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Oxidized and Free Whole Blood Thiols in Preeclampsia

From the Departments of Gastroenterology and Obstetrics and Gynecology, University Hospital St. Radboud, Nijmegen, Netherlands.

Address reprint requests to: Wilbert H. M. Peters, PhD Department of Gastroenterology University Hospital St. Radboud P.O. Box 9101 6500 HB Nijmegen Netherlands E-mail: w.peters{at}gastro.azn.nl

	Abstract

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Objective: To measure levels of oxidized and free thiols in whole blood of normotensive pregnant and preeclamptic women and evaluate the role of oxidative stress.

Methods: We measured whole blood oxidized and free levels of cysteine, homocysteine, cysteinylglycine, and glutathione by high performance liquid chromatography in women with normotensive pregnancies (n = 50), preeclampsia (n = 29), and preeclampsia complicated by the hemolysis, elevated liver enzymes, low platelets (HELLP) syndrome (n = 16).

Results: Oxidized and free levels (median [range], µmol/L) of cysteine and homocysteine were higher in women with preeclampsia than normotensive pregnancies (45 [27–81] versus 29 [9–91], P < .001, and 98 [57–193] versus 69 [33–215], P < .001; 0.8 [0.2–4.4] versus 0.4 [0.01–1.6], P < .001, and 2.1 [0.7–9.4] versus 1.2 [0.2–21.2], P < .01; respectively). The ratios of free to oxidized cysteine, homocysteine, and cysteinylglycine were lower in preeclampsia than normotensive pregnancy (2.2 [1.3–3.0] versus 2.4 [1.7–4.3], P < .001; 2.3 [0.5–5.4] versus 2.9 [1.1–24], P < .001; 4.1 [2.3–11.6] versus 5.4 [2.6–24.3], P < .02, respectively), indicating a shift in favor of the oxidized form of those thiols. In HELLP syndrome, levels of oxidized and free cysteine and levels of oxidized homocysteine were higher than normal (44 [33–63] versus 29 [9–91], P < .001, and 102 [82–133] versus 69 [33–215], P < .001; 1.0 [0.3–2.9] versus 0.4 [0.01–1.6], P < .001, respectively). No significant differences were found in oxidized glutathione levels in women with preeclampsia (22 [5–49] versus 17 [2–60], P = .06) or free levels in preeclamptic women with HELLP syndrome (757 [624–993] versus 842 [539–1516], P = .09) as compared with normotensive pregnant women. The ratios of free to oxidized cysteinylglycine and glutathione were higher in women with HELLP syndrome than in those with preeclampsia (5.4 [3.3–12.7] versus 4.1 [2.3–11.6], P = .02, and 56 [28–124] versus 45 [16–166], P = .02, respectively).

Conclusion: Significantly lower ratios of free to oxidized cysteine, homocysteine, and cysteinylglycine in preeclampsia might indicate oxidative stress.

Preeclampsia is one of the most frequent (5–7%) complications of pregnancy, leading to fetal growth restriction, preterm delivery, and maternal and fetal morbidity and mortality.¹ The hemolysis, elevated liver enzymes, and low platelets (HELLP) syndrome complicates preeclampsia in approximately 10% of cases. Although the etiologies of those disorders remain obscure, increasing evidence indicates that dysfunction of the vascular endothelium might result in increased vasoconstriction leading to maternal hypertension² and reduced uteroplacental blood flow.³ Endothelial dysfunction can be mediated by oxidative stress, defined as disturbance of the redox-balance, caused by increased amounts of oxidants such as cytokines or free radicals or a deficiency of antioxidants, resulting in an overall oxidant insult.^3–7 In women with preeclampsia, lipid peroxidation products are elevated^3,5,7 and might contribute to the etiology of preeclampsia.⁸ In normal pregnancy, lipid peroxidation increases progressively with gestational age and antioxidant capacity, vitamins A, C, and E, and erythrocyte levels of thiols increase in parallel.^3,5,7 Red blood cells contain high concentrations (up to 2 mM) of reduced glutathione accounting for almost 98% of the total blood content.^7,9 In addition to their detoxifying function of conjugating with noxious compounds, glutathione and other thiols maintain the redox balance of cells, thereby preventing oxidative damage.⁷ Glutathione maintains the redox balance with the use of glutathione peroxidases by reducing organic peroxides or (oxygen) free radicals and oxidizing glutathione.^10,11 Glutathione reductase–catalyzed reaction converts the inactive oxidized glutathione back into the active reduced form¹⁰ (Figure 1).

View larger version (10K): [in this window] [in a new window]   Figure 1. Glutathione cycling in detoxification of reactive oxygen species. Reactive oxygen species (RO·), hydrogen peroxide (H2O2), and organic peroxides (ROOH) are detoxified by glutathione peroxidase (GPX) with reduced glutathione (GSH) as a substrate, yielding the oxidized form of glutathione (GSSG) and relatively harmless ROH. Oxidized glutathione is regenerated to GSH by glutathione reductase (GR), which consumes nicotinamide adenine dinucleotide phosphate (NADPH).

	Materials and Methods

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The experimental protocol was approved by the Medical Ethical Review Committee of the University Hospital St. Radboud, Nijmegen. Preeclampsia was defined according to the standards of the International Society for the Study of Hypertension in Pregnancy as pregnancy-induced hypertension (diastolic blood pressure above 90 mmHg on two or more consecutive occasions, each more than 4 hours apart) and proteinuria (ratio above 0.3 g of protein to 10 mM of creatinine). HELLP syndrome was defined as hemolysis (lactic dehydrogenase above 600 IU/L), elevated liver enzymes (aspartate aminotransferase and alanine aminotransferase levels above 70 IU/L), and low platelets (thrombocyte count below 100 x 10⁹ per liter). After written informed consent was provided, blood samples of women with normotensive pregnancies (n = 50, 40% nulliparous), preeclampsia (n = 29, 40% nulliparous), and pre-eclampsia complicated by HELLP syndrome (n = 16,70% nulliparous) were collected before anti–hypertensive treatment was started. Patients and control subjects were studied between July 1998 and November 1999. All women were white. Women with diabetes mellitus or essential hypertension were excluded from the study.

N-ethylmaleimide, 3-[N-morpholino]-propanesulfonic acid and bathophenanthrolinedisulfonic acid were obtained from Sigma Chemical Co. (St. Louis, MO). All other chemicals were analytic grade and obtained from Merck (Darmstadt, Germany). Whole blood was collected into sterile vacutainers containing ethylenediaminetetra-acetic acid (Sherwood Medical, Ballymore, Northern Ireland) and processed within 1 hour after collection. For assay of free thiol levels (reduced and oxidized nonprotein bound thiols), proteins were precipitated by adding 500 µL whole blood to 500 µL 12% perchloric acid containing 2.0 mM bathophenanthro-linedisulfonic acid. For assay of oxidized free thiols, another 500 µL whole blood was added to 500 µL 12% perchloric acid containing 2.0 mM bathophenanthrolinedisulfonic acid and 40 mM N-ethylmaleimide. After thorough mixing and centrifugation at 16,000g for 20 minutes at 4C, supernatants were collected and stored at -80C until analysis. Samples were analyzed within 2 weeks. Just before analysis of the oxidized and free thiols, excess N-ethylmaleimide was removed by adding 70 µL KOH (2.0 M) followed by 60 µL HCl (0.01 M in 0.3 M 3-[N-morpholino]-propanesulfonic acid buffer) to 100 µL of sample. Samples were analyzed for levels of cysteine, homocysteine, cysteinylglycine, and glutathione by high performance liquid chromatography with fluorescent detection.¹⁵ Data were analyzed with the Chromeleon chromatography data system (Gynkotek, Munich, Germany) and the ratios of free to oxidized thiols were calculated for each subject.

To detect a difference in whole blood glutathione concentration of 250 µmol/L, a sample of at least 15 women in each group was needed, assuming = .05 and ß = .95. Kruskal–Wallis analysis of variance and, when appropriate, Mann–Whitney U tests were performed to assess statistical significance in thiols between groups; P < .05 was considered significant.

	Results

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Table 1 shows the characteristics of the women studied. The control women did not differ from those in the preeclampsia and HELLP groups regarding age, gestational age, and hemoglobin and hematocrit levels. Median values of whole blood free levels of cysteine, homocysteine, cysteinylglycine, and glutathione are shown in Table 2. Levels of cysteine and homocysteine were significantly higher in the preeclamptic group, whereas cysteine alone was significantly higher in the HELLP group compared with corresponding control values. No significant differences for cysteinylglycine and glutathione were found between the control group and those with preeclampsia with or without HELLP syndrome.

As reported by Ueland et al¹⁶ and Droge,¹⁷ the ratio of free to oxidized thiol measures the balance between oxidants and antioxidants, so changes in this ratio can be used to measure oxidative stress. These ratios were calculated for each thiol in every subject. Median ratios of free to oxidized cysteine, homocysteine, cysteinylglycine, and glutathione are listed in Table 2. In the preeclamptic group, those ratios were significantly lower for cysteine, homocysteine, and cysteinylglycine compared with the corresponding values in the control group, whereas no significant difference was found for glutathione. No significant differences in those ratios were found between the HELLP and control groups. Free to oxidized ratios of cysteinylglycine and glutathione were significantly higher in the HELLP group than in the preeclamptic group.

	Discussion

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Values for free glutathione and cysteine in whole blood were well documented and data in normotensive pregnancies as measured in the present study are comparable with those of nonpregnant controls reported elsewhere.^9,18,19 However, no whole blood reference values are known for free and oxidized cysteinylglycine and homocysteine levels.

We previously reported that whole blood ratios of glutathione to hemoglobin in women with preeclampsia or HELLP syndrome were lower than in those with normotensive pregnancies, whereas whole blood glutathione levels tended to be lower but only weakly significant (P = .05).¹⁴ In this more extensive study, we found that free whole blood glutathione levels tended to be lower in the women with HELLP syndrome, whereas values were not different in women with preeclampsia compared with those with normotensive pregnancies. Those data contrasted with our previous findings. There are two possible reasons for that discrepancy. 1) In the previous report, other patient and control populations and smaller groups were studied. The size of the study groups might be important particularly because whole blood values for glutathione vary between 500 and 1500 µmol/L.^9,14,18,19 In the present study, the control values showed a similar range (539–1516 µmol/L). 2) We used monobromobimane as a fluorescent probe to quantify glutathione in the previous study¹⁴ and we estimated glutathione and other thiols simultaneously by using 7-fluorobenzofurazane-4-sulfonic acid in the present study.¹⁵ The method using 7-fluorobenzofurazane-4-sulfonic acid is more specific because of the absence of breakdown products of monobromobimane and lesser background peaks, and the run-time is considerably shortened.

Free and oxidized levels of cysteine and homocysteine are higher in preeclampsia than in normotensive pregnancy. The ratios of free to oxidized thiol are significantly lower for cysteine, cysteinylglycine, and homocysteine and indicates a shift in redox balance toward more oxidized levels of cysteine, cysteinylglycine, and homocysteine in women with preeclampsia. As reviewed by Davidge⁴ and Walsh,³ preeclampsia is characterized by an imbalance between oxidants and antioxidants. In those studies, oxidative stress was only indirectly demonstrated by assessment of lipid peroxidation products or antioxidant levels^3,4; in the present study, we reported the direct measurement of oxidative stress. Chappell et al²⁰ showed that oxidative stress might be overcome in part by supplementation with vitamins C and E early in pregnancy, resulting in lower incidence of preeclampsia in women at risk.

Thiols are an important component of the total redox buffer system.²¹ Reduced glutathione in particular is a well-recognized endogenous scavenger of free radicals and peroxides because it maintains the redox potential and is very protective against oxidative stress.^10,18,22 In deactivating free radicals and reactive oxygen species, the reduced form of glutathione and other thiols is oxidized, thereby changing the ratio of free to oxidized thiol. We anticipated that this ratio for glutathione would be lower in women with preeclampsia, which we did not find. A possible explanation is that the oxidized form of glutathione is rapidly reduced by glutathione reductase, thereby consuming nicotinamide adenine dinucleotide phosphate¹⁰ (Figure 1), whereas such an efficient reducing system is not present for the other thiols. In addition, when increasing amounts of glutathione are oxidized, some of the oxidized glutathiones undergo renal degradation by gammaglutamyl transpeptidase, resulting in an irreversible loss of glutathione.²¹ As a result of one or both of these mechanisms, the ratio of free to oxidized thiol remains unchanged.

Decreased ratios of free to oxidized cysteine, homocysteine, and cysteinylglycine were expected in preeclamptic women with HELLP syndrome. Although oxidized levels of cysteine and homocysteine were significantly higher, significantly decreased ratios were not found, indicating that the reduced levels of those thiols were elevated in parallel with the oxidized levels and that oxidative stress is probably absent. Hemolysis and especially impairment of liver function might influence whole blood levels of the thiols because the liver plays an important role in thiol metabolism.

Reduced forms of thiols, specifically cysteine and homocysteine, might combine with the common active nitric oxide radical (NO^·) to produce S-nitrosothiol intermediates, which may be more functional in vasodilatation than NO^· itself.¹⁰ Although free levels of cysteine and homocysteine are high in women with preeclampsia, levels of the reduced form of both thiols are low because free to oxidized ratios are elevated in this group. These lower levels of reduced thiols might lead to a lower nitric oxide–mediated vasodilatation resulting in elevation of blood pressure as seen in the women in this study. In addition, lower levels of reduced cysteine and homocysteine and increased biosynthesis of NO^·23 might result in elevation of oxidative species through reaction of NO^· with superoxide, leading to the formation of peroxynitrite. Peroxynitrite is a strong oxidant that initiates lipid peroxidation,³ which was reported to be elevated in placentas from women with preeclampsia compared with those from normotensive pregnant women.⁴

In this study, whole blood thiol levels in normotensive pregnancies and those complicated by preeclampsia with or without HELLP syndrome were investigated for a better understanding of the pathophysiology of both diseases. Some reports suggested that preeclampsia and HELLP syndrome are different clinical syndromes with the same underlying pathology. Our results confirm this view because these complications of pregnancy exert pressure on the thiol detoxification system. Oxidized levels of cysteine and homocysteine were significantly higher in women with HELLP syndrome. Ratios of free to oxidized cysteine, homocysteine, and cysteinylglycine were significantly lower in women with preeclampsia compared with those with normotensive pregnancies but were not found changed in women with preeclampsia with HELLP syndrome. Ratios of free to oxidized cysteinylglycine and glutathione were significantly lower in women with preeclampsia than in those with preeclampsia with HELLP syndrome. The results suggested that elevated oxidative stress or disturbance in detoxification processes might influence the etiology or maintenance of preeclampsia or HELLP syndrome.

	Footnotes

 
This study was supported by grant 28-2801.1 from the Praeventiefonds, The Hague, Netherlands.

PII S0029-7844(00)01127-3

Received June 14, 2000. Received in revised form October 5, 2000. Accepted October 12, 2000.

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