A STUDY ON STATUS OF LIPID METABOLISM IN

PREGNANCY INDUCED HYPERTENSIVE WOMEN

Thesis submitted in partial fulfillment for the degree of

Doctor of Philosophy in Medical Biochemistry

By

J.VISALA SREE

Under the guidance of

Dr. PHILIPS ABRAHAM

Vinayaka Missions University

(Vinayaka Missions Research Foundation, Deemed University)

Ariyanoor, Salem - 636308

Tamilnadu, India

MARCH, 2017

Dr. Philips Abraham, Ph.D Place:

Associate Professor, Date:

Department of Biochemistry

Vinayaka Missions Kirupananda Variyar Medical College

Salem

CERTIFICATE

I, Dr. Philips Abraham, certify that the thesis entitled “ A

Study on status of lipid metabolism in pregnancy induced

hypertensive women” submitted by Ms. J.Visala sree, for the

award of the degree of Doctor of Philosophy in Medical

Biochemistry is the record of research work carried out by her

during the period July 2012 to March 2017 under my

guidance and supervision and that this has not been formed the

basis for the award of any other degree, diploma,

associateship, fellowship or any other similar titles in this or

any other institution of higher learning.

Signature & Official seal of the guide

DECLARATION

I, J. Visala sree, declare that the thesis entitled “A Study

on status of lipid metabolism in pregnancy induced

hypertensive women” submitted by me for the award of Doctor

of Philosophy in Medical Biochemistry is the record of

research work carried out by me during the period July 2012 to

March 2017 under the guidance of Dr. Philips Abraham and

that has not formed the basis for the award of any other degree,

diploma, associateship, fellowship or another similar titles in

this or any other institution of higher learning.

Place: Salem Signature of the candidate

Date

ACKNOWLEDGEMENT

I thank god for the wisdom and perseverance that he has been bestowed

upon me during this research project, and indeed, throughout my life:

I take this opportunity to express my most humble and sincere gratitude and

indebtedness to my PhD supervisor Dr. Philips Abraham, Associate Professor,

Department of Biochemistry, Vinayaka Mission Kirupananda Variyar Medical

College, Salem, for his constant support, guidance, encouragement and

valuable advice in this work.

I would like to place on record my humble and sincere acknowledgement to

the honourable Chancellor of Vinayaka Missions University, Dr.Mohan, Dean,

Dr.P.M.Subramanyam, former Dean, Dr. B.Usha, Vice principal., AMCH for

their kind concurrence to fulfil my assignment in this prestigious institution. I

extend my grateful thanks to them.

I thank Dr.Manu Ghatikesh, HOD, Biochemistry, Dr.Kanmani, Professor,

Biochemistry, Dr.K.Ponsuganthi, Associate professor., Biochemistry,

Dr.S.Ramya, Assistant professor., Biochemistry, Dr.N.Saravanan., HOD

Obstetrics&Gynaecology., Dr.Vadivoo natarajan., Associate professor,

Microbiology, Dr.Meizhagan, HOD, Forensic medicine, Dr.Paramesh, HOD ,

Pharmacology, Dr.Aruna, HOD, Anatomy, Dr.Illango, Dept. of surgery,

Annapoorna Medical college & Hospital for their support.

I would also like to thank Dr.Manoharan.P.S, Dean, Dr.Evanageline Jones,

HOD, Biochemistry, Dr.Jeypal, former Dean, Dr.Rajesh, Prof, Microbiology,

VMKV Medical College and hospital for their constant support in all official

formalities.

With great sense of gratitude, I would like to thank Dr.K. Shanthi Naidu, HOD,

Clinical Laboratory Medicine, CARE Hospitals, Banjara hills, Hyderabad for

building up the confidence levels in me.

From the bottom of my heart, I would like to express my special appreciation

to my husband Mr. G. Amar kumar, Tutor, Department of Microbiology,

AMC&H and also to my daughter A.V .Havisha sree for their constant support.

I honestly thank Dr.P.Sivaprasad, Asst professor., Biochemistry, AMC&H for

his valuable support in completing my thesis work.

I also thank Dr.Saravanan, Mr.Vignesh, Dept.of Microbiology, and AMC&H for

their valuable suggestions in writing my thesis.

I sincerely thank Dr.Vishal Babu, Dr.Desigamani, Dr.Viswa kalyan.K,

Dr.Suresh.P for their guidance in building up my hypothesis

I am deeply grateful to, Dr. K. Sugendran, Dr. Sachu Philip, for their indirect

support to complete my thesis work.

I express my blessing to my student Dr. Adithya for her support in sample

collection.

I owe my special thanks to the lab technicians Ms.Priscilla, Ms.Sujata,

Ms.Ruba, Ms.Nandini, Ms.Sivagami, Mr.Laxmikanthan, Mr.Franklin and also

to the computer operator Ms.Swathi for their continuous technical support.

I am very grateful and deeply indebted to my parents, sisters for their faith,

moral support and encouragement for fulfilling my work.

The successful completion of this manuscript was made possible through the

invaluable contribution of number of people.

CONTENTS

S.No TITLE Page No

1 INTRODUCTION 1-38

2 REVIEW OF LITERATURE 39-60

3 NEED OF THE STUDY 61-62

4 OBJECTIVES AND HYPOTHESIS 63

5 MATERIALS AND METHODS 64-109

6 RESULTS AND DISCUSSION 110-145

7 SUMMARY 146-148

8 CONCLUSION 149

9 LIMITATIONS 150

10 FUTURE PROSPECTIVES 151

11 BIBLIOGRAPHY 152-207

12 PUBLICATIONS/ETHICAL

CLEARANCE

208-212

LIST OF THE CONTENTS

S.No TITLE PAGE No

1.0 INTRODUCTION

1.1 Pregnancy induced hypertension 1

1.2 Prevalence of PIH 1-2

1.3 Classifications of hypertensive disorders in

pregnancy

3

1.3.1 Preeclampsia 3

1.3.2 Chronic hypertension 4

1.3.3 Superimposed preeclampsia 4

1.3.4 Pregnancy induced hypertension 4

1.4 Etiology of PIH 5

1.4.1 Abnormal placentation 5-6

1.4.2 Vasculopathy and inflammatory changes 7

1.4.3 Immunological factors 7-8

1.5 Pathophysiology of PIH 8

1.5.1 Pathogenesis of PIH 8-9

1.5.2 Vasospasm 9-10

1.5.3 Endothelial dysfunction 10-12

1.6 Pathological changes in various organs 13

1.6.1 Brain 13

1.6.2 Eye 13

1.6.3 Kidneys 13-14

1.6.4 Liver 14

1.6.5 Cardiovascular 14

1.6.6 Lungs 14

1.6.7 Hematological changes 15

1.7 ED- A major culprit in PIH 15

1.7.1 The vascular endothelium and its functions 15-17

1.8 Endothelium in pregnancy 17

1.8.1 Endothelium and regulation of vascular tone in

normal pregnancy

17-18

1.8.2 Endothelial dysfunction in PIH 19

1.9 Factors associated with ED 21

1.9.1 Vascular endothelial growth factor 21-22

1.9.2 Placental growth factor 22

1.9.3 Soluble fms like tyrosine kinase receptor-1 23-24

1.9.4 Cytokines 24-27

1.9.5 Circulating lipids and lipoproteins 27-28

1.10 Maternal changes in pregnancy 28

1.10.1 Carbohydrate metabolism in pregnancy 28-29

1.10.2 Lipid changes in normal pregnancy 29

1.11 Maternal changes in PIH 30

1.11.1 Carbohydrate metabolism in PIH 30

1.11.2 Lipid metabolism in PIH 31

1.11.3 Amino acid metabolism in PIH 32

1.12 Oxidative stress- A predominant toxic factor in

pathogenesis of PIH

32-33

1.12.1 ROS and free radicals 33-34

1.12.2 MDA 34

1.12.3 Antioxidant 34-35

1.12.4 Oxidative stress in pregnancy 35-36

1.12.5 Oxidative stress in preeclampsia 36-38

2.0 REVIEW OF LITERATURE

2.1 Lipids 39-41

2.1.1 Apolipoproteins 42-44

2.2 Atherogenic index 45

2.3 Oxidative stress 46

2.3.1 MDA 46-47

2.3.1 FRAP 48

2.4 Inflammation 48

2.4.1 Inflammatory cytokines 49

2.4.2 TNF-α 49-50

2.4.3 IL-6 50-52

2.4.4 HsCRP 52-54

2.5 Angiogenic and anti angiogenic factors 54

2.5.1 VEGF 55-56

2.5.2 PlGF 56-57

2.5.3 Antiangiogenic factor (sFlt-1) 57-59

2.6 Endothelial dysfunction 59

2.6.1 Nitric oxide 59-60

3.0 NEED OF THE STUDY 61-62

4.0 OBJECTIVES AND HYPOTHESIS 63

5.0 MATERIALS AND METHODS

5.1 Selection of patients 64

5.2 Study groups 65

5.3 Specimen collection and processing 66-67

5.4 Measurement of BMI 67-68

5.5.1 Estimation of plasma cholesterol 68-69

5.5.2 Estimation of Triglycerides 70-71

5.5.3 Estimation of HDL by Immunoinhibition method 72-73

5.5.4 Determination of VLDL by calculation method 73

5.5.5 Determination of LDL by calculation method 73

5.5.6 Estimation of apoA-1 74-76

5.5.7 Estimation of apoB 76-78

5.5.8 Estimation of Lp(a) 79-81

5.5.9 Atherogenic index calculation 81

5.5.10 Estimation of MDA 81-83

5.5.11 Estimation of Nitric oxide as nitrite 83-86

5.5.12 FRAP 87-89

5.5.13 Estimation of TNF-α 89-93

5.5.14 Estimation of IL-6 93-96

5.5.15 Estimation of sFlt-1 96-100

5.5.16 Estimation of VEGF 100-103

5.5.17 Estimation of PlGF 103-105

5.5.18 Estimation of Hemoglobin 106-107

5.5.19 Estimation of urine proteins 108-109

5.6 Anthropometric measurements 109

5.6.1 Statistical analysis 109

6.0 RESULTS AND DISCUSSION 110

6.0 Sociodemographic characters 111-113

6.1 Objective :1 To determine the serum lipid

and lipoprotein levels in PIH women

114

6.1.1 Comparison of lipid profile, lipoproteins,

apolipoproteins in PIH and control women

115-120

6.2 Objective: 2 To assess the Antioxidant

capacity, lipid peroxidation in PIH women

120

6.2.1 Comparison of MDA and FRAP between PIH

and control women

121-124

6.3 Objective: 3 To assess the levels of

inflammatory markers, angiogenic and anti

angiogenic factors in PIH women.

125

6.3.1 Comparison of inflammatory markers between

controls and PIH women

126-130

6.3.2 Comparison of angiogenic and anti angiogenic

factors between controls and PIH women

131-136

6.4 Objective :4 To assess the endothelial

dysfunction and atherogenic index (AI) in

PIH women

137

6.4.1 Comparison of NO & AI between Controls, PIH

women

137-139

6.5 Objective: 5 Correlation of lipids and

lipoproteins with NO, angiogenic and anti

angiogenic factors.

140-145

7.0 SUMMARY 146-148

8.0 CONCLUSION 149

9.0 LIMITATIONS 150

10.0 FUTURE PROSPECTIVES 151

11.0 BIBLIOGRAPHY 152-207

12.0 PUBLICATIONS/ ETHICAL CLEARANCE 208-213

ABBREVATIONS

ADMA Asymmetric dimethylarginine

AIP (AI) Atherogenic index of plasma

AMPA α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor

ApoA-1 Apolipoprotein A-1

ApoB Apolipoprotein B

AT-1 AA Angiotensin type II receptor agonistic autoantibodies

AT1 Angiotensin receptor II type I

B2 Bradykinin receptors

BMI Body mass index

BMR Basal metabolic rate

CAT Catalase

cGMP Cyclic guanosine monophosphate

CHOD Cholesterol oxidase

CE Cholesterol esterase

CMC-EQAS Christian medical college external quality assurance scheme

CM Chylomicrons

CRP C- reactive protein

DBP Diastolic blood pressure

DHEA Dehydroepiandrosterone

DHAP Di hydroxy acetone phosphate

DNA Deoxy ribonucleic acid

E Eclampsia

EC Endothelial cell

ED Endothelial dysfunction

EDTA Ethylene diamine tetra acetic acid

ELISA Enzyme linked immune sorbent assay

eNOS endothelial nitric oxide synthase

ET-1 Endothelin-1

FFA Free fatty acid

Flt-1 fms like tyrosine kinase-1

FRAP Ferric reducing ability of plasma

G6PDH Glucose-6- phosphate dehydrogenase

GABAA γ-aminobutyric acid receptor

GDM Gestational diabetes mellitus

GH Gestational hypertension

GK Glycerol kinase

GPO Glycerol phosphate oxidase

GPx Glutathione peroxidase

GR Glutathione reductase

GSH Gluthathione

Hb Hemoglobin

HDL High density lipoprotein

HELLP Hemolysis elevated liver enzymes, low platelets

HIF Hypoxia-inducible factor

HNE 4-hydroxynonenal

HRP Horseradish peroxidase

HsCRP High sensitive C- reactive protein

HSP Heat shock proteins

IDL Intermediate-density lipoprotein

IERD Institutional Ethical Research Board

IL-1β Interleukin 1β

IL-6 Interleukin -6

IL-8 Interleukin-8

IQC Internal quality control

ISSHP International Society for the Study of Hypertension in Pregnancy

IUGR Intra uterine growth restriction

KDR Kinase insert domain region

LDL Low density lipoprotein

LJ charts Levey-Jenning charts

Lp (a) Lipoprotein (a)

LPL Lipoprotein lipase

LRP Lipoprotein receptors

MCP-1 Monocyte chemotactic protein-1

MDA Malondialdehyde

MLCK Myosin light chain kinase

MLP Myosin light chain phosphatase

MMR Maternal mortality rate

mRNA messenger ribonucleic acid

MTTP Microsomal triglyceride transfer protein

NF-κβ Nuclear factor κβ

NHBPEP National high blood pressure education programme

NO Nitric oxide

NOD-like receptor 3

NLRP3- Nucleotide-binding oligomerization domain-like receptors.

OS Oxidative stress

Ox-LDL Oxidized LDL

PAI-1 Plasminogen activator inhibitor-1

PDGF Platelet- derived growth factor

PDIA2 Protein disulfide Isomerase family A member 2

PE Pre-eclampsia

PGI2 Prostacyclin

PIH Pregnancy induced hypertension

PKA Protein kinase A

PKG Protein kinase G

PlGF Placental growth factor

POD Peroxidase

PON-1 Paraoxonase-1

PUFA Polyunsaturated fatty acids

RAS Renin-angiotensin system

RClS Reactive chlorine species

RCT Reverse cholesterol transport

RNS Reactive nitrogen species

ROS Reactive oxygen species

RPM Revolutions per minute

RT-PCR Reverse transcriptase PCR

SBP Systolic blood pressure

Sd-LDL Small dense LDL

sFlt-1 Soluble fms like tyrosine kinase-1

SGA Small gestational age

SICAM Soluble cell adhesion molecule

SOD Superoxide dismutase

TAC Total antioxidant capacity

TBARS Thiobarbituric acid reactive substances

TBA Thiobarbituric acid

TC Total cholesterol

TCA Trichloroacetic acid

TGL Triglycerides

Th1 T helper cell 1

Th2 T helper cell 2

TMP Tetramethoxy propane

TNF- α Tumour necrosis factor-alpha

TXA2 Thromboxane A2

VCAM-1 Vascular cell adhesion molecule -1

VCAM-1 Vascular cell adhesion molecule -1

VEGF Vascular endothelial growth factors

VEGFR Vascular endothelial growth receptor

VLDL Very low density lipoprotein

WHO World health organization

1

1. INTRODUCTION

1.1 Pregnancy induced hypertension (PIH)

Hypertensive disorders during pregnancy are the common medical

disorders in pregnancy. It effects both on expectant mother and foetus [Vata,

P.K et al., 2015, Ananth, C.V et al., 2013]. Hypertensive pregnancies are

associated with increased risk of adverse foetal, ne1onatal and maternal

outcomes, including preterm birth, intrauterine growth restriction (IUGR),

perinatal death, acute renal or hepatic failure, antepartum haemorrhage,

postpartum haemorrhage and maternal death [Sajith, M et al., 2014].

1.2 Prevalence of PIH

In developing countries all over the world, about 8-10% of pregnant

women suffers with preeclampsia (PE) [Asghari, E et al., 2016]. The research

findings solidly confirm that the maternal mortality rate (MMR) is 400 maternal

deaths out of 100,000 live births. In Africa 1:16 life time risk is recorded which

is the highest, compared to the Western nations (1:2800). Eclampsia (E)

accounts for 12 % of such deaths [Shaheen, A et al., 2016]. Approximately 12

to 25% of foetal growth restriction, small for gestational age infants as well as

15 to 20% of all preterm births are attributable to preeclampsia [Jeyabalan, A.,

2013]. In developing countries, the PE & E are leading threats to safe

motherhood where a woman is seven times more likely to develop these

conditions. In such sceneries, it is assessed that 10–25% of these cases (an

estimated ∼40 000 women) lead to maternal deaths annually [Agrawal, S et

2

al., 2016]. The national incidence of PIH is 15.2% in India, while it is four times

higher in primipara women than in multipara [Saxena, S et al., 2014].

In pregnancy there are various categories of hypertensive disorders

which include pregnancy induced hypertension (gestational hypertension),

preeclampsia, eclampsia and chronic hypertension. Pregnancy induced

hypertension is appearance of hypertension of greater than 140/90 mm of Hg

after 20 weeks of gestation. If hypertension is associated with significant

proteinuria it is termed preeclampsia. Complication of preeclampsia along with

seizures is called eclampsia. Hypertension originating before pregnancy is

known as chronic hypertension. Chronic hypertension might be superimposed

with preeclampsia or eclampsia [Watanabe, K et al., 2013].

Pregnancy induced hypertension is a multiple organ system disease

which is exclusive to pregnancy and can cause maternal complications like

eclampsia, Hemolysis, Elevated Liver enzymes, low platelets (HELLP)

syndrome, acute renal failure, cerebrovascular accidents etc. It can effect the

foetus like foetal growth restriction, oligohydramnios, foetal distress etc.

During pregnancy the priority concerning hypertension is in making the correct

diagnosis inorder to distinguish the pre-existing (chronic) from pregnancy

induced (gestational hypertension). It is also important to distinguish the blood

pressure levels as either mild (140/90 to 159/109 mm of Hg) or severe (≥

160/110 mm of Hg) rather than as stages [Backes, C. H et al., 2011].

3

1.3 Classification of hypertensive disorders in pregnancy

There are various classifications for hypertensive disorders in

pregnancy based on diagnostic criteria [WHO Study Group on the

Hypertensive Disorders of Pregnancy, 1987; Helewa, M.E et al., 1997;

NHBPEP, 2000]. The extensively accepted classification presently is

International Society for the Study of Hypertension in Pregnancy (ISSHP)

[Brown, M. A et al., 2014]. According to this classification there are four

categories

Pre-eclampsia

Chronic hypertension – essential or secondary

Pre-eclampsia superimposed on chronic hypertension

Pregnancy induced hypertension- also referred as Gestational

hypertension (GH)

1.3.1 Pre-eclampsia (PE)

As per ISSHP classification preeclampsia is defined as new onset

hypertension of more than 140/90 mm of Hg after 20 weeks gestation,

proteinuria of more than 300mg/day [Hladunewich, M et al., 2007]. This

definition is for the research purposes. But, if there is any evidence of foetal

growth restriction or end organ damage without proteinuria, this condition is

branded clinically as pre-eclampsia as per ISSHP. This condition occurs in 5

to 8% of all pregnancy.

4

1.3.2 Chronic hypertension

Chronic hypertension is defined as BP > 140/90 mm of Hg before

pregnancy or before 20 weeks gestation. It complicates 3% of pregnancies.

Findings of proteinuria more than 300 mg/day or evidence of foetal growth

restriction in chronic hypertensive cases is termed as pre-eclampsia

superimposed on chronic hypertension.

1.3.3 Superimposed preeclampsia

Superimposed preeclampsia is diagnosed in the following three cases,

New onset proteinuria (≥300 mg/24 hours) in hypertensive women who

had not exhibited proteinuria before 20 weeks gestation.

Documented hypertension and proteinuria prior to pregnancy or

detected before 20 weeks gestation, one or both of which developing

after 20 weeks gestation.

Documented renal disease with proteinuria former to pregnancy or

detected before 20 weeks of gestation, which is escorted with new

onset hypertension after 20 weeks gestation.

1.3.4 Pregnancy induced Hypertension

During pregnancy, the woman is diagnosed as gestationally

hypertensive when the blood pressure touches ≥140/90 mmHg for the first

time after 20 weeks gestation without proteinuria. The Blood pressure usally

normalizes by 12 weeks postpartum.

5

1.4 Etiology of Pregnancy induced Hypertension

Pregnancy induced hypertensive disorder can be triggered by various

etiological factors. It is a disorder of theory and condition which involves all

organs in the body. The foremost causes of pregnancy induced hypertension

are [Kintiraki, E et al., 2015]

Abnormal placentation

Vasculopathy and inflammatory changes

Immunological factors

Genetic factors

1.4.1 Abnormal placentation

During normal pregnancy, the placental bed spiral arterioles undergo a

sequence of physiological changes. The endovascular trophoblast invades the

placental bed spiral arterioles by breaking down the endothelium, internal

elastic lamina and muscular coat of the vessel, substituting with fibrinoid

material. These modifications occurs in two waves, the invasion of decidual

segments of spiral arterioles occurs in the first trimester and myometrial

segments, by a following wave in the second trimester. These biological

changes acclimatize the vessels supplying the placenta from muscular end

arteries to wide mouth sinusoids, which become unresponsive to vasoactive

substances. The vascular supply is thus renovated into low pressure high flow

system inorder to meet the needs of the foetus and placenta [Furuya, M et al.,

2008, Hladunewich, M et al., 2007].

6

In PIH, because of inadequate maternal vascular response to

placentation, the above changes are limited to the decidual segments of the

uteroplacental arteries, the principal trophoblast invasion is partially impaired,

and therby subsequent wave of trophoblastic invasion also fails to occur.

Therefore, the myometrial segments of spiral arterioles are left with their

musculoelastic design, and there by responding to hormonal substances

[Hladunewich, M et al., 2007]. This limitation of normal physiological changes,

results into restricted placental flow and also becomes more critical with

progressing gestation. The Intra myometrial segments of spiral arterioles

display changes like endothelial damage, insudation of plasma constituents

into vessel wall, proliferation of lipid laden myointimal cells and medial

necrosis named acute atherosis [Kim, J.Y et al., 2015, Kim, Y.M et al., 2015].

The affected vessels with atherosis develop aneurysmal dilatation and may

also impair placental blood flow by obstructing the lumen. These changes

diminish the placental blood flow pathologically which leads to infarcts, patchy

necrosis, intracellular damage to the synctiotrophoblast and obliterative

endarteritis of foetal stem arteries. The incomplete development of foetal

microvasculature associated with foetal growth restriction has been suggested

in pregnancy induced hypertension [Abdo, I et al., 2014].

7

1.4.2 Vasculopathy and inflammatory changes

In PIH, various noxious substances are released from the placenta and

decidua in response to ischemic changes. These constituents assist as

mediators to aggravate endothelial injury. Cytokines such as tumour necrosis

factor-alpha (TNF-α) and interleukins contributes to the oxidative stress

characterized by the release of reactive oxygen species (ROS) and free

radicals which lead to the formation of lipid peroxides. These peroxides in turn

can generate vastly toxic radicals that can injure the endothelial cells, modify

the nitric oxide production and can also cause prostaglandin imbalance [Tayal,

D et al., 2014]. Oxidative stress can lead to the production of lipid laden

macrophage foam cells appreciated in atherosis [Hubel, C.A et al., 1999],

stimulation of micro vascular coagulation seen in thrombocytopenia and

enhanced capillary permeability observed in oedema and proteinuria

[Granger, D et al., 2010].

1.4.3 Immunological factors

Immunological factors play an important role in the expansion of

pregnancy induced hypertension. This scenario includes deficiency of blocking

antibodies, reduced cell mediated immune responses, activation of neutrophils

and participation of cytokines. An abnormal immune reaction between foetal

trophoblast and placental bed maternal tissue is a central factor in the

aetiology of PIH. This is supported by the findings that the women with first

pregnancy were mostly complicated by this syndrome. The change of partner

8

and pregnancy after birth control methods which prevent sperm exposure

might increase the incidence. Women who develop pregnancy induced

hypertension have documented reduced helper T cells (Th 1) proportion in

early second trimester, compared with normotensive pregnant women.The

imbalance between Th 1/Th 2 might be mediated by adenosine which is found

in higher concentrations in pregnancy induced hypertensive women. The

helper lymphocytes promote implantation by secreting cytokines and therefore

their dysfunction might lead to pregnancy induced hypertension [Laresgoiti-

Servitje, E et al., 2012].

1.5 Pathophysiology of PIH

1.5.1 Pathogenesis

PIH is characterized by vasospasm, endothelial dysfunction which

results into triggering of coagulation system [Lyall, F et al., 1996; Alladin, A. A

et al., 2012].

9

Figure 1: Pregnancy induced hypertension – pathogenesis [Peter von

Dadelszen et al., 2002]

1.5.2 Vasospasm

A decline in synthesis of vasodilator nitric oxide (NO) and amplification

in the production of endothelin by the vascular endothelium could account for

distinguishing vasospasm and also for activation of circulating platelets in PIH

women. Accompanying endothelial damage might cause interstitial leakage

through which blood constituents like platelets and fibrinogen are dropped sub

endothelially with diminished blood flow because of maldistribution; ischemia

of neighboring tissues would lead to necrosis, haemorrhage and other end

10

organ disturbances which are the characteristics of this syndrome [Alladin, A.

A et al., 2012].

1.5.3 Endothelial dysfunction

The harmful placental factors released by ischemic variations and toxic

radicals produced by oxidative stress cause activation and dysfunction of

vascular endothelium. Integral endothelium reduces responsiveness of

vascular smooth muscles to agonists by releasing nitric oxide and it also have

anticoagulant properties. Damage endothelium discharges substances which

promote coagulation and increases the sensitivity to vasopressors. The

markers of endothelila dysfuction like amplified circulating fibronectin, factor

VIII antigen and thrombomodulin are told in pregnancy induced hypertension/

preeclampsia [Yuan, H. T et al., 2005].

Enhanced pressor responses

Normal pregnant women are noncompliant to infused vasopressors like

angiotensin II. But, the women who are destined to develop pregnancy

induced hypertension/pre eclampsia have enhanced vascular reactivity to

angiotensin II. This amplified sensitivity heads the onset of hypertension.

Autoantibodies are thought to activate angiotensin receptor II type I (AT1)

receptors and increased angiotensin II sensitivity. Up regulation of bradykinin

receptors (B2) leads to hetero dimerization with ATI receptors. ATI/B2

receptors have been shown to increase responsiveness to angiotensin II in-

vitro [Shah, D. M. et al., 2005].

11

Prostaglandins

A decrease in the production of endothelial prostacyclin (PGI2), a

vasodilator produced by the mediation of phospholipase A2 is documented in

pregnancy induced hypertension/pre eclampsia. [Walsh, S. W et al., 1986].

Nitric oxide

Nitric oxide, a potent vasodilator synthesized from L-arginine by

endothelial cells. It helps in maintaining the normal low pressure of feto

placental circulation in humans whereas PIH women were associated with

decreased endothelial nitric oxide synthesis [Var, A et al., 2003].

Endothelin

Endothelin-1, a potent vasoconstrictor and is the primary isoform

produced by human endothelium. However, pregnancy induced hypertensive

women are associated with higher endothelin levels in response to endothelial

activation [Taylor, R. N., 1990].

Circulating angiogenic factors

Vascular endothelial growth factors (VEGF) are the endothelial specific

growth factors which play a major role in stimulating angiogenesis; placental

growth factor (PlGF) is another member of VEGF family which is made

predominantly by placenta. The VEGF action is mediated by interaction with

two high affinity receptor tyrosine kinases namely,

Kinase insert domain region (KDR) and

12

Fms like tyrosine kinase-1 (Flt-1)

These receptors are expressed on the endothelial surface. The

alternative splicing of Flt-1 gene results in the production of sFlt-1 which

cannot attach to the cell membranes and is directly secreted in to the maternal

blood circulation. It can act by antagonizing the actions of VEGF and PlGF by

binding to it and thereby preventing its interaction with endogenous receptors.

Excess sFlt-1 production is observed in pregnancy induced hypertension/pre

eclamptic placentas, which creates an antiangiogenic state and also plays a

contributory role in the pathogenesis of maternal syndrome in pregnancy

induced hypertension/pre eclampsia. VEGF stimulates angiogenesis as well

as promotes vasodilation by increasing the production of nitric oxide and

prostacyclin, which are signalling molecules that are decreased in pregnancy

induced hypertension/pre eclampsia. PLGF is essential for vasculogenesis

and control of microvascular permeability [McMahon, K et al., 2013, Hoeben,

Ann et al., 2004, Qiong Zhou et al., 2010].

13

1.6 Pathological changes in various organs

Vasospasm, endothelial cell activation with subsequent platelet

activation and aggregation accounts for most of the pathological changes that

are observed in PIH.

1.6.1 Brain

Vasospasm and cerebral edema were involved in the cerebral

manifestations of pregnancy induced hypertension/preeclampsia. It has been

observed that small haemorrhages were scattered throughout its substance.

Death may occur due to massive haemorrhage in the brain. Along with there

may be cerebral oedema, increased intracranial tension, cerebral

haemorrhage, seizures and hyperaemia [Cipolla, M.J et al., 2007].

1.6.2 Eye

Hypertensive encephalopathy is characterisec by retinal haemorrhage,

exudates and papilledema which are considered as rare in pregnancy induced

hypertension. Occipital lobe vasospasm is the common cause of temporary

blindness which is found some times in severe preeclampsia [Cunningham,

F.G et al., 1995].

1.6.3 Kidneys

The kidneys are characterized by lesion called glomeruloendotheliosis

which consists of endothelial and mesangial cell swelling, basement

membrane inclusions but little disruption of renal endothelial podocytes. There

14

are proteinuria, decreased glomerular filtration rate and decreased urate

excretion [Al-Jameil, N et al., 2014].

1.6.4 Liver

Sub endothelial fibrin deposition is associated with elevated liver

enzymes. These are associated with haemolysis and low platelet count due to

platelet consumption constituting the HELLP syndrome (haemolysis, elevated

liver enzymes, low platelets) [Pandey, C.K et al., 2015]. There could be

periportal haemorrhagic necrosis and sub capsular hematoma. The epigastric

pain and liver tenderness probably arise from capsule distention [Martin, J.N

Jr et al., 1998].

1.6.5 Cardiovascular

In initial phase, cardiac output is high with low peripheral resistance, but

as the disease progresses this changes to low cardiac output and high

peripheral resistance.The central venous pressure and pulmonary wedge

pressure are reduced. Generalized vasospasm is considered as basic factor.

Cardiac arrhythmia, failure and pulmonary oedema can occur due to the

disease or drugs used. Rarely peripartum cardiomyopathy was also reported

in preeclampsia women after delivery [Melchiorre, K et al., 2011].

1.6.6 Lungs

Pathological changes in lungs results into adult respiratory distress

syndrome, bronchopneumonia and airway obstruction.

15

1.6.7 Haematological

Platelet activation and coagulopathy, reduced plasma volume, amplified

blood viscosity [Juan, P et al., 2011].

1.7 Endothelial Dysfunction- A major culprit in PIH

1.7.1 The Vascular Endothelium and Its Functions

The arterial wall has 3 layers: the intima, including the endothelium, the

media, and the adventitia (Figure 2). Each of these layers has individual roles

in the systemic circulation. The endothelium is a monolayer of cells on blood

and lymphatic vessels. The thin squamous type of epithelium was virtually

invisible in light microscopy and initially considered as a nonessential

cellophane-like sheet

Fig 2: Structure of an arterial wall

16

The major functions of the endothelium are

Regulation of vascular tone

Regulation of vascular permeability

Pro- and anticoagulant activity

Contribution for the balance of pro- and anti-inflammatory mediators

Generation of new blood vessels

Interaction with circulating blood cells

Figure 3: Major functions of endothelium; eNOS endothelial nitric oxide

synthase, NO nitric oxide, cGMP cyclic guanosine monophosphate, NF-κβ

nuclear factor κβ, VCAM-1 vascular cell adhesion molecule -1, MCP-1

monocyte chemotactic protein-1 [Van der Oever, et al. 2010].

17

Endothelial cells are heterogenic; the phenotype varies according to the

requirements of the individual organ [Arid, W.C et al., 2012]. Endothelial cells

receive and respond to signals from both surrounding cells and tissues and

flowing blood. The response to a given stimulus may vary dramatically from

one vascular bed to another. Quiescent endothelial cells display the

thromboresistant, anti-adhesive and vasodilatory phenotype, whereas

activated endothelial cells have procoagulant, proadhesive, and

vasoconstricting properties. The normal relatively dilated state of the vascular

wall is maintained mainly by nitric oxide [Rajendran, P et al., 2013]. The

principal physiologic stimulus for endothelial NO synthesis is blood flow-

induced shear stress. This process is called flow mediated vasodilation.

1.8 Endothelium in Pregnancy

1.8.1 Endothelium and regulation of vascular tone in normal pregnancy

Normal pregnancy is characterized by vasodilation resulting in reduction

of peripheral vascular resistance. Blood pressure begins to decrease early in

the first trimester and reaches its nadir by 20 to 24 weeks' gestation. The

physiological changes are not completely understood, but the human

chorionic gonadotropin– induced increased production of relaxin by the corpus

luteum may facilitate vasodilation in normal pregnancy. Relaxin up-regulates

vascular gelatinase activity thereby contributing to the dilation of blood vessels

and reduction in myogenic reactivity of small arteries by activating the

endothelial endothelin B receptor–nitric oxide pathway [Failli, P et al., 2005].

18

The amount of Nitric oxide (NO) produced by endothelial NO synthase

(eNOS) is determined by the maximum capacity of the cell (eNOS expression

levels), the eNOS phosphorylation state, and the intracellular [Ca2+]

concentration in response to circulating hormones or physical forces [Boeldt,

D .S et al. 2011]. In early pregnancy, the scale of endothelium-dependent flow

mediated brachial artery dilation is determined in part by carriage of eNOS

gene polymorphism Asp298 variant [Savvidou, M.D et al. 2001]. Angiogenic

factors like vascular endothelial growth factor (VEGF) also may have an

important function in the increased production of NO and prostacyclin in

pregnancy via pathways involving phospholipase C, mitogen-activated protein

kinase, and protein kinase C. The balance between vasodilatory (NO,

prostacyclin) and vasoconstrictive (thromboxane A2, endothelin) substances,

and in parallel the balance between angiogenic and anti-angiogenic factors,

are speculated to be important determinants of blood pressure in pregnancy

[Possomato-Vieira, J.S et al. 2016]. In pregnancy, vascular nitric oxide (NO)

production is increased in the systemic and more so in the uterine

vasculature, thereby supporting maximal perfusion of the uterus [Boeldt, D.S

et al. 2011]. Increased activity of the NO vasodilatory mechanism occurs in

the maternal systemic vasculature in general and this is even more

pronounced in the uterine vasculature. Several studies have described

improvements in endothelial function in terms of brachial artery flow mediated

dilation during normal pregnancy with some alterations between the three

19

trimesters [Savvidou et al., 2001; Saarelainen, H et al., 2006; Saarelainen, H

et al., 2012].

1.8.2 Endothelial Cell Dysfunction in Pregnancy induced hypertension

Endothelial cell dysfunction or activation is a term used to define an

altered state of endothelial cell differentiation, typically induced as a result of

cytokine stimulation [Sprague, A.H, et al., 2009]. It represents an inflammatory

response to sublethal injury of these cells and is proposed to play a major role

in the pathophysiology of hypertension. Endothelial cell activation in

preeclampsia may result from a variety of circulating factors, angiogenic

factors, metabolic factors, and inflammatory mediators.

Endothelial dysfunction manifest biochemically by the synthesis and

secretion of a variety of endothelial cell products including prostanoids,

endothelin-1 (ET-1), platelet derived growth factor (PDGF), fibronectin,

selectins, and other molecules that influence vessel tone and remodeling

[Possomato-Vieira, J.S et al., 2016]. When these factors are elaborated in

response to acute mechanical or biochemical endothelial cell damage they

facilitate efficient wound healing. However, when activated by a chronic

pathological process, such as preeclampsia, these responses can create a

vicious circle of vasospasm, microthrombosis, and disruption of vascular

integrity, creating serious physiologic disturbances, which persist until the

inciting factor is eliminated [Rajendran, P et al., 2013].

20

Fig 4: Mechanism of Endothelial dysfunction [Rajendran, P et al., 2013]

Some insights into the vascular pathophysiology of PIH are derived

from clinical experience with thrombotic microangiopathic disorders occurring

during pregnancy. It has been noted that, while rare, pregnancy appears to

predispose or exacerbate the development of these syndromes of

microvascular thrombosis [Radhi, M et al., 2012].Thrombotic

thrombocytopenic purpura (TTP) is characterized by thrombocytopenia,

microangiopathic hemolytic anemia, renal involvement, neurological

symptoms, and fever. Like PIH, the signs of TTP during pregnancy most

commonly manifest in the late second trimester [Stavrou, E et al., 2009].

21

1.9 Factor associated with endothelial dysfunction

1.9.1 Vascular endothelial growth factor

The vascular endothelial growth factors are secreted as dimeric

glycoproteins which are involved in vasculogenesis and angiogenesis. In

humans this growth factor family includes VEGF-A and placental growth

factor. VEGF is a pro-angiogenic factor which promotes the proliferation,

survival of endothelial cells and induces vascular permeability. PlGF is a

VEGF homolog released by the placenta which also has pro-angiogenic

activity [Shibuya, M 2011]. VEGF is produced by many cell types including

tumor cells, macrophages, platelets, keratinocytes, and renal mesangial cells

[Duffy, A.M et al., 2013]. In adult mice VEGF is expressed by cell types

located adjacent to fenestrated endothelia including the epithelial cells of the

choroid plexus, renal podocytes, and hepatocytes [Maharaj, A.S et al., 2006].

Hypoxic cells can also induce VEGF-A production. Through the stimulation of

hypoxia-inducible factor (HIF) which can stimulate the release of VEGF-A

[Coppe, J.P et al., 2006].

The receptors of VEGF family which are present on vascular endothelial

cells include Flt-1 (VEGFR-1) and KDR (VEGFR-2, murine Flk-1). VEGF can

bind to both Flt-1 and KDR receptors whereas PlGF homodimers bind

exclusively to Flt-1. KDR is thought to be primarily responsible for VEGF's

action on endothelial cells [Shen, B.Q et al., 1998]. More recent work done by

Chappell et al suggest that the expression of anti angiogenic protein sFlt-1 by

22

Flt1 gene is regulated by the local VEGF availability [Chappell, J.C et al.,

2009].

VEGF induces vasodilation in the systemic vasculature by up regulating

the nitric oxide and prostacyclin levels in vascular endothelial cells [He, H et

al., 1999] in order to increase the uterine blood flow to the feto placental unit

[Itoh, S et al., 2002] and also to control the systemic blood pressure

[Facemire, C.S et al., 2009].

1.9.2 Placental growth factor

PlGF, which has approximately 53% homology with VEGF, is expressed

at high levels by the human placenta throughout all stages of gestation. PlGF

homodimers do not bind to the KDR receptor, but do bind to the Flt-1 receptor

with high affinity. PlGF has weak mitogenic activity and potentiates the actions

of VEGF [Park, JE et al., 1994]. PlGF plays a role in angiogenesis in

pathological settings [Luttun A, et al., 2002]. It controls trophoblast growth and

differentiation dilates uterine vessels, promotes EC growth, vasculogenesis

and placental development [De Falco, S 2012]. During pregnancy, the

placenta releases PlGF at high amounts into the maternal circulation. Levels

increase beginning in the 2nd trimester, peak during weeks 29–32 and decline

thereafter [Autiero M, et al., 2003, Powe, C.E et al., 2007]. In preeclampsia, it

was stated that the levels of serum PlGF are reduced in the second and third

trimesters of gestation [Livingston, J.C et al., 2000].

23

1.9.3 Soluble Fms-like tyrosine kinase (sFlt1) / Soluble vascular

endothelial growth factor receptor 1 (sVEGFR1)

sFlt-1 is a tyrosine kinase protein that acts by disabling the proteins that

cause blood vessel growth. It is produced by alternative splicing of Flt-1 gene

[Khalil, A et al., 2008]. The transmembrane and cytoplasmic domains of Flt-1

are lacked in sFlt-1. It to all isoforms of VEGF-A and placenta growth factor

(PlGF) with high affinity and inhibits their action [Kendall, R.L et al., 1996].

In normal pregnancies sFlt-1 prevents damage to the placenta and fetus

by inhibiting excess VEGF signalling, which would otherwise leads to

excessive placental invasion and catastrophic hemorrhage at delivery. Thus in

normal pregnancies VEGF levels are tightly controlled at the maternal-fetal

interface in order to regulate placental invasion and facilitate detachment of

placenta after delivery of the fetus, through the modulation by sFlt-1

[McMahon, K et al., 2014]. Studies indicate that up-regulated level of sFlt-1

along with decreased free VEGF & PlGF was observed in the circulation of

preeclamptic women [Maynard, S.E et al., 2003, Varughese, B et al., 2010].

24

Fig 5: Protein structure of Flt-1 and sFlt-1 [Davison, J. M et al., 2004]

1.9.4 Cytokines

Cytokines are small secreted proteins which have a specific effect on

the interactions and communications between cells [Zhang, J.M et al., 2007].

Cytokines include tumor necrosis factors, interleukins, lymphokines,

monokines, interferons, colony stimulating factors, and transforming growth

factors. They are produced by immune cells like macrophages, B

lymphocytes, T lymphocytes and mast cells, endothelial cells, fibroblasts, and

various stromal cells in response to the potent inflammatory stimuli like

infectious agents, mechanical factors, oxygen radicals, immune complexes,

angiotensin II (AngII), inflammasomes, heat shock proteins (HSP), cellular

micro particles, adipokines, platelet products and coagulation factors

[Sprague, A.H et al., 2009]. They are involved in cellular and humoral immune

responses, initiation of inflammatory responses, regulating hematopoiesis,

25

controlling cellular proliferation and differentiation, stimulation of wound

healing [Werner, S et al., 2015].

The maternal immune system activation plays an important role in the

development of preeclampsia [Ahn, H, et al., 2011, Saito, S et al., 1999].

Excessive inflammation is central to this response and is believed to be a

mediator of maternal endothelial dysfunction in preeclampsia [Redman CW et

al., 1999]. Many cytokines, particularly TNF-α, are known mediators of

endothelial activation and dysfunction [Goodwin, B. L et al., 2007].

Tumor necrosis factor-α

TNF-α is a cell signalling protein involved in systemic inflammation and

is one of the cytokines that make up the acute phase reaction. It is produced

chiefly by activated macrophages, monocytes, T-cells, smooth muscle cells,

adipocytes, and fibroblasts (Popa, C et al., 2007). It alters the balance

between endothelium-derived vasoconstrictors and vasodilators and impairs

endothelium dependent relaxation, in part, by activation of NAD (P) H oxidase,

leading to production of superoxide anions that can scavenge NO [Matsubara,

K et al., 2015]. It stimulates mitochondrial production of free radicals and it

also upsurges the expression of endothelial adhesion molecules such as

ICAM-1 and tissue factor, inhibits the thrombomodulin / protein C

anticoagulation pathway, and blocks fibrin dissolution by stimulation of PAI-1

[Hung, T.H et al., 2004]. Several studies have reported higher serum

26

concentrations of TNF-α in preeclamptic women compared to normal pregnant

women that normalize after delivery [Conrad, K. P et al., 1997].

Interleukin-6

IL-6 (Interleukin-6) is a proinflammatory cytokine produced by

mononuclear phagocytes, endothelial cells, fibroblasts and T cells is involved

in immune activation, vascular wall function and modulation of TNF-α

production [Afshari, J.T et al., 2005]. IL-6 may increase the permeability of

endothelial cells by changing the cell shape and rearrangement of intracellular

actin fibers [Maruo, N et al., 1992]. IL-6 also increase the thromboxane A2 to

prostacyclin ratio, reduces prostacyclin (PG I2) synthesis by inhibiting the

cyclooxygenase enzyme and stimulating the platelet-derived growth factor.

This cytokine could also trigger the neutrophil activation, expression of von

Willebrand factor, and cell adhesion on endothelium which results into

vascular damage [Lockwood, C.J et al., 2008]. IL-6 levels were said to be

increased in the circulation from preeclamptic women [Benyo, D.F et al.,

2000]. The endothelial production of IL-6 can be induced by oxygen free

radicals, leading to the reduction in NO synthesis, prostaglandin imbalance

and endothelial damage in preeclamptic women [Afshari, J.T et al., 2005].

27

C - reactive protein

It is a highly sensitive nonspecific marker of inflammation which

contributes to the inflammatory response seen in preeclampsia. It is an acute

phase reactant produced by the liver in response to pro inflammatory

cytokines, especially IL-6 and TNF-α [Ali, Z et al., 2013].

It was reported that the CRP levels of serum are higher in healthy

pregnant women as compared to non pregnant women because even normal

pregnancy is accompanied by mild systemic inflammatory response [Qiu, C et

al., 2004]. It was reported that the levels of HsCRP were significantly high in

the preeclamptic group and correlated with severity of the disease [Hwang,

H.S et al., 2002, Ertas, I. E et al., 2010].

1.9.5 Circulating Lipids and Lipoproteins

Endothelial cell dysfunction may be caused by an imbalance between

circulating VLDL particles or other lipids and a protective, basic isoform of

plasma albumin (TxPA) [Arbogast, B. W et al., 1994]. Triglycerides are a

major feature of metabolic syndrome, which is increasing due to the rising

number of reproductive-aged women who are obese [Hadjiyannakis, S et al.,

2005]. Normotensive pregnancies are characterized by progressive increases

in serum LDL levels accompanied by parallel increases in serum triglyceride

levels followed by sharp fall post-partum [Choi, J. W et al., 2000]. Increased

triglyceride content of LDL results in smaller but denser LDL particles. These

small dense LDL particles are more atherogenic and are more susceptible to

28

oxidative modification [Rajman, I et al., 1999]. Reactive oxygen-derived free

radicals may promote LDL oxidation in the vascular wall and attenuate

endothelium-dependent vasodilation (Engler, M. M et al., 2003).

1.10 Metabolic changes in Pregnancy

Maternal metabolic changes occur during pregnancy. Early gestation is

an anabolic state in which the nutrients are stored in order to meet the feto-

placental and maternal demands of late gestation and lactation. In contrast,

late pregnancy is a catabolic state which results in the increase of maternal

glucose and free fatty acid concentrations, allowing for greater substrate

availability for foetal growth [Lain, K. Y et al., 2007].

1.10.1 Carbohydrate Metabolism in Pregnancy

During the early stage of pregnancy (20 weeks) there will be a little or

no change in the Basal metabolic rate (BMR), however it rises exponentially

over the pre-pregnancy baseline during the second half of pregnancy. The

sequence of biological changes can be divided into those occurring in early (1

- 20 weeks) and late (21 - 40 weeks) pregnancy. Early pregnancy is

characterized by enhanced first-phase insulin release, normal or slightly

increased peripheral insulin sensitivity, normal or slightly enhanced glucose

tolerance and maternal fat accumulation.

29

Late pregnancy is characterized by insulin resistance and accelerated

starvation. Hormones such as human placental lactogen, estrogen,

progesterone, cortisol and growth hormone cause insulin resistance [Butte, N.

F et al., 2000]. Moreover, during late pregnancy, the glucose requirement of

the foetus is increased. In order to meet the demand, hepatic glucose

production is increased [Rao, P.S et al., 2013]. Lipids become the source of

hepatic gluconeogenesis through the production of glycerol and free fatty

acids [Herrera, E 2000]. Obesity is one of the most common causes of insulin

resistance [Kahn, B.B et al., 2000] which might lead to hyperlipidemia [Miccoli,

R et al., 2008].

1.10.2 Lipid and Lipoprotein Changes in Normal Pregnancy

Generally, concentrations of all lipoprotein fractions increase over

progress during pregnancy. The increase in lipoprotein subclass

concentrations represent either increased synthesis or reduced catabolism.

The decrease in lipoprotein lipase (LPL) activity and enhanced hepatic

production of VLDL triglycerides [Butte, N.F 2000] are the reasons for

hypertriglyceridemia seen during gestation. The LDL and HDL are also

specifically enriched with triglycerides [Alvarez, J.J et al., 1996). These

changes are caused by insulin- resistant condition that normally takes place at

this stage of pregnancy [Munilla, M.A et al., 2000].

30

1.11 Metabolic changes in Pregnancy induced hypertension

1.11.1 Carbohydrate Metabolism in PIH

Several studies have reported that insulin resistance is more casual

among women with preeclampsia, as well as evident among women who later

develop preeclampsia [Hauth, J.C et al., 2011]. The cause of the increase in

insulin resistance among women who later develop preeclampsia as well as

women with the syndrome has not been fully explained. However, based on

the pathophysiology of the syndrome several possibilities exist including

increased inflammation, the metabolic syndrome and obesity. Studies have

reported elevations in inflammatory cytokines (TNF alpha and interleukin-6)

and markers (C reactive protein) during normal pregnancy and a further

elevation in these markers among women with preeclampsia [Bhagat, K. et

al., 1997]. Inflammation is a powerful biological mediator and has been clearly

demonstrated to contribute to insulin resistance [Wellen, K.E et al., 2005]. The

metabolic syndrome is characterized by a group of metabolic risk factors

including: abdominal obesity, dyslipidemia (high triglycerides and LDL

cholesterol, and low HDL cholesterol), insulin resistance, pro-thrombotic state,

and pro-inflammatory state. The dominant underlying risk factors for the

metabolic syndrome were obesity and insulin resistance. Since the

inflammation, metabolic syndrome, insulin resistance, hyperlipidemia, and a

pro-thombotic phenotype are all associated with obesity. It is likely that obesity

is also contributing significantly to the presence of these conditions in

preeclampsia [Rafeeinia, A et al., 2014].

31

1.11.2 Lipid metabolism in PIH

In pregnancy, lipolysis of TG-rich lipoproteins is reduced because of

decreased lipolytic activities of the mother, whereas placental VLDL receptors

are up-regulated [Wittmaack, F. M et al., 1995]. This results in a rerouting of

TG-rich lipoproteins to the fetoplacental unit. However, in PIH, the

vascularization of the fetoplacental unit may be impaired, resulting in yet-

undefined compensatory mechanisms that may further increase synthesis of

maternal TG levels (A). In addition, the decreased catabolism of TG -rich

lipoproteins by reduced placental uptake (B) and the putative concomitant

decrease of lipoprotein lipolysis (C) results in the accumulation of TG- rich

remnant lipoproteins in the maternal circulation. Remnant lipoproteins may

induce platelet activation and endothelial dysfunction, thus leading to the

major clinical symptoms of PIH [Winkler, K et al., 2002].

Fig 6: Lipid metabolism in pregnancy induced hypertension

32

1.11.3 Amino acids metabolism in PIH

In general, there have been relatively few studies investigating amino

acids in preeclampsia. Most studies that have investigated differences in

amino acids in preeclampsia have focused on single amino acids, primarily L-

arginine, homocysteine and asymmetric dimethylarginine (ADMA), because of

their potential to influence maternal vascular function via alterations in nitric

oxide synthase activity. Most of these studies have reported finding higher

maternal concentrations of homocysteine and ADMA and lower

concentrations of L-arginine. In some cases these differences have been

associated with differences in maternal vascular function, markers of vascular

dysfunction or insulin resistance [Laskowska, M et. al., 2013, Speer, P. D

et.al., 2008, Holden, D.P et.al.,1998].

1.12 Oxidative stress: Predominant toxic element in PIH

Oxidative stress was postulated to be an important factor in the

development of PIH. Oxidative Stress is a general term used to describe the

steady state level of oxidative damage in a cell, tissue, or organ, caused by

the ROS. It is caused by an imbalance between the production of ROS and a

biological system's ability to readily detoxify the reactive intermediates or

easily repair the resulting damage. Under normal conditions, ROS are cleared

from the cell by the action of body‘s antioxidant systems which includes

superoxide dismutase (SOD), catalase (CAT), or glutathione peroxidase

(GPx). The main damage to cells by oxidative stress results from the ROS-

33

induced alteration of macromolecules such as polyunsaturated fatty acids in

membrane lipids, essential proteins, and DNA [Sharma, P et al., 2012].

1.12.1 Reactive Oxygen Species (ROS) and Free radicals

ROS includes a number of oxygen and free radical derived chemically

reactive molecules. The ROS includes Superoxide radical (O2.-), Hydrogen

peroxide (H2O2), Hydroxyl Radical (.OH), Singlet oxygen (1O2). They

participate in several physiological functions, and form an integral part of the

organism‘s defense against invading microbial agents. They are known

mediators of intracellular signaling cascades. They accelerate aging and

contribute to the development of many diseases, including cardiovascular,

neurosensory disorders, inflammation, cancer [Lamina, S et al., 2013].

Most free radicals come from the endogenous sources as by-products

of normal and essential metabolic reactions, such as energy generation from

mitochondria or the detoxification reactions involving the liver cytochrome P-

450 enzyme system. Exogenous sources include exposure to cigarette

smoke, environmental pollutants such as emission from automobiles and

industries, consumption of alcohol in excess, asbestos, exposure to ionizing

radiation, and bacterial, fungal or viral infections. It can cause tissue damage

by reacting with nucleotides in DNA, sulfhydryl groups in proteins and cross-

linking/fragmentation of ribonucleoproteins [Lobo, V et al., 2010]. Free radicals

affects polyunsaturated fatty acids (PUFA), because they contain multiple

double bonds in between which lie methylene bridges (-CH2-) that possess

34

especially reactive hydrogen atoms. This process is called lipid peroxidation

[Spiteller, G, 2005]. The end products of lipid peroxidation are reactive

aldehydes, such as malondialdehyde (MDA) and 4-hydroxynonenal (HNE)

[Ayala, A et al., 2004].

1.12.2 Malondialdehyde (MDA):

MDA is one of the low-molecular-weight end products formed via the

decomposition of certain primary and secondary lipid peroxidation products. At

low pH and elevated temperature, MDA readily participates in nucleophilic

addition reaction with thiobarbituric acid (TBA), generating a red, fluorescent

1:2 MDA: TBA adduct [Janero, D.R 1999. It is one of the most frequently used

indicators of lipid peroxidation and a potential biomarker for oxidative stress

Nielsen, F et al., 1997].

1.12.3 Antioxidant

To prevent damage to cellular components, there are numerous

enzymatic antioxidant defenses designed to scavenge ROS in the cell.

Antioxidants terminate oxidative damage by scavenging free radical

intermediates, and inhibit other oxidation reactions by being oxidized

themselves. Hence, antioxidants are said to be ―free radical scavengers.

They provide the necessary defense against the OS generated by ROS.

Different classes of antioxidants that scavenge ROS:

35

Enzymatic antioxidants

• Superoxide dismutase

• Catalase

• Glutathione peroxidase/Glutathione reductase

Non-enzymatic antioxidants

• Vitamins C

• Vitamin E

• Vitamin A

• Proteins like Albumin, Transferrin, and Heptoglobulin

• Gluthathione (GSH) [Nimse, S.B et al., 2015].

The ferric reducing ability of plasma which is called as ―FRAP‖ in short is

useful as a measure of total antioxidant capacity [Benzie, I.F et al., 1996].

1.12.4 Oxidative stress in pregnancy

Pregnancy is characterized by dynamic changes in multiple body

systems resulting in increased basal oxygen and energy consumption in

different organs including the fetoplacental unit. Initially the placenta has a

hypoxic environment but with maturity, its vascularization develops which

changes it to an oxygen rich environment [Sies, H 1997]. The placenta is rich

in mitochondria, highly vascular, consumes about 1% of the basal metabolic

36

rate of the pregnant woman and is exposed to high maternal oxygen partial

pressure, therefore resulting in increased production of reactive oxygen

species [Liochev, S. I et al., 1997, Dotsch, J et al., 2001].

Nitric oxide (NO) is also locally produced by the placenta and together

with other reactive nitrogen species (RNS) contributes to potential oxidative

stress in presence of transition metals. Placenta which is rich in macrophages

also favours the local production of free radicals like reactive chlorine species

(RClS) by autocatalysis in presence of iron [Wisdom SJ et al., 1991]. The

body on account of susceptibility to oxidative insult is naturally provided with

an efficient antioxidant system which gets enhanced as the pregnancy

progresses. Normal pregnant women exhibit an increase in lipid peroxidation

and oxidative stress compared with non-pregnant women [Patil, S. B et al.,

2008, Kaur, G et al., 2008]. The ratio of prostacyclin to thromboxane favours

prostacyclin, suggesting an effective defense system against oxidative stress

in normal pregnancy [Wang, Y et al., 1991, Romero, R et al., 2003].

The role of vitamins A, C and E in preventing free radical damage is

well documented and their nutritional adequacy is important in pregnancy

[Yoshioka T et al., 1990, Dordević, N. Z et al., 2007].

1.12.5 Oxidative stress in Preeclampsia

Several studies relate the development of PIH with the inadequate

invasion of the trophoblast and uterine artery remodelling due to the abnormal

regulation of cell– cell and cell–matrix interaction [Hung, T. H et al., 2006,

37

Myatt, L 2002]. This results in reduced uteroplacental perfusion, placental

ischemia and placental tissue damage [Van Asselt, K et al., 1998]. The

steadiness of placental perfusion acts as an important factor than the rate of

blood flow, because both the foetus and the placenta extract considerable

quantities of oxygen during mid to late gestation. So, the placental tissues will

soon become locally hypoxic during periods of vasoconstriction. When the

maternal blood flow is re-established there will be a rapid increase in tissue

oxygenation, and such fluctuations in oxygen tension could provide the basis

for an ischemic/reperfusion (I/R) type phenomenon. Depending on severity

and frequency of I/R insults the result might range from minor oxidative stress

till severe tissue damage. Ischemia-reperfusion injury is mediated principally

through the generation of cytotoxic reactive oxygen species (ROS) which

leads to infarction and syncytial necrosis [Hung, T.H et al., 2001]. In PIH

women, maternal circulating levels, placental tissue levels and production rate

of lipid peroxides are increased and several antioxidants are markedly

decreased [Serdar, Z et al., 2003, Orhan, H. et al., 2001].

Another source of oxidative stress in preeclamptic women is activation

of leukocytes in circulation. It has been conveyed that in PIH, maternal

circulating neutrophils and monocytes are activated, which generate

superoxides (O2) by the activity of NADPH oxidase and hence cause

oxidative stress. Activated neutrophils also produce cytokines such as Tumor

necrosis factor (TNF-α), Interleukin-6 (IL-6) and vascular adhesion molecule

VCAM-1, indicating leukocyte-endothelial attachment and activation

38

[Aris, A et al., 2009]. This activation of endothelial cells produces local

damage and dysfunction of the cells leading to lipid peroxidation of the cell

membrane [O'Riordan, M. N et al., 2003].

Hyperlipidaemia stimulate oxidative stress and inflammatory cytokine

release leading to ED. At the molecular level, poor trophoblast invasion and

uteroplacental artery remodeling described in PE, increases reactive oxygen

species (ROS), hypoxia and ED. Despite all research efforts performed so far,

still the etiology of the disease is not known.

39

2. REVIEW OF LITERATURE

Preeclampsia, a clinical syndrome is a leading cause of maternal

mortality with only known remedy i.e delivery of the placenta. In developed

countries preeclampsia is an important cause of premature delivery indicated

for the benefit of the mother which results in infant morbidity and substantial

excess health care expenditure. In spite of the considerable morbidity and

mortality, the cause of preeclampsia has remained mysterious [Powe, C.E,

2011]. However, it is proposed that widespread endothelial dysfunction is the

hallmark feature of PE [Rohra, D.K et al., 2012]. The mechanisms involved in

the induction of endothelial dysfunction (ED) are poorly understood. ED can

be caused by several conditions like diabetes or metabolic syndrome,

hypertension, smoking, and physical inactivity [Rajendran, P et al., 2013].

Altered lipids and lipoproteins may also provoke the spectrum of endothelial

changes in PIH women [Adiga, U et al., 2007].

2.1 Lipids:

Various patterns of dyslipidemias have been observed by different

authors in PIH women. In a cross sectional study conducted by Musa, A.H et

al., 2014 in which 100 pregnant women were recruited (40 pre-eclamptic, 20

eclamptic and 40 normotensive), the mean serum TGL concentrations were

significantly high in PE & E women compared to normal controls whereas the

mean serum TC and HDL-Cholesterol concentration were significantly low in

eclamptic women when compared with control group. The study exhibited

40

that, though hyperlipidemia is associated with normal pregnancy, this is

exaggerated in both preeclampsia and eclampsia. The increase is also related

with severity of disease.

Furthermore, in a study conducted by Gohil, J. T et al., 2011, a

significantly decreased HDL concentration and significantly increased TC,

LDL, VLDL & TGL concentrations were conspicuously evident in subjects of

preeclampsia as compared to non-pregnant, normotensive pregnant and

postpartum subjects.

In a case control study done at M.A.G. Osmani Medical College, Sylhet,

to evaluate the association of lipid profile with pre- eclampsia and eclampsia,

40 normotensive pregnant women and 60 already diagnosed preeclamptic &

eclamptic women were included. According to that study the preeclampsia

was associated with a significant rise in TGL and fall in HDL cholesterol

concentration while eclamptic women showed significant reduction in HDL

cholesterol and rise in LDL cholesterol as compared to normal pregnant

women. The study has concluded that the increased triglycerides levels and

decreased HDL-cholesterol levels along with delayed triglycerides clearance

and high blood pressure are associated with development of preeclampsia

and eclampsia [Islam, N.A.F et al. 2011].

In contrast to the above studies, a prospective case-control study was

conducted by Enaruna, N.O et al., 2014 had stated that lipid profile

41

abnormalities were not associated with increased frequency of complications

in preeclampsia.

Furthermore, only HDL-C was significantly higher in the gestational

hypertensive group especially with a rising SBP & DBP using Pearson‘s

correlation coefficient and no correlation was found with other lipid fractions in

a study done by Irinyenikan, T.A et al., 2013. In this study all the lipid levels

were in normal range among normotensive pregnant group. The study

concluded that gestational hypertension is not associated with hyperlipidemia.

In a study done by Hentschke, M.R et al., 2013 placental biopsies were

collected. Maternal and umbilical serum samples from 27 normotensive and

24 preeclamptic women were evaluated for LDL, HDL, total cholesterol, and

triglycerides. The study had also quantified placental mRNA expression of

lipoprotein receptors/transporters using quantitative RT-PCR. Placental mRNA

expression of all genes except paraoxonase-1 (PON-1), microsomal

triglyceride transfer protein (MTTP), and protein disulfide Isomerase family A

member 2 (PDIA2) were observed. No differences for any lipoprotein

receptors (LRP) /transporters were found between groups; however, in the

preeclamptic group placental LRP-1 expression was lower in small gestational

age (SGA) delivering mothers. These findings have not support a role of

altered lipid metabolism in preeclampsia, but may be involved in fetal growth.

42

2.1.1 Apolipoproteins:

ApoB is a large protein in the plasma that occurs in two isoforms,

apoB48 and apoB100. ApoB48 is expressed by the intestine and is also

present on chylomicrons and chylomicron remnants. ApoB100 is largely

expressed in liver and on all atherogenic lipoprotein particles like very-low-

density lipoprotein (VLDL), intermediate-density lipoprotein (IDL), LDL and

lipoprotein a (Lp (a)) [Visser, M.E et al., 2010].

Apolipoprotein A-1 is an exclusive component of anti - atherogenic

lipoprotein HDL. It initiates the reverse cholesterol transport (RCT) process in

peripheral tissues. It is also involved in anti-inflammation, anti-oxidation, anti-

infectious activity, anti-protease activity, anti-apoptotic, and anti-thrombotic

functions. Additionally, apoA-I can initiate the endothelial production of nitric

oxide which is vital in vasodilation. Elevated apo B and reduced levels of apo

A-I are associated with increased cardiac events [Walldius, G 2004]. Lp (a) a

variant of carries one copy of a protein apo (a) joined to apo B100, by

disulphide linkage. It is a circulating lipoprotein particle and is found to

enhance blood coagulation by competing with plasmalogen for its binding

sites on fibrin clots & endothelial cells. It is a variant of LDL that is highly

correlated with atherosclerosis [Nicholls, S.J et al., 2010].

In a study done by Timur, H et al., 2016 with 48 PE and 48

normotensive pregnancies which are matched for gestational age, maternal

age, parity, obstetrical complications like intrauterine growth restriction and

43

gestational diabetes mellitus , serum apo A-1 and apo B-100 levels, and apo

B-100/apo A-1 ratio were paralleled. Preeclamptic patients had lower Apo A-1

levels and higher apoB-100/ApoA-1 ratio but comparable Apo B-100 levels.

Mean Apo A-1 and apo B-100 levels and apo B-100/apo A-1 ratio were

parallel in patients with severe PE, HELLP syndrome, IUGR, and patients

requiring antihypertensive therapy compared to PE patients who was not

having these complications . This study concluded that apo A-1 and apo B-

100/apo A-1 ratio might be useful markers in patients with PE.

A cross sectional study by Nazli, R et al., 2013 in three tertiary care

hospitals of Peshawar including 110 women with eclampsia and 90 healthy

pregnant women has demonstrated a significantly higher mean SBP/DBP, TC,

TG, VLDL-C and Lipoprotein(a) (Lp (a)) levels in eclamptics compared to

normotensive women. The apoA-1 levels were lower significantly and apoB

levels were lower non-significantly in eclamptic women compared to

normotensive women. The study has concluded that serum lipids may be

helpful in early assessment and prevention of complications in the eclampsia

patients. But, in this study the serum lipoproteins and apo-lipoproteins were

measured in non-fasting state.

A significant elevation in the levels of serum Lp (a) was observed in PE

women compared to the normotensive women. 20 normal healthy pregnant

control, 25 mild PE& 28 severe PE 30 PIH cases of the same trimester were

included in the study and were evaluated for TC, TG, HDL-C, LDL-C, VLDL-C,

ApoB, ApoA & Lp (a) were evaluated.The study has concluded that the Lp (a),

44

Lipid peroxidation, LDL, TGL are the risk factors for atherosclerosis and needs

to be evaluated in PIH [Bayhan, G et al., 2005]. The high levels of Lp (a)

significantly correlated with blood pressure and proteinuria in pre-eclamptic

women [Parvin, S et al., 2010].

In a detailed longitudinal investigation done by Sattar, N et al., 2000

alterations in plasma Lp (a) concentrations were examined in 10 PE women

and 10 normotensive women together with changes in other lipid parameters.

A significant increase in Lp (a) levels was observed in all subjects with

increasing gestation .But the study had not found any significant difference in

Lp (a) levels in pre-eclampsia, compared with normotensive pregnant women

and thus the study was concluded that the Lp (a) is unlikely to play a role in

the pathology of PE. A cross-sectional design study by Manten, G.T.R et al.,

2005, Bukan, N et al., 2012 showed a decreased level of Lp (a) in subjects

with severe pre-eclampsia and it was due to more extensive endothelial

damage and higher consumption since it is an acute-phase protein.

The reports regarding lipids and lipoproteins were inconsistent. Some of

the studies were done using nonfasting serum samples. Even though some of

the studies stated that the concentrations of lipid profile parameters rises

during PE & E, the pattern of dyslipidemia and its association with severity of

disease is still doubtful.

45

2.2 Atherogenic index of plasma (AIP)

AIP is a tough marker inorder to predict risk of atherosclerosis and

coronary heart diseases [Nwagha, U et al., 2010, Dobiasov, M et al., 2001]. It

reveals the true relationship between the protective and atherogenic

lipoproteins. It is also associated with size of pre- and anti- atherogenic

lipoprotein particle [Dobiasova, M et al., 2011]. An AIP value of <0.1 is

associated with lower risk of CVD; the values between 0.1 to 0.24 and upper

than 0.24 are associated with medium and increased risks, respectively

[Dobiasova, M 2006]. In PIH women several studies had stated a significant

rise in AI compare to the normal pregnant women [Singh, M et al., 2015,

Aksonova, A et al., 2016, Herrara-Villalobos, J.E et al., 2012]. A study done by

Ahenkorah, L et al., 2008 had revealed a positive correlation between AI and

malondialdehyde (MDA) levels in PIH women. On the other hand in a study

done by Emokpae, M.A et al., 2012 among sickle cell nephropathy subjects,

the AIP was negatively correlated with antioxidant enzymes and positively

correlated with MDA.

46

2.3 Oxidative stress

Pregnancy is a period of oxidative stress [Fialova, L et al., 2006].

Hyperlipidemia is one of the factors that increases oxidative stress [Yang, R.L

et al., 2008]. It has been considered as one of the important underlying

mechanisms in pathogenesis of ED. The significant role of oxidative stress in

causing PE has been demonstrated in different studies [Negi, R et al., 2011,

Guerby, P et al., 2015, De Lucca, L et al., 2015]. An increased level of

oxidants and decreased antioxidant status resulting in oxidative stress can

contribute to the high risk of preeclampsia [Gupta, S et al., 2009].

2.3.1 MDA

Malondialdehyde is a stable product of lipid peroxidation [Ayala, A et al.,

2014, Grotto, D et al., 2009]. Bayhan G et al had conducted a cross sectional

study on 20 normal pregnant women, 25 women with mild preeclampsia and

28 women with severe PE in the third trimester. Serum lipids, lipoproteins,

apolipoproteins and MDA were estimated. Placental tissue MDA levels were

also estimated. This study has shown an increased serum levels of MDA,

Lp(a), l TC, TGL, LDL-C and placental MDA whereas HDL-C and Apo A-I

levels were significantly lower, in severely preeclamptic and mildly

preeclamptic women than in the normal pregnant women, but there was no

difference found in Apo B among groups. The study also found a positive

correlation between Lp (a) & BMI, serum MDA & SBP in severely preeclamptic

women. It has also reported that lipids and lipid peroxidation are the important

47

risk factors for atherosclerosis among preeclamptic women [Bayhan, G et al.,

2005]. Across the world several studies have reported the important role of

oxidative stress in the pathogenesis of preeclampsia [Guerby, P et al., 2015,

Raijmakers, M. T et al., 2004, Agarwal, A et al., 2015].

A study done with 30 preeclamptic women, 30 normotensive women, 30

non pregnant women has reported an increased lipid peroxidation (MDA) in

PE women when compared to other 2 groups and there was only a moderate

rise in the levels of MDA in normotensive pregnant women compared to the

non-pregnant women. The study has also reported that the elevated blood

pressure had returned to normal after delivery, but lipid peroxidation and urine

protein excretion remained higher than normal, suggesting that factors other

than oxidative stress might contribute to development of hypertension in

preeclamptic women. Oxidative stress, however, plays an important role in the

causation of target tissue damage such as renal injury [Gowda, V MN et al.,

2009]. Another study had reported that an increased level of oxidation and

reduced antioxidant level may be the important factors in the pathogenesis of

preeclampsia [Begum, R 2012]. These reports are thus accentuating the

importance of measuring oxidant and anti-oxidant status in preeclamptic

women [Trivedi, D.J et al., AlGubory, K. H et al., 2010, Matsubara, K et al.,

2010].

48

2.3.2 Ferric Reducing Ability of Plasma (FRAP)

FRAP is a measure to assess the antioxidant capacity of plasma

[Koushik, A et al., 2012]. An earlier study had shown a significantly lower level

of FRAP in preeclamptic women compared to the normotensive women

[Gupta, A et al., 2016]. A very strong positive correlation between TAC

measured by FRAP assay with the severity of preeclampsia was

demonstrated by a study done by Hermawan, M et al., 2011, Priyamvada, R.P

et al., 2016 and the same study had demonstrated a significant increase in the

levels of MDA in preeclamptic women. It has been stated by Biswas, S.K 2015

that a number of ROS can initiate intracellular signaling cascade that

enhances proinflammatory gene expression. On the other side, inflammatory

cells also liberate a number of reactive species at the site of inflammation

leading to exaggerated oxidative stress. Thus, inflammation and oxidative

stress are closely related pathophysiological events that are tightly linked with

one another.

2.4 Inflammation

Inflammation is a host response triggered by noxious stimuli arising

during infection, tissue injury. Normal pregnancy is characterized by a mild

systemic inflammation, but the inflammation may become damaging if

dysregulated, as seen in preeclampsia [Ann-Charlotte, I et al 2013].

49

2.4.1 Inflammatory cytokines

Cytokines are regulators of host responses to infection, immune

responses, inflammation, and trauma. Proinflammatory cytokines act to make

the disease worse [Dinarello, C.A 2000].

2.4.2 Tumor Necrosis Factor-alpha (TNF-α)

It is produced by monocytes and macrophages in response to inflammatory

stimuli and upsurges the release of other cytokines, chemokines, growth

factors, and acute phase proteins. It is a pleiotropic cytokine which can exert

multiple effects. Increased TNF-α production can lead to oxidative stress and

it may also cause generalized endothelial dysfunction. Serum Levels of TNF-α

and IL-6 Are Associated With Pregnancy-Induced Hypertension [Li, Y et al.,

2016]. In a study done by Gupta, M et al., 2015, TNF-α was evaluated along

with other cytokines like IL-1β, IL-6 and IL-8 with progression of normal

pregnancy and development of PE. In this study 40 primigravidas with

uncomplicated normal pregnancies in first trimester were followed till the last

trimester (control) and 35 primigravidas who developed PE (study group) in

the third trimester were selected by random sampling. The levels of TNF-α, IL-

6 and IL-8 were significantly elevated in PE compared with the levels in

normal healthy controls and normal pregnant females in the third trimester.

There was a significant progression in the levels of TNF-α from the first

trimester in females who subsequently developed PE. The study concluded

50

that the measurement of TNF-α early in pregnancy can be of used to predict

the progression of PE.

A prospective longitudinal study was done by Serin, Y.S et al 2002 with

120 pregnant women. In this study plasma TNF-alpha levels were higher in

preeclamptic patients than normotensive women in the third trimester of

pregnancy and there was no difference found between these groups in the

first and second trimesters. The study concluded that plasma TNF-alpha

levels are not useful as a specific marker for prediction of preeclampsia in the

first and second trimesters. But it might be useful for the prediction in the early

third trimester.

A study by Vahid Roudsari, F et al., 2009 had stated that the TNF-alpha

concentration was not statistically different between the PE women (mild,

severe) and control normotensive groups. The study concluded that the serum

TNF-α is not significantly associated with preeclampsia.

2.4.3 Interleukin-6 (IL-6)

It is a peptide which has many actions in relation with immune response

to infection, injury including lymphocyte proliferation and activation, antibody

production, and hepatic production of acute-phase proteins [Kauma, S.W et

al., 1995].

A study by Xiao, J. P et al., 2012 had demonstrated significant high

levels of IL-6 in both early onset and late onset women with preeclampsia

compared to gestationally matched health pregnant women. The study

51

concluded that there is an excessive maternal inflammatory response in

preeclampsia.

On the other hand, a study by Ozler, A et al., 2012 did not observe any

significant difference among mean serum TNF-alpha and IL-6 levels among

four groups i.e. 3 groups of preeclamptic women i.e. (mild, severe, HELLP

syndrome) and 1 normotensive pregnant woman.

A prospective study was performed by Vitoratos, N et al., 2010 to

evaluate maternal TNF-alpha and IL-6 plasma levels in normotensive

pregnant women, PE women in university of Athens. The study had also

examined the temporal changes in their levels from the antepartum to the

postpartum period. In this study during antepartum period the levels of TNF-

alpha were significantly higher compared to controls, however no statistical

significance was found in IL-6 levels. The TNF-alpha levels were significantly

high in preeclamptics when compared to the normotensive controls long after

delivery. Nonetheless, there was no difference in the levels observed between

before and after delivery levels. No difference was noticed regarding IL-6

levels in women of normotensive group long after delivery compared to that

before delivery. Long after delivery IL-6 levels were statistically significant

higher in preeclamptic women compared to normal controls. The study

concluded that the preeclamptic women remain under a status of increased

inflammatory stress up to 12-14 weeks postpartum despite the fact that all the

other signs of preeclampsia are resolved.

52

Afshari, J.T et al., 2005 had evaluated IL-6 & TNF-alpha in serum of 24

PE women and 18 normotensive pregnant women. The study has found

significantly high IL-6 levels in preeclamptic women compared to the

normotensive pregnant women. But, there was no significant change found in

the concentration of TNF-alpha among preeclamptic women. These findings

suggest that serum TNF-alpha level is not associated with preeclampsia.

2.4.4 High sensitive C- reactive protein (HsCRP)

It is a protein measured by using labeled (enzyme / fluorescent/

polystyrene beads) antibodies and has been suggested to be more sensitive

in confirmation of inflammation than conventional measurement of CRP.

Serum HsCRP can be used as biomarker for identifying women at risk of

preeclampsia [Mandal, K.K et al., 2016].

A cross-sectional study done with normal pregnant (n=40), mild

preeclamptic (n=37), and severe preeclamptic women (n=38) had

demonstrated a significant difference in the means of serum HsCRP between

normal pregnant women and mild preeclamptic women. Serum concentration

of HsCRP was significantly higher in severe preeclampsia (P<0.05) than

normal pregnancy. The study had also observed a significant difference in

HsCRP levels between mild and severe preeclamptics. The levels of HsCRP

increases with severity of the disease [Farzadnia, M et al., 2012].

Another study by Onuegbu, A. J et al., 2015 has demonstrated a

significant rise in the levels of HsCRP, Total cholesterol, TGL in PE women

53

compared to the normotensive women. HDL values were less in the case

group than in control group whereas LDL levels were non-significant between

the 2 groups. The study concluded that high levels of serum HsCRP in

preeclampsia could be due to an exaggerated systemic inflammation and

abnormal lipid profiles may have a role in promotion of preeclampsia.

A cross sectional study with mild pre-eclampsia (N=40), severe pre-

eclampsia (N=40) and chronic hypertension (N=40) and normal pregnant

subjects as control (N=60) at Shiraz medical university affiliated hospitals had

measured highly sensitive C-Reactive protein. The study had not observed

any statistically significant changes in HsCRP levels among the four groups

and concluded that highly sensitive C - reactive protein assessment is not

advised for the prediction of preeclampsia [Tavana, Z et al., 2010].

Furthermore, in a cross sectional study done by including 26 normal

pregnant women, 25 PE women and 21 non pregnant women the levels of C-

reactive protein, TNF-alpha, IL-6 were significantly higher in PE women

compared to the other groups. The study concluded that it was not possible to

determine whether the increase in the concentrations of CRP and other pro-

inflammatory cytokines were a cause or consequence of disease [Teran, E et

al., 2001].

In a study done inorder to determine the levels of TNF α, IL-6, and C

reactive protein in women with severe preeclampsia (n=50) compared with

gestational age- matched normotensive pregnant women (n=50), the

54

inflammatory cytokines, IL6, TNF α and CRP are elevated in severe

preeclampsia. This study also reported a significant association between CRP

and systolic blood pressure, diastolic blood pressure [Udenze, I et al., 2015]. It

was stated by Lamarca, B.D et al., 2007 that the blood pressure regulatory

systems (eg: renin-angiotensin system (RAS) and sympathetic nervous

system) interact with proinflammatory cytokines, affect angiogenic and

endothelium-derived factors regulating endothelial function. The study had

concluded that the inflammatory cytokines elevate blood pressure during

pregnancy by activating multiple neurohumoral and endothelial factors.

Previous reports on the involvement of proinflammatory cytokines like

TNF- alpha, IL-6 and HsCRP in preeclampsia were conflicting. A systematic

study is required in larger population.

2.5 Angiogenic and anti angiogenic factors

Angiogenesis is a process of development of new blood vessel from

pre-existing vasculature which involves endothelial cell division, selective

degradation of the basement membrane and the surrounding extracellular

matrix, endothelial cell migration, and the formation of a tubular structure. It is

controlled by proangiogenic and antiangiogenic factors [Hoeben, A et al.,

2004]. Several studies had demonstrated an imbalance in angiogenic factors

in preeclampsia [Maynard, S.E et al., 2003, Bdolah, Y et al., 2004, Furuya, M

et al., 2011].

55

2.5.1 Vascular endothelial growth factor (VEGF)

It is widely distributed throughout adult and fetal tissues. Even though there

are numerous angiogenic factors associated with fetal development, VEGF

appears to be one of the most potent factors associated with fetal and

placental vascular growth [Vonnahme, K. A et al., 2005].

A cross-sectional study was done by Baker, P. N et al., 1999 on 78

nulliparous women which were subdivided into preeclampsia (n = 27),

nonproteinuric pregnancy-induced hypertension (n = 15), and normal pregnant

women (n = 36). In the same study, in addition to samples taken before

delivery, serum samples were obtained in early pregnancy (before clinical

disease) and 24-48 hours postpartum from 12 of the patients with

preeclampsia, 12 of those with pregnancy-induced hypertension with out

proteinuria, and 12 of the normal pregnant subjects. Umbilical cord blood was

also sampled from 14 of the preeclamptic and 16 of the normal pregnant

subjects. All these samples were analysed for VEGF levels. The study had

suggested that VEGF plays a role in endothelial cell activation which occurs in

the disease.

A significant rise in the levels of VEGF was observed in the serum

samples collected from 10 patients with preeclampsia and 10 gestation-

matched normotensive controls in a study done by Brockelsby, J.C et al.,

2009. The study concluded that the increased VEGF may be involved in the

endothelial dysfunction, pathophysiology of the disease.

56

The VEGF levels were reported to be significantly higher in PIH women

than normotensive pregnant and normal non pregnant women [Tandon, V et

al., 2017].

In disparity with the above studies, VEGF levels were reduced in

preeclamptics which may contribute to the development of generalized ED, a

characteristic of maternal syndrome in the disease [Molvarec, A et al., 2010].

In the above reports, the disparity that was observed regarding VEGF

levels can be elucidated by the fact that VEGF-protein complexes are

undetectable by the sandwich-type ELISA because there is a substantial

increase in circulating VEGF binding proteins during pregnancy. All prior

studies reporting on decreased VEGF have used an ELISA kit, which

measures free (unbound) VEGF whereas all studies reporting on an increased

VEGF in preeclampsia used either a radioimmunoassay or an ELISA system

measuring total (bound and unbound) VEGF.

2.5.2 Placental Growth Factor (PlGF)

It is an angiogenic protein secreted by the placenta. It has been shown

that PlGF is necessary for the proper functioning of the endothelial cells during

pregnancy by acting in synergy with VEGF .It is known that oxygen is the main

regulator of the balance between the function of PlGF and VEGF [Deiana, S.

F et al., 2014].

A study by Schmidt, M et al., 2009 with serum samples of 61 women

between 15 to 18 weeks of pregnancy in correlation with outcomes of

57

pregnancy stated that the PlGF levels were significantly low in 7 women who

developed preeclampsia later among 61 women enrolled. This study

concluded that among women with suspected preeclampsia or Intra uterine

growth restriction (IUGR), PlGF helps to identify women who will experience

an adverse outcome within a time period of 15 days.

Deiana, S. F et al., 2014 had stated that the PlGF levels were

significantly lower than cut off values in all women with preterm delivery

without known causes.

Patients with a medical history of hypertensive disorders and low PIGF

levels in early second trimester were reported to have an increased risk of

preeclampsia [Dover, N et al., 2012].

2.5.3 Antiangiogenic factor (sFlt-1)

Soluble fms-like tyrosine kinase -1 (sFlt-1 or sVEGFR-1) is a tyrosine

kinase protein that disables proteins that cause blood vessel growth. It is a

splice variant of VEGF receptor 1 (Flt-1). It binds and reduces free circulating

levels of the proangiogenic factors VEGF and PlGF thereby blunts the

beneficial effects of these proangiogenic factors on maternal endothelium,

with consequent maternal hypertension and proteinuria [Troisi, R et al., 2008].

Several studies have reported a significant high level of sFlt-1 in preeclamptic

women [Hertig, A et al., 2004, Yuan, H.T et al., 2005, Wang, A et al., 2009].

58

The ratio of sFlt-1 to PlGF was reported to be elevated in pregnant

women before the clinical onset of preeclampsia [Zeisler, H et al., 2016,

Hassan, M.F et al., 2013, Kim, S.Y et al., 2007].

Varughese, B et al., 2010 had done a case-control study using serum

samples obtained from 40 pre-eclamptic women and 40 normotensive, non-

proteinuric pregnant women to ascertain whether pre-eclampsia is associated

with changes in serum concentrations of VEGF, PIGF and sFlt-1 in Indian

patients. The study concluded that an increase in sFlt-1 levels and a

simultaneous decrease in free VEGF and PIGF levels in pre-eclampsia

compared with normotensive pregnant women could have an important role in

the pathology of pre-eclampsia.

Furthermore Lee, E.S et al., 2007 had measured total VEGF, free

VEGF and soluble Flt-1 (sFlt-1) concentrations in 20 patients with

preeclampsia and 20 normotensive gestationally matched women. In this

study the serum concentrations of total VEGF were significantly increased in

preeclamptic women compared to the women with uncomplicated

pregnancies. But, the serum concentration of free VEGF was reduced in the

patients with PE. The study also demonstrated a positive correlation between

the levels of serum total VEGF and sFlt-1 with SBP/DBP, respectively and a

negative correlation between the serum free VEGF levels and SBP/DBP. A

strong negative correlation between free VEGF and sFlt-1 concentrations was

observed in this study. The study concluded that VEGF and sFlt-1 were

related to the pathogenesis of preeclampsia. Reduced concentrations of free

59

VEGF might interfere with endothelial cell function and survival because it has

been shown to induce the release of NO from human vascular endothelial

cells [Shen, B.Q et al., 1999].

2.6 Endothelial dysfunction:

2.6.1 Nitric oxide

The maternal vascular endothelium is an important target of the factors

triggered during PE. Both endothelium-derived relaxing and contractile factors

plays an important role in regulation of arterial compliance, vascular

resistance and blood pressure [LaMarca, B et al., 2012]. The pre-eclamptic

maternal syndrome arises from a generalized maternal inflammatory systemic

response including a substantive component of endothelial cell dysfunction.

Un like pre-eclampsia, ED does not resolve post-partum which might increase

risk of CVD in later life [Poston, L. et al., 2005]. The increase in the estrogen

levels during pregnancy up regulates the nitric oxide level, which in turn

mediates endothelium-dependent vasodilatation. NO mediates its effect by

guanosine 3', 5‘-cyclic monophosphate (cGMP) produced by soluble guanylyl

cyclase. cGMP activates protein kinase A (PKA) and protein kinase G (PKG)

which induces smooth muscle relaxation through the attenuation of myosin

light chain kinase (MLCK)activity and augmentation of myosin light chain

phosphatase (MLP) activity, there by dephosphorylating the 20-kDa,

regulatory, myosin light chain [Matsubara, K et al., 2015]. But, in PE several

studies had reported a reduction in the levels of NO [Seligman, S.P et al.,

60

1994, Granger, J. P et al., 2001, Var A et al., 2003] which leads to an increase

in endothelial permeability. This might be associated with a reduction in eNOS

expression and its activity in endothelial cells [Wang, Y et al., 2004]. Abnormal

changes in lipid metabolism may contribute towards the endothelial lesions

observed in preeclampsia [Lima, V.J et al., 2011].

Preeclampsia is associated with placental hypoxia, the putative culprit

initiating the cascade of events that ultimately results into impaired

angiogenesis which leads to maternal manifestations of the disease

[Dobierzewska, A et al., 2016]. The existing evidence suggests that the

reason for impaired angiogenesis is sFlt-1 which inhibits VEGF signaling in

the endothelium [Goel, A et al., 2013] and thereby reduces the expression of

eNOS [Shen, B.Q et al., 1999]. It was also stated by Zechariah, A et al., 2012

that the hyperlipidemia also attenuates VEGF induced angiogenesis, impairs

cerebral blood flow and disturbs stroke recovery. It has been shown in

previous study done by Duan, J et al., 2000 that hypercholesterolemia impairs

angiogenesis in vivo by inducing oxidative stress in blood vessels and also by

decreasing the activity of the L-arginine/NO pathway in the ischemic tissues,

there by leading to ED, blood brain barrier disturbances . It had also been

stated that the native form of LDL have anti-angiogenic activity which

attenuates the endothelial angiogenesis by down regulating the Hypoxia

inducible factors (HIFs) through increasing HIF hydroxylation / proteasome

activity there by disrupting the HIF pathway [Yao, G et al., 2015].

61

3. NEED OF THE STUDY

Even though abnormal placentation is considered as major causative

factor for PIH, endothelial dysfunction plays a pivotal role in the genesis of the

multisystem disorder that develops in pre eclampsia and eclampsia.

Dyslipidemia is one of the major factor to cause endothelial dysfunction in

various diseases .Since the earlier reports were inconsistent , A systematic

study is required to analyze the changes in lipids and lipoprotein levels among

PIH women.

Since hyperlipidemia is major cause for the generation of ROS, there is

a need to analyze whether there is any imbalance in the levels of oxidative

stress marker (MDA) & antioxidant capacity (FRAP) which might lead to

inflammation and ED in mother.

The analysis of inflammatory markers (TNF-alpha, IL-6, HsCRP) and

ED marker (NO) is also needed.

sFlt-1 is an anti angiogenic factor which is involved in ED by inhibiting

the actions of angiogenic factors (VEGF, PlGF) among PIH women. As of our

knowledge there are no studies on angiogenic and anti angiogenic factors

among PIH women in south India. A study is needed to assess the levels of

sFlt-1, VEGF, PlGF in south Indian PIH women but, these are costly

investigations which cannot be done in all the clinical laboratories.

Since abnormal lipid metabolism can influence the angiogenesis, an

association between lipid parameters and angiogenic, anti angiogenic factors

62

might give us an idea whether the simple measurement of serum lipid

concentrations can be of use to predict the onset and progression of the

disease rather than placing the burden of costly investigations on the patients.

63

4. OBJECTIVES

The present study was taken up with the following objectives:-

1) To determine the serum lipid and lipoprotein levels in PIH women

2) To assess the Antioxidant capacity (AOC), lipid peroxidation in PIH

women

3) To assess the levels of inflammatory markers, angiogenic and anti

angiogenic factors in PIH women.

4) To assess the endothelial dysfunction and atherogenic index (AI) in PIH

women

5) Correlation of lipids and lipoproteins with NO, angiogenic and anti

angiogenic factors.

HYPOTHESIS:

Pre-eclampsia is characterized by endothelial dysfunction.

Hyperlipidemia is one of the factors which can lead to ED by aggravating the

oxidative stress, inflammation, angiogenic and anti angiogenic imbalance. So,

the estimation of the same might help us to find out whether a regular

monitoring of lipid profile in pregnant women can be of any use to predict the

disease in early stages.

64

5. MATERIALS AND METHODS

5.1 Selection of the patients

The proposed case control study was conducted in Annapoorana

medical college and hospital, Salem, Tamilnadu, India. A total of 300 pregnant

women were included. 100 pregnant women with preeclampsia and 100

eclamptic pregnant women who were admitted in the Gyneac and Obstetrics

unit were selected randomly and were screened at the outpatient prenatal

visits, labor and delivery. 100 healthy pregnant gestationally matched women

were taken as controls. Informed consent was obtained from each subject for

the participation in the study after explaining my aims and objectives. The

study was ethically approved by the Institutional Ethical Research Board

(IERD) at Annapoorana Medical College and Hospital, Salem, Tamilnadu,

India

Exclusion criteria:

Women with a history of hypertensive blood pressure, diabetes, renal

diseases, and cardiovascular diseases were excluded from our study.

Study population:

Pregnancy induced hypertension was defined as occurrence of

hypertension after 20 weeks of gestation with or without proteinuria and

edema with SBP > 140 mmHg and DBP > 90 mmHg on repeated readings.

65

Categories of PIH:

Pregnancy Induced Hypertension was sub divided into 3 groups as under:

Pre-eclampsia (PE) was defined by persistent hypertension and

proteinuria of 2+ or greater on dipstick testing according to International

Society for the Study of Hypertension in Pregnancy.

Eclampsia was defined by women presenting with convulsions/coma

during or soon after pregnancy, described by pathological edema,

hypertension, and proteinuria.

Gestational hypertension or transient hypertension. : PIH was occurring

without proteinuria

5.2 Study Groups:

The study subjects were divided into 3 groups of pregnant women, with

two patient and one control group as under:

Group1: Women having normal uncomplicated pregnancy without

hypertension

Group 2: Women with pre-eclamptic toxemia (PE)

Group 3: Toxemic women with eclampsia.

66

5.3 Specimen Collection & Processing

8ml of venous blood samples were collected from both cases and

controls after overnight fasting from antecubital vein applying aseptic

technique and tourniquet for as short a time as needed. 4 ml of blood was

transferred to an EDTA vacutainer for plasma. 2.0 ml of blood was transferred

to thrombin activator tube for serum. 2.0 ml was transferred in to heparin

vacutainer. All specimens were transferred over ice cubes to Clinical

Biochemistry Laboratory; AMC&H. A clear and cell free serum / plasma were

separated by centrifugation at 3000 rpm for 10 minutes. The serum/ plasma

samples were isolated with proper labeling.

Biochemical analysis of lipids (total cholesterol, triglycerides),

lipoprotein (HDL), apolipoproteins (apoA-I, apo-B), Lp (a) were done in semi

auto-analyzer by using commercially available standard kits (Agappe

Diagnostics Ltd). MDA, FRAP, NO were analysed using spectrophotometric

manual methods in the hospital clinical laboratory on the same day of sample

collection. Hemoglobin (Hb) was measured by using hematology auto

analyzer. For the estimation of remaining special parameters serum/plasma

samples were separated into 5 aliquots and stored at -200C deep freezer.

Spot urine samples were collected into a sterile urine container and it

was analysed for proteins.

The accuracy and precision of the results were maintained with utmost

care. The AMC&H clinical biochemistry laboratory maintains internal quality by

67

using commercially available BIORAD- level I & II as internal quality control

(IQC) which mainly verifies the stability of laboratory estimation at the time of

testing and also controls the imprecision and inaccuracy [Westgard J.O &

Barry P.L 1986]. In addition, our clinical lab also participates in Christian

medical college external quality assurance scheme (CMC-EQAS)

[Ref.No.3516] which can contribute to improve laboratory performance and

accuracy (Matson, P.L et al., 1998). The Levey-Jenning (LJ charts) charts

were plotted and maintained on daily basis which can detect all kinds of

analytical errors [Karkalousos, P & Evangelopoulos, A 2011].

5.4 Measurement of BMI

Body Mass Index is an index of weight-for-height which is commonly

used to classify underweight, overweight and obesity in adults. It is calculated

as weight in kilograms divided by height in meters square (kg/m2)

[Rasmussen, K.M et al., 2009].

68

5.5.1 ESTIMATION OF SERUM CHOLESTEROL

Method: Cholesterol oxidase peroxidase or CHOD-POD Method.

Principle: The enzyme cholesterol esterase (CE) is used to hydrolyze the

cholesterol esters present in the serum to cholesterol and fatty acids. The

enzyme cholesterol oxidase (CO) in the presence of oxygen oxidizes the

cholesterol to cholesten-3one and hydrogen peroxide. Hydrogen peroxide

oxidizes phenol and 4-aminoantipyrine to produce red color that can be

measured spectrophotometrically.

Cholesterol Ester + H2O Cholesterol + Fatty acids

Cholesterol + O2 Cholesten-3-one + H2O2

2 H2O2 + Phenol +4-Aminoantipyrine Quinoneimine + 4 H2O

Prepregnancy Weight

Category

Body Mass Index

( kg/m2)

Recommended range of total

weight (lb)

Recommended Rates

of Weight Gain in the 2nd, 3rd

trimesters (lb) (Mean range lb/wk)

Underweight <19.8 28–40 ~1 (0.5 kg/wk)

Normal Weight 19.8–26.0 25–35 1 (0.4 kg/wk)

Overweight 26–29.0 15–25 0.6 (0.3 kg/wk)

Obese (includes all classes)

>29 and greater >15 Not specified

Cholesterol Esterase

(CE)

Cholesterol oxidase

(CO)

Peroxidase

(PO)

69

The intensity of the pink color is proportional to cholesterol concentration in

the sample.

Procedure: The reagents and samples were brought to room temperature.

Clean dry test tubes were labeled as Blank (B), Standard (S) and Test (T).

Pipette into Test

tubes Blank Standard Test

Reagent 1 ml 1 ml 1 ml

Distilled water 10µl - -

Standard - 10µl -

Sample(serum) - - 10µl

Mixed well and incubated for 10 minutes at 37°C. The absorbance of sample

and standard was measured within 60 minutes against reagent blank at

500nm wavelength [Allain, C.C et al., 1974].

Calculation:

OD of Test Cholesterol (mg/dl) = --------------- X Concentration of STD (200mg/dl)

OD of STD

70

5.5.2 ESTIMATION OF TRIGLYCERIDES

PHOTOMETRIC DETERMINATION OF TRIGLYCERIDE BY GPO-POD

METHOD.

Method: Lipoprotein lipase, Glycerol phosphate oxidase and Peroxidase.

Principle: Glycerol released from hydrolysis of triglycerides by lipoprotein

lipase (LPL) is converted by glycerol kinase (GK) into glycerol-3-phosphate

which is oxidized by glycerol phosphate oxidase (GPO) to dihydroxyacetone

phosphate and hydrogen peroxide. In presence of peroxidase (PO), hydrogen

peroxide oxidizes phenolic chromogen to a red colored compound.

Triglycerides Fatty acids + Glycerol

Glycerol + ATP Glycerol-3-phosphate + ADP

Glycerol-3-phosphate + O2 DHAP+ H2O2

H2O2 +Phenolic chromogen Red colored compound

Lipoprotein lipase (LPL)

Glycerol kinase (GK)

Glycerol

phosphate

oxidase (GPO)

Peroxidase

(PO)

71

Procedure:

Pre warm at room temperature the required amount of reagent

Pipette into Test

tubes Blank Standard Test

Reagent 1ml 1ml 1ml

Standard - 10µl -

Sample - - 10µl

Mixed well and incubated for 10 minutes at 37°C. After incubation the

absorbance was measured against blank at 510nm [Fossati, P et al., 1982].

Calculation:

OD of test Triglycerides (mg/dl) = --------------- X Concentration of STD (200mg/dl)

OD of STD

72

5.5.3 ESTIMATION OF HDL CHOLESTEROL BY IMMUNOINHIBITION

METHOD

Method: Immuno-inhibition, 2 reagent method.

Principle: In this assay system, only HDL (high density lipoproteins) is

solubilized by a special detergent; other lipoproteins such as low density

lipoprotein, very low density lipoprotein (VLDL) and chylomicrons (CM) are not

disrupted. After HDL is selectively disrupted, HDL cholesterol is measured

enzymatically. Consequently, only HDL cholesterol is measured.

HDL, LDL, VLDL, CM HDL (Disrupted)

Disrupted HDL cholesterol 4 Cholestenon + H2O2

H2O2 + 4-Aminoantipyrine + DSBmT Reddish purple compound

DSBmT = N, N-bis(4-sulfobutyl)-m-toludine disodium salt.

Detergent

Cholesterol Esterase

(CE) & Cholesterol

oxidase (CO)

Peroxidase

(PO)

73

Procedure:

3μl of sample and 300μl of reagent-I was taken in a test tube, and then

incubated it for 5 minutes at 37°C. After 5 min. 100μl of reagent-II was added

and incubated for 5 min. at 37°C. The absorbance was measured at 700/600

before and after addition of reagent-II against reagent blank.

Calculation:

HDL Cholesterol conc. = Difference in absorbance‘s between 700nm &

600nm [Williams, P et al., 1979].

5.5.4 DETERMINATION OF VLDL BY CALCULATION METHOD

The value of VLDL-cholesterol can be calculated as follows. If the value of

triglycerides is known, VLDL-cholesterol can be calculated based on

Friedwald‘s equation.

VLDL-cholesterol (mg/dl) = Triglycerides / 5 [Warnick, G.R et al., 1990].

5.5.5 DETERMINATION OF LDL BY CALCULATION METHOD

The value of LDL-cholesterol can be calculated as follows. If the values

of total cholesterol, VLDL and HDL are known, LDL-cholesterol can be

calculated based on Friedwald‘s equation.

LDL-cholesterol (mg/dl) = Total Cholesterol – (VLDL+HDL) [Warnick, G R et

al., 1990].

74

5.5.6 ESTIMATION OF APOA-I BY TURBIDIMETRIC IMMUNOASSAY

Method: Turbidometric immune assay

Principle: Anti-human apo A-1 antisera when mixed with human serum

containing apo A-1, react to cause an absorbance change, which is measured

by immune turbidometric principle. The change in the absorbance can be

interpolated in a calibration curve prepared with different known

concentrations of calibrator.

Reagents:

Activation buffer (R1) - buffer

Reagent (R2) - Anti apoA-I antibody

Calibrator: lyophilized preparation of plasma equivalent to the stated amount

of apoA-1 on an mg/dl basis, when hydrated appropriately.

Preparation of calibration curve:

The Agappe apoA-I calibrator was reconstituted exactly with 1 ml of

distilled water, and allowed to stand for 10 minutes. The vials were gently

swirled till the solution attains homogeneity at room temperature. Once

reconstituted it was ready to use for apoA-I test. The high concentrated

calibrator was diluted 1/10 using normal saline (1ml calibrator + 9 ml normal

saline) and this is used for the preparation of calibration curve. The

concentration of the apo A-1 calibrator was multiplied by the corresponding

75

factors stated in the table below to obtain the apo A-1 concentration of each

dilution.

Dilution 1 2 3 4 5 6

1/10 Dil. Cali. (μl) - 10 10 20 50 100

Normal saline (μl) - 150 70 60 50

Dil. Factor 0 0.0625 0.125 0.25 0.5 1.0

Procedure:

Reagents Calibrator Sample

apo A-1 R 1 450µl 450µl

Dil.Calibrator - 5 µl -

Dil.Sample/control - 5 µl

apo A-1 R 2 75 µl 75 µl

After mixing well, the absorbance (A1) was measured immediately after

addition of apo A-1 R2 and the second absorbance (A2) was taken exactly

after 300 sec at 340 nm.

76

The ∆ absorbance (DA = A2−A1) was calculated for all the calibrators

and the standard curve was plotted. The concentration of controls and

samples were read using the calibration curve [Ziegenhagen, G et al., 1983].

Linearity: This reagent is linear upto 300 mg/dl.

5.5.7 Estimation of apo-B by turbidometric immunoassay

Method: Turbidometric immune assay

Principle: Anti-human apo- B antisera when mixed with human serum

containing apo- B, react to cause an absorbance change, which is measured

by immune turbidometric principle. The change in the absorbance can be

interpolated in a calibration curve prepared with different known

concentrations of calibrator.

Reagents:

Activation buffer (R1) - buffer

Reagent (R2) - Anti apo- B antibody

Calibrator: lyophilized preparation of plasma equivalent to the stated amount

of apo-B on an mg/dl basis, when hydrated appropriately.

Preparation of calibration curve:

The Agappe apo-B calibrator was reconstituted exactly with 1 ml of

distilled water, and allowed to stand for 10 minutes. The vials were gently

swirled till the solution attains homogeneity at room temperature. Once

77

reconstituted it was ready to use for apo-B test. The high concentrated

calibrator was diluted 1/10 using normal saline (1ml calibrator + 9 mL normal

saline) and this is used for the preparation of calibration curve. The

concentration of the apo-B calibrator was multiplied by the corresponding

factors stated in the table below to obtain the apo-B concentration of each

dilution.

Dilution 1 2 3 4 5 6

1/10 Dil.

Cali. (μl) - 10 10 20 50 100

Normal

saline (μl) - 150 70 60 50 -

Dil. Factor 0 0.0625 0.125 0.25 0.5 1.0

Procedure:

Reagents Calibrator Sample

apo B R 1 450µl 450µl

Dil.Calibrator - 15 µl -

Dil.Sample/control - 15 µl

apo B R 2 75 µl 75 µl

78

After mixing well, the absorbance A1 was measured immediately after

addition of apo- B R2 and the second absorbance (A2) was taken exactly after

300 sec at 340 nm.

The ∆ absorbance (DA = A2−A1) was calculated for all the calibrators

and the standard curve was plotted. The concentration of controls and

samples were read using the calibration curve [Ziegenhagen, G et al., 1983].

Linearity: This reagent is linear up to 330 mg/dl.

5.5.8 ESTIMATION OF Lp (a)

Method: Immunoturbitometric method

Principle: The test sample was mixed with Lp (a) antibody and activation

buffer containing reagents, they were allowed to react. Presence of Lp (a) in

the test sample results in the formation of an insoluble complex resulting in an

increase in turbidity, which is measured at wavelength 340 nm. Increased

turbidity is corresponding to the concentration of Lp (a) in the sample.

Reagents:

Activation buffer (R1): Buffer

Reagent (R2): Anti- Lp (a) antibody

Calibrator: lyophilized preparation of plasma equivalent to the stated amount

of fibrinogen on an mg/dl basis, when hydrated appropriately.

79

Preparation of Lp (a) curve:

The Quantia-Lp (a) calibrator was reconstituted exactly with 1 ml of

distilled water, and allowed to stand for 10 minutes. The vials were gently

swirled till the solution attains homogeneity at room temperature. Once

reconstituted it was ready to use for Lp (a) test.

Lp (a) working standard (1 ml of 80 mg/dl):

The working standard from the reconstituted calibrator was prepared. It was

stable for 8 hours at 2-300C.

Preparation of Calibration curve:

Test tube No 1 2 3 4 5

Working std dilution No

D1 D2 D3 D4 D5

Volume of working std

400 200D1 200D2 200D3 200D4

Volume of saline

- 200µl 200 µl 200 µl 200 µl

Conc. Of Lp(a) in mg/dl

80 40 20 10 5

The above five dilutions of the calibrator including the highest 80 mg/dl

(D1) and lowest 5 mg/dl concentrations of measuring range were used for the

preparation of the calibration curve.

80

Procedure:

1. 400 μl of R1 and 100 μl of R2 were added in the measuring cuvette. Mixed

well and incubated for 5 minutes at 370C.

2. 50 μl of D1 was added. The stop watch was started immediately.

3. The absorbance (A1) was taken exactly at the end of 10 seconds

4. The 2nd absorbance (A2) was taken exactly at the end of five minutes

5. Steps no.1-4 were repeated for each diluted working standard (D2 to D5)

for preparing calibration curve.

6. Calculate ΔA (A2-A1) for each dilution of the working standard (D1 to D5).

Plot a graph ΔA versus concentration of Lp (a) on the graph paper provided

with the kit.

Test procedure: Determination of Lp (a) concentration in the test sample,

dilute the test sample 1:2 with normal saline.

1. The steps 1-4 were followed as mentioned in above procedure for

calibration curve using the diluted test sample in place of the working

standard.

2. ΔA (A2-A1) was calculated for the diluted test sample.

Calculation:

1. ΔA of the diluted test sample on the calibration curve was interpolated and

the Lp (a) concentration ‗C‘ of the diluted test sample was obtained.

81

2. The Lp (a) concentration ‗C‘ was multiplied with the dilution factor (F) of the

test sample for obtaining the concentration of Lp (a) in the neat test sample.

Concentration of Lp (a) in the neat test sample in mg/dl = CxF

(Where ‗F‘ is the dilution factor of the test sample) [Isser, H. S et al., 2001]

5.5.9 ATHEROGENIC INDEX OF PLASMA (AIP) CALCULATION:

AIP = log (TG/HDL) [Dobiasova, M et al., 2001]

5.5.10 Estimation of serum malondialdehyde (MDA)

Method: MDA is determined as Thiobarbituric acid reactive substances

(TBARS)

Principle: Free MDA, as a measure of lipid peroxidation, was measured

spectrophotometrically as TBA reactive substances after precipitating the

proteins with trichloroacetic acid (TCA).

Reagent preparation:

1. Normal saline: 0.9%

2. TBA reagent: TBA with a concentration of 8 mg/ ml was prepared using

distilled water. Required reagent was prepared fresh for each assay.

3. n-Butanol

Standard: Tetramethoxy propane (TMP) was used as standard. Sigma grade

chemical was procured. It was diluted with distilled water to yield a working

82

calibrator of 15μmol/L concentration. This working calibrator was diluted as

follows to yield standards with various concentrations for standard curve:

Standard

Concentration

Working

standard Distilled water

standard1 12μmol/L 2.4 ml 0.6 ml

standard2 6 μmol/L 1.5 ml of Std1 1.5 ml

standard3 3 μmol/L 1.5 ml of Std2 1.5 ml

standard4 1.5 μmol/L 1.5 ml of Std3 1.5 ml

standard5 0.75 μmol/L 1.5 ml of Std4 1.5 ml

standard6 0 μmol/L - 3 ml

Sample – Serum

Procedure:

500 μl of plasma and 500 μl of saline were taken in a test tube. 1 ml of

24% TCA was added and mixed. The mixture was centrifuged at 2000 rpm for

20minutes. 1.0ml of the supernatant [protein free filtrate] was transferred into

a test tube. To 1 ml of the above protein free filtrate and to the standards, 250

μl of TBA reagent was added. The test tubes were covered with rubber cork

and kept in boiling water bath for 1hour. The tubes were then removed and

cooled under tap water. 500 μl of n-Butanol was added and vortexed for 1

minute. Later the mixture was centrifuged and the upper butanol layer was

83

read at 532 nm using Perkin Elmer Lamda 1.2 spectrophotometer. The

concentrations of the samples were derived from the calibration curve.

Calculation:

Concentration of MDA= OD of Test – OD of Blank _____________________ X Conc. Of STD

OD of STD – OD of Blank

Concentration of Standard = 5 μmol/L [Lefevre, G et al., 1997].

5.5.11 ESTIMATION OF NITRIC OXIDE (AS NITRITE)

Method: Kinetic cadmium reduction.

Principle:

Nitrate, the stable product of nitric oxide is reduced to nitrite by

cadmium reduction method after deproteinisation of sample by Somogyi

reagent. The nitrite produced is determined by diazotization of sulphanilamide

and coupling with napthylethylene diamine.

Reagent preparation:

1. Glycine – NaOH buffer (pH -9.7): 7.5 gm of glycine was dissolved in 200

ml. of deionized water, then adjusted to pH 9.7 by 2 M NaOH. The

solution was finally adjusted to 500 ml. by deionised water.

84

2. Sulphanilamide: 2.5 gm. of sulphanilamide was dissolved in 250 ml. of

warm 3 mol/L HCl solution, and then allowed to cool. This reagent is

stable for one year at room temperature.

3. N-Naphthylethylene diamine: 50 mg of N-Naphthylethylene diamine was

dissolved in deionised water and volume was adjusted up to 250 ml.

4. Standard (NaNO2) – Sodium nitrite

5. Stock standard – (100Mmol/L) 690 mg of NaNO2 was dissolved in 100

ml of 10 mmol/L sodium borate solution.

6. Working standard - (10 u mol/L): 10 ml of stock was diluted to 100 ml

with 10mmol/L solution of sodium borate.

5). ZnSO4 solution (75mmol/L)

6) NaOH solution (55mmol/L)

7) H2SO4 solution (0.1mol/L)

8) CuSO4 solution (5mmol/L): 125 mg of CuSO4 was dissolved in 100

ml. of glycine – NaOH buffer.

Sample - Heparinized Plasma

Procedure:

A) Deproteinization –

In a clean, dry centrifuge tube 0.5ml of plasma was taken and 2.0 ml of

75mmol/L ZnSo4 solution and with mixing 2.5 ml of 55mmol/L NaOH reagent

was added, mixed well and centrifuged for 10 minutes.

85

B). Activation of cadmium granules –

1) Cadmium granules were stored in 0.1 mol/L H2SO4 solution.

2) The acid from granules was rinsed three times with deionised

water at the time of assay. .

3) Then the granules were swirled in 5mmol/L CuSO4 solution for 1-

2 minutes.

4) These copper coated granules were drained & washed with

glycine NaOH buffer.

5) These activated granules were used within 10 minutes after

activation.

6) The granules after use were washed with deionized water and

stored in 0.1mol/L, H2SO4 and subsequently the same procedure

for activation was followed.

C) Nitrite Assay:

1) Three Erlenmeyer flasks were taken and labeled as test, standard

and blank.

2) To each of the flasks 1 ml glycine – NaOH buffer was added.

3) To these test tube labeled as blank, test and standard; deionised

water, deproteinized sample and working standard solution, 1 ml

each was added respectively.

86

4) With a spatula, 2.5 to 3 gm of freshly activated cadmium granules

was added to each flask.

5) All the flasks were stirred for swirling the granules.

6) After 90 minutes, the mixture in all the three flasks was diluted to

4 ml with deionized water.

7) 2 ml of this above diluted solution was pipetted out in clean, dry

test tubes labeled as blank, test and standard respectively.

8) 1 ml. sulphanilamide was added to each of the tube.

9) 1 ml. of N-Naphthylethylene diamine solution was added to each

tube.

10) The contents of the three test tubes were mixed well and after 20

minutes absorbances were read against blank by using 540 nm

filter on colorimeter [Cortas, N.K et al., 1990].

Calculation:

OD of Test – OD of Blank Plasma Nitrite (µ mol/L) = ----------------------------------- X 100

OD of Std. – OD of Blank

87

5.5.12 FERRIC REDUCING ABILITY OF PLASMA (FRAP)

Method: The ferric reducing ability of plasma as a measure of Antioxidant

power.

Principle: Antioxidant capacity converts ferric into ferrous ion. Reduction at

low pH causes a colored ferrous tripyridyltriazine complex to form Ferric

Reducing Ability of Plasma values are obtained by comparing the absorbance

change at 593 nm in mixture (test), with those containing ferrous ion in known

concentration (standard).

Reagent Preparation:

1. FeCl3.6H2O (20mmol/L) - 270mg of Fecl3. 6H2O was dissolved in

50 ml of D.W.

2. 1% Na2CO3: 500mg was dissolved in 50 ml of DW.

3. Acetate Buffer (PH 3.6): 0.31 gm of sodium acetate was

dissolved in 50 ml DW

1.6 ml glacial acetic acid was added to the above and PH was

adjusted to 3.6 using 1% Na2CO3 .The volume was made up to

100 ml with DW

4. 0.04M HCl: 348 µl of Concentrated HCl was added to DW and the

volume was made up to 100 ml

88

5. Ferrozine: 49 mg Ferrozine was dissolved in 0.04M HCl and

made up to 10 ml.

6. Ferrous Standard (Stock): mM 0.0139 gm of FeSo4. 7H2O was

dissolved in 25 ml of DW & made up to 50 ml with DW.

Sample - Heparinized Plasma

Procedure:

Chemicals

Test

(l)

Control

(l)

Standard

(l)

Blank

(l)

Acetate

Buffer 400 440 400 400

FeCl3 40 - 40 40

Ferrozine 40 40 40 40

Sample 60 - - -

Std - - 60 -

DW - - - 60

The test tubes were incubated at 37°C for 30 min.

Sample - 60 - -

All the test tubes were incubated at 37°C for 30 min & OD read at 593 nm

[Benzie, I F et al 1996].

89

Calculation:

T-C TAC (μmol/L) ---- X 1000 S-B

Concentration Std DW

1 100 µmol/l 50 µl + 450 µl

2 200 µmol/l 100 µl + 400 µl

3 400 µmol/l 200 µl + 300 µl

4 800 µmol/l 400 µl + 100 µl

5 1000 µmol/l Directly add from stock std. to the std

test tube.

Reference Range: 0.6-1.6mmol/L

5.5.13 Estimation of TNF-α:

Method: Quantitative sandwich enzyme immunoassay technique (ELISA)

using Quantikine ELISA kit.

Principle: A specific monoclonal antibody for TNF-α was pre-coated onto a

microplate. Standards and samples were pipetted into the wells. TNF-α

present in the samples and standards were bound by the immobilized

antibody. After washing the unbound substances, another TNF-α specific

enzyme-linked polyclonal antibody was added to the wells. The unbound

90

antibody-enzyme reagent was removed by wash. A substrate solution is

added to the wells. The color was developed in proportion to the amount of

TNF-α bound in the first step. The color development is stopped and intensity

of color is measured.

Reagents:

Anti TNF- α coated micro plate

Polyclonal antibody conjugated to horseradish peroxidase

TNF-α STD

Assay diluent

RD1F- buffered protein base

Calibrator diluent- RD6-35 animal serum

Wash buffer concentrate-

Color Reagent A - stabilized hydrogen peroxide.

Color Reagent B - stabilized chromogen (tetramethylbenzidine).

Stop Solution - 2 N sulfuric acid.

Reagent preparation:

1 Wash Buffer- 20 ml of wash buffer concentrate was diluted with

deionized water to prepare 500 ml of wash buffer.

2 Calibrator Diluent RD6-35 - 10 ml of Calibrator diluent was added to 40

ml of deionized water to yield 50 ml of diluted calibrator diluent RD6-35.

3 Substrate Solution - Color Reagents A and B are mixed together in

equal volumes.

91

4 TNF-α Standard - TNF-α standard was reconstituted with deionized

water which had produced a stock solution of 10,000 pg/ml. The

standard was allowed to sit for a minimum of 15 minutes with gentle

agitation.

5 100 μl of 10000 pg/ml standard was pipetted into 5ml test tube. 900 μL

of calibrator diluent RD6-35 was pipetted into a 1000 pg/mL tube. 500 μl

of the calibrator diluent was pipietted into the remaining six (S2-S7)

tubes. Using the stock solution the dilution series were made and mixed

thoroughly. The 1000 pg/ml standard serves as a high standard. The

appropriate calibrator diluent served as a zero standard (S0=0

pg/ml).The concentrations of the standards (S1-S7) are as below.

Standard Concentration ( pg/ml)

S1 1000 pg/ml

S2 500 pg/ml

S3 250 pg/ml

S4 125 pg/ml

S5 62.5 pg/ml

S6 31.2 pg/ml

S7 15.6 pg/ml

92

Assay procedure:

1. 50 μl of assay diluent was added to each well.

2. 200 μl of Standard, sample, or control per well was added and

covered with the adhesive strip.

3. Incubated for 2 hours at room temperature.

4. Aspirated each well and washed, repeated the process three times

for a total number of four washes using a squirt bottle.

5. After the last wash, removed any remaining wash buffer by

aspirating or decanting. Inverted the plate and blotted it against

clean paper towels.

6. 200 μl of TNF-α Conjugate was added to each well.

7. Incubated for 2 hours at room temperature.

8. Repeated the aspiration /wash as in step 4.

9. 200 μl of substrate solution was added to each well. Incubated for 20

minutes at room temperature. Protected from light.

10. 50 μl of stop solution was added to each well and mixed well.

11. Determined the optical density of each well using a microplate

reader set to 450 nm.

12. The OD of standard, control, and sample was subtracted from zero

standard OD.

13. A standard curve was plotted with the mean absorbance for each

standard on the y-axis against the concentration on the x-axis and a

best fit curve through the points on the graph was drawn.

93

14. The concentrations of the samples were derived from the calibration

curve [Idriss, H .T et al., 2000].

5.5.14 Estimation of IL-6:

Method: Quantitative sandwich enzyme immunoassay technique (ELISA)

using Quantikine ELISA kit

Principle: A specific monoclonal antibody for IL-6 was pre-coated onto a

microplate. Standards and samples were pipetted into the wells. IL-6 present

in the samples and standards were bound by the immobilized antibody. After

washing the unbound substances, another IL-6 specific enzyme-linked

polyclonal antibody was added to the wells. The unbound antibody-enzyme

reagent was removed by wash. A substrate solution is added to the wells. The

color was developed in proportion to the amount of IL-6 bound in the first step.

The color development is stopped and intensity of color is measured.

Reagents:

1. Anti IL-6 coated micro plate

2. Human IL-6 standard

3. Polyclonal antibody specific for human IL-6 Conjugated to horse radish

peroxidase

4. Assay Diluent RD1W

5. Calibrator Diluent RD5T

6. Calibrator Diluent RD6F

7. Wash Buffer Concentrate:

94

8. Color Reagent A: stabilized hydrogen peroxide.

9. Color Reagent B: stabilized chromogen (tetramethylbenzidine)

10. Stop Solution: 2 N sulfuric acid.

Reagent preparation

1. All reagents were brought to the room temperature before use.

2. 20 ml of wash buffer concentrate was added to the to deionized water to

prepare 500 ml of wash buffer.

3. Substrate Solution - Color Reagents A and B were mixed together in

equal volumes within 15 minutes of use.

4. Human IL-6 Standard –Human IL-6 standard was reconstituted with

calibrator Diluent RD6F which had produced a stock solution of 300

pg/ml.

5. Pipetted 333 μl of standard from 300 pg/ml stock standard solution and

667 μl of the calibrator diluent RD6F into a test tube which gave

100pg/ml concentration working standard (S1).

6. Marked the test tubes from S2- S6 and pipetted 500 μl of diluent into

each tube. Used S1 to produce a dilution series. Mixed each tube

thoroughly before the next transfer. The undiluted standard served as

the high standard (300pg/ml). The appropriate calibrator diluent served

as the zero.

95

7. The concentrations of S1 –S6 were as below

Standard Concentration (pg/ml)

S0 0

S1 100

S2 50

S3 25

S4 12.5

S5 6.25

S6 3.13

ASSAY PROCEDURE:

1. 100 μl of Assay Diluent RD1W was added to each well.

2. 100 μl of standard, sample, or control per well was added. Covered

with the adhesive strip.

3. Incubated for 2 hours at room temperature.

4. Aspirated each well and washed, repeated the process three times

for a total of four washes.

5. Washed by filling each well with wash buffer (400 μl) using a squirt

bottle

6. After the last wash, removed any remaining wash buffer by

aspirating or decanting. Inverted the plate and blotted it against

clean paper towels.

96

7. 200 μl of human IL-6 conjugate was added to each well. Covered

and incubated for 2 hours at room temperature.

8. Repeaedt the aspiration/wash

9. 200 μl of substrate solution was added to each well. Incubated for 20

minutes at room temperature. Protected from light.

10. 50 μl of stop solution was added to each well.

11. Determined the optical density of each well within 30 minutes, using

a microplate reader set to 450 nm.

12. The OD of standard, control, and sample was subtracted from zero

standard OD.

13. A standard curve was plotted with the mean absorbance for each

standard on the y-axis against the concentration on the x-axis and a

best fit curve through the points on the graph was drawn.

The concentrations of the samples were derived from the calibration

curve [Mansell, A et al., 2013].

5.5.15 Estimation of sFlt-1:

Method: Quantitative sandwich enzyme immunoassay.

Principle: A specific monoclonal antibody for sVEGFR1 was pre-coated onto

a microplate. Standards and samples were pipetted into the wells. sVEGFR1

present in the samples and standards were bound by the immobilized

antibody. After washing the unbound substances, another sVEGFR1 specific

enzyme-linked polyclonal antibody was added to the wells. The unbound

97

antibody-enzyme reagent was removed by wash. A substrate solution is

added to the wells. The color was developed in proportion to the amount of

sVEGFR1 bound in the first step. The color development is stopped and

intensity of color is measured.

Reagents:

1. Monoclonal antibody coated with human VEGF R1 microplate

2. Human VEGF R1 Standard

3. Human VEGF R1 Conjugate

4. Assay Diluent RD1-68

5. Calibrator Diluent RD6-10

6. Wash buffer concentrate

7. Color Reagent A: stabilized hydrogen peroxide.

8. Color Reagent B: stabilized chromogen (tetramethylbenzidine).

9. Stop Solution: 2 N sulfuric acid.

Reagent preparation:

1. All reagents were brought to the room temperature.

2. 20 ml of wash buffer concentrate was added to the deionized water to

prepare 500 ml of wash buffer.

3. Substrate solution - Color reagents A and B were mixed together in

equal volumes within 5 minutes of use.

4. Human VEGF R1 Standard - Reconstituted the human VEGF R1

Standard with deionized water which produced a stock solution of

98

20,000 pg/ml. Mixed the standard to ensure complete reconstitution and

allowed the standard to sit for a minimum of 15 minutes with gentle

agitation prior to making dilutions.

5. Pipetted 100 μl of stock standard and 900 μl of calibrator diluent RD6-

10 into a test tube and mixed it properly. This served as working

standard (S1) with 2000pg/ml concentration.

6. Pipetted 500 μL of calibrator diluent RD6-10 into the remaining tubes

(S2-S7). Used the S1 solution to produce a dilution series. Mixed each

tube thoroughly before the next transfer. The 2000 pg/mL standard

served as the high standard. Calibrator Diluent RD6-10 served as the

zero standard.

7. The concentrations of S1- S7 were as below:

Standard Concentration (pg/ml)

S1 2000

S2 1000

S3 500

S4 250

S5 125

S6 62.5

S7 31.3

99

ASSAY PROCEDURE:

1. 100 μL of assay diluent RD1-68 was added to each well.

2. 100 μL of standard, control, or sample was added per well. Incubated

for 2 hours at room temperature on a horizontal orbital microplate

shaker set at 500 rpm ± 50 rpm.

3. Aspirated each well and washed, repeating the process three times for

a total of four washes.

4. Washed by filling each well with wash buffer (400 μl) using a squirt

bottle.

5. After the last wash, removed any remaining wash buffer by aspirating or

decanting. Inverted the plate and blotted it against clean paper towels.

6. 200 μl of Human VEGF R1 Conjugate was added to each well. Covered

it.

7. Incubated for 2 hours at room temperature on the shaker.

8. Repeated the aspiration/wash.

9. 200 μl of substrate solution was addedto each well. Incubated for 30

minutes at room temperature on the bench top. Protected from light.

10. 50 μl of stop solution was added to each well.

11. Determined the optical density of each well within 30 minutes, using a

microplate reader set to 450 nm.

12. The OD of standard, control, and sample was subtracted from zero

standard OD.

100

13. A standard curve was plotted with the mean absorbance for each

standard on the y-axis against the concentration on the x-axis and a

best fit curve through the points on the graph was drawn.

14. The concentrations of the samples were derived from the calibration

curve [Shibuya, M et al., 1990].

5.5.16 Estimation of VEGF

Method: Quantitative sandwich enzyme immunoassay technique (ELISA)

using Quantikine ELISA kit

Principle: A specific monoclonal antibody for VEGF was pre-coated onto a

microplate. Standards and samples were pipetted into the wells. VEGF

present in the samples and standards were bound by the immobilized

antibody. After washing the unbound substances, another VEGF specific

enzyme-linked polyclonal antibody was added to the wells. The unbound

antibody-enzyme reagent was removed by wash. A substrate solution is

added to the wells. The color was developed in proportion to the amount of

VEGF bound in the first step. The color development is stopped and intensity

of color is measured.

Reagents:

1. Monoclonal antibody coated against VEGF microplate

2. VEGF Standard

3. VEGF Conjugate:

101

4. Assay Diluent RD1W

5. Calibrator Diluent RD6U

6. Wash buffer concentrate:

7. Color Reagent A: Hydrogen peroxide.

8. Color Reagent B: Stabilized chromogen (tetramethylbenzidine).

9. Stop solution: 2 N sulfuric acid.

Reagent preparation

1. All the reagents were brought to the room temperature before use.

2. 20 ml of wash buffer concentrate was added to deionized water to

prepare 500 ml of wash buffer.

3. Substrate solution - Color Reagents A and B are mixed together in

equal volumes within 15 minutes of use.

4. VEGF standard -VEGF standard was reconstituted with calibrator

diluent RD6U. Mixed well before use. This reconstitution produced a

stock solution of 2000 pg/ml which served as high standard (S1).

Allowed the standard to sit for a minimum of 15 minutes with gentle

agitation prior to making dilutions.

5. Pipetted 500 μl of stock solution and calibrator diluent RD6U into a test

tube. This gave us a concentration of 1000pg/ml (S2). Used this

solution to produce a dilution series (S3-S7). Mixed each tube

thoroughly before the next transfer. Calibrator diluent RD6U served as

zero standard (0pg/ml). The concentrations of S1- S7 were as below:

102

Standard Concentration (pg/ml)

S1 2000

S2 1000

S3 500

S4 250

S5 125

S6 62.5

S7 31.3

Assay procedure:

1. 100 μl of assay diluent RD1W was added to each well.

2. 100 μl of Standard, control, or sample was added per well.

3. Covered and incubated for 2 hours at room temperature.

4. Aspirated each well and washed, repeating the process twice for a total

of three washes. Washed by filling each well with wash buffer (400 μl)

using a squirt bottle. After the last wash, removed any remaining wash

buffer by aspirating or decanting. Inverted the plate and blotted it

against clean paper towels.

5. 200 μl of VEGF conjugate was added to each well. Covered it and

incubated for 2 hours at room temperature.

6. Repeated the aspiration/wash.

7. 200 μl of substrate solution was added to each well. Protected from

light.

103

8. Incubated for 25 minutes at room temperature.

9. 50 μl of stop solution was added to each well

10. Determined the optical density of each well within 30 minutes, using a

microplate reader set to 450 nm.

11. The OD of standard, control, and sample was subtracted from zero

standard OD.

12. A standard curve was plotted with the mean absorbance for each

standard on the y-axis against the concentration on the x-axis and a

best fit curve through the points on the graph was drawn.

13. The concentrations of the samples were derived from the calibration

curve [Leung, D. W et al., 1989].

5.5.17 Estimation of PlGF

Method: Quantitative sandwich enzyme immunoassay technique (ELISA)

using Quantikine ELISA kit

Principle: A specific monoclonal antibody for PlGF was pre-coated onto a

microplate. Standards and samples were pipetted into the wells. PlGF present

in the samples and standards were bound by the immobilized antibody. After

washing the unbound substances, another PlGF specific enzyme-linked

polyclonal antibody was added to the wells. The unbound antibody-enzyme

reagent was removed by wash. A substrate solution is added to the wells. The

color was developed in proportion to the amount of PlGF bound in the first

step. The color development is stopped and intensity of color is measured.

104

Reagents:

1. Monoclonal antibody specific to human PlGF coated microplate

2. Human PlGF standard

3. Human PlGF Conjugate

4. Assay Diluent RD1-22

5. Calibrator Diluent RD6-11

6. Wash buffer concentrate

7. Color Reagent A: Stabilized hydrogen peroxide.

8. Color Reagent B: Stabilized chromogen (tetramethylbenzidine).

9. Stop Solution: 2 N sulfuric acid.

Reagent preparation:

1. All the reagents were brought to the room temperature before use.

2. 20 ml of wash buffer concentrate was added to deionized water to

prepare 500 ml of wash buffer.

3. Color reagents A and B were mixed together in equal volumes within 15

minutes of use.

4. Human PlGF Standard was reconstituted with with calibrator diluent

RD6-11. This reconstitution produced a stock solution of 1000 pg/ml

which served as high standard (S1). The appropriate calibrator diluent

served as the zero standard (S0)

105

5. Pipetted 500 μl of the appropriate calibrator diluent into each tube. Used

the stock solution (S1) to produce a dilution series. The concentrations

of S1- S7 were as below:

Standard Concentration (pg/ml)

S1 1000

S2 500

S3 250

S4 125

S5 62.5

S6 31.3

S7 15.6

Assay procedure

1. 100 μl of assay diluent RD1-22 was added to each well.

2. 100 μl of standard, control, or sample was added per well. Covered it.

Incubated for 2 hours at room temperature.

3. Aspirated each well and washed, repeating the process three times for

a total of four washes. Washed by filling each well with wash buffer

(400μl) using a squirt bottle.

4. After the last wash, removed any remaining wash buffer by aspirating or

decanting. Inverted the plate and blotted it against clean paper towels.

5. 200 μl of human PlGF conjugate was added to each well. Covered it.

Incubated for 2 hours at room temperature.

106

6. Repeated the aspiration/wash

7. 200 μl of substrate solution was added to each well. Incubated for 30

minutes at room temperature.

8. 50 μl of stop solution was added to each well.

9. Determined the optical density of each well within 30 minutes, using a

micro plate reader set to 450 nm.

10. The OD of standard, control, and sample was subtracted from zero

standard OD.

11. A standard curve was plotted with the mean absorbance for each

standard on the y-axis against the concentration on the x-axis and a

best fit curve through the points on the graph was drawn.

12. The concentrations of the samples were derived from the calibration

curve [Li, X et al., 2003].

5.5.18 ESTIMATION OF HEMOGLOBIN

Method: Cyanmethemoglobin method

Principle: The principle of this method is that when blood is mixed with a

solution containing potassium ferricyanide and potassium cyanide, the

potassium ferricyanide oxidizes iron to form methemoglobin. The potassium

cyanide then combines with methemoglobin to form cyanmethemoglobin,

which is a stable color pigment read photometrically at a wave length of

540nm.

107

Reagents:

DRABKINS solution:

Potassium ferricyanide = 200 mg

Potassium cyanide = 50 mg

Potassium dihydrogen phosphate = 140 mg

Non-ionic detergent = 1 ml

Distilled water = Make up to 1000 ml (1 L)

Standard: 12g/dl

Procedure:

Take 20µl of blood and 4ml of Drabkin solution .Mix well. Read within 6 hours

of mixing on filter 540. Read against blank of drabkin solution. Also read the

standard solution with the same dilution like test sample.

Hb of test sample = (OD of test /OD of STD) X conc.of STD

[Balasubramaniam, P et al., 1982].

108

5.5.19 ESTIMATION OF URINE PROTEIN

Method: Pyrogallol Red Method

Principle: Pyrogallol Red is combined with molybdenum acid at a low pH.

When the complex is combined with protein, a bluepurple color is formed. The

increase in absorbance at 600 nm is directly proportional to the protein

concentration in the sample.

Reagents:

Microprotein reagent: Contains buffer, Pyrogallol red 0.067 mmol/L,

sodium molybdate stabilizer 0.153 mmol/L, surfactants, and preservative.

Protein standard solution: Contains Albumin 50 mg/dl in saline with

sodium azide 0.05% as a preservative

Procedure:

1. Three test tubes were labeled as ―Blank‖, ―Standard‖, ―Control‖, and

―Samples‖

2. Pipette 1.0ml of Microprotein reagent to each tube.

3. Allow the tubes to warm to 37°C.

4. Pipette 20µl of deionized water, standard, controls, and samples to the

appropriately labeled tubes.

5. Allow tubes to incubate at 37°C for 5 minutes.

109

6. After 5 minutes, set the spectrophotometer to 600nm and zero the

instrument with the BLANK tube.

7. Absorbances of B, S, and T were read at 600nm in spectrophotometer

[Andresen, B. D. et al., 1986].

Calculation:

Protein (mg/dl) = OD of Test --------------- X Conc. of STD (50 mg/dl)

OD of STD

5.6 Anthropometric Measurements:

Height: using vertical scale

Weight: using accurate physical balance

Blood pressure: Sphygmomanometer

5.6.1 Statistical analysis

Statistics analysis was done by using SPSS software version 16.0.

Quantitative variables were demonstrated as Mean±Standard deviation.

ANOVA, Pearson‘s correlation analysis were performed to compare the

quantitative variables. A ‗p‘ value <0.05.was considered as statistically

significant.

110

6. RESULTS AND DISCUSSION

Pregnancy-Induced Hypertension (PIH) continues to be a main obstetric

problem in present-day healthcare practice. It affects not only maternal health

but also puts fetal development at risk [Kim, S.Y et al., 2007]. It complicates

almost 10% of all pregnancies. Approximately 30% of hypertensive disorders

of pregnancy were due to chronic hypertension while 70% of the cases were

diagnosed as gestational hypertension/preeclampsia in a multi-center study

[Sajith, M et al 2014]. Eclampsia is a very serious complication of pregnancy

which is responsible for high maternal and perinatal mortality. It accounts for

50,000 maternal deaths annually worldwide. In spite of several global and

regional interventions and initiatives from governments and other concerned

agencies, maternal mortality is still very high in India, with eclampsia as a

major cause [Das, R et al., 2015]. In our study we analysed the levels of lipids,

lipoproteins, oxidative stress, inflammatory markers, angiogenic,

antiangiogenic factors and the extent of endothelial dysfunction in both

normotensive and PIH (PE & E) women. The correlation status between lipids,

lipoproteins, apolipoproteins and angiogenic, antiangiogenic factors was

analysed in this study.

111

Sociodemographic details of the study subjects:

This study was conducted on the pregnant women attending the

Gynecology and obstetrics department of Annapoorana medical college and

hospital, Salem, Tamilnadu, India. A total of 300 pregnant women at

gestational age of > 20weeks were registered in the study after taking

informed consent. Out of these, 200 women were randomly selected and

compared with a control group of women for age, BMI and other biochemical

parameters.

The study subjects were divided into three groups

Group 1: Healthy women having normal uncomplicated pregnancy without

hypertension (100).

Group 2: Women with pre-eclampsia (100)

Group 3: Women with eclampsia (100)

The socio demographic and reproductive characteristics of the two

patients groups and control subjects investigated are summarized in Table

number 2. The significant difference was not observed in the level of maternal

age, gestational age, Hb between three groups. The Mean SBP/DBP was

gradually increasing with the increase in the severity of disease as in

eclamptic group .Urine proteins were significantly high in PE & E women

compared to the control group. Eclamptic women had shown significant

proteinuria compared PE women.

112

Table No: 2 Comparison of socio demographic characters in PIH women and controls

Parameters

Controls

Preeclampsia

P value

Eclampsia

P value

P value between PE & E

Mean SD Mean SD Mean SD

Age ( Years)

23.97 3.30 24.62 4.07 NS 25.71 3.71 <0.01 NS

BMI (Kg/m2) 24.37 1.80 28.1 6.08 <0.01 30.2 5.45 <0.01 <0.01

Gestational age (weeks)

31.57 2.67 32.42 3.12 NS 31.05 2.90 NS NS

SBP (mm Hg)

116 5.45 162.18 18.26 <0.01 170 15.52 <0.01 <0.01

DBP (mm Hg)

75 5.99 107.5 11.35 <0.0.1 112.28 10.59 <0.05 <0.01

Hb (g/dl) 10.23 1.74 10.34 1.57 NS 10.68 1.58 NS NS

Urine albumin (mg/day)

150.9 33.4 436 96 <0.01 432 101 <0.01 <0.01

*Data expressed as Mean±SD. A p value of <0.05 is considered as significant.

**Data expressed as Mean±SD. A p value of <0.01 is considered as highly significant.

Table No: 2 shows high levels of SBP, DBP, BMI and Urine albumin between

Normotensive women and PE, E women. The above levels were high in

eclamptic women than preeclampsia.

113

BODY MASS INDEX:

In our study a significantly high level of BMI was observed in PE & E

groups compared to the control group. Eclamptic women (30.2 ± 5.45kg/m2)

were found to be more obese in contrast to preeclamptics (28.1±6.08 kg/m2).

Two different studies that determined BMI has reported that the risk of severe

and mild preeclampsia is greater in obese and overweight women [Roberts,

J.M et al, 2011, De Melo Dantas, E.M et al., 2013]. One retrospective cohort

study demonstrated a strong relationship between the occurrence of

preeclampsia / eclampsia and excess maternal body weight prior to

pregnancy and weight gain during pregnancy [Lewis, F et al ., 2014].The risk

of preeclampsia doubles with each 5 to 7 Kg/m2 increase in maternal BMI [El-

Makhzangy, I et al., 2010]. According to a Meta analytical study [Poorolajal, J

et al., 2016] the BMI is significantly associated with the risk of PE and

therefore, overweight and obesity can be considered as a predictor of

preeclampsia. Hyperlipidemia and inflammation are two mechanisms that are

hypothesized for BMI related preeclampsia [Bodnar, L.M et al., 2005].

114

6.1 Objective 1: To determine the serum lipid and lipoprotein levels in

PIH women

Pregnancy is known to affect biochemical metabolic processes involved

in carbohydrate, protein, lipid and lipoprotein metabolism. These metabolic

changes have evolved to meet the metabolic demands of the growing fetus.

However, in some cases, such metabolic changes particularly lipid and

lipoprotein are found to be exaggerated which may lead to ED [Musa, A.H et

al. 2014]. A study by Chiang na etal demonstrated two to three times rise in

serum TG in normal pregnancy as compared to nonpregnant women, which

may be as high as two to three folds in the third trimester [An-Na, C et al

1995]. Two case control studies and a comparative observational study had

demonstrated a consistent significant rise in the levels of TC, TGLs, LDL,

VLDL and consistent significant reduction in the levels of HDL and ApoA-1

from 1st trimester towards 3rd trimester [Anjum, R et al.; 2013, Nayan, S et

al.; 2014, Deshpande, H et al.; 2016]. In addition, it has been stated that the

dyslipidemia may impair trophoblast invasion thus contributing to a cascade of

pathophysiologic events that lead to the development of preeclampsia. This

hypothesis is supported by a report that the triglyceride accumulation in the

endothelial cells is associated with decreased release of prostacyclin

[Sharami, S.H et al., 2012].

115

Table No. 6.1.1 Comparison of lipid profile, lipoproteins, apolipoproteins

in PIH and control women

Parameters

Controls Pre-

eclampsia P

value

Eclampsia P

value

P value

between

PE & E Mean SD Mean SD Mean SD

TC

(mg/dl) 209.7 34.90 223.59 39.46 <0.05 244.14 43.56 <0.01 <0.01

TGL

(mg/dl) 203.6 37.31 246.53 34.29 <0.01 318.48 78.39 <0.01 <0.01

HDL

(mg/dl) 44.02 7.71 35.65 7.64 <0.01 29.97 4.61 <0.01 <0.01

LDL

(mg/dl) 124.95 34.30 138.63 34.66 <0.05 150.47 46.0 <0.01 <0.01

VLDL

(mg/dl) 40.72 7.46 49.30 6.85 <0.01 63.69 15.67 <0.01 <0.01

ApoB

(mg/dl) 129.22 16.69 152.85 26.6 <0.01 182.14 16.17 <0.01 <0.01

ApoA-1

(mg/dl) 174.39 26.22 144.65 32.05 <0.01 131.18 23.72 <0.01 <0.01

Lp(a)

(mg/dl) 48.86 11.21 72.92 13.44 <0.01 75.76 11.63 <0.01 NS

*Data expressed as mean±SD. A p value of <0.05 is considered as significant.

**Data expressed as Mean±SD. A p value of <0.01 is considered as highly

significant.

In the present study a significant high levels of triglycerides, LDL, VLDL,

Apo B were observed in PE & E groups compared to the controls. In eclamptic

women the above levels were significantly higher compared to the PE women.

116

Graph: 6.1.1 Comparison of lipid profile, lipoproteins, apolipoproteins in

PIH and control women

*Data expressed as Mean±SD. A p value of <0.05 is considered as significant.

**Data expressed as Mean±SD. A p value of <0.01 is considered as highly

significant.

The observed hypertriglyceridemia in PIH women might be due to

hypoestrogenaemia and insulin resistant visceral fat that induces hepatic

biosynthesis of TGs [Sahu, S et al., 2009]. Lipogenesis is encouraged in early

pregnancy in order to prepare for the rapid fetal growth in late pregnancy. But,

in late pregnancy as a result of insulin resistance the lipolysis is increased,

leading to increased flux of fatty acids to the liver via portal vein [Agarwal, V et

al.,2014] with two consequences: hepatic steatosis due to an increase in TGL

synthesis and increase in the blood VLDL [Soca, P.E.M et al., 2013].

Observed hypertriglyceridemia could also be due to low activity of LPL, an

117

insulin-dependent endothelial enzyme. Because of the decrease in the activity

of LPL, the removal of chylomicrons and VLDL from circulation is low in insulin

resistant patients. VLDL-C level elevates in PIH as found in present study is in

agreement with other researchers [Sattar, N et al., 1997, Yadav, S et al.,

2013]. Thus VLDL remains in the plasma for a longer time and accumulates.

In normal pregnancy the placental VLDL receptors are up regulated which

results in rerouting of TGL rich lipoproteins to feto-placental unit. However, the

state of hypoestrogenemia in preeclampsia leads to decreased expression of

VLDL receptors in the placenta. This results in reduced transport of VLDL to

fetal compartment, which might be the reason for maternal

hypertriglyceridemia.

The increase in the LDL levels observed in our study might be due to

the reduced fetoplacental perfusion and reduced uptake by the fetus for the

synthesis of Dehydroepiandrosterone (DHEA) [Pushparaj, J.L et al., 2012].

The increased maternal TGL are expected to be deposited in the predisposed

vessels, like uterine spiral arteries contributing to an activation of endothelial

cells and thus leading to the production of placental derived factors thereby

probably contributing to the pathogenesis of PIH [Phalak, P et al., 2012,

Kaloti, A.S et al., 2015].

It is well known that APO-B100 is a protein component of a variety of

lipoproteins which are LDL-C, VLDL-C, IDL-C and lipoprotein (a). The

significant higher values of Apo B-100 found in our study was in consistent

with other studies [Cekmen, M.B et al., 2003, Lei, Q et al., 2011]. In most

118

conditions more than 90% of all ApoB in blood is found in LDL [Visser, M.E et

al., 2010]. It has been observed that in the cases where LDL C is in the

normal/low range, high ApoB levels indicate an increased number of small

dense LDL (sd-LDL) particles, which are the most atherogenic particles

[Walldius, G et al., 2012].

Other studies had demonstrated that the higher Lp (a) levels were

associated with severity of disease [Wang, J et al., 1998, Bayhan, G et al.,

2005, Nazil. R et al., 2013]. In our study also the Lp (a) levels were

significantly high in eclamptic women compared to the controls and PE

women but there is no significant difference between PE & E women. This

increase might be due to more widespread endothelial cell damage,

necessitating increased levels of Lp(a) to act both as an acute phase protein

and as a vehicle for cholesterol deposition at the site of dysfunction

[Fanshawe, A. E et al., 2013]. However some studies had demonstrated no

change in the level of Lp (a), this might be due to the differences in the

method used, study design, sample sizes and ethnicity of study populations

[Cesur, M et al., 2006, Isildak, M.Y et al., 2008]. It was stated that the

association between Lp (a) and cardiovascular outcomes may differ by

race/ethnicity [Banerjee, D et al., 2011].

In the present study the HDL & apoA –I levels were significantly lower in

PE & E groups compared to the normotensive women. Eclamptic women had

significantly low HDL & ApoA-1 levels than PE women. Our results were in

consistent with studies from other populations Demir, B et al., 2013, Timur et

119

al., 2016). The reason for reduced HDL-C might be due to the increased TGL

which plays a part in decreasing the HDL-cholesterol [Ekhator, C.N et al.,

2012]. The triglyceride enrichment of HDL is a common metabolic

consequence in hypertriglyceridemia and may play an important role in the

decline of HDL. The primary mechanisms leading to reduced plasma HDL

cholesterol levels and HDL particle number in hypertriglyceridemic states may

be due to any one or a combination of the following possibilities: (1) small HDL

particles, which are the product of the intravascular lipolysis of triglyceride-

enriched HDL, may be cleared more rapidly from the circulation, (2) The

lipolytic process of triglyceride-enriched HDL may lower HDL particle number

by shedding the loosely bound apo A-I from the HDL particles and clearing

from the circulation, (3) a dysfunctional lipoprotein lipase or reduced LPL

activity may contribute to the lowering of HDL levels by reducing the

availability of surface constituents of triglyceride-rich lipoproteins that are

necessary for the formation of nascent HDL particles [Lamarche, B et al.,

1999]. On the other hand, the reduced apo-A1 levels that has been displayed

in our study can be originated from of apo-A1 polymorphism of HDL-C or

functional disorder of HDL-C [Nazli, R et al., 2013].

In the current study, the total cholesterol concentrations were

significantly higher in the pregnancy induced hypertensive women (PE & E)

and the levels were significantly higher in eclamptics than preeclamptics. Our

results were in consistency with the findings from other studies [Singh, A et

al., 2016, Nasr, G.M et al., 2010, Yadav, K et al, 2014]. One function of HDL

120

cholesterol is to facilitate reverse cholesterol transport by carrying excess,

potentially harmful cholesterol from peripheral tissues to the liver, where it can

be metabolized. Low levels of HDL-Cholesterol might be the reason for the

displayed higher TC levels in our study [Gohil J. T et al., 2011].

It has been hypothesized by several investigators that there is a

relationship between a disordered lipid profile, endothelial cell and oxidative

stress is of major importance to the pathophysiology of PIH [Raijmakers, M. T

et al., 2004, Anita, C etal. 2015]. PIH is a widespread inflammatory state

where a number of plasma factors that control endothelial functions are

changed [Sibai, B., 2004]. Lipid-mediated oxidative stress is likely to

contribute endothelial hyperstimulation finally leading to severe endothelial

dysfunction which results in distributed micro angiopathic disease with hyper

coagulation and vasospasm [Gratacós, E etal., 2000].

6.2 To assess the Antioxidant capacity (AOC), lipid peroxidation in PIH

women

Lipid peroxidation is an oxidative process that normally occurs at low

levels in all cells and tissues. Under normal conditions a variety of antioxidant

mechanisms serve to control this peroxidative process [Chowdeswari, N et al.,

2016]. In disease states such as toxemia of pregnancy, an imbalance between

lipid peroxidation and antioxidant mechanisms is recorded as an important

causative factor for pathogenesis of pre-eclampsia [Mohanthy, S et al., 2006]

and it could also impair normal endothelial function [Anitha, C et al., 2015]. An

121

earlier study had demonstrated a significant rise in the lipid peroxidation

product levels and a significant fall in the levels of antioxidants levels in PIH

women [Patil, S.B et al., 2007]. This observation holds true in our study also.

Table No: 6.2.1 Comparison of MDA and FRAP between PIH and control

women

Parameters Controls Pre-eclampsia

P value

Eclampsia P value

P value between PE & E

Mean SD Mean SD Mean SD

MDA (μmol/L)

1.08 0.86 4.49 1.75 <0.01 5.50 1.97 <0.01 <0.01

FRAP (μmol/L)

2.21 0.89 0.67 0.42 <0.01 0.40 0.38 <0.01 <0.01

*Data expressed as Mean±SD. A p value of <0.05 is considered as significant.

**Data expressed as Mean±SD. A p value of <0.01 is considered as highly

significant.

In the present study the MDA levels were significantly high in PE & E

women compared to the normotensive pregnant women and eclamptic women

had shown significantly higher MDA levels compared with the PE women. Our

results were in agreement with other studies [Sahu, S et al., 2009, Gupta, S et

al., 2009]. Malondialdehyde (MDA) is the lipid peroxidation end product that

reflects the oxidative status of biological system [Meera, K.S et al., 2010]. The

122

reason for the increase in MDA levels might be due to the placental oxidative

stress. The shallow trophoblast invasion which occurs during early stages of

placentation in PE results into incomplete remodeling of spiral arteries. This

phenomenon leads to intermittent, more pulsatile, blood flow giving rise to

ischaemia /reperfusion type injury along with following increases in ROS

[Hung, T.H et al., 2006]. a) On exposure to ROS, cell membranes predisposes

the occurrence of lipid peroxidation, thus contributing to cell damage for

promoting change in the physical properties and structural organization of

membrane components. Lipid peroxidation ROS attack the polyunsaturated

fatty acids (PUFA) on cell membranes there by leading to cell disintegration

[Ghate, J et al., 2011]. b) Increased lipid peroxidation leads to decrease in

prostacyclin (PGI2): thromboxane A2 (TXA2) ratio causes the vasospastic

phenomenon in kidney, uterus, placenta and brain as seen in PIH [Sahu, S et

al., 2009]. Moreover, increased oxidative stress may also lead to LDL

oxidation. The oxidized LDL is taken up by macrophages via scavenger

receptors and form foam cells resulting in atherogenesis contributing to

endothelial injury and, in turn, dysfunction in preeclampsia [De Lucca, L et al.,

2015].

123

Graph No: 6.2.1 Comparison of MDA and FRAP between PIH and control

women

*Data expressed as Mean±SD. A p value of <0.05 is considered as significant.

**Data expressed as Mean±SD. A p value of <0.01 is considered as highly

significant.

The Ferric reducing ability of plasma (FRAP) value is a marker for

antioxidant capacity [Benzie, I.F et al., 1996]. In our study the antioxidant

statuses as measured by FRAP is significantly lower in PE & E women

compared to the controls. The FRAP levels were significantly lower in

eclamptic women than in pre eclamptic women. These results were in

consistent with findings reported by other studies [Gupta, A et al., 2016,

Bosco, C et al., 2010]. The FRAP assay is recently developed, direct test of

―total anti-oxidant power‖. Other tests of total anti-oxidant power are indirect

methods of measuring anti-oxidative power. In contrast to other tests of total

124

anti-oxidant power, the FRAP assay is simple, fast, inexpensive and robust

[Gupta, A et al., 2016]. It can measure the antioxidant capacity of all the

antioxidants in a biological sample and not just the antioxidant capacity of a

single compound [Rao, P.S et al., 2013]. This reduction in the FRAP levels

might be due to the utilization of antioxidants to a greater extent in order to

counteract free radical mediated cellular changes [Kashinakunti, S.V .et al

2010]. Several studies had demonstrated an significant increase in MDA

levels and reduced antioxidant vitamin E levels [Phalak, P et al, 2012, Begum,

R et al., 2011]. It has been stated that the main placental antioxidant enzymes

such as Superoxide dismutase (SOD), catalase (CAT), Glutathione

peroxidase, glutathione reductase, glutathione S-transferase and glucose-6-

phosphate dehydrogenase (G6PDH) activities were decreased in women with

preeclampsia [De Lucca, L et al., 2015]. Furthermore, obesity, insulin

resistance and hyperlipidemia act as risk factors by stimulating the

inflammatory cytokine release and oxidative stress leading to endothelial

dysfunction [Sanchez-Aranguren, L.C et al., 2014].

125

6.3 To assess the levels of inflammatory markers, angiogenic and anti

angiogenic factors in PIH women.

In the first trimester of pregnancy an inflammatory microenvironment is

needed for successful implantation and tissue remodeling [Mor, G et al.2010].

This inflammatory reaction is characterized by an upregulation of cytokines,

chemokines and their receptors. Increased inflammatory changes during

pregnancy may be explained by different stimuli occurring at different phases

of pregnancy such as implantation, monocyte/macrophage production

stimulated by interleukin-6(IL-6) and the necrotic process associated with

placental ageing [Dhok, A.J et al., 2011]. Added to these reasons, in PE

because of the placental hypoperfusion, ROS and cytokines are released from

the placenta which may induce oxidative stress, inflammatory response and

endothelial cell dysfunction in mother [Catarino, C et al., 2014]. An excessive

inflammatory reaction has been associated with recurrent miscarriage or other

pregnancy complications such as pre-eclampsia or premature labor [Martínez

Varea, A et al., 2014]. In this respect our study displayed significant higher

levels of TNF-α, IL-6, HsCRP in PE & E women compared to the

normotensive pregnant women. But, only TNF- α levels were significantly

higher in eclamptic women compared to pre-eclamptics whereas IL-6 & Hs

CRP were not significant.

126

Table No: 6.3.1 Comparison of inflammatory markers between controls

and PIH women

Parameters

Controls Pre-

eclampsia P value

Eclampsia P

value

P value between PE & E

Mean SD Mean SD Mean SD

TNF-α (pg/ml)

10.57 3.00 22.17 8.04 <0.01 27.50 14.07 <0.01 <0.01

IL-6

(pg/ml) 2.64 0.73 9.57 3.26 <0.01 10.13 3.25 <0.01 NS

HsCRP

(mg/L) 2.05 0.58 7.51 1.67 <0.01 7.59 2.89 <0.01 NS

*Data expressed as Mean±SD. A p value of <0.05 is considered as significant.

**Data expressed as Mean±SD. A p value of <0.01 is considered as highly

significant.

TNF-α and IL-6 are some of the pro-inflammatory cytokines playing an

important role in activation of immune system among pre-eclamptic women

and are associated with disease severity [Vitoratos, N et al., 2010]. TNF-alpha

is produced by monocytes, induces apoptosis, and inhibits proliferation of

trophoblast cells in preeclampsia [Seki, H et al., 2007]. Pathologically secreted

TNF-α damage the vascular endothelial cells by causing occlusion of vessels

there by reducing the regional blood flow leading to the increase in the

permeability of endothelium [Vahid Roudsari, F et al., 2009]. The displayed

high levels of TNF – alpha in our study can be explained as a consequence of

127

placental hypoxia which exists in PIH [Conrad, K.P et al., 1997]. The ROS

generated by hypoxia /reoxygenation insults of placenta might also play a

central role in placental expression and production of TNF-alpha by direct

activation of p38 MAP kinase (mitogen activated kinase) and NF-KB (Nuclear

factor) [Hung, T.H et al. 2004]. Furthermore, in preeclampsia the placenta

derived factors might stimulate monocytes and neutrophils to produce TNF-α

that lead to endothelial disturbances [Bakheet, M.S et al., 2016]. Generally the

monocytes are the main reservoir for proinflammatory cytokines and therefore

can be good candidates for excessive TNF-α synthesis in preeclampsia

[Lockwood.C.J et al., 2008].

Unlike most cytokines, circulating TNF-α can cross the blood brain

barrier (BBB) through receptor-mediated endocytosis [Pan, W et al., 2003].

The binding of TNF-α to its receptors on the BBB upsurges paracellular

permeability that can promote vasogenic edema [Pan, W et al., 2007].

Furthermore, peripheral inflammation has been exposed to cause TNF-α

dependent microglial activation that increases neuronal excitability in the

brain. TNF-α upregulates endothelial cell adhesion molecules such as E-

selectin, ICAM-1 and VCAM-1 that facilitate passage of leukocytes into the

brain. Leukocyte infiltration of the BBB has been revealed to be seizure

provoking by activating microglia that can then produce TNF-α. In the brain,

the production of TNF-α can both lower the seizure threshold and cause

seizure via effects on AMPA (α-amino-3-hydroxy-5-methyl-4-

128

isoxazolepropionic acid receptor ) and GABAA (γ-aminobutyric acid receptor)

[Cipolla, M.J et al., 2011].

Many reports indicated that the plasma of pre-eclamptic patients contain

elevated levels of IL-6, a multifunctional cytokine that regulates hematopoiesis

along with acute phase reaction. It controls both pro- and anti-inflammatory

events. In the present study elevated IL-6 levels might be due to the

characteristic decidual secretion .TNF-alpha, markedly up-regulates the IL-6

mRNA and its protein expression by the resident decidual cells [Lockwood,

C.J et al., 2008]. More over plasma from pre-eclamptic women activates

vascular endothelial cells through an NK-κB- mediated mechanism, these

cells could be a potential source of increased circulating IL-6 that is seen in

this disease [Swellam, M et al., 2009]. Elevated IL-6 interferes with

endothelial cell function by increasing the endothelial cell permeability by

changing the cell shape and rearrangement of intracellular actin fibers [Lau,

S.Y et al., 2013, Tosun, M et al., 2010, Conrad, K.P et al., 1998, Teran, E et

al., 2001]. It increases the thromboxane A2 to prostacyclin ratio; reduce

prostacyclin (PG I2) synthesis by inhibiting the cyclooxygenase enzyme and

stimulating the platelet derived growth factor. It could also trigger the

neutrophil activation, expression of von Willebrand factor and cell adhesion on

the endothelium resulting in vascular damage [Mihu, D et al., 2010, Lockwood,

C.J et al., 2008]. The inflammatory mediators TNF-α, IL-8 and IL 1-β

synergizes with elevated plasma IL-6 levels to promote systemic vascular

129

damage, particularly in the kidney, that results into a characteristic proteinuria

and hypertension of the maternal syndrome of PE [Gupta, M et al., 2015].

Graph No: 6.3.1 Comparison of inflammatory markers between controls

and PIH women

*Data expressed as Mean±SD. A p value of <0.05 is considered as significant.

**Data expressed as Mean±SD. A p value of <0.01 is considered as highly

significant.

C-reactive protein (CRP) is an acute phase protein which is increased in

systemic inflammation [Chandrashekara, S., 2014]. During the challenges like

severe tissue injury, microbial infections, systemic autoimmune disease and

malignant tumors it is mainly synthesized by hepatocytes. Our results

regarding HsCRP were in consistent with several studies [Can, M et al., 2011,

Catarino, C et al., 2012, Behboudi-Gandevani,S et al., 2012, Dhok, A.J et al.,

2011].The reason might be due to the regulation of production by the

130

correspondent gene located on the long arm of chromosome 1, induced at the

transcriptional level by IL-6 & tumor necrosis factor-alpha (TNF-α) which are

produced predominantly by macrophages as well as adipocytes [Swellam, M

et al., 2009, Tavana, Z et al., 2010, Dhok, A et al., 2010]. The cytokines exert

their biological effects on CRP by signalling through their receptors on hepatic

cells there by activating different kinases and phosphatases. This leads to the

translocation of various transcription factors on the CRP gene promoter and

the production of CRP. The concentration of CRP doubles for every 8 hours

and peaks at 36-50 hours, while it depends on the stimulus and its severity.

CRP concentration can increase above 500 mg/l and this amounts to as much

as a 1000-fold or more concentration variation in response to a inflammatory

insult. If preeclampsia is accompanied by superimposed infection then the

level of CRP increases more [Mandal, K.K et al., 2016]. CRP, in agreement

with its proposed function, may play a role in eliciting the inflammatory

response characteristics of preeclampsia [Nanda, K, Sadanand, G et al.,

2012].

131

Table No: 6.3.2 Comparison of angiogenic and anti angiogenic factors

between controls and PE, E women:

Parameters

Controls Pre-eclampsia

P

value

Eclampsia

P

value

P value

between

PE & E Mean SD Mean SD Mean SD

sFlt-1

(pg/ml) 1271.22 365.22 3854.12 741.97 <0.01 7827.57 1841.29 <0.01 <0.01

VEGF

(pg/ml) 274.05 36.15 179.12 18.87 <0.01 131.48 36.93 <0.01 <0.01

PlGF

(pg/ml) 682.97 212.19 225.56 56.46 <0.01 141.63 121.74 <0.01 <0.01

*Data expressed as Mean±SD. A p value of <0.05 is considered as significant.

**Data expressed as Mean±SD. A p value of <0.01 is considered as highly

significant.

The development of placenta is facilitated by a coordinated and

complex processes of vasculogenesis and angiogenesis which occurs prior to

implantation and throughout gestation. Impairment of these processes may

result in restricted blood flow to the fetus, increased maternal blood pressure

and premature delivery [Arroyo, J.A et al., 2008, Lam, C et al., 2005]. sFlt-1, a

soluble form of VEGFR-1 can bind and reduce the free circulating levels of

proangiogenic factors (VEGF and PlGF). Thus it blunts the beneficial effects

132

of these factors on maternal endothelium, with consequent maternal

hypertension and proteinuria [Roberts, J.M et al., 2009].

In normal pregnancies sFlt-1 prevents damage to the placenta and fetus

by inhibiting excess VEGF signaling, which would otherwise leads to

excessive placental invasion. This may lead to catastrophic hemorrhage at

delivery. Thus in normal pregnancies VEGF levels are tightly controlled at the

maternal-fetal interface inorder to regulate placental invasion and facilitate

detachment of placenta after delivery of the fetus, through the modulation by

sFlt-1 [McMahon, K et al., 2014]. In vitro sFlt1 is involved in vasoconstriction

and endothelial dysfunction, mimicking the effects of plasma from pre-

eclamptic women. [Maynard, S.E et al., 2005]. In our study the sFlt-1 levels

were significantly high in PE & E women compared to the normotensive

pregnant women. Eclamptic women had displayed significantly higher levels

compared to the PE women. Our results were in consistent with different

studies [Levine, R.J et al., 2004, De Vivo, A et al., 2009, Reddy, A et al.,

2009].

A critical balance exists between endothelium-derived relaxing and

contracting factors to maintain vascular homeostasis. When the balance in

these factors is disturbed, the vasculature is predisposed to vasoconstriction,

leukocyte adhesion, mitogenesis, pro-oxidation and vascular inflammation

[Cines, D.B et al 1998].

133

Vascular endothelial growth factor (VEGF) is a highly specific mitogen

for micro- and macro vascular endothelial cells derived from arteries, veins,

and lymphatics. It has five members in its family, VEGF-A, -B, -C, and -D and

Placental Growth Factor (PlGF). The most widely studied form, VEGF-A (or

simply VEGF) is the dominant angiogenic molecule in physiological and

pathological angiogenesis and its production is induced by hypoxia/ischemia

[Ferrara, N et al., 2013].. It was stated that during pregnancy the maternal

plasma concentrations of VEGF were increased because the nutritional

provision to the fetoplacental unit depends partly on the uterine blood flow. So,

the increased concentrations of VEGF induces vasodilation through

production of NO and PGI2 in order to increase the uterine blood flow to the

feto placental unit [Itoh, S et al., 2002].

PlGF, a member of VEGF family has 42% homology with VEGF. The

name refers to placenta since it was cloned from a human placental cDNA

library. PIGF is highly expressed in placenta throughout all stages of

gestation. It controls trophoblast growth and differentiation dilates uterine

vessels, promotes EC growth, vasculogenesis and placental development. It

has weak mitogenic activity and potentiates the actions of VEGF. PlGF

stimulates angiogenesis in different physiological and pathological conditions

[De Falco, S et al., 2012].

134

Graph No: 6.3.2 Comparison of angiogenic and anti angiogenic factors

between controls and PE, E women:

*Data expressed as Mean±SD. A p value of <0.05 is considered as significant.

**Data expressed as Mean±SD. A p value of <0.01 is considered as highly

significant.

Preeclampsia is associated with placental hypoxia, which is said to be

the putative culprit in initiating the cascade of events that ultimately results in

the maternal manifestations of the disease [Cohen, J. M et al., 2015]. Usually

hypoxic conditions stimulates the production of VEGF & PlGF levels, but our

study showed a significant decrease in free VEGF & PlGF levels in PE & E

women as compared with controls. However, studies on circulating levels of

VEGF in preeclampsia have been inconsistent, with reports of both increased

and decreased levels. This discrepancy could be explained by the fact that

VEGF-protein complexes are undetectable by the sandwich-type ELISA

because there is a substantial increase in circulating VEGF binding proteins

during pregnancy. All prior studies reporting on decreased VEGF have used

135

an ELISA kit, which measures free (unbound) VEGF whereas all studies

reporting on an increased VEGF in preeclampsia used either a

radioimmunoassay or an ELISA system measuring total (bound and unbound)

VEGF [Lee, E.S et al., 2007]. In our study we estimated free VEGF & PlGF

levels. The reduction in VEGF levels might be a reason for observed

hypertension and proteinuria among PE and E women because VEGF is

important in regulation of blood pressure and maintaining the integrity of

glomerular filtration barrier. It also has a role in glomerular healing. Alterations

in the VEGF bioavailability might have resulted in endothelial as well as

podocyte damage [Muller Deile, J et al 2011]. Our study also displayed a

significantly low free VEGF & PlGF levels in eclamptic women than

preeclamptic women. This might be the reason for the disruption of endothelial

cells by disrupting the endothelial cells that maintains blood-brain barrier

and/or endothelial cells lining the choroid plexus of the brain thus leading to

cerebral edema and seizures seen in eclampsia [Karumanchi, S.A et al,

2015].

The signal transduction of VEGFs involves binding to VEGFR1,

VEGFR2 and VEGFR3, tyrosine kinase receptors .VEGF-A binds to both

VEGFR1 and VEGFR2, whereas PlGF binds only to VEGFR1 [Hoeben, A et

al 2004]. Soluble Flt-1 (sFlt-1) is a splice variant of VEGFR1 (Flt-1) which is

produced by a variety of tissues. It contains the extracellular ligand-binding

domain but lacks the trans membrane and cytoplasmic portions of VEGFR1

[McMahon, K et al., 2014]. Our study showed significantly high levels of sFlt-1

136

in PE & E women than controls. The sFlt-1 levels were significantly high in

eclamptic women than PE women. This may be due to the TNF-alpha

mediated up regulatory mechanism. A study by Sydney etal had stated that

TNF-alpha may stimulate sFlt-1 production through an indirect mechanism,

possibly mediated by the Angiotensin type II receptor agonistic autoantibodies

(AT1-AA). Alternatively, under chronic conditions TNF-alpha can directly

stimulate the sFlt-1 production [Murphy, S.R et al., 2012]. This high level of

sFlt-1 might be a reason for reduced level of free VEGF & PlGF in our study.

This phenomenon of sFlt-1-VEGF complex formation substantiates the ironical

observation in PE ―Low circulatory VEGF in conditions of high VEGF mRNA

expression‖ [Zhou, Q et al 2010].

137

6.4 Objective: 4 To assess the levels of Nitric oxide & Atherogenic index

(AI) in PIH women

Table 6.4.1: Comparison of NO & AI between Controls and PIH women

Parameter

Controls Pre-eclampsia P

value

Eclampsia P

value

P value between PE & E

Mean SD Mean SD Mean SD

NO

(μmol/L) 117.37 14.77 43.8 6.13 <0.01 38.6 9.94 <0.01 <0.01

AIP 0.30 0.09 0.48 0.08 <0.01 0.56 0.12 <0.01 <0.01

*Data expressed as Mean±SD. A p value of <0.05 is considered as significant.

**Data expressed as Mean±SD. A p value of <0.01 is considered as highly

significant.

NO is an endothelium derived factor responsible for vasodilatation and

platelet activation inhibition. It is involved in various stages of pregnancy

including implantation, maintenance of uterine acquiescence during

pregnancy, control of uterine contractions and relaxation, basic physiological

adaption for successful gestation and regulation of blood pressure [Saha, T et

al., 2013]. The hallmark of ED is impaired NO bioavailability [Sena, C.M et al.

2013]. Our study displayed significantly low levels of NO in PE & E women

than in controls. The levels of NO were significantly low in eclamptic women

than in preeclamptic women. Our results were in consistent with other studies

138

[Seligman, S.P et al., 1994, Sandrim, V. C et al., 2008, Choi, J.W et al., 2002,

El-Said, R et al., 2015]. This observed low level of NO can be explained as a

consequence of reduced level of free VEGF as it plays a major role in the

expression of eNOS [Shen, B.Q et al 1999]. The inflammatory cytokines can

also inhibit eNOS, causing reduction in NO levels leading to vasoconstriction

in the peripheral circulation [Matsubara, K et al., 2014]. It has also been stated

that arginine deficiency can also reduce NO and increase superoxide

formation, leading to NO degradation and excess peroxynitrite formation

[Huang, L.T et al., 2012].

Graph No: 6.4.1 To assess the levels of Nitric oxide & Atherogenic index

(AI) in PIH women

*Data expressed as Mean±SD. A p value of <0.05 is considered as significant.

**Data expressed as Mean±SD. A p value of <0.01 is considered as highly

significant.

139

In the present study the atherogenic index of plasma (AIP) was

calculated. The mean of AIP in PE and E was significantly higher than that of

control group .In eclamptic women the AIP was significantly higher than PE

women. The AIP was calculated as log (TG / HDL) has been proposed as a

marker of plasma atherogenicity [Dobiasova, M et al 2011]. Our findings were

in agreement with other studies [Aragon-Charris, J et al., 2014, Rohita, et al.

2014, Herrera-Villalobos, J. E et al., 2012]. A positive significant correlation

between AIP and systolic blood pressure was observed by Singh, M et al.,

2015 .This might be due to elevated TC level, reduced HDL-C level and ROS

which may lead to the fatty deposition in the vessel walls. This may

predispose the patients for coronary heart disease in future. Our results can

be interpreted in such a way that plasma lipids in normal pregnancy are at

atherogenic levels and were more abnormal in pre-eclampsia and eclampsia.

These abnormalities in lipid profile turn out to be a risk factor for

cardiovascular complications. Evaluation of the atherogenic indices during

pregnancy may help to prevent this risk.

140

6.5 Correlation of lipids and lipoproteins with angiogenic and anti

angiogenic factors & NO

Parameter

VEGF

(pg/ml)

PlGF

(pg/ml)

sFlt-1

(pg/ml)

NO

(µmol/L)

‘r’ value ‘r’ value

‘r’ value

‘r’ value

TC (mg/dl) -0.357** -0.332** 0.425** -0.319**

TGL (mg/dl) -0.633** -0.559** 0.624** -0.545**

HDL (mg/dl) 0.540** 0.538** -0.583** 0.563**

LDL (mg/dl) -0.264** -0.264** 0.347** -0.261**

VLDL (mg/dl) -0.633** -0.559** 0.624** -0.545**

ApoA (mg/dl) 0.410** 0.515** -0.328** 0.493**

ApoB (mg/dl) -0.633** -0.562** 0.626** -0.614**

Lp(a) (mg/dl) -0.672** -0.659** 0.643** -0.689**

** Correlation is significant at the 0.01 level (2-tailed)

*. Correlation is significant at the 0.05 level (2-tailed)

141

In the present study the TC, TGL, LDL, VLDL, apoB, LP (a) were

positively correlated with sFlt-1 and negatively correlated with VEGF, PlGF,

and NO however HDL and apoA-1 were positively correlated with VEGF,

PlGF, NO and negatively correlated with sFlt-1. It might be due to the

hyperlipidemia which can attenuates VEGF induced angiogenesis, impairs

cerebral blood flow and disturbs stroke recovery [Zechariah, A et al., 2013]. It

was also stated that the hypercholesterolemia impairs angiogenesis in vivo by

inducing oxidative stress in blood vessels and also by decreasing the activity

of the L-arginine/NO pathway in the ischemic tissues, there by leading to ED,

blood brain barrier disturbances [Duan, J et al., 2000]. In addition, the native

form of LDL have anti-angiogenic activity which attenuates the endothelial

angiogenesis by down regulating the Hypoxia inducible factors (HIFs) through

increasing HIF hydroxylation / proteasome activity there by disrupting the HIF

pathway [Yao, G et al., 2015]. Our correlation analysis can be understood in a

way that abnormal lipid metabolism along with principally low HDL-C and high

TGL concentrations, may add to the promotion of vascular dysfunction seen in

pregnancy induced hypertensive women. Lipid profile and lipoproteins can act

as indices for assessing endothelial and vascular function in PIH women.

Regular monitoring of this cost effective investigations might guide to assess

the onset and progression of the disease.

Pre-eclampsia is complex pathophysiological process which is known to

be associated with abnormal placentation and impaired placental perfusion. It

has lead to the developing concept that maternal predisposing factors must

142

combine with the placental disorder to result in pre-eclamptic maternal

syndrome [Phalak, P et al., 2012, Gawande, M.S et al., 2016]. Maternal

endothelial dysfunction is considered as a classic hallmark of preeclampsia.

The severity of both hypertension and proteinuria reflects the degree of

endothelial damage. Lipid deposits are known to get accumulated in the

glomeruli of preeclamptic patients causing glomerular endotheliosis.

Glomerular lesions are accompanied with proteinuria, a predictive indicator

and marker of disease severity. Furthermore, the possible correlation between

the altered lipid profile and the severity of renal lesions, as reflected by

proteinuria, may contribute to clarify the complex pathophysiology of

preeclampsia. Abnormal lipid metabolism might play an important role in the

elevation of oxidative stress and vascular dysfunction seen in the

preeclamptics women. Evidence regarding the oxidative stress can be seen

both in the maternal circulation and in the placenta [Al-Jameil, N et al., 2014].

Oxidative stress can induce inflammation and the inflammatory process can in

turn induce oxidative stress, through activation of multiple pathways. The

reactive hydrogen peroxide species can induce inflammation through

activation of transcription factor NF-κB, which regulates the gene expression

of proinflammatory cytokines, chemokines, inflammatory enzymes, adhesion

molecules, receptors, and microRNA [Anderson, M.T et al., 1994].

Furthermore, The ROS that are released from damaged mitochondria

play an important role in activation of NOD-like receptor 3 inflammasome

(NLRP3- nucleotide-binding oligomerization domain-like receptors ) [Zhou, R

143

et al., 2011, Shimada, K et al., 2012, Zhou, R et al., 2010] leading to IL-1β

secretion and localized inflammation [Zhou, R et al., 2011]. The ROS-induced

DNA base modification has also been shown to induce inflammation [Scholz,

H et al., 2003].

On the other side, during inflammatory process the activated phagocytic cells

like neutrophils and macrophages produce large amounts of ROS, reactive

nitrogen and chlorine species including superoxide, hydrogen peroxide,

hydroxyl free radical, nitric oxide, peroxynitrite, and hypochlorous acid to kill

the invading agents. Under pathological inflammatory conditions there may be

exaggerated generation of reactive species and some of those reactive

species diffuse out of the phagocytic cells and there by inducing localized

oxidative stress and tissue injury [Fialkow, L et al., 2007]. However, apart from

the direct production of reactive species by the professional phagocytic cells,

the non-phagocytic cells can also produce reactive species in response to

proinflammatory cytokines [Wu, Y et al., 2013, Li, J et al., 2015]. Due to their

highly reactive properties, they can cause structural, functional damage to

cellular DNA, proteins and cell membranes.

It may also lead to oxidation of LDL. The oxidized LDL is taken up by

macrophages via scavenger receptors and form foam cells resulting in

atherogenesis contributing to endothelial injury and dysfunction in

preeclampsia [De Lucca, L et al., 2015], a key mechanism in the proposed

pathophysiology of PE. ROS alters endothelial metabolism by blocking

144

mitochondrial electron transport, oxidizing the proteins and initiating the lipid

peroxidation [Hansson, S.R et al., 2014].

In pre-eclampsia, fibrinoid necrosis of the vessel walls along with

accumulation of lipid-laden foam cells is considered as the hallmark of

oxidized LDL. Increased triglycerides found were likely to be deposited in the

predisposed vessels, such as uterine spiral arteries and contributes to the

endothelial dysfunction, both directly and indirectly through generation of small

dense LDL. Increased triglycerides may also be associated with

hypercoagulability [Al Jameil, N et al., 2014]. Impaired uteroplacental blood

flow or hypoperfusion can affect the foeto–placental unit, causing intrauterine

growth restriction, intrauterine fetal demise, oligohydramnios, or placental

abruption [Hutter, D et al., 2010]. Uteroplacental ischemia could also induce

the shedding of placental microparticles into maternal circulation. These

particles might trigger inflammation and vascular damage. Restricted placental

blood flow might also increase placental production of sFlt-1 towards the

pathological levels and thus initiating the maternal syndrome [Nagamatsu, T et

al., 2004]. The placental TNF-alpha can also stimulate sFlt-1 in respone to

hypoxia through an indirect mechanism, possibly mediated by the Angiotensin

type II receptor agonistic autoantibodies (AT1-AA). Alternatively, under

chronic conditions TNF-α can directly stimulate the sFlt-1 production [Murphy,

S.R et al., 2012] which inhibits the actions of free VEGF, PlGF there by

inhibiting the angiogenesis [Powe, C.E et al., 2011]. Circulating sFlt-1 deprives

the vasculature of kidney, liver, brain and other organs required for essential

145

survival and maintenance of signals, ultimately eliciting the maternal vascular

dysfunction in pre-eclampsia [Noris, M et al., 2005]. There is an evidence that,

in addition to the angiogenesis, VEGF also induces vasodilation by enhancing

the endothelial synthesis of NO and PGI2 [Itoh, S et al., 2002]. As a

consequence, low circulating levels of VEGF increases the vascular tone and

causes hypertension which is a hallmark of maternal syndrome.

146

7. SUMMARY

Preeclampsia is a pregnancy specific disorder that adversely affects

mother and fetus. Maternal endothelial dysfunction is the key event resulting

into diverse clinical manifestations of preeclampsia. Both feto-placental and

maternal factors interact in manifesting endothelial cell dysfunction and its

clinical manifestations. It is associated with a distinct pathological lesion of the

decidual arterioles known as acute atherosis which bears a striking

resemblance to atherosclerotic lesions of coronary arteries. Hence, the study

has been designed to delineate the involvement of lipids and its association

with the severity of disease in PIH women there by, to find out whether the

regular monitoring of lipids can be of any use in assessing the onset and

progression of disease.

The present study was done on 300 subjects among which 100 subjects

were preeclamptic women, 100 subjects were eclamptic women and

remaining 100 normotensive pregnant women served as controls. The blood

samples of all the subjects were analysed for lipid profile, lipoproteins,

apolipoproteins, oxidative stress, total antioxidant capacity, inflammation,

angiogenic and antiangiogenic factors, endothelial dysfunction and

atherogenic index.

The present study has identified an abnormal increase in the levels of

TC, TGL, VLDL, LDL, apoB, Lp (a) and decreased HDL, apoA-1 in PE & E

women. Abnormalities in lipid levels were more prominent in eclamptic women

147

compared to the preeclamptics. These observations reveal the fact that the

lipid abnormalities are spiking along with severity of the disease.

This study has also observed a high level of lipid peroxidation marker

(MDA) and low total antioxidant capacity (TAC) in PE & E women compared to

the controls. The lipid peroxidation is high and TAC is low in eclamptics

compared to preeclamptics. This can be understood in a way that, as the lipid

levels increases, the lipid peroxidation also upturns which might cause

inflammation.

In this study the inflammatory markers (TNF- α, IL-6, HsCRP) were high

in PE & E women compared to the controls. The TNF-α levels were high in

eclamptics than preeclamptics whereas IL-6 and HsCRP were not. These

observations put out a fact that a significant level of inflammation exists in PIH

which might also be involved in the progression of the disease towards

severely higher side. Furthermore the proinflammatory cytokines can

aggravate the release of antiangiogenic factors from placenta.

In this study the antiangiogenic factor (sFlt-1) levels were high in PIH

(PE & E) women compared to controls and the same levels were high in

eclamptic women compared to the preeclamptics. In contrast, the angiogenic

factor concentrations (VEGF, PlGF) were low in PE & E women compared to

controls. The VEGF, PlGF levels were lower in eclamptics than preeclamptics.

These observations reveals the fact that the imbalance between angiogenic

148

and antiangiogenic factors might play an important role in promoting the ED

among PIH women and the imbalance increases with the severity of disease.

The present study has also observed a reduced NO levels & increased

AI in PE & E women compared to the controls. The NO levels were low and AI

is high in eclamptics than preeclamptics. These results indicate that the ED is

involved in progression of disease and as the disease progresses; the women

are more prone to atherogenesis and vascular diseases.

Our correlation analysis exposed a fact that the hyperlipidemia in PIH

women might promote towards vascular diseases by encouraging the

imbalance between angiogenic and anti angiogenic factors.

149

8. CONCLUSION

Our study suggest that an abnormal lipid metabolism may enhance the

generation of vascular dysfunction, oxidative stress, inflammation, angiogenic

and antiangiogenic imbalance seen in pregnancy induced hypertensive

women. It is consequently essential to measure and monitor blood lipid

concentrations in pregnant women in addition to the clinical assessment

during antenatal care. It might be useful in revealing and prevention of

dangerous obstetric complication like eclampsia in early stages. Moreover,

lipid profile is inexpensive than other investigations and can be measured in

all clinical laboratories.

150

9. LIMITATIONS

1) The placental histopathological studies could not be done.

2) The patients were not followed ante partum since it is not included in the

study

151

10. FUTURE PROSPECTIVES

I want to do a follow up molecular study from the first trimester which

may give a better understanding about the molecular involvement in the

abnormal upsurge of lipids among PIH women.

It has been stated in studies conducted among other diseases that the

hyperlipidemia attenuates VEGF. In addition, our study had also found a

negative correlation between hyperlipidemia and VEGF but, the mechanism

has not yet been explored so far in PIH women.

152

11. BIBLIOGRAPHY

Abdo, I., George, R. B. Farrag, M., Cerny, V., & Lehmann, C. (2014).

Microcirculation in pregnancy. Physiological Research, 63(4), 395.

Adiga, U., D'souza, V., Kamath, A., & Mangalore, N. (2007). Antioxidant

activity and lipid peroxidation in preeclampsia. Journal of the Chinese Medical

Association, 70(10), 435-438.

Afshari, J. T., Ghomian, N., Shameli, A., Shakeri, M. T., Fahmidehkar, M. A.,

Mahajer, E., & Emadzadeh, M. (2005). Determination of Interleukin-6 and

Tumor Necrosis Factor-alpha concentrations in Iranian-Khorasanian patients

with preeclampsia. BMC pregnancy and Childbirth, 5(1), 14.

Agarwal, A., Tvrda, E., & Mulgund, A. (2015). Oxidative Stress in

Preeclampsia.

Agarwal, V., Gupta, B. K., & Vishnu, A. (2014). Association of lipid profile and

uric acid with pre-eclampsia of third trimester in nullipara women. Journal of

clinical and diagnostic research: JCDR, 8(7), CC04.

Agrawal, S., & Fledderjohann, J. (2016). Hypertensive disorders of pregnancy

and risk of diabetes in Indian women: a cross-sectional study. BMJ open, 6(8),

e011000.

Ahn, H., Park, J., Gilman‐Sachs, A., & Kwak‐Kim, J. (2011). Immunologic

characteristics of preeclampsia, a comprehensive review. American journal of

reproductive immunology, 65(4), 377-394.

153

Ahenkorah, L. (2009). Metabolic Syndrome, Oxidative Stress and Putative

Risk Factors amongst Ghanaian Women Presenting with Pregnancy-Induced

Hypertension (Doctoral dissertation, Kwame nkrumah university of science &

technology).

Aird, W. C. (2003). Endothelial cell heterogeneity. Critical care medicine,

31(4), S221-S230.

Aksonova, А., Ventskovskaya, I., & Yuzvenko, T. (2016). Atherogenic

parameters and cardiac indices in prognostication of development of pre-

eclampsia and its cardiometabolic consequences. eureka: Health Sciences,

(6), 30-36.

Al-Gubory, K. H., Fowler, P. A., & Garrel, C. (2010). The roles of cellular

reactive oxygen species, oxidative stress and antioxidants in pregnancy

outcomes. The international journal of biochemistry & cell biology, 42(10),

1634-1650.

Ali, Z., Zaki, S., Tauseef, A., & Akmal, A. (2013). C Reactive Protein levels are

elevated in the Third Trimester in Preeclamptic pregnant Women. Pakistan

Journal of Medical and Health Sciences, 7(1).

Al-Jameil, N., Tabassum, H., Ali, M. N., & Qadeer, M. A. (2014). Lipid profile

and its effect on kidney in pregnancy-induced preeclampsia: A prospective

case-controlled study on patients of Riyadh, Saudi Arabia. Biomedical

Research, 25(4).

154

Alladin, A. A., & Harrison, M. (2012). Preeclampsia: systemic endothelial

damage leading to increased activation of the blood coagulation cascade.

Journal of Biotech Research, 4, 26-43.

Allain, C. C., Poon, L. S., Chan, C. S., Richmond, W. F. P. C., & Fu, P. C.

(1974). Enzymatic determination of total serum cholesterol. Clinical chemistry,

20(4), 470-475.

Alvarez, J. J., Montelongo, A., Iglesias, A., Lasuncion, M. A., & Herrera, E.

(1996). Longitudinal study on lipoprotein profile, high density lipoprotein

subclass, and postheparin lipases during gestation in women. Journal of lipid

research, 37(2), 299-308.

Ananth, C. V., Keyes, K. M., & Wapner, R. J. (2013). Pre-eclampsia rates in

the United States, 1980-2010: age-period-cohort analysis. Bmj, 347, f6564.

Anderson, M. T., Staal, F. J., Gitler, C., & Herzenberg, L. A. (1994).

Separation of oxidant-initiated and redox-regulated steps in the NF-kappa B

signal transduction pathway. Proceedings of the National Academy of

Sciences, 91(24), 11527-11531.

Andresen, B. D. (1986). Textbook of clinical chemistry (Vol. 486). N. W. Tietz

(Ed.). Philadelphia et al.: Saunders.

Anita, c.,bhagyalakshmi, a., Rekha, c., & Rani, a. s. A study of

malondialdehyde and lipid profile in pregnancy induced hypertension.

155

Ann-Charlotte, I. (2013). Inflammator mechanisms in preeclampsia.

Pregnancy Hypertension: An International Journal of Women's Cardiovascular

Health, 3(2), 58.

Anjum, R., Zahra, N., Rehman, K., Alam, R., Parveen, A., Tariq, M., & Akash,

M. S. H. (2013). Comparative analysis of serum lipid profile between

normotensive and hypertensive Pakistani pregnant women. J Mol Genet Med,

7(64), 1747-0862.

An-Na, C., Man-Li, Y., Jeng-Hsiu, H., Pesus, C., Shin-Kuo, S., & Heung-Tat,

N. (1995). Alterations of serum lipid levels and their biological relevances

during and after pregnancy. Life sciences, 56(26), 2367-2375.

Aragon-Charris, J., Reyna-Villasmil, E., Guerra-Velasquez, M., Mejia-Montilla,

J., Torres-Cepeda, D., Santos-Bolívar, J., & Reyna-Villasmil, N. (2014).

Atherogenic index of plasma in patients with preeclampsia and in healthy

pregnant women. Medicina clinica, 143(3), 104-108.

Aris, A., Benali, S., Ouellet, A., Moutquin, J. M., & Leblanc, S. (2009).

Potential biomarkers of preeclampsia: inverse correlation between hydrogen

peroxide and nitric oxide early in maternal circulation and at term in placenta

of women with preeclampsia. Placenta, 30(4), 342-347.

Arbogast, B. W., Leeper, S. C., Merrick, R. D., Olive, K. E., & Taylor, R. N.

(1994). Which plasma factors bring about disturbance of endothelial function

in pre-eclampsia?. The Lancet, 343(8893), 340-341.

156

Arroyo, J. A., & Winn, V. D. (2008, June). Vasculogenesis and angiogenesis in

the IUGR placenta. In Seminars in perinatology (Vol. 32, No. 3, pp. 172-177).

WB Saunders.

Asghari, E., Faramarzi, M., & Mohammmadi, A. K. (2016). The Effect of

Cognitive Behavioural Therapy on Anxiety, Depression and Stress in Women

with Preeclampsia. Journal of Clinical and Diagnostic Research: JCDR,

10(11), QC04.

Autiero, M., Waltenberger, J., Communi, D., Kranz, A., Moons, L.,

Lambrechts, D., & Kliche, S. (2003). Role of PlGF in the intra-and

intermolecular cross talk between the VEGF receptors Flt1 and Flk1. Nature

medicine, 9(7), 936-943.

Ayala, A., Muñoz, M. F., & Argüelles, S. (2014). Lipid peroxidation: production,

metabolism, and signaling mechanisms of malondialdehyde and 4-hydroxy-2-

nonenal. Oxidative medicine and cellular longevity, 2014.

Backes, C. H., Markham, K., Moorehead, P., Cordero, L., Nankervis, C. A., &

Giannone, P. J. (2011). Maternal preeclampsia and neonatal outcomes.

Journal of pregnancy, 2011.

Baker, P. N., Krasnow, J., Roberts, J. M., & Yeo, K. T. (1995). Elevated serum

levels of vascular endothelial growth factor in patients with preeclampsia.

Obstetrics & Gynecology, 86(5), 815-821.

157

Bakheet, M. S., Seddik, M. I., Abdelzaher, M. H., & Abdelshakor, M. (2016).

Evaluation of TNF–α, Nitric Oxide, Lipid Profile, Urea and Creatinine Serum

Levels for the Prediction of Preeclampsia. International Journal, 27.

Balasubramaniam, P., & Malathi, A. (1992). Comparative study of hemoglobin

estimated by Drabkin's and Sahli's methods. Journal of Postgraduate

Medicine, 38(1), 8.

Banerjee, D., Wong, E. C., Shin, J., Fortmann, S. P., & Palaniappan, L.

(2011). Racial and ethnic variation in lipoprotein (a) levels among Asian Indian

and Chinese patients. Journal of lipids, 2011.

Bayhan, G., Koçyigit, Y., Atamer, A., Atamer, Y., & Akkus, Z. (2005). Potential

atherogenic roles of lipids, lipoprotein (a) and lipid peroxidation in

preeclampsia. Gynecological endocrinology, 21(1), 1.

Bdolah, Y., Sukhatme, V. P., & Karumanchi, S. A. (2004, November).

Angiogenic imbalance in the pathophysiology of preeclampsia: newer insights.

In Seminars in nephrology (Vol. 24, No. 6, pp. 548-556). WB Saunders.

Begum, R. (2012). Lipid peroxidation and antioxidant status in preeclampsia.

Journal of Enam Medical College, 1(2), 56-59.

Behboudi-Gandevani, S., Moghadam, N. A., Mogadam-banaem, L.,

Mohamadi, B., & Asghari, M. (2012). Association of high-sensitivity C-reactive

protein serum levels in early pregnancy with the severity of preeclampsia and

fetal birth weight.

158

Benyo, D. F., Smarason, A., Redman, C. W., Sims, C., & Conrad, K. P.

(2001). Expression of Inflammatory Cytokines in Placentas from Women with

Preeclampsia 1. The Journal of Clinical Endocrinology & Metabolism, 86(6),

2505-2512.

Benzie, I. F., & Strain, J. J. (1996). The ferric reducing ability of plasma

(FRAP) as a measure of ―antioxidant power‖: the FRAP assay. Analytical

biochemistry, 239(1), 70-76.

Bhagat, K., & Vallance, P. (1997). Inflammatory cytokines impair endothelium-

dependent dilatation in human veins in vivo. Circulation, 96(9), 3042-3047.

Biswas, S. K. (2016). Does the interdependence between oxidative stress and

inflammation exp Ann-Charlotte, I. (2013). Inflammatory mechanisms in

preeclampsia. Pregnancy Hypertension: An International Journal of Women's

Cardiovascular Health, 3(2), 58. lain the antioxidant paradox?. Oxidative

medicine and cellular longevity, 2016.

Bodnar, L. M., Ness, R. B., Harger, G. F., & Roberts, J. M. (2005).

Inflammation and triglycerides partially mediate the effect of prepregnancy

body mass index on the risk of preeclampsia. American journal of

epidemiology, 162(12), 1198-1206.

Boeldt, D., Yi, F., & Bird, I. M. (2011). Pregnancy Adaptive Programming of

Capacitative Entry Responses Alters NO Output in Vascular Endothelium:

New Insights Into eNOS Regulation through Adaptive Cell Signaling. Journal

of Endocrinology, JOE-11.

159

Bosco, C., Buffet, C., Diaz, E., Rodrigo, R., Morales, P., Barja, P., ... & Parra-

Cordero, M. (2010). VEGF in the muscular layer of placental blood vessels:

immuno-expression in preeclampsia and intrauterine growth restrictrion and its

association with the antioxidant status. Cardiovascular & Hematological

Agents in Medicinal Chemistry (Formerly Current Medicinal Chemistry-

Cardiovascular & Hematological Agents), 8(2), 87-95.

Brockelsby, J. C., Wheeler, T., Anthony, F. W., Wellings, R., & Baker, P. N.

(1998). Increased circulating levels of vascular endothelial growth factor in

preeclampsia. Hypertension in Pregnancy, 17(3), 283-290.

Brown, M. A., Lindheimer, M. D., de Swiet, M., Assche, A. V., & Moutquin, J.

M. (2001). The classification and diagnosis of the hypertensive disorders of

pregnancy: statement from the International Society for the Study of

Hypertension in Pregnancy (ISSHP).

Bukan, N., Kandemir, O., Nas, T., Gulbahar, O., Unal, A., & Cayci, B. (2012).

Maternal cardiac risks in pre-eclamptic patients. The Journal of Maternal-Fetal

& Neonatal Medicine, 25(7), 912-914.

Butte, N. F. (2000). Carbohydrate and lipid metabolism in pregnancy: normal

compared with gestational diabetes mellitus. The American journal of clinical

nutrition, 71(5), 1256s-1261s.

Can, M., Sancar, E., Harma, M., Guven, B., Mungan, G., & Acikgoz, S. (2011).

Inflammatory markers in preeclamptic patients. Clinical chemistry and

laboratory medicine, 49(9), 1469-1472.

160

Catarino, C., Santos-Silva, A., Belo, L., Rocha-Pereira, P., Rocha, S., Patrício,

B., ... & Rebelo, I. (2012). Inflammatory disturbances in preeclampsia:

relationship between maternal and umbilical cord blood. Journal of pregnancy,

2012.

Cekmen, M. B., Erbagci, A. B., Balat, A., Duman, C., Maral, H., Ergen, K., ... &

Kuskay, S. (2003). Plasma lipid and lipoprotein concentrations in pregnancy

induced hypertension. Clinical biochemistry, 36(7), 575-578.

Cesur, M., Ozbalkan, Z., Temel, M. A., & Karaarslan, Y. (2007). Ethnicity may

be a reason for lipid changes and high Lp (a) levels in rheumatoid arthritis.

Clinical rheumatology, 26(3), 355-361.

Chandrashekara, S. (2014). C-reactive protein: An inflammatory marker with

specific role in physiology, pathology, and diagnosis. Internet Journal of

Rheumatology and Clinical Immunology, 2(S1).

Chappell, J. C., Taylor, S. M., Ferrara, N., & Bautch, V. L. (2009). Local

guidance of emerging vessel sprouts requires soluble Flt-1. Developmental

cell, 17(3), 377-386.

Choi, J. W., & Pai, S. H. (2000). Serum lipid concentrations change with

serum alkaline phosphatase activity during pregnancy. Annals of Clinical &

Laboratory Science, 30(4), 422-428.

161

Choi, J. W., Im, M. W., & Pai, S. H. (2002). Nitric oxide production increases

during normal pregnancy and decreases in preeclampsia. Annals of Clinical &

Laboratory Science, 32(3), 257-263.

Chowdeswari, N., Menon, M. G., Jaya, N., & Ramarao, B. V. Biochemical

Profile of women with pregnancy induced hypertension.

Cines, D. B., Pollak, E. S., Buck, C. A., Loscalzo, J., Zimmerman, G. A.,

McEver, R. P., ... & Barnathan, E. S. (1998). Endothelial cells in physiology

and in the pathophysiology of vascular disorders. Blood, 91(10), 3527-3561.

Cipolla, M. J. (2007). Cerebrovascular function in pregnancy and eclampsia.

Hypertension, 50(1), 14-24.

Cipolla, M. J., & Kraig, R. P. (2011). Seizures in women with preeclampsia:

mechanisms and management. Fetal and maternal medicine review, 22(02),

91-108.

Cohen, J. M., Kramer, M. S., Platt, R. W., Basso, O., Evans, R. W., & Kahn, S.

R. (2015). The association between maternal antioxidant levels in

midpregnancy and preeclampsia. American journal of obstetrics and

gynecology, 213(5), 695-e1.

Conrad, K. P., & Benyo, D. F. (1997). Placental cytokines and the

pathogenesis of preeclampsia. American journal of reproductive immunology,

37(3), 240-249.

162

Conrad, K. P., Miles, T. M., & Benyo, D. F. (1998). Circulating levels of

immunoreactive cytokines in women with preeclampsia. American Journal of

Reproductive Immunology, 40(2), 102-111.

Coppé, J. P., Kauser, K., Campisi, J., & Beauséjour, C. M. (2006). Secretion

of vascular endothelial growth factor by primary human fibroblasts at

senescence. Journal of Biological Chemistry, 281(40), 29568-29574.

Cortas, N. K., & Wakid, N.W. (1990). Determination of inorganic nitrate in

serum and urine by a kinetic cadmium-reduction method. Clinical chemistry,

36(8), 1440-1443.

Cunningham, F. G., Fernandez, C. O., & Hernandez, C. (1995). Blindness

associated with preeclampsia and eclampsia. American journal of obstetrics

and gynecology, 172(4), 1291-1298.

Das, R., & Biswas, S. (2015). Eclapmsia: the major cause of maternal

mortality in eastern india. Ethiopian journal of health sciences, 25(2), 111-116.

Davison, J. M., Homuth, V., Jeyabalan, A., Conrad, K. P., Karumanchi, S. A.,

Quaggin, S., & Luft, F. C. (2004). New aspects in the pathophysiology of

preeclampsia. Journal of the American Society of Nephrology, 15(9), 2440-

2448.

De Falco, S. (2012). The discovery of placenta growth factor and its biological

activity. Experimental & molecular medicine, 44(1), 1-9.

163

De Lucca, L., Gallarreta, F. M. P., & de Lima Gonçalves, T. (2015). Oxidative

Stress Markers in Pregnant Women with Preeclampsia. American Journal of

Medical and Biological Research, 3(3), 68-73.

De Melo Dantas, E. M., Pereira, F. V. M., Queiroz, J. W., de Melo Dantas, D.

L., Monteiro, G. R. G., Duggal, P., & Araújo, A. C. P. F. (2013). Preeclampsia

is associated with increased maternal body weight in a northeastern Brazilian

population. BMC pregnancy and childbirth, 13(1), 159.

De Vivo, A., Baviera, G., Giordano, D., Todarello, G., Corrado, F., & D'anna,

R. (2008). Endoglin, PlGF and sFlt‐1 as markers for predicting pre‐eclampsia.

Acta obstetricia et gynecologica Scandinavica, 87(8), 837-842.

Deiana, S. F., Atzeni, I., Meloni, A., Soddu, S., Parodo, G., Puddu, M., &

Melis, G. B. (2014). Placental growth factor and placental perfusion. Journal of

Pediatric and Neonatal Individualized Medicine (JPNIM), 3(2), e030248.

Demir, B., Demir, S., Atamer, Y., Guven, S., Atamer, A., Kocyigit, Y., &

Toprak, G. (2011). Serum levels of lipids, lipoproteins and paraoxonase

activity in pre-eclampsia. Journal of International Medical Research, 39(4),

1427-1431.

Deshpande, H., Madkar, C., Vishnoi, P., Suryarao, P., Patel, N., &

Katokdhand, S. (2016). Study of Serum Lipid Profile in Pregnancy Induced

Hypertension. Indian Journal of Applied Research, 6(1).

164

Dhok, A. J., Daf, S., Mohod, K., & Kumar, S. (2011). 'Role of Early Second

Trimester High Sensitivity C-Reactive Protein for Prediction of Adverse

Pregnancy Outcome'.

Dinarello, C. A. (2000). Proinflammatory cytokines. Chest Journal, 118(2),

503-508.

Dobiasova, M. (2006). AIP--atherogenic index of plasma as a significant

predictor of cardiovascular risk: from research to practice. Vnitrni lekarstvi,

52(1), 64-71.

Dobiásová, M., & Frohlich, J. (2001). The plasma parameter log (TG/HDL-C)

as an atherogenic index: correlation with lipoprotein particle size and

esterification rate inapoB-lipoprotein-depleted plasma (FER HDL). Clinical

biochemistry, 34(7), 583-588.

Dobierzewska, A., Palominos, M., Sanchez, M., Dyhr, M., Helgert, K.,

Venegas-Araneda, P., & Illanes, S. E. (2016). Impairment of angiogenic

sphingosine kinase-1/sphingosine-1-phosphate receptors pathway in

preeclampsia. PloS one, 11(6), e0157221.

Ðorđević, N. Z., Babić, G. M., Marković, S. D., Ognjanović, B. I., Štajn, A. Š.,

Žikić, R. V., & Saičić, Z. S. (2008). Oxidative stress and changes in

antioxidative defense system in erythrocytes of preeclampsia in women.

Reproductive Toxicology, 25(2), 213-218.

165

Dötsch, J., Hogen, N., Nyúl, Z., Hänze, J., Knerr, I., Kirschbaum, M., &

Rascher, W. (2001). Increase of endothelial nitric oxide synthase and

endothelin-1 mRNA expression in human placenta during gestation. European

Journal of Obstetrics & Gynecology and Reproductive Biology, 97(2), 163-

167.

Dover, N., Gulerman, H. C., Celen, S., Kahyaoglu, S., & Yenicesu, O. (2013).

Placental growth factor: as an early second trimester predictive marker for

preeclampsia in normal and high-risk pregnancies in a Turkish population. The

Journal of Obstetrics and Gynecology of India, 63(3), 158-163.

Duan, J., Murohara, T., Ikeda, H., Katoh, A., Shintani, S., Sasaki, K. I., &

Imaizumi, T. (2000). Hypercholesterolemia inhibits angiogenesis in response

to hindlimb ischemia. Circulation, 102 (suppl 3), Iii-370.

Duffy, A. M., Bouchier-Hayes, D. J., & Harmey, J. H. (2004). Vascular

endothelial growth factor (VEGF) and its role in non-endothelial cells:

autocrine signalling by VEGF. VEGF and Cancer, 133-144.

Ekhator, C. N., & Ebomoyi, M. I. (2012). Blood glucose and serum lipid

profiles during pregnancy. African Journal of Diabetes Medicine, 20(1).

El-Makhzangy, I., Moeity, F., & Anwer, M. (2010). Relationship between

maternal obesity and increased risk of preeclampsia. Alexandria Journal of

Medicine, 46(2), 207-212.

166

El‐Said, R., Karkor, T., Elsaway, M., & Abdel‐Hafez, H. (2015). OC15. 06:

Study of nitric oxide level in pre‐eclampsia and the effect of nitroglycerine on

Doppler changes in fetal and maternal vessels. Ultrasound in Obstetrics &

Gynecology, 46(S1), 33-33.

Emokpae, M. A., & Uadia, P. O. (2012). Association of oxidative stress

markers with atherogenic index of plasma in adult sickle cell nephropathy.

Anemia, 2012.

Enaruna, N. O., Idemudia, J. O., & Aikoriogie, P. I. (2014). Serum lipid profile

and uric acid levels in preeclampsia in University of Benin Teaching Hospital.

Nigerian medical journal: journal of the Nigeria Medical Association, 55(5),

423.

Engler, M. M., Engler, M. B., Malloy, M. J., Chiu, E. Y., Schloetter, M. C., Paul,

S. M., & Ridker, P. M. (2003). Antioxidant vitamins C and E improve

endothelial function in children with hyperlipidemia. Circulation, 108(9), 1059-

1063.

Ertas, I. E., Kahyaoglu, S., Yilmaz, B., Ozel, M., Sut, N., Guven, M. A., &

Danisman, N. (2010). Association of maternal serum high sensitive C‐reactive

protein level with body mass index and severity of pre‐eclampsia at third

trimester. Journal of Obstetrics and Gynaecology Research, 36(5), 970-977.

167

Facemire, C. S., Nixon, A. B., Griffiths, R., Hurwitz, H., & Coffman, T. M.

(2009). Vascular endothelial growth factor receptor 2 controls blood pressure

by regulating nitric oxide synthase expression. Hypertension, 54(3), 652-658.

Failli, P., Nistri, S., Mazzetti, L., Chiappini, L., & Bani, D. (2005). Effects of

relaxin on vascular smooth muscle and endothelial cells in normotensive and

hypertensive rats. Annals of the New York Academy of Sciences, 1041(1),

311-313.

Fanshawe, A. E., & Ibrahim, M. (2013). The current status of lipoprotein (a) in

pregnancy: A literature review. Journal of cardiology, 61(2), 99-106.

Farzadnia, M., Ayatollahi, H., Hasan-Zade, M., & Rahimi, H. R. (2013). A

comparative study of vascular cell adhesion molecule-1 and high-sensitive C-

reactive protein in normal and preeclamptic pregnancies. Interventional

Medicine and Applied Science, 5(1), 26-30.

Ferrara, N., & Davis-Smyth, T. (1997). The biology of vascular endothelial

growth factor. Endocrine reviews, 18(1), 4-25.

Fialkow, L., Wang, Y., & Downey, G. P. (2007). Reactive oxygen and nitrogen

species as signaling molecules regulating neutrophil function. Free Radical

Biology and Medicine, 42(2), 153-164.

Fialova, L., Malbohan, I., Kalousova, M., Soukupova, J., Krofta, L., Štípek, S.,

& Zima, T. (2006). Oxidative stress and inflammation in pregnancy.

Scandinavian journal of clinical and laboratory investigation, 66(2), 121-128.

168

Fossati, P., & Prencipe, L. (1982). Serum triglycerides determined

colorimetrically with an enzyme that produces hydrogen peroxide. Clinical

chemistry, 28(10), 2077-2080.

Furuya, M., Ishida, J., Aoki, I., & Fukamizu, A. (2008). Pathophysiology of

placentation abnormalities in pregnancy-induced hypertension. Vasc Health

Risk Manag, 4(6), 1301-1313.

Furuya, M., Kurasawa, K., Nagahama, K., Kawachi, K., Nozawa, A.,

Takahashi, T., & Aoki, I. (2011). Disrupted balance of angiogenic and

antiangiogenic signalings in preeclampsia. Journal of pregnancy, 2011.

Gawande, M. S., & Joshi, S. A. (2016). Lipid profile in patients of

preeclampsia: A comparative study. Panacea Journal Of Medical Sciences,

6(3), 155-158.

Ghate, J., Choudhari, A. R., Ghugare, B., & Ramji, S. (2011). Antioxidant role

of Vitamin C in normal pregnancy. Biomedical Research, 22(1).

Goel, A., & Rana, S. (2013). Angiogenic factors in preeclampsia: potential for

diagnosis and treatment. Current opinion in nephrology and hypertension,

22(6), 643.

Gohil, J. T., Patel, P. K., & Gupta, P. (2011). Estimation of lipid profile in

subjects of preeclampsia. The Journal of Obstetrics and Gynecology of India,

61(4), 399.

169

Goodwin, B. L., Pendleton, L. C., Levy, M. M., Solomonson, L. P., & Eichler,

D. C. (2007). Tumor necrosis factor-alpha reduces argininosuccinate synthase

expression and nitric oxide production in aortic endothelial cells. American

journal of physiology, 293(2), H1115.

Gowda, V., Aroor, A. R., & Krishna, L. (2010). Studies on Oxidative stress in

Preeclampsia. Biomedical Research, 21(1).

Granger, D., Rodrigues, S. F., Yildirim, A., & Senchenkova, E. Y. (2010).

Microvascular responses to cardiovascular risk factors. Microcirculation, 17(3),

192-205.

Granger, J. P., Alexander, B. T., Llinas, M. T., Bennett, W. A., & Khalil, R. A.

(2001). Pathophysiology of hypertension during preeclampsia linking placental

ischemia with endothelial dysfunction. Hypertension, 38(3), 718-722.

Gratacós, E. (2000). Lipid-mediated endothelial dysfunction: a common factor

to preeclampsia and chronic vascular disease. European journal of obstetrics

& gynecology and Reproductive biology, 92(1), 63-66.

Grotto, D., Maria, L. S., Valentini, J., Paniz, C., Schmitt, G., Garcia, S. C., ... &

Farina, M. (2009). Importance of the lipid peroxidation biomarkers and

methodological aspects for malondialdehyde quantification. Quimica Nova,

32(1), 169-174.

170

Guerby, P., Vidal, F., Garoby-Salom, S., Vayssiere, C., Salvayre, R., Parant,

O., & Negre-Salvayre, A. (2015). Oxidative stress and preeclampsia: A review.

Gynecologie, obstetrique & fertilite, 43(11), 751-756.

Gupta, A., Kant, S., Gupta, S. K., Prakash, S., Kalaivani, M., Pandav, C. S., ...

& Misra, P. (2016). Serum FRAP Levels and Pre-eclampsia among Pregnant

Women in a Rural Community of Northern India. Journal of Clinical and

Diagnostic Research: JCDR, 10(10), LC12.

Gupta, M., & Chari, S. (2015). Predictive value of inflammatory cytokines in

preeclampsia. International Journal of Biomedical and Advance Research,

6(4), 334-338.

Gupta, S., Aziz, N., Sekhon, L., Agarwal, R., Mansour, G., Li, J., & Agarwal, A.

(2009). Lipid peroxidation and antioxidant status in preeclampsia: a systematic

review. Obstetrical & gynecological survey, 64(11), 750-759.

Hadjiyannakis, S. (2005). The metabolic syndrome in children and

adolescents. Paediatrics & child health, 10(1), 41.

Hansson, S. R., Nääv, Å., & Erlandsson, L. (2015). Oxidative stress in

preeclampsia and the role of free fetal hemoglobin. Frontiers in physiology, 5,

516.

Hassan, M. F., Rund, N. M. A., & Salama, A. H. (2013). An elevated maternal

plasma soluble fms-like tyrosine kinase-1 to placental growth factor ratio at

171

midtrimester is a useful predictor for preeclampsia. Obstetrics and gynecology

international, 2013.

Hauth, J. C., Clifton, R. G., Roberts, J. M., Myatt, L., Spong, C. Y., Leveno, K.

J., ... & Peaceman, A. M. (2011). Maternal insulin resistance and

preeclampsia. American journal of obstetrics and gynecology, 204(4), 327-e1.

He, H., Venema, V. J., Gu, X., Venema, R. C., Marrero, M. B., & Caldwell, R.

B. (1999). Vascular endothelial growth factor signals endothelial cell

production of nitric oxide and prostacyclin through flk-1/KDR activation of c-

Src. Journal of Biological Chemistry, 274(35), 25130-25135.

Helewa, M. E., Burrows, R. F., Smith, J., Williams, K., Brain, P., & Rabkin, S.

W. (1997). Report of the Canadian Hypertension Society Consensus

Conference: 1. Definitions, evaluation and classification of hypertensive

disorders in pregnancy. Canadian Medical Association Journal, 157(6), 715-

725.

Hentschke, M. R., Poli-de-Figueiredo, C. E., da Costa, B. E. P., Kurlak, L. O.,

Williams, P. J., & Mistry, H. D. (2013). Is the atherosclerotic phenotype of

preeclamptic placentas due to altered lipoprotein concentrations and placental

lipoprotein receptors? Role of a small-for-gestational-age phenotype. Journal

of lipid research, 54(10), 2658-2664.

Hermawan, M. (2016). Correlation of Total Antioxidant Capacity Measured by

Ferric Reducing Ability of Plasma (FRAP) Assay with the Severity of

172

Preeclampsia. Indonesian Journal of Obstetrics and Gynecology (INAJOG),

35(4).

Herrera, E. (2000). Metabolic adaptations in pregnancy and their implications

for the availability of substrates to the fetus. European journal of clinical

nutrition, 54(S1), S47.

Herrera-Villalobos, J. E., Jaimes, P. A. S., & González, F. M. P. (2012). Índice

aterogénico como factor de riesgo para el síndrome de preeclampsia.

CorSalud, 4(4), 261-265.

Hertig, A., Berkane, N., Lefevre, G., Toumi, K., Marti, H. P., Capeau, J., ... &

Rondeau, E. (2004). Maternal serum sFlt1 concentration is an early and

reliable predictive marker of preeclampsia. Clinical chemistry, 50(9), 1702-

1703.

Hladunewich, M., Karumanchi, S. A., & Lafayette, R. (2007). Pathophysiology

of the clinical manifestations of preeclampsia. Clinical Journal of the American

Society of Nephrology, 2(3), 543-549.

Hoeben, A., Landuyt, B., Highley, M. S., Wildiers, H., Van Oosterom, A. T., &

De Bruijn, E. A. (2004). Vascular endothelial growth factor and angiogenesis.

Pharmacological reviews, 56(4), 549-580.

Holden, D. P., Fickling, S. A., Whitley, G. S. J., & Nussey, S. S. (1998).

Plasma concentrations of asymmetric dimethylarginine, a natural inhibitor of

173

nitric oxide synthase, in normal pregnancy and preeclampsia. American

journal of obstetrics and gynecology, 178(3), 551-556.

Huang, L. T., Hsieh, C. S., Chang, K. A., & Tain, Y. L. (2012). Roles of nitric

oxide and asymmetric dimethylarginine in pregnancy and fetal programming.

International journal of molecular sciences, 13(11), 14606-14622.

Hubel, C. A. (1999). Oxidative stress in the pathogenesis of preeclampsia.

Experimental Biology and Medicine, 222(3), 222-235.

Hung, T. H., & Burton, G. J. (2006). Hypoxia and reoxygenation: a possible

mechanism for placental oxidative stress in preeclampsia. Taiwanese Journal

of Obstetrics and Gynecology, 45(3), 189-200.

Hung, T. H., Charnock-Jones, D. S., Skepper, J. N., & Burton, G. J. (2004).

Secretion of tumor necrosis factor-α from human placental tissues induced by

hypoxia-reoxygenation causes endothelial cell activation in vitro: a potential

mediator of the inflammatory response in preeclampsia. The American journal

of pathology, 164(3), 1049-1061.

Hung, T. H., Skepper, J. N., & Burton, G. J. (2001). In vitro ischemia-

reperfusion injury in term human placenta as a model for oxidative stress in

pathological pregnancies. The American journal of pathology, 159(3), 1031-

1043.

174

Hutter, D., & Jaeggi, E. (2010). Causes and mechanisms of intrauterine

hypoxia and its impact on the fetal cardiovascular system: a review.

International journal of pediatrics, 2010.

Hwang, H. S., Kwon, J. Y., Kim, M. A., Park, Y. W., & Kim, Y. H. (2007).

Maternal serum highly sensitive C-reactive protein in normal pregnancy and

pre-eclampsia. International Journal of G Arbogast, B. W., Leeper, S. C.,

Merrick, R. D., Olive, K. E., & Taylor, R. N. (1994). Which plasma factors bring

about disturbance of endothelial function in pre-eclampsia?. The Lancet,

343(8893), 340-341. ynecology & Obstetrics, 98(2), 105-109.

Idriss, H. T., & Naismith, J. H. (2000). TNFalpha and the TNF Receptor

Superfamily: Structure-Function Relationship (s). Microscopy research and

technique, 50(3), 184-195.

Irinyenikan, T. A., Roberts, O. A., & Arowojolu, A. (2013). Serum lipid level in

pregnant normotensive and gestational hypertensive women in Ibadan,

Nigeria. Ann Biol Res, 4, 204-8.

Işildak, M. Y., Yildirmak, S. T., Doğan, S., Çakmak, M., & Özakin, E. (2009).

Serum Homocysteine and Lipoprotein (a) Levels in Preeclamptic Pregnants.

Balkan Medical Journal, 2009(3).

Islam, N. A. F., Chowdhury, M. A. R., Kibria, G. M., & Akhter, S. (2010). Study

of serum lipid profile in pre-eclampsia and eclampsia. Faridpur Medical

College Journal, 5(2), 56-59.

175

Isser, H. S., Puri, V. K., Narain, V. S., Saran, R. K., Dwivedi, S. K., & Singh, S.

(2000). Lipoprotein (a) and lipid levels in young patients with myocardial

infarction and their first-degree re

Itoh, S., Brawley, L., Wheeler, T., Anthony, F. W., Poston, L., & Hanson, M. A.

(2002). Vasodilation to vascular endothelial growth factor in the uterine artery

of the pregnant rat is blunted by low dietary protein intake. Pediatric research,

51(4), 485-491.

Janero, D. R. (1990). Malondialdehyde and thiobarbituric acid-reactivity as

diagnostic indices of lipid peroxidation and peroxidative tissue injury. Free

Radical Biology and Medicine, 9(6), 515-540.

Jeyabalan, A. (2013). Epidemiology of preeclampsia: impact of obesity.

Nutrition reviews, 71(suppl 1), S18-S25.

Juan, P., Stefano, G., Antonella, S., & Albana, C. (2011). Platelets in

pregnancy. Journal of prenatal medicine, 5(4), 90.

Kahn, B. B., & Flier, J. S. (2000). Obesity and insulin resistance. The Journal

of clinical investigation, 106(4), 473-481.

Kaloti, A. S., Kaur, C., Goel, R. K., Jha, S., & Budhera, G. (2013). Study of

lipid profile trends in women of pregnancy induced hypertension cases in a

rural setup. Journal of Evolution of Medical and Dental Sciences, 2(13), 2024-

31.

176

Karumanchi, S. A. (2015). U.S. Patent No. 8,969,322. Washington, DC: U.S.

Patent and Trademark Office.

Kashinakunti, S. V., Sunitha, H., Gurupadappa, K., Shankarprasad, D. S.,

Suryaprakash, G., & Ingin, J. B. (2010). Lipid peroxidation and antioxidant

status in preeclampsia. Al Ameen J Med Sci, 3(1), 38-41.

Kauma, S. W., Wang, Y., & Walsh, S. W. (1995). Preeclampsia is associated

with decreased placental interleukin-6 production. Journal of the Society for

Gynecologic Investigation, 2(4), 614-617.

Kaur, G., Mishra, S., Sehgal, A., & Prasad, R. (2008). Alterations in lipid

peroxidation and antioxidant status in pregnancy with preeclampsia. Molecular

and cellular biochemistry, 313(1-2), 37-44.

Kaushik, A., Jijta, C., Kaushik, J. J., Zeray, R., Ambesajir, A., & Beyene, L.

(2012). FRAP (Ferric reducing ability of plasma) assay and effect of Diplazium

esculentum (Retz) Sw.(a green vegetable of North India) on central nervous

system.

Kendall, R. L., Wang, G., & Thomas, K. A. (1996). Identification of a natural

soluble form of the vascular endothelial growth factor receptor, FLT-1, and its

heterodimerization with KDR. Biochemical and biophysical research

communications, 226(2), 324-328.

177

Khalil, A., Muttukrishna, S., Harrington, K., & Jauniaux, E. (2008). Effect of

antihypertensive therapy with alpha methyldopa on levels of angiogenic

factors in pregnancies with hypertensive disorders. PLoS One, 3(7), e2766.

Kim, J. Y., & Kim, Y. M. (2015). Acute atherosis of the uterine spiral arteries:

clinicopathologic implications. Journal of pathology and translational medicine,

49(6), 462-471.

Kim, S. Y., Ryu, H. M., Yang, J. H., Kim, M. Y., Han, J. Y., Kim, J. O., ... &

Kim, D. J. (2007). Increased sFlt-1 to PlGF ratio in women who subsequently

develop preeclampsia. Journal of Korean medical science, 22(5), 873-877.

Kim, Y. M., Chaemsaithong, P., Romero, R., Shaman, M., Kim, C. J., Kim, J.

S., & Hassan, S. S. (2015). Placental lesions associated with acute atherosis.

The Journal of Maternal-Fetal & Neonatal Medicine, 28(13), 1554-1562.

Kintiraki, E., Papakatsika, S., Kotronis, G., Goulis, D. G., & Kotsis, V. (2015).

Pregnancy-induced hypertension. Hormones (Athens), 14(2), 211-223.

Karkalousos, P., & Evangelopoulos, A. (2011). Quality control in clinical

laboratories. INTECH Open Access Publisher.

Lain, K. Y., & Catalano, P. M. (2007). Metabolic changes in pregnancy.

Clinical obstetrics and gynecology, 50(4), 938-948.

Lam, C., Lim, K. H., & Karumanchi, S. A. (2005). Circulating angiogenic

factors in the pathogenesis and prediction of preeclampsia. Hypertension,

46(5), 1077-1085.

178

LaMarca, B. (2012). Endothelial dysfunction; an important mediator in the

Pathophysiology of Hypertension during Preeclampsia. Minerva ginecologica,

64(4), 309.

LaMarca, B. D., Ryan, M. J., Gilbert, J. S., Murphy, S. R., & Granger, J. P.

(2007). Inflammatory cytokines in the pathophysiology of hypertension during

preeclampsia. Current hypertension reports, 9(6), 480-485.

Lamarche, B., Rashid, S., & Lewis, G. F. (1999). HDL metabolism in

hypertriglyceridemic states: an overview. Clinica Chimica Acta, 286(1), 145-

161.

Lamina, S., Ezema, C. I., Theresa, A. I., & Anthonia, E. U. (2013). Effects of

free radicals and antioxidants on exercise performance. Oxidants and

Antioxidants in Medical Science, 2(2), 83-91.

Laresgoiti-Servitje, E., Gómez-López, N., & Olson, D. M. (2010). An

immunological insight into the origins of pre-eclampsia. Human reproduction

update, 16(5), 510-524.

Laskowska, M., Laskowska, K., Terbosh, M., & Oleszczuk, J. (2013). A

comparison of maternal serum levels of endothelial nitric oxide synthase,

asymmetric dimethylarginine, and homocysteine in normal and preeclamptic

pregnancies. Medical Science Monitor, 19, 430-437.

Lau, S. Y., Guild, S. J., Barrett, C. J., Chen, Q., McCowan, L., Jordan, V., &

Chamley, L. W. (2013). Tumor necrosis factor‐alpha, interleukin‐6, and

179

interleukin‐10 levels are altered in preeclampsia: a systematic review and

meta‐analysis. American Journal of Reproductive Immunology, 70(5), 412-

427.

Lee, E. S., Oh, M. J., Jung, J. W., Lim, J. E., Seol, H. J., Lee, K. J., & Kim, H.

J. (2007). The levels of circulating vascular endothelial growth factor and

soluble Flt-1 in pregnancies complicated by preeclampsia. Journal of Korean

medical science, 22(1), 94-98.

Lefevre, G., Beljean-Leymarie, M., Beyerle, F., Bonnefont-Rousselot, D.,

Cristol, J. P., Therond, P., & Torreilles, J. (1997, December). Evaluation of

lipid peroxidation by measuring thiobarbituric acid reactive substances. In

Annales de biologie clinique (Vol. 56, No. 3, pp. 305-319).

Lei, Q., Lv, L. J., Zhang, B. Y., Wen, J. Y., Liu, G. C., Lin, X. H., & Niu, J. M.

(2011). Ante-partum and post-partum markers of metabolic syndrome in pre-

eclampsia. Journal of human hypertension, 25(1), 11-17.

Leung, D. W., Cachianes, G., Kuang, W. J., Goeddel, D. V., & Ferrara, N.

(1989). Vascular endothelial growth factor is a secreted angiogenic mitogen.

Science, 246(4935), 1306.

Levine, R. J., Maynard, S. E., Qian, C., Lim, K. H., England, L. J., Yu, K. F., &

Sibai, B. M. (2004). Circulating angiogenic factors and the risk of

preeclampsia. New England Journal of Medicine, 350(7), 672-683.

180

Lewis, F. (2014). Excess Maternal Body Weight and Preeclampsia/Eclampsia

Risk among Women in San Ber-nardino County, 2007-2008. J Fud Nutr, 1, 1-

6.

Li, J., Lan, T., Zhang, C., Zeng, C., Hou, J., Yang, Z., & Liu, B. (2015).

Reciprocal activation between IL-6/STAT3 and NOX4/Akt signalings promotes

proliferation and survival of non-small cell lung cancer cells. Oncotarget, 6(2),

1031.

Li, X., & Eriksson, U. (2003). Novel PDGF family members: PDGF-C and

PDGF-D. Cytokine & growth factor reviews, 14(2), 91-98.

Li, Y., Wang, Y., Ding, X., Duan, B., Li, L., & Wang, X. (2016). Serum Levels

of TNF-α and IL-6 Are Associated With Pregnancy-Induced Hypertension.

Reproductive Sciences, 23(10), 1402-1408.

Lima, V. J. D., Andrade, C. R. D., Ruschi, G. E., & Sass, N. (2011). Serum

lipid levels in pregnancies complicated by preeclampsia. Sao Paulo Medical

Journal, 129(2), 73-76.

Liochev, S. I., & Fridovich, I. (1997). How does superoxide dismutase protect

against tumor necrosis factor: a hypothesis informed by effect of superoxide

on ―free‖ iron. Free Radical Biology and Medicine, 23(4), 668-671.

Livingston, J. C., Chin, R., Haddad, B., McKinney, E. T., Ahokas, R., & Sibai,

B. M. (2000). Reductions of vascular endothelial growth factor and placental

181

growth factor concentrations in severe preeclampsia. American journal of

obstetrics and gynecology, 183(6), 1554-1557.

Lobo, V., Patil, A., Phatak, A., & Chandra, N. (2010). Free radicals,

antioxidants and functional foods: Impact on human health. Pharmacognosy

reviews, 4(8), 118.

Lockwood, C. J., Yen, C. F., Basar, M., Kayisli, U. A., Martel, M., Buhimschi,

I., & Schatz, F. (2008). Preeclampsia-related inflammatory cytokines regulate

interleukin-6 expression in human decidual cells. The American journal of

pathology, 172(6), 1571-1579.

Luttun, A., Tjwa, M., Moons, L., Wu, Y., Angelillo-Scherrer, A., Liao, F., &

Compernolle, V. (2002). Revascularization of ischemic tissues by PlGF

treatment, and inhibition of tumor angiogenesis, arthritis and atherosclerosis

by anti-Flt1. Nature medicine, 8(8), 831-840.

Lyall, F., & Greer, I. A. (1996). The vascular endothelium in normal pregnancy

and pre-eclampsia. Reviews of reproduction, 1(2), 107-116.

Maharaj, A. S., Saint-Geniez, M., Maldonado, A. E., & D'Amore, P. A. (2006).

Vascular endothelial growth factor localization in the adult. The American

journal of pathology, 168(2), 639-648.

Mandal, K. K., Das, A., Devi, H. L., Omita, N., Singh, N. N., & Singh, W. G. ‟

Serum high sensitivity C-reactive Protein as predictor of Preeclampsia‖. IOSR

Journal of Dental and Medical Sciences (IOSR-JDMS), 1(15), 26-31.

182

Mansell, A., & Jenkins, B. J. (2013). Dangerous liaisons between interleukin-6

cytokine and toll-like receptor families: a potent combination in inflammation

and cancer. Cytokine & growth factor reviews, 24(3), 249-256.

Manten, G. T. R., van der Hoek, Y. Y., Sikkema, J. M., Voorbij, H. A.,

Hameeteman, T. M., Visser, G. H., & Franx, A. (2005). The role of lipoprotein

(a) in pregnancies complicated by pre-eclampsia. Medical hypotheses, 64(1),

162-169.

Martin, J. N., Rinehart, B. K., May, W. L., Magann, E. F., Terrone, D. A., &

Blake, P. G. (1999). The spectrum of severe preeclampsia: comparative

analysis by HELLP (hemolysis, elevated liver enzyme levels, and low platelet

count) syndrome classification. American journal of obstetrics and gynecology,

180(6), 1373-1384.

Martínez-Varea, A., Pellicer, B., Perales-Marín, A., & Pellicer, A. (2014).

Relationship between maternal immunological response during pregnancy

and onset of preeclampsia. Journal of immunology research, 2014.

Maruo, N., Morita, I., Ishizaki, Y., & Murota, S. I. (1992). Inhibitory effects of

interleukin 6 on prostaglandin I2 production in cultured bovine vascular

endothelial cells. Archives of biochemistry and biophysics, 292(2), 600-604.

Matson, P. L. (1998). Internal quality control and external quality assurance in

the IVF laboratory. Human Reproduction, 13(suppl 4), 156-165.

183

Matsubara, K., Higaki, T., Matsubara, Y., & Nawa, A. (2015). Nitric oxide and

reactive oxygen species in the pathogenesis of preeclampsia. International

journal of molecular sciences, 16(3), 4600-4614.

Matsubara, K., Matsubara, Y., Hyodo, S., Katayama, T., & Ito, M. (2010). Role

of nitric oxide and reactive oxygen species in the pathogenesis of

preeclampsia. Journal of Obstetrics and Gynaecology Research, 36(2), 239-

247.

Maynard, S. E., Min, J. Y., Merchan, J., Lim, K. H., Li, J., Mondal, S., &

Epstein, F. H. (2003). Excess Placental Soluble fms-like Tyrosine Kinase 1

(sFlt1) Could Contribute to Endothelial Dysfunction, Hypertension, and

Proteinuria in Preeclampsia. Obstetrical & gynecological survey, 58(9), 564-

565.

McMahon, K., Karumanchi, S. A., Stillman, I. E., Cummings, P., Patton, D., &

Easterling, T. (2014). Does soluble fms-like tyrosine kinase-1 regulate

placental invasion? Insight from the invasive placenta. American journal of

obstetrics and gynecology, 210(1), 68-e1.

Meera, K. S., Maitra, S., & Hemalatha, R. (2010). Increased level of Lipid

peroxidation in preeclamptic pregnancy; a rela-tionship with paraoxanase 1

(PON1) activity. Biomedical Research, 21(4).

Melchiorre, K., Sutherland, G. R., Liberati, M., & Thilaganathan, B. (2011).

Preeclampsia is associated with persistent postpartum cardiovascular

impairment. Hypertension, 58(4), 709-715.

184

Metkari, S. B., Tupkari, J. V., & Barpande, S. R. (2007). An estimation of

serum malondialdehyde, superoxide dismutase and vitamin A in oral

submucous fibrosis and its clinicopathologic correlation. Journal of Oral and

Maxillofacial Pathology, 11(1), 23.latives. Indian heart journal, 53(4), 463-466.

Miccoli, R., Bianchi, C., Penno, G., & Del Prato, S. (2008). Insulin resistance

and lipid disorders.

Mihu, D., Costin, N., Sabau, L., Oancea, M., Ciortea, R., & Malutan, A. (2010).

Interleukin-6-a Marker of the Inflammatory Syndrome in Preeclampsia.

GINECO RO, 6(4), 230-234.

Mohanty, S., Sahu, P. K., Mandal, M. K., Mohapatra, P. C., & Panda, A.

(2006). Evaluation of oxidative stress in pregnancy induced hypertension.

Indian Journal of clinical biochemistry, 21(1), 101-105.

Molvarec, A., Ito, M., Shima, T., Yoneda, S., Toldi, G., Stenczer, B., & Saito,

S. (2010). Decreased proportion of peripheral blood vascular endothelial

growth factor–expressing T and natural killer cells in preeclampsia. American

journal of obstetrics and gynecology, 203(6), 567-e1.

Mor, G., & Cardenas, I. (2010). Review article: the immune system in

pregnancy: a unique complexity. American journal of reproductive

immunology, 63(6), 425-433.

Müller-Deile, J., & Schiffer, M. (2010). Renal involvement in preeclampsia:

similarities to VEGF ablation therapy. Journal of pregnancy, 2011.

185

Munilla, M. A., & Herrera, E. (2000). Maternal hypertriglyceridemia during late

pregnancy does not affect the increase in circulating triglycerides caused by

the long-term consumption of a sucrose-rich diet by rats. The Journal of

nutrition, 130(12), 2883-2888.

Murphy, S. R., LaMarca, B. B. D., Parrish, M., Cockrell, K., & Granger, J. P.

(2013). Control of soluble fms-like tyrosine-1 (sFlt-1) production response to

placental ischemia/hypoxia: role of tumor necrosis factor-α. American Journal

of Physiology-Regulatory, Integrative and Comparative Physiology, 304(2),

R130-R135.

Musa, A. H., Mairiga, A. G., Jimeta, A. A., Ahmed, A., & Daja, A. (2014). Lipid

profile pattern of pre-eclamptic and eclamptic patients attending University of

Maiduguri Teaching Hospital. Gynecol Obstet (Sunnyvale), 4(217), 2161-0932

Myatt, L. (2002). Role of placenta in preeclampsia. Endocrine, 19(1), 103-111.

Nagamatsu, T., Fujii, T., Kusumi, M., Zou, L., Yamashita, T., Osuga, Y., ... &

Taketani, Y. (2004). Cytotrophoblasts up-regulate soluble fms-like tyrosine

kinase-1 expression under reduced oxygen: an implication for the placental

vascular development and the pathophysiology of preeclampsia.

Endocrinology, 145(11), 4838-4845.

Nanda, K., Sadanand, G., Krishna, M., & Mahdevappa, K. L. (2012). C-

reactive protein as a predictive factor of pre-eclampsia. Int J Biol Med Res,

3(1), 1307-1310.

186

Nasr, G. M., Nasr, A. M., & Ibrahim, D. Vascular changes as a predictor of

pregnancy induced hypertension and its association with lipid profile.

Nayan, S., Meena, M. L., Hooja, N., Fatima, A., Singh, N., & Aseri, S. (2014).

A Study of Comparison of Serum Lipid Profile of Women with Pregnancy

Induced Hypertension and Normal Pregnancy. Sch. Acad. J. Biosci, 2(11),

834-836.

Nazli, R., Khan, M. A., Akhtar, T., Lutfullah, G., Mohammad, N. S., Ahmad, J.,

... & Aslam, H. (2013). Abnormal Lipid levels as a risk factor of eclampsia,

study conducted in tertiary care Hospitals of Khyber Pakhtunkhwa Province-

Pakistan. Pakistan journal of medical sciences, 29(6), 1410.

Negi, R., Pande, D., Karki, K., Khanna, R. S., & Khanna, H. D. (2011).

Oxidative stress and preeclampsia. Advances in Life Sciences, 1(1), 20-23.

Nicholls, S. J., Tang, W. W., Scoffone, H., Brennan, D. M., Hartiala, J.,

Allayee, H., & Hazen, S. L. (2010). Lipoprotein (a) levels and long-term

cardiovascular risk in the contemporary era of statin therapy. Journal of lipid

research, 51(10), 3055-3061.

Nielsen, F., Mikkelsen, B. B., Nielsen, J. B., Andersen, H. R., & Grandjean, P.

(1997). Plasma malondialdehyde as biomarker for oxidative stress: reference

interval and effects of life-style factors. Clinical chemistry, 43(7), 1209-1214.

187

Nimse, S. B., & Pal, D. (2015). Free radicals, natural antioxidants, and their

reaction mechanisms. RSC Advances, 5(35), 27986-28006.

Noris, M., Perico, N., & Remuzzi, G. (2005). Mechanisms of disease: pre-

eclampsia. Nature clinical practice Nephrology, 1(2), 98-114.

Nwagha, U. I., Ikekpeazu, E. J., Ejezie, F. E., Neboh, E. E., & Maduka, I. C.

(2010). Atherogenic index of plasma as useful predictor of cardiovascular risk

among postmenopausal women in Enugu, Nigeria. African health sciences,

10(3).

Onuegbu, A. J., Olisekodiaka, J. M., Udo, J. U., Umeononihu, O., Amah, U.

K., Okwara, J. E., & Atuegbu, C. (2015). Evaluation of high-sensitivity C-

reactive protein and serum lipid profile in southeastern Nigerian women with

pre-eclampsia. Medical Principles and Practice, 24(3), 276-279.

Orhan, H., Önderoglu, L., Yücel, A., & Sahin, G. (2003). Circulating

biomarkers of oxidative stress in complicated pregnancies. Archives of

gynecology and obstetrics, 267(4), 189-195.

O'Riordan, M. N., & Higgins, J. R. (2003). Haemostasis in normal and

abnormal pregnancy. Best Practice & Research Clinical Obstetrics &

Gynaecology, 17(3), 385-396.

Ozler, A., Turgut, A., Sak, M. E., Evsen, M. S., Soydinc, H. E., Evliyaoglu, O.,

& Gul, T. (2012). Serum levels of neopterin, tumor necrosis factor-alpha and

188

Interleukin-6 in preeclampsia: relationship with disease severity. Eur Rev Med

Pharmacol Sci, 16(12), 1707-12.

Pahune, P. P., Choudhari, A. R., & Muley, P. A. (2013). The total antioxidant

power of semen and its correlation with the fertility potential of human male

subjects. J Clin Diagn Res, 7(6), 991-5.

Pan, W., & Kastin, A. J. (2007). Tumor necrosis factor and stroke: role of the

blood–brain barrier. Progress in neurobiology, 83(6), 363-374.

Pan, W., Csernus, B., & Kastin, A. J. (2003). Upregulation of p55 and p75

receptors mediating TNF-α transport across the injured blood-spinal cord

barrier. Journal of Molecular Neuroscience, 21(2), 173-184.

Pandey, C. K., Karna, S. T., Pandey, V. K., & Tandon, M. (2015). Acute liver

failure in pregnancy: Challenges and management. Indian journal of

anaesthesia, 59(3), 144.

Park, J. E., Chen, H. H., Winer, J., Houck, K. A., & Ferrara, N. (1994).

Placenta growth factor. Potentiation of vascular endothelial growth factor

bioactivity, in vitro and in vivo, and high affinity binding to Flt-1 but not to Flk-

1/KDR. Journal of Biological Chemistry, 269(41), 25646-25654.

Parvin, S., Samsuddin, L., Ali, A., Chowdhury, S. A., & Siddique, I. (2010).

Lipoprotein (a) level in pre-eclampsia patients. Bangladesh Medical Research

Council bulletin, 36(3), 97-99.

189

Patil, S. B., Kodliwadmath, M. V., & Kodliwadmath, S. M. (2006). Role of lipid

peroxidation and enzymatic antioxidants in pregnancy-induced hypertension.

Clinical and experimental obstetrics &Sahu, S., Abraham, R., Vedavalli, R., &

Daniel, M. (2009). Study of lipid profile, lipid peroxidation and vitamin E in

pregnancy induced hypertension. gynecology, 34(4), 239-241.

Patil, S. B., Kodliwadmath, M. V., & Kodliwadmath, S. M. (2008). Correlation

between lipid peroxidation and non-enzymatic antioxidants in pregnancy

induced hypertension. Indian Journal of Clinical Biochemistry, 23(1), 45-48.

Phalak, P., & Tilak, M. (2012). Study of lipid profile in pre-eclampsia. Indian

Journal of Basic & Applied Medical Research, 5(2), 405-409.

Poorolajal, J., & Jenabi, E. (2016). The association between body mass index

and preeclampsia: a meta-analysis. The Journal of Maternal-Fetal & Neonatal

Medicine, 29(22), 3670-3676.

Popa, C., Netea, M. G., Van Riel, P. L., van der Meer, J. W., & Stalenhoef, A.

F. (2007). The role of TNF-α in chronic inflammatory conditions, intermediary

metabolism, and cardiovascular risk. Journal of lipid research, 48(4), 751-762.

Possomato-Vieira, J. S., & Khalil, R. A. (2016). Chapter Eleven-Mechanisms

of Endothelial Dysfunction in Hypertensive Pregnancy and Preeclampsia.

Advances in Pharmacology, 77, 361-431.

Poston, L. (2006). Endothelial dysfunction in pre-eclampsia. Pharmacol Rep,

58(Suppl), 69-74.

190

Powe, C. E., Levine, R. J., & Karumanchi, S. A. (2011). Preeclampsia, a

disease of the maternal endothelium. Circulation, 123(24), 2856-2869.

Priyamvada, R. P., Patange, R. P., Patil, S. K., & Patil, Y. S. (2016). To

assess the magnitude of oxidative stress and antioxidant defense in

preeclampsia. journal of evolution of medical and dental sciences-jemds,

5(57), 3898-3902.

Program, N. H. B. P. E. (2000). Report of the national high blood pressure

education program working group on high blood pressure in pregnancy.

American journal of obstetrics and gynecology, 183(1), s1-s22.

Pushparaj, J. L., & Subramanyam, G. (2012). Dyslipidemia in pre-eclampsia-

risk factor for future maternal cardiovascular diseases. Journal of Evolution of

Medical and Dental Sciences, 1(4), 487-95.

Qiu, C., Sorensen, T. K., Luthy, D. A., & Williams, M. A. (2004). A prospective

study of maternal serum C‐reactive protein (CRP) concentrations and risk of

gestational diabetes mellitus. Paediatric and perinatal epidemiology, 18(5),

377-384.

Radhi, M., & Carpenter, S. L. (2012). Thrombotic microangiopathies. ISRN

hematology, 2012.

Rafeeinia, A., Tabandeh, A., Khajeniazi, S., & Marjani, A. (2014). Metabolic

Syndrome in Preeclampsia Women in Gorgan. The open biochemistry journal,

8, 94.

191

Raijmakers, M. T., Dechend, R., & Poston, L. (2004). Oxidative stress and

preeclampsia. Hypertension, 44(4), 374-380.

Rajendran, P., Rengarajan, T., Thangavel, J., Nishigaki, Y., Sakthisekaran, D.,

Sethi, G., & Nishigaki, I. (2013). The vascular endothelium and human

diseases. Int J Biol Sci, 9(10), 1057-69.

Rajman, I., Eacho, P. I., Chowienczyk, P. J., & Ritter, J. M. (1999). LDL

particle size: an important drug target?. British journal of clinical

pharmacology, 48(2), 125-133.

Rao, P. S., Shashidhar, A., & Ashok, C. (2013). In utero fuel homeostasis:

Lessons for a clinician. Indian journal of endocrinology and metabolism, 17(1),

60.

Rasmussen, K. M., Catalano, P. M., & Yaktine, A. L. (2009). New guidelines

for weight gain during pregnancy: what obstetrician/gynecologists should

know. Current opinion in obstetrics & gynecology, 21(6), 521.

Reddy, A., Suri, S., Sargent, I. L., Redman, C. W., & Muttukrishna, S. (2009).

Maternal circulating levels of activin A, inhibin A, sFlt-1 and endoglin at

parturition in normal pregnancy and pre-eclampsia. PloS one, 4(2), e4453.

Redman, C. W., Sacks, G. P., & Sargent, I. L. (1999). Preeclampsia: an

excessive maternal inflammatory response to pregnancy. American journal of

obstetrics and gynecology, 180(2), 499-506.

192

Roberts, J. M., & Rajakumar, A. (2009). Preeclampsia and soluble fms-like

tyrosine kinase 1.

Roberts, J. M., Bodnar, L. M., Patrick, T. E., & Powers, R. W. (2011). The role

of obesity in preeclampsia. Pregnancy Hypertension: An International Journal

of Women's Cardiovascular Health, 1(1), 6-16.

Rohita, B., Keerti, M., Deepak, S., & Manisha, S. (2014). The Relationship

between Oxidative Stress and Atherogenic Index in Preeclampsia. Sch. J App

Med Sci, 2(6D), 3092-96.

Rohra, D. K., Zeb, A., Qureishi, R. N., Azam, S. I., Khan, N. B., Zuberi, H. S.,

& Sikandar, R. (2012). Prediction of pre-eclampsia during early pregnancy in

primiparas with soluble fms-like tyrosine kinase-1 and placental growth factor.

Romero, R., Chaiworapongsa, T., & Espinoza, J. (2003). Micronutrients and

intrauterine infection, preterm birth and the fetal inflammatory response

syndrome. The Journal of nutrition, 133(5), 1668S-1673S.

Saarelainen, H., Kärkkäinen, H., Valtonen, P., Punnonen, K., Laitinen, T.,

Heiskanen, N., & Heinonen, S. (2012). Flow-mediated vasodilation is not

attenuated in hypertensive pregnancies despite biochemical signs of

inflammation. ISRN obstetrics and gynecology, 2012.

Saarelainen, H., Valtonen, P., Punnonen, K., Laitinen, T., Raitakari, O. T.,

Juonala, M., & Heinonen, S. (2008). Subtle changes in ADMA and l‐arginine

concentrations in normal pregnancies are unlikely to account for

193

pregnancy‐related increased flow‐mediated dilatation. Clinical physiology and

functional imaging, 28(2), 120-124.

Saha, T., Halder, M., Das, A., & Das, S. K. (2013). Role of nitric oxide,

angiogenic growth factors and biochemical analysis in preeclampsia.

Sahu, S., Abraham, R., Vedavalli, R., & Daniel, M. (2009). Study of lipid

profile, lipid peroxidation and vitamin E in pregnancy induced hypertension.

Saito, S., Umekage, H., Sakamoto, Y., Sakai, M., Tanebe, K., Sasaki, Y., &

Morikawa, H. (1999). Increased T‐helper‐1‐type immunity and decreased

T‐helper‐2‐type immunity in patients with preeclampsia. American Journal of

Reproductive Immunology, 41(5), 297-306.

Sajith, M., Nimbargi, V., ModI, A., Sumariya, R., & Pawar, A. (2014). Incidence

of pregnancy induced hypertension and prescription pattern of

antihypertensive drugs in pregnancy. Age (years), 23, 4-0.

Sánchez-Aranguren, L. C., Prada, C. E., Riaño-Medina, C. E., & Lopez, M.

(2014). Endothelial dysfunction and preeclampsia: role of oxidative stress.

Frontiers in physiology, 5, 372.

Sandrim, V. C., Palei, A. C., Metzger, I. F., Gomes, V. A., Cavalli, R. C., &

Tanus-Santos, J. E. (2008). Nitric oxide formation is inversely related to serum

levels of antiangiogenic factors soluble fms-like tyrosine kinase-1 and soluble

endogline in preeclampsia. Hypertension, 52(2), 402-407.

194

Sattar, N., Bendomir, A., Berry, C., Shepherd, J., Greer, I. A., & Packard, C. J.

(1997). Lipoprotein subfraction concentrations in preeclampsia: pathogenic

parallels to atherosclerosis. Obstetrics & Gynecology, 89(3), 403-408.

Sattar, N., Clark, P., Greer, I. A., Shepherd, J., & Packard, C. J. (2000).

Lipoprotein (a) levels in normal pregnancy and in pregnancy complicated with

pre-eclampsia. Atherosclerosis, 148(2), 407-411.

Savvidou, M. D., Vallance, P. J., Nicolaides, K. H., & Hingorani, A. D. (2001).

Endothelial nitric oxide synthase gene polymorphism and maternal vascular

adaptation to pregnancy. Hypertension, 38(6), 1289-1293.

Saxena, S., Srivastava, P. C., Thimmaraju, K. V., Mallick, A. K., Dalmia, K., &

Das, B. (2014). Socio-demographic profile of pregnancy induced hypertension

in a tertiary care centre. Sch J App Med Sci, 2(6D), 3081-6.

Schmidt, M., Dogan, C., Birdir, C., Kuhn, U., Gellhaus, A., Kimmig, R., &

Kasimir-Bauer, S. (2009). Placental growth factor: a predictive marker for

preeclampsia?. Gynäkologisch-geburtshilfliche Rundschau, 49(2), 94-99.

Scholz, H., Yndestad, A., Damås, J. K., Wæhre, T., Tonstad, S., Aukrust, P.,

& Halvorsen, B. (2003). 8-isoprostane increases expression of interleukin-8 in

human macrophages through activation of mitogen-activated protein kinases.

Cardiovascular research, 59(4), 945-954.

195

Seki, H., Matuoka, K., Inooku, H., & Takeda, S. (2007). TNF‐α from monocyte

of patients with pre‐eclampsia‐induced apoptosis in human trophoblast cell

line. Journal of Obstetrics and Gynaecology Research, 33(4), 408-416.

Seligman, S. P., Buyon, J. P., Clancy, R. M., Young, B. K., & Abramson, S. B.

(1994). The role of nitric oxide in the pathogenesis of preeclampsia. American

journal of obstetrics and gynecology, 171(4), 944-948.

Sena, C. M., Pereira, A. M., & Seiça, R. (2013). Endothelial dysfunction—a

major mediator of diabetic vascular disease. Biochimica et Biophysica Acta

(BBA)-Molecular Basis of Disease, 1832(12), 2216-2231.

Serdar, Z., Gür, E., Çolakoðullarý, M., Develioðlu, O., & Sarandöl, E. (2003).

Lipid and protein oxidation and antioxidant function in women with mild and

severe preeclampsia. Archives of gynecology and obstetrics, 268(1), 19-25.

Serin, Ý. S., Özçelik, B., Bapbuoð, M., Kýlýç, H., Okur, D., & Erez, R. (2002).

Predictive value of tumor necrosis factor alpha (TNF-α) in preeclampsia.

European Journal of Obstetrics & Gynecology and Reproductive Biology,

100(2), 143-145.

Shah, D. M. (2005). Role of the renin-angiotensin system in the pathogenesis

of preeclampsia. American Journal of Physiology-Renal Physiology, 288(4),

F614-F625.

196

Shaheen, A., Khan, R. N., Fatima, S., Ali, R., Khan, I., & Khattak, S. (2016).

Adipokine Serum visfatin level in pregnancy induced hypertension and

uncomplicated pregnancy.

Sharami, S. H., Tangestani, A., Faraji, R., Zahiri, Z., & Azam, A. (2012). Role

of dyslipidemia in preeclamptic overweight pregnant women. International

Journal of Reproductive BioMedicine, 10(2), 105-112.

Sharma, P., Jha, A. B., Dubey, R. S., & Pessarakli, M. (2012). Reactive

oxygen species, oxidative damage, and antioxidative defense mechanism in

plants under stressful conditions. Journal of Botany, 2012.

Shen, B. Q., Lee, D. Y., & Zioncheck, T. F. (1999). Vascular endothelial

growth factor governs endothelial nitric-oxide synthase expression via a

KDR/Flk-1 receptor and a protein kinase C signaling pathway. Journal of

Biological Chemistry, 274(46), 33057-33063.

Shen, B. Q., Lee, D. Y., Gerber, H. P., Keyt, B. A., Ferrara, N., & Zioncheck,

T. F. (1998). Homologous up-regulation of KDR/Flk-1 receptor expression by

vascular endothelial growth factor in vitro. Journal of Biological Chemistry,

273(45), 29979-29985.

Shibuya, M. (2011). Vascular endothelial growth factor (VEGF) and its

receptor (VEGFR) signaling in angiogenesis: a crucial target for anti-and pro-

angiogenic therapies. Genes & cancer, 2(12), 1097-1105.

197

Shibuya, M., Yamaguchi, S., Yamane, A., Ikeda, T., Tojo, A., Matsushime, H.,

& Sato, M. (1990). Nucleotide sequence and expression of a novel human

receptor-type tyrosine kinase gene (flt) closely related to the fms family.

Oncogene, 5(4), 519-524.

Shimada, K., Crother, T. R., Karlin, J., Dagvadorj, J., Chiba, N., Chen, S.,. &

Rentsendorj, A. (2012). Oxidized mitochondrial DNA activates the NLRP3

inflammasome during apoptosis. Immunity, 36(3), 401-414.

Sibai, B., Dekker, G., & Kupferminc, M. (2005). Pre-eclampsia. The Lancet,

365(9461), 785-799.

Sies, H. (1997). Oxidative stress: oxidants and antioxidants. Experimental

physiology, 82(2), 291-295.

Singh, A., Khambra, P., Rani, K. U., & Mandal, A. K. (2016). Assessment of

serum βhCG, lipid profile and uric acid levels in early second trimester as

predictors of pregnancy induced hypertension. Annals of Pathology and

Laboratory Medicine, 3(3), A157-161.

Singh, M., Pathak, M. S., & Paul, A. (2015). A study on atherogenic indices of

pregnancy induced hypertension patients as compared to normal pregnant

women. Journal of clinical and diagnostic research: JCDR, 9(7), BC05.

Soca, P. E. M., Ojeda, L. S., & del Toro, I. C. Dyslipidemia in preeclampsia

syndrome. Santiago, 175, 6.

198

Speer, P. D., Powers, R. W., Frank, M. P., Harger, G., Markovic, N., &

Roberts, J. M. (2008). Elevated asymmetric dimethylarginine concentrations

precede clinical preeclampsia, but not pregnancies with small-for-gestational-

age infants. American journal of obstetrics and gynecology, 198(1), 112-e1.

Spiteller, G. (2005). The relation of lipid peroxidation processes with

atherogenesis: a new theory on atherogenesis. Molecular nutrition & food

research, 49(11), 999-1013.

Sprague, A. H., & Khalil, R. A. (2009). Inflammatory cytokines in vascular

dysfunction and vascular disease. Biochemical pharmacology, 78(6), 539-552.

Stavrou, E., & McCrae, K. R. (2009). Immune thrombocytopenia in pregnancy.

Hematology/oncology clinics of North America, 23(6), 1299-1316.

Swellam, M., Samy, N., Abdl Wahab, S., & Ibrahim, M. S. (2009). Emerging

role of endothelial and inflammatory markers in preeclampsia. Disease

markers, 26(3), 127-133.

Tandon, V., Hiwale, S., Amle, D., Nagaria, T., & Patra, P. K. (2017).

Assessment of Serum Vascular Endothelial Growth Factor Levels in

Pregnancy-Induced Hypertension Patients. Journal of Pregnancy, 2017.

Tavana, T. Z., & Madadi, G. (2011). The relationship between maternal serum

highly sensitive C-reactive protein, leptin and hypertensive disorders of

pregnancy. Internet J Endocrinol, 6, 1.

199

Tayal, D., Goswami, B., Patra, S. K., Tripathi, R., & Khaneja, A. (2014).

Association of inflammatory cytokines, lipid peroxidation end products and

nitric oxide with the clinical severity and fetal outcome in preeclampsia in

Indian women. Indian Journal of Clinical Biochemistry, 29(2), 139-144.

Taylor, R. N., Varma, M., Teng, N. N., & Roberts, J. M. (1990). Women with

preeclampsia have higher plasma endothelin levels than women with normal

pregnancies. The Journal of Clinical Endocrinology & Metabolism, 71(6),

1675-1677.

Teran, E., Escudero, C., Moya, W., Flores, M., Vallance, P., &

Lopez‐Jaramillo, P. (2001). Elevated C‐reactive protein and pro‐inflammatory

cytokines in Andean women with pre‐eclampsia. International Journal of

Gynecology & Obstetrics, 75(3), 243-249.

Timur, H., Daglar, H. K., Kara, O., Kirbas, A., Inal, H. A., Turkmen, G. G., &

Uygur, D. (2016). A study of serum Apo A-1 and Apo B-100 levels in women

with preeclampsia. Pregnancy Hypertension: An International Journal of

Women's Cardiovascular Health, 6(2), 121-125.

Tosun, M., Celik, H., Avci, B., Yavuz, E., Alper, T., & Malatyalioğlu, E. (2010).

Maternal and umbilical serum levels of interleukin-6, interleukin-8, and tumor

necrosis factor-α in normal pregnancies and in pregnancies complicated by

preeclampsia. The Journal of Maternal-Fetal & Neonatal Medicine, 23(8), 880-

886.

200

Trivedi, D. J., Trivedi, C. D., & Sagre, A. (2013). Oxidant and antioxidant

imbalance as root cause in preeclampsia. Transworld Medical Journal, 1(2),

33-6.

Troisi, R., Braekke, K., Harsem, N. K., Hyer, M., Hoover, R. N., & Staff, A. C.

(2008). Blood pressure augmentation and maternal circulating concentrations

of angiogenic factors at delivery in preeclamptic and uncomplicated

pregnancies. American journal of obstetrics and gynecology, 199(6), 653-e1.

Udenze, I., Amadi, C., Awolola, N., & Makwe, C. C. (2015). The role of

cytokines as inflammatory mediators in preeclampsia. Pan African Medical

Journal, 20(1).

Vahid Roudsari, F., Ayati, S., Ayatollahi, H., Esmaeily, H., Hasanzadeh, M.,

Shahabian, M., & Pour Ali, L. (2009). Comparison of maternal serum Tumor

Necrosis Factor-alpha (TNF-α) in severe and mild preeclampsia versus

normal pregnancy. International Journal of Reproductive BioMedicine, 7(4),

153-158.

Van Asselt, K., Gudmundsson, S., Lindqvist, P., & Marsal, K. (1998). Uterine

and umbilical artery velocimetry in pre-eclampsia. Acta obstetricia et

gynecologica Scandinavica, 77(6), 614-614.

Var, A., Yildirim, Y., Onur, E., Kuscu, N. K., Uyanik, B. S., Goktalay, K., &

Guvenc, Y. (2003). Endothelial dysfunction in preeclampsia. Gynecologic and

obstetric investigation, 56(4), 221-224.

201

Varughese, B., Bhatla, N., Kumar, R., Dwivedi, S. N., & Dhingra, R. (2010).

Circulating angiogenic factors in pregnancies complicated by pre-eclampsia.

Vata, P. K., Chauhan, N. M., Nallathambi, A., & Hussein, F. (2015).

Assessment of prevalence of preeclampsia from Dilla region of Ethiopia. BMC

research notes, 8(1), 816.

Van den Oever, I. A., Raterman, H. G., Nurmohamed, M. T., & Simsek, S.

(2010). Endothelial dysfunction, inflammation, and apoptosis in diabetes

mellitus. Mediators of inflammation, 2010.

Visser, M. E., Akdim, F., Tribble, D. L., Nederveen, A. J., Kwoh, T. J.,

Kastelein, J. J., & Stroes, E. S. (2010). Effect of apolipoprotein-B synthesis

inhibition on liver triglyceride content in patients with familial

hypercholesterolemia. Journal of lipid research, 51(5), 1057-1062.

Visser, M. E., Kastelein, J. J., & Stroes, E. S. (2010). Apolipoprotein B

synthesis inhibition: results from clinical trials. Current opinion in lipidology,

21(4), 319-323.

Vitoratos, N., Economou, E., Iavazzo, C., Panoulis, K., & Creatsas, G. (2010).

Maternal serum levels of TNF-alpha and IL-6 long after delivery in

preeclamptic and normotensive pregnant women. Mediators of inflammation,

2010.

Vonnahme, K. A., Wilson, M. E., Li, Y., Rupnow, H. L., Phernetton, T. M.,

Ford, S. P., & Magness, R. R. (2005). Circulating levels of nitric oxide and

202

vascular endothelial growth factor throughout ovine pregnancy. The Journal of

physiology, 565(1), 101-109.

Von Dadelszen, P., Magee, L. A., Lee, S. K., Stewart, S. D., Simone, C.,

Koren, G., ... & Russell, J. A. (2002). Activated protein C in normal human

pregnancy and pregnancies complicated by severe preeclampsia: a

therapeutic opportunity?. Critical care medicine, 30(8), 1883-1892.

Walldius, G. (2012). The apoB/apoA-I ratio is a strong predictor of

cardiovascular risk. lipoproteins in health and diseases, 95-148.

Walldius, G., & Jungner, I. (2004). Apolipoprotein B and apolipoprotein A‐I:

risk indicators of coronary heart disease and targets for lipid‐modifying

therapy. Journal of internal medicine, 255(2), 188-205.

Walsh, S. W., & Parisi, V. M. (1986). The role of arachidonic acid metabolites

in preeclampsia. Semin Perinatol, 10(4), 334-355.

Wang, A., Rana, S., & Karumanchi, S. A. (2009). Preeclampsia: the role of

angiogenic factors in its pathogenesis. Physiology, 24(3), 147-158.

Wang, J., Mimuro, S., Lahoud, R., & Trudingera, B. (1998). Elevated levels of

lipoprotein (a) in women with preeclampsia. American journal of obstetrics and

gynecology, 178(1), 146-149.

Wang, Y., Walsh, S. W., Guo, J., & Zhang, J. (1991). Maternal levels of

prostacyclin, thromboxane, vitamin E, and lipid peroxides throughout normal

203

pregnancy. American journal of obstetrics and gynecology, 165(6), 1690-

1694.

Wang, Y., Gu, Y., Zhang, Y., & Lewis, D. F. (2004). Evidence of endothelial

dysfunction in preeclampsia: decreased endothelial nitric oxide synthase

expression is associated with increased cell permeability in endothelial cells

from preeclampsia. American journal of obstetrics and gynecology, 190(3),

817-824.

Warnick, G. R., Knopp, R. H., Fitzpatrick, V., & Branson, L. (1990). Estimating

low-density lipoprotein cholesterol by the Friedewald equation is adequate for

classifying patients on the basis of nationally recommended cutpoints. Clinical

Chemistry, 36(1), 15-19.

Watanabe, K., Naruse, K., Tanaka, K., Metoki, H., & Suzuki, Y. (2013). Outline

of definition and classification of ―pregnancy induced hypertension (PIH)‖.

Hypertension Research in Pregnancy, 1(1), 3-4.

Wellen, K. E., & Hotamisligil, G. S. (2005). Inflammation, stress, and diabetes.

The Journal of clinical investigation, 115(5), 1111-1119.

Werner, S., & Grose, R. (2003). Regulation of wound healing by growth

factors and cytokines. Physiological reviews, 83(3), 835-870.

WHO Study Group on the Hypertensive Disorders of Pregnancy. (1987). The

hypertensive disorders of pregnancy: report of a WHO study group (Vol. 758).

World Health Organization.

204

Williams, P., Robinson, D., & Bailey, A. (1979). High-density lipoprotein and

coronary risk factors in normal men. The Lancet, 313(8107), 72-75.

Winkler, K., Wetzka, B., Hoffmann, M. M., Friedrich, I., Kinner, M., Baumstark,

M. W., & Marz, W. (2003). Triglyceride-rich lipoproteins are associated with

hypertension in preeclampsia. The Journal of Clinical Endocrinology &

Metabolism, 88(3), 1162-1166.

Wisdom, S. J., Wilson, R., McKillop, J. H., & Walker, J. J. (1991). Antioxidant

systems in normal pregnancy and in pregnancy-induced hypertension.

American journal of obstetrics and gynecology, 165(6), 1701-1704.

Wittmaack, F. M., Gåfvels, M. E., Bronner, M., Matsuo, H., McCrae, K. R.,

Tomaszewski, J. E., & Strauss 3rd, J. F. (1995). Localization and regulation

of the human very low density lipoprotein/apolipoprotein-E receptor:

trophoblast expression predicts a role for the receptor in placental lipid

transport. Endocrinology, 136(1), 340-348.

Wu, Y., Lu, J., Antony, S., Juhasz, A., Liu, H., Jiang, G., & Roy, K. (2013).

Activation of TLR4 is required for the synergistic induction of dual oxidase 2

and dual oxidase A2 by IFN-γ and lipopolysaccharide in human pancreatic

cancer cell lines. The Journal of Immunology, 190(4), 1859-1872.

Westgard, J. O., & Barry, P. L. (1986). Cost-effective quality control: managing

the quality and productivity of analytical processes. AACC press.

205

Xiao, J. P., Yin, Y. X., Gao, Y. F., Lau, S., Shen, F., Zhao, M., & Chen, Q.

(2012). The increased maternal serum levels of IL-6 are associated with the

severity and onset of preeclampsia. Cytokine, 60(3), 856-860.

Yadav, K., Aggarwal, S., & Verma, K. (2014). Serum βhCG and lipid profile in

early second trimester as predictors of pregnancy-induced hypertension. The

Journal of Obstetrics and Gynecology of India, 64(3), 169-174.

Yadav, S. (2013). Serum lipid profile in early pregnancy as a predictor of

preeclampsia. International Journal of Medical Research and Review, 1(02).

Yang, R. L., Shi, Y. H., Hao, G., Li, W., & Le, G. W. (2008). Increasing

oxidative stress with progressive hyperlipidemia in human: relation between

malondialdehyde and atherogenic index. Journal of clinical biochemistry and

nutrition, 43(3), 154-158.

Yao, G., Zhang, Q., Doeppner, T. R., Niu, F., Li, Q., Yang, Y., & Dai, Y.

(2015). LDL suppresses angiogenesis through disruption of the HIF pathway

via NF-κB inhibition which is reversed by the proteasome inhibitor BSc2118.

Oncotarget, 6(30), 30251.

Yoshioka, T., Ando, M., Taniguchi, K., Yamasaki, F., & Motoyama, H. (1990).

Lipoperoxidation and antioxidant substances in the human placenta during

gestation. Nihon Sanka Fujinka Gakkai Zasshi, 42(12), 1634-1640.

206

Yuan, H. T., Haig, D., & Karumanchi, S. A. (2005). Angiogenic factors in the

pathogenesis of preeclampsia. Current topics in developmental biology, 71,

297-312.

Zechariah, A., ElAli, A., Hagemann, N., Jin, F., Doeppner, T. R., Helfrich, I., &

Hermann, D. M. (2013). Hyperlipidemia Attenuates Vascular Endothelial

Growth Factor–Induced Angiogenesis, Impairs Cerebral Blood Flow, and

Disturbs Stroke Recovery via Decreased Pericyte Coverage of Brain

Endothelial CellsSignificance. Arteriosclerosis, thrombosis, and vascular

biology, 33(7), 1561-1567.

Zeisler, H., Llurba, E., Chantraine, F., Vatish, M., Staff, A. C., Sennström, M.,&

Dilba, P. (2016). Predictive value of the sFlt-1: PlGF ratio in women with

suspected preeclampsia. New England Journal of Medicine, 374(1), 13-22.

Zhang, J. M., & An, J. (2007). Cytokines, inflammation and pain. International

anesthesiology clinics, 45(2), 27.

Zhou, Q., Liu, H., Qiao, F., Wu, Y., & Xu, J. (2010). VEGF deficit is involved in

endothelium dysfunction in preeclampsia. Journal of Huazhong University of

Science and Technology [Medical Sciences], 30(3), 370-374.

Zhou, R., Tardivel, A., Thorens, B., Choi, I., & Tschopp, J. (2010).

Thioredoxin-interacting protein links oxidative stress to inflammasome

activation. Nature immunology, 11(2), 136-140.

207

Zhou, R., Yazdi, A. S., Menu, P., & Tschopp, J. (2011). A role for mitochondria

in NLRP3 inflammasome activation. Nature, 469(7329), 221-225.

Ziegenhagen, G., & Drahovsky, D. (1983). Klinische bedeutung des c-

reaktiven proteins. Med klin, 78, 24-35.

208

12. PUBLICATIONS

Jammalamadaga, V. S., Abraham, P., & Sivaprasad, P. (2016).

ApoB/ApoA-1 ratio and nitric oxide levels in pregnancy induced

hypertensive women. International Journal of Research in

Medical Sciences, 4(5), 1329-1334.

Jammalamadaga, V. S., & Abraham, P. (2016). Spectrum of

Factors Triggering Endothelial Dysfunction in PIH. Journal of

Clinical & Diagnostic Research, 10(12).

209

210

211

PATIENT PROFORMA

NAME

ADDRESS

DATE

HOSPITAL:IP/OP NO:

AGE

SEX

OCCUPATION

BLOOD GROUP

HEIGHT

WEIGHT

BMI

BLOOD PRESSURE (SBP/DBP)

MARRIED AGE

GESTATIONAL AGE (WEEKS)

NO: OF PRENATAL VISITS

PARITY

GRAVIDITY

EXPECTING TWINS OR TRIPLETS

FAMILY HISTORY OF PIH

H/O BP

H/O PIH

H/O DIABETES

212

H/O DYSLIPIDEMIA

H/O RENAL DISORDERS

H/O ABORTIONS

H/O CARDIOVASCULAR DISEASES

213

CONSENT FORM

உறுதிமொறி படிலம

NAME: AGE/SEX :

HOSPITAL NO.OP NO: ID NO :

ADDRESS:

I,______________________________ am given to understand that I have the change of

developing vascular complication problems. Which need to be studied for the benefit of

improving scientific knowledge . I Here by agree to give my blood and urine sample for bio

chemical analysis. It was clearly explained to me that my identity shall not be disclosed. With

this condition, I have no objection to the usage of the data obtained from the experiments

performed with my blood and urine sample for research purpose. I am giving this consent

whole heartedly without any pressure or compulsion.

நொன , ___________________________________ எனககு இதத நொரம மதொடரபொன

ககொரொறுகள உருலொக லொயபபுளரதொகவும, அதனன கலும அமிநது ஆொயநதொல

அமிலில லரரசசிககு உதவும எனவும புொிநதுமகொணகடன. உிொிகலதில ஆொயசசிககொக

நொன எனது இததம றறும சிறுநர மகொடுகக லிருபபம மதொிலிககிகமன எவலிதததிலும என

சுலிலஙகள மலரிலொது எனறு மதரிலொக எடுததுனககபபடடுளரது. இதன

அடிபபனடில எனது இததம றறும சிறுநொில கணடமிபபடும லிலஙகனர

ஆொயசசிககொக பனபடுததிகமகொளர லிருபபம மதொிலிககிகமன. எவலித நிறபநதமும

லறபுறுததலும, இலயொல இதறகு நொன ஒபபுதல அரிககிகமன.

கததி :

கநொொரிின மபர :

கநொொரிின னகமொபபம :

சொடசிின மபர :

சொடசிின னகமொபபம :