Disease mechanisms and markers for non-alcoholic steatohepatitis in a population with non-alcoholic fatty liver disaese: a prospective cohort study with biobank

Non-alcoholic fatty liver disease (NAFLD) is with 20-30% the most prevalent
liver disorder in Western society and is associated with overweight and obesity
in 50-100%. NAFLD has been described as the hepatic component of the metabolic
syndrome. The majority of patients have simple steatosis, in about 15-30%
non-alcoholic steatohepatitis (NASH) develops, which leads to an overall
increase in morbidity and mortality due to the progression to fibrosis,
cirrhosis and hepatocellular carcinoma (HCC). The term NAFLD comprises both
simple steatosis and NASH. Most patients with NAFLD have no or few, mainly
aspecific symptoms; and generally there is a silent progression of simple
steatosis to NASH and in the end liver-related morbidity and mortality.
NASH is a chronic systemic inflammatory disease of multifactorial origin. In
the pathogenesis of simple steatosis to NASH, oxidative stress plays a crucial
role in the initiation and the progression of the inflammatory cascade. Genetic
susceptibility, activated white adipose tissue with adipocytokine secretion,
altered intestinal microbiota and an increased intestinal permeability are also
involved, but their exact contribution and interaction in the occurrence of
NASH still has to be determined. Further insight in factors contributing to
the initiation of NASH in patients with simple steatosis and early diagnosis
are essential for identifying future therapeutic options and to limit the risk
of complicated NASH (i.e. fibrosis, and cirrhosis with portal hypertension)
HCC, liver-related mortality and extrahepatic morbidity.

Objective: The objective of the prospective NAFLD cohort study with biobank is:
1. In a cross-sectional design (a) to determine which factors are associated
with the presence of NASH in a population of NAFLD patients and (b) to identify
and validate non-invasive markers to identify patients with NASH. 2. In a
longitudinal design (a) to define factors contributing to the development and
progression of NASH in patients with simple steatosis, and (b) to identify and
validate non-invasive markers to identify patients with NASH.
Therefore, the role of metabolic, systemic and local inflammatory, intestinal
(i.e. microbiota and barrier function) and/or lifestyle factors will be studied
both cross-sectionally and longitudinally in a cohort of NAFLD patients.
Furthermore, non-invasive diagnostic tools and/or markers associated with NASH
will be investigated.

Study design: A cohort study, with both a cross-sectional and a longitudinal
part, of obese subjects with proven NAFLD based on liver biopsy and/or imaging
(any form). Eligible subjects will be included via the MUMC+ outpatient clinic
and second line obesity clinic, Zuderland MC and general practitioners in
South-Limburg. The majority of eligible subjects will undergo/have undergone
imaging or liver biopsy for clinical reasons. It is to be expected that about
33% of subjects will be asked to undergo a MRI for study purpose only. Subjects
with a suspected diagnosis of NAFLD (no prior imaging or biopsy) need to
undergo a CAP measurement to confirm the diagnosis, before the other
measurements are scheduled.
All participants will be asked to complete several questionnaires (i.e.
demographics, clinical data, SF-36, GAD-7 and PHQ-9, FFQ, SQUASH, and Baecke),
to undergo anthropometric measurements. Furthermore, blood, urine, faeces and
exhaled air will be collected and a fibroscan and DEXA-scan will be performed.
This will be the standard to be collected data set. Additionally, participants
will be asked to participate in a multi-sugar test for intestinal permeability.
After 5 and 10 years, participants will be invited to undergo in the same study
procedures, data and sample collection to study the factors responsible for the
development of NASH in the group with simple steatosis at baseline, and the
development of (extra)hepatic complications in the group with NASH at baseline.
The time load for the different investigations is estimated 5 hours, consisting
of completing the questionnaires at home and one visit to the hospital to
collect to collect the anthropometric data, blood/stool/urine/air samples,
perform a fibroscan and DEXA-scan. In case of participating in the intestinal
permeability test, the time load will be another 24 hours, for collecting
urine.

Nature and extent of the burden and risks associated with participation,
benefit and group relatedness: The measurements, data and sample collections
will be planned with the participant. Anthropometric data collection, hand grip
strength, blood, urine and faeces collection, exhaled air (VOC), and fibroscan
will be performed during study visit 1 (after a pre-visit for signing the
informed consent). The participant can fill out the questionnaires at home and
bring the forms when visiting the research physician. The DEXA-scan will be
planned in agreement with the participant. MRI for study purposes only, is
needed in a subset of subjects. CAP to confirm a suspected diagnosis is needed
in a subset of patients. The DEXA-scan and, if possible, also the MRI will be
combined with study visit 1. The multiple sugar permeability test will be
performed at home by participants who consented (and signed informed consent)
for this.

No side effects of the investigations are expected, apart from a small bruise
of taking the blood samples. Three tubes (21 ml) will be collected for research
purposes; if possible this will be combined with regular blood sampling. If
participants are not regular patients at the outpatient clinic, a maximum of 4
extra tubes (max.17,5 ml in total) will be collected to determine (part of) the
standard clinical laboratory investigations (e.g. liver function tests,
cholesterol) for research purposes only.
Radiation exposure during the DEXA scan is 0.01 mSV per procedure. No side
effects are expected with this minimum dose of radiation exposure.
The time burden associated with participating in the research is: 90 minutes to
fill out the questionnaires, 2 hours for the first visit to collect samples and
anthropometry and perform a fibroscan. The DEXA-scan will take approximately
15-30 minutes. If an MRI is needed, this will take 45-60 minutes.
Subjects participating in the intestinal permeability test will have to collect
their urine for 24 hours. In case of unexpected findings, awareness of normally
unknown pathology may affect a person*s perception of his own health condition
negatively. On the other hand, early detection is likely to have favourable
effects on disease progression and enable early intervention