The effects of plant stanol esters on intestinal mucosal gene expression profiles and microbiota composition in healthy human subjects.

Plant sterols and stanols are components that are naturally present in plants.
Their biological function in plants is comparable with these of cholesterol in
animals. They are structurally related to cholesterol, but are absorbed by
enterocytes to a much lesser extent. It is generally accepted that they inhibit
intestinal cholesterol absorption and consequently lower serum low-density
lipoprotein (LDL) cholesterol concentrations up to 10% at daily intakes of 2.5
g. The exact underlying mechanism of the plant sterol/stanol mediated reduction
in intestinal cholesterol absorption is still unknown. It has been suggested
that they lower the activity of sterol uptake transporters like Niemann-Pick C1
like 1 protein (NPC1L1) in enterocytes, otherwise several studies indicated
that these compounds could activate the liver X receptor (LXR) in enterocytes,
thereby activating the ABC transporters involved in the intestinal cholesterol
metabolism, whereas recently suggestions have been made that plant sterols and
stanols activate transintestinal cholesterol excretion (TICE). This is the
direct cholesterol secretion from the blood into the intestinal lumen, in which
the enterocytes play a central role. None of these assumptions have so far been
evaluated in humans.

The major objective of the present study is to examine the acute effects of
dietary plant stanol esters on the intestinal mucosal gene expression profiles
in intestinal biopsies in healthy volunteers. Two minor objectives are (1) to
investigate whether semi-long-term use (3 weeks) of plant stanol esters have an
effect on microbiota composition.

If the volunteers agree, we will take 2 extra jejunal biopsies. These will then
be used as a control group to examine the expression of nutrient receptors.

A randomized, double-blind, placebo-controlled cross-over design. The total
study duration will be 12 weeks, consisting of 2 test periods of 4 weeks in
which the volunteers will use a plant sterol/stanol poor diet for 1 week
followed by the consumption of the investigational products for 3 weeks.
Directly after the plant sterol/stanol poor diet period, at day one of the 3
weeks intervention period all volunteers will participate in a postprandial
test. In this test volunteers will consume a meal with or without added plant
stanol esters. Five hours after the breakfast meal with or without plant stanol
esters, we will take a small intestinal biopsy to determine the acute changes
in intestinal mucosal gene expression profiles. The day before the beginning of
the plant sterol/stanol poor diet as well as during the last two days of the 3
week plant stanol ester or placebo consumption, we will sample stool for
microbiota analysis.

During each test period, the volunteers will use a plant sterol/stanol poor
diet for 1 week followed by the consumption of the investigational products
(margarines with or without added plant stanol esters (3 g/day) for 3 weeks.
During the 4-week wash-out period, they will return to their normal eating
habits. For the postprandial tests at the start of the two experimental periods
(week 2 and 10), the subjects will consume a high fat breakfast enriched with
or without 4.0 gram plant stanols. Total follow-up during the postprandial
period is 5.5 hours.

Before the start of the study, subjects will be screened to determine
eligibility. The study itself includes two times 1 postprandial test day;
duration of both days will be approximately 5.5 hours. On both days, subjects
may not have any food or drinks other than water. During the postprandial test
day, 8 blood samples and 8 intestinal biopsies will be taken, i.e. 4 duodenal
and 4 in proximal jejunum.
Fasting blood samples will be drawn on 8 different occasions in a time frame of
12 weeks with a total amount of 139 mL. During the screening procedure, 2 mL
blood will be sampled. Furthermore, subjects will be asked to fill out a food
frequency questionnaire three times at the end of both experimental periods.
Blood samples might cause bruises or haematoma. Obtaining small intestinal
biopsies by standard flexible gastroscopy induces local discomfort in the
pharynx only during the procedure, which takes about 15 minutes, and causes a
theoretical risk for perforations (1:1000) and an infection risk of
1:1.800.000. Healthy subjects will have a lower risk.