BIOLOGICAL TRACE ELEMENT RESEARCH 4, 233-239 (1982)

Selenium Concentration and Glutathione Peroxidase Activity in Cow's Milk YASUJI HoJO

Department of Food Science, Faculty of Living Science, Kyoto Prefectural University, Shimogamo, Kyoto, 606, Japan Received and Accepted April 13, 1982

Abstract Samples of raw or pasteurized cow's milk and infant formula were assayed for glutathione peroxidase (GSH-Px) activity, selenium (Se), and total protein. GSH-Px activity was detected in raw milk, but not in market-pasteurized milk or infant formula. The correlation between GSH-Px activity and Se levels was significant, even when the influence of protein level was removed. This result implies a role of GSH-Px as one of biologically active forms of Se in raw milk. Absence of GSH-Px activity in pasteurized milk and infant formula result from the heating process in these productions, because the heating of raw milk gave an irreversible inactivation of GSH-Px. Both GSH-Px activity and Se levels had significant correlation with protein level, but not so when the respective influences of Se and GSH-Px levels were removed. In raw cow's milk, Se content was 23 ng/mL and GSH-Px activity was 20 U/mL. About 12% of Se was bound to GSH-Px, and 0.003% of protein was GSH-Px. Raw milk obtained in July contained higher levels of Se, GSH-Px, and protein than that in November. Data for cow's milk were discussed in relation to those for human milk and those in New Zealand. Index Entries: Selenium; glutathione peroxidase; raw cow's milk; pasteurized cow's milk; infant formula; infant nutrition; active forms of selenium; correlation between glutathione peroxidase and selenium levels.

Introduction Selenium (Se), an element once known only for its toxic effects, is now generally accepted as an essential micronutrient for mammals, birds, and several bacteria 9 1982 by The Humana Press Inc. All rights of any nature whatsoever reserved. 0163~.984/82/6900-0233502.00

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(1), and plays positive and negative roles in environmental health (2, 3). In several mammalian tissues, Se is found as a Se-dependent enzyme, glutathione peroxidase (GSH-Px), although some glutathione S-transferases are known to exhibit the GSH-Px activity independent of Se (4, 5). GSH-Px not only contributes to a number of cellular defense mechanisms essential for the survival of animals, but also has several possible regulatory functions (1). The erythrocytes of newborn infants, especially premature infants, show increased sensitivity to oxidant stress, probably as a result of reduced GSH-Px activities, suggesting that they may be vulnerable to Se deficiency (6). Recently cow's milk has been in daily use as a breast feeding substitute and can be a major source of nutrients for the infants. However, the chemically and biologically active forms of Se in cow's milk have never been investigated. Practically all of Se in milk is present in the protein-bound forms, and 74% of Se in dialyzed skim milk is found in the casein fraction, 14% in the albumin fraction, and 5% in the globulin fraction (7). The author has recently found the Se-dependent GSH-Px activity in human milk (8), which suggested a role of GSH-Px as a biologically active form of Se in human milk. The purpose of the present study was to investigate biologically active form of Se in cow's milk by measuring GSH-Px activity and its relationship with the Se level.

Materials and Methods Raw mature milk samples were collected directly from 21 Holstein cows at a pasture in Kyoto City in July and November. Five pasteurized milk and five infant formulas were purchased from local food stores in Kyoto City. Pasteurization of milk was carried out by heating at 85~ for 7 min and thereafter at 140~ for 2 s. All infant formulas were of cow's milk-based powder types and were prepared in a concentration of 14% with deionized distilled water before assay. All samples were analyzed for GSH-Px activity, and Se and protein contents were determined in those samples that exhibited GSH-Px activity. Selenium was determined by the fluorimetric method (9). Protein was measured by the method of Lowry et al. (10) using crystalline bovine serum albumin (Sigma Chemical Company, St. Louis) as standard protein. The Se-dependent GSH-Px activity was assayed by the method of Hafeman et al. (11) using hydrogen peroxide as substrate (12). Organic hydroperoxides were not used because they are substrates for total GSH-Px activity, that is, the sum of Se-dependent and Seindependent GSH-Px activities (12). The level of Se (ng/mL) bound to GSH-Px was estimated by multiplying GSH-Px activity level (U/mL) in milk sample by 0.131, which is the ratio of Se level (ng/mL) to GSH-Px activity level (U/mL) in a solution of the crystalline GSH-Px isolated from bovine blood (13). The enzyme preparation from bovine blood was assumed to be identical to the enzyme in milk. The GSH-Px content (ng/mL) was calculated by multiplying the Se level (ng/mL) bound to GSH-Px in the milk sample by 265.8, which is the ratio of total molecular

SE AND GSH-Px IN COW'SMILK

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weight to weight of Se for 1 mol of GSH-Px. The data obtained were tested statistically using the Student's t-test and by simple linear regression analysis.

Results and Discussion GSH-Px activity was detected for the first time in raw cow's milk, with levels between 12 and 32 U/mL (Table 1). The GSH-Px activity level correlated significantly with Se concentration (Fig. 1) and its correlation was significant even when influence of protein level on correlation coefficient was removed (r = 0.639). This suggests that the activity found in the milk originates from Se and that GSH-Px is one of the biologically active forms of Se in raw cow's milk. No activity of GSH-Px could be detected in market-pasteurized milk or infant formulas. This may result from the heating processes undergone in these productions, because heating of raw cow's milk at 80~ for 10 min brought about an irreversible inactivation of GSH-Px in this study. Thus, raw milk with GSH-Px activity may be better as an infant food than pasteurized milk and infant formula without GSH-Px activity, because GSH-Px exhibits cellular defense function through decomposition of hydroperoxides (1). Both Se concentration and GSH-Px activity correlated significantly with protein concentration (Figs. 2 and 3), but these correlations were not significant when the respective influences of GSH-Px activity and Se concentration on the correlation coefficients were removed (r = 0.434 and 0.128). Table 1 shows the levels of various parameters related to Se and GSH-Px in raw cow's milk together with those in human milk (8). The Se level (ng/mL) was similar in cow's and human milk. This is different from earlier studies, in which human milk was found to contain approximately twice as much Se as cow's milk (14, 15). The protein level (mg/mL) in cow's milk was significantly higher than that in human milk. In contrast, cow's milk contained only half the activity (U/mL) of human milk in spite of the close correlation of GSH-Px activity with Se and protein concentrations in both cow's and human milk. The GSH-Px activity (U/mg protein) and the proportion of GSH-Px content to total protein content (%) for human milk were 2.68 and 0.0093, respectively, and were more than thrice those for cow's milk, 0.84 and 0.0029. The proportion of GSH-Px content to total protein content for cow's milk, 0.0029%, is much lower than that for liver, 0.25%. Liver is regarded as one of the richest source of GSH-Px (1). About 12% of total Se in cow's milk was bound to GSH-Px. This value is lower than that in human milk, 27%, is intermediate between those in rat erythrocyte, 100% (16), and human blood plasma, 1.5% (17), and suggests the presence of other forms of Se besides GSH-Px in raw cow's milk. In New Zealand, where widespread areas of Se deficiency are well known to exist, the Se contents of cow's and human milk, 5 and 13 ng/mL, respectively (15), are less than those in Table 1,23 and 23 ng/mL. In contrast, the protein contents of cow's and human milk in New Zealand, 41 (18) and 21 (15) mg/mL, are higher than those in Table 1,24 and 15 mg/mL. Therefore, the Se levels (ng/mg protein) of cow's and human milk in New Zealand, 0.12 and 0.62, respectively, are much

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Selenium concentration and glutathione peroxidase activity in cow's milk.

Samples of raw or pasteurized cow's milk and infant formula were assayed for glutathione peroxidase (GSH-Px) activity, selenium (Se), and total protei...
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