The effects of plant sterol and plant stanol ester enriched foods on biopsy proven liver inflammation in NAFLD patients - a proof-of-concept pilot study -

As the prevalence of obesity is reaching epidemic proportions, the prevalence
of non-alcoholic fatty liver disease (NAFLD), including non-alcoholic
steatohepatitis (NASH), increases concomitantly and becomes a major global
health hazard. Successful pharmacological interventions to treat or prevent
NASH are not available and so far only weight loss has clear benefits, but
sustained weight-loss is difficult to achieve on the longer-term. We recently
demonstrated in mice that plant sterol and stanol ester consumption inhibited
the development of liver inflammation, which needs to be validated in humans in
a translational approach. Cathepsin-D is a plasma lysosomal enzyme that can be
used as a novel non-invasive marker to predict the presence or absence of liver
inflammation. The sensitivity of cathepsin-D as a marker to follow the efficacy
of an intervention needs however further study. In the current proposed
proof-of-concept study, the effect of consuming plant sterol or plant stanol
esters on biopsy proven liver inflammation will be investigated in NAFLD
patients. In addition, the use of cathepsin-D concentrations as a non-invasive
marker of liver inflammation will be investigated in NAFLD patients.

To assess the effect of consuming plant sterol or plant stanol esters (3
grams/day) for 12 months on biopsy proven liver inflammation in NAFLD patients.

This study is a randomized, placebo-controlled pilot study with a run-period of
2 weeks, an intervention period of 12 months and a wash-out period of 1 month.

All subjects will start a run-in period of two weeks during which they consume
daily 20 grams of control margarine after which they will be randomly allocated
to consume 20 grams control margarine or plant sterol or plant stanol enriched
margarine on a daily basis for a period of 12 months.

During a screening visit, body weight, body height and blood pressure are
determined, an electrocardiogram will be obtained and a blood sample (2 mL)
will be drawn. During the run-in period of two weeks, subjects will receive 20g
control margarine and during the intervention period of 12 months they will
receive at random, control, plant sterol ester or plant stanol ester margarine.
On 11 occasions a fasting blood sample will be drawn (with a total of 354 mL)
and exhaled breath will be sampled for VOC s analysis. In addition, at baseline
(week 2), in week 26 and 52, subjects will participate in a two-step
hyperinsulinemic-euglycemic clamp (417 mL blood sample) including a ventilated
hood measurement for indirect calorimetry and taking muscle biopsies, and we
will use MRS imaging and a FibroScan to measure liver fat, liver inflammation
and liver fibrosis and samples for VOCs analysis will be taken. Finally,
subjects will undergo a second liver biopsy in week 52. All subjects will be
asked to fill out a food frequency questionnaire and a physical activity
questionnaire three times and to keep a diary throughout the study and body
weight and blood pressure will be assessed on five occasions.
Plant sterol and plant stanol enriched products are commercially available and
we therefore do not foresee any risks related to the consumption of these food
products. Venipuncture and insertion of a cannula can cause discomfort and
possibly a local haematoma or bruise. There is a minor chance of a hypoglycemic
reaction during the hyperinsulinemic-euglycemic clamp. Indirect calorimetry
might evoke claustrophobic reactions, but there are no physical risks involved.
No ionizing radiation is used for the MRI/MRS scans. If an unexpected medical
condition is revealed, which the radiologist considers potentially relevant for
the subjects* health, the subject will be informed and the treating physician
of the subject will also be informed.If Sampling skeletal muscle tissue
biopsies is performed under local anaesthesia and muscle pain can occur due to
invasive method for taking muscle tissue biopsy. In principle, all measurements
are routine in our metabolic research unit (MRUM) and are not expected to lead
to physical side effects. A liver biopsy is a safe test. Complications may
occur in some cases (less than 5%). The complications that can occur are
bleeding in the liver, pain in the right shoulder or in the right upper abdomen
or a bleeding of the skin.Total time investment spread-out over the one-year
study participation will be approximately 45 hours, excluding travel time.