A Study on Significance of Non-HDL Cholesterol and Its relation with Cardiovascular Risk in Diabetic Population at GVMCH, Vellore

A STUDY ON SIGNIFICANCE OF NON-HDL CHOLESTEROL AND ITS RELATION WITH CARDIOVASCULAR RISK IN

DIABETIC POPULATION AT GVMCH , VELLORE

A DISSERTATION SUBMITTED TO

THE TAMILNADU DR.M.G.R MEDICAL UNIVERSITY

In partial fulfillment of the regulations for the award of the degree of

M.D. GENERAL MEDICINE

–

BRANCH I

DEPARTMENT OF GENERAL MEDICINE GOVERNMENT VELLORE MEDICAL COLLEGE AND

HOSPITAL

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

APRIL 2017

CERTIFICATE

This is to certify that the dissertation titled

“

A STUDY ON SIGNIFICANCE OF NON-HDL CHOLESTEROL AND ITS RELATION WITH CARDIOVASCULAR RISK IN DIABETIC POPULATION AT GVMCH , VELLORE

” is a genuine work done by

Dr.ASWINTH.R

,

Post Graduate student (2014

–

2017) in the Department of General Medicine, Government Vellore Medical College, Vellore under the guidance of Prof. Dr.S.P.Kumaresan M.D.,

Prof. Dr.S.P.Kumaresan M.D., Guide and Chief, Medical Unit- III,

Department of General Medicine, Govt. Vellore Medical College.

Prof. Dr. J. Philomena, M.D., Head of the Department,

Department of General Medicine, Govt. Vellore Medical College.

Prof. Dr. Usha Sadasivan, M.D., The Dean,

Government Vellore Medical College.

ETHICS COMMITTEE CERTIFICATE

TURNITIN DIGITAL RECEIPT

PLAGIARISM SCREEN SHOT

DECLARATION

I, DR. ASWINTH R solemnly declare that this dissertation titled

“A

STUDY ON SIGNIFICANCE OF NON-HDL CHOLESTEROL AND ITS RELATION WITH CARDIOVASCULAR RISK IN DIABETIC POPULATION AT GVMCH , VELLORE

”

is a bonafide work done by me in Department of General Medicine, Government Vellore Medical College and Hospital, Vellore under the guidance and supervision of Prof. Dr.S.P.Kumaresan M.D.,Guide and Head of Department, General Medicine.

This dissertation is submitted to The Tamilnadu Dr. M.G.R. Medical University, Chennai in partial fulfilment of the university regulations for the award of M.D., Degree in General Medicine (Branch

–

I)

DATE : DR.ASWINTH .R

ACKNOWLEDGEMENT

It gives me immense pleasure me to thank everyone who has helped me during the course of my study and in preparing this dissertation.

My sincere thanks to Prof. Dr. Usha sadasivan M.D., the Dean, Govt. Vellore Medical College for permitting me to conduct the study and use the resources of the College.

I am very thankful to the chairman of Ethical Committee and members of Ethical Committee, Government Vellore Medical College and hospital for their guidance and help in getting the ethical clearance for this work.

I am deeply indebted to my esteemed teacher, chief, head of department and guide Prof. Dr.S.P.Kumaresan M.D., for his active involvement at all times. I feel it was my good fortune to have had Prof.

Dr.S.P.Kumaresan M.D., as my guide and teacher. He has been a source of constant inspiration and encouragement to accomplish this work.

With a deep sense of gratitude I acknowledge the guidance rendered to me by him.

I express my sincere gratitude to Prof. Dr.Philomina MD Prof.

Dr. D.Anbarasu, M.D. and Prof. Dr. Govindarajalu, M.D., for their

valuable inputs and support.

I express my deepest sense of thankfulness to my Assistant Professors Dr. B.Gowrishankar, M.D., Dr.P.S.Ramesh M.D., for their valuable inputs and constant encouragement without which this dissertation could not have been completed.

I am particularly thankful to my fellow postgraduate colleagues Dr. M.Lavanya, Dr. Aravind.C.S and Dr.Prathibha.P for their

valuable support in the time of need throughout the study.

I thank my junior Post Graduate Dr.Ashwini metry and Dr.Aravinda kumar who supported me in completing the dissertation.

It is my earnest duty to thank my parents, friends and crris without whom accomplishing this task would have been impossible.

I am extremely thankful to my patients who consented and participated to make this study possible.

LIST OF ABBREVIATIONS USED

ADA American Diabetes Association ATP Adult Treatment Panel

CVD Cardio Vascular Disease CAD Coronary Artery Disease DM Diabetes Mellitus

DBP Diastolic Blood Pressure FBS Fasting Blood Sugar HDLc High Density Lipoprotein LDLc Low Density Lipoprotein PPBS Post Prandial Blood Sugar SBP Systolic blood pressure

VLDLc Very Low Density Lipoprotein IR Insulin Resistance

IDL Intermediate Density Lipoprotein TGLs Triglycerides

OGTT Oral Glucose Tolerance Test

NCEP National Cholesterol Education Program

METS Metabolic Syndrome

TABLE OF CONTENTS

S.NO TITLE PAGE NO

1 INTRODUCTION 1

2 AIM 3

3 REVIEW OF LITERATURE 4

4 MATERIALS AND METHODS 48

5 RESULTS AND ANALYSIS 52

6 DISCUSSION 68

7 CONCLUSION 75

8 BIBLIOGRAPHY

9 ANNEXURES PROFORMA MASTER CHART CONSENT FORM

ABSTRACT

Background :

Cardiovascular disease is the most leading cause for mortality globally. It is estimated that around 17.5 million died due to CVD and its about 31% of all death globally. The risk of CVD and its mortality seems to be significantly raised in patients having type 2 diabetes mellitus when compared with normal individuals. A major contributor to the increased CVD among with type 2 diabetes is dyslipidaemia. NCEP ATP III emphasis the role of LDL-C in the pathogenesis of cardiovascular disease, but it has been recently suggesting that raised levels non-HDLc may be better in predicting risk associated with cardiovascular disease .

Aims and objectives

The primary objective of this study is to assess the significances of non-hdl cholesterol in the diabetic population and its relation with cardiovascular risk among patients attending diabetic clinic.

Methods

This is the study conducted on diabetic population who are all diagnosed as type 2 diabetes mellitus as per ADA 2015 criteria and attending diabetic clinic at Government Vellore Medical College Hospital, vellore. This cross sectional study on significance of non-HDL cholesterol in diabetic population and its relation to cardiovascular risk which calculated through ASCVD risk scoring system.

Results

This study is conducted on diabetic population attending diabetic clinic at government vellore medical college , involves totally 100 subjects of which 54 were males and 46 were females. Most of them were in the age ranging from 50 to 60 years which includes 49 subjects and with mean age was 55 years. Among those 100 subjects , elevated 10 year risk for cardiovascular disease calculated through ASCVD risk assessment was present in 68 subjects. In this study of 100 diabetic population, 58 subjects had non- HDL cholesterol above their target . In these 58 subjects , 56 subject has elevated 10 year risk for cardiovascular disease as calculated through ASCVD risk scoring system. They are statistically significant with P value of 0.0001 (<0.05).This study shows results after dividing the study population based on the TGLs concentration above and below 200 mg /dl. The results shows, in population with TGLs concentration less than 200 mg / dl both LDLc and non- HDL cholesterol was good in assessing CVD risk while in population with TGLs concentration more than 200 mg/dl only non-HDL cholesterol was very good in assessing CVD risk but LDLc was not significant in assessing CVD risk. Thus in both groups non –HDL cholesterol was very good in anticipating 10 year risk for cardiovascular disease.

Conclusion:

The result of this study suggest that non-HDL cholesterol may be superior in predicting CVD especially in people with diabetes where TGLs are elevated.

Keywords:

type 2 diabetes mellitus, non – HDL cholesterol , cardiovascular disease

1

INTRODUCTION

Cardiovascular disease is the most leading in the cause for mortality in global census. It is estimated that around 17.5 million died due to CVD and its about 31% of all death globally ^1,2. The underlying mechanism for CVD is atherosclerosis and may be caused by high blood pressure , diabetes, smoking , physical inactivity, obesity, excessive alcohol consumption, improper diet, increased blood cholesterol³.

The risk of CVD and its mortality seems to be significantly raised in patients having type 2 diabetes mellitus when compared with normal individuals ^4,5. A major contributor to the raised CVD among with type 2 diabetes is dyslipidaemia.

Dyslipidaemia in type 2 diabetes were not restricted only quantitative values , but it was also qualitative with kinetic in nature⁴. Many factors contribute to abnormality in lipid metabolism associated with type 2 diabetes mellitus which includes increased insulin resistance , relative deficiency in insulin levels , role of adipo cytokines like adiponectin, and hyperglycaemia itself ⁵.

The characteristic quantitative abnormalities includes hypertriglyceridaemia, prolonged postprandial hyperlipidaemia, raised levels in the remnant lipo protein particles and reduced HDL-cholesterol levels³.

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The qualitative nature of abnormalities which is atherogenic includes raise in large VLDLc particle size , a increase in proportion of small , dense LDLc particle , susceptibility nature of LDLc to the oxidation , an raise in triacyl glycerol content in both LDLc and HDLc. So LDLc levels may be normal among type 2 diabetes patients ⁵.

National Cholesterol Education Program (NCEP) Adult Treatment Panel (ATP) III emphasis the role of LDL-C in the pathogenesis of cardiovascular disease, but it has been recently suggesting that raised levels non-HDLc may be better in predicting risk associated with cardiovascular disease¹ .

Non –HDLc includes all potential atherogenic lipo protein particles like VLDLc, IDLc, LDLc, and LIPO PROTEIN (A) . LDLc is calculated using friedwald equation which is considered as less accurate with increasing TGL level as in diabetes patient¹.

Advantage in using non – HDL cholesterol as a screening tool includes

- Its measurement can be done in non fasting sample as its estimation requires only total cholesterol and HDLc values.

- LDL –C requires fasting sample.

- In diabetic population its value is not altered in presence of elevated TGL.

- It includes all atherogenic lipoprotein like VLDL ,HDL, IDL, and LIPOPROTEIN (a)

3

So our study is focused on assessing significance of non-hdl cholesterol in type 2 diabetes population in vellore district and its relation with cardiovascular risk using ASCVD risk score³⁸.

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AIM OF THE STUDY

1. The aim of the study is to determine the significance of non- HDL cholesterol in diabetic population and to find out its relation to cardiovascular risk .

5

REVIEW OF LITERATURE

STRUCTURE OF LIPIDS AND LIPOPROTEINS

Lipids plays important role in normal physiologic functions of human body. It is source of energy for normal physiological function and integral part of cell structure. Lipids components includes fatty acids, Triglycerides, cholesterol, and lipoproteins

⁷

.

Cholesterol is essential for stabilizing cell wall and helps in transporting particles across cell membranes

⁵

. It is also required for synthesis in various hormones , precursor of adrenal and sex hormones, synthesis of bile acids which is essential for absorption of fat in diet from intestine.

Triglycerides (TGLs) are important for storage and production of energy .Through lipolysis TGLs are converted in fatty acid and glycerol

⁶

. Fatty acid is a vital source of energy for cardiac and skeletal muscles. In muscles and liver, fatty acids are converted to glucose by gluconeogenesis

³

.

Lipid molecules are hydrophobic in nature and hence it is

difficult for transportation in the blood . Therefore , they usually get

attached to some specific proteins synthesized in the body called apo

6

lipoprotein to form a soluble complexes consisting of lipids and proteins called lipoproteins

⁸

. Lipoproteins in plasma are composed with TGLs and cholesterol esters in the core of complex making it hydrophobic inside , whereas phospholipid and apo lipoproteins will be present in its outermost layer making it hydrophilic

⁸

. Hence it becomes soluble form and easily transported in the plasma . There are many lipo protein complexes in the plasma and classified as

TYPES OF LIPOPROTEINS IN PLASMA

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LDL – CHOLESTEROL

LDLc is an important atherogenic causing lipoprotein^1,8. It is identified as primary target for lipid lowering therapy according to lipid guidelines proposed by National Cholesterol Education Program –Adult Treatment Panel III (NCEP ATP III 2002). It also consititute about 60 to 70% of total serum cholesterol. It has apo B-lOO as a single apo lipoprotein¹.

HDL CHOLESTEROL

HDLc is called good cholesterol as there is lot of evidence showing its protective action in the development of atherosclerosis cholesterol¹⁰. It constitutes about 20 to 30% of the total cholesterol in serum. It has apo A-I

8

and apo A-II as its apo lipoprotein ^7,10.The level of HDLc has inverse relationship with CVD risk.

VLDL CHOLESTEROL

The VLDLc is produced in liver which is precursor of LDLc. VLDLc is degraded by losing TGLs to form VLDL remnant. VLDL remnant is rich in cholesterol ester and atherogenic. VLDLc forms 10 -15% of total serum cholesterol. VLDLc has apoB-100, apo CI, apo CII, apo CIII, and apo E as predominant apolipoprotein⁸.

9

CHYLOMICRONS

Chylomicron is rich in triglyceride. It is formed from dietary fat in the small intestine epithelium⁴. It has same lipoprotein like VLDL except apo B- 48 is present for of apo B-100 in VLDLc. Chylomicron degrade to form as chylomicron remnant which is more atherogenic.

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LIPID TRANSPORT PATHWAY

Lipids are mainly transported through following three pathways.

THE EXOGENOUS PATHWAY

This pathway includes transport of lipids, absorbed from the intestine following meal intake and transported to the liver⁶. Dietary fats absorbed from intestine organized to form chylomicrons. They are made up of TGLs , cholesterol, and apo lipoproteins⁹. They transport TGLs from the intestine to the muscle and adipose tissue for storage and also dietary cholesterol is transported to the liver.

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In adipose tissue and muscle, those transported TGLs undergo hydrolysis by enzyme lipoprotein lipase to form fatty acids and which are released for energy⁷ . The remaining part of chylomicrons left in circulation are collectively named as chylomicron remnants and which are taken up by LDL receptors in the liver¹¹.

THE ENDOGENOUS PATHWAY

The role of endogenous pathway includes synthesis and transporting lipo proteins from liver to the target tissue . TGLs and cholesterol in the liver are packed to form VLDL particles¹⁰. They are released from liver in to circulation and transported various tissues. lipoprotein lipase in the tissues hydrolyze VLDL to form fatty acid and glycerol¹³. Those fatty acids are taken by adipose tissue and muscle storage and energy production respectively. The remaining particles after hydrolysis is organized as VLDL remnant. They are taken back by liver through LDL receptors²¹. The remnant remaining in circulation after transport of VLDL remnant in to liver become IDL. Some IDL particles are absorbed by LDL receptors into liver. Other IDL particle are hydrolyzed by hepatic triglyceride lipase to form lesser dense LDL particle⁷. As TGLs are lossed in each step of hydrolysis LDL contains cholesterol predominantly. By these mechanism TGLs are transferred from liver to tissues. LDL which is rich in cholesterol acts primary carrier of cholesterol to peripheral tissues.

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LDLc in the circulation are cleared by uptake of LDL particles by LDL receptor in the liver and also by scavenger pathway in the cells. During this process free cholesterol is released and accumulated within the cells¹⁰. LDL concentrations in the blood is regulated by uptake by LDL receptor .By regulating the synthesis of hydroxyl -3 - methyl glutaryl coenzyme A reductase, suppressing new LDL receptors formation in the cells, and activating acyl - coenzyme A cholesterol Acyltransferase LDL concentration can be titrated¹⁰.

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THE REVERSE CHOLESTEROL PATHWAY

In this pathway cholesterol from tissue is transported and taken back to the liver where they are excreted via bile into feces⁷. the third pathway is the reverse cholesterol pathway. HDL is mainly involved in reverse cholesterol pathway. In this pathway cholesterol which are released after cell death and turnover are transferred to core of HDL particle. HDL is formed by maturation process after series of conversion to attract those released cholesterol along with precursor particle secreted from liver and intestine¹⁰.

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In plasma after absorbption into HDL particle, they are esterified by lecithin cholesterol acyl transferase with long chain fatty acid and transferred by cholesteryl ester transfer protein into VLDL OR IDL and which are taken back to liver¹¹. Through above mechanism cholesterol is recovered, transported and excreted.

LIPID AS RISK FACTOR

Lipid is important risk factor for cardiovascular disease which proved by many clinical trials⁸. Among various lipoproteins LDLc which is rich in cholesterol content is demonstrated by many animal , pathological, clinical and genetic studies as major athergenic lipoprotein¹¹. So LDLc is Primary Target for lipid lowering therapy as proposed by many standard guidelines like NECP ATP¹ .

The role of LDLc in causing CVD has been supported by lot of controlled clinical trials. Animal based trial shows that LDLc levels below 80mg/dl does not develop atherosclerosis. LDLc levels as low as 25 to 60 mg/dL is sufficient for normal physiological living. Some of reputed studies like Framingham Heart Study and Lipid Research Clinics trials¹² showed there is significant association with raised LDLc and development of CVD in males and postmenopausal women. In patient with known cardiovascular disease there is direct relationship with elevated LDLc with recurring CV events. Similarly Studies performed on migrating population from different regions with variable prevalence of serum cholesterol level showed significant

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relation with LDLc and CVD . adolesants with increased LDLc level due inherited dyslipidemia also showed significant relation with CVD⁵. In some individuals , in the absence of other risk factors individuals develop CVD with isolated elevation of LDL cholesterol. Thus the above trials strongly suggest LDLc as a major atherogenic lipoprotein and considered as primary target for lipid lowering therapy.

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Most commonly LDL is estimated using Friedewald formula because of complexity and cost involved in direct measurement of LDLc levels¹². So by this method of LDL estimation has certain limitations in individuals with high TGLc levels²⁵. Non-HDL cholesterol may overcome the limitations of LDL cholesterol. The cut-offs for LDL cholesterol levels are presented below.

Low HDL Cholesterol is another important risk factor for CVD³¹. Many clinical and epidemiological studies have shown that decreased HDLc levels act as an independent risk factor for CVD² .Similarly animal models have also demonstrated high HDL levels have been to protective against atherosclerosis . HDLc has anti -oxidant and anti -inflammatory property,

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which helps in preventing atherosclerosis²² . It has been shown that there is less risk for CHD in population with high HDLc level in famous Framingham Heart Study. Even lipid guidelines proposed by NCEP ATP III suggest to remove one risk factor from risk scoring ³⁸.

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NON - HDL CHOLESTEROL

Non - HDL cholesterol can be calculated by doing subtraction of HDLc from total cholesterol which are measured by standard technique. It includes all the atherogenic remnant lipids in the plasma³². Eventhough there are many methods of measuring these remnant lipoproteins by various methods like electrophoresis , ultracentrifuge and other immunological methods they are not routinely recommended by standard guidelines³. In individuals with TGLs level more than 200 mg/dl there will be elevated VLDLc. VLDLc is one of important lipoprotein representing remnant lipoproteins which are atherogenic. In some studies VLDLc is combined with LDLc to assess risk for CVD for those individuals with raised TGLs more than 200mg/dl ²⁵.

Non-HDL cholesterol which is representing all those atherogenic lipoprotein can be measured easily and thereby CVD risk can be assessed and they are also highly related to total amount of apo- B levels. Apo B is the most important apo lipoprotein for all known atherogenic lipo proteins²¹. In individual with TGLs level less than 200 mg/ dl , increase in both LDLc and VLDLc are associated with more risk for CVD . However, when TGLs level more than 200 mg/ dl , VLDLc level will be increased , and LDLc levels are not significantly correlated with CVD than non-HDL levels^21,22.

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So , LDLc levels as single parameter cannot predict risk caused due to elevated levels atherogenic lipo proteins. Hence , non -HDLc can be considered as very good marker for predicting average levels of elevated lipo protein that has atherogenic potential when compared with using only LDLc single parameter³² .

21

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APPLICATIONS OF NON-HDL CHOLESTEROL

 Non - HDLc includes all atherogenic lipo proteins like LDLc , VLDLc etc, and also reflects total cholesterol present in lipo protein containing apoB

 Among individual with raised TGLs level , non - HDL cholesterol will be cost effective tool to assess risk for developing cardiovascular related disease. Its because when TGLs levels are raised , VLDLc level will raise as the composition TGLs in VLDLc is always high. So in these condition LDLc alone cannot predict associated risk for CVD ²². Therefore , its better to use non - HDLc as marker for elevated cardiovascular risk.

 For estimation of non – HDL c patient need not be in fasting for sample collection . It can be taken at any time of the day ⁴.

 For above reasons and also its cost – effectiveness in estimating non - HDLc , it is considered and suggested by many standard lipid guidelines as target for assessing and planning intervention to prevent cardiovascular related disease.

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NON - LIPID RISK FACTORS

There are many non lipid risk factors which mainly contribute to the CVD risk. These factors either individually or along with lipid abnormalities may accelerate the atherosclerotic process. Thereby lead to development of cardiovascular related disease¹⁷. These risk factors can be grouped broadly in modifiable and non- maodifiable factors. So when there is associated lipid abnormalities aggressive lipid lowering medications are required to modify the disease process.

Modifiable Risk Factors

The primary aim of assessing and finding modifiable risk factor is mainly for introducing an early intervention²² . Thereby development of cardiovascular related disease can be prevented.

Hypertension

According to seventh joint national committee on blood pressure , the hypertension is defined when there is raise in systolic blood pressure of 140 mmHg and diastolic blood pressure of 90 mmHg or with control blood pressure due to intake of anti hypertensive medications³¹. There are lot of studies conducted on hypertension and its relation with cardiovascular related disease . Its one of risk factor which can cause CVD independently. So adequate blood pressure control is needed to modify its risk involvement ²⁶. it is also advised to add lipid lowering medications to achieve goal effectively.

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Cigarette Smoking

Smoking is one of very important modifiable risk factor that can be independently related to cardiovascular related disease¹⁸ . The relationship between smoking and CVD is dose dependent . With cessation of smoking or reduction in the amount of smoking , there is significant reduction in risk for cardiovascular related disease . since there is concrete evidence which was proved by many clinical trial , it is advisable to quit smoking to prevent them from CVD¹⁴.

Diabetes mellitus

Diabetes mellitus is another major independent risk factor for development of cardiovascular related disease . These risk was associated with both type 2 and also type 1 diabetes²⁰ . The guidelines on cholesterol states that presence of diabetes mellitus itself equivalent to having 10 year risk for development of cardivacular related disease.

The presence high blood sugar for chronic period of time itself has potential to cause CVD irrespective of presence other risk factors. But in the presence other risk factors like elevated lipid levels and hypertension doubles the risk of CVD ¹⁹. So other risk factors should be addressed to reduce risk for CVD. For example , UKPDS study group suggest lowering blood pressure to target levels proved to cause reduction in risk for CVD . Hence another lipid abnormalities should be addressed in diabetes population ³⁵.

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Metabolic Syndrome

The metabolic syndrome is caused due to sequence of environmental triggers on genetically pre disposed individual . this syndrome has strong association for development of cardiovascular related disease²⁴. The mechanism for this metabolic syndrome is described below.

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With increase in prevalence of obesity due to sedentary activity and overnutrition , there is raise in prevalence of metabolic syndrome .therefore , this complex syndrome requires multidomain approach for prevention of those individual from development of cardiovascular related disease³³. The diagnostic criteria for metabolic syndrome as follows

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Overweight-Obesity

The (body mass index) BMI values based on the Indian standards BMI 18-22.9kg/m² = normal

BMI 23-24.9 kg/m² = overweight BMI > 25kg/m² = obesity

Individual having BMI range suggestive of overweight and obesity most commonly have others CVD risk factors like abnormal lipid composition , type 2 diabetes due insulin resistance and hypertension³⁶ . so those individuals are highly predisposed for development of CVD and have high mortality.

Physical Inactivity

Physical inactivity is one life style modifiable factor which predispose to sequence of events leading many complications . those complication will be associated with elevated risk for development of CVD³⁷ . on other hand physical activity will augment sensitivity for insulin , reduces blood pressure and reduces LDLc ,TGLs and raises HDLc . So it advisable to have atleast moderate physical activity to prevent individual from development CVD risk factors¹².

Atherogenic Diet

Diet is one of the important modifiable risk factor . Diet that are rich in trans fatty acids and saturated fats predispose to obesity and thereby lead to

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development of CVD. On other hand , patients consuming foods including fruits , vegetables, whole grains are protective in nature. Foods containing high sodium content will predipose to hypertension¹⁶.

Non-Modifiable Risk Factors

As name suggest non modifiable cannot be modified . hence individual with associated lipd abnormalities should aggressively treated .

Age

As age of individual increases , they are more prone to develop atherosclerotic vascular disease . This age difference for this predisposition to CVD varies among both males and females⁹ . At any given cholesterol value there is significance difference in predisposition for CVD among younger and older adults. The reason behind this risk factor there will continous deposition of cholesterol in the vessel wall leading to increase in thickness of atherosclerotic plaque as age advances ¹⁰. As this Plaque grows its thickness increases and leads to obstruction of vessel wall which leads to decreased perfusion to target organ . otherwise when these plaque ruptures will lead to complete occlusion of predisposed vessels leading to vascular related hypoperfusion .according to NCEP guidelines male with age of 45 years and female of 55 years to be considered as high risk for CVD ¹.

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Male Sex

Male gender has higher risk for cardiovascular related illness when compared with female at the same age group. The reason behind these difference was not clear . it is considered that generally female is 10 to 15 years behind in development of CVD¹⁴. One explanation can be offered is difference in hormone , where these hormone give protection to female and that is reason postmenopausal women have raised risk for for CVD²⁴ . but both gender respond to lipid lowering therapy equally .

Family History of Premature CHD

As we have discussed earlier in this review , when individual with genetic predisposition tend to develop cardiovascular related disease when they are exposed to environmental risk factors ¹⁵. This may be the one of the reason behind occurrence of CVD in families at different stages of life. There was also lot case report sudden cardiac death run in families at particular decade of life .especially in age group less than 55 years and among first degree relatives . similarly studies on monozygotic twins also proved association of genetic factor.

So it is wise to treat aggressively and take primary prevention for these individuals.

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PATHOGENESIS OF ATHEROSCLEROSIS

Atherosclerosis related disease is the leading cause for both mortality and morbidity in all over the world^3,6,8 . So pathogenesis of this atherosclerotic process should be completely understood for better intervention and primarily for prevention . There is lot of studies and awareness program going on to prevent this process and thereby prevent morbidity and lead healthy life.

By definition atherosclerosis is a chronic process which starts much earlier in the life. This process continues slowly over many years silently and comes notice only when disease manifest unless some diagnostic evaluation done²¹. This chronic process can be accelerated when co existing risk factors present. So those risk factor should be addressed for prevention and slowing the process .

The risk factors which accelerates this atherosclerotic process includes all modifiable and non modifiable factors as dicussed in earlier literature . Dyslipidaemia is one among those risk factor which plays major role in these atherosclerotic process ²³. lipid abnormalities which specifically leads to this process include raised LDLc , reduced HDLc , elevation of other atherogenic lipo proteins like VLDLc , VLDLc remnant , and TGLs . raised non- HDLc includes all atherogenic lipo protein.

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Various stages of deposition are described below

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Atherosclerosis is a chronic degenerative process which occurs mainly in the initimal layer of arteries of medium and large size³⁵ . This process starts early in life with deposition of different particles like lipids , carbohydrate substances , some components of blood , and waste products from cellular substances , these all leads to fibrous formation and intimal calcification . These deposition and calcification in the presence lipid mplecules gradually narrows the vessel lumen and reducing the supply of nutrients to distal target organ. Sometimes ther will distruption plaque which leads to complete occlusion.

During early stages of process of atherosclerosis there is only functional impairment in the endothelium ⁸. Then during the process when these arterial wall gets injured. Macrophages enters these injured endothelial wall and activate further inflammation . Thus process of atherosclerosis begins structurally by more expression of adhesion molecules in the vascular cell . These in the presence of abnormal lipids will cause increased amount of lipids in the vessel wall²⁴. This early structural abnormality will not cause obstruction to vessels

The important lipo proteins which are deposited in the vessels wall include LDLc , remnant lipo proteins and lipo protein (a) . after their deposition in the intimal layer , they undergo series of chemical reactions such as oxidation and taken up by concern cells²¹ .

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These potential lipids once deposited in the vessel wall , they may be transferred and processed either of three possible routes . Those LDLc particle some will move back to blood depends on favourable circumstances . other route is it may be oxidized by free radicals and leukocytes, or it will be taken by macrophages and converted in to foam cells . LDLc which is filled in the macrophages is called as foam cells^21,35 . scavenger receptors helps in binding oxidized LDLc to the macrophages. Binding of these LDLc to the scavenger receptor prevent from mobilizing it othersites. Thereby it causes accumulation lipids in the vessel wall .

Once foam cells are formed , they start secreting mediators of inflammation and cytokines which initiates chronic inflammatory process³². There is also proliferation of smooth muscle cells . so by these process lot of inflammatory mediators and new foam cells are recruited and vessel wall thickens leading to obstruction as well as endothelial dysfunction²³.

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ROLE OF INSULIN IN LIPOPROTEIN METABOLISM

Insulin is important anabolic hormone in the regulation of many metabolic pathways. Lipo protein metabolism is one such pathway which is regulated mainly by insulin in the body ¹⁶. So any variation in the level of insulin due to either deficiency or raised insulin secretion due to insulin resistance causes changes in lipoprotein metabolism

36

In the adipose tissue , insulin normally acts as inhibitory to lipolysis.

This is carried out by inhibiting hormone sensitive lipase in the adipose tissue²¹ . This action leads to increase in triacylglycerol storage and deacrese in secretion of NEFA realesed from the adipose tissue. This above described action mainly occurs effectively during postprandial raise in insulin secretion

31,8.

37

In the hepatocyte , insulin action primarily causes decrease in VLDLc production thereby atherogenicity is reduced. Some data shows , with normal functioning of insulin hormone , it causes about 66 precent decrease in production of VLDL/triacylglycerol production and VLDL/Apo B production decrease by 53 percent.

VLDL production can be reduced by insulin through several mechanism . Insulin by suppressing the release and production NEF , it indirectly reduces supply of substrates for VLDL formation. It also exhibit direct inhibitory action for production of VLDL in the liver²¹. Another pathway involved in the suppression of VLDL production is phosphatidylinositol kinase 3( PI 3K). This Pathway normally begins with binding of insulin to its tyrosine kinase receptor which in turn activates PI 3K through this pathway it proceeds and finally acts by inhibiting maturation process thereby transfer of lipids to VLDL formation decreased³⁵.

Other action insulin on lipo proteins includes , it acts by inhibiting Apo B mRNA translation which inturn reduces Apo B synthesis. It accelerates the catabolism of triacylglycerol rich lipo proteins through activation of enzyme lipoproptein lipase²⁷.

In addition, it helps in the clearance of chylomicron remnants by promoting its transport across the plasma membrane by relocating the LRP to the cell surface . it also facilitates the uptake of LDLc by the LDL receptor

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in the liver . therefore during insulin deficiency there increase in the LDLc cholesterol concentration which promotes atherogenicity³² . but action insulin on hepatic lipase is still controversial .

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TYPE 2 DIABETES MELLITUS AND LIPID ABNORMALITIES

Type 2 diabetes mellitus is very commonly associated lipid abnormalities. The prevalence of dyslipidaemia in type 2 diabetes was found through many studies ranges from 70 to 85 percent¹⁴. With high prevalence of lipid abnormalities all diabetic population have elevated risk for cardiovascular related disease.

The lipid abnormalities in diabetes exhibit both quantitative as well as qualitative defects in lipoprotein metabolism which are more kinetic in nature mainly due altered response of insulin²³. The well known quantitative abnormalities include raised levels of triglycerides and reduced HDLc.

Other lipid abnormalities observed include mor production of VLDLc due insulin deficiency as we have discussed earlier. Reduction in catabolism of chylomicrons , thereby leads to raised production of chylomicrons³³.

The lipid abnormalities in type 2 diabetes population would have been present well before the diagnosis . This is because of fact that insulin resistance plays major role in development of these abnormalities¹³. So only abnormalities in lipid was include as part of metabolic syndrome. The insulin resistance when inherited in families can present with abnormal lipid values even without presence of diabetes²¹.

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DIABETIC DYSLIPIDEMIA AND INSULIN RESISTANCE

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CHOLESTEROL SYNTHESIS AND ABSORPTION

In individual with type 2 diabetes mellitus there will be decrease campesterol level and raised lathosterol level . They are marker of cholesterol absorption and synthesis respectively. It is also demonstrated by administration of isotopes per orally . Mechanism for this abnormality is not clearly understood . But it is proposed that it may be due to alteration in the expression of SREBP -2 which plays vital role in absorption and synthesis of cholesterol .

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POSTPRANDIAL HYPER LIPIDEMIA AND CHYLOMICRON

In diabetic individual with insulin resistance , an elevated chylomicron production and post prandial elevated TGLs are commonly noted. The reason for these peculiar abnormalities were already discussed in the previous heading under lipid abnormalities associated with insulin resistance²² . so all these changes would lead elevated levels of atherogenic remnants particles which raises risk for CVD²⁷.

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LIPID TRANSFERING PROTEIN

In individual with type 2 diabetes mellitus , the qualitative abnormalities in lipo protein was observed . Especially more TGLs content of LDLc and HDLc was mainly due to increase in activity of CETP . The activity of CETP is increased due to increased level of VLDLc which in turn caused by insulin resistance³⁵.

44

ROLE OF ADIPOCYTOKINES DIABETIC DYSLIPIDAEMIA Adiponectin

Adiponectin which was secreted from adipose tissue has property of acting against atherogenic substance in the body. It helps in increasing insulin sensitivity ²¹. The adiponectin level are very low in the diabetic population.

So their cardioprotective effectsare reduced in diabetic individual .

Retinol binding protein 4

The retinol binding protein is a adipokine , which was secreted by adipocyte and liver . Its level is raised in diabetic population . they have been related to raised level of triglycerides⁹ .

45

ATHEROSCLEROTIC CARDIOVASCULAR RISK SCORE (ASCVD )

This risk calculating guideline was framed by American Heart association[AHA] for the purpose of assessing cardiovascular risk for 10 year based on several parameters³⁸. This calculator includes following parameters.

46

INTERPRETATION OF ASCVD RISK SCORE

47

OBJECTIVES

The primary objective of this study is to assess the significances of non- hdl cholesterol in the diabetic population and its relation with cardiovascular risk among patients attending diabetic clinic.

48

MATERIALS AND METHODS

This is the study conducted on diabetic population who are all diagnosed as type 2 diabetes mellitus as per ADA 2015 criteria and attending diabetic clinic at Government Vellore Medical College Hospital, vellore. This cross sectional study on significance of non-HDL cholesterol in diabetic population and its relation to cardiovascular risk which calculated through ASCVD risk scoring system.

Study design:

cross sectional study Sample size:

100 Study period:

september 2015 - august 2016 Study tool:

Self structured questionnaire Inclusion criteria:

People with diagnosed as type 2 diabetes mellitus of age more than 40years and less than 79 years

49

Exclusion criteria:

People with known thyroid disorder

People with known hereditary lipid abnormalities Acute illness/infection

People taking anti lipid drugs

People not willing to participate in the study

Methodology:

People who were diagnosed as type 2 diabetes mellitus and attending diabetic clinic in the outpatient department of Government vellore medical college hospital were included in this study. After obtaining the written and informed consent from above study population individually , the detailed history as per mentioned in the proforma was elicited and physical examination was performed.

Venous blood was drawn from the subjects separately and sample was sent for blood sugar , Total cholesterol , HDL cholesterol , LDL cholesterol and triglycerides level estimation . Patient details regarding the general information, clinical findings and investigation results are all filled in specially designed proforma for this study.

50

Ethical clearance:

This study was approved by the ethical committee of Government Vellore Medical College, Vellore.

Statistical analysis:

Statistical analysis was done by using SPSS 16 software trial version.

Quantitative data was expressed in mean, median, mode and standard deviation. Qualitative data was expressed by Chisquare test . The difference was considered statistically significant when p value < 0.05

Operational guidelines:

1. Diagnosis of diabetes mellitus was based on ADA criteria:

HbA1C > 6.5%

or

Fasting blood sugar > 126mg/dl or

2 hour plasma glucose > 200mg/dl during an oral glucose tolerance test or

Symptoms of hyperglycemia and a casual plasma glucose > 200mg/dl 2. National cholesterol educational program adult treatment panel III Total cholesterol > 200mg/dl

Triglycerides > 150mg/dl HDL cholesterol ≤ 35 mg / dl

51

Non – HDL cholesterol = TC – HDL cholesterol LDL cholesterol ≥ 100 mg / dl

3. Joint national committee ( JNC – 8 )

Systolic blood pressure ≥ 140 mm Hg Diastolic blood pressure ≤ 90 mm Hg

4. Atherosclerotic Cardiovascular Risk score (ASCVD risk score) system proposed by American Heart Association (AHA) /American College Of Cardiology ( ACC)

This scoring system estimates 10 year risk for atherosclerotic cardiovascular disease using following parameters

Age Sex Race

Smoking present or not

Drugs intake for hypertension present or not Diabetes present or not

Systolic blood pressure Diastolic blood pressure Total cholesterol

HDL cholesterol

52

RESULTS AND ANALYSIS

In this study totally 100 patients diagnosed as type 2 diabetes mellitus attending diabetic clinic at Government Vellore Medical College were studied and analyzed during period between sep 2015 and august 2016. Results are analyzed using SPSS trial version software. The following data shows observation in this study.

AGE DISTRIBUTION IN THIS STUDY POPULATION.

Age group

Sex

Total

Male Female

40-50 years 17 12 29

51-60 years 27 22 49

61-70years 10 11 21

Above 70 0 1 1

Total 54 46 100

53

29

49

21

1 0

10 20 30 40 50 60

40 -50 years 51-60years 61-70years above 70 years

AGE DISTRIBUTION

age distribution

54 46

SEX DISTRIBUTION

MALE FEMALE

54

AGE WISE DISTRIBUTION OF SEX IN THIS STUDY

This study which is conducted among 100 diabetic population of age ranging from 40 years to 79 years with mean age as 55. They are distributed as 29 patients in the age group of 40 to 50 years with 17 males and 12 females, 49 patients in the age group of 51 to 60 years with 27 males and 22 females, 21 patients in the age group of 61 to 70 years with 10 males and 11 females and 1 female patient above 70 year age group.

0 5 10 15 20 25 30 35 40 45 50

40 -50 YEARS 51-60YEARS 61-70YEARS ABOVE 70 YEARS 17

27

10

0 12

22

11

1

FEMALE MALE

55

The results of individual parameters and its relation with cardiovascular risk assessed by using ASCVD RISK calculator.

SMOKING AND ITS RELATION WITH ASCVD RISK SCORE

SMOKING LOW RISK ELEVATED RISK TOTAL P VALUE

YES 6 28 34 0.027

NO 26 66 66

32 68 100

The observation in this study shows , 34 subjects smokes among 100 diabetic population studied . All 34 subjects who were smoking in this study population belongs to male gender and 28 among them have elevated cardiovascular risk. Female subjects in this study were not smoking .The above data were statistically analysed and P value were found as 0.027 which is statistically not significant (P>.005)

0 10 20 30 40 50 60 70 80 90 100

PRESENT ABSENT

ELEVATED RISK LOW RISK

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FASTING BLOOD SUGAR RELATION TO ASCVD RISK

FASTING

BLOOD SUGAR

LOW RISK ELEVATED RISK

TOTAL P VALUE

<130 4 16 20 0.210

>130 28 52 80

TOTAL 32 68 100

In this study among 100 diabetic population studied , 20 subjects had fasting blood sugar less than 130 mg / dl and remaining 80 subjects is having blood sugar more than 130 mg / dl . 16 subjects in the former group and 52 subjects in the latter group had elevated cardiovascular risk calculated by ASCVD risk scoring system. The Statistical analysis of above data shows data is not statistically significant 0.210 (P<0.05).

0 10 20 30 40 50 60 70 80

FBS<130 FBS >130

ELEVATED RISK LOW RISK

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POST PRANDIAL SUGAR RELATION TO ASCVD RISK

POST PRANDIAL

BLOOD SUGAR LOW RISK ELEVATED RISK TOTAL P VALUE

<200 2 2 4 0.424

>200 30 66 96

TOTAL 32 68 100

In this study among 100 diabetic population studied , 4 subjects had post prandial blood sugar less than 200 mg / dl and remaining 96 subjects is having blood sugar more than 200 mg / dl . 2 subjects in the former group and 66 subjects in the latter group had elevated cardiovascular risk calculated by ASCVD risk scoring system. The Statistical analysis of above data shows data is not statistically significant 0.424 (P<0.05).

0 10 20 30 40 50 60 70 80 90 100

PPBS <200 PPBS > 200

ELEVATED RISK LOW RISK

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SYSTOLIC BLOOD PRESSURE RELATION TO ASCVD RISK

SYSTOLIC BP LOW RISK ELEVATED RISK TOTAL P value

<140 29 36 65 0.001

> 140 3 32 35

TOTAL 32 68 100

In this study of 100 diabetic population, systolic blood pressure more than 140 mmhg were considered to be hypertensive and statistically analyzed . Results were showing 35 patients among 100 study population were hypertensive and 32 patients among them had elevated cardiovascular risk . This results were found to be statistically significant 0.001 (P<0.05)

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

SBP <140 SBP>140

ELEVATED RISK LOW RISK

59

DIASTOLIC BLOOD PRESSURE RELATION TO ASCVD RISK

DIASTOLIC BP LOW RISK ELEVATED RISK TOTAL P value

<90 30 41 71 0.002

>90 2 27 29

TOTAL 32 68 100

In this study of 100 diabetic population, diastolic blood pressure more than 140 mmhg were considered to be hypertensive and statistically analyzed.

Results were showing 29 patients among 100 study population were hypertensive and 27 patients among them had elevated cardiovascular risk . This results were found to be statistically significant 0.002 (P<0.05)

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

DBP <90 DBP <90

ELEVATED RISK LOW RISK

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HDL CHOLESTEROL RELATION TO ASCVD RISK

HDL

CHOLESTEROL LOW RISK ELEVATED

RISK TOTAL P value

NORMAL 31 50 81 0.005

ABNORMAL 1 18 19

TOTAL 32 68 100

In this study among 100 diabetic population studied ,19 patients had low HDLc level (<35mg/dl) and remaining 81 patients had HDLc level above 35mg/dl . Among 19 patients , 18 patients haved elevated CVD risk calculated by ASCVD risk calculator. Statistical analysis of above data shows it is statistically significant with P value 0.005 (P<0.05)

0 10 20 30 40 50 60 70 80 90

NORMAL ABNORMAL

ELEVATED RISK LOW RISK

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NON HDL CHOLESTEROL IN RELATION TO ASCVD RISK

NON HDL CHOLESTEROL

LOW RISK ELEVATED RISK TOTAL P value

<130 30 12 42 0.0001

>130 2 56 58

TOTAL 32 68 100

This study group of 100 diabetic population were divided into two groups and statistically analysed based on NCEP ATP III guidelines which has suggest non-HDL cholesterol as secondary target after LDL cholesterol in subjects with raised TGLs more than 200mg/dl. Non-HDL cholesterol target was LDL cholesterol plus 30 which is calculated as 130 mg/dl. So among 100 diabetic population studied 58 subjects had non-HDL cholesterol more than 130 mg / dl and remaining 42 subjects had levels below 130 mg/ dl. Elevated 10 year cardiovascular risk calculated through ASCVD risk scoring system were present in 56 subjects among 58 subjects with raised non –HDL levels which is also statistically significant with P value of 0.0001(<0.005)

0 10 20 30 40 50 60

NONHDL<130 NONHDL>130

ELEVATED RISK LOW RISK

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LDL VS NON

–

HDL CHOLESTEROL IN RELATION WITH ASCVD RISK

This study population which includes 100 diabetic subjects were divided into two groups based on their triglycerides value . First group includes subjects with TGLs more than 200 and TGLs less than 200 has been included in second group. After that each group is statistically analyzed for significant relation between LDLc with elevated ASCVD risk and non- HDL cholesterol with ASCVD risk score.

GROUP A

[ TGLs<200]

GROUP B [TGLs>200]

TOTAL

NO OF PERSONS 33 67 100

33

67

TRIGLYCERIDES LEVELS

< 200

>200

63

GROUP A ( TRIGLYCERIDES < 200 )

RELATIONSHIP OF LDL CHOLESTEROL WITH ASCVD RISK

LOW RISK ELEVATED

RISK

TOTAL P VALUE LDLc < 100 24 3 27

0.001 LDLc > 100 1 5 6

TOTAL 25 8 33

In group A population [ TGLs < 200] which has 33 subjects , 6 subjects have high LDL cholesterol (>100 mg/dl) .Among them 5 subjects have elevated CVD risk calculated by ASCVD risk calculator. Statistical analysis of above data shows it is statistically significant with P value 0.001 (P<0.05)

0 5 10 15 20 25 30

LDLc <100

LDLc >100 24

1 3

5

HIGH RISK LOW RISK

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RELATIONSHIP OF NON-HDL CHOLESTEROL WITH ASCVD RISK

NON- HDLc LOW RISK ELEVATED

RISK

TOTAL P VALUE <130 24 3 27

0.001 >130 1 5 6

TOTAL 25 8 33

In this group A population [ TGLs < 200] which has 33 subjects , 6 subjects have high non-HDL cholesterol (>130 mg/dl) . Among them 5 subjects have elevated CVD risk calculated by ASCVD risk calculator.

Statistical analysis of above data shows it is statistically significant with P value 0.001 (P<0.05)

0 5 10 15 20 25 30

NON-HDL <130 NON-HDL >130

HIGH RISK LOW RISK

65

LDL VS NON – HDL CHOLESTEROL IN RELATION WITH ASCVD RISK AMONG GROUP A SUBJECTS.

{ TGLs <200 }

In group A population with triglyceride level less than 200 mg /dl, both LDL cholesterol and non- HDL cholesterol were statistically significant in assessing elevated 10 year risk cardiovascular disease calculated through ASCVD risk score.

LOW RISK HIGH RISK 0

5 10 15 20 25

LDL <100

LDL >100

LDL <130

LDL >130

LOW RISK HIGH RISK

66

GROUP B ( TRIGLYCERIDES > 200 )

RELATIONSHIP OF LDL CHOLESTEROL WITH ASCVD RISK

LOW RISK ELEVATED

RISK

TOTAL P VALUE LDLc < 100 4 45 49

0.061 LDLc > 100 5 13 18

TOTAL 9 58 67

In group B population [ TGLs >200] which has 67 subjects , 18 subjects have high LDL cholesterol (>100 mg/dl) .Among them 13 subjects have elevated CVD risk calculated by ASCVD risk calculator. Statistical analysis of above data shows it is statistically not significant with P value 0.06 (<0.05)

0 5 10 15 20 25 30 35 40 45 50

LDLc <100 LDLc >100

4 5

45

13

HIGH RISK LOW RISK