2

PREVALENCE OF AND RISK FACTORS FOR NON

ALCOHOLIC FATTY LIVER DISEASE IN NIGERIANS WITH

TYPE 2 DIABETES MELLITUS IN LAGOS UNIVERSITY

TEACHING HOSPITAL.

BY

DR. OLUSANYA TITILOLA OSAWAYE

A DISSERTATION SUBMITTED TO THE NATIONAL POSTGRADUATE

MEDICAL COLLEGE OF NIGERIA IN PARTIAL FULFILMENT OF

THE REQUIREMENTS FOR THE AWARD OF THE FELLOWSHIP

OF THE FACULTY OF INTERNAL MEDICINE (SUBSPECIALTY:

GASTROENTEROLOGY AND HEPATOLOGY).

MAY 2012

DECLARATION

3

I hereby declare that this work is original unless otherwise acknowledged and that

it has not been presented to any other college for fellowship nor has it been

submitted elsewhere for publication.

_______________________ DATE: ____________________

Olusanya Titilola Osawaye.

SUPERVISION

4

We, the undersigned supervised the execution and writing of the study contained in

this Dissertation.

____________________ DATE: _________________

DR O.A LESI

Gastroenterology and Hepatology Unit,

Department of Medicine

Lagos University Teaching Hospital,

Lagos.

_______________________ DATE: _________________

DR A.A ADEYOMOYE

Department of Radio-diagnosis,

Lagos University Teaching Hospital,

Lagos.

HEAD OF DEPARTMENT’S CERTIFICATION

5

I certify that this work was done by Dr Olusanya Titilola Osawaye under the

supervision of Dr O.A Lesi of the Department of Medicine and Dr A.A

Adeyomoye of Department of Radio-diagnosis Lagos University Teaching

Hospital, Idi – Araba, Lagos, Nigeria.

................................................................. .....................

Prof M.O Kehinde. Date

Professor and Head,

Department of Medicine,

Lagos University Teaching Hospital, Idi – Araba

DEDICATION.

6

This book is dedicated to my Parents for all the love and care you gave me and for instilling in

me the determination to succeed against all odds. To my Siblings, for all your unwavering

support and love, I love you all. To my Darling Husband who has been a pillar of support and

love at all times, I could not have done this without you and to my darling son I am a better

person because of you.

ACKNOWLEDGEMENTS

7

I would like to acknowledge God Almighty, for being there at every stage of this Journey called

Life. I give you all the Thanks and Praise.

I am extremely indebted to my supervisor and teacher, Dr O.A Lesi. Thank you for your

understanding and support throughout my residency.

My sincere gratitude goes to my supervisor Dr Adeyomoye. Thank you for your willingness to

help at all times. Your selflessness has been an inspiration to me.

I would like to extend my sincere gratitude to Dr Kehinde, present Head of Department, who is

like a father to me. My sincere appreciation goes to all members of the Gastroenterology Unit

and all Residents in the Department of Medicine as a whole.

I would also like to acknowledge other academic and non- academic staff of the Department of

Medicine, LUTH for their role during my training.

Finally to all the patients I have come across so far in my career, serving you has been an

enriching experience. Thank you.

LIST OF ABBREVIATIONS.

8

ALD: Alcoholic Liver Disease.

ALT: Alanine Transaminase.

AST: Aspartate Transaminase.

BCG: Bromocresol Green Method

BMI: Body Mass Index (BMI),

CAD: Coronary Artery Disease.

ChREBP: Carbohydrate Regulatory Element Binding Protein.

CI: Confidence Interval.

CKD: Chronic Kidney Disease

CT: Computerized Tomography

CVD: Cardiovascular Disease

DBP: Diastolic blood pressure.

DM: Diabetes Mellitus

DNL: De novo lipogenesis.

ER: Endoplasmic Reticulum

FBS: Fasting Blood Sugar.

FFA: Free Fatty Acid.

FPG: Fasting Plasma Glucose.

HAART: Highly Active Anti Retroviral Therapy.

HbA1c: Glycated heamoglobin

HBV: Hepatitis B Virus.

HBsAg: Hepatitis B Surface Antigen.

HC: Hip Circumference

HCV: Hepatitis C Virus.

HDL-C: High Density Lipoprotein Cholesterol.

Iκκβ- IkappaB kinase-Beta

9

IL6- Interleukin 6

IGT: Impaired Glucose Tolerance

LDL-C: Low Density Lipoprotein Cholesterol

LUTH: Lagos University Teaching Hospital.

mRNA: Messenger Ribonucleic Acid.

MRI: Magnetic Resonance Imaging.

NAFLD: Non Alcoholic Fatty liver Disease.

NASH: Non Alcoholic Steatohepatitis.

NFκβ: Nuclear Factor kappa B.

SBP: Systolic Blood Pressure.

SREBP1c: Sterol Regulatory Element Binding protein 1c

TAG- Triacylglycerol

TCHOL: Total Cholesterol

TNFα- Tumour Necrosis Factor alpha

TG: Triglyceride

T2DM: Type 2 Diabetes Mellitus

UK: United Kingdom

US: United States.

USA: United States of America.

USS: Ultrasound Scan.

VLDL: Very low density lipoprotein.

WC: Waist circumference,

WHR: Waist to Hip Ratio

WHO: World Health Organisation

LIST OF TABLES

10

Table 1: Socio-demographic features distribution of cases and controls. 32

Table 2: Distribution of risk factors for chronic liver disease between diabetic 34

cases and non-diabetic controls.

Table 3: Distribution of Metabolic risk factors between diabetic cases and 36

non diabetic controls

Table 4: Distribution of Clinical measurements between diabetic cases and non 38

diabetic controls

Table 5: Distribution of Biochemical and Viral serological parameters 40

between diabetic cases and non diabetic controls.

Table 6: Distribution of NAFLD between diabetic cases and non diabetic controls. 42

Table7: Distribution of Clinical and Serological parameters among 46

diabetics based on NAFLD status.

Table 8: Distribution of Biochemical Parameters among cases based on 48

NAFLD status.

Table 9: Distribution of Associated risk factors for NAFLD among Diabetics. 50

LIST OF FIGURES.

11

Figure1: Pathogenesis of NAFLD. 13

Figure 2: Bar Chart showing distribution of NAFLD among cases and controls. 43

TABLE OF CONTENTS.

12

DECLARATION ii

SUPERVISION iii

HEAD OF DEPARTMENT’S CERTIFICATION iv

DEDICATION v

ACKNOWLEDGEMENT vi

LIST OF ABBREVIATIONS vii

LIST OF TABLES ix

LIST OF FIGURES x

TABLE OF CONTENTS xi

SUMMARY xii

CHAPTER 1.0 INTRODUCTION 1 - 4

1.1 JUSTIFICATION FOR THE STUDY 5

1.2 AIMS AND OBJECTIVES 6

CHAPTER 2.0 LITERATURE REVIEW 7 – 21

CHAPTER 3.0 STUDY DESIGN AND METHODS 22-30

CHAPTER 4.0 RESULTS 31 - 50

CHAPTER 5.0 DISCUSSION 51 - 60

CHAPTER 6.0 CONCLUSIONS AND RECOMMENDATIONS 61-62

REFERENCES 63-74

APPENDICES 75-85

SUMMARY

13

Background: Non Alcoholic Fatty Liver Disease (NAFLD) describes a spectrum of diseases

ranging from simple steatosis to non alcoholic steatohepatitis with or without fibrosis to cirrhosis

and liver failure. It is a common cause of Chronic Liver Disease and its presence is associated

with increased morbidity and mortality. In view of the increase in the number people with known

risk factors for NAFLD such as Type 2 Diabetes Mellitus (T2DM) and Obesity worldwide

including Sub-Saharan Africa, it is rapidly emerging as a public health issue. It has also been

implicated in increasing both microvascular and macrovascular complications in people with

Type 2 Diabetes Mellitus. Radiological investigations such as ultrasound scans can aid the

diagnosis of NAFLD. Prevalence of NAFLD in T2DM varies worldwide and certain risk factors

for developing NAFLD in T2DM such as Obesity and Dyslipidemia have been well documented

in world Literature but not much work has been done with regards to Nigeria and Africa as a

whole. This study aims to address this paucity of data concerning Prevalence of and associated

risk factors for NAFLD in patients with T2DM in this part of the world.

Objectives: To determine prevalence of NAFLD as well as associated risk factors for NAFLD in

patients with T2DM and also to compare some biochemical parameters such as fasting lipid

profile and Liver function tests in diabetics based on their NAFLD status.

Research Design and Methodology: This is a prospective case control study of 336 subjects

comprising of 168 cases with T2DM and 168 non diabetic controls. This study was carried out at

Lagos University Teaching Hospital. Consecutive patients who satisfied the study criteria were

recruited and an investigator administered questionnaire was filled to determine risk factors for

liver disease and other metabolic conditions. Physical examination was carried for signs of liver

14

disease and anthropometric measurements were done. Fasting blood samples were taken for lipid

profile, liver enzymes, and serology for Hepatitis B and C. Abdominal ultrasound scan was done

in order to determine radiological evidence of fatty liver using Aloka Pro-sound 3500 machine

equipped with a with 3.75MHz probe after a 6 hour fast by the subjects.

Results: A higher prevalence of fatty liver was found in patients with T2DM compared with the

non diabetic controls (16.7% versus 1.2% P=0.00). The odds of developing NAFLD were

increased 16.6 fold in patients with type 2 DM, odds ratio 16.6 (CI 95%, 3.9-70.9). The mean

transaminases, TCHOL, LDL-C and TG, though higher in diabetics with NAFLD than those

without was not statistically significant. Only 2 diabetics with NAFLD had elevated

transaminases. Obesity including Central obesity using the waist circumference was significantly

higher in diabetics with NAFLD compared with those without. Male diabetics with NAFLD had

lower HDL-C than those without NAFLD.

Conclusions: The prevalence of NAFLD in type 2 DM, though not as high as in studies from the

western world was still very significant, as people with T2DM were 16.6 times more likely to

develop NAFLD compared to non-diabetics. Biochemical parameters like fasting lipid profile

and liver biochemistry were not significantly different in T2DM with regard to their NAFLD

status. The presence of NAFLD in T2DM was associated with Obesity including Central Obesity

and low HDL-C in males.

CHAPTER ONE

15

INTRODUCTION

Nonalcoholic fatty liver disease (NAFLD) is rapidly becoming a worldwide public health

problem and is the most common cause of chronic liver disease in the Western World.1,2 It is

characterized by insulin resistance and hepatic steatosis, in the absence of other identifiable

causes of fat accumulation such as alcohol abuse, viral hepatitis, autoimmune hepatitis, alpha-1

antitrypsin deficiency, medications like corticosteroids and estrogens, and other conditions.3

Although previously considered a disease of exclusion, NAFLD should now be suspected in all

patients with associated metabolic risk factors such as hypertension, type 2 diabetes (T2DM),

hyperlipideamia, and truncal obesity.4,5 It is the most common cause of cryptogenic cirrhosis.4,5

Hepatic steatosis may range from a 'benign' indolent deposition of fat to severe lipotoxicity-

induced steatohepatitis with necroinflammation known as nonalcoholic steatohepatitis (NASH)

which can progress further to end stage liver disease.6 Many clinical symptoms of fatty liver are

nonspecific or silent with hepatomegaly being the only physical finding in most patients.7,8

Usually, mild to moderate elevation of serum aspartate or alanine aminotransferase activities

may be the only laboratory abnormality.7 NAFLD is however the commonest cause of

unexplained persistent elevation of these enzymatic activities.9 Radiological investigations such

as Ultrasound Scan which is available and affordable can also aid diagnosis and has specificity

and sensitivity of 93% and 89% respectively in detecting hepatic steatosis.10 Liver biopsy is

however the gold standard as it can differentiate between Hepatic Steatosis and NASH.

The perception of nonalcoholic fatty liver disease as an uncommon and benign condition is

rapidly changing.6 In the United States of America (USA), current estimates are that 20% of the

general populations and 33% of potential live liver donors undergoing liver biopsy are affected

with NAFLD. 1 The prevalence in the morbidly obese population is as high as 75%-92%.1 At

16

present it is estimated that six million individuals in the US general population have progressed

from hepatic steatosis to nonalcoholic steatohepatitis and about 600,000 to NAFLD- related

cirrhosis.1 Thus the number of individuals at risk for end stage liver disease, development of

primary liver cell cancer, and those potentially eligible for liver transplant is large.1 In Europe,

the Dionysos study reported the prevalence rate of NAFLD to be 25% of the general population

in Italy. 11 In Asia, the prevalence rate of NAFLD is 12%-24% depending on age, gender,

locality and ethnicity.12

In Africa, Researchers in Western Cape, South Africa showed that 48% of Overweight/obese

patients that were screened had histologically confirmed NAFLD. 13 In a study in Lagos, Nigeria,

the prevalence rate of Non-alcoholic fatty liver disease among patients with type 2 diabetes

mellitus was 9.5%.14 Another study done in Lagos, Nigeria, showed the prevalence rate of

hepatic steatosis in HIV positive people on Highly Active Anti Retroviral Therapy (HAART)

was 13.3%.15 There however appears to be paucity of data in Africa.

Globally the prevalence of NAFLD appears to be increasing in part due to the increasing

number of adults with various components of the metabolic syndrome such as truncal obesity,

T2DM and Hyperlipideamia (which are risk factors for developing NAFLD).1 Of particular

interest is Diabetes Mellitus (DM).

Diabetes Mellitus is a chronic metabolic disorder of carbohydrate, protein and lipid metabolism

characterized by chronic hyperglycemia due to absolute or relative insulin deficiency/ insulin

resistance. The prevalence of Diabetes mellitus (DM) is reaching pandemic proportions as it

affects more than 120 million people world-wide, and it is estimated that it will affect 370 million by

the year 2030.16 In 2007, the estimated prevalence of DM in the USA was 7.8% of which almost

one third of the cases were undiagnosed.17 More than 90% of cases of diabetes are T2DM. 17

17

T2DM is less common in non-western countries where the diet contains less calories and

caloric expenditure on a daily basis is higher. However as people in these countries adopt

western lifestyle; there will be an increase in weight and T2DM.18, 19 T2DM also has a strong

genetic component with various studies showing family history as a significant risk factor.20-22

The prevalence rate of diabetes is 2.2% and is the most important non communicable disease in

Nigeria.23 Gender distribution of T2DM varies in Africa with no discernible pattern which is in keeping

with global findings. 24, 25 T2DM is a major cause of morbidity and mortality because of its

insidious onset and late recognition in resource poor nations like Nigeria. 26

In USA, liver disease is one of the leading causes of death in persons with T2DM and the

standardized mortality rate for death from liver disease is greater than that for cardiovascular

disease.27 The spectrum of liver disease in T2DM ranges from Nonalcoholic Fatty Liver Disease

to Cirrhosis and Hepatocellular Carcinoma. The incidence of Hepatitis C and acute liver failure

is also on the increase.27 Fatty liver is considered to occur commonly in T2DM, with estimates of

prevalence ranging from 21% -78%. 7, 28, 29 The prevalence of NAFLD among people with

T2DM is 55.8% in Tehran, Iran. 30 While some authors in Brazil, South America put the

prevalence of NAFLD in T2DM at 69.4%. 31

Obesity, insulin resistance, and increased concentrations of plasma fatty acids are considered to

increase the risk for fatty liver. 32, 33 Each of these metabolic factors is also characteristic of

T2DM.32,33 Approximately 70% of persons with T2DM have hepatic steatosis which follows a

more aggressive course with necroinflammation and fibrosis.6

The consequences of NAFLD are such that subjects with NAFLD have been shown to have

significant decrement in quality of life compared with US populations with and without other

chronic illnesses.34 Recent studies have also suggested that NAFLD may be a marker and

18

mediator of coronary artery disease.35 To further support this, some authors in Italy, showed a

high prevalence of NAFLD amongst T2DM; and its association with a higher prevalence of

cerebrovascular disease, coronary artery disease (CAD) as well as peripheral vascular disease

compared with subjects with T2DM without NAFLD.36 The risk of Hepatocellular Carcinoma is

also increased in patients with T2DM and NASH.37 NAFLD may also be associated with a

greater risk of renal disease in patients with T2DM.38

To the best of my knowledge, one study has been done locally on the prevalence of NAFLD in

type 2 DM, 14 but none has assessed associated risk factors for NAFLD in patients with T2DM in

Lagos Nigeria. This current study seeks to establish the prevalence as well as identify associated

risk factors for NAFLD in patients with T2DM in view of the paucity of data from Nigeria and

Africa as a whole.

19

1.1 JUSTIFICATION FOR THE STUDY.

Liver disease is a major cause of morbidity and mortality in people with DM. 25 Liver

abnormalities in DM patients may be discovered incidentally while they are being investigated

for other conditions. NAFLD is one of the many hepatic diseases commonly seen in T2DM and

is almost a universal finding in diabetics that are obese.25 Most patients with liver steatosis or

NASH are asymptomatic and have no signs of liver disease at the time of diagnosis. 7,8, Even

when present, symptoms and findings are nonspecific and do not correlate well with the severity

of the disease. 7,8,

Abdominal Ultrasound Scan has been found to be an effective radiological tool in diagnosing

hepatic steatosis and is of paramount importance in resource poor settings as it is affordable and

available in comparison to other radiological modalities. 10

Unfortunately, people with DM are more likely to have other risk factors for developing

NAFLD, such as obesity and hyperlipideamia and are more likely to progress to NASH. 6,32,33

The diagnosis of NAFLD in T2DM has been found to be associated with the risk of both macro-

and microvascular complications like coronary artery disease (CAD) and DM nephropathy

respectively. 36,38 Hence in view of the large number of people affected with T2DM worldwide,

coupled with the high prevalence of NAFLD in them, a large group of people are at risk of

developing these complications which will have a negative impact on both the individual and

society at large. 7,16,28,29.

This current study will help fill a gap in local literature and identify associated risk factors for

NAFLD in patients with T2DM and NAFLD. It will also highlight a group of patients that need

20

to be closely followed up in view of the association between NAFLD and increased risk of

coronary artery disease (CAD) and chronic kidney disease (CKD) in order to reduce the

occurrence of these events. 36,38

1.2 AIMS AND OBJECTIVES.

AIM

To determine the prevalence and risk factors of NAFLD in Type 2 DM.

OBJECTIVES

To determine the prevalence of NAFLD in T2DM and controls.

To compare some biochemical parameters (e.g. liver function, lipid profile) in T2DM

based on NAFLD status.

To identify associated risk factors for NAFLD such as Dyslipidemia and Obesity

including Central Obesity in T2DM.

.

.

21

CHAPTER TWO.

LITERATURE REVIEW.

2.1.1 DIABETES MELLITUS: GLOBAL TREND IN ITS PREVALENCE.

DM affects more than 120 million people world-wide, and it is estimated that it will affect 370

million by the year 2030.16 In the second half of the twentieth century, it became obvious that a

relentless increase in T2DM affecting the economically affluent countries, is also gradually

afflicting the developing world. Presently, the highest prevalence of DM which is more than 13%

is in Saudi Arabia.39 The prevalence of DM is very high, with over 10% of adults in the USA,

Switzerland and Austria affected.39 Prevalence is low in Norway, China and in Iceland.39 T2DM

is less common in non-western countries where the diet contains fewer calories and caloric

expenditure on a daily basis is more. However as people in these countries adopt western

lifestyle, there will be an increase in weight and T2DM.18,19

Predictions of epidemiologists for the first third of the twenty-first century claim up to 2.5

times increase in DM in the Middle East, Sub-Saharan Africa, India, rest of Asia and in the Latin

America.39 In China the number of patients with DM will double but in the economically

advanced countries that experienced rise in DM in the twentieth 20th century, the increase will be

only about 50%.39 Remarkably, the lowest increase in DM is expected in the countries that

22

formerly belonged to the Soviet Union.39 The prevalence rate of diabetes in Nigeria is 2.2% and

it is the most important non communicable disease.23

2.1.2 THE LINK BETWEEN DIABETES AND NON ALCOHOLIC LIVER DISEASE.

The spectrum of liver disease in T2DM ranges from nonalcoholic fatty liver disease to cirrhosis

and Hepatocellular carcinoma. The incidence of hepatitis C and acute liver failure is also on the

increase in T2DM.27 In the USA, liver disease is one of the leading causes of death in persons

with T2DM and the standardized mortality rate for death from liver disease is greater than that

for cardiovascular disease.27

Diabetes Mellitus is a chronic metabolic disorder of carbohydrate, protein and lipid metabolism

characterized by chronic hyperglycemia due to absolute or relative insulin deficiency/ insulin

resistance. The hallmark of type 2 diabetes is insulin resistance which has also been implicated in the

pathogenesis of NAFLD. Insulin Resistance causes a compensatory hyperinsulinemia which if persistent

can eventually lead to T2DM. Hyperinsulinemia and insulin resistance increase both hepatic

glucose production and adipose tissue free fatty acid (FFA) release, respectively. These

circulating FFA are taken up by the liver and undergoes either β-oxidation or is esterified with

glycerol to form triglycerides, which then causes accumulation of fat in the liver.8

Fatty liver is presently viewed as the hepatic component of the Metabolic Syndrome, a

syndrome which also includes central obesity, dyslipidemia, hypertension and insulin

resistance/impaired glucose tolerance. The relationship between Diabetes Mellitus and NAFLD

is such that they share common risk factors such as obesity and insulin resistance.

23

2.2 NAFLD: HISTORICAL PERSRECTIVE &EPIDEMIOLOGY.

Nonalcoholic fatty liver disease (NAFLD) represents a spectrum of conditions characterized

histologically by macrovesicular hepatic steatosis and occurs in those who do not consume

alcohol in amounts generally considered to be toxic to the liver.10 NAFLD includes both Hepatic

steatosis and NASH with or without varying degrees of fibrosis and cirrhosis. It is rapidly

becoming a worldwide public health problem.1 More than half the world's population are at risk

of developing NAFLD or in extreme cases non-alcoholic steatohepatitis or cirrhosis in the

coming decades because of the increasing prevalence of obesity, coupled with T2DM,

dyslipidemia, hypertension and ultimately metabolic syndrome which are risk factors for

developing NAFLD.12

Hepatologists actually recognized what is now called NASH in the 1970's, but it was

described under different names. In 1980, Researchers at Mayo Clinic noted that certain

individuals with common features like DM, Obesity, and Hyperlipideamia had a liver disease

that closely resembled alcoholic liver disease (ALD).40 However, these patients drank no alcohol

hence the term nonalcoholic steatohepatitis (NASH). For many years, however, NASH was

thought to be a totally harmless condition. Or, it was considered an innocent bystander that was

associated with other liver diseases especially viral hepatitis. In those years, simple fatty liver

(steatosis) and NASH were considered to be the same. A conceptual division of NAFLD into

‘big’ and ‘little’ NASH was proposed at a consensus conference in 1998. It was also noted at that

time that there was a spectrum of disorders which appropriately fell under the broad term ‘fatty

liver disease’. Since then this group has published a refined classification of NAFLD.41 .

In the USA, current estimates are that 20 % of the general population is affected with NAFLD.1

The prevalence in the morbidly obese population has been estimated to be as high as 75%-92%.1

24

At present it is estimated that six million individuals in the US general population have

progressed from fatty liver to nonalcoholic steatohepatitis and about 600,000 to NAFLD- related

cirrhosis.1 Thus the number of individuals at risk for end stage liver disease, development of

primary liver cell cancer, and those potentially eligible for liver transplant is large.1 The

Dionysos study which was done in two towns in Northern Italy reported the prevalence rate of

NAFLD in the general population to be 25%.11

Generally in Asia, the prevalence rate of NAFLD is estimated to be 12%-24%.12 Hepatic

steatosis is highly prevalent in China and it is more often linked to obesity than to alcoholism.

Among more affluent regions of China, the community prevalence of non-alcoholic fatty liver

disease (NAFLD) is approximately 15%.42 With the increasing pandemic of obesity, the

prevalence of NAFLD has approximately doubled in the past decade in China.42 In India, a

population based study was carried out and it was suggested that the prevalence of NAFLD in

India is comparable to the West.43

In Africa, researchers in Western Cape, South Africa showed that 48% of Overweight patients

that were screened had histologically confirmed NAFLD; all patients had insulin resistance or

were found to have DM. This study was first of its kind in South Africa.13 In Nigeria, a study

was done on NAFLD and the metabolic syndrome and it was observed that though the

prevalence rate of Non-alcoholic fatty liver disease among patients with type 2 diabetes mellitus

was found to be higher than the control group (9.5 vs. 4.5, p=0.2) it was not statistically

significant and that though NAFLD was present in Africans, it is less than American and

European studies.14 Another study done in Nigeria showed the prevalence rate of hepatic

steatosis in HIV positive people on Highly Active Anti Retroviral Therapy (HAART) was

13.3%.15 Generally there appears to be paucity of data in Africa. A study that was done in a

25

racially diverse country like the USA in order to determine the racial and ethnic distribution of

NAFLD showed in decreasing order of prevalence of NAFLD (Hispanics > Asians > Black

Americans >other races). These racial differences may reflect genetic susceptibility to visceral

adiposity.44

Prevalence of NAFLD appears to be increasing due to the increasing number of adults who

have various components of the metabolic syndrome such as Insulin resistance/ T2DM, Truncal

Obesity and Hyperlipideamia, (all risk factors for development of NAFLD).1

NAFLD is very common in T2DM with an estimated prevalence rate ranging from 9.5% -

78%.7,14,28,29 A study done in Turkey showed 14.81% of four thousand five hundred and thirty

six patients attending gastroenterology clinic were found to have hepatic steatosis of which

27.5% had DM.45 In Tehran, Iran the prevalence rate of people with T2DM who had abdominal

ultrasound evidence of NAFLD was 55.8% and it was concluded that it was a common finding

among T2DM.30 In India, the prevalence rate of NAFLD in T2DM is 62.3%.10 In a cross

sectional study done in Brazil, South America, 69.4% of 180 patients with T2DM had ultrasound

features of NAFLD and it was observed that NAFLD in T2DM patients will progress

independent of the diabetes progression itself.31

As a rule, the prevalence of NAFLD is higher in males, increases with age, and varies

depending on the diagnostic method used and the characteristics of the population, especially

lifestyle habits. Population-based studies provide better estimates of the prevalence of NAFLD.11

The diagnosis of NAFLD in population studies is usually obtained by ultrasonography.11 The

point prevalence of NAFLD at each of its different stages: simple steatosis, NASH and cirrhosis

remains poorly defined throughout the world. 46-49

26

2.3 PATHOGENESIS OF NAFLD.

Initial theories for the pathogenesis of NAFLD were based on a ‘2-hit hypotheses

The ‘first hit’, hepatic triglyceride accumulation, or steatosis, increases susceptibility of the

liver to injury mediated by ‘second hits’, such as inflammatory cytokines/adipokines,

mitochondrial dysfunction and oxidative stress, which in turn lead to steatohepatitis and/or

fibrosis.50, 51 However, there is increasing recognition of the role that free fatty acids (FFA) play

in directly promoting liver injury, which has led to modification of this theory. In obesity and

Insulin resistance there is an increased influx of FFA to the liver. These FFA either undergo β-

oxidation or are esterified with glycerol to form triglycerides, leading to hepatic fat

accumulation.52

There is now substantial evidence that FFA can directly cause toxicity by increasing oxidative

stress and by activation of inflammatory pathways.53 Therefore hepatic triglyceride accumulation

may be a protective mechanism by preventing the toxic effects of unesterified FFA.54

Additionally, a further component, or ‘third-hit’ has been added to reflect inadequate hepatocyte

proliferation.55 In the healthy liver, cell death stimulates replication of mature hepatocytes which

replace the dead cells and reconstitute normal tissue function.55 However oxidative stress, a

central feature of NAFLD pathogenesis, inhibits the replication of mature hepatocytes which

results in expansion of the hepatic progenitor cell (oval cell) population.56 These cells can

differentiate into hepatocyte-like cells, and both oval cell and intermediate hepatocyte-like cell

numbers are strongly correlated with fibrosis stage, suggesting that cumulative hepatocyte loss

promotes both accumulation of progenitor cells and their differentiation towards hepatocytes.56

In chronic liver injury, the development of fibrosis/cirrhosis is dependent on the efficacy of

27

hepatocyte regeneration, and therefore cell death with impaired proliferation of hepatocyte

progenitors represents the proposed ‘third hit’ in NAFLD pathogenesis.55

FIGURE1: PATHOGENESIS OF NAFLD. 52

28

29

TAG- Triacylglycerol Iκκβ- IkappaB kinase-Beta NFκβ- Nuclear Factor kappa B

TNFα- Tumour Necrosis Factor alpha IL6- Interleukin 6 FFA- Free Fatty Acid

ER- Endoplasmic Reticulum

(a) The traditional 2-hit hypothesis: steatosis represents the ‘first hit’, which then sensitises the

liver to injury mediated by ‘second hits’, such as inflammatory cytokines, adipokines, oxidative

stress and mitochondrial dysfunction, leading to steatohepatitis and fibrosis. The presence of

high levels of oxidative stress reduces the ability of mature hepatocytes to proliferate, resulting

in reduced endogenous liver repair. 52

(b) Modified 2-hit hypothesis: the accumulation of FFA alone has been suggested to be sufficient

to induce liver damage, without recourse for a second hit. Indeed, rather than being harmful,

triglyceride accumulation in the form of steatosis may actually be protective by preventing FFA-

induced inflammation and oxidative stress. 52

30

(c) The 3-hit hypothesis: oxidative stress reduces the ability of mature hepatocytes to proliferate,

resulting in the recruitment of other pathways of liver regeneration, such as Hepatocyte

Progenitor Cells. These cells have the capability of differentiating into both cholangiocytes and

hepatocytes and contributing to liver repair. It has been suggested that an inability to mount such

a ductular response, as is seen in patients transplanted for NASH who have denervated livers,

may be responsible for a more progressive pattern of liver damage. Thus, impaired proliferation

of hepatocyte progenitors represents the proposed ‘third hit’ in NAFLD pathogenesis.55

NAFLD is characterized by the accumulation of triglycerides, which are formed from the

esterification of FFA and glycerol within the hepatocyte. FFAs arise in the liver from three

distinct sources; lipolysis (the hydrolysis of FFA and glycerol from triglyceride) within adipose

tissue, dietary sources, and de novo lipogenesis (DNL).57 In contrast, FFA may be utilized either

through β-oxidation, re-esterification to triglycerides and storage as lipid droplets, or packaged

and exported as very low density lipoprotein (VLDL). Hence hepatic fat accumulation can occur

as a result of increased fat synthesis, increased fat delivery, decreased fat export, and/or

decreased fat oxidation. 52

Human hepatocytes incubated with unsaturated fatty acids accumulate large amounts of

triglyceride without harm, but saturated fatty acids readily cause endoplasmic reticulum stress

and apoptosis, as they are poorly incorporated into triglyceride.58 Fatty acids impair insulin

signaling and activate proinflammatory serine/threonine kinases and promote the accumulation

of diacylglycerol (DAG) , and enhance the expression of cytokines such as TNF-α and IL-1β.59

Unsaturated fatty acids prevent palmitate-induced apoptosis by channeling palmitate into less

harmful triglyceride pools and away from apoptotic pathways.60 Saturated fatty acids induce

31

mitochondrial dysfunction and oxidative stress by mechanisms dependent on lysosomal

disruption and activation of cathepsin B.61

Among the metabolic defects, chronic hyperinsulinemia and hyperglycemia as observed in

T2DM are of paramount importance as they promote lipogenesis by upregulating hepatic sterol

regulatory element binding protein 1c (SREBP1c) and carbohydrate regulatory element binding

protein (ChREBP) activity, respectively. 6

High carbohydrate/fructose-based diets promote de novo lipogenesis and activate harmful

inflammatory pathways with hepatocyte apoptosis.62 Increased consumption of dietary

carbohydrates is common in patients with NAFLD and has recently been associated with

increased hepatic mRNA expression of fructokinase, a key enzyme for fructose metabolism, and

in fatty acid synthase, an important lipogenic enzyme.63 Restriction of dietary carbohydrates

reduces hepatic fat and elevated ALT in obese patients with NAFLD. 64,65

In T2DM excessive rates of lipolysis from insulin-resistant adipose tissue is also a driving

force for the development of steatosis.59,66,67 Adipocytes account for approximately 60-70% of

the free fatty acid (FFA) used for hepatic triglyceride synthesis and very low-density lipoprotein

(VLDL) secretion.68

2.4 RISK FACTORS FOR DEVELOPING NAFLD IN TYPE 2 DM.

NAFLD is believed to be the hepatic component of the metabolic syndrome of which insulin

resistance/DM is one of its components. Other components of the metabolic syndrome include

abdominal obesity, hypertension and dyslipidemia which are pathological conditions frequently

associated with NAFLD and their coexistence in the same individual increases the likelihood of

having more advanced forms of liver disease.6

32

Of all the documented risk factors for NAFLD, Obesity has the strongest association with

NAFLD. In obesity there is an increased influx of FFA to the liver which either undergoes β-

oxidation or is esterified with glycerol to form triglycerides, leading to hepatic fat

accumulation.52

It is important to note that these same components of metabolic syndrome as earlier mentioned

also occur commonly in T2DM. A high prevalence of hypertension in people with DM has been

reported worldwide 69-72 as insulin resistance may be responsible for the occurrence of

hypertension in people with diabetes mellitus, coupled with the occurrence of Diabetes Mellitus

Nephropathy a microvascular complication of diabetes mellitus. Obesity is also very common in

people with T2DM as the risk of diabetes increases exponentially as BMI increases above 25kg/m2.

Dyslipidemia another component of the metabolic syndrome and a known risk factor for NAFLD

is frequently seen in people with T2DM. 73-77

A substantial proportion of the mortality in patients with type 2 diabetes mellitus (T2DM) is

related to non-alcoholic fatty liver disease (NAFLD) and its complications.31 Hence it is of vital

importance to identify associated risk factors for developing NAFLD in patients with T2DM in

order for preventive and curative measures to be put in place.

Studies have been done to assess risk factors for developing hepatic steatosis in patients with

established T2DM. A study done in Brazil, South America, observed that the presence of

NAFLD in T2DM was associated with obesity mainly abdominal, Hypertriglyceridaemia and

high or normal Alanine transferase (ALT) levels.31

Some authors in India however had different findings as the presence of central obesity did not

predict the presence or severity of NAFLD or fibrosis in patients with type 2 DM.10 They

33

however went further to show that a family history of DM, duration of DM and glycaemic

control also did not predict its presence. 10

Similarly another group of authors in India evaluated risk factors for developing NASH in

T2DM and concluded that risk factors such as central obesity, dyslipidemia and family history of

chronic liver disease did not predict the development of NASH in T2DM.78

The Edinburgh Type 2 Diabetes Study observed that people with T2DM who had hepatic

steatosis were significantly younger, had a shorter duration of diabetes and had higher body mass

index (BMI), waist circumference, Glycated heamoglobin (HbA1c), Triglyceride level and

significantly lower High Density Lipoprotein (HDL) level and use of Metformin was also more

common in them. 79

Researchers in Iran observed that people with T2DM and NAFLD also had higher BMI and

triglyceride levels than controls without NAFLD and that the degree of insulin resistance was not

predictive of fatty liver amongst them. 30

Researchers in USA showed that fatty liver is relatively common in overweight and obese

people with T2DM. It was also found to be common in people with T2DM and Dyslipidemia. 80

In Pakistan, Obesity, Dysglycemia, Dyslipidemia, higher BMI and elevated liver enzymes

were seen more commonly in fatty liver than non fatty liver type 2 diabetic patients. 81

2.5 CLINICAL FEATURES AND DIAGNOSIS OF NAFLD.

Most patients with NAFLD are asymptomatic and have no sign of liver disease at time of

diagnosis. . 7,8 They may however complain of persistent fatigue, malaise or upper abdominal

discomfort. Hepatomegaly may be the only physical finding in most patients. 7,8 Other findings

suggestive of chronic liver disease such as ascites may point to the presence of cirrhosis due to

34

progressive NASH. Diagnosis is one of exclusion, the absence of alcohol abuse, viral,

autoimmune, metabolic, heredity or other known causes of liver pathology must be ascertained.82

Generally absence of alcohol abuse or consumption of less than 20grams/day and negative

serological tests for hepatitis B and C should raise the suspicion of NAFLD. 82

Mild to moderate elevation of serum aspartate or alanine aminotransferase activity is usually

the only laboratory abnormality, and NAFLD is the commonest cause of unexplained persistent

elevation of these enzymatic activities, once other known etiologies of chronic liver disease have

been ruled out.10 The ratio of aspartate aminotransferase to alanine aminotransferase is usually

less than 1, but it increases with progression of fibrosis, leading to a loss of diagnostic value in

patients with cirrhosis. 7, 8 Serum alkaline phosphatase or gamma-glutamyltransferase activities

may be mildly elevated. Presence of antinuclear antibodies in the serum, the significance of

which is unknown, has been reported in 10% - 25% of patients.10 Thirty to fifty percent of

patients with NASH have diabetes or glucose intolerance.9,83-85 Twenty to Eighty percent of

patients with NAFLD have hypertriglyceridemia.9,83,84,86 Other abnormalities, including

hypoalbuminemia, prolonged prothrombin time and hyperbilirubinemia, can be seen in patients

with cirrhosis.

Radiological imaging can be used to diagnose NAFLD. However they do not distinguish

between fatty liver, steatohepatitis and steatohepatitis with fibrosis. Ultrasound Scan,

Computerized Tomography Scan, Magnetic Resonance Imaging and Photon Magnetic

Resonance Spectroscopy have all been used. A recent study showed that ultrasound, CT scan and

MRI have similar diagnostic accuracy for quantifying the severity of steatosis when fat

deposition is more than 33% of the liver volume.87 While photon magnetic resonance

spectroscopy has greater sensitivity than the other three modalities and has been shown to detect

35

as little as 5% fat deposition in the liver.88 USS is however the most common modality used

because of its availability and its affordability, factors which must be considered especially in

resource poor settings. It has specificity and sensitivity of identifying fatty liver of 93% and 89%

respectively.10 Findings on USS include increased parenchymal echoes, hepatorenal contrast and

attenuation of the diaphragm. 82

Liver biopsy is however the gold standard in diagnosing NAFLD and has the advantages of

being able to distinguish fatty liver, steatohepatitis and steatohepatitis with fibrosis. Histological

findings in NAFLD are similar to alcoholic liver disease. These histological changes range from

simple macrovesicular steatosis to NASH. They also include ballooning degeneration, acute,

chronic or mixed inflammation, peri-sinusiodal fibrosis and Mallory hyaline bodies.82

A non invasive system was postulated which identifies liver fibrosis in patients with NAFLD

using six parameters; age, Body mass index (BMI), Impaired glucose tolerance (IGT)/Diabetes,

ratio of Aspartate transaminase (AST) to Alanine transaminase (ALT), platelet count and serum

albumin levels in which their findings showed that advanced fibrosis could be accurately

diagnosed using this score thereby rendering liver biopsy as unnecessary in a large proportion of

patients.89

2.6 CLINICAL CONSEQUENCES OF HEPATIC STEATOSIS.

The burden of NAFLD cannot be over emphasized; simple hepatic steatosis may progress to

NASH most especially in individuals with more than one metabolic risk factor for NAFLD.

NASH in itself is frequently associated with fibrosis and approximately 10% of patients with it

will develop cirrhosis.6 Hence with the growing number of people being affected globally,

morbidity and mortality from NAFLD- related complications are expected to be on the increase.

36

The consequences of NAFLD are such that subjects with NAFLD have significant decrement

in quality of life compared with US populations with and without other chronic illnesses.34

Recent studies have also suggested that NAFLD may be a marker and mediator of coronary

artery disease.35 Diabetes, dyslipidemia, hypertension, and cardiovascular disease (CVD) occur

more frequently in individuals with NAFLD.90 Up to 75% of patients with T2DM and over 50%

of patients with a history of myocardial infarct have NAFLD.91-93 To further support this, some

authors in Italy, also showed a high prevalence of NAFLD amongst T2DM and its association

with a higher prevalence of cerebrovascular disease, coronary artery disease as well as peripheral

vascular disease compared with subjects with T2DM without NAFLD.36

On a larger scale, in the UK, the health and economic burden from NAFLD is expected to rise

in parallel to the increasing prevalence of T2DM and childhood obesity.94-97 Health care costs

have long been suspected to be higher in NASH patients, a finding recently confirmed in a large

cohort study from Western Germany.98 Hence a diagnosis of NAFLD is not only a burden on the

individual but also on the society at large.

Approximately 70% of T2DM Patients with NAFLD will have hepatic steatosis which follows

a more aggressive course that may end up with NASH. 6 NASH is an overlooked complication of

T2DM that if missed may carry serious long-term consequences.6 The risk of Hepatocellular

carcinoma is also increased in patients with T2DM and NASH.37 Unfortunately studies have

shown that patients with NAFLD are significantly more likely to die or suffer transplant failure

during the first thirty days after Liver transplant than patients without NAFLD and of the co

morbidities frequently associated with NAFLD, DM was associated with worse outcomes and

was independently associated with poorer survival at three years after transplant.99 NAFLD may

also be associated with a greater risk of renal disease in patients with T2DM, the implication of

37

which may highlight a subgroup of people with T2DM and NAFLD that will need more

intensive therapy to decrease the risk of CKD and CVD events. 38

In conclusion, a diagnosis of NAFLD is associated with an increased risk of overall death

among patients with diabetes mellitus.100

CHAPTER THREE

METHODOLOGY.

3.1 DESCRIPTION OF STUDY AREA.

The study was carried out at the Endocrinology and other medical out-patients clinics and wards

of the Lagos University Teaching Hospital (LUTH), Idi-Araba, Surulere in Lagos state, South-

West, Nigeria. LUTH is a tertiary referral centre providing both medical and surgical care to the

general population of Lagos State.

3.2 STUDY DESIGN.

This is a prospective case control point prevalence study in which subjects with established

T2DM who fulfil the inclusion criteria were recruited in the study. The controls were not

previously known to be diabetic or known to have symptoms suggestive of diabetes mellitus and

had a Fasting Blood Sugar level less than 126mg/dl on screening. 101

3.3 SAMPLING METHOD

38

3.3.1 STUDY POPULATION.

The subjects were consecutively consenting patients attending the Endocrinology Out- Patient

Clinic, Lagos University Teaching Hospital Idi-Araba Lagos, Nigeria, who had established

T2DM according to American Diabetes Association (ADA) diagnostic criteria, 101

i.e.

Symptoms of diabetes plus random plasma glucose concentration ≥200 mg/dl (11.1

mmol/l). Random was defined as any time of day without regard to time since last meal.

The classic symptoms of diabetes include polyuria, polydipsia, and unexplained weight

loss.

Or

Fasting Plasma Glucose (FPG) ≥126 mg/dl (7.0 mmol/l). Fasting was defined as no

caloric intake for at least 8 hours.

In the absence of symptoms, the test was repeated on another day.

The recruitment was carried between December 2011 and February 2012.

3.3.2 SELECTION OF CONTROLS.

Controls were consecutively consenting non- diabetic patients who had been screened for the

absence of diabetes using history (absence of polyuria, polydipsia, unintentional weight loss) and

fasting blood sugar level less than 126mg/dl using a glucometer (Accu-chek glucometer, Roche

diagnostics). They were selected from medical Out-Patient Clinics and medical wards of the

same hospital. The recruitment was carried between December 2011 and February 2012.

39

3.3.3 CONSENT

Informed consent (written and verbal) was obtained from all participating subjects prior to

enrollment into the study. (Appendix I)

3.4 INCLUSION CRITERIA.

The subjects recruited into this study were consecutive consenting adults older than 18 years, of

either sex who had established T2DM, and were on oral anti diabetic agents with or without

Insulin or were being controlled with diet.

The controls were consecutive consenting adults older than 18 years who were not previously

known to have diabetes mellitus or symptoms suggestive of diabetes mellitus and had a Fasting

Blood Glucose level less than 126mg/dl on screening for DM.

3.5 EXCLUSION CRITERIA FOR SUBJECTS.

Exclusion criteria were

History of alcohol intake in excess of 20 grams per day.

History of jejunoileal bypass or extensive small bowel resection

Usage of drugs known to cause secondary steatosis like corticosteroids, methotrexate,

amiodarone.

Severe Co- morbidities including Malignancies and severe chronic disorders like

Biventricular heart failure, Chronic kidney disease, Decompensated chronic liver

disease.

Pregnant women.

3.6 SAMPLE SIZE.

40

3.6.1 STUDY SAMPLE SIZE.

Minimal sample size was determined using Fisher’s statistical formula. 102

Sample size (N) = Z2PQ

D2

Z = 1.96, that is normal standard deviation at 95% confidence interval

P = Prevalence rate of hepatic steatosis in type 2 DM.

Q = 1-P

D = margin of acceptable error or measure of precision= 5% or 0.05

The prevalence (P) of hepatic steatosis in type 2 DM which would be used is 21%

based on the findings of Angulo P. 7 The study population was estimated to be 160.

N = (1.96)2 x 0.21 x 0.5 =160

(0.05)2

3.6.2 CONTROL SUBJECTS.

The control subjects were individuals without DM. The ratio of study subjects to control was 1:1.

Thus the number of control subjects was 160.

3.6.3 TOTAL STUDY POPULATION.

Total study population was estimated to be 320 (study subjects and control). To make up for

attrition, 5% was added to the total study population; hence the final study population was 336

comprising of 168 cases and 168 controls.

3.7 ETHICAL CONSIDERATON.

41

Ethical approval was obtained from the Ethics and Research committee of Lagos University

Teaching Hospital Idi- Araba, Lagos Nigeria prior to commencement of the study. Pre study

counselling was done and written informed consent was obtained from all patients prior to

enrolment. See Appendix 2&3.

3.8 CLINICAL METHODS

3.8.1 MATERIALS.

Sterile bottles (Lithium Heparin bottles)

Disposable gloves.

Cotton wool.

Plaster.

Sterile syringes and needles, 5ml and 10ml.

Measuring tape (Butterfly Brand, China).

3.8.2 EQUIPMENT

Aloka Pro-sound 3500 Ultrasound machine equipped with 3.75MHz probe.

Surgifriend Medicals Weighing scale (England)

Stadiometer (Avery, England)

3.8.3 METHOD.

After obtaining written and verbal informed consent, subjects and controls that satisfied the study

criteria were recruited from medical out-patient clinics and medical wards.

42

A structured questionnaire was administered to each subject by the researcher which included

bio-data, and questions on symptoms suggestive of NAFLD such as fatigue, malaise or upper

abdominal pain or discomfort, duration of DM, drugs used for treatment of DM as well as family

history of Diabetes and Liver Disease. Questions on intake of alcohol were also asked as well as

other risk factors for chronic liver disease. Another questionnaire was administered to the control

group in which symptoms suggestive of DM were added (polyuria, polydipsia and unintentional

weight loss) See Appendix 4 and 5 for details.

All patients were examined physically by the researcher with particular emphasis on features

suggestive of liver pathology such as jaundice, ascites and hepatosplenomegaly.

Each patient was weighed using a weighing scale (surgifriend,England) ensuring that there was

no zero error. Weight was recorded in kilogrammes (Kg) with subjects wearing light clothes.

Height was measured using a stadiometer (Avery, England) with the patient standing erect with

back against the height meter rule such that the occiput, back and heels made contact with it. It

was recorded in Metre (m). All these were done by the researcher.

The Body Mass Index (BMI) was calculated using the following formula-

BMI=weight (kg)/height (m2) and classified based on World Health Organisation (WHO).103

Criteria as follows;

Normal BMI-18.5-24.9

Overweight- BMI 25-29.9.

Obese Class I- BMI 30- 34.9.

Obese Class II- BMI 35-39.9.

43

Obese Class III- BMI ≥ 40

Other anthropometric measurements included waist circumference and Hip circumference. Waist

circumference (WC) was measured using a flexible, tension-sensitive, nonstretching tape

measure (Butterfly Brand, China) placed directly on the skin. Participants stood relaxed, with

arms at the side and feet together. Restrictive clothing was removed. Measurement was made at

the end of normal expiration; special attention was paid to ensure the tape was positioned

perpendicular to the long axis of the body and parallel to the floor. Measurements was taken

from the right side in the mid-axillary line by the researcher using the midway between the lower

margin of the least palpable rib and the top of the iliac crest (highest point of the hip bone on the

right side) as the point of reference.88 It was measured in centimetres (cm). Increased WC was

defined as more than 88cm in females and more than 102cm in males.104 Hip circumference

measurement (HC) in centimetres was done at the point of widest circumference of the buttocks

with the tape parallel to the floor.104 Both waist and hip circumference was taken twice and the

average was calculated. The same tape measure was used and all measurements were done by the

researcher. The Waist to Hip Ratio (WHR) was calculated. Central obesity was defined by a

waist to hip ratio ≥ 0.85 and ≥0.90 in females and males respectively. 104

Three Blood Pressure readings were taken by the researcher using an Accoson branded mercury

sphygmomanometer from each patient after they had rested for 5 minutes to eliminate anxiety.

While sitting comfortably with feet on the floor, the cuff was placed on the well supported arm,

free of any constricting clothing which was at the level of the heart and inflated, while

simultaneously palpating for the radial pulse. It was inflated to 20mmHg above the reading in

44

which the radial pulse was no more palpable. A Littmann’s stethoscope was placed over the area

of the brachial artery and the cuff was gradually deflated at a rate of 2mmHg per second until the

1st Korotkoff sound was heard which corresponded to the systolic blood pressure. The point

where the sound disappeared was the 5th korotkoff sound and corresponded to the diastolic blood

pressure. 105 Both arms were measured and the arm with the higher blood pressure was used. The

average of all three readings was calculated. Hypertension was considered at a blood pressure

reading of more than or equal to 140/90mmHg or current intake of anti hypertensive or

both. 105

3.8.4 COLLECTION OF BLOOD SAMPLES.

A tourniquet was applied to the arm and 10 millilitres of blood withdrawn from the antecubital

vein under aseptic conditions by the researcher and placed into appropriate sample bottles

(Lithium Heparin). The samples were sent to Lagos University Teaching Hospital laboratory for

evaluation of serum levels of Alanine Transaminase (ALT), Aspartate Transaminase (AST),

Fasting Lipid Profile (FLP) ; Total Cholesterol(TCHOL), High density lipoprotein cholesterol

(HDL-C) , Triglyceride (TG).

ALT and AST was done manually using a Spectrum LAB 23A Spectrophotometer (Gulfex

Medical and Scientific England) and Hydrazone method which is a kinetic method. Serum

Albumin was done using Bromocresol Green Method (BCG) which is a direct method.

TG was determined using a kit employing enzymatic hydrolysis with lipases. TCHOL was

determined using a kit employing enzymatic hydrolysis and oxidation. HDL Cholesterol was

done using phosphotungstic acid precipitation method and reagent used was BIOLABO (Maizy,

45

France). Low density lipoprotein cholesterol (LDL-C) was calculated using Friedwald formulae;

106

LDL= (TCHOL-HDL-C) –TG/5 When the value of TG was less than 400mg/dl

Randox Laboratory Limited kit (UK) was used for ALT, AST, Serum Albumin, TCHOL and

TG. Samples were also be analysed for detection of anti-hepatitis C virus and Hepatitis B surface

antigen using Diaspot rapid diagnostic kit (USA). This was done using Immunochromatography

method. All tests were free of any charges for the subjects.

Dyslipidemia was defined as anyone with one or more of the following: LDL-C ≥100

mg/dL, total cholesterol ≥200 mg/ dL, triglycerides ≥150 mg/dL, or HDL-C < 40 mg/dL in

males and <50 mg/dL in females. 107

3.8.5 ABDOMINAL ULTRASOUND SCAN.

Subjects had abdominal ultrasound scan (USS) done, which was performed by the researcher

under the supervision of a consultant radiologist and findings were reported. Aloka Pro-sound

3500 machine was used equipped with 3.75MHz probe. This was also done at no cost to the

patients and it has no known side effects and is safe at diagnostic frequency. Subjects were

required to fast for at least 6 hours before USS. The subjects were placed in supine position;

ultrasound gel was spread over the right hypochondrium as this helps with transmission of sound

waves. The transducer was then placed on the same region ensuring that both the liver and the

right kidney were visualised for comparison. Findings suggestive of hepatic steatosis include

‘bright liver’ with increased echogenicity and hepatorenal contrast. 82

46

3.8.6 FUNDING/COST OF THE STUDY.

Expenses were incurred in the following areas.

Procurement of Stationeries.

Blood investigations (HBsAg, Anti HCV, ALT, AST, Fasting Lipid Profile) for 336

patients.

Abdominal ultrasound Scan for 336 patients.

Sources of funding were personal. All costs related to the study was the responsibility of the

researcher.

3.8.7 STATISTICAL ANALYSES

The data obtained was analyzed using the statistical software SPSS Version 20 (SPSS Inc,

Chicago, IL USA). Frequency distribution was generated using nominal variables. Various

measures of central tendency and of dispersion were computed for quantitative variables.

Proportions were evaluated using odds ratio and statistical significance was taken as P < 0.05.All

confidence levels were calculated at the 95% level.

Analytical statistical methods employed included student t test to compare continuous variables

and chi-square test to compare categorical variables.

47

CHAPTER FOUR

RESULTS.

SOCIO-DEMOGRAPHIC CHARACTERISTICS OF STUDY POPULATION.

The study population comprised of a total of 336 people who fulfilled the study criteria i.e. 168

cases with T2DM were recruited for the study and 168 controls that had been screened for

diabetes mellitus with normal FBS result and had no history suggestive of T2DM.

The mean ages were comparable in both cases 53.17±8.6 years and non-diabetic controls which

was 52.01±8.9 (P=0.22). The diabetic cases had 59 males (35.1%) and 109 females (64.9%) while

the non-diabetic controls had 71 males (42.3%) and 97 females (57.7%) which was not

statistically significant P=0.22.

There was no significant difference in the level of education in both cases and controls with

more than 97% having some form of formal education P= 0.08.

There was also no statistical difference in ethnicity between both cases and controls; however the

Igbo ethnicity was the highest among the cases while Yoruba ethnicity was the highest among

the controls P=0.06.

Seventy three percent of both cases and controls earn less than =N=50,000 monthly, however

there was no statistical difference in level of income among cases and controls P=0.10.

All variables in the socio-demographic data of both cases and controls were all comparable.

See table 1.

48

TABLE 1: SOCIO-DEMOGRAPHIC FEATURES DISTRIBUTION OF DIABETIC CASES AND

NON DIABETIC CONTROLS.

VARIABLES

CASES

(DIABETICS)

N=168(%)

CONTROL

(NON DIABETICS)

N=168(%)

P VALUE

AGE MEAN (SD) 53.17±8.6 52.01±8.9 0.22

AGE GROUP(Years)

20-30 2(1.2) 2(1.2)

31-40 12(7.1) 19(11.3)

41-50 50(29.8) 52(31)

51-60 74(44.0) 70(41.7)

>60 30(17.9) 25(14.8)

GENDER

Females 109 (64.9) 97 (57.7) 0.22

Males 59 (35.1) 71 (42.3)

LEVELOF EDUCATION

None 4 (2.4) 3 (1.8) 0.08

Primary 43 (25.6) 28 (16.7)

Secondary 58 (34.5) 73 (43.5)

Tertiary* 63 (37.5) 64 (38.1)

ETHNICITY

Hausa 0 (0) 1 (0.6) 0.06

Yoruba 62 (36.9) 81(56.6)

Igbo 71 (42.3) 56 (33.3)

Others** 35 (20.8) 30 (17.9)

INCOME/MONTH(=N=)

Less than 10,000 65 (38.7) 62 (36.9) 0.10

10,000-50,000 58 (34.5) 60 (35.7)

50,000-100,000 25 (14.9) 27 (16.1)

100,000-200,000 16 (9.5) 8 (4.8)

49

*Inclusive of University, Polytechnic, Teacher training college. **Inclusive of Edo, Ibibio, Efik.

RISK FACTORS FOR CHRONIC LIVER DISEASE BETWEEN DIABETIC CASES

AND NON DIABETIC CONTROLS.

Amongst both diabetic cases and non-diabetic controls there was no significant difference in risk

factors for developing chronic liver disease. They also did not have any history of use of any

drug that can cause secondary steatosis such as amiodarone, tamoxifen or steroids.

See Table 2

More than 200,000 4 (2.4).

11 (6.5)

50

TABLE 2: DISTRIBUTION OF RISK FACTORS FOR CHRONIC LIVER DISEASE BETWEEN

DIABETIC CASES AND NON-DIABETIC CONTROLS.

VARIABLES CASES

(DIABETICS)

N=168 (%)

CONTROLS

(NON-DIABETICS)

N=168 (%)

P

VALUE

Past history of jaundice 6 (3.6) 2(1.2) 0.28

Scarification marks 24(14.3) 29 (17.3) 0.55

Multiple sexual partners 3(1.8) 1(0.6) 0.62

Sharing of

needles/sharps

0(0) 0 (0)

Iv drug use 0(0) 0(0)

Blood transfusion 0(0) 0(0)

Family history of liver

disease.

2(1.2) 0(0) 0.50

Alcohol use <20g/day 0(0) 4(2.4) 0.12

The questions were answered as either Yes or No, the percentages of Yes for each risk factor was

recorded in this table.

51

DISTRIBUTION OF METABOLIC RISK FACTORS BETWEEN DIABETIC CASES

AND NON-DIABETIC CONTROLS.

There was a statistically significant difference in a history of hypertension among cases and

controls, as out of 117 (34.8%) subjects with a history of hypertension, 80 (47.6%) were diabetic

cases while 37 (22.0%) were non-diabetic controls P<0.01

The mean SD duration of hypertension was longer for diabetic cases 87.48±80.9 months than

in non-diabetic controls 46.05±43.34months. This difference was also statistically significant

P<0.01. Ninety-two subjects (27.4%) had a family history of DM of which 74 (44%) were cases

and 18 (10.7%) were controls. This was statistically significant P<0.01.

See table 3.

52

TABLE 3: DISTIBUTION OF METABOLIC RISK FACTORS BETWEEN CASES AND

CONTROLS

VARIABLES

CASES

(DIABETICS)

N=168 (%)

CONTROLS

(NON

DIABETICS)

N=168 (%)

P

VALUE

Hypertension(history) 80(47.6) 37(22.0) <0.01

Duration of hypertension (months) 87.48±80.9 46.05±43.34 <0.01

Family history of DM 74(44.0) 18(10.7) <0.01

53

CLINICAL MEASUREMENTS OF SUBJECTS

The difference in mean BMI between the cases and controls was statistically significant

P=0.04. Both cases and controls had more subjects with abnormal BMI (>25kg/m2) and there

was no statistical difference in the distribution of subjects with normal (18.5-24.9kg/m2) and

abnormal BMI (≥25kg/m2) in both cases and controls P=0.07. However when abnormal BMI is

divided into subgroups there was a significant difference between cases and controls in class I

Obesity P=0.02. Both cases and controls however had most people in the overweight category

(BMI-25-29.9 kg/m2).

The difference in the mean waist circumference between cases and controls was statistically

significant P<0.01. Among the females, cases appeared to have a mean WC higher than the

controls 96.9 (11.9) vs. 94 (11.1) also 77.1% of the cases had a WC>88cm compared to 71.1% of

the controls. Both measures however were not of statistical significance. Among the males

54

however, the mean WC was significantly greater in cases than in controls 97.2 (12.4) vs.

91.7(11.7), P<0.01. Waist Circumference of >102cm was similarly higher in cases than in

controls this however was not statistically significant P=0.08.

Waist to Hip ratio was noted to be statistically significantly higher in subjects with DM than in

controls. The significance remained true for both males and females.

The mean SBP was significantly higher among the cases compared with the controls P<0.01

while the difference in the mean DBP was not statistically significant P=0.61.

See Table 4.

TABLE 4: DISTRIBUTION OF CLINICAL MEASUREMENTS BETWEEN DIABETIC CASES

AND NON DIABETIC CONTROLS

VARIABLES

CASES

(DIABETIC)

N=168(%)

CONTROLS

(NON

DIABETIC)

N=168(%)

P VALUE

BMI (kg/ m2) Mean (SD) 28.47±4.39 27.42±5.09 0.04

Normal BMI(18.5-24.9kg/m2) 40(23.8) 56(33.3) 0.07

Abnormal BMI (≥25 kg/ m2) 128(76.2) 112(66.7)

Overweight(25-29.9) 66(39.3) 68(40.5) 0.28

Class I Obesity(30-34.4) 53(31.5) 36(21.4) 0.02

Class II Obesity(35-39.9) 7(4.2) 5(3.0) 0.36

Class III Obesity(≥40) 2(1.2) 3(1.8) 1.00

Waist circumference (cm) Mean

(SD)

97.04±12.08

93.29±11.39 <0.01

Females

55

WC (cm) Mean (SD) 96.9± 11.9 94±11.1 0.05

WC >88cm 84(77.1) 69(71.1) 0.34

Males

WC (cm) Mean (SD) 97.2±12.4 91.7±11.7 <0.01

WC >102cm 21(35.6) 15(21.1) 0.08

Hip circumference (cm) Mean (SD) 104.05±10.67 104.58±9.86 0.63

Waist/Hip ratio Mean (SD) 0.93±0.06 0.89±0.07 <0.01

Females

WHR Mean (SD) 0.93±0.07 0.89±0.07 <0.01

WHR ≥ 0.85 93(85.3) 67(69.1) <0.01

Males

WHR Mean (SD) 97.2±12.4 91.7±11.7 <0.01

WHR≥ 0.90 43(72.9) 34(47.9) <0.01

Systolic BP (mmHg)Mean (SD) 130.41±18.99 123.05±17.74 <0.01

Diastolic BP (mmHg) Mean (SD) 80.59±12.87 79.92±11.54 0.61

DISTRIBUTION OF BIOCHEMICAL AND VIRAL SEROLOGY PARAMETERS

BETWEEN DIABETIC CASES AND NON DIABETIC CONTROLS

Serum Transaminases levels appear to be similarly distributed in both cases and controls P=0.10

and 0.35 for both AST and ALT respectively. The mean AST/ALT ratio was also not statistically

significant P=037. Although Serum Albumin appears significantly higher in cases than controls

both levels were within normal limits.

Diabetic cases appeared to have more dyslipidemia107 than controls. This was particularly so

with TCHOL and LDL-C. Mean TCHOL was 179.49(40.89) compared with 170(31.69) in

controls P<0.01. TCHOL more than 200mg/dl was noted in 27.4% of cases vs. 17.9% of controls

56

although it was not statistically significant P=0.05. Mean LDL-C Levels and LDL>100mg/dl

were significantly more elevated in diabetics than none diabetics P=0.00. Values of mean HDL-

C and TG were not significantly different, P=0.29 and 0.20 respectively in both cases and

controls.

Seven (2.1%) of the 336 subjects were positive for HBsAg. There was no statistical difference in

prevalence among cases and controls (0.6% vs. 3.6%, P=0.12).

No subject tested positive for Hepatitis C.

See Table 5.

TABLE 5: DISTRIBUTION OF BIOCHEMICAL AND VIRAL SEROLOGY PARAMETERS

BETWEEN DIABETIC CASES AND NON DIABETIC CONTROLS.

VARIABLES

CASES

(DIABETIC)

N=168(%)

CONTROL

(NON DIABETIC)

N=168 (%)

P VALUE

AST (IU/L) Mean (SD) 5.86± 3.70 6.49±3.41 0.10

ALT (IU/L) Mean SD 8.43±7.59 9.07± 4.34 0.35

AST/ALT 0.74 ±0.20. 0.72±0.23 0.37

Serum Albumin (g/l) 45.42±8.85 43.40±6.96 0.02

57

Mean (SD)

TCHOL (mg/dl) Mean(SD) 179.49±40.89 170 ±31.69. <0.01

TCHOL ≥ 200mg/dl 46(27.4) 30(17.9) 0.05

HDL-C(mg/dl) Mean (SD) 71.07 ±25.25 73.89±23.32 0.29

Males<40mg/dl 6(10.2) 4(5.6) 0.51

Females<50mg/dl 30(27.2) 20(20.6) 0.26

LDL-C(mg/dl)Mean (SD) 94.46±40.57 81.26±32.94 <0.01

LDL-C ≥100mg/dl 76(45.2) 46(27.4) <0.01

Triglyceride(mg/dl) Mean

(SD)

75.22±37.96 80.07±31.47 0.20

TG≥150mg/dl 10(6) 5(3) 0.29

HBsAg 1(0.6) 6(3.6) 0.12

Anti-HCV 0 0

THE PREVALENCE OF FATTY LIVER AMONGST DIABETICS AND NON

DIABETICS.

Of the total number of subjects (336), 30 (8.9%) of persons had NAFLD. 28 (16.7%) were

diabetic cases while 2 (1.2%) were non- diabetic controls. This was statistically significant

(p=<0.01). See Table 6.

58

TABLE 6: DISTRIBUTION OF NAFLD AMONG DIABETIC CASES AND NON

DIABETIC CONTROLS.

VARIABLES

CASES

(DIABETIC)

N=168(%)

CONTROLS

(NON DIABETIC)

N=168(%)

X2 95%CI

O.R

P

VALUE

NAFLD Positive 28 (16.7%) 2 (1.2%) 24.74 16.6(3.9-70.9) <0.01

59

NAFLD

Negative

140 (83.3%) 166 (98.8%)

FIGURE 2: BAR CHART SHOWING DISTRIBUTION OF NAFLD AMONG CASES

AND CONTROLS.

60

P<0.01

CHARACTERISTICS OF SUBJECTS WITH NAFLD.

61

There were 30 subjects with NAFLD in this study of which 28 were Diabetic cases and 2 were

non-diabetic controls. The 2 NAFLD controls were both males in the 5th decade of life, both

were hypertensive with abnormal BMI (one was overweight while the other had class III

Obesity), central obesity, normal transaminases and dyslipidemia. Further comparison of the

characteristics of both cases and controls with NAFLD were not done due to the small number of

controls with NAFLD.

CLINICAL AND SEROLOGICAL PARAMETERS OF DIABETICS BASED ON NAFLD

STATUS.

The mean age difference between NAFLD positive and NAFLD negative cases was not

statistically different P=0.45. There were 12 males and 16 females with NAFLD among the cases

(M: F – 1.1.3) while there were 47 males and 93 females without NAFLD among the cases (M: F

-1:1.9).

Eighty (47.6%) of the diabetic subjects had a history of hypertension and 74(44%) of them had a

family history of Diabetes. There was no statistically significant difference in these parameters

based on NAFLD status. Cases with NAFLD had a shorter duration of diabetes 70.43±62.55

months compared with those without NAFLD which was 94.56±83.08 months and this was not

statistically significant (P=0.15).

None of the symptoms of NAFLD such as fatigue, malaise or upper abdominal discomfort/pain

were statistically significant P=0.21, 0.79, 0.31 respectively among the cases with or without

NAFLD.

62

The distribution of risk factors for chronic liver disease such as family history of liver disease,

past history of jaundice, multiple sexual partners or scarification marks were similar in both

cases with or without NAFLD.

The mean weight was statistically higher in cases with NAFLD compared with those without

NAFLD P<0.01.

Only one diabetic was positive for HBsAg and NAFLD. The subject was a middle aged man,

who was hypertensive, had a family history of DM, obese (BMI=33.9 kg/m2) and had central

obesity (WC=110cm, W/H ratio= 0.98)

See Table 7.

63

TABLE 7: DISTRIBUTION OF CLINICAL AND SEROLOGICAL PARAMETERS AMONG DIABETICS

BASED ON NAFLD STATUS.

VARIABLES

NAFLD Positive

N=28 (%)

NAFLD Negative

N=140 (%)

P VALUE

Age Mean (SD) 53.86±8.9 54.05±8.9 0.45

Sex (M:F) 1:1.3 1:1.9 0.39

History of HTN* 14(50) 66(47.1) 0.84

Family history of DM* 14(50) 60(42.9) 0.54

Duration OF Diabetes

(months) (SD)

70.43±62.55 94.56±83.08 0.15

History of Fatigue* 3 (10.7) 31 (22.1) 0.21

History of Malaise* 4 (14.3) 27 (19.3) 0.79

History of upper abdominal

discomfort*

4 (14.3)

12( 8.6)

0.31

Past history of Jaundice* 1(3.6) 5(3.6) 1.00

Family history of Liver

Disease*

0(0) 2(1.4) 1.00

Scarification Marks* 5(17.9) 19(13.6) 0.56

Multiple Sexual Partners* 1(3.6) 2(1.4) 0.42

Weight Mean (SD) 83.89±13.21 74.28±13.65 <.0.01

HBsAg positive 1(3.6) 0(0) 0.17

Anti-HCV 0(0) 0(0)

*The questions were answered as either Yes or No, the percentages of Yes for each symptom was

recorded in this table.

64

DISTRIBUTION OF BIOCHEMICAL PARAMETERS AMONG DIABETICS BASED

ON NAFLD STATUS .

The mean transaminases were higher in diabetics with NAFLD compared with those without

NAFLD. Also two (7.1%) cases with NAFLD and 6(4.3%) without NAFLD had elevated AST

above upper limit of normal (12IU/L) while two (7.1%) cases with NAFLD and 13(9.3%)

without NAFLD had elevated ALT above upper limit of normal (12IU/L). All these were not of

statistical significance. The differences in mean AST/ALT and Serum albumin were also not

statistically significant P=0.15 and 0.53 respectively in the diabetics with or without NAFLD

Diabetics with NAFLD appeared to have more dyslipidemia than those without NAFLD.

However the differences in each of the lipid parameters (TCHOL, HDL-C, LDL-C and TG) were

not statistically significant. P= 0.80, 0.25, 0.19 and 0.86 respectively.

See table 8.

65

TABLE 8: DISTRIBUTION OF BIOCHEMICAL PARAMETERS AMONG DIABETIC CASES BASED

ON NAFLD STATUS.

VARIABLES

NAFLD

Positive

N=28(%)

NAFLD

Negative

N=140(%)

P VALUE

AST Mean (SD) (IU/L) 6.64±6.14 5.70±3.00 0.22

AST>12IU/L 2(7.1) 6(4.3) 0.62

ALT Mean (SD) (IU/L) 10.18±16.52 8.08±3.93 0.51

ALT > 12IU/L 2(7.1) 13(9.3) 1.00

AST/ALT 0.79±0.20 0.73±0.21 0.15

Serum Albumin Mean

(SD) (g/L)

46.39±10.57 45.23±8.50 0.53

TCHOL Mean (SD)

(mg/dl)

181.25±37.88 179.14±41.59 0.80

HDL-C Mean (SD)

(mg/dl)

66.04±26.93 72.08. ±24.89. 0.25

LDL-C Mean (SD)

(mg/dl)

103.68±40.40 92.62±40.50 0.19

TG Mean (SD) (mg/dl) 76.36±44.55 74.99±36.68 0.86

66

DISTRIBUTION OF ASSOCIATED RISK FACTORS FOR NAFLD AMONG

DIABETICS.

Diabetic subjects with NAFLD were significantly more likely to be Obese than subjects without.

Two cases with NAFLD and 38 cases without NAFLD had normal BMI (18.5-24.9 kg/ m2) while

26 cases with NAFLD and 102 without had abnormal BMI (>25 kg/ m2).

There was no significant difference in central obesity (using WHR) in diabetics with or without

NAFLD. Twenty–six cases (92.6%) with NAFLD and 110(78.6%) without NAFLD had central

obesity (WHR ratio of ≥0.85 or for females and ≥ 0.90 for males). 85 When divided among

gender, there was still no statistical difference. Using the WC however as a measure of central

obesity, the mean Waist circumference was significantly higher in cases with NAFLD than those

without NAFLD P= 0.02. However this difference was due to the males as 75% of those with

NAFLD had WC>102cm compared with 25.5% of those without NAFLD P<0.01

Five (17.9%) cases with NAFLD and 41(29.3%) without NAFLD had hypercholesterolemia of

≥200mg/dl which was not statistically significant P=0.25. Low HDL-C of <40mg/dl was more

prevalent in male diabetics with NAFLD compared with those without NAFLD (44.4% vs. 8.7%,

P<0.01) While there was no significant difference among female diabetics based on their

NAFLD status with regards to low HDL-C of <50mg/dl.

67

The prevalence of elevated LDL-C was higher in diabetics with NAFLD when compared with

those without NAFLD although it was not statistically significant (53.6% vs. 43.6%, P=0.41).

Similarly, there was no significant difference in prevalence of Hypertriglyceridaemia in diabetics

with or without NAFLD (14.3% vs.4.3%, P=0.06).

See Table 9.

TABLE 9: DISTRIBUTION OF ASSOCIATED RISK FACTORS FOR NAFLD AMONG DIABETICS.

VARIABLES

NAFLD Positive

N=28 (%)

NAFLD Negative

N=140 (%)

P VALUE

BMI 87 (kg/ m2)

Normal (18.5-24.9 kg/ m2) 2(7.1) 38(27.1)

0.03 Abnormal BMI(>25 kg/ m2 ) 26(92.9) 102(72.9)

Increased Waist: Hip Ratio88 26 (92.6) 110 (78.6) 0.11

Female≥0.85 15(93.8) 78(83.9) 0.46

Male ≥ 0.90 11(91.7) 32(68.1) 0.15

WC Mean (SD) 103.64±9.67 95.72±12.11 <0.01

Increased Waist Circumference88 23(82.1) 82(58.6) 0.02

Females>88cm 14(87.5) 70(75.3) 0.35

Males >102cm 9(75) 12(25.5) <0.01

Dyslipidemia*107

(mg/dl)

16(57.1) 80(57.1) 1.00

T-CHOL ≥ 200mg/dl 5(17.9) 41(29.3) 0.25

HDL

Female<50mg/dl 5(55.6) 21(91.3) 0.53

Male<40mg/dl 4(44.4) 2(8.7) <0.01

68

LDL≥ 100mg/dl 15(53.6) 61(43.6) 0.41

TG≥150mg/dl 4(14.3) 6(4.3) 0.06

*One or more of the following-Total cholesterol≥200mg/dl, LDL-C-≥100mg/dl, HDL-C<40mg/dl in males

and<50mg/dl in females, TG≥150mg/dl. 107

CHAPTER FIVE

DISCUSSION.

This study set out to determine the prevalence of NAFLD amongst Nigerians with T2DM, to

compare some biochemical parameters (e.g. liver function, lipid profile) between diabetics with

NAFLD and those without NAFLD and to identify associated risk factors for NAFLD such as

Dyslipidemia and Obesity including central obesity in T2DM.

CHARACTERISTICS OF SUBJECTS

The mean age of subjects with T2DM was 53.17±8.6, with 91.7% being above the age of 40

years. This is in keeping with the occurrence of T2DM in people usually above the age of

30years.16 Insulin resistance which is the hallmark of T2DM worsens with age. There was female

preponderance amongst the diabetics with male: female ratio of 1:1.8. This is similar to a study

done in south west Nigeria, where obesity and insulin resistance, risk factors for diabetes

mellitus were commoner in women.108 However gender distribution of T2DM varies in Africa

with no discernible pattern and this is also in keeping with global findings. 24,25 Ninety-seven

percent of all subjects had some form of formal education which is due to the research centre

69

being situated in an urban setting with easy accessibility to schools. Igbo ethnicity was the

largest group in case subjects, reflecting the cosmopolitan nature of Lagos State which is situated

in South-west Nigeria, an area predominantly inhabited by the Yoruba ethnic group. The reason

for Igbo ethnicity predominance in T2DM in this study remains unapparent and would require

further epidemiological studies to elucidate.

Slightly more than a third of cases and controls earn less than =N=10,000 monthly. This may be

a reflection of the economic situation in Nigeria where 83.9% of people earn less than $2/day. 109

Also LUTH being a public hospital is affordable to low income earners compared with private

hospitals.

There was a significantly higher prevalence of hypertension among diabetics compared with non

diabetics in this study. Diabetic patients also had a longer mean duration of hypertension than

non-diabetics. This finding is in keeping with local studies as well as studies from other parts of

the world which have shown a high prevalence of hypertension among people with DM. 69-72 There is

increasing evidence that insulin resistance and hyperinsulinemia may play a key role in the

pathogenesis of hypertension in T2DM and also developing nephropathy may also be

contributory. In this study, more diabetics had a family history of diabetes compared with non

diabetics in this study which was significant and is thus consistent with findings from Sudan20,21

and rural South Africa 22 and many other parts of the world, where a positive family history has

been reported as a risk factor for developing T2DM. 16 T2DM has a strong genetic component

which may be responsible for this.

70

Three- quarters of the diabetic cases in this study were overweight or obese with BMI of

25kg/m2 and above, the mean BMI of diabetic cases was also significantly higher compared with

the non-diabetic controls. Studies have shown that the risk of diabetes increases as BMI

increases above 25kg/m2. 73 Diabetic cases in this study also had central obesity with both the

mean WC and WHR being significantly higher compared with the non diabetic controls. The

pathophysiology of this relationship between obesity (including central) and T2DM can be

explained as adipocytes secrete biological products like FFA, leptin which may contribute to

insulin resistance, which in turn leads to compensatory hyperinsulinemia. As insulin resistance

and hyperinsulinemia continue to progress, Diabetes may eventually develop.

Current findings have shown that the typical lipid disorder in patients with diabetes is

characterized by increased numbers of small, dense LDL particles, elevated triglycerides and low

levels of HDL cholesterol. 74,75 Our findings were consistent with regard to the elevated LDL-C

in diabetics as the mean LDL-C was significantly higher in diabetics than non diabetics.

However there were no differences regarding TG and HDL-C in diabetics and non diabetics in

this study. The prevalence of Hypercholesterolemia was 27.4% in the diabetic cases and 17.9%

in non diabetics also; the mean TCHOL was significantly higher in diabetics compared with non

diabetics.

No subject was positive for Hepatitis C thus reflects the low prevalence in this environment

while 7 subjects were positive for the surface antigen of Hepatitis B.

CHARACTERISTICS OF DIABETICS WITH NAFLD.

The mean age of diabetics with NAFLD compared with those without NAFLD was not

significantly different. The mean age for diabetics with NAFLD was 53.86±8.9. This is

71

consistent with it being found mostly in the 5th to 7th decade of life and although it can occur at

any age, its prevalence increases with age.30 There was slight female gender preponderance in

this study as male to female ratio was 1:1.2. However findings differ as some authors have

reported it occurring more in males 12 while others have found no gender preponderance.30

This study also showed that the association between obesity (including central) and NAFLD was

significant as has been well documented in literature. 1, 13 The mean waist circumference was

significantly higher in diabetics with NAFLD compared with those without NAFLD. When

distributed based on gender, both males and female diabetics with NAFLD had a higher

prevalence of central obesity when compared with those without NAFLD however this

difference was significant only in male diabetics. To further buttress the association between

obesity (including central obesity) and NAFLD in this study, the only 2 non-diabetics with

NAFLD in this study were obese with central obesity. The underlying pathophysiology of this

association can be explained as there is an increased influx of FFA to the liver in obesity. These

FFA either undergo β-oxidation or are esterified with glycerol to form triglycerides, leading to

hepatic fat accumulation.52

In this study, none of the symptoms of NAFLD was significantly different amongst diabetics

with NAFLD compared with those without NAFLD. This can be explained as the symptoms of

NAFLD are non specific and can be often found in the general population. The commonest

symptom however was abdominal pain /discomfort and malaise. This is in keeping with other

literature where abdominal pain/discomfort appears to be the commonest symptom when

present.8 Abdominal pain may be as a result of stretching of the capsule of the liver as it

enlarges. All subjects however had no findings suggestive of liver disease. This is also in

72

keeping with previous literature where most cases are asymptomatic and have no sign of liver

disease at the time of diagnosis. 7,8

Although the mean duration of diabetes with NAFLD was shorter than those without NAFLD it

was not statistically significant. This was comparable to the study done in Iran where the mean

duration of diabetes for NAFLD was shorter than those without NAFLD. 30 The Edinburgh study

also showed that T2DM with NAFLD had a shorter duration of diabetes compared with those

without NAFLD. 79 One possible explanation is that the greater degree of hyperinsulinemia in

early type 2 diabetes drives uptake of free fatty acids by hepatocytes.79

With time fat accumulation tends to disappear with lifestyle modification and treatment for

diabetes and its co-morbid conditions like dyslipidemia.

No subject had any history of use of drugs that could cause secondary steatosis such as

amiodarone, steroids or tamoxifen. One diabetic was positive for HBsAg and NAFLD. The

subject was a middle aged man, who was hypertensive, had a family history of DM, obese and

had central obesity all metabolic risk factors for NAFLD. This finding is similar to preliminary

studies in HBV which suggests that hepatic steatosis in these patients may be related to host

factors rather than viral factors. 110-112

PREVALENCE OF NAFLD AMONG STUDY POPULATION.

The prevalence rate of NAFLD in this study was higher amongst the diabetic cases compared

with the non diabetics (16.7% vs. 1.2 %,) and this was statistically significant (P=0.00,

O.R=16.6: 95% CI 3.9-70.9). The odds for developing NAFLD are 16.6 times more in diabetics

73

compared with non-diabetics. This finding of a high prevalence of NAFLD in T2DM has been

noted by several other researchers both locally and internationally. 14,30,31 The reason for this may

be that in T2DM, there is excessive rate of lipolysis from insulin-resistant adipose tissue which

acts as a driving force for the development of steatosis.59,66,67. Another contributory factor may

be the frequent co-existence of Obesity and T2DM in the same individual, two well documented

risk factors for developing NAFLD.

The prevalence of NAFLD in diabetics in this study as well as another also done in Lagos,

Nigeria in which it was 9.5%14 appears to be low compared with other studies from other parts of

the world. In Iran the prevalence rate of NAFLD amongst T2DM was 55.8% 30, while in Turkey

it was 27.5%.45 India and Brazil also had higher prevalence rate of 62.3% 10 and 69.4% 31

respectively.

These differences in prevalence may be due to differences in study population. A study that was

done in a racially diverse country like the USA in order to determine the racial and ethnic

distribution of NAFLD showed in decreasing order of prevalence of NAFLD (Hispanics >

Asians >other races > Black Americans ). These racial differences may reflect genetic

susceptibility to visceral adiposity.44 To further buttress the role of race in developing NAFLD;

black Americans had the highest BMI yet had the lowest prevalence of NAFLD in this study.

Diet may also be contributory to the higher prevalence, in Brazil for example; there has been an

increase in adoption of Western-style diet which is rich in simple carbohydrates, saturated fat,

and highly processed food stuffs which have been shown to increase the risk of developing

74

NAFLD. 113 When this is coupled with sedentary lifestyle, caloric imbalance can occur, resulting

in increased weight gain in most individuals. This can be compared with this environment where

the diet has less calories and caloric expenditure is more.

COMPARING THE BIOCHEMICAL PARAMETERS (E.G. LIVER FUNCTION, LIPID

PROFILE) IN STUDY SUBJECTS.

This study showed that diabetics had higher prevalence of dyslipidemia than non diabetics

though it failed to reach statistical significance. This is still a crucial finding as diabetic

dyslipidemia has more atherogenic potential than dyslipidemia in non diabetic subjects. The high

prevalence of dyslipidemia in T2DM has been well documented in literature. 75,76 Insulin

resistance which is the underlying pathology in T2DM causes an increase in Triglycerides, Low

Density Lipoprotein cholesterol (LDL-C) and reduces High density Lipoprotein Cholesterol

(HDL-C) in T2DM.

The Fasting Lipid profile (T-CHOL, HDL-C, LDL-C, and TG) was also compared between

diabetics with NAFLD and without NAFLD. Though the mean values for TCHOL, LDL-C and

TG were higher while HDL-C was lower they were not significantly different in diabetics with

NAFLD compared with those without. A study done in Lagos, Nigeria also showed no

significant difference in any of the lipid profile parameters among subjects with NAFLD and

those without NAFLD. 14

Similarly in the study from Iran, the mean TCHOL and LDL-C and TG were also higher and

HDL-C was lower in diabetics with NAFLD compared with those without NAFLD. However in

Iran, while the differences in mean of TCHOL, LDL-C and HDL-C were not significant in

75

diabetics based on their NAFLD status which is similar to this study, the mean TG was however

significant in diabetics with NAFLD compared with those without NAFLD.30 The Iranian study

also had a higher mean TG level among diabetics with NAFLD compared with this study

(223.4±120.1mg/dl vs. 76.36±44.55mg/dl). 30

The reason for the significantly higher mean triglyceride level in the study from Iran compared

with this study may be due to differences in study population as triglyceride levels from a study

done in a racially diverse country like USA were found to be lowest in blacks compared with

other races (Asians >White >Hispanics> Blacks.). 44 Also, the Iranian diet is high in

hydrogenated fat, which can lead to increased triglyceride levels. 114

In this study, the liver function parameters were AST, ALT, AST/ALT and Serum Albumin. The

mean AST and ALT though higher in diabetics with NAFLD compared with diabetics without

NAFLD was not statistically significant. The mean difference in serum albumin was also not

statistically significant. This present study was also similar to a study done in Iran in which the

mean AST and ALT levels in diabetics with NAFLD compared with those without NAFLD was

also not statistically significant. 30 In a local study done here in Lagos, Nigeria, AST and ALT

levels of subjects with NAFLD and those without NAFLD when compared were also higher

which is similar to this study however, it however differed in the mean ALT which was

statistically significant between the two groups. 14

The reason why diabetics with NAFLD have higher levels of transaminases compared with

those without NAFLD in this study may be due to inflammation of the liver as diabetics with

NAFLD were significantly more obese than those without NAFLD and one of the sources of free

fatty acids (FFA) in the liver is adipose tissue which produces pro inflammatory cytokines, such

76

as tumor necrosis factor-α and interleukin-6 which all contribute to inflammation of the liver

with injury of the hepatocytes and subsequent elevation of liver enzymes. 57

In this study only 2 cases with NAFLD had elevated transaminases. This is similar to another

study done locally in Lagos, Nigeria in which only one diabetic with NAFLD had elevated

transaminases14. This study is in keeping with findings of normal levels in most people with

NAFLD. 30

ASSOCIATED RISK FACTORS FOR NAFLD IN T2DM.

This study was also done to identify associated risk factors for NAFLD in T2DM. Well

documented risk factors for NAFLD include obesity including central obesity, insulin

resistance/DM and dyslipidemia and which are parts of the metabolic syndrome.

This study compared diabetics with NAFLD with those without NAFLD in order to identify

associated risk factors for NAFLD in T2DM. Three parameters were assessed and these were;

overweight /obesity (using BMI), Central Obesity (using Waist circumference and W/H ratio)

and Dyslipidemia. The relationship between Obesity (using BMI) and NAFLD in T2DM was

significant. This means the presence of NAFLD in T2DM was associated with Obesity in this

study. This finding was similar to another study done in Brazil, 31 Edinburgh79 and Iran 30 where

the presence of NAFLD in T2DM was also associated with Obesity. NAFLD is almost a

universal finding in diabetics that are obese.25 In USA, the prevalence of NAFLD in the

morbidly obese population is as high as 75%-92%.1 Obesity can be considered a low-grade

chronic inflammatory condition and obesity-related cytokines such as interleukin-6 (IL-6) and

77

tumor necrosis factor (TNF) α play important roles in the development of NAFLD. 8 In obesity

there is also an increase influx of free fatty acids into the liver. 52

Central Obesity using the waist circumference was significantly different between diabetics with

and without NAFLD however, using WHR there was no statistical significance between them.

The majority of current studies agree that waist circumference is probably a better indicator of

abdominal fatness than waist-to-hip ratio115-119 By requiring a single measurement as opposed to

the ratio of two measures; it is less susceptible to measurement and calculation errors. 115

The association between Dyslipidemia107 and NAFLD in T2DM was not significant. The

association between individual parameters like TCHOL, LDL-C, TG and NAFLD in T2DM

were also not significant. However when HDL-C was divided into gender, the association

between diabetic males with low HDL-C and NAFLD was significant while that of diabetic

females with low HDL-C was not. This was also similar to a study done in Lagos, Nigeria where

there was no significant difference in dyslipidemia and NAFLD in T2DM. The Edinburgh study

however differed as people with T2DM and NAFLD had significantly higher triglyceride levels

and significantly lower HDL Cholesterol levels. 79 A study in Iran and Brazil also showed higher

Triglyceride levels in those with NAFLD compared with those without NAFLD in T2DM.30, 31

The studies from Iran,30 Brazil31 and Edinburgh,79 all had a larger sample size than this study and

the local study done in Lagos, Nigeria14 which may account for the significantly higher

triglyceride levels in T2DM with NAFLD compared with those without NAFLD. Hence a study

78

with a larger sample size in this environment is recommended in order to ascertain if a true

relationship exists between the presence of NAFLD in T2DM and Hypertriglyceridaemia.

The mean TG levels were also higher in the study from Edinburgh79 and Iran30 in comparison to

this study and the reason may be due to differences in study population. Triglyceride levels have

been found to be lowest in Blacks and highest in Asians. 44 Also Diet may play a role in these

differences, in Edinburgh for example, the diet is rich in saturated fat and processed food which

may account for the higher triglyceride than what was seen in this study. 1

In summary this study has shown that diabetics middle aged with both general and central

obesity, a significant history of both hypertension and a family history of diabetes. They also

have dyslipidemia. It has also shown that NAFLD is 16.6 times more common in diabetics than

non diabetics and its presence in T2DM is associated with obesity including central obesity using

(WC) and low HDL-C levels in males.

CHAPTER SIX.

CONCLUSIONS, LIMITATIONS AND RECOMMENDATIONS.

79

CONCLUSIONS.

The prevalence of NAFLD in this study is lower than in other parts of the

world.

The odds of developing NAFLD if diabetic are 16.6 times that of non-

diabetics.

The presence of NAFLD in T2DM was associated with Obesity including

central obesity.

Low HDL-C in males with T2DM is an associated risk factor for NAFLD.

Biochemical tests like AST and ALT is normal in most people with NAFLD.

STUDY LIMITATIONS.

Some of the study’s limitations include:

Reliance on patient’s estimate of their alcohol consumption which may be inaccurate.

Diagnosis of fatty liver was by ultrasonography however a liver biopsy is the gold

standard. It is however invasive and not ethically acceptable for this kind of study.

This study has its own inherent weakness which includes: difficulty in interpretation of

the association between the outcome and the exposure.

80

RECOMMENDATIONS

In view of the significant relationship between NAFLD and T2DM,

individuals with type 2 DM should be screened for NAFLD.

Abdominal ultrasound scan should be used as a screening tool for NAFLD.

Individuals who are obese with or without DM should be encouraged to lose

weight.

Individuals with diabetes mellitus should have a lipid profile as part of their

management and Dyslipidemia should be corrected.

.

81

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APPENDIX 1

INFORMED CONSENT

94

TITLE OF RESEARCH: PREVALENCE OF AND RISK FACTORS FOR NON

ALCOHOLIC FATTY LIVER DISEASE IN NIGERIANS WITH TYPE 2 DIABETES

MELLITUS IN LAGOS UNIVERSITY TEACHING HOSPITAL.

NAME OF RESEARCHER: DR T.O. OLUSANYA.

HEALTH RESEARCH COMMITTEE ASSIGNED NO: ADM/DCST/HREC/APP/144

DURATION OF HREC APPROVAL 20-07-2011 TO 19-07-2012

You are invited to participate in a research titled above. I hope to learn the number of people

with diabetes mellitus and fatty liver as well as risk factors for developing it.

I would ask you a few questions, collect your bio data and measure your height, weight and

blood pressure. I would also be taking some blood samples which would be sent to the laboratory

in order to check your liver function and cholesterol level.

I would be performing ultrasonography of your liver. This does not have any known side effects

and is safe at diagnostic frequency. All these will take 15-20 minutes of your time.

Your participation is entirely voluntary and you can decide to withdraw at any stage of the study.

Your decision to participate or not will not prejudice you or your medical care.

All information obtained in this study will be strictly confidential and will be given code

numbers which cannot be linked to you in anyway and your name or any identifier will not be

used in any publication or reports from this study.

However all tests will be done free of any charges and results will be communicated to your

doctors.

95

Statement of person obtaining informed consent:

I have fully explained this research to .................................................................................. and

have given sufficient information, including risks and benefits, to make an informed decision

DATE:.............................................SIGNATURE:...............................................................

NAME:......................................................................................................................

Statement of person giving consent:

I have read the description of the research or have had it translated into a language i understand. I

have also talked with the doctor to my satisfaction. I understand that my participation is

voluntary. I know enough about the purpose, methods, risks and benefits of the research study to

judge that I want to take part in it. I understand that I may freely stop being part of this study at

any time. I have received a copy of this consent form and additional information sheet to keep

for myself.

DATE:..........................................SIGNATURE...................................................................

NAME:..........................................................................................................

WITNESS NAME

........................................................................................................................

Thank you.

Dr T.O. Olusanya.

Department of Medicine,

LUTH.

08023286534.

96

APPENDIX 2

ETHNICAL APPROVAL

97

98

APPENDIX 3

STUDY SUBJECT DATA

BIODATA

Serial Number .............. Initials................ Phone No ........................

(1) Age ..................

(2) Gender (a) Male (b) Female

(3) Ethnicity ...........................

(4) Level of educational (a) Nil (b) Primary (c) Secondary (d) tertiary or post secondary

(5) Occupation ………………….

(6) Income Bracket per month (a) < #10,000 (b) #10,000 - #50,000 (c) #50,000 - #100,000(d)

#100,000 - #200,000 (e) > #200,000

MEDICAL HISTORY

(1) History of Fatigue? (a) Yes (b) No

(2) History of Malaise? (a) Yes (b) No

(3) History of Upper Abdominal Discomfort? (a) Yes (b) No

(4) History of Diabetes Mellitus? (a) Yes (b) No

(5) If (4) is Yes what is the duration? ..............................

(6) History of Jaundice/ Hepatitis? (a) Yes (b) No

(7) If (6) is Yes, what type of Hepatitis? if known ……………………

(8) History of Hypertension? (a)Yes (b) No

(9) If (8) is Yes, what is the duration? .........................

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DRUG HISTORY

(1) What medication/ treatment do you use for managing your diabetes? (a) Sulphonyureas

(b) Biguanides c)Thiazolidinediones d) Alpha glucosidase inhibitors d) Others (please

specify).............

(2) Are you on any other routine medication(s) (a) Antihypertensives (b) Amiodarone (c)

Chemotherapy drugs d) Corticosteriods (e) Others please specify…………….

FAMILY HISTORY.

(1) Any family history of Diabetes Mellitus? (a) Yes (b) No.

(2) Any family history of liver cancer or chronic liver disease? (a) Yes (b) No

SOCIAL HISTORY

(1) History of alcohol intake? (a) Yes (b) No

(2) If (1) is yes, what type (a) Beer (b) Hard Liquor (c) Wine (d) All of the above

(3) If (1) is yes, how many bottles/shots per day? …………………….

(4) Scarification marks? (a) Yes (b) No

(5) Multiple sexual partners? (a) Yes (b) No

(6) Sharing of needles/sharps? (a) Yes (b) No

(7) Intravenous drug abuse? (a) Yes (b) No

(8) Blood Transfusion? (a) Yes (b) No

CLINICAL PARAMETERS

PHYSICAL FINDINGS

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1) Weight (Kg) ______________

2) Height (m) _______________

3) BMI (Kg/m2) _______________

4) Waist Circumference (cm): First reading: ___________________________

Second reading: ________________________

Average reading: ________________________

5) Hip Circumference(cm): First reading: ___________________________

Second reading:__________________________

Average reading:_________________________

6) Waist: Hip Ratio: _________________________

7) Blood Pressure (mmHg): First Recording _____________

Second Recording _________________

Third Recording_________________

Mean Recording ___________

8) Jaundice a) Yes b) No

9) Hepatomegaly a) Yes b) No

10) Splenomegaly a) Yes b) No

11) Ascites a) Yes b) No

ANXILLARY INVESTIGATIONS

LIVER FUNCTION TEST.

AST(IU/L):__________________

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ALT(IU/L):_____________________

AST/ALT(IU/L):________________

SERUM ALBUMIN(g/dl):_____________________

FASTING LIPID PROFILE (mg/dl)

TOTAL CHOLESTEROL (TCHOL)-_________________________

HIGH DENSITY LIPOPROTEIN CHOLESTEROL (HDL-C)-____________

LOW DENSITY LIPOPROTEIN CHOLESTEROL (LDL-C)-_______________

TRIGLYCERIDES (TG)-___________________

VIRAL MARKERS.

ANTI-HCV (a) positive (b) negative

HBsAg (a) positive (b) negative

ABDOMINAL ULTRASOUND FINDINGS.

……………………………………………………………………………………………

……………………………………………………………………………………………

………………………………………………………………………………………………

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APPENDIX 4

CONTROL SUBJECT DATA

BIODATA

Serial Number .............. Initials................ Phone No ........................

(1) Age ..................

(2) Gender (a) Male (b) Female

(3) Ethnicity ...........................

(4) Level of educational (a) Nil (b) Primary (c) Secondary (d) tertiary or post secondary

(5) Occupation ………………….

(6) Income Bracket per month (a) < #10,000 (b) #10,000 - #50,000 (c) #50,000 - #100,000(d)

#100,000 - #200,000 (e) > #200,000

MEDICAL HISTORY

(1) History of Fatigue? (a) Yes (b) No

(2) History of Malaise? (a) Yes (b) No

(3) History of Upper Abdominal Discomfort? (a) Yes (b) No

(4) History of Diabetes Mellitus? (a) Yes (b) No

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(5) If (4) is yes what is the duration? ..............................

(6) History of excessive urination? (a) Yes (b) No

(7) History of excessive thirst? (a) Yes (b) No

(8) History of unintentional weight loss (a) Yes (b) No

(9) History of Jaundice/ Hepatitis? (a) Yes (b) No

(10) If (9) is Yes, what type of Hepatitis? if known ……………………

(11) History of Hypertension? (a)Yes (b) No

(12) If (11) is Yes, what is the duration? .........................

DRUG HISTORY

(1) Are you on any other routine medication(s) (a) Antihypertensives (b) Amiodarone (c)

Chemotherapy drugs d) Corticosteriods (e) Others please specify…………….

FAMILY HISTORY.

(1) Any family history of Diabetes Mellitus? (a) Yes (b) No.

(2) Any family history of liver cancer or chronic liver disease? (a) Yes (b) No

SOCIAL HISTORY

(1) History of alcohol intake? (a) Yes (b) No

(2) If (1) is yes, what type (a) Beer (b) Hard Liquor (c) Wine (d) All of the above

(3) If (1) is yes, how many bottles/shots per day? …………………….

(4) Scarification marks? (a) Yes (b) No

(5) Multiple sexual partners? (a) Yes (b) No

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(6) Sharing of needles/sharps? (a) Yes (b) No

(7) Intravenous drug abuse? (a) Yes (b) No

(8) Blood Transfusion? (a) Yes (b) No

CLINICAL PARAMETERS

PHYSICAL FINDINGS

8) Weight (Kg) ______________

9) Height (m) _______________

10) BMI (Kg/m2) _______________

11) Waist Circumference (cm): First Reading:____________________

Second Reading:____________________

Average Reading:______________________

12) Hip Circumference(cm): First reading: _________________

Second reading: _________________

Average reading:____________________

13) Waist: Hip Ratio: ___________________

14) Blood Pressure (mmHg): First Recording _____________

Second Recording _________________

Third Recording_________________

Mean Recording ___________

8) Jaundice a) Yes b) No

9) Hepatomegaly a) Yes b) No

10) Splenomegaly a) Yes b) No

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11) Ascites a) Yes b) No

ANXILLARY INVESTIGATIONS

LIVER FUNCTION TEST.

AST(IU/L): ______________

ALT(IU/L): ___________________

AST/ALT(IU/L): ____________________

SERUM ALBUMIN(g/dl): _________________

FASTING LIPID PROFILE (mg/dl)

TOTAL CHOLESTEROL (TCHOL)- ___________________

HIGH DENSITY LIPOPROTEIN CHOLESTEROL (HDL-C)-___________

LOW DENSITY LIPOPROTEIN CHOLESTEROL (LDL-C)-_______________

TRIGLYCERIDES (TG)- ____________________

FASTING BLOOD SUGAR (MG/DL)…………

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VIRAL MARKERS.

ANTI-HCV (a) positive (b) negative

HBsAg (a) positive (b) negative

ABDOMINAL ULTRASOUND FINDINGS.

……………………………………………………………………………………………

……………………………………………………………………………………………

………………………………………………………………………………………………