( SeY)/g of salt mix. The SeY was from Alltech, Inc. (Nicholasville, KY) and was composed largely of selenomethionine (10). Estimated dietary intakes of Se were 2.4, 4.7, 8.7, and 4.8 mg/d, respectively. Cows were fed experimental diets (Table 2 ) from d 90 prepartum until 3 mo postpartum. Blood samples used in this experiment were taken from cows at –90 and 90 d postpartum. Selenium-dependent GSHpx activity in blood was assayed according to the method of Paglia and Valentine (13). Determinations of Se in whole blood and in serum were by neutron activation analysis on lyophilized samples. Feed was analyzed for Se by neutron activation analysis, and other minerals were determined by atomic absorption spectrophotometry, except P, which was determined colorimetrically. Dry feed samples were analyzed for CP according to the AOAC ( 1 ) . Neutral detergent fiber and ADF were determined according to the method of Goering and Van Soest ( 9 ) . Serum proteins (GSHpx, albumin, and selenoprotein P ) were separated using two columns (1.5 × 6.0 cm; 7-ml volume) linked together according to the procedure described by Deagen et al. ( 8 ) . The first column contained heparin-sepharose Cl-6B (Pharmacia LKB Biotechnology, Piscataway, NJ), and the second column contained reactive blue
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SELENIUM DISTRIBUTION AMONG SERUM PROTEINS TABLE 3. Effect of dietary Se supplementation for 20 wk on the distribution of Se among serum proteins in young Holstein heifers. Dietary Se Fraction Serum Albumin GSHpx1 Selenoprotein P 1Glutathione
Adequate
Excessive
Se (ng/ml) 115.3 287.2 Se in serum ( % of total) 34.1 36.0 23.6 20.6 37.4 44.1
SE
P
0.1 ppm of Se. In rats that were deficient in Se, concentrations of selenoprotein P increased sharply when the diets were supplemented with 0.04 ppm of Se (20). There was no difference in 75Se labeling patterns between rats fed diets that were deficient or adequate in Se. Cows used as controls in the present study were marginally deficient in Se according to Puls (14); it was not possible to determine whether changes in the distribution of Se among serum proteins occurred when cows that were severely deficient in Se received supplemental Se. The chemical form of Se in the diet may affect the relative distribution of Se within protein fractions in
TABLE 7. Distribution of Se among fractions of albumin, glutathione peroxidase (GSHpx), and selenoprotein P in serum of cows given free access to salt containing three amounts and two chemical forms of Se. Na Selenite 60 mg/g of SeY2
Serum fraction
20 mg/g1
60 mg/g
Albumin GSHpx Selenoprotein P
29.3 14.6 53.6
( % of Se in protein fraction) 20.1 11.8 15.6 17.1 6.0 9.3 73.0 73.9 75.2
1Amount
120 mg/g
SE 2.7 3.5 4.8
of Se in salt. yeast.
2Selenized
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serum. Selenocysteine is the chemical form of Se in selenoprotein P and GSHpx, and selenomethionine is associated with the albumin fraction ( 7 ) . Butler et al. ( 5 ) reported a significant increase in the percentage of Se in the albumin fraction of plasma in monkeys supplemented with Se as selenomethionine compared with supplementation of Se as selenite. In this study, selenomethionine was the main form of Se in alfalfa hay and grain, and selenite was the form of supplemental Se for all treatments except that for cows fed salt with 60 mg/g of Se as SeY. However, because of possible chemical conversion in the rumen of compounds containing Se, direct comparison of dietary forms of Se between ruminants and nonruminants is not possible. The concentrations of Se in serum were positively correlated with the percentage of albumin in serum ( r = 0.62; P < 0.05), and GSHpx in serum was positively correlated with GSHpx activity in blood ( r = 0.65; P < 0.05). Positive correlations also were found between Se concentrations in selenoprotein P and GSHpx activity ( r = 0.72; P < 0.01). CONCLUSIONS Selenite and SeY as a dietary Se supplement for calves and cows did not change the pattern of Se distribution in serum among albumin, GSHpx, and selenoprotein P. The distribution of Se among protein fractions in the serum of cattle was similar to that reported for other species. Selenoprotein P contained the highest percentage of Se and was the only serum protein that was positively correlated with concentrations of Se in serum. This study confirms the presence of selenoprotein P in the sera of calves and mature cows. Further work needs to be done on the distribution of Se among serum proteins in cows that are deficient in Se and on the efficacy of different chemical forms of Se as dietary Se supplements. ACKNOWLEDGMENTS The authors thank Dave Dostal and Dan Coonrad for care of the experimental cows and calves. REFERENCES 1 Association of Official Analytical Chemists. 1990. Vol. I. Official Methods of Analysis. 15th ed. AOAC, Arlington, VA.
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2 Avissar, N., J. C. Within, P. Z. Allen, I. S. Palmer, and H. J. Cohen. 1989. Antihuman plasma glutathione peroxidase antibodies: immunologic investigations to determine plasma glutathione peroxidase protein and selenium content in plasma. Blood 73:318–323. 3 Beckett, G. J., S. E. Beddows, P. C. Morrice, F. Nicol, and J. R. Arthur. 1987. Inhibition of hepatic deiodination of thyroxin is caused by selenium deficiency in rats. Biochem. J. 248:443–447. 4 Burk, R. F., and K. E. Hill. 1993. Regulation of selenoproteins. Annu. Rev. Nutr. 13:65–82. 5 Butler, J. A., P. D. Whanger, A. J. Kaneps, and N. M. Patton. 1990. Metabolism of selenite and selenomethionine in the Rhesus monkey. J. Nutr. 120:751–759. 6 Chittum, H. S., S. Himeno, K. E. Hill, and R. F. Bruck. 1996. Multiple forms of selenoprotein P in rat plasma. Arch. Biochem. Biophys. 325:124–128. 7 Deagen, J. T., M. A. Berlstein, and P. D. Whanger. 1991. Chemical forms of selenium in selenium containing proteins from human plasma. J. Inorg. Biochem. 41:261–268. 8 Deagen, J. T., J. A. Butler, B. A. Zachara, and P. D. Whanger. 1993. Determination of the distribution of selenium between glutathione peroxidase, selenoprotein P, and albumin in plasma. J. Anal. Biochem. 208:176–184. 9 Goering, H. K., and P. J. Van Soest. 1970. Forage Fiber Analyses (Apparatus, Reagents, Procedures, and Some Applications). Agric. Handbook No. 379, ARS-USDA, Washington, DC. 10 Kelly, M. P., and R. F. Power. 1995. Fractionation and identification of the major selenium containing compounds in selenized yeast. J. Dairy Sci. 78(Suppl. 1):237.(Abstr.) 11 National Research Council. 1989. Nutrient Requirements of Dairy Cattle. 6th rev. ed. Natl. Acad. Sci., Washington, DC. 12 Nicholson, J.W.G., R. E. McQueen, and R. S. Bush. 1991. Response of growing cattle to supplementation with organically bound or inorganic sources of selenium or yeast cultures. Can. J. Anim. Sci. 71:803–811. 13 Paglia, D. E., and W. N. Valentine. 1967. Studies on the quantitative and qualitative characteristics of erythrocyte peroxidase. J. Lab. Clin. Med. 70:158–169. 14 Puls, R. 1988. Mineral Levels in Animal Health. Sherpa Int., Clearbrook, BC, Canada. 15 Rotruck, J. T., A. L. Pope, H. E. Ganther, A. B. Swanson, D. G. Hafeman, and W. G. Hoekstra. 1973. Selenium: biochemical role as a component of GSHPx. Science (Washington, DC) 170: 588–590. 16 SAS User’s Guide. Statistics. Version 5 Edition. 1989. SAS Inst., Inc., Cary, NC. 17 Wichtel, J. J., A. L. Craigie, D. A. Freeman, H. Varela-Alvarez, and N. B. Williamson. 1996. Effect of selenium and iodine supplementation on growth rate and on thyroid and somatotropic function in dairy calves at pasture. J. Dairy Sci. 79:1865–1872. 18 Wilson, K., and J. M. Walker. 1994. Principles and Techniques of Practical Biochemistry. 4th ed. Cambridge Univ. Press, New York, NY. 19 Xia, Y., X. Zhao, L. Zhu, and P. D. Whanger. 1992. Metabolism of selenate and selenomethionine by a selenium-deficient population of men in China. J. Nutr. Biochem. 3:202–210. 20 Yang, J. G., K. E. Hill, and R. F. Burk. 1989. Dietary selenium intake controls rat plasma selenoprotein P concentration. J. Nutr. 119:1010–1012.
