Effects of Vitamin E in non alcoholic fatty liver disease

EFFECTS OF VITAMIN E IN

NON ALCOHOLIC FATTY LIVER DISEASE

Dissertation submitted to

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

in partial fulfillment of the

regulations for the award of the degree of

M.D. (PHARMACOLOGY) BRANCH – VI

GOVT. STANLEY MEDICAL COLLEGE & HOSPITAL THE TAMIL NADU DR. M.G.R. MEDICAL UNIVERSITY

CHENNAI, INDIA

MAY 2018

CERTIFICATE

This is to certify that this dissertation entitled “Effects of Vitamin E in Non Alcoholic Fatty Liver Disease” by the candidate Dr.R.Divakar, for M.D. (Pharmacology) is a bonafide record of the research work done by him under the guidance of Dr. G.Hemavathy, M.D., Professor, Department of Pharmacology, Government Stanley Medical College, during the period of study (2015- 2018), in the Department of Pharmacology, Government Stanley Medical College,Chennai-01.

I also certify that this dissertation is the result of the independent work on the part of the candidate.

Dr.S.Ponnambala Namasivayam, M.D., D.A., DNB Dean

Govt.Stanley Medical College Chennai.

Dr.G.Hemavathy, M.D., Professor,

Department of Pharmacology Govt.Stanley Medical College.

Dr.M.Kulandaiammal, D.G.O., M.D., Professor & Head of the Department

Department of Pharmacology Govt.Stanley Medical College.

DECLARATION

I hereby declare that this dissertation entitled in “Effects of Vitamin E in Non Alcoholic Fatty Liver Disease” was written by me in the Department of Pharmacology, Government Stanley Medical College and Hospital, Chennai under the guidance and supervision of Dr.G.Hemavathy, M.D., Professor, Department of Pharmacology, Government Stanley Medical College, Chennai – 600 001.

This dissertation is submitted to The Tamilnadu Dr. M.G.R.

Medical University, Chennai in partial fulfillment of the university regulations for the award of Degree of M.D., Pharmacology(Branch -VI) Examination to be held in May 2018.

Date:

Place: Chennai Dr.R.Divakar

ACKNOWLEDGEMENT

I express my sincere gratitude to Dr.S.Ponnambala Namasivayam M.D., D.A., DNB., Dean, Govt. Stanley Medical College for permitting me to undertake this research work as a part of my MD curriculum.

I would like to convey my gratitude and indebtedness to Dr.

M.Kulandaiammal D.G.O., M.D., Professor and Head of the Department of Pharmacology, Govt. Stanley Medical College for her sincere advice, unfailing support and attention throughout the study.

I owe my sincere thanks and appreciation to my guide Dr.G.Hemavathy M.D., Professor, Department of Pharmacology, Govt. Stanley Medical College, Chennai for her inspirational guidance and encouragement with which the dissertation has been prepared.

I would like to convey my gratitude to Dr.P.Vasanthi., M.D., Professor and Head of the Department of Medicine, Govt.Stanley Medical College for permitting me to carry out this study in Medicine outpatient department, Govt.Stanley medical college.

I express my sincere thanks to Dr.A.R.Venkateswaran,M.D., D.M., Professor and Head of the Department of Medical Gastro Enterology, Stanley Medical College for his advice and encouragement.

I express my sincere thanks to my Professors Dr. R. Jeyalalitha M.D., and Dr.R.Sivagami, M.D., Department of Pharmacology for their constant support and advice.

I am thankful to Dr. M.Mohana Lakshmi D.G.O.,M.D., Dr.B.

Kalaimathi. M.D., Dr.N.Asvini M.D., Dr. M. Prakash M.D., Dr.T.Meenakshi M.D., Dr.M.Sangavai M.D., Dr.B.Pushpa M.D., Dr.K.Thamayanthi M.D., Dr. C. R.

Anuradha D.G.O., M.D., Dr. J. Sam Anbu Sahayam D.L.O., M.D., and Lecturers Mr.J.Jeyasuresh M.Pharm, Mr.R.Selvakumar M.Pharm, Mr.K.Arunprasad M.Pharm, for their unconditional co-operation and help.

I thank Dr.S.P.Subahan, Dr.A. Preethi, Dr.G.vanitha, Dr.R.Punitha my fellow postgraduates for their help throughout this study.

I wish to place on record my gratitude to my parents and my family members for creating a congenial atmosphere and support when it was needed.

I thank all the staffs of the Department of pharmacology, Stanley medical college, for their cooperation in the completion of my study.

Finally I thank all my patients for willingly submitting themselves for this study.

CERTIFICATE-II

This is to certify that this dissertation work titled

“Effects of Vitamin E in Non Alcoholic Fatty Liver Disease”

of the candidate

Dr.R.Divakar

with registration number

201516051 for the award of M.D. Pharmacology

in the branch of

VI. I personally verified the

urkund.com website for the purpose of plagiarism check. I found that the uploaded thesis file contains from introduction to conclusion pages and result shows 3 percentage of plagiarism in the dissertation.

Guide & Supervisor sign with Seal

CONTENTS

S.No Title Page No

1. Introduction 1

2. Review of literature 5

3. Aim and Objectives 52

4. Methodology 54

5. Results 62

6. Discussion 72

7. Conclusion 78

8. Bibliography 9. Annexures

ABBREVIATIONS

ALT - Alanine Amino Transferase

AST - Aspartate Amino Transferase

CREBP - Carbohydrate Response Element Binding Protein

ER - Endoplasmic reticulum

HCC - Hepato Cellular Cancer

HDL - High Density Lipoprotein

LDL - Low Density Lipoprotein

MTTP - Microsomal Triglyceride Transfer Protein

NAFLD - Non-Alcoholic Fatty Liver Disease

NAS - NAFLD Activity Score

NASH - Nonalcoholic Steatohepatitis

OSA - Obstructive sleep apnoea.

PAT protein - Perilipin-adipose differentiation protein-tail interacting protein

PNPLA3 - Patatin-like phospholipase domain containing 3

PPAR - Peroxisome Proliferators-Activated Receptor

PUFA - Poly unsaturated fatty acid

ROS - Reactive Oxygen Species

SCOS - suppressor of cytokine signaling

SREBP - Sterol Regulatory Element Binding Protein

T2DM - Type 2 Diabetes Mellitus

TASH - Toxicant-Associated Steatohepatitis

TC - Total Cholesterol

TG - Triglyceride

TGF-β - Transforming Growth Factor-β

TLR - Toll like receptor

TNF - Tumour Necrosis Factor

TPN - Total Parenteral Nutrition

VLDL - Very Low Density Lipoprotein

INTRODUCTION

Non-Alcoholic Fatty Liver Disease (NAFLD) is a growing health problem around the world. NAFLD includes all cases of fatty liver disease from simple steatosis to cirrhosis, without excessive alcohol intake, use of steatogenic medication or hereditary disorders.

The overall prevalence of NAFLD in western countries from 15-40%

and in Asian countries from9-40%.¹ In India also, NAFLD is developing as an important cause of liver disease. Studies have shown the prevalence of NAFLD to be in the range of 9-32% in general Indian population, with overweight/obese and diabetic/ prediabetic patients contributing to bulk of the casses.² There is a strong association of NAFLD with insulin resistance and the metabolic syndrome.

NAFLD is considered to be a part of the metabolic syndrome comprising diabetes, or pre-diabetes, overweight or obese, elevated blood lipids such as cholesterol and triglycerides, and high blood pressure.

Nonalcoholic fatty liver disease is a spectrum of liver disease which includes hepatic steatosis, steatohepatitis and cirrhosis. Hepatic steatosis is a benign form, but nonalcoholic steatohepatitis (NASH) is a progressive disease which can progress to cirrhosis and liver failure.

Based on several observational studies, reviews, and meta-analyses, it is currently believed that patients with NAFLD have higher overall mortality

and patients with Non Alcoholic Steato Hepatitis have higher mortality due to liver pathology, when compared to the general population_.³ Hence it is important to screen the patients with metabolic syndrome for features of NAFLD and treat them.

Increased waist circumference is the best single clinical indicator for underlying insulin resistance and the presence of NAFLD. In type 2 diabetes mellitus (T2DM) patients, the prevalence of NAFLD is 40–70%.

About 90% of patients with NAFLD have one or more features of the metabolic syndrome and 30–40% of patients have the full syndrome (three or more of the following):- central obesity, impaired fasting glucose, hypertriglyceridaemia, low serum HDL cholesterol and hypertension. The severity of NAFLD is also associated with the severity of the metabolic syndrome.

The pathophysiology of NAFLD is best explained by the “multiple hit”

hypothesis. This proposes first “hit” as the development of hepatic macrosteatosis as a result of increased lipolysis and free fatty acid levels. The increase in the reduction of free fatty acid oxidation with insulin resistance leads to fatty acid accumulation. Several possible “second hits” may be oxidative stress from reactive oxygen species in the mitochondria and cytochrome P450 enzymes, endotoxins, cytokines, adipokines and environmental factors. Therefore, antioxidant and anti-inflammatory agents may have a role in the prevention of this disease.

Population screening for NAFLD is not currently recommended, subjects with obesity, metabolic syndrome and T2DM are at very high risk of having NASH, so clinicians should have a high index of suspicion for the diagnosis in these subjects, even in the presence of normal liver function tests.

The majority of patients with NAFLD are asymptomatic and so are diagnosed following an incidental finding of abnormal LFTs or fatty liver on imaging. Where present, the typical biochemical abnormalities found in NAFLD are mildly elevated transaminases (alanine amino transferase (ALT)

>aspartate amino transferase (AST)) and/or gamma-glutamyl transferase.

Lipid peroxidation and secondary cellular injury are the dominant mechanism in the transition from relatively stable hepatic steatosis to potentially progressive steatohepatitis in nonalcoholic fatty liver disease.⁴ Hence Nonalcoholic Steatohepatitis (NASH) can be viewed as a component of systemic lipotoxicity.⁵ Poorly controlled oxidation of excessive fatty acids generates free radicals (reactive oxygen species [ROS]) that bind with various cellular components, ultimately causing the formation of end-stage low energy molecules such as lipofuschin (a conjugate of lipid and protein formed from malondialdehyde and a Schiff base intermediary)⁶ and stimulation of cell signaling pathways leading to activation of stellate cells and collagen deposition.⁷

Abnormalities of mitochondrial form and function are one of the major associations of uncontrolled lipid peroxidation.⁸ Mitochondrial changes

observed in fatty liver have been associated with activation of cell death pathways such as the cytochrome c-induced apoptosis pathways.⁹

Vitamin E is a chain-breaking antioxidant in free radical reactions that is of particular importance in lipid peroxidation and membrane stabilization due to its lipid solubility. Vitamin E stabilizes biologic membranes by protecting unsaturated fatty acids from lipid peroxidation.¹⁰ Possible roles beyond direct antioxidant effects include attenuating cytokine stimulation of stellate cells by decreasing transforming growth factor-β (TGF-β) levels.¹¹

Animal studies have demonstrated that vitamin E improves fibrosis, reduces mitochondrial lipid peroxidation, and corrects oxidative stress in experimental models of liver disease associated with oxidative injury.¹² These antioxidant and possibly antifibrotic properties of vitamin E form a theoretical basis for its use in the treatment of NAFLD.

Vitamin E therapy, as compared with placebo, was associated with a significantly higher rate of improvement in nonalcoholic steatohepatitis , Serum alanine and aspartate aminotransferase levels were reduced with vitamin E as compared with placebo, associated with reductions in hepatic steatosis and lobular inflammation.¹³ This study is undertaken with the aim to find the effects of vitamin E supplementation in NAFLD patients.

REVIEW OF LITERATURE

Definition :

Non-alcoholic fatty liver disease is defined as fatty infiltration affecting greater than 5% of hepatocytes in the absence of excessive alcohol consumption(>20 g day for women and >30 g/day for men).¹⁴

Nonalcoholic fatty liver disease represents a spectrum of liver disease ranging from hepatic steatosis to steatohepatitis and cirrhosis. Hepatic steatosis is generally thought to be benign condition, nonalcoholic steatohepatitis is a progressive disease that can lead to cirrhosis and liver failure.

Epidemiology :

Non-alcoholic fatty liver disease is one of the most common liver disorders . The prevalence of NAFLD is as high as 38% depending on criteria and region studied.¹⁵ NAFLD accounts for about one third of cases of chronic liver disease in primary care.¹

Most of the cases of NAFLD is discovered during the fourth to sixth decade of life, but nowadays there is a increase in incidence among adolescents and children due to an increase in occurrence of obesity and overweight in them.

Recent studies have shown that NAFLD is more common in men than women, but women show peak prevalence in later part of life, due to influence of sex hormones and menopause.

About 60 to 76 % of diabetics are associated with NAFLD and it is seen as a hepatic manifestation of the metabolic syndrome. Metabolic syndrome is defined as by the presence of 3 or more of the following

 Abdominal obesity

 Hypertriglyceridemia

 Low high density lipoprotein levels

 Hypertension

 Elevated fasting plasma glucose levels

Histological features :

Macro vesicular fat accumulation in more than 5 % of hepatocytes is characteristic feature of NAFLD. Most of the patients with NAFLD have isolated fatty liver in which there is hepatic steatosis without inflammation or fibrosis.

NASH is characterised by ballooning degeneration of hepatocyte and lobular inflammation of mixed inflammatory cells. it is also associated with mallory bodies, mega mitochondria, kupffer cells, vacuolated nuclei in periportal hepatocytes, pericellular and perisinusoidal fibrosis called as chicken wire fibrosis is seen in acinar zone 3.

The NAFLD activity score (NAS) combines scores of steatosis, lobular inflammation and hepato cellular ballooning. A score of 0 to 2 suggests not- NASH, and a score of 5 or more suggests NASH .

The biopsy of NAFLD shows only minimal inflammation and no fibrosis.

Steatosis

5 % 1

5 % - 33% 2

33% - 66% 3

Ballooning

None 0

Few 1

Many 2

Lobular Inflammation

Mild 1

Moderate 2

Severe 3

Total score

0-2 Likely not NASH

3-4 Intermediate

5- 8 Likely NASH

NAFLD Activity score on liver biopsy specimen

CAUSES OF FATTY LIVER DISEASE

Acquired Metabolic Disorders

Diabetes mellitus Dyslipidemia

Kwashiorkor and Marasmus Obesity

Rapid weight loss Starvation

Cytotoxic and Cytostatic Drugs L-Asparginase

Bleomycin Cisplatin 5-Flurouracil Methotrexate Tetracyclines

Other Drugs and Toxins Amiadarone

Camphor Chloroform Cocaine Ethanol

Ethyl bromide

Estrogens Glucocorticoids Griseofulvin

HAART(Zidovudine, Stavudine, Diadanosine) Nifedipine

Nitrofurantoin

NSAIDs (Piroxicam, Ibuprofen, Indomethacin, Sulindac) Tamoxifen

Valproic acid

Metals Antimony Barium Salts Chromates Mercury Phosphorus

Inborn Errors of Metabolism Abetalipoproteinemia

Familial hepatosteatosis Galactosemia

Glycogen storage disease Hereditary fructose intolerance

Homocystinuria

Systemic carnitine deficiency Tyrosinemia

Weber-Christian syndrome Wilson disease

Surgical Procedures Biliopancreatic diversion

Extensive small bowel resection Jejunoileal bypass

Miscellaneous Conditions Irritable bowel disease Total Parenteral Nutrition Starvation

Industrial exposure to petro chemicals

Jejunal diverticulosis with bacterial over growth Partial lipodystrophy

Other conditions associated with NAFLD(“secondary” NAFLD) Jejunoileal bypass

Historically, older forms of bariatric surgery played a role in the recognition of NAFLD due to a severe form of NASH following jejunoileal bypass.¹⁷ Stimulation of TNF by bacterial overgrowth and endotoxin production, micronutrient deficiency, and rapid weight loss were conditions etiologically implicated. Modern forms of bariatric surgery are much less likely to exacerbate NASH and may indeed ameliorate the condition.

Medication-induced

A number of medications have been implicated as causes of steatohepatitis. Steatosis has been associated with exposure to anabolic steroids among body builders in the absence of obesity¹⁸ and amiodarone has long been associated with phospholipidosis and steatohepatitis, although this seems less common now possibly due to lower dosing.¹⁹

Acquired lipodystrophy, insulin resistance, and steatosis has been well described with therapy for human immunodeficiency virus (HIV) infection.²⁰ Obesity also appears to increase the risk of chemotherapy-associated steatohepatitis in colorectal cancer liver metastatic disease .²¹

Parenteral nutrition, malnutrition, and celiac disease.

Liver disease, often with macro- and microvesicular steatosis, is a potentially severe side effect of total parenteral nutrition (TPN).²² Both the

amount and composition of infused lipid appear to affect the expression of liver disease.²³ Choline deficiency may play a role in some patients.

At the opposite end of the spectrum, steatosis in kwashiorkor results from impaired hepatic lipid export due to protein malnutrition (diminished apolipoprotein B).²⁴ Celiac disease can be seen in up to 3–4% of NASH patients even in the absence of significant weight loss.²⁵ In this situation, celiac disease is postulated to exacerbate intrahepatic inflammation.

Solvents and industrial agents

A variety of toxins have been implicated in the development of fatty liver diseases.²⁶ Better described agents include carbon tetrachloride (now rarely used), dimethylformamide, perchloro-ethylene, and more recently petrochemical derivatives .²⁷ These forms of steatohepatitis have been termed TASH (toxicant-associated steatohepatitis) and can also be seen with high exposures to vinyl chloride.²⁸

Wilson disease and other inherited metabolic diseases

Macro- and microvesicular steatosis are well known features of Wilson disease.²⁹ It should be considered particularly with steatohepatitis in a younger individual. It is not known how often the carrier state for mutations in the copper-transporting ATPase gene could influence NASH.³⁰

Macrovesicular steatosis is seen in other inherited metabolic diseases, most of which present in childhood. These include glycogen storage diseases, galactosemia, tyrosinemia, heterozygous hypobetalipoproteinemia, and

abetalipoproteinemia. These disorders involve impaired formation of VLDL due to decreased synthesis of apolipoprotein B.³¹

Lipid storage diseases (cholesterol ester, Niemann–Pick, Tay–Sachs, and Gaucher disease) can have fatty infiltration of the liver with cholesterol esters, sphingolipids, phospholipids, sphingomyelin, gangliosides, or glucocerebrosides. Presentation in infancy and distribution in the reticuloendothelial cells distinguish the lipid storage disorders.³²

Pathogenesis

Nonalcoholic fatty liver disease can be viewed as conditions within the spectrum of the metabolic syndrome and systemic lipotoxicity.³³ Steatosis and oxidative injury known as the “two hit” hypothesis encapsulates the consistent role played by oxidative injury.³⁴ A cascade of events leads eventually to cellular ballooning, cell death, organ fibrosis, and cirrhosis. Although fat necrosis may occur from the direct release of fat from swollen hepatocytes in NAFLD,³⁵ cell injury results predominantly from the indirect effects of intracellular lipid peroxidation and the toxicity of fatty acids on organelles.

Lipid peroxidation , cell injury and the role of anti oxidants

Local cellular factors Steatosis

Steatosis exists when fat stores exceed 5–10% of the organ by weight.³⁶ Fat is derived from the uptake of plasma fatty acids (nonesterified fatty acids (NEFAs)) released by lipolysis of adipose tissue, the uptake of VLDL-derived LDL remnants or circulating dietary chylomicron remnants, and from de novo lipogenesis from carbohydrate precursors.³⁷ Both triglycerides (mostly as unsaturated fatty acids) and free fatty acids (mostly as saturated fatty acids) are increased in steatosis.³⁸

The disposal of fatty acids proceeds by several routes:

 incorporation into triglyceride in cytoplasmic lipid droplets,

 export as lipoproteins such as VLDL,

 oxidation especially via mitochondrial β-oxidation, or

 formation of phospholipids as membrane components.

 Recycling through autophagy.

The regulation of these pathways depends on energy homeostasis influenced by peroxisome proliferators-activated receptor (PPAR) activity and the adrenergic nervous system.³⁹ The transcription factors sterol regulatory element binding protein (SREBP), governed by insulin and dietary fatty acids, and carbohydrate response element binding protein (CREBP), governed by glucose levels, regulate lipid metabolism.⁴⁰

SREBP and CREBP stimulate nuclear transcription of the enzymes responsible for fatty acid synthesis and their incorporation into triglyceride in lipid droplets or exported as VLDLs.

De novo lipogenesis from carbohydrates

Adenosine triphosphate-dependent synthesis of the 16-carbon palmitic acid begins with translocation of carbohydrate-derived acetyl-coenzyme A (acetyl-CoA) subunits which pass through the mitochondrial membrane to the cytosol as citrate.

Acetyl-CoA carboxylase then activates the formation of malonyl-CoA from acetyl-CoA. Importantly, Acetyl-CoA carboxylase is activated by insulin and inactivated by epinephrine, glucagons, and long chain fatty acids.

Through a series of repetitive cytosolic condensations catalyzed by fatty acid synthase , molecules of malonyl-CoA are assembled into palmitic acid. As a control, malonyl-CoA inhibits fatty acid β-oxidation by blocking carnitine, which shuttles fatty acids into the mitochondrion.

Once formed, palmitic acid undergoes elongation in the endoplasmic reticulum to longer chain fatty acids or desaturation and esterification to glycerol to form mono-, di-, and triglycerides. The triglycerides is incorporated in the ER via MTTP into VLDLs for export in association with apolipoprotein B100.⁴¹ Triglyceride synthesis in human NAFLD results from the uptake of adipose-derived Non Esterified Fatty Acids (59%), De Novo

Lipolysis (26%), and dietary fat (15%) based on nutrition studies with radio labeled precursors.⁴²

Lipid composition (lipidomics)

Incorporation of non esterified fatty acids into droplet triglyceride depends on the activity of diacylglycerol acyltransferase 1 (Dgat1).⁴³ Lipidomic analysis shows a stepwise increase from normal to NASH in the triacylglycerol to diacylglycerol ratio, indicating a relative increase in triacylglycerol in more advanced disease.⁴⁴

The ratio of n6 : n3 polyunsaturated fatty acid is higher in NASH possibly due to the diminished triacylglycerol content of eicosapentanoic acid and docosahexanoic acid. Free fatty acids were not different but phosphatidylcholine was decreased NASH, while the free cholesterol to phosphotidylcholine ratio was increased.

Ceramide, a toxic lipid intermediary, has been detected more in obese patients with fatty liver but its role is uncertain.⁴⁵

Lipoproteins

Although lipid trafficking and lipoprotein metabolism are important factors , no dominant pattern of fasting lipoproteins is established in NAFLD.

In some cases, primary lipoprotein disorders might constitute a separate and/or overlapping entity. VLDL secretion is increased in human NAFLD but plateaus at a hepatic triglyceride content of 10%, indicating limited compensation for increased uptake of Non esterified Fatty Acids.

The secretion of apolipoprotein B100, a component of VLDL, is insufficient and may promote the secretion of a larger VLDL particle with greater triglyceride content.⁴⁶

Lipid peroxidation

The most enduring finding that distinguishes NASH from NAFLD is lipid peroxidation. This process is evident as the accumulation of oxidative by-products such as 4-HNE(4-hydroxynonenal), MDA (malondialdehyde), nitrotyrosine, and 8-hydroxydeoxyguanine, and systemically as adducts of oxidized phospholipids and 11-HETE (hydroxyeicosatetraenoic acid), a by- product of arachidonic acid oxidative injury.⁴⁷

Lipid peroxidation is a branching, chain reaction attack on unsaturated fatty acids that produces another free radical and a lipid hydroperoxide. It is sparked by superoxide radicals derived especially from mitochondrial oxidative phosphorylation in the electron transport chain.

The superoxide radical is metabolized via superoxide dismutase to hydrogen peroxide, which in the presence of Fe2+ (Fenton or Haber–Weiss reaction) decays to hydroxyl radicals. Hydroxyl radicals catalyze peroxidation of fatty acids damaging membranes, cellular proteins, and DNA .⁴⁸

Oxidation of the phospholipid monolayer of fat droplets (including PAT proteins (perilipin-adipose differentiation protein-tail interacting protein)) and constituents of the endoplasmic reticulum may be particularly relevant to cellular ballooning, impaired disposal of fatty acids, and hepatic

insulin resistance.⁴⁹ The speed of these pathologic reactions in vivo, whether in seconds, minutes, hours, days or longer, is uncertain.

Lipid droplets, VLDL lipidation, and lipophagy

Lipid droplets are composed of a hydrophobic core of triglycerides and lesser amounts of cholesterol esters surrounded by a phospholipid monolayer . They form at the site of fatty acid acyl transferases in the ER in association with the cytoskeleton .

Lipid droplets are associated with lipoprotein-like proteins known as PAT proteins which govern lipase activity and are themselves regulated by PPAR-γ agonists . Their growth appears to occur predominantly by fusion .

Because oxidative injury to the droplet surface alters PAT protein expression, it is likely that secretion and fusion is disturbed in NASH .⁵⁰ The smallest droplets in alcoholic steatohepatitis resemble precursors of VLDLs.

Endoplasmic reticulum dysfunction

Experimentally, small droplet accumulation is associated with the formation of cholesterol-rich “Apo-B crescents” at sites in the ER . This is directly related to dysfunction of the ER. endoplasmic reticulum dysfunction is evident in human NASH as the unfolded protein response (“ER stress”), which activates proinflammatory pathways such as interleukin 8, nuclear factor κB (NF-κB), and c-jun N-terminal kinase (JNK) .

Thus, impaired Apo- B100 interaction with lipid droplets at the Endoplasmic reticulum provides a link between oxidative stress, droplet accumulation, altered VLDL secretion, the ER stress reaction and activation of proinflammatory pathways.⁵¹

Mitochondrial dysfunction and adenosine triphosphate homeostasis

An association between steatosis and diminished ATP was observed over 50 years ago and later confirmed using 31P magnetic resonance spectroscopy in vivo.

The evolutionary history of the mitochondrion places it centrally in critical pathways including fatty acid synthesis and oxidation, oxidative- phosphorylation, and apoptosis signaling.⁵² Changes in permeability cause the release of cytochrome c and apoptosis signaling. Mitochondrial cholesterol content may contribute to changes in permeability.

Cell death, fibrosis, and the ductal reaction

Cell death results from a combination of apoptosis signaling and focal necrosis resulting in a process best described as “necroapoptosis” or

“apoptonecrosis” .

Excessive free fatty acids due to diminished fatty acid-binding protein alter autophagosome permeability, leading to the release of cathepsins, which then alter mitochondrial permeability, leading to the release of cytochrome c and activation of proapoptotic caspases.

Caspase 3 in particular causes fragmentation of keratin 18, possibly contributing to the formation of Mallory–Denk bodies and to the release of keratin 18 fragments detectable in serum.⁵³ Although apoptotic pathways are diffusely activated, necrosis appears to dominate histologically, possibly as a result of an ATP deficit, fat droplet accumulation, and cytoskeletal injury – all of which appear to contribute to cellular ballooning.

Cytochrome P450 and peroxisomal metabolism

Induction of cytochrome P450 2E1 (CYP2E1) has been described as a possible source of oxidative stress through microsomal beta-oxidation of fatty acids . Expression of CYP2E1 is influenced by dietary fat and co localizes to markers of lipid peroxidation. Its role is uncertain as experimental overexpression of CYP2E1 is protective against oxidative injury and decreases apoptosis but increases the risk of necrosis induced by fatty acid exposure .

Peroxisomes are involved in numerous potentially relevant lipid-related metabolic pathways and morphologic abnormalities have been described in human fatty liver.⁵⁴ It is possible that genetic abnormalities, nutritional abnormalities, or adaptive changes in the peroxisome may contribute in some patients.

Ballooned cells

Hepatocyte ballooning is a major histologic feature of NASH evident in all scoring systems. Defined at the light level based on hematoxylin and

eosin (H&E) staining as cellular enlargement 1.5–2 times the normal diameter (normal up to 30µm) with rarefied cytoplasm .

Recent advances have shown that ballooning is associated with injury to the cytoskeleton evident as diminished cytokeratin 8 and 18, the presence of Mallory–Denk bodies, and the detection of cytokeratin 18 fragments (M30), suggesting impairment of cell size regulation.

There is association between the accumulation of small and medium fat droplets and morphologic changes in the ER. These studies show cytoskeletal injury also in smaller cells, suggesting that this pattern exists as a spectrum. Experimentally, abnormal keratin metabolism may also play a role in mitochondrial dysmorphology.

Systemic factors

Insulin resistance and systemic inflammatory changes

Although insulin resistance is neither sufficient nor essential for NAFLD, it is present in the majority of NAFLD patients and is intertwined with disease pathogenesis. Insulin resistance (evident in adipose tissue, skeletal muscle, and liver) is mediated by lipid accumulation, free fatty acids, and altered mitochondrial metabolism.⁵⁵

Manifestations include decreased muscle utilization of glucose, mobilization of fatty acids from adipose tissue (lipolysis), and unrestrained hepatic glucose output. Biochemically, insulin resistance is characterized by a shift from tyrosine phosphorylation in the insulin receptor substrate to serine

phosphorylation resulting in diminished insulin-stimulated activity in metabolic pathways (mediated by phosphatidylinositol 3-kinase (PI3-kinase), Akt, and mTOR) and mitogenic pathways (mediated by Ras, Raf, and mitogen-activated protein or MAP-kinase).

Inflammatory changes in adipose tissue are characterized by ER stress and JNK activation, which stimulates the production of systemically active cytokines, which contribute to the shift from tyrosine to serine phosphorylation. Tumor necrosis factor α (TNF-α) and interleukins 6 and 8 act through their effects on a modulator of cytokine activity known as SOCS (suppressor of cytokine signaling).

Adipocytokines and lipokines

Adipose-derived “adipokines” (adiponectin, leptin, resistin, visfatin) modulate insulin signaling. Adiponectin is the most abundant protein in the adipocyte and participates in glucose homeostasis via receptors in muscle (adipoR1) and liver (adipoR2) where it activates adenosine monophosphate (AMP) kinase and promotes fatty acid oxidation .

Depressed production of adiponectin has a prominent role in NAFLD and successful therapy is often associated with the recovery of normal levels.

Recently, adipose-derived “lipokines,” including C16:1n7-palmitoleate, have been proposed as additional systemic modulators of hepatic fat and insulin activity in skeletal muscle .

Other systemic changes

Alterations in small bowel permeability and associated bacterial overgrowth in NAFLD patients have been implicated in the activation of inflammatory mediators through exposure to substances such as endotoxin in portal blood .⁵⁶ The endotoxins may partly explain the previously reported association between celiac disease and NAFLD due to increased gut permeability in celiac disease.

Energy homeostasis

Steatosis can be viewed as a component of integrated systems involved with energy (and thermoregulatory) homeostasis including the adrenergic system, the adipose organ, the thyroid axis, and insulin/glucagon signaling.

Adrenergic modulation of hepatic inflammation has been demonstrated in animal models and may play a role in human fatty liver.

Human genetic factors

In 2008, two genome-wide association (GWAS) studies linked the rs738409 polymorphism (IL48M) of patatin-like phospholipase domain containing 3 (PNPLA3) with hepatic fat content and ALT levels.⁵⁷ A recent meta-analysis have also corroborated such association between the IL48M polymorphism and NAFLD in almost all ethnic groups, and in adults, children and adolescents.

Further studies suggest that the IL48M variant is an important risk factor for accumulation of hepatic steatosis in particular when additional

factors are present such as free fatty acid release, insulin resistance, visceral obesity, increase in lipogenesis, and changes in lipid metabolism.

IL48M polymorphism also predisposes to cirrhosis and hepatocellular carcinoma. All recent data suggest that the 148M PNPLA3 polymorphism favours hepatic carcinogenesis in steatohepatitis as well as in other liver diseases, and the mechanism is partly independent of the predisposition towards fibrogenesis and cirrhosis.

The influences of visceral ectopic fat accumulation, adipose tissue inflammation, type 2 diabetes, diet and intestinal dysbiosis to promote the development of progressive liver disease in NAFLD.

Microbiome

The gut microbiota, now also called the gut microbiome, is involved in the pathophysiology of non-alcoholic fatty liver disease as well as in obesity and the metabolic syndrome. All the metabolic products generated by the intestinal microbiome first enter the liver. It has also been shown recently by many investigators that the microbiome differs between obese and lean animals and between obese and lean humans.

A recent study proposed that the altered microbiome in obesity might produce more ethanol and might thereby contribute to the development of NASH.⁵⁸ Another recent paper shows that inflammasome or interleukin-18 deficiency enhances the progression of NASH and obesity by inducing microbiome dysbiosis . This dysbiosis-induced inflammation enters into the portal circulation through the influx of toll like receptor (TLR) 4 and TLR9 agonists and thereby leads to an increase in tumour necrosis factor (TNF) .

Recent metagenome-wide association (MGWAS) studies of gut microbiota showed patients with type 2 diabetes were characterised by a moderate degree of gut microbial dysbiosis, a decrease in butyrate-producing bacteria and an increase in various opportunistic pathogens, as well as an enrichment of other microbial functions conferring sulphate reduction and oxidative stress resistance in type 2 diabetes.⁵⁹

Studies have shown that bacteroides abundance was independently associated with NASH and ruminococcus with fibrosis . These results suggest that NAFLD severity associates with gut dysbiosis and a shift in metabolic function of the gut microbiota.⁶⁰ In particular bacteroides may be associated with NASH and ruminococcus with significant fibrosis.

Natural history

In the general population, the risk for liver-related death in NAFLD appears to be associated mainly with age, insulin resistance, and histological evidence of hepatic inflammation and fibrosis.⁶¹ Probably around 10% of NAFLD patients will progress to NASH over a period of 10 years Cirrhosis later develops in 5–25% of patients with NASH and 30–50% of these patients die from liver-related causes over a 10-year period.⁶²

.

Cirrhosis in patients with NASH can also decompensate into subacute liver failure, progress to hepatocellular cancer (HCC), and recur after liver transplantation . Steatosis alone is reported to have a more benign clinical course, with cirrhosis developing in only 1–3% of patients. Patients with NASH and fibrosis also have a significant risk for hepatocellular carcinoma.

The incidence and prevalence of NAFLD and NASH are increasing in almost all industrialised countries. Some recent papers suggest that NASH may soon be the leading cause of cirrhosis, hepato cellular carcinoma and liver transplantation.

Clinical Features

NAFLD is usually discovered incidentally because of elevated liver biochemical test level or the finding of hepatic steatosis on imaging. Most patients with NAFLD are asymptomatic but some describe vague right upper quadrant pain, fatigue and malaise.

Hepatomegaly is commonly seen but is difficult to appreciate due to obesity signs of chronic liver disease like splenomegaly, spider telangiectasias and ascites are seen in patients with cirrhosis.

Clinical Associations

Clinically associated comorbidities of NAFLD and NASH are numerous and include

 Cerebro Vascular Disease,

 Diabetes,

 Obstructive Sleep Apnoea

 Colonic adenomas,

 Hyperuricemia,

 Vitamin D deficiency,

 Hyperferritinemia,

 Pancreatic steatosis,

 Hypothyroidism, and

 Polycystic Ovarian Syndrome

Diagnosis

NAFLD and NASH require valid reporting about alcohol consumption.

The threshold of a daily alcohol intake of 20 g for women and 30 g for men.

The workup of NAFLD and NASH also includes

 Assessment of drug use,

 Exclusion of HBVand HCV infections,

 Haemochromatosis,

 Autoimmune liver disease and,

 In younger patients, Wilson's disease.

In practice, NAFLD is often diagnosed by combining elevated levels of ALT and gamma GT with the sonographic appearance of an increase in echogenicity of the liver.

But in some patients with NAFLD and even with NASH and fibrosis have normal serum liver enzymes In these cases, ALT is usually higher than AST unless there is already severe fibrosis or cirrhosis.

Common laboratory features of NAFLD

 2- 4 fold elevation of serum ALT and AST levels

 ALT/AST ratio < 1 in most patients

 Serum alkaline phosphatase slightly elevated in one third of patients

 Normal serum bilirubin, serum albumin and prothrombin time

 Elevated serum ferritin level

Fasting serum glucose should be checked in all patients with NAFLD and NASH; one will also often find elevated serum insulin, insulin resistance, and/or diabetes.

Metabolic Syndrome

We also have to routinely look for metabolic syndrome which is diagnosed when three of the following features are seen:⁶³

 Central obesity (waist circumference ≥94 cm or ≥80 cm for men and women, respectively),

 Impaired fasting glucose (>100mg/dl),

 Hypertriglyceridaemia (>150 mg/dl),

 Low serum high density lipoprotein cholesterol (<40 mg/dl for men or

<50 mg/dl) and

 Hypertension (>135/85 mm Hg).

The severity of NAFLD is also associated with the severity of the metabolic syndrome.

Ultrasound

Ultrasound of the liver has a high sensitivity and specificity (both approaching 90%) for detection of fatty infiltration but does not allow assessment for the presence or degree of inflammation and fibrosis. Therefore, diagnosis of fat in the liver is easily made by ultrasound but diagnosis of NAFLD or NASH cannot be made without a liver histology.

Grades of fatty liver

Ultrasound image shows (a) Normal liver echogenicity

(b) Grade 1 fatty liver with increased liver echogenicity

(c) Grade 2 fatty liver with the echogenic liver obscuring the echogenic walls of the portal venous branches

(d) Grade 3 fatty liver in which the diaphragmatic outline is obscured

Since NAFLD is a very frequent but also relatively benign disease, our aim is to identify risk factors for NASH in order to avoid doing liver biopsies in all NAFLD patients. Risk factors for NASH include older age, excessive obesity, diabetes mellitus, other hepatotoxins, and clinical, laboratory or sonographic signs suggesting severe liver disease.

Liver biopsy remains the gold standard for characterising liver histology in patients with NAFLD. However, it is expensive and carries some morbidity and a small mortality risk. Thus, it should be performed in those patients who benefit most from diagnostic, therapeutic and prognostic perspectives. Liver biopsy should be considered in patients with NAFLD who are at increased risk to have steatohepatitis and advanced fibrosis.

The presence of metabolic syndrome and the NAFLD Fibrosis Score may be used for identifying patients who are at risk for steatohepatitis and advanced fibrosis.

Some of the non-invasive methods to identify fibrosis in patients with NAFLD ⁶⁴ – are

 The NAFLD Fibrosis Score,

 Enhanced Liver Fibrosis (ELF) panel, and

 Transient elastography (“Fibroscan”).

The NAFLD Fibrosis Score is based on six readily available variables (age, BMI, hyperglycaemia, platelet count, albumin, AST/ALT ratio). In a meta-

analysis of 13 studies consisting of 3064 patients, NAFLD Fibrosis Score was useful for predicting advanced fibrosis or cirrhosis.

A recent meta-analysis showed that transient elastography (“Fibroscan”) has a high sensitivity and specificity for identifying fibrosis in NAFLD but it has a high failure rate in individuals with a higher BMI .

Experimental and animal models Small animal models

Classic models include the ob/ob mouse, which has a congenital deficiency of leptin, the FA/FA rat, which has an impaired leptin receptor, and the methionine-choline deficient (MCD) diet rodent. While often helpful, the models often have limitations such as absence of insulin resistance in the MCD models . Recent studies have focused on dietary manipulations such as trans fat or fructose content or high fat with toxin exposure and others have utilized the zebra fish as a model of fat metabolism.⁶⁵

Large animal steatosis

Fatty liver is seen in cows, hens, cats, and some pigs . It is a common disorder in cats . Seasonal variation in steatosis is seen in wild deer.

Palmipedes (migratory geese) develop fatty liver before migration and utilize fat as a preferred source of energy for muscle metabolism. This has been exploited in the production of foie gras wherein geese are fed a corn based diet resulting in an increase in liver size in as little as 2 weeks from 100 to 800 g.

Mixed micro- and macrosteatosis is evident with increased susceptibility to accidental mycotoxin exposure. Steatosis involves increased synthesis and altered very low-density lipoprotein (VLDL) secretion. These relationships illustrate the role of the liver in the evolution of adipose and energy metabolism and thermoregulation.

Treatment

Treatment strategies for NAFLD have revolved around

(1)identification and treatment of associated metabolic conditions such as diabetes and hyperlipidaemia;

(2) improving insulin resistance by weight loss, exercise, or pharmacotherapy;

(3) using hepato-protective agents such as antioxidants to protect the liver from secondary insults.

Treatment of associated metabolic conditions

The metabolic syndrome and its features of central obesity, glucose intolerance, hyper triglyceridaemia, low HDL cholesterol and hypertension are associated with cardiovascular morbidity and mortality.⁶⁶

These features are commonly present in subjects with NAFLD, with 67%–71% being obese,

12%–37% having impaired fasting glycaemia, 57%–68% having disturbed lipid profiles, and 36%–70% being hypertensive.

Therefore, patients with newly diagnosed NAFLD should be screened for these conditions and appropriate treatment started in order to reduce the vascular risk as well as to improve NAFLD.

Weight loss and exercise

Moderate amounts of weight loss as well as exercise are associated with improvement in insulin sensitivity and thus are logical treatment modalities for patients with NAFLD who are overweight or obese.⁶⁷

Weight reduction may be achieved by caloric restriction from dieting, physical exercise, and/or pharmacotherapeutic agents as well as bariatric surgery in those patients with morbid obesity. Energy restriction of about 25–

30 kcal/kg/day seems reasonable with a target weight loss of about 10% of body weight over six months.

Patients with NAFLD seem more likely to have a diet high in saturated fats and cholesterol and low in fibre and antioxidants.⁶⁸ Mono and poly- unsaturated fats may potentially improve insulin resistance and may be beneficial in improving hepatic steatosis.

Most trials have used a diet similar to that recommended by the American Heart Association with energy restriction and energy intake composed of 40%–50% carbohydrates, 15%– 20% protein, and 25%–40%

predominately unsaturated fats.⁶⁹

Insulin sensitising drugs

It is well established that insulin resistance is a common association with patients with NAFLD and plays an important part in lipid accumulation within the liver and perhaps its progression to NASH.⁷⁰

Insulin resistance is predictive of the necroinflammatory form of NAFLD and conditions associated with insulin resistance such as obesity and diabetes are associated with the presence of advanced fibrosis among subjects with NASH.⁷¹ This had provided the impetus to trial insulin sensitising drugs such as metformin and the thiozoladinediones in NAFLD.

Metformin is a biguanide antihyperglycaemic agent. In animal models of fatty liver, metformin improved hepatic steatosis, which was accompanied by down-regulation of TNFα and lipid transcription factors. Small pilot trials of four to six months’ duration using doses of 1–1.5 g/day, have showed improvement in ALT levels compared with baseline.⁷²

The thiozoladinediones bind to the peroxisome proliferator activated receptor γ (PPAR) resulting in improved insulin sensitivity and redistribution of adipose tissue. In animal models, PPARγ agonists also have a protective effect against liver fibrosis by inhibiting activation of hepatic stellate cells.⁷³

Two well designed pilot trials using pioglitazone (30 mg daily) and rosiglitazone (4 mg twice daily) showed improvement in ALT, hepatic steatosis, and features of hepatic inflammation compared with baseline.⁷⁴ Weight gain with fat redistribution from the central/truncal area to the lower

body was the most common side effect occurring in 67%–72% of subjects taking pioglitazone or roziglitazone. Potential hepatotoxicity in the setting of liver disease remains a concern.

Antioxidants

Vitamin E refers to a family of tocopherols and tocotrienols that exhibit antioxidant activity. Available forms usually contain only α-tocopherol. A 6- month, placebo-controlled combination study (vitamin E 1,000 IU plus vitamin C 1,000mg daily) showed improvement in fibrosis although no improvement in inflammation or necrosis was seen.⁷⁵

These results were supported in a recent 2-year controlled trial of 247 nondiabetic adults, comparing α-tocopherol (800 IU/day) with placebo and pioglitazone. Significantly greater improvement was seen in key parameters, including steatosis, inflammation, and ballooning, compared with placebo.⁷⁶ Toxicity has not been reported in these studies although there remains some theoretical concern regarding higher doses .

Hepato-protective agents

A variety of hepato-protective agents used in other liver disease have been evaluated in patients with NAFLD . Pentoxifylline inhibits TNF α and has been shown to improve short term survival in severe alcoholic hepatitis.

Betaine, a methyl donor that protects against hepatic lipid accumulation, lowered aminotransaminase levels and also improved steatosis, inflammation, and liver fibrosis in a pilot trial.⁷⁷

Angiotensin II promotes insulin resistance and hepatic fibrosis in animal models. Losartan is an antagonist against the angiotensin II receptor that improved aminotransaminases, serum markers of fibrosis, and levels of profibrotic cytokine transforming growth factor β1 in a pilot trial of seven subjects with NASH.⁷⁸

Lipid lowering drugs

As hyper triglyceridaemia and low HDL cholesterol levels are a manifestation of insulin resistance and common among subjects with NAFLD, several investigators have used lipid lowering drugs to treat NAFLD . The use of statin drugs is currently contraindicated in the presence of active liver disease or persistent unexplained increases of aminotransaminases.

Recent evidence, however, shows that patients with raised liver enzymes may not be at increased risk of serious hepatotoxicity with standard doses of these drugs.⁷⁹ Subsequently two small pilot trials have shown improvement of liver enzymes with atorvastatin.

Caspase inhibitors

Because of the role of apoptosis pathway activation, a great deal of interest has centered on caspase inhibition as a possible therapeutic avenue in experimental models.⁸⁰ However, the effects on necrosis pathways, the relative risk–benefit with chronic therapy, and the costs of this approach are uncertain.

VITAMIN E

History

A fat soluble dietary constituent was found to be essential for the prevention of fetal deaths and sterility in rats in the year of 1922. This was originally called as 'Factor S' and 'ant sterility factor 'and named as Vitamin E in 1936 when it was isolated from wheat germ oil. Vitamin E is called as 'tocopherol' from the greek words tokos and pherein , meaning ' to bring forth children'.

There are now known to be several forms of tocopherols and the term Vitamin E is often used to denote any mixture of biologically active tocopherols.

Chemical structure of Vitamin E

There is an alpha, beta, gamma, and delta form of both tocopherols and tocotrienols, determined by the number of methyl groups on the chromanol ring.

Sources of Vitamin E:

 Unrefined vegetable oil (palm oil, olive oil, sunflower oil, canola oil)

 Nuts

 Sunflower seeds

 Wheat germ

 Whole grain

 Fish

 Green leafy vegetables

Commercial vitamin E supplements can be classified into several distinct categories.

1. Fully synthetic vitamin E, “dl – alpha tocopherol” the most inexpensive, most commonly sold supplement form usually as the acetate ester.

2. Semi – synthetic “natural source” vitamin E ester, the natural source form.

3. Less fractionated “natural mixed tocopherols.

As tocopherols and tocotrienols are readily oxidized, commercial preparations of vitamin E are often protected by acetylation and succinylation.

The resultant esters are hydrolyzed by pancreatic enzymes to yield the biologically active free tocopherols.

Anti oxidant role of Vitamin E

Vitamin E is an example of a phenolic anti – oxidant, such molecules readily donate the hydrogen from the hydroxyl group on the ring structure to free radicals, which then become un reactive.

Vitamin E has a major biological role in protecting polyunsaturated fats and other components of cell membrane from oxidation by free radicals and is therefore, primarily located within the phospholipid bilayer of cell membranes. Vitamin E is particularly effective in preventing lipid peroxidation, a series of chemical reactions involving the oxidative deterioration of polyunsaturated fatty acids.(PUFA:H)

Lipid peroxidation is a process of oxidative decomposition of omega 3 and omega 6 PUFA of membrane phospholipids, leading to formation of lipid hydroperoxides and aldehydic end products like melondialdehyde (MDA) and 4 hydroxynonenol. This process may cause disruption of cell structure and function and thus play an important role in the etiology of many disease.

Initiation and propagation of lipid peroxidation are mediated by free radicals.

Removal of the hydrogen from PUFA:H by a free radical R^* can initiate chain reaction.

PUFA : H + Rˉ PUFA˙ + RH PUFA˙ + O2 PUFAOO˙

PUFAOO˙ + PUFA : H PUFAOOH + PUFA˙

Further self propagation of the process can occur because of hemolytic fission of the lipid hydroperoxide (PUFAOOH).

PUFAOOH PUFAO˙ + OH˙

The above auto catalytic process may progress to the formation of more than 60 end products, which are cytotoxic. However , in biological systems, the peroxidative cascade is more likely to be terminated by vitamin E, which is present in the cell membrane.

Tocopherol – OH + PUFAOOˉ tocopherol - Oˉ +PUFAOOH.

On denoting the hydrogen, vitamin E becomes a relatively unreactive free radical (tocopheroxyl radical) which loses its antioxidant property and is unable to attack adjacent fatty acids. Vitamin C has a sparing effect on vitamin E and regenerates tocopherol from tocopheroxyl radical.

Other Functions of Vitamin E

Vitamin E may also play an important role in other biological progress other than its antioxidant function. These include:

1. Structural roles in the maintenance of cell membrane integrity 2. Anti – inflammatory action – vitamin E inhibits phospholipase A2

activity preventing release of arachidonic acid, thus inhibiting both cyclo oxygenase and lipoxygenase pathways. With prolonged therapy, it has been shown to decrease the release of proinflammatory cytokines, the chemokine IL- 8 and plasminogen activator inhibitor(PAI - 1) as well as decrease the adhesion of monocytes to the endothelium. In addition, it decrease the CRP level.

Membrane phospholipids

Phospholipase A2 Vitamin E

Arachidonic acids

3. In stimulating the immune response.

4. In the regulation of intercellular signaling and cell proliferation through modulation of protein kinase C.

5. Analgesic action – central analgesic effect mediated by a suppressive action on nitric oxide, which is implicated in central pain processing. NO reduces the threshold in the periphery , lowers

the threshold of nociceptors and facilitates nociceptive transmission within central pathways. Vitamin E also inhibits protein kinase C, which plays an important part in signal transduction triggered by neurotransmitters and cellular stimuli.

Effects of Vitamin E on Cholesterol Metabolism

Tocotrienols (analog of tocopherols) decrease the hepatic cholesterol production and reduce plasma cholesterol levels in mammalian cells by post- transcriptional suppression of HMGCo A reductase.⁸¹

Gamma delta tocotrienols suppresses the upstream regulators of lipid homeostasis genes (DGAT2, APOB100, SREBP1/2 and HMGCR) leading to the suppression of triglycerides, cholesterol & VLDL biosynthesis; also enhances LDL efflux through induction of LDL receptor expression in animal models.⁸²

Vitamin E deficient rabbits with signs of muscular dystrophy showed accumulation of cholesterol in muscle as well as elevation of plasma cholesterol (LDL, VLDL).⁸³

Pharmacokinetics

Absorption

Vitamin E is taken up by the enterocytes in the small intestine by passive diffusion. Following this vitamin E is incorporated into the chylomicrons, which enter systemic circulation. Here the majority of chylomicrons undergo lipolysis by lipoprotein lipase, which is present in the

capillary endothelium. The resultant chylomicron remnants enter the liver.

Here vitamin E is repackaged and secreted in VLDL. Following VLDL secretion into the plasma, lipolysis results in transfer of vitamin E to HDL &

LDL, which deliver vitamin E to the peripheral tissue.

Metabolism and Excretion

Vitamin E is metabolised by – oxidation by CYP4F2 and CYP3A to carboxyethyl hydroxychroman (CEHC), which is excreted after glucuronidation and sulfation. The metabolites are excreted in urine and bile.

Unabsorbed vitamin E is excreted unchanged in the faeces.

Storage and Degradation of Vitamin E

Storage of vitamin E is limited. The liver briefly stores vitamin E but only in small quantities. Adipose tissue and the adrenal gland store vitamin E as well. Adipose slowly accumulates vitamin E and then in time, slowly releases it as well. Vitamin E is extensively metabolized in the liver, the metabolites are called simon metabolites—tocopheronic acid and

tocopheronolactone. Both metabolites are excreted in the urine as glucuronides or sulphates.

Preparations

1. α – tocopherol acetate (B.P) – a clear, yellow or greenish yellow, odourless oily liquid unstable in alkaline solution.

2. d – α – tocopherol – this is considered to be the form of vitamin E found in nature.

3. dl – α – tocopherol – it is a permitted antioxidant for food.

4. Vitamin E capsule (U.S.P) – contain either d or dl a – tocopherol.

This preparation should be stored in an atmosphere of inert gas protected from light.

5. Vitamin E suspension (B.P) – contains a – tocopherol acetate 500mg/5ml. Excipients used are benzoic – a – tocopherol acid, castor oil, sorbic acid and polyethylene glycol.

DEFICIENCY Causes

(i) Malabsorption

 Cystic fibrosis. .

 Exocrine pancreatic insufficiency

 Congenital biliary atresia.

 Advanced liver disease.

 Abetalipoproteinemia

 Untreated celiac disease

 Intestinal resection (ii) Genetic defects

 Familial isolated vitamin E deficiency.

(iii) Increased oxidative damage

 Premature infants.

 Pre- eclampsia.

 Atherosclerosis.

 Smoking.

 Aging

 Neoplastic conditions

 Exposure to environmental pollutions

 Neurodegenerative diseases

Symptoms

Vitamin E deficiency causes damage to cell membrane and leakage of cell contents, resulting in myopathies, neuropathies and liver necrosis. Early diagnostic signs of deficiency include leakage of muscle enzyme such as creatinine kinase into plasma, increased level of lipid peroxidation products in plasma and increased erythrocyte hemolysis. Suboptimal levels of vitamin E may be linked with atherogenesis, coronary heart disease, cancer, arthritis, and other degenerative and neurological disorders.

Severe vitamin E deficiency leads to neuromuscular abnormalities characterised by,

 Loss of deep tendon reflexes.

 Opthalmoplegia and retinitis pigmentosa.

 Cerebellar ataxia.

 Dysarthria.

 Mental retardation.

 Hemolytic anemia in premature infants

 Diminished proprioception and vibratory sense.

Adverse Reactions

In large population human studies, oral vitamin E supplementation resulted in few side effects even at doses as high as 3200mg/day.

With Therapeutic Dose

 Nausea.

 Flatulence.

 Diarrhoea.

 Fatigue.

 Hypertension

 Myopathy

 Thrombophlebitis

With Toxic Dose (>1200mg / Day)

 --- Necrotising enterocolitis in premature infants.

 --- Bleeding tendency due to decreased vitamin K absorption.

 --- Increased triglyceride.

Drug Interactions

 Iron binds with vitamin E and inactivates it.

 Synergistic action with selenium.

 High dose can impair intestinal absorption of vitamin A and K.

 Vitamin E may impair vitamin K function at the level of prothrombin formation and thus potentiate the effect of warfarin.

Uses

--- Nutritional deficiency. --- Muscular dystrophy.

--- Atherosclerosis. --- Dupuytren’s contracture.

--- Hemolytic anemia in newborn. --- Fibrosis.

--- Retrolental fibroplasia. --- Lupus erythematosus.

--- Nocturnal muscle cramps. --- Purpura.

--- Infertility. --- Scleroderma.

--- Fibrocystic breast disease. --- Diabetes mellitus.

--- Arthritis. --- Habitual abortion.

--- Menopausal syndrome. --- Healing wounds.

--- Senile vaginitis. --- Parkinsons disease.

--- Intermittent claudication --- Interstitial keratitis⁸⁴

AIM

To study the effect of vitamin E supplementation in patients with Non Alcoholic Fatty Liver Disease.

Primary Objective:

 To determine the changes in the grade of fatty infiltration of liver in ultra sonogram in NAFLD patients supplemented with vitamin E.

Secondary Objective

:

 To monitor biochemical parameters.(serum transaminases)

 To monitor subjective improvement(upper abdominal discomfort, pain) .

 To monitor adverse effects