@Copyright 1986 by The Humana Press Inc. All rights of any nature whatsoever reserved. 0163M984/86/1003-0175502.6(}

Glutathione Peroxidase Activity, Selenium, and Lipid Peroxide Concentrations in Blood from a Healthy Polish Population I. Maternal and Cord Blood BRONISEAWA. ZACHARA,*"WOJCIECH W,~SOWlCZ,2 JOLANTA GROMADZIIqSKA,2 MARIA SKILODOWSKA,2 AND GRZEGORZ KRASOMSKP

1Department of Biochemistry, hqedical Academy, Bydgoszcz, Poland, and Department of Biochemistry, Institute of Agriculture, Bydgoszcz, Poland; 2Department of Biochemistry, Medical Academy, lddd~., Poland; and 3Institute of Gynecology and Obstetrics, l~edical Academy, lddd2, Poland Received April 4, 1985; Accepted December 29, 1985

ABSTRACT Selenium (Se) concentrations in whole blood and plasma of 19 nonpregnant women, 14 mothers at delivery, 14 neonates, and 13 infants, aged 2-12 mo, were evaluated. The activity of glutathione peroxidase (GSH-Px) in erythrocytes and plasma and the level of lipid peroxides in plasma were also analyzed. Selenium concentrations in whole blood and plasma in mothers at delivery were significantly lower compared to nonpregnant women. Selenium concentrations in cord blood components were lower compared to mothers, but the differences were not significant. The concentration of the element decreased in the first few months of life. Glutathione peroxidase activity in erythrocytes differed only slightly in the examined groups. In plasma, however, the enzyme activity was significantly lower in *Author to whom all correspondence and reprint requests should be addressed. Biological Trace Element Research

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pregnant compared to nonpregnant women and in neonates compared to their mothers. Lipid peroxide concentrations in plasma differed only slightly in the examined groups. The results obtained are discussed in terms of the observations of other investigators. Index Entries: Selenium, in whole blood; selenium, in plasma; glutathione peroxidase, in erythrocytes; glutathione~peroxidase, in plasma; lipid peroxides, in plasma; mothers at delivery, selenium, glutathione peroxidase, and lipid peroxide, concentrations of; neonates, selenium, glutathione peroxidase, and lipid peroxides, concentrations of; infants, selenium, glutathione peroxidase, and lipid peroxides, concentrations of.

INTRODUCTION Selenium (Se) is one of the micronutrients that have received considerable attention in the last few years. It is n o w well established that this essential dietary trace element is both beneficial and harmful to various animal species and m a n (1-3). Such effects may result from either insufficient or excessive amounts of Se in the organism. Wide geographic variations in Se concentrations have been demonstrated in soils (4,5), forage crops (6-8), and foods (9,10). As a result of the a m o u n t of Se intake with foods and feeds, the concentration of this element in milk (10-12) and blood (7,13,14) differs significantly. In 1973, a physiological role of Se was established following the discovery that Se is an essential structural c o m p o n e n t of the only k n o w n mammalian enzyme, glutathione peroxidase (GSH-Px; glutathione: H202 oxidoreductase, E.C. 1.11.1.9) (15,16). A high correlation was found between Se concentrations and GSH-Px activities in whole blood in a large n u m b e r of N e w Zealand residents aged 2-92 yr (17). It was assumed that GSH-Px protected hemoglobin against oxidative denaturation by hydrogen peroxide (18). Little and O'Brien (19) found that the e n z y m e reduced in the liver cells not only h y d r o g e n peroxide, but a variety of organic hydroperoxides, including lipid peroxides; it may thus be thought to protect the cells from the deleterious effects of peroxides. The red blood cells of newborn infants show increased sensitivity to oxidant stress, most likely as a result of lowered Se concentration and decreased GSH-Px activity (20). Erythrocyte abnormalities in these infants are the result of m e m b r a n e damage secondary to increased lipid peroxidation, which is e n h a n c e d by the high polyunsaturated fatty acid content (13). It is n o w an established fact that the populations of two countries have an inadequate alimentary intake of Se resulting in low blood levels of that element. The countries in question are Finland (8,10,14) and N e w Zealand (13,17,21). Lombeck et al. (22) have examined the Se concentration and GSH-Px activity in the blood of Germany's population, and Verlinden et al. (23,24) have recently carried out a similar investigation in Biological Trace Element Research

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Belgium (a wide spectrum of age groups). Thorough investigations of various age groups in other European countries have yet to be undertaken. Verlinden et al. (23) suggest that research into the normal values of blood Se concentration in any particular country is essential. Therefore, the purpose of our study was to investigate in the paired samples of maternal and cord blood the Se and lipid peroxide concentrations and the GSH-Px activities. Simultaneously, the results obtained were compared with adult, nonpregnant females and with infants aged 2-12 too. The results reported here are the first ever obtained for the Polish population.

MATERIALS AND METHODS Paired samples of maternal and cord blood were obtained, with maternal consent, at the time of delivery, from 14 subjects, residents of E6d~., aged 23-38 yr (mean, 27 yr). Venous blood was also taken from 19 females, aged 17-52 yr (mean, 36 yr, and 13 healthy, well-nourished infants, aged 2-12 mo (mean, 5 mo), whose blood were aliquots of that for routine control analysis. A few days after birth the infants were admitted to the nursery. With every precaution being taken to avoid contamination, specimens were collected into carefully cleaned, dried, heparinized tubes. The blood samples were divided into two portions, one of which was stored at 4~ and the second centrifuged at 4~ and the plasma removed and stored for further analysis. The buffy coat was removed, and the remaining red cells were washed twice with an excess of chilled 0.154 mol/L NaCI solution. After the final centrifugation, the saline solution was discarded, and 1 mL of the red blood cells was used for Se determination. The remaining cells were hemolyzed by two cycles of freezing and thawing. Hemoglobin concentration in whole blood and hemolysates was measured by the cyanmethemoglobin method. The GSH-Px activity of the red-cell hemolysates and plasma was assayed using the Hopkins and Tudhope modification (25) of a coupledenzyme assay of Paglia and Valentine (26), with t-butylhydroperoxide as the acceptor substrate. Enzyme activity was measured at 25~ in a Unicam SP 500 series 2 spectrophotometer fitted with a constanttemperature cell housing. The GSH-Px activity was defined as ~mol NADPH oxidized/min/g Hb (27,28). Selenium concentration was assayed by the fluorometric method of Watkinson (29). When sample size allowed it, duplicate determinations were made, but in most cases only single determinations were possible. Samples were wet digested by an HNOB/HCIO4 mixture. 2,3Diaminonaphthalene was used as the complexing reagent and cyclohexane as the extracting solvent for the 4,5-benzpiazselenol formed. The fluorescence was measured in an Aminco-Bowman spectrofluorometer. The Se content was expressed as ng/mL of whole blood, erythrocytes, or Biological Trace Element Research

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plasma. The Se content in erythrocytes was also calculated according to the hematocrit and Se contents of whole blood and plasma (30). Lipid peroxides were measured in blood plasma by the fluorometric method, using thiobarbituric acid (TBA), described by Yagi (31). Plasma lipids containing lipid peroxides were precipitated with phosphotungstic acid, followed by the reaction with TBA. The reaction produced was measured fluorometrically and the lipid peroxide concentrations were expressed, in terms of malondialdehyde, in nmol/mL plasma. The GSH-Px activities and lipid peroxide concentrations were determined on the day of blood sampling and Se concentrations within 2 d of blood collection. The data obtained were expressed as means and standard deviations and were subjected to statistical analysis by the Student's t-test. Statistically significance was set at p < 0.05. The results were occasionally subjected to correlation and linear regression analyses.

RESOLTS The mean whole blood, red blood cells, and plasma Se concentrations, the red blood cells and plasma GSH-Px activities, and the plasma lipid peroxide levels of adult female control subjects, mothers at delivery, cord blood, and infants are shown in Table 1. It was found that the Se concentrations in whole blood and plasma of pregnant women were significantly lower (p < 0.001) than those of nonpregnant females (97 and 55 vs 125 and 95 ng/mL, respectively). The GSH-Px activity in plasma was also significantly lower (p K 0.02) in maternal blood compared with the female controls (0.187 and 0.235 IU/mL, respectively). Pregnant women, in comparison with nonpregnant females, exhibited reduced Se concentrations in red blood cells (145 and 174 ng/mL, respectively), but the differences were not statistically significant. The levels of plasma lipid peroxides were slightly higher in pregnant women than those in control females, but the differences were not significant either. Mean Se concentrations in newborn infants (in whole blood, red blood cells, and plasma) were lower than those in mothers at delivery. The differences are not statistically significant. They are, however, significantly lower (p < 0.001) when compared with adult females. The GSH-Px activities of red blood cells and plasma in newborn infants were significantly lower when compared with both pregnant and nonpregnannt women. It is noticeable that when each mother's red cell and plasma enzyme activity was compared with that of her own neonate's, the mother's GSH-Px activities were---with one exception only (in this case the plasma enzyme activity was exactly at the same level)--consistently higher than that of her newborn (Fig. 1). The correlation coefficients are: red blood cells, r = 0.866 (p < 0.001) (Fig. 2A); and plasma, r = 0.370 (p, NS). Lipid peroxide concentrations in plasma of maternal blood, cord blood, and infants were nearly at the same level. There was, however, a high, positive correlation Biological Trace Element Research

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Fig. 1. The GSH-Px activity in erythrocytes (A) and plasma (B) of 14 paired maternal and cord blood samples. In erythrocytes and plasma, the GSH-Px activities were significantly lower in cord blood compared with maternal blood (p < 0.05 and p < 0.01, respectively). b e t w e e n paired samples of maternal and cord blood plasma (r = 0.748; p < 0.005) (Fig. 2B). In infants aged 2-12 mo, the Se concentrations of whole blood and blood cells are significantly lower (p < 0.01) than those of neonates. The slightly lower m e a n value of plasma Se concentrations is statistically nonsignificant b e t w e e n these two groups. The m e a n GSH-Px activities in red blood cells and plasma of cord blood and infants were similar. No significant correlations between Se concentrations and GSH-Px activities were observed a m o n g all groups studied (adult females, pregnant w o m e n , neonates, and infants). Similarly, in no case were there any significant correlations found b e t w e e n plasma GSH-Px activities and lipid peroxide concentrations.

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