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
3
.
1
. Subject characteristics The subjects’ ages ranged from 48 to 63 years, with a
mean of 54.2 9 2.9 mean 9 S.D. years. The mean BMI was 26.0 9 2.5 kgm
2
and the average waist-to-hip ratio was 0.80 9 0.05. The subjects had been postmenopausal
on an average for 5.5 9 3.2 years. The high FSH levels 65 9 22 I.U.l and the low values for estradiol 95 9
150 pmoll and estrone 198 9 116 pmoll verified the menopause in all the subjects.
3
.
2
. Plasma lipids and lipoproteins and LDL metabolism
Among all the subjects the mean plasma total choles- terol was 6.26 mmoll range, 3.9 – 8.80 mmoll, LDL
cholesterol 4.09 mmoll 2.20 – 6.15 mmoll, HDL cholesterol 1.58 mmoll 0.85 – 2.56 mmoll, and total
triglycerides 1.31 mmoll 0.61 – 3.26 mmoll. The FCR values for LDL apo B ranged from 0.183 to 0.418
poolsday with a mean value of 0.300 poolsday, and the LDL apo B production rates ranged from 7.3 to
20.2 mgkg per day with a mean value of 12.5 mgkg per day. Cholesterol absorption efficiency varied among
the subjects from 16.1 to 82.6 with an average value of 50.4, and the mean absolute absorption of choles-
terol was 2.0 mgkg per day, ranging from 0.4 to 4.9 mgkg per day.
In the high LDL cholesterol group the mean LDL cholesterol was 4.76 9 0.58 mmoll compared with
3.41 9 0.51 mmoll in the low cholesterol group Table 1. BMI, total, VLDL and IDL cholesterol, triglyce-
rides, total and LDL apo B and Lpa were higher, whereas HDL and HDL2, but not HDL3, cholesterol
were lower, in the high LDL cholesterol group com- pared with the low LDL cholesterol subjects Table 1.
Also, the production of LDL apo B was significantly higher and the FCR value for LDL apo B lower in the
high LDL cholesterol group than in the low LDL cholesterol subjects. No differences were noticed in the
fractional and absolute absorption of cholesterol be- tween the high and low cholesterol groups.
Overall, the fractional catabolic rate FCR for LDL apo B was strongly negatively associated with the total
and LDL cholesterol levels and less markedly with VLDL and IDL cholesterol values Table 2. The LDL
apo B production rate was also associated with all the
measured lipid values. Plasma triglycerides and VLDL cholesterol, but not LDL or HDL cholesterol values,
were negatively associated with fractional cholesterol absorption Table 2. No relationship was observed
between absolute cholesterol absorption and any lipid or lipoprotein value data not shown.
In this study population the lipid and lipoprotein levels were not significantly associated with smoking,
alcohol consumption or physical activity, except the concentrations of HDL cholesterol which correlated
with the amount of alcohol consumption r = 0.312, P B 0.01. The dietary intakes and habits for smoking,
Table 1 BMI, plasma lipids, lipoproteins, cholesterol absorption and FCR and production rate values for LDL apo B among subjects with low and high
LDL cholesterol
a
Mean difference 95 CI of difference LDL\4.05
LDLB4.05 n = 40
n = 39 54.1 2.9
54.3 3.0 −
0.2 −1.5;1.1 Age years
BMI kgm
2
− 1.2 −2.4;−0.1
26.7 2.4 25.4 2.5
69.9 7.0 −
2.7 −6.0;0.6 67.2 7.8
Weight kg −
0.02 −0.04;0.01 Waist-to-hip ratio
0.81 0.05 0.79 0.05
Plasma cholesterol mmoll Total
− 1.32 −1.63;−1.01
6.91 0.70 5.59 0.69
0.31 0.16 0.53 0.28
VLDL −
0.21 −0.31;−0.11 0.31 0.13
− 0.13 −0.18;−0.08
0.19 0.09 IDL
− 1.34 −1.59;−1.10
3.41 0.51 LDL
4.76 0.58 1.68 0.37
HDL 1.47 0.32
0.21 0.06;0.37 0.88 0.35
HDL2 1.16 0.45
0.29 0.11;0.47 −
0.01 −0.06;0.04 HDL3
0.57 0.11 0.56 0.13
Plasma triglycerides mmoll −
0.43 −0.66;−0.21 1.53 0.63
Total 1.10 0.30
0.47 0.20 0.70 0.40
− 0.23 −0.37;−0.09
VLDL 0.08 −0.09;0.25
1.96 0.38 Plasma apo A1 gl
1.88 0.38 −
0.35 −0.46;−0.24 0.91 0.20
Plasma apo B gl 1.26 0.28
− 9.8−17.3;−2.2
24.4 20.4 Lpa mgdl
14.6 12.4 0.043 0.026;0.061
Fractional cholesterol absorption 50 12
5116 2.1 1.1
Absolute cholesterol absorption mgkg per day 1.8 0.9
− 0.3 −0.7;0.2
0.327 0.039 0.284 0.036
0.043 0.026;0.061 FCR for LDL apo B poolsday
11.2 2.3 13.6 2.5
Production rate of LDL apo B mgkg per day −
2.5 −3.6;−1.3 −
32.3 −41.9;−22.6 108.4 24.6
76.1 14.0 LDL apo B mgdl
a
The values for the LDL subgroups are expressed as mean S.D.. PB0.05.
PB0.01. PB0.001.
Table 2 Correlation coefficients r-values between plasma lipids and lipoproteins and cholesterol absorption, LDL catabolism and LDL production rate
LDL apo B production mgkg Fractional catabolic rate of LDL apo B
Fractional cholesterol per day
absorption poolsday
Plasma cholesterol mmoll
0.008 Total
− 0.752
0.439 −
0.252 −
0.232 0.466
VLDL −
0.155 IDL
− 0.366
0.483 0.531
0.010 LDL
− 0.757
0.205 HDL
0.026 −
0.473 Plasma triglycerides
mmoll 0.509
Total −
0.222 −
0.324 VLDL
0.460 −
0.295 −
0.204 PB0.05.
PB0.01. PB0.001.
Table 3 BMI, plasma lipids, lipoproteins, cholesterol absorption and the FCR and production rate values for LDL apo B among subjects with low and
high BMI
a
BMI\26 BMIB26
Mean difference 95 CI of difference n = 39
n = 40 BMI kgm
2
28.1 1.6 24.1 1.6
− 3.9 −4.7;−3.2
54.5 3.2 53.9 2.6
0.58 −0.72;1.89 Age years
63.8 5.4 Weight kg
73.2 6.3 9.4 6.8;12.0
0.78 0.04 Waist-to-hip ratio
0.82 0.06 0.04 0,02;0,06
Plasma cholesterol mmoll Total
6.62 0.83 5.89 0.94
0.73 0.34;1.13 0.50 0.28
0.34 0.20 0.16 0.05;0.26
VLDL 0.22 0.12
IDL 0.29 0.13
0.07 0.01;0.12 3.79 0.9
LDL 4.39 0.73
0.61 0.24;0.97 1.54 0.39
1.61 0.32 −
0.77 −0.24;0.08 HDL
0.96 0.44 HDL2
− 0.12 −0.31;0.07
1.07 0.40 0.57 0.13
0.57 0.11 0.002 −0.052;0.056
HDL3 Plasma triglycerides mmoll
1.08 0.37 Total
1.54 0.59 0.46 0.24;0.68
0.71 0.37 0.46 0.25
0.26 0.12;0.40 VLDL
Plasma apo A1 gl 1.91 0.33
1.92 0.43 0.01 −0.16;0.18
1.20 0.31 0.97 0.24
0.23 0.11;0.36 Plasma apo B gl
18.5 16.8 Lpa mgdl
20.5 18.3 2.0 -5,9;9,9
54.5 13.6 Fractional cholesterol absorption
46.3 13.5 −
8.2 −14.4;−2.0 1.7 0.9
2.3 1.0 −
0.6 −1.1;−0.2 Absolute cholesterol absorption mgkg per day
0.298 0.040 −
0.011 −0.032;0.009 FCR for LDL apo B poolsday
0.309 0.047 13.3 2.8
11.5 2.3 1.8 0.6;3.0
Production rate of LDL apo B mgkg per day 85.4 24.9
LDL apo B level mgdl 100.925.0
15.5 3.8;27.3
a
The values for the BMI subgroups are expressed as mean S.D.. PB0.05.
PB0.01. PB0.001.
alcohol consumption and physical activity did not dif- fer between the high and low LDL cholesterol groups
data not shown.
3
.
3
. Effect of obesity on lipids, lipoproteins and metabolic factors
Since BMI was positively associated with total cholesterol r = 0.288, P B 0.01, VLDL r = 0.385, P B
0.01, IDL r = 0.354, P B 0.001 and LDL r = 0.265, P B 0.05 cholesterol and both total r = 0.497, P B
0.001 and VLDL r = 0.424, P B 0.001 triglycerides, the study population was divided into two groups with
BMI B 26 kgm
2
and BMI ] 26 kgm
2
Table 3. The obese women had higher plasma total, VLDL, IDL and
LDL cholesterol, total and VLDL triglyceride and apo B levels than the non-obese subjects. No significant
differences between the two groups were observed for HDL, HDL2, HDL3, apo A1 and Lpa concentra-
tions. The obese subjects also had a higher LDL apo B production rate and lower fractional and absolute ab-
sorption of cholesterol than the non-obese individuals, but no differences were observed in the FCR values for
LDL apo B between the two groups.
3
.
4
. Effects of apo E phenotype The women with apo E phenotype 2 E 22, 23
and 24 n = 4 tended to have lower total 5.93 9 1.11 mmoll and LDL cholesterol 3.54 9 1.21 mmol
l and higher VLDL 0.53 9 0.21 mmoll, IDL 0.34 9 0.10 mmoll and HDL 1.81 9 0.34 mmoll
cholesterol levels than the women with the phenotypes E 33 n = 49 or E 4 E 43, 44 n = 26, while the
women with the phenotype E 4 tended to have the highest total 6.5 9 0.88 mmoll and LDL 4.27 9
0.77 mmoll cholesterol and the lowest HDL choles- terol 1.47 9 0.39 mmoll levels. Also, an increasing
trend in the fractional 42 9 8; 50 9 14; 52 9 14, apo E phenotype 2, 3, 4, respectively and absolute ab-
sorption of cholesterol 1.4 9 0.5; 1.9 9 1.0; 2.2 9 1.1 mgkg per day and the LDL apo B production rate
10.3 9 4.1; 12.6 9 2.5; 12.6 9 2.9 mgkg per day and a decreasing trend in the FCR values for LDL apo B
0.322 9 0.084; 0.308 9 0.037; 0.293 9 0.049 poolsday could be observed between the apo E phenotypes 2, 3
and 4, respectively. However, these differences be- tween the phenotype groups were not statistically sig-
nificant.
3
.
5
. Effects of EcoRI, XbaI and
7
a -hydroxylase
polymorphisms Fifty-three subjects had the EcoRI genotype + +
presence of cutting site, 24 were heterozygous + − and 2 homozygous − − , absence of cutting site. For
the XbaI polymorphism 13 had the XbaI genotype + +
, 45 were heterozygotes + − and 21 had the XbaI genotype − − . The EcoRI and XbaI polymor-
phisms of the apo B gene had no significant effect on cholesterol metabolism in these subjects data not
shown.
No significant differences in cholesterol metabolism in relation to the polymorphism of 7a-hydroxylase were
observed between the subjects homozygous for the A allele AA, n = 21 compared with those having only
one AC, n = 40 or none of this allele CC, n = 18 data not shown.
4. Discussion
