Validation of cholesterol absorption as measured by the dual isotope method

Intestinal cholesterol absorption varies considerably in the general
population. Previous studies have suggested a classification of subjects with
high or low cholesterol absorption, the so-called *high and low absorbers*. The
high absorbers are thought to have elevated cholesterol levels due to high
absorption. On the other hand, low absorbers have elevated levels based on high
synthesis, which suggests a negative association between absorption and
synthesis. In most studies, levels of plant sterols have been used as markers
for cholesterol absorption. However, whether high and low absorbers indeed can
be identified based on plasma plant sterol levels has never been directly
verified by means of actual cholesterol absorption measurements. Besides the
fact that the validity of these markers may be questionable, they also do not
provide any indication regarding the quantity of cholesterol that is absorbed.

To this end, we measured actual cholesterol absorption rates by means of the
dual isotope method in 80 mildly hypercholesterolaemic subjects, who were
predefined as high or low absorbers based on their plasma campesterol/TC ratios
(Dahlia-2a study, METC 09/204). To our surprise, cholesterol absorption rates
did not correlate with plasma campesterol/TC ratios in these 80 subjects. This
implies that plasma campesterol/TC ratios are not valid markers of cholesterol
absorption, in contrast to what was previously suggested and which has been
used throughout literature ever since. However, absorption rates varied between
5% and 46% in our study, which is considerably lower than the overall range of
20-70% described in literature. This was not due to technical differences in
the measurement of the samples. A possible explanation might be the method of
administration of the oral D7-cholesterol tracer. In order for cholesterol to
be absorbed, it needs to be incorporated into so-called micelles; particles
consisting of bile salts (BS) and phospholipids (PL), with a hydrophilic outer
layer and hydrophobic centre. BS and PL are secreted by the liver into the bile
and induce formation of these micelles. Our standard breakfast might not have
been fatty enough to allow for the maximum secretion of bile salts and
phospholipids, which may have affected the absorption of the D7-cholesterol. In
addition, in certain cholesterol absorption studies, the oral cholesterol
tracers are dissolved in oil prior to administration, in order to promote
micelle formation. Hence, the lower absorption rates measured in our previous
study might be explained by a lower absorption of the oral D7-cholesterol
tracer. Therefore, we will investigate whether the method of D7-cholesterol
administration might explain our findings.

To study whether the lower cholesterol absorption rates of the Dahlia-2a study
were due to lower bioavailability of the oral D7-cholesterol tracer.

This is a cross-over study, in which 7 mildly hypercholesterolaemic subjects,
who previously participated in the Dahlia-2a study, will undergo three
measurements of plasma D7-cholesterol enrichment after three different methods
of oral D7-cholesterol administration. These methods include: 1. Standard
breakfast as in Dahlia-2a study + capsule containing 50mg D7 cholesterol in
powder; 2. Standard breakfast + 30g margarine + capsule containing 50mg D7
cholesterol in powder; 3. Standard breakfast + capsule containing 50mg
D7-cholesterol dissolved in sunfloweroil. The three measurements will be
performed in a random order and will take 4 days each, with 4-week
time-intervals between measurements.

At the first study visit, subjects will attend the AMC in the morning fasting
state. We will explain the procedure and informed consent will be obtained.
Subsequently, a blood sample will be collected for the measurement of lipid and
lipoprotein levels. Additionally, a physical examination will be performed. In
case subjects are still eligible based on the in- and exclusion criteria, a
second study visit will be planned one week after study visit 1.
At the second study visit, subjects will visit the AMC in the morning fasting
state. The first blood sample will be collected, after which participants will
ingest 50mg of D7 cholesterol in one of the three forms, as described above.
After breakfast, subjects return to their homes. Additional blood samples will
be collected at 24h, 48h and 72h after infusion. To this end, subjects will
return to the AMC in the morning fasting state in the three days after
ingestion of the D7 cholesterol. After a wash-out period of four weeks,
subjects return to the AMC for the same procedures during four days and for the
last measurement, after another period of 4 weeks.

Hardly any risks are involved in this study. At screening, a single blood
sample will be obtained. At the second visit this will be repeated, followed by
three additional blood drawings in the following three days. These blood
drawings will be repeated twice, with 4-week time intervals. The venepunctures
can cause a hematoma at the site of puncture.
Furthermore, study subjects will ingest 50mg of D7-cholesterol three times
throughout the study. This a so-called stable cholesterol isotope and is not
harmful, since it behaves as its natural substrate.