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The Effect of Estrogen Replacement Therapy on Plasma Nitric Oxide and Endothelin-1 Levels in Postmenopausal Women
- Patricia J.M. Best, MD;
- Peter B. Berger, MD;
- Virginia M. Miller, PhD; and
- Amir Lerman, MD
- From the Mayo Clinic and Mayo Foundation, Rochester, Minnesota. Grant Support: By grants HL07111-21D and HL51736 from the National Institutes of Health, the Miami Heart Research Institute, the Rappaport Program in Vascular Biology, and the Mayo Foundation. Requests for Reprints: Amir Lerman, MD, Department of Internal Medicine and Cardiovascular Diseases, Mayo Clinic, 200 First Street SW, Rochester, MN 55905. Current Author Addresses: Drs. Best, Berger, and Lerman: Division of Cardiovascular Diseases and Internal Medicine, Mayo Clinic, 200 First Street SW, Rochester, MN 55905.
Abstract
Background: Estrogen replacement therapy (ERT) in postmenopausal women decreases cardiac mortality and improves endothelial function. The endothelium regulates vascular tone and growth by releasing such factors as nitric oxide and endothelin-1.
Objective: To determine whether ERT alters the balance between the total oxidized products of nitric oxide and endothelin-1.
Design: Single-arm, before–after clinical trial.
Setting: Outpatient clinical research center of an academic medical center.
Patients: 15 postmenopausal women.
Intervention: Treatment with 17β-estradiol for 6 months and a 10-day course of methoxyprogesterone every 3 months.
Measurements: Plasma nitric oxide and endothelin-1 levels were measured at baseline and after 6 months of ERT.
Results: The mean baseline nitric oxide level was 27.5 nmol/mL (range, 20.3 to 34.8 nmol/mL) and increased by a mean of 7.2 nmol/mL (range, 0.2 to 14.1 nmol/mL) (P = 0.04). The mean baseline plasma endothelin-1 level was 16.4 pg/mL (range, 12.0 to 20.8 pg/mL) and decreased by a mean of 3.9 pg/mL (range, 0.4 to 7.8 pg/mL) (P = 0.04). The mean baseline ratio of nitric oxide to endothelin-1 was 2.0 (range, 1.3 to 2.8) and increased by 1.2 (range, 0.1 to 2.2) (P = 0.03).
Conclusion: ERT results in an increased ratio of nitric oxide to endothelin-1. This may be one mechanism by which ERT provides cardiovascular benefit.
Cardiovascular disease is the leading cause of illness and death in women. Estrogen replacement therapy (ERT) in postmenopausal women decreases total mortality and cardiovascular mortality [1]. This therapy increases coronary vasodilatation in response to acetylcholine and decreases angiographically detectable coronary artery disease [2, 3]. Alterations in nitric oxide and endothelin-1 levels have been hypothesized to be one of the mechanisms by which ERT provides cardiovascular benefit [4].
Nitric oxide and endothelin-1 are endothelium-derived vasoactive factors that are important in cardiovascular disease [5, 6]. Nitric oxide causes vasodilatation, inhibits platelet aggregation, suppresses smooth-muscle proliferation, and acts as an anti-atherogenic factor [5, 7]. Continuous release of nitric oxide from the endothelium maintains basal vascular tone, and alterations in nitric oxide release allow autoregulation of blood flow [8]. Both endogenous substances and physical forces, such as shear stress, stimulate production of nitric oxide. Alternatively, endothelin-1 opposes the effects of nitric oxide. It causes potent vasoconstriction of the systemic and coronary vasculature through the endothelin receptors, increases monocyte adhesion, activates macrophages, and promotes vascular smooth-muscle cell proliferation and migration [9].
In addition to the opposing effects of nitric oxide and endothelin-1 on vascular tone and smooth-muscle cell proliferation, the regulatory mechanisms of these vasoactive factors interact. Nitric oxide inhibits the production of endothelin-1 and modulates both the number and affinity of the endothelin-A receptors. Nitric oxide synthase is functionally coupled to the endothelin-B receptor [10]. In such disease states as hypercholesterolemia, atherosclerosis, and congestive heart failure, imbalances between nitric oxide and endothelin-1 are detected and may contribute to both vasomotor abnormalities and vascular remodeling [9]. We tested the hypothesis that ERT increases the ratio between plasma total oxidized products of nitric oxide and circulating endothelin-1.
Methods
Participants
After the institutional review board approved our study, participants were recruited by local announcements. All participants gave informed consent. Participants were healthy, ambulatory women who were 40 to 65 years of age, had no contraindications to ERT, had not undergone surgically induced menopause, had not had a menstrual period in the preceding year, and had a follicle-stimulating hormone level greater than 50 IU/L and a serum 17β-estradiol level of 100 pmol/L or less. At the beginning of the study, a complete history, physical examination, laboratory evaluation (chemistry and hematology panel), and electrocardiography were done at the Mayo Clinic for each participant. Exclusion criteria were clinical or laboratory abnormalities that suggested cardiovascular, hepatic, or renal disorders; coagulopathy; use of oral or transdermal estrogen, progestin, androgen, or other steroids in the preceding year; smoking habit of more than 10 cigarettes per day; or therapy with cholesterol-lowering or cardiovascular medications. All patients agreed not to alter their diet and exercise regimens during the study period.
Study Protocol
All participants had a baseline cholesterol profile, and plasma nitric oxide and endothelin-1 were measured on samples obtained while participants were fasting to minimize dietary effects. Participants received estradiol-17β, 2 mg daily for 6 months, and a 10-day course of methoxyprogesterone, 5 mg daily every 3 months. To minimize the effects of the methoxyprogesterone, repeated daily laboratory measurements were done before the second course of methoxyprogesterone on samples obtained while participants were fasting.
Assays
Plasma endothelin was measured by using an endothelin-1,2[I125] assay system (Nycomed Amersham, Bucks, United Kingdom) as described elsewhere [6]. Blood was drawn, and plasma was separated. The efficacy of the extraction procedure was 81%; interassay variations were 9%, and intra-assay variations were 5%. In this assay, cross-reactivities were less than 5% for endothelin-2, less than 3% for endothelin-3, and less than 37% for proendothelin [6]. Previously established normal values (±SD) for plasma endothelin-1 are 7.1 ± 0.1 pg/mL [11].
Nitric oxide was measured by chemiluminescence with a Sievers Nitric Oxide Analyzer, model 280 (Boulder, Colorado) [12]. Blood samples were centrifuged at 2500 rpm for 20 minutes at 10°C. The supernatant was removed and stored at −70°C.The nitric oxide assay was standardized by a calibration curve using known concentrations of nitrate (0.01 to 100 µmol/L) obtained from sodium nitrate. For each measurement, a 4-µL sample was placed in a reducing vessel with 5 mL of 0.1 mol of vanadium III chloride per L, 1 mol of hydrochloric acid per L, and 100 µL of antifoaming agent (Sievers) at 90°C. Each standard was analyzed three times, and each plasma sample was analyzed at least five times. The mean value was used for all subsequent analysis.
Total cholesterol, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, and triglyceride levels were measured in the Mayo Clinic ImmunoChemistry Laboratory (Roche Diagnostic Systems, Branchburg, New Jersey).
Statistical Analysis
Data are given as the mean ±SD with 95% CIs and ranges. Comparisons before and after ERT were based on paired sample t-tests. A P value of 0.05 or less was considered statistically significant. Correlations were determined by using the Pearson correlation equation.
Results
The mean age of the 15 women who received ERT was 53 years (range, 46 to 58 years). Two patients were current smokers of 10 cigarettes per day, 3 patients had a history of smoking, and 2 patients had a substantial family history of coronary artery disease. The mean baseline level of follicle-stimulating hormone was 79.1 ± 21.0 IU/L (range, 43.8 to 120.0 IU/L). The data on nitric oxide, endothelin-1, and cholesterol are summarized in the Table 1, and individual patient data are shown in the (Figure 1). The mean nitric oxide level was 27.5 nmol/mL (range, 20.3 to 34.8 nmol/mL) at baseline; this increased by a mean of 7.2 nmol/mL (range, 0.2 to 14.1 nmol/mL) (P = 0.04) after 6 months of ERT. The mean plasma endothelin-1 level was 16.4 pg/mL (range, 12.0 to 20.8 pg/mL) at baseline; this decreased by 3.9 pg/mL (range, 0.4 to 7.8 pg/mL) (P = 0.04) after 6 months of ERT. Before ERT began, endothelin-1 levels were significantly higher than the control value of 7.1 ± 0.1 pg/mL. With ERT, total and low-density lipoprotein cholesterol levels decreased (P = 0.005 and P = 0.004, respectively) and high-density lipoprotein cholesterol levels increased (P = 0.003). Use of ERT did not change systolic blood pressure, diastolic blood pressure, or heart rate (P < 0.2 for all comparisons). No correlation existed between estradiol levels and nitric oxide (r = 0.04) or endothelin-1 (r = −0.18)or between plasma nitric oxide and endothelin-1 concentrations (r = 0.05). The ratio of nitric oxide to endothelin-1 increased from 2.0 (range, 1.3 to 2.8) by a mean of 1.2 (range, 0.1 to 2.2) after ERT (P = 0.03).
Discussion
Our study shows that in postmenopausal women, ERT increases plasma nitric oxide levels and decreases plasma endothelin-1 levels, thereby increasing the ratio of nitric oxide to endothelin-1. A previous study by Roselli and coworkers [13] showed that ERT in postmenopausal women increases levels of circulating nitrate and nitrite. Ylikorkala and colleagues [14] reported that ERT in postmenopausal women decreases plasma endothelin-1 levels. Our study extends these observations and shows the effect of ERT on nitric oxide and endothelin-1 levels in the same study group. It also shows that these changes are associated with improvement in the lipid profile.
However, a subset of patients in whom ERT does not increase the ratio of nitric oxide to endothelin-1 may exist. The therapy failed to improve the ratio of nitric oxide to endothelin-1 in two patients. Our results may be limited by the small sample size and the large variability in responses to ERT among study participants.
Our study did not address the mechanism by which ERT alters these vasoactive substances, but we can suggest several possibilities. The therapy may regulate nitric oxide by increased production of nitric oxide synthase [15], and additional evidence suggests that estrogen can directly increase the release of nitric oxide [16].
Another possible mechanism for the increase in nitric oxide seen with ERT is the beneficial effects of estrogen on lipid metabolism. The therapy lowers total cholesterol and low-density lipoprotein cholesterol levels and increases high-density lipoprotein cholesterol levels; all of these reductions reduce the risk for cardiovascular events. Although the beneficial effects of estrogen on cardiovascular disease are mediated through this lipid-lowering effect, not all of the benefits of estrogen relate to cholesterol [17]. Because hypercholesterolemia is associated with impaired vascular tone (probably secondary to decreased bioavailability of nitric oxide), decreased cholesterol levels may increase vascular nitric oxide and may be another mechanism by which estrogen exerts its effect [18].
Through its antioxidant effects, estrogen may also decrease nitric oxide degradation. This potent inhibitor of lipid peroxidation may hinder the accelerated degradation of nitric oxide that results from the increased production of oxide free radicals associated with hypercholesterolemia [19]. Thus, estrogen may increase systemic nitric oxide levels through multiple mechanisms, improving endothelium function and maintaining the critical balance between nitric oxide and endothelin-1.
In our study, endothelin-1 concentrations decreased in response to ERT and returned to levels considered by previous studies to be in the normal range after ERT [11]. The mechanism by which estrogen may affect endothelin-1 concentrations remains a matter of speculation. Hormone levels have clearly been shown to alter endothelin-1. Pregnant women have lower endothelin-1 levels than nonpregnant women [20]. In cross-sex hormone therapy, treatment of men with estrogen and cyproterone decreases endothelin-1 levels and treatment of female-to-male transsexual patients with testosterone increases endothelin-1 levels [20]. Thus, altered endothelin-1 after hormonal manipulation is consistent with our study results, which show reduced endothelin-1 levels after ERT.
In conclusion, the ratio between nitric oxide and endothelin-1 increases after ERT in postmenopausal women. These interactions between nitric oxide and endothelin-1 may be one mechanism for the cardiovascular benefit provided by ERT.
Dr. Miller: Department of Surgical Research, Mayo Clinic, 200 First Street SW, Rochester, MN 55905.
- Copyright ©2004 by the American College of Physicians
