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Glutathione and Glutathione-related Enzymes in Decidua and Placenta of Controls and Women with Pre-eclampsia
Placenta (1999), 20, 541–546
Glutathione and Glutathione-related Enzymes in Decidua and Placenta of Controls and Women with Pre-eclampsia M. F. C. M. Knapena, W. H. M. Petersb,c, T. P. J. Mulderb, H. M. W. M. Merkusa, J. B. M. J. Jansenb and E. A. P. Steegersa Departments of a Obstetrics/ Gynaecology and Paper accepted 26 February 1999
b
Gastroenterology, University Hospital St Radboud, Nijmegen, The Netherlands
Pre-eclampsia is a major complication of pregnancy with high morbidity and mortality rates. The aetiology is still unclear but impaired detoxification or enhanced levels of reactive (oxygen) metabolites may contribute to the development or maintenance of pre-eclampsia. Glutathione and glutathione-related enzymes, as one of the major detoxificating and free-radical scavenging systems, may play a role in controlling the disease. Seventeen normotensive pregnant women and 24 pre-eclamptic women were investigated prospectively with respect to placental and decidual levels of total glutathione (GSH), glutathione S-transferase activity (GST), selenium-dependent glutathione peroxidase (SeGPX) and total glutathione peroxidase activity (TGPX, both selenium- and non-selenium-dependent GPX). Decidual levels of glutathione and related enzymes were compared with placental levels, and the investigated parameters in pre-eclampsia were compared with those in normotensive pregnancy by the Mann–Whitney U-test. Clinical data were correlated with biochemical parameters by Spearman’s correlation test. Glutathione levels were significantly higher in decidua as compared with placenta. Glutathione levels were elevated in pre-eclampsia and HELLP (haemolysis, elevated liver enzymes, low platelets) as compared to normotensive pregnancy for decidua and in the placenta of patients with pre-eclampsia only. Glutathione S-transferase activity was not different between the two groups. In the placenta of patients with pre-eclampsia+HELLP, total glutathione peroxidase activity was elevated versus controls. Selenium-dependent glutathione peroxidase activity was higher in decidua versus placenta and in decidua of pre-eclamptic versus control subjects. Enhanced glutathione concentrations and glutathione peroxidase activities were often found in placenta and decidua in pre-eclampsia, probably as a compensatory mechanism to prevent further damage by peroxides, (oxygen) radicals or other toxins in the placenta or in the feto–placental interface. 1999 Harcourt Publishers Ltd Placenta (1999), 20, 541–546
INTRODUCTION Hypertension complicates 6–20 per cent of all pregnancies and ranks amongst the four most common causes of maternal as well as perinatal mortality in the world (Turnbull, 1987). The aetiology of this gestational disorder is largely unknown, but increasing evidence suggests impairment of endothelial cell function, possibly mediated by (oxygen) free-radicals, lipid peroxides or other toxins (Hubel et al., 1989; Roberts et al., 1989). Impaired placental perfusion or increased activity of the decidual lymphoid tissue due to immunological maladaptation could lead to excessive production of oxygen free-radicals and/or lipid peroxides acting locally or being spilled into the maternal circulation. Lipid peroxides activate cyclo-oxygenase c
To whom correspondence should be addressed at: Department of Gastroenterology, University Hospital Nijmegen, PO Box 9101, 6500 HB Nijmegen, The Netherlands. Fax: +31-243540103; E-mail: [email protected] 0143–4004/99/070541+06 $12.00/0
and impair endothelial prostacyclin synthetase, leading to decreased prostacyclin levels (Higgs and Vane, 1983). In normal conditions a variety of antioxidant mechanisms serve to control peroxidative processes (Sies, 1985), but in preeclampsia an imbalance between lipid peroxidation and antioxidant mechanisms may impair normal endothelial function (Frank and Massaro, 1980). Glutathione and glutathione-related enzymes are involved in the metabolism and detoxification of cytotoxic and carcinogenic compounds as well as reactive (oxygen) species. Glutathione can act either as a substrate in the cytosolic GSH-redox cycle or directly inactivate reactive oxygen species, such as the oxygen radicals O2 · and OH · . Reduced glutathione is present in most cells at high concentrations (0.5–10 m) (Sies and Akerboom, 1984). Glutathione S-transferases (GSTs) are enzymes catalysing the nucleophilic addition of glutathione to electrophilic centres of a wide variety of compounds. They have 1999 Harcourt Publishers Ltd
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non-selenium-dependent glutathione peroxidase activity and can also serve as transport proteins for a broad range of lipophilic compounds, such as bilirubin, bile acids, steroid hormones and various xenobiotics (Beckett and Hayes, 1993; Hayes and Pulford, 1995). Glutathione peroxidases (GPXs) are enzymes that catalyse the reduction of organic hydroperoxides (lipid hydroperoxides, DNA hydroperoxides) and hydrogen peroxide. Two major types of GPX have been found, of which one contains selenium (SeGPX) which is active with both organic hydroperoxides and hydrogen peroxide. The second type of GPX consists of proteins that do not depend on selenium and have negligible activity with hydrogen peroxide. This class comprises mainly glutathione S-transferases (Mannervik, 1985). In this study, levels of glutathione and activities of glutathione S-transferase and glutathione peroxidase were estimated in the placenta and decidua of women with pre-eclamptic and normotensive pregnancies.
MATERIALS AND METHODS Study populations Two groups of women were studied: 17 normotensive pregnant and 24 pre-eclamptic women, 10 of whom developed pre-eclampsia complicated by the HELLP (haemolysis, elevated liver enzymes, low platelets) syndrome. The experimental protocol was approved by the institutional review board of the University Hospital Nijmegen, and written informed consent was obtained from each patient. Except for two women in the pre-eclamptic group, all women were delivered by caesarian section (SC); elective SC due to cephalopelvic disproportion in the control group and emergency SC in the pre-eclamptic group, for either deteriorating maternal or fetal condition. Pre-eclampsia was defined as pregnancy-induced hypertension (diastolic blood pressure >90 mm Hg on two or more consecutive occasions, each more than 4 h apart) and concordant proteinuria (urinary protein >0.3 g/l) (Davey and MacGillivray, 1988). The HELLP syndrome was defined as: haemolysis, lactate dehydrogenase activity (LDH) >600 IU/l; elevated liver enzymes, aspartate aminotransferase (ASAT) >70 IU/l and alanine aminotransferase (ALAT) >70 IU/l; and low platelets, a platelet count <100109/l (Sibai, 1990). Blood pressure (BP) was taken in a sitting position with a sphygmomanometer. Diastole was recorded at phase IV Korotkoff sound. Urinary protein was determined in 24-h urine samples.
Tissue preparation and biochemical analysis One representative fragment of placenta (infarcted sites were avoided) and one fragment of decidua were excized during caesarian section and frozen within 20 min. For placenta, McRobie, Glover and Tracy (1996) have recently
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demonstrated that levels of GST enzyme activity did not differ in samples taken at different sites. Tissue fragments (20– 100 mg) were thawed and homogenized in 10 volumes ice-cold homogenizing buffer (250 m sucrose, 20 m Tris-HCl, 1 m dithriothreitol, pH 7.4) with 10 strokes in small glass–glass homogenizers. The homogenates were centrifuged at 150 000 g at 4C for 1 h. Supernatants were frozen in liquid nitrogen and stored at 20C in small portions. Protein contents were determined using the method of Lowry et al. (1951) with bovine serum albumin as standard. Total glutathione was determined by high-performance liquid chromatography after reaction with monobromobimane as described previously (Nijhoff et al., 1995). In short, the experimental samples (cytosolic fractions) were diluted with 10 per cent w/v trichloroacetic acid and centrifuged for 2 min in an Eppendorf microcentrifuge in order to remove denatured protein. The clear supernatant was used as follows; 33 ìl sample, 167 ìl sodium borohydride solution (2.5 mg/ml in 100 m 4-ethyl-morpholine, pH=8.0), 100 ìl monobromobimane solution (2 m in acetonitril) were added together for 30 min, and the reaction was stopped by adding 20 ìl 10 per cent trichloroacetic acid. Standard samples of glutathione (50 ì stored in 1 per cent acetic acid) were treated in parallel with the experimental samples. Twenty ìl of the solutions were injected on a 2003 mm Chromsep HPLC column with Chromspher 5C18 cartridge. Separation and detection was carried out on a Separations High Precision Pump model 480 (HI Ambacht, The Netherlands) equipped with a Marathon model autosampler and a Jasco 821-FP model spectrofluorometric detector. The elution protocol with linear gradient (flow rate 0.35 ml/min at room temperature) was as follows. Zero min, 100 per cent solvent A (10 per cent v/v methanol, 0.25 per cent v/v acetic acid, 10 m tetrabutyl-ammonium hydrogen sulphate, adjusted to pH 3.5 with sodium hydroxide), 0 per cent solvent B (100 per cent v/v methanol, 0.25 per cent v/v acetic acid); 20 min, 100 per cent solvent B; 22 min, 100 per cent solvent A; 36 min, reinjection. Cytosolic glutathione S-transferase activity was determined by the method of Habig, Pabst and Jakoby (1974) using 1-chloro-2,4-dinitrobenzene (CDNB) as substrate. Cytosolic glutathione peroxidase enzyme activities with hydrogen peroxide (selenium-dependent activity, SeGPX; Rotruck et al., 1973; Mannervik, 1985) and t-butylhydroperoxide (both selenium- and non-selenium-dependent activity; TGPX; Prohaska and Ganther, 1977) as substrates were measured essentially as described by van Lieshout et al. (1998) as follows: reduction in absorption at 340 nm (37C) was recorded on a Lambda 12 spectrophotometer (Perkin Elmer, Germany) at intervals of 1 min and a total analysis time of 5 min. The assay solution contained 60 m Tris-HCl buffer pH 7.6, 0.12 m EDTA, 1 m sodium azide, 0.33 m NADPH, 1.3 m reduced glutathione and 1.3 U glutathione reductase. The reaction was started with the addition of substrate and the final concentrations of the substrates were 1.2 m for t-butylhydroperoxide and 0.6 m for hydrogen peroxide. All
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Table 1. Study group characteristics
Parameter
Normotensive pregnancy (n=17)
HELLP+ pre-eclampsia (n=10)
Pre-eclampsia only (n=14)
Maternal age (years) Gestational age (weeks) Parity Diastolic blood pressure (mmHg) Hb (mmol/l) Ht (l/l) Platelet count (109/l) Serum creatinine (ìmol/l) Serum uric acid (mmol/l) Serum ASAT (IU/l) Serum ALAT (IU/l) Serum LDH (IU/l) Proteinuria (g/l)
33 (22–42) 38 +5 (37 +4–41 +3) 1 (0–3) 75 (58–86) 7.4 (6.7–8.4) 0.36 (0.32–0.41) 217 (150–301) 60 (54–83) 0.28 (0.17–0.36) 10 (5–14) 7 (4–11) 208 (182–302) None
31 (26–39) 31 +5b (26 +3–39) 0b (0–0) 108b (95–120) 7.5 (6.6–9.0) 0.35 (0.30–0.40) 59b (31–93) 74b (62–108) 0.32a (0.22–0.44) 124b (101–510) 128b (80–333) 822b (600–1607) 3.3b (0.3–10.5)
28a (22–33) 34 +2b (28 +5–38 +2) 0a (0–2) 110b (100–120) 7.5 (6.5–8.7) 0.36 (0.30–0.40) 161a (55–305) 80b (52–96) 0.41b (0.24–0.55) 23b (7–73) 19a (5–82) 360b (206–783) 9.0b (0.3–27.6)
Values are given as median (range). Hb, haemoglobin; Ht, haematocrit. a P<0.05 versus normotensive pregnancy. b P<0.01 versus normotensive pregnancy.
enzyme measurements were performed in triplicate. The within and between batch coefficients of variation for the enzyme activity measurements were below 5 and 10 per cent, respectively. GSH levels and activities of GST, TGPX and SeGPX were calculated per mg total cytosolic protein.
Statistical evaluations Comparisons of differences between placental and decidual tissues on the one hand and pre-eclamptic women and normotensive controls on the other were performed by the Mann–Whitney U-test. Correlations between the parameters were tested with the Spearman’s rank correlation test. Differences were considered significant if the P-value was below 0.05.
RESULTS The characteristics of the women involved in this study are depicted in Table 1. Decidual GSH levels were significantly higher than placental levels in both normotensive and pre-eclamptic pregnancies (Table 2). GSH levels were significantly higher in decidua in pre-eclampsia compared with normotensive pregnancy, and in placentae of patients with pre-eclampsia only as compared to normotensives (Table 2). Decidual and placental GSH levels correlated significantly with several haematological and biochemical parameters reflecting the severity of the disease (Table 3). GST activities in decidua and placenta were not significantly different. Decidual GST levels correlated significantly with several parameters reflecting the severity of the disease (Table 3).
Decidual TGPX activity was significantly higher than the corresponding placental activity in the control group. Placental TGPX activity, in contrast to that of decidua, was significantly higher in pre-eclampsia complicated with HELLP as compared with normotensive pregnancies. Decidual SeGPX activity in the pre-eclamptic group was significantly higher than the corresponding placental activity and was significantly higher in pre-eclampsia than in normotensive pregnancy (Table 2). TGPX activity correlated significantly with SeGPX activity in both decidua and placenta (r=0.67, P<0.0001; r=0.60, P<0.0001, respectively).
DISCUSSION An imbalance between lipid peroxides or (oxygen) free-radicals on the one hand and detoxicating and scavenging substances on the other might contribute to the aetiology or pathophysiology of pre-eclampsia (Walsh, 1985; Hubel et al., 1989). Most organisms are equipped with enzymatic and nonenzymatic defence mechanisms against oxidants and other toxic compounds. Important antioxidant and detoxificating enzymes are superoxide dismutase (SOD), catalase, glutathione peroxidase and glutathione S-transferase. Nonenzymatic detoxification is achieved by many different agents, such as transferrin, ceruloplasmin, lactoferrin, vitamin E, vitamin C and uric acid, as well as thiols such as glutathione, cysteamine and cysteine. For example, activities of CuZnSOD and tissue levels of vitamin E were shown to be significantly lower in placentae of pre-eclamptic as compared with those of normotensive pregnant women (Wang and Walsh, 1996). Since the glutathione system in a quantitative sense is one of the most important protective systems in
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Table 2(A). Glutathione levels, glutathione S-transferase, total and selenium-dependent glutathione peroxidase activities in decidua of pre-eclampsia and normotensive pregnancies Decidua
GSH (nmol/mg protein) GST (nmol/min/mg protein) TGPX (nmol/min/mg protein) SeGPX (nmol/min/mg protein)
Normotensive pregnancy (n=17)
HELLP and pre-eclampsia (n=8)
Pre-eclampsia (n=14)
115b (13–240) 217 (111–518) 55a (24–101) 119 (106–169)
184b,c (98–354) 243 (111–365) 70 (51–90) 137a,c (127–167)
185b,d (59–306) 291 (130–458) 64 (35–167) 140a,c (107–173)
Results are presented as median (range). a P<0.05, bP<0.0001, decidual versus placental tissue. c P<0.05, dP<0.01, pre-eclamptic versus normotensive pregnancy.
Table 2(B). Glutathione levels, glutathione S-transferase, total and selenium-dependent glutathione peroxidase activities in placenta of pre-eclampsia and normotensive pregnancies Placenta
GSH (nmol/mg protein) GST (nmol/min/mg protein) TGPX (nmol/min/mg protein) SeGPX (nmol/min/mg protein)
Normotensive pregnancy (n=17)
HELLP and pre-eclampsia (n=10)
Pre-eclampsia (n=14)
25 (12–49) 202 (80–318) 46 (30–75) 106 (78–171)
26 (9–111) 182 (118–449) 66a (37–94) 113 (97–170)
39a (21–77) 195 (107–458) 54 (29–116) 119 (85–160)
Results are presented as median (range). a P<0.01, versus normotensive pregnancy.
Table 3. Correlations between glutathione, glutathione S-transferase, glutathione peroxidases and several parameters reflecting the severity of the disease in pre-eclamptic pregnancy
Glutathione (GSH) GSH versus BP GSH versus proteinuria GSH versus platelet count GSH versus creatinine GSH versus urate GSH versus ASAT GSH versus ALAT GSH versus LDH Glutathione S-transferase (GST) GST versus BP GST versus Hb GST versus creatinine Glutathione peroxidase (TGPX) TGPX versus LDH
Decidua
Placenta
r=0.57, P=0.0001 r=0.43, P=0.047 r= 0.36, P=0.03 r=0.40, P=0.01 r=0.42, P=0.0078 r=0.46, P=0.005 r=0.43, P=0.0064 r=0.45, P=0.0069
r=0.39, P=0.01 n.s. n.s. n.s. r=0.34, P=0.03 n.s. n.s. n.s.
r=0.41, P=0.0097 r=0.33, P=0.04 r=0.36, P=0.03
n.s. n.s. n.s.
n.s.
r=0.34 , P=0.04
humans, we focused our attention on glutathione and glutathione-related enzymes in both placenta and decidua. Glutathione levels were much higher in decidua as compared with placenta in both pre-eclampsia and normotensive pregnancy. In fact the decidual GSH values appeared to be
much higher than in any other human tissue examined before (Peters et al., 1993; Mulder et al., 1995; Nijhoff et al., 1995). This high decidual level may point at a pronounced protective role of this tissue, either protecting the mother against toxins such as peroxides or oxygen free-radicals produced by the
Knapen et al.: Glutathione in Women with Pre-eclampsia
feto-maternal interface or the placenta (Wang, Walsh and Kay, 1992), or protecting the fetus against such harmful compounds (Kobayashi et al., 1995). We found considerably higher levels of glutathione in the placenta and decidua of pre-eclampsia than in normotensive pregnancy. Enhanced lipid peroxidation may be involved in the foam-cell formation of decidua and in the pathogenesis of pre-eclampsia (Branch et al., 1994). GSH can inhibit lipid peroxidation via membrane-bound GPX (Sies, 1991). The high GSH levels found, in accordance with a previous report by Gu¨lmezoglu, Oosthuizen and Hofmeyr (1996), may point at a compensating mechanism in order to prevent excessive lipid peroxidation. The glutathione assay as performed by us quantifies total glutathione levels and does not discriminate between oxidized and reduced glutathione. Therefore, a high glutathione level does not necessarily reflect a high tissue oxidative state. Enhancement of GSH and GST levels is seen very often as a reaction to chemical stress (Hayes and Pulford, 1995). Few data so far are available with respect to oxidative stress; however, repeated exposure of mammary adenocarcinoma cells to the anthracycline drug adriamycin, which is highly cytotoxic due to oxidative damage, enhanced levels of GSH, GST and GPX (Lee, Sciandra and Siemann, 1989). Decidual glutathione S-transferase (GST) activity was slightly enhanced in pre-eclampsia compared with normotensive pregnancy, but the difference was not statistically significant. There is a possibility of a yet unknown toxin produced by the feto–maternal interface, which may be detoxified in a reaction with glutathione, catalysed by GSTs. Until now only one paper on placental GST activity in pre-eclampsia has been published where no difference between pre-eclamptic and normotensive pregnancy was found (Poranen et al., 1996), which is in agreement with our results. We noticed significantly higher SeGPX activity in the decidua and significantly higher TGPX activities in the placenta of pre-eclamptic than in normotensive pregnancies. In normotensive pregnancies, TGPX activity in the decidua was significantly higher than in the placenta, whereas in preeclamptic pregnancies SeGPX activity was significantly higher in the decidua as compared to the placenta. This may point at a differential upgrading of peroxidases in these tissues. In pre-eclampsia, relative upgrading of SeGPX is higher in the decidua and TGPX upgrading is higher in the placenta. TGPX activity correlated significantly with SeGPX activity in
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both the decidua and the placenta, indicating that GPX activity in both tissues is mainly involved in the detoxification of organic hydroperoxides and not of H2O2. Glutathione peroxidase enzyme activity (Takehara, Yoshioka and Sasaki, 1990; Walsh and Wang, 1993) as well as messenger RNA levels of glutathione peroxidase (Wang and Walsh, 1996) were reported to be lower in the placenta of pre-eclamptic than in normotensive pregnancies, whereas levels of lipid peroxides were higher (Wang, Walsh and Kay, 1992). The discrepancy between our results and those reported in the literature could possibly be caused by the differences in severity of disease of the patients studied. We may have studied a more severe form of pre-eclampsia, resulting in a more enhanced compensatory mechanism; however, a direct comparison of patients is difficult since several important clinical parameters are given in different entities. There seems to be an interaction between lipid peroxides, the prostaglandin/thromboxane system and glutathione peroxidases. Lipid peroxides which are elevated in the maternal circulation of hypertensive pregnant women (Maseki et al., 1981) may stimulate prostaglandin H2 synthase and increase thromboxane and oxygen radical production, which subsequently may enhance formation of lipid peroxides (Walsh and Wang, 1993). Cyclooxygenase activity not only generates thromboxane but also oxygen radicals (Kukreja et al., 1986). Glutathione peroxidases inactivate peroxides, thereby diminishing peroxide-mediated stimulation of prostaglandin synthase. Inhibition of glutathione peroxidase activity in normal placenta resulted in a dose-dependent increase in the production of both lipid peroxides and thromboxane without affecting prostacyclin, so that the ratio of thromboxane to prostacyclin progressively increased (Walsh and Wang, 1993). The ratios of thromboxane/prostacyclin and lipid peroxide/prostacyclin were shown to be three-fold higher in pre-eclamptic than in normal placenta (Wang, Walsh and Kay, 1992). In this respect the increased peroxidase activities we observed may act as a compensatory mechanism, both in the placenta and the decidua. In conclusion, we found enhanced levels of glutathione and activities of glutathione peroxidase in the placenta and the decidua in pre-eclampsia, which correlated significantly with severity of the disease. This most probably represents a compensatory mechanism to prevent damage to the fetus or the mother by lipid peroxides, (oxygen) free-radicals or other toxins.
ACKNOWLEDGEMENTS This study was supported by the Dutch ‘Praeventiefonds’ (grant no. 28-2801).
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Glutathione and Glutathione-related Enzymes in Decidua and Placenta of Controls and Women with Pre-eclampsia M. F. C. M. Knapena, W. H. M. Petersb,c, T. P. J. Mulderb, H. M. W. M. Merkusa, J. B. M. J. Jansenb and E. A. P. Steegersa Departments of a Obstetrics/ Gynaecology and Paper accepted 26 February 1999
b
Gastroenterology, University Hospital St Radboud, Nijmegen, The Netherlands
Pre-eclampsia is a major complication of pregnancy with high morbidity and mortality rates. The aetiology is still unclear but impaired detoxification or enhanced levels of reactive (oxygen) metabolites may contribute to the development or maintenance of pre-eclampsia. Glutathione and glutathione-related enzymes, as one of the major detoxificating and free-radical scavenging systems, may play a role in controlling the disease. Seventeen normotensive pregnant women and 24 pre-eclamptic women were investigated prospectively with respect to placental and decidual levels of total glutathione (GSH), glutathione S-transferase activity (GST), selenium-dependent glutathione peroxidase (SeGPX) and total glutathione peroxidase activity (TGPX, both selenium- and non-selenium-dependent GPX). Decidual levels of glutathione and related enzymes were compared with placental levels, and the investigated parameters in pre-eclampsia were compared with those in normotensive pregnancy by the Mann–Whitney U-test. Clinical data were correlated with biochemical parameters by Spearman’s correlation test. Glutathione levels were significantly higher in decidua as compared with placenta. Glutathione levels were elevated in pre-eclampsia and HELLP (haemolysis, elevated liver enzymes, low platelets) as compared to normotensive pregnancy for decidua and in the placenta of patients with pre-eclampsia only. Glutathione S-transferase activity was not different between the two groups. In the placenta of patients with pre-eclampsia+HELLP, total glutathione peroxidase activity was elevated versus controls. Selenium-dependent glutathione peroxidase activity was higher in decidua versus placenta and in decidua of pre-eclamptic versus control subjects. Enhanced glutathione concentrations and glutathione peroxidase activities were often found in placenta and decidua in pre-eclampsia, probably as a compensatory mechanism to prevent further damage by peroxides, (oxygen) radicals or other toxins in the placenta or in the feto–placental interface. 1999 Harcourt Publishers Ltd Placenta (1999), 20, 541–546
INTRODUCTION Hypertension complicates 6–20 per cent of all pregnancies and ranks amongst the four most common causes of maternal as well as perinatal mortality in the world (Turnbull, 1987). The aetiology of this gestational disorder is largely unknown, but increasing evidence suggests impairment of endothelial cell function, possibly mediated by (oxygen) free-radicals, lipid peroxides or other toxins (Hubel et al., 1989; Roberts et al., 1989). Impaired placental perfusion or increased activity of the decidual lymphoid tissue due to immunological maladaptation could lead to excessive production of oxygen free-radicals and/or lipid peroxides acting locally or being spilled into the maternal circulation. Lipid peroxides activate cyclo-oxygenase c
To whom correspondence should be addressed at: Department of Gastroenterology, University Hospital Nijmegen, PO Box 9101, 6500 HB Nijmegen, The Netherlands. Fax: +31-243540103; E-mail: [email protected] 0143–4004/99/070541+06 $12.00/0
and impair endothelial prostacyclin synthetase, leading to decreased prostacyclin levels (Higgs and Vane, 1983). In normal conditions a variety of antioxidant mechanisms serve to control peroxidative processes (Sies, 1985), but in preeclampsia an imbalance between lipid peroxidation and antioxidant mechanisms may impair normal endothelial function (Frank and Massaro, 1980). Glutathione and glutathione-related enzymes are involved in the metabolism and detoxification of cytotoxic and carcinogenic compounds as well as reactive (oxygen) species. Glutathione can act either as a substrate in the cytosolic GSH-redox cycle or directly inactivate reactive oxygen species, such as the oxygen radicals O2 · and OH · . Reduced glutathione is present in most cells at high concentrations (0.5–10 m) (Sies and Akerboom, 1984). Glutathione S-transferases (GSTs) are enzymes catalysing the nucleophilic addition of glutathione to electrophilic centres of a wide variety of compounds. They have 1999 Harcourt Publishers Ltd
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non-selenium-dependent glutathione peroxidase activity and can also serve as transport proteins for a broad range of lipophilic compounds, such as bilirubin, bile acids, steroid hormones and various xenobiotics (Beckett and Hayes, 1993; Hayes and Pulford, 1995). Glutathione peroxidases (GPXs) are enzymes that catalyse the reduction of organic hydroperoxides (lipid hydroperoxides, DNA hydroperoxides) and hydrogen peroxide. Two major types of GPX have been found, of which one contains selenium (SeGPX) which is active with both organic hydroperoxides and hydrogen peroxide. The second type of GPX consists of proteins that do not depend on selenium and have negligible activity with hydrogen peroxide. This class comprises mainly glutathione S-transferases (Mannervik, 1985). In this study, levels of glutathione and activities of glutathione S-transferase and glutathione peroxidase were estimated in the placenta and decidua of women with pre-eclamptic and normotensive pregnancies.
MATERIALS AND METHODS Study populations Two groups of women were studied: 17 normotensive pregnant and 24 pre-eclamptic women, 10 of whom developed pre-eclampsia complicated by the HELLP (haemolysis, elevated liver enzymes, low platelets) syndrome. The experimental protocol was approved by the institutional review board of the University Hospital Nijmegen, and written informed consent was obtained from each patient. Except for two women in the pre-eclamptic group, all women were delivered by caesarian section (SC); elective SC due to cephalopelvic disproportion in the control group and emergency SC in the pre-eclamptic group, for either deteriorating maternal or fetal condition. Pre-eclampsia was defined as pregnancy-induced hypertension (diastolic blood pressure >90 mm Hg on two or more consecutive occasions, each more than 4 h apart) and concordant proteinuria (urinary protein >0.3 g/l) (Davey and MacGillivray, 1988). The HELLP syndrome was defined as: haemolysis, lactate dehydrogenase activity (LDH) >600 IU/l; elevated liver enzymes, aspartate aminotransferase (ASAT) >70 IU/l and alanine aminotransferase (ALAT) >70 IU/l; and low platelets, a platelet count <100109/l (Sibai, 1990). Blood pressure (BP) was taken in a sitting position with a sphygmomanometer. Diastole was recorded at phase IV Korotkoff sound. Urinary protein was determined in 24-h urine samples.
Tissue preparation and biochemical analysis One representative fragment of placenta (infarcted sites were avoided) and one fragment of decidua were excized during caesarian section and frozen within 20 min. For placenta, McRobie, Glover and Tracy (1996) have recently
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demonstrated that levels of GST enzyme activity did not differ in samples taken at different sites. Tissue fragments (20– 100 mg) were thawed and homogenized in 10 volumes ice-cold homogenizing buffer (250 m sucrose, 20 m Tris-HCl, 1 m dithriothreitol, pH 7.4) with 10 strokes in small glass–glass homogenizers. The homogenates were centrifuged at 150 000 g at 4C for 1 h. Supernatants were frozen in liquid nitrogen and stored at 20C in small portions. Protein contents were determined using the method of Lowry et al. (1951) with bovine serum albumin as standard. Total glutathione was determined by high-performance liquid chromatography after reaction with monobromobimane as described previously (Nijhoff et al., 1995). In short, the experimental samples (cytosolic fractions) were diluted with 10 per cent w/v trichloroacetic acid and centrifuged for 2 min in an Eppendorf microcentrifuge in order to remove denatured protein. The clear supernatant was used as follows; 33 ìl sample, 167 ìl sodium borohydride solution (2.5 mg/ml in 100 m 4-ethyl-morpholine, pH=8.0), 100 ìl monobromobimane solution (2 m in acetonitril) were added together for 30 min, and the reaction was stopped by adding 20 ìl 10 per cent trichloroacetic acid. Standard samples of glutathione (50 ì stored in 1 per cent acetic acid) were treated in parallel with the experimental samples. Twenty ìl of the solutions were injected on a 2003 mm Chromsep HPLC column with Chromspher 5C18 cartridge. Separation and detection was carried out on a Separations High Precision Pump model 480 (HI Ambacht, The Netherlands) equipped with a Marathon model autosampler and a Jasco 821-FP model spectrofluorometric detector. The elution protocol with linear gradient (flow rate 0.35 ml/min at room temperature) was as follows. Zero min, 100 per cent solvent A (10 per cent v/v methanol, 0.25 per cent v/v acetic acid, 10 m tetrabutyl-ammonium hydrogen sulphate, adjusted to pH 3.5 with sodium hydroxide), 0 per cent solvent B (100 per cent v/v methanol, 0.25 per cent v/v acetic acid); 20 min, 100 per cent solvent B; 22 min, 100 per cent solvent A; 36 min, reinjection. Cytosolic glutathione S-transferase activity was determined by the method of Habig, Pabst and Jakoby (1974) using 1-chloro-2,4-dinitrobenzene (CDNB) as substrate. Cytosolic glutathione peroxidase enzyme activities with hydrogen peroxide (selenium-dependent activity, SeGPX; Rotruck et al., 1973; Mannervik, 1985) and t-butylhydroperoxide (both selenium- and non-selenium-dependent activity; TGPX; Prohaska and Ganther, 1977) as substrates were measured essentially as described by van Lieshout et al. (1998) as follows: reduction in absorption at 340 nm (37C) was recorded on a Lambda 12 spectrophotometer (Perkin Elmer, Germany) at intervals of 1 min and a total analysis time of 5 min. The assay solution contained 60 m Tris-HCl buffer pH 7.6, 0.12 m EDTA, 1 m sodium azide, 0.33 m NADPH, 1.3 m reduced glutathione and 1.3 U glutathione reductase. The reaction was started with the addition of substrate and the final concentrations of the substrates were 1.2 m for t-butylhydroperoxide and 0.6 m for hydrogen peroxide. All
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Table 1. Study group characteristics
Parameter
Normotensive pregnancy (n=17)
HELLP+ pre-eclampsia (n=10)
Pre-eclampsia only (n=14)
Maternal age (years) Gestational age (weeks) Parity Diastolic blood pressure (mmHg) Hb (mmol/l) Ht (l/l) Platelet count (109/l) Serum creatinine (ìmol/l) Serum uric acid (mmol/l) Serum ASAT (IU/l) Serum ALAT (IU/l) Serum LDH (IU/l) Proteinuria (g/l)
33 (22–42) 38 +5 (37 +4–41 +3) 1 (0–3) 75 (58–86) 7.4 (6.7–8.4) 0.36 (0.32–0.41) 217 (150–301) 60 (54–83) 0.28 (0.17–0.36) 10 (5–14) 7 (4–11) 208 (182–302) None
31 (26–39) 31 +5b (26 +3–39) 0b (0–0) 108b (95–120) 7.5 (6.6–9.0) 0.35 (0.30–0.40) 59b (31–93) 74b (62–108) 0.32a (0.22–0.44) 124b (101–510) 128b (80–333) 822b (600–1607) 3.3b (0.3–10.5)
28a (22–33) 34 +2b (28 +5–38 +2) 0a (0–2) 110b (100–120) 7.5 (6.5–8.7) 0.36 (0.30–0.40) 161a (55–305) 80b (52–96) 0.41b (0.24–0.55) 23b (7–73) 19a (5–82) 360b (206–783) 9.0b (0.3–27.6)
Values are given as median (range). Hb, haemoglobin; Ht, haematocrit. a P<0.05 versus normotensive pregnancy. b P<0.01 versus normotensive pregnancy.
enzyme measurements were performed in triplicate. The within and between batch coefficients of variation for the enzyme activity measurements were below 5 and 10 per cent, respectively. GSH levels and activities of GST, TGPX and SeGPX were calculated per mg total cytosolic protein.
Statistical evaluations Comparisons of differences between placental and decidual tissues on the one hand and pre-eclamptic women and normotensive controls on the other were performed by the Mann–Whitney U-test. Correlations between the parameters were tested with the Spearman’s rank correlation test. Differences were considered significant if the P-value was below 0.05.
RESULTS The characteristics of the women involved in this study are depicted in Table 1. Decidual GSH levels were significantly higher than placental levels in both normotensive and pre-eclamptic pregnancies (Table 2). GSH levels were significantly higher in decidua in pre-eclampsia compared with normotensive pregnancy, and in placentae of patients with pre-eclampsia only as compared to normotensives (Table 2). Decidual and placental GSH levels correlated significantly with several haematological and biochemical parameters reflecting the severity of the disease (Table 3). GST activities in decidua and placenta were not significantly different. Decidual GST levels correlated significantly with several parameters reflecting the severity of the disease (Table 3).
Decidual TGPX activity was significantly higher than the corresponding placental activity in the control group. Placental TGPX activity, in contrast to that of decidua, was significantly higher in pre-eclampsia complicated with HELLP as compared with normotensive pregnancies. Decidual SeGPX activity in the pre-eclamptic group was significantly higher than the corresponding placental activity and was significantly higher in pre-eclampsia than in normotensive pregnancy (Table 2). TGPX activity correlated significantly with SeGPX activity in both decidua and placenta (r=0.67, P<0.0001; r=0.60, P<0.0001, respectively).
DISCUSSION An imbalance between lipid peroxides or (oxygen) free-radicals on the one hand and detoxicating and scavenging substances on the other might contribute to the aetiology or pathophysiology of pre-eclampsia (Walsh, 1985; Hubel et al., 1989). Most organisms are equipped with enzymatic and nonenzymatic defence mechanisms against oxidants and other toxic compounds. Important antioxidant and detoxificating enzymes are superoxide dismutase (SOD), catalase, glutathione peroxidase and glutathione S-transferase. Nonenzymatic detoxification is achieved by many different agents, such as transferrin, ceruloplasmin, lactoferrin, vitamin E, vitamin C and uric acid, as well as thiols such as glutathione, cysteamine and cysteine. For example, activities of CuZnSOD and tissue levels of vitamin E were shown to be significantly lower in placentae of pre-eclamptic as compared with those of normotensive pregnant women (Wang and Walsh, 1996). Since the glutathione system in a quantitative sense is one of the most important protective systems in
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Table 2(A). Glutathione levels, glutathione S-transferase, total and selenium-dependent glutathione peroxidase activities in decidua of pre-eclampsia and normotensive pregnancies Decidua
GSH (nmol/mg protein) GST (nmol/min/mg protein) TGPX (nmol/min/mg protein) SeGPX (nmol/min/mg protein)
Normotensive pregnancy (n=17)
HELLP and pre-eclampsia (n=8)
Pre-eclampsia (n=14)
115b (13–240) 217 (111–518) 55a (24–101) 119 (106–169)
184b,c (98–354) 243 (111–365) 70 (51–90) 137a,c (127–167)
185b,d (59–306) 291 (130–458) 64 (35–167) 140a,c (107–173)
Results are presented as median (range). a P<0.05, bP<0.0001, decidual versus placental tissue. c P<0.05, dP<0.01, pre-eclamptic versus normotensive pregnancy.
Table 2(B). Glutathione levels, glutathione S-transferase, total and selenium-dependent glutathione peroxidase activities in placenta of pre-eclampsia and normotensive pregnancies Placenta
GSH (nmol/mg protein) GST (nmol/min/mg protein) TGPX (nmol/min/mg protein) SeGPX (nmol/min/mg protein)
Normotensive pregnancy (n=17)
HELLP and pre-eclampsia (n=10)
Pre-eclampsia (n=14)
25 (12–49) 202 (80–318) 46 (30–75) 106 (78–171)
26 (9–111) 182 (118–449) 66a (37–94) 113 (97–170)
39a (21–77) 195 (107–458) 54 (29–116) 119 (85–160)
Results are presented as median (range). a P<0.01, versus normotensive pregnancy.
Table 3. Correlations between glutathione, glutathione S-transferase, glutathione peroxidases and several parameters reflecting the severity of the disease in pre-eclamptic pregnancy
Glutathione (GSH) GSH versus BP GSH versus proteinuria GSH versus platelet count GSH versus creatinine GSH versus urate GSH versus ASAT GSH versus ALAT GSH versus LDH Glutathione S-transferase (GST) GST versus BP GST versus Hb GST versus creatinine Glutathione peroxidase (TGPX) TGPX versus LDH
Decidua
Placenta
r=0.57, P=0.0001 r=0.43, P=0.047 r= 0.36, P=0.03 r=0.40, P=0.01 r=0.42, P=0.0078 r=0.46, P=0.005 r=0.43, P=0.0064 r=0.45, P=0.0069
r=0.39, P=0.01 n.s. n.s. n.s. r=0.34, P=0.03 n.s. n.s. n.s.
r=0.41, P=0.0097 r=0.33, P=0.04 r=0.36, P=0.03
n.s. n.s. n.s.
n.s.
r=0.34 , P=0.04
humans, we focused our attention on glutathione and glutathione-related enzymes in both placenta and decidua. Glutathione levels were much higher in decidua as compared with placenta in both pre-eclampsia and normotensive pregnancy. In fact the decidual GSH values appeared to be
much higher than in any other human tissue examined before (Peters et al., 1993; Mulder et al., 1995; Nijhoff et al., 1995). This high decidual level may point at a pronounced protective role of this tissue, either protecting the mother against toxins such as peroxides or oxygen free-radicals produced by the
Knapen et al.: Glutathione in Women with Pre-eclampsia
feto-maternal interface or the placenta (Wang, Walsh and Kay, 1992), or protecting the fetus against such harmful compounds (Kobayashi et al., 1995). We found considerably higher levels of glutathione in the placenta and decidua of pre-eclampsia than in normotensive pregnancy. Enhanced lipid peroxidation may be involved in the foam-cell formation of decidua and in the pathogenesis of pre-eclampsia (Branch et al., 1994). GSH can inhibit lipid peroxidation via membrane-bound GPX (Sies, 1991). The high GSH levels found, in accordance with a previous report by Gu¨lmezoglu, Oosthuizen and Hofmeyr (1996), may point at a compensating mechanism in order to prevent excessive lipid peroxidation. The glutathione assay as performed by us quantifies total glutathione levels and does not discriminate between oxidized and reduced glutathione. Therefore, a high glutathione level does not necessarily reflect a high tissue oxidative state. Enhancement of GSH and GST levels is seen very often as a reaction to chemical stress (Hayes and Pulford, 1995). Few data so far are available with respect to oxidative stress; however, repeated exposure of mammary adenocarcinoma cells to the anthracycline drug adriamycin, which is highly cytotoxic due to oxidative damage, enhanced levels of GSH, GST and GPX (Lee, Sciandra and Siemann, 1989). Decidual glutathione S-transferase (GST) activity was slightly enhanced in pre-eclampsia compared with normotensive pregnancy, but the difference was not statistically significant. There is a possibility of a yet unknown toxin produced by the feto–maternal interface, which may be detoxified in a reaction with glutathione, catalysed by GSTs. Until now only one paper on placental GST activity in pre-eclampsia has been published where no difference between pre-eclamptic and normotensive pregnancy was found (Poranen et al., 1996), which is in agreement with our results. We noticed significantly higher SeGPX activity in the decidua and significantly higher TGPX activities in the placenta of pre-eclamptic than in normotensive pregnancies. In normotensive pregnancies, TGPX activity in the decidua was significantly higher than in the placenta, whereas in preeclamptic pregnancies SeGPX activity was significantly higher in the decidua as compared to the placenta. This may point at a differential upgrading of peroxidases in these tissues. In pre-eclampsia, relative upgrading of SeGPX is higher in the decidua and TGPX upgrading is higher in the placenta. TGPX activity correlated significantly with SeGPX activity in
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both the decidua and the placenta, indicating that GPX activity in both tissues is mainly involved in the detoxification of organic hydroperoxides and not of H2O2. Glutathione peroxidase enzyme activity (Takehara, Yoshioka and Sasaki, 1990; Walsh and Wang, 1993) as well as messenger RNA levels of glutathione peroxidase (Wang and Walsh, 1996) were reported to be lower in the placenta of pre-eclamptic than in normotensive pregnancies, whereas levels of lipid peroxides were higher (Wang, Walsh and Kay, 1992). The discrepancy between our results and those reported in the literature could possibly be caused by the differences in severity of disease of the patients studied. We may have studied a more severe form of pre-eclampsia, resulting in a more enhanced compensatory mechanism; however, a direct comparison of patients is difficult since several important clinical parameters are given in different entities. There seems to be an interaction between lipid peroxides, the prostaglandin/thromboxane system and glutathione peroxidases. Lipid peroxides which are elevated in the maternal circulation of hypertensive pregnant women (Maseki et al., 1981) may stimulate prostaglandin H2 synthase and increase thromboxane and oxygen radical production, which subsequently may enhance formation of lipid peroxides (Walsh and Wang, 1993). Cyclooxygenase activity not only generates thromboxane but also oxygen radicals (Kukreja et al., 1986). Glutathione peroxidases inactivate peroxides, thereby diminishing peroxide-mediated stimulation of prostaglandin synthase. Inhibition of glutathione peroxidase activity in normal placenta resulted in a dose-dependent increase in the production of both lipid peroxides and thromboxane without affecting prostacyclin, so that the ratio of thromboxane to prostacyclin progressively increased (Walsh and Wang, 1993). The ratios of thromboxane/prostacyclin and lipid peroxide/prostacyclin were shown to be three-fold higher in pre-eclamptic than in normal placenta (Wang, Walsh and Kay, 1992). In this respect the increased peroxidase activities we observed may act as a compensatory mechanism, both in the placenta and the decidua. In conclusion, we found enhanced levels of glutathione and activities of glutathione peroxidase in the placenta and the decidua in pre-eclampsia, which correlated significantly with severity of the disease. This most probably represents a compensatory mechanism to prevent damage to the fetus or the mother by lipid peroxides, (oxygen) free-radicals or other toxins.
ACKNOWLEDGEMENTS This study was supported by the Dutch ‘Praeventiefonds’ (grant no. 28-2801).
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