Mannose Binding Lectin as a Diagnostic Marker for Coronary Artery Disease in Hypertensive Patients

MANNOSE BINDING LECTIN AS A DIAGNOSTIC

MARKER FOR CORONARY ARTERY DISEASE IN

HYPERTENSIVE PATIENTS

Dissertation submitted for

M.D. BIOCHEMISTRY BRANCH – XIII

DEGREE EXAMINATION

THE TAMILNADU DR.M.G.R.MEDICAL UNIVERSITY

CHENNAI – 600 032

TAMILNADU

BONAFIDE CERTIFICATE

This is to certify that this dissertation work entitled “MANNOSE BINDING

LECTIN AS A DIAGNOSTIC MARKER FOR CORONARYARTERY DISEASE IN HYPERTENSIVE PATIENTS” is the original bonafide work done by

DR.M.DILAGAMARI, Post Graduate Student, Institute of Biochemistry,

Madras Medical College, Chennai under our direct supervisionand guidance.

Prof. Dr. V.Amuthavalli, MD., (Guide)

Professor,

Institute of Biochemistry Madras Medical College, Chennai – 600003.

Prof. Dr. K.Ramadevi. MD.,Ph.D Director & Professor,

Institute of Biochemistry Madras Medical College Chennai-600 003.

Dean

Madras Medical College and

DECLARATION

I, Dr. M. DILAGAMARI, Post Graduate , Institute of Biochemistry, Madras Medical College, solemnly declare that the dissertation titled

“MANNOSE BINDING LECTIN AS A DIAGNOSTIC MARKER FOR CORONARY ARTERY DISEASE IN HYPERTENSIVE PATIENTS” is the

bonafide work done by me at Institute of Biochemistry, Madras Medical College

under the expert guidance and supervision of Prof. Dr.V.AMUTHAVALLI,

M.D., Institute of Biochemistry, Madras Medical College. The dissertation is

submitted to the Tamil Nadu Dr. M.G.R Medical University towards partial

fulfillment of requirement for the award of M.D., Degree (Branch XIII) in

Biochemistry.

Place: Chennai

SPECIAL ACKNOWLEDGEMENT

The author gratefully acknowledges and sincerely thanks

Dr.R .JAYANTHI ,MD, FRCP (Glasg ) , Dean, Madras Medical College and Rajiv Gandhi Government General Hospital, Chennai, for granting her

ACKNOWLEDGEMENT

The author expresses her warmest respects and profound gratitude to

Dr. K. Ramadevi, M.D.,Ph.D., Director and Professor, Institute of Biochemistry, Madras Medical College, Chennai, for her able guidance, constant

encouragement, support and valuable time but for which this dissertation could

not have been made possible.

The author expresses her heartfelt gratitude to her guide and supervisor

Dr.V.Amuthavalli, M.D., Professor, Institute of Biochemistry, Madras Medical College, Chennai, for her constant and valuable guidance, unfailing support,

encouragement and inspiration throughout the period of her study.

The author in particular, is extremely thankful to Prof. Dr. S.Mayilvahanan

M.D., Director of General Medicine & Professor Dr.N.Swaminathan M.D.,DM,

Director of Cardiology, Rajiv Gandhi Government General Hospital, Chennai, for

granting permission to obtain blood samples from the patients.

The author expresses her sincere gratitude to the Professors Dr.R.Chitraa

M.D., Dr.K.Pramila M.D., Dr.Sumathy.S. M.D. & Dr. Chelladurai, M.D,

Institute of biochemistry, Madras Medical College, for their guidance and support.

The author expresses her warm respects and sincere thanks to her co-guide,

Madras Medical College for her guidance regarding the practical issues of

research which is beyond the scope of textbooks.

The author expresses her respects and sincere thanks to all other Assistant

Professors, Institute ofBiochemistry, Madras Medical College, for their guidance and support.

The author expresses warm respects to the members of the Institutional

Ethics committee for approving the study.

The author is indebted to the patients and persons from whom blood

samples were collected for conducting the study.

The author expresses her special thanks to Biochemistry Laboratory Staff,

for their timely help and cooperation during sample collection.

The author expresses her special thanks to her colleagues for their

cooperation and genuine support.

The author gratefully acknowledges the help rendered by Mr. Albert, for

the statistical analysis of the study.

The author expresses her special thanks to her parents for the moral support

CONTENTS

SI.

NO TITLE PAGE No.

1 INTRODUCTION 1

2 REVIEW OF LITERATURE 3

3 AIMS & OBJECTIVES 45

4 MATERIALS & METHODS 46

5 RESULTS 62

6 STATISTICAL ANALYSIS 63

7 DISCUSSION 79

8 SUMMARY & CONCLUSION 86

10 SCOPE FOR FURTHER STUDIES 87

11 BIBLIOGRAPHY 88

ABBREVIATIONS

CAD - Coronary artery disease

NFK - Nuclear factor kappa.

SRB - Scavenger rceptor class B

VCAM - Vascular cell adhesion molecule.

ICAM - Intercellular adhesion molecule.

PG - Prostaglandin

CRP - C Reactive protein.

hs-CRP - High sensitive C Reactive protein.

NO - Nitricoxide.

MBL - Mannose Binding lectin

MASP - MBL associated serine proteases

eNOS - Endothelial nitric oxide synthase

ROS - Reactive oxygen species

PDGF - Platelet derived growth factor

FGF - Fibroblast growth factor

TNF α - Tumour necrosis factor alpha

PeCAM - Pericellular cell adhesion molecule

BMI - Body Mass Index

TGL - Triglycerides

PAF-AH - Platelet Activating Factor – Acetyl Hydrolase

ER - Endoplamic reticulum

ANG II - Angiotensin II

MCP - Monocyte Chemoattractant Protein

M-CSF - Monocyte colony stimulating factor

IL - Interleukin

HDL - High density lipoprotein

LDL - Low density lipoprotein

CHD - Coronary Heart Disease

LVH - Left Ventricular Hypertrophy

HMG-CoA - 3-hydroxy-3-methyl glutaryl coenzyme A

ACAT - acylCoA:cholesterol acyl transferase

LCAT - lecithin:cholesterol acyl transferase

CRD - C- terminal carbohydrate recognition domain

CL - Collectin

I/R - Ischemia –reperfusion injury

C1-INH - C1esterase inhibitor

BOS - Bronchiolitis obliterans syndrome

SLE - Systemic Lupus Erythematosus

sMAP - small MBL associated protein

Map - MBL associated protein

DIC - Disseminated intravascular coagulation

MI - Myocardial Infarction

HT - Hypertension

1

INTRODUCTION

Cardiovascular disease is one of the leading cause of mortality and

morbidity worldwide. Epidemiological investigations pointed out that

hypertension is a powerful cardiovascular risk factor. Elevated blood pressure

levels have been found to be highly predictive of cardiovascular events including

ischemic coronary disease, stroke and peripheral arterial disease. Relationship

between elevated blood pressure and risk of cardiovascular events is continuous,

consistent and independent of other risk factors(1).

The cardiovascular events have a predominant vascular origin resulting

from atherosclerosis. Factors contributing to atherosclerosis are multiple and

complex. Multiple theories including role of dyslipidemia , hypercoagulability ,

oxidative stress, inflammation, endothelial dysfunction have been put forth.

Inflammation plays a major role in all phases of atherogenesis from plaque

initiation to plaque rupture(2). Several inflammatory markers such as C-reactive protein (CRP), Secretory phospholipase A2, interleukin (IL)-6 have been

associated with increased risk of atherosclerosis(3,4).In addition , the markers of

innate immunity have been shown to predict the development of coronary artery

disease.

Mannose - binding lectin (MBL) also known as Mannan – binding lectin or

Mannan – (Mannose - ) binding protein (MBP) is an important component of the

2

associated serine proteases (MASPs) has the ability to activate complement via

the lectin pathway. Since innate immunity has been implicated in atherogenesis ,

MBL has been suggested to play a role in the formation of atherosclerotic

plaque(5,6).

However studies examining the relations between serum levels of MBL

and coronary artery disease risk have reported equivocal results.At present , no

conclusive data are available about the relationship between serum MBL levels

and coronary artery disease risk in hypertensive patients. Hence it is proposed to

study the association of serum levels of MBL in the development of coronary

artery disease in hypertensive patients .

HYPOTHESIS

MBL is an important component of innate immune system. Elevated serum

MBL levels are associated with increased risk of future coronary artery disease in

3

REVIEW OF LITERATURE

Hypertensive patients are more prone for cardiovascular disease. MBL

being a component of innate immune system can be used as a marker of

cardiovascular risk in hypertension. This study was conducted in 90 patients to

study the correlation between the serum MBL levels and other risk factors of

coronary artery disease and hypertension.

HYPERTENSION:

Hypertension is one of the most common chronic disease. In India the

prevalence rate is 29.8% . About 23% of men and 22.6% of women above 25

years suffer from hypertension .Coronary Artery Disease is a leading cause of

morbidity and mortality in hypertensive patients.

Hypertension is a multifactorial disorder that involves pathological changes

in neuronal, renal, genetic, hormonal and vascular mechanism. Genetic alterations

may initiate the cascade to permanent hypertension. Genetic contribution have

been estimated to range from 30% to 60%. Polymorphisms of certain genes

involving rennin – angiotensin system , aldosterone synthesis and adrenergic

receptors are more common in hypertensives than normotensives(7).

Hypertension is known to act on the arterial wall to promote both vascular

remodelling and atherosclerosis resulting in diminished arterial wall compliance

and elevated stiffness. Clinical and experimental investigations have shown that

4

AETIOLOGY:

It is of two types;

1. Primary / Idiopathic / Essential

About 90 -95% of hypertension is idiopathic .The pathogenesis of essential

hypertension is not clearly understood. Different investigators have proposed that

the kidney, the peripheral resistant vessels and sympathetic nervous system as the

seat of primary abnormality.

2. Secondary causes

Of the remaining 5 to 10% is secondary to renal disease or less often to

narrowing of renal artery ,usually by an atheromatous plaque (Renal vascular

hypertension). Infrequently , secondary hypertension is the result of adrenal

disorders such as primary aldosteronism, Cushing’s syndrome and

pheochromocytoma.(9)

Regulation of Normal Blood Pressure:

The magnitude of the arterial pressure depends on two fundamental

hemodynamic variables: Cardiac Output and Total Peripheral Vascular

Resistance.

BLOOD PRESSURE = CARDIAC OUTPUT X PERIPHERAL RESISTANCE

Indeed, the blood pressure level is a complex trait that is determined by the

interaction of multiple genetic ,environmental , and demographic factors that

5

Cardiac output is highly dependent on blood volume which itself is greatly

influenced by the whole body sodium homeostasis. Peripheral vascular resistance

is determined mainly at the level of arterioles and is affected by neural and

hormonal factors.

Pathogenesis of Hypertension:

The multiple mechanism of hypertension constitute aberrations of the

normal physiological regulation of blood pressure. Arterial hypertension occurs

when the relationship between cardiac output and total peripheral resistance is

altered.

Genetics of Hypertension :

In studies of twins and family members in which the degree of familial

aggregation of blood pressure levels is compared with the closeness of genetic

sharing, the contributions have been estimated to range from 30 to 60 percent.(7) Single gene disorders cause relatively rare and severe form of hypertension

eg., Gene defects in enzymes involved in aldosterone metabolism and mutations

in proteins like α - adducin that affect sodium reabsorption.

Inherited variations in blood pressure may also depend on the cumulative

effects of allelic forms of several genes that affect blood pressure.eg., variations in

6

Reduced renal sodium excretion in the presence of normal arterial pressure

may well be the key initiating event in essential hypertension and a final common

pathway for the pathogenesis of hypertension. Decreased sodium excretion

might lead sequentially to an increase in fluid volume, increased cardiac output,

and peripheral vascular constriction , thereby elevating blood pressure.(Fig.1)

Figure 1 : Scheme of pathogenesis of essential hypertension(9)

An alternative hypothesis implicates vasoconstrictive influences either the

factors that induce functional vasoconstriction or stimuli that induce direct

7

Environmental factors :

Environmental factors could modify expression of the genetic determinants

of increased pressure.

 Stress

 Obesity

 Smoking

 Physical inactivity

 Increased intake of salt

have all been implicated as exogenous factors in hypertension.

Essential hypertension results from the combined effect of mutations or

polymorphisms at several gene loci that influence blood pressure, interacting with

a variety of environmental factors. Thus, environmental factors affect the

variables that control blood pressure in the genetically predisposed individual.

Hypertension and Oxidative stress :

Hypertension is considered as a state of oxidative stress(10)

Excessive production of ROS (Reactive Oxygen Species), outstripping

antioxidant mechanism, decreased bioavailability of NO (Nitric Oxide) in the

vasculature and kidneys and ROS mediated cardiovascular remodelling plays an

important pathophysiological role in development of hypertension. Biomarkers of

systemic oxidative stress such as isoprostanes, malondialdehyde and nitrotyrosine

8

Increased ROS bioactivity leads to endothelial dysfunction , increased

contractility, vascular smooth muscle cell growth, monocyte invasion, lipid

peroxidation, inflammation and increased deposition of extracellular matrix

proteins(11), important factors in hypertension mediated vascular damage (Fig.2). An excessive activity of rennin – angiotensin - aldosterone system leads

to the production of ROS and also contributes to endothelial dysfunction, vascular

inflammation and thrombosis.(12,13)

NO and Hypertension:

Impaired endothelium – mediated vasodilation in hypertension has been

linked to decreased NO bioavailability. This may be secondary to decreased NO

synthesis or to increased NO degradation because of its interaction with

superoxide anion (O2

-

) to form peroxynitrite (ONOO- ).

9

Dyslipidemia and Hypertension :

Recent experimental data suggests marked similarities between the effects

of hypertension and hypercholesterolemia on the arterial intima. Both conditions

exert proinflammatory effects on the artery, resulting in the recruitment of

monocytes into the intima. These effects may be due to the production of free

radicals , which in turn stimulate the genes involved in the recruitment of

inflammatory cells into the arterial wall(15)

Endothelial dysfunction is in cross road between hypertension and

dyslipidemia. The key feature in this disorder is the reduced availability of nitric

oxide (NO) due to both decrease in synthesis as well as enhanced degradation.

Hypercholestrolemia plays an important role, as oxidised LDL diminishes the

expression of endothelial NO synthase(12)

Role of complement in hypertension:

Hypertension and hypertensive end organ damage are not only mediated by

hemodynamic but also by innate and adaptive immune responses. Complement

activation may drive the pathology of hypertension through its impact on innate

immunity. Inflammatory responses mediate the development of perivascular

fibrosis and heart dysfunction induced by hypertension. In hypertension induced

cardiac damage, the most important component is the inflammatory

microenvironment including T cells, macrophages, neutrophils, and the

chemokines and cytokines released from these cells which could affect the degree

10

The role of C5a in hypertension has recently been examined. Zhang et

al.(17) reported increased levels of C5a in humans with high blood pressure. Infusion of ANG II-causing arterial hypertension lead to increased systemic

anaphylatoxin generation in mice. C5aR1-deficient mice exhibited markedly

reduced cardiac remodeling and inflammation after ANG II infusion.

Hypertension stimulates structural arterial remodeling, which is

characterized by vascular smooth muscle cell (VSMC) hyperplasia and infiltration

of inflammatory cells. Recent findings demonstrate a key role for complement

C1q induced activation of β-catenin signalling is implicated in VSMC

proliferation during intimal thickening after vascular injury . β-catenin signalling pathway regulates the proliferation and differentiation of smooth muscle cells.

Complement activation causes hypertensive arterial remodelling.(18)

CORONARY ARTERY DISEASE:

Coronary artery disease has been defined as impairment of function of

heart due to inadequate blood supply to the heart compared to its needs. Its being

a multifactorial disease has a complex etiology. Many genetic and environmental

factors act in combination to determine an individual’s risk of developing

coronary artery disease.(19) A large number of studies such as The Framingham heart study(20), The lipid research clinic’s coronary primary prevention trial, The Helsenki heart study, have been conducted to examine the role of risk factors for

11

The risk factors identified by these epidemiological studies include;

Epidemiological risk factor

1. Positive family history

2. Age

3. Gender

4. Cigarette smoking

5. Hypertension

6. Hyperlipidemia

7. Coronary atherosclerosis prone personality

8. Obesity

9. Diabetes Mellitus

Pathological risk factor

1. Atherosclerosis

2. Platelet dysfunction

3. Endothelial dysfunction

Biochemical risk factors

1. Total cholesterol > 240 mg / dL

2. HDL < 35 mg / dL

12

ATHEROSCLEROSIS:

Atherosclerosis is a disease affecting arterial blood vessels. It is a chronic

inflammatory response in the walls of arteries, in large part due to the

accumulation of macrophages promoted by low density lipoproteins (LDL)

without adequate removal of fats and cholesterol from the macrophages by

functional high density lipoproteins (HDL). It is commonly referred to as a

“hardening” or “ furring” of the arteries.(21)

The lesions of atherosclerosis occur principally within the innermost layer

of the artery wall, the intima.(22) They include

 Fatty streak

 Fibrous plaque

 Complicated lesion

 Plaque disruption

 Atherothrombosis

Fatty streak

The process of atherogenesis begins in childhood with the development of

lipid rich lesions called fatty streaks. They are also found to contain macrophages,

T lymphocytes , smooth muscle cells – each of these cells are found to contain

deposits of cholesterol and cholesterol oleate. The lesions are yellowish and

sessile in appearance and they cause little or no obstruction of the affected artery

13

Fibrous plaque

The fibrous plaques are derived from fatty streaks that continue the process

of cell proliferation , lipid accumulation , and connective tissue formation and the

deep core of lipid and cell debris results from inadequate blood supply,

inflammation, and cell necrosis.

There is a lesion that is accepted as a forerunner of the fibrous plaque –

that is known as fibromusculoelastic or intermediate lesion of the intima, which

consists of proliferated smooth muscle cells surrounded by connective tissue and

contains little or no lipid.

A fully blown fibrous plaque consists of numerous smooth muscle cells

surrounded by connective tissue matrix often intermixed with numerous

macrophages. This cap covers a deep layer of macrophages filled with lipid that

are often intermixed with variable number of T lymphocytes.

Advanced lesions – Plaque disruption and Atherothrombosis

The typical advanced, complicated lesion contains a large necrotic core

with a fibrous core, loaded with macrophages . The macrophages can form

numerous proteolytic enzymes, including metalloproteinases – these enzymes

cause the removal of fibrous cap – thus plaque disruption is found to happen at the

shoulder of the lesion where the cap is thin and concentration of macrophages is

the greatest. The plaque disruption allow the lesion to get involved in thrombotic

14

Two major effects of atherosclerosis are

1. The atheromatous plaques , though compensated by artery enlargement,

eventually lead to plaque rupture and stenosis of the artery and therefore an

insufficient blood supply to the organ it feeds

2. If the compensating artery enlargement process is excessive , then a net

aneurysm results.

These complications are chronic, slowly progressive and cumulative. Most

commonly , soft plaque suddenly ruptures , causing the formation of a thrombus

that will rapidly slow or stop blood flow, leading to death of the tissues fed by the

artery in approximately 5 minutes. This catastrophic event is called an infarction.

The clinical scenarios of this catastrophic event depend on which artery is

affected.

1. Coronary artery – Myocardial Infarction

2. Carotid artery and its branches – Stroke or Transient Ischemic attack

3. Peripheral artery disease

Theories of Atherogenesis:

Long ER et al. has discussed the development of clinicopathological

correlations that evolved during the era when autopsy examination permitted the

formulation of a hypothesis relating the degree of atherosclerosis to the incidence

15

Virchow proposed the idea that some form of injury to the arterial wall

associated with the inflammatory response resulted in the degenerative lesion of

atherosclerosis(25). This idea was subsequently modified by Anitschkow (26) and further included the role of platelets and thrombogenesis in atherosclerosis as

expanded by Duguid(27).

John French noted that the structural integrity of endothelial lining of the

artery represented a key element in the maintenance of normal arterial function(28)

and that alteration in endothelial integrity might precede a sequence of events that

would lead to the various forms of the lesions of atherosclerosis.

Response to endothelial injury:

In normal artery , the endothelial cells form a continuous monolayer that

regulates the passage of substances from the plasma to the underlying wall(29,30), forms a thromboresistance surface that promotes the continuous flow of blood

throughout the vascular tree. Production of certain cytokines by the endothelial

cells regulates the migration and proliferation of smooth muscle cells .

Endothelial cells are capable of transporting plasma lipoproteins of into the

arterial wall (31,32). The endothelium exhibits thromboresistant characters by production of three factors

1. Surface glycoproteins and proteoglycans

2. Prostacyclin

16

Prostacyclins and nitric oxide are potent vasodilatory agents and inhibitors

of platelet aggregation. Injury to the endothelium results in structural and

functional alterations in the endothelial cells, in such a way that they would permit

plasma constituents such as lipoproteins and inflammatory cells to have a ready

access to the arterial wall(33).

Leukocyte adhesion:

The endothelial dysfunction is associated with overexpression of E, L, P

selectin that appear to play a role in inducing rolling and attachment of monocytes

and T lymphocytes to endothelium. This rolling is facilitated by the upregulation

of ICAM 1 and VCAM 1 also. PeCAM 1 , another molecule formed by

endothelium has been involved in interendothelial migration by the adherent

leukocyte into the subendothelial space or intima of the artery. Thus, the earliest

phase of chronic inflammatory response is represented by leukocyte adhesion due

to the formation of these attachment and adherence molecules on the surface of

the endothelium and the leukocytes(34–36).

Transmigration of lipoproteins:

Second event accompanying endothelial dysfunction is transmigration of

lipoproteins particularly LDL particles, this transmigration places the LDL in the

subendothelial space which is virtually devoid of any antioxidant properties of the

circulation , hence it gets oxidised. Oxidised LDL can act as one of the

chemotactic factor and can also induce the endothelial cells and the underlying

17

Chemoattractant Protein 1 ( MCP – 1 ) and a colony stimulating factor (M - CSF).

In this scenario , the monocyte gets activated to macrophages , which express

SR-B1 causing unregulated uptake of LDL particles , forming foam cells. Such a

lesion with foam cells ,activated inflammatory cells is called as fatty streak.

Formation of stable atherosclerotic plaque:

Oxidised LDL, foam cells, the activated macrophages, T- cells produce

various cytokines like IL-1 and TNF-α . Under the influence of these cytokines,

endothelium, macrophages, and T cells produce PDGF and FGF(37).

 PDGF stimulates smooth muscle cell migration and proliferation.

 FGF stimulates vascular smooth muscle cell to produce collagen and the

various components of extracellular matrix together they form the fibrous

cap.

 TNF – α induces apoptosis of foam cells causing exocytosis of its lipid

content, which forms the lipid core.

The lesion with lipid core , surrounded by activated T-cells, macrophages,

platelets lined by a fibrous cap is called as a stable atherosclerotic plaque.

Thus oxidised LDL is not only toxic to the endothelium and the

surrounding cells in the intima but also chemotactic for monocytes and can

activate monocyte derived macrophages to produce growth factors and cytokines.

18

If the injury to the endothelium were a self – limited event and the

endothelial functions were restored , the proliferative lesions may regress and

would be clinically silent. If the injury at focal sites in the artery wall is of long

standing, the lesion would continue to progress .

The atherosclerotic plaque does not only have smooth muscle cells but also

macrophages , which are capable of producing metalloproteinases and TNF α , both of which cause necrosis and digestion of fibrous cap . The loss of fibrous cap

is called plaque rupture.

The plaque rupture exposes the subendothelial extracellular matrix to the

factors of coagulation in the circulation initiating the intrinsic pathway of

coagulation – this is responsible for atherothrombosis.

Effect of Hypertension on Atherosclerosis:

Elevated systolic and diastolic blood pressure have a strong, positive and

graded relationship to CHD (38,39). The risk imposed by hypertension is heightened

substantially when other risk factors are present.

Hypertension clusters with

 Insulin resistance

 Hyperinsulinemia

 Glucose Intolerance

19

 Left Ventricular Hypertrophy and

 Obesity

and occurs in isolation in fewer than 20% of individuals(40).

The potential mechanism by which hypertension may cause endothelial

dysfunction include

1. Increased endothelial permeability to lipoproteins,

2. Increased adherence of leukocytes,

3. Increased oxidative stress, and

4. Hemodynamic stress that may trigger acute plaque rupture,

All these are mediated by the activation of NF-kB pathway and

inactivation of eNOS enzyme.

Obesity:

Obesity promotes insulin resistance, hyperinsulinemia, hypertriglyceridemia,

low HDL cholesterol and LVH(41,42). Many observational studies have found that

obesity strongly and positively correlates with the risk of CHD in univariate

analysis. In multivariate analysis , when controlling statistically for risk factors

such as hypertension, diabetes, and dyslipidemia, obesity is not found to be an

20

21

The adverse consequences of obesity are mediated through resultant

metabolic risk factors acting as pathological links in the causal pathway. In

general the greater the degree of overweight , the higher the risk of coronary

mortality(44,45). The central distribution of body fat predicts CHD in men independently of body-mass index and other major risk factors(46). Weight loss improves insulin sensitivity and glucose disposal, reduces blood pressure,

triglycerides and LVH, and increases HDL cholesterol(41,42).

Dyslipidemia:

Plasma cholesterol may arise from the diet or from endogenous

biosynthesis. Cholesterol is transported between tissues in combination with

proteins and phospholipids as lipoproteins. Abnormal levels of plasma lipids is

called dyslipidemia.

Plasma lipoproteins :

Lipoproteins are composed of a neutral lipid core containing

triacylglycerol (TAG) and cholesteryl esters surrounded by a shell of amphipathic

apolipoproteins, phospholipids and unesterified cholesterol. The lipoprotein

particles include

1. Chylomicrons

2. Very low density lipoproteins ( VLDLs )

3. Low density lipoprotein ( LDLs )

22

Metabolism of Chylomicrons(47):

Chylomicrons are assembled in intestinal mucosal cells and carry dietary

TAG, cholesterol, fat soluble vitamins and cholesterol esters to the peripheral

tissues. TAGs account for 90% of lipids in chylomicron. Apo B-48, Apo C-II and

Apo E are the specific apolipoproteins of chylomicrons.

1) Intestinal mucosal cells secrete nascent TAG – rich chylomicrons produced

from dietary lipids.

2) Apo C-II and Apo E are transferred from HDL to the nascent chylomicron.

3) Extracellular lipoprotein lipase , activated by Apo C-II , degrades the TAG

in chylomicrons resulting in the formation of free fatty acids and glycerol.

Insulin enhances lipoprotein lipase synthesis and its translocation to the

luminal surface of capillary.

4) Apo C-II is returned to HDL forming chylomicron remnant.

5) CE – rich chylomicrons remnants bind through apo E to specific receptors

on the liver and are endocytosed.

6) The cholesteryl esters and leftover TGL are metabolised by hepatic lipase.

Metabolism of VLDL and LDL(47):

They are produced in liver. VLDL composed predominantly of endogenous

TAG and their function is to carry this lipid from liver to the peripheral

tissue.LDL particles composed of high concentration of cholesterol and

cholesteryl esters. Their primary function is to provide cholesterol to the

23

1. Liver secretes nascent , TAG – rich VLDL particles.

2. Apo C-II and Apo E are transferred from HDL to the nascent VLDL.

3. Extracellular lipoprotein lipase , activated by Apo C-II, degrades the TAG in

VLDL resulting in the formation of free fatty acids, glycerol and VLDL

remnant. This action of lipoprotein lipase decreases the lipid content of both

Chylomicrons and VLDL by 70-90%. VLDL remnants are also called as IDL.

4. Most of the IDL undergoes conversion to LDL after hydrolysis of all TGL.

LDL principally contains cholesterol and cholesterol esters.

5. LDL binds to LDL receptors specific for Apo B-100 on hepatic and

extrahepatic tissues and are endocytosed.

Regulation of LDL receptor:

LDL (apoB-100, apo-E) receptors are cell surface receptors that are coated

by a protein called clathrin on the cytosolic side of the membrane. It is a

glycoprotein receptor. It spans the membrane with B-100 binding domain at the

extracellularly exposed amino terminal. After binding, LDL is endocytosed. The

endocytosed vesicle fuses with lysosome to form endosome with the receptor

being returned to the surface. The cholesterol esters and apoproteins are

hydrolysed and cholesterol is translocated into the cell. This cholesterol influx

downregulates the transcription of genes encoding HMGCoA synthase, HMGCoA

reductase and other enzymes of cholesterol synthetic pathway. It also inhibits

transcription of genes coding for LDL receptors. It stimulates ACAT enzyme

24

Metabolism of HDL(47)

HDL is synthesized in both liver and intestine.

1. Nascent HDL are disc shaped particles composed mainly of

phosphatidylcholine and apolipoproteins A-1, C and E.

2. They take up unesterified cholesterol with the help of ABCA1 ( ATP-

binding cassette transporters A ) from nonhepatic tissue.

3. Cholesterol is esterified by LCAT ( lecithin: cholesterol acyltransferase )

bound to nascent HDL which is activated by Apo- A1.

4. CETP moves some of the cholesteryl esters from HDL to VLDL in

exchange for TAG, relieving the product inhibition of LCAT.

5. Cholesteryl esters rich HDL binds to liver leading to selective transfer of

cholesteryl esters into the cells mediated by SR-B1 ( scavenger receptor

class B type 1)

6. Then HDL transports cholesterol to liver where it is excreted through bile.

This is called reverse cholesterol transport.

Total Cholesterol and LDL cholesterol in atherosclerosis:

Numerous prospective studies have identified a continuous, graded and

direct relationship between serum cholesterol and CHD incidence(48). The level of

total cholesterol and LDL cholesterol interacts with other risk factors to multiply

25

Elevated LDL cholesterol levels appear to be involved in all stages of

atherogenesis – endothelial dysfunction, plaque formation and growth and plaque

instability and disruption. Elevated cholesterol levels in the plasma lead to an

increased retention of LDL particles in the arterial wall , their oxidation and the

secretion of various inflammatory mediators and chemoattractants(51).

LDL is also a potent mitogen for smooth muscle cells ; progressive growth

of atherosclerotic plaques with a large lipid core and numerous lipid filled

macrophages are prone to rupture(52). Thus the epidemiological evidence strongly

supports LDL- Cholesterol’s role in atherosclerosis.

Small dense LDL are felt to be more atherogenic(53). The two possible reasons for this are

1) When a person has more of small LDL particles, for a given cholesterol

content, the number of LDL particles will be more and an LDL receptor

can accept only one LDL particle at a time and hence the rate of

metabolism of LDL is decreased , causing accumulation of LDL in the

plasma.

2) The endothelium will be more permeable to small LDL particle when

compared to normal LDL.

Triglycerides:

The relationship between triglycerides and CHD has been less clear. In

men , univariate analysis have demonstrated a direct dose-response relationship.

26

HDL cholesterol , obesity and diabetes(54). Hypertriglyceridemia however has been found to be an independent risk factor in women(55).

Several mechanisms have been proposed to explain the triglyceride- CHD

association.

 Hypertriglyceridemia have a predominance of small , dense LDL particles

 Fasting hypertriglyceridemia may be a marker of exaggerated postprandial

hyperlipidemia, which may promote the uptake of atherogenic triglyceride

rich lipoprotein remnants by endothelial cells(56).

 Serum triglyceride levels are strongly related to fibrinogen and factor VII

in numerous epidemiological studies(57).

Therefore , a number of mechanisms act as direct and indirect link

serum triglycerides and CHD.

HDL cholesterol :

Numerous prospective epidemiological studies have demonstrated a

continuous, inverse relationship between HDL cholesterol levels and the

incidence of CHD. The total cholesterol to HDL cholesterol ratio is better

predictor of CHD than the HDL cholesterol level alone(58). Two important

mechanisms by which HDL is thought to play a protective role against

atherosclerosis are;

1. Reverse cholesterol transport and

27

Normal HDL contains several enzymes such as

o Paraoxonase ( PON )(60)

o Lecithin : cholesterol acyltransferase(61)

o Glutathione selenoperoxidase(62)

o Platelet activating factor – Acetyl Hydrolase ( PAF – AH )(63)

that can potentially prevent the formation of oxidized LDL and also

inactivate the inflammatory LDL-derived oxidized phospholipids.

MANNOSE BINDING LECTIN:

Mannose binding lectin is a pattern recognition molecule of the innate

immune system. It is a part of complement cascade and plays an important role in

the first line of defense against pathogenic microorganisms(64,65). MBL has opsonic activity . In association with MBL-associated serine proteases (MASPs) ,

have the ability to activate complement via the lectin pathway(66).

MBL belongs to the collection family, a group of C-type lectin also

possessing a collagen –like helical domain. Human MBL exists as a series of

oligomers of 2-6 subunits, built up from three identical polypeptide chains

(24 KDa, 228 aminoacids each). The apparent molecular weight of these

oligomers range from approximately 1,50,000 to 4,50,000. It forms a bouquet-

28

Four characteristic regions of MBL are

1. The short N- terminal cysteine –rich domain - responsible for arrangement

of subunits in the oligomer , dependent on disulphide bonds ; this region

consist of 21 aminoacids including three Cys residues.

2. The collagen like region interacts with MASPs ; it consists of 59 amino

acids (among them 19 Gly-X-Y triplets); this domain is glycosylated.

3. An α- helical neck region consists of 30 aminoacids stabilizes the polypeptide chain within a structural subunit.

4. The C- terminal carbohydrate recognition domain ( CRD ) is responsible

for pattern recognition and consists of 118 aminoacids(65,67–69)

MBL recognizes sugar patterns on the surface of many pathogens,

phospholipids, immune complexes and apoptotic cells. MBL binds with highest

affinity to D-mannose , N- acetyl – D – glucosamine and L – fucose which allows

the specific recognition of numerous polysaccharides and glycoconjugates like

bacterial lipopolysaccharides, capsular polysaccharides and fungal mannans (70,71). The LPS structure exerts a major influence on MBL attachment to bacteria.

Mannan- binding lectin is synthesized by hepatocytes and secreted into the

blood in an oligmeric form . Moreover , specific mRNA has been found in bone

marrow, fetal lung , small intestine, and testis (72). Its synthesis is controlled by the MBL2 gene located on chromosome 10 (10q11.2) and regulated in a similar

manner to acute phase proteins. The normal serum level of MBL is about 400 –

29

Figure 4 :schematic representation of MBL 2 gene and its genetic polymorphisms(73)

Figure 5 : Structure of tetrameric human mannose binding lectin(74)

30

The MBL2 gene contains 4 exons and 3 introns(75).

1. Exon 1 of the MBL2 gene encodes the signal peptide , the cysteine – rich

domain, and part of glycine rich collagen like region.

2. Exon 2 encodes the remaining part of collagen like region.

3. Exon 3 encodes the neck region and

4. Exon 4 encodes CRD.

Most mammals synthesize two forms of MBL : A and C. Human MBL

resembles the C form . The MBL1 pseudogene ( not expressed) , corresponding to

A form , has been found in the human genome(76).

Polymorphism of MBL gene :

Structural mutation in exon 1 of the human MBL 2 gene at codon 52

(allele D), codon 54 (allele B) and codon 57 (allele C) reduce the functional MBL

levels by disrupting the collagenous structure. B,C,D alleles are collectively

known as ‘O’ allele. Several nucleotide substitution at the promoter region like

H/L polymorphism, X/Y polymorphism and P/Q polymorphism affect the serum

MBL concentration(77–79).

MBL dependent complement activation:

MBL forms a complex with MBL-associated serine proteases (MASPs).

This complex becomes enzymatically active and activates the complement. This

facilitates complement - dependent opsonisation and subsequent uptake and

31

MBL was believed to be the sole collectin able to activate the lectin

pathway (LP) of complement. However , several reports indicate that MBL shares

that property not only with ficolins (another family of collagen – related lectins)

but also with the so called “novel collectins” like collectin 11 ( CL-11 or collectin

–kidney 1, CL-K1) and collectin 10 ( CL-10 or collectin – liver 1, CL-L1)(81,82). After binding of the MBL-MASPs complex to the target structure,

conformational changes lead to the activation of MASPs which in consequence

makes the cleavage of C4 and C2 possible and thus the initiation of the

complement cascade.

In spite of the different initiation mechanism , the lectin pathway resembles

the classical pathway (CP), forming the same convertases for C3 and C5

components(83,84).

MASP

Three proteases

1. MASP - 1

2. MASP - 2

3. MASP - 3

MASP – 2 is believed to be the key enzyme responsible for LP activation

as its proteolytic activity against C4 and C2 significantly exceeds the activity of

32

33

MASP – 1 is believed to upregulate lectin pathway activation. However,

recently its crucial role in MASP – 2 activation has been postulated(88,89).

MASP – 3 and nonenzymatic proteins – MAp19 (sMAP ) and Map44 are

believed to play a regulatory role in this process(84).

Involvement of MBL in Coagulation and Kallikrein-Kinin system:

MBL plays an important role in other systemic processes including

coagulation , inflammation and tissue injury. MASP – 1 and – 2 may participate in

activation of the coagulation system.

MASP – 1 – cleaves fibrinogen , factor XIII , and thrombin – activate fibrinolysis

inhibitor (TAFI )(90)

MASP – 2- cleaves prothrombin(91)

Megyeri et al found MASP – 1 to interact with protease activated

receptor-4, a mediator of inflammation and platelet activation(93). Later, Dobo et al. found high molecular weight kininogen to be its additional substrate(94). This activity like that of kallikrein enables release of bradykinin, a highly proinflammatory

mediator of the kinin – kallikrein system. Although MASP – 2 cleaves kininogen,

no bradykinin is released during this process.

The involvement of MBL – MASP complexes in haemostatic processes

was later demonstrated in an animal model by Takahashi et al.(95) who found that

MBL – null mice infected with staphylococcus aureus were predisposed to the

34

Figure 7 : Role of MBL in complement pathway , coagulation system & kinin system(92)

MBL and disease association:

MBL deficiency and increased susceptibility to disease:

1. Infectious disease especially extracellular pathogens(96,97)

2. Autoimmune disease e.g. SLE(98)

MBL deficiency and protection against disease:

35

MBL and modulation of disease severity

1. Infectious disease e.g. HIV , Hepatitis B and C , pulmonary disease in

cystic fibrosis(100,101)

2. Autoimmune disease e.g. Rheumatoid arthritis(102)

Inappropriate activation of MBL – MASP pathway

1. Lectin pathway activation in renal disease(103–105) e.g. Lupus nephropathy

Membranoproliferative glomerulonephritis

Post – streptococcal glomerulonephritis

Henoch – schonlein purpura nephritis

2. Lectin pathway activation on vascular endothelium following oxidative

stress

e.g. Myocardial reperfusion injury(106)

Factors that increase the concentration of MBL :(107–109)

 Thyroid hormone

 Growth hormone

 Dexamethasone

Low levels of MBL(110,111) are seen in

 Breast cancer

36

MBL in ischaemic – reperfusion injury:

Innate immune response is a “double – edge sword”, sometimes beneficial

and at other times disastrous for the host. MBL and MBL- dependent complement

activation have been found to be involved in ischaemia-reperfusion (I/R) injury

associated with numerous clinical condition such as graft rejection and other

pathological processes in the gastrointestinal tract or central nervous system.

In kidney:

Significant deposition of MBL - MASP 2 complexes was found in porcine

kidney after I/R injury. Moreover, colocalization of C4d with MBL was

observed(112). Administration of C1esterase inhibitor ( C1-INH ) resulted in inhibition of apoptosis of tubular epithelial cells and tubular damage. Therefore, a

pathological role of both lectin and classical pathway in I/R renal injury was

implicated.

In Van der Pol et al.(113) Rodent model study therapeutic inhibition of MBL was protective against tubular damage , preventing accumulation of

macrophages and neutrophils as well as expression of proinflammatory cytokines

and chemokines . Following reperfusion , MBL was internalized into tubular

epithelial cells, including rapid cell death. Therefore it was concluded that MBL

-mediated cytotoxicity preceded complement activation and was the primary

reason for tubular injury.

MBL deposition in ischaemically injured human kidney was first

37

capillaries and tubular epithelial cells. Berger et al. demonstrated a beneficial

effect of low MBL concentrations and low MBL variants of the corresponding

gene in recipients of kidney or combined pancreas – kidney transplants on graft

and patient survival. Low levels of circulating MBL correlated with improved

long term graft survival(115,116).

Damman and Seelen (117) suggested that under moderate graft injury, lectin

pathway activation may be beneficial due to participation in the clearance of

dying cells. However, under severe injury, MBL might contribute to the renal

tubular epithelium damage. According to Osthoff et al.(118) MBL deficiency could

offer some protection from I/R radiocontrast –induced kidney injury.

MBL in Cerebral Ischaemia:

Several reports demonstrated an association of MBL with cerebral

ischaemia. Ducruet et al. observed deposition of MBL in murine ishaemic

endothelium(119). Even in MBL – null mice, they observed C3 deposition in the cerebral hemisphere during reperfusion, suggesting involvement of other

complement activation pathways in the pathology.

Later, Orsini et al. working with mice confirmed MBL deposition in

ischaemic vessels and a protective effect of genetically determined MBL

deficiency ; they also found an increase in circulating MBL – MASP – 2 complex

38

Elvington et al. demonstrated the involvement of the alternative pathway of

complement activation(121). Interestingly C6 deficiency had no effect, indicating the lack of significance of the common pathway and thus formation of the MAC

(membrane attack complex). Osthoff et al. (122) determined MBL concentrations in 353 patients with ischaemic stroke observed that MBL – deficient patients had

significantly lower risk of unfavourable outcome and showed smaller lesion

volumes. They suggested that inhibition of lectin pathway may be a promising

strategy for reducing I/R associated cerebral damage.

In a recent clinical study Wang et al.(123) assessed serum levels of MBL in patients with acute ischaemic stroke and found that the MBL levels were

significantly higher in stroke patients compared with healthy controls and

increased with increasing severity of stroke. The authors postulated that elevated

MBL levels could be an independent risk factor for stroke.

MBL in Lung transplantation:

Several papers have focused on the role of MBL in lung transplantation. Its

higher levels in plasma of recipients were associated with development of

bronchiolitis obliterans syndrome (BOS) and poorer long- term outcome(124,125). Immunohistochemistry revealed the presence of MBL in lung tissue from patients

with BOS and at the time of ischaemia. Moreover , Carroll et al. observed a

significant increase of MBL concentration in plasma at 3,6 and 12 months after

39

after transplant was associated with later BOS development(126). Low MBL

concentrations were associated with a longer survival.

Gastrointestinal ischaemia:

Zhang et al. found that IgM bound to antigens in ischaemic mesenterium ,

provided a binding site for MBL resulting in complement activation(127).This finding was further confirmed by Lee et al. who detected MBL complexed to

antigen and natural IgM in the intestinal I/ R injury(128). Schwaeble et al. postulated MASP-2 deficiency to protect mice from gastrointestinal ischaemia –

reperfusion damage(129).

Cardiovascular disease:

Involvement of MBL in coagulation and ischaemia – reperfusion injury is

potentially harmful in the development of cardiovascular disease, especially

coronary artery disease and myocardial infarction, as well as in the rejection of a

heart transplant.

Ischaemia changes the expression of surface molecules and leads to

formation of neoantigens. Paradoxically, reperfusion causes harmful

inflammatory response that can counteract the beneficial effects of improved

blood flow. During reperfusion, myocardial cells become the targets of innate

immunity; promoting the release of inflammatory mediators, neutrophil

40

Mechanism causing myocardial ischaemia – reperfusion (MI/R ) injury

was provided by Busche et al. Using MBL and IgM – null mice , they found that

myocardial tissue injury following MI/R associated with complement activation

dependent on both MBL and IgM antibodies(131).

Pesonen et al. found lower serum concentrations of C3, higher levels of

MBL, and a higher frequency of high MBL level – associated MBL2 genotypes in

a cohort of patients with unstable angina pectoris or acute myocardial infarction

compared with healthy controls(132). Similarly , Haahr-Pedersen et al. observed significantly higher MBL and lower soluble C5b-9 (complement membrane attack

complex, MAC) concentrations in sera of patients with ST – elevation myocardial

infarction (STEMI) , undergoing primary percutaneous intervention with left

ventricular ejection fraction (LVEF) <35 % compared with those of LVEF

≥ 35%(133).

Keller et al. noted that an elevated MBL level is a risk factor for future

coronary artery disease (CAD) in apparently healthy men but not in women(2). Later , Schoos et al. have found high plasma levels of MBL and ficolin – 2

(L-ficolin ) to be synergistically associated with increased postinfarct left ventricular

end systolic and diastolic volumes (ESV, EDV) in STEMI patients(134).

Trendelenburg et al. reported that MBL functional deficiency contributed

to the significant reduction of 90- day mortality in patients with acute STEMI,

41

MBL and activation of lectin pathway of complement may be protective

against the development of atherosclerotic lesions by

 clearance of apoptotic cells and cell debris from atherosclerotic plaques or

 protection from Chlamydia pneumonia infection , known to be associated

with development of atherosclerosis(136).

On the other hand MBL may take part in I/R injury and enhance

thrombosis. The resultant effect may depend on diverse factors, specific for the

individual , including accompanying diseases , life style , age and sex.

C-REACTIVE PROTEIN:

It is an acute phase protein of hepatic origin(137). CRP was so named because it was first identified as a substance in serum of the patients with acute

inflammation that reacted with the somatic – C polysaccharide antigen of the

capsule of pneumococcus.

CRP is a member of Pentraxin family consisting of five identical, non

glycosylated polypeptide subunits, non covalently linked to form a disk shaped

cyclic polymer with a molecular weight of 115 k Da. Apart from the

polysaccharides in many bacteria, fungi and protozoal parasites CRP also binds to

phosphoryl choline such as lecithin and polyanions like nucleic acids in the

presence of calcium ions. In the absence of calcium ions , it binds to polycations

42

FUNCTIONS OF CRP:

 C- reactive protein activates classical complement pathway of immune

system via C1q .

 Initiates opsonisation, phagocytosis and lysis of invading cells and plays

an important role in body’s defence mechanism

 Detoxification of toxic autogenous substances(140).

FACTORS THAT INCREASE THE CONCENTRATION OF CRP:

1) Obesity

2) Smoking

3) Metabolic syndrome

4) Dyslipidemia

5) Steroids

6) Hypertension.

Major elevations of CRP occur in

 Myocardial infarction

 Infection

 Inflammation

 Stress

 Trauma

43

FACTORS DECREASING THE CONCENTRATION OF CRP:

1) Weight loss

2) Medications:-Statins, Fibrates, Niacin, Aspirin, NSAIDs, Moderate

alcohol consumption.

ROLE OF CRP IN ATHEROGENESIS :

 CRP released by action of IL-6, binds directly with oxidised LDL and is

present within Lipid laden plaques(141).

 It causes Monocyte adhesion & trans migration into the vessel wall leads to

polarisation of Macrophages, which is a pro inflammatory trigger in plaque

deposition in atherosclerotic lesions

 Decreases the NO synthesis by inhibiting endothelial Nitric oxide

synthases, and promotes chemotaxis(142).

 Increases the expression of tissue factor which acts as a procoagulant

resulting in DIC.

 Production of Plasminogen activating inhibitor inhibits tissue plasminogen

activator, decreases fibrinolysis and results in atherogenesis (143).

High sensitivity CRP:

CRP assays are used to predict recurrent ischemia in patients with unstable

angina(3). These standard assays were unable to identify those patient at risk for acute myocardial infarction, as they lacked low – end specificity. Development of

44

mg / L are referred to as high sensitivity (hs ) or ultrasensitive assays. The value

of hs-CRP as a marker of cardiovascular disease risk is recognized and accepted.

Routine CRP measurements – over the range of 3 mg / L

High sensitivity CRP measurement – including the range upto 3 mg / L

Applications of hs – CRP:

1. Diagnostic and prognostic marker in acute coronary syndrome

2. Predictor of future coronary events(144)

Inflammation plays a major role in all phases of atherogenesis and the role

45

AIM OF THE STUDY

1. To evaluate the risk of coronary artery disease in recently diagnosed

hypertensive individuals by estimating serum Mannose Binding Lectin

levels.

2. To correlate the Mannose Binding Lectin level with CRP for predicting

cardiovascular risk in hypertensive patients

OBJECTIVES

1. To estimate the level of serum mannose binding lectin in hypertensive

patients with coronary artery disease.

2. To estimate the level of serum mannose binding lectin in age and sex

matched healthy subjects.

3. To estimate the level of C-Reactive Protein(CRP) in all the study groups.

4. To correlate the level of serum mannose binding lectin and

 C-Reactive Protein (CRP)

 Lipid profile

 BMI and

46

MATERIALS AND METHODS

The study protocol was approved by the Institutional Ethics Committee of

Madras Medical College, Chennai.

STUDY DESIGN : Cross sectional case- control study

STUDY PERIOD : August 2017 - February 2018

SUBJECT SELECTION :

90 subjects were selected for the study. They were divided into three

groups as follows.

 Group A - 30 Recently diagnosed hypertensive patients

 Group B- 30 Hypertensive patients who had myocardial infarction recently

 Group C - 30 Age and sex matched healthy controls

Inclusion Criteria: GROUP A:-

 Recently diagnosed hypertensive patients of less than 6 months duration.

 Age – 30 years and above

 Outpatients attending hypertension clinic in Rajiv Gandhi Government

General Hospital, Chennai

 Hypertension was considered to be present if an individual had a history of

hypertension and was on antihypertensive agents or if the systolic blood

pressure exceeded 140mmHg or the diastolic pressure exceeded 90

47

GROUP B :-

 Hypertensive patients who had Myocardial Infarction recently.( < 7 days

duration)

 Age – 30 years and above

 Inpatients admitted in the cardiology department in Rajiv Gandhi

Government General Hospital, Chennai.

 The diagnosis of myocardial infarction was based on

a) a history of characteristic prolonged chest pain or chest discomfort

b) Electro cardiographic evidence of new Q wave or abnormal ST segment

c) Elevated levels of known cardiac biochemical markers like CK-MB.

GROUP C:-

 Age and sex matched apparently healthy subjects who were staffs of

Madras Medical College, their relatives and friends.

Exclusion criteria :

 Patients with Diabetes mellitus

 Patients with renal disorders.

 Patients with Liver diseases.

 Patient with acute illness / infection.

 Chronic Smokers and alcoholics.

48

Blood collection :

5 mL of blood sample was collected from all subjects after overnight

fasting and transferred to serum tubes. The blood was allowed to clot and the

serum was separated after centrifugation at 3000 RPM for 15 minutes. The serum

levels of CRP , total cholesterol , triglycerides , HDL –C, urea , creatinine & uric

acid were measured immediately. About 0.5 mL of serum was stored in eppendrof

at – 20oC for the analysis of mannose binding lectin.

BIOCHEMICAL INVESTIGATIONS:

Analytes Methods

Serum Mannose Binding Lectin ELISA

Serum C-Reactive Protein(CRP) Immunoturbidimetry

Serum Urea , Creatinine & Uric acid Spectrophotometer

Serum Lipid Profile Spectrophotometer

ESTIMATION OF SERUM MANNOSE BINDING LECTIN:

METHOD:

Enzyme linked immunosorbent assay – non competitive , sandwich

PRINCIPLE:

 MBL in standard and test samples are captured by anti-MBL

49

 Second biotinylated antibody to human MBL is added which binds with

the immobilized MBL thus forming a sandwich of MBL between two anti-

MBL antibodies.

 Streptavidin conjugated with enzyme horse radish peroxidase is added.

 Streptavidin binds avidly with biotin in the detection antibody.

 Substrate to the enzyme horse radish peroxidase is added resulting in the

formation of a coloured complex.

The intensity of colour produced is directly proportional to concentration

of MBL in the sample. The absorbance is measured at 450 nm in an automated

microplate reader.

REAGENTS :

1) Pre coated ELISA plate

2) Standard solution – MBL – 3200 ng / mL

3) Standard diluents

4) Biotin conjugate Anti – human MBL antibody

5) Streptavidin – HRP

6) Wash buffer concentrate

7) Substrate solution A

8) Substrate solution B

9) Stop solution

REAGENT PREPARATION:

 All reagents should be brought to room temperature before use.

50

The various concentrations of standard are prepared as follows

Standard 5 1600 ng / mL 120 μL original standard + 120 μL standard diluents

Standard 4 800 ng / mL 120 μL standard 5 + 120 μL standard diluents

Standard 3 400 ng / mL 120 μL standard 4 + 120 μL standard diluents

Standard 2 200 ng / mL 120 μL standard 3 + 120 μL standard diluents

Standard 1 100 ng / mL 120 μL standard 2 + 120 μL standard diluents

ng/ml 3200 1600 800 400 200 100

 Wash buffer

Mix the wash concentrate thoroughly until no visible crystals are present.

Then dilute 20 mL of wash concentrate in 480 mL of deionized water to make

500mL of wash buffer.

PROCEDURE:

1. The reagents and samples are brought to room temperature ( 18oC – 25oC)

before use.

2. Label strips as appropriate for the assay.

51

5. Then add 10 μL of anti- MBL antibody to sample wells.

6. Add 50 μL of streptavidin – HRP to sample wells and standard wells. 7. Mix well. Cover the plate with a sealer and incubate at 37oC for 60

minutes.

8. Discard the solution. Wash each well 5 times with 350 μL of reconstituted wash buffer solution in an auto washer. Invert and blot dry the plate against

clean filter paper.Add 50 μL of substrate solution A to each well and then add 50 μL of substrate solution B to all wells. Cover the plate with a new sealer and incubate at 37oC in the dark for 10 minutes.

9. Add 50 μL of stop solution to each well. The blue color will change into

yellow immediately. The absorbance is read at 450 nm within 30 minutes

after adding the stop solution.

STANDARD CURVE :

The standard curve is plotted with standard concentration in ng/mL along

x- axis and absorbance along y- axis. The line of best fit is drawn through the

standard points. The line has to be linear. The concentration of each sample is

calculated from the standard curve.

S. No

Concentration of Std ( ng/mL )

Absorbance (OD)

1 0 0.106

2 100 0.280

3 200 0.450

4 400 0.700

5 800 1.19