2. Subjects and methods
2
.
1
. Subjects Seventy-nine postmenopausal caucasian women seek-
ing hormone substitution therapy for climacteric symp- toms volunteered to participate in the metabolic
studies. Only hysterectomized women were included, because our subsequent study focused on unopposed
estrogen replacement therapy. Written informed con- sent was obtained from all the subjects, and the study
was approved by the Ethical Committees of the two hospitals involved.
Seventy-nine women in the age range 45 – 65 years were recruited. A previous hysterectomy with at least
one remaining ovary, serum follicle-stimulating hor- mone FSH over 30 I.U.l, fasting blood glucose less
than 6.7 mmoll and body mass index BMI less than 30 kgm
2
were used as inclusion criteria. None of the subjects were on any lipid-lowering drugs or had any
clinical evidence of familial hypercholesterolemia. The latter possibility was also excluded by determining the
two most common LDL receptor gene mutations in Finland among the subjects whose total cholesterol was
over 7.0 mmoll. None of the women were on post- menopausal hormone therapy.
Smoking habits, alcohol consumption and physical activity were documented using a standardized ques-
tionnaire. Smoking was presented as the number of cigarettes smoked per day and alcohol consumption as
grams of absolute alcohol consumed in a week. To estimate physical activity, both occupational and leisure
time activities were documented by an interview and scored on a four-point scale, where one indicated a
sedentary and four a highly active lifestyle. Sixteen participants were current smokers, and 63 subjects were
ex-smokers or non-smokers. The mean alcohol intake of the subjects reporting some alcohol consumption was
26 gweek. Eighteen subjects were teetotalers. All the subjects continued their regular home diet and custom-
ary physical activities during the study period.
2
.
2
. Clinical characterization and hormone analyses Height, weight, BMI weight kgheight
2
m
2
and waist and hip circumference were determined. Serum
FSH was measured by the immunofluorometric method and serum estrone and estradiol by radioimmunogical
assays in the laboratory of the Oulu Deaconess Institute.
2
.
3
. Lipid and lipoprotein analyses Blood samples for assays of cholesterol metabolism
were drawn into EDTA-containing tubes between 07:00 and 09:00 after an overnight fast. The plasma was
separated by centrifugation at 1200 × g 2600 rpm for 15 min + 4°C. Total plasma cholesterol and triglyce-
rides were determined by enzymatic colorimetric meth- ods kits from Boehringer Diagnostica, Mannheim,
Germany. Very low density lipoprotein VLDL, inter- mediate density lipoprotein IDL, LDL, HDL2 and
HDL3 cholesterol were isolated by repeated ultracen- trifugations according to their densities [10]. HDL
cholesterol was determined from VLDL-free plasma after precipitation of low density lipoproteins with hep-
arin-manganese. LDL cholesterol was also calculated by the Friedewald formula [11], and these values, which
turned out similar to the values obtained after ultracen- trifugation, were used in the analyses. For the lipid and
lipoprotein analyses, blood samples were taken at the first visit and three times during the subsequent LDL
turnover study. The mean value of the four measure- ments was used in the final analyses. The protein
content of the lipoproteins was measured by the method of Lowry et al. [12] and the plasma LDL apo B
level was determined with isopropanol precipitation [13]. Plasma total apo A1 and B concentrations were
determined by immunoprecipitation methods. Plasma lipoprotein a Lpa concentration was measured
with a solid-phase two-site immunoradiometric assay Pharmacia Diagnostica, Uppsala, Sweden. The coeffi-
cients of variation within and between assays were 5.0 and 6.6, respectively.
2
.
4
. Clearance and production of LDL apo B Seventy-two women volunteered to participate in the
LDL turnover study, which was carried out as previ- ously described [14,15]. Briefly, 100 ml of fasting blood
was drawn for the isolation of LDL d = 1.019 – 1.060 gml. LDL protein was labeled with
125
I by the iodine monochloride method of McFarlane [16] as modified
by Bilheimer et al. [17]. Radiolabeled LDL was injected in the morning and blood samples were collected at 0,
15 and 30 min and 1, 2 and 3 h and thereafter three times a week for 14 days after the injection. The
radioactivity of total plasma was measured from each sample. The fractional catabolic rate FCR was calcu-
lated from the plasma decay curves using the Matthews method as previously described [15,18]. The production
rate of LDL apo B was calculated from the FCR of LDL, pool volume and LDL apo B concentration, and
expressed as milligrams of LDL apo B produced per day per kilogram of body weight.
2
.
5
. Cholesterol absorption Cholesterol absorption efficiency was measured in 76
subjects by the peroral double-isotope continuous-feed- ing method described by Crouse and Grundy [19]. The
subjects received [
14
C]cholesterol and [
3
H]b-sitosterol
or [
3
H]b-sitostanol three times a day with the major meals for 7 days, keeping a dietary record during the
same time. Stool collections were performed on the last 3 days. The dietary records were analysed by a dietician
using the Finnish Food Database Program, Nutrica [20]. Absolute cholesterol absorption was calculated by
multiplying the daily dietary cholesterol intake with the cholesterol absorption percentage and expressed as mil-
ligrams per kilogram of body weight.
2
.
6
. Determination of the apo E phenotype and polymorphisms of the apo B and
7
a -hydroxylase genes
The apo E phenotype was determined after delipida- tion with isoelectric focusing and immunoblotting tech-
niques [21,22] using commercial antibodies. The EcoRI and XbaI polymorphisms of the apo B gene were
determined by the PCR method as previously described [23]. The polymorphism of the gene encoding 7a-hy-
droxylase CYP7 was also determined by PCR [24].
2
.
7
. Statistical analysis Power analysis was initially calculated for the estro-
gen replacement study. In the retrospective power cal- culation the power of the study was 95 to find the
difference in LDL cholesterol levels, FCR for LDL apo B and the LDL apo B production rate between high
and low LDL groups. Twenty-four subjects turned out to be sufficient to show the observed differences in
LDL cholesterol a = b = 0.05, 48 subjects to show the observed differences in the FCR values for LDL apo B
and 64 subjects in the production of LDL apo B a = b = 0.05. However, for the polymorphism studies
the power of our study design was lower.
The data analyses were performed with the Statistical Package for Social Sciences SPSSPC + statistical soft-
ware. The results for continuous variables are pre- sented as mean 9 S.D. of the mean. Before the
statistical analyses, natural logarithmic transformations were performed for plasma triglycerides, Lpa and the
values of absolute cholesterol absorption because of the skewed distribution.
Because the main objective was to elucidate the regu- lation of the LDL cholesterol level, the study popula-
tion was divided into two groups according to the LDL cholesterol concentration. The cut point to high and
low LDL cholesterol was 4.05 mmoll. The effect of obesity was estimated by comparing subjects having
BMI equal or higher than 26 with those whose BMI was lower than 26. The significance of the differences
between the two groups was tested by Student’s two- tailed t-test. The mean differences and their 95 confi-
dence intervals are presented in the Tables 1 and 3. Pearson’s correlation coefficients were calculated to in-
dicate the relationships between lipids, lipoproteins, diet, cholesterol absorption and LDL kinetic parame-
ters. A one-way ANOVA test was used to compare the effects of apo E and B and 7a-hydroxylase polymor-
phisms. P B 0.05 was considered to indicate statistical significance.
3. Results
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