QUANTIFICATION OF CHOLESTEROL FLUXES IN HUMANS: Assessment of hepatic cholesterol excretion, intestinal absorption and excretion and faecal sterol loss using quadruple stable isotope technique (STORC EXTRA)

Dylipidemia is one of the most important risk factors for cardiovascular
disease. Roughly, the cholesterol that is present in the human body can be
divided into 'good' HDL-cholesterol (HDL-C) and 'bad' LDL-cholesterol (LDL-C).
In order to increase cardiovascular disease prevention, plasma levels of the
bad LDL-C should remain low, whereas the good HDL-C should be as high as
possible. The anti-atherogenicity of HDL-C is mainly attributed to its pivotal
role within the reverse cholesterol transport (RCT) process. This is the
transport of excess cholesterol from the periphery (including the arterial
wall) to the liver for excretion into the faeces out of the body. We have
recently found that this net cholesterol excretion is significantly diminished
in patients with hereditary decreased HDL-C levels.
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, recent evidence in mice suggests that the
intestine is also an important secretory organ for cholesterol. In fact, direct
trans-intestinal cholesterol excretion (TICE) accounted for 33% of total fecal
sterol excretion in mice. It is unknown whether TICE is present in humans.
However, sterols of non-dietary origin were found in fecal samples of patients
with biliary obstruction, suggesting that TICE is present in these patients.
TICE might be a novel therapeutic target for cholesterol excretion and
cholesterol-lowering, as evidence in mice suggests that TICE can be stimulated
by LXR-agonists. The latter are novel cholesterol lowering compounds which will
soon become available for use in humans.
To date, no technique has been described to reliably estimate cholesterol
homeostasis in humans. The latter requires simultaneous assessment of reverse
cholesterol transport, cholesterol absorption, biliary cholesterol excretion
and, as final common pathway, faecal sterol excretion. Daily biliary
cholesterol output is the most difficult flux to assess non-invasively in
humans. The purpose of the current study is to optimize a method in humans for
combined assessment of the above mentioned cholesterol fluxes, in one single
protocol by use of cholesterol and bile acid kinetic tracers. Moreover, this
protocol will enable us to quantify TICE for the first time in humans. If
valid, this experimental protocol can be used to estimate net-cholesterol
efflux in patients following administration of novel pharmacological
interventions, such as LXR-agonists.

To estimate reference values for reverse cholesterol transport, cholesterol
absorption, hepatic cholesterol excretion and direct trans-intestinal
cholesterol excretion in humans.

The design of the study is cross-sectional cohort study, which comprises a
single measurement of cholesterol fluxes in a period of 15 days.
Visit 1 (day -7, morning fasting state)
At the screening visit written informed consent will be obtained. Subsequently,
a blood sample will be collected for the measurement of lipoprotein levels and
safety parameters, such as liver and renal function. Additionally, a physical
examination will be performed for a general health evaluation. In case subjects
are eligible, they will start with a cholesterol-restricted diet, which they
will monitor in a food diary until the end of study. In addition, subjects
receive a box with 3mg 2H4-sitostanol capsules, which subjects will start using
3 times daily from day -2 for a period of 8 days.
Visit 2 (day 0, 06.00pm)
Subjects return to the AMC and ingest a standardized mealt, together with a
single bolus of two bile acid tracers (dissolved in fruit juice), which are
used to measure daily biliary output rate and enterohepatic cycling of
cholesterol and bile acids.
Visit 3 (day 1, 09.00am)
The next day two cholesterol isotopes will be administered. A plastic
intravenous (IV) catheter will be placed in the antecubital fossa of the
forearm, which will be used for a blood draw. Subsequently, this same catheter
will be used for the infusion of a single dose of 50mg 13C2-cholesterol.
Immediately thereafter, subjects will have a standardized breakfast, with which
they will ingest a capsule containing 50mg of another cholesterol-marker
(2H7-cholesterol). One and four hours after this meal, blood will be drawn and
subjects return to their homes. At 06.00pm they return to the AMC for an
additional blood draw. Finally, subjects receive the encapsulated
Enterotest-thread which will be used for the bile sampling the next day, as
described in section E6. Subjects will ingest this capsule before going to bed
and the upper end of the thread is taped to the corner of the mouth.
Visit 4 (day 2, 08.00am-08.00pm)
This studyvisit comprises a 12-hour admission to the hospital ward for a single
bile sample and in order to study bile acid and cholesterol kinetics. Again, a
plastic intravenous (IV) catheter will be placed in the antecubital fossa of
the forearm. This will be used to administer IV bolus of 0.05*g/kg
cholecystokinin (Cerulotide, Takus®), which will induce gallbladder
contraction. One hour thereafter, the Enterotest-thread is withdrawn. The
catheter is then used for frequent bloodsampling (1ml every 30min) for the
rest of the day. Starting from this visit, subjects will be asked to collect
daily faecal samples, using a special specimen collection system, until the end
of the study (day 8). At 08.00pm subjects return to their homes, where they
continue to use the oral tracer capsules, keep the food diary and collect the
faecal samples until day 8.
Visit 5 and 6 (day 3 and 4, 09.00am and 06.00pm)
Subjects will return twice to the AMC at day 3 and day 4 for a blood sample.
Visit 7 (day 8, morning fasting state)
At day 8, a blood sample is obtained and the diaries, faecal samples and
remaining capsules will be collected at the subjects* homes.

At screening a blood sample will be obtained, as well as a medical history,
aimed at dietary habits and cardiovascular riskfactors, combined with a general
physical examination. In addition, subjects are asked to ingest a single
solution of stable bile acid isotopes (dissolved in fruit juice), a single
capsule of 50mg 2H7-cholesterol and to maintain a cholesterol restricted diet
during the subsequent eight days, together with the use of 3mg sitostanol
capsules three times daily.

In addition, subjects are asked to visit the AMC for frequent bloodsampling,
including once during a 12-hour admission period. Other invasive actions
include the administration of an iv dose of cholesterol tracer
(13C2-cholesterol), the iv administration of cholecystokinin for the bile
sample to be obtained. Finally, participants are asked to collect daily faecal
samples in a special collection system for a period of 7 days.

Hardly any health risks are involved in this study. Stable isotopes of
cholesterol, sitostanol and bile acids behave like their natural substrates and
therefore carry no health risks. The dose of isotopes administered to the
subjects is small compared to the amount already present in the body. A
hematoma can occur at the site of venepuncture. Ethanol can cause burning at
the site of infusion, which usually disappears quickly. Administration of
Takus® can cause slight transient nausea. In rare cases, the synthetic
cholecystokinin (Takus®) can induce an allergic reaction. Withdrawal of the
Enterotest® during bile sampling can cause transient nausea as well.