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Associations among Oral Estrogen Use, Free Testosterone Concentration, and Lean Body Mass among Postmenopausal Women¹

University of Alabama at Birmingham, Department of Nutrition Sciences (B.A.G.), Division of Physiology and Metabolism, and Clinical Nutrition Research Center, and Department of Physiology and Biophysics (L.N.), Birmingham, Alabama 35294-3360

Address all correspondence and requests for reprints to: Barbara A. Gower, Ph.D., Department of Nutrition Sciences, University of Alabama at Birmingham, 427 Webb Building, 1675 University Boulevard, Birmingham, Alabama 35294-3360. E-mail: bgower{at}uab.edu

	Abstract

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Circulating concentrations of sex hormone-binding globulin (SHBG) are increased by use of oral estrogen. The objective of this study was to determine whether postmenopausal women who used oral estrogen had higher serum concentrations of SHBG and lower serum concentrations of free testosterone (T) than nonusers, and whether free T was associated with lean body mass, particularly skeletal muscle mass. Subjects were 70 postmenopausal women, 46–55 yr old, 46 of whom used oral estrogen. Total and regional body composition were determined by dual-energy x-ray absorptiometry. Serum concentrations of SHBG, total T, and estradiol (E₂) were determined by RIA. Free T was calculated from concentrations of total T and SHBG. Hormone users had higher serum concentrations of E₂ and SHBG (182.0 ± 58.5 vs. 82.9 ± 41.1 nmol/L, mean ± SD, P < 0.001) and lower concentrations of free T (3.7 ± 2.2 vs. 7.9 ± 4.1 pmol/L, mean ± SD, P < 0.001); total T did not differ. Total lean mass and leg lean mass were significantly correlated with free, but not total T [r values of 0.29 (P < 0.05) and 0.31 (P < 0.01) for total and leg lean mass, respectively, vs. free T]; arm lean mass was not correlated with either measure of T. Serum E₂ was significantly correlated with SHBG (r = 0.50, P < 0.001) and free T (r = -0.33, P < 0.01). These observations imply that, by reducing the concentration of bioavailable T, oral estrogen therapy may accelerate or augment lean mass loss among postmenopausal women. This conclusion awaits confirmation by longitudinal observation.

	Introduction

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ORAL ESTROGEN THERAPY, because of the first pass effect, results in exposure of the liver to supraphysiological concentrations of estrogen. One of the consequences of this exposure is an alteration in the production of several hepatic proteins. Concentrations of sex hormone-binding globulin (SHBG) (1) and apolipoprotein A-I (Apo A-I) (2) are higher, and insulin-like growth factor (IGF)-I are lower (3), in women who use oral estrogen therapy, when compared with nonusers.

The physiological ramifications of altered hepatic protein production with estrogen use are not entirely clear. This is particularly true with respect to the elevation in SHBG. SHBG binds to circulating testosterone (T). In premenopausal women, approximately 62% of circulating T is bound to SHBG; another 36–37% is bound to albumin, and approximately 1.5% is unbound (4). It is assumed that the unbound (or free) fraction is the physiologically active, or bioavailable, fraction. Although SHBG binds to other steroid hormones, its affinity for other hormones is lower than that for T, and it accounts for a lower percentage of bound hormone [e.g. in women, 45–46% of estradiol (E₂) (4) and 6.63% of androstenedione (5) circulate bound to SHBG]. Thus, an increase in SHBG with estrogen use could lower circulating concentrations of bioavailable T.

T promotes deposition and maintenance of skeletal muscle. Administration of T to elderly men increases the fractional synthetic rate of muscle protein, as well as muscle strength (6). Among normal male subjects, exogenous T increases muscle mass and muscle protein synthesis, and it decreases leucine oxidation (7, 8). By contrast, pharmacologic gonadal suppression in normal young men causes a decrease in whole-body protein synthesis in conjunction with losses of fat-free mass and leg muscle strength (9). Among women with polycystic ovary syndrome, serum T is correlated with lean body mass (10). The decline of T (11, 12, 13) and its precursors (14) with age may be one of the factors that contribute to sarcopenia in older men and women.

An age-related loss of muscle mass has been observed in women, and it seems to increase in rate at the time of menopause (15, 16). Loss of muscle mass, particularly in the lower extremity, may lead to loss of physical function, which could increase risk for fall-related injury and other disabilities (17, 18). In addition, the lower total-body energy requirements associated with loss of muscle could increase risk for obesity and associated diseases (15, 19). Thus, factors that accelerate or augment the normal age-related decline in skeletal muscle would be undesirable.

The present cross-sectional study was conducted to determine whether postmenopausal women who used estrogen had higher serum concentrations of SHBG and lower serum concentrations of free T than nonusers, and whether free T was associated with lean body mass, particularly skeletal muscle mass.

	Subjects and Methods

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Experimental subjects

Subjects were: 70 postmenopausal women, 46–55 yr old (62 Caucasian and 8 African-American). Only women who experienced a natural menopause, with the time of menopause known to occur at least 6 months before contact, were recruited. Both women using hormone replacement therapy (HRT) and women not using HRT were recruited. Among hormone users, only subjects using an oral estrogen preparation were selected (n = 46). Most used conjugated equine estrogens (0.625 mg/day) in combination with medroxyprogesterone acetate (2.5 mg/day), although several women used other types of estrogen or other doses of conjugated equine estrogens. Six women used unopposed estrogen. "No HRT use" (n = 24) was defined as no current use, and no use within the past 6 months. Twenty-one of 24 nonusers had never used HRT. Data were collected over a 27-h period during an in-patient visit to the Department of Nutrition Sciences and the General Clinical Research Center at the University of Alabama at Birmingham (UAB). The protocol was approved by the Institutional Review Board for Human Use at UAB, and all subjects signed an informed consent before testing.

Protocol

Subjects arrived at the Department of Nutrition Sciences, at approximately 0900 h, in the fasted condition (12-h fast). Body composition was determined by dual-energy x-ray absorptiometry (DXA). Three fasting blood samples were taken, over a 40-min period on the following morning, at UAB’s General Clinical Research Center at approximately 0700 h. Samples were allowed to clot, then were centrifuged. Sera were aspirated, pooled, aliquoted, and stored at -85 C until used for hormone assay.

Body composition

Total and regional (leg, arm) body composition (fat mass and lean body mass) were measured by DXA using a DPX-L densitometer (Lunar Corp., Madison, WI). Subjects were scanned in light clothing while lying flat on their backs with arms at their sides. DXA scans were performed and analyzed with adult software version 1.5 g. Leg and arm tissue masses were determined by manually placing delimiting lines at specific landmarks on the computer-generated image. The legs were indicated to be all soft tissue below the triangle formed by drawing a horizontal line across the top of the pelvis, and two angled lines (one through each femoral neck). To delineate the arms, lines were drawn between the torso and the arms, from the top of the arm socket to the phalange tips, avoiding contact with the rib and pelvic areas.

Hormone assay

Serum E₂ was measured using a double-antibody RIA (Diagnostic Products, Los Angeles, CA). Sera were first extracted in diethyl ether. Assay sensitivity was determined to be 15.42 pmol/L, intraassay coefficient of variation to be 5.3%, and interassay coefficient of variation to be 6.0%. Serum total T was measured with a coated-tube RIA (Diagnostic Products); assay sensitivity was 0.409 nmol/L, intraassay coefficient of variation was 7.7%, and interassay coefficient of variation was 8.2%. For measurement of SHBG, sera were first diluted 1:101, then assayed in duplicate 25-µL aliquots using an immunoradiometric assay (Diagnostic Systems Laboratories, Inc., Webster, TX); the lower limit was 5 nmol/L, the intraassay coefficient of variation was 8.2%, and the interassay coefficient of variation was 8.2%. Free T (pmol/L) was calculated from serum concentrations of total T and SHBG using the method of Sodergard et al. (4). This method is based on the concentration of albumin, the binding capacity of SHBG, and the association constants of T for SHBG and albumin, as determined in a sample of normal men and women.

Statistics

For all analyses, values for body composition variables and serum analytes were log-transformed to produce a normal distribution. ANOVA was used to compare measurements between hormone users and nonusers. Pearson correlation analysis was used to examine associations among serum hormone concentrations and between regional lean mass and serum hormone concentrations. All data were analyzed with SAS for Windows version 6.12. Differences and associations were considered significant if P was less than 0.05.

	Results

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Descriptive statistics are shown in Table 1. Hormone users and nonusers did not differ, with respect to age, weight, or lean mass (total and regional). Nonusers had a higher total body fat mass and percent body fat. Hormone users had higher serum concentrations of E₂ and SHBG and lower concentrations of free T; total T did not differ (Fig. 1; A, B, C, and D, respectively).

View larger version (19K): [in this window] [in a new window]   Figure 1. A, Serum E2 concentration (pmol/L) in users and nonusers of HRT; ***, P < 0.01. B, Serum SHBG concentration (nmol/L) in users and nonusers of HRT; ***, P < 0.01. C, Serum free T concentration (pmol/L) in users and nonusers of HRT; ***, P < 0.01. D, Serum total T concentration (nmol/L) in users and nonusers of HRT; means were not significantly different.

View larger version (13K): [in this window] [in a new window]   Figure 2. Leg lean mass (kg) vs. serum free T concentration (pmol/L) in all women combined; P < 0.01, R2 = 0.10. Open circles indicate nonusers and filled circles indicate users, respectively, of HRT.

View larger version (15K): [in this window] [in a new window]   Figure 3. A, Serum SHBG concentration (nmol/L) vs. serum E2 concentration (pmol/L) in all women combined; P < 0.001, R2 = 0.24. B, Serum free T concentration (pmol/L) vs. serum E2 concentration (pmol/L) in all women combined; P < 0.01, R2 = 0.11. Open circles indicate nonusers and filled circles indicate users of HRT, throughout this figure.

	Discussion

Top Abstract Introduction Subjects and Methods Results Discussion References

As reported previously with use of oral contraceptives (20), use of oral estrogen among postmenopausal women in this study was associated with a 2-fold greater concentration of circulating SHBG, relative to women not using hormone therapy. Presumably as a result of this difference in SHBG concentration, the concentration of free T was 50% lower in hormone users vs. nonusers. It is generally assumed that only the free fraction of circulating T is biologically active. Unbound T can cross the cell membrane and affect gene transcription. Increased messenger RNA for the anabolic hormone IGF-I and decreased expression of IGF binding protein-4, a protein that inhibits the mitogenic action of IGF-I, were observed in skeletal muscle after T administration (6). Thus, activation of the intramuscular IGF-I system is one mechanism through which T may increases protein synthesis. The possibility that lower free T limits muscle growth or maintenance in women using oral estrogen is a topic that has received little attention.

In this study, the concentration of free T was positively correlated with total lean mass and leg lean mass but not arm lean mass. Limb lean mass, as assessed by DXA, is a reasonable surrogate for appendicular skeletal muscle mass (21). The positive association of T with lean mass is logical if T has a positive influence on skeletal muscle accrual or retention. However, in this study, only leg lean mass was associated with serum free T. This association might be explained by the observation that the anabolic effects of T on skeletal muscle are augmented when the muscle is simultaneously stressed by use (22). Thus, it would seem most likely that free T be correlated with those muscles that are used most frequently and experience the greatest loading or force. Whereas leg muscles are used regularly for walking, arm muscles are likely to experience less resistance on a day-to-day basis in individuals who do not engage in resistance training or other activities that involve lifting heavy objects with the arms.

Synthesis of T declines with age in women (12). This decline is marked between the years of 20–30 and 50–60 and is more gradual thereafter (14). A further, transient decline is observed at the time of menopause (23). Thus, at the time of menopause, women are likely to be experiencing a phase of accelerated decline in circulating anabolic hormones. This phenomenon may explain the significant age-independent decline in fat-free mass observed longitudinally in women who traverse the menopause (16).

Many women also initiate use of hormone replacement therapy during the perimenopausal years. The decline in free T associated with oral estrogen use could potentially accelerate the loss of muscle mass that is already occurring because of age- and menopause-related declines in serum T. Loss of muscle mass may be associated with a perceived inability to engage in physical activity, thus resulting in a vicious cycle of inactivity and worsening sarcopenia.

Associations between lean body mass and physical activity have been observed in women. Thirty-nine percent of the decline in fat-free mass with age in women is attributable to a change in maximal oxygen consumption (an index of physical fitness) (15). In addition, leisure time physical activity, as determined by questionnaire, decreases in conjunction with lean body mass during the perimenopausal period (16). Although the cause-and-effect nature of these relationships is not known, it is possible that it could be bidirectional; decreases in muscle mass attributable to hormonal factors could precipitate a decrease in physical activity that, in turn, would lead to further loss of skeletal muscle.

In conclusion, use of oral estrogen therapy was associated with higher SHBG and lower free T among postmenopausal women. Free, but not total, T was associated with total and leg lean body mass. These results imply that use of oral estrogen could accelerate the decline in skeletal muscle mass that occurs with age in women. However, longitudinal observation will be required to determine whether the hypothesis is correct.

	Acknowledgments

 
We acknowledge Kangmei Ren for hormone determinations, Tena Hilario for subject recruitment, Jill Foster and ola Oguntolu for physician coverage, Stephen J. Winters for the spreadsheet for calculating free T, and the help of the GCRC staff.

	Footnotes

 
¹ Supported by NIA K01-AG-00740 (to B.A.G.) and General Clinical Research Center Grant M01-RR-00032.

Received May 22, 2000.

Revised August 10, 2000.

Accepted August 18, 2000.

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This Article Abstract Full Text (PDF) Submit a related Letter to the Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Copyright Permission Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Gower, B. A. Articles by Nyman, L. Search for Related Content PubMed PubMed Citation Articles by Gower, B. A. Articles by Nyman, L.