Abstract
Background
Pre-eclampsia is a multisystem pregnancy-related disorder with multiple theories regarding its aetiology resulting in lack of reliable screening tests and well-established measures for primary prevention. However, oxidative stress is increasingly being implicated in the pathogenesi of pre-eclampsia although conflicting findings have been reported.
Aim
To determine and compare the levels of oxidative stress in early and late onset pre-eclampsia by measuring urinary excretion of isoprostane and total antioxidant power (TAP) in a cohort of pre-eclamptic women at Korle Bu Teaching Hospital
Methodology
This was a cross-sectional study conducted at Korle-Bu Teaching Hospital, Accra, Ghana involving pre-eclamptic women between the ages 18 and 45 years who gave written informed consent. Urinary isoprostane levels were determined using an enzyme-linked immunosorbent assay (ELISA) kit whereas the Total Anti-oxidant Power in urine samples was determined using Total Antioxidant Power Colorimetric Microplate Assay kit. The data obtained were analyzed using MEGASTAT statistical software package.
Results
We included 102 pre-eclamptic women comprising 68 (66.7%) and 34 (33.3%) with early-onset and late-onset pre-eclampsia respectively. There were no statistically significant differences between the mean maternal age, haematological indices, serum ALT, AST, ALT, albumin, urea, creatinine uric acid and total protein at the time of diagnosis. The mean gestational age at diagnosis of early and late onset pre-eclampsia were 31.65 ± 0.41 and 38.03 ± 0.21 respectively (p ˂ 0.001). Also, there were statistically significant differences between the diastolic blood pressure (BP), systolic BP and mean arterial pressure (MAP) at diagnosis of pre-eclampsia in the two categories. The mean urinary Isoprostane excretion was significantly higher in the early onset pre-eclamptic group (3.04 ± 0.34 ng/mg Cr) compared to that of the late onset pre-eclamptic group (2.36 ± 0.45 ng/mg Cr), (p=0.019). Urinary total antioxidant power (TAP) in early onset PE (1.64 ± 0.06) was lower but not significantly different from that of late onset PE (1.74 ± 0.09) with p = 0.369.
Conclusion
Significantly increased urinary isoprostane excretion was detected in early onset pre-eclampsia compared to late onset pre-eclampsia, suggestive of increased oxidative stress in the former. However, there was no significant difference in total anti-oxidant power between the two categories of pre-eclampsia women although there was a tendency of reduced total antioxidant power in the women with early onset pre-ecalmpsia.
Keywords: Pre-eclampsia, oxidative stress, isoprostane, total anti-oxidant power
Introduction
Pre-eclampsia is a complex multisystem disorder and a leading cause of maternal and fetal morbidity and mortality worldwide and more especially in low resource settings1. In Ghana hypertensive disorders in pregnancy including pre-eclampsia are the leading cause of institutional maternal mortality due to severe complications such as acute renal failure, intra-cerebral haemorrhage and pulmonary edema2,3. At Korle Bu Teaching Hospital in Accra, Ghana where the current study was conducted, significant adverse maternal outcomes of pre-eclampsia/eclampsia have recently been reported and most of these complications are preventable3. Characterized as early- and late onset disease5, oxidative stress is considered pivotal in the pathophysiology of this syndrome6. Early onset pre-eclampsia is considered as a severe form of the disease with different underlying pathophysiology and worse perinatal and maternal outcomes compared with late onset type7,8. Vascular defect is thought to represent the main aetiology of oxidative stress because early onset pre-eclampsia is associated with incomplete transformation of the spiral arteries of the myometrial segments of the arteries mainly9.
Until recently the primary problem in pre-eclampsia was thought to be mainly due to poorly perfused placenta. The inability of the spiral arteries to remodel in pre-eclampsia results in their ability to retain their vasoreactivity to vaso-active substances in the plasma. Inadequate conversion of the highly contractile myometrial segments of the spiral arteries results in intermittent perfusion of the placenta, and a low grade ischaemia-reperfusion type of injury leading to generation of reactive oxygen species (ROS) within the trophoblast and endothelial cells10.
It is currently suggested that there is intermittent perfusion of the intervillous space instead of the original notion of chronically hypoxic placental blood supply11. This leads to a fluctuating level of oxygenation to the placenta often described as hypoxia–reoxygenation or ischaemia-reperfusion injury. Increased oxidative stress has been demonstrated in placentas exposed to hypoxia–reoxygenation from in vitro studies in contrast to placentas exposed to chronic hypoxia which did not show an increase in oxidative stress12. The hypoxia–reoxygenation theory has also been demonstrated to be associated with an increase in placental apoptosis resulting in increased oxidative stress levels11. Late onset pre-eclampsia is thought to have a minimal influence on placental villous and vascular morphology compared with early-onset pre-eclampsia which is associated with placental dysfunction marked by a reduction in placental weights, volume or the intervillous space, terminal villous volumes and surface area13.
Oxidative stress resulting from placental ischemia reperfusion injury is increasingly implicated in the pathogenesis of pre-eclampsia via lipid peroxidation and endothelial cell dysfunction. However, causal relationship between increased lipid peroxidation and the pathogenesis of pre-eclampsia is not universally supported as a result of conflicting findings in previous studies.
In Ghana, data on oxidative stress in early and late onset pre-eclampsia is limited although such information may be very useful in clinical management and scientific discourse. In this study, we set out to determine and compare the level of oxidative stress in early and late onset pre-eclampsia by measuring urinary excretion of isoprostane in a cohort of pre-eclamptic women at Korle Bu Teaching Hospital.
Materials & Methods
This was a cross-sectional study conducted between December 2006 and May 2007 at maternity unit of Korle-Bu Teaching Hospital in Accra Ghana. Korle-Bu Teaching Hospital is the largest teaching hospital in Ghana with about 11,000 deliveries annually and pre-eclampsia is the leading cause of maternal death in the facility2. The hospital provides 24-hour maternity services and the National health insurance free delivery scheme covers all the pregnant women delivering at the facility.
In this study, pre-eclamptic women between the ages 18 and 45 years, and who gave written informed consent were included the study after the diagnosis of pre-eclampsia had been made. This cohort of women were selected based on simple random sampling following the initial diagnosis and their blood samples were taken at the time of diagnosis, prior to commencement of treatment. Specific exclusion criteria consisted of women with chronic hypertension, diabetes mellitus, sickle cell disease, seizure disorders, alcohol abuse or any other chronic medical disorders. The Ethical and Protocol Review committee of the University of Ghana Medical School approved the study and a written informed consent was obtained from all study participants. The potential risks associated with the participation in the study were thoroughly explained to the participants. The study participants were informed that their participation in the study was voluntary and that failure to be included in the study would not affect the normal management of their medical conditions in any way.
Pre-eclampsia is defined as new onset of hypertension and proteinuria after the gestational age of 20 weeks14,15. Hypertension was defined as systolic blood pressure of 140mm Hg or more and/or diastolic blood pressure of 90mm Hg or more. Proteinuria of +1 or more, determined by a urine dipstick, was considered significant. Also, early- and late onset pre-eclampsia were defined as the development of pre-eclamptic syndrome before and at/or after 34 weeks of gestation5,7, respectively.
DETERMINATION OF BIOCHEMICAL AND HEMATOLOGICAL PARAMETERS
The pre-eclamptic women included in the study had their blood samples obtained from the veins at the antecubital fossae following an application of a tourniquet. Serum urea, uric acid levels and liver function tests were determined using a Microlab 300 Chemistry Semi-automated Analyzer. A Sysmex Hematology Analyzer was employed to assess the full blood count parameters including white cell differentials. Urinary albumin levels were determined with the use of Albumin Blue Fluorescent Assay kit (Active Motif, USA). Serum creatinine levels were determined by the alkaline picrate method. Creatinine level in urine was measured with a commercial kit (No. CR01; Oxford Biomedical Research, Oxford, MI, USA) strictly based on the manufacturer’s instructions.
ASSESSMENT OF URINARY ISOPROSTANE AND TOTAL ANTIOXIDANT POWER
Urinary isoprostane levels were determined using a 15- Isoprostane- F2t enzyme-linked immunosorbent assay (ELISA) kit (Oxford Biomedical Research, Oxford, MI, USA) based strictly on the instructions of the manufacturer. The total antioxidant power in urine samples were determined using Total Antioxidant Power Colorimetric Microplate Assay kit (No. TA 01; Oxford Biomedical Research) based strictly on the manufacturer’s advice.
STATISTICAL ANALYSIS
The analysis of data obtained was performed with MEGASTAT statistical software package.
Descriptive statistics were performed and the results were presented in means (±SD) and bar
graphs where appropriate. The independent Student t-test was used to compare the
continuous variables and statistically significant difference was considered for a p-value of less
than 0.05.
Results
During the study duration, we enrolled 102 pre-eclamptic women comprising 68 (66.7%) and 34 (33.3%) early-onset and late-onset pre-eclampsia respectively. There were no statistically significant differences between early and late onset pre-eclampsia regarding the mean maternal age, haematological indices, serum ALT, AST, ALT, albumin, urea, creatinine uric acid and total protein at the time of diagnosis (Table 1). The mean gestational age (in weeks) at diagnosis of early and late onset pre-eclampsia were 31.65 ± 0.41 and 38.03 ± 0.21 respectively (p ˂ 0.001). Also, there were statistically significant differences between the two subtypes of pre-eclampsia with respect to the diastolic BP, systolic BP and MAP at diagnosis of pre-eclampsia in the categories (Table 1). The mean urinary Isoprostane excretion was significantly higher in the early onset pre-eclamptic group (3.04 ± 0.34 ng/mg Cr) compared to that of the late onset pre-eclamptic group (2.36 ± 0.45) with p=0.019 (figure 1). Urinary total antioxidant power (TAP) in early onset pre-eclampsia (1.64±0.06) was lower but not significantly different from that of late onset pre-eclampsia (1.74±0.09) with p=0.369 (figure 2).
Table 1. Table 1: Demographic and laboratory data of pregnant women with early and late onset pre-eclampsia .
| Parameter | Early onset pre-eclampsia (n=68) | Late onset pre-eclampsia(n=34) | P value |
| Maternal age (Years) | 30.15 ± 0.71 | 30.31 ± 0.86 | 0. 886 |
| Gestational age (weeks) | 31.65 ± 0.41 | 38.03 ± 0.21 | ˂ 0.001 |
| Systolic BP (mmHg) | 169.78 ± 2.82 | 157.14 ± 2.34 | 0.004 |
| Diastolic BP (mmHg) | 111.54 ± 1.67 | 104.43 ± 1.57 | 0.007 |
| MAP (mmHg) | 130.56 ± 1.94 | 122.10 ± 1.55 | 0.024 |
| Haemoglobin (g/dl) | 11.36 ± 0.21 | 11.04 ± 0.25 | 0.298 |
| Haematocrit (%) | 35.34 ± 0.56 | 34.84 ± 0.73 | 0.503 |
| Total WBC (× 10-9) | 8.30 ± 0.36 | 8.45 ± 0.60 | 0.885 |
| Platelets (× 10-9) | 193.54 ± 9.66 | 188.29 ± 10.95 | 0.230 |
| ALT (µ /l) | 24.18 ± 1.13 | 26.35 ± 1.56 | 0.674 |
| AST (µ/L) | 31.00 ± 1.38 | 29.60 ± 1.23 | 0.148 |
| ALP (µ /l) | 321.98 ± 20.00 | 298.53 ± 17.69 | 0.237 |
| Gamma GT (µ /L) | 34.94 ± 1.34 | 31.44 ± 1.20 | 0.040 |
| Serum albumin (g/L) | 38.07 ± 1.35 | 35.37 ± 1.57 | 0.222 |
| Urea (mg/dL) | 22.14 ± 1.12 | 24.81 ± 1.74 | 0.183 |
| Serum Cr (mg/dl) | 1.12 ± 0.02 | 1.07 ± 0.03 | 0.191 |
| Serum uric acid (mg/dL) | 6.07 ± 0.27 | 5.69 ± 0.33 | 0.393 |
| Serum total protein (g/l) | 76.18 ± 1.12 | 76.57 ± 1.97 | 0.852 |
| Microalbuminuria (g/l) | 161.44 ± 99.96 | 142.67 ± 95.68 | 0.1809 |
Figure 1. Urinary 15-F2t-Isoprostane excretion in Early- and Late-onset pre-eclampsia .
Figure 2. Total antioxidant power (TAP) in early-onset (LO) and late-onset (LO) pre-eclampsia .
The results in the table are presented as mean ± standard deviation (SD), ALT= Alanine transaminase, AST= aspartate aminotransferase, ALP= Alkaline phosphatase, WBC= White blood cells, GGT= Gamma-glutamyl transferase, Cr= Creatinine, BP= Blood pressure
Discussion
Oxidative stress is considered a central feature in the pathophysiology of pre-eclampsia although conflicting results have been determined in previous studies. In this study, we sought to determine the level of oxidative stress in early and late onset pre-eclampsia to buttress the purported role of oxidative stress in pre-eclampsia. Our study showed a statistically significant increase in urinary isoprostane excretion in the women with early-onset pre-eclampsia compared with the late-onset disease (figure 1). Generally, early-onset pre-eclampsia is considered a more severe form of the disease associated with different etiopathogensis and subsequently, worse multi-organ derangement5. Oxidative stress is thought to be indispensable in the pathogenesis of early onset disease via endothelial cell dysfunction mediated by the release of toxic substances from the injured stressed placenta. The finding of significant increase in excretion of urinary isoprostane in early-onset compared to late-onset PE suggests worse oxidative stress levels in the former and this may have significant effects on the both the maternal and perinatal outcomes.
However, Wilkrom et al determined no significant differences between the median urinary concentrations of isoprostane 8-iso-PGF2α in early-onset and late-onset pre-eclampsia. However, they found a higher median concentration of isoprostane 8-iso-PGF2α in placental tissue among women with early-onset pre-eclampsia compared to that of late onset disease and in their normotensive pregnant controls16. In the current study, however, placental tissue concentration of isoprostane was not determined and normotensive pregnant controls were not included.
Interestingly, there was no statistically significant difference in Total Oxidative Power between women with early and late-onset pre-eclampsia although the level of TAP was lower in the former as shown in figure 2. This finding was unexpected because with the significantly increased urinary isoprostane excretion in early-onset pre-eclampsia the significant reduction in the TAP was anticipated to complete the picture to provide better understanding of the pathophysiology of the disease. This might be attributed to smaller numbers recruited in this study and, perhaps, the use of a larger sample size would have resulted in statistical difference in the results, given the fact that a decreased level of TAP is seen in early-onset subjects.
In the current study, there was a significantly higher diastolic, systolic and mean arterial pressures in the early-onset pre-eclampsia compared to late-onset disease (Table 1)and this buttresses the fact that the former is a more severe disease. It is therefore not surprising that urinary isoprostane excretion was higher in the early onset disease mirroring high plasma and placental concentrations of this potent biomarker of oxidative stress. Although we did not determine the plasma and placental levels of isoprostanes the significantly higher urinary level determined in our study is suggestive of increased plasma concentration with spillage into the urine by exceeding its renal threshold. Therefore the higher blood pressures in early onset disease might be partly attributed to the high levels of isoprostanes, biologically active biomarkers of oxidative stress. This assertion is supported by the fact that, isoprostanes generally have a potent vasoconstrictive effect on the placental vasculature in addition to maternal systemic circulation by stimulating increased endothelial production of endothelin-16,17,18.
Recently, we demonstrated an increased urinary excretion of isoprostanes and a decreased total antioxidant power among pre-eclamptics compared to normotensive pregnant women suggestive of an imbalance between the maternal pro-oxidants and antioxidants in favour of excess oxidation19. Other studies have also determined an increase in oxidative stress in pre-eclampsia by measurement of serum isoprostane levels indicative of significant oxidative stress20,21. In addition, Baden et al found a significantly lower urinary 8-iso-prostane excretion in pre-eclamptics compared with women with normotensive pregnancies and they attributed it to impaired renal clearance of 8-iso-prostane in pre-eclampsia21. The significantly elevated urinary isoprostane excretion determined in the current study is not consistent with the low levels reported by Barden et al who attributed it to renal impairment in the midst of higher concentrations of free plasma isoprostanes in pre-eclampsia. In our study, we did not find any significant differences in parameters of renal function such as serum urea, creatinine and uric acid between the two subtypes of pre-eclampsia. This suggests that the significantly higher urinary excretion of isoprostane determined in early onset disease might be related to high isoprostanemia with spillage into the urine.
Generally, Isoprostanes are generated by random peroxidation of the phospholipid tissue by oxygen free radicals and increased levels may be found in conditions associated with high levels of oxidative stress such as pre-eclampsia. Significant elevation of serum 8-isoprostane is indicative of deficient antioxidant mechanisms in conditions associated with increased oxidative stress such as pre-eclampsia20. In this context, timely administration of antioxidants to susceptible women in early pregnancy may effectively prevent and or delay the onset of pre-eclampsia and avert the major complications attributable to pre-eclampsia syndrome. However conflicting results have been determined in large randomized trials regarding the effectiveness of these antioxidant vitamins and this buttresses the fact that the etiology of pre-eclampsia remains enigmatic and its pathophysiology not well understood22.
Some studies have determined the oxidative stress at the placental levels by measuring the serum level of isoprostane in the placenta which provides a direct reflection of placental pathology in pre-eclampsia16,23. However, in the current study the oxidative stress at the placental level was not determined and urinary isoprostane excretion is considered a surrogate for the serum levels which is also indicative of the placental oxidative stress level.
The limitation of our study include the fact that dietary considerations were not taken into account in determining the level of total antioxidant as dietary preferences could have influenced the pro-oxidant and anti-oxidant balance24. Also, fetal outcomes of early and late onset pre-eclampsia were not included in the study and this would have thrown more light into the effect of increased oxidative stress on fetal wellbeing. The strength of the current study, however, hinges on the fact that this is one of the first studies to determine the oxidative stress in early and late onset pre-eclampsia especially in Sub-Saharan Africa (SSA). This should serve as a springboard for further investigations into the subject, especially in SSA.
Conclusions
In conclusion, the current study has demonstrated significantly increased urinary isoprostane excretion in early onset pre-eclampsia compared to late onset disease suggestive of increased oxidative stress in the former. However, there was no significant difference in Total Anti-oxidant Power between the two categories of pre-eclampsia syndrome. There is the need for more in-depth studies in relating the relevance of oxidative stress to the pathophysiology of early and late onset pre-eclampsia especially in SSA where the disease burden is huge with significant adverse perinatal and maternal morbidity. Selective administration of essential vitamins with anti-oxidant properties to prospective mothers with risk factors for the development of early onset or severe pre-eclampsia in early pregnancy might prevent and or delay the onset of the disease and its complications.
Footnotes
Competing Interests: The authors have declared that no competing interests exist.
Grant support: None
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