Oral N-acetylcysteine administration does not stabilize the process of severe preeclampsia

AbstractAim: To stabilise the disease process in women with early onset severe preeclampsia and/or hellp syndrome by enhancing maternal antioxidants effects of glutathione.

Material and methods: In a randomised, double-blind, placebo-controlled trial women with severe preeclampsia and/or hellp syndrome received oral N-acetylcysteine administration. Primary outcome measures were: disease stabilization expressed as treatment-to-delivery interval and biochemical assessment of glutathione and parameters of oxidative stress. Secondary outcome measures were maternal complications, rate of caesarean section, stay at intensive care unit, postpartum hospital stay and neonatal morbidity and mortality. Analyses were done by intention to treat using Wilcoxons two-sample test and regression analysis.

Results: Median treatment-to-delivery interval was not significantly different between the N-acetylcysteine and placebo group. The whole blood and plasma levels of glutathione and other thiols were not affected by N-acetylcysteine administration, except for plasma homocysteine concentrations, which were lower in the N-acetylcysteine group. There were no differences found in maternal nor neonatal secondary outcome measures between both groups.

Conclusion: Oral N-acetylcysteine administration does not stabilize the disease process of early onset severe preeclampsia and/or help syndrome.

INTRODUCTIONSevere preeclampsia and hellp (haemolysis, elevated liver enzymes, low platelets) syndrome belong to the most serious complications of pregnancy and contribute substantially to maternal and perinatal morbidity and mortality. In the management of preeclamptic patients there are two options being either temporising management or immediate delivery. In the past, randomised controlled trials of expectant management demonstrated improved perinatal outcome without significantly compromising the maternal condition.Preeclampsia is associated with an imbalance of increased oxidative stress vs. a deficiency of antioxidant protection. The deficiency in antioxidant defence in women with preeclampsia is expressed by decreased levels of several antioxidants, such as superoxide dismutase, glutathione peroxidase, erythrocyte glutathione, ceruloplasmin, -tocopherol, and ascorbic acid.Glutathione is one of the antioxidants probably involved in the pathophysiology of preeclampsia and hellp syndrome. Human cells are protected against reactive oxygen species and other noxious compounds by a combined action of glutathione and the enzymes glutathione S-transferase and glutathione peroxidase. Several other diseases such as acetaminophen intoxication, sepsis, adult respiratory distress syndrome (ards), and acquired immune deficiency syndrome (aids) are related to a deficiency of the intracellular antioxidant glutathione. In these diseases N-acetylcysteine has been used as a precursor in the synthesis of glutathione and as a direct radical scavenger. N-acetylcysteine, which is able to pass through biological membranes, enhances thiol levels in cells exposed to oxidising agents.These observations led us to hypothesise that recovery of the lowered levels of glutathione by N-acetylcysteine might stabilise the underlying disease process of severe, early onset preeclampsia and may result in prolongation of pregnancy benefiting neonatal survival. We therefore performed a randomised, double-blind, placebo-controlled trial of oral administration with N-acetylcysteine in women with severe preeclampsia and/or hellp syndrome.

MATERIAL AND METHODSThis double-blind, placebo-controlled trial was conducted at the University Medical Center Nijmegen between January 1999 and October 2001. The study protocol was approved by the Medical Ethical Review Committee of the University Medical Center Nijmegen, and informed consent was obtained from each patient.All patients with early onset severe preeclampsia and/or hellp syndrome between 25 and 33 weeks gestation and a singleton pregnancy were invited to participate. Severe preeclampsia was defined as a diastolic blood pressure of 110 mmHg or higher, measured at least two times with an interval of at least four hours, and a proteinuria of at least 0.3 g/L, according to the definitions of the International Society for the Study of Hypertension in Pregnancy (isshp). The hellp syndrome was biochemically characterised as lactate dehydrogenase concentration 600 iu/l, aminotransferases 70 iu/l and a platelet count < 100 x 109/L.Primary outcome measures were: 1. disease stabilisation expressed as treatment-to-delivery interval; 2. biochemical assessment of glutathione and other parameters of oxidative stress. Secondary outcome measures were: maternal complications, rate of caesarean section, stay at intensive care unit, postpartum hospital stay, and neonatal morbidity and mortality.

Hypothesizing that oral N-acetylcysteine administration would result in prolongation of pregnancy for seven days, it would be necessary to enrol 35 patients in each arm of the study, either N-acetylcysteine or placebo (estimated standard deviation 8 days, power 95%, significancelevel 0.05). Before start of the trial, all medication boxes were assigned with a number by a block wise randomisation protocol. In ascending order of the numbers the medication boxes were used for each following patient included in the study. Participants were randomly assigned to Nacetylcysteine or placebo administration.A safety board was installed comprising of a paediatrician, pulmonary specialist and intensive care specialist, monitoring the safety of the study medication for both mother and child. After the first trial year, maternal and perinatal morbidity and mortality rates were evaluated by this safety board. No striking aberrant clinical features were detected.Effervescent tablets containing 600 mg N-acetylcysteine as well as similar looking and tasting tablets without N-acetylcysteine (placebo medication) were provided at a dosage schedule of three tablets every eight hours, starting from time of inclusion until delivery. Placebo tabletswere flavoured in order to have the same taste as the N-acetylcysteine tablets.

CLINICAL FOLLOW-UPDepending on their condition, patients were either monitored at the intensive care unit or at the obstetrical ward. Antihypertensives were administered either orally as alpha-methyldopa or nifedipin, or intravenously as ketanserin or nifedipin, and all participants received intravenous magnesium sulphate for seizure prophylaxis. Plasma volume expansion was not used as a standard procedure. All patients received corticosteroids for foetal lung maturation. All patients underwent venapunction before start of trial medication and every morning as part of the daily routine. At delivery, arterial and venous umbilical cord blood was taken in preheparinised syringes immediately after clamping of the cord. Maternal blood was sampled again 24 hours and six weeks after delivery.In order to promote and assess therapy compliance medication use was checked daily, whereas after delivery, the tablets remaining in the medication box were counted.

BIOCHEMICAL ASSAYSBlood samples were collected into sterile evacuated blood collection tubes containing ethylenediaminetetra-acetic acid (edta) (Sherwood Medical, Ballymore, Northern Ireland) and in sterile collection tubes for serum analyses. Standard blood parameters were determined at the clinical chemistry laboratory of our hospital.Venous blood samples were processed within one hour for the determination of free (sum of reduced and oxidised non-protein bound) and oxidised thiol levels and stored at 80oC. Remaining blood was centrifuged (1500x g for 15 minutes) and plasma was stored at 30oC formeasurement of plasma thiols and ferric reducing ability of plasma (frap). Both whole blood and plasma levels of the thiols cysteine, homocysteine, cysteinylglycine, glutathione and N-acetylcysteine were analysed by high performance liquid chromatography (hplc) as described by Raijmakers et al [10,11]. frap levels were measured spectrophotometrically essentially as described for plasma by Benzie et al. and values were expressed as nmol Fe2+ equivalent/ml.

STATISTICAL ANALYSISThe analyses were performed on an intention-to-treat basis. The difference in treatment-to-delivery interval between the nac and the placebo group has been analysed using Wilcoxons two-sample test. Also a 95% confidence interval for the population difference, using Conoversmethod was computed [23].The time course of all biochemical parameters were modelled as piecewise linear with a breakpoint at one day after start of treatment, since the largest change due to N-acetylcysteine administration would be expected during the first day. Only values of patients from whom measurements at start and one day after start of treatment were obtained, were included in the computation of the first part of the time course. For the n-acetylcysteine as antioxidant 73 second part of the time course only values of patients with at least three sampling points, after start of the intervention, including day one, were included. The time courses were computed separately for each patient using regression analysis. Differences between the nac and placebo groups for each of the three coefficients involved, e.g. the intercept and two slopes, were tested using the two-sample t-test. When no significant difference existed between the two treatment groups (all three p > 0.05), one common time course was estimated for both treatment groups. Otherwise a different time course was estimated for each of the treatments. Estimation was done within a repeated measurement model, with treatment (if applicable), time (days after start of treatment) and their interaction as fixed factors; the model was completed with random intercepts and slope(s) for each patient, specifying an unstructured covariance matrix. The SAS procedure MIXED was used.In addition, for both groups all biochemical parameters measured at 24 hours and six weeks post partum were compared with pre-treatment values using the Wilcoxon-Signed-Rank test. The differences of biochemical parameters at 24 hours compared to pre-treatment values and at six weeks compared to pre-treatment values were compared between the N-acetylcysteine and placebo group using the unpaired Wilcoxon- Mann-Whitney-U test. Pre-treatment frap levels were compared with those after three study days using the Wilcoxon-Signed-Rank test. Secondary parameters of maternal and neonatal outcome were compared using Wilcoxon-Mann-Whitney-U test or Chi-square analysis, as appropriate.All analyses were performed either with sas version 6.12 statistical software (sas Institute, Inc, Cary, nc) or with spss version 9.0 statistical software and significance was assessed at a two-sided level with p < 0.05. All tests with regard to the clinical and biochemical parameters were explorative in nature.

RESULTS.From the 59 women with severe preeclampsia, who were invited to participate, 38 women were enrolled in this study. Reasons for not participating in the trial were emotional distress at admission to the hospital, fear for the use of extra medication during pregnancy or planned delivery within a couple of hours after admission.All patients were randomly assigned to N-acetylcysteine medication or a placebo, resulting in 19 patients in each group. Clinical characteristics of both study groups were comparable, as summarised in Table 1.

PRIMARY OUTCOME MEASURESMedian treatment-to-delivery interval in the N-acetylcysteine and placebo groups was 6 (range 0-21) and 5 (range 0-17) days, respectively (p = 0.85). The corresponding 95% confidence interval for the difference between the two time intervals was 3 to 3 days.N-acetylcysteine administration resulted in median plasma N-acetylcysteine concentrations of 20.0 mol/L (range 1.1-65.9) on the first treatment day, remaining stable thereafter.Pre-treatment levels of whole blood and plasma thiols were similar to those levels in earlier studies from our group in women with severe preeclampsia and/or hellp syndrome (Table 2a and 2b).During treatment the course of whole blood thiol levels were equal in both groups. Despite a peak of free and oxidised cysteine levels and of the ratio of free to oxidised glutathione during the first study day, there were no significant fluctuations. Plasma homocysteine concentrations significantly increased (p