Validation of plasma plant sterol levels as markers of cholesterol absorption

Intestinal cholesterol absorption varies considerably in the general
population, ranging between 20 and 80%. Previous studies have suggested a
classification of subjects with high or low cholesterol absorption. In omost
studies, levels of non-cholesterol sterols have been used as markers of
cholesterol absorption and synthesis respectively. Bssed on these markers, a
classification of subjects with high or low cholesterol absorption, the
so-called high and low absorbers, has been suggested. The high absorbers are
thought to have elevated cholesterol levels due to high absorption, whereas the
low absorbers have elevated levels based on high synthesis. Subsequently, it
has been suggested that high absorbers do not or hardly benefit from statin
treatment alone, either with respect to cholesterol reduction and the
recurrence of coronary hear disease. Therefore, the high absorbers may benefit
form the addition of cholesterol absorption inhibitors.

This underscores the need to identify high absorbers in order to treat them
accurately. Therefore, easy accessible markers are essential in clinical
practice. However, whether high and low absorbers indeed can be identified
based on plasma levels of non-cholesterol sterols has bever been verified by
means of actual cholesterol absoption 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.

In a previous study, we investigated the actual cholesterol absorption rates by
means of stable isotope methods, in 12 mildly hypercholesterolemic subjects,
who were predefined as high or low absorbers, based on their rplasma
campesterol/TC ratios (Dhalia-2 study). We expected to find a difference of 20%
between the so-called high and low absorbers. Although we found a broad range
of cholesterol absorption (12-98%), to our surprise, cholesterol absorption
rates did not correlate with plasma campesterol/TC ratios in these 12 subjects.
This implies that plasma campesterol/TC ratios are not valid markers of
cholesterol absorption, in contrast to what was previously suggested based on
only 80 subjects and which has been used throughout literature ever since.
Another explanation might be that the Dahlia-2 study was underpowered to show
the expected results. Therefore, we will expand our study population by
investing cholesterol absorption rates in a larger population of 80 mildly
hypercholesterolemic subjects. Data of the present Dahlia-2a study will be
combined with those of the previous Dahlia-2 study.

What is the difference in fractional cholesterol absorption, measured by means
of the dual isotope method, between mildly hypercholesterolemic subjects with
high plasma campesterol/TC ratios compared to subjects with low plasma
campesterol/TC ratios?

This is a cross-sectional single measurement of cholesterol absorption, by
means of the dual isotope method in 34 subjects with high campesterol/TC ratios
and 34 subjects with low campesterol/TC ratios. These results will be combined
with previous results of the Dahlia-2 study in 12 hypercholesterolemic
subjects.

First, subjects will attend a screening visit, consisting of a medical history,
physical examination and blood sampling to determine lipid profile. At the
second study visit, the actual cholesterol absorption measurement will start,
consisting of a blood sample with ensuing single intravenous administration of
13C2 cholesterol and an oral administration of 2H7 cholesterol via a
stardardized breakfast. Subjects will return to their homes and will attend the
AMC in the subsequent three mornings for additional blood sampling. Percent
intestinal cholesterol absorption will be calculated by dividing the plasma
ratio of 13C2 and 2H7 enrichment by the ratio of 13C2 and 2H7 cholesterol
species administered to the subjects.

To rule out that the method of cholesterol absorption measurement might be of
influence on our study question, we will repeat a cholesterol absorption
measurement in 20 subjects according to a diffent method. These subjects will
attend the AMC for an additional measurement, in which cholesterol absorption
will be measured in fecal samples instead of in blood samples.

This substudy will take a week, in which participants will attend the AMC twice
(day ) and 8). On Day 0, subjects will start taking two sets of capsules three
times daily for a period of 7 days. These capsules contain 3mg 2H7 cholesterol
and 3mg 2H4 sitostanol respectively. During the last 4 days, subjects will be
asked to collect daily stool samples. Percent absorption is calculated from the
fecal ratio of 2H7 cholesterol and 2H4 sitostanol, divided by their ratio which
was administered to the subjects.

Hardly any risks are involved in this study. At screening, a single blood
sample will be obtained. At the second visit, an intravenous catheter will be
inserted, followed by four blood drawings, during the following three days.
This may lead to a hematoma at the site of venepuncture. Furthermore, two kinds
of cholesterol markers will be administered to the study subjects, both orally
and intravenously. These so-called stable isotopes are not harmful, as they
behave as their natural substrates. This also applies to the oral sitostanol
which is used in the substudy. In this substudy, no blood samples are obtained,
however, subjects are asked to collect stool samples for a period of 4 days.
Finally, we do not expect any unfavorable effects of discontinuation of any
possible cholesterol lowering medication, neither from a possible 8-week
cessation of fish oil or fibrates.