ing that of HDL particles [6]. Other studies also demonstrated, however, that estrogen stimulates the
hepatic production of very low-density lipoprotein VLDL which, like LDL, is considered an atherogenic
lipoprotein particle [7]. In addition, estrogen induces hypertriglyceridemia, which is associated with an in-
creased incidence of coronary heart disease [8]. Plasma triglyceride TG levels are closely related to the size of
the LDL particles [9]. Thus, the smaller, denser LDL particles are associated with an increased risk of coro-
nary heart disease [10]. We previously reported that estrogen-induced hypertriglyceridemia may lead to a
reduction in the size of LDL particles and an increased prevalence of LDL subclass pattern B [11], which con-
sists of particles having diameters less than 25.5 nm and which is strongly associated with an increased risk for
atherosclerosis [10]. Campos et al. reported [12] that estrogen therapy decreases the proportion of large, but
not of small, LDL particles.
The National Cholesterol Education Program [13] and the Japanese Atherosclerosis Society [14] have
stated the indications and goals of dietary or drug therapy for hypercholesterolemia based on the patients’
plasma levels of total-Ch and LDL-Ch. The estrogen- induced reduction in plasma LDL-Ch is less than 20
[6].
We previously
demonstrated that
in post-
menopausal women with moderate to severe hyperc- holesterolemia, it may be difficult to achieve target
levels of total-Ch or LDL-Ch in the plasma by estrogen therapy alone [15], especially since the estrogen-induced
reduction in LDL-Ch is less than 20.
Simvastatin, a 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitor, effectively reduces the plasma
concentrations of VLDL and intermediate-density lipo- protein IDL, as well as of LDL [16]. Simvastatin has
also been shown to reduce plasma total-TG levels in patients with hypertriglyceridemia [17]. Simvastatin
slightly increases the plasma levels of HDL-Ch [18]. Combination with estrogen plus simvastatin may
provide
additional favorable
effects on
lipid metabolism.
The present study investigated the effects of estrogen and simvastatin, alone and in combination, on the
plasma lipid levels and the activities of enzymes in- volved in lipoprotein metabolism in postmenopausal
women with type IIa hypercholesterolemia.
2. Materials and methods
2
.
1
. Subjects Between April 1, 1995 and March 31, 1996, we
evaluated 45 postmenopausal Japanese women with type IIa hypercholesterolemia with plasma total-Ch
levels of 220 mgdl or greater and plasma total-TG levels of less than 150 mgdl, as defined by Japanese
Atherosclerosis Society [14]. Their mean age was 55 years range, 46 – 64 years; their mean body mass index
was 23.1 9 2.0 kgm
2
range, 19.7 – 25.8 kgm
2
. Subjects with familial hypercholesterolemia FH, as defined by
the Ministry of Health Welfare [14], were excluded from the study. None of the women had had a men-
strual period for at least 1 year. None had a history of coronary heart disease or of risk factors for coronary
heart disease, such as hypertension, diabetes mellitus, or low HDL cholesterolemia. None of the subjects
smoked, used caffeine or alcohol, had a history of thyroid disease, liver disease, or was currently taking
any
medication known
to influence
lipoprotein metabolism. Written informed consent was obtained
from each subject before admission to the study. The study design was approved by the Ethics Committee of
Kochi Medical School.
2
.
2
. Treatment protocol Each subject had received dietary counseling by nu-
tritionists at our institution before drug therapy. In none of the patients did the plasma total-Ch levels
decrease to less than 220 mgdl after 6 months of this dietary regimen. The 45 patients were then randomly
assigned in open, parallel-group fashion to the three treatment groups. Subjects in the estrogen group n =
15 received 0.625 mg oral conjugated equine estrogen daily, those in the simvastatin group n = 15 received 5
mg simvastatin daily, and those in the combination group n = 15 received both agents daily for 3 months.
After they had signed informed consent forms, the subjects were randomized by means of a random num-
ber generator. Endometrial biopsies and blood samples were obtained from each subject before and after
treatment.
2
.
3
. Blood collection and lipoprotein isolation Blood samples were drawn between 08:00 and 10:00
am following a 12-h fast and centrifuged immediately at 1500 × g for 20 min at 4°C to obtain the plasma.
Subsequently, heparin 10 Ukg body weight was ad- ministered intravenously, followed by 5.0 ml saline
infusion to flush the line. Ten minutes after heparin administration, blood was collected and postheparin
plasma was obtained by centrifugation at 1500 × g, for 20 min at 4°C to determine LPL and hepatic triglyce-
ride lipase H-TGL activities.
VLDL d \ 1.006 gml, IDL 1.006 B d B 1.019 g ml, LDL subfractions LDL1, 1.019 B d B 1.045 gml;
LDL2, 1.045 B d B 1.063 gml, and HDL subfractions HDL2, 1.063 B d B 1.125 gml; HDL3, 1.125 B d B
1.21 gml were subsequently separated by ultracen- trifugation,
as described
by Havel
et al.
[19].
Concentrations of total-Ch and total-TG in the plasma, as well as the Ch and TG levels in the lipoproteins,
were measured enzymatically [20]. Plasma concentra- tions of apolipoproteins apo A-I, A-II, B, C-II, C-III,
and E were determined by a turbidimetric im- munoassay [21]. Assays of plasma lipids were per-
formed within 24 h of storing the samples at 4°C. Assays of plasma apolipoproteins, lipoprotein lipids,
and enzyme activities were performed within 7 days of storing the samples at − 80°C.
2
.
4
. Enzyme assays To determine LPL activity, 0.49 ml assay mixture 50
mmoll glyceryl trioleate, 15 gum arabic emulsion, 0.2 moll Tris – HCl at pH 8.2, 5 bovine serum albumin,
0.1 NaCl, 140 ml serum was incubated for 80 min at 37°C. Postheparin plasma and 100 mmoll sodium lau-
ryl sulfate mixture was incubated for 60 min at 26°C. The reaction was started by adding 0.01 ml postheparin
plasma-sodium lauryl sulfate mixture to the assay mix- ture and incubation at 28°C for 60 min. Subsequently,
2.5 ml of an extraction mixture containing 40:10:1 isopropyl alcohol, n-heptane and 1 N H
2
SO
4
was added to the incubation mixture and shaken vigorously. After
standing for 10 min, the mixture was separated into two phases, and each phase was centrifuged. An aliquot 1
ml of the upper phase was dried with nitrogen and dissolved with 500 ml 5 Triton X. Fatty acid levels in
the eluate were determined enzymatically [3].
To determine H-TGL activity, the reaction was started by adding 0.01 ml of a mixture of postheparin
plasma and 0.2 moll Tris – HCl at pH 8.8 to 0.49 ml assay mixture 50 mmoll glyceryl trioleate, 15 gum
arabic emulsion, 0.2 moll Tris – HCl at pH 8.8, 5 bovine serum albumin, 0.75 moll NaCl. After incuba-
tion for 60 min at 28°C, fatty acid levels were measured as already described [3].
Plasma lecithin cholesterol acyltransferase LCAT activity was measured using a commercially available
enzymatic kit Nippon Shoji, Osaka. Cholesterol ester transfer protein CETP activity
was quantified as the capacity of the samples to pro- mote the transfer of radiolabeled cholesteryl esters from
a tracer amount of labeled HDL3 to apo B-containing lipoproteins. Serum samples 25 ml were incubated with
radiolabeled HDL3, 2.5 nmol cholesterol and 75 nmol iodoacetate in a final volume of 50 ml at 37°C for 3 h.
A negative control for each sample was carried out by incubating the mixture at 0°C. Of the incubation mix-
ture, 45 ml were then added to 2 ml potassium bromide solution d = 1.07 gml and centrifuged at 260,000 × g
for 4 h at 4°C. Both the supernatant which contained VLDL, IDL, and LDL fractions, and the subnatant,
which contained the HDL fraction, were recovered, and radioactivities were measured in both fractions. The
results were expressed as nanomoles of radiolabeled cholesterol esters transferred from HDL3 to apo B-con-
taining lipoproteins per minute [22].
2
.
5
. Statistical analysis All data are expressed as the mean 9 standard devia-
tion S.D.. Treatment-induced changes were analyzed by a paired t-test. Differences in the lipid levels among
the three groups before and after treatment were ana- lyzed by one-way analysis of variance ANOVA. If the
ANOVA indicated a significant difference, a multiple comparison procedure was performed by Fisher’s pro-
tected least-significant difference. A level of P B 0.05 was accepted as statistically significant.
3. Results
