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
3
.
1
. Patient characteristics No significant differences among the groups were
found in mean age estrogen, 54.9 9 4.7 years; simvas- tatin, 53.5 9 7.1 years; combination, 55.8 9 4.7 years
and body mass index estrogen, 22.8 9 2.1 kgm
2
; sim- vastatin, 23.2 9 1.6 kgm
2
; combination, 23.3 9 2.3 kg m
2
. The mean body mass index did not change significantly during the study period. Histologic analy-
sis of endometrial biopsy specimens from all patients did not detect hyperplasia in any of the patients before
or after treatment. Baseline levels of lipids, apolipo- proteins, and the enzyme activities were not signifi-
cantly different among the three groups.
3
.
2
. Changes in cholesterol le6els We initially investigated the effects of treatment with
estrogen and simvastatin, alone or in combination, on cholesterol levels in the various lipoprotein subfrac-
tions. Estrogen treatment reduced the plasma levels of total-Ch by 11.2 and LDL1-Ch by 21.0, increased
the plasma level of HDL2-Ch by 10.6, and did not significantly alter the concentrations of VLDL-Ch,
IDL-Ch, LDL2-Ch, and HDL3-Ch Table 1. With simvastatin treatment, the plasma levels of total-Ch
decreased by 22.3, VLDL-Ch by 20.0, IDL-Ch by 40.6, and LDL1-Ch by 33.5. Simvastatin had no
significant effect, however, on the concentrations of LDL2-Ch, HDL2-Ch, and HDL3-Ch. Combination
therapy reduced the plasma levels of total-Ch by 27.5, VLDL-Ch by 31.5, IDL-Ch by 17.6, LDL1-Ch by
41.9, and LDL2-Ch by 15.4. Conversely, HDL2-Ch plasma levels increased by 9.4 after combination ther-
apy. For total-Ch and LDL1-Ch levels, the percentage decreases were significantly greater after simvastatin
and combined therapies than after estrogen therapy.
3
.
3
. Changes in triglyceride le6els In addition to cholesterol levels, we evaluated the
effects of each treatment on triglyceride levels in the various lipoprotein subfractions. Estrogen treatment
resulted in significant increases in the plasma levels of total-TG, an increase of 22.5, IDL-TG 46.6,
HDL2-TG 69.1, and HDL3-TG 47.7. No signifi-
Table 1 Changes in cholesterol levels in the lipoproteins in hypercholesterolemic postmenopausal women treated with estrogen, simvastatin, or a
combination of estrogen plus simvastatin
a
Combination n = 15 Cholesterol
Estrogen n = 15 Simvastatin n = 15
266.5 9 28.9 269.1 9 33.7
268.6 9 35.7 Before
Total mgdl 237.4 9 27.7
205.6 9 23.2 193.2 9 19.5
After Change
− 27.5 9 7.5†††
− 22.3 9 10.0††
− 11.2 9 10.6
36.9 9 12.9 39.3 9 12.3
Before 31.3 9 10.5
VLDL mgdl 33.3 9 11.0
23.7 9 8.8 25.3 9 8.4
After −
20.0 9 30.0 −
31.5 9 24.2† −
5.6 9 29.0 Change
13.6 9 4.4 14.4 9 5.2
15.5 9 5.1 Before
IDL mgdl 11.0 9 4.3
After 12.9 9 5.3
9.3 9 5.2 −
40.6 9 25.2†† −
17.6 9 36.9 3.6 9 60.9
Change 135.3 9 29.3
135.7 9 21.3 132.9 9 30.5
LDL1 mgdl Before
105.0 9 17.8 89.1 9 15.1
75.6 9 14.1 After
− 33.5 9 12.1††
− 41.9 9 11.5†††
Change −
21.0 9 13.2 25.5 9 6.7
24.8 9 6.2 23.7 9 8.6
Before LDL2 mgdl
19.4 9 6.3 After
22.6 9 6.6 20.9 9 9.0
− 6.6 9 78.4
− 15.4 9 40.0
− 4.6 9 37.2
Change 39.5 9 10.2
41.1 9 10.5 38.8 9 10.7
HDL2 mgdl Before
44.9 9 9.5 45.2 9 13.8
43.1 9 9.5 After
9.4 9 17.8 6.3 9 17.9
10.6 9 20.2 Change
18.3 9 1.5 19.2 9 2.1
18.4 9 1.7 HDL3 mgdl
Before After
18.7 9 2.2 17.5 9 2.4
18.7 9 3.4 3.2 9 25.3
− 7.4 9 16.8
2.7 9 11.6 Change
a
Data are expressed as mean 9 S.D. PB0.05, PB0.01, PB0.001 versus before treatment as determined by paired t-test. † PB0.05, †† PB0.01, ††† PB0.001 versus estrogen treatment as determined by multiple comparison analysis.
Table 2 Changes in triglyceride levels in the lipoproteins in hypercholesterolemic postmenopausal women treated with estrogen, simvastatin, or a
combination of estrogen plus simvastatin
a
Triglyceride Simvastatin n = 15
Combination n = 15 Estrogen n = 15
109.9 9 22.3 99.5 9 26.5
Total mgdl 106.6 9 23.0
Before 108.3 9 25.4
After 128.4 9 39.8
89.2 9 31.0 −
5.8 9 33.6† 1.4 9 25.9
Change 22.5 9 35.5
47.7 9 21.2 VLDLmgdl
Before 42.5 9 21.6
42.1 9 23.2 43.3 9 19.2
33.9 9 21.7 50.9 9 30.9
After 42.2 9 89.7
14.1 9 90.4 −
2.4 9 60.5 Change
Before 9.3 9 3.43
9.0 9 3.5 IDL mgdl
8.2 9 2.9 7.6 9 3.91
10.0 9 4.0 After
11.3 9 4.2 46.6 9 62.0
20.9 9 62.8 −
13.3 9 40.1†† Change
32.5 9 7.6 Before
33.8 9 7.6 28.0 9 7.2
LDL1 mgdl 24.5 9 8.8
After 26.2 9 6.8
33.5 9 10.1 −
13.5 9 35.3 Change
2.0 9 35.4 −
8.2 9 38.0 5.1 9 1.8
5.7 9 2.1 Before
LDL2 mgdl 6.5 9 1.9
8.6 9 5.08 6.7 9 2.8
5.2 9 1.9 After
36.1 9 81.6 43.9 9 91.8
12.8 9 68.9 Change
10.2 9 2.6 Before
10.7 9 3.4 10.8 9 3.5
HDL2 mgdl 13.0 9 2.6
After 15.7 9 4.0
17.2 9 4.8 52.6 9 34.4
32.3 9 46.4 69.1 9 49.5
Change 4.8 9 1.1
Before 4.3 9 1.3
4.7 9 1.15 HDL3 mgdl
5.1 9 1.7 6.9 9 3.0
After 6.5 9 2.4
8.2 9 37.5 Change
47.7 9 51.6‡ 61.0 9 66.1‡‡
a
Data are expressed as mean 9 S.D. PB0.05, PB0.01, PB0.001 versus before treatment as determined by paired t-test. † PB0.05, †† PB0.01 versus estrogen treatment and ‡ PB0.05, ‡‡ PB0.01 versus simvastatin treatment as determined by multiple comparison analysis.
Table 3 Changes in apolipoprotein levels in hypercholesterolemic postmenopausal women treated with estrogen, simvastatin, or a combination of estrogen
plus simvastatin
a
Estrogen n = 15 Apolipoprotein
Simvastatin n = 15 Combination n = 15
A-I mgdl 132.2 9 20.8
Before 136.5 9 20.2
130.3 9 20.9 150.8 9 20.6
142.8 9 24.8 After
152.4 9 16.3 15.3 9 14.7
4.9 9 11.6 Change
18.7 9 16.1‡ 35.1 9 4.1
35.5 9 4.1 Before
35.3 9 4.4 A-II mgdl
37.9 9 5.6 After
35.2 9 3.5 38.7 9 5.5
8.0 9 10.0‡ −
0.1 9 10.7 9.7 9 10.4‡
Change 120.9 9 17.9
115.8 9 13.6 B mgdl
120.5 9 18.0 Before
106.2 9 16.2 87.6 9 14.5
After 86.5 9 12.6
− 11.6 9 10.7
− 24.0 9 11.0††
Change −
27.2 9 12.7††† 4.1 9 1.1
4.61 9 1.11 Before
4.4 9 1.1 C-II mgdl
3.6 9 1.0 4.1 9 1.2
3.0 9 0.8 After
− 9.6 9 21.0
− 11.5 9 16.4
− 29.8 9 22.7††‡
Change 11.6 9 3.0
11.2 9 3.1 C-III mgdl
12.3 9 3.4 Before
12.6 9 2.6 10.1 9 2.6
After 11.4 9 3.0
Change 12.3 9 25.1
− 6.6 9 24.8
− 3.8 9 28.4
Before E mgdl
5.8 9 1.5 5.8 9 1.5
5.7 9 1.7 4.6 9 1.1
5.1 9 1.3 After
3.9 9 1.3 Change
− 18.5 9 15.6
− 10.6 9 16.2
− 31.6 9 15.4††‡‡
a
Data are expressed as mean 9 S.D. PB0.05, PB0.01, PB0.001 versus before treatment as determined by paired t-test. †† PB0.01, ††† PB0.001 versus estrogen treatment and ‡ PB0.05, ‡‡ PB0.01 versus simvastatin treatment as determined by multiple comparison analysis.
cant changes occurred in LDL-TG levels Table 2. Simvastatin treatment, in contrast, significantly reduced
the plasma level of total-TG by 5.8, but did not significantly affect the plasma levels of IDL-TG, LDL-
TG, and HDL3-TG. Simvastatin did, however, signifi- cantly increase the concentration of HDL2-TG by
32.3. Finally, combination therapy significantly in- creased the levels of HDL2-TG by 52.6 and of
HDL3-TG by 61.0. When comparing the three treat- ment approaches, the post-treatment levels of total-TG
and IDL-TG were significantly lower in the simvastatin group than in the estrogen group.
3
.
4
. Apolipoprotein changes We also assessed the effects of estrogen, simvastatin,
and combination therapy on the patients’ apolipo- protein levels. These analyses found that estrogen treat-
ment significantly increased the plasma levels of apo A-I, an increase of 15.3, and A-II 8.0, and signifi-
cantly reduced the plasma levels of apo B, a decrease of 11.6, apo C-II 9.6 and apo E 18.5 Table 3.
Simvastatin treatment also resulted in significant reduc- tions in the plasma levels of apo B decrease of 24.0,
apo C-II 11.5, and apo E 10.6, but did not significantly alter the levels of apo A-I and A-II. With
combination therapy, the plasma levels of apo A-I were elevated by 18.7 and apo A-II by 9.7, whereas the
plasma levels of apo B were significantly reduced by 27.2, apo C-II by 29.8, and apo E by 31.6.
Comparison of the three groups found that the percent decreases in the levels of apo B, apo C-II and apo E
were significantly greater in the combination group than in the estrogen group.
3
.
5
. Enzymatic changes Finally, we evaluated the effects of estrogen and
simvastatin, individually or in combination, on the activities of enzymes involved in lipoprotein metabo-
lism. Estrogen treatment significantly inhibited the ac- tivities of H-TGL by 27.7, and LCAT by 11.6. The
activities of LPL and CETP did not change significantly Table 4. Simvastatin caused no significant changes in
the activities of any of these enzymes tested. Combina- tion therapy, in contrast, significantly suppressed the
activities of LPL by 17.2, H-TGL by 44.8, and LCAT by 9.8, but had no effect on CETP activity.
4. Discussion