Clinical Chemistry Link to Randox Laboratories Web Site
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Clinical Chemistry 52: 474-481, 2006. First published December 29, 2005; 10.1373/clinchem.2005.052126
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
clinchem.2005.052126v1
52/3/474    most recent
Right arrow Submit an electronic Letter to
the Editor about this paper
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (7)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Giton, F.
Right arrow Articles by Raynaud, J.-P.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Giton, F.
Right arrow Articles by Raynaud, J.-P.
Related Collections
Right arrow Endocrinology and Metabolism
(Clinical Chemistry. 2006;52:474-481.)
© 2006 American Association for Clinical Chemistry, Inc.

Endocrinology and Metabolism

Serum Bioavailable Testosterone: Assayed or Calculated?

Frank Giton1, Jean Fiet1,2,a, Jérôme Guéchot3, Fidaa Ibrahim4, Françoise Bronsard5, Dominique Chopin1 and Jean-Pierre Raynaud6

1 Emi INSERM 03-37, Centre de Recherches Chirurgicales, CHU Henri Mondor, Faculté de Médecine, Créteil, France.
2 Laboratoire de Biochimie, Faculté de Pharmacie, Paris, France.
3 Laboratoire d’hormonologie, Hôpital St. Antoine, Paris, France.
4 Laboratoire d’hormonologie, Hôpital St. Louis, Paris, France.
5 Institut Universitaire de Technologie de Cachan, Cachan, France.
6 Université Pierre et Marie, 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 fiet{at}univ-paris12.fr.


   Abstract
Top
 Abstract
Introduction
 Participants and Methods
 Results
 Discussion
 References
 
Background: Bioavailable testosterone (BT), circulating testosterone not bound to sex hormone–binding globulin (SHBG), is thought to easily penetrate cells. We compared BT measurements obtained by assays with those obtained by calculation with different testosterone association constants.

Methods: We obtained sera from 2 groups of hypogonadal men [group 1 (G1), 1421 samples; group 2 (G2), 170 samples] and a group of healthy men [group 3 (G3), 109 samples]. We added minute doses of [3H]testosterone to the sera, precipitated the SHBG-bound fraction of testosterone with ammonium sulfate (50% saturation), and then assayed serum BT (ABT) as %BT x total. Calculated BT (CBT) was determined with theoretical association constants of testosterone for SHBG (Ks = 1 x 109 L/mol) and albumin (Ka = 3.6 x 104 L/mol) and paired optimal Ks and Ka values obtained by use of Microsoft Excel software.

Results: CBT calculated with theoretical constants differed from ABT by >30% in 85.7% (G1), 84.1% (G2), and 77.9% (G3) of samples, and the mean CBT/ABT ratios were 1.57 (G1), 1.85 (G2), and 1.50 (G3) in spite of fairly good correlations. CBT calculated with paired optimal Ks and Ka differed from ABT by <30% in 87.4% (G1), 87.5% (G2), and 97.5% (G3) of samples, and mean CBT/ABT ratios were 0.95–1.04.

Conclusions: To obtain CBT values as close as possible to ABT, optimal paired association constants determined for each studied population must be used instead of the theoretical association constants. Considering the uncertainty of calculating BT, however, use of the ammonium sulfate precipitation method for determining BT is advisable.


   Introduction
Top
 Abstract
 Introduction
Participants and Methods
 Results
 Discussion
 References
 
Bioavailable testosterone (BT), 1 circulating testosterone not bound to sex hormone–binding globulin (SHBG), is a biological marker of androgenicity in men. In older men, BT is related to physiologic changes, decreased muscle strength, bone density, and depressed mood (1)(2)(3)(4)(5). Serum BT is widely assayed in testosterone replacement programs in older hypogonadal men (6)(7). BT is usually measured after removal of SHBG from serum by ammonium sulfate precipitation (50% saturation) and calculation of the percentage of non–SHBG-bound testosterone by use of tracer-binding methods (8)(9) or direct measurement of testosterone in supernatant that contains free and albumin-bound testosterone (10). Both free testosterone and BT may be calculated by measuring total testosterone, SHBG, and albumin concentrations in serum and using the equilibrium binding constants of testosterone to SHBG and albumin given in published equations (11).

Calculation of free testosterone by use of the association constants of testosterone for albumin (Ka = 3.6 x 104 L/mol) and SHBG (Ks = 1 x 109 L/mol) produced calculated results that corresponded to those obtained by equilibrium dialysis (11), but in some cases (n = 24) led to higher calculated BT than assayed BT. Because several theoretical SHBG association constant values (range of Ks values, 0.27 x 109 to 1.9 x 109 L/mol) have been reported (11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22), we compared assayed BT (ABT) concentrations measured by 2 different laboratories for 2 large populations of hypogonadal men (n = 1421 and 170), and 109 apparently healthy men with calculated BT (CBT) concentrations obtained with different testosterone association constants.


   Participants and Methods
Top
 Abstract
 Introduction
 Participants and Methods
Results
 Discussion
 References
 
Total testosterone, BT, and SHBG were assayed in fasting blood samples drawn from untreated and testosterone patch–treated hypogonadal men (group G1; 1421 samples) in laboratory 1 (Emi INSERM 03-37, Centre de Recherches Chirurgicales, CHU Henri Mondor, Créteil, France), from untreated hypogonadal men (group G2; 170 samples) in laboratory 2 (Laboratoire d’Hormonologie, Hôpital St. Antoine, Paris, France), and from untreated healthy men (group G3; 109 samples) in laboratory 1. The healthy participants were recruited in a health center (IRSA, Tours, France). The procedure was in accordance with the Helsinki Declaration, and each participant gave informed consent. Albumin was measured only in group G1. In groups G1 and G3, we performed total testosterone assays by time-resolved fluoroimmunoassay (TR-FIA) after extraction and then chromatography on Celite (23)(24). Interassay CVs were 4.9%, 5.1%, 4.2%, 4.6%, 3.6%, and 2.2%, respectively, for the following concentrations of serum controls: 2.60 (75), 5.20 (150), 8.66 (250), 10.40 (300), 13.86 (400), and 20.80 (600) nmol/L (ng/dL). Compared with gas chromatography–mass spectrometry (GCMS), the correlation coefficient (r) was 0.99, and the equation for the regression curve was as follows: TR-FIA = 1.0644(GCMS) – 3.58 ng/dL. Nonparametric paired comparative tests showed no significant difference. In group G2, total testosterone was assayed with a commercial reagent set (Beckman Immunotech; Ref. IM 1087) after solvent extraction. The highest cross-reactivity of the anti-testosterone antibody (used in the reagent set) was 10% with dihydrotestosterone. Interassay CVs were 5%–10%. To measure BT in serum samples from the 3 groups, we added minute doses of freshly purified tritiated testosterone to serum at 37 °C; we then precipitated the SHBG by adding a saturated ammonium sulfate solution previously equilibrated at 37 °C (8)(9). Samples were immediately centrifuged for 15 min at 3000g in a Jouan KR 422 centrifuge previously equilibrated at 37 °C. Duplicate 0.2-mL amounts were taken from the supernatant and placed in minivials. Ultima Gold scintillating fluid (Packard) for radioactivity counting (Tricard 2300 TR; Packard) was added to all vials. We deducted the percentage of non–SHBG-bound tritiated testosterone, or BT, from the radioactivity measurements and calculated the concentrations of serum BT by multiplying %BT by the serum total testosterone concentrations. The interassay CVs of %BT calculated from 3 serum pools measured in each assay run were 4.5%, 3.8%, and 10%, respectively, for mean serum %BT values of 48.9%, 30.4%, and 18.9% (n = 50 runs). SHBG was measured in all sera by an RIA method (SHBG-RIACT reagent set) purchased from Cisbio International/Schering. Interassay CVs were 7.6%, 8.5%, and 5.9% for serum SHBG at mean concentrations of 13, 38, and 72 nmol/L, respectively (n = 45 runs).

We compared results obtained with the SHBG-RIACT reagent set with those obtained with the DELFIA SHBG (Ref. A070-101; Wallac) and the SHBG IRMA ORION (Ref 68563; Orion Diagnostica). The correlation coefficients (r) were 0.983 and 0.985, respectively (n = 40 in duplicate for each comparison). There was no significant difference between the paired SHBG-RIACT and DELFIA SHBG results or between paired SHBG-RIACT and ORION IRMA results. Moreover, although BT measurement with ammonium sulfate precipitation is widely used, we checked the efficiency of the separation of albumin from SHBG after SHBG precipitation with 50% saturated ammonium sulfate. We assayed albumin concentrations in the supernatant after SHBG precipitation and centrifugation, and in the serum sample. We performed the assays with a nephelometric method with a kinetic reaction on the Array Beckman Analyzer. The albumin concentrations in the supernatant (obtained by adding 1 volume of saturated ammonium sulfate to 1 volume of serum) were one half the concentrations of albumin in the pure serum samples, indicating that no albumin had been precipitated in the assay conditions. The assays were carried out on 12 samples.

We also assayed SHBG concentrations (IRMA; Cisbio International/Schering) in the supernatant after adding saturated ammonium sulfate to the serum samples and performing centrifugation at 37 °C for 15 min at 3000g. The assays were performed on 12 samples from group G1, whose SHBG concentrations were 8.5–39 nmol/L. We found no detectable SHBG in the supernatants of the 12 samples. In addition, an assay was performed on the serum of a diethylstilbestrol-treated patient with prostate adenocarcinoma whose SHBG concentration was very high (310 nmol/L). After addition of the saturated ammonium sulfate and centrifugation, we measured the SHBG concentration in the undiluted supernatant and in 1:2, 1:4, 1:8, 1:16, and 1:32 dilutions of the supernatant. SHBG was undetectable in the undiluted and diluted supernatant samples (the detection limit of the method was <0.5 nmol/L), indicating that no SHBG was present and that, consequently, all of the SHBG had been precipitated by ammonium sulfate. We assayed albumin in samples from group G1 with the bromcresol green dye-binding method on a Hitachi 911 automated analyzer, whereas we considered albumin concentrations in groups G2 and G3 to be constant and equal to 43 g/L in each serum sample.

Using the assay results for total testosterone, SHBG, and albumin, we determined the CBT according to the formulas of Vermeulen et al. (11), applying various association constants (Ks) between 0.6 x 109 L/mol and 2 x 109 L/mol and various Ka values. We then compared the CBT with the ABT concentrations.

We based our comparison of ABT and CBT on calculation of the correlation coefficients between CBT and ABT, the CBT/ABT ratio for each sample, and the number of samples for which CBT differed from ABT by less than 10%, 20% and 30%. For this purpose, we determined the (CBT-ABT)/ABT ratio (negative, positive, and absolute ratio), termed the relative difference (RD). These comparisons were carried out with Microsoft Excel software. We also performed global variance analysis and post-ANOVA Bonferroni/Dunn tests to compare ABT and CBT results.


   Results
Top
 Abstract
 Introduction
 Participants and Methods
 Results
Discussion
 References
 
We compared CBT concentrations calculated with the theoretical association constants Ks = 1 x 109 L/mol and Ka = 3.6 x 104 L/mol (11) with ABT concentrations. In group G1 (1421 samples), the correlation coefficient between CBT and ABT was 0.9720 and the mean (SD) CBT/ABT ratio was 1.576 (0.292), indicating that the CBT was higher than the ABT. The number of samples displaying absolute RDs <0.10, <0.20, and <0.30 were only 45, 107, and 204, respectively. Thus, 1217 (1421 – 204) samples (85.6%) exhibited an absolute RD between CBT and ABT greater than 0.30. These results are reported in Table 1 . We obtained similar results for group G2 (170 samples): r = 0.9575; CBT/ABT = 1.85. The numbers of samples with an absolute RD <0.10, <0.20, and <0.30 were 8, 14, and 27, respectively. In the group of 109 healthy persons (G3), the correlation coefficient was 0.9312, the CBT/ABT ratio was 1.4945, and the number of samples with an absolute RD <0.10, <0.20, and <0.30 were 0, 9, and 24, respectively. For all 3 groups, CBT values obtained with the theoretical association constants were higher than the ABT values.


View this table:
[in this window]
[in a new window]
  		Table 1. Comparison between ABT and CBT in 1421 samples from hypogonadal men (group G1), obtained with 14 optimal Ks/Ka pairs and 1 nonoptimal pair.1

For Ka = 3.6 x 104 L/mol, the number of CBT values that were nearly identical to the ABT values increased when the Ks value increased (Fig. 1 ). For Ks = 2.9 x 109 L/mol, the absolute RDs were <0.10, <0.20, and <0.30, respectively, for 560, 995, and 1243 samples, corresponding to 39.4%, 70%, and 87.4% of the 1421 samples. The absolute RD decreased for higher Ks values, and there were paired optimal Ks (2.9 x 109) and Ka (3.6 x 104) values for which close correspondence of CBT to ABT values was maximal. In group G2, the optimal Ks was 3 x 109 L/mol for a Ka of 3.6 x 104 L/mol. With these paired optimal Ks and Ka values, 40%, 68%, and 85% of the 170 samples from group G2 had an absolute RD <0.10, <0.20, and <0.30, respectively.


Figure 1
View larger version (41K):
[in this window]
[in a new window]
  		Figure 1. Variation in the number of samples (in group G1; 1421 samples) with an absolute RD <0.10 (Figure 1), <0.20 ({square}), and <0.30 ({blacksquare}) for Ka = 3.6 x 104 L/mol with increasing Ks (from 0.60 x 109 L/mol to 4.40 x 109 L/mol).

For Ks = 1 x 109 L/mol in group G1, the number of CBT values nearly identical to the ABT values increased when the Ka increased, and an optimal value of Ka was reached for Ka = 1.1 x 104 L/mol (Fig. 2 ). For Ka values >1.1 x 104 L/mol, the number of CBT values nearly identical to the ABT values decreased.


Figure 2
View larger version (34K):
[in this window]
[in a new window]
  		Figure 2. Variation in the number of samples (in group G1; 1421 samples) with absolute RD <0.10 (Figure 2), <0.20 ({square}), and <0.30 ({blacksquare}) for Ks = 1 x 109 L/mol with increasing Ka (from 0.10 x 104 L/mol to 3.90 x 104 L/mol).

We determined the optimal Ka values in group G1 for several published Ks values (0.6 x 109 to 1.9 x 109 L/mol; Table 1Up ) and the absolute, negative, and positive RDs, the r values, and the CBT/ABT ratios [mean (SD)] for each optimal pair of Ks and Ka association constants. The optimal correlation coefficient (r) was 0.9790–0.9792, the CBT/ABT ratio was 1.0136–1.0404, and the numbers of samples with an absolute RD <0.10, <0.20, and <0.30 were maximal and nearly the same regardless of the optimal paired Ks and Ka values (Table 1Up ). Thus, for an absolute RD <0.30, the number of CBT values differing by <30% from ABT values was 1242–1245. In comparison, with the theoretical association constants, the number of samples with CBT values that differed by <30% from the ABT values was only 204 (Table 1Up ). Moreover, for the same samples, the CBT values did not change regardless of the optimal paired association constants Ks and Ka used for calculation.

We obtained similar results for group G2 with optimal paired Ks and Ka values (Ks = 0.6 x 109 to 1.9 x 109 L/mol, corresponding to a Ka of 0.5 x 104 to 2 x 104 L/mol). The percentages of samples with an absolute RD <0.10, <0.20, and <0.30 were 35.2%–37.6%, 66.4%–69.4%, and 86.4%–87.5%, respectively. These percentages are close to those reported for group G1. For Ks = 1 x 109 L/mol, the optimal Ka was 1.1 x 104 L/mol, as in group G1 (r = 0.9597–0.9610; CBT/ABT ratios = 0.96–1.0).

On the basis of the correlation coefficients, optimal Ks/Ka pairs were those yielding the greatest correlation coefficient (r = 0.9793). For this correlation coefficient, in group G1, the optimal paired Ks and Ka values were 0.6 x 109 and 0.8 x 104 L/mol, 0.8 x 109 and 1 x 104 L/mol, 1 x 109 and 1.4 x 104 L/mol, 1.2 x 109 and 1.6 x 104 L/mol, 1.8 x 109 and 2.4 x 104 L/mol, and 1.9 x 109 and 2.6 x 104 L/mol. Although these optimal Ks and Ka pairs were not exactly the same as those obtained from the RD determination (Table 1Up ), this optimization approach led to practically the same results as those obtained by counting samples with absolute RD values <0.10, <0.20, and <0.30. In group G1, the optimal Ks/Ka pairs that yielded a CBT/ABT ratio as close as possible to 1 were 0.6 x 109 and 0.6 x 104 L/mol, 0.8 x 109 and 0.8 x 104 L/mol, 1 x 109 and 1.1 x 104 L/mol, 1.2 x 109 and 1.3 x 104 L/mol, 1.4 x 109 and 1.6 x 104 L/mol, 1.6 x 109 and 1.8 x 104 L/mol, 1.8 x 109 and 2 x 104 L/mol, and 1.9 x 109 and 2.2 x 104 L/mol, which were very similar to those reported in Table 1Up .

Whatever the mode of optimization, we found that use of optimal paired Ks and Ka values yielded a greater number of samples with the CBT close to the ABT, in contrast to the results obtained with the theoretical Ks = 1 x 109 L/mol and Ka = 3.6 x 104 L/mol, as illustrated in Fig. 3 .


Figure 3
View larger version (21K):
[in this window]
[in a new window]
  		Figure 3. Regression curves between CBT and ABT (in group G1; 1421 samples).

Correlation coefficients (r) and mean CBT/ABT ratios were obtained with either optimal Ks and Ka pair (Ks = 1 x 109 L/mol; Ka = 1.1 x 104 L/mol; blue) or the nonoptimal Ks = 1 x 109 L/mol and Ka = 3.6 x 104 L/mol (red).

The calculated CBTs and the corresponding ABTs are shown on the same axis in Fig. 3Up . The CBTs were calculated based on 2 paired Ks and Ka values: Ks = 1 x 109 L/mol with Ka = 1.10 x 104 L/mol (one of the optimal Ks/Ka pairs; Table 1Up ), and Ks = 1 x 109 L/mol with Ka = 3.6 x 104 L/mol [association constants applied by Vermeulen et al. (11)]. The results show that the CBT values obtained from the association constants of Vermeulen et al. (11) were well above the CBT obtained from optimal paired Ks and Ka values.

Using global variance analysis and a post-ANOVA Bonferroni/Dunn test, we found a significant difference between ABT and CBT values obtained with the formulas of Vermeulen et al. (11) but no significant difference between ABT and one set of the optimal paired Ks and Ka values reported in Table 1Up .

In group G3, for the same Ks, the optimal Ka values were higher than in groups G1 and G2 (Table 2 ). The absolute percentage of CBT values obtained from each pair of optimal Ks and Ka values that differed by <30% from the ABT values was 97.5% (Table 2 ). In this group, the mean %BT of the 34 young men (20–39 years of age) was 39% for a mean total testosterone of 16.8 nmol/L and a mean ABT of 6.38 nmol/L.


View this table:
[in this window]
[in a new window]
  		Table 2. Comparison between ABT and CBT in 109 samples from healthy individuals (group G3), obtained with 15 optimal Ks/Ka pairs and 1 nonoptimal pair.1


   Discussion
Top
 Abstract
 Introduction
 Participants and Methods
 Results
 Discussion
References
 
Vermeulen et al. (11) reported that free testosterone concentrations could be calculated with the association constants Ks = 1 x 109 L/mol and Ka = 3.6 x 104 L/mol. The authors noticed, however, that CBT concentrations were 23-fold and ABT concentrations 20-fold higher than free testosterone concentrations, indicating higher CBT than ABT in a small group of individuals (n = 24) (11). Using the same association constants, we also found, in a rather large sample group of 1421 samples (group G1), that the choice of these 2 numeric values (Ks = 1 x 109 L/mol and Ks = 3.6 x 104 L/mol) yielded CBT results that were clearly much higher than the ABT results Even higher or similar mean CBT/ABT ratios have also been reported by others [CBT/ABT = 2.2 in 700 patients (Dechaud et al., unpublished data) and CBT/ABT =1.50 in control and hypogonadal men (Tremblay et al., unpublished data)].

Although different values of Ks have been published (11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22), we found that by recalculation of the CBT to obtain a greater number of samples with CBT results close to the ABT, a Ka of 3.6 x 104 L/mol led to optimal Ks values of 2.9 x 109, 3 x 109, and 2.3 x 109 L/mol for our sample groups G1, G2, and G3, respectively. The Ks values of 2.9 x 109 and 3 x 109 L/mol, however, were higher than the upper value of the previously published Ks value of 1.9 x 109 L/mol (11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22). On the basis of the hypothesis that Ka = 3.6 x 104 L/mol was not the exact association constant of testosterone for albumin in serum, our recalculation of the optimal paired Ks and Ka values for various Ks values of 0.6 x 109 to 1.9 x 109 L/mol showed that the corresponding Ka values were 0.60 x 104 to 2.29 x 104 L/mol (group G1), 0.5 x 104 to 1.97 x 104 L/mol (group G2), and 0.8 x 104 to 3 x 104 L/mol (group G3), lower than the Ka (3.6 x 104 L/mol) applied by Vermeulen et al. (11).

In group G2, the optimal paired Ks and Ka values were slightly different from those obtained in group G1, as were the correlation coefficients and CBT/ABT ratios. These slight differences may be related to the methods used to assay total testosterone, which were not identical, and to the choice of the same arbitrary concentration of albumin (43 g/L) in all G2 patients for the calculation of BT. However, the percentages of samples with an RD <0.10, <0.20, and <0.30 were practically the same in groups G1 and G2. In group G3, we observed for one theoretical published Ks (1.9 x 109 L/mol) (12) that the corresponding optimal Ka (3 x 104/mol) was only a little lower than the theoretical Ka. It is probable that the theoretical Ka (3.6 x 104 L/mol) determined on pure human albumin (25) is higher than the true Ka in serum, which could partly explain an ABT lower than the CBT. Vermeulen et al. (11) hypothesized the presence of lipids to explain why CBT was lower than ABT, and free fatty acids in serum have been reported to change albumin-bound steroids (26). It is possible that the higher optimal Ka determined in samples from healthy men (group G3) compared with groups G1 and G2 could be attributable to lower serum concentrations of free fatty acids. The mean percentage of BT that we found in 34 young men (20–39 years of age) among the 109 healthy men of group G3 was 39%. This value was between the extreme mean values for BT reported previously (20%–50%) in different, rather small populations of young healthy men (10)(27)(28)(29)(30)(31)(32), which were obtained by similar, but not strictly identical, BT assay methods (method differences concerning incubation temperature, use or not of a tritiated testosterone tracer, and purification of tritiated testosterone, frequently not reported in the BT assay methods). However, this dispersion in the published assayed percentage of BT of healthy young men does not explain the much lower ABT we measured in the 3 populations compared with the CBT obtained with Ks = 1 x 109 L/mol and Ka = 3.6 x 104L/mol.

Recently, Emadi-Konjin et al. (33), applying the formulas given by Vermeulen et al. (11), with Ks = 1 x 109 L/mol and Ka = 3.6 x 104 L/mol, found systematic differences between CBT and ABT in samples from a group of almost 400 men. These authors reported "implausibly" higher CBT than ABT, and most of the percentage CBT values were in the 30%–70% range, whereas the corresponding measured %BT results were in the range 10%–40%. On the basis of the best correlation coefficients, these authors empirically adjusted the Ks and Ka association constants and found optimal paired Ks and Ka values of 1.4 x 109 L/mol and 1.3 x 104 L/mol, respectively. These reported results (33) can be compared with ours: for the same Ks (1.4 x 109 L/mol), we found optimal Ka values of 1.60 x 104 L/mol in group G1, 1.40 x 104 L/mol in group G2, and 2.1 x 104 L/mol in group G3. We do not think that lower concentrations of ABT compared with CBT (with the association constants Ks = 1 x 109 L/mol and Ka = 3.6 x 104 L/mol) that we and others (Dechaud et al. and Tremblay et al., unpublished data) have found can be explained by a methodologic problem, although differences in methodologies exist. To our knowledge, no such comparisons of CBT and ABT [except by Emadi-Konjin et al. (33)] in large numbers of patients and healthy men have been reported. The CBT largely depends on the Ks and Ka values chosen. Numerous theoretical Ks values have been reported in the past, and the exact Ks and Ka values in serum are not well known. Moreover, as suggested recently (34)(35), the Ks value could vary with age.

In conclusion, by calculating optimal pairs of Ks and Ka, we were able to determine CBT values that better agreed with the ABT values than CBT values determined with theoretical association constants. Using optimal Ks/Ka pairs, we found in our population of untreated and treated hypogonadal nonfasting patients that 30% of the CBT results differed from ABT by at least 20%, whereas in the population of fasting healthy men, CBT obtained with optimal Ks/Ka pairs led to CBT values close to the ABT values in 97% of samples. Considering the uncertainty of calculating BT, ABT obtained with ammonium sulfate precipitation seems to be a better method than CBT. It would be wise, however, to thoroughly standardize the BT ammonium sulfate precipitation assay method and to determine BT reference values in men.


   Acknowledgments
 
We thank Dr. Gérard Brilman (IUT Cachan) for his assistance in using the EXCEL software; Professor Jacques Callebert (Hôpital Lariboisière, Paris) for his expertise in statistics; Dr. Noah Hardy, an American scientist, for assistance with correcting the English in the manuscript; and Dr. Monique Pressac (Hôpital Trousseau, Paris) for her expertise in measuring albumin.


   Footnotes
 
1 Nonstandard abbreviations: BT, bioavailable testosterone; SHBG, sex hormone–binding globulin; ABT, assayed bioavailable testosterone; CBT, calculated bioavailable testosterone; and RD, relative difference between calculated bioavailable testosterone and assayed bioavailable testosterone.


   References
Top
 Abstract
 Introduction
 Participants and Methods
 Results
 Discussion
 References
 

  1. Guéchot J, Vaubourdolle M, Ballet F, Giboudeau J, Darnis F, Poupon R. Hepatic uptake of sex steroids in men with alcoholic cirrhosis. Gastroenterology 1987;92:203-207.[Medline] [Order article via Infotrieve]
  2. Pardridge WM. Transport of protein-bound hormones into tissues in vivo. Endocr Rev 1981;2:103-123.[ISI][Medline] [Order article via Infotrieve]
  3. Guéchot J, Loric S, Vaubourdolle M, Chretien Y, Giboudeau J, Poupon R. Effect of protein binding on testosterone extraction by human cirrhotic liver: evidence for a dissociation-limited uptake. J Clin Endocrinol Metab 1989;69:200-203.[Abstract]
  4. Van Den Beld AW, De Jong FH, Grobbee DE, Pols HAP, Lamberts SWJ. Measures of bioavailable serum testosterone and estradiol and their relationships with muscle strength, bone density, and body composition in elderly men. J Clin Endocrinol Metab 2000;85:3276-3282.[Abstract/Free Full Text]
  5. Barrett-Connor E, Von Mühlen DG, Kritz-Silverstein D. Bioavailable testosterone and depressed mood in older men: the Rancho Bernardo Study. J Clin Endocrinol Metab 1999;84:573-577.[Abstract/Free Full Text]
  6. Hajjar RR, Kaiser FE, Morley JE. Outcomes of long-term testosterone replacement in older hypogonadal males: a retrospective analysis. J Clin Endocrinol Metab 1997;82:3793-3796.[Abstract/Free Full Text]
  7. Sih R, Morley JE, Kaiser FE, Perry HM, Patrick P, Ross C. Testosterone replacement in older hypogonadal men: a 12-month randomized controlled trial. J Clin Endocrinol Metab 1997;82:1661-1667.[Abstract/Free Full Text]
  8. Tremblay RR, Dube JY. Plasma concentrations of free and non-TeBG bound testosterone in women on oral contraception. Contraception 1974;10/6:599-605.
  9. Loric S, Guéchot J, Duron F, Aubert P, Giboudeau J. Determination of testosterone in serum not bound by sex-hormone-binding globulin: diagnostic value in hirsute women. Clin Chem 1988;34:1826-1829.[Abstract/Free Full Text]
  10. Déchaud H, Lejeune H, Garoscio-Cholet M, Mallein R, Pugeat M. Radioimmunoassay of testosterone not bound to sex-steroid-binding protein in plasma. Clin Chem 1989;35:1609-1614.[Abstract/Free Full Text]
  11. Vermeulen A, Verdonck L, Kaufman JM. A critical evaluation of simple methods for the estimation of free testosterone in serum. J Clin Endocrinol Metab 1999;84:3666-3672.[Abstract/Free Full Text]
  12. Burke CW, Anderson DC. Sex hormone binding globulin is an estrogen amplifier. Nature 1982;240:38-40.
  13. Lutz RA, Lutz-Ewan L, Weder HG. Further studies on the temperature dependence of the binding of testosterone to human pregnancy plasma proteins. Steroids 1973;21:423-431.[CrossRef][ISI][Medline] [Order article via Infotrieve]
  14. Rosner W, Smith RN. Isolation and characterization of the testosterone-estradiol-binding globulin from human plasma: use of a novel affinity column. Biochemistry 1975;14:4813-4820.[CrossRef][Medline] [Order article via Infotrieve]
  15. Vermeulen A. Transport and distribution of androgens at different ages. Martini L Motta M eds. Androgens and antiandrogens 1997:53-65 Raven Press New York. .
  16. Nisula BC, Dunn JF. Measurement of the testosterone binding parameters for both testosterone-estradiol binding globulin and albumin in individual serum samples. Steroids 1979;34:771-791.[CrossRef][ISI][Medline] [Order article via Infotrieve]
  17. Dunn JF, Nisula BC, Rodbard D. Transport of steroid hormones: binding of 21 endogenous steroids to both testosterone-binding globulin and corticosteroid-binding globulin in human plasma. J Clin Endocrinol Metab 1981;53:58-68.[Abstract]
  18. Pugeat M, Dunn JF, Nisula BC. Transport of steroid hormones: interaction of 70 drugs with testosterone-binding globulin and corticosteroid-binding globulin in human plasma. J Clin Endocrinol Metab 1981;53:69-75.[Abstract]
  19. Siiteri PK, Murai JT, Hammond GL, Nisker JA, Raymoure WJ, Kuhn RW. The serum transport of steroid hormones. Recent Prog Horm Res 1982;38:457-503.[ISI][Medline] [Order article via Infotrieve]
  20. Södergard R, Bäckström T, Shanbhag V, Carstensen H. Calculation of free and bound fractions of testosterone and estradiol-17ß to human plasma proteins at body temperature. J Steroid Biochem 1982;16:801-810.[CrossRef][ISI][Medline] [Order article via Infotrieve]
  21. Strel’chenok OA, Survilo LI, Tsapelik GZ, Sviridov OV. Purification and physicochemical properties of the sex steroid-binding globulin of human blood plasma. Biokhimiia 1983;48:756-762.[Medline] [Order article via Infotrieve]
  22. Westphal U. Steroid-protein interactions II. Monogr Endocrinol 1986;27:1-603.[Medline] [Order article via Infotrieve]
  23. Fiet J, Gosling JP, Soliman H, Galons H, Boudou P, Aubin P, et al. Hirsutism and acne in women: coordinated radioimmunoassays for eight relevant plasma steroids. Clin Chem 1994;40:2296-2305.[Abstract/Free Full Text]
  24. Fiet J, Giton F, Ibrahim F, Valleix A, Galons H, Raynaud JP. Development of a highly sensitive and specific new testosterone time-resolved fluoroimmunoassay in human serum. Steroids 2004;69:461-471.[CrossRef][ISI][Medline] [Order article via Infotrieve]
  25. Vermeulen A. Testosterone in plasma: a physiopathological study. Verh K Acad Geneeskd Belg 1973;35:95-180.[Medline] [Order article via Infotrieve]
  26. Reed MJ, Cheng RW, Beranek PA, Few JD, Franks S, Ghilchik MW, et al. The regulation of biologically available fractions of oestradiol and testosterone in plasma. J Steroid Biochem 1986;24:317-320.[CrossRef][ISI][Medline] [Order article via Infotrieve]
  27. O’Connor S, Baker HW, Dulmanis A, Hudson B. The measurement of sex steroid binding globulin by differential ammonium sulphate precipitation. J Steroid Biochem 1973;4:331-339.[CrossRef][ISI][Medline] [Order article via Infotrieve]
  28. Winters SJ, Kelley DE, Goodpaster B. The analog free testosterone assay: are the results in men clinically useful?. Clin Chem 1998;44:2178-2182.[Abstract/Free Full Text]
  29. Nahoul K, Roger M. Age-related decline of plasma bioavailable testosterone in adult men. J Steroid Biochem 1990;35:293-299.[CrossRef][ISI][Medline] [Order article via Infotrieve]
  30. Korenman SG, Morley JE, Mooradian AD, Danis SS, Kaiser FE, Silver AJ, et al. Secondary hypogonadism in older men: its relation to impotence. J Clin Endocrinol Metab 1990;71:963-969.[Abstract]
  31. Morley JE, Patrick P, Perry HM, III. Evaluation of assays available to measure free testosterone. Metabolism 2002;51:554-559.[CrossRef][ISI][Medline] [Order article via Infotrieve]
  32. Tremblay RR, Massè J. Usefulness and limitation of bioavailable testosterone in assessment of androgenicity during the process of aging in men. Aging Male 1999;2:16-21.
  33. Emadi-Konjin P, Bain J, Bromberg IL. Evaluation of an algorithm for calculation of serum "bioavailable" testosterone (BAT). Clin Biochem 2003;36:591-596.[CrossRef][ISI][Medline] [Order article via Infotrieve]
  34. Morley JE, Perry HM, III. Androgen treatment of male hypogonadism in older males. J Steroid Biochem Mol Biol 2003;85:367-373.[CrossRef][ISI][Medline] [Order article via Infotrieve]
  35. Haren M, Nordin BEC, Pearce CEM, O’Loughlin P, Chapman I, Morley JE, et al. The calculation of bioavailable testosterone. Robaire B Chemes H Morales CR eds. Proceedings of the VII International Congress of Andrology 2001:209-213 Medimond Medical Publications Englewood, NJ. .



The following articles in journals at HighWire Press have cited this article:


Home page
 Clin. Chem.Home page
F. Giton, S. Urien, C. Born, J. Tichet, J. Guechot, J. Callebert, F. Bronsard, J. P. Raynaud, and J. Fiet
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
Clin. Chem., December 1, 2007; 53(12): 2160 - 2168.
[Abstract] [Full Text] [PDF]

Home page
 J. Clin. Endocrinol. Metab.Home page
P. Y. Takahashi, P. Votruba, M. Abu-Rub, K. Mielke, and J. D. Veldhuis
Age Attenuates Testosterone Secretion Driven by Amplitude-Varying Pulses of Recombinant Human Luteinizing Hormone during Acute Gonadotrope Inhibition in Healthy Men
J. Clin. Endocrinol. Metab., September 1, 2007; 92(9): 3626 - 3632.
[Abstract] [Full Text] [PDF]

Home page
 Clin. Chem.Home page
H. Dechaud, A. Denuziere, S. Rinaldi, J. Bocquet, H. Lejeune, and M. Pugeat
Age-Associated Discrepancy between Measured and Calculated Bioavailable Testosterone in Men
Clin. Chem., April 1, 2007; 53(4): 723 - 728.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
clinchem.2005.052126v1
52/3/474    most recent
Right arrow Submit an electronic Letter to
the Editor about this paper
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (7)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Giton, F.
Right arrow Articles by Raynaud, J.-P.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Giton, F.
Right arrow Articles by Raynaud, J.-P.
Related Collections
Right arrow Endocrinology and Metabolism

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS