The Role of microbial produced ethanol in etiology of Non-Alcoholic Steatohepatitis (ETHANASH trial)

Changes in the composition of the gut microbiota have been associated with
alterations in host metabolism and recent evidence suggest that gut microbiota
might also be involved in the development of Non-Alcoholic Fatty Liver Disease
(NAFLD) and subsequent Non-alcoholic steatohepatitis (NASH). So far, however,
causality has not been demonstrated. Among many gut microbial metabolites,
endogenous intestinally produced ethanol has gained interest in the past decade
for its involvement in the development of NAFLD. Ethanol is produced by
intestinal bacteria and it has been suggested that ethanol might play a role in
the development of NAFLD, especially in the transition from NAFLD to NASH. When
produced in significant amounts, hepatic ethanol metabolism inhibits
beta-oxidation of fatty acids which will induce storage of lipids in the liver.
Endogenously produced ethanol reaches the liver via the portal vein and is then
rapidly removed from the circulation via extremely efficient hepatic
mechanisms, leaving almost untraceable concentrations in the peripheral plasma
if liver function is uncompromised. Several studies have showed that subjects
with NASH (known to have a compromised liver function) have increased
peripheral concentrations of ethanol, however these concentrations are so low
that one might argue whether this is clinical relevant regarding the
development of NASH. The first step in ethanol catabolism is the oxidation of
ethanol to acetaldehyde using NAD+, mainly via the hepatic enzyme alcohol
dehydrogenase. Fomepizole (4-methylpyrazole) is a specific inhibitor of the
enzyme alcohol dehydrogenase. In a previous study, a significant elevation of
peripheral plasma ethanol concentrations were observed in lean subjects who
were treated with fomepizole after intake of lingonberry juice. Since subjects
with NASH might have more ethanol producing bacteria, we anticipate to find
increased concentrations of ethanol in subjects with NASH compared to healthy
control subjects during a mixed meal test after the infusion of
fomepizole.Moreover, when intestinal microbiota is temporarily eradicated by a
short term oral antibiotic course, we expect to see no increase in peripheral
plasma ethanol levels upon fomipizol infusion in all subjects.

To investigate whether microbial produced ethanol plays a role in the
development of NASH.

cross sectional cohort study

Subjects will be recruited from subjects that visited the outpatient clinic of
internal medicine at AMC in the last year and in whom NASH was proven by
biopsy. After informed consent, biological samples (including saliva, blood and
feces sample) will be collected. In addition, subjects will undergo an
ultrasonography of the liver. Prior to the mixed meal tolerance test, an
infusion with fomepizole will be given to suppress the alcohol dehydrogenase
enzyme in the liver. Repetitive blood samples will be drawn after the intake
of the mixed meal. In total, 8 blood samples will be drawn during the 5-hour
mixed meal tolerance test (total duration of study 15 h). The burden consists
of the extra time invested in the measurements. This study will further
investigate the role of endogenously produced ethanol in the development of
NASH and more importantly, the relation with liver histology (already available
from the biopsy taken at the outpatient clinic). We therefore believe that the
scientific insight of our findings will outweigh the minimal risks for the
participating subjects in this study. Total amount of blood taken is 70 ml per
visit mixed meal test and 1x clinical lab (20ml) including hba1c, lipid
profile, liver function 20ml) thus 230ml for all participants.