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Clinical Chemistry 53: 2160-2168, 2007. First published October 11, 2007; 10.1373/clinchem.2007.087312
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(Clinical Chemistry. 2007;53:2160-2168.)
© 2007 American Association for Clinical Chemistry, Inc.

Endocrinology and Metabolism

Determination of Bioavailable Testosterone [Non–Sex Hormone–Binding Globulin (SHBG)-Bound Testosterone] in a Population of Healthy French Men: Influence of Androstenediol on Testosterone Binding to SHBG

Frank Giton1,2,a, Saïk Urien3, Catherine Born4, Jean Tichet4, Jérôme Guéchot5, Jacques Callebert6, Françoise Bronsard7, Jean Pierre Raynaud8 and Jean Fiet2

1 Assistance Publique-Hôpitaux de Paris (AP-HP), Biological Center of Investigations, University Hospital Group, Sud Henri Mondor, Faculté de Médecine, Créteil, France.
2 Institut National de la Santé et de la Recherche Médicale Unité 841 Eq07, Centre Hospitalier Universitaire Henri Mondor, Faculté de Médecine, Créteil, France.
3 Unité de Recherche Clinique Paris Centre, Hôpital Tarnier, Paris, France.
4 Institut Inter-Régional pour la Santé, La Riche, France.
5 AP-HP, Laboratoire d’Hormonologie, Hôpital St. Antoine, Paris, France.
6 AP-HP, Laboratoire de Biochimie, Hôpital Lariboisière, Paris, France.
7 Institut Universitaire de Technologie de Cachan, Cachan, France.
8 Université Paris VI, Pierre et Marie Curie, Paris, France.

aAddress correspondence to this author at: Centre de Recherches Chirurgicales, Faculté de Médecine, 8 rue du Général Sarrail, 94010 Créteil Cedex, France. Fax 33-1-49-81-35-52; e-mail giton{at}univ-paris12.fr.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Background: Bioavailable testosterone (BT) is measured [assayed BT (aBT)] or calculated (cBT) in the diagnosis of hypogonadism in men. The cBT depends, however, on the values of the association constants of total testosterone (TT) for sex hormone–binding globulin (SHBG; Ks) and albumin (Ka), and its use therefore remains controversial.

Methods: In 503 selected, untreated healthy men, 20–74 years old, we measured TT, dihydrotestosterone (DHT), and androstenediol (5-diol) by GC-MS, SHBG by RIA, and BT after ammonium sulfate precipitation or by calculation according to the law of mass action.

Results: A slight decrease in TT, significant decreases in BT and 5-diol, no variation in DHT, and an increase in SHBG were observed with age. In young males (≤39 years), the lower normal limits were between 2.30 and 2.72 nmol/L for aBT and 8.50 nmol/L for TT. For Ks = 1 x 109 L/mol and Ka = 3.6 x 104 L/mol, the lower cBT limit was found to be 2-fold higher than for aBT. With optimized Ks = 1.9 x 109 L/mol and Ka = 2.45 x 104 L/mol, cBT values close to aBT were obtained. When 5-diol was included in the model as a competitive SHBG inhibitor, the correlation between cBT and aBT was better and the cBT:aBT ratios vs 5-diol were less biased.

Conclusion: Lower normal serum aBT concentration in normal men appears to be between 2.30 and 2.72 nmol/L. Much higher serum cBT concentrations are associated with use of different association constants that may be inappropriate. When using the optimized binding constants, taking age-related 5-diol values into consideration slightly improves prediction of cBT.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Bioavailable testosterone (BT)1 appears to provide a more accurate view of testosterone status than total testosterone (TT)(1). According to recommendations of the International Society for the Study of the Aging Male (ISSAM)(2), the most reliable and widely accepted parameters for confirming hypogonadism are measurement of BT or, alternatively, calculating free testosterone and BT. However, the lower limits for demonstrating testosterone deficiency in both young and aging men have not been established using reference methods in a sufficiently large population.

Various normal lower limits have been proposed for measured TT and BT, usually based on studies carried out in rather small groups of normal men. In addition, the use of a simplified formula for calculating BT [calculated BT (cBT)] using specific association constants of testosterone for sex hormone–binding globulin (SHBG) and for albumin(3) seems to yield higher calculated than measured BT [assayed BT (aBT)](4). This formula takes into account only binding of testosterone to albumin and SHBG in determining BT, although other steroids with nonnegligible serum concentrations in males, such as dihydrotestosterone (DHT) and androstenediol (5-diol), also bind to SHBG.

To take into account the interference of DHT and 5-diol, we used reference analytical methods to determine TT, DHT, aBT, 5-diol, and SHBG in a selected group of 503 men age 20 to 74 years. We then calculated cBT and compared it with aBT. To demonstrate that BT did not depend only on TT and SHBG, we compared aBT in pairs of young and older men in whom the SHBG concentrations were identical and the TT concentrations were nearly identical.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
population studied
Volunteer men age 20 to 74 years and representing the general French population were recruited in a health center in the city of Tours, France, the Institut Inter-Régional pour la Santé. The recruitment procedure was in accordance with the Helsinki Declaration, and each participant provided prior written informed consent. All volunteers were excluded who were taking drugs known to influence the hypothalamic-pituitary-gonadal axis function, had a body mass index ≥29 kg/m2, or were suffering from chronic disease. Blood was drawn between 0800 and 1000 AM after a 12-h overnight fast. Serum for hormone study was isolated from the blood, divided into several samples, and frozen at –20 °C. Samples of the sera of the 539 men retained for the study were sent to our hormone biochemistry laboratory for analysis, and we measured their glucose, creatinine, triglycerides, {gamma}-glutamyl transferase, alanine aminotransferase (ALT), and aspartate aminotransferase (AST). All volunteers exhibiting abnormal biochemical values were excluded from the study.

hormone assays
TT, DHT, and 5-diol.
Serum TT, DHT, and 5-diol were measured by GC-MS according to Labrie et al.(5). Briefly, 0.5 mL serum was extracted after adding deuterated steroid internal standards. The organic extracts were purified on conditioned Bond Elut-Si (Varian, ref. 12102037). After derivatization with pentafluorobenzoylchloride (Aldrich, ref. 10 377-2), TT, DHT, and 5-diol analytes were separated on a GC system (6890N Agilent Technologies) using a 50% phenylmethylpolysiloxane capillary column (J&W Scientific, ref. 122-1831). An HP5973 (Agilent Technologies) quadrupole mass spectrometer was used for detection.

The interassay CVs (n = 23) were 6.0%, 2.8%, and 2.0% for TT (2.50, 10.47, and 20.59 nmol/L, respectively); 1.9%, 2.7%, and 3.7% for DHT (0.86, 3.55, and 8.26 nmol/L); and 13.7%, 8.2%, and 6.0% for 5-diol (0.90, 3.37, and 6.85 nmol/L). The lower limits of quantification for TT, DHT, and 5-diol were 0.17, 0.069, and 0.34 nmol/L, respectively.

BT assay.
We measured the percentage BT (%BT) as previously described(4). Briefly, after adding minute doses of purified tritiated testosterone to serum samples, followed by incubation at 37 °C, we used a solution of saturated ammonium sulfate to precipitate the SHBG-bound testosterone. The precipitate was then centrifuged, and we deduced the percentage of SHBG-unbound tritiated testosterone (BT) from the radioactivity measurements. We then multiplied the %BT by the serum TT concentration and calculated the concentration of bioavailable serum testosterone.

SHBG assay.
We used the Schering SHBG-RIACT radioimmunometric kit to measure SHBG. The interassay CVs for SHBG were 7.1%, 8.2%, and 5.6% (15.8, 46.3, and 68.2 nmol/L).

Gonadotropins and albumin assays.
We measured follicle-stimulating hormone and luteinizing hormone using radioimmunometric kits (Beckman Coulter luteinizing hormone IM 1381; follicle-stimulating hormone IM 2125). We used the bromcresol green method (Olympus AU 800) to assay the albumin concentration.

calculations
Calculation of BT.
cBT was calculated according to Vermeulen’s formula(3), but applying association constants of TT for SHBG (Ks) varying between 0.6 x 109 and 2 x 109 L/mol and association constants for albumin (Ka) varying between 0.7 x 104 and 3.6 x 104 L/mol. The cBT was then compared with the aBT. Comparison of aBT and cBT was based on counting the number of samples in which the cBT differed from the aBT by <10%, 20%, and 30%(4). The (cBT-aBT):aBT ratio (relative difference between cBT and aBT) was determined for each sample. We also calculated the cBT:aBT correlation coefficients (r) and cBT:aBT ratios for each sample.

We also carried out an alternative calculation that took into account the possible inhibitory effect of 5-diol on testosterone binding to SHBG, according to published equations(6). Accordingly, new equations were derived for the determination of cBT.

cBT correction for the presence of 5-diol as a competitive inhibitor.
Protein-bound testosterone (B) and protein-bound inhibitor (Ib) concentrations in the presence of 5-diol were calculated from the following equations(6):

Formula 1(1)

Formula 2(2)
in which F, If, SHBG, and A stand for serum concentrations of free testosterone, free inhibitor, total SHBG, and albumin, respectively. Ks and Ksi are the respective association constants of TT and 5-diol for SHBG(7). Ka is the proportional binding constant of testosterone and 5-diol to albumin. The binding constants were Ks = 1.9 x 109 L/mol, Ksi = 1.5 x 109 L/mol, and Ka = 2.45 x 104 L/mol.

The total plasma concentrations of testosterone (TT) and inhibitor (It) were as follows:

Formula 3(3)

Formula 4(4)
Given the binding constants for testosterone and inhibitor and the TT and It concentrations, the free concentrations of the ligands F and If were determined by iterative minimization of the function f:

Formula 5(5)
This determination was done using the Nelder–Mead algorithm implemented in the R program (R Development Core Team). Free testosterone concentrations were then used to determine cBT.

Simplified solution for cBT determination using the TT and 5-diol concentrations.
The free 5-diol fraction in the 503 patients was characterized by a median of 2.42%, with an interquartile range of 1.94%–2.92%. This narrow range of free fraction variation suggested that the free 5-diol concentration could be approximated as follows:

Formula 6(6)
Using this approximation, an analytical solution to F can be derived for Eqs. 1Up and 3Up :

Formula 7(7)
with

Formula 8(8)

Formula 9(9)

Formula 10(10)

Formula 11(11)

Formula 12(12)
then

Formula 13(13)

Comparison of aBT in young and older men with the same serum SHBG concentrations and nearly identical TT concentrations.
Classically, BT is considered to depend principally on TT and SHBG (normal slight physiological variations in the albumin concentration do not influence cBT results). Thus, sera with the same SHBG and TT will theoretically have the same BT. We hypothesized that BT could also depend on factors other than TT and SHBG that are not taken into account in calculating the cBT. We compared aBT in pairs of young and aging men in whom the SHBG was identical and the TT not more than 10% different.

Measurement of aBT in charcoal-treated sera overloaded with TT and 5-diol or dehydroepiandrosterone.
We measured BT in 2 pools of charcoal-treated sera containing the same concentrations of testosterone (13.7 nmol/L) and either 45.5 nmol/L or 17.7 nmol/L of SHBG. Each pool was divided into samples overloaded with increasing quantities of 5-diol or of dehydroepiandrosterone (DHEA). We then measured BT as previously described (n = 6).

statistics
We used the nonparametric paired Wilcoxon and Spearman regression tests.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Of the 539 individuals age 20 to 74 years, 36 with abnormally high or low serum gonadotropin concentrations were excluded. The remaining 503 serum samples were then stratified into groups of increasing age: 20–39 (n = 142); 40–49 (n = 100); 50–59 (n = 135); and 60–74 years (n = 126).

normal tt, bt, dht, and 5-diol values and shbg variation expressed in percentiles by age group (table 1 )
The percentiles of total participants and age-stratified subgroups are reported in Table 1 . In the group of young men (20–39), the 1st and 5th percentiles of TT and BT were 8.49 and 9.67 nmol/L and 2.70 and 3.95 nmol/L, respectively, whereas in the overall population studied they were 7.45 and 9.33 nmol/L and 2.08 and 2.60 nmol/L. We observed significant age-related decreases in BT and 5-diol (P <0.0001) and TT (P <0.0048), as well as a significant increase in SHBG (P <0.0001), but no significant variation in DHT with age (P = 0.4499).


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  		Table 1. Normal serum values of TT, BT, DHT, 5-diol, SHBG, and albumin in untreated healthy men according to age.

comparison of ABT and CBT
We calculated cBT according to the association constants of testosterone for SHBG, 1 x 109 L/mol, and for albumin, 3.6 x 104 L/mol (values in the ISSAM website)(3). The values obtained for cBT were higher (P <0.0001) than for aBT (Table 2 ), although aBT and cBT were correlated (r = 0.831; mean cBT:aBT ratio 1.63; Table 3 ). Moreover, the cBT:aBT ratio significantly increased with age, according to the regression equation cBT:aBT = 0.009 x years + 1.208 (r = 0.598).


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  		Table 2. aBT and cBT values according to Vermeulen formula and using association constants for SHBG and albumin (respectively, Ks = 1 x 109 L/mol, Ka = 3.6 x 104 L/mol) and optimal bioavailable testosterone calculated according to paired optimal Ks and Ka as reported in Table 3Up for each age group.


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  		Table 3. Optimization and comparison between aBT and cBT in 503 samples of normal subjects (full population) and groups of increasing age, using optimal paired and nonoptimal Ks and Ka.

optimization of KS and KA association constants
Using optimal paired association constants, the number of cBT values differing by <30% from the aBT was between 421 and 428 (among the 503 normal participants), whereas applying the constants used in the ISSAM website(3), only 67 of the cBT values differed by <30% from the aBT (Table 3Up ).

We show that for the same Ks = 1.9 x 109 L/mol, the corresponding optimal Ka values decreased according to age range, as reported in Table 3Up .

In our overall normal population, the optimal cBT values (reported as a percentile curve in Fig. 1 ) could be superimposed over the aBT values, whereas cBT values calculated from the Ks and Ka used in the ISSAM website formula led to a higher shift in the cBT curve (Fig. 1 ).


Figure 1
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  		Figure 1. Percentiles of BT normal values in 503 untreated healthy men (whole population) age 20–74 years.

BT was measured ({circ} = aBT) or calculated according to paired values of optimized association constants Ks and Ka [x = opt (optimized) BT] and nonoptimized (ISSAM website, + = cBT) association constants.

influence of 5-diol on testosterone binding
To find out whether BT depended only on TT and SHBG, we compared BT values in young and aging individuals in whom the SHBG values were identical and the TT values were nearly identical (Table 4 ).


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  		Table 4. Comparison of aBT, TT, DHT, and 5-diol in pairs of samples from young and older subjects exhibiting the same SHBG values and TT in older individuals equal to TT in young individuals ±10%.

Comparison of SHBG and TT among those in the 20–39 and 40–49 age groups yielded 55 sample pairs with identical SHBG and TT values in the latter group that were <10% different from those in the former group (Table 4Up ). The mean aBT (5.83 nmol/L) in the older group was significantly lower (P <0.0001) than the mean aBT (6.7 nmol/L) in the younger group. Comparisons of pairs of individuals in other groups yielded similar observations (Table 4Up ).

The mean DHT was not significantly different in pairs of younger and aging participants with the same SHBG and nearly identical TT (Table 4Up ). Conversely, under these conditions, a significant decline in mean 5-diol was observed in the aging compared with younger participants.

relation between in vitro 5-diol and bt concentrations
For the same TT (13.87 nmol/L) and SHBG concentrations in charcoal-treated pooled serum (older men, 45.5 nmol/L; younger men, 17.7 nmol/L), measurement of BT in sera containing increasing concentrations of 5-diol (2.13, 4.27, 8.54, and 17.11 nmol/L) revealed an increase in serum aBT concentrations compared with sera with no 5-diol (Fig. 2 ; aging men, 7%, 11%, 20%, and 38%; younger men, 0%, 2%, 6%, and 16%.) Overloading with 3.47, 6.93, 13.87, and 27.74 nmol/L DHEA revealed an increase in aBT of 1%, 2%, 3%, and 5% in the aging men’s pooled sera and no increase in younger men. Overloading with the same concentrations of 5-diol plus DHEA as above led to an increase in aBT of 6%, 12%, 21%, and 41% in the sera of the aging men, and 1%, 1%, 6%, and 15% in younger men.


Figure 2
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  		Figure 2. aBT gain (%) in charcoal-treated sera overloaded with a constant testosterone concentration and overloaded with increasing amounts of 5-diol and/or DHEA [n = 6 (SD)].

Inset, cBT:aBT ratio in 503 serum samples as a function of 5-diol concentration; 2.5th and 97.5th percentile lines did not show any obvious trend (slope approximately –0.002) when the 5-diol concentration was taken into account in the cBT:aBT ratio, whereas there was a clear trend (slope approximately –0.02) when this was not taken into account (plot not shown).

influence of 5-diol concentrations on the serum CBT and CBT:ABT ratio
We also determined the cBT(5-diol; BT calculated taking into account the 5-diol plasma concentration), assuming competitive inhibition between testosterone and 5-diol on SHBG (see Eqs. 1–4Up Up Up Up in Calculations of BT. The cBT(optimal) vs aBT correlation was 0.876, whereas the cBT(5-diol) vs aBT correlation was 0.888. In addition, the cBT(5-diol):aBT ratio vs the 5-diol plasma concentration (Fig. 2Up ) did not show any bias (regression equation: y = 1.09 – 0.002x; theoretical: y = 1), whereas the cBT:aBT ratio vs the 5-diol plasma concentration showed a significant bias. Indeed, the cBT:aBT ratio rapidly decreased below 1 when the 5-diol concentration was >5 nmol/L. However, the exact determination of cBT in the presence of 5-diol requires an iterative process. Therefore, assuming a constant median free fraction of plasma 5-diol (2.4%), we were able to derive an analytical solution for the calculation of free testosterone allowing determination of cBT(5-diol; see Eqs. 6–13Up Up Up Up Up Up Up Up in Calculations of BT). Correlation between the analytical cBT(5-diol) and the exact BT(5-diol) was 0.999.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
normal limits of tt, bt, dht, shbg, and 5-diol
Normal or usual hormone concentration values depend on several factors, such as accuracy of assay methods used, the population studied and its age, and choice of statistical expression of normal limits. Because assayed hormone plasma concentrations do not have a gaussian distribution, we preferred to express the results of serum concentrations in percentile form.

As reported(8)(9)(10)(11)(12), we found a slight but significant decrease with age in TT and a steeper decrease in BT, with a significant concomitant increase in SHBG. As others have, we found no significant variation in DHT(13)(14)(15) and a significant decrease in 5-diol(14)(16) with age.

The aim of this study was to define the limits of normal serum BT values obtained from the results of accurate GC-MS steroid assay in a population of untreated healthy French men, to best identify a state of hypogonadism. In a group of 142 young men (<40 years), we observed a mean TT concentration of 9.67 nmol/L, corresponding to the 5th percentile (Table 1Up )—that is, <11 nmol/L, considered the lower limit of TT calculated from the mean –2.5 SD obtained in a population of healthy young men in whom the population distribution was considered to be normal after log transformation(2)(17). These TT limits are interesting, because a clinically symptomatic threshold for androgen deficiency corresponding to a TT of approximately 10 nmol/L measured before reimplantation of a testosterone depot product has recently been reported in a population of testosterone-treated hypogonadal men(18).

Morales and Lunenfeld(2) considered BT measurement to be the most reliable and widely acceptable parameter for confirming hypogonadism. In our reference population of 142 healthy young men, the 1st 2 individual lower BT values were 2.32 and 2.72 nmol/L. Therefore the 1st percentile of our reference population of healthy young men was between 2.32 and 2.72 nmol/L. This agrees with a cutoff point of 2.50 nmol/L(19) or 2.32 nmol/L(20) for hypogonadism, established as a value not found in eugonadal young males. Nelson et al.(21) reported that BT concentrations <2.43 nmol/L warrant androgen treatment.

The interquartile BT values (3.26–5.17 nmol/L; n = 126) we reported in the aging group (60–74 years) may be compared with interquartile BT values (2.77–4.09 nmol/L) published for a large group of 547 aging men between 59 and 89 years(22). As we have, 3 other authors measured BT by the SHBG ammonium sulfate precipitation method, adding minute amounts of 3H-testosterone(4)(23)(24). Our median DHT concentration (1.69 nmol/L; Table 1Up ) was similar to that reported by these 3 authors, and a stable normal mean value, whatever the age, has already been reported(13)(14)(16). Median 5-diol concentrations in the younger (<40 years; 6.06 nmol/L) and aging (60–74 years; 3.86 nmol/L) groups are similar to those described by others in smaller groups(14)(25).

ABT and CBT
Using the Vermeulen formula(3) and association constants (Ks = 1 x 109 L/mol and Ka = 3.6 x 104 L/mol) in 503 healthy normal men, cBT was found to be higher than aBT (Table 2Up ). These higher cBT than aBT values(4)(26)(27) are supported by several recent validation experiments in which the free testosterone concentration calculated using the ISSAM website calculator was found to be higher than the measured free testosterone(28)(29)(30)(31).

In young men, the lower normal aBT limit and the mean normal (7 nmol/L) and median (6.59 nmol/L) aBT values we found were very similar to those obtained by other authors using the same ammonium sulfate SHBG precipitation for BT measurement(19)(20)(32). However, these aBT results contrast with recently published lower normal cBT limits of 4.85 nmol/L(33) and 5.27 nmol/L(34) and a mean cBT of 13.28 nmol/L(35). These same recently published normal cBT values (obtained from the ISSAM website) are much higher than widely published normal limits for measured BT in men and lead to confusion when used in diagnosing hypogonadism.

optimization of KS and KA association constants
Results for cBT obtained using the Vermeulen formula largely depend on the association constant values used for calculation. A wide range of association constants of testosterone for SHBG have been published(3)(36)(37)(38)(39)(40). We determined optimal paired Ks and Ka(4), which, when used with the Vermeulen formula, yielded cBT values that were very close to the aBT values.

After optimization, 83% to 85% of our cBT values differed by <30% from the aBT. In addition, the cBT:aBT ratio was nearly 1 (Table 3Up ) and did not vary with age. In contrast, using the ISSAM website, only 13.3% had cBT values similar to aBT values, and the mean cBT:aBT ratio was 1.63 and increased with age.

We showed that factors other than TT and SHBG contribute to the decrease in the aBT in aging men relative to younger men. We hypothesized that 5-diol, whose association constant for SHBG (1.5 x 109 L/mol)(7) is practically the same as that of testosterone for SHBG (1.6 x 109 L/mol)(7), could modulate SHBG testosterone binding in men. Therefore, besides the well-known age-related variations in TT and SHBG, the reduction in 5-diol with age further decreases aBT.

We demonstrate in vitro that aBT increases as 5-diol increases and that this increase is relatively higher in serum with a higher SHBG concentration than in serum with a lower SHBG concentration (Fig. 2Up ). In addition, we show that an overload of serum with DHEA increases the aBT rather moderately in serum with a high SHBG and not at all in serum with a low SHBG. This moderate interference of DHEA in the aBT compared with 5-diol is to be expected with the lower association constant of DHEA for SHBG (66 x 106 L/mol)(7).

The significant effect of 5-diol concentrations on the binding of testosterone to SHBG was confirmed by the bias observed when the cBT:aBT ratio was plotted against the 5-diol concentration. This clearly showed that cBT:aBT ratios decreased more rapidly below 1 as 5-diol concentrations increased. When the inhibiting effect of 5-diol on SHBG was included in the testosterone-binding model (see Calculation of BT), the cBT (5-diol):aBT ratio vs 5-diol concentration was significantly improved, with no further evidence of bias. In Calculation of BT, we propose a cBT determination based on the total concentrations of testosterone, 5-diol, SHBG, and albumin.

In conclusion, using reference assay methods, the lower normal limits we found in the younger men in our study were 8.50 nmol/L for TT and between 2.30 and 2.72 nmol/L for BT, similar to values reported by most other authors, whereas according to the ISSAM website calculator, the corresponding cBT was much higher. Using optimized association constants of testosterone for SHBG and albumin and including 5-diol levels in the calculation of BT improved the correlation between cBT and aBT. An alternative calculation that takes into account a possible inhibitory effect of 5-diol on testosterone binding to SHBG was also carried out, using previously published equations(6). We therefore derived new equations for use in determining cBT (see Calculation of BT).


   Acknowledgments
 
Grant/funding support: None declared.

Financial disclosures: None declared.

Acknowledgments: We thank Dr. Noah Hardy for editing the manuscript and René Bérubé for expert technical assistance.


   Footnotes
 
1 Nonstandard abbreviations: BT, bioavailable testosterone; TT, total testosterone; ISSAM, International Society for the Study of the Aging Male; cBT, calculated BT; SHBG, sex hormone–binding globulin; aBT, assayed BT; It, inhibitor testosterone; DHT, dihydrotestosterone; 5-diol, androstenediol; DHEA, dehydroepiandrosterone.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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