Fatty Acids, Genes and Microbiota in Fatty Liver

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

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.

Study Overview

• Status: Completed
• Conditions: Nonalcoholic Fatty Liver Disease; Steatosis; Nonalcoholic Steatohepatitis

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.

• Study Type: Observational
• Enrollment (Actual): 205

Contacts and Locations

Study Locations

• Canada
  • Ontario
    • Toronto, Ontario, Canada, M5G 1Z5
      • Toronto General Hospital

Participation Criteria

Eligibility Criteria

• Ages Eligible for Study: 18 years to 70 years (Adult, Older Adult)
• Accepts Healthy Volunteers: No
• Genders Eligible for Study: All

• Sampling Method: Non-Probability Sample

Study Population

Healthy living liver donors from the Multiorgan transplant program at the University Health Network Patients with nonalcoholic fatty liver disease (SS or NASH on liver biopsy) or patients with elevated liver enzymes but no significant findings on liver biopsy.

Description

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

Study Plan

How is the study designed?

Design Details

• Observational Models: Cohort
• Time Perspectives: Cross-Sectional

Number of groups / cohorts

4

Cohorts and Interventions

Group / Cohort
Healthy controls; Healthy living liver donors with healthy liver on imaging and/or liver histology
Simple steatosis; Patients with non-alcoholic fatty liver disease confirmed by liver biopsy with a diagnosis of simple steatosis
Nonalcoholic steatohepatitis; Patients with non-alcoholic fatty liver disease confirmed by liver biopsy with a diagnosis of steatohepatitis
Minimal findings; 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

Other Outcome Measures

Outcome Measure	Measure Description	Time Frame
Hepatic phospholipid composition	Gas chromatography	Baseline
Red blood cell fatty acid and phospholipid composition	Gas chromatography	Baseline
Plasma fatty acid composition	Gas chromatography	Baseline
Plasma lipid peroxides	Test kit	Baseline
Plasma antioxidant vitamins	Vitamin C colorimetric, alpha- and gamma-tocopherol and beta-carotene by high-performance liquid chromatography	Baseline
Serum antioxidant power	Test kit	Baseline
TNF-alpha in the liver	Enzyme linked immunosorbent assay	Baseline
Immunohistochemistry	Staining for malondialdehyde, alpha-smooth muscle actin, transforming growth factor beta	Baseline
Free choline in serum	liquid chromatography/electrospray ionization-isotope dilution mass spectrometry (LC/ESI-IDMS)	Baseline
Bacterial DNA in plasma	Quantitative polymerase chain reaction for bacterial 16S rDNA	Baseline
Insulin resistance	Fasting glucose and insulin to calculate insulin resistance (HOMA-IR), C-peptide, hemoglobin A1c, all by standard laboratory methods	Baseline
Plasma ethanol	standard laboratory measurement	Baseline
Anthropometry	Weight, height, skinfolds, bioelectrical impedance analysis	Baseline
Food intake	7-day food records	Baseline
Physical activity	7 day activity logs	Baseline
Factors influencing intestinal microbiota	Environmental questionnaire	Baseline
Liver function tests	Alanine transaminase, aspartate transaminase, alkaline phosphatase, standard laboratory tests	Baseline

Collaborators and Investigators

• Sponsor: Johane Allard
• Collaborators: Canadian Institutes of Health Research (CIHR); Canadian Liver Foundation; American College of Gastroenterology
• Investigators: Principal Investigator: Johane Allard, MD,FRCPC, University Health Network, Toronto

Publications and helpful links

General Publications

• Schwenger KJ, Allard JP. Clinical approaches to non-alcoholic fatty liver disease. World J Gastroenterol. 2014 Feb 21;20(7):1712-23. doi: 10.3748/wjg.v20.i7.1712.
• Monteiro J, Leslie M, Moghadasian MH, Arendt BM, Allard JP, Ma DW. The role of n - 6 and n - 3 polyunsaturated fatty acids in the manifestation of the metabolic syndrome in cardiovascular disease and non-alcoholic fatty liver disease. Food Funct. 2014 Mar;5(3):426-35. doi: 10.1039/c3fo60551e.
• Mouzaki M, Allard JP. The role of nutrients in the development, progression, and treatment of nonalcoholic fatty liver disease. J Clin Gastroenterol. 2012 Jul;46(6):457-67. doi: 10.1097/MCG.0b013e31824cf51e.
• Mouzaki M, Allard J. Non-alcoholic steatohepatitis: the therapeutic challenge of a global epidemic. Ann Gastroenterol. 2012;25(3):207-217.
• Da Silva HE, Arendt BM, Noureldin SA, Therapondos G, Guindi M, Allard JP. A cross-sectional study assessing dietary intake and physical activity in Canadian patients with nonalcoholic fatty liver disease vs healthy controls. J Acad Nutr Diet. 2014 Aug;114(8):1181-94. doi: 10.1016/j.jand.2014.01.009. Epub 2014 Mar 14.
• Arendt BM, Ma DW, Simons B, Noureldin SA, Therapondos G, Guindi M, Sherman M, Allard JP. Nonalcoholic fatty liver disease is associated with lower hepatic and erythrocyte ratios of phosphatidylcholine to phosphatidylethanolamine. Appl Physiol Nutr Metab. 2013 Mar;38(3):334-40. doi: 10.1139/apnm-2012-0261. Epub 2012 Oct 15.
• Mouzaki M, Comelli EM, Arendt BM, Bonengel J, Fung SK, Fischer SE, McGilvray ID, Allard JP. Intestinal microbiota in patients with nonalcoholic fatty liver disease. Hepatology. 2013 Jul;58(1):120-7. doi: 10.1002/hep.26319. Epub 2013 May 14.
• Allard JP, Aghdassi E, Mohammed S, Raman M, Avand G, Arendt BM, Jalali P, Kandasamy T, Prayitno N, Sherman M, Guindi M, Ma DW, Heathcote JE. Nutritional assessment and hepatic fatty acid composition in non-alcoholic fatty liver disease (NAFLD): a cross-sectional study. J Hepatol. 2008 Feb;48(2):300-7. doi: 10.1016/j.jhep.2007.09.009. Epub 2007 Nov 20.
• Arendt BM, Comelli EM, Ma DW, Lou W, Teterina A, Kim T, Fung SK, Wong DK, McGilvray I, Fischer SE, Allard JP. Altered hepatic gene expression in nonalcoholic fatty liver disease is associated with lower hepatic n-3 and n-6 polyunsaturated fatty acids. Hepatology. 2015 May;61(5):1565-78. doi: 10.1002/hep.27695. Epub 2015 Feb 27.
• Pettinelli P, Arendt BM, Schwenger KJP, Sivaraj S, Bhat M, Comelli EM, Lou W, Allard JP. Relationship Between Hepatic Gene Expression, Intestinal Microbiota, and Inferred Functional Metagenomic Analysis in NAFLD. Clin Transl Gastroenterol. 2022 Jul 1;13(7):e00466. doi: 10.14309/ctg.0000000000000466. Epub 2022 Feb 10.

Study record dates

Study Major Dates

• Study Start: October 1, 2003
• Primary Completion (Actual): August 1, 2015
• Study Completion (Actual): August 1, 2015

Study Registration Dates

• First Submitted: May 23, 2014
• First Submitted That Met QC Criteria: May 23, 2014
• First Posted (Estimate): May 28, 2014

Study Record Updates

• Last Update Posted (Estimate): May 12, 2016
• Last Update Submitted That Met QC Criteria: May 10, 2016
• Last Verified: May 1, 2016

More Information

Terms related to this study

• Keywords: Nonalcoholic fatty liver disease; Steatosis; Nonalcoholic steatohepatitis
• Additional Relevant MeSH Terms: Digestive System Diseases; Liver Diseases; Fatty Liver; Non-alcoholic Fatty Liver Disease
• Other Study ID Numbers: 03-0505-A

Drug and device information, study documents

• Studies a U.S. FDA-regulated drug product: No
• Studies a U.S. FDA-regulated device product: No
• product manufactured in and exported from the U.S.: No