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A Critical Evaluation of Simple Methods for the Estimation of Free Testosterone in Serum

Laboratory for Hormonology and Department of Endocrinology, University Hospital Ghent, 9000 Ghent, Belgium

Address all correspondence and requests for reprints to: Dr. J. M. Kaufman, University Hospital, 9K12 IE Endocrinology, De Pintelaan 185, 9000 Ghent, Belgium. E-mail: jean.kaufman{at}rug.ac.be

The free and nonspecifically bound plasma hormone levels generally reflect the clinical situation more accurately than total plasma hormone levels. Hence, it is important to have reliable indexes of these fractions. The apparent free testosterone (T) concentration obtained by equilibrium dialysis (AFTC) as well as the fraction of serum T not precipitated by 50% ammonium sulfate concentration (non-SHBG-T; SHBG, sex hormone-binding globulin), often referred to as bioavailable T, appear to represent reliable indexes of biologically readily available T, but are not well suited for clinical routine, being too time consuming. Several other parameters have been used without complete validation, however: direct immunoassay of free T with a labeled T analog (aFT), calculation of free T (FT) from total T and immunoassayed SHBG concentrations (iSHBG), and the free androgen index (FAI = the ratio 100T/iSHBG). In the view of substantial discrepancies in the literature concerning the free or bioavailable T levels, we compared AFTC, FT, aFT, FAI, and non-SHBG-T levels in a large number of sera with SHBG capacities varying from low, as in hirsute women, to extremely high as in hyperthyroidism. All these indexes of bioavailable T correlated significantly with the AFTC concentration; AFTC and FT values were almost identical under all conditions studied, except during pregnancy. Values for aFT, however, were only a fraction of either AFTC or FT, the fraction varying as a function of SHBG levels. Also, the FAI/AFTC ratio varied as a function of the SHBG levels, and hence, neither aFT nor FAI is a reliable index of bioavailable T.

The FT value, obtained by calculation from T and SHBG as determined by immunoassay, appears to be a rapid, simple, and reliable index of bioavailable T, comparable to AFTC and suitable for clinical routine, except in pregnancy. During pregnancy, estradiol occupies a substantial part of SHBG-binding sites, so that SHBG as determined by immunoassay overestimates the actual binding capacity, which in pregnancy sera results in calculated FT values that are lower than AFTC. The nonspecifically bound T, calculated from FT, correlated highly significantly with and was almost identical to the values of non-SHBG-T obtained by ammonium sulfate precipitation, testifying to the clinical value of FT calculated from iSHBG.

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				M. Rachmiel, S. Kives, E. Atenafu, and J. Hamilton Primary Amenorrhea as a Manifestation of Polycystic Ovarian Syndrome in Adolescents: A Unique Subgroup? Arch Pediatr Adolesc Med, June 1, 2008; 162(6): 521 - 525. [Abstract] [Full Text] [PDF]

				P. E. Knapp, T. W. Storer, K. L. Herbst, A. B. Singh, C. Dzekov, J. Dzekov, M. LaValley, A. Zhang, J. Ulloor, and S. Bhasin Effects of a supraphysiological dose of testosterone on physical function, muscle performance, mood, and fatigue in men with HIV-associated weight loss Am J Physiol Endocrinol Metab, June 1, 2008; 294(6): E1135 - E1143. [Abstract] [Full Text] [PDF]

				S. A. Hall, A. B. Araujo, G. R. Esche, R. E. Williams, R. V. Clark, T. G. Travison, and J. B. McKinlay Treatment of Symptomatic Androgen Deficiency: Results From the Boston Area Community Health Survey Arch Intern Med, May 26, 2008; 168(10): 1070 - 1076. [Abstract] [Full Text] [PDF]

				S. Loves, J. Ruinemans-Koerts, and H. de Boer Letrozole once a week normalizes serum testosterone in obesity-related male hypogonadism Eur. J. Endocrinol., May 1, 2008; 158(5): 741 - 747. [Abstract] [Full Text] [PDF]

				T. Scalvini, P. R. Martini, A. Gambera, R. Tardanico, L. Biasi, F. Scolari, G. Gregorini, and E. Agabiti Rosei Spermatogenic and Steroidogenic Impairment of the Testicle Characterizes the Hereditary Leucine-75-Proline Apolipoprotein A-I Amyloidosis J. Clin. Endocrinol. Metab., May 1, 2008; 93(5): 1850 - 1853. [Abstract] [Full Text] [PDF]

				M. Grossmann, M. C. Thomas, S. Panagiotopoulos, K. Sharpe, R. J. MacIsaac, S. Clarke, J. D. Zajac, and G. Jerums Low Testosterone Levels Are Common and Associated with Insulin Resistance in Men with Diabetes J. Clin. Endocrinol. Metab., May 1, 2008; 93(5): 1834 - 1840. [Abstract] [Full Text] [PDF]

				J. S. Lee, A. Z. LaCroix, L. Wu, J. A. Cauley, R. D. Jackson, C. Kooperberg, M. S. Leboff, J. Robbins, C. E. Lewis, D. C. Bauer, et al. Associations of Serum Sex Hormone-Binding Globulin and Sex Hormone Concentrations with Hip Fracture Risk in Postmenopausal Women J. Clin. Endocrinol. Metab., May 1, 2008; 93(5): 1796 - 1803. [Abstract] [Full Text] [PDF]

				J. A. Talbot, R. Sheldrick, H. Caswell, and S. Duncan Sexual function in men with epilepsy: How important is testosterone? Neurology, April 15, 2008; 70(16): 1346 - 1352. [Abstract] [Full Text] [PDF]

				M. Mohlig, M. O Weickert, E. Ghadamgahi, A. M Arafat, J. Spranger, A. F H Pfeiffer, and C. Schofl Retinol-binding protein 4 is associated with insulin resistance, but appears unsuited for metabolic screening in women with polycystic ovary syndrome. Eur. J. Endocrinol., April 1, 2008; 158(4): 517 - 523. [Abstract] [Full Text] [PDF]

				L. J. Shaw, C. N. Bairey Merz, R. Azziz, F. Z. Stanczyk, G. Sopko, G. D. Braunstein, S. F. Kelsey, K. E. Kip, R. M. Cooper-DeHoff, B. D. Johnson, et al. Postmenopausal Women with a History of Irregular Menses and Elevated Androgen Measurements at High Risk for Worsening Cardiovascular Event-Free Survival: Results from the National Institutes of Health--National Heart, Lung, and Blood Institute Sponsored Women's Ischemia Syndrome Evaluation J. Clin. Endocrinol. Metab., April 1, 2008; 93(4): 1276 - 1284. [Abstract] [Full Text] [PDF]

				A. Mueller, L. J. Gooren, S. Naton-Schotz, S. Cupisti, M. W. Beckmann, and R. Dittrich Prevalence of Polycystic Ovary Syndrome and Hyperandrogenemia in Female-to-Male Transsexuals J. Clin. Endocrinol. Metab., April 1, 2008; 93(4): 1408 - 1411. [Abstract] [Full Text] [PDF]

				K. Allvin, C. Ankarberg-Lindgren, H. Fors, and J. Dahlgren Elevated Serum Levels of Estradiol, Dihydrotestosterone, and Inhibin B in Adult Males Born Small for Gestational Age J. Clin. Endocrinol. Metab., April 1, 2008; 93(4): 1464 - 1469. [Abstract] [Full Text] [PDF]

				E. Elaut, G. De Cuypere, P. De Sutter, L. Gijs, M. Van Trotsenburg, G. Heylens, J.-M. Kaufman, R. Rubens, and G. T'Sjoen Hypoactive sexual desire in transsexual women: prevalence and association with testosterone levels Eur. J. Endocrinol., March 1, 2008; 158(3): 393 - 399. [Abstract] [Full Text] [PDF]

				M. Das, O. Djahanbakhch, B. Hacihanefioglu, E. Saridogan, M. Ikram, L. Ghali, M. Raveendran, and A. Storey Granulosa Cell Survival and Proliferation Are Altered in Polycystic Ovary Syndrome J. Clin. Endocrinol. Metab., March 1, 2008; 93(3): 881 - 887. [Abstract] [Full Text] [PDF]

				O. P. Almeida, B. B. Yeap, G. J. Hankey, K. Jamrozik, and L. Flicker Low Free Testosterone Concentration as a Potentially Treatable Cause of Depressive Symptoms in Older Men Arch Gen Psychiatry, March 1, 2008; 65(3): 283 - 289. [Abstract] [Full Text] [PDF]

				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]

				K. S. Fritz, A. J.S. McKean, J. C. Nelson, and R. B. Wilcox Analog-Based Free Testosterone Test Results Linked to Total Testosterone Concentrations, Not Free Testosterone Concentrations Clin. Chem., March 1, 2008; 54(3): 512 - 516. [Abstract] [Full Text] [PDF]

				E. Hormones and Prostate Cancer Collaborative Group Endogenous Sex Hormones and Prostate Cancer: A Collaborative Analysis of 18 Prospective Studies J Natl Cancer Inst, February 6, 2008; 100(3): 170 - 183. [Abstract] [Full Text] [PDF]

				H. F. Escobar-Morreale, R. Sanchon, and J. L. San Millan A Prospective Study of the Prevalence of Nonclassical Congenital Adrenal Hyperplasia among Women Presenting with Hyperandrogenic Symptoms and Signs J. Clin. Endocrinol. Metab., February 1, 2008; 93(2): 527 - 533. [Abstract] [Full Text] [PDF]

				M. H. Emmelot-Vonk, H. J. J. Verhaar, H. R. Nakhai Pour, A. Aleman, T. M. T. W. Lock, J. L. H. R. Bosch, D. E. Grobbee, and Y. T. van der Schouw Effect of Testosterone Supplementation on Functional Mobility, Cognition, and Other Parameters in Older Men: A Randomized Controlled Trial JAMA, January 2, 2008; 299(1): 39 - 52. [Abstract] [Full Text] [PDF]

				C. A. Allan, B. J. G. Strauss, H. G. Burger, E. A. Forbes, and R. I. McLachlan Testosterone Therapy Prevents Gain in Visceral Adipose Tissue and Loss of Skeletal Muscle in Nonobese Aging Men J. Clin. Endocrinol. Metab., January 1, 2008; 93(1): 139 - 146. [Abstract] [Full Text] [PDF]