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Endocrinology and Metabolism |
1 Department of Biochemistry, Loma Linda University School of Medicine, Loma Linda, CA; 2 Departments of Internal Medicine and Pathology, Loma Linda University School of Medicine, Loma Linda, CA.
aAddress correspondence to this author at: Department of Biochemistry, Loma Linda University School of Medicine, Mortensen Hall, Room 209, Loma Linda, CA 92350. Fax (909) 558-7916; e-mail bwilcox{at}llu.edu.
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Methods: An analog-based free testosterone immunoassay and a total testosterone immunoassay were applied to well-defined fractions of serum testosterone. First, they were applied to the 2 fractions (retentate and dialysate) of normal male serum obtained by equilibrium dialysis. Second, they were applied to covaried concentrations of SHBG and total testosterone. Third, they were applied to decreasing concentrations of SHBG and protein-bound testosterone, offset by increasing concentrations of protein-free testosterone, while total testosterone was held constant.
Results: The analog-based free testosterone assay and the total testosterone assay detected and reported serum testosterone test results from serum retentate, whereas neither assay detected the free testosterone in serum dialysate. Test results reported by the analog-based free testosterone assay followed varied concentrations of SHBG and total testosterone. When total testosterone was held constant, however, analog-based free testosterone test results did not follow varied concentrations of serum proteins or of free testosterone.
Conclusion: An analog-based free testosterone immunoassay reported free testosterone test results that were related to total testosterone concentrations under varied experimental conditions. This alleged free testosterone assay did not detect serum free testosterone (the test results it reported were nonspecific) and should not be used for this purpose.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Analog-based free testosterone immunoassays are the most widely used free testosterone methods. They are the only automated free testosterone methods. Multiple studies report that analog-based free testosterone test results are proportional to SHBG concentrations (1)(2)(3)(4)(5)(6)(7). It has also been asserted that they are proportional to total testosterone concentrations (1)(7)(8). This led us to question whether these analog-based free testosterone test results are more closely related to the concentrations of total testosterone or SHBG.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We measured total testosterone using a total testosterone immunoassay (Coat-A-Count; Siemens). This manual method also uses radiolabeled (125I) free testosterone, immobilized testosterone antibody, and a single incubation, following a 21-fold dilution of the sample with a single aqueous reagent.
We applied each assay to the same serum-based testosterone solutions (Fig. 1
). We also used a well-documented tracer dialysis free testosterone assay to verify free testosterone concentrations in these same-serum based testosterone solutions (see below in Free Testosterone by Tracer Dialysis). Each assay was performed according to manufacturer’s instructions. Gamma radiation was detected and quantified using a Gamma 4000 multiwell automated 
counter (Beckman-Coulter). Each testosterone measurement reported is a mean of triplicate determinations, and each experiment was repeated for confirmation.
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normal human serum
We obtained serum from 16 healthy male volunteers, age 21–55 years. Serum collection was approved by the institutional review board, and serum samples were given anonymous identifiers. These sera were pooled. In the pool, the total testosterone of 21.6 nmol/L (Coat-A-Count; in-house), free testosterone of 232 pmol/L (by dialysis), total protein of 7.4 g/dL, serum albumin of 4.4 g/dL, and SHBG of 26 nmol/L were within their respective reference intervals (Quest Diagnostics).
testosterone
Testosterone and ethanol were obtained from Sigma Aldrich. Testosterone was dissolved at room temperature in 95% ethanol to produce a stock solution containing 6.9 mmol/L. This stock solution was diluted with a well-characterized dialysate buffer (10) to a concentration of 0.35 µmol/L testosterone.
preparative equilibrium dialysis
Dialysis devices and dialysate buffer were obtained from Antech Diagnostics. The dialysis device uses 200 µL sample retentate and 2400 µL dialysis buffer. The chemical composition of this buffer has been reported (10). Serum samples were dialyzed for 18 h at 37 °C in an Isotemp Incubator, model 630D (Fisher Scientific) (11)(12)(13). A moisture-saturated atmosphere was maintained during dialysis by enclosing dialysis devices in closed containers with open water reservoirs. The pH values of serum dialysate and retentate were controlled to 7.4 (±0.1) during equilibrium dialysis at 37 °C by HEPES acid in the dialysate buffer (14). At equilibrium, the final HEPES ion concentration was calculated to be 54 mmol/L.
free testosterone by tracer dialysis
We determined free testosterone concentrations by use of a previously reported tracer dialysis method (11)(15) and total testosterone by use of the Coat-A-Count total testosterone immunoassay described above. A 1-µL aliquot (approximately 13 pmol) of stock 3H-testosterone (PerkinElmer) was added to 1 mL of each sample, incubated for 1 h at 37 °C, and dialyzed as described above. Radioactivity was determined in retentates and dialysates using a liquid scintillation counter (LS7500, Beckman). We calculated the fraction of free testosterone at equilibrium as 3H-testosterone in dialysate/3H-testosterone in serum. We then calculated serum free testosterone by multiplying the fraction of free testosterone by total testosterone concentration.
matrix effects
It is well documented that sample and assay reagent matrices can play an important role in the performance of free hormone immunoassays. As previously mentioned, the analog-based free testosterone and total testosterone assays each use a 21-fold sample dilution before incubation and quantification. Both of these assays add 1 mL of a "proprietary" radioactive buffer solution to 50 µL sample. Multiple attempts to determine the constitution of this proprietary radioactive buffer solution have been thwarted by the assay’s manufacturer. Therefore, for the purpose of this study, it is assumed that both the 21-fold sample dilution as well as the components of this radioactive buffer will minimize any differences in sample matrices between a serum retentate and serum dialysate.
testosterone adsorption
Testosterone can be adsorbed onto solid surfaces from protein-free aqueous solutions. We tested the borosilicate glassware used in this study (Fisher Scientific) for testosterone adsorption: <2% of testosterone in serum dialysate was adsorbed in the absence of serum proteins.
experimental strategies
Fig. 1
shows the experiment design. In the first experiment, preparative equilibrium dialysis was applied to the normal adult male serum as described above (200 µL retentate vs 2400 µL dialysate). The analog-based free testosterone assay and the total testosterone assay were applied to the resulting serum retentate and serum dialysate. The tracer dialysis method was also applied to the normal adult male serum to verify free testosterone concentrations. A total testosterone assay is expected to detect the concentrations of total testosterone in serum retentate, whereas a free testosterone assay is expected to detect the concentrations of free testosterone in serum dialysate.
In the second experiment, concentrations of serum proteins, including SHBG, protein-bound testosterone, and total testosterone, were covaried. This was accomplished by progressively diluting serum retentate with serum dialysate (obtained using the equilibrium dialysis method described above). Normal serum retentate was diluted with normal serum dialysate to obtain serum protein and total testosterone concentrations of 100%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, and 20% of normal levels. The analog-based free testosterone assay and the total testosterone assay were applied to these solutions. The tracer dialysis method was also applied to these solutions to verify dialyzable free testosterone concentrations. A total testosterone assay is expected to track the decreasing total testosterone concentrations, whereas a free testosterone assay is not expected to track serum protein or total testosterone concentrations.
In the third experiment, decreasing concentrations of SHBG and protein-bound testosterone were offset by increasing concentrations of free testosterone, while total testosterone was held constant. This was accomplished by measuring the total testosterone in serum retentate and then adding testosterone to an aliquot of serum dialysate until the total testosterone concentration was equal in both retentate and dialysate (21.6 nmol/L). The serum retentate was then progressively diluted with the protein-free, testosterone-enriched dialysate from 1- to 1000-fold. This procedure varied free testosterone concentrations in the opposite direction of SHBG and protein-bound testosterone concentrations. The analog-based free testosterone assay and the total testosterone assay were applied to these solutions. The tracer dialysis method was also applied to these solutions to verify dialyzable free testosterone concentrations. A total testosterone assay is expected to track the constant total testosterone concentrations, whereas a free testosterone assay is expected to track the increasing free testosterone concentrations.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In the second experiment, when concentrations of serum protein, including SHBG, protein-bound testosterone, and total testosterone, were progressively decreased, analog-based free testosterone test results ranged from 56 to 6.2 pmol/L (Fig. 2
). Total testosterone test results ranged from 21.2 to 6.6 nmol/L. (Fig. 2
). Tracer dialysis free testosterone test results ranged from 304 to 169 pmol/L (Fig. 2
). Total testosterone test results and analog-based free testosterone test results correlated more closely (r2 0.97; P <0.001) than analog-based free testosterone test results and tracer dialysis free testosterone test results; (r2 0.90; P <0.001).
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In the third experiment, when decreasing concentrations of serum protein, including SHBG and protein-bound testosterone, were offset by increasing concentrations of protein-free testosterone while total testosterone was held constant, analog-based free testosterone test results [51.8 (1.3) pmol/L] paralleled total testosterone test results [22.4 (0.5) nmol/L] (Fig. 3
). Tracer dialysis test results, or free testosterone concentration, increased from 376 to 798 pmol/L (Fig. 3
).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and 3
). Based on our conclusions, the experimental strategies applied to this analog-based free testosterone assay should be applied to other, as yet untested, commercially available analog-based free testosterone assays.
The design and results of this study differ from previous reports (1)(2)(3)(4)(5)(6)(7)(8). Previous studies have contributed to our understanding by applying these assays to patient samples. The present study used well-defined solutions prepared with the aim of elucidating to which form of serum testosterone an analog-based free testosterone assay would respond. Also, one of the more recent studies (1) found that SHBG was an important determinant in this analog-based free testosterone assay and that this assay appeared to measure a constant fraction of the total testosterone in adult male plasma, leading the authors to conclude that this analog-based free testosterone assay provides essentially the same information as a total testosterone assay when applied to healthy adult males. These previously published assertions are supported by the results of our second and third experiments (Figs. 2
and 3
).
There is a hypothetical explanation that might account for the nonspecificity observed in this assay. Nonspecificity would occur if serum protein testosterone complexes bind to testosterone antibody, leading to a 3-way competition between free testosterone, testosterone complexes, and testosterone conjugates (analogs) for binding to the same antibody. This competition would explain the lack of specificity and would confound calibration. The data from the second and third experiments in this study are consistent with this hypothesis (Figs. 2
and 3
). Similar characteristics have recently been reported in an analog-based free thyroxine immunoassay (17). These data are now sufficient to warrant further testing of this hypothesis. Until the characteristics of assays such as this have been fully accounted for, they should not be confused with free hormone assays that are sensitive, specific, and gravimetrically calibrated with an analytical balance to a scientifically acceptable mass standard. There is no traceability when specificity is absent and there is no specificity when covariables have not been accounted for. Since the analog-based assay in this study does not detect or quantify free testosterone, it should not be used as a free testosterone assay.
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Acknowledgments |
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Financial Disclosures: J.C.N. is currently a consultant to Antech Diagnostics. He was formerly Senior Medical Director of Quest Diagnostics Nichols Institute, San Juan Capistrano, and has no current affiliation with Quest Diagnostics.
Acknowledgments: We wish to acknowledge the expert technical assistance provided by Rene Weiss.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The following articles in journals at HighWire Press have cited this article:
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  G. Sartorius, L. P Ly, K. Sikaris, R. McLachlan, and D. J Handelsman Predictive accuracy and sources of variability in calculated free testosterone estimates Ann Clin Biochem, March 1, 2009; 46(2): 137 - 143. [Abstract] [Full Text] [PDF]
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 R. S. Swerdloff and C. Wang Free Testosterone Measurement by the Analog Displacement Direct Assay: Old Concerns and New Evidence Clin. Chem., March 1, 2008; 54(3): 458 - 460. [Full Text] [PDF]
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