Paraoxonase and Arylesterase Activities in Patients with Preeclampsia

Abstract

Objective:

Paraoxonase is a high density lipoprotein-associated anti-oxidant enzyme. The aim of this study was to investigate the serum paraoxonase and arylesterase activities and associations between these enzymes and lipid hydroperoxide (LOOH) in preeclamptic patients.

Materials and Methods:

The study included 47 preeclamptic (25 severe and 22 mild preeclampsia cases) and 20 normotensive healthy pregnant women. Serum paraoxonase and arylesterase activities and LOOH levels were measured.

Results:

While serum paraoxonase and arylesterase activities were significantly lower in both severe and mild preeclamptic patients (P<0.05 for both) than those in normal pregnant subjects, LOOH levels were significantly higher in severe (P<0.01) and mild preeclampsia cases (P<0.05) than in normotensive pregnant subjects. When all subjects were evaluated, there was a negative correlation between paraoxonase and LOOH (r=−0.24, P<0.05) and between arylesterase and LOOH (r=−0.31, P<0.01).

Conclusion:

Our results show that serum paraoxonase and arylesterase activities decrease in preeclampsia, and this situation may be associated with increased LOOH in these patients.

Introduction

Preeclampsia is a common disease of human pregnancy and a leading cause of both maternal morbidity and neonatal mortality. Proteinuria and hypertension with or without edema are associated with preeclampsia. Numerous theories on the pathophysiological mechanisms have been investigated; nevertheless, the etiology and pathogenesis of preeclampsia is not yet fully understood [1].

Endothelial dysfunction and inflammation play a role in preeclampsia pathogenesis. Additionally, a reduced invasion of the trophoblast into the uterus and placental hypoxia lead to placental oxidative stress. If the reactive oxygen species (ROS) levels exceed the antioxidant capacity or if the antioxidant levels fall in preeclampsia, then generalized oxidative stress will occur. ROS cause oxidation of unsaturated fatty acids and thus increase the levels of lipid peroxidation products. Lipid peroxidation may cause peroxidative damage to the vascular endothelium, and this situation may be associated with clinical symptoms in preeclampsia [2].

Paraoxonase is associated in human serum with high density lipoprotein (HDL), and its arylesterase/paraoxonase activities are present. Paraoxonase protects low density lipoprotein and HDL from oxidation. This protection is probably related to the ability of paraoxonase to hydrolyze some oxidized phospholipids [3] and/or cholesteryl linoleate hydroperoxides [4]. Serum paraoxonase activity was found to be reduced in a number of pathological conditions, including myocardial infarction, diabetes and hypercholesterolemia.

Previous studies showed decreased serum paraoxonase activity in cases of preeclampsia [5–7]. In this study, we aimed to investigate serum paraoxonase, arylesterase activities in severe and mild preeclamptic patients and to compare these with the activities in healthy pregnant patients. Additionally, we evaluated the associations between these enzymes and lipid hydroperoxide (LOOH).

Materials and Methods

A total of 47 preeclamptic patients (25 severe preeclampsia and 22 mild preeclampsia cases) and 20 healthy pregnant subjects were studied. Following the American College of Obstet-ricians and Gynecologists recommendations, preeclampsia was defined as a persistent elevated diastolic blood pressure (≥ 90mm Hg), proteinuria greater than 300 mg in a 24-h urine sample and the presence of edema [8]. Mild preeclampsia was diagnosed if a blood pressure of 140/90 mmHg was observed on at least two occasions 6 h apart with proteinuria (>2+) and edema. Severe preeclampsia was diagnosed when the following criteria were present: (1) a systolic blood pressure of 160 mmHg or greater or a diastolic blood pressure of 110 mmHg or greater on two occasions at least 6 h apart, with the patients resting in bed and (2) a proteinuria of 5 g or greater in a 24-h urine collection or of 3+ or greater on a dipstick in at least 2 random clean-catch samples at least 4 h apart. Healthy pregnant subjects were selected randomly and matched to the preeclamptic patients on the basis of maternal age and gestational age. None of the women were current smokers or had multiple pregnancies, pre-gestational hypertension or diabetes mellitus.

Written informed consent was obtained from all enrollees, according to the criteria of the Ethical Committee of Medical Faculty, Ataturk University. Blood samples were withdrawn from the preeclamptic group after the diagnosis and from normal pregnant women at their routine prenatal visits. The blood was collected into dry tubes, and the serum was separated. A serum aliquot was stored at −70°C.

Paraoxonase activity was measured using diethyl-p-nitrophenylphosphate as a substrate as previously described [9]. Briefly, 40 µL of serum (diluted 1:2 with water) was incubated with reaction mixture at 37°C for 5 min, and then the enzymatic reaction was started by the addition of a substrate solution. The basal assay mixture contained 1.2 mM paraoxon, 1 mM CaCl₂ and 50 mM Tris-HCl (pH 6.8). The increase in A₄₀₅ was recorded. The amount of p-nitrophenol formed per minute was calculated from the molar absorption coefficient at pH 6.8 (7480 M⁻¹.cm⁻¹). A quantity of 1 U/mL of paraoxonase was defined as 1 nmol of p-nitrophenol formed per minute. Arylesterase activity was determined as the rate of phenyl acetate hydrolysis catalyzed by serum in a cuvette with 4.4 mM phenyl acetate in 50 mM Tris/acetate (pH 7.8) containing 20 mM CaCl₂ at 37°C. The hydrolysis of phenyl acetate was monitored at 270 nm with a molar absorption coefficient at 270 nm of 1310 M⁻¹.cm⁻¹.

Serum LOOH levels were measured by the method as previously described [10]. The method is based on the principle of the oxidation of Fe²⁺ to Fe³⁺ by lipids.

Statistical Analysis

Results were given as mean±SD. According to the Kolmogorov Smirnov test, because the distribution of all analytes was normal, one-way ANOVA followed by the LSD test was used to assess the differences between groups. Correlation analyses were performed using Pearson’s correlation test. A p value <0.05 was accepted as significant.

Results

Clinical and demographic data from the preeclamptic and control groups are summarized in Table 1. The mean maternal age was not different among the groups. As expected from the definition criteria, systolic and diastolic blood pressures were significantly higher in the severe and mild preeclampsia groups than in the normotensive group.

Table 1.

Clinical characteristics of the study groups

	Severe preeclampsia (n=25)	Mild preeclampsia (n=22)	Normal (n=20)
Maternal age (years)	21.96±2.41	22.31±2.62	22.65±2.73
Gestational age (weeks)	34.48±2.40	34.86±1.49	35.40±2.01
Systolic blood pressure (mmHg)	162.80±19.041	147.27±7.031	111.00±12.52
Diastolic blood pressure (mmHg)	101.20±7.261	94.09±5.901	65.25±10.70

Results are given as mean±SD,

¹P<0.0001 compared to normal pregnancy group

In the present work, the serum paraoxonase and arylesterase activities were significantly lower in both preeclampsia groups (P<0.05 for all, Table 2). LOOH levels were significantly higher in severe (P<0.01) and mild preeclampsia cases (P<0.05) than in normotensive pregnant subjects. There were no differences with regard to these enzyme activities between the severe and mild preeclampsia groups. When evaluating all patients and controls, a negative association between serum paraoxonase and LOOH was found (r =−0.24, P<0.05). There was also a significant inverse correlation between serum arylesterase and LOOH (r=−0.31, P<0.01).

Table 2.

Paraoxonase and arylesterase activities and LOOH levels in the preeclamptic and normal pregnant women

	Severe preeclampsia (n=25)	Mild preeclampsia (n=22)	Normal (n=20)
Paraoxonase (U/L)	276.26±60.971	280.57±68.331	325.54±75.56
Arylesterase (U/L)	147.71±20.901	148.14±20.511	162.06±23.75
LOOH (μmol/L)	6.51±1.672	6.22±2.161	4.92±1.63

Results are given as mean±SD,

¹P<0.05 compared to normal pregnancy group,

²P<0.01 compared to normal pregnancy group

Discussion

Although the pathogenesis of preeclampsia is not understood completely, in recent studies oxidative stress was considered in the pathogenesis of preeclampsia [11, 12]. Oxidative stress results from the imbalance between increased reactive oxygen species and defects in antioxidant activities. Wiktor et al. [13] investigated oxidative DNA damage in the placentas of preeclamptic pregnancies. They found that 8-hydroxy-2-deoxyguanosine levels were higher in preeclamptic pregnancies than in the healthy control groups. Also, we have previously found that levels of the placental lipid peroxidation product, malondialdehyde (MDA), were significantly higher in women with severe and mild preeclampsia than in healthy pregnant women [14].

Llurba et al. [15] measured indicative markers of lipoperoxidation and protein oxidation as well as changes in antioxidant defense systems in women with preeclampsia. In their study, erythrocyte glutathione peroxidase (GPx) activity, superoxide dismutase (SOD) and total plasma lipid hydroperoxide levels were significantly enhanced in preeclamptic pregnancies than in normal pregnancies. In our study, we found there to be higher lipid hydroperoxide levels in preeclamptic patients consistent with the study by Llurba et al. [15]. Wu [16] reported increased MDA levels in preeclamptic pregnancies.

Paraoxonase is an antioxidant enzyme located on HDL that protects lipoproteins from being oxidized and is responsible for the antioxidant activity of HDL. Sarandol et al. [7] studied the levels of serum MDA and the activities of paraoxonase and arylesterase in the serum of preeclamptic pregnancies. They found that serum MDA was significantly higher in the preeclampsia group than in the normal pregnant group, and serum paraoxonase and arylesterase activities were not different between the two groups. Our findings support those of Kumru et al. [6]. They performed a study on the changes of serum paraoxonase and arylesterase activities in severe preeclamptic women. In their study, HDL-cholesterol levels and the paraoxonase and arylesterase activities were found to be lower in the severe preeclamptic women than in the controls. They detected a moderately positive correlation between the serum levels HDL-cholesterol and paraoxonase and between HDL-cholesterol and arylesterase. They concluded that an abnormal lipid profile and decreases in paraoxonase and arylesterase activities might play a role in the pathogenesis of preeclampsia.

Since paraoxonase is entirely complexed to HDL in serum, the reduction in paraoxonase activity may be due to a reduction in the HDL-Cholesterol concentration in preeclampsia. Although we did not measure HDL-Cholesterol levels, in some studies it was reported that serum HDL-Cholesterol levels were decreased in preeclampsia cases [6,17]. The other explanation is that increased oxidative stress may lead to a low activity of paraoxonase in preeclampsia. Indeed, it has been shown by Aviram et al. [18] that paraoxonase is inactivated in the presence of lipid peroxides. Also there was a negative correlation between paraoxonase and LOOH in our study. Therefore, decreased paraoxonase activity in preeclampsia can be partly explained by the lower increased oxidative stress. In our study, there were no significant differences in serum paraxonase and arylesterase activities or LOOH levels between mild and severe preeclampsia cases.

Contrast to the reports of Sarandol et al. [7], in the present study it was found that serum paraoxonase and arylesterase activities were significantly lower in preeclamptic patients than in normal pregnant patients. Bayhan et al. [17] found that the serum levels of lipid peroxide were significantly increased in women with severe and mild preeclampsia. The study of Ilhan et al. [19] revealed that the MDA level was significantly increased and the SOD activity was significantly decreased in preeclamptic patients.

In conclusion, serum paraoxonase and arylesterase activities decrease in preeclampsia, and this situation may be one result of increased LOOH in such patients. Antioxidant support might be helpful to eliminate oxidative stress in preeclampsia. Further investigations are necessary to define the association between antioxidant activities and preeclampsia.