Influence of Sex Hormones on Plasma Endothelin Levels

Atherosclerosis and hypertension are more common in men than in women, a difference that may be due, in part, to the actions of their respective sex hormones. Men have more atherogenic lipid profiles than women, and sex hormones play an important role in the development of this difference [1-3]. In addition, some evidence has emerged that androgens can induce insulin resistance [3, 4]; several case reports describe cardiovascular events in young persons after the use of anabolic steroids [5, 6]. Sex hormone-associated differences in lipid profiles and insulin sensitivity may partly explain why premenopausal women are relatively protected against cardiovascular disease. It remains to be determined, however, whether the difference between men and age-matched women in the incidence of atherosclerosis can be explained by differences in lipid profiles and insulin sensitivity alone or whether other mechanisms might be involved. Endothelial cells synthesize many active substances that regulate local blood pressure and maintain the fluidity of blood and the patency of blood vessels [7]. These include vasodilators such as endothelium-derived relaxing factor and prostacyclin (which also inhibit platelet adhesion and aggregation), vasoconstrictors such as endothelin, and larger molecules such as fibronectin. Endothelin may also have mitogenic properties [7, 8]. The release of these substances affects the local environment in the blood vessel; endothelin may also have a systemic function [7]. Some evidence has emerged that endothelin may be involved in the pathogenesis of hypertension [7, 9] and atherosclerosis [10]. Elevated plasma levels of endothelin have been observed in patients with myocardial infarction and diabetes. Whether sex hormones have direct effects on the endothelium is not known. We found that plasma endothelin levels tended to be higher in men than in women and lower in pregnant women than in nonpregnant controls. These considerations prompted us to compare endothelin levels in healthy young men and women and to assess the effects of long-term sex hormone therapy in male-to-female and female-to-male transsexuals on plasma endothelin levels. Methods We measured endothelin levels in 29 healthy young women (18 to 31 years of age; mean age, 24.1 years), 20 pregnant women (20 to 32 years of age; mean age, 26.3 years), and 23 healthy men (23 to 33 years of age; mean age, 24.7 years). To assess the effects of sex hormone therapy on plasma levels of endothelin, we measured endothelin levels before and during sex hormone therapy in 12 male-to-female transsexual patients (17 to 33 years of age; mean age, 28.4 years) and 13 female-to-male transsexual patients (17 to 26 years of age; mean age, 24.4 years). Informed consent was obtained from all participants, and the study was approved by the hospital ethics committee. All patients were within 10% of their ideal body weight (Metropolitan Life Insurance Tables, 1959). None had a personal or family history of diabetes or hypertension or had evidence of cardiovascular disease on routine examination (medical history, physical examination, and electrocardiogram). No hormone preparations (such as oral contraceptives) had been used by any of the participants in the 6 months before the study. All women had a regular menstrual cycle (28 to 31 days) before hormone treatment; blood samples were drawn during the follicular phase of the menstrual cycle (days 3 to 5). Female-to-male transsexuals received intramuscular injections of testosterone esters (Sustanon, Organon, Oss, the Netherlands), 250 mg every 2 weeks. Male-to-female transsexuals received oral ethynylestradiol (Lynoral, Organon), 0.1 mg/d, and cyproterone acetate (Androcur, Schering, Weesp, the Netherlands), 100 mg/d, to counteract the effects of testosterone. Plasma endothelin levels were measured before therapy and after 4 months of hormone use. Blood samples were drawn between 0900 hours and 0930 hours after an overnight fast, patients having rested in the supine position for at least 30 minutes. Samples were immediately placed in ice. Testosterone levels were determined 10 to 14 days after injection, and blood samples were drawn simultaneously with those in which plasma endothelin levels were determined. Plasma was separated within 1 hour and then stored at 20C until assayed. Plasma endothelin was measured by radioimmunoassay (Nichols Institute [formerly ITS], Wijchen, the Netherlands) after extraction on Sep-Pak C18 cartridges (Waters, Milford, Massachusetts), as described previously [17]. Recovery rate for this assay is 92.4%. Intra-assay and interassay coefficients of variation according to the manufacturer are 2.4% and 4.2%, respectively. (We found these values to be slightly higher: 3.6% and 5.1%, respectively.) Sensitivity of the assay is 1 pg/mL; cross-reactivity with endothelin-2 is 52%; with endothelin-3, 96%; and with big endothelin, 7%. Intra-assay variation ranged from 2% to 8%, and interassay variation ranged from 4% to 9%. In all patients, blood pressure was determined during a 2-hour period using an automatic sphygmomanometer; patients remained at rest during this period. Blood pressure measurements were done every 5 minutes, and the results were averaged. Results are expressed as mean SD. The Student two-tailed t-test for paired data was used to compare measurements within the same group before and during hormone therapy. The Student two-tailed unpaired t-test was used for between-group comparisons. A P value of less than 0.05 was considered statistically significant. Results Plasma endothelin levels were significantly higher in men than in women (5.9 1.2 compared with 4.17 0.67 pg/mL; P < 0.01). Endothelin levels were lower in pregnant women than in nonpregnant controls (2.19 0.73 compared with 4.17 0.67 pg/mL; P < 0.01), suggesting that high levels of estradiol and progesterone found during pregnancy are associated with low endothelin levels (Figure 1). Figure 1. Plasma endothelin levels in 23 men, 29 women, and 20 pregnant women. P P In biological women treated with intramuscular injections of testosterone esters, basal testosterone levels increased from 1.25 0.66 nmol/L to 24.8 13.0 nmol/L (P < 0.01). No significant change was seen in levels of estradiol-17 (247 207 compared with 185 94 pmol/L). Biological men were treated with oral ethynylestradiol, 0.1 mg/d, and cyproterone acetate, 100 mg/d. Testosterone levels decreased from 18.4 8.6 to 1.1 0.4 nmol/L in this group (P < 0.01). Levels of estradiol-17 and ethynylestradiol were not assessed. The biological effects of sex hormone therapy were manifest in both groups. Effects of sex hormone therapy on endothelin levels in both groups are shown in Figure 2. Endothelin levels before treatment tended to be higher in men than in women (8.1 3.1 compared with 6.2 1.1 pg/mL; P = 0.06), confirming our earlier observations. In biological women treated with testosterone, average endothelin levels increased from 6.2 1.1 to 7.8 1.2 pg/mL (P < 0.01) after 4 months of therapy. In men treated with estradiol and cyproterone acetate, endothelin levels decreased from 8.1 3.0 to 5.1 2.0 pg/mL (P < 0.01). Thus, the decrease in endothelin levels in men was greater than the increase in women treated with testosterone (37% and 26%, respectively); antiandrogenic actions of cyproterone acetate may have contributed to the decrease in endothelin levels in men. Figure 2. Effects of cross-gender sex hormone treatment on plasma endothelin levels in transsexual patients. Left panel. P Right panel. Blood pressure was measured in all participants (at rest) during a 2-hour period before and during hormone therapy. No significant alterations were seen; mean systolic and diastolic pressures were unchanged. To investigate the variability of endothelin plasma levels, we measured endothelin levels twice within a period of 2 months in 10 normal men. Endothelin levels were 5.90 0.85 pg/mL at the first measurement and 5.85 1.05 pg/mL at the second measurement (P 0.05), the coefficient of variation being 16.2%. These data suggest that endothelin levels are relatively stable over a period of several months. Discussion To our knowledge, this is the first study to compare plasma endothelin levels in healthy men and women. Our observations suggest that endothelin levels are higher in men than in women and that this difference is mediated by sex hormones. Atherosclerosis is characterized by endothelial injury and the proliferation of intimal smooth-muscle cells, which may be a result of the release of growth factors from the vessel wall [11]. The evidence suggests that endothelin, which is a strong vasoconstrictor, also has mitogenic properties [7, 8]. A correlation between endothelin levels and atherosclerosis has been described [10], suggesting that endothelin might participate in atherogenesis. Thus, if sex hormones affect levels of endothelin, this might be one of the mechanisms by which sex hormones influence the risk for cardiovascular disease. We speculate that a sex-associated difference in levels of endothelin may be one of the mechanisms underlying the difference in the incidence of cardiovascular disease between men and women. In our patients, blood pressure did not change during hormonal treatment. Asscheman and colleagues [12] assessed blood pressure in 425 transsexual patients (303 biological men and 122 biological women) receiving long-term sex hormone therapy. High blood pressure developed in 10 biological men treated with estrogens (2.4% of the total group of 425 patients) and in none of the women treated with testosterone. Studies of the effect of estrogen therapy on blood pressure in postmenopausal women [13, 14] and in women using oral contraceptives [15] have produced conflicting results. Thus, the effect of sex hormone therapy on blood pressure remains unclear. In healthy men, the intravenous administration of endothelin induces an increase in blood pressure and serum p