antenatal glutathione and superoxide dismutase …
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ANTENATAL GLUTATHIONE AND SUPEROXIDE DISMUTASE
ESTIMATION FOR PREDICTION OF PRE-ECLAMPSIA
DISSERTATION SUBMITTED BY
DR. OBEHIOYE ONOMEN ENABOR
MBBS (IBADAN),
DEPARTMENT OF OBSTETRICS AND GYNAECOLOGY
UNIVERSITY COLLEGE HOSPITAL, IBADAN.
TO
THE NATIONAL POSTGRADUATE MEDICAL COLLEGE OF NIGERIA
IN PART FULFILLMENT OF THE REQUIREMENT FOR THE PART
TWO FELLOWSHIP EXAMINATION IN THE FACULTY OF
OBSTETRICS AND GYNAECOLOGY.
NOVEMBER 2013
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DECLARATION
I hereby declare that this work is the original project conducted by me and has neither been
presented to any college before now for the conferment of any fellowship award nor has been
submitted elsewhere for publication.
--------------------------------------------
DR. OBEHIOYE ONOMEN ENABOR
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TABLE OF CONTENTS
Pages
Title Page i
Declaration ii
Table of contents iii
Supervisor’s Endorsement iv
Dedication vii
Acknowledgement viii
Summary 1
Introduction 3
Justification 4
Literature Review 6
Objectives 20
Study Hypothesis 20
Material and Methods 21
Results 28
Discussion 41
Conclusion and Recommendations 45
References 46
Appendices 56
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SUPERVISOR’S ENDORSEMENT
I hereby declare that the contents of this dissertation were developed after
problem identification and discussed with me by the candidate. I also
supervised the conduct of the study, analysis and write up of the final
report.
----------------------------------------------------
Prof.A.O.AROWOJOLU
FWACS, FMCOG (Nig), FRCOG, MFFP, MNIM
Professor, Consultant Obstetrician & Gynaecologist
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SUPERVISOR’S ENDORSEMENT
I hereby declare that the contents of this dissertation were developed after
problem identification and discussed with me by the candidate. I also
supervised the conduct of the study, analysis and write up of the final
report.
----------------------------------------------------
Dr. A.O. Fawole.
FWACS, FMCOG (Nig), Cert. Spec. Rep. Med/Biol(Geneva)
Consultant Obstetrician & Gynaecologist.
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DEDICATION
This book is dedicated to the persistent search for answers to problems in women’s health.
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ACKNOWLEDGEMENT
I wish to thank God for bringing me this far and I would like to acknowledge my preceptor- Prof.
Arowojolu, my supervisor, Dr. Fawole and the consultant staff of the Department. I appreciate
the help and encouragement of fellow residents, house officers and nursing staff during the
course of this study. In addition, I want to appreciate the assistance of Prof. Arinola, Prof.
Olaleye, Prof. Olayemi, Mr. Oyewole, Mr. Ifeanyi, Mr. Paul and Mr. Bolu Popoola. Special
thanks to Drs. Anayochukwu Ugwu and Adesanya for their persistent encouragement and
contributions. Lastly, I want to thank my family for their support and their prayers. God bless
you all.
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SUMMARY
Study Title: Antenatal glutathione and superoxide dismutase estimation for prediction of pre-
eclampsia
Introduction: Pre-eclampsia is still a significant cause of foeto-maternal morbidity and mortality
in our environment. Exaggerated oxidative stress is a characteristic of this disease and studies
have shown altered levels of antioxidants in patients with pre-eclampsia. Current research is
being directed towards identification of effective screening tools and predictors. The role of
antioxidants in predicting the onset of pre-eclampsia among Nigerian women is therefore being
evaluated in this dissertation.
Objectives:
The primary objective was to determine the value of plasma glutathione and superoxide
dismutase estimation for prediction of clinical onset of pre-eclampsia in women with pregnancies
less than 20 weeks. The specific objectives were as follows:
1. To determine the mean plasma glutathione and superoxide dismutase levels among
women presenting for booking at less than 20 weeks gestation in Ibadan.
2. To evaluate the change between plasma glutathione and superoxide dismutase levels in
the first/early second trimester and at onset of pre-eclampsia or delivery.
3. To determine the plasma glutathione and superoxide dismutase levels at which pre-
eclampsia occurs.
Methods:
This was a prospective cohort study conducted in the antenatal clinic and labour ward of the
University College Hospital, Ibadan. One hundred and sixty six women with pregnancies <20
weeks of gestation were recruited at booking for antenatal care. A structured proforma was
utilised to extract socio-demographic, obstetric, medical and drug history. Their plasma
antioxidant levels- glutathione (GLU) and superoxide dismutase (SOD) were determined and this
cohort was followed longitudinally. A repeat assessment of these enzymatic antioxidants was
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done on presentation in labour, before caesarean delivery or at the point of clinical evidence of
pre-eclampsia, whichever came first. Data entry and analysis was done using the Statistical
Package for Social Sciences software (SPSS), version 16. The level of significance for
significant associations was p<0.05.
Results
The mean age of the cohort and mean gestational age at recruitment were 30.51±4.10 (SD) years
and 17.3±8.9 (SD) weeks respectively. Mean plasma GLU was 0.424±0.303µM (SD) while
mean plasma SOD at recruitment was 0.172±0.082U/ml (SD). Only 4 patients out of 166 (2.4%)
developed pre-eclampsia while 16 (9.6%) of the cohort had gestational or post partum
hypertension (HDP). The mean plasma GLU at delivery in all participants was 0.007±0.028 µM
(SD) while the mean reduction in antioxidant levels was 0.418±0.307 µM (SD). Mean plasma
SOD at delivery in all participants was 0.047±0.014U/ml (SD) while the mean decrease in
antioxidant levels was 0.124±0.084U/ml (SD). There was no significant difference in reduction
of plasma GLU and SOD between participants with pre-eclampsia, HDP and normal women.
Plasma GLU values of ≤ 0.442 µM had a sensitivity of 25%, specificity of 29%, positive
predictive value of 1% and negative predictive value of 94% for pre-eclampsia. A cut off value
of 0.442 µM and below had 81% sensitivity, 30% specificity, positive predictive value of 11.3%
and a negative predictive value of 94% for HDP. An SOD value of ≤0.110U/ml at recruitment
had a sensitivity value of 50%, specificity value of 75%, PPV of 4.8% and NPV of 98.3% for the
prediction of pre-eclampsia. When applied to HDP, a value of ≤0.110U/ml had a sensitivity
value of 12.5%, specificity value of 74.0%, PPV of 5% and NPV of 88.5%. These values were
however not significant for prediction of either entity.
Conclusion
Plasma glutathione and plasma superoxide dismutase values before 20 weeks gestation may not
be useful in predicting clinical onset of pre-eclampsia among low risk women in Ibadan. Further
community based research is needed to confirm these findings.
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INTRODUCTION
Hypertension is the commonest medical disease in pregnancy worldwide with a spectrum that
includes chronic hypertension (detected before 20 weeks gestation), chronic hypertension with
super-imposed pre-eclampsia, pregnancy induced hypertension, pre-eclampsia and eclampsia 1, 2.
.Together, pre-eclampsia and eclampsia are responsible for more than 50,000 maternal deaths per
year in developing countries where prompt detection of symptoms and complications is hindered
by the delays described by Thaddeus and Maine 3-6.
Pre-eclampsia is peculiar to pregnancy and characterised by new onset hypertension (blood
pressure ≥140/90mmHg from multiple readings taken 4-6hours apart) and significant proteinuria
(>300mg/24hrs in 2 random specimens taken 4-6hours apart) in previously normotensive women
after 20 weeks gestation. Pre-eclampsia occurs in 5-8% of pregnancies while the incidence of
eclampsia- convulsions in women with pre-eclampsia- varies in resource poor countries from
1/100-1/1700; both disorders continue to be a significant contributor to maternal mortality in
Nigeria 7-11.
It has been previously established that the pathogenesis of pre-eclampsia is closely linked to the
development of the placenta. This is supported by its unique association with pregnancy,
resolution of disease following delivery and development in the absence of the foetus i.e.: molar
pregnancy12. Early studies have also associated the occurrence of pre-eclampsia with defective
invasion of uterine vasculature by the placenta. In normal pregnancies, trophoblastic invasion is
completed by the 20th week of gestation with conversion of the uterine spiral arteries to high
capacitance, low resistance vessels that expedite feto-maternal exchange of nutrients and waste.13
However, in pre-eclampsia, trophoblastic invasion is patchy and subsequently followed by
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vasospasm, hypoxia, excessive free radical generation and endothelial damage14. Endothelial
dysfunction is the trigger for multi-systemic involvement in pre-eclampsia with features akin to
an extreme systemic inflammatory syndrome15.The disease is often complicated by
cerebrovascular accidents, pulmonary oedema, oliguric renal failure and coagulopathy which
can easily lead to maternal death, fetal growth restriction or fetal demise.
The severity of complications and associated mortality rates particularly in developing countries
have made pre-eclampsia and eclampsia prevention a major concern. Currently, the most widely
used method of prevention of pre-eclampsia consists largely of antenatal risk assessment and
vigilance to ensure identification of danger signs 16. Unfortunately, these measures have been
unsuccessful in reducing incidence rates of pre-eclampsia as its onset and complications remain
difficult to determine in each patient.
Because of this, the goal of current research continues to be the identification of predictive
investigations; various methods have been evaluated-mean arterial blood pressure measurement,
severity of proteinuria, uric acid measurement and Doppler studies of the uterine arteries- with
limited success17-20. Several biomarkers including serum soluble fms-like tyrosine kinase
1(sFLT-1), soluble endoglin, plasma protein 13 and pregnancy associated plasma protein A
(PAPP-A) have also been found to be altered significantly in pre-eclampsia but none is presently
accepted for daily clinical use 12.
Oxidative stress has been described as an imbalance between free radical generation and the
production of endogenous antioxidants21. This imbalance has been observed in normal pregnancy
but in pre-eclampsia it is usually exaggerated particularly in early onset disease 22-24. Excessive free
radical generation commonly follows the hypoxia-ischemia sequence generated by inadequate
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trophoblastic invasion with the production of several compounds including superoxide (O2-.),
peroxylnitrite (ONOO-), hydroxyl (OH-) and nitric oxide (NO.) ions. These free radicals or reactive
oxygen species (ROS) influence secondary messenger signalling pathways in normal physiology 25
but are highly toxic to proteins, lipids and DNA if they are not limited by endogenous antioxidants.
The presence of oxidative stress is usually evident in decreased levels of antioxidants in blood and
tissues while there is increased production of lipid peroxidants e.g.: malondialdehyde (MDA) and
isoprostanes.26
Antioxidants prevent the effects of oxidative stress by catalyzing the conversion of ROS to
hydrogen peroxide and water 23. Micronutrients such as selenium are an important part of this
process suggesting that polymorphisms of antioxidant enzymes and dietary restriction of
micronutrients can predispose to an increase in oxidative stress and pregnancy complications 27, 28.
These findings have led to further study of antioxidant supplementation using Vitamins C and E to
prevent pre-eclampsia with disappointing results29-32.
Pre-eclampsia continues to contribute significantly to maternal mortality and fetal deaths
worldwide. Unfortunately, there is no consensus yet on the most suitable screening method for this
disease 33. Oxidative stress may therefore be the key to indentifying suitable screening methods and
biomarkers which can supplant the use of antenatal risk assessment. In order to determine the utility
of antioxidants in screening, the value of glutathione and superoxide dismutase in women less than
20 weeks gestation in predicting the onset of pre-eclampsia is examined in this dissertation.
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LITERATURE REVIEW
Pre-eclampsia –definition, classifications & disease burden
Pre-eclampsia is a syndrome peculiar to human pregnancy and characterised by hypertension and
proteinuria. The National High Blood Pressure Education Program Working group criteria for
diagnosis are a systolic blood pressure of ≥140mmHg and diastolic pressure of ≥90mmHg in a
previously normotensive pregnant woman after 20weeks gestation34. Blood pressure
measurement is usually in the left lateral, recumbent or sitting position with the cuff at the level
of the heart using conventional mercury sphygmomanometers or validated electronic devices.
The fifth Korotkoff sound is utilised for determination of diastolic blood pressure because of its
reproducibility. White coat hypertension is excluded by ensuring a period of rest before
measurement and repeating the reading 4-6 hours later. Hypertension is regarded as severe with
blood pressure ranges of ≥160/110mmHg or 170/110mmHg.
Proteinuria is determined using a 24 hour estimate but in practice, visual estimation by urine
dipsticks is more common. A value of 0.3g or greater of protein in a 24 hour urine collection or
a value of at least 1+ (ph<8.0, specific gravity < 1.030 is regarded as significant). Other methods
of assessing proteinuria include an assessment of the ratio of urinary protein to creatinine with a
cut off value of 30mg/mmol35. Heavy proteinuria has been defined as 1g/L (equivalent to 2+ on
dipstick) or 3-5g/day by 24 hour urinary protein estimation35.
Based on severity of hypertension and proteinuria, pre-eclampsia is now classified into mild and
severe forms36. Mild pre-eclampsia is characterised by blood pressure levels of at least
140/90mmHg but less than 160/110mmHg with proteinuria more than 0.3g/24-hours but <5g/24-
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hours. Severe pre-eclampsia is characterised by blood pressure ≥160/110mmHg and heavy
proteinuria; the clinical features of severe pre-eclampsia are vomiting, persistent headaches,
blurring of vision, epigastric pain and symptoms consistent with pulmonary oedema 36. Severe
pre-eclampsia may also be complicated by the HELLP syndrome which is characterised by
microangiopathic haemolysis, elevated liver enzymes-alanine aminotransferase (ALT) and
aspartate transaminase (AST) with low platelet counts. These disorders are usually ameliorated
following delivery by the placenta and foetus.
Pre-eclampsia occurs in 5-8% of all pregnancies worldwide 37, 14, 38. It has been estimated that
60,000 maternal deaths occur per year in developing countries while global estimates of perinatal
and neonatal mortality are put at 10%-23.6%39-41. Analysis of maternal deaths worldwide suggest
that hypertensive disorders are second only to haemorrhage as causes of maternal mortality42.
Risk factors for pre-eclampsia include extremes of maternal age, nulliparity, race, multiple
gestation, pre-eclampsia in a previous pregnancy, obesity, diabetes mellitus, pregnancies
conceived by assisted reproduction, limited sperm exposure, chronic hypertension and renal
disease 43,44.
Overview of clinical presentation and management
The diagnosis of pre-eclampsia and determination of severity require clinical evaluation for
symptoms, blood pressure measurement, urinalysis or 24 hour urinary protein estimation.
Additional information from history taking such as gestational age, change in sexual partner,
background hypertension or other co-morbidities may assist in determining prognosis. It has
been observed that the clinical presentation is more severe in patients with early onset disease
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(<34weeks gestation) while pre-eclampsia in the late third trimester and at term usually has a
milder presentation.7 In general however, the severity and progression of disease in each patient
is highly unpredictable.
The principles of management in pre-eclampsia are hinged on maternal stabilisation: control of
blood pressure, prevention of seizures, anticipating and monitoring for complications suggesting
end organ damage e.g. oliguria, cerebral oedema, HELLP syndrome and assessment of the fetal
condition to determine the best time for delivery14. Blood pressure control is largely achieved by
parenteral or oral antihypertensive medication-common drugs include alpha-methyl dopa,
nifedipine, labetalol and hydralazine with combination therapy commonly required for optimal
blood pressure control45.
The state of the foetus is determined by assessment of fetal weight, viability and liquor volume
using ultrasound. Cardiotocography (CTG) is also used to determine fetal health. Management
and delivery is determined by several factors including the gestational age, fetal condition,
presence of maternal complications, labour and favourability of the cervix. In some patients who
are remote from term, conservative management can be instituted with serial monitoring of
maternal vital signs, full blood count, liver function tests, electrolytes, urea, uric acid &
creatinine, clotting profile, 24-hour urinary protein, ultrasound and CTG 46. Critical unit care
may be required to avoid and manage aspiration pneumonitis, cerebrovascular haemorrhage,
pulmonary oedema and renal failure.47 Following delivery, neonatal intensive care admission
may be required because of birth asphyxia or complications associated with prematurity and
growth restriction. Women admitted with a diagnosis of pre-eclampsia must be counselled on the
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possibility of progression to eclampsia, the importance of the post- natal visit, risk of recurrence
and the development of hypertension and renal impairment later in life48.
Pre-eclampsia- patho-physiology and screening
The exact reasons for the development of pre-eclampsia are unknown and the causes of the
disease are ascribed to several theories 44. The two stage model of pre-eclampsia however
remains a common explanation for some of the clinical symptoms and sign49. These stages are
abnormal placentation and the maternal syndrome. Cytotrophoblastic invasion has been found to
consist of two phases: invasion of the decidual segments of the spiral arteries around 10-12
weeks gestation and the myometrial portions of the spiral arteries by 15-16 weeks 50. In pre-
eclampsia, this process is said to be characterised by patchy placentation and failed
vasculogenesis of extra villous cytotrophoblast which leads to failure of spiral artery remodelling
and conversion to high capacitance vessels 51, 44. This process leads to placental ischemia-
reperfusion and release of various substances-cytokines, antiangiogenic factors and
syncytiotrophoblast particles into the maternal circulation 44.
The release of these ‘toxic’ substances is contributory to the maternal syndrome. Endothelial
dysfunction and a severe maternal inflammatory response are characteristic of this stage;
endothelial damage is characterised by an imbalance between thromboxane A2 and prostacyclin
in favour of the former with increased activation of leukocytes, macrophages, complement
system with various cytokines including Tumour necrosis factor–α (TNF- α, IL-6 and C-reactive
protein 52-55.
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Despite the widespread acceptance of the two stage model, there is considerable controversy
concerning the factors that link these two models as some women with placental ischemia and
intrauterine growth restriction do not exhibit features of pre-eclampsia 44 This has stimulated
research on the role of genetics and immunology in pre-eclampsia. Early studies suggest a role
for several modifier genes including genes encoding angiotensinogen, superoxide dismutase and
endothelial nitric oxide synthase51. Further studies have shown a 31% and 20% heritability for
pre-eclampsia 56 and gestational hypertension respectively in addition to pre-eclampsia loci on
genomic studies 57. These findings however are unable to explain majority of cases of pre-
eclampsia.
Primiparternity and sperm exposure have also been found to predispose to pre-eclampsia and
studies have suggested that sperm in the female tract causes mucosal allo-imunization
accompanied by a post-mating inflammatory reaction under the influence of seminal vesicle
derived Transforming growth factor β158. Repeated sexual intercourse has been found to increase
exposure to this cytokine and lead to partner specific mucosal tolerance.58
The patho-physiology of pre-eclampsia remains complex and this has turned the direction of
research towards prediction and screening.Several methods have been utilised in attempt to
identify women with significant risk of development of pre-eclampsia; uterine artery Doppler has
been used as a measure of placental resistance in order to indirectly assess the extent of placental
remodelling59,60. Doppler studies of the uterine artery at 20-23 weeks gestation were found to
have an 80% detection rate of pre-eclampsia requiring delivery before 34 weeks 61, 62.
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Mean arterial blood pressure has been evaluated in combination with maternal variables in
women in the first trimester; combination testing was found to detect 62.5% of women who
would develop pre-eclampsia with a false positive rate of 10% 63 Biomarkers have also been
used for screening; values of placenta derived peptides such as pregnancy associated plasma
protein A (PAPP-A, plasma protein 13(PP13) have been found to be reduced in women in the
first trimester though their sensitivity when used alone for screening has been found to be low60
Combined screening panels including biomarkers, maternal characteristics and uterine artery
Doppler were found to be more promising in predicting pre-eclampsia 64, 65. Homocysteine,
isoprostanes and antioxidant status have also been assessed to determine the role of oxidative
stress in screening for pre-eclampsia60
Oxidative stress and pre-eclampsia
Oxidative stress has been described as the presence of active oxygen species in excess of
available antioxidant buffering capacity; it has been considered a component of the patho-
physiology of pre-eclampsia for decades 66, 67, 59. This release of free radicals has been found to
predispose to endothelial cell damage, placental apoptosis and release of synctiotrophoblast
particles suggesting that oxidative stress may be the link between placenta ischemia and the
maternal syndrome. 68
Antioxidants may be enzymatic or non-enzymatic; superoxide dismutase (SOD), glutathione
peroxidase (GPx), catalase and glutathione reductase (GR) are examples of enzymatic
antioxidants while ascorbic acid (Vitamin C) and Vitamin E are non-enzymatic antioxidants.
Usually, the enzymatic antioxidants are intrinsic while the non-enzymatic are generated from
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exogenous sources e.g: diet. Studies in women with pre-eclampsia have shown significant
alterations in endogenous antioxidants; Mehendale et al compared blood levels of ascorbic acid
and α-tocopherol in patients with a diagnosis of pre-eclampsia and normotensive healthy
pregnant women with significantly lower levels of these antioxidants in the pre-eclamptic
patients.21 Haque compared levels of erythrocyte markers of oxidative stress- superoxide
dismutase, catalase and glutathione peroxidase in women with pre-eclampsia and normotensive
pregnancy and established greater suppression of antioxidant enzyme activity in different age
groups 69
Sharma et al compared the levels of glutathione peroxidase, superoxide dismutase,
malondialdehyde, vitamin C and lycopene in 50 women with pre-eclampsia and 50 pregnant
controls70. The mean levels of markers of lipid peroxidation were higher in patients with pre-
eclampsia with marked increases in patients with severe disease while plasma levels of
antioxidants were significantly lower in the women with pre-eclampsia. As part of the
recommendations, it was advocated that further study of markers of oxidative stress and markers
in pre-eclampsia was required. Other case control studies or cross sectional surveys have
evaluated the levels of superoxide dismutase, endothelin-1 and total antioxidant status in sera and
placentae of patients with pre-eclampsia with evidence of increased free radical scavenging and
antioxidant consumption 71-74.
Glutathione and glutathione related enzymes are involved in detoxification of reactive oxygen
species. Glutathione can act as a substrate in the cytosolic glutathione redox cycle or can
inactivate reactive oxygen species directly e.g: superoxide (O2-) and hydroxyl (OH -) anions75.
Synthesis of glutathione is highlighted below in Figure 176. Gamma-glutamyl cysteine and
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cystein with the enzymatic action of glutathione synthetase are involved in production of
glutathione which is subsequently metabolized by oxidation or reduction for the purpose of
detoxification and as part of the redox cycle. Glutathione transferase catalyzes conjugation
between glutathione and toxic compounds before excretion76. Because of the diverse pathways
for glutathione metabolism, there are oxidised, reduced and protein bound fractions.
Total glutathione represents the sum of these fractions. Red blood cells and hepatocytes are an
important source of glutathione apart from extracellular fluid. In addition to its role in oxidative
stress, glutathione also helps maintain exogenous antioxidants such as vitamins C and E.
Glutathione is involved with the breakdown of peroxides77. It has a role in regulating the nitric
acid cycle. Glutathione can directly bind many inorganic and organic xenobiotic (foreign
chemicals) and carcinogenic compounds, such as the heavy metals mercury and arsenic. It is
important to the proper function and maximum effect of the immune system. In addition,
glutathione is fundamentally involved with many metabolic and biochemical mechanisms such
as protein and prostaglandin synthesis, DNA synthesis and repair, maintenance of disulfide
bonds in proteins, enzyme activation and amino acid transport across cell membranes76.
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FIGURE 1: SYNTHESIS AND METABOLISM OF GLUTATHIONE76
Reduced glutathione activity has been found in pre-eclamptic patients in comparison with
healthy controls. Knapen et al75 compared total whole blood glutathione levels in 23 women with
pregnancies complicated by HELLP/pre-eclampsia and 22 normotensive pregnant women. The
median value of the patients with complicated pregnancies was much lower than in normotensive
women (p=0.05). Mean glutathione/haemoglobin ratios were significantly lower in women with
pre-eclampsia or HELLP. Another study however showed no significant difference in oxidized
or free glutathione was found between women with pre-eclampsia or HELLP and normotensive
women; there was however a lower free to oxidized glutathione level among those with these
complications 76. Total plasma glutathione levels were compared in 10 non-pregnant women, 10
women with normotensive pregnancies and 20 women with pre-eclampsia at delivery76. Reduced
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plasma values of glutathione were found in pre-eclampsia in comparison with normotensive
pregnant women (p<0.05).
Superoxide dismutase (SOD) is an intrinsic enzymatic antioxidant which is found in all tissues
and it is a significant component of the human body’s response to generation of reactive oxygen
species (ROS). Superoxide dismutases constitute an enzyme family that catalyses conversion of
superoxide to hydrogen peroxide; they are often differentiated by virtue of the metal binding ion-
copper(Cu), zinc(Zn), iron(Fe) or Manganese (Mn) into extracellular(ec-SOD/SOD3),
mitochondrial (Mn-SOD/SOD2) and cytosolic (CuZn-SOD/SOD1) variants78,79.SOD acts as the
first line of defense in the antioxidant response by converting 2 molecules of superoxide(O2-) to
hydrogen peroxide and molecular oxygen. (figure 2)
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FIGURE 2: ANTIOXIDANT CYCLE OF SUPEROXIDE DISMUTASE80
SOD activity in pregnancy exceeds that of non-pregnant women with increased activity as the
gestation progresses; however studies have shown decreased SOD activity in placental tissue and
trophoblast cells of women with pre-eclampsia79, 81, 82. Decreased SOD activity has also been
associated with other pathological conditions in pregnancy including gestational diabetes
mellitus, small for gestational age babies and miscarriages. 79There is limited information
concerning the role of SOD in predicting pre-eclampsia though SOD has frequently been
compared in pre-eclampsia and normal pregnancy. Mahadik and Sina found significantly lower
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levels of serum SOD in pre-eclamptic and eclamptic women in comparison with normal controls;
a cut off value of 0.52U/ml was found to have a sensitivity of 68.5% and negative predictive
value of 78.6%83. Majority of research has confirmed the association of reduced SOD activity
with pre-eclampsia though these studies have been primarily cross-sectional or case control
studies.
Local research on antioxidants and pre-eclampsia
Prospective studies in the evaluation of markers of oxidative stress are limited. Local research on
the effect of oxidative stress on pre-eclampsia is also limited in Nigeria and sub-Saharan Africa.
In Nigeria, a preliminary report by Idogun et al identified reduced total antioxidant capacity in 20
pregnant women when compared with healthy non-pregnant controls84. Ademuyiwa et al studied
97 primigravidae and 20 healthy non-pregnant controls; samples were obtained and
plasma/erythrocyte levels of superoxide dismutase, manganese superoxide dismutase, catalase,
glutathione, glutathione transferase and aminoleuvilinic acid dehydratase were determined85.
Findings in this study include reduction in activity of total superoxide dismutase and amino
leuvilinic acid dehydratase in pre-eclamptic patients. A study done in Sudan compared
antioxidant status in women with pre-eclampsia and healthy pregnant women; a significantly
negative correlation was found between glutathione peroxidase and diastolic blood pressure but
plasma total antioxidant capacity was found to be similar between both groups of women. 86
More recently, Adedapo et al evaluated total antioxidant status in 580 pregnant women
presenting for booking with follow up and repeat sampling.87 There was a significant difference
between total antioxidant status levels in the women who subsequently developed pre-eclampsia
-35% while there was a 19% difference in women who did not develop hypertension. Other
25
studies in this environment have focused on the role of urinary micro-albumin, uric acid, albumin
and creatinine88, 89 .Another study done in Irrua showed significant reduction in plasma vitamin
C and E in patients with pre-eclampsia in comparison with controls90. There are limited studies
concerning glutathione and pre-eclampsia from Nigeria. Ademuyiwa’s study showed no
significant differences in glutathione or glutathione transferase between pregnant women,
controls and women with pre-eclampsia85.
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RATIONALE FOR THE STUDY
Hypertensive disorders are responsible for about 20% of maternal deaths in Nigeria91 and they
are also associated with significant fetal compromise-up to 28% of foetuses in patients with pre-
eclampsia experience fetal growth restriction92. Pre-eclampsia commonly progresses to
eclampsia if pregnancy is not terminated and the placenta is removed; several studies from
Nigeria have highlighted the contribution of eclampsia to maternal mortality-fatality rates of
15.6%, 10.7% and 8.5% were found in Enugu, Benin and Abuja93, 94, 5
The literature is replete with research on the possible cause of pre-eclampsia but at present, no
conclusive reason for its occurrence has been found. Screening however has been attempted by
several investigators; the benefits of screening would include identification of women with
potential for development of significant hypertension and proteinuria in pregnancy. Despite the
findings of the systematic review of screening tests conducted by the WHO95, the benefit of
continued research and success in this approach cannot be overstated.
Oxidative stress, though present in pregnancy, is exaggerated in patients with pre-eclampsia and
eclampsia. This is characterised by profound increases in markers of oxidative stress and low
levels of enzymatic antioxidants. It is anticipated that screening for this disease using markers of
oxidative stress can be utilised singly or as a part of multi- tiered screening using different
markers. Another potential benefit is the identification of a critical level above which patients are
likely to develop pre-eclampsia. This may aid obstetricians, in addition to clinical assessment,
early detection and prevention of complications.
27
OBJECTIVES
General Objective:
The primary objective was to determine the value of plasma glutathione and superoxide
dismutase estimation for prediction of clinical onset of pre-eclampsia in women with pregnancies
less than 20 weeks.
Specific Objectives
1. To determine the mean plasma glutathione and superoxide dismutase levels among women
presenting for booking at less than 20 weeks gestation in Ibadan.
2. To evaluate the change between plasma glutathione and superoxide dismutase levels in the
first/early second trimester and at onset of pre-eclampsia or delivery.
3. To determine the plasma glutathione and superoxide dismutase levels at which pre-eclampsia
occurs.
Null Hypothesis
Glutathione and Superoxide dismutase plasma levels are not useful in predicting the
development of pre-eclampsia in pregnant women below 20 weeks of gestation.
Alternate Hypothesis
Glutathione and Superoxide dismutase plasma levels are useful in predicting the development of
pre-eclampsia in pregnant women below 20 weeks of gestation.
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MATERIALS AND METHODS
Study design
The study was a prospective cohort study aimed at evaluating the predictive value of plasma
glutathione and superoxide dismutase levels in pregnant women below 20 weeks gestation for
clinical onset of pre-eclampsia.
Study site
The study was conducted in the Antenatal Clinic of the Department of Obstetrics and
Gynaecology, University College Hospital, Ibadan, Oyo State. The University College Hospital
is a tertiary teaching hospital located in Ibadan North Local Government of Oyo State, Nigeria; it
serves as a major referral centre for primary and secondary health care facilities in South western
Nigeria and other parts of the country.
The Department of Obstetrics and Gynaecology is staffed by 21 consultants and over 30
residents in five units-Feto-Maternal, Assisted Conception, Fertility & Reproductive
Endocrinology, Gynaecologic Oncology and Urogynaecology. The Obstetric Units run 5
antenatal clinics and 1 booking clinic every week. At these clinics, women are booked to begin
antenatal care. In the year 2010, the average weekly antenatal client number for each unit was
55 while the total number of obstetric admissions for that year was 8,819. The total number of
new antenatal clients(all units) was 1864 and the total number of deliveries was 2643. The
incidence rate for pre-eclampsia in UCH, Ibadan has previously been quoted as 9.7%88
29
Study duration
The study was conducted between June 23rd and December 23rd, 2012.
Study procedure
Pregnant women below 20 weeks of gestation (see inclusion and exclusion criteria) were
selected by convenience sampling. Parity was defined as number of pregnancies ≥28 weeks’
gestation irrespective of outcome and gestational ages in completed weeks were calculated from
the last menstrual period or first trimester ultrasound. The patients were informed about the
research and asked to give written informed consent. Their antenatal information, bio data,
obstetric and past medical history was extracted using a structured proforma (see Data
collection).
The study participants were asked to fast overnight and have a fasting and two hour post prandial
blood sugar sample taken to screen for diabetes mellitus. A 5mls sample of venous blood was
obtained from the patient without the use of a tourniquet and sent to the laboratory for analysis of
levels of plasma superoxide dismutase (SOD). The women were counselled about symptoms
suggestive of pre-eclampsia and advised to come to the hospital promptly in the event of any of
these symptoms. The participants’’ estimated frequency of use of multivitamin supplements was
determined at entry to the study ,they were then advised to use only routine haematinics- ferrous
sulphate/fumarate and folate (which is usually provided the UCH Pharmacy) in addition to
ensuring good nutrition. The participants were instructed to avoid use of Vitamin C and Vitamin
E tablets for the duration of the study. The only means of ensuring compliance with avoidance of
these vitamins was by enquiry at each antenatal visit.
30
Once the initial sample was taken, the patient continued her antenatal visits as instructed by her
doctors-four weekly visits up to 28 weeks, fortnightly visits to 36 weeks and weekly till delivery.
Women who required more frequent visits were not excluded from the study unless the exclusion
criteria were met. On admission to the labour ward or lying in ward, the patient’s systolic blood
pressure, diastolic blood pressure, urinalysis, gestational age at delivery and mode of delivery
will also be noted. Repeat SOD sampling was done at delivery or onset of pre-eclampsia,
gestational hypertension or post partum hypertension.
Pre-eclampsia was defined in this study as the presence of blood pressure ≥140/90mmHg from
two consecutive readings 4-6 hours apart or a single diastolic blood pressure reading of
≥110mmHg with 24-hour urinary protein more than 300mg or ≥1+ on conventional dipstick
from two urine specimens 4-6hours apart. A single blood pressure reading ≥160/110mmHg with
24-hour urinary protein >5g or 2+ on dipstick was regarded as severe pre-eclampsia. Once
diagnosed with pre-eclampsia, patients was admitted to the labour ward and managed according
to the departmental protocol. Irrespective of the proposed line of management, a repeat sample
was obtained from the patient.
31
Inclusion criterion
All pregnant women booked in the UCH for antenatal care and delivery whose
gestational age was <20 weeks.
Exclusion criteria
Women with one or more of the following conditions:
Chronic hypertension
Previous pre-eclampsia
Pre-gestational or Gestational diabetes mellitus
Human immunodeficiency virus infection (HIV) infection
Hepatitis B or C infection
Chronic medical diseases e.g: antiphospholipid syndrome, systemic lupus erythrematosus
Multiple gestation.
32
Measurement of enzymatic antioxidants
Measurement of total glutathione 96
The OxiSelectTM Total Glutathione Assay kit is a quantitative assay for measuring total
glutathione content (GSH/GSSG). Glutathione reductase reduces oxidized glutathione (GSSG)
to reduced glutathione in the presence of NADPH. The chromogen then reacts with the thiol
group of GSH to produce a colored compound that absorbs at 405nm (Figure 3). The total
glutathione content is determined by comparison with the predetermined glutathione standard
curve. The rate of chromophore production is proportional to the concentration of glutathione
within the sample. The rate can be determined from the absorbance change over time.
Metaphosphoric acid is utilised in the assay technique to remove interfering proteins or enzymes
from samples.
FIGURE 3: PRINCIPLE FOR TOTAL GLUTATHIONE DETERMINATION
The samples were collected and centrifuged at 12,000 rpm for 10minutes to remove insoluble
particles and the supernatant was collected and stored in batches at -80oC.
33
Table 1: DISTRIBUTION OF STOCK AND BUFFER SOLUTION FOR
GLUTATHIONE STANDARD PREPARATION
Standard Tubes 1µM GSSG standard
(µL)
0.5% MPA in Assay
Buffer (µL)
GSSG
(µM)
1 500 500 0.5
2 500 tube 1 500 0.250
3 500 tube 2 500 0.125
4 500 tube 3 500 0.0625
5 500 tube 4 500 0.03125
6 500 tube 5 500 0.0156
7 500 tube 6 500 0.0078
8 0 500 0
Calculation of results
The results were calculated by determining the average of the absorbance readings for each
GSSG standard and samples. The average absorbances of each standard and sample were
graphed against incubation time and the slope for each standard or sample was determined from
the linear portion of each curve (Figure 4). These slopes were then subtracted from the
background slopes. The net slopes were then plotted against glutathione concentration (Figure
5). The glutathione value of each sample was then intrapolated from the concentration. The kit
has a detection sensitivity limit of 8nM.
34
FIGURE 4: GRAPH OF STANDARD ABSORBANCES VERSUS INCUBATION TIME
FIGURE 5: GRAPH OF NET SLOPES OF STANDARDS VERSUS CONCENTRATION
35
Determination of superoxide dismutase97
Superoxide dismutase detoxifies the superoxide radical (O2.) with the production of hydrogen
peroxide and molecular oxygen.
O2. + O2. + 2H+ SOD→ O2 + H20
The analytical method employed was from an assay developed by Cayman Chemicals-xanthine
and xanthine oxidase (XOD) were used to generate superoxide radicals which reacted with 2-(4-
iodophenyl)-3-(4 nitrophenol)-5-phenyltetrazolium chloride (INT) to form a red formazan dye.
SOD activity was then measured by the degree of inhibition of this reaction. One unit of SOD
causes a 50% inhibition of the rate of reduction of INT under the conditions of the assay. The
unit of measurement for SOD with this assay is U/ml.
Sample collection
The blood samples were collected using heparinized bottles and centrifuged at 700-1000g for
10minutes at 4 0 C. The plasma was then separated from the erythrocytes using a micropipette
and micropipette tips and stored at -80 0C.
Procedure
Standard preparation
The SOD standard was diluted- 20µL with 1.98mls of the sample buffer to obtain the stock
solution and this stock solution was then added to seven clean glass test tubes marked A-G. The
distribution of stock and buffer solution with SOD activity is outlined in the table below. This
table was utilized to distribute the stock and sample buffer solutions in the standard wells.
36
Table 2: DISTRIBUTION OF STOCK AND BUFFER SOLUTION FOR SOD
STANDARD PREPARATION
Performance of assay
A microplate reader with standard wells corresponding to tubes A-G and sample wells was used
with each kit analyzing assay 98 samples(including standard). For the standard wells, 200µl of
the radical detector (diluted) and 10µL of the standard solution were added in the designated
wells A-G. For the sample wells, 200µL of diluted radical detector and 10µl of sample were
added to the sample wells. The plasma was diluted (1:5) with sample buffer before assay. The
reaction was initiated by the addition of 20µL of xanthine oxidase to all the wells. The exact time
at which the additions were made was as precise as possible. The well plate was shaken carefully
for a few seconds to mix and covered with a plate cover. The plate was then incubated on a
shaker for 20 minutes at room temperature and the absorbance read at 440-460nm using an
ELISA plate reader.
Tube SOD Stock (µL) Sample buffer(µL) Final SOD Activity(U/ml)
A 0 1,000 0
B 20 980 0.025
C 40 960 0.05
D 80 920 0.10
E 120 880 0.15
F 160 840 0.20
G 200 800 0.25
37
Calculation of SOD value
The absorbance data of samples and standards were entered in a Microsoft Excel 2010
spreadsheet. The standard absorbance A was then divided by itself and by other standards and
samples absorbance values to yield the linearized rate. This is illustrated below as an example:
LR Standard A = Standard A
Standard A
LR Standard B = Standard A
Standard B
LR Sample 1 = Standard A
Sample 1
The linearised rates of the standards were used to plot a standard curve with the final SOD
activity on the x-axis (Figure 6).
FIGURE 6: STANDARD CURVE FOR SUPEROXIDE DISMUTASE
38
The SOD activities of the samples were then calculated from the linear regression of the standard
curve substituting the linearized rate of each sample. Again, one unit of SOD activity is the
amount of enzyme required to cause 50% dismutation of the superoxide radical. The equation is
highlighted below:
SOD (U/ml) = [(sample LR- y-intercept) x 0.23 mls] x sample dilution
Slope 0.01mls
The intra-assay coefficient of variation of the assay kit has been listed at 3.2% for 60 samples
analysed over one day while an inter-assay coefficient of variation of 3.7% for 60 samples
analysed over 5 days under the same experimental conditions. The dynamic range of the kit
under the standardized conditions of the assay has been put at 0.025-0.25 U/ml of SOD.
Outcome Measures
The main outcome measures of the trial were:
1. Pre-eclampsia
2. Mean reduction in antenatal antioxidant levels at delivery or onset of pre-eclampsia.
3. Antioxidant level cut-off for development of pre-eclampsia.
Data Collection
The data was collected by the principal investigator and one trained research assistant in the
antenatal clinic with the aid of a pre-designed proforma which was interviewer administered. At
the point of recruitment, eligible women were interviewed to determine their socio-demographic,
obstetric, drug and social history. The proforma was divided into four sections: Bio data,
39
Obstetric history, Drug history and Delivery outcomes. The proforma sheets were kept in the
custody of the principal investigator.
Blood samples were obtained by the principal investigator and a trained phlebotomist. A 5mls
venous blood sample was obtained from an ante-cubital vein without the use of a tourniquet and
collected in a heparinised bottle. The blood sample was centrifuged as indicated in the section
for measurement of antioxidants. The plasma sample was quickly transported to a freezer and
stored at -800C. Estimation of enzymatic antioxidants was done using the procedures described
above. The values obtained following measurement were entered into the structured proforma.
Follow up
The participants’ mobile phone numbers were obtained with their consent. They were asked for
permission to periodically remind them about the study protocol by SMS or periodic phone calls.
They were counseled to inform the principal investigator in the event of any danger signs
suggesting severe pre-eclampsia i.e. marked progressive pedal/ generalized oedema, persistent
headaches associated with blurring of vision and upper abdominal pain. They were also informed
periodically about the need to adhere to good diet, and avoid daily use of prescription vitamin C
and vitamin E.
The women were given a contact phone number in the event of any problems or if they desired
any information about the study protocol. Finally, the participants were asked to inform the
principal investigator once they were admitted to hospital for any reason. The same process was
used for repeat sampling in patients at delivery or if pre-eclampsia occurred.
40
Sample size estimation
Formula for calculating sample size
To determine the sample size for this study, the following formula was used98
N = Z1-α/2 2 (1-P)/ ε2 P
Z1-α/2 = standard normal deviate corresponding to 5% level of significance (2 sided test)
= 1.96
P = prevalence=0.10
ε = relative precision = 50%
A minimum sample size of 138 was obtained; with a 20% rate of loss to follow up and drop out,
the total sample size for the study was 165.
41
ETHICAL CONSIDERATIONS:
The study protocol was approved by the University of Ibadan/University College Hospital
Ibadan Institutional Review Committee (UI/UCH IRC). All patients were adequately counselled
and written informed consent obtained before their inclusion in the study.
Voluntary participation
The purpose of the study was explained to all participants. Participation was strictly voluntary
and participants were asked to sign informed consent forms. They were told that they could
refuse to take part in the study or withdraw at any time without change to their usually expected
standard of care.
Confidentiality
All information including history, physical findings and results obtained from the patients were
kept confidential. The patients were assured that their identity and medical history would be kept
in confidence by the investigators.
Beneficence
There were no additional financial requirements or demands apart from those necessary for
routine prenatal and intra-partum care. The results obtained from the study would be a useful part
of ongoing worldwide research for an effective screening tool that will enable prevention of pre-
eclampsia and eclampsia.
Non-maleficience
Blood samples were taken by a trained phlebotomist and physician to prevent undue discomfort
or blood loss.
42
Justice
The method of patients’ selection was by consecutive sampling of women who met the inclusion
criteria in order to ensure fairness.
Data management
Data from the pre-designed proforma was entered into a Microsoft Excel 2010 spreadsheet,
required slopes, y-intercepts and graphs were drawn using this software. Other data were
cleaned and extracted for entry into a data sheet using the Statistical Package for Social Sciences
for Windows (SPSS©, Inc., Chicago, IL, USA ) v.16.
Variables were analysed using frequency tables and descriptive statistics as appropriate. Levels
of enzymatic antioxidant were presented using mean ± standard deviation (SD) or mean±
standard error (SE). Mean blood pressure was calculated from systolic and diastolic pressures
using the sum of diastolic pressure and a third of pulse pressure. Data from different groups
were compared using student’s t-test and one-way ANOVA. Cut-off values were determined by
cross tabulation (2x2) using the lower limit of 1SD from the mean. Where the standard error was
used, this was indicated. The level of statistical significance was set at 0.05.
43
RESULTS
Two hundred and five patients were recruited for participation in the study and their blood
samples were taken at recruitment; 189 patients were followed up but only 166 participants were
available and suitable for repeat sampling. The flow chart of patient selection from this cohort is
highlighted in figure 4.
FIGURE 7: FLOW CHART OF COHORT FROM RECRUITMENT TO ANALYSIS
205 patients recruited 16 patients lost to follow up-
(could not be contacted, no
record of delivery in UCH)
189 patients delivered
Data from 166
patients available for
analysis
2 patients refused
repeat sampling-
(normotensive,
vaginal delivery)
13 patients excluded- study protocol violation
(exclusion criteria e.g: HIV (2), chronic HTN
(8), DM (2), TWINS (1) identified at delivery,
repeat sample not taken)
8 patients-delivered in hospital
but were identified beyond
interval for sampling (no pre-
eclampsia or hypertension,
excluded from analysis)
44
Analysis of study findings was completed with 166 patients who constituted the study
population. The mean age of participants was 30.51±4.10years while the predominant age groups
were 26-30years (46.2%) and ≥31 years (46.2%). Majority of the women were professionals
(28.4%) and traders (22.8%) (figure 8), while most of them had tertiary education (81.9%,
n=136), 14.5 %(n=24) had secondary education while only 2 patients had primary education
(1.2%).
FIGURE 8: DISTRIBUTION OF OCCUPATION AMONG TOTAL POPULATION
45
There was no personal or family history of diabetes mellitus in the cohort. Sixteen percent of
patients had a family history of hypertension but no personal history of hypertension Four
patients had gestational hypertension which developed about 2-4 years before the study. All the
cases were diagnosed at term and the range of diagnosis was 2-4 years before recruitment.. When
asked about multivitamin intake, 53.1% of respondents (n=113) reported use of multivitamins in
the index pregnancy, 42.2% of women (n=92) stated that they had used these multivitamins
daily.
There was no history of smoking, alcohol intake or use of prescription medication at the time of
recruitment. Urinalysis was negative for albumin in 80.5% of women; there was no glycosuria
in the population. Twenty three patients had mild, moderate albuminuria while 2 patients had
severe albuminuria. These patients had negative urine microscopy and culture results and were
treated with oral nitrofurantoin 100mg tds for 1 week. The albuminuria subsequently resolved.
Analysis of obstetric variables revealed that the median number of previous pregnancies was 2
while the median number of parous experiences and miscarriages was 1 and 0 respectively.
There were only 6 antenatal admissions (3.6%), 4 intrauterine foetal deaths (2.4%) and 1
spontaneous miscarriage. There were 89 vaginal deliveries (53.6%) and 77 caesarean sections
(46.4%). The mean birth weight was 3.12±0.52kg, while the median Apgar score and duration of
hospital stay were 10(0-10) and 4(2-140) respectively.
The mean gestational ages of the cohort at booking and sampling were 14.8±3.8weeks and
17.3±8.9 weeks respectively while the mean fasting & 2-hour post prandial blood sugar values
were 81.2mg/dl and 91.8mg/dl.(N=15). The mean gestational ages at presentation for planned
delivery and in labour were 38.3±2.5 weeks and 38.5±2.3 weeks respectively. The mean arterial
46
blood pressures in the whole population at booking, on presentation in labour and pre-operatively
were 81.28±8.54mmHg, 91.17±12.69mmHg and 94.35±16.06mmHg respectively. Mean arterial
blood pressure at discharge was 89.59±8.37mmHg.
The primary outcome was pre-eclampsia and the incidence was 2.4%; two patients had mild pre-
eclampsia in labour at term and they were eventually delivered by caesarean section for poor
progress in labour secondary to cephalo- pelvic disproportion. Two patients had severe pre-
eclampsia at term- and they also had caesarean delivery. Fourteen patients (8.4%) had
gestational hypertension (PIH) while two patients (1.2%) had hypertension post partum (HTN).
The incidence of hypertensive disorders in pregnancy (HDP) was 9.6%.
Socio-demographic and obstetric variables were compared among normotensive patients, women
with gestational hypertension or post partum hypertension and women with pre-eclampsia; only
the mean number of days of admission was significantly different (p=0.001) (Table 3).
Comparison of mean arterial blood pressure readings between groups showed significantly
higher values in women with pre-eclampsia, PIH or HTN than normotensive women at booking,
in labour, pre-operatively and at discharge (Table 4, 5).
47
TABLE 3: COMPARISON OF SOCIO-DEMOGRAPHIC & OBSTETRIC VARIABLES
AMONG GROUPS
Variable Mean (SD) 95%CI (mean) F value p-value
Age
Normal 30.42(4.01) (29.76-31.09) 0.527 0.591
Pre-eclampsia 30.00(9.24) (15.30-44.70)
PIH or HTN 31.50(4.10) (29.74-33.26)
Gestational age at blood sampling
Normal 17.23(9.32) (15.68-18.77) 0.128 0.880
Pre-eclampsia 19.00(1.16) (17.16-20.84)
PIH or HTN 18.00(2.13) (16.87-19.13)
Gestational age in labour
Normal 38.45(2.63) (37.98-39.82) 0.149 0.862
Pre-eclampsia 39.00(0.00) (39.00-39.00)
PIH or HTN 38.25(1.13) (37.43-38.82)
Birth weight
Normal 3.11(0.53) (3.02-3.20) 0.608 0.543
Pre-eclampsia 3.42(0.35) (2.85-3.95)
PIH or HTN 3.09(0.50) (2.83-3.36)
Apgar score in 5 minutes
Normal 9.42(1.65) (9.14-9.70) 1.537 0.218
Pre-eclampsia 10.00(0.00) (10.00-10.00)
PIH or HTN 8.62(3.38) (6.82-10.43)
Number of days on admission
Normal 4.10(3.06) (3.46-4.74) 7.28 0.001
Pre-eclampsia 4.50(0.58) (3.58-5.42)
PIH or HTN 23.20(49.46) (-5.27-51.84)
*Parity Nulliparous(n/%) Multiparous(n/%)
Normal 68(47.9) 74(52.1) 0.727
Pre-eclampsia 2(50) 2(50)
PIH or HTN 6(37.5) 10(72.5)
*: cross tabulation
48
TABLE 4: MEAN ARTERIAL BLOOD PRESSURE: COMPARISON BETWEEN GROUPS
MABP Mean (SD) 95%CI (mean) F-value P
Booking
Normal 80.01( 7.99) (78.88-81.33) 15.26 0.00
Pre-eclampsia 88.33(5.77) (79.15-97.52)
PIH or HTN 90.83( 7.05) (87.08-94.59)
Presentation/delivery
Normal 87.65( 8.08) (86.30-89.00) 121.68 0.00
Pre-eclampsia 134.33(12.70) (114.12-154.54)
PIH or HTN 111.12(5.66) (108.11-114.14)
Pre-operatively
Normal 89.52( 9.87) (87.21-91.82) 55.80 0.00
Pre-eclampsia 132.17(25.98) (90.83-173.51)
PIH or HTN 119.50(4.32) (115.89-123.11)
Discharge
Normal 87.44( 6.64) (86.06-88.83) 33.55 0.00
Pre-eclampsia 91.67(1.92) (88.60-94.73)
PIH or HTN 102.95(7.13) (98.84-107.07)
49
TABLE 5: COMPARISONS OF MEAN ARTERIAL BLOOD PRESSURES AMONG
DIFFERENT GROUPS
MABP (mmHg) Mean SD T (df) P
Booking
HDP 90.33 6.75 6.26(27.11) 0.00
Nil 80.01 7.99
Labour/delivery
HDP 115.77 11.90 10.24(21.57) 0.00
Nil 87.65 8.08
Pre-operatively
HDP 123.72 15.32 7.48(12.54) 0.00
Nil 89.52 9.87
Discharge
HDP 100.44 7.93 6.52(21.96) 0.00
Nil 87.44 6.64
The mean plasma glutathione level in the whole population at recruitment was 0.424±0.303µM
while the mean levels at delivery or onset of pre-eclampsia were 0.007±0.028 µM. The mean
difference in antioxidant levels was 0.418±0.307 µM. The following table illustrates the mean
difference between normal women, those who had pre-eclampsia and hypertension (Table 6).
Mean GLU values (delivery/onset of pre-eclampsia) were lower in the women who developed
pre-eclampsia in comparison with normal women but this was not significant (p=0.915). In
comparison to normotensive women, mean difference in antioxidant values at repeat sampling
were higher in women with pre-eclampsia were higher while values in those with hypertension
alone were lower but this was also not significant. (p=0.591).
50
TABLE 6: COMPARISON OF MEAN PLASMA GLUTATHIONE LEVELS AMONG
GROUPS:
GLU (booking) Mean (SD) 95% CI (mean) F P
Normal 0.428( 0.321) (0.375-0.480) 0.563 0.571
Pre-eclampsia 0.525( 0.083) (0.393-0.657)
PIH or HTN 0.363( 0.075) (0.322-0.403)
GLU (outcome)
Normal 0.007(0.031) (0.002-0.012) 0.088 0.915
Pre-eclampsia 0.006(0.006) (0.004-0.016)
PIH or HTN 0.004(0.006) (0.001-0.007)
Mean GLU difference
Normal 0.422(0.325) (0.368-0.475) 0.527 0.591
Pre-eclampsia 0.519(0.080) (0.390-0.648)
PIH or HTN 0.358(0.075) (0.318-0.398)
Mean plasma glutathione at booking, outcome and mean glutathione difference were not
significantly different in women with HDP in comparison to normo-tensive women (Table 7).
The mean glutathione level in women with pre-eclampsia was 0.525±0.083 µM (Table 6). A cut
off level 1 SD below the mean value was calculated (0.525-0.083 µM) and the result- 0.442 µM
used in a 2x2 table to calculate the sensitivity, specificity, positive predictive and negative
predictive values.(Table 8)
51
TABLE 7: COMPARISON OF MEAN PLASMA GLUTATHIONE LEVELS AMONG
GROUPS
GLU (booking). Mean (SD) T (DF) Sig (2-tailed)
Normal 0.428(0.321) -.947(87.30) 0.346
HDP 0.395(0.100)
GLU (outcome)
Normal 0.007(0.031) -.969(151.77) 0.334
HDP 0.005(0.006)
Mean GLU diff.
Normal 0.422(0.326)
HDP 0.390(0.099) -.894(91.47) 0.374
TABLE 8: NUMBER OF WOMEN WITH PLASMA GLUTATHIONE ≤0.442 µM
VERSUS THOSE WITH PLASMA GLUTATHIONE WITH >0. 442 µM
GLU cutoff Pre-eclampsia (n/%) Normal (n/%) p
≤0.442 µM 1(25) 115(71) 0.050
>0. 442 µM 3(75) 47(29)
Total 4(100) 162(100)
HDP Normal
≤0.442 µM 13(81.2) 102(69.9) 0.341
>0.442 µM 3(18.8) 44(30.1)
Total 16(100) 146(100)
52
A cut off value of 0.442 µM and below had a sensitivity of 25%, specificity of 29%, and positive
predictive value of 1% and negative predictive value of 94% for pre-eclampsia. The p value was
0.048. Likelihood ratio for the test being positive was 0.135. A cut off value of 0.442 µM and
below had 81% sensitivity, 30% specificity, positive predictive value of 11.3% and a negative
predictive value of 94% for HDP. Likelihood ratio for the test being positive was 1.16.
Mean plasma superoxide dismutase (SOD) at booking in the whole population was
0.172±0.082U/ml while mean SOD at delivery or onset of pre-eclampsia was 0.047±0.014U/ml.
The mean decrease in antioxidant levels for the whole population was 0.124±0.084U/ml.
Table 9 illustrates the mean reduction in antioxidants between normal women, those who had
pre-eclampsia, PIH and post partum HTN. Mean SOD values were lower in the women who
had pre-eclampsia in comparison with normal women but this was not significant (p=0.180).
Mean antioxidant values at repeat sampling were also not significantly different in pre-
eclampsia and hypertensive women in comparison with normal women (p=0.050). There was
no significant variation in mean SOD reduction between the three groups using analysis of
variance(ANOVA, F: 2.466, P=0.088). Mean plasma SOD at booking, outcome and average
difference in booking and outcome values were not significantly different in women with HDP
in comparison to normotensive women on analysis using student’s T-test(Table 10).
53
TABLE 9: COMPARISON OF MEAN PLASMA SUPEROXIDE DISMUTASE LEVELS
AMONG GROUPS:
SOD (booking) Mean (SD) 95% CI (mean) F P
Normal 0.169( 0.080) (0.156-0.183) 1.733 0.180
Pre-eclampsia 0.131( 0.042) (0.064-0.199)
PIH or HTN 0.103( 0.089) (0.155-0.250)
SOD (outcome)
Normal 0.048(0.014) (0.046-0.502) 3.132 0.050
Pre-eclampsia 0.045(0.005) (0.047-0.064)
PIH or HTN 0.040(0.010) (0.035-0.046)
Mean SOD difference (SOD booking-SOD outcome)
Normal 0.121(0.084) (0.108-0.135) 2.466 0.088
Pre-eclampsia 0.076(0.048) (0.000-0.152)
PIH or HTN 0.063(0.082) (0.119-0.206)
54
TABLE 10: COMPARISON OF MEAN PLASMA SUPEROXIDE DISMUTASE LEVELS
AMONG GROUPS
SOD (booking). Mean (SD) T (DF) Sig (2-tailed)
Normal 0.188(0.086) 0.942(23.187) 0.356
HDP 0.076(0.048)
SOD (outcome)
Normal 0.043(0.011) -1.705( 27.548) 0.100
HDP 0.040(0.014)
Mean SOD difference (SOD booking-SOD outcome)
Normal 0.145(0.083)
HDP 0.121(0.084) 1.207(24.618) 0.239
The mean SOD value at recruitment for women with pre-eclampsia was 0.131 ±0.021 U/ml (SE).
One standard error below the mean was taken as the lowest limit of normal (0.131U/ml-
0.021U/ml=0.110U/ml). This value was used as a predictor value and used in a cross tabulation.
The standard errors were subtracted from the mean values and used as cut off values Values at
or below the cut offs were regarded as predictive of the disease and used in a cross tabulation
(2x2 table) to determine the sensitivity, specificity, positive predictive (PPV) and negative
predictive values(NPV) (Table 11).
An SOD value of 0.110U/ml and at recruitment had a sensitivity value of 50%, specificity value
of 75%, PPV of 4.8% and NPV of 98.3% for the prediction of pre-eclampsia. When applied to
HDP, a value of 0.110U/ml had a sensitivity value of 12.5%, specificity value of 74.0%, PPV of
5% and NPV of 88.5%. This value was however not significant for prediction of either entity..
55
TABLE 11: NUMBER OF WOMEN WITH PLASMA SOD ≤ 0.110U/ml vs. WOMEN
WITH PLASMA SOD ≥0.110U/ml.
SOD cutoff Pre-eclampsia (n/%) Normal (n/%) p
≤0.110U/ml 2(50) 40(24.7) 0.250
Above 0.110U/ml 2(50) 122(75.3)
Total 4(100) 162(100)
HDP Normal
≤0.110U/ml 2(12.5) 38(26) 0.234
Above 0.110U/ml 14(87.5) 108(74)
Total 16(100) 144(100)
Further evaluation of the patients who used multivitamins during the course of the study showed
an increased usage of multivitamins among those that eventually developed HDP or pre-
eclampsia. There was no significant difference in the number of women who used multivitamins
and those who did not ( p= 0.179 & 0.327).
56
DISCUSSION
Oxidative stress has been established as a component of the patho-physiology of pre-eclampsia
and various studies have identified a greater reduction in individual antioxidants and total
antioxidant status in comparison to normal pregnancy. 90, 99, 100 The age group distribution of the
participants was similar to a previous study on antioxidant status and pre-eclampsia and majority
of the women were within the reproductive age group87. For this study, mean arterial blood
pressures were used to compare findings in different groups and this form of measurement has
been found to be more useful in diagnosis and prediction of hypertensive disorders particularly in
the first or mid second trimester. 101 Mean arterial blood pressure readings were higher in the
women with pre-eclampsia and hypertension even at booking. This finding merits consideration
and further research as an additional means of predicting hypertension and pre-eclampsia in
Nigerian women.
Only 2.4% of the cohort developed the primary outcome (pre-eclampsia). This was lower than
previous studies by Adedapo et al, Basbug - 5%, 8.9% 87, 102. At the University College Hospital,
Ibadan, Salako et al obtained a prevalence of 9.7%88 .Possible contributions to this low yield for
pre-eclampsia include loss to follow up, decision to deliver in another facility and seasonal
variations in occurrence of pre-eclampsia which have been described. 103 The greater number of
patients with gestational hypertension and post partum hypertension alone (16) was the reason
for further analysis under the group- hypertensive disorders of pregnancy (HDP) which also
contribute significantly to maternal morbidity and mortality104.
57
There was a significant reduction in the mean glutathione values from the point of recruitment to
delivery in all groups consistent with the antioxidant depletion seen in other cross sectional
studies. 81, 82. A local study, however by Ademuyiwa did not confirm any significant change in
glutathione across trimesters of pregnancy. 87 The mean value of plasma glutathione at booking
was greater in the pre-eclampsia group possibly because the number of women in this group was
fewer. This may also be responsible for the greater average drop in plasma SOD seen in women
with hypertension in comparison with normotensive women. Again, the general trend appeared
consistent with the reduction of intrinsic antioxidant enzyme levels caused by oxidative stress in
pre-eclampsia 74, 70 but statistical significance was not reached.
A search of the literature did not identify the distinct use of plasma glutathione in a prospective
cohort setting for prediction purposes; Ademuyiwa’s study appears to be the closest to this
model. The only identified study using SOD as a predictor measured serum SOD with fetal death
and eclampsia as outcomes80.
Though the study outcomes are different, the dissimilarities may have been influenced by
differing diets in the study populations as studies have suggested that diet does influence the
balance between free radicals and antioxidants and perinatal outcome.96, 101,102 Since pre-
eclampsia and hypertension in pregnancy are major causes of pregnancy complications, a high
sensitivity and positive predictive value are ideal. The values obtained in this study, however,
have shown limited predictive power, particularly in sensitivity. There is very limited
information in the literature for comparison of mean plasma SOD or glutathione values below 20
weeks as most studies have been either cross sectional or case control studies conducted by
estimating serum or erythrocyte SOD or glutathione26,88, 105. In addition, no previous
58
standardized range of values for superoxide dismutase or glutathione in normal women and pre-
eclamptic patients or mean differences in SOD was identified in the literature. These are
identified gaps in knowledge which are potential areas for further research.
For this study, the use of multivitamins was determined by the patients’’ subjective assessment.
Previous studies have examined the role of antioxidant or multivitamin supplementation in the
prevention of pre-eclampsia and their conclusions suggest there may be no place for this
intervention106-108. A more recent study has suggested that antioxidant supplementation in
women with low antioxidant status might reduce the risk of pre-eclampsia109.
The presence of conflicting evidence made it important to exclude a possible contribution of
multivitamin intake to the primary outcome in this study. The analysis did identify an increased
frequency of multivitamin use in 20% of women with HDP. In addition, a large proportion of
women with normal pregnancies also stated that they used multivitamins frequently during the
period. There was no statistical significance and it was not clear whether there was protection or
increased predisposition to HDP or pre-eclampsia with multivitamin use. This aspect of the
results should therefore be interpreted with caution and merits further study.
Limitations
The study outcomes may have been influenced by the small number of women with the primary
outcome in spite of the sample size calculation. Also, the patients were recruited consecutively
without randomization. Majority of the women refused screening for diabetes mellitus and the
predominant reason was aversion to multiple needle pricks apart from laboratory investigations
59
for their routine antenatal care. The diagnosis of proteinuria was made using dipsticks because of
the additional cost of performing 24hour urinary protein measurements.
Industrial actions accounted for some of the attrition –most of them could not be reached until
several days after their delivery. Other women notified the investigator of their deliveries but this
was done several days after delivery. These women, if included, may have improved statistical
significance. Some patients might have adhered to the instruction to abstain from multivitamins;
it may be difficult to tell whether the use of multivitamins contributed to the occurrence of pre-
eclampsia or hypertensive disorders of pregnancy (HDP).
60
CONCLUSION AND RECOMMENDATIONS
In conclusion, plasma glutathione and superoxide dismutase values before 20 weeks gestation
may not be useful for prediction of pre-eclampsia in low risk women in Ibadan. Larger hospital
or community based studies are required to confirm these findings. Additional research may also
be required to identify the influence of multivitamin use before pregnancy on the likelihood of
developing pre-eclampsia. Continued research in the form of cohort studies is also required to
determine the most suitable antioxidant marker for prediction of pre-eclampsia and hypertensive
disorders of pregnancy.
61
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75
APPENDIX 1
ETHICAL APPROVAL
76
APPENDIX II
LETTER OF ATTESTATION
77
APPENDIX III
DEPARTMENT OF OBSTETRICS & GYNAECOLOGY, UNIVERSITY COLLEGE
HOSPITAL, IBADAN
ANTENATAL SUPEROXIDE DISMUTASE ESTIMATION FOR PREDICTION OF PRE-
ECLAMPSIA
Serial:................. Hospital number...............................
Biodata:
1. Age(years)........................
2. Occupation: a. Unemployed b. Semi-skilled c. Skilled d. Highly skilled
3. Education: a. Nil b. Primary c. Secondary d. Tertiary
Booking parameters & Obstetric history
4. Parity:...........................................................................................................
5. Booking weight (kg):...................................................................................
6. Booking gestational age(GA) ( )
7. GA at sampling ( )
8. Booking BP(mmHg)....................................................................................
9. Mean arterial blood pressure:......................................................................
10. Urinalysis:...................................................................................................
11. Previous history of hypertensive disorder: yes( ) no( )
12. Type: a. PE( ) b. ECL( ) c. CHTN( ) d. CHTN+ SUPERIMPOSED( ) e. PIH( )
13. Gestational age at diagnosis: ( )
14. Year of diagnosis( )
15. Pregnancy number( )
16. Delivery outcome: a. SVD ( ) b. IOL + SVD c. IVD( ) d. ABD( ) e. Destructive ( )
f. ELSCS ( ) g. EMLSCS ( )
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Past Medical & Family history & Drug history:
17. Family history of hypertension: yes( ) no( )
18. If yes to 15, in which relative:......................................................................
19. Previous history of diabetes mellitus yes( ) no( )
20. Type: GDM( ) pre-gestational( )
21. Use of haematinics yes( ) no( )
22. Use of multivitamin supplements yes( ) no( )
23. How frequently have you used these multivitamin supplements in the past 3 months?
a. Daily b. Weekly c. Fortnightly d. When I remember e. Not at all
24. Alcohol intake yes( ) no( )
25. Smoking yes( ) no( )
26. Any other drugs being used(please state):.................................................................
27. History of bleeding disorders or coagulopathy.........................................................
28. If yes, please state type:...................................................................................................
29. Blood sugar at recruitment........................................................................................
30. Antenatal antioxidant estimation:
SOD:.......................................................
To be filled following when presenting in labour or following diagnosis of primary
outcome:
1. Gestational age at presentation( )
2. Gestational age in labour ( )
3. BP in labour...........................BP pre-op................................................
4. Urinalysis..............................................................................................................
5. Mode of delivery: a. SVD ( ) b. IOL + SVD ( ) c. ABD ( ) d. IVD ( ) e.
Destructive ( ) f. ELSCS ( ) g. EMLSCS( )
6. Indication for Emergency Cesarean section....................................................................
7. Diagnosis of hypertensive disorder a. yes( ) b. No ( )
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8. Type of disorder: a. PE ( ) b. ECL ( ) c. CHTN( ) d. CHTN+ SUPERIMPOSED( )
e. PIH( )
9. Time of diagnosis: antepartum( ) intrapartum( ) post partum( )
10. State number of days post partum............................................................
11. Maternal outcome: a. Satisfactory b. Admission to ICU c. Death d.
Others:.......................................................................................................
12. Baby’s weight: ( ), Gender( ), Apgar: 1minute( ) 5minute( )
13. Admission to SCBU yes( ) no( )
14. Indication for admission to SCBU:...........................................................
15. Repeat antioxidant values:
a. SOD:.............................. b. GPx:...............................
16. Number of days on admission ( )
17. BP before discharge ( ) Mean arterial blood pressure ( )
18. Urinalysis before discharge( )
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APPENDIX IV
INFORMED CONSENT
IRB Research Approval number:
This approval will lapse on:
Title of research: Antenatal superoxide dismutase estimation for prediction of pre-eclampsia
Researcher: This study is being conducted by Dr. O. O. Enabor of the Department of Obstetrics
and Gynaecology, University College Hospital, Ibadan. The study is self sponsored.
Purpose: The purpose of this research is to find out whether antioxidant enzymes are useful in
predicting whether a pregnant woman will develop pre-eclampsia.
Proceedure of research: Women who are pregnant with gestational age less than 20weeks and
are coming to register for antenatal care will be asked to participate in the study once they
consent. They will be asked some questions in the antenatal clinic regarding any previous
pregnancies, illnesses, surgeries and use of drugs particularly multivitamins. They will be asked
to provide a blood sample for estimation of antioxidants and they will be tested for diabetes
mellitus. They will be allowed to continue their antenatal care until the end of their pregnancies
or in the event that they develop hypertension and protein in their urine. A repeat sample of
antioxidants will then be requested at this time. Those who participate will be asked to avoid
multivitamin preparations for the period of the study though iron and folic acid tablets can be
used. The study is expected to last for the duration of the participant’s pregnancy. There are no
risks expected from participating in this study apart from minor discomfort during blood
sampling. This will not differ from the patient’s experience during routine antenatal blood
investigations. The participation will not cost you anything.
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The objective of the study is to find out whether these substances can be used to find out whether
a woman is at risk of developing pre-eclampsia. Pre-eclampsia is a major cause of problems in
pregnancy and can lead to eclampsia, a major contributor to maternal death. This information can
be used to help the participants and other pregnant women.
All information collected in the study will be coded and the patient’s names will not be used.
However, institutions associated with proper conduct of research e.g: NAFDAC, NHREC and
ethics regulators may require these records. Your participation in this research is entirely
voluntary and if you choose not to participate this will not affect your treatment in this hospital
in any way. You will be compensated for lost wages and cost of transport to and from the
research site but you will not be paid any fees for participation in this research.
You can also choose to withdraw from the research at anytime. Please note that some of the
information that has been obtained may be used for reports and publication. Injury during this
study is unlikely but any injury occurred in the process of this research will be taken care of at
the University of Ibadan Teaching Hospital. The research will bear the cost of this treatment. The
researchers will inform you of the outcome of the research through a news bulletin or journal
article. During the course of the research, you will be informed about any information that may
affect your continued participation or your health. There is no plan yet to contact participants
about commercial products arising from this research. In conclusion, there are no conflicts of
interest. The researchers have no affiliation with the company manufacturing the kits used to
analyse the blood samples.
Statement of person obtaining informed consent:
I have fully explained this research to:
and have given sufficient information, including the risks and benefits to make an informed
decision.
DATE: _______________________________________SIGNATURE ____________________
NAME:_______________________________________
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Statement of person giving consent:
I have read the description of the research or have had it translated into language I understand. I
have also talked it over with the doctor to my satisfaction. I understand that my participation is
voluntary. I know enough about the purpose, methods, risks and benefits of the research study to
judge that I want to take part in it. I understand that I may freely stop being part of this study at
any time. I have received a copy of this consent form and additional information sheet to keep
for myself.
DATE: __________________________________ SIGNATURE_________________________
NAME: ___________________________________
WITNESS’ SIGNATURE:____________________
WITNESS’ NAME: _______________________
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The following individuals can be contacted concerning any questions about this research:
Chairman: UI/UCH Ethics Committee, Biode Building, 2nd Floor
` Room T10, IMRAT, College of Medicine
University of Ibadan.
E-mail: [email protected]
The Principal Investigator:
Name: Dr Obehioye .O. Enabor
Dept: Obstetrics & Gynaecology.
Phone no: 08036088220
E-mail: [email protected]
PLEASE KEEP A COPY OF THE SIGNED INFORMED CONSENT.