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Fatty Acids, Genes and Microbiota in Fatty Liver

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ClinicalTrials.gov Identifier: NCT02148471
Recruitment Status : Completed
First Posted : May 28, 2014
Last Update Posted : May 12, 2016
Sponsor:
Collaborators:
Canadian Liver Foundation
Canadian Institutes of Health Research (CIHR)
American College of Gastroenterology
Information provided by (Responsible Party):
Johane Allard, University Health Network, Toronto

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.

Condition or disease
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.


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Study Type : Observational
Actual Enrollment : 205 participants
Observational Model: Cohort
Time Perspective: Cross-Sectional
Official Title: Non-alcoholic Steatohepatitis Versus Simple Hepatic Steatosis: Is There a Difference in the Nutritional Factors Influencing Lipid Perioxidation and Inflammation?
Study Start Date : October 2003
Actual Primary Completion Date : August 2015
Actual Study Completion Date : August 2015


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.



Primary Outcome Measures :
  1. Hepatic fatty acid composition in total lipids in liver biopsy [ Time Frame: Baseline ]
    Gas chromatography

  2. Hepatic gene expression [ Time Frame: Baseline ]
    mRNA by microarray

  3. Intestinal microbiota composition [ Time Frame: Baseline ]
    Illumina 16S technology


Secondary Outcome Measures :
  1. Lipid peroxides in the liver [ Time Frame: Baseline ]
    Test kit

  2. Hepatic liver antioxidant power [ Time Frame: Baseline ]
    Test kit

  3. Hepatic microRNA expression in the liver [ Time Frame: Baseline ]
    NanoString

  4. Intestinal microbiota - specific organisms and groups [ Time Frame: Baseline ]
    Quantitative real-time polymerase chain reaction

  5. Intestinal microbiome on a genetic level [ Time Frame: Baseline ]
    Illumina sequencing technology

  6. Short-chain fatty acids in stool [ Time Frame: Baseline ]
    Gas chromatography

  7. Plasma endotoxin [ Time Frame: Baseline ]
    Limulus assay


Other Outcome Measures:
  1. Hepatic phospholipid composition [ Time Frame: Baseline ]
    Gas chromatography

  2. Red blood cell fatty acid and phospholipid composition [ Time Frame: Baseline ]
    Gas chromatography

  3. Plasma fatty acid composition [ Time Frame: Baseline ]
    Gas chromatography

  4. Plasma lipid peroxides [ Time Frame: Baseline ]
    Test kit

  5. Plasma antioxidant vitamins [ Time Frame: Baseline ]
    Vitamin C colorimetric, alpha- and gamma-tocopherol and beta-carotene by high-performance liquid chromatography

  6. Serum antioxidant power [ Time Frame: Baseline ]
    Test kit

  7. TNF-alpha in the liver [ Time Frame: Baseline ]
    Enzyme linked immunosorbent assay

  8. Immunohistochemistry [ Time Frame: Baseline ]
    Staining for malondialdehyde, alpha-smooth muscle actin, transforming growth factor beta

  9. Free choline in serum [ Time Frame: Baseline ]
    liquid chromatography/electrospray ionization-isotope dilution mass spectrometry (LC/ESI-IDMS)

  10. Bacterial DNA in plasma [ Time Frame: Baseline ]
    Quantitative polymerase chain reaction for bacterial 16S rDNA

  11. Insulin resistance [ Time Frame: Baseline ]
    Fasting glucose and insulin to calculate insulin resistance (HOMA-IR), C-peptide, hemoglobin A1c, all by standard laboratory methods

  12. Plasma ethanol [ Time Frame: Baseline ]
    standard laboratory measurement

  13. Anthropometry [ Time Frame: Baseline ]
    Weight, height, skinfolds, bioelectrical impedance analysis

  14. Food intake [ Time Frame: Baseline ]
    7-day food records

  15. Physical activity [ Time Frame: Baseline ]
    7 day activity logs

  16. Factors influencing intestinal microbiota [ Time Frame: Baseline ]
    Environmental questionnaire

  17. Liver function tests [ Time Frame: Baseline ]
    Alanine transaminase, aspartate transaminase, alkaline phosphatase, standard laboratory tests


Biospecimen Retention:   Samples With DNA
DNA - samples retained, with potential for extraction of DNA from at least one of the types of samples retained (e.g., frozen tissue, whole blood


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Ages Eligible for Study:   18 Years to 70 Years   (Adult, Older Adult)
Sexes Eligible for Study:   All
Accepts Healthy Volunteers:   Yes
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.
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

Information from the National Library of Medicine

To learn more about this study, you or your doctor may contact the study research staff using the contact information provided by the sponsor.

Please refer to this study by its ClinicalTrials.gov identifier (NCT number): NCT02148471


Locations
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Canada, Ontario
Toronto General Hospital
Toronto, Ontario, Canada, M5G 1Z5
Sponsors and Collaborators
Johane Allard
Canadian Liver Foundation
Canadian Institutes of Health Research (CIHR)
American College of Gastroenterology
Investigators
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Principal Investigator: Johane Allard, MD,FRCPC University Health Network, Toronto

Publications of Results:

Other Publications:
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Responsible Party: Johane Allard, Prof. Dr., University Health Network, Toronto
ClinicalTrials.gov Identifier: NCT02148471     History of Changes
Other Study ID Numbers: 03-0505-A
First Posted: May 28, 2014    Key Record Dates
Last Update Posted: May 12, 2016
Last Verified: May 2016

Keywords provided by Johane Allard, University Health Network, Toronto:
Nonalcoholic fatty liver disease
Steatosis
Nonalcoholic steatohepatitis

Additional relevant MeSH terms:
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Liver Diseases
Fatty Liver
Non-alcoholic Fatty Liver Disease
Digestive System Diseases