The Journal of Clinical Endocrinology & Metabolism Vol. 84, No. 3 985-989
Copyright © 1999 by The Endocrine Society
Relationship between Serum Estradiol Levels and the Increases in High-Density Lipoprotein Levels in Postmenopausal Women Treated with Oral Estradiol1
Brian W. Walsh2,
Donna Spiegelman,
Mary Morrissey and
Frank M. Sacks
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
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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.
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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.
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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.
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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.
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Figure 1. Correlation between plasma estradiol levels
measured twice, 1 week apart, in postmenopausal women given oral
estradiol.
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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.
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Lipoprotein concentrations
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.
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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.
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The baseline levels of HDL and LDL cholesterol, and of lipoprotein
(a), were not predictive of absolute or percent changes in those
lipoproteins with estrogen treatment. Subjects with HDL cholesterol
below the median (i.e. 60 mg/dL) had similar increases in
HDL-cholesterol in comparison to subjects with HDL levels greater than
the median. Similarly, subjects with LDL-cholesterol levels above
vs. below the median showed comparable decreases in LDL
levels. There was no correlation among the magnitudes of the increases
in HDL-cholesterol, increases in plasma triglyceride levels, and
decreases in LDL-cholesterol levels.
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).
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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.
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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.
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Discussion
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We found that women treated with 1 mg estradiol daily had
submaximal increases in HDL-cholesterol levels if their serum estradiol
levels were less than the median value of 53 pg/mL. Without exception,
they demonstrated further increases in HDL levels when their estrogen
dose was increased. In contrast, women who had estradiol levels greater
than 53 pg/mL did not show appreciable elevations in HDL levels by
increasing their estrogen dose. It therefore appears that measurement
of a morning estradiol level in women maintained on 1 mg estradiol may
predict who may benefit from an increase in their estrogen dose.
Because HDL cholesterol is the most powerful predictor of heart disease
in women (8), this approach may allow the clinician to identify which
women would derive additional benefit by increasing their estrogen
dose, which could maximize cardiovascular benefit.
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.
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Acknowledgments
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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.
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Footnotes
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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.
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Abstract
Introduction
