Effect of short-term hormone therapy on oxidative stress and endothelial function in African American and Caucasian postmenopausal women

FERTILITY AND STERILITY威 VOL. 79, NO. 5, MAY 2003 Copyright ©2003 American Society for Reproductive Medicine Published by Elsevier Inc. Printed on acid-free paper in U.S.A.

Effect of short-term hormone therapy on oxidative stress and endothelial function in African American and Caucasian postmenopausal women Raymond W. Ke, M.D.,a Diane Todd Pace, Ph.D.,b and Robert A. Ahokas, Ph.D.a University of Tennessee Health Science Center, Memphis, Tennessee

Received June 6, 2002; revised and accepted November 13, 2002. Supported in part by The University of Tennessee Health Science Center. Reprint requests: Raymond W. Ke, M.D., Division of Reproductive Endocrinology, Department of Obstetrics and Gynecology, University of Tennessee Health Science Center, 853 Jefferson Avenue, Rm E-102, Memphis, Tennessee 38163 (FAX: 901-4484701); E-mail: rke@utmem. edu). a Department of Obstetrics and Gynecology. b College of Nursing. 0015-0282/03/$30.00 doi:10.1016/S0015-0282(03) 00153-5

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Objective: In postmenopausal women (PMW), the effect of a short-term course of estrogen/progestin HT on free radical oxidative stress was evaluated. In addition, HT’s effect on plasma nitric oxide (NO) activity was determined as a measure of vascular endothelial function. We investigated the relationship of these markers and HT across race and the cardiovascular risk factors of smoking, diabetes and hypertension. Design: A prospective, observational study comparing preintervention and postintervention. Setting: Academic research center. Patient(s): Twenty-seven (14 African American and 13 Caucasian) PMW volunteers. Intervention(s): Six weeks of continuous, combined estrogen/progestin HT. Main Outcome Measure(s): Plasma concentrations of free 8-epi-prostaglandin F2␣ (8-isoprostane) before and after HT were compared as a measure of oxidative stress. Nitrite, the stable oxidation metabolite of NO, was measured by the Greiss reaction after nitrate reduction to nitrite with cadmium. Results: Plasma levels of free 8-isoprostane decreased significantly after 6 weeks of HT. Although almost all subjects benefited from the reduction in free 8-isoprostane, PMW with at least one cardiovascular risk factor (n ⫽ 19) demonstrated higher free 8-isoprostane than did subjects with no risk factors. Plasma levels of nitrite increased after 6 weeks of HT, but the difference was not statistically significant. Caucasian PMW demonstrated a greater increase in plasma levels of nitrite after 6 weeks of HT as compared with African American subjects, who exhibited almost no change. Conclusion(s): Short-term administration of HT significantly reduces oxidative stress in PMW and is consistent across race. However, there was an observed racial difference in endothelial NO response to HT between African American and Caucasian PMW. (Fertil Steril威 2003;79:1118 –22. ©2003 by American Society for Reproductive Medicine.) Key Words: Hormone therapy, oxidative stress, nitric oxide, menopause, African-American women, Caucasian women

Correction of estrogen deficiency at time of menopause appears to exert a protective effect against coronary heart disease (CHD) in postmenopausal women (PMW) (1). For women who retain their uterus, HT is comprised of a combination of an estrogen along with a progestin to protect against endometrial neoplasia. Proposed mechanisms for HT’s protective action against CHD include a favorable influence upon lipoproteins which may retard vascular atherosclerosis. It would also appear that estrogen acts as an antioxidant to buffer the actions of oxygen free radicals such as hydroxyl radicals (⫺OH), hydrogen peroxide (H2O2) and

superoxide radicals (O⫺ 2 ) (2). These reactive oxygen species attack cell membrane phospholipids and circulating low-density lipoproteins (LDL), which contribute to endothelial damage and promote the formation of foam cells by monocytes/macrophages. Collectively, the damage imparted by oxygen free radicals is termed oxidative stress. In addition, in both humans and animals, estrogen acts directly on the endothelium to promote acute vasodilatation through a mechanism involving release of nitric oxide (NO) and/or prostacyclin (3). A rising awareness of relatively low usage among postmenopausal African Americans had

led health care providers to direct more attention to prescribing HT to all minority women (4). Unfortunately, the purported benefits of postmenopausal HT are almost exclusively derived from data obtained from Caucasian subjects, despite evidence that African American women suffer a higher morbidity and mortality from CHD (5). In a review of 30 studies of HT and CHD, only 173 of 148,437 subjects (0.1%) were African American (6). The assumption that the conclusions from these studies apply to any other racial group other than Caucasian postmenopausal women may be erroneous. In this preliminary study, we evaluated the effect of a short-term course of combination estrogen/progestin HT on both free and total plasma 8-epi-prostaglandin F2␣ (8-isoprostane), a specific metabolite of the oxidation of arachadonic acid bound in cell membrane phospholipids, as a measure of oxidative stress. We also measured the effect of HT on nitrate/nitrite, a stable metabolite of plasma NO and a marker of vascular endothelial function. We investigated the relationship of these markers and HT across race and cardiovascular risk factors, such as smoking, diabetes, and hypertension.

MATERIALS AND METHODS Postmenopausal women were recruited from inner city primary care clinics and asked to participate if they were between ages 45 and 60 years, had an intact uterus with self-reported amenorrhea, had FSH level of ⱖ30 ␮IU/mL and serum estradiol of ⱕ30 pg/mL, and had a willingness to consent to the 6-week study. This study was approved by the institutional review board of the University of Tennessee, Memphis. Upon enrollment, the patients had 20 –30 mL of venous blood drawn after a 12-hour fast. All women were then started on combined, continuous HT consisting of 0.625 mg of conjugated equine estrogens with 2.5 mg of medroxyprogesterone acetate (Prempro, Wyeth Ayerst, Philadelphia, PA), daily for 6 weeks. Venous blood was again collected on the last day of HT immediately before their last tablet and after a 12-hour fast. The plasma was separated and stored at ⫺70°C until analysis.

8-Epi-Prostaglandin F2␣ Assay

Thawed plasma was deproteinated by alcohol precipitation and centrification. Free plasma 8-isoprostane was measured in an aliquot of the supernatant by first acidifying to pH ⬍4.0 with HCl and passed through a Supelclean C-18 SPE cartridge (Supelco, Bellefonte, PA) activated with methanol and water. Total plasma 8-isoprostane was determined in another aliquot of the supernatant after incubation with an equal volume of 15% KOH at 40°C for 1 hour. The hydrolysate was then acidified to pH ⬍4.0 and passed through an activated SPE cartridge. The 8-isoprostane was eluted from the SPE cartridges with 1% methanol in ethyl acetate and evaporated to dryness under a steam of dry FERTILITY & STERILITY威

nitrogen. After redissolving with buffer, the 8-isoprostane concentration was determined by enzyme immunoassay (EIA; Cayman Chemical, Ann Arbor, MI). Recovery of extracted 8-isoprostane was 69%, as determined with 3H-8isoprostane, and the interassay coefficient of variation was 18.8%.

Nitrite Assay The biological half-life of NO is extremely short as it is metabolized in vivo in a few seconds. Monitoring of NO levels is therefore impractical, so indirect methods (e.g., nitrite/nitrate) are the current standard. Nitrite, the stable oxidation metabolite of NO, was measured by the Griess method after cadmium reduction of nitrate to nitrite was performed. Thawed plasma was deproteinated with ZnSO4 and centrifuged. The supernatant was incubated overnight with granular cadmium beads washed with 0.1 M HCl, followed by 0.1 M NH4OH. After centrifugation, the supernatant was mixed with an equal volume of Griess reagent (1% sulfanilamide, 0.1% naphthalene, ethylene diamine dihydrochloride, and 5% H3PO4) and immediately mixed. After 5 minutes, the optical density was read at 550 nm. Statistical analysis was performed with nonpaired and paired Student’s t test.

RESULTS We studied 27 PMW, 14 African American and 13 Caucasian. Nineteen women reported a current history of at least one cardiovascular risk factor consisting of smoking, diabetes mellitus, or hypertension. Eleven of these 19 PMW were African American, and 8 were Caucasian. None of the subjects had experienced a cardiovascular disease event before enrollment. All 27 subjects completed the study. The demographic characteristics of the group are detailed in Table 1. Plasma levels of both total and free 8-isoprostane decreased significantly after 6 weeks of HT (Fig. 1). Mean level of free 8-isoprostane before HT in this sampling of 27 postmenopausal women was 286.0 ⫾ 227.5 pg/mL (mean ⫾ SD). This decreased to a mean of 145.7 ⫾ 103.6 pg/mL after 6 weeks of HRT (P⫽.002, paired t test). There was no difference in baseline levels of 8-isoprostane between African American and Caucasian subjects. The suppression of free and total 8-isoprostane was consistent across race and presence of cardiovascular risk factors (Table 2). Postmenopausal women with at least one cardiovascular risk factor had higher levels of total 8-isoprostane at baseline measurement (314.6 ⫾ 244.3 pg/mL), as compared with the case of eight PMW who reported no risk factors (218.1 ⫾ 176.9, P⫽.06, nonpaired t test). Plasma nitrate/nitrite increased after 6 weeks of HT, but the difference did not reach statistical significance (Fig. 2). Mean nitrite level for all women before HT was 28.1 ⫾ 9.9 ␮M, which increased to 33.1 ⫾ 15.1 ␮M after 6 weeks of HT. Baseline nitrite levels were similar in all subjects across 1119

TABLE 1 Demographic characteristics and cardiovascular risk factors by race. Characteristic

All subjects (N ⫽ 27)

African American (N ⫽ 14)

Caucasian (N ⫽ 13)

54.3 ⫾ 3.5 26.1 ⫾ 5.2 17 (63.0) 18 (66.7) 49.4 ⫾ 5.1 5.6 ⫾ 5.4 10 (37.0) 11 (40.7) 8 (29.6)

54.2 ⫾ 4.2 27.7 ⫾ 4.1 7 (50.0) 10 (71.4) 48.4 ⫾ 4.2 6.0 ⫾ 4.7 5 (35.7) 9 (64.2) 5 (35.7)

54.6 ⫾ 4.5 24.6 ⫾ 5.5 10 (77.0) 8 (61.5) 50.2 ⫾ 6.5 7.9 ⫾ 5.1 5 (38.5) 2 (15.4) 3 (23.1)

Mean age (yr ⫾ SD) Mean BMI (⫾ SD) Completed high school (%) Employed (%) Mean age at menopause (yr ⫾ SD) Years of menopause (⫾ SD) Smoking (%) Hypertension (%) Diabetes (%) Ke. Influence of race on response to HT. Fertil Steril 2003.

race and cardiovascular risk factors. However, a significant racial difference was observed after 6 weeks of HT as Caucasian subjects demonstrated an increase in mean nitrite level of 7.7 ␮M (27.4%), compared with essentially no change in African American subjects (Table 3). This difference persisted even when subjects with at least one cardiovascular risk factor were excluded.

DISCUSSION In this study, short-term administration of continuous, combined conjugated equine estrogen, and medroxyprogesterone acetate to PMW resulted in a dramatic reduction in oxidative stress as measured by 8-isoprostane, the oxidative metabolite of membrane-bound arachadonic acid. This reduction was consistent in both African American and Caucasian subjects. The formation of isoprostanes in vivo seems to reflect primarily, if not exclusively, a nonenzymatic process of lipid peroxidation (7). Isoprostanes are a family of prostaglandin F2␣ isomers that have resulted from free radical attack on arachidonic acid bound to cell membrane phospholipids. Their formation results in alterations in the fluidity and the integrity of cellular membranes. Formation of isoprostanes on the cell membranes of LDL leads to their uptake by monocytes/macrophages, resulting in the formation of foam cells. In addition, 8-isoprostane is a potent vasoconstrictor and modifies platelet function such as adhesive reactions (7). All these factors contribute to the proliferation of atherosclerotic disease. Our findings would support the concept that conjugated estrogens, progestin, or both act as an antioxidant in vivo. Estrogen structures contain a phenol ring that effectively scavenges hydroxyl radicals, giving rise to hydroxylated products (8). In vitro, certain components of conjugated equine estrogens such as ⌬8,9-dehydroestrone, 17␤-dihydroequilenin and 17␣-dihydroequilenin contain unsaturated ring B structures and are particularly potent inhibitors of LDL oxidation exceeding that of 17␤-estradiol and vitamin E (9). Ex vivo, Bhavnani et al. (10) demonstrated that serum from 1120 Ke et al.

Influence of race on response to HT

PMW treated with ⌬8,9-dehydroestrone was able to prolong the lag time to oxidation of LDL. However, it is not clear whether the antioxidant effect is limited to only exogenous, pharmacological forms of estrogen. In serum from both premenopausal women and PMW, varying endogenous estradiol levels did not correlate with rate of LDL oxidation. Only at extremely high levels of endogenous estradiol (⬎2,000 pg/mL), obtained from women undergoing fertility therapy, was LDL oxidation inhibited (11). Morrow et al. (12) measured free 8-isoprostane from fresh plasma of 12 normal female volunteers and found a mean level of 35 ⫾ 6 pg/mL, N ⫽ 12 (12). In comparison, our population of older PMW exhibited relatively high baseline levels of oxidative stress (286.0 ⫾ 227.5 pg/mL). This may reflect their age or their relative estrogen deficiency as PMW. Our study population also exhibited a high incidence of obesity, smoking, and diabetes. Persistently enhanced

FIGURE 1 Change in 8-isoprostane for all subjects after 6 weeks of HT.

Ke. Influence of race on response to HT. Fertil Steril 2003.

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TABLE 2 Free and total 8-isoprostane before and after 6 weeks of HT. Free 8-isoprostane (pg/mL) Parameter African American (N ⫽ 14) Caucasian (N ⫽ 13) No CV risksb (N ⫽ 8) One or more CV risks (N ⫽ 19)

Total 8-isoprostane (pg/mL)

Baseline

After HT

Change

Baseline

After HT

Change

291.3 ⫾ 197.0 280.4 ⫾ 264.6 218.1 ⫾ 176.9 314.6 ⫾ 244.3

153.6 ⫾ 107.3 136.6 ⫾ 103.1 116.0 ⫾ 63.5 156.7 ⫾ 114.4

⫺137.7a ⫺143.8a ⫺102.1a ⫺157.9a

1025.4 ⫾ 1228.4 1004.2 ⫾ 1304.0 643.0 ⫾ 451.6 1,171.8 ⫾ 1,434.2

420.7 ⫾ 356.6 482.4 ⫾ 351.9 429.9 ⫾ 470.1 456.3 ⫾ 308.2

⫺604.6a ⫺521.8a ⫺216.7 ⫺715.5a

Note: CV ⫽ cardiovascular. a Baseline vs. after HT: P⬍.05, paired t test. b CV risk factors: a current history of smoking, diabetes mellitus, or hypertension. Ke. Influence of race on response to HT. Fertil Steril 2003.

formation of isoprostanes has been reported in association with these risk factors as well as hypercholesterolemia (7). Our results confirmed that patients with one or more of these cardiovascular risk factors exhibit higher levels of total 8-isoprostane as compared with patients without risk factors. We did not observe a difference in baseline 8-isoprostane levels between African Americans and Caucasians. Our results revealed a small increase in NO metabolites after 6 weeks of combined estrogen and progestin therapy that did not reach statistical significance. This may reflect inadequate power for our sample size or alternatively, combined therapy with medroxyprogesterone acetate may have attenuated the expected estrogen response. Both oral and transdermal estrogen therapy alone increase NO activity, as measured by nitrate/nitrite levels, with transdermal 17-␤ estradiol producing a significant effect as early as 24 hours after administration (13, 14). In vivo, estrogen administration promotes vasodilatation in part by stimulating NO synthesis in the endothelium and inducing the enzyme that

catalyzes its formation (3, 14). In 12 postmenopausal subjects, acute administration of conjugated equine estrogens augmented both NO-mediated and non–NO-mediated forearm vasodilatation (15). However, Imthurn et al. (16) found the increase in NO levels with estradiol treatment was not uniform in all PMW, with only 13 of 26 subjects increasing nitrite/nitrate levels by ⬎30%. Furthermore, the induction of NO activity by long-term oral estrogen therapy could be nullified by concomitant progestin administration. The mechanism is unclear but is additional support for the detrimental effect of progestin therapy on the cardiovascular system in PMW. Our inability to demonstrate a large effect on NO activity may reflect the racial makeup of our population. We observed that although Caucasian subjects demonstrated a highly significant 27.4% increase in NO metabolites with HT, this response was absent in African American subjects. The discrepancy in NO response was consistent even after excluding subjects with cardiovascular risk factors. The explanation for such a divergent response is unknown. It may reflect a difference in diet or physical activity, both of which

FIGURE 2 TABLE 3

Change in nitrate/nitrite after 6 weeks of HT for all subjects.

Nitrite/nitrate levels before and after 6 weeks of HT. Nitrate/nitrite (␮M) Parameter African American (N ⫽ 14) Caucasian (N ⫽ 13) No CV risksb (N ⫽ 8) One or more CV risksb (N ⫽ 19)

Baseline

After HT

Change

28.0 ⫾ 11.9 28.2 ⫾ 7.7 24.6 ⫾ 6.8 29.5 ⫾ 10.8

27.6 ⫾ 12.2a 39.0 ⫾ 16.0a 30.3 ⫾ 9.3 34.2 ⫾ 17.0

⫺0.4 7.7 3.1 3.7

Note: CV ⫽ cardiovascular. Post-HT values for African American vs. Caucasian: P⫽.04, nonpaired t test. b CV risk factors: a current history of smoking, diabetes mellitus, or hypertension. a

Ke. Influence of race on response to HT. Fertil Steril 2003.

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Ke. Influence of race on response to HT. Fertil Steril 2003.

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could modulate nitrite/nitrate determination (17). Alternatively, our findings may provide evidence of a racial or genetic difference in endothelial response to HT. In summary, we demonstrated that estrogen/progestin replacement therapy in PMW resulted in a beneficial reduction in oxidative stress after 6 weeks of treatment. However, we could not conclusively confirm that HT had a similar protective effect on increasing NO activity. This may be due to an observed racial discrepancy, as African American subjects showed no increase in measured NO metabolites after HT as opposed to Caucasian subjects demonstrating the expected increase. We intend to expand our study of African American endothelial response in a larger study. References 1. Stampfer MJ, Willet WC, Colditz JA, Rosner B, Speizer FE, Hennekens CH. A prospective study of postmenopausal estrogen therapy and coronary heart disease. N Engl J Med 1985;313:1044 –9. 2. Subbiah MTR, Kessel B, Agrawal M, Rajan R, Abplanalp W, Rymaszewski Z. Antioxidant potential of specific estrogens on lipid peroxidation. J Clin Endocrinol Metab 1993;77:1095–7. 3. Guetta V, Quyyumi AA, Prasad A, Panza JA, Waclawiw M, Cannon RO. The role of nitric oxide in coronary vascular effects of estrogen in postmenopausal women. Circulation 1997;96:2795–801. 4. Nabulsi AN, Folsom AR, White A, Patsch W, Heiss G, Wu KK, et al. Association of hormone-replacement therapy with various cardiovascular risk factors in postmenopausal women. N Engl J Med 1993;328: 1069 –75. 5. Lenfant C. Report of the NHLBI working group on research in coronary heart disease in blacks. Circulation 1994;90:1613–23. 6. Nicholson WK, Brown AF, Gathe J, Grumbach K, Washington AE, Perez-Stable EJ. Hormone replacement therapy for African American women: missed opportunities for effective intervention. Menopause 1999;6:147–55.

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