Selenium levels, thiobarbituric acid-reactive substance concentrations and glutathione peroxidase activity in the blood of women with gestosis and imminent premature labour† Jolanta Gromadzinska*a, Wojciech Wasowicza, Grzegorz Krasomskib, Danuta Broniarczykb, Michal Andrijewskic, Konrad Rydzynskia and Piotr Wolkanind a Department of Toxicology and Carcinogenesis, Nofer Institute of Occupational Medicine, 90-950 Lodz, Poland b Clinical Division of Pathologic Pregnancy, Polish Mother Memorial Hospital, 90-338 Lodz, Poland c Department of Toxicology Military Medical Academy, 90-759 Lodz, Poland d Department of Ultrastructural Pathology Military Medical Academy, 90-759 Lodz, Poland The aim of the study was to investigate antioxidant status, monitored by selenium and thiobarbituric acid-reactive substance concentrations in blood plasma, and glutathione peroxidase activity in erythrocytes and blood plasma in women with gestosis (n = 26), imminent premature labour (n = 48) and normal pregnancy (n = 23) during 19–38 weeks of pregnancy.Selenium concentrations in blood plasma were significantly higher in women with pathological pregnancies than in normal (45.5 ± 10.5 mg l21, p < 0.01 and 44.1 ± 11.6 mg l21, p < 0.05 vs. 38.6 ± 8.3 mg l21, respectively). In all groups of pregnant women Se concentrations were extremely low as compared with non-pregnant females. Glutathione peroxidase (GSH-Px) activity in blood plasma was significantly higher in complicated pregnancies than in healthy ones.There were no significant differences in thiobarbituric acid-reactive substance concentrations between all groups of pregnant women. Statistically significant correlations were found between blood plasma Se concentrations and GSH-Px activity in healthy pregnant (r = 0.53, p < 0.01), imminent premature labour (r = 0.39, p < 0.01), and non-pregnant females (r = 0.56, p < 0.001). Keywords: Pregnancy; gestosis; premature labour; selenium; glutathione peroxidase; thiobarbituric acid-reactive substances High risk pregnancy may lead to perinatal mortality of foetuses and neonates and poses a threat to the mother, contributing to frequent occurrence of complications which may sometimes be fatal.A typical example of high risk pregnancy is primary or secondary gestosis. The symptoms of gestosis occur in about 20% of high risk pregnancies in Poland. The aetiology of gestosis remains unclear.Its most common symptoms are rapid increase of body mass and oedema of lower limbs, in advanced forms accompanied by proteinuria and hypertension. If not treated, the most severe forms easily turn into preeclampsia and eclampsia. Premature birth is the most common cause of foetal and neonatal mortality accounting for about 80% of perinatal deaths. About 13% of pregnancies in the Lodz region and about 7% in Poland are at risk of premature birth. It is believed that high incidence of premature birth, especially in industrial areas, may result not only from the motherAs predisposition, but also from her lifestyle and pollution of the environment.Life in an oxygen medium requires establishment of mechanisms of protection of vital cells from oxygen-induced damage that could result from oxygen free-radical species produced during metabolism. A disturbance of the balance between formation of active oxygen metabolites and the tempo at which they are scavenged by enzymatic and non-enzymatic antioxidants is referred to as oxidative stress.1 Oxidative stress has been suggested to play a role in some physiological conditions and in many disease processes, including ageing, pregnancy and its complications, carcinogenesis, hypertension, atherosclerosis and diabetes, etc.1,2 Several investigators have reported that in normal pregnancy, peroxidation and antioxidant reactions are increased as compared with non-pregnancy.3,4 Selenium (Se) is an essential trace element necessary for maintaining optimal activity of glutathione peroxidase (GSHPx), an enzyme which may play an important role in protecting polyunsaturated fatty acid biological membranes from oxidative damage.5 There is evidence that Se is particularly important in the nutrition of pregnant women whose requirement for this element is significantly increased during pregnancy and puerperium.6 The present knowledge of the role of Se in pregnancy and in neonatal life is sparse.Dietary Se deficiency has been reported to result in abnormal foetal and postnatal development in laboratory animals.7 Selenium deficiency in preterm infants has been associated with increased haemolysis in babies exposed to oxidant stress8 and the increased risk of pulmonary damage in preterm infants treated with oxygen for respiratory distress syndrome.9 In our previous study we discovered that Se concentrations in whole blood and blood plasma and GSH-Px activity in the blood plasma of delivering women were significantly lower than in the non-pregnant female.10,11 The level of Se in the blood of the residents of our country is relatively low12 and it is estimated that dietary selenium intakes are less than the minimum doses considered safe and adequate. 13 Imbalance of increased lipid peroxides and a decrease in a net antioxidant activity have been reported, not only in normal pregnancies but also in cases of gestosis3, habitual abortion14 and in premature infants.15 The aim of the present study was to examine antioxidant/ oxidant status, monitored by the concentration of blood plasma Se and thiobarbituric acid reactive substances (TBARS, as marker of peroxidative processes) and activity of blood plasma and erythrocyte GSH-Px in women with gestosis, imminent premature labour and normal pregnancy.† Presented at The Sixth Nordic Symposium on Trace Elements in Human Health and Disease, Roskilde, Denmark, June 29–July 3, 1997.Analyst, January 1998, Vol. 123 (35–40) 35Experimental Studies were carried out on 97 women, patients of the Clinical Division of Pathologic Pregnancy Polish Mother Memorial Hospital in Lodz, Poland, in 1995. Twenty-three healthy pregnant women, 26 pregnant women with gestosis and 48 women with imminent premature labour aged from 21 to 39 years in the second and third trimester of pregnancy were chosen for this study. A group of 64 aged-matched nonpregnant healthy women were investigated as controls.Gestosis was defined as: the onset of hypertension, blood pressure > 140/90 mm Hg; proteinuria, > 3.0 g protein d21; and oedema persisting after a night’s rest. The women with diagnosed gestosis were given drugs to lower blood pressure and improve blood supply. Imminent premature labour was diagnosed on the basis of premature uterus contractions (over 100 Montevideo units) and a change of the uterine cervix configuration.Drugs of the gestagen, spasmolytic and psychopharmacological groups were administered to all women with imminent premature labour to stop uterus contractions. Blood samples were collected in heparinized tubes free of trace elements. After centrifugation, blood plasma was removed and used for further analysis. Red blood cells were washed three times with 0.9% NaCl solution and lysed by freezing and thawing. Stroma was removed by centrifugation and haemoglobin concentrations in haemolysates were measured by the cyanmethaemoglobin method.The protocol of this study was approved by the Ethical Committee of the Medical Academy in Lodz. At the time of blood collection, the patients selected for the examination completed a questionnaire concerning their living conditions, lifestyle, dietary habits and medication received. The respondents defined their material status as good, consumed products typical of Polish diet, did not smoke during pregnancy and drank alcohol only occasionally.The patients who received medication containing antioxidants were excluded from the investigation. Selenium concentrations were assayed by the fluorimetric method of Watkinson.16 A detailed description of the method of Se determination has been published elsewhere.17 Lyophilised human reference serum samples of the Seronorm (batch number 010017) from Nycomed Pharma AS (Oslo, Norway) were used to assess the accuracy of the methods.The mean Se concentrations for eight determinations of human serum were 94.5 ± 2.1 mg l21, as compared with the recommended value of 96 mg l21. The RSD for plasma was 3.8% (n = 8). The recovery of Se from reference materials was 92–104% (98% average). GSH-Px activities in lysed red blood cells and blood plasma were measured by the method of Paglia and Valentine18 with tert-butyl hydroperoxide as substrate. One unit of the enzyme activity was defined as micromoles of NADPH oxidised per minute per gram of haemoglobin or millilitre of blood plasma. TBARS concentrations in blood plasma were determined by the optimised fluorimetric procedure as modified by Wasowicz et al.19 The data were expressed as mean ± s and subjected to statistical analysis using StudentAs t-test, analysis of variance, and calculation of correlation coefficients.Statistical significance was set on p < 0.05. Results At the time of examination, blood pressure of all gestosis patients was stable (diastolic 86.2 ± 14.6 mm Hg and systolic 138.7 ± 14.6 mm Hg) and pressure stabilising drugs were administered to them. Biochemical indicators of renal function (uric acid, urea and creatinine concentrations in blood plasma) were normal.Blood plasma protein concentrations were 58.1 ± 6.6 g l21. In the other groups of pregnant women the above mentioned parameters were normal. Considering gestational age, ultrasonography of the foetuses showed growth retardation only in one case, in a woman with gestosis. Table 1 presents blood plasma Se concentrations and glutathione peroxidase activity in all investigated groups of pregnant women and non-pregnant controls. It was found that blood plasma Se concentrations in normal pregnancy were significantly lower than in non-pregnant females (38.6 ± 8.3 mg l21 vs. 60.7 ± 10.5 mg l21, p < 0.001). Selenium concentrations in women with gestosis and imminent premature labour were significantly higher than in healthy pregnancy (p < 0.01 and p < 0.05, respectively).GSH-Px activity in blood plasma was significantly higher in complicated pregnancies than in healthy ones. No differences were observed in erythrocyte GSH-Px between the examined groups of pregnant women. Both erythrocyte and plasma GSHPx activities were significantly lower in healthy pregnant women than in non-pregnant women (11.1 ± 3.9 U g21 Hb vs. 15.9 ± 3.3 U g21 Hb, p < 0.001 and 0.113 ± 0.045 U ml21 vs. 0.179 ± 0.033 U ml21, p < 0.0001). Blood plasma TBARS concentrations were determined in all groups of pregnant women. The differences between them were not statistically significant. However, the concentration of TBARS in the patients with imminent premature labour was about 20% higher than in the group of healthy pregnant women (1.22 ± 0.45 mmol l21 vs. 1.02 ± 0.37 mmol l21). In 20% of the patients with gestosis (5/26) and in 30% of those with imminent premature labour (15/48), TBARS concentrations were found to be higher than mean + s for healthy pregnant women (Fig. 1). In healthy pregnant women, imminent premature labour and in the group of non-pregnant female statistically significant correlations were found between Se concentrations and GSHPx activity in the blood plasma (Fig. 2). Besides, in the group of healthy pregnant women and women with imminent premature labour, a significant relationship with high correlation coefficients were observed between blood plasma Se concentrations and erythrocyte GSH-Px activity (r = 0.51, p < 0.01 and r = 0.45, p < 0.001, respectively). Furthermore, in the group of patients with gestosis, statistically significant correlation was found between TBARS concentration in blood plasma and GSH-Px activity in erythrocytes (r = 0.46, p < 0.02, Fig. 3). As the examination encompassed women at different stages of pregnancy, starting with the 19th week, Fig. 4 presents pregnancy age-dependent changes of the investigated parameters in normal pregnancy and in patients with gestosis or imminent premature labour.Statistically significant decrease in Table 1 Plasma Se concentrations and GSH-Px activities in blood plasma and erythrocytes in controls and pregnant women in the second and third trimester (mean ± s) Plasma Erythrocyte Plasma GSH-Px/ GSH-Px/ Patients n Se/mg l21 U ml21 U g21 Hb Non-pregnant 64 60.7 ± 10.5 0.179 ± 0.033 15.9 ± 3.3 Healthy pregnant 23 38.6 ± 8.3 0.113 ± 0.045 11.1 ± 3.9 p < 0.001* p < 0.0001* p < 0.001* Imminent premature labour 48 44.1 ± 11.6 0.140 ± 0.038 12.5 ± 3.2 p < 0.05† p < 0.01† Gestosis 26 45.5 ± 10.5 0.146 ± 0.048 12.4 ± 4.4 p < 0.01† p < 0.01† * Statistical significance as compared with non-pregnant women.† Statistical significance as compared with healthy pregnant women. 36 Analyst, January 1998, Vol. 123Se concentration correlated with the age of pregnancy was observed in patients with gestosis (r = 20.45, p < 0.05, Fig. 4A). In the last trimester of pregnancy, a statistically significant decrease of Se concentration and GSH-Px activity in erythrocytes and blood plasma correlated with the stage of pregnancy was observed in women with imminent premature labour (r = 20.29, r = 20.28, r = 20.29, respectively, p < 0.05 in all cases). In healthy pregnant women, gradual increase of TBARS was observed in the third trimester of pregnancy. The increase was 47% although it was not statistically significant (Fig. 4D). In the groups of patients with pathological pregnancy, TBARS concentrations were nearly the same, irrespective of the stage of pregnancy. Discussion Selenium concentrations, and thus GSH-Px activity in human blood, present wide regional variations. The mean Se concentration in blood plasma of adult inhabitants of Poland is lower than that found in inhabitants of Western Europe and comparable with the levels in the blood of inhabitants of Eastern Europe.12 In the present study, plasma Se concentrations in healthy pregnant women are extremely low (38.6 ± 8.3 mg l21), as compared with Se levels of healthy non-pregnant females (Table 1).According to available studies on Se concentration in the blood of pregnant women, these results are among the lowest in the world.20 In women with pathological pregnancies the concentration of Se in blood plasma is about 25% higher than in healthy pregnant women, although it remains one of the lowest reported in the world, comparable with the data of the female residents of New Zealand.21 The concentration of Se in plasma decreases as the pregnancy develops and reaches the lowest level just before delivery.Concentration of Se in blood plasma of women in advanced pregnancy has been demonstrated to be lower than in non-pregnant women in studies of many other centres.20,22,23 In the present work we did not find a pregnancy age-dependent decrease of blood plasma Se concentration in healthy pregnant women.The decrease of the concentration of the microelement in blood plasma observed as the pregnancy develops is sometimes connected with the increase of the volume of plasma and reduced concentration of albumins in the blood of pregnant women. However, the concentrations of many components of blood plasma, including some microelements (e.g., Cu), increase during pregnancy.24 This fact suggests that there are mechanisms which control changes of concentration of microelements in the organism during pregnancy.Korpela et al.6 demonstrated that Se concentrations in placenta and amniotic membranes must be subjected to particular control. Selenium concentrations in the placenta of Finnish pregnant women is the same as in Americans (2.28 ± 0.32 mmol kg21 vs. 2.24 ± 0.20 mmol kg21) whereas the ratio of its concentration in placenta and blood is 3 and 1.5, respectively. This may indicate that placenta and foetal membranes are being provided primarily with sufficient amounts of selenium and explain the reason of differences between the concentration of the microelement in non-pregnant and healthy pregnant women. In our investigation, the drop of Se concentration in the blood plasma of healthy pregnant women was accompanied by decreased activity of plasma and erythrocyte GSH-Px activity.Data published by other authors are not so explicit; Behne and Wolters23 reported decreased blood plasma GSH-Px activity, without changes in the activity of the enzyme in erythrocytes.Butler et al.25 and Rudolph and Wong26 observed an increase of erythrocyte GSH-Px activity. Zachara et al.22 found a gradual Fig. 1 Plasma TBARS concentrations in pregnant women (IPL; imminent premature labour). Full line represents mean TBARS level for each group of patients. Fig. 2 Relationship between blood plasma Se concentration and GSH-Px activity in non-pregnant female (A) and pregnant women (B, healthy; C, gestosis; D, imminent premature labour).Analyst, January 1998, Vol. 123 37decrease of the activity of the enzyme in blood plasma and erythrocytes during pregnancy. These differences in erythrocyte/ plasma GSH-Px activity may result from different concentrations of Se in blood/blood plasma during pregnancy. In female USA residents Se concentrations in blood were about 110 mg l21 at the beginning of pregnancy and dropped to about 70 mg l21 in the final weeks,25 whereas in healthy Polish women in the first weeks of pregnancy they were 75.8 ± 16.7 mg l21 and decreased to 49.5 ± 9.5 mg l21 in the final weeks before delivery.22 Blood plasma TBARS concentrations in normal pregnancy are higher than in non-pregnant women.3 Our studies showed that in normal pregnancy, the concentration of TBARS increases in the last trimester.Unfortunately, due to lack of analyses of blood plasma of non-pregnant women, it is not possible to compare these data with the population of healthy women.Wang et al.27 demonstrated that serum levels of lipid peroxides remained relatively stable throughout normal gestation. Analysing also concentrations of plasma vitamin E, the same authors suggested an increase of antioxidative activity of blood plasma over peroxidation with advancing gestation in normal pregnancy. Little is known about oxidant status in pathological pregnancies. It has been shown that concentrations of placental lipid peroxides are abnormally increased in preeclampsia and eclampsia.Walsh3 postulates that high levels of lipid peroxides initiate the arachidonic acid cascade, causing imbalance between thromboxane and prostacycline production. Thromboxane and unsaturated fatty acids peroxides formed in the course of its synthesis may be an important factor leading to blood vessel damage and disturbances first of placental circulation and then the motherAs circulation, which in consequence contributes to the development of different stages of gestosis.Sane et al.14 pointed out that maximum increase of concentration of lipid peroxides in blood plasma occurs immediately before spontaneous abortion. As the aetiology of imminent premature labour is unclear, an attempt was made to determine oxidant/antioxidant status in both types of abnormal pregnancies. At the time of the examination all women with pathologic pregnancy received medication, so TBARS level was affected not only by the disorders in the pregnancy, but also by the use of therapeutic agents.Therefore the increase of TBARS concentration in blood plasma of women with pathologic pregnancy should be considered an outcome of combined action of the administered drugs and disorders of the normal metabolism of the organism. In both examined pathologic conditions Se concentration and GSH-Px activity were significantly higher than in healthy pregnancy (Table 1).Uotila et al.28,29 demonstrated that the concentration of blood Se in the third trimester of pregnancy in women with different stages of gestosis does not differ from that observed in healthy pregnant females. Erythrocyte and plasma GSH-Px activities, however, were significantly higher in women with severe gestosis than in healthy pregnancies. In another work Uotila et al.2 demonstrated that total peroxyl radical-trapping capacity is significantly higher in blood plasma Fig. 3 Relationship between plasma TBARS concentration and erythrocyte (RBC) GSH-Px activity in gestosis women. Fig. 4 Pregnancy age-dependent changes in: blood plasma Se level (A); erythrocyte (B); and plasma (C) GSH-Px activity; and TBARS concentration (D). IPL, imminent premature labour; 19–24, 25–29, 30–34, 35–40 weeks of pregnancy. 38 Analyst, January 1998, Vol. 123and cerebrospinal fluid of women with gestosis than in normal pregnancy. In their investigation Nicotra et al.30 did not find differences in TBARS and vitamin E concentrations or GSH-Px activity between patients with habitual abortion 3–6 months after the most recent abortion and non-pregnant women.The relatively low number of publications on antioxidants in pathologic pregnancy does not allow extensive discussion of our results. GSH-Px is one of the primary antioxidants present in tissues that limits the concentration of lipid peroxides. It has been shown that GSH-Px activity in placentas of pre-eclamptic women is significantly lower than in placentas obtained from healthy pregnant women.31 Deficiency of GSH-Px in the placenta may be one of the factors contributing to the development of gestosis.The cause of the low GSH-Px activity in the placenta of women with gestosis is unknown. GSH-Px activity in the tissues depends on the availability of Se, and first of all on its dietary intake. However, it is probably not the dietary factors that cause the decrease of GSH-Px activity in the placenta.If its drop were due to Se deficiency in the diet, it would lead to its decrease in all tissues, and not only in the placenta. The increase of erythrocyte and blood plasma GSH-Px activity may be a protective mechanism compensating for the increase of TBARS concentration in women with gestosis. Moreover, disturbances in the normal function of kidneys in gestosis patients may result in increased blood plasma GSH-Px activity. Blood plasma GSH-Px protein originates from proximal tubular cells of the kidney.32 The studies demonstrated statistically significant relation between plasma Se concentration and erythrocyte and plasma GSH-Px activity. Since in people with appropriate concentrations of Se in blood plasma (over 100 mg l21) only a small portion of Se (10–15%) is incorporated in erythrocyte and plasma GSH-Px,25 such correlations are found only in populations with very low Se levels.Selenium in blood plasma is bound with GSH-Px, selenoprotein P and albumin.The percentage of Se content in this protein fraction depends on its availability, so its concentration in blood may also vary considerably in people.33 In Se deficiency the amount of the microelement bound with GSH-Px in erythrocytes and plasma may be as high as about 60%.33 A linear correlation between Se concentration and GSH-Px is then observed. High correlation coefficients of the statistically significant relations between Se concentration and GSH-Px activity in blood plasma may indicate that the saturation of the organism with GSH-Px has not been achieved.In women with pathologic pregnancy Se concentration in blood plasma was also higher than in healthy pregnant women. The concentration of Se in the organism depends primarily on its dietary supply. The consumption of Se in Poland is estimated to be about 40 mg d21.12 The amount which is appropriate for pregnant women is not known.High correlation coefficients between Se concentration and GSH-Px activity in blood plasma in all groups of examined women may indicate that the consumption of Se in Poland is too low to cover the demand. There are also other factors which may influence the concentration of selenium in blood; supply of antagonistic microelements (e.g. fluorine),34 smoking, alcohol consumption35 or exposure to industrial pollution.36 Moreover, it depends on accumulation in tissues in certain physiological and pathological conditions6 and on other than ‘normal’ excretion in urine.6,37 As all the examined patients lived in the same geographic region, the average dietary intake in all the groups is probably the same.Also the lifestyle, dietary habits and clinical status reported by the patients were similar. At the time of examination, the basic morphological and biochemical indices of the blood of patients with complicated pregnancies were normal. In women with gestosis, blood pressure was stabilised and oedema was eliminated.In all groups of examined women, ultrasonography showed that the development of foetuses was normal, considering gestational age. This provided grounds for the assumption that the increase of plasma volume in all examined groups of women was comparable. Therefore it can be speculated that increased Se concentration in blood plasma of women with gestosis and imminent premature labour results from metabolic changes caused by pathologic pregnancy.It might also explain the increased activity of plasma GSH-Px. Barrington et al.38 demonstrated significant decrease of serum Se concentration in women in the first trimester of pregnancy at risk of nonrecurrent miscarriage. Mask and Lane39 found out that GSH-Px activity in plateletrich plasma in women with imminent premature labour is significantly higher than in healthy pregnant women and comparable with non-pregnant women. They did not observe differences in the concentration of plasma Se in the examined groups of women but at the time of labour it was about four times higher than in the groups of patients examined in our study (130 ± 30 mg l21 vs. 40.5 ± 4.2 mg l21 in healthy pregnant women and 120 ± 30 mg l21 vs. 36.5 ± 13.2 mg l21 in imminent premature labour).39 Uotila et al.,29 who examined women with hypertensive complications of pregnancy, did not find differences in their blood Se concentration in comparison with the control group.However, it can be supposed that the clinical status of the two groups of examined women differed considerably. The comparison of Se concentration in foetal blood or umbilical blood of babies born by mothers with gestosis and healthy women was very interesting. The decrease of GSH-Px activity in erythrocytes and platelets of umbilical blood of a neonate born by a mother with gestosis may suggest accumulation of antioxidants in maternal organism as a kind of mechanism protecting from oxidative stress.29 The same authors also demonstrated that significantly increased plasma or platelet GSH-Px activity is associated with poor outcome of pregnancy.29 The results of the present study and the analysis of data by other authors indicate that changes of plasma Se concentration and erythrocyte and plasma GSH-Px activity during pregnancy depend in general on the concentration of Se in the whole population of a given area.When Se concentration in blood of non pregnant women is high, the changes of Se concentration and of Se-dependent GSH-Px activity during pregnancy do not involve significant decrease of concentration/activity of these antioxidants.However, when the concentrations are low, physiological transfer of Se and GSH-Px between tissues causes much more drastic decrease of these antioxidants in blood plasma. In conclusion, our results indicate that pregnancy influences blood Se status and GSH-Px activity.Complications of pregnancy are accompanied by lipid peroxidation and elevated levels of antioxidants, selenium concentrations and GSH-Px activity. It would be advisable to continue studies on the balance of TBARS, Se, GSH-Px and other antioxidants in the foetus, placenta and mother in pathological pregnancies. References 1 Papas, A. M., Lipids, 1996, 31, S77. 2 Uotila, J. T., Kirkkola, A. L., Rorarius, M., Tuimala, R. J., and Metsa- Ketela, T., Free Radicals Biol. Med., 1994, 5, 581. 3 Walsh, S. W., Hypertens. Pregnancy, 1994, 13, 1. 4 Walsh, S. W., in World Review of Nutrition and Dietetics, ed. Simopoulos A. P., and Karger, S., Basel, Switzerland, 1994, pp. 114–118. 5 Neve, J., Experientia, 1991, 47, 187. 6 Korpela, H., Loneniva, R., Yrjanheikki, E., and Kauppila, A., Int. J. Vitam. Nutr. Res., 1984, 54, 257. 7 Smith, A. M., and Picciano, M. F., J. Nutr., 1986, 116, 1068. Analyst, January 1998, Vol. 123 398 Gross, S., Semin. Hematol., 1976, 13, 187. 9 Kim, H. Y., Picciano, M. F., Wallig, M. A., and Milner J. A., Pediat. Res., 1991, 29, 440. 10 Zachara, B. A., Wasowicz, W., Gromadzinska, J., Sklodowska, M., and Krasomski, G., Biol. Trace Elem. Res., 1986, 19, 175. 11 Wasowicz, W., Wolkanin, P., Bednarski, M., Gromadzinska, J., Sklodowska, M., and Grzybowska, K., Biol. Trace Elem. Res., 1993, 38, 205. 12 Wasowicz, W., Proceedings of the Fifth International Symposium on Uses of Selenium and Tellurium, ed. Carapella, S. C., Olfield, J. E., and Palmieri, Y., Bibliotheque Royale de Belgique `a Bruxelles, Brussels, 1994, pp. 163–170. 13 Levander, O. A., J. Am. Diet. Assoc., 1991, 91, 1572. 14 Sane, A. S., Shobha, A., Mishra, V. V., Barad, D. P., Shah, V. C., and Nagpal, S., Gynecol. Obstet. Invest., 1994, 31, 172. 15 Kelly, F. J., Brit. Med. Bull., 1993, 49, 668. 16 Watkinson, J. H., Anal. Chem., 1966, 38, 92. 17 Wasowicz, W., and Zachara, B. A., J. Clin. Chem. Clin. Biochem., 1987, 25, 402. 18 Paglia, D.E., and Valentine, W. N., J. Lab. Clin. Med., 1967, 70, 158. 19 Wasowicz, W., Neve, J., and Peretz A., Clin. Chem., 1993, 39, 2522. 20 Bro, S., Berendtsen, H., Norgaard, J., Host, A., and Jorgensen, P. J., J. Trace Elem. Electrolytes Health Dis., 1988, 2, 165. 21 Thomson, C. D., and Robinson, M. F., Am. J. Clin. Nutr., 1980, 33, 303. 22 Zachara, B. A., Wardak, C., Didkowski, W., Maciag, A., and Marchaluk, E., Gynecol. Obstet. Invest., 1993, 35, 12. 23 Behne, D., and Wolters, W., J. Clin. Chem. Clin. Biochem., 1979, 17, 133. 24 Campbell, D. M., Proc. Nutr. Soc., 1988, 47, 45. 25 Butler, J., Whanger, P. D., and Trip, M. J., Am. J. Clin. Nutr., 1982, 36, 15. 26 Rudolph, N., and Wong, S. L., Pediatr. Res., 1978, 12, 789. 27 Wang, Y., Walsh, S. W., Guo, J., and Zhang, J., Am. J. Obstet. Gynecol., 1991, 165, 1690. 28 Uotila, J. T., Tuimala, R. J., Aarnio, T. M., Pyykko, K. A., and Ahotupa, M. O., Br. J. Obstet. Gynaecol., 1993, 100, 270. 29 Uotila, J., Tuimala, R., Pyykko, K., and Ahotupa, M., Gynecol. Obstet. Invest., 1993, 36, 153. 30 Nicotra, M., Muttinelli, C., Sbracia, M., Rolfi, G., and Passi, S., Gynecol. Obstet. Invest., 1994, 38, 223. 31 Walsh, S. W., and Wang, Y., Am. J. Obstet. Gynecol., 1993, 169, 1456. 32 Avissar, N., Ornt, D. B., Yagil, Y., Horowitz, S., Watkins, R. H., Kerl, E. A., Takashaki, K., Palmer, I. S., and Cohen, H. J., Am. J. Physiol, 1994, 266, C367. 33 Xia, Y., Zhao, X., Zhu, L., and Whanger, P. D., J. Nutr. Biochem., 1992, 3, 211. 34 Wasowicz, W., Golebiowska, M., and Chlebna-Sokol, D., Trace Elem. Med., 1988, 5, 43. 35 Alfthan, G., and Neve, J., J. Trace Elem. Med. Biol., 1996, 10, 77. 36 Gromadzi�nska, J., W�asowicz, W., Sk�lodowska, M., Bulikowski, W., and Rydzy�nski, K., Environ. Health Perspect., 1996, 104, 1312. 37 Swanson, C. A., Reamer, D. C., Veillon, C., King, J. C., and Levander, O. A., Am. J. Clin. Nutr., 1983, 38, 169. 38 Barrington, J. W., Lindsay, P., James, D., Smith, S., and Roberts, A., Brit. J. Obstet. Gynaecol., 1996, 103, 130. 39 Mask, G., and Lane, H. W., Nutr. Res., 1993, 13, 901. Paper 7/05396G Received July 25, 1997 Accepted October 22, 1997 40 Analyst, January 1998,