AIM AND OBJECTIVES

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A STUDY OF PARAOXONASE GENE POLYMORPHISMS IN CORONARY ARTERY DISEASE – UNDERSTANDING THE BIOCHEMICAL AND GENETIC BASIS OF CORONARY ARTERY

DISEASE.

Dissertation Submitted in

Partial fulfilment of the regulations required for the award of M.D.DEGREE

In

BIOCHEMISTRY- BRANCH XIII

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

PSG INSTITUTE OF MEDICAL SCIENCES AND RESEARCH COIMBATORE.

MAY-2020.

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CERTIFICATE

This is to certify that the dissertation titled ‘_A study of Paraoxonase gene Polymorphisms in Coronary artery disease – Understanding the Biochemical and Genetic basis of Coronary artery disease’is an original work done by Dr.J.FathimaNasreen, Postgraduate student, during the period of her post-graduation in Biochemistry in our institution. This work is done under the guidance of Dr.B.Gayathri, Professor, Department of Biochemistry, PSG Institute of Medical Sciences and Research, Coimbatore.

DR.G.JEYACHANDRAN MD., Dr.B. GAYATHRI MD., Professor & Head, Guide and Professor,

Department of Biochemistry Department of Biochemistry,

PSG IMS&R PSG IMS&R.

Coimbatore. Coimbatore.

DR.S.RAMALINGAM MD., Dean,

PSG IMS&R.

Coimbatore.

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DECLARATION

I hereby declare that this dissertation entitled “A study of Paraoxonase gene Polymorphisms in Coronary artery disease – Understanding the Biochemical and Genetic basis of Coronary artery disease” was prepared by me under the guidance and supervision of Dr.B.Gayathri, Professor, Department of Biochemistry, PSG Institute of Medical Sciences and Research.

This dissertation is submitted to The Tamilnadu Dr. MGR Medical University in fulfillment of the university regulations for the award of an MD degree in Biochemistry - Branch XIII examinations to be held in May 2020.

Place : Coimbatore.

Date : Dr.J. FATHIMA NASREEN

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ACKNOWLEDGEMENT

I express my sincere thanks to Dr.S.Ramalingam ,Dean ,PSG Institute of Medical Sciences and Reseach for granting me permission to conduct this study and utilize the facilities required for the study.

I convey with great pleasure my heartful thanks to DR.G.Jeyachandran , Professor and Head ,Department of Biochemistry for his guidance and support throughout my study period.

I render my immense pleasure in expressing my heartful and sincere gratitude to my guide Dr.B.Gayathri , Professor ,Department of Biochemistry for the sustained motivation and invariable support given by her all the way through my study period .It is her dynamic support and valuable suggestions that made this study a possible one.

I express my sincere thanks to Dr.D.Vijaya , Professor , Department of Biochemistry for the support in this endeavour. I wish to thank Dr.S.Kavitha , Associate Professor for her help during my study. I render my grateful and sincere thanks to Dr.G.Sumitra , Associate Professor of our department for the unstinting support and constant motivation she gave during my study period.

I extend my thanks to Assistant Professors Dr.A.S.MeenakshiSundaram, Dr.R.Sujatha , Dr.D.Saranya for their help in my study.I wish to thank Mrs.Aruna Lecturer for her help in my study.

I deliver to grateful and sincere thanks to Dr.G.Rajendran , Professor and Head , Department of Cardiology for permitting me to collect the samples .

I convey with immense pleasure my heartful thanks to Dr.Sudha Ramalingam , Professor , PSG centre for Molecular Medicine and

Therapeutics , for her persistant support , unrelenting motivation and timely suggestion throughout the course of the study.

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I extend my thanks to Dr.M.S.Karthikeyan , Associate Professor , Department of Community Medicine for his help in my study.

I express my gratitude for my seniors Dr.B. Dhanalakshmi , Dr.S.Zinnia for their support in my study period.

With a deep sense of gratitude , I thank Ms.Meenu , Mr.Raghavan , Ms.Hema , Ms.Susipriya , Research fellows , PSG centre for Molecular Medicine and Therapeutics.

I express my thanks to technicians and other workers in the department of Biochemistry , Cardiology , Master Health check-up , PSG centre of Molecular Medicine and Therapeutics for their help in my study.

I wholeheartedly thank my husband Dr.I .Syed Ummar for his relentless support in all my work during my study period.

I also thank my parents , in-laws and my children for their support in all my work during the study period .

I am extremely thankful for all my patients who consented to be a part of my study without whom the whole study would have been impossible. I wish them all good health and long life !

Dr.J. FATHIMA NASREEN

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ABBREVIATIONS

CAD Coronary artery disease CHD Coronary heart disease

PON Paraoxanase

BMI Body Mass Index

HDL-c High Density Cholesterol LDL-c Low Density Cholesterol

TC Total Cholesterol

CR Creatinine

SGOT/AST Aspartate Transaminase SGPT/ALT Alanine Transaminase ALP Alkaline Phosphatase

GGT Gamma Glutamyl Transferase TOT.P Total Protein

SNP Single Nucleotide Polymorphism T2DM Type 2 Diabetes Mellitus.

WHO World Health Organization.

CVD Coronary Vascular Disease.

DNA Deoxy Nucleic Acid.

IL-1 Interleukin 1.

TNF-α Tumor Necrosis Factor Alpha.

VCAM Vascular Cell Adhesion Molecule – 1.

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ICAM Intercellular Adhesion Molecule – 1.

ROS Reactive Oxygen Species.

PCR Polymerase Chain Reaction.

EDTA Ethylene Diamine Tetra Aceticacid.

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TABLE OF CONTENTS

S.NO CONTENTS PAGE.NO.

1 INTRODUCTION 1

2 AIMS AND OBJECTIVES 4

3 REVIEW OF LITERATURE 5

4 MATERIALS AND METHODOLOGY 23

5 STATISCAL ANALYSIS 62

6 RESULTS 63

7 DISCUSSION 99

8 CONCLUSION 104

9 BIBILIOGRAPHY 10 ANNEXURE

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1

INTRODUCTION

Coronary heart disease (CHD), also called coronary artery disease, occurs when plaque builds up in the coronary arteries .Plaque narrows the coronary arteries and reduces blood flow to the heart muscle. Blood clots can partially or completely block blood flow.

The complications of CAD have become one of the leading causes of mortality and morbidity worldwide. Even though epidemiological studies have shown that levels of HDL -c are inversely associated to the risk of coronary artery disease and its thrombotic complications, it is becoming increasingly evident that HDL particle functionality is as important as HDL -c levels. This is because, HDL particles not only promote reverse cholesterol transport but also exert anti-oxidative and anti-inflammatory activities.

Heightened oxidative stress in the form of oxidation of lipids and proteins by reactive oxidant species adversely contributes to disease progression in cardiovascular disease .⁽¹⁾

Serum Paraoxonase (PON) is an arylesterase synthesized in the liver and is a HDL -c associated enzyme which is responsible for the antioxidant properties of HDL -c ^(2,3). This enzyme plays an importance role in preventing low density lipoprotein cholesterol (LDL-c) oxidation, it is considered to protect against the development of coronary heart disease ⁽⁴⁾. The serum HDL - c concentration is inversely correlated with atherosclerosis risk ⁽⁵⁾. Many

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2 studies showed an association between activity of serum paraoxonase and CHD

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PON1 activity is inversely related to the risk of developing an atherosclerotic lesion, which contains cholesterol-loaded macrophage foam cells. Although experimental studies have demonstrated the reduction in PON1 activity due to oxygen frees radicals in ischaemia and reperfusion ⁽⁷⁾.

Q192R is a substitution of Glutamine/Arginine at position 192 of amino acid sequence posing a substrate dependent manner with respect to lipid peroxides ⁽⁸⁾. The PON1 R192 isoform hydrolyses paraoxon rapidly as compared to PON1 Q192 isoform, whereas Q192 isoform hydrolyses diazoxon, sarin and soman rapidly as compared to R192 isoform . However, phenylacetate hydrolysis i.e. AREase activity is not affected by Q192R polymorphism and has been shown to correspond with PON1 levels determined by immunological methods. This heterogeneous function results in calling Gln192 as low activity isoform and Arg192 as high activity isoform . Also, the R allele of this polymorphism has been correlated with T2DM regardless of PON1 activity^(9,10)

The Leucine/Methionine substitution at position 55 (L55M) is another important polymorphism of PON1 in case of enzyme characteristics . Formerly, L55M polymorphism was believed not to affect the activity of PON1 but recent studies claim to explore such effect ,even then there is a suggestion on the alterations of PON1 concentration by L55M polymorphism⁽¹¹⁾ .

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3 Numerous studies on paraoxonase polymorphisms have been conducted to determine the differences concerning the hydrolytic activities of polymorphic forms and subsequent susceptibility to the development of certain diseases. It has been shown that the polymorphism of PON1Q192R is less effective in inhibiting the oxidation of LDL-c, because it hydrolyzes lipid peroxides to a lesser degree ⁽¹²⁾ . A similar result was obtained by analysing the differences between Q and R allo enzymes using three different methods one referring to their inhibitory action on LDL-c oxidation, one assessing the formation of lipid peroxides, and one that makes use of recombinant PON1 192R and PON1 192Q ⁽¹³⁾ .

The obtained results clearly indicate the higher hydrolytic and antioxidant efficiencies of PON1 192Q. It is also proven that the genotype PON1 192QQ plays an important role in preventing the formation of atherosclerotic lesions, while PON1 192RR is associated with an increased risk of coronary heart disease ⁽¹⁴⁾. Similar results were obtained in studies conducted on patients suffering ischemic strokes, in which the PON1 192RR genotype was correlated with the risk of stroke⁽¹⁵⁾ . In contrast, other studies do not provide evidence of these PON1 changes, showing no relation between the Q192R polymorphism and the risk of cardiovascular diseases ⁽¹⁶⁾. However, recent studies have again shown a correlation between coronary heart disease and the PON1 192RR genotype^(17,18) .

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4

AIM AND OBJECTIVES

AIM:

To determine the genetic polymorphisms of PON1 gene Q192R and L55M in coronary artery disease (CAD) patients.

OBJECTIVES:

1. To study the genotype and allele frequencies of PON1 gene polymorphism (PON1 gene ; Q192R and L55M) in CAD patients and healthy individuals.

2. To find out the association between PON1 levels and polymorphisms in CAD patients and healthy individuals.

3. To find out the association between PON1 levels and polymorphisms and serum HDL levels in CAD patients and healthy individuals.

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5

REVIEW OF LITERATURE

EPIDEMIOLOGY OF CORONARY ARTERY DISEASE(CAD)

Cardiovascular diseases (CVDs), have contributed to a major epidemic proportions worldwide. Globally, CVD led to 17.5 million deaths in 2012.⁽¹⁾ Nearly 78% of these deaths reported to be in developing countries, whereas in developed countries it is rapidly declining. Industrialization and related lifestyle changes known as epidemiological transition affected the developed world, including countries of Europe and North America, in the early 20th century and spread to developing countries 50 years later.⁽²⁾ In India high mortality rates, premature coronary heart disease(CHD), and increasing burden are the striking features of CVD epidemiology ⁽³⁾ The mortality varies from

<10% in rural areas to >35% in more developed urban regions⁽⁴⁾. Based on WHO report on the epidemiology of CVDs, the South Asian region has one of the highest cardiovascular mortality rates in the world⁽¹⁾.

The Framingham Heart Study show that, both high-density lipoprotein (HDL -c) and low-density lipoprotein (LDL-c) cholesterol levels are important in determining risk for coronary artery disease (CAD). Decreased high-density lipoprotein and increased low-density lipoprotein cholesterol levels are associated with an increase in coronary artery disease . These relations are independent of the usual risk factors for coronary artery disease, such as

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6 cigarette use , hypertension, genetic causes, etc. A 1% greater low-density lipoprotein value is associated with slightly more than a 2% increase in coronary artery disease over 6 years; a 1% lower high-density lipoprotein value is associated with a 3 to 4% increase in coronary artery disease . Even at total cholesterol levels less than 200 mg/dL, lower high-density lipoprotein levels are associated with increased myocardial infarction rates in both men and women.

ATHEROSCLEROSIS PATHOGENESIS

In contrast to the homeostasis that exists between the endothelium and smooth muscle cells of healthy vessels, inflammatory activation of vascular cells in diseased vessels corrupts their normal functions and favours processes that contribute to the atherosclerotic plaque development. Consequences of inflammatory activation in vascular cells include disruption in the endothelial cells permeability barrier, increased production of inflammatory cytokines (e.g.

IL-1, TNF-α) and recruitment of leukocyte adhesion molecules (e.g. VCAM-1, ICAM-1, E- selectin, P- selectin).Areas of yellow discoloration on artery’s inner surface called fatty streak formation occurs, which allows entry and modification of lipids in the vessel wall layer (sub intima); these lipids then serve as pro-inflammatory mediators that initiate leukocyte recruitment and foam cell formation .Any injury to the arterial endothelium like increased production of reactive oxygen species (ROS) e.g. shear stress ,triggers

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7 increased endothelial permeability and also the entry of LDL-c into the vessel intima layer. LDL-c binds to proteoglycans in the extracellular matrix and becomes trapped which allows lipoprotein modificaions. Further oxidation of the lipoproteins by local ROS derived from endothelial cells or macrophages that penetrate the vessel wall attracts chemokines and recruits smooth muscle cells, finally formation of a plague and plague progression. Initially, cholesterol esters accumulate within foam cells, but as the lesions become more fibrous and necrotic, large amounts of extracellular cholesterol estersare found. These plagues limit the blood flow resulting in tissue ischemia and cell death ,which manifests as angina pectoris or claudication in peripheries. There is increasing evidence that the local production of ROS has a pivotal role in atherosclerosis, specifically in the diabetic milieu. Increased vascular superoxide production in aortas from atherosclerotic apo E ( mice model) has been demonstrated ^[6] . These changes were mediated by increased NAD(P)H oxidase (Nox) activity in the aorta. ROS can activate NADPH oxidase, and more specifically, the p47phox subunit of NADPH oxidase .

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8 ATHEROSCLEROTIC PLAGUE- PATHOGENESIS

The McGraw-Hill Companies .Harrison's Internal Medicine 20^THEDITION

The McGraw-Hill Companies.Harrison's Internal Medicine 20^TH EDITION

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9 Although a low level of HDL -c is a major risk factor for coronary heart disease (CHD) in westernized societies, the genetic control of HDL -c levels is only partly understood. HDLs are macromolecular complexes of proteins and lipids that range in diameter from 70 to 100 A . They sediment in the density region 1.063 to 1.21 g/ml. Analytical ultracentrifugation reveals lighter and heavier particles in this region, called HDL2 and HDL3, respectively.

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10 HDL -C

The lipid in HDL -c is 32 percent cholesterol ester, 5 percent free cholesterol, 55 percent phospholipid, and 8 percent triglyceride. The protein consists principally of apo A-I (70 percent) and apo A-II (20 percent). HDL-2 is relatively lipid-rich and protein-poor compared with HDL-3. HDL -c heterogeneity in apo lipoprotein composition also exists with apo A-I-only, apo A-I plus apo A-II, and apo E-rich particles. The apo A-I-only particles contain four apo A-Is per particle and float predominantly in the HDL-2 region. The apo A-I plus apo A-II particles contain two of each of these apolipoproteins and are enriched in the HDL-3 region. The distribution of HDL -c levels in the population was determined by the Lipid Research Clinic Prevalence Study .For men ages 45 to 49 years, the average HDL -c level is 45 mg/dL, with the bottom decile below 33 mg/dL and the top decile above 60 mg/dL. In women, the average is 56 mg/dL, with the bottom decile below 39 mg/dL and the top decile above 78 mg/dL.

In a prospective study, the Framingham Study reported a highly significant inverse relationship between coronary heart disease incidence and HDL -c levels based on a 12-year follow-up.⁽⁷⁾

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11 High-density lipoprotein (HDL) provides a pathway for the passage of lipid peroxides and lysophospholipids to the liver via hepatic scavenger receptors.

Furthermore, it is becoming increasingly evident that HDL particle functionality is at least as important as HDL -c levels since HDL particles not only promote reverse cholesterol transport from the periphery (mainly macrophages) to the liver but also exert pleiotropic effects on inflammation, haemostasis and apoptosis.HDL actually metabolizes lipid hydroperoxides preventing their accumulation on low-density lipoprotein (LDL), thus impeding its atherogenic structural modification. A meta-analysis^(8), including 302.430 subjects from 68 long-term prospective studies, supported the importance of HDL -c measurement in the risk assessment for CAD .

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12 HDL may exert anti-atherogenic effects at multiple steps^(9).These include (1) inhibition of monocyte chemotaxis; (2) inhibition of monocyte adhesion to endothelial cells; (3) decreased retention or aggregation of LDL in the arterial wall; (4) decreased oxidation of LDL; (5) increased cholesterol efflux macrophages; and (6) increased endothelial synthesis of prostacyclin (PGI2).

The McGraw-Hill Companies. Harrison's Internal Medicine 20^THEDITION

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13 ANTIOXIDANT, ANTI-INFLAMMATORY, AND

ANTIAGGREGATORY EFFECTS OF HDL

Several of the antiatherogenic properties of HDL may be related to its antioxidant properties.⁽¹⁰⁾In a co-culture system of endothelial cells on smooth muscle cells, the migration of monocytes into the subendothelial space is promoted by minimally oxidized LDL; this process is inhibited by HDL ⁽¹¹⁾ There are several mechanisms underlying the antioxidant properties of HDL.

HDL may sequester oxidized lipids away from LDL, transport lipid hydroperoxides to the liver, and bind pro-oxidant transition metals. This activity may supplement the activity of the selenium-dependent plasma glutathione peroxidase activity, and could be particularly important in pathophysiological states where the latter activity is reduced. A number of candidates have been suggested to be responsible for HDL's antioxidant function, with paraoxonase-1 (PON1) perhaps the most prominent. The enzyme paraoxonase (PON) circulates bound to HDL -c, and promotes the breakdown of the oxidized phospholipids that accumulate in minimally oxidized LDL.

In humans, the relationship of paraoxonase to the protective effect of HDL is complex. Common genetic variation in the coding sequence of paraoxonase leads to altered activity toward the artificial substrate paraoxon.

An allelic association between paraoxonase variants and coronary heart disease was found in six of nine cross-sectional studies.⁽¹²)

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14 Paraoxonase-1 (PON1)

Paraoxonase-1 (PON1) is a high-density lipoprotein-associated esterase and is speculated to play a role in several human diseases including diabetes mellitus and atherosclerosis. Low PON1 activity has been associated with increased risk of major cardiovascular events, therefore a variety of studies have been conducted to establish the cardioprotective properties and clinical relevance of PON1.

Human serum paraoxonase-1 (PON1) is a calcium-dependent hydrolytic enzyme that is found in a variety of mammalian species. Abraham Mazur⁽¹³⁾ and Norman Aldridge⁽¹⁴⁾played a pivotal role in the identification and classification of PON1 in the mid-1940s to early 1950s. Initially, the enzymes were referred to as “A”-esterases, but later became universally known as paraoxonases due to their ability to detoxify the organophosphate compound paraoxon which is the toxic metabolite of parathion, a commonly used agricultural insecticide.¹⁵

PON1 belongs to a family of three serum paraoxonases, including PON2 and PON3; however, PON1 remains the most popular member of this family.

This is largely due to the elegant studies by Mackness et al.that described the role of high-density lipoprotein (HDL)-associated PON1 in decreasing lipid peroxide accumulation on low-density lipoprotein (LDL).^(16-18)

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15 Structure of PON1

Overall structure of RePON1 in complex with the inhibitor 2-hydroxyquinoline (2HQ) obtained at pH 6.5 (PDB ID 3SRG). Here the flexible loop (residues 70–81) is highlighted in red, the three surface helices are highlighted in salmon, the Ca²⁺ ions are highlighted in green, and 2HQ is highlighted in orange⁽¹⁸⁾

PON1 consisting of 354 amino acids has a molecular mass of 43 kDa.

Structural analysis using X-ray crystallography revealed the six-bladed β- propeller structure of PON1 with a central tunnel that houses two calcium ions.⁽²⁰⁾ Each calcium ion, depending on its location within the enzyme, plays an important part in the activity of PON1⁽²²⁾.

In a study by W.H. Wilson Tang et al ,it was established analytically, with robust high-throughput assays for serum paraoxonase and arylesterase activities , measured in 3668 stable subjects undergoing elective coronary

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16 angiography without acute coronary syndrome , were prospectively followed for major adverse cardiovascular events (MACE= death, myocardial infarction, stroke) over 3 years. Low serum arylesterase and paraoxonase activities were both associated with increased risk for MACE, with arylesterase activity showing greatest prognostic value (quartile 4 versus quartile 1; hazard ratio 2.63; 95% CI, 1.97–3.50; p <0.01). Arylesterase remained significant after adjusting for traditional risk factors, C-reactive protein, and creatinine clearance (hazard ratio, 2.20; 95% CI, 1.60–3.02; p <0.01), predicted future development of MACE in both primary and secondary prevention populations, and reclassified risk categories incrementally to traditional clinical variables.

A genome-wide association study identified distinct single nucleotide polymorphisms within the PON-1 gene that were highly significantly associated with serum paraoxonase (1.18×10^-303) or arylesterase (4.99×10⁻¹¹⁶) activity but these variants were not associated with either 3-year MACE risk in an angiographic cohort (n = 2136) or history of either coronary artery disease or myocardial infarction in the Coronary Artery Disease Genome-Wide Replication and Meta-Analysis consortium (n ≈ 80,000 subjects).It was concluded that diminished serum arylesterase activity, but not the genetic determinants of PON-1 functional measures, provides incremental prognostic value and clinical reclassification of stable subjects at risk of developing

MACE⁽¹⁹⁾

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17 Ahmed M. A. Akabawy et al , in his study on cardio-protective effect of paraoxonase , included 200 clinically diagnosed type 2 diabetic patients (sub divided according to presence of cardiovascular complication). In addition, 60 healthy controls were selected with comparable socioeconomic, age, body mass index, and sex distribution. Glycemic control indices, lipid profile, Paraoxonase-1, MCP-1, ICAM-1, VCAM-1, and Lp (a) were measured. At the end of the study, serum levels of Paraoxonase-1 (↓), MCP-1 (↑), ICAM-1 (↑), VCAM -1 (↑), and Lp(a) (↑) were altered significantly in both diabetic cohorts compared to healthy controls. Also, Paraoxonase-1 (↓↓), MCP-1 (↑↑), ICAM-1 (↑↑), VCAM-1 (↑↑) levels showed significant alteration in type 2 diabetic patients with cardiovascular disease compared to those without complication.

Additionally, with respect to prediction of cardiovascular disease risk, results predict that Paraoxonase-1 has higher and better diagnostic accuracy index as indicated by ROC analysis. It was concluded that Paraoxonase-1 is a sensitive biomarker in type 2 diabetic with anti-atherogenic cardio-protective properties; its circulating levels may be a valuable future tool for early diagnosis and/or assessment of cardiovascular disease risk associated with type 2 diabetic ^(19).

In a study by Anna Wysocka ,71 patients aged 43–76 years with confirmed coronary heart disease (CHD). Established risk factors of coronary

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18 heart disease such as hypertension, elevated total cholesterol and LDL cholesterol (LDL-c), low HDL cholesterol (HDL -c), diabetes mellitus, obesity, smoking and premature CHD in family history were assessed. PON1 genotype for −108C/T promotor region was determined by polymerase chain reaction- restriction fragments length polymorphism (PCR–RFLP) method. Paraoxonase activity towards paraoxon and arylesterase activity towards phenyl acetate were measured spectro photometrically. Significant correlations between diabetes mellitus and paraoxonase activity (R = −0.264, p = 0.026) and between the premature coronary heart disease in family history and PON1 activity (R =

−0.293, p = 0.013) were found.

Genetics of PON1

The human PON1 gene is a member of a multigene family consisting of three members in total. PON1, PON2 and PON3 are located next to each other on chromosome 7 and share extensive structural homology. The most widely and accurately described Paraoxonase polymorphism is the single nucleotide polymorphism, which impacts the conversion of glutamine to arginine and has the effect of altering the hydrolytic activity of the Paraoxonase-1 form. Each Paraoxonase form plays an important role in the human body, and they exhibit antioxidant, anti-atherosclerotic, and anti-inflammatory influences. The PON 1(192RR) genotype was associated with lower HDL levels and higher LDL

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19 levels. Each PON form plays an important role in the human body, and they

exhibit antioxidant, anti-atherosclerotic, and anti-inflammatory influences ⁽²⁰⁾. In multivariate analysis, PON1 paraoxonase activity was independently

of confounding factors associated with diabetes (OR = 0.985; p = 0.024) and premature CHD in family history (OR = 0.983; p = 0.027). PON1 activity towards aryl acetate positively correlated with HDL -c level⁽²¹⁾

Paraoxonase activity is under both genetic and environmental influences and varies widely among individuals . The human serum PON gene shown to have two common polymorphisms: Q or R at position 191 (glutamine or arginine, respectively) and M or L at position 54 (methionine or leucine, respectively). PON Q and PON R qualitatively differ in their enzymic activities to hydrolyze various organophosphates. It has been studied that the Q allele more abundant than the R allele, is responsible for the anti-oxidant effect of PON against atherosclerosis reported to be related to the risk for coronary heart disease⁽²²⁾ .

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20 Cytogenetic Location: 7q21.3, which is the long (q) arm of chromosome 7 at position 21.3

In a systematic review and meta-analysis of 64 case-control studies on effects of paraoxonase 1 gene polymorphisms on heart diseases , 64 studies involving a total of 19,715 cases and 33,397 controls were included in the meta-analysis. It was found that the L55M polymorphism showed a significant association with heart diseases in Europeans (OR 1.44, 95%CI 1.33–1.56) and Asians(OR 1.18, 95%CI 1.03–1.35). This meta-analysis also showed a protective association of Q192R polymorphism with HD in Asian (OR0.49, 95%CI 0.37–0.66) and African populations (OR 0.67, 95%CI 0.53–0.84).

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21 The 192R allele significantly decreased the risk of myocardial infarction (OR 0.75, 95%CI 0.57–0.99) and coronary artery disease (OR 0.91, 95%CI 0.84–0.98); however, individuals with 192Q allele had a markedly increased risk of coronary artery disease development (OR 1.38, 95%CI 1.22–1.56).It was demonstrated that the genetic risk for heart diseases is associated with the PON1gene polymorphisms.L55M polymorphism was a risk factor and Q192R polymorphism is protective for coronary artery disease in certain populations.⁽²⁶⁾

Human epidemiological studies have shown that polymorphisms of the human PON1 gene are correlated with coronary artery disease, indicating a genetic association between PON1 and coronary artery disease^{(23) .}Mice lacking serum PON1 exhibit increased LDL-c oxidation in vivo and are more susceptible to atherosclerosis than are wild-type mice^(24,25)

High density lipoprotein inhibits LDL-c modification ^(36,37) and abolishes the induction of monocyte chemotactic protein-1 (MCP-1) in arterial wall cells, thus preventing monocyte transmigration⁽³⁸⁾. Paraoxonase-1 (PON1) is one of several enzymes that possesses anti-atherogenic properties associated with HDL -c. PON1 Paraoxonases belong to a family of mammalian serum and liver enzymes with aryldialkylphosphatase activity.It is a calcium-dependent hydrolase that is tightly associated with apoA-I in HDL -c⁽³⁹⁾ . Preparations of purified PON1 prevents oxidation of LDL-c in vitro,⁽⁴⁰⁾ and treatment of mildly

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22 oxidized LDL-c (MM-LDL) with purified PON1 significantly decreases the ability of mildly oxidized LDL-c (MM-LDL) to induce monocyte-endothelial interactions.⁽⁴¹⁾

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23

MATERIALS AND METHODS

The study was conducted at PSG Institute Of Medical Sciences and Research, Coimbatore. Ethical clearance for conducting the study was acquired from the Institutional Human Ethics Committee. An informed consent was acquired from the patients (cases and controls) before obtaining blood samples from them.

The study design is a case control study in which patients angiographically proved coronary artery disease attending cardiology outpatient department in PSG IMSR, Coimbatore, India were selected as cases .Patients satisfying inclusion and exclusion criteria were explained about the study before collecting their blood samples.

Patients as controls were selected based on the inclusion and exclusion study criteria from the master health check up department. The blood samples collected were processed and stored for analysis. The required patient data namely height, weight ,age and other data were retrieved from their case sheet with their consent.

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24 INCLUSION CRITERIA:

• Males and females

• Age > 30 years

• Angiographically proved coronary artery disease .

EXCLUSION CRITERIA:

 Malignancy

 Genetic malformation

 Acute illness

 Pregnancy

 Other valvular dysfunctions

 Other Endocrine abnormalities

Collection of blood samples:

The blood sample of both cases and controls was collected in sodium fluoride vacutainer for fasting and post-prandial plasma glucose estimation , in EDTA vacutainer for estimation of HbA1c and DNA extraction. Red topped serum tubes were used for lipid profile and estimation of urea and creatinine.

The sample for DNA extraction was transferred to sterile cryovial ,labelled and stored at -80 degree celsius.

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25 The methods used for estimation of biochemical parameters are given below.

PLASMA GLUCOSE Method:

Enzymatic reference method with Hexokinase.

Principle:

Enzymatic reference method with hexokinase. Hexokinase catalyzes the phosphorylation of glucose by ATP to form glucose-6-phosphate and ADP. To follow the reaction, a second enzyme, glucose-6-phosphate dehydrogenase (G6PDH) is used to catalyze oxidation of glucose-6- phosphate by NADP+ to form NADPH.

Reaction : Hexokinase

D-glucose + ATP D-glucose-6-phosphate + ADP G6PDH

D-glucose-6-phosphate + NADP⁺ D-6-phosphogluconate + NADPH+ H⁺

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26 The concentration of the NADPH formed is directly proportional to the glucose concentration. It is determined by measuring the increase in absorbance at 340 nm.

Test definition:

Measuring Mode Absorbance

Abs. calculation mode Endpoint Reaction

Reaction mode Reaction direction

Reaction direction Increase

Wavelength A/B 340/652 nm

Calu . first / last 33/69

Unit mmol/L

Conversion factor: mmol/L x 18.02 = mg/dL GLYCOSYLATED HEMOGLOBIN (HbAlc) : Method :

Turbimetric inhibition immunoassay Principle:

Total Hb and HbAlc concentrations are determined after hemolysis of the anticoagulated whole blood specimen. Total Hb is measured colorimetrically.

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27 HbAlc is determined immunoturbidimetrically. The ratio of both concentrations yields the final precent HbAlc result [HbAlc (%)]. The anticogulated whole blood specimen is hemolyzed automatically on Cobas Integra Systems with HbAlc hemolysis reagent in the predilution cuvette.

Erythrocytes are lysed by low osmotic pressure. The released Hb is proteolytically degraded by pepsin, to make the β-N-terminal structures more accessible for the immunoassay. Additionally, the heme portions are oxidized for the Hb assay. Total Hb is determined on COBAS INTEGRA systems in the hemolysate using a cyanide free colormetric method based on the formation of a brownish - green chromophore (alkaline hematin D-575) in alkaline detergent solution. The color intensity is proportional to the Hb concentration in the sample and is determined by monitoring the increase in absorbance at 552nm.

The result ia calculated using a fixed factor determined from the primary calibrator chlorohemin. HbAlc is measured on Cobas Integra systems using monoclonal antibodies attached to latex particles. The antibodies bind the β-N- terminal fragments of HbAlc.

Glycopeptides + antibody latex Bound glycopeptides Remaining free antibodies are agglutinated with a synthetic polymer carrying multiple copies of the β-N-terminal structure of HbAlc. The change in turbidity is inversely related to the amount of bound glycopeptides and is measured turbidimetrically at 552 nm.

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28 A synthetic polypeptide comprising the N-terminal structure of HbAlc is used for calibration. The final result is expressed as percent HbAlc and is calculated from the HbAlc / Hb ratio as follows:

Protocol 1 (according to IFCC):

HbAlc (%) = (HbAlc / Hb) x 100

Protocol 2 (according to DCCT / NGSP):

HbAlc (%) = (HbAlc / Hb) x 87.6 + 2.27 Test definition:

Reaction mode D-R2-S

Wavelength A/B 552 / 659 nm

Test range 50 - 400 μmol/L (81 - 644 mg/dL)

Unit mmol/L

SERUM TOTAL CHOLESTEROL Method:

Enzymatic, colorimetric method.

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29 Principle:

Cholesterol esters are cleaved by the action of cholesterol esterase (CE) to yield free cholesterol and fatty acids. Cholesterol oxidase (CHOD) then catalyzes the oxidation of cholesterol to cholest-4-en-3-one and hydrogen peroxide. In the presence of peroxidase (POD) , the hydrogen peroxide formed effects the oxidative coupling of phenol and 4-aminoantipyrine to form a red quinone-imine dye.

Reaction :

CE

Cholesterol esters + H₂O₂ Cholesterol + RCOOH CHOD

Cholesterol + O₂ Cholest-4-en-3-one+ H₂O₂ POD

2 H₂O₂ + 4- AAP + phenol quinone-imine dye + 4 H₂O The color intensity of the dye formed is directly proportional to the cholesterol concentration. It is determined by measuring the increase in absorbance at 512 nm.

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30 Test definition:

Reaction mode R-S

Test range 0 – 20.7 mmol/L

mmol/L

Conversion factor: mmol/L x 38.66 = mg/dL SERUM TRIGLYCERIDES

Method:

Enzymatic, colorimetric method (GPO/PAP) with glycerol phosphate oxidase and 4-aminophenazone.

Principle:

Triglycerides are hydrolyzed by lipoprotein lipase (LPL) to glycerol and fatty acids. Glycerol is then phosphorylated to glycerol and fatty acids.

Glycerol is then phosphorylated to glycerol-3-phosphate by ATP in a reaction catalyzed by glycerol kinase (GK). The oxidation of glycerol-3-phosphate is catalyzed by glycerol phosphate oxidase (GPO) to form dihydroxyacetone phosphate and hydrogen peroxide (H₂O₂).

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31 LPL

Triglycerides glycerol + fatty acids GK

Glycerol + ATP glycerol-3-phosphate +ADP GPO

Glycerol-3-phosphate +O₂ dihydroxacetone phosphate + H₂O₂ POD

2H₂O₂ + 4 aminophenazone quinoneimine dye + 4 H₂O + 4- chlorophenol

In the presence of peroxidase (POD), hydrogen peroxide effects the oxidative coupling of 4-chlorophenol and 4-aminophenazone to form a red- coloured quinoneimine dye, which is measured at 512 nm. The increase in absorbance is directly proportional to the concentration of triglycerides in the sample.

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Reaction mode R-S

Test range 0-10 mmol/L (0-885 mg/dL)

Unit mmol/L

Conversion factor: mmol/L x 38.66 = mg/dL SERUM HDL -CHOLESTEROL

Method :

Homogeneous enzymatic colorimetric test.

Principle:

In the presence of magnesium ions, dextran sulfate selectively forms water soluble complexes with LDL, VLDL and chylomicrons which are resistant to PEG – modified enzymes. The cholesterol concentration of HDL cholesterol is determined enzymztically by cholesterol esterase and cholesterol oxidase coupled with PEG to the amino groups (approx 40%). Cholesterol esters are broken down quantitatively into free cholesterol and fatty acids by cholesterol esterase.

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33 PEG – cholesterol esterase

HDL Cholesterol esters + H₂O HDL – Cholesterol + RCOOH In the presence of oxygen, cholesterol is oxidized by cholesterol oxidase to ∆4 cholestenone and hydrogen peroxide.

PEG – cholesterol oxidase

HDL Cholesterol + O₂ ∆4 cholestenone + H₂O₂ In the presence of peroxidase, the hydrogen peroxide generated reacts with 4-amino-antipyrine and HSDA to form a purple blue dye. The color intensity of this dye is directly proportional to the cholesterol concentration and is measured photometrically.

POD

2H₂O₂ + 4-aminoantipyrine+HSDA*+H⁺+H₂O Purple blue pigment + 5 H₂O

*HSDA = Sodium N ( 2 - hydroxy - 3- sulfopropyl) – 3,5-dimethoxyaniline

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Reaction mode R1-S-SR

Test range 0-3.12 mmol/L (0-120 mg/dL)

Unit mmol/L

Conversion factor: mmol/L x 38.66 = mg/dL SERUM LDL-CHOLESTEROL

Calculation:

VLDL = Triglycerides /5

LDL cholesterol = Tot cholesterol – (HDLc + VLDLc) SERUM TOTAL BILIRUBIN

Method :

Colorimetric Assay . Principle:

Total bilirubin, in the presence of a suitable solubilizing agent, is coupled with a diazonium ion in a strongly acidic medium.

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35 Acid

Bilirubin + diazonium ion azobilirubin

The color intensity of the red azo dye formed is directly proportional to the total bilirubin and can be determined photometrically.

Assay type 2-point End

Reaction time/assay points 10 / 10-25 (STAT 4 /10-25)

Wavelength(sub/main) 600 / 546 nm

Units μmol/L (mg/dL, mg/L)

Conversion factor: mmol/L x 38.66 = mg/dL SERUM DIRECT BILIRUBIN

Method :

Colorimetric Assay Principle:

Conjugated bilirubin and δ– bilirubin (direct bilirubin) react directly with 3,5 – dichlorophenyl diazonium salt in acid buffer to form the red – colored azobilirubin.

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36 Acid

Bilirubin + + 3,5 – DPD azobilirubin

The color intensity of the red azo dye formed is directly proportional to the direct (conjugated) bilirubin and can be determined photometrically.

Reaction time/assay points 10 / 10-13

Units μmol/L (mg/dL, mg/L)

Conversion factor: mmol/L x 38.66 = mg/dL SERUM ALANINE AMINO TRANSFERASE (ALT) Principle & Method:

Method according to the International Federation of Clinical Chemistry (IFCC), but without pyridoxal-5 -phosphate. ALT catalyzes the reaction between L-alanine and 2-oxoglutarate. The pyruvate formed is reduced by NADH in a reaction catalyzed by lactate dehydrogenase (LDH) to form L- lactate and NAD+.

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37 ALT

L-Alanine + 2-oxoglutarate pyruvate + L-glutamate LDH

Pyruvate + NADH + H⁺ L-lactate + NAD⁺

The rate of the NADH oxidation is directly proportional to the catalytic ALT activity. It is determined by measuring the decrease in absorbance at 340 nm.

Assay type Rate A

Reaction time/assay points 10 / 18 - 46

Wavelength(sub/main) 700 /340 nm

Reaction direction Decrease

Units U/L (μkat/L)

SERUM ASPARTATE AMINO TRANSFERASE (ALT) Principle & Method:

Method according to the International Federation of Clinical Chemistry (IFCC), but without pyridoxal-5’-phosphate. AST in the sample catalyzes the transfer of an amino group between L-aspartate and 2-oxoglutarate to form

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38 oxaloacetate and L-glutamate. The oxaloacetate then reacts with NADH, in the presence of malate dehydrogenase (MDH), to form NAD+.

AST

L-Aspartate + 2-oxoglutarate oxaloacetate + L-glutamate MDH

Oxaloacetate + NADH + H⁺ L-malate + NAD⁺

The rate of the NADH oxidation is directly proportional to the catalytic AST activity. It is determined by measuring the decrease in absorbance at 340 nm.

Assay type Rate A

Reaction time/assay points 10 / 18 - 46

Reaction direction Decrease

Units U/L (μkat/L)

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39 SERUM ALKALINE PHOSPHATASE (ALP)

Principle & Method:

Colorimetric assay in accordance with a standardized method In the presence of magnesium and zinc ions, p-nitrophenyl phosphate is cleaved by phosphatases into phosphate and p-nitrophenol.

ALP

P-nitrophenyl phosphate + H2O phosphate + p-nitrophenol The p-nitrophenol released is directly proportional to the catalytic ALP activity. It is determined by measuring the increase in absorbance at 409 nm.

Assay type Rate A

Reaction time/assay points 10 / 19 – 48

Units U/L (μkat/L)

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40 SERUM GAMMA GLUTAMYL TRANSFERASE ( GGT)

Principle & Method :

Enzymatic colorimetric assay. Gamma-glutamyltransferase transfers the γ- glutamyl group of L-γ glutamyl 1-3-carboxy-4-nitroanilide to glycylglycine.

GGT

L-γ-Glutamyl-3-carboxy-4-nitroanilide L-γ Glutamyl-glycylglycine +glycylglycine +5 amino-2 nitrobenzoate

The amount of 5-amino-2 nitrobenozate liberated is proportional to the GGT activity in the sample. It is determined by measuring the increase in absorbance at 409 nm.

Assay type Rate A

Units U/L

Conversion factor: U/L x 0.0167 = μ kat/L

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41 SERUM TOTAL PROTEINS

Method :

Colormetric assay.

Principle:

Divalent copper reacts in alkaline solution with protein peptide bonds to form the characteristic purple-coloured biuret complex. Sodium potassium tartrate prevents the precipitation of copper hydroxide and potassium iodide prevents auto reduction of copper.

Alkaline pH

Protein +Cu²⁺ Cu-protein complex

The color intensity is directly proportional to the protein concentration. It is determined by measuring the increase in absorbance at 552 nm.

Reaction time/assay points 10 / 10 – 34(STAT 5/10-34)

Units g/L(g/dL)

Conversion factor: g/L x 0.1 = g/dL

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42 SERUM ALBUMIN

Method :

Colormetric assay with endpoint method.

Principle:

At a pH value of 4.1, albumin displays a sufficiently cationic character to be able to bind with bromcresol green (BCG), an anionic dye, to form a blue-green complex. pH 4.1 Albumin + BCG Albumin - BCG complex The color intensity of the blue-green color is directly proportional to the albumin concentration in the sample. It is determined by monitoring the increase in absorbance at 583 nm.

Units g/L((μmol/L ,g/dL)

Conversion factors: g/L x 0.1 = g/dL g/dL x 10 = g/L

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43 SERUM CREATININE

Method :

Colormetric assay.

Principle:

The enzymatic method is based on the established determinationof hydrogen peroxide after conversion of creatinine with the aid of creatininase, creatinase, and sarcosine oxidase. The liberated hydrogen peroxide reacts with 4- aminophenazone and HTIB to form a quinone imine chromogen.

Creatininase

creatinine + H₂O creatine creatinase

creatine + H₂O sarcosine + urea SOD

sarcosine + O₂ + H₂O glycine + HCHO + H₂O₂

POD

H2O2+4-aminophenazone+HTIB^a quinone imine chromogen+H2O+HI a-2,4,6-triiodo-3-hydroxybenzoic acid

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44 The color intensity of the quinone imine chromogen formed is directly proportional to the creatinine concentration. It is determined by measuring the increase in absorbance at 552 nm.

Abs. calculation mode Endpoint

Calu . first / last 35 / 65

Reaction Mode R1- S-SR

Test range 0 – 2700 μmol/L (0 – 30.5 mg/dL)

with postdilution 0 - 27 000 μmol/L(0 – 305 mg/dL)

Postdilution factor 10 recommended

Conversion factor: μmol/L x 0.0113 = mg/dL

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45 SERUM UREA

Method :

Kinetic test with urease and glutamate dehydrogenase.

Principle:

Urea is hydrolyzed by urease to form ammonium and carbonate. In the second reaction 2-oxoglutarate reacts with ammonium in the presence of glutamate dehydrogenase (GLDH) and the coenzyme NADH to produce L- glutamate. In this reaction two moles of NADH are oxidized to NAD for each mole of urea hydrolyzed.

Urease

Urea + 2 H₂O 2 NH₄⁺ + CO₃^2-

GLDH

NH₄⁺ + 2-oxoglutarate + NAD L-glutamate + NAD⁺ + H₂O

The rate of decrease in the NADH concentration is directly proportional to the urea concentration in the specimen. It is determined by measuring the absorbance at 340 nm.

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Abs. calculation mode kinetic

Reaction direction decrease

Calu . first / last 23 / 28

Reaction Mode R1- S

Test range 0 – 40 mmol/L (0 – 240 mg/dL)

with postdilution 0 - 400 mmol/L(0 – 2402 mg/dL)

Postdilution factor 10 recommended

Unit mmol/L

Conversion factor: mmol/L x 6.006 = mg/dL urea PON 1 GENE POLYMORPHISM

DNA Extraction:

The blood samples were sorted based on their codes as cases and

controls.The samples were thawed and the DNA extraction was done with the Macherey Nagel- Genomic DNA purification Nucleospin Blood kit.The basic

principle applied –with the Nucleo spin Blood method ,genomic DNA is prepared from whole blood.Lysis is achieved by incubation of whole blood in a solution containing large amounts of chaotropic ions in the presence of

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47 Proteinase -K. Appropriate conditions for binding of DNA to the silica membrane of the corresponding Nucleo Spin Blood Columns are achieved by addition of ethanol to the lysate. The binding process is reversible and specific to nucleic acids. Washing steps efficiently remove contaminations. With the NucleoSpin Blood Quick Pure kit,contaminations are removed by a single wash step. Pure genomic DNA is finally eluted under low ionic strength conditions in a slightly alkaline elution buffer.

Kit contents

Lysis Buffer BQ1 13 mL

Wash Buffer BQ2(Concentrate) 7 mL

Elution Buffer BE 13 mL

Proteinase K (lyophilized) 30 mg Proteinase Buffer PB 1.8 mL NucleoSpin BloodQuickPure Columns Collection Tubes (2 mL)

In addition to the above mentioned kit components, the reagents required were 96-100% ethanol,Phosphate-buffered saline (PBS) for some samples and 1.5mL microcentrifuge tubes for sample lysis and DNA elution.

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48 Equipment:

 Manual pipettors

 Centrifuge for microcentrifuge tubes (NucleoSpin® Blood), with a swing-bucket rotor

 Vortex mixer

 Thermal heating block (NucleoSpin® Blood / QuickPure) or water bath

 Personal protection equipment (lab coat, gloves, goggles)

During storage, especially at low temperatures, a white precipitate may form in Buffer T1, B3, or BQ1. Such precipitates can be easily dissolved by incubating the bottle at 70 °C before use. Before starting any NucleoSpin®

Blood protocol the following are prepared:

 Wash Buffer B5 (NucleoSpin Blood): The indicated volume of ethanol (96–100 %) is added to Wash Buffer B5 Concentrate and the bottle is labelled to indicate that ethanol was added. It is stored at room temperature (18–25 °C).

 Proteinase K: The indicated volume of Proteinase Buffer PB is added to dissolve lyophilized Proteinase K. Proteinase K solution is stable at - 20°C .

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49 Protocols for DNA purification from whole blood:

Incubator or water bath is set to 70 °C.

Elution Buffer BE is preheated to 70 °C.

200 μL blood

+ 25 μL Proteinase K

+ 200 μL B3

Mix -vortex the mixture vigorously (10–20 s).

Incubate samples at 70 °C for 10–15 min.

(The lysate should become brownish during incubation with Buffer B3)

+ 210 μLethanol

Mix- vortex the mixture vigorously (10–20 s).

Load lysate

Centrifuge 11,000 x g for 1 min

+ 500 μL BW(wash buffer) Centrifuge 11,000 x g for 1 min

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50 + 600 μL B5

Discard flow-through and reuse Collection Tube.

+ 100 μL BE (70 °C)

Incubate at room temperature for 1 min.

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51 The obtained DNA is ready-to-use for subsequent reactions like PCR, or any kind of enzymatic reactions.

Technology Silica-membrane technology

Format Mini spin columns

Sample material 5–200 µL whole blood

Fragment size 200 bp–approx. 50 kbp

Typical yield 4–6 µg (200 µL blood)

A260/A280 1.6–1.9

Typical concentration 40–100 ng/µL

Elution volume 60–200 µL

Preparation time 30 min/prep

DNA can be purified successfully from blood samples treated with EDTA, citrate,or heparin. For the samples with leukocyte rich materials( like buffy coat), smaller volumes of the samples taken and diluted the samples with sterile PBS (by dissolving 8 g NaCl, 0.2 g KCl,1.44 g Na₂HPO₄, and 0.24 g KH₂PO₄ in 800 mL H₂O and adjusting the pH to 7.4 with HCl.Finally adding H₂O to make it upto 1 liter.

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52 Polymerase Chain Reaction (PCR):

A standard polymerase chain reaction (PCR) setup consists of four steps:

1. Add required reagents or mastermix and template to PCR tubes.

2. Mix and centrifuge.

(Add mineral oil to prevent evaporation in a thermal cycler without a heated lid).

3. Amplify per thermo cycler and primer parameters.

4. Evaluate amplified DNA by agarose gel electrophoresis followed by ethidium bromide staining.

Readymix PCR Reaction

Amount Component Final Concentration

25 µL Readymix (R2523 or P4600) w µL Forward primer

(typically 15-30 bases in length) 1 µM x µL Reverse primer

(typically 15-30 bases in length) 1 µM y µL Template DNA (typically 10 ng) 300 pg/µL

z µL Water

µL Total reaction volume

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53 Typical Cycling Parameters

35 cycles of amplification are recommended.

Temperature °C PCR Step Duration

94 °C Denature template 1 min 51-60 °C Anneal primers 2 min

72 °C Extension 3 min

4. The amplified DNA can be evaluated by agarose gel electrophoresis and subsequent ethidium bromide staining.

Reagents for Nucleic Acid Electrophoresis:

 Agarose (precast gels, powder, etc.)

 Buffer such as MOPS-EDTA-sodium acetate, tris-acetate-EDTA (TAE) or tris-borate-EDTA (TBE)

 Gel loading solution and sample loading buffer for RNA

 Electrophoresis stain or dye such as ethidium bromide

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54 Figure: Thermocycler

Photograph showing PCR products of SNP L55M with 172bp

There are four classes of restriction endonucleases: types I, II,III and IV.

All types of enzymes recognise specific short DNA sequences and carry out the endonucleolytic cleavage of DNA to give specific double-stranded fragments

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55 with terminal 5'-phosphates. They differ in their recognition sequence, subunit composition, cleavage position, and cofactor requirements. AlwI recognises GGATC,N.There are several key factors to consider when setting up a restriction endonuclease digest. By definition, 1 unit of restriction enzyme will completely digest 1 μg of substrate DNA in a 50 μl reaction in 60 minutes. A

"Typical" Restriction Digest.

Restriction Enzyme 10 units generally 1 µL is used

DNA 1 µg

10X NEBuffer 5 µL (1X)

Total Reaction Volume 50 µL

Incubation Time 1 hour

Incubation Temperature Enzyme dependent

Enzyme:

 Store in the freezer (-20degree)

 Should be the last component added to reaction

 Mix components by pipetting the reaction mixture up and down, or by

"flicking" the reaction tube..

 In general, 5–10 units of enzyme per µg DNA, and 10–20 units for genomic DNA in a 1 hour digest.

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56

 AlwIII enzyme for SNP Q192R

 NlaIII enzyme for SNP L55M Reaction Volume:

 A 50 µl reaction volume is recommended for digestion of 1 µg of substrate

 Enzyme volume should not exceed 10% of the total reaction volume to prevent star activity due to excess glycerol

 Additives in the restriction enzyme storage buffer (e.g., glycerol, salt) as well as contaminants found in the substrate solution (e.g., salt, EDTA, or alcohol) can be problematic in smaller reaction volumes. The following guidelines can be used for techniques that require smaller reaction volumes.

Incubation Time:

 Incubation time is 1 hour

 Can often be decreased by using an excess of enzyme, or by using one of our Time-Saver Qualified enzymes.

 It is possible, with many enzymes, to use fewer units and digest for up to 16 hours. For more information, visit

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57

 Storage at -20°C is recommended for most restriction enzymes. For a few enzymes, storage at -80°C is recommended for periods longer than 30 days.

SNP Q192R:

• PCR product of SNP Q192R was digested with AlwIII enzyme.

• Incubation at 37⁰C for two hours.

• No inactivation required.

SNP L55M

• PCR product of SNP L55M was digested with NlaIII enzyme.

• Incubation at 37⁰C for one hour and 30 minutes.

• Inactivation at 65^oC for 20 minutes.

Identification of Genotypes:

Three genotypes were identified for each of single nucleotide polymorphisms.

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58 Q192R:

• Restriction did not occur when A allele was present , yielding a fragment of 238bp.

• Restriction occured when G allele was present , yielding fragments of 175bp and 63bp.

• Restriction occured when both A allele and G allele were present , yielding fragments of 238bp, 175bp and 63bp.

Figure: Photograph showing RFLP products of SNP Q192R

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59 L55M

 Restriction did not occur when T allele was present , yielding a fragment of 172bp.

 Restriction occured when A allele was present , yielding fragments of 106bp and 66bp.

 Restriction occured when both T allele and A allele were present , yielding fragments of 172 bp, 106 bp and 66 bp.

Figure: Photograph showing RFLP products of SNP L55M

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60

 Serum Paraoxanase Level (PON)

The RayBio® Human PON1 ELISA kit , an in vitro enzyme-linked immunosorbent assay ,has been used for the quantitative measurement of human PON1 in serum. This assay employs an antibody specific for human PON1 coated on a 96-well plate. Standards and samples are pipetted into the wells and PON1 present in a sample is bound to the wells by the immobilized antibody. The wells are washed and biotinylated antihuman PON1 antibody is added. After washing away unbound biotinylated antibody, HRPconjugated streptavidin is pipetted to the wells. The wells are again washed, a TMB substrate solution is added to the wells and color develops in proportion to the amount of PON1 bound. The Stop Solution changes the color from blue to yellow, and the intensity of the color is measured at 450 nm.

ASSAY PROCEDURE

1. All reagents and samples are brought to room temperature (18 - 25ºC) before use. It is recommended that all standards and samples be run at least in duplicate.

2. Label removable 8-well strips as appropriate for your experiment.

3. 100 µl of each standard (see Reagent Preparation step 3) is added and sample into appropriate wells and incubate for 2.5 hours at room temperature with gentle shaking.

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61 4. The solution is discarded and wash 4 times with 1X Wash Solution.

Wash by filling each well with Wash Buffer (300 µl) using a multi- channel Pipette or autowasher. Complete removal of liquid at each step is essential to good performance. After the last wash, remove any remaining Wash Buffer by aspirating or decanting. Invert the plate and blot it against clean paper towels.

5. 100 µl of 1X prepared biotinylated antibody (Reagent Preparation step 6) to each well is added and incubate for 1 hour at room temperature with gentle shaking.

6. The solution is discarded and wash 4 times with 1X Wash Solution 7. 100 µl of prepared Streptavidin solution (see Reagent Preparation step

7) to each well is added and incubate for 45 minutes at room temperature with gentle shaking.

8. The solution is discarded and wash 4 times with 1X Wash Solution 9. 100 µl of TMB One-Step Substrate Reagent (Item H) to each well is

added and incubate for 30 minutes at room temperature in the dark with gentle shaking.

10. 50 µl of Stop Solution (Item I) to each well is added and read at 450 nm immediately.

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62

STATISTICAL ANALYSIS

The data obtained , after computing BMI , lipid profile , serum paraoxanase levels and genotype analysis were statistically analysed using IBM SPSS software version 24.

The data distribution, and comparisons between cases and controls were displayed using histogram , bar diagram and Pie chart. Independent t-test was used to find the statistically significant difference in the distribution of demographic, biochemical parameters among the study groups. The dependence of categorical variable was tested with Chi-Square test. p value <

0.05 was considered as statistically significant.