Clinical Chemistry 54:3 512 516 (28) Endocrinology and Metabolism Analog-Based Free Testosterone Test Results Linked to Total Testosterone Concentrations, Not Free Testosterone Concentrations Kristofer S. Fritz, 1 Alastair J.S. McKean, 1 Jerald C. Nelson, 2 and R. Bruce Wilcox 1* BACKGROUND: Analog-based free testosterone test results, sex hormone binding globulin (SHBG) concentrations, and total testosterone concentrations are somehow related. This study used new experiments to clarify these relationships. 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. 28 American Association for Clinical Chemistry Serum free testosterone concentrations in vivo are thought to be under the control of the hypothalamicpituitary-gonadal negative feedback regulatory system. Thus the amount of serum luteinizing hormone (LH) secreted is determined by the negative feedback effects of the circulatory free (unbound) testosterone concentrations. The circulatory concentrations of LH directly affect the amount of testosterone secreted by the testes. Serum total testosterone concentrations vary in proportion to varied free testosterone concentrations and in proportion to varied sex hormone binding globulin (SHBG) concentrations. 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 7). It has also been asserted that they are proportional to total testosterone concentrations (1, 7, 8). This led us to question whether these analogbased free testosterone test results are more closely related to the concentrations of total testosterone or SHBG. Materials and Methods TESTOSTERONE IMMUNOASSAYS The analog-based free testosterone immunoassay studied (Coat-A-Count; Siemens Medical Solutions Diagnostics) is a manual method that uses a radiolabeled ( 125 I) testosterone analog (a conjugated form of testosterone), immobilized testosterone antibody, and a sin- 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. * Address correspondence to this author at: Department of Biochemistry, Loma Linda University School of Medicine, Mortensen Hall, Room 29, Loma Linda, CA 9235. Fax (99) 558-7916; e-mail bwilcox@llu.edu. Received July 12, 27; accepted November 23, 27. Previously published online at DOI: 1.1373/clinchem.27.9487 512
Free Testosterone Assay Tracks Total Testosterone Fig. 1. Illustration of experiments. A tracer dialysis free testosterone (Te) assay, an analog-based free Te assay, and a total Te assay were applied to 3 sets of solutions. (A), The solutions in the first experiment were serum retentate and serum dialysate. (B), In the second experiment, serum retentate was progressively diluted with serum dialysate, diluting total Te from 1% to 2%. (C), In the third experiment, an aliquot of serum dialysate was enriched with Te to match the total Te concentration in the serum retentate. The serum retentate was then progressively diluted with the Te-enriched dialysate. SHBG Te, SHBG-bound Te. gle incubation, following a 21-fold dilution of the serum sample with a single aqueous reagent. It is critical to note that the calibration range of this analog-based free testosterone assay is 1.9 to 173 pmol/l; generally accepted reference ranges for free testosterone are approximately 17 to 73 pmol/l for adult males (9). We measured total testosterone using a total testosterone immunoassay (Coat-A-Count; Siemens). This manual method also uses radiolabeled ( 125 I) 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 welldocumented tracer dialysis free testosterone assay to verify free testosterone concentrations in these sameserum 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 4 multiwell automated counter (Beckman-Coulter). Each testosterone measurement reported is a mean of triplicate determinations, and each experiment was repeated for confirmation. 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 (1) to a concentration of.35 mol/l testosterone. PREPARATIVE EQUILIBRIUM DIALYSIS Dialysis devices and dialysate buffer were obtained from Antech Diagnostics. The dialysis device uses 2 L sample retentate and 24 L dialysis buffer. The chemical composition of this buffer has been reported (1). Serum samples were dialyzed for 18 h at 37 C in an Isotemp Incubator, model 63D (Fisher Scientific) (11 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 (.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- L aliquot (approximately 13 pmol) of stock 3 H-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 (LS75, Beckman). We calculated the fraction of free testoster- Clinical Chemistry 54:3 (28) 513
one at equilibrium as 3 H-testosterone in dialysate/ 3 H- 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 5 L 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 (2 L retentate vs 24 L 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 1%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, and 2% 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 1-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. Results In the first experiment, the analog-based free testosterone assay and the total testosterone assay detected the nondialyzable fraction of serum testosterone, that is, the testosterone in the serum retentate. The total testosterone assay reported mean 21.6 (SD 1.) nmol/l in the serum retentate. The analog-based free testosterone assay reported 56 (4.5) pmol/l in the serum retentate. Neither assay detected dialyzable concentrations of serum testosterone. The tracer dialysis free testosterone assay reported 347 pmol/l of testosterone in serum dialysate. 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 34 to 169 pmol/l (Fig. 2). Total testosterone test results and analog-based free testosterone test results correlated more closely (r 2.97; P.1) than analog-based free testosterone test results and tracer dialysis free testosterone test results; (r 2.9; P.1). 514 Clinical Chemistry 54:3 (28)
Free Testosterone Assay Tracks Total Testosterone ) 6 A ) 1 A Dialyz abl e Te test results (pmol/l 5 4 3 2 1 Dialyz abl e Te test results (pmol/l 8 6 4 2 Analog-based free Te test results (pmol/l ) Total Te te st re sults (nm ol /L ) 6 5 4 3 2 1 25 2 15 1 5 B C 1 8 6 4 Percentage of native serum protein and total testosterone concentration 2 Analog-based free Te test results (pmol/l ) Total Te te st re sults (nm ol /L ) 1 8 6 4 2 4 3 2 1 B C 1 1 1 1 Dilution of protein-bound testosterone with free testosterone (dilution factors) Fig. 2. Analog-based free testosterone assay tracks serum protein and total testosterone concentrations. Tracer dialysis free testosterone test results (A), analogbased free testosterone test results (B), and total testosterone test results (C) when these assays were applied to progressive dilutions of serum retentate with serum dialysate. Te, testosterone. Fig. 3. Analog-based free testosterone assay tracks total testosterone concentrations. Tracer dialysis free testosterone test results (A), analogbased free testosterone test results (B), and total testosterone test results (C) when these assays were applied to progressive dilutions of serum retentate with testosteroneenriched serum dialysate. Total testosterone was held constant. Te, testosterone. 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 (.5) nmol/l] (Fig. 3). Tracer dialysis test results, or free testosterone concentration, increased from 376 to 798 pmol/l (Fig. 3). Discussion The Endocrine Society recently reported a review of the evidence that analog-based free testosterone immunoassays should be avoided because of problems with accuracy and sensitivity (16). The data obtained in the present study document nonspecificity associated with insensitivity and incorrect calibration in one of these assays (Figs. 2 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 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 Clinical Chemistry 54:3 (28) 515
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 References 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. Grant/funding Support: This work was supported by the Loma Linda University School of Medicine and the Mortensen Chair. No extramural funds were used for this study. 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. 1. Winters SJ, Kelley DE, Goodpaster B. The analog free testosterone assay: are the results in men clinically useful? Clin Chem 1998;44:2178 82. 2. 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 72. 3. Tibblin G, Adlerberth A, Lindstedt G, Bjorntorp P. The pituitary-gonadal axis and health in elderly men: a study of men born in 1913. Diabetes 1996;45:165 9. 4. Jowett T, Chu F, Ekins R. Validity of current analog-based free hormone immunoassays. Steroids 1988;52:365 6. 5. Haffner SM, Shaten J, Stern MP, Smith GD, Kuller L. Low levels of sex hormone-binding globulin and testosterone predict the development of noninsulin-dependent diabetes mellitus in men. Am J Epidemiol 1996;143:889 97. 6. Slaats EH, Kennedy JC, Kruijswijk H. Interference of sex-hormone binding globulin in the Coat-A- Count testosterone no-extraction radioimmunoassay. Clin Chem 1987;33:3 2. 7. Phillips GB, Jing T-Y, Resnick LM, Barbagallo M, Laragh JH, Sealey JE. Sex hormones and hemostatic risk factors for coronary heart disease in men with hypertension. J Hypertens 1993;11: 699 72. 8. Van Uytfanghe K, Stockl D, Kaufman JM, Fiers T, De Leenheer A, Thienpont LM. Validation of 5 routine assays for serum free testosterone with a candidate reference measurement procedure based on ultrafiltration and isotope dilution-gas chromatography-mass spectrometry. Clin Biochem 25;38:253 61. 9. Greenspan FS, Gardner DG [eds]. Basic and Clinical Endocrinology. 7th ed. New York: McGraw- Hill, 24. 1. Nelson JC, Tomei RT. Direct determination of free thyroxin in undiluted serum by equilibrium dialysis/radioimmunoassay. Clin Chem 1988;34: 1737 44. 11. Miller KK, Rosner W, Lee H, Hier J, Sesmilo G, Schoenfeld D, Neubauer G, Klibanski A. Measurement of free testosterone in normal women and women with androgen deficiency: comparison of methods. J Clin Endocrinol Metab 24;89: 525 33. 12. Sinha-Hikim I, Arver S, Beall G, Shen R, Guerrero M, Sattler F, Shikuma C, Nelson JC, Landgren BM, Mazer NA, Bhasin S. The use of a sensitive equilibrium dialysis method for the measurement of free testosterone levels in healthy, cycling women and in human immunodeficiency virus-infected women. J Clin Endocrinol Metab 1998;83: 1312 8. 13. Umstot ES, Baxter JE, Andersen RN. A theoretically sound and practicable equilibrium dialysis method for measuring percentage of free testosterone. J Steroid Biochem 1985;22:639 48. 14. Wilcox RB, Nelson JC. Time course of ph regulation in free thyroxin determinations in serum. Clin Chem 1991;37:1298 3. 15. Bhasin S, Swerdloff RS, Steiner B, Peterson MA, Meridores T, Galmirini M, Pandian MR, Goldberg R, Berman N. A biodegradable testosterone microcapsule formulation provides uniform eugonadal levels of testosterone for 1 11 weeks in hypogonadal men. J Clin Endocrinol Metab 1992; 74:75 83. 16. Rosner W, Auchus RJ, Azziz R, Sluss PM, Raff H. Utility, limitations, and pitfalls in measuring testosterone: an Endocrine Society position statement. J Clin Endocrinol Metab 27;92:45 13. 17. Fritz KS, Wilcox RB, Nelson JC. A direct free T 4 immunoassay with the characteristics of a total T 4 immunoassay. Clin Chem 27;53:911 5. 516 Clinical Chemistry 54:3 (28)