Original Contributions |
From the Center for Clinical & Basic Research, Ballerup (P.A., J.H., I.S., H.L., C.C.); and Novo Nordisk A/S, Clinical Department, Gynecological Pharmaceuticals, Bagsvaerd (M.S.), Denmark.
Correspondence to Peter Alexandersen, MD, Center for Clinical & Basic Research, Ballerup Byvej 222, DK-2750 Ballerup, Denmark.
| Abstract |
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Key Words: atherosclerosis prevention rabbits estradiol NETA
| Introduction |
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| Methods |
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Rabbit Chow
Each rabbit was fed 100 g of chow per day throughout the
entire study. The chow was prepared by first dissolving the E2,
E2+low-dose NETA, or E2+high-dose NETA (NovoNordisk A/S), in ethanol
(96%; 0.50 mL per animal per day), then mixing with maize oil
(Unikem). Another mixture was prepared by dissolving
cholesterol (SIGC-8503, Bie & Berntsen A/S) in maize oil by
slow heating. The two solutions containing maize oil (total daily
intake of maize oil was 8 mL per animal) were then mixed manually
together with the pellets (Altromin 2123, Brogaarden). For the placebo
group, the chow was prepared as described; however, no hormone was
added. For logistic reasons, the chow for a period of 5 weeks was
produced, labeled, and stored at -20°C in daily portions. Food
consumption was monitored by weighing any remaining chow each week. All
animals had free access to water.
Blood Samples
Blood samples were taken in weeks 4, 13, 19, 25, 32, 40, 48, and
55, and always after a 24-hour fast.
Serum Lipids and Lipoproteins
Total serum cholesterol and
triglycerides were measured enzymatically by means of
kinetic colorimetric methods (Cobas Mira). To determine
serum lipoproteins (HDL, density
1.063 g/mL; LDL, 1.019 g/mL
density
<1.063 g/mL; IDL, 1.006 g/mL
density <1.019 g/mL; VLDL, density
<1.006 g/mL), three aliquots from the serum samples were adjusted to
specific densities, using solutions of NaCl and NaBr. The aliquots were
then sealed and ultracentrifuged at 4°C at
1.58x108 g/min for 14.5 hours in a Beckman
50.4 Ti rotor (Beckman Instruments, Inc). The top and bottom fractions
of each aliquot were divided by tube slicing, and the
cholesterol levels of the fractions were then measured
enzymatically. The lipoproteins were calculated from the fractions
after correction for recovery and dilution.
Serum E2
Serum levels of E2 were measured in weeks 4 and 55 by
radioimmunoassay, with an intra-assay imprecision of 8.5%, an
interassay imprecision of 12.3%, and a detection limit of 0.010
nmol/L.11
Aortic Cholesterol Content
At the end of the study (week 59), the rabbits were killed with
an intravenous injection of 1 to 2 mL of mebumal
(pentobarbital) 20% solution. The thoracic aorta (just above the
aortic valves to the level of the diaphragm) was dissected free, and
the connective tissue adhering to the adventitia was then carefully
removed under running saline. The aorta was cut longitudinally and the
luminal surface rinsed with saline. The vessel was fixed at the corners
with pins onto a piece of paper on a cork board. The aortic surface
area was determined and the tissue was separated in two parts (a
proximal and a distal part) at the level of the first intercostal
arteries. The proximal part was used to strip the luminal layer
containing the intima and part of the media from the underlying
media/adventitia. The proximal part was weighed and stored at -20°C
until analyzed.
For analysis, the luminal layer of the aortic tissue was minced and the lipids were extracted chemically with chloroform and methanol (2:1, vol/vol) over 24 hours. The lipids were separated from the proteins.12 The total aortic cholesterol content in the tissue specimens was measured enzymatically after the fraction containing cholesterol had been taken to dryness by heating and then dissolved in 1.0 mL of 2-propanol. The amount of protein in the aorta was measured as described by Lowry et al.13 The weight of the heart was recorded.
The Uterus
The bicorn uterus was cut at the level of the vagina and
beginning of the salpinges, respectively, removed, and the weight was
determined.
Body Weight
Body weight was determined every 4 weeks throughout the study on
the same scales.
Statistical Analysis
The average levels of serum cholesterol and
lipoproteins during the treatment period were calculated as the area
under the curve divided by the length of the intervention period (38
weeks). ANOVA was performed for the baseline values (Table
1), the average serum lipids and lipoprotein levels, aortic
cholesterol content, and uterine weight. If ANOVA indicated
statistical significance, a t test was used to compare
groups two by two using the Bonferroni correction for multiple
comparisons. The relation between aortic accumulation of
cholesterol and the averaged serum total
cholesterol (and lipoprotein) level was determined by
correlation analysis. The influence of baseline total serum
cholesterol and triglyceride levels, average
serum cholesterol, triglycerides, and
lipoprotein levels, and treatment (the independent variables) on
aortic cholesterol accumulation (the dependent
variable) was adjusted by ANCOVA. All statistical analyses
were performed with the Statistical Analysis System (SAS) with
5% as the level of significance.14
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| Results |
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Figure 1
shows the changes in total serum
cholesterol during the study. Serum values peaked in week
19, ie, at the end of the atherosclerosis-induction
period. Table 2
shows the average serum total
cholesterol and lipoprotein levels during the treatment
period. For the two estradiol/NETA groups, the averaged total serum
cholesterol, triglycerides, IDL
cholesterol, and VLDL cholesterol values were
lower than those for the placebo and estradiol groups (ANOVA
P<0.05). There was no statistically significant difference
in these parameters between the two estradiol/NETA groups
or between the placebo and the estradiol group. Serum estradiol was low
and remained unchanged in the placebo group (0.17±0.04 versus
0.12±0.03 nmol/L, baseline versus week 55; mean±SEM) but increased
significantly in the E2 group (from 0.10±0.01 to 0.21±0.04 nmol/L,
P=0.02) and the estradiol/NETA groups (E2NETA1, from
0.11±0.01 to 0.22±0.03 nmol/L, and E2NETA3, from 0.08±0.01 to
0.16±0.03 nmol/L, P<0.01 for both). The uterine weight in
the placebo group was 6.2±0.7 g (mean±SEM) and was significantly and
equally higher in both estradiol/NETA groups (17.5±1.4 g for E2NETA1
and 19.6±2.1 g for E2NETA3), although lower than in the E2 group
(28.3±1.8 g) (ANOVA P<0.0001). Thus, an increase in the
NETA dose does not apparently add further to the reduction in uterine
weight.
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Figure 2
visualizes the accumulation of
cholesterol in the luminal proximal layer of the thoracic
aorta for the four groups, unadjusted (A), adjusted for the
corresponding aortic protein content (B), adjusted for aortic wet
weight (C), or adjusted for the aortic area (D). The differences in
aortic cholesterol accumulation were highly statistically
significant (ANOVA P<0.0001). When adjusted for the aortic
area, the estrogen group had accumulated 75.7% of that found in the
placebo group, and this was significantly more (P<0.01)
than in the E2NETA1 and E2NETA3 groups, which had accumulated
respectively only 38.2% and 24.6% of the aortic
cholesterol in the placebo group. Aortic accumulation of
cholesterol was significantly related to the averaged serum
total cholesterol, VLDL cholesterol, IDL
cholesterol, and LDL cholesterol
(.54
r
0.71, P<0.0001 for these correlations)
but not to HDL cholesterol (r=-0.05). ANCOVA
was performed, with the aortic cholesterol content as the
dependent variable and baseline and average total serum
cholesterol, triglyceride, and lipoprotein
levels, and treatment as covariates. This analysis showed
that only treatment, LDL cholesterol, and VLDL
cholesterol were significant (respectively,
P=0.006, P=0.045, and P=0.011)
independent predictors of aortic
atherosclerosis. Compared with placebo, the estimates
(mean±SEM) were as follows: with E2NETA1:-3.02±0.95
µmol/cm-2 (P<0.01); with E2NETA3:
-2.90±0.97 µmol/cm-2
(P<0.01); with E2: -0.56±0.97
µmol/cm-2 (NS); and for LDL
cholesterol: 0.46±0.22
µmol/cm-2 (P<0.05) and VLDL
cholesterol: 0.24±0.09
µmol/cm-2 (P<0.05). The amount of
aortic cholesterol accumulation after correction for LDL
cholesterol and VLDL cholesterol is depicted in
Figure 3
. In addition, each of the two
E2/NETA groups had statistically significantly less aortic
cholesterol accumulation than the E2 group
(P<0.01 for both comparisons). Comparison of the two
E2/NETA groups showed that the aortic cholesterol
accumulation (when adjusted for aortic protein or surface area) tended
to be lower in the high-dose NETA group than in the low-dose NETA group
(P<0.1) (Figure 2
). However, after correction for
lipoproteins, the cholesterol content in the two groups was
similar, which indicates a lipid-mediated, dose-response effect (Figure 3
).
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| Discussion |
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The possible mechanism by which NETA could enhance one or more of the
beneficial estrogenic effects is, however, largely speculative. It has
been suggested that accumulation and/or metabolism of LDL
cholesterol in the arterial wall may be reduced
during E2 therapy,24 which, theoretically, could
be explained by a potent estrogenic antioxidant
capacity.25 Recent data suggest that metabolites
of NETA (3ß,5
-NETA and 3
,5
-NETA, respectively) may exert an
estrogenic (and perhaps an antioxidant) effect through binding to the
estrogen receptor.26 Indeed, some data indicate
that NETA in vivo is also metabolized to ethinyl estradiol, which in
turn exerts the estrogenic effect via the estrogen
receptor.27 In addition, the progestogenic effect
of NETA is mediated via interaction of NETA with the progesterone
receptor,27 whereas other studies in the rabbit
uterus have shown that MPA does not bind to the estrogen
receptor.28 In previous studies we found that
NETA alone29 and in combination with
E29 had a neutral effect on early atherogenic
processes. It may therefore be speculated that NETA primarily has a
synergistic beneficial effect together with estradiol in established
and more advanced atherogenic stages.
Two recent epidemiological studies investigated the role of estradiol combined with progestogens on hard cardiovascular end points. One study30 showed that postmenopausal women treated with an estrogen/progestogen combination (predominantly a 19-nortestosterone derivative other than NETA, namely levonorgestrel) tended to have fewer cardiovascular events (ie, myocardial infarctions) than women receiving E2 monotherapy, who in turn had fewer events than women not taking hormone replacement therapy. Very recently, data from the Nurses' Health Study31 indicated that the current use of MPA combined with oral conjugated estrogens was associated with markedly less risk of a major coronary heart disease condition than when hormone replacement therapy was never used. The relative risk was even lower than that of current estrogen use. These risk estimates were, however, based on a very small number of subjects.
If the estrogen-enhancing effect of NETA reported here is confirmed in future studies, it will undoubtedly have a great impact on future considerations regarding estrogen/progestogen therapy in the (secondary) prevention of CVD in postmenopausal women.
| Selected Abbreviations and Acronyms |
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Received September 26, 1997; accepted December 8, 1997.
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