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Bioavailable estradiol in man: Relationship with age and testosterone
Clinica Chimica Acta 398 (2008) 145–147
Contents lists available at ScienceDirect
Clinica Chimica Acta j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / c l i n c h i m
Short communication
Bioavailable estradiol in man: Relationship with age and testosterone Laurence Dolomie-Fagour a, Blandine Gatta b, Thi Diem Tien Nguyen a, Jean-Benoît Corcuff a,⁎ a b
Department of Nuclear Medicine, University Hospital and University of Bordeaux, France Department of Endocrinology, University Hospital and University of Bordeaux, France
a r t i c l e
i n f o
Article history: Received 30 July 2008 Received in revised form 28 August 2008 Accepted 3 September 2008 Available online 14 September 2008 Keywords: Bioavailable testosterone Bioavailable estradiol Aging
a b s t r a c t Sex hormones undergo decreases in aging men. Several studies have shown the association of low levels of bioavailable estradiol with osteoporosis in man. To allow a better approach of sex hormones influences, we evaluated bioavailable estradiol concentrations in men and its correlation with age and testosterone. We show that bioavailable estradiol decreases significantly with age. We provide reference values in men with normal testosterone levels. © 2008 Elsevier B.V. All rights reserved.
1. Introduction
2. Patients and methods
With age in men, sex hormones undergo changes even though an abrupt reduction in endogenous sex hormone production is not experienced. Indeed, a slow and progressive decrease of total testosterone (TT) occurs [1]. The bioavailable fraction of testosterone (free and non sex hormone-binding globulin-bound) decreases more rapidly mainly because of the marked age-related increase in serum SHBG levels [2]. Bioavailable testosterone (BT) measurement would thus allow a better appreciation of the hormonal status in elder patients [3]. Importantly, sex hormones concentrations have been linked in male and females to various diseases such as breast or prostate cancer, cardiovascular disease and metabolic syndrome or osteoporosis. The role of total estradiol (TE2) in male skeleton has also recently become appreciated [4]. Similarly to TT and BT expected roles in androgen action, bioavailable estradiol (BE2) may reflect more accurately the clinical potency of estradiol compared to TE2. The proposed rational is that, because the albumin-bound hormone is easily dissociated, the biologically active hormone includes the free and the albumin-bound hormone which can diffuse from circulation to tissues and is available for target cells [3]. Thus, BE2 could represent a better indicator of estrogenic status than TE2. Several studies have shown the association of low levels of BE2 with osteoporosis and vertebral fracture in older men [5,6]. In order to be able to investigate sex hormones abnormalities, it appears necessary to explore physiological sex steroid profiles in men. The aim of this study is to determine in men without gonad insufficiency reference values of BE2 concentrations related to age.
2.1. Patients Sera from 185 male in-patients from an endocrinology department were analyzed for measurement of TE2 and of BE2. All displayed normal TTand BTconcentrations and took no estrogens nor androgens therapy. All subjects were in-patients for various diseases hence the determination of “reference values” for BE2 in our hospital (as opposed to “normal values” that to be established would require recruitment of proven normal subjects). The patients were investigated for: pituitary or hypothalamic tumor (31%), non pituitary tumor (16%), metabolic investigations (12%), rheumatology (10%), fertility and gynecomastia (5%), various endocrine investigations (26%). Patients BMI was calculated. 2.2. Methods Estradiol and testosterone concentrations were assayed with commercial solidphase RIA (ESTRADIOL-2 Clinical Assays and Spectria TESTOSTERONE RIA, Orion Diagnostica, respectively). Testosterone was measured after diethylether extraction. Coefficients of variation for estradiol assay were 9 and 6% at 150 and 400 pmol/L, respectively. Coefficients of variation for testosterone assay were 9.5 and 9.2 % at 2.5 and 11.2 nmol/L, respectively. For BT or BE2 determination serum was equilibrated with tritiated hormones. SHBG and SHBG-bound steroids were precipitated by adding an equal volume of a saturated aqueous solution of ammonium sulfate. Separation of the precipitate was performed by centrifugation for 15 min at 4 °C. Tritiated hormone in the supernatant (not SHBG-bound) was quantified, related to the total amount of tritiated hormone and the percentage was applied to TE2 or TT concentrations to provide BT or BE2 concentrations [7,8]. Coefficients of variation for bioavailable assays were 5.7 and 5.3% for BT and BE2, respectively. 2.3. Statistics Patient's data were log-transformed when appropriate. Patient's groups were compared by Student t-test. Relationships between data were assessed by the Spearman test or by multiple regression analysis.
3. Results ⁎ Corresponding author. Service de Médecine Nucléaire, Hôpital Haut-Lévêque, 33604 Pessac, France. E-mail address: [email protected] (J.-B. Corcuff). 0009-8981/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.cca.2008.09.005
Population median [5th percentile–95th percentile] age, BMI, TT, BT, TE2 and BE2 values are presented in Table 1.
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L. Dolomie-Fagour et al. / Clinica Chimica Acta 398 (2008) 145–147
Table 1 Hormonal results of the patients (whole population and classified groups according to age)
All subjects Sub group b 55 yr (N = 114) Sub group N 55 yr (N = 71)
Age (yr)
BMI kg/m2
[BE2] pmol/L
[E2] pmol/L
[BT] nmol/L
[T] nmol/L
49.0 [21.2–69.8] 41.0 [18.7–53.4] 59.0 [56.0–73.5]
28.0 [20.1–41.8] 29.4 [19.6–43.7] 27.2 [23.0–37.1]
32.2 [15.2–74.9] 34.6 [18.1–72.5] 30.2 [13.0–82.4]
73.2 [38.5–170.2] 75.9 [41.5–165.6] 68.9 [29.0–175.3]
3.4 [1.6–6.8] 3.8⁎ [1.9–6.8] 3.1 [1.5–5.8]
13.2 [7.6–29] 13.3 [7.7–30.5] 13.0 [7.5–27.2]
Median [5th percentile–95th percentile]. ⁎ p b 0.0001 between normal men below or above 55 yr.
BT and BE2 significantly decreased with age (p b 0.0001 and p = 0.017, respectively) ; conversely TT and TE2 were not modified by age. The distribution of data according to age is displayed in Fig. 1. Because previously published work describes a stability of testosterone concentration before 55 yr and a possible decrease after 55 yr [9] we split the subjects in 2 groups according to this age: BT was the only significantly different parameter between groups (p b 0.0001) (see Table 1); specifically BE2 was not significantly different. BE2 was significantly correlated with TT (p = 0.004), BT (p b 0.0001) and TE2 (p b 0.0001). Multiple regression analysis showed that when BE2 relationship was assessed with age and BT the only significantly related parameter was BT (p b 0.001). BE2/BT ratio increases significantly with age (p b 0.001). TT was correlated with BMI (p b 0.0008); conversely neither BT, TE2 nor BE2 was significantly correlated with BMI. As our population included morbidly obese subjects less frequently seen outside specialized departments, we reprocessed the data excluding subjects with a BMIN35 kg/m2. No modification of significance of any test was found (data not shown). 4. Discussion This study shows a significant decrease of BE2 (and BT) with age in men. We propose to use [18.1–72.5] and [13.0–82.4] pmol/l as reference ranges for BE2 in men younger and older 55 yr, respectively. The age-associated increase in sex hormone binding capacity would explain the greater reduction in bioavailable sex hormones. Moreover, it has recently been shown that in combination with the change of SHBG-bound fraction, age-related decrease of BE2 could be also affected by decreasing serum albumin levels [10]. This argues for the use of BE2 assay in elder patients to appreciate estrogenic impregna-
tion, e.g. low BE2 levels :b13 pmol/L (5th percentile when N55 yr). Similar results are reported by Vermeulen et al. [11] whereas 26 pmol/L is the 25th percentile determined in the elderly by Khosla et al. [12]. However, the latter find the threshold concentration associated with a higher rate of bone loss as the median value (40 pmol/L). Although BE2 assay is not difficult it rests on some fundamentals. Technically, to assay estradiol in men obviously requires a sensitive assay [13]. Secondly, our technique requires the use of tritiated estradiol which is not largely available. Direct assay of estradiol in the supernatant after ammonium sulfate has been proposed to avoid isotope use but i/ interferences due to ammonium sulfate salts have been described [14] and ii/ automated assays may not be sensitive enough to directly measure the low level of estradiol in the supernatant. The decrease of BE2 seems at least partly related to the reduction of BT as shown by the multiple analysis. Indeed, testosterone is the main substrate for male estradiol production: most of the circulating androgens in the male comes from peripheral aromatization of androgens precursors in different tissues via the aromatase enzyme system [4]. In agreement with other authors [5,11], we did not observe any significant influence of age on TE2 level. This study finds no relationship between BE2 or BT and BMI; only TT is significantly correlated with BMI. This seems to demonstrate that age-related BE2 decrease is independent of BMI although epidemiological studies of elder men found BMI as a determinant of sex hormones concentration including BT and BE2 [15,1]. The relationship between SHBG and BMI and estradiol (and testosterone) is known and complex [2,9]. On one hand BE2 concentrations are obviously linked to those of SHBG (linked to BMI). On another hand TE2 concentrations in men depend on testosterone aromatization in adipose tissue (and thus of BMI). In agreement with the hypothesis that ageing is associated with endocrine changes related to aromatase activity BE2/BT ratio
Fig. 1. Sex hormone variations according to age. TT total testosterone, BT bioavailable testosterone, E2 total estradiol, BE2 bioavailable estradiol.
L. Dolomie-Fagour et al. / Clinica Chimica Acta 398 (2008) 145–147
increases significantly with aging (and in fat mass [11]). In this study however a decreased ratio cannot be more attributed to an increased aromatase activity than to the age-related decrease of BT. Direct measurement of body fat could help to better discriminate these two factors. This study confirms that BE2 concentrations decrease with age in man in parallel with BT decrease. Serum BE2 concentration b13 pmol/L after 55 yr may be considered as low. Whether this threshold has to be taken into account for risk assessment independently of BT remains to be determined by epidemiological studies. Should BE2 be proven useful, detailed comparison of assays using ammonium sulfate precipitation versus calculation using SHBG concentrations will be required as stressed by comparative studies conducted for bioavailable testosterone [8]. References [1] Muller M, den Tonkelaar I, Thijssen JH, Grobbee DE, van der Schouw YT. Endogenous sex hormones in men aged 40–80 years. Eur J Endocrinol 2003;149:583–9. [2] Field AE, Colditz GA, Willett WC, Longcope C, McKinlay JB. The relation of smoking, age, relative weight, and dietary intake to serum adrenal steroids, sex hormones, and sex hormone-binding globulin in middle-aged men. J Clin Endocrinol Metab 1994;79:1310–6. [3] Pardridge WM, Landaw EM. Tracer kinetic model of blood-brain barrier transport of plasma protein-bound ligands. Empiric testing of the free hormone hypothesis. J Clin Invest 1984;74:745–52.
147
[4] Gennari L, Nuti R, Bilezikian JP. Aromatase activity and bone homeostasis in men. J Clin Endocrinol Metab 2004;89:5898–907. [5] Khosla S, Melton 3rd LJ, Atkinson EJ, O'Fallon WM, Klee GG, Riggs BL. Relationship of serum sex steroid levels and bone turnover markers with bone mineral density in men and women: a key role for bioavailable estrogen. J Clin Endocrinol Metab 1998;83:2266–74. [6] Szulc P, Munoz F, Claustrat B, et al. Bioavailable estradiol may be an important determinant of osteoporosis in men: the MINOS study. J Clin Endocrinol Metab 2001;86:192–9. [7] Tremblay RR, Dube JY. Plasma concentrations of free and non-TeBG bound testosterone in women on oral contraceptives. Contraception 1974;10:599–605. [8] Giton F, Fiet J, Guechot J, et al. Serum bioavailable testosterone: assayed or calculated? Clin Chem 2006;52:474–81. [9] Vermeulen A, Kaufman JM, Giagulli VA. Influence of some biological indexes on sex hormone-binding globulin and androgen levels in aging or obese males. J Clin Endocrinol Metab 1996;81:1821–6. [10] Hayashi T, Yamada T. Association of bioavailable estradiol levels and testosterone levels with serum albumin levels in elderly men. Aging Male 2008;11:63–70. [11] Vermeulen A, Kaufman JM, Goemaere S, van Pottelberg I. Estradiol in elderly men. Aging Male 2002;5:98–102. [12] Khosla S, Melton 3rd LJ, Atkinson EJ, O'Fallon WM. Relationship of serum sex steroid levels to longitudinal changes in bone density in young versus elderly men. J Clin Endocrinol Metab 2001;86:3555–61. [13] Taieb J, Benattar C, Birr AS, Lindenbaum A. Limitations of steroid determination by direct immunoassay. Clin Chem 2002;48:583–5. [14] Davies R, Collier C, Raymond M, Heaton J, Clark A. Indirect measurement of bioavailable testosterone with the Bayer Immuno 1 system. Clin Chem 2002;48: 388–90. [15] Ferrini RL, Barrett-Connor E. Sex hormones and age: a cross-sectional study of testosterone and estradiol and their bioavailable fractions in community-dwelling men. Am J Epidemiol 1998;147:750–4.
Contents lists available at ScienceDirect
Clinica Chimica Acta j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / c l i n c h i m
Short communication
Bioavailable estradiol in man: Relationship with age and testosterone Laurence Dolomie-Fagour a, Blandine Gatta b, Thi Diem Tien Nguyen a, Jean-Benoît Corcuff a,⁎ a b
Department of Nuclear Medicine, University Hospital and University of Bordeaux, France Department of Endocrinology, University Hospital and University of Bordeaux, France
a r t i c l e
i n f o
Article history: Received 30 July 2008 Received in revised form 28 August 2008 Accepted 3 September 2008 Available online 14 September 2008 Keywords: Bioavailable testosterone Bioavailable estradiol Aging
a b s t r a c t Sex hormones undergo decreases in aging men. Several studies have shown the association of low levels of bioavailable estradiol with osteoporosis in man. To allow a better approach of sex hormones influences, we evaluated bioavailable estradiol concentrations in men and its correlation with age and testosterone. We show that bioavailable estradiol decreases significantly with age. We provide reference values in men with normal testosterone levels. © 2008 Elsevier B.V. All rights reserved.
1. Introduction
2. Patients and methods
With age in men, sex hormones undergo changes even though an abrupt reduction in endogenous sex hormone production is not experienced. Indeed, a slow and progressive decrease of total testosterone (TT) occurs [1]. The bioavailable fraction of testosterone (free and non sex hormone-binding globulin-bound) decreases more rapidly mainly because of the marked age-related increase in serum SHBG levels [2]. Bioavailable testosterone (BT) measurement would thus allow a better appreciation of the hormonal status in elder patients [3]. Importantly, sex hormones concentrations have been linked in male and females to various diseases such as breast or prostate cancer, cardiovascular disease and metabolic syndrome or osteoporosis. The role of total estradiol (TE2) in male skeleton has also recently become appreciated [4]. Similarly to TT and BT expected roles in androgen action, bioavailable estradiol (BE2) may reflect more accurately the clinical potency of estradiol compared to TE2. The proposed rational is that, because the albumin-bound hormone is easily dissociated, the biologically active hormone includes the free and the albumin-bound hormone which can diffuse from circulation to tissues and is available for target cells [3]. Thus, BE2 could represent a better indicator of estrogenic status than TE2. Several studies have shown the association of low levels of BE2 with osteoporosis and vertebral fracture in older men [5,6]. In order to be able to investigate sex hormones abnormalities, it appears necessary to explore physiological sex steroid profiles in men. The aim of this study is to determine in men without gonad insufficiency reference values of BE2 concentrations related to age.
2.1. Patients Sera from 185 male in-patients from an endocrinology department were analyzed for measurement of TE2 and of BE2. All displayed normal TTand BTconcentrations and took no estrogens nor androgens therapy. All subjects were in-patients for various diseases hence the determination of “reference values” for BE2 in our hospital (as opposed to “normal values” that to be established would require recruitment of proven normal subjects). The patients were investigated for: pituitary or hypothalamic tumor (31%), non pituitary tumor (16%), metabolic investigations (12%), rheumatology (10%), fertility and gynecomastia (5%), various endocrine investigations (26%). Patients BMI was calculated. 2.2. Methods Estradiol and testosterone concentrations were assayed with commercial solidphase RIA (ESTRADIOL-2 Clinical Assays and Spectria TESTOSTERONE RIA, Orion Diagnostica, respectively). Testosterone was measured after diethylether extraction. Coefficients of variation for estradiol assay were 9 and 6% at 150 and 400 pmol/L, respectively. Coefficients of variation for testosterone assay were 9.5 and 9.2 % at 2.5 and 11.2 nmol/L, respectively. For BT or BE2 determination serum was equilibrated with tritiated hormones. SHBG and SHBG-bound steroids were precipitated by adding an equal volume of a saturated aqueous solution of ammonium sulfate. Separation of the precipitate was performed by centrifugation for 15 min at 4 °C. Tritiated hormone in the supernatant (not SHBG-bound) was quantified, related to the total amount of tritiated hormone and the percentage was applied to TE2 or TT concentrations to provide BT or BE2 concentrations [7,8]. Coefficients of variation for bioavailable assays were 5.7 and 5.3% for BT and BE2, respectively. 2.3. Statistics Patient's data were log-transformed when appropriate. Patient's groups were compared by Student t-test. Relationships between data were assessed by the Spearman test or by multiple regression analysis.
3. Results ⁎ Corresponding author. Service de Médecine Nucléaire, Hôpital Haut-Lévêque, 33604 Pessac, France. E-mail address: [email protected] (J.-B. Corcuff). 0009-8981/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.cca.2008.09.005
Population median [5th percentile–95th percentile] age, BMI, TT, BT, TE2 and BE2 values are presented in Table 1.
146
L. Dolomie-Fagour et al. / Clinica Chimica Acta 398 (2008) 145–147
Table 1 Hormonal results of the patients (whole population and classified groups according to age)
All subjects Sub group b 55 yr (N = 114) Sub group N 55 yr (N = 71)
Age (yr)
BMI kg/m2
[BE2] pmol/L
[E2] pmol/L
[BT] nmol/L
[T] nmol/L
49.0 [21.2–69.8] 41.0 [18.7–53.4] 59.0 [56.0–73.5]
28.0 [20.1–41.8] 29.4 [19.6–43.7] 27.2 [23.0–37.1]
32.2 [15.2–74.9] 34.6 [18.1–72.5] 30.2 [13.0–82.4]
73.2 [38.5–170.2] 75.9 [41.5–165.6] 68.9 [29.0–175.3]
3.4 [1.6–6.8] 3.8⁎ [1.9–6.8] 3.1 [1.5–5.8]
13.2 [7.6–29] 13.3 [7.7–30.5] 13.0 [7.5–27.2]
Median [5th percentile–95th percentile]. ⁎ p b 0.0001 between normal men below or above 55 yr.
BT and BE2 significantly decreased with age (p b 0.0001 and p = 0.017, respectively) ; conversely TT and TE2 were not modified by age. The distribution of data according to age is displayed in Fig. 1. Because previously published work describes a stability of testosterone concentration before 55 yr and a possible decrease after 55 yr [9] we split the subjects in 2 groups according to this age: BT was the only significantly different parameter between groups (p b 0.0001) (see Table 1); specifically BE2 was not significantly different. BE2 was significantly correlated with TT (p = 0.004), BT (p b 0.0001) and TE2 (p b 0.0001). Multiple regression analysis showed that when BE2 relationship was assessed with age and BT the only significantly related parameter was BT (p b 0.001). BE2/BT ratio increases significantly with age (p b 0.001). TT was correlated with BMI (p b 0.0008); conversely neither BT, TE2 nor BE2 was significantly correlated with BMI. As our population included morbidly obese subjects less frequently seen outside specialized departments, we reprocessed the data excluding subjects with a BMIN35 kg/m2. No modification of significance of any test was found (data not shown). 4. Discussion This study shows a significant decrease of BE2 (and BT) with age in men. We propose to use [18.1–72.5] and [13.0–82.4] pmol/l as reference ranges for BE2 in men younger and older 55 yr, respectively. The age-associated increase in sex hormone binding capacity would explain the greater reduction in bioavailable sex hormones. Moreover, it has recently been shown that in combination with the change of SHBG-bound fraction, age-related decrease of BE2 could be also affected by decreasing serum albumin levels [10]. This argues for the use of BE2 assay in elder patients to appreciate estrogenic impregna-
tion, e.g. low BE2 levels :b13 pmol/L (5th percentile when N55 yr). Similar results are reported by Vermeulen et al. [11] whereas 26 pmol/L is the 25th percentile determined in the elderly by Khosla et al. [12]. However, the latter find the threshold concentration associated with a higher rate of bone loss as the median value (40 pmol/L). Although BE2 assay is not difficult it rests on some fundamentals. Technically, to assay estradiol in men obviously requires a sensitive assay [13]. Secondly, our technique requires the use of tritiated estradiol which is not largely available. Direct assay of estradiol in the supernatant after ammonium sulfate has been proposed to avoid isotope use but i/ interferences due to ammonium sulfate salts have been described [14] and ii/ automated assays may not be sensitive enough to directly measure the low level of estradiol in the supernatant. The decrease of BE2 seems at least partly related to the reduction of BT as shown by the multiple analysis. Indeed, testosterone is the main substrate for male estradiol production: most of the circulating androgens in the male comes from peripheral aromatization of androgens precursors in different tissues via the aromatase enzyme system [4]. In agreement with other authors [5,11], we did not observe any significant influence of age on TE2 level. This study finds no relationship between BE2 or BT and BMI; only TT is significantly correlated with BMI. This seems to demonstrate that age-related BE2 decrease is independent of BMI although epidemiological studies of elder men found BMI as a determinant of sex hormones concentration including BT and BE2 [15,1]. The relationship between SHBG and BMI and estradiol (and testosterone) is known and complex [2,9]. On one hand BE2 concentrations are obviously linked to those of SHBG (linked to BMI). On another hand TE2 concentrations in men depend on testosterone aromatization in adipose tissue (and thus of BMI). In agreement with the hypothesis that ageing is associated with endocrine changes related to aromatase activity BE2/BT ratio
Fig. 1. Sex hormone variations according to age. TT total testosterone, BT bioavailable testosterone, E2 total estradiol, BE2 bioavailable estradiol.
L. Dolomie-Fagour et al. / Clinica Chimica Acta 398 (2008) 145–147
increases significantly with aging (and in fat mass [11]). In this study however a decreased ratio cannot be more attributed to an increased aromatase activity than to the age-related decrease of BT. Direct measurement of body fat could help to better discriminate these two factors. This study confirms that BE2 concentrations decrease with age in man in parallel with BT decrease. Serum BE2 concentration b13 pmol/L after 55 yr may be considered as low. Whether this threshold has to be taken into account for risk assessment independently of BT remains to be determined by epidemiological studies. Should BE2 be proven useful, detailed comparison of assays using ammonium sulfate precipitation versus calculation using SHBG concentrations will be required as stressed by comparative studies conducted for bioavailable testosterone [8]. References [1] Muller M, den Tonkelaar I, Thijssen JH, Grobbee DE, van der Schouw YT. Endogenous sex hormones in men aged 40–80 years. Eur J Endocrinol 2003;149:583–9. [2] Field AE, Colditz GA, Willett WC, Longcope C, McKinlay JB. The relation of smoking, age, relative weight, and dietary intake to serum adrenal steroids, sex hormones, and sex hormone-binding globulin in middle-aged men. J Clin Endocrinol Metab 1994;79:1310–6. [3] Pardridge WM, Landaw EM. Tracer kinetic model of blood-brain barrier transport of plasma protein-bound ligands. Empiric testing of the free hormone hypothesis. J Clin Invest 1984;74:745–52.
147
[4] Gennari L, Nuti R, Bilezikian JP. Aromatase activity and bone homeostasis in men. J Clin Endocrinol Metab 2004;89:5898–907. [5] Khosla S, Melton 3rd LJ, Atkinson EJ, O'Fallon WM, Klee GG, Riggs BL. Relationship of serum sex steroid levels and bone turnover markers with bone mineral density in men and women: a key role for bioavailable estrogen. J Clin Endocrinol Metab 1998;83:2266–74. [6] Szulc P, Munoz F, Claustrat B, et al. Bioavailable estradiol may be an important determinant of osteoporosis in men: the MINOS study. J Clin Endocrinol Metab 2001;86:192–9. [7] Tremblay RR, Dube JY. Plasma concentrations of free and non-TeBG bound testosterone in women on oral contraceptives. Contraception 1974;10:599–605. [8] Giton F, Fiet J, Guechot J, et al. Serum bioavailable testosterone: assayed or calculated? Clin Chem 2006;52:474–81. [9] Vermeulen A, Kaufman JM, Giagulli VA. Influence of some biological indexes on sex hormone-binding globulin and androgen levels in aging or obese males. J Clin Endocrinol Metab 1996;81:1821–6. [10] Hayashi T, Yamada T. Association of bioavailable estradiol levels and testosterone levels with serum albumin levels in elderly men. Aging Male 2008;11:63–70. [11] Vermeulen A, Kaufman JM, Goemaere S, van Pottelberg I. Estradiol in elderly men. Aging Male 2002;5:98–102. [12] Khosla S, Melton 3rd LJ, Atkinson EJ, O'Fallon WM. Relationship of serum sex steroid levels to longitudinal changes in bone density in young versus elderly men. J Clin Endocrinol Metab 2001;86:3555–61. [13] Taieb J, Benattar C, Birr AS, Lindenbaum A. Limitations of steroid determination by direct immunoassay. Clin Chem 2002;48:583–5. [14] Davies R, Collier C, Raymond M, Heaton J, Clark A. Indirect measurement of bioavailable testosterone with the Bayer Immuno 1 system. Clin Chem 2002;48: 388–90. [15] Ferrini RL, Barrett-Connor E. Sex hormones and age: a cross-sectional study of testosterone and estradiol and their bioavailable fractions in community-dwelling men. Am J Epidemiol 1998;147:750–4.