Fatty Acids, Genes and Microbiota in Fatty Liver

Study Overview
=================
Brief Summary
-----------------
The first aim of this study is to assess oxidative stress and nutritional status in patients with elevated liver enzymes who were found to have either simple steatosis (SS) or nonalcoholic steatohepatitis (NASH) or normal histological findings on liver biopsy by measuring liver lipid peroxides and tumor necrosis factor (TNF)-α, liver pathology and immunohistochemistry, liver function tests, liver and red blood cell membrane fatty composition, insulin resistance (IR) parameters, plasma lipid peroxides, plasma antioxidant vitamins and antioxidant power, lipid profile, subject demographics, medical history and medication use. The second aim is to detect differences in hepatic gene expression (messenger RNA, mRNA) and epigenetic regulation (micro RNA, miRNA) between patients with SS or NASH and healthy controls, in addition to determine in patients with non-alcoholic fatty liver disease (NAFLD = SS+NASH combined) whether there is an association between hepatic n-3 PUFA content and gene expression. The third aim is to determine the intestinal microbiome (microbial composition and metagenome) in patients with SS or NASH and healthy controls.

Detailed Description
-----------------
NASH is associated with obesity, diabetes and hyperlipidemia. Fat accumulation in the liver is likely due to variable degrees of disordered fatty-acid metabolism and insulin resistance (IR). Liver steatosis, especially polyunsaturated fatty acids (PUFA) in the liver, increases lipid peroxidation and is associated with a reduction in the antioxidant defense system. This oxidative stress can lead to increased production of pro-inflammatory cytokines (TNF-α, transforming growth factor-beta) contributing to the development of steatohepatitis and fibrosis.TNF-α - may further contribute to IR. In addition, changes in fatty acid composition within the liver may influence lipid metabolism and inflammation. In particular, n-3 PUFA have an effect on the insulin sensitivity, transcription of antioxidant genes, inflammatory response and production of reactive oxygen species. Differences might be seen on the gene expression level (mRNA) and also in epigenetic regulation (miRNA). Microbiota composition might influence energy metabolism, and inflammatory tone and IR through increased endotoxemia and therefore could also play a role in the development of NAFLD.

Official Title
-----------------
Non-alcoholic Steatohepatitis Versus Simple Hepatic Steatosis: Is There a Difference in the Nutritional Factors Influencing Lipid Perioxidation and Inflammation?

Conditions
-----------------
Nonalcoholic Fatty Liver Disease, Steatosis, Nonalcoholic Steatohepatitis

Participation Criteria
=================
Eligibility Criteria
-----------------
Inclusion criteria: Male and female patients, age >18 y A liver biopsy with a diagnosis of SS or NASH OR No signs of steatosis, fibrosis or any other kind of liver disease on histology (minimal findings) OR For healthy control subjects, those with normal liver enzymes and normal liver imaging on ultrasound alcohol consumption (<20g of ethanol per day); absence of any other possible cause for liver dysfunction. Exclusion criteria: any other liver disease apart from NAFLD anticipated need for liver transplantation in one year or complications of liver disease; any reasons contraindicating a liver biopsy (patients) or liver donation (healthy donors) chronic gastrointestinal diseases, previous gastrointestinal surgery modifying the anatomy, patients with diabetes requiring insulin. medications known to precipitate steatohepatitis (corticosteroids, high dose estrogens, methotrexate, amiodarone, spironolactone, sulfasalazine, perhexiline maleate, diethylamino- ethoxyhexestrol (DH), tamoxifen, diethylstilbestrol, naproxen or oxacillin) or regular intake of non-steroidal anti-inflammatory drugs (except for low dose aspirin), use of ursodeoxycholic acid or any experimental drug in the 6 months prior to entry. regular intake of prebiotics, probiotics, antibiotics, or laxatives; in the 3 months prior to study entry Pregnant or lactating

Ages Eligible for Study
-----------------
Minimum Age: 18 Years
Maximum Age: 70 Years

Sexes Eligible for Study
-----------------
All

Accepts Healthy Volunteers
-----------------
Accepts Healthy Volunteers

Study Plan
=================
How is the study designed?
-----------------

Arms and Interventions

| Participant Group/Arm | Intervention/Treatment |
| --- | --- |
| Healthy controls<br>Healthy living liver donors with healthy liver on imaging and/or liver histology | |
| Simple steatosis<br>Patients with non-alcoholic fatty liver disease confirmed by liver biopsy with a diagnosis of simple steatosis | |
| Nonalcoholic steatohepatitis<br>Patients with non-alcoholic fatty liver disease confirmed by liver biopsy with a diagnosis of steatohepatitis | |
| Minimal findings<br>Patients undergoing liver biopsy because of suspected fatty liver but nonspecific findings on liver histology. This group was initially used as a control group. Later in the study, this group was replaced by healthy donors as true healthy controls. | |

What is the study measuring?
-----------------
Primary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| Hepatic fatty acid composition in total lipids in liver biopsy | Gas chromatography | Baseline | 
| Hepatic gene expression | mRNA by microarray | Baseline | 
| Intestinal microbiota composition | Illumina 16S technology | Baseline | 

Secondary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| Lipid peroxides in the liver | Test kit | Baseline | 
| Hepatic liver antioxidant power | Test kit | Baseline | 
| Hepatic microRNA expression in the liver | NanoString | Baseline | 
| Intestinal microbiota - specific organisms and groups | Quantitative real-time polymerase chain reaction | Baseline | 
| Intestinal microbiome on a genetic level | Illumina sequencing technology | Baseline | 
| Short-chain fatty acids in stool | Gas chromatography | Baseline | 
| Plasma endotoxin | Limulus assay | Baseline | 

 Terms related to the study
=================
Keywords Provided by Centre Hospitalier Valida
-----------------
Nonalcoholic fatty liver disease, Steatosis, Nonalcoholic steatohepatitis