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Relationship between Serum Estradiol Levels and the Increases in High-Density Lipoprotein Levels in Postmenopausal Women Treated with Oral Estradiol¹

Departments of Obstetrics and Gynecology (B.M.W.) and Medicine (F.M.S.), Brigham and Women’s Hospital, and the Departments of Nutrition (F.M.S.), Epidemiology (D.S., M.M.), and Biostatistics (D.S., M.M.), Harvard School of Public Health, Boston, Massachusetts 02115

Address all correspondence and requests for reprints to: Brian M. Walsh, Department of Obstetrics and Gynecology, Brigham and Women’s Hospital, 75 Francis Street, Boston, Massachusetts 02115. E-mail: bwwalsh{at}bics.bwh.harvard.edu

	Abstract

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Postmenopausal women are prescribed a standard dose of estrogen, which is optimal for a population but not for all individuals. We wished to identify if an individual’s estradiol level can indicate the minimum effective dose of estrogen which maximally increases high-density lipoprotein (HDL) levels, which could be cardioprotective. We performed a prospective, double-blind crossover study in 19 healthy postmenopausal women, receiving three treatments in random order for 9 weeks each: a) placebo, b) 1 mg oral estradiol daily, and c) 2 mg oral estradiol daily. Lipoprotein and estradiol (E₂) levels were measured 10–12 h after pills were taken. E₂ levels with 1 mg estradiol were positively correlated with the increases in HDL levels (r = 0.70, P < 0.01). Only the eight subjects who had E₂ levels < 50 pg/mL after 1 mg estradiol treatment demonstrated further increases in HDL levels by increasing the daily dose to 2 mg (by 3 ± 5% with 1 mg estradiol and by 13 ± 7% with 2 mg). The other 11 subjects who had E₂ levels > 50 pg/mL with 1 mg estradiol had no additional benefit from increasing the estradiol dose (HDL increased by 13 ± 9% with 1 mg, and by 17 ± 10% with 2 mg). Thus, measurement of an E₂ level the morning after taking 1 mg estradiol at bedtime identifies who may benefit from improvement in HDL levels by increasing to a 2-mg dose.

	Introduction

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IN CLINICAL PRACTICE, the doses of most drugs are titrated within individual patients to achieve a desired clinical effect. Optimal therapeutic benefit may therefore be obtained without incurring unnecessary risks or toxicities. The only exceptions are drugs which have wide therapeutic windows, such as antibiotics. Most hormonal drugs prescribed for endocrine gland failure, in contrast, are carefully titrated. For example, patients with diabetes mellitus are given varying doses of insulin to achieve euglycemia; patients with hypothyroidism undergo adjustments in their thyroxin dose to obtain physiologic thyroid-stimulating hormone levels. The notable exception in hormonal treatment is the dosing of postmenopausal estrogen replacement. At present, most women are prescribed standard doses of estrogen, despite the fact that estrogen pharmacokinetics varies among individuals. For example, postmenopausal women treated with estrogen achieve lower circulating estrogen levels if they smoke cigarettes (1) but higher levels if they consume alcohol (2). In addition, thin postmenopausal women have lower estrogen levels than do obese women (3), so that they typically experience more menopausal symptoms (4) and have a higher incidence of osteoporosis (5). They may thus require treatment with higher doses of estrogen to achieve a therapeutic level.

The current practice of prescribing standard doses of estrogen may cause many women to be undertreated, and to not accrue maximal benefit, whereas others may be overtreated and be at undue risk for conditions such as breast and endometrial cancer. Titrating estrogen dose for each individual could be advantageous, particularly because millions of women currently take estrogens for several years. However, it is currently unclear how to titrate the dose because estrogens act on multiple end-organs that differ in their dose-response relationships. It may be necessary to select which effect of estrogen is paramount, and to adjust dose to optimize that particular effect. The most beneficial effect of estrogens may cardioprotection: estrogen users have as little as half the incidence of cardiovascular disease compared with nonusers (6). This may be mediated by estrogen’s ability to raise high-density lipoprotein (HDL) levels and to lower low-density lipoprotein (LDL) levels (7). Because HDL is the most powerful inverse predictor of heart disease among women (8), it may be useful to identify the blood estrogen level required to achieve a maximal increase in HDL levels. We thus performed a dose-response study to identify the mean changes in the levels of HDL, LDL, and other lipids in postmenopausal women given oral estradiol, and to identify if the estrogen levels achieved by the estradiol are related to the magnitude of the increase in HDL levels. This could identify the minimum effective dose of estrogen that maximally increases HDL levels. A secondary objective was to determine the time course of these effects, to establish how long a patient would need to take a given dose of estradiol before having lipids measured.

	Materials and Methods

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Subjects

Healthy postmenopausal women were eligible if they were amenorrheic for at least 1 yr, had a body-mass index (BMI) between 18 and 31 kg/m², and had serum FSH levels greater than 40 IU/L. To eliminate confounding factors, which may alter lipoprotein levels, we excluded women who smoked, consumed more than 28 mL of ethanol daily, had hypertension requiring medical treatment, or had diabetes mellitus. We excluded women with a history of breast or uterine cancer or of thrombophlebitis, or had screening LDL-cholesterol levels greater than 160 mg/dL, because they may require pharmacological treatment. Subjects were required to have not taken sex hormones for at least 2 months before entry. Twenty subjects ages 44 to 69, were enrolled and gave their informed consent. This study was approved by the institutional review board of Brigham and Women’s Hospital. One subject was unable to comply with the protocol and was dropped; the other 19 subjects completed this study.

Protocol

This study was a randomized, double-blind, placebo-controlled, cross-over trial with three treatment periods of 9 weeks each: micronized estradiol (Estrace, Mead Johnson, Evansville, IN) 2 mg orally daily; estradiol, 1 mg orally daily; and matching placebo. Subjects were instructed to take their study drug at bedtime, with no specific time specified. Subjects were randomly assigned, in blocks of 6, to one of six sequences of treatment. All treatment periods were immediately preceded by medroxyprogesterone acetate (Provera, Upjohn, Kalamazoo, MI) 10 mg daily for 10 days to induce shedding of any preexisting proliferated endometrium. Treatment periods were separated by 2 weeks without study drug.

Lipoprotein and hormone measurement

Blood for lipoprotein measurement was obtained in the morning after a 12-h overnight fast six times during each treatment period: twice during the third week of treatment, twice during the sixth week of treatment, and twice during the 9th week of treatment. Plasma was immediately extracted by centrifugation and stored at -80 C. The blood samples from each subject were analyzed in one batch at the end of the study in the Lipid Research Laboratory of the Nutrition Department, Harvard School of Public Health. This study is standardized for cholesterol and HDL measurements by the Centers for Disease Control and the National Heart, Lung, and Blood Institute. Very low-density lipoprotein (VLDL) was separated from plasma by overlaying 0.5 mL of 0.9 percent sodium chloride over 0.5 mL of plasma, and spinning in a Beckman Coulter, Inc. Type 25 rotor (Beckman Coulter, Inc. Instruments, Palo Alto, CA) at 25,000 rpm for 16 h. The VLDL fraction was separated from the LDL and HDL fractions by tube slicing. HDL and HDL₃ were sequentially separated by precipitation with dextran sulfate and magnesium chloride (9). Cholesterol and triglyceride were measured with enzymatic reagents and quantified photometrically using a COBAS Mira Plus auto-analyzer (Roche Diagnostics Systems, Belleville, NJ). Cholesterol was measured in whole plasma, VLDL, the combined LDL and HDL fractions, HDL, and HDL₃. Triglycerides and apoB were measured in whole plasma and in the VLDL fraction. Apo B, apo A-I, and Lp (a) were measured by immunoturbidimetry with rabbit antiserum (10) obtained from INCSTAR Corp. (Stillwater, MN), by the Comprehensive Biological Analysis System (COBAS) Mira Plus auto-analyzer. The coefficients of variation for blinded control specimens were as follows: 2.4% for cholesterol, 3.3% for HDL cholesterol, 2.8% for HDL₃ cholesterol, 2.2% for apo AI, 2.0% for apoB, and 3.0% for Lp (a).

Serum estradiol and estrone levels were measured in two specimens obtained during the 9th week of each treatment. Estradiol was measured with the immunofluorescent Delfia system (Wallach, Gaithersburg, MN). The lower limit of sensitivity of this assay is 13.6 pg/mL. The intraassay and interassay coefficients of variation are both 4.5%. The cross-reactivity with estrone is less than 1%. Estrone was measured by RIA (11) following methylene chloride extraction, using a Wein Laboratories estrone test set (Succasunna, NJ). This antibody is sensitive to 15 pg/mL and has 4% cross-reactivity with estradiol at 1000 pg/mL. Its interassay coefficient of variation is 15% at 268 pg/mL, and 7% at 780 pg/mL.

Statistical analysis

The treatment effect was defined as the difference in plasma lipoprotein concentrations measured during the ninth week of placebo treatment and during the ninth week of each estrogen treatment. The differences were analyzed using analysis of variance with treatment, estradiol dose, time, and baseline lipid values as part of the model. Logarithmic transformations were used when the data was skewed. Two-tailed P values were used throughout.

	Results

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Patient characteristics

Eleven percent of subjects had previously undergone a hysterectomy, and 89% had previously used estrogen after the menopause. The mean ± SD age of the subjects was 55 ± 7 yr, time since menopause was 8 ± 7 yr, BMI 27.5 ± 2.5 kg/m², systolic blood pressure 118 ± 20 mm Hg, and diastolic blood pressure 68 ± 9 mm Hg.

Estrogen concentrations

Estrogen concentrations measured twice during the final week of each treatment were found to be quite consistent (Fig. 1), with a correlation coefficient of 0.96 (P < 0.0001). Thus, both biological and methodological variation in estradiol measurement appears to be quite low. Mean ± SD estradiol concentrations during treatment with 1 mg and 2 mg oral estradiol daily were 54 ± 16 pg/mL (198 ± 59 pmol/L) and 98 ± 31 pg/mL (360 ± 114 pmol/L), respectively. All subjects had estradiol levels less than the lower limit of the assay during placebo treatment. As shown in Fig. 2, there was considerable overlap in serum estradiol levels achieved by the two different estradiol doses. Estradiol concentrations during treatment with 1 mg estradiol for 9 weeks were found to be positively correlated with the subject’s BMI (r = 0.48, P = 0.036). Mean ± SD estrone levels were 351 ± 119 pg/mL (1289 ± 437 pmol/L) and 743 ± 257 pg/mL (2728 ± 943 pmol/L) during treatment with 1 mg and 2 mg estradiol, respectively.

View larger version (20K): [in this window] [in a new window]   Figure 1. Correlation between plasma estradiol levels measured twice, 1 week apart, in postmenopausal women given oral estradiol.

View larger version (35K): [in this window] [in a new window]   Figure 2. Frequency distribution of serum estradiol levels in 19 postmenopausal women treated with oral estradiol, 1 mg daily (shaded bars), and 2 mg daily (clear bars). As shown, there is considerable overlap in estradiol levels achieved by the two estradiol doses.

The effects of treatment with 1 mg and 2 mg estradiol for 9 weeks are shown in Table 1. With the exception of LDL-cholesterol, all lipoprotein changes were found to be at maximum change at 6 weeks, with nearly 85% of the change occurring by 3 weeks (Fig. 3). In contrast, LDL-cholesterol levels progressively declined over the 9-week treatment periods, with 62% of the decrease occurring by 3 weeks, and 73% of the decrease occurring by 6 weeks. There was found to be no carryover effects from one treatment to the next: treatment sequence did not influence any observed treatment effects.

View larger version (20K): [in this window] [in a new window]   Figure 3. Effects of 3, 6, and 9 weeks of oral estradiol treatment, 2 mg daily, on plasma HDL, LDL, lipoprotein (a), and triglyceride levels. Values shown are the mean percent changes; the bars show the standard errors for the mean percent changes.

Relationship between estrogen and HDL concentrations

The serum estradiol levels produced by treatment with 1 mg estradiol for 9 weeks were positively correlated with the percent increases in HDL cholesterol levels (r = 0.70, P < 0.01), as shown in Fig. 4. In fact, subjects with estradiol levels below the median (i.e. 53 pg/mL) taken as a group had negligible increases in HDL levels with the 1-mg dose, with a mean change of only 3% (Fig. 5). In contrast, subjects with estradiol levels greater than the median exhibited a 13% increase in HDL levels (P < 0.001). There thus appears to be a critical threshold for estradiol levels because those subjects who showed estradiol levels greater than 53 pg/mL on 1 mg of oral estradiol had minimal further increases in HDL cholesterol levels when given 2 mg of oral estradiol: the mean (± SD) increase in HDL levels changed from 13 ± 9% (on 1 mg) to only 17 ± 10% (on 2 mg), P = n.s. Subjects with estradiol levels less than 53 pg/mL on 1 mg oral estradiol demonstrated significant increases in HDL-cholesterol level when given a 2-mg estradiol dose, with the mean (± SD) increase being 13 ± 7% (P < 0.001).

View larger version (12K): [in this window] [in a new window]   Figure 4. Effect of estradiol treatment, 1 mg daily for 9 weeks, on serum estradiol levels and the corresponding percent increase in serum HDL-cholesterol levels (vs. placebo value). Each point represents individual data from 19 postmenopausal women.

View larger version (32K): [in this window] [in a new window]   Figure 5. Percent increase in HDL-cholesterol levels with treatment with 1 mg and 2 mg estradiol daily for 9 weeks. The subjects are divided according to the estradiol levels achieved by treatment with 1 mg estradiol daily. Those subjects who had estradiol levels less than 53 pg/mL are shown in the left panel; those who were greater than 53 pg/mL are shown in the right panel. As indicated, only those subjects who had estradiol levels less than the median, 53 pg/mL, showed significant additional increases in HDL levels when given a 2-mg estradiol dose. *, Compared with placebo, P < 0.001; #, compared with 1 mg, P < 0.001.

	Discussion

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This apparent threshold of approximately 50 pg/mL necessary to raise HDL levels is similar to the estradiol levels needed to relieve dyspareunia (12) and to induce superficial vaginal squamous cells seen on vaginal cytology (13). In contrast, prevention of osteoporosis may require somewhat higher estradiol levels, in the range of 70–80 pg/mL. Slemenda et al. (14) prospectively followed 84 peri- and newly post-menopausal women for 3 yr, measuring the bone densities of the radius as well as estradiol levels every 4 months. None lost more than 1% of their bone mass of the radius over 3 yr if their mean estradiol level exceeded 70 pg/mL. Studd et al. (15) found that effective treatment of postmenopausal women with estradiol implants required estradiol levels greater than 80 pg/mL. Out of 15 women given 25 mg estradiol implants, the two who lost bone density of the lumbar spine, and the one who lost bone density at the hip, all had estradiol levels less than 80 pg/mL.

The estradiol levels achieved by administering 1-mg and 2-mg doses of oral estradiol had wide ranges and substantial overlap. This suggests that there are considerable differences in estradiol pharmacokinetics between individuals. Some of this difference may be explained by body weight: our heaviest subjects tended to have the highest estradiol levels with estrogen treatment. This is not surprising because obese women are known to be less hypoestrogenic: they have more fat to aromatize adrenal androgens to estrogen. This explains why obese women have fewer menopausal symptoms and less osteoporosis but are more likely to develop breast and endometrial cancers, which are estrogen dependent. This observation would argue for prescribing a lower starting dose to obese women.

By individually titrating estrogen dose to the minimum effective levels, the risks of breast and uterine cancer caused by estrogen treatment could be minimized. The risks of both of these malignancies may be dose dependent. Rubin et al. (16) found that the relative risk of endometrial cancer among women who took at least 1.25 mg of conjugated estrogens daily was 3.8 (95% CI: 1.7–8.5), whereas the risk was 1.2 (95% CI: 0.5–2.7) for women who took 0.625 mg or less. Weiss et al. (17) found that the relative risk of endometrial cancer was 8.8 (95% CI: 5.0–12.7) with average daily doses of 0.6 mg or greater, compared with 2.5 (95% CI: 1.1–5.3) with an average estrogen daily dose of 0.5 mg or less. A relationship between estrogen dose and breast cancer risk is less clear, however. Ross et al. (18) found that women prescribed 1.25 mg of conjugated estrogens had the greatest relative risk (1.8) of developing breast cancer, whereas women treated with 0.625 mg or less had no such increase in risk. Other investigators were unable to demonstrate a dose-response relationship between estrogen dose and breast cancer risk (19).

Measurement of estradiol levels in postmenopausal women treated with oral estradiol can be both reliable and convenient. Timing of estradiol measurement would not be critical: our subjects were only instructed to take their estrogen tablet at their usual bedtime, and to have blood drawn in the morning at no particular time: the correlation coefficient of specimens measured on different mornings within the same week was 0.90. This approach may enable the clinician to individually titrate estrogen dosing to optimize benefit while possibly lowering the risk of estrogen replacement.

	Acknowledgments

 
We wish to thank Louise Greenberg, M.Ed., for her diligent work as our research coordinator, Helena Judge for expert technical assistance in the lipid laboratory, and most of all to the dedicated patients in this study.

	Footnotes

 
¹ Supported by grants from NIH (National Heart Lung and Blood Institute no. HL-34980), Bristol-Myers/Mead Johnson Laboratories, and from the outpatient Clinical Research Center (NIH Grant NCRR-GCRC-M01-RR-02635).

² Recipient of a Clinician Scientist Award in Lipoprotein Metabolism from the American Heart Association/Parke-Davis.

Received May 15, 1998.

Revised December 23, 1998.

Accepted January 5, 1999.

	References

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