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Clinical Studies |
Clinic of Endocrinology (B.I., M.R., P.J.K.), Department of Gynecology and Obstetrics, University Hospital Zurich; and Schering (Schweiz) AG (A.E.J.), Zurich, Switzerland; and Department of Medicine, Center for Clinical Pharmacology (R.K.D.), University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania 15213
Address all correspondence and requests for reprints to: Raghvendra K. Dubey, Center for Clinical Pharmacology, Department of Medicine, 200 Lothrop Street, 623 Scaife Hall, University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania 15213-2582.
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Abstract |
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 Top Abstract IntroductionSubjects and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Although it is now well accepted that in women estrogen induces cardioprotective effects, the mechanism(s) of estrogen-induced cardioprotective effects remains unclear. Therefore, we hypothesize that estrogen-induced cardioprotection is mediated in part by increased synthesis of nitric oxide (NO). Several observations provide a strong rationale for this hypothesis including: 1) NO exerts a number of cardioprotective actions, e.g. vasodilation, inhibition of platelet aggregation, and vascular smooth muscle cell growth (5, 6, 7). 2) Treatment of cultured human endothelial cells with 17ß-estradiol stimulates constitutive NO synthase activity (8). 3) Aortic rings from female vs. male rabbits release more NO, and this enhanced release correlates with circulating estradiol levels (9). 4) Pharmacological blockade of NO synthase attenuates estradiol-induced vasodilation of the uterine circulation (10). 5) In humans, circulating levels of NO increase with follicular development and correlate with 17ß-estradiol levels (11). To test our hypothesis, we recently examined in PMW the effects of estrogen replacement therapy using transdermal patches on circulating levels of nitrite/nitrate (metabolites of NO; 12). These studies provided the first clinical evidence that estrogen replacement therapy increases NO production in PMW.
Our previous study, however, raises several important questions: 1) Is the effect of estrogen on NO production caused by a direct action of estrogen on NO synthase or caused by an indirect effect? In this regard, low-density lipoprotein (LDL) is known to inhibit NO synthase (13, 14) and is increased in PMW (2, 15). Because estrogen replacement therapy reduces LDL levels in PMW (2, 3, 16), it is possible that estrogen increases NO production via reducing LDL levels. 2) Is the effect of estrogen on NO production attenuated by concomitant treatment with a progestin? In our previous study (12), increases in circulating NO levels in response to 17ß-estradiol were substantially diminished while PMW were taking the progestin norethisterone acetate. 3) Does only transdermal delivery of estrogen increase NO synthesis or is orally administered estrogen also effective? Previous studies (17) suggest that the therapeutic effects of estrogen may depend on the route of administration. The goal of the current study was to further investigate the role of NO synthase in estrogen-induced cardioprotection by addressing each of the three aforementioned question. To achieve this objective, we examined the effects of oral administration of estradiol valerate on serum levels of nitrite/nitrate and LDL in PMW with and without concomitant administration of two chemically distinct progestins.
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Subjects and Methods |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Thirty-two healthy, caucasian PMW participated in this study. Inclusion criteria were as follows. Participants 1) were 45–55 yr of age; 2) were more than 1 yr after menopause; 3) exhibited symptoms requiring hormone replacement therapy (HRT) for therapeutic or preventive reasons; 4) were never treated with HRT or, if ever on HRT, had a wash-out period of at least 4 weeks (on conjugated estrogen at least 12 weeks) before to the beginning of the trial; 5) had FSH serum levels more than 40 IU/L; 6) had estradiol serum levels less than 100 pmol/L; and 7) had a body weight within the range of standard weight ± 15%. The women enrolled in the study had similar lifestyles and dietary habits and were instructed to avoid any changes in dietary habits during the investigation. All subjects gave written informed consent to the study, which was approved by the Institutional Medical Research Committee on Clinical Investigation.
Protocol
HRT was applied in a sequentially combined fashion. The estrogen used was estradiol valerate, which is immediately metabolized to estradiol, the natural human estrogen. Estradiol valerate (2 mg daily; Progynova, Schering, Switzerland) was administered orally for 21 days. Subjects were randomly assigned to receive with the last 10 tablets of Progynova in each cycle either 1 mg of cyproterone acetate (CPA) or 5 mg of medroxyprogesterone acetate (MPA). At the end of each 21-day treatment cycle, there was a 7-day treatment-free period. Blood samples for serum NO2-/NO3- levels were drawn at baseline (i.e. before HRT was initiated) and then again at 6 and 12 months into HRT. The samples were withdrawn on one of the last 3 days of unopposed estradiol intake and on the last 3 days of the combined estradiol-progestin treatment. All blood samples were taken in the morning after a 12-h fast period, and immediately after the patient’s arrival. Serum was separated by centrifuging the samples at 800 x g for 10 min. The samples were stored at -20 C until analysis.
Nitrite/nitrate analysis
Serum NO2-/NO3- levels were measured by reacting the samples with the Griess reagent, by our previously described method (12) and a commercially available kit (Alexis Corp., Laeufelingen, Switzerland). Briefly, aliquots (40 µL) of serum in duplicate were incubated at room temperature with enzyme cofactors and nitrate reductase for 1 h to convert NO3- to NO2-. Total NO2- was then analyzed by reacting the samples with 50 µL of Griess reagent component. Amounts of NO2- in serum were estimated from a standard curve measuring the absorbance at 540 nm.
Hormone analysis
Serum 17ß-estradiol levels were analyzed by RIA (Diagnostics Products Corp., Los Angeles, CA). Serum FSH levels were estimated by means of microbead enzyme immunoassay (IMX, Abbott Park, IL).
LDL and high-density lipoprotein (HDL) analysis
Serum levels of LDL cholesterol were measured by the methods of Burstein et al. (18) and Friedwald et al. (19) using a commercially available kit (bioMerieux, MarcyI’Etolle, France). Briefly, the LDL fraction in 50-µl aliquots of serum was precipitated by addition of 1 mL of amphophillic polymers [anionic-polycyclic activator (0.4 mg/mL), anionic-polycondensated activator (0.6 mg/mL), and polysubstituted dioxan (12.4 mmol/L) in an imidazole-buffer (25 mmol/L), pH 6.1]. After incubation for 30 min at 2–8 C, the samples were centrifuged at 4 x 103 rpm. The supernatant was discarded, and the pellet resuspended in 0.5 mL trisodium citrate (0.15 mol/L) containing NaCl (0.11 mol/L). Aliquots (50 µL) of this suspension were reacted with an enzyme solution (provided in the kit), and the levels of LDL measured spectrophotometrically at 546 nm. The amounts of LDL were estimated from a standard curve of LDL run in parallel.
Serum HDL levels were measured spectrophotometrically at 500 nm by the methods of Burstein et al. (18) and Lopes-Virella et al. (20) and by using a commercially available kit (Boehringer-Mannheim, Mannheim, Germany).
Statistical analyses
The person performing the LDL, HDL, and NO2-/NO3- assay was unaware of whether the sample was from a subject in the CPA or MPA group. Baseline (before HRT) measurements of serum LDL, HDL, and NO2-/NO3- levels were obtained from all 32 individuals. Five of the 32 subjects (1 in the MPA, 4 in the CPA group) retrospectively showed serum estradiol levels higher than 100 pmol/L, and therefore these individuals were excluded from the statistical analyses. Two subjects (1 in the MPA, 1 in the CPA group) had incomplete data, missing the sample of the combined estradiol-progestin application at treatment month 6. Out of the 27 subjects with complete sets of data, one had significantly high levels of nitrite/nitrate (60 µmol/L vs. 20.1 ± 1.58 averaged from 26 subjects) and was an outlier according to the box plot statistical analysis and was excluded. Statistical analysis was performed using ANOVA and paired Student’s t test, as appropriate. The criterion of significance was a value of P < 0.05. Data are presented as mean ± SEM.
To study whether estradiol valerate administration altered serum NO2-/NO3-, LDL, and HDL levels with the time of treatment, the 26 subjects with complete data sets were analyzed by making between group comparisons by ANOVA, and Fisher’s least significant test was used to detect differences between specific groups. The effects of the two different progestins (CPA and MPA) on the NO2-/NO3- levels were compared using ANOVA. To evaluate whether sequential treatment with the progestins (CPA and MPA) modulate the effects of estradiol valerate on NO synthesis, only the data from the PMW who responded to estradiol valerate with an increase in NO2-/NO3- levels were analyzed. This analysis was conducted using two-factor ANOVA with repeated measures (factor A: treatment group; factor B: time period) and Friedmans repeated measure ANOVA on ranks; all pairwise multiple comparisons were by Newman Keuls method. A response to estradiol of greater than a 30% increase in NO2-/NO3- levels compared with baseline was used to define subjects as responders and a scatter plot was constructed to show a bimodal distribution. Linear regression analysis was performed to analyze whether there was a correlation between the levels of NO2-/NO3- and LDL in responders and nonresponders.
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Results |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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). However, in samples collected after 12 months of
treatment, serum NO2-/NO3- concentrations
increased significantly and were 30.0 ± 3.7 µmol/L
(P < 0.01 vs. baseline and
P > 0.05 vs. 6 month).
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). A detailed analysis
demonstrated that out of 26 subjects, only 13 individuals had an
increase of greater than 30% in serum
NO2-/NO3- levels, i.e. from
20.5 ± 3.4 µmol/L at baseline to 39.3 ± 5.5 µmol/L in
samples collected after 12 months of HRT (P = 0.007
vs, baseline; Fig. 2, A and B), and these subjects were
classified as responders. In the remaining 13 subjects serum
NO2-/NO3- serum levels either increased less
than 30% or decreased or remained unchanged after 12 months of HRT
(baseline: 22.8 ± 2.5 µmol/L vs. after 12 months of
HRT: 19.8 ± 2.8 µmol/L; P > 0.05) and these
subjects were classified as nonresponders (Fig. 2, A and B). Although
differential increases in serum NO2-/NO3-
levels were observed in PMW treated for 12 months with estradiol
valerate, there were no differences in serum
NO2-/NO3- levels in samples taken after 6
months of substitution, which were 22 ± 2 µmol/L in responders
and 23.7 ± 4.2 µmol/L in nonresponders (P >
0.05). The subjects in these two sets, classified as responders
(increase in NO2-/NO3- levels) and
nonresponders (no change or decrease in
NO2-/NO3- levels) did not have any significant
difference with respect to age, baseline serum FSH, estradiol, and
NO2-/NO3- levels.
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). In contrast, the serum
NO2-/NO3- levels in samples collected while
subjects were taking estradiol plus CPA or MPA (after 12 months of HRT)
were not significantly different from the serum
NO2-/NO3- levels observed at baseline and were
17.6 ± 3.6 at baseline vs. 19 ± 3 µmol/L
(P > 0.05) in the CPA-treated group and 17.4 ±
1.5 at baseline vs. 22.5 ± 4.0 µmol/L
(P > 0.05) in MPA-treated group. However, when
12-month samples were included in the statistical analysis regardless
of collection with respect to estradiol valerate and the type of
progestin used (CPA and MPA combined), the increase in serum
NO2-/NO3- levels was significant compared with
the levels before the initiation of the HRT (25.4 ± 1.58 µmol/L
after 12 months of HRT vs. 20.1 ± 1.6 µmol/L at
baseline; P < 0.05).
To investigate the effects of CPA and MPA on estradiol-induced NO
synthesis, only the samples collected at 12 months from the responders
group taking either MPA or CPA were considered for statistical
analysis. Serum NO2-/NO3- levels were
significantly increased (P < 0.05 vs.
baseline in MPA group and P < 0.05 vs.
baseline in CPA) in samples collected while the subjects were taking
estradiol valerate alone (Fig. 3); in contrast, the
serum NO2-/NO3- levels were decreased to basal
levels in samples collected while the subjects were taking estradiol
valerate with CPA or MPA (Fig. 3). However, when all of the 12-month
samples were included in the statistical analysis in the responders
group, regardless of collection with respect to estradiol valerate with
or without CPA or MPA treatment, the change in
NO2-/NO3- levels in HRT-PMW receiving CPA or
MPA was significantly increased and was 26.5 ± 5.4 µmol/L
(P < 0.05 vs. baseline 17.6 ± 35.4
µmol/L) and 37.4 ± 5 µmol/L (P < 0.002
vs. baseline 17.4 ± 1.9 µmol/L), respectively (Fig. 4). Finally, when all the samples in the responders
group, regardless of the type of progestin used (CPA and MPA combined),
were included in the statistical analysis, the increase in serum
NO2-/NO3- levels at 12 months was significant
compared with the levels before the initiation of the HRT (33.2 ±
4.2 µmol/L after 12 months of HRT vs. 17.5 ± 1.63
µmol/L at baseline; P = 0.002; Fig. 4).
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). Compared with the group of PMW in which no
increases in NO synthesis were observed after HRT (nonresponders), the
basal levels of LDL were substantially higher in the group of PMW in
which increases in NO synthesis were observed after HRT (responders).
Furthermore, the levels of LDL decreased significantly from baseline
4.1 ± 0.2 mmol/L to 3.7 ± 0.2 mmol/L (64 10% decrease;
P < 0.05) after HRT in the responders but not in the
nonresponders (P > 0.05). Nonparameteric regression
analysis between the NO2-/NO3- levels and LDL
levels showed a significant negative correlation in the responders’
group (r2 = 0.17; P < 0.05; Fig. 5B) but
not in the nonresponders’ group (Fig. 5C). In contrast to LDL, HRT
increased the circulating HDL levels in PMW receiving HRT for 12 months
by 6% (P = 0.051 compared with levels of HDL before
initiation of HRT, which were 1.7 ± 0.1 mmol/L. In contrast to
LDL, the increases in HDL levels after HRT were not differential in
responders (P = 0.25) and nonresponders
(P = 0.13).
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Discussion |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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In the current study, changes in circulating levels of NO2-/NO3- were used as an index for changes in NO synthesis. An important issue therefore is whether this methodological approach is valid. In this regard, NO is a labile molecule (5) that decomposes into nitrite and nitrate within seconds of its release. Several in vivo studies have shown that endogenous changes in NO synthesis induced by either L-nitroarginine methyl ester or endotoxin can be measured by analyzing changes in circulating or urinary levels of NO2-/NO3- (11, 12, 22, 23). More importantly, stoichometric metabolic tracer studies by Hibbs et al. (24) using L-[guanidino-15N2] arginine as a substrate for NO synthesis demonstrate that in humans increased nitrate production in serum is derived from NO generated from the terminal guanidino nitrogen atom of the labeled L-arginine.
Although the above studies suggest that NO is the primary source for circulating NO2-/NO3-, dietary nitrates must also be considered. However, it is extremely unlikely that the changes in circulating levels of NO2-/NO3- measured in the present study were caused by dietary factors. This conclusion is based on two lines of reasoning. First, dietary nitrates are excreted in the urine within 18 h of ingestion (25, 26). Because in the present study serum NO2-/NO3- levels were measured approximately 14 h after the last meal (overnight fast), most of the dietary nitrates would have been eliminated at the time of blood sampling. Second, because each subject served as her own control, dietary habits would be the same before and during HRT, thus nullifying any effect of dietary nitrates.
The mechanism(s) by which estradiol induces NO synthesis are unknown. Presumably the effects are receptor mediated because estrogens are well known to interact with high-affinity estrogen receptors in the nucleus and thereby direct the expression of numerous target genes. The findings that estrogen stimulates constitutive nitric oxide synthase activity in cultured human vascular smooth muscle cells (8), and that estrogen-induced increases in blood flow are blocked by L-nitroarginine methyl ester (10) suggest that estrogen receptors might directly and/or indirectly regulate constitutive nitric oxide synthase gene expression. Another possibility is that estrogen-induced changes in lipoprotein levels indirectly result in increased production of NO. For instance, estrogen-induced reductions in LDL and oxidized LDL (2, 3, 27, 28) and increases in HDL (3) should improve overall endothelial/vascular function and diminish vascular disease and thereby augment vascular NO production. Indeed, this latter hypothesis fits well with the slow time course of enhanced NO levels (i.e. >6 months) observed in the present study.
Our finding that serum NO2-/NO3- levels were increased after 12 but not 6 months of treatment indicate that effects of estrogen on NO levels are not rapid. In contrast, short-term increases in estrogen levels in premenopausal women during the menstrual cycle increases NO levels dynamically (11). Estrogen increases transcription of NO synthase in male guinea pigs, but with a marked delay compared with females (29). It has been suggested that these differential vasodilatory effects in males and females are receptor dependent, and that the lag phase in NO synthase transcription in male guinea pigs could be caused by the presence of fewer estrogen receptors. Estrogen receptors within the blood vessels of PMW are lost or decreased (30), and estrogen up-regulates its own receptors (31). Therefore, it is possible that the effects of estrogen are receptor operated, and that the delayed effects of estrogen on NO levels are caused by the initial lack of and subsequent up-regulation of estrogen receptors in PMW; whereas, in premenopausal women the receptors are continuously expressed and primed because of the constant presence/generation of estrogen, thus leading to a rapid receptor mediated increase in NO levels. Alternatively, because increases in NO2-/NO3- levels are accompanied with a decrease in LDL levels, it is feasible that the delayed effects of estrogen replacement on NO levels/production also are indirect and may in part be caused by the effects of estrogen on LDL, free radical generation, and oxidative stress-induced endothelial injury.
An important observation of the present study is that only half of the investigated subjects showed an increase in serum NO2-/NO3- levels during the treatment period. This observation suggests the existence of responders and nonresponders to postmenopausal estrogen treatment with respect to improvement in endogenous NO levels/production; however, the explanation for the dichotomonus response remains obscure. Similar to this finding, is the finding that a heterogeneous response of HRT on LDL previously has been observed (28). One reason for the different reaction of these two groups on estrogen treatment could be the inability of nonresponders to reestablish the estrogen receptors normally lost in postmenopause (30). It has been shown that PMW lose estrogen receptors in the vascular endothelium (30), hence subjects with a widely damaged endothelium, as in atherosclerosis, may be unable to reconstitute estrogen receptors, and therefore there is no increase in NO levels in response to estrogen. Another possibility is that reductions in LDL mediate the increases in NO levels/production. In this regard in our study, serum LDL levels decreased significantly in responders (P = 0.01) but not in nonresponder (P > 0.05) after 12 months HRT compared with baseline values. Linear regression analysis showed a significant negative correlation between LDL and NO2-/NO3- levels in the responders but not in the nonresponders. Although, there is a significant correlation between LDL and NO2-/NO3- in responders, it is not strong enough to establish a cause-and-effect relationship (correlation coefficient value of r2 = 0.185; P = 0.03). These findings suggest that although lowering of LDL does increase NO levels, it is not the sole factor influencing NO levels/production. Hence, based on the above information it could be argued that apart from the largely receptor-mediated increases in NO levels, the increases in NO2-/NO3- in PMW may in part be caused by the lowering of LDL. Furthermore, the above finding implies that the cardiovascular benefit of HRT is higher for a responder than for a non-responder.
Progestins have been reported to impair the cardioprotective effects of
estrogen (32), as well as enhance the vascular damage associated with
hypertension (33). We have shown previously that luteal, but not
follicular, increases in progesterone levels in hormonally stimulated
and in spontaneous menstrual cycles decrease serum
NO2-/NO3- levels, even in the presence of high
concentrations of 17ß-estradiol (11). Furthermore, we have shown
recently that norethisterone acetate (NETA), a commonly used progestin
for HRT in Europe, prevented 17ß-estradiol-induced increases in
circulating NO2-/NO3- levels during HRT (12).
Combined therapy of estrogen with MPA has been shown recently to have
better cardioprotective effects even compared with estrogen alone (3).
NETA is a testosterone-derived progestin with androgenic effects,
whereas MPA and CPA are derivatives of 17
-OH-progesterone.
Therefore, we speculated that compared with NETA, MPA and CPA may have
a different influence on circulating NO2-/NO3-
levels. Similar to our observation with NETA, in contrast to unopposed
estrogen treatment, no significant increase in serum
NO2-/NO3- levels were observed when estradiol
valerate was coadministered with MPA or CPA. Our data suggests that
progestins, whether natural or artificial and whether
17-OH-progesterone or testosterone derivatives, attenuate
estrogen-induced NO levels/production. In this regard, progesterone
inhibits estrogen-induced endothelium-dependent responses associated
with the production of NO (34). However, the exact mechanism(s) by
which progesterone decreases estradiol-induced NO levels is
unclear.
Because of the rapid clearance of orally administered estradiol valerate, the levels of estradiol in the serum were not measured, and this raises the issue as to whether the differential responses observed in the PMW were real or caused by noncompliance. To address this issue, we reevaluated the data from our previous study (12) in which PMW were substituted with transdermal patches, and both estradiol and NO2-/NO3- levels were measured. Similar to the present study, estrogen replacement differentially increased NO2-/NO3- levels in PMW (66% responders and 34% nonresponders), yet circulating estradiol levels in both responders and nonresponders were similar and not statistically different. These findings strongly suggest that the phenomena of differential increases in NO levels in response to estradiol replacement observed in the present study is real.
In conclusion, we provide the first clinical evidence that oral administration of estradiol valerate in PMW for HRT increases circulating NO2-/NO3- levels and lowers LDL levels. Furthermore, the progestins MPA and CPA attenuate estrogen-induced increases in circulating NO2-/NO3- levels. Finally, our data confirm our previous finding and strengthens our contention that the cardioprotective effects of estradiol are mediated at least in part through estradiol-induced NO synthesis. Additionally, these data suggest that irrespective of the route of administration and chemical form of estradiol used, HRT increases endogenous NO activity. Finally, our data also provides evidence for the existence of responders and nonresponders to postmenopausal estrogen treatment with respect to improvement of endogenous NO levels, suggesting that a significant number, but not all, of the hormonally substituted PMW profit fully from the beneficial properties of a HRT.
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Acknowledgments |
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Footnotes |
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2 Visiting scientist at the Department of Gynecology and Obstetrics,
University Hospital, Zurich, Switzerland.
Received June 7, 1996.
Revised October 8, 1996.
Accepted November 7, 1997.
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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, P < 0.05 compared with levels of
nitrite/nitrate in samples collected when subjects were taking
estradiol valerate (E) alone.
