Blood selenium and glutathione peroxidases in miscarriage

British Journal of Obstetrics and Gynaecology March 2001, Vol. 108, pp. 244±247

Blood selenium and glutathione peroxidases in miscarriage Bronisøaw A. Zachara a,*, Waldemar DobrzynÂski b, Urszula Tra®kowska a, Wiesøaw SzymanÂski b Objective To investigate the selenium glutathione and glutathione peroxidase (glutathione-Px) levels in blood in women who experience miscarriage. Design An observational study. Participants Forty women with miscarriage occurring in the ®rst and second trimesters. Methods Concentrations were measured in whole blood and plasma. Glutathione was measured in red cells and glutathione-Px activity was measured in the red cells and plasma. The results were compared with 36 women in the same period of viable pregnancy and 28 age-matched, healthy, non-pregnant controls. Results The selenium concentrations in whole blood and plasma of women following abortion were the same as in viable pregnancy, but were signi®cantly lower compared with controls. The glutathione levels were significantly higher in women with miscarriage compared with viable pregnancies and with non-pregnant women. Red cell and plasma glutathione-Px activities of women who had had a miscarriage were signi®cantly lower than in both normal pregnancies and the control group. Conclusion The decreased activities of the antioxidant enzymes, red cell and plasma glutathione-Px, may play an important role in the aetrology of spontaneous abortion.

INTRODUCTION Selenium, as an integral component of the enzyme glutathione peroxidase (glutathione-Px), plays an important role during pregnancy. We have shown previously 1,2 that the selenium concentrations in whole blood and plasma, as well as the activity of glutathione-Px in red cells and plasma, are signi®cantly lower in women at delivery, compared with non-pregnant women. Similar ®ndings have been published by others 3,4. We have shown 2 an almost linear decline in these parameters from the ®rst trimester to parturition. Lowest values were found at delivery. Smith and Picciano 5 have demonstrated that during pregnancy the requirement for selenium is increased as a result of the transport of this element to the growing fetus. It is believed that de®ciency of selenium in humans and animals may be the cause of some health complications 6,7. In the past few years some authors 8±10 have suggested that miscarriage is related to selenium de®ciency. Our study was undertaken to determine the selenium levels in whole blood and plasma as well as glutathione concentration in red cells and glutathionePx activities in red cells and plasma in women with

a

Department of Biochemistry, The Ludwik Rydygier Medical University, Bydgoszcz, Poland b Depertment of Gynaecology and Obstetrics, The Ludwik Rydygier Medical University, Bydgoszcz, Poland * Correspondence: Professor B. A. Zachara, Department of Biochemistry, The Ludwik Rydygier Medical University, Bydgoszcz, Poland. q RCOG 2001 British Journal of Obstetrics and Gynaecology PII: S03 06-5456(00)0003 0-9

miscarriage during the ®rst and second trimester of pregnancy. METHODS Studies were carried out on 40 women with miscarriage (six to 23 weeks of pregnancy; 17 were in the ®rst trimester and 23 in the second trimester of pregnancy) (mean, 12.5 weeks) aged 17 - 40 years (mean, 26.4 years). The majority of women were pregnant for the ®rst time (n ˆ 22), 10 of the total group gave birth to one child and did not have miscarriages, two were delivered of two live births, and two others three children. Four women had given birth to one child previously, and among them three had had a miscarriage once, and one twice. Blood samples were drawn into plastic tubes with lithium heparin (Vacuette, Geiner Labourtechnik, Austria) just before, or within 12 hours after uterine curettage. Part of the blood was centrifuged, plasma was harvested and red cells were washed with an excess of 0.9% NaCl solution. The selenium concentration in whole blood and plasma was measured ¯uorometrically with 2,3-diaminonaphthalene 11. The accuracy of the method was checked with whole blood and serum reference materials produced by Seronorm, Nycomed, Oslo, Norway. The mean level of selenium in the reference whole blood was 81.7 ^ 2.5 ng/mL and in serum 84.5 ^ 2.4 ng/mL, while the certi®ed concentrations were 83 and 86 ng/mL, respectively. The glutathione level in red cells was assayed by the method of Beutler 12, and glutathione-Px activity in red cells and plasma was measured spectrophotometrically by the method of Paglia www.bjog-elsevier.com

SELENIUM AND GLUTATHIONE PEROXIDASES IN MISCARRIAGE 245

and Valentine 13 with t-butyl hydroperoxide as substrate. Details of the methods have been published elsewhere 14. The results were compared with the values obtained from 36 pregnant women (6 to 23 weeks of pregnancy (mean, 11.8 weeks) aged 19 - 40 years (mean, 26.4 years) and with 28 healthy, age-matched (21 -37, mean, 28.4 years) non-pregnant women. The study protocol was approved by the University Ethics Committee for Medical Research. The nature and purpose of the study was explained to the patients and they gave written consent. All statistical analyses and the simple linear regression analyses were carried out using Statgraphics software. Student's t test was used to determine statistical signi®cance which was set at P , 0.05. RESULTS The mean values and standard deviations of selenium levels, glutathione concentration and glutathione-Px activities in the blood components of women with miscarriage, normal pregnancy and healthy non-pregnant controls are presented in the Table 1. Inpatients with miscarriage and normal pregnancy, the selenium levels in whole blood as well as in plasma did not differ between these two groups, but were signi®cantly lower (0.0001 , P , 0.001) compared with non-pregnant healthy controls. In one healthy woman the selenium level in plasma was as low as 16.3 ng/mL (conversion factor to SI units, 0.0127). glutathione concentration in the red cells of pregnant women was not signi®cantly different from that of the controls. In women with miscarriage the glutathione concentration was signi®cantly (P , 0.01) higher compared with the values of both the other groups. On the contrary, red cell as well as plasma glutathione-Px activities were the same in pregnant women and the control group, but were signi®cantly lower (0.0001 , P , 0.01) in women who had had a miscarriage. DISCUSSION Selenium concentrations in blood components are generally related to diet 15,16. The geographical distribution of selenium in the soil varies considerably and thus plant

and animal products will re¯ect these concentrations. It is generally accepted that the normal selenium concentrations in the serum/plasma of healthy subjects are about 100 ng/mL. In some European countries, including Belgium, Finland, Holland and the United Kingdom, these levels were in the range of 100 to 126 ng/mL, while in some others, such as Austria, Germany, Yugoslavia and Greece, they were about half that level 17±19. In our study the mean plasma selenium concentration in healthy women was 66.1 $ 13.1 ng/mL. Studies performed in 1990 by Debski 20, and based on selenium content in cow's milk, grass and liver of hares showed that 75% of Poland is selenium de®cient and only 25% is adequate. Rayman 21has shown that in the last few years, due to the drop in imports of selinium-rich wheat from North America (particularly from Canada) 22, the selenium intake in the UK has progressively declined and consequently the selenium concentrations in blood and plasma dramatically decreased. In another study Rayman et al. 23 have shown that in the serum of pregnant women (mean, 33 weeks of pregnancy) the median selenium level was only 48.5 ng/ mL. In our present study plasma selenium levels in normal pregnancy (mean, 11.8 weeks of pregnancy) were 54.6 ng/ mL, which did not differ from the levels of aborted women (56.0 ng/mL), but were signi®cantly lower compared with non-pregnant controls (66.1 ng/mL). In farm animals selenium de®ciency may be the cause of infertility, abortion and placental retention 24±26. Naziroglu et al. 27 have shown that selenium, vitamin E, copper (both of which are antioxidants) magnesium and phosphorus are signi®cantly lower in the plasma of aborted ewes compared with non-aborted animals. The administration of selenium before mating and throughout the pregnancy increased selenium levels and the activity of glutathione-Px 26,28, and dramatically reduced the incidence of barren ewes, while substantially increasing the percentage of lamb crop 29,30. In humans the problem with reproductive performance is not so evident. Iwanier and Zachara 31 supplemented a group of sub-fertile men with selenium and have shown that the selenium levels and glutathione-Px activities in their blood increased but the quality of the spermatozoal characteristics did not change. Barrington et al. 8 showed a signi®cantly higher risk of ®rst trimester non-recurrent miscarriages in

Table 1. Selenium and glutathione concentrations and glutathione peroxidase activities in blood components of women with spontaneous abortion, normal pregnancy and the control group. Values are shown as mean (SD) or [median]*. Selenium levels (ng/ml) Groups Abortion (n ˆ 40) Normal pregnancy (n ˆ 36) Control (n ˆ 28)

Glutathione peroxidase activities

Whole blood

Plasma

U/g Hb

U/l plasma

74.9 (9.7) b [71.1] 74.1 (11.6) b [71.9] 90.5 (11.2) [91.5]

56.0 (8.77) a [54.7] 54.6 (11.1) a [54.6] 66.1 (13.1) [65.1]

15.3 (2.96) [14.9] 18.4 (6.0) c [17.6] 18.3 (3.18) d [18.0]

169 (30.7) [162] 231 (102) d [180] 230 (54.1) e [210]

GSH level (mmol/l) 2.60 (0.32) [2.52] 2.33 (0.25) c [2.32] 2.39 (0.25) c [2.40]

* Statistical signi®cances compared with the control group: (a) P , 0.001; (b) P , 0.0001, and with the group of aborted women: (c) P , 0.01; (d) P , 0.001 and (e) P , 0.0001.

q RCOG 2001 Br J Obstet Gynaecol 108, pp. 244±247

246 B.A. ZACHARA ET AL.

women with low serum selenium concentrations. In their study the mean selenium values (54.7 ^ 16.7 ng/mL) and the number of patients (n ˆ 40) were exactly the same as in our study. However, the mean selenium level in the ®rst trimester of viable pregnancy was signi®cantly higher (65.3 ^ 16.3 ng/mL; P , 0.01) than ours and the level in serum of non-pregnant volunteers was even higher (81.1 ^ 16.0). In a later study, Barrington et al. 9 have shown that in women with recurrent miscarriage, serum selenium concentration is also signi®cantly lower compared with non-pregnant women, but signi®cantly higher than that in non-recurrent miscarriage. There are very few reports on antioxidant activity in women who had had miscarriage. In our study the selenium concentrations in whole blood and in plasma did not differ between aborted women from that of viable pregnancy. However, the glutathione-Px activities both in red cells and in plasma were signi®cantly lower in nonrecurrent miscarriage compared with normal pregnancy as well as with the control group. Although oxygen is essential for all aerobic organisms, its metabolism leads to the formation of harmful free radicals and other reactive oxygen species. Survival in the presence of oxygen is thus dependent on the prevention of oxidative damage by enzymes. In general, trace elements function as prosthetic groups of various enzymes 32. Selenium is incorporated into the active site of glutathione-Px 33±34. Copper, zinc and manganese are constituents of superoxide dismutase 35, which plays a major role in catalysing the transformation of superoxide radical anion to less harmful H2O2 36. Glutathione-Px catalyses the reduction of H2O2 and organic hydroperoxides, thus preventing lipid peroxidation of cell membranes and acting as a free radical scavenger 32. Oxidative stress, of which lipid peroxidation represents a major manifestation, is involved aetiologically in a variety of clinical conditions including pregnancy and miscarriage 37. Glutathione-Px reduces almost every hydroperoxide, (i.e. it is non-speci®c for the hydroperoxide, but highly speci®c for glutathione which is the only substrate for this enzyme 38). The lower activities of glutathione-Px in women who had had miscarriage compared with viable pregnancy, despite unchanged selenium concentrations in both groups, are unclear and do not allow further conclusions to be drawn at this stage. Con¯icting results are available on the blood glutathione-Px activity of pregnant women. One group reported that red cell enzyme activity was not different 4, another group found non-signi®cantly lower activity 2, and a third group found signi®cantly higher activities in pregnant compared with non-pregnant women 39. Plasma glutathione-Px activity decreased 2,4 or increased 39 during pregnancy. Nicotra et al. 40 have shown that red cell glutathione-Px and plasma lipoperoxides in women with habitual abortion were not signi®cantly different from those of the controls. Unchanged

plasma glutathione-Px activity in habitual abortion has also been found by Simsek et al. 37. In addition these authors found that glutathione and lipid peroxide levels in the plasma of aborted women were signi®cantly higher than in healthy controls, while other small molecule antioxidants, such as vitamins A and E, beta carotene and bilirubin, were signi®cantly lower in habitual abortion. It is likely that, due to lower glutathione-Px activities found in our study, the increased concentration of glutathione participates in the elimination of free radicals which are higher in aborted women 37,41. Glutathione is the most important non-enzymatic thiol of the cells and functions as an essential redox compound 42. It is well known that glutathione, as an effective reductant, plays an important role in a variety of detoxi®cation processes. It readily neutralizes the hydroxyl radicals, which are considered a major source of free radical damage 43. In conclusion, our study shows that selenium concentrations in the whole blood and plasma of women who who had had a miscarriage was not signi®cantly different from those of viable pregnancy, but was signi®cantly lower than in non-pregnant women. The activity of glutathione-Px in red cells and plasma was signi®cantly lower, while glutathione level in red cells was signi®cantly higher in miscarriage patients compared with normal pregnancy and the control group. As there is little information available about the antioxidant system in women who had had a miscarriage, further studies are needed to give a more precise answer to this problem. References 1. Zachara BA, Wasowicz W, Gromadzinska J, Sklodowska M, Krasomski G. Glutathione peroxidase activity, selenium, and lipid peroxide concentrations in blood from a healthy Polish population I. Maternal and cord blood. Biol Trace Elem Res 1986;10:175±187. 2. Zachara BA, Wardak C, Didkowski W, Maciag A, Marchaluk E. Changes in blood selenium and glutathione concentrations and glutathione peroxidase activity in human pregnancy. Gynecol Obstet Invest 1993;35:12±17. 3. Rudolph N, Wong SL. Selenium and glutathione peroxidase activity in maternal and cord plasma and red cells. Pediat Res 1978;12:789±792. 4. Behne D, Wolters W. Selenium content and glutathione peroxidase activity in the plasma and erythrocytes of non-pregnant and pregnant women. J Clin Chem Clin Biochem 1979;17:133±135. 5. Smith AM, Picciano MF. Evidence for increased selenium requirement for the rat during pregnancy and lactation. J Nutr 1986;116:1068±1079. 6. Lockitch G. Selenium: Clinical signi®cance and analytical concepts. Crit Rev Clin Lab Sci 1989;27:483±541. 7. Bedwal RS, Nair N, Sharma MP, Mathur RS. Selenium: Its biological perspectives. Med Hypotheses 1993;41:150±159. 8. Barrington JW, Lindsay P, James D, Smith S, Roberts A. Selenium de®ciency and miscarriage: a possible link? Br J Obstet Gynaecol 1996;103:130±132. 9. Barrington JW, Taylor M, Smith S, Bowen-Simpkins P. Selenium and recurrent miscarriage. J Obstet Gynaecol 1997;17:199±200. 10. Kocak I, Aksoy E, Ustun C. Recurrent spontaneous abortion and selenium de®ciency. Int J Gynecol Obstet 1999;65:79±80. 11. Watkinson JH. Fluorometric determination of selenium in biological material with 2,3-diaminonaphthalene. Anal Chem 1966;38:92±97.

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