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EVALUATION OF LIVER ENZYMES, LIPID PROFILE AND

GLYCEMIC STATUS AMONG PATIENTS WITH NON-ALCOHOLIC FATTY LIVER DISEASE – A CROSS SECTIONAL STUDY

Dissertation submitted to

THE TAMILNADU DR .M .G .R. MEDICAL UNIVERSITY In partial fulfillment of the regulations

For the award of the degree

M.D. BIOCHEMISTRY BRANCH-XIII

Registration Number: 201723401 DEPARTMENT OF BIOCHEMISTRY

TRICHY SRM MEDICAL COLLEGE HOSPITAL AND RESEARCH CENTRE

IRUNGALUR, TRICHY – 621 105.

Affiliated To

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

MAY 2020

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CERTIFICATE

This is to certify that the dissertation titled “Evaluation of Liver Enzymes, Lipid Profile and Glycemic Status among Patients with Non-Alcoholic Fatty Liver Disease –A Cross Sectional Study” by Dr.S.Santhini, Postgraduate in Biochemistry (2017-2020), is a Bonafide research work carried out under our direct supervision and guidance and is submitted to The Tamil Nadu Dr. M.G.R. Medical University, Chennai, for M.D Degree Examination in M.D Biochemistry (Branch XIII), to be held in May 2020.

Guide: Professor and Head:

Dr.V.R. Prakash,MD., Dr.V.R. Prakash,MD.,

Professor and Head, Professor and Head,

Department of Biochemistry, Department of Biochemistry, Trichy SRM Medical College Trichy SRM Medical College Hospital and Research Centre. Hospital and Research Centre.

Dean:

Dr.A.Jesudoss M.S.,

Trichy SRM Medical College Hospital and Research Centre,

Irungalur, Trichy - 621105

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GUIDE CERTIFICATE

GUIDE:

Prof. Dr. V.R.Prakash M.D., Professor and Head,

Department of Biochemistry,

Trichy SRM Medical College Hospital and Research Centre, Irungalur, Trichy-621105.

CO-GUIDE:

Dr. K. Sridharan M.D., DM., Head of the Department,

Department of Gastroenterology

Trichy SRM Medical College Hospital and Research Centre, Irungalur, Trichy-621105.

Remarks of the Guide:

The bonafide work done by Dr.S.Santhini, on “Evaluation of Liver Enzymes, Lipid Profile and Glycemic Status among Patients with Non-Alcoholic Fatty Liver Disease – A Cross Sectional Study” is under my supervision and I assure that this candidate has abided by the rules of the Ethical Committee.

GUIDE: Prof. Dr. V.R.PRAKASH M.D., Professor and Head,

Department of Biochemistry,

Trichy SRM Medical College Hospital and Research Centre, Irungalur,

Irungalur, Trichy-621105

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DECLARATION

I, Dr.S.Santhini, hereby solemnly declare that the dissertation entitled

“Evaluation of Liver Enzymes, Lipid Profile and Glycemic Status among Patients with Non-Alcoholic Fatty Liver Disease – A Cross Sectional Study” was done by me at Trichy SRM Medical College Hospital and Research Centre, Irungalur, Trichy under the supervision and guidance of my Professor and Head of the Department Dr.V.R.Prakash, M.D.,This dissertation is submitted to The Tamil Nadu Dr. M.G.R. Medical University, Chennai, towards partial fulfillment of the regulations for the award of the Degree M.D Biochemistry (Branch XIII).

Place: Irungalur,Trichy Signature of the Candidate

Date: (Dr.S.Santhini)

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INSTITUITIONAL ETHICS COMMITTEE CERTIFICATE

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ANTI-PLAGIARISM- ORIGINALITY REPORT

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PLAGIARISM-ANALYSIS REPORT

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CERTIFICATE II

This is to certify that this dissertation work titled “Evaluation of Liver Enzymes, Lipid Profile and Glycemic Status among Patients with Non-Alcoholic Fatty Liver Disease – A Cross Sectional Study” is of the candidate Dr.S.Santhini, with Registration Number 201723401 for the award of M.D.DEGREE in the Branch–XIII BIOCHEMISTRY. I personally verified the urkund.com website for the purpose of plagiarism check. I found that the uploaded dissertation file contains from introduction to limitations pages and result shows 11% plagiarism in the dissertation.

Guide & Supervisor:

Prof. Dr. V.R.PRAKASH MD., Professor and Head,

Department of Biochemistry,

Trichy SRM Medical College

Hospital and Research Centre,

Irungalur, Trichy-621105.

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ACKNOWLEDGEMENT

First of all, I am grateful to God for the good health and wellbeing that were necessary to complete this book.

I wish to express my sincere thanks to the Chairman Dr.R.Shivakumar M.D., and the Dean Dr.A.Jesudoss M.S., DLO, of Trichy SRM Medical College Hospital and Research Centre, Irungalur for providing me with all the necessary facilities for the research.

I am deeply grateful to my Guide Prof.Dr.V.R.Prakash M.D., Professor and Head of the Department, Department of Biochemistry, Trichy SRM Medical College Hospital and Research Centre, for his invaluable guidance, constructive comments and warm encouragement to complete the study.

I take this opportunity to express my gratitude to Prof. Dr. Kalavathy Ponniraivan M.D., former Head of the Department of Biochemistry for her immense support from the beginning of the study.

I am thankful to my Co-Guide Dr. K. Sridharan M.D., DM. Head of the Department , Department of Gastroenterology, Trichy SRM Medical College Hospital and Research Centre, for his invaluable support and guidance to complete this study.

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My special thanks to Dr.R.Thamarai M.D., Associate Professor, Department of Biochemistry, Trichy SRM Medical College Hospital and Research Centre, for giving me insightful comments and suggestions throughout my study.

I am thankful to Dr.A.Velayutharaj M.D., Associate Professor, Department of Biochemistry, Trichy SRM Medical College Hospital and Research Centre, for his generous support.

I want to thank Dr. M. Rasheed khan M.D., and Dr.T.M.Moonishaa M.D., Assistant Professors, Department of Biochemistry, Trichy SRM Medical College Hospital and Research Centre, for their encouragement, suggestions and comments about the study.

I extend my heartfelt thanks to Dr.K.Hemalatha M.D., Associate Professor, Department of Community Medicine, Trichy SRM Medical College Hospital and Research Centre, for helping me in statistical analysis.

I am thankful to my seniors Dr.T. Jayakala M.D., Dr. B. Ramya M.D., Tutor Mr. A. Venkatesan M.Sc., for their valuable guidance and my friend Dr.S.Kalavathy, junior colleagues Dr.K.Durga Sowmithri, Dr.M.Muthu Uma Maheshwari, and Dr.P.Nivedha for their great moral support.

I am thankful to all the laboratory staffs of Biochemistry and Pathology, and the outpatient department staffs who helped me during the study. I also want to thank

staffs in the Radiology Department who helped me a lot during the study.

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Most importantly, none of this could have happened without my family. Words are

not enough to thank my family. I am deeply grateful to my life partner Mr. S. Jeevanandam M.E., who is an inspiration to me in all aspects and who takes

care of me in all my struggles.

My special thanks to my father in law Mr. S. Saravanan and my mother Mrs. S. Julia for their unconditional support to complete the study.

Without thanking my kids J. Sanjeevan and J. Akilan, it would be incomplete.

Without their sacrifice, love and affection, it would not have been possible for me to complete this dissertation and my postgraduation.

This dissertation is dedicated to the memory of my father A. Sankaran who showed me this world and provided great moral support in every step of my life.

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CONTENTS

SL.NO TITLE PAGE NO.

THESIS

1.

Introduction

1

2.

Aims and objectives

3

3.

Review of literature

4

4.

Materials and Methods

39

5.

Statistical Analysis and Results

72

6.

Discussion

82

7.

Summary

87

8.

Conclusion

89

9.

Limitations

90

ATTACHMENTS 10.

Bibliography

11.

Annexure I – Master Chart

Annexure II- Case Proforma

Annexure III-Consent Forms

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ABBREVIATIONS

NAFLD Non-Alcoholic Fatty Liver Disease NAFL Non-Alcoholic Fatty Liver

NASH Non-Alcoholic SteatoHepatitis T2DM Type 2 Diabetes Mellitus TG Triglycerides

HDL-C High Density Lipoprotein-Cholesterol LDL-C Low Density Lipoprotein-Cholesterol VLDL-C Very Low -Density Lipoprotein-Cholesterol AST Aspartate aminotransferase

ALT Alanine aminotransferase ALP Alkaline Phosphatase

GGT Gamma-Glutamyl Transferase USG Ultrasonogram

CT Computerized Tomography MRI Magnetic Resonance Imaging TE Transient Elastography NK-T Natural Killer -T cells

AFLD Alcoholic Fatty Liver disease WHO World Health Organization

NHANES National Health and Nutritional Examination Survey BMI Body Mass Index

FFA Free Fatty Acids SAM S-Adenosyl Methionine

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CETP Cholesteryl Ester Transfer Protein SHBP Steroid Hormone Binding Protein TSH Thyroid Stimulating Hormone PCOS Polycystic Ovary Syndrome

PNPLA-3 Patatin- like Phospho Lipase containing domain 3 TM6SF2 Transmembrane 6 Super Family member 2

SREBP 1c Sterol Regulatory Binding Protein 1c

PPAR-γ Peroxisome Proliferator Activated Receptor-γ ChREBP Carbohydrate Responsive Element Binding Protein NF-kβ Nuclear Factor kappaβ

TNF-α Tumour Necrosis Factor α IL-6 Interleukin- 6

IL-1β Interleukin- 1β

SOCS 3 Signalling Cytokine Suppressor 3 ER stress Endoplasmic Reticulum stress ROS Reactive Oxygen Species DAGs Diacylglycerols

SNP Single Nucleotide Polymorphism CVD Cardiovascular Disease

CKD Chronic Kidney Disease IQR Inter Quartile Range kPa kilopascal

MHz Mega Hertz

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1

INTRODUCTION

Non -alcoholic Fatty Liver Disease (NAFLD) is defined as accumulation of lipids in hepatocytes which exceeds 5% of the weight of the liver in the absence of alcohol consumption and without hepatitis B virus or hepatitis C virus infections.1

NAFLD is a spectrum of diseases and comprises of Non-Alcoholic Fatty Liver (NAFL), Non -Alcoholic SteatoHepatitis (NASH), fibrosis and cirrhosis.2 Hepatic Steatosis is the first recognizable stage. When hepatic inflammation occurs, it results in Non- Alcoholic Steato Hepatitis (NASH), which carries a higher risk of progression to fibrosis, cirrhosis or hepatocellular carcinoma.

With a worldwide prevalence of approximately 24% among general population, NAFLD is now becoming a global burden. 3 The real burden of NAFLD is under-estimated due to lack of awareness among the people, long natural history for the development of fibrosis and mortality being not related to liver. It is predicted that NAFLD will be the leading cause of end stage liver disease by the year 2020.4

Central obesity increases the risk of NAFLD and is a key determinant of its pathogenesis.5 Central Obesity is a component of metabolic syndrome which is associated with increased risk for the development of Diabetes mellitus. NAFLD is considered as the hepatic component of the metabolic syndrome.6

Liver enzymes like Alanine amino transferase (ALT), Aspartate amino transferase (AST) and Gamma glutamyl transferase (GGT), which act as markers of hepatocellular injury, have been shown to be elevated in NAFLD.7 Particularly, elevation of ALT, two to three times the normal limit is observed in NAFLD.

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2

NAFLD has been found to be associated with dyslipidemia in 60 -70% of patients.8 Imbalance between increased fatty acid synthesis and reduced delivery of fatty acids by VLDL results in steatosis

Impaired glucose tolerance and/or impaired fasting glucose are precursors for NAFLD and its further progression to fibrosis. T2DM is an independent risk factor for the development of NAFLD.9 Risk of microvascular complications of DM like nephropathy, retinopathy gets increased in patients with NAFLD.10 Glycated haemoglobin (HbA1c) level also gets increased with the progression of fibrosis.11 On the other hand, the presence of NAFLD is associated with increased risk of development of DM.

NAFLD can be diagnosed by clinical history, clinical examination, laboratory investigations and ultrasonogram (USG) abdomen.12 Liver biopsy is the gold standard for assessing the stages of NAFLD. Transient Elastography (TE) – Fibroscan, a non-invasive method can also be used to assess the stages of hepatic fibrosis.13

Studies on NAFLD among our population are lacking. Estimation of the prevalence will give an idea about the burden of the disease. Measurement of anthropometric measures and evaluation of various biochemical parameters among NAFLD patients will help in the identification of the risk factors, early diagnosis and treatment and helps to prevent progression.

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3

AIM OF THE STUDY:

To evaluate the association of obesity, liver enzymes, lipid profile and glycemic status with Non -alcoholic Fatty Liver Disease (NAFLD).

OBJECTIVES:

1. To find the prevalence of NAFLD among our population.

2. To find out the association between Obesity and NAFLD.

3. To find out the association between Liver enzymes and NAFLD.

4. To find out the association between Lipid Profile and NAFLD.

5. To find out the association between Glycemic status and NAFLD.

6. To evaluate the stages of NAFLD by Fibroscore using Transient Elastography.

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4

REVIEW OF LITERATURE Anatomy of Liver:

Liver is the second largest organ of the human body. It weighs about 1.5 kg. It is a peritoneal organ located in the right hypochondrium and epigastric region. It is dark reddish brown in colour having a smooth external surface.

Falciform ligament divides the liver into two lobes, the larger right lobe and the smaller left lobe.14Two accessory lobes namely caudate lobe and quadrate lobe are seen on the visceral surface of the right lobe. Caudate and quadrate lobes are separated by a deep transverse fissure called porta hepatis through which all the blood vessels, nerves and ducts enter and leave the liver except for hepatic veins.

Liver is a highly vascular organ which receives 25 % of cardiac output at rest. It has dual blood supply – about 25 % to 30% supplied by hepatic artery and 70 % - 75 % supplied by portal vein. Arterial and portal blood mixes in the hepatic sinusoids and then drains into the systemic circulation through hepatic venous system.

Figure 1:15 Anatomy of Liver

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5

The fundamental functional unit of the liver is the lobule.16 It is hexagonal in shape and comprises of

1. Hepatocytes

2. Hepatic macrophages 3. Branch of the hepatic artery 4. Branch of the portal vein 5. Tributary of the bile duct 6. Bile canaliculi

7. Sinusoids 8. Central vein

Figure 2: 17 Liver Lobule – Functional unit

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6

Functions of Liver :

The functions of liver are best grouped as secretory , metabolic , hematologic , detoxification, immunologic and storage function.18

Secretory function:

Liver secretes bile which contains bile salts, bilirubin, cholesterol, electrolytes and water. Bile salts help in the digestion and absorption of lipids.

Metabolic function:

Bilirubin metabolism - Bilirubin is a breakdown product of heme. Liver conjugates bilirubin with glucuronic acid which transforms it into a water soluble that gets excreted in bile.

Liver plays a central role in carbohydrate metabolism by synthesizing and degrading glycogen. Liver also synthesizes triacylglcerol, cholesterol, phospholipids, all plasma proteins except immunoglobulins, enzymes like aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase and lactate dehydrogenase.

Hematologic function:

Due to its high vascularity, liver holds large volume of blood. Liver synthesizes prothrombin, fibrinogen and several other clotting factors contributing to hemostasis.

Detoxification:

Liver converts both exogenous and endogenous substances like alcohol, drugs like steroids, barbiturates and hormones like aldosterone, anti-diuretic hormones etc. into less toxic and less biologically active components facilitating their intestinal and renal excretion.

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7

Immunological function:

Liver is a hematopoietic organ and is involved in both innate and adaptive immunity through kupffer cells and Natural Killer-T (NK-T) cells.

Storage function:

Liver stores fat soluble vitamins like A, D, E, K, water soluble vitamin B 12 and minerals like iron and copper.

Liver Diseases:

Liver diseases are classified according to the etiology as19

 Viral Liver Diseases

 Alcoholic Liver Diseases

 Non-Alcoholic Liver Diseases

 Metabolic Liver Diseases

 Autoimmune Liver Diseases

 Cholestatic Liver Diseases

 Tumours and

 Drug Induced Liver Diseases

According to the duration of illness liver diseases can be classified as20

 Acute Liver Diseases ( < 6 months duration)

 Chronic Liver Diseases ( > 6 months duration)

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8

Fatty Liver Diseases:

Excessive accumulation of lipids, particularly triglycerides in liver results in fatty liver disease. Defects in the metabolism of fatty acids underlie the development of fatty liver disease. Hepatic accumulation of fat may result when the amount of fatty acids taken up from the blood and the amount that gets synthesized de novo far exceeds the amount that gets oxidized or secreted via VLDL. Studies have shown in patients with NAFLD that the large bulk of hepatic fat (59%) is derived from free fatty acids released from adipose tissue, 26 % derived from fatty acid synthesis de novo and 15% from diet.21

In general, there are two types of fatty liver disease.

Alcoholic Fatty Liver Disease (AFLD) Occurs in chronic alcoholics

Non - Alcoholic Fatty Liver Disease (NAFLD)

Occurs in the absence of alcohol consumption or with an intake of alcohol < 20 g/day in females and < 30 g/day in males.22

Stages of NAFLD:

Histopathologically, NAFLD comprises of four stages as follows23

 Steatosis

 Steatohepatitis

 Fibrosis

 Cirrhosis

(23)

9

Figure 5:24 Stages of NAFLD

Normal Liver Fatty Liver NASH ± Cirrhosis Fibrosis

Steatosis:

It is the benign condition in which lipid accumulation occurs in 5% or more of hepatocytes. It comprises of macro vesicular steatosis and micro vesicular steatosis. In macro vesicular steatosis, large lipid droplet occupies the cytoplasm and displaces the nucleus to one side whereas in micro vesicular steatosis, innumerable minute lipid droplets are present with nucleus in the original position.25 Initially these lipid droplets comprise of a central core of triacylglycerols with or without cholesterol esters and a monolayer of phospholipids peripherally. It has been shown that inactive PNPLA3 accumulates on the lipid droplet surface and gives rise to macro vesicular steatosis.26

Steatohepatitis:

Steatosis with inflammation results in ballooned hepatocytes.12 Inflammation occurs due to oxidative stress, endoplasmic reticulum stress, loss of intermediate filament cytoskeleton and retention of fluids. Glycogenated nuclei, mega mitochondria, acinar lipogranulomas, and acidophilic bodies are seen in NASH. No single feature is entirely specific for the diagnosis of NASH.

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10 Fibrosis:

It is characterized by perisinusoidal, pericellular and periportal deposition of fibrous tissue.12 Collagen fibres can be seen encircling hepatocytes with more advanced lesions.

Progression of the disease is assessed by the presence or absence of bridging fibrosis which is a connective tissue septum linking the portal tracts and central veins across lobules.

Cirrhosis:

Cirrhosis, the end stage of chronic liver disorder is characterized by diffuse distribution of fibrosis and nodularity in the parenchyma.12 Cirrhosis is diagnosed histologically by the absence of central portal relationship, bridging fibrosis and nodules due to hepatic regeneration. Cryptogenic cirrhosis is a condition which presents without the features of NASH.

NASH clinical research network’s NAFLD activity score (NASH CRN-NAS), Brunt staging system and steatosis, activity, and fibrosis (SAF) are histological scoring systems currently used for staging NASH.27

Pathogenesis:

Development of Steatosis:

Steatosis arises from the interaction between genetic factors, gut microbiota, diet and de novo lipogenesis by upregulation of lipogenic transcription factors such as sterol regulatory element binding protein-1c (SREBP-1c), peroxisome proliferator-activated receptor gamma (PPAR-γ) and carbohydrate -responsive element binding protein (chREBP).

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11

SREBP-1c is a membrane bound transcription factor. It activates all genes needed for lipogenesis. It also activates Acetyl CoA carboxylase 2 that forms malonyl CoA. Malonyl CoA inhibits Carnitine Palmitoyl Transferase 1(CPT-1) resulting in reduced entry of fatty acids into mitochondria for beta oxidation. Hence, overexpression of SREBP-1c can result in fatty liver. Transcription of SREBP is stimulated by insulin even when peripheral resistance to its action is present.28

PPAR-γ is a member of the super family of nuclear hormone receptors needed for normal differentiation of adipocytes. Nuclear receptor’s activating ligand gets stimulated by SREBP-1c results in transcriptional activation of PPAR-γ. PPAR-γ expression is enhanced in animal models with fatty liver.29

ChREBP mediates the lipogenic effects of glucose. Its translocation from cytosol to nucleus and its binding to DNA is activated by glucose. Glucose activates binding of ChREBP to the promoter region of pyruvate kinase gene in the Liver. Pyruvate kinase is a key glycolytic enzyme that converts phosphoenol pyruvate to pyruvate. Pyruvate can enter the TCA cycle after conversion to acetyl CoA and generate citrate – the main source of acetyl CoA used for the synthesis of fatty acids.30

Patatin-like phospholipase domain containing 3 (PNPLA-3), involved in the hydrolysis of triacylglycerols in the adipocytes has been found to play an important role in the causation of NAFLD. The genetic polymorphism, in PNPLA3-I148M (rs 738409C/G) is strongly associated with NAFLD.31 Genetic variant of transmembrane 6 super family member-2 (TM6SF2) gene encoding E167 K (rs58542926 C/T) results in increased deposition of triglycerides in liver.32

(26)

12

Figure: 3

33

Pathogenesis of NAFLD

This figure shows the pathogenesis of NAFLD and its progression to NASH due to various factors like oxidative stress, ER stress, Cytokines production etc.

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13 NAFLD – Progression:

Three mechanisms underlie the progression of NAFLD. They are steatosis, lipotoxicity and inflammation.

The surplus fat in the liver causes lipotoxicity and leads to mitochondrial dysfunction and endoplasmic reticulum stress (ER stress).34 Dysfunctional mitochondria develop a high capacity to oxidize fatty acids resulting in production of ROS. This leads to oxidative stress because of an imbalance between the production of protective oxidants and ROS which ends up with hepatocytes death.35

The transcription factor nuclear factor kappa β (NF-kβ) signalling gets enhanced by steatosis which results in the production of proinflammatory mediators like tumour necrosis factor α (TNF-α), interleukin- 6 (IL-6) and interleukin- 1β (IL-1β). Kupffer cells get activated by these cytokines and result in inflammation in NASH.36 It has been noted that TNF-α and IL-6 play a part in hepatic insulin resistance by upregulating signalling cytokine suppressor 3 (SOCS 3).37

Gut microbiota plays an important role in both pathogenesis of NAFLD and its progression. Ethanol producing bacteria Escherichia coli increase the gut permeability.

Dysbiosis of gut microbiota results in lymphocytes loss from intestinal mucosa leading to intestinal inflammation. Intestinal inflammation, impaired immune function and increased intestine permeability together mediate pathogenesis and progression of NAFLD.38

Thus, NAFLD’s pathogenesis appears to be a vicious cycle of steatosis, lipotoxicity and inflammation resulting in complex changes in the liver’s histopathological and biochemical characteristics.

(28)

14

Figure 4

39

:

Pathogenesis of NASH

This figure shows carbohydrate and fatty acid substrates overload promoting the generation of lipotoxic species like diacylglycerols (DAGs), ceramides, lysophosphatidyl cholines that lead to mitochondrial dysfunction, ER stress, hepatocellular injury, inflammation and apoptosis resulting in NASH, fibrosis and hepatocellular carcinoma.

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15

Epidemiology of NAFLD:

The prevalence of NAFLD is approximately 25% worldwide with the highest levels recorded in Arabia (32%) and in South America (31%) followed by Asia (27%), USA (24%) and Europe (23%). NAFLD is less prevalent in Africa (14%).40

National Health and Nutrition Examination Survey (NHANES ) reports have shown that, between 1999 and 2008, the prevalence of chronic liver diseases due to NAFLD rose from 47% to 75%.41

Among T2DM patients, the prevalence of NAFLD ranges from 70 %-75% whereas the prevalence of T2DM among NAFLD patients was reported to be 43%.42

The incidence of NAFLD may exceed 95% among obese individuals. Patients with NAFLD are observed with co-morbid illnesses such as hyperlipidemia (69%) particularly hypertriglyceridemia (41%), metabolic syndrome (43%) and hypertension (39%).28

A population based research revealed that Hispanics had a higher incidence of NAFLD/NASH (58.3%) compared to Whites (44%) and Blacks (35.1%).43 The difference in racial and ethnic incidence may depend on socioeconomic, behavioural and genetic variables.

Studies show that the prevalence of NAFLD in India is approximately 16 –32%

among urban population and 9 –16% among rural population.44 The mean age of occurrence of NAFLD among Indian population is around 35-50 years.45 NAFLD’s incidence increases with age. Higher incidence of NAFLD among females is noticed due to loss of protective effects of estrogen after 50 years of age.46

The incidence of NAFLD in children and adolescents has risen significantly in current decades due to childhood obesity.NAFLD has a prevalence of up to 70% in overweight and obese youth compared to 7% among those with normal weight.47

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16

Classification of NAFLD:

48

1. Primary Insulin Resistant NAFLD 2. Primary Non-Insulin Resistant NAFLD 3. Secondary NAFLD

1.Primary Insulin Resistant NAFLD:

 Metabolically Healthy Obesity (MHO) ( Visceral Obesity)

 Metabolically Obesity Normal Weight (MONW)

 Type 2 Diabetes Mellitus (T2DM)

 Congenital Lipodystrophy

 Lysosomal Acid Lipase Deficiency (LALD) (Non Obese Liver Disease) 2.Primary Non-Insulin Resistant NAFLD:

 Genetic Causes (PNPLA & TM6SF2 genes)

 Hypobetalipoprotein Syndrome

 Cryptogenic (Unknown Cause) 3.Secondary NAFLD:

 Associated with endocrine disorders (PCOS, Hypothyroidism)

 Environmental ( High fat diet, High fructose diet)

 Drug related (Amiadarone, Methotrexate, Tamoxifen, Corticosteroids)

 Associated with other hepatic diseases (Viral, Autoimmune)

 Total parenteral nutrition, Starvation

 Jejunal bypass

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17

Risk factors for NAFLD:

14

 Genetics

 Ethnicity

 Gender and Age

 Obesity

 Diabetes Mellitus

 Dyslipidemia

 Metabolic Syndrome

 Polycystic ovary syndrome

 Hypothyroidism

 Sleep apnoea syndrome

Genetics and Ethnicity:

A genetic background is highlighted by the ethnic variations in NAFLD and NASH incidence. A study of three family cohorts (FRAM, Fam HS, Amish) based on CT measurement has shown that hepatic steatosis is heritable (26%-27%).49 The first genome wide associations study (GWAS) performed in NAFLD patients described the most coherent association with patatin -like phospholipase domain containing 3 (PNPLA-3) or adiponutrin.31

A study on multi-ethnic population revealed a strong association between single nucleotide polymorphism (SNP), rs 738409 with substitution of cytosine by guanine and hepatic fat content and serum aminotransferases levels. In Hispanics, this single nuclear polymorphism (SNP) was more common followed by European-Americans and less frequent in African Americans, like racial variations in NAFLD prevalence.18

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18

Various genetic associations proposed by GWAS and verified by case-control candidate gene studies are (SNP Details)

 Neurocan (NCAN)– an adhesion molecule with hepatis steatosis (rs 2228603).50

 Glucokinase regulatory gene (GCKR) with steatosis and NASH (rs 780094).36

 Lysophospholipase-like 1 (LYPLAL1) – associated with sex-specific pattern of fat distribution. SNP results in increased waist -hip ratio (rs 12137855). 36

 Protein phosphatase-1 regulatory subunit 3b (PPP1R3B) – involved in synthesis of glycogen. Studies show that Minor rs4841132 A allele reduces risk of fibrosis and hepatocellular carcinoma.36

 Angiotensin II receptor-1 variants- 5 variants (rs 3772622, rs 3772627, rs 3772630, rs 3772633, rs 3772636) stimulates JAK-2 leading to hepatic stellate cells activation which results in hepatic fibrosis. 51

 Polymorphisms in promoter of TNF gene -238 A allele – associated with inflammation and progression of the disease.52

Gender and Age:

Recent studies revealed that males are highly prone for the development of fatty liver disease (31%) compared to females (16%).53There is increased incidence of NAFLD in young to middle aged men and a decline is observed after 50-60 years of age. But in women, increased incidence of NAFLD is noticed above 50 years with the peak around 60-69 years of age.54 In elderly patients with NAFLD, not only the incidence but also the probability of disease progression is more.

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19 Obesity and NAFLD:

World Health Organization (WHO) describes overweight and obesity as unusual or excessive accumulation of fat that may impair health.55 Obesity and its accompanying illnesses are among the world’s major causes of death. The 2014 survey from the WHO Global Health Observatory shows that worldwide obesity occurs in 15% of females and 11%

of males among the age group 18 years and above.

Obesity is defined by WHO as a body mass index (BMI) greater than 30 kg/m2. BMI is calculated by the formula weight in kg / height in square meter.

Table 1:

International classification of Body Mass Index by WHO

There are significant variations in genetics, lifestyle and body structure between Asian and Western population. Even with a lower BMI, Asians are more probable to have central fat deposition. Hence, WHO recommends different cut off points for overweight and obesity among Asian population.56

Classification International

Underweight <18.5 kg/m2

Normal weight 18.5 – 24.9 kg/m2

Overweight 25 – 29.9 kg/m2

Obese Class I 30 -34.9 kg/m2 Obese Class II 35 -39.9 kg/m2 Obese Class III >40 kg/m2

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20 Table 2:

Classification of BMI for Asians

Obesity is associated with co-morbidities like T2DM, hypertension, hyperlipidemia, NAFLD, cardiovascular disease, chronic kidney disease, osteoarthritis, obstructive sleep apnoea and malignancies. Moreover, higher all-cause mortality is observed among obese individuals.57

Increasing trends in obesity worldwide result in increased prevalence of NAFLD as well as increased severity of progression of NAFLD to NASH which may advance to cirrhosis and hepatocellular cancer.58

In conditions like obesity and lipodystrophies, the excess energy storing capacity of adipose tissue gets reduced.59 Enhanced adipocyte dysfunction and insulin resistance leads to lipolysis.

Classification Asians

Underweight <18.5 kg/m2

Normal weight 18.5 – 22.9 kg/m2

Overweight 23 – 24.9 kg/m2

Obese Class I 25 – 29.9 kg/m2 Obese Class II >30 kg/m2

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21

Increased circulation of free fatty acids (FFA), leptin and decreased levels of adiponectin contribute to intrahepatic fat accumulation in the form of triglycerides (Simple Steatosis). There will also be increased intake of high fat diet and carbohydrate diet leading to increase de novo lipogenesis.

If obesity is not managed at the stage of simple steatosis, immune cells infiltrate the liver resulting in chronic, low grade intrahepatic inflammation. Prolonged inflammation leads to liver injury. Normally if liver injury occurs, immune cells and wound healing cells like activated endothelial cells, myofibroblasts and progenitor cells work together for tissue regeneration. In obesity, this mechanism of tissue degeneration in response to liver injury fails resulting in fibrosis. The risk of development of cirrhosis is also increased with obesity.

Approximately 3-15% of obese NASH patients advance to cirrhosis and approximately 4-27% of cirrhotics progress to hepatocellular carcinoma.60 Hepatocellular carcinoma may develop very early in patients with NAFLD or it may occur even without cirrhosis.

In response to severe and persistent liver cell injury, Ito cells get activated to produce abnormal extracellular matrix in its quantity and composition. Myofibroblasts increases portal venous resistance and abnormal matrix density. This is the initiation step of progression to fibrosis. Regenerating hepatocytes tries to repair the injured cells and alters the hepatic architecture and its functions. This results in progression of fibrosis to cirrhosis.

(36)

22 Figure6:61

NAFLD Pathophysiology driven by Obesity

This figure shows the dysfunctional adipose tissue prone for inflammation and pathophysiology in progression of NAFLD.

(37)

23 Diabetes Mellitus and NAFLD:

Diabetes mellitus (DM) is a public health issue and is regarded as the epidemic of the century.62 Around 415 million individuals suffer from DM globally and as per WHO estimate, in 2030 diabetes will be the seventh major cause of death.

DM is a group of metabolic diseases characterized by hyperglycemia caused by insulin secretion defects, insulin action or both. According to American Diabetes Association (ADA), based on etiopathogenesis, diabetes mellitus is classified into four types namely

 Type 1 diabetes mellitus (T1DM)

 Type 2 diabetes mellitus (T2DM)

 Gestational diabetes mellitus (GDM) and

 Specific types of diabetes due to other causes

(Maturity Onset Diabetes of the Young (MODY), diseases of exocrine pancreas, drug induced etc).63

Incidence of T1DM is about 5%-10% of DM occurrences and is due to autoimmune pancreatic β cell destruction and complete insulin deficiency. Incidence of T2DM is about 90%-95% of diabetic population and is due to insulin resistance and relative insulin deficiency.

The incidence of NAFLD in individuals with T2DM has risen and is estimated at around 70% to 75%. India is now the world’s diabetic capital and NAFLD has emerged as a major cause of liver disease. Several studies show that T2DM patients with NAFLD are highly prone for progressive forms of NAFLD like NASH, liver fibrosis, hepatocellular carcinoma development and liver related mortality.

(38)

24

On the other hand, NAFLD patients have a high prevalence of prediabetes and the presence of NAFLD predicts the T2DM development.64 NAFLD was associated with higher microalbuminuria rates, lower glomerular filtration rate and retinopathy in patients with T1DM and T2DM. In non-diabetic patients with NAFLD, family history of diabetes may provide additional risk stratification.65 Diabetes and NAFLD have shared pathogenetic processes. Genetic and environmental variables may interact with metabolic derangements to speed up the development of NAFLD in diabetic patients.

Pathophysiology of NAFLD in DM

Insulin resistance tends to be the most important in NAFLD pathogenesis.66 Insulin resistance by homeostasis model of assessment for insulin resistance ( HOMA-IR) has been linked with increased fibrosis and also with increased rates of hepatic complications.67 Insulin resistance results in hyperglycemia and thereby increased insulin secretion &

enhanced lipase activity in adipose tissue. Hyperglycemia and hyperinsulinemia upregulate several important lipogenic transcription factors like sterol regulatory element-binding protein 1c (SREBP1c) and carbohydrate response element binding protein (ChREBP) thereby promoting hepatic lipid synthesis or de novo lipogenesis.43

Diabetic patients with NAFLD have greater circulating inflammation markers than non-diabetic patients with NAFLD.68 In patients with NAFLD, serum concentrations of adiponectin, an anti-inflammatory insulin sensitizer and possibly hepatoprotective adipokine is in reduced levels.69 Fall in adiponectin levels among prediabetes and T2DM reveals a link between adipocyte dysfunction and NASH.70

(39)

25

Figure 7:

71

Pathophysiology of NAFLD in DM

This figure shows insulin resistance in diabetes mellitus which leads to increased gluconeogenesis, glycogenolysis, de novo lipogenesis and results in the development of NAFLD.

(40)

26 Dyslipidemia and NAFLD:

Fatty acids undergo elongation, desaturation and re-esterification for synthesis of complex lipids like phospholipids (PL), diglycerides or triglycerides. Some of the re- esterified triglycerides are combined with apo-lipoprotein-B in very low- density lipoproteins and exported into circulation. This process is controlled by microsomal triglyceride transfer protein and assisted by encapsulation of the neutral lipid nucleus with a phospholipid monolayer enriched with phosphatidyl choline molecules containing poly unsaturated fatty acids like arachidonic acid, docosahexaenoic acid72.

S–Adenosyl Methionine (SAM) is the methyl group donor in all methyl transfer reactions occurring in cells. SAM deficiency leads to impaired synthesis and release of VLDL-TG resulting in triglyceride, diglyceride and fatty acid accumulation. Due to concurrent oxidative stress, steatosis develops and progresses to non-alcoholic steatohepatitis.

A curvilinear relationship exists between intrahepatic triglyceride levels and very low- density lipoprotein (VLDL) – triglyceride (TG) secretion rate. In hepatic steatosis, VLDL-TG secretion reaches a plateau irrespective of the amount of intra hepatic triglycerides.

(41)

27 Figure 8:73

Lipid Metabolism

This figure shows the lipid metabolism. FA-fatty acid, DG-diglyceride, TG- triglyceride, MTTP-microsomal triglyceride transport protein, APOB- apo lipoprotein B, PC- PUFA-phosphatidyl choline poly unsaturated fatty acid, SAM-S adenosyl methionine, VLDL-TG -very low-density lipoprotein triglyceride

(42)

28

NAFLD and dyslipidemia have a strong and interdependent relationship. Up to 60%- 70% NAFLD patients are present with dyslipidemia.43 NAFLD patients have higher frequency of cardiovascular disease (CVD) compared to those without NAFLD. NAFLD is associated with increased levels of triglycerides, low high- density lipoprotein (HDL-C) and dense, small low- density lipoprotein (LDL-C) which predisposes an individual for CVD.

Insulin resistance and high liver fat content result not only in overproduction of very low-density lipoprotein (VLDL) particles but also increase in VLDL size and triglyceride content and number of apo-B 100 particles.74 Hypertriglyceridemia in NAFLD is linked with reduced peripheral clearance of triglycerides by lipoprotein lipase.

Cholesteryl ester transfer protein (CETP) gets stimulated by increased VLDL levels leading to enhanced exchange of triglycerides and cholesterol between VLDL and LDL-C particles which results in production of triglyceride rich LDL-C. These TG-rich particles are in turn transformed to small and dense LDL-C which is related to increased cardiovascular risk.75

Similarly, VLDL triglycerides are exchanged for HDL cholesteryl esters by CETP resulting in the formation of triglyceride rich HDL-C. Modification of TG-rich HDL-C particles by hepatic lipases leads to increased catabolism of HDL-C and renal clearance of its significant protein component, apo A-1. This results in low HDL-C levels which in turn predisposes to cardiovascular disease. Circulating HDL-2 and HDL-3 functions also get altered leading to atherogenesis. High levels of oxidized forms of LDL-C in NASH are also atherogenic.

(43)

29 Figure 9:76

Pathophysiology mechanism of dyslipidemia in NAFLD

This figure shows production of TG-rich LDL-C and HDL-C leading to increased lipid peroxidation and increased renal clearance of HDL-C.

(44)

30 Polycystic ovarian syndrome and NAFLD:

Increased incidence of NAFLD is observed among females suffering from PCOS which is frequently associated with obesity and insulin resistance leading to increased incidence of NAFLD among those patients.77

Hypothyroidism and NAFLD:

Obesity and insulin resistance play an important role in the development of NAFLD among subclinical or overt hypothyroidism patients.78

Obstructive sleep apnoea:

Obstructive sleep apnoea (OSA) is characterized by total or partial obstruction of the airway due to pharyngeal collapse that occurs during sleep. The incidence of OSA which is about 4% among general population has risen to 35%-45% among obese individuals.79 Repetitive hypoxemic and hypercapnic episodes (chronic intermittent hypoxia) results in production of proinflammatory cytokines, oxidative stress, endothelial dysfunction, metabolic dysregulation and ultimately insulin resistance prone to the development of NAFLD.

Liver Enzymes in NAFLD:

Abnormal levels of liver enzymes can indicate damage to the liver or changes in bile flow. While tests that evaluate serum liver enzymes are frequently referred to as liver function tests, they tend to represent hepatocyte integrity or cholestasis rather than function of the liver. Liver enzymes are categorized into two types namely

1. Enzymes reflecting hepatocyte integrity 2. Enzymes reflecting cholestasis.80

(45)

31 1. Enzymes reflecting hepatocytes integrity:

 Aspartate aminotransferase (AST)

 Alanine aminotransferase (ALT)

 Glutathione S transferase

 Glutamate dehydrogenase 2. Enzymes reflecting cholestasis:

 Alkaline phosphatase (ALP)

 Gamma glutamyl transferase (GGT)

 5 ́- Nucleotidase

AST:

AST is an intracellular enzyme which is present in cytoplasm (20% of total activity) as well as in mitochondria (80% of total activity).81 It is widely distributed in all tissues of the body particularly in cardiac muscle, liver, skeletal muscle and kidney. It catalyses the transfer of α-amino group from aspartate to α-ketoglutarate to produce oxaloacetate which enters citric acid cycle and glutamate.

Normal turnover of tissue cells contributes to the serum or plasma AST levels.

Elevated level of AST is observed in tissue damage especially hepatic inflammation or necrosis, cardiac muscle damage (mostly ischemic injury) and skeletal muscle damage (rhabdomyolysis). Lack of tissue specificity is the major disadvantage of AST, as an indicator of liver cell damage.

(46)

32 ALT:

ALT is an intracellular enzyme present particularly in cytoplasm of liver cells.

Minimal concentration of ALT is present in skeletal muscle and kidney. ALT catalyse the transfer of α-amino group from alanine to α-ketoglutarate to produce pyruvate and glutamate which contributes to citric acid cycle.

Though both AST and ALT require pyridoxal phosphate as coenzyme for their activity, pyridoxal phosphate deficiency has greater impact on ALT activity than AST activity.82 This is the reason behind increased AST/ALT ratio among alcoholic liver disease patients who are highly prone for pyridoxal deficiency. ALT is liver specific and is measured in liver disorders and in people at risk of developing liver diseases.

ALP:

Alkaline phosphatases are a group of isoenzymes situated on the cell membrane. They hydrolyse organic phosphate esters present in extracellular space. They require Zn and Mg as cofactors. Alkaline phosphatases are categorized into two types namely tissue -specific and tissue-nonspecific types. ALP in germinal tissues, placenta and intestine are tissue- specific whereas the bone, liver and renal forms are tissue-non-specific.83

The enzyme present in blood originates primarily from the liver and bone. It may also originate from other tissues. Normal individuals have varying levels of serum ALP according to the age. Because of bone development and growth, ALP levels are high among children and adolescents. There will be a decline in ALP levels between 15-50 years of age group and an increase is seen again in old age. Men have higher levels of ALP compared to women.

Elevated levels of ALP are observed primarily in bone disorders and cholestasis. 84

(47)

33 GGT:

GGT is found in hepatocytes, biliary epithelial cells, kidney tubules, intestines and pancreas. GGT helps in the transport of amino acids through gamma glutamyl cycle. It is involved in the metabolism of glutathione, the most important antioxidant in humans, metabolism of leukotrienes, xenobiotics and glutaminase action.85

GGT is a marker of liver dysfunction, bile duct obstruction or injury and alcohol use.

Consumption of even small amount of alcohol may result in alteration in GGT levels.86 Several studies showed an association of increased GGT levels with risk of development of coronary heart disease, type 2 DM and stroke. GGT along with ALP helps to differentiate diseases of liver pathology or bone pathology. Both GGT and ALP get elevated in liver diseases whereas only ALP gets elevated in bone diseases.

In recent times, serum levels of liver enzymes like AST and ALT are used as surrogate markers to assess the severity of NAFLD.87 One of the prevalent factors for patients visiting gastroenterology or hepatology clinics is high concentrations of aminotransferases like AST and ALT. Aminotransferases are good indicators of progression of NAFLD.88

Based on the magnitude of altered aminotransferases level, it is classified into three categories namely

 Mild (<5 times the upper reference limit)

 Moderate (5-10 times the upper reference limit) and

 Marked (>10 times the upper reference limit)

(48)

34

The biochemical picture of NAFLD consists of slightly increased activity of amino transferase enzymes (not more than 4 times the upper reference value). In NAFLD, GGT concentrations can be increased up to 3 times the upper reference limit in 50% of patients with NAFLD lacking alcohol consumption.89 Elevated GGT level is associated with increased severity of NAFLD and with increased mortality. ALP levels may get elevated in non-alcoholic steatohepatitis. It may increase up to 2-3 times the upper reference range.

About 78% of patients with NAFLD may have normal liver enzymes.

Diagnosis of NAFLD:

Liver Biopsy:

The gold standard investigation to diagnose NAFLD is Liver biopsy.90 Diagnosis of fatty liver or steatosis is made if more than 5% of hepatocytes in a liver biopsy have ectopic lipid droplets. Steatohepatitis requires the presence of lobular inflammation and hepatocyte lesion in the form of hepatocellular ballooning with or without Mallory-Denk bodies in addition to steatosis. Due to invasiveness and expensive procedure associated complications like hemorrhage, liver biopsy is not used routinely for the diagnosis of NAFLD.

Imaging Modalities:

 Ultrasound Abdomen

 Computed Tomography

 Magnetic Resonance Imaging

 Controlled Attenuation parameter

(49)

35

Ultrasound Abdomen

Abdominal ultrasound is the first technique to be used in the clinical practice to diagnose NAFLD. It is non- invasive, commonly accessible and has reasonable precision in the diagnosis of hepatic steatosis with 60%-94% sensitivity and 66%-97% specificity91. Its sensitivity for mild steatosis reduces dramatically, being only reliable for steatosis above 30%92. The existence of fibrosis, edema, necrosis and extra-hepatic adipose tissue affects its specificity.

Normal liver echotexture and mild, moderate or severe steatosis is defined by a semi- quantitative grading system as Grade I – mild, Grade II – moderate and Grade III – severe steatosis. 93One of the most important limitations of USG for evaluation of NAFLD is limited diagnostic accuracy for mild hepatic steatosis. Intra- and inter observer variability is the other major drawback.

Computerized Tomography

Non enhanced CT helps to evaluate the severity of NAFLD based on inverse relationship between hepatic attenuation and hepatic steatosis. Attenuation value of liver is 50-65 HU which is 8-10 HU higher than spleen. In steatosis, the attenuation value of liver gets reduced to < 40 HU. More than 30 % of steatosis can be diagnosed with 100%

specificity and 82% sensitivity using non-enhanced CT. Contrast-enhanced CT is another modality used which reduces the radiation exposure of non-enhanced CT.94

(50)

36

Magnetic Resonance Imaging

MRI-derived proton density fat fraction (MRI-PDFF), a non -invasive MRI based method determines hepatic steatosis by differences in signal intensity on opposed phase.

It uses MRI visible protons combined with liver fat to quantify steatosis by dividing up all protons in the liver. It is superior to other imaging modalities since its performance is not affected by obesity and is used to monitor the treatment efficacy.95

Controlled Attenuated Parameter (CAP)

The Fibroscan measures CAP, a parameter based on ultrasonic signals. Fibroscan is a non-invasive tool that uses the transient elastography method to assess the rigidity of the liver. By evaluating the velocity of a vibration wave (shear wave) produced on the skin, liver rigidity is assessed. The velocity of the shear wave is determined by evaluating the time the vibration wave takes to move within the liver to a specific depth96.

Since the fibrous tissues are harder than normal liver texture, liver rigidity can infer the degree of hepatic fibrosis. Reliability of the test is improved by taking a minimum of 10 readings with 60% success rate and inter quartile range (IQR) ≤ 30% of the median value.97

The results are expressed as kilopascals (kPa). Certain condition in which fibrosis is over estimated are acute hepatitis, biliary obstruction, tumour in the liver and hepatic congestion due to heart failure. A European study reveals that inaccurate results are obtained in about 20% of patients with BMI >30 kg/m2, features of metabolic syndrome, older age and the presence of ascites.98 M probe (3.5 MHz) is used normally whereas XL probe is used in obese individuals of BMI ≥ 28 kg/ m2 (99).

(51)

37

Serum biomarkers and indices NAFLD:

 Fatty Liver Index (FLI) – based on BMI, waist circumference, triglycerides and gamma glutamyl transferase. Score varies between 0-100. Value of 30 rules out NAFL.100

 Hepatic Steatosis Index (HSI) – based on BMI, diabetes and ALT/AST ratio. Reduced accuracy in obesity.101

 Steato Test – includes 10 biochemical tests, age, gender and BMI.102

 NAFL screening score– based on age, BMI, fasting plasma glucose, triglyceride, ALT/AST ratio and uric acid.103

NASH:

 Cytokeratin-18 (CK18), CXCL-10.104

 Fibroblast growth factor 21 (FGF 21).105

 Adipocytokines - adiponectin, leptin, resistin.106

 Serum Ferritin.107

 NASH test.87 and NASH Clin Lip Met score.108 Fibrosis:

 AST-to-Platelet Ratio Index (APRI).109

 FIB-4 – includes age, platelet, AST and ALT.110

 NAFLD Fibrosis Score (NFS) – age, BMI, hyperglycemia, AST/ALT ratio, platelets and albumin.111

 BARD score – BMI, aldosterone-renin activity score and presence of T2DM.112

 Serum DNA methylation of PPAR-γ.113

(52)

38 Figure 14:19

Extrahepatic Complications of NAFLD

(53)

39

MATERIALS AND METHODS Study Protocol:

This cross-sectional observational study was carried out in the Department of Biochemistry, Trichy SRM Medical College Hospital and Research Centre, Irungalur, Trichy over a period of one year from June 2018 to May 2019.

The study participants included both males and females of age ≥ 18 years, attending the outpatient department (OPD) with general complaints and without the history of alcoholism. Persons with history of alcoholism, hepatic illness, chronic illness, critical illness, on drugs which alter liver enzymes levels and pregnant individuals were excluded. The study was approved by the Institutional ethical committee and conforms to the Helsinki declaration.

Informed consent was obtained from the participants.

After registering the patient for this study, a brief history of presenting illness, past illness, co-morbidities like diabetes, hypertension, personal history such as smoking, substance abuse and treatment history were taken.

Clinical Examination:

Anthropometric measurements like Height (cm), Weight (kg) were measured and body mass index (BMI) was calculated. With the help of stadiometer, height was measured in centimetres. The person was told to stand upright barefoot, heels held close together with back against the backboard kept vertically and eyes looking forward. Weight was measured by electronic weighing scale with light garments on a horizontal surface.

BMI was calculated by dividing weight in kg with height in metre square. Patients were grouped into following categories based on BMI according to WHO recommendations for Asian population.

(54)

40 Table 3:

Underweight <18.5 kg/m2 Normal weight 18.5 – 22.9 kg/m2

Overweight 23 – 24.9 kg/m2

Obese ≥25 kg/m2

Pulse rate was noted and by using mercury sphygmomanometer, blood pressure was recorded by making the patient be seated comfortably. According to JNC-7 guidelines, subjects with BP ≥140/90 mmHg were considered as hypertensive patients along with the patients on anti-hypertensive drugs. Systemic examination - cardiovascular system, respiratory system, central nervous system and abdomen was done.

Strictly on aseptic precautions, 5 ml of venous blood was obtained after an overnight fasting for biochemical assessment of plasma glucose, lipid profile and liver enzymes in a clot activator tube and for HbA1c in EDTA tube. Samples were centrifuged immediately and assayed. All parameters were assayed using the fully automated analyser Mindray BS 420.

HbA1c was assayed using the fully automated analyser Cobas C 311.

Ultrasonogram Abdomen:

USG - abdomen was done by using GE – Model LOGIQ P9 device. Liver lobes were scanned through a sub-costal approach using standard protocols of imaging. To identify normal echotexture or steatosis, a semi quantitative system of grading was used.

(55)

41 Grading of Fatty liver by USG

Normal liver echotexture:

Liver parenchyma and cortex of right kidney have similar echogenicity. We can visualize the intra-hepatic vasculature clearly and well depicted posterior aspects of liver can be seen.

Grade I Fatty liver:

Mild steatosis with slightly increased liver echogenicity is present when compared to renal cortex.

Grade II Fatty liver:

Moderate steatosis in which there will be a clearly increased echogenicity of the liver parenchyma, visualization of intra- hepatic vasculature is impaired, and echogenicity of renal cortex is decreased.

Grade III Fatty liver:

Severe steatosis with markedly increased liver echogenicity and poor visualization of diaphragm and intra-hepatic vasculature with markedly decreased echogenicity of renal cortex

(56)

42 Figure 10: 93

Normal Liver Echotexture

This figure shows similar echogenicity of liver parenchyma and renal cortex

Figure 11: 93

Grade I Fatty liver

This figure shows increased echogenicity of liver parenchyma compared to renal cortex

(57)

43 Figure 12: 93

Grade II Fatty liver

This figure shows decreased visualization of hepatic vasculature Figure 13: 93

Grade III Fatty liver

This figure shows poor visualization of diaphragm and markedly decreased echogenicity of renal cortex.

(58)

44 TRANSIENT ELASTOGRAPHY – FIBROSCAN:

Patients who were diagnosed as having NAFLD underwent transient elastography procedure for assessing the stages of fibrosis. FibroScan® (Echo Sens) – the most popular device was used to assess the liver stiffness non – invasively. An ultrasound probe is placed on the skin above the liver area in the right mid-axillary line with the patient lying in supine posture. Vibration waves were generated on the skin whose velocities were measured and this gave the liver stiffness values. Every time when a vibration wave is generated by the probe, the patients felt a gentle ‘flick’. It took 10 minutes for the performance of the test...

FibroScan® results were interpreted from a guide developed by Echo Sens. Results ranged from 1 kPa to 75 kPa. About 95 % of healthy people without any liver pathology will have the liver stiffness value < 7 kPa.

Figure:14

Interpretation of Fibroscan

Table 4:

F0 – F1 F -2 F- 3 F-4

< 7 kPa 7.5 – 8.5 kPa 8.6 – 10.5kPa > 10.5 kPa

Fatty Liver NASH Fibrosis Cirrhosis

(59)

45

METHODOLOGY Biochemical Investigations:

Table 5: Estimated Parameters

Parameter Method

Glucose Glucose oxidase peroxidase

HbA1c Turbidimetric Inhibition Immunoassay

Total Cholesterol Cholesterol oxidase peroxidase

Triglycerides GPO method- Trinder’s reaction

High Density Lipoprotein-cholesterol (HDL-c) Direct Immuno inhibition method Low Density Lipoprotein-cholesterol (LDL-c) Selective Direct Single Measurement Aspartate aminotransferase (AST) IFCC recommended procedure Alanine aminotransferase (ALT) IFCC recommended procedure Alkaline phosphatase (ALP) IFCC recommended procedure Gamma glutamyl transferase (GGT) Method according to Szasz/Persijn

Calculated Parameter:

Very Low- Density Lipoprotein (VLDL) = Triglycerides / 5

(60)

46

PLASMA GLUCOSE ESTIMATION

Method:

Enzymatic method by Glucose Oxidase – Peroxidase (GOD-POD) enzymes Principle:

The enzyme glucose oxidase oxidizes glucose present in plasma to gluconic acid. Hydrogen peroxide which is liberated in this reaction is converted into water and nascent oxygen by the action of peroxidase enzyme. An oxygen acceptor, 4-aminoantipyrine accepts the oxygen and along with phenol produces a pink coloured chromogen whose absorbance is measured at 505 nm and is proportional to the concentration of the glucose.

Glucose + O2 Gluconic acid + H2O2

2H2O2 + 4-Aminoantipyrine + Phenol Red quinoneimine complex + H2O

Table 6: Reagents

250 mmol/L of phosphate buffer at pH 7.5 5 mmol/L of Phenol

0.5 mmol/L of 4- Aminoantipyrine

≥ 15 kU/L of Glucose Oxidase

≥ 1 kU/L of Peroxidase

Standard Concentration: 100

Glucose oxidase

Peroxidase

(61)

47 Procedure:

Reagent Volume: 300 µL Sample Volume: 3 µL

Incubation time: 10 min Calculation:

Analyte concentration is automatically calculated by the analyzer Conversion factors:

mmol / L x 18.02 = mg / dL mg / dL x 0.0555 = mmol / L Measuring Range:

1 – 400 mg/dl Reference Value:

Fasting Glucose Level : 70 – 100 mg/dl Post prandial Glucose Level : < 140 mg/dl

References

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