Quantification of trans-intestinal cholesterol excretion in patients with familial hypobetalipoproteinemia

Reverse cholesterol transport (RCT) is an important anti-atherogenic mechanism,
as it mediates the elimination of excess cholesterol out of the body into the
faeces. Recently, the concept of RCT has been revisited. In the *classical*
concept, the liver is considered to be the only organ capable to eliminate
cholesterol via excretion into the bile. However, evidence from animal models
suggests that the intestine is also an important secretory organ for
cholesterol 1-4. In fact, direct trans-intestinal cholesterol excretion (TICE)
accounted for 33% of total faecal sterol excretion in mice 4 and activation of
the Liver X Receptor (LXR) has been found to induce a 6-fold increase in
trans-intestinal cholesterol secretion in mice 4. This suggests that TICE might
be a novel therapeutic target for cholesterol excretion and thereby for
cardiovascular disease prevention.
Previous human perfusion studies suggested that secretion of cholesterol in the
small intestine also occurs in humans 5. In order to establish the existence of
such a direct intestinal cholesterol-excreting pathway, we studied the
contributions of various cholesterol fluxes to total fecal sterol elimination
in healthy humans and found that approximately one third of total fecal sterol
excretion in healthy mildly hypercholesterolemic men was eliminated via TICE
(Storc-Extra study, METC 10/092).
The next step in elucidating the mechanisms involved in human trans-intestinal
cholesterol excretion is the evaluation of differences in TICE across
populations. From animal studies it can be hypothesized that triglyceride-rich
particles in the VLDL-range are likely to be the so-called *donor particles*
for TICE 4,6. These lipoproteins are thought to deliver the plasma-derived
cholesterol to the enterocyte for elimination via TICE. Individuals lacking
such particles would exhibit impaired TICE as compared to healthy unaffected
controls. Therefore, we propose to investigate the role of VLDL-particles as a
determinant of human TICE by studying the contribution of TICE to fecal sterol
loss in patients with familial hypobeta-lipoproteinemia (FHBL).
Familial hypobetalipoproteinemia (FHBL) is a rare disorder of lipoprotein
metabolism (estimated prevalence of 1 in 500 - 1 in 1000) and is characterized
by LDL-cholesterol and total apolipoprotein B (apoB) levels below the 5th
percentile 7,8. Approximately 50% of FHBL subjects are carriers of a mutation
in the apoB gene7 leading to the formation of a dysfunctional form of apoB.
Since apoB is the main component of VLDL, mutations in the apoB gene give rise
to a defective VLDL-export system. As a consequence, plasma VLDL-concentrations
are extremely low in these patients. In case blood-derived cholesterol destined
for elimination via TICE, is truly mediated via VLDL-particles, patients with
FHBL should exhibit a significant reduction in TICE, as compared to unaffected
age, gender and BMI-matched controls.

The purpose of the current study is to evaluate the difference in TICE in
FHBL-patients as compared to unaffected controls, according to the previously
developed stable isotope method with minor modifications (METC 10/092). This
way we can prove the hypothesis dat VLDL is the donor particle for TICE in
humans.

It is a cross-sectional cohort study. Both patients with FHBL as healthy
controls will be recruited. The study starts with a screening visit

Day -7, AMC, morning: obtaining informed consent; fasting blood sample
(lipoproteins, liver-, renal- function); physical examination

Day -2, at home: start taking sitostanol capsules (1 capsule per meal, 3 meals
per day); start cholesterol-restricted diet; start dietary record; collecting
feces sample as control sample

Day 0, AMC, 09.00u: baseline blood sample withdrawal

Day 0, AMC, 06.00pm: start study; standardized meal with 2 hours hereafter
ingestion of two bile acid isotopes

Day 1, AMC, 09.00am: insertion of venous catheter and fasting blood sample
(bile acid isotopes); admission of intravenous cholesterol isotope; ingestion
of oral cholesterol isotope togetehr with breakfast; two hours after breakfast
blood sample (bile acid isotopes); start collecting feces samples
Day 1, AMC, 06.00pm: fasting blood sample (cholesterol isotopes); standardized
meal; 2 hours hereafter blood sample (bile acid isotopes); providing enterotest
(to be taken in before going to sleep)

Day 2, AMC, 08.00am: admission in the hospital; fasting blood sample (bile acid
isotopes) every 30 minutes until 01.00pm; administration of 0,05ug/kg
cholecystokinin intravenously to contract gallbladder at 08.00am; 1 hour
hereafter withdrawal of enterotest and analysis of enrichment with bile acids;
standardized lunch at 01.00pm; blood sample (bile acid isotopes) at 03.00pm

Day 3, AMC, 09.00am: fasting blood sample (cholesterol isotopes); hereafter
standardized breakfast and blood sample again at 11.00am (bile acid isotopes)
Day 3, AMC, 06.00pm: fasting blood sample (cholesterol isotopes); hereafter
standardized meal and blood sample again at 08.00pm (bile acid isotopes)

Day 4: identical to day 3

Day 6, AMC, 09.00am: fasting blood sample (cholesterolisotopes)

Day 8: identical to day 6

Day 9: last day of ingesting sitostanol capsules

Day 10: last day of cholesterol-restricted diet, recording diet and collecting
feces

Day 11, at home: collecting feces samples, dietary records etc. End of study

The total study will take place within a period of two weeks time. It will
include 11 visits to the hospital and one visit at home. This will be preceded
by a screening visit to the hospital. Total duration of all visits is
approximately 25 hours. During the study, participants will need to follow a
cholesterol-restricted diet, keep a dietary record and collect their feces
samples.

Administration of the three isotopes expectedly will not have any side effects,
as the isotopes behave like their natural (endogenous) counterparts, without
causing harm to their 'hosts'. Infusion of cholecystokinin can cause mild
transient nausea or abdominal discomfort. In rare cases, it can lead to an
allergic reaction.

We don't expect any AEs or SAEs