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Journal of Clinical Endocrinology & Metabolism, doi:10.1210/jc.2006-0284
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The Journal of Clinical Endocrinology & Metabolism Vol. 91, No. 11 4387-4394
Copyright © 2006 by The Endocrine Society

Circulating Bioactive Androgens in Midlife Women

Jiangang Chen, MaryFran R. Sowers, Francisco M. Moran, Daniel S. McConnell, Nancy A. Gee, Gail A. Greendale, Cheryl Whitehead, Sidika E. Kasim-Karakas and Bill L. Lasley

Center for Health and the Environment (J.C., F.M.M., N.A.G., C.W., B.L.L.) and Department of Internal Medicine (S.E.K.-K.), University of California, Davis, Davis, California 95616; Department of Epidemiology (M.R.S., D.S.M.), School of Public Health, University of Michigan, Ann Arbor, Michigan 48104; and Department of Medicine/Geriatrics (G.A.G.), David Geffen School of Medicine at University of California, Los Angeles, Los Angeles, California 90095

Address all correspondence and requests for reprints to: B. L. Lasley, Ph.D., University of California, Center for Health and the Environment, One Shields Avenue, Davis, California 95616. E-mail: bllasley{at}ucdavis.edu.


   Abstract
Top
 Abstract
Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
Context: It is important to characterize the biological activity of circulating androgenic steroid hormones during the menopausal transition because these appear to impact the metabolic and cardiovascular health risk factors of women.

Objective: The objective of the study was to develop and characterize a cell-based bioassay that measures the androgen receptor-mediated signal transduction in serum.

Design: This was a clinically relevant experimental study nested in a sample population of a longitudinal cohort study.

Setting: The study was conducted at a university laboratory.

Methods: A receptor-mediated luciferase expression bioassay based on HEK 293 cells that were stably cotransfected with plasmids containing the human androgen receptor and luciferase gene was developed. In 49 samples from menstruating women aged 42–52 yr, total testosterone (T) and SHBG concentrations were measured by immunoassay; free T concentrations were calculated from the total T and SHBG concentrations.

Results: Mean total T concentration of the sample was 1.15 nM (SD 0.46, range 0.57–3.86 nM). The mean bioactive androgen detected was 1.00 nM (SD 0.24, range 0.53–1.60 nM). Calculated free T (mean 0.0156 nM) was significantly lower than the levels of bioactive androgens measured by receptor-mediated bioassay. There was significant positive correlation between bioactive androgen levels and total T values in young women and polycystic ovarian disorder patients, whereas no correlation was found between the two values in middle-aged women.

Conclusions: An androgen receptor-mediated bioassay can provide additional information in the evaluation of total bioactive androgens in midlife women. Our data suggest that levels of circulating SHBG may have a significant impact on the levels of total circulating bioavailable androgens.


   Introduction
Top
 Abstract
 Introduction
Subjects and Methods
 Results
 Discussion
 References
 
RECENT STUDIES INDICATE androgen levels during the menopausal transition are associated with increased cardiovascular and metabolic health risk factors (1, 2). The evolving role of androgens in women’s health and an established concern about the impact of hypogonadism in men (3) have generated an increasing interest in the ability to assess lower levels of circulating androgens, identify their functional concentrations, and better understand the relative importance of various forms of androgens (4, 5). Until recently, methods that accurately measured circulating androgens were better suited for concentrations above those found in hypogonadal men and women, particularly women after midlife with minimal ovarian steroid production. Although the prevailing dogma is that testosterone (T) and androstenedione (A4) are the primary circulating androgens in women, the potential contribution from the peripheral conversion of weaker adrenal steroid hormones makes the capacity to assess these weaker androgens important (6, 7, 8, 9).

There is controversy regarding the biological activity of circulating androgenic steroid hormones that are bound for transport with the plasma proteins, albumin (Ka = 3.6 x 104 liters/mol) or SHBG (Ka = 1 x 109 liters/mol). Whereas it is the consensus that the biologically active androgenic component resides primarily in the non-SHBG-bound portion (10), there is debate surrounding the accuracy in estimating proxy values of biologically active androgens (4). Some suggest considering only total T measurements (11), whereas others advocate the application of approaches such as the measurement of free T both directly and indirectly with gas chromatography and mass spectrometry (GCMS) and celite chromatography. However, these methodologies are impractical for clinical or epidemiological investigations because they are expensive and labor intensive to implement and/or demand strict control of the assay environment (4, 11, 12). Vermeulen et al. (4) compared different approaches to estimate bioactive circulating androgens and demonstrated that calculated free androgen indices are appropriate under some physiological conditions; however, calculated values may be less accurate when sex hormones and/or SHBG concentrations are at high or low extremes.

The accurate estimation of androgen activity among perimenopausal women may be particularly challenging because some women experience an increase in adrenal androgen production (8, 9, 13) and body size, which is associated with a downward shift in SHBG concentration. All of these conditions may contribute to the increase of the bioavailable androgen component.

Current analytic methods may not be capable of accurately assessing the contribution of androgenic steroids other than T, and the formulas for estimating free androgen, based on SHBG and albumin, may fail to adjust appropriately for the changes of SHBG binding affinity (14) or the nonlinear effects of low or high carrier protein concentrations. These limitations may contribute to erroneous levels of bioavailable androgens and explain the poor correlation between androgen-dependent syndromes and measured circulating androgen values or free androgen index in some women (9, 15).

One approach to addressing these limitations has been the development of in vitro transcriptional assays such as receptor-mediated luciferase expression assays (16, 17). This technology has allowed investigators to evaluate the signal transduction activity of multiple steroid/protein hormones and their precursors. These assays have been used to study different steroid hormones and have deepened our understanding of their relative biological potencies.

In this report, we describe the development and application of a novel transcriptional androgen receptor-mediated assay system for evaluating circulating bioactive androgens in midlife women.


   Subjects and Methods
Top
 Abstract
 Introduction
 Subjects and Methods
Results
 Discussion
 References
 
Materials

Human embryonic kidney (HEK 293) cells and MDA-kb2 were obtained from the American Type Culture Collection (Manassas, VA). DMEM, L-15 (Leibovitz) medium, fetal bovine serum (FBS), blasticidin, neomycin (G418), pCDNA6 vector, and T4 DNA ligase were purchased from Invitrogen (Carlsbad, CA). 17ß-Hydroxy-5{alpha}-androstan-3-one (DHT), 17ß-hydroxy-4-androsten-3-one (T), 5-androstene-3, 17ß-diol (A5), 4-androstene-3, 17-dione (A4), 4-androstene-3, 17ß-diol (4-diol), 5{alpha}-androstane-3,17ß-diol (5{alpha}-diol), 3ß-hydroxy-5-androsten-17-one (DHEA), DHEA sulfate, 17{alpha}-ethynyl-17ß-hydroxy-19-nor-4-androsten-3-one (norethindrone), 6{alpha}-methyl-17{alpha}-hydroxy-4-pregnene-3,20-dione (medroxyprogesterone), 1,3,5(10)-estratriene-3,17ß-diol [estradiol (E2)], 11ß,17,21-trihydroxy-4-pregnene-3,20-dione (cortisol), and 4-pregnene-3,20-dione [progesterone (P4)] were purchased from Steraloids (Newport, RI). Phenol red free DMEM was obtained from Sigma Chemical (St. Louis, MO). All steroid compounds were dissolved in 100% ethanol before addition to cell cultures. Dextran-coated charcoal-treated (DCC) FBS was purchased from Hyclone (Logan, UT). Restriction enzymes were supplied by New England Biolabs (Beverly, MA). Serum matrix (from castrated elephants) lacking strong sex steroid binding proteins (18) was obtained under University of California, Davis, Animal Use protocols.

Assay development

Cell line selection. The androgen receptor-mediated assay was developed from HEK 293 cells stably cotransfected with a human androgen receptor (hAR) plasmid and a luciferase reporter gene under the control of mouse mammary tumor virus (MMTV) promoter. HEK 293 was chosen because of its human origin and lack of endogenous androgen, estrogen, or progesterone receptors (19, 20, 21). This cell line lacks endogenous steroidogenic enzymes such as P450 aromatase, 3ß-hydroxysteroid dehydrogenase, 5{alpha}-reductase, 17ß-hydroxysteroid dehydrogenase, and steroid 17{alpha}-hydroxylase/17, 20-lyase (22, 23, 24), obviating potential concerns of interconversion among steroid hormones and metabolism of androgens within the cells. HEK 293 also expressed adenoviral E1 gene products, which can superactivate the cytomegalovirus promoter in the hAR plasmid (25, 26), assuring the continuous expression of the hAR in transfected cells.

HEK 293 cells were grown in 10% FBS-DMEM, supplemented with 2.0 mM glucose and 100 U penicillin/streptomycin at 37 C and 5% CO2. MDA-kb2, a breast cancer cell line with endogenous androgen receptors (27), was used as a positive control for the HEK 293 subcloning.

Plasmid construction. PSG5 AR plasmid (28) was a gift from Dr. C. S. Chang (University of Rochester, Rochester, NY). MMTV-LUC.neo was generously provided by Dr. V. S. Wilson, United States Environmental Protection Agency (USEPA) and has G418 resistance as the selection marker (27). To construct the hAR plasmid with blasticidin resistance as the selection marker, PSG5 AR and pCDNA6 vectors were double digested with NheI and BamHI and the linearized fragments ligated by T4 DNA ligase. The resulting construct with both hAR and blasticidin genes was confirmed by qualitative restriction digests and DNA sequencing.

Cell transfection. Cell transfection and positive colony selection were performed according to the manufacturer’s recommendations (Superfect transfection reagent; QIAGEN, Valencia, CA). The colony that gave the highest signal to noise ratio as determined by functional assay was named 2933Y and used in all subsequent assays.

RNA isolation and real-time quantitative PCR. To quantify the expression of hAR in 2933Y, total RNAs were extracted using TRIzol reagent (Invitrogen). RNAs extracted from MDA-kb2 were used as a positive control. Amplification of a human housekeeping gene RNA, glyceraldehyde 3-phosphate dehydrogenase, was used in the reaction as an internal control. Gene-specific mRNA was subsequently normalized to glyceraldehyde 3-phosphate dehydrogenase RNA. Levels of hAR mRNA were expressed as a fold difference from the MDA-kb2 cell line.

Serum samples

Serum samples were obtained from the Study of Women’s Health Across the Nation (SWAN), a multisite, longitudinal cohort study. Participants were 42–52 yr old, had an intact uterus, had at least one ovary, did not currently use estrogens or other medications known to affect ovarian function, and had at least one menstrual period in the 3 months before screening. Cohort recruitment and enrollment have been previously described (29). Forty-nine samples from midlife women with a wide range of T (mean 1.15 nM, range 0.57–3.86 nM) were selected to evaluate the receptor-mediated bioassay. In addition, 50 serum samples (mean immunoreactive T 4.26 nM, range 3.07–8.36 nM) from the upper quartile of circulating T concentrations in SWAN participants and 50 samples (range < 0.18–0.39 nM) from the lowest quartile were selected to create two serum pools to assess the relationship between SHBG and measured bioactive androgen. To determine the correlation between levels of circulating bioactive androgens and biomarkers of health outcomes (cardiovascular risk factors and carbohydrate metabolism), 168 serum samples with different levels of SHBG were also randomly selected from the SWAN repository.

Circulating bioactive androgens in serum samples from cycling women, pregnant women, postmenopausal women, postmenopausal oophorectomized women, polycystic ovarian disorder (PCOD) women, and men were also measured to demonstrate the relative range of levels (Table 1Go). Informed consent was obtained from all participants and institutional review board approval was obtained.


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  		TABLE 1. Levels of bioactive androgens (nM) in human samples

 
Androgen bioassay

2933Y cells were cultured in DMEM with 10% FBS. When cells reached 80% confluence, they were trypsinized, and equal numbers of cells (density 25,000 in 50 µL/well) were added into 96-well tissue culture plates containing 150 µl/well of phenol red free DMEM with 10% DCC-FBS. The following day, the media were removed and replaced with fresh media. On d 3, media were removed, and 200 µl phenol red free cell culture media containing 20 µl T standards at concentrations of 10–8 to 10–11 M or 20 µl of serum samples were added (final standard concentrations 10–9 to 10–12 M). If required, serial dilutions of serum samples were prepared in DCC-FBS. To compensate for any matrix effects in the serum, the same proportion (10%) of charcoal-stripped FBS was added to the standard preparation. The final content of alcohol in assay medium was 0.1%. Cells were cultured for 16 h and harvested, and then cell lysates were measured for luciferase activity. Concentrations of other steroids used for cross-reactivity evaluation are designated in the corresponding figures.

Immunoassays

Circulating total T was evaluated with the ACS-180 (Bayer Diagnostics, Tarrytown, NY) using a modified protocol to increase precision in the low ranges (assay sensitivity 0.18 nM) (13). To address the concern of the accuracy of the T measurement by immunoassay with the ACS-180, a human serum pool was obtained from the Central Ligand Assay Satellite Services Laboratory at the University of Michigan. Measurements of T were then compared between the ACS-180 assay and celite chromatography/RIA (30). Similar results were obtained by both assays (0.413 ± 0.032 and 0.425 ± 0.016 nM for the ACS-180 and celite chromatography/RIA, respectively). This pool was also used to assess the linearity of the receptor-mediated androgen bioassay by spiking with T standards. SHBG was measured using commercially available kits from Diagnostic Systems Laboratories (Webster, TX).

Total T was indexed to SHBG to calculate the free androgen index [FAI; 100 x T (nM)/SHBG (nM)]. To calculate free T (FT), an albumin concentration of 43 g/liter (6.2 x 10–4 mol/liter) was used (4), assuming that there is no change of albumin concentrations during the menopausal transition (31). Cortisol levels were determined using the ACS-180.

GCMS

To compare the levels of bioactive androgens measured by bioassay with a gold standard method, testosterone measurements by GCMS were performed by Dr. Linda Theinpont (University of Ghent, Belgium). The isotope dilution-GCMS method has been previously described (32). Assays were performed on each sample in triplicate in three independent runs using the preset analytical performance criteria (32, 33, 34). Calibration mixtures were taken from three independently prepared working solutions. The expanded uncertainty for the specified measurement conditions was estimated to be 2.5%.

The effect of different SHBG concentrations on androgen detection by bioassay

Two pools of serum with different levels of T and SHBG were created to investigate the relationship between SHBG concentration and bioactive androgen levels. Pool 1 was comprised of aliquots from participants with lower total T levels (n = 50, range < 0.180–0.39 nM), and pool 2 was comprised of aliquots from participants with higher levels of total T (n = 50, range 3.07–8.36 nM). Each serum pool was spiked with T, resulting in an exogenous T concentration of 10 nM. One hundred microliters of T-spiked serum were added to 900 µl of medium and allowed to equilibrate at room temperature for 7 h. Then 200 µl of the sample/medium mixture (final content of 0.1% alcohol and 1 nM T) was taken to the cell-based bioassay and incubated at 37 C with 5% CO2 for an additional 16 h before the cells were lysed for luminometer measurement. Serum lacking strong sex steroid binding proteins from castrated elephants was used as a control. All spiking experiments were performed using the same protocol.

Data analysis

Data are presented as means ± SD. Wilcoxon signed rank tests or paired t tests were used to evaluate the difference between FT approximated by T and SHBG values (4) and concentrations of bioactive androgens. Spearman correlation analysis was used to show the strength and direction of associations between measured and calculated androgen variables as well as the correlation between levels of bioactive androgens and biomarkers of health outcomes. The significance level had an alpha of 0.05. Analyses were performed using SigmaStat (SPSS, Chicago, IL).


   Results
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
Discussion
 References
 
The expression of hAR in 2933Y cells

As shown in Fig. 1Go, the expression of hAR mRNA from 2933Y was more than 5-fold higher than MDA-kb2 cells. The lower limit of detection with the receptor-mediated assay was 15 pM T in cell culture medium (corresponding to 150 pM in serum, blank +3 SD) with intra- and interassay coefficients of variation of 7.4 and 7.5% at a T level of 0.25 nM and 4.9 and 6.4% at a T level of 0.03 nM, respectively. The signal response of 2933Y cells to T remained stable for more than 60 passages and after multiple freeze-thaw cycles.


Figure 1
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  		FIG. 1. Real-time PCR quantification of the expression of hAR in HEK293 and 2933Y cells. The MDA-kb2, which expresses endogenous hAR and is derived from breast cancer cells, was used as positive control. The expression of hAR from MDA-kb2 was set as unity. Data are presented as mean ± SD of triplicates.

 
The dose response of 2933Y to androgens and its cross-reactivity to other steroid hormones

As shown in Fig. 2AGo, when the relative androgenic potencies of the different ligands were compared, DHT was the most potent steroid tested, followed by T, 4-diol, A4, 5{alpha}-diol, and A5. Two synthetic progestogens, norethindrone and medroxyprogesterone, also exhibited androgenic potential, with norethindrone having an androgenic potency of one tenth that of T. No significant transcriptional activity was induced by DHEA up to a concentration of 10–6 M (Fig. 2AGo). E2, P4, and cortisol were also tested. E2 and P4 did not induce significant bioassay activity at concentrations less than 10–9 M (Fig. 2BGo), whereas no significant luciferase activity was induced by cortisol less than 10–8 M. No significant induction of transcriptional activity was detected for DHEA sulfate at 10–4 M.


Figure 2
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  		FIG. 2. A, The androgen receptor-mediated transcriptional activation of the hAR in 2933Y by different ligands: DHT ({circ}), T (•), 4-diol ({blacksquare}), A4 ({triangledown}), 5{alpha}-diol ({square}), A5 ({blacktriangledown}), and DHEA ({diamondsuit}). B, The comparison of transcriptional activity between T (•), 17{alpha}-ethynyl-17ß-hydroxy-19-nor-4-androsten-3-one (norethindrone, {blacksquare}), 6{alpha}-methyl-17{alpha}-hydroxy-4-pregnene-3,20-dione (medroxyprogesterone, {square}), P4 ({triangledown}), E2 ({blacktriangledown}), and cortisol ({circ}). Data are presented as mean ± SD of triplicates. RLU, Relative light unit.

 
Linearity of the receptor-mediated androgen bioassay

The linearity of the receptor-mediated bioassay was evaluated by spiking the same serum pool used for ACS-180/celite chromatography RIA comparison. The serum pool was spiked with T standards ranging from 0.031 to 1.0 nM. The endogenous total T concentration of the pool was 0.413 nM. The bioassay demonstrated a linear dose response with increasing levels of T from the spikes (bioactive androgens = 0.024 + 0.38T, R2 = 0.996, P < 0.001, Fig. 3Go).


Figure 3
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  		FIG. 3. The linearity of receptor-mediated bioassay after spiking with different concentrations of T. Data are presented as mean ± SD of triplicates.

 
The levels of bioactive androgens in serum samples of subjects with different age, gender, and pathological/reproductive status

Table 1Go reports the ranges of bioactive androgens in subjects of different age, gender, and pathological/reproductive status. The relatively small sample size (n) in each category may introduce a bias; however, the levels of bioactive androgens in men and PCOD patients are considerably higher. In addition, a good correlation was found between levels of bioactive androgens and immunoreactive T (n = 11, r = 0.80, P < 0.05) in younger women and PCOD patients (n = 22, r = 0.70, P < 0.05).

Comparison of androgen measurements by cell-based bioassay and GCMS

Table 2Go shows an important comparison of androgen measurements by cell-based bioassay and GCMS in 15 samples from adult women and men. Comparison indicates a high degree of correlation between the two assay values (r = 0.99 for all subjects, and r = 0.81 for 11 adult women, respectively).


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  		TABLE 2. Comparison of androgen measurements (nM) by bioassay and GCMS

 
Total T, calculated FT, and levels of bioactive androgens in the serum of middle-aged women

The mean total T concentration of 49 serum samples was 1.15 nM (SD 0.46, range 0.57–3.86 nM), whereas the mean bioactive androgen level detected was 1.00 nM (SD 0.24, range 0.53–1.60 nM). The mean calculated FAI was 2.40, and the mean calculated FT was 0.0156 nM, values significantly lower than those measured by the receptor-mediated bioassay (Wilcoxon signed rank test, P < 0.001). There was no correlation between the concentrations of bioactive androgen levels measured by receptor-mediated assay and total T measured by ACS-180 (r = –0.2, P > 0.05).

Bioactive androgens and serum SHBG in midlife women

The bioactive androgens in 49 samples were negatively associated with SHBG, although the association was not statistically significant. The level of bioactive androgens measured in pool 1 (low total T), with an SHBG mean level of 105.5 nM, was 0.04 nM T equivalent; the level in pool 2 (high total T), with an SHBG mean level of 53.4 nM, was 0.22 nM T equivalent. After the addition of a 1.0 nM T spike to each pool, the level of bioactive androgens detected in pool 1 was only 0.269 nM T equivalent (23% recovery) but was 0.997 nM in pool 2 (78% recovery). By comparison, 98% of the 1.0 nM T spike was recovered in castrated elephant serum, which lacks strong sex hormone binding proteins (Fig. 4AGo). To further explore the potential impact of SHBG in serum on bioactive androgen measurements, randomly selected individual human serum samples from the SWAN repository with varying concentrations of SHBG were spiked with a concentration of 2.5 nM of T. As shown in Fig. 4BGo, again an inverse relationship between the concentration of SHBG and percent of T recovered from spiked samples was observed.


Figure 4
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  		FIG. 4. A, The levels of androgens measured by receptor-mediated assay in the low T pool (n = 50, pool 1), high T pool (n = 50, pool 2), and the castrated elephant serum (ES) before (solid box) and after a spiking of 1.0 nM T (open box). The levels of androgens measured by receptor-mediated assay are expressed as nM of equivalent T. Data are presented as mean ± SD of triplicates. *, Lower than 15 pM. B, The effects of endogenous SHBG concentrations on the recovery of spiked T.

 
Bioactive androgens and biomarkers of health outcomes in midlife women

To investigate whether bioactive androgen levels provide additional information on health outcomes in midlife women, the strength and direction of the correlation between the levels of bioactive androgens and biomarkers of health outcome were analyzed. Compared with immunoreactive T, the levels of bioactive androgens demonstrated a stronger correlation with circulating triglycerides (biomarker of cardiovascular risk factor) when endogenous SHBG levels were relatively higher (Table 3Go). In addition, the levels of circulating immunoreactive T failed to reveal statistically significant correlations with biomarkers of carbohydrate metabolism (HOMA IR, insulin) (Table 3Go).


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  		TABLE 3. Spearman correlations of serum androgens with markers of cardiovascular risk factors and carbohydrate metabolism in high SHBG tertile1

 
Bioactive androgens and circulating cortisol

We investigated whether physiological levels of cortisol in serum could contribute significantly to the receptor-mediated assay results. The mean circulating total cortisol concentration in 49 samples was 13.01 µg/dl (range 4.53–23.51 µg/dl). As shown in Fig. 5Go, there was no statistically significant correlation between individual circulating levels of cortisol and the concentrations of bioactive androgens assessed by bioasssay (r = 0.185, P > 0.05).


Figure 5
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  		FIG. 5. The relationship between circulating total cortisol (µg/dl) and measured bioactive androgens by receptor-mediated assay (nM equivalent of T) in 49 samples.

 

   Discussion
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
References
 
Several recent population-based longitudinal studies monitoring women during the menopausal transition have challenged the long-standing dogma that most perimenopausal symptoms and physical changes are only the direct result of decreased circulating E2. Indeed, studies suggest the importance of considering androgens as major contributors to health status in perimenopause. In both cross-sectional and longitudinal examinations, hemostatic (1, 35) and other cardiovascular (CV) risk factors (36) have only poor associations to circulating E2 levels but significant positive correlations to T. Surprisingly, SHBG had the single strongest association with CV risk factors followed by a weaker, but still statistically significant, association with the FAI, an estimate for non-SHBG-bound T (36). The failure to correlate phenotypic changes with circulating E2 concentrations and the apparent stronger association of CV risk factors to the inhibition of SHBG production has promoted an investigation of androgen action as the primary risk factor. A logical extension is to address androgen measurement, recognizing that low circulating levels of androgens and the range of SHBG concentrations present challenges in the measurement of analytes and calculation of bioavailability.

This receptor-mediated assay is appropriate for the measurement of circulating of androgens in midlife women in whom androgen concentrations are often near the limit of sensitivity for standard clinical assays. Treatment of these cells with 1.0 nM T resulted in a 50-fold induction of luciferase over the background. More importantly, this assay will account for total androgen bioactivity in the serum. Stably transfected cell-based bioassays in robust cell lines such as HEK 293 are relatively easy to standardize and exhibit modest intra- and interassay variations.

This receptor-mediated assay can detect values as low as 150 pM T equivalent in serum. The ability of the assay to respond to all biologically active free circulating androgen ligands makes it applicable to most, if not all, samples. Furthermore, this assay is formatted for 96-well plates, enhancing the likelihood that both economy-of-scale and high throughput would make it appropriate for screening purposes or implementation in large population studies.

We detected higher levels of bioactive androgens using the androgen receptor-mediated assay, compared with the long-standing dogma or calculated FT derived from standard formulas. Raivio et al. (16) demonstrated that the addition of T to an SHBG-containing serum pool from prepubertal boys yielded androgen bioactivity values that were approximately one fourth the T concentration measured by RIA. Our results are consistent with reports that indicate that circulating free T represents only a portion of total bioactive androgens available to target tissues. It is more likely the sum of free T, albumin-bound T, and other free androgens that contribute to the total circulating bioavailable androgen levels. This, in part, may explain why T and its derived FAI do not correlate to the androgen-mediated phenomena as well as androgen-controlled SHBG (1, 35, 36). In fact, compared with immunoreactive T, we observed a better correlation between circulating bioactive androgens and certain biomarkers of health outcomes when the SHBG levels are relatively higher (Table 3Go).

We demonstrated that extreme conditions influenced the bioactive androgen component of serum using T-spiked serum pools from women with low and high circulating T and SHBG concentrations. Seventy-eight percent of added T was recovered in the high T/low SHBG pool, but only 23% was recovered in the low T/high SHBG pool. In contrast, 98% T was recovered from the same spike in elephant serum, which lacks strong sex steroid binding proteins (Fig. 4AGo). This was further confirmed when individual human serum samples with varying concentrations of SHBG were spiked with T and measured for recovery (Fig. 4BGo). Thus, the acute dynamics of increased circulating sex steroids may depend on the ratio of T (or other SHBG ligands) to SHBG. The presence of relatively higher levels of SHBG may act to attenuate the bioavailability of an acute addition of T. In contrast, relatively lower SHBG levels may allow acute increases in T to exert a transiently greater increase of androgenicity.

The increased bioactivity detected in the higher T pool demonstrated the potential nonlinear function in the relationship between bound and nonbound T, and thus, a nonlinear relationship of T to directly measured bioactivity might be expected. In this report, we demonstrate that the variability of serum protein binding is the main factor associated with differences between bioactive androgen and total T. This explanation does not, however, negate the possibility that androgens other than T have a greater contribution to the bioactive pool (direct contribution to bioactivity, competition with T for SHBG binding sites, or changes in the binding affinity of T for SHBG) when T concentrations are high as opposed to when they are low (8, 9, 14). This also does not negate the possibility that higher levels of SHBG may prolong the half-life of T in circulation. Additional studies will be required to identify factors, specifically SHBG binding, that contribute to the increased androgenicity.

To our knowledge, few cell-based bioassays have been developed and applied to study human samples. The first cell-based bioassay measuring androgen bioactivity of human serum used a reporter COS-1 cell line (16). The sensitivity of the assay was 1.0 nM but required transient transfection for each analysis. The second cell-based bioassay was developed using a Chinese hamster ovary cell line with a sensitivity of less than 1.0 nM (17). However, this stable cell line exhibited endogenous 5{alpha}-reductase activity as well as 17ß-hydroxysteroid dehydrogenase and 3ß-hydroxysteroid dehydrogenase activities (17). Enzymes that could metabolize androgens are absent from the HEK293 cells. There was no evidence of cross-reactivity with other steroids such as E2 and P4, which, only at supraphysiological concentrations, are able to bind the AR and stimulate the expression of androgen response element reporter genes (37, 38).

HEK cells have generally been considered glucocorticoid receptor deficient (39) until the identification of glucocorticoid receptor-{alpha} in wild-type HEK 293 cells (40). We observed no significant signal response to cortisol levels up to 10–8 M in serum. Furthermore, there was no correlation between circulating cortisol and androgen concentrations measured by receptor-mediated assay. Additional studies of very high or supraphysiological cortisol levels may be required to clarify the contribution of free cortisol to this androgen receptor-mediated assay.

Both the receptor-mediated assay for androgens and immunoassay for total T used in this investigation are appropriate for the measurement of circulating of androgens in midlife women in whom androgen concentrations are often near the limit of sensitivity for standard clinical assays. The immunoassay for total T was modified to achieve a sensitivity of 0.18 nM (13). Several recent reports have expressed concern about the reliability of direct immunoassay for measuring low circulating T levels (41, 42), but the assay reported here has excellent correspondence to direct T measurements as well as T measurements derived after organic solvent extraction and celite chromatography separation.

We found no correlation between bioactive androgens and immunoreactive testosterone in middle-aged women. In contrast, a good correlation was found between these two measurements (n = 11, r = 0.80, P < 0.05) in younger women. Furthermore, a positive correlation between measured bioassay values and immunoreactive T values was also revealed in PCOD patients (n = 22, r = 0.70, P < 0.05). Therefore, our data demonstrate that the lack of correlation between bioactive androgens and immunoreactive T may be limited to middle-aged women based on current data. This lack of strong correlation could contribute to the relatively large variability in circulating SHBG between individual women as they move through the menopausal transition (range of SHBG in midlife women: 17–295 nM; young women: 50–101 nM; and PCOD patients: 17–114 nM). In addition, the increase in body size (which is associated with a downward shift in SHBG concentration) and/or an increase in adrenal androgen production (8, 9, 13) in midlife women may also contribute to the increase of the bioavailable androgen component. Given the concern that the SHBG levels influence the fraction of androgens that are bioavailable, it is clear that the interaction between bioactive androgens and SHBG across the menopausal transition deserves further investigation.

In middle-aged women, specifically in women that are approaching the menopausal transition, nearly all bioactive androgens are converted locally in peripheral target tissues from DHEA. In fact, all enzymes required for bioactive androgen and estrogen conversion are expressed in a cell-specific fashion, thus permitting local control of steroid formation and action (43). This concept is known as intracrinology. Whereas the measurements of circulating bioactive androgens provide additional information concerning androgens in circulation, they may not necessarily reflect the local steroid production scenario. Further investigations regarding the potential relationship between circulating bioactive androgens and local androgen production in steroid hormone-dependent tissue are warranted.

In summary, the present data illustrate the ability of a stably transfected cell line to provide a direct measure of total androgen-mediated signal transduction in the sera of midlife women. Theoretically such receptor-mediated measurements of androgens capture not only non-SHBG-bound testosterone but all other circulating non-SHBG-bound bioactive androgens as well. This method may obviate the need for the simultaneous measurements of steroid binding proteins that are required to calculate FAI or FT. This approach may also alleviate many concerns related to nonlinear interactions between steroids and protein binding when circulating protein concentrations are at their extremes (4). Clearly additional studies are needed to characterize fully the nature and sites of action of all biological androgens that are known or speculated to be present in pre- and perimenopausal women and determine the degree to which these direct measurements of androgen activity can explain the variation in health outcomes.


   Acknowledgments
 
We thank the study staff at each site and all the women who participated in SWAN. The authors also gratefully acknowledge the help of Dr. James Overstreet for assisting in the preparation of the manuscript, Dr. C. S. Chang (University of Rochester, Rochester, NY) for providing PSG5 AR plasmid, Dr. V. S. Wilson (United States Environmental Protection Agency) for providing MMTV-LUC.neo plasmid, and Dr. Sybil Crawford for statistical advice.

Clinical centers: University of Michigan, Ann Arbor, MI, MaryFran Sowers, principal investigator; Massachusetts General Hospital, Boston, MA, Robert Neer, principal investigator 1994–1999; Joel Finkelstein, principal investigator, 1999 to present; Rush University, Rush University Medical Center, Chicago, IL, Lynda Powell, principal investigator; University of California, Davis/Kaiser, CA, Ellen Gold, principal investigator; University of California, Los Angeles, CA, Gail Greendale, principal investigator; University of Medicine and Dentistry, New Jersey Medical School, Newark, NJ, Gerson Weiss, principal investigator 1994–2004, Nanette Santoro, principal investigator 2004 to present; and University of Pittsburgh, Pittsburgh, PA, Karen Matthews, principal investigator.

NIH program office: National Institute on Aging, Bethesda, MD, Marcia Ory, 1994–2001, Sherry Sherman, 1994 to present; National Institute of Nursing Research, Bethesda, MD, program officers.

Central laboratory: University of Michigan, Ann Arbor, MI, Daniel McConnell; Central Ligand Assay Satellite Services.

SWAN repository: University of Michigan, Ann Arbor, MI, MaryFran Sowers.

Coordinating center: New England Research Institutes, Watertown, MA, Sonja McKinlay, principal investigator 1995–2001; University of Pittsburgh, Pittsburgh, PA, Kim Sutton-Tyrrell, principal investigator, 2001 to present.

Steering Committee: Chris Gallagher, Chair; Susan Johnson, Chair.


   Footnotes
 
The Study of Women’s Health Across the Nation (SWAN) has grant support from the National Institutes of Health, Department of Health and Human Services, through the National Institute on Aging, the National Institute of Nursing Research, and the NIH Office of Research on Women’s Health (Grants NR004061, AG012505, AG012535, AG012531, AG012539, AG012546, AG012553, AG012554, AG012495) and the SWAN Repository (AG017719).

This research was supported by the Superfund Basic Research Program (P42ES04699) and NIEHS (P01ES06198 and P30ES005707).

Disclosure statement: J.C., F.M.M., D.S.M., N.A.G., C.W., S.E.K.-K., and B.L.L. have nothing to disclose. M.R.S. consults for Wyeth and owns stock in Wyeth. G.A.G. consults for Pfizer.

First Published Online August 29, 2006

Abbreviations: A4, Androstenedione; A5, 5-androstene-3, 17ß-diol; CV, cardiovascular; DCC, dextran-coated charcoal-treated; DHEA, 3ß-hydroxy-5-androsten-17-one; DHT, 17ß-hydroxy-5{alpha}-androstan-3-one; 4-diol, 4-androstene-3, 17ß-diol; 5{alpha}-diol, 5{alpha}-androstane-3,17ß-diol; E2, estradiol; FAI, free androgen index; FBS, fetal bovine serum; FT, free T; GCMS, gas chromatography and mass spectrometry; hAR, human androgen receptor; HEK, human embryonic kidney; MMTV, mouse mammary tumor virus; P4, progesterone; PCOD, polycystic ovarian disorder; SWAN, Study of Women’s Health Across the Nation; T, testosterone.

Received February 8, 2006.

Accepted August 23, 2006.


   References
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
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