Maarten T.M. Raijmakers

Oxidative Stress and Preeclampsia: Rationale for Antioxidant Clinical Trials

Preeclampsia remains a frequent and potentially dangerous complication of pregnancy. The cause remains largely unknown, but oxidative stress and a generalized inflammatory state are features of the maternal syndrome. The placenta appears to be the principal source of free radical synthesis but maternal leukocytes and the maternal endothelium are also likely contributors. Recent reports have suggested an important role for placental trophoblast NAD(P)H oxidase in free radical generation in preeclampsia. The antioxidant vitamin E is now known to have multiple actions in addition to prevention of lipid peroxidation (ie, inhibition of NAD(P)H oxidase activation and the inflammatory response). In view of the abnormally low plasma vitamin C concentrations in preeclampsia, a combination of vitamins C and E is a promising prophylactic strategy for prevention of preeclampsia. Several multicenter randomized clinical trials are now underway. The potential use of antioxidants and the recognized, albeit modest, benefit of low-dose aspirin prophylaxis have heightened the need for a reliable predictive test for preeclampsia. A combination test involving several relevant biomarkers is likely to provide the best predictive potential.

Association of human liver bilirubin UDP-glucuronyltransferase activity with a polymorphism in the promoter region of the UGT1A1 gene

BACKGROUND/AIMS Gilberts syndrome is a benign form of a deficiency in bilirubin glucuronidation. It is associated with a homozygous polymorphism, A(TA)7TAA instead of A(TA)6TAA, in the TATA-box of the promoter region of the bilirubin UDP-glucuronyltransferase gene. In this study the correlation between this promoter region polymorphism and in vitro human liver bilirubin UDP-glucuronyltransferase enzyme activity was investigated. METHODS Liver samples from organ transplant donors n=39) and two known Gilberts syndrome patients were used for measuring bilirubin UDP-glucuronyltransferase enzyme activity and for isolation of DNA followed by detection of the promoter region polymorphism by polymerase chain reaction. Genotypes were assigned as follows; 6/6: homozygous for the A(TA)6TAA-allele, 7/7: homozygous for the A(TA)7TAA-allele, and 6/7: heterozygous with one of each alleles. RESULTS Seventeen out of 39 subjects (44%) had the homozygous 6/6 genotype, 18 subjects (46%) had the heterozygous 6/7 genotype, whereas four individuals (10%) and the two individuals with Gilberts syndrome had the 7/7 genotype correlated with Gilberts syndrome. This resulted in an allele frequency of 0.33 for the A(TA)7TAA-allele. The median bilirubin UDP-glucuronyltransferase enzyme activity of the 17 subjects with the 6/6 genotype (1565 nmol/g liver/h) was significantly higher than the activity of the 18 subjects with the 6/7 genotype (985 nmol/g liver/h; p<0.05) and the six individuals with the 7/7 genotype (749 nmol/g liver/h; p<0.005). No significant differences in enzyme activity were found between the 6/7 and the 7/7 genotype groups. CONCLUSIONS The results indicate a close association between the promoter region genotype and the expression of hepatic bilirubin UDP-glucuronyltransferase enzyme activity. Subjects who have a 7/7 genotype have the lowest enzyme activity, whereas subjects with the heterozygous 6/7 genotype have an intermediate enzyme activity.

Plasma thiol status in preeclampsia

Objective To measure plasma thiol levels in women with normal pregnancies, women with preeclampsia, and non-pregnant controls to define plasma thiols effect on glutathione homeostasis and pathophysiology of preeclampsia. Methods Total plasma cysteine, γ-glutamylcysteine, homocysteine, cysteinylglycine, and glutathione levels were measured in ten nonpregnant women, ten women with normotensive pregnancies, and 20 women with preeclampsia at delivery. Results Median total plasma levels of all thiols in normotensive pregnant women were significantly lower than in nonpregnant women. Median total plasma cysteine and homocysteine levels in women with preeclampsia were significantly higher compared with pregnant controls (254 versus 190 μmol/L, P < .001; and 13.3 versus 8.4 μmol/L, P < .02, respectively), whereas glutathione levels were significantly lower in women with preeclampsia compared with those in pregnant controls (5.1 versus 6.3 μmol/L, P < .05). Conclusion In women with preeclampsia, homocysteine and cysteine levels, which are lowered in normotensive pregnancy, were comparable to levels in nonpregnant women, whereas glutathione levels were lower. Those results suggest that in women with preeclampsia, glutathione use is higher or its synthesis is disturbed. Therefore, glutathione might affect pathophysiology of preeclampsia.

Homocysteine, cysteine, and glutathione in human colonic mucosa: elevated levels of homocysteine in patients with inflammatory bowel disease

The present study was performed to evaluate the levels of the amino thiols cysteine, homocysteine, and glutathione in the colonic mucosa of patients with various intestinal diseases, especially chronic inflammatory bowel disease. Colonic biopsies of macroscopically normal mucosa out of a proximal and distal segment were collected from 187 patients with various intestinal diseases. Protein was assayed in duplicate by the method of Lowry et al (1951), using bovine serum albumin as standard. Total glutathione, cysteine, and homocysteine were quantified by high performance liquid chromatography (HPLC) with fluorescent detection. Only in patients with inflammatory bowel disease were the homocysteine levels in the large bowel mucosa significantly elevated compared with the concentrations in patients with normal mucosa. No significant differences were seen for glutathione and cysteine concentrations in colonic mucosa among the different groups of diseases. No correlation was found between the age of the patients and levels of the amino thiols investigated. GSH content and concentrations of cysteine and homocysteine were similar in male and female subjects. In our study markedly elevated concentrations of homocysteine in the colonic mucosa were observed in patients suffering from ulcerative colitis and Crohns disease. This finding has been reported already in the literature for plasma homocysteine levels. Increased homocysteine levels in the colonic mucosa and plasma of patients with inflammatory bowel disease may play a role in the pathogenesis of Crohns disease and ulcerative colitis.

Hyperhomocysteinaemia: a risk factor for preeclampsia?

Preeclampsia represents one of the most frequent complications of pregnancy, however, little is known about its aetiology. Damage of the endothelial layer lining the blood vessel wall is thought to play an important role in the pathophysiology of preeclampsia, accordingly, mild hyperhomocysteinaemia has been reported to be more prevalent among preeclamptic women. Therefore, we investigated the role of hyperhomocysteinaemia in preeclampsia by measuring plasma levels of homocysteine and studying the prevalence of the 677(C-->T) polymorphism in the 5,10-methylenetetrahydrofolate reductase (MTHFR) gene, which may lead to reduced MTHFR enzyme activity and subsequently to higher plasma homocysteine levels. Plasma samples of 10 healthy non-pregnant women, 10 normotensive pregnant women, and 20 women with preeclampsia were analysed for total homocysteine levels by high performance liquid chromatography. Furthermore, 167 Dutch non-pregnant women previously hospitalised for preeclampsia and 403 population-based controls were analysed for the 677(C-->T) polymorphism by polymerase chain reaction followed by restriction fragment length polymorphism analysis (PCR/RFLP). In normotensive pregnancy homocysteine levels were lower compared with levels in healthy non-pregnant controls (8.4 versus 13.7micromol/l, P<0.001). Women with preeclampsia showed higher concentrations than women during normotensive pregnancy (13.3 versus 8.4micromol/l, P<0.02). However, levels of homocysteine in preeclampsia were comparable to those found in healthy non-pregnant women. PCR/RFLP showed no significant difference in the incidence of the 677(C-->T) polymorphism in the MTHFR gene between preeclamptic women with or without HELLP syndrome and controls (13 and 9% homozygous for the less common T-allele, respectively; OR 1.5, 95% CI 0.8-2.6, P=0.17). In contrast with previous reports, we cannot confirm that mild hyperhomocysteinaemia is a risk factor for preeclampsia. Pregnancy induced hyperhomocysteinaemia found in preeclampsia might better be explained by fluctuations in plasma volume than by the presence of the 677(C-->T) polymorphism in the MTHFR gene.

Oxidized and free whole blood thiols in preeclampsia.

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.

Glutathione and glutathione S-transferases A1-1 and P1-1 in seminal plasma may play a role in protecting against oxidative damage to spermatozoa

OBJECTIVE To study the levels of glutathione, glutathione S-transferase A1-1, and glutathione S-transferase P1-1 in seminal fluid of fertile and subfertile men. DESIGN Retrospective case-control study. SETTING Departments of gastroenterology, obstetrics and gynecology, and epidemiology and biostatistics in a university medical center. PATIENT(S) Twenty-five subfertile men visiting the fertility clinic and 25 fertile men from midwife practices were recruited. INTERVENTION(S) Collection of semen of subfertile and fertile men. MAIN OUTCOME MEASURE(S) Plasma levels of glutathione and glutathione S-transferases A1-1 and P1-1 in relation to seminal characteristics. RESULT(S) Glutathione, glutathione S-transferase A1-1, as well as glutathione S-transferase P1-1 were found in considerable amounts in seminal fluid of subfertile and fertile men. No differences between groups were found for glutathione S-transferases A1-1 and P1-1. Also, no associations with sperm count, motility, or morphology could be detected. Fertile men had significantly higher glutathione levels as compared with the case of subfertile men. Associations of glutathione with sperm motility quality (r(s) = 0.321) and abnormal sperm morphology (r(s) = -0.496) were found. CONCLUSION(S) The presence of glutathione S-transferases A1-1 and P1-1 in seminal fluid suggests a role in the protection against (oxidative) damage of spermatozoa, whereas glutathione may play a role in male fertility.

Low plasma levels of oxidized low density lipoprotein in preeclampsia.

Background.  Markers of lipid peroxidation are commonly used to assess oxidative stress in preeclampsia. The aim of this study was to assess the concentration of oxidized low density lipoprotein (oxLDL), a novel marker for lipid peroxidation, and that of the thiobarbituric acid reactive substances (TBARS) in the pathogenesis of severe preeclampsia and to investigate the influence of gestational age on these parameters.

Paternal contribution to the risk for pre-eclampsia

Pre-eclampsia is a major cause of fetal and maternal morbidity and mortality with a still obscure aetiology. A major feature in pre-eclampsia is placental maladaptation, probably because of inadequate invasion of fetal trophoblast cells in the myometrium and spiral arteries that might be related to local oxidative stress. Reactive oxygen species (ROS), lipid peroxides, and other toxic compounds are metabolised by biotransformation enzymes in scavenging and detoxifying processes. Increasing evidence suggests an important function of antioxidants and detoxification enzymes in pre-eclampsia. We recently proposed that the 105Ile→Val polymorphisms in the glutathione S-transferase P1 gene ( GSTP1 ), associated with lower enzyme detoxification capacity, enhanced maternal susceptibility to pre-eclampsia.1 Glutathione S-transferase P1-1 (GSTP1-1) is an important detoxification enzyme and is the main GST isoform in placenta and decidua.2 The GSTP1-1 level was found to be lower in placental and decidual tissue of pre-eclamptic women as compared with corresponding tissues of normal pregnant women.2 Since placenta is of fetal origin and therefore characterised by both maternal and paternal contribution, the risk for pre-eclampsia might be modified by maternal as well as paternal genetic variations in detoxification activities. We therefore studied GSTP1 polymorphisms in a cohort …

Haptoglobin and its association with the HELLP syndrome

Haptoglobin (Hp) is an acute phase α2-sialoglycoprotein, which is characterised by molecular heterogeneity.1 Owing to a genetic polymorphism, different Hp phenotypes exist of which Hp1-1, Hp1-2, and Hp2-2 are the three major isoforms in humans. Hp consists of two different polypeptide chains, the heavy β chain, which is identical in all haptoglobins, and the light α chain, consisting of two α1 chains and a α2 chain, modifications of which result in the different Hp phenotypes.2 The most important function of Hp is capturing haemoglobin, thereby preventing iron loss and subsequent oxidative damage generated by free iron in the vascular system of the kidneys. Binding of haemoglobin to Hp is beneficial for the human body in several other ways. Hp is protective against cell damage by scavenging free radicals, such as the hydroxyl radical, the formation of which is promoted by the presence of free haemoglobin. Furthermore, the Hp-haemoglobin complex inhibits the vasodilatory effect of nitric oxide and provides a non-specific defence against bacterial invasion, since free haem iron is necessary for bacterial growth. Furthermore, Hp itself was identified as a serum angiogenic factor and plays a role in proliferation and differentiation of vascular endothelium. Hp2-2 has stronger angiogenic functionality than Hp1-1, whereas Hp1-1 has the highest affinity for haemoglobin and is therefore associated with the antioxidant capacity of Hp.1 Pre-eclampsia, which is characterised by pregnancy induced hypertension and concurrent proteinuria, can be complicated by the haemolysis, elevated liver enzymes, and low platelets (HELLP) syndrome, which may also occur alone.3 The pathogenesis of pre-eclampsia and HELLP is largely unknown, although it is postulated that maladaptation of trophoblast invasion may result in poor placental perfusion and local oxidative stress,4 which could subsequently affect maternal circulation. …