Noha H. Farag, M.D., Bruce A. Barshop, M.D., Ph.D., and Paul J. Mills, Ph.D.

Transcription

1 MENOPAUSE FERTILITY AND STERILITY VOL. 79, NO. 2, FEBRUARY 2003 Copyright 2003 American Society for Reproductive Medicine Published by Elsevier Science Inc. Printed on acid-free paper in U.S.A. Effects of estrogen and psychological stress on plasma homocysteine levels Noha H. Farag, M.D., Bruce A. Barshop, M.D., Ph.D., and Paul J. Mills, Ph.D. Departments of Psychiatry and Pediatrics, University of California, San Diego, San Diego, California Objective: To investigate the effects of estrogen (E) and psychological stress on plasma total homocysteine levels in relation to menopausal status. Design: Double-blind, randomized, placebo-controlled study. Setting: The General Clinical Research Center of a university hospital. Patient(s): Thirty-six postmenopausal women and 26 premenopausal women. Both samples were healthy nonsmokers. Intervention(s): Both premenopausal and postmenopausal women were subjected to a 6-minute psychological stressor. Postmenopausal women were randomized to one of three treatment arms: 2 mg of E 2 or2mg of E 2 5 mg of medroxyprogesterone acetate (MPA), or a placebo, all of which were given orally for 3 months. The psychological stressor was readministered after the 3-month regimen. Main Outcome Measure(s): Plasma total homocysteine levels were measured before and after the psychological stressor on one occasion for premenopausal women and before and after hormone replacement or placebo for postmenopausal women. Result(s): There were no significant differences in homocysteine levels between premenopausal ( mol/l; mean SD) and postmenopausal women ( ; mean SD). There was no effect of stress or hormone replacement on homocysteine levels. Conclusion(s): Psychological stress, menopausal status, and oral hormone replacement therapy (HRT) do not affect plasma total homocysteine levels in women with normal basal homocysteine levels. (Fertil Steril 2003; 79: by American Society for Reproductive Medicine.) Key Words: Homocysteine, menopause, estrogen, progesterone, stress Received March 8, 2002; revised and accepted September 4, Supported by grants AG , MO1-RR00827, and HL from the National Institutes of Health and an American Psychosomatic Society General Clinical Research Center Award. Reprint requests: Noha H. Farag, M.D., University of California, San Diego, Department of Psychiatry, 9500 Gilman Drive, DEPT 0804, La Jolla, California (FAX: ; nfarag@ucsd.edu) /03/$30.00 PII S (02) Homocysteine (thcy) is an amino acid that has been implicated in the development and progression of vascular disease (1 3). Although the specific mechanisms are not definitive, evidence suggests that the increased risk of vascular disease with elevated plasma thcy levels is due to direct damage of the vascular endothelium through the induction of atherosclerotic lesions (4, 5). Exaggerated responses to psychological stress may also contribute to the development of arteriosclerosis by inducing damage to the vascular endothelium (6). The leading cause of death among women in developed nations is coronary heart disease (CHD), the incidence of which increases substantially after menopause (7). Observational studies have demonstrated a lower risk of CHD among postmenopausal women using estrogen (E) supplements compared with nonusers (8 10). The potential beneficial effect of E on CHD risk is multifactorial, including changes in lipid profile, direct effects on the arterial wall, and a decrease in stress reactivity (11, 12), to name a few. It has been suggested that thcy levels may be influenced by E (13). Previous studies examining the differences in thcy concentrations between premenopausal and postmenopausal women have yielded equivocal results (13 15). Findings of clinical trials examining the effects of hormone replacement therapy (HRT) on thcy levels have also been inconsistent (16, 17). Aside from dietary and hormonal modifications, no other behavioral factors have been implicated as potential determinants of plasma thcy. Stoney (18) was the first to test for an effect of acute psychological stress on thcy levels in a sample of women between the ages of 40 and 63 years. In their study, they demonstrated that acute psychological stress in- 256

2 duced rapid and significant elevations in plasma thcy concentrations that were coincident with elevations in blood pressure and heart rate. The purpose of this study was twofold: first, we examined the effects of E on thcy levels, and second, we tested for the effect of psychological stress on thcy levels. To do so, we compared thcy levels in premenopausal and postmenopausal women. We also examined the effects of oral E or E combined with medroxyprogesterone acetate (MPA) therapy on thcy levels in the sample of postmenopausal women. To examine the effects of stress on thcy, we measured thcy levels before and after a standardized psychological stressor in the two study groups. MATERIALS AND METHODS Forty-three postmenopausal women (age range, years; mean, 55; SD 4) and 26 premenopausal women (age range years, M 37; SD 9) were recruited through advertisement and referral. The final sample size for the postmenopausal group was 36 because three subjects were dropped from the analysis due to noncompliance and four subjects dropped out before the follow-up visit. Subjects were studied after obtaining written informed consent that was approved by the University of California San Diego Institutional Review Board. All women were normotensive (BP 140/90 mm Hg). Blood pressure was measured while subjects were sitting upright after a 15-minute rest. Blood pressure was measured using a Dinamap Vital Signs Monitor (Critikon Inc., 845XT, Tampa, FL). A cuff of appropriate size for each subject was used by ensuring that the bladder length was at least 80% and the bladder width was at least 40% of the circumference of the subject s midupper arm. Three blood pressure readings were taken at 1-minute intervals on two separate visits at least 2 weeks apart. The average of the six readings was used to assess normotensive status. All subjects were nonsmokers. Postmenopausal women were eligible to participate if they met the following criteria: [1] absence of a menstrual period for the past 12 months; [2] no HRT for the past 6 months; and [3] FSH levels in the postmenopausal range ( 40 miu/ml). Premenopausal women were not using oral contraceptives (OC) and were tested during days 3 8 of their menstrual cycle. Each participant underwent a physical examination. They were excluded if they had history or displayed current evidence of heart disease, high blood pressure, liver or renal disease, diabetes, psychosis, severe asthma, cerebrovascular disease, or cancer. Subjects were not taking any medications at the time of the study. Procedures Qualified subjects were admitted to the General Clinical Research Center of the UCSD Medical Center. After a light noncaffeine-containing lunch, testing started at 1:00 P.M. An indwelling intravenous cannula was placed in the antecubital vein. After instrumentation subjects were allowed to sit quietly for 30 minutes for habituation to the instrumentation and testing environment. A venous blood sample was drawn at the end of the habituation period. After the 30-minute resting period, subjects were given instructions for performing a public speaking task (19). The task involved preparing and presenting a speech in response to one of two situations, being accused of shoplifting or an injustice at work. The order of the topics was randomly presented across subjects. Subjects were told that the performance would be videotaped and rated by experts on poise and articulation. A video camera was displayed prominently during the procedure. Subjects were given 3 minutes to prepare and 3 minutes to deliver the speech. If subjects stopped talking before the end of the speech, they were reminded to continue the talk by reiterating or summarizing the main points. A second blood sample was drawn at the end of the speech. All venous blood samples were collected in vacutainers containing 7.5% tri-potassium EDTA and placed on ice. Samples were then centrifuged at 4 C for 10 minutes at 3,000 rpm. After centrifugation the plasma was separated from blood cells and stored at 80 C until the thcy determination. Total plasma thcy was determined in anonymous, coded samples by the use of tandem mass spectrometry, as previously reported by Magera et al. (20). For postmenopausal women, after the first testing, subjects were assigned in a double-blind, randomized, placebo controlled fashion, to one of three treatment arms: E 2 2 mg/day (ERT), E 2 2 mg/day MPA 5 mg/day (HRT), or a placebo, which they took orally for 3 months. The randomization process was computer-generated using an SAS program. A random number table was used to assign treatment group randomly within blocks, with a block size of 15. The behavioral stressor was repeated following the 3-month treatment period. Statistical Analysis One-way ANOVA was used to test for statistical differences in age, body mass index (BMI), systolic blood pressure, diastolic blood pressure, heart rate, and basal plasma thcy levels between premenopausal and postmenopausal women and among the three treatment arms of postmenopausal women. To test for the effects of stress and treatment, the data were analyzed using 2 (premenopausal, postmenopausal) 2 (rest, speech) 3 (placebo, ERT, HRT) repeated measures ANOVAs (SPSS for Windows 9.0; SPSS Inc., Chicago, IL). Bonferroni corrections were applied to pair-wise comparisons. Subjects with missing data were dropped from the analysis and the degrees of freedom were adjusted accordingly. FERTILITY & STERILITY 257

3 TABLE 1 Baseline characteristics of premenopausal and postmenopausal women. Baseline Characteristic Premenopausal (n 26) Postmenopausal (n 36) P Value Age (yr) Body mass index (kg/m 2 ) Systolic blood pressure (mm Hg) Diastolic blood pressure (mm Hg) Heart rate (beats/min) Plasma FSH (miu/ml) Plasma E 2 (pg/ml) Basal total homocysteine ( mol/l) Poststress total homocysteine ( mol/l) Values are means SD. P values are obtained from one-way ANOVA. Farag. Homocysteine, menopause, and stress. Fertil Steril RESULTS There were no significant differences between premenopausal and postmenopausal women with regard to BMI, systolic blood pressure, diastolic blood pressure, heart rate, or basal thcy levels (Table 1). There were no significant differences among the three treatment arms in our postmenopausal sample with regard to age, BMI, heart rate, or basal thcy levels (Table 2). The placebo group had higher systolic blood pressure and diastolic blood pressure, which were statistically significant (P.01 and.02, respectively) as compared to the ERT and HRT groups (Table 2). In the repeated measures ANOVA, there was no statistically significant effect of stress on plasma thcy in premenopausal or postmenopausal women (F 1,60 2.1; P.2) (Table 1). When the ANOVA was repeated for postmenopausal women by the three treatment groups there was no effect of stress or treatment on plasma thcy (F 2,29.2; P.8) (Table 2). There was no correlation between age and plasma thcy (Pearson correlation 0.1, P.5). DISCUSSION Menopause is associated with a significant increase in cardiovascular disease in women (7). The use of HRT has been found to reduce cardiovascular morbidity and mortality in postmenopausal women (8 10). Because hyperhomocysteinemia has been found to be a factor involved in cardiovascular disease (1 3), it is possible that the cardioprotective effect of HRT may involve a reduction in thcy levels. However, the findings of previous research have been inconsistent in this regard (13 17). To control for factors that may TABLE 2 Characteristics of postmenopausal women by treatment group. Treatment Group Placebo (n 11) ERT (n 14) HRT (n 13) P Age (yr) Body mass index (kg/m 2 ) Systolic blood pressure (mm Hg) Diastolic blood pressure (mm Hg) Heart rate (beats/min) Pretreatment E 2 (pg/ml) Post-treatment E 2 (pg/ml) Pretreatment basal total homocysteine ( mol/l) Pretreatment poststress total homocysteine ( mol/l) Post-treatment basal total homocysteine ( mol/l) Post-treatment poststress total homocysteine ( mol/l) Values are means SD. P values are obtained from one-way ANOVA. Farag. Homocysteine, menopause, and stress. Fertil Steril Farag et al. Homocysteine, menopause, and stress Vol. 79, No. 2, February 2003

4 have confounded prior thcy research, we studied only healthy, nonobese, nonsmoking women. In this study there were no differences between premenopausal and postmenopausal women in terms of plasma thcy levels. These findings are consistent with those of previous studies that have also been unable to detect differences in thcy levels between pre- and postmenopausal women (21 24). de Bree and colleagues (14) observed an age-related increase in thcy, but were unable to detect an effect of menopause. In our sample there was no correlation between age and thcy levels. We were unable to detect an effect of HRT in the form of E only or E combined with MPA on thcy levels. In previous studies, researchers were able to detect a decrease in thcy levels after hormone replacement only in women with high pretreatment values of thcy (thcy levels, 13.8 mol 1 ) (25, 26). Our sample of postmenopausal women had thcy values in the lower range (range, 5 14 mol 1 ; mean, 7.9; SD 2.1). Perhaps an effect of hormone replacement may have been evident in a sample with higher baseline thcy concentrations. In a sample of the postmenopausal estrogen/progestin intervention (PEPI) trial, hormone replacement had a modest but transient impact on plasma thcy levels during 36 months of follow-up (27). Evio et al. (16) were also unable to detect a change in thcy levels in postmenopausal women after 1 year of treatment with sequential oral or transdermal E 2 plus norethisterone acetate. Aside from dietary and oral hormone modifications, no other behavioral factors have been implicated as potential determinants of plasma thcy. Stoney (18) was the first to detect an effect of brief psychological stress on thcy levels, whereby there was an elevation in plasma thcy levels after acute psychological stress. Stoney suggests that the increase in thcy concentration in response to acute stress is sympathetically mediated. We were unable to replicate such findings. In our sample of pre- and postmenopausal women, there was no significant effect of stress on thcy levels. We used a 6-minute stressor (3 minutes of preparation and 3 minutes of speech). On the other hand, Stoney used a 10-minute stressor period during which two stressors were administered back to back. It is possible that our stressor was too short in duration to induce an effect on thcy levels. Some issues of our study need to be addressed. The blood samples were not collected after a standard 12-hour fast. However, previous studies have shown that there is no significant difference between fasting and nonfasting thcy levels (14, 28, 29). It may have been useful to have information on nutritional factors, as thcy elevations are caused by nutritional deficiencies in vitamin cofactors (30). However, it is not likely that food supplement intake was distributed differently between premenopausal and postmenopausal women or among the three postmenopausal treatment groups; therefore, we doubt that the lack of information on nutritional factors affected the validity of the study results. In conclusion, in this study we were unable to demonstrate a significant difference in thcy concentration between premenopausal and postmenopausal women at rest or in response to a brief psychological stressor. There was also no effect of 3 months of HRT on thcy levels in postmenopausal women. This is not entirely surprising in light of the findings of epidemiological studies that were unable to detect a significant association between thcy and risk of vascular disease among strata of premenopausal and postmenopausal women (15, 31). The hypothesis that thcy mediates the potential protective benefit of E on the cardiovascular system requires further evaluation. 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