Tackling the Testosterones: Total, Free, and Bioavailable

When a patient gets their “testosterone test” at the doctor to assess their libido, do they really know what they’re getting? Does your lab test for testosterone, and are you confused about which of these confusingly-named tests are in-house versus send-out? Do you need a refresher on the types of testosterone tests out there and the clinical significance of each?

A Primer on Testosterone

Testosterone, being a fairly hydrophobic member of the steroid-ring family, is the major androgen in males. Apart from its well-known function in promoting the development of primary male reproductive organs and secondary male sex characteristics, it also has important anabolic effects in maintaining muscle mass, bone maturation, regulation of the hypothalamic-pituitary-adrenal axis under stress, and even in promoting platelet aggregation through enhancing platelet thromboxane A2 expression.1 In females, testosterone increases sexual arousal, and is in fact used clinically as treatment for female sexual arousal disorders. So, clearly an important member of the steroid family.

Being hydrophobic, much of the testosterone in the human body is not freely available, but rather bound. Total testosterone signifies the total pool of testosterone available in the human body, and is largely encompassed by the majority of bound testosterone with a small (usually 1.5-2.0%) proportion of free testosterone, which is biologically active. The bound testosterone can further be subdivided into testosterone bound to sex-hormone binding globulin (SHBG), a small glycoprotein that strongly binds various androgens and estrogens, and testosterone bound toalbumin, which is a relatively weak interaction.

Recently, the concept of bioavailable testosterone has been defined,2 based on the understanding that testosterone bound to SHBG (around 2/3rd of the bound proportion) is relatively inaccessible, while testosterone bound to albumin is weakly interacting, and thus potentially bioactive. Therefore, the definition of bioavailable testosteroneincludes both free and albumin-bound testosterone, which comprise the non-SHBG bound proportion.

How is testosterone measured?

Conventionally, total testosterone is measured through either immunoassays (both radioimmunoassays, or more commonly, chemiluminescent immunoassays) or mass spectrometry coupled with gas chromatography (GC/MS) or liquid chromatography (LC-MS/MS). Isotope dilution mass spectrometry (IDMS) is the reference method for testosterone measurement,3 but due to cost and convenience, most labs utilize immunoassays. Sex hormone binding globulin (SHBG) is commonly measured through chemiluminescent immunoassays, and also available for many platforms.4

There are two main approaches to the measurement of free testosterone, which is significantly more challenging. The gold standard for free testosterone measurement is equilibrium dialysis (see inset), a time consuming, expensive, and laborious assay that uses semi-permeable membranes to measure antibody-bound fractions of testosterone. Moreover, results can vary with pH, temperature, and methods of dilution.5 Due to these complications, calculated free testosterone is an attractive alternative used by many laboratories.

What is equilibrium dialysis? Equilibrium dialysis and ultrafiltration are reference methods used to determine true free testosterone calculation. Briefly, a relatively large quantity of serum (500 to 1000 uL) is placed in one chamber of an equilibrium dialysis apparatus, which is comprised of two fluid chambers separated by a semi-permeable membrane. Free-labeled testosterone passes through the membrane, while testosterone bound to SHBG does not. The radioactivity in the free chamber is quantified as a proportion of the total testosterone level, as measured by another assay, such as LC/MS-MS.

What is calculated free testosterone, and how is it calculated?

Recognizing the difficulty of performing equilibrium dialysis on large volumes of testosterone specimens, several researchers have looked into devising good approximations of free testosterone through mathematical expressions modeling the distribution of testosterone among its various compartments. One of the most popular approximations, the Vermeulen equation developed by Dr. Alex Vermeulen,6 models the distribution of testosterone among the SHBG-bound, albumin-bound, and free component through association constants of testosterone among these compartments, and can be modeled by the equation in Figure 1, which depends on the total testosterone, SHBG concentration, and concentration of albumin (although this will be discussed below). The overall concordance of this method with apparent free testosterone obtained through equilibrium dialysis (AFTC), the reference method, is very good, with a correlation coefficient of 0.987 and mean values well within the SEM between the two methods.6

Figure 1. The Vermeulen equation for calculated free testosterone.

In studies of the variation of calculated free testosterone values to the albumin concentration, Vermeulen et al. demonstrated that between “normal” albumin concentrations ranging from 5.8–7.2 × 10−4 mol/L (40 to 50 g/L), the mean calculated free testosterone varied from 340 ± 40.9 pmol/L assuming an albumin concentration of 40 g/L, to 303 ± 35.4 pmol/L assuming a concentration of 50 g/L albumin. Moreover, the concordance of calculated FT results to AFTC concentrations remained very good (correlation coefficient of 0.992) when an intermediate fixed albumin concentration (43 g/L) was used in this calculation, compared to actual albumin levels. Overall, these calculations suggest that for healthy individuals without marked abnormalities in plasma protein composition, such as in nephrotic syndrome or cirrhosis of the liver, or pregnant patients, a fixed albumin concentration could be used without significantly affecting calculated FT results. Of course, in individuals with marked changes in plasma proteins, the actual albumin concentration should be accounted for.

Willem de Ronde et al5 compared five different algorithms for calculating free or bioavailable, which includes the Vermeulen and Sodergard method (which use similar parameters), as well as methods by Emadi-Konjin et al, Morris et al, and Ly et al. In general, there was high concordance between the Vermeulen and Sodergard methods (r=0.98) for measuring free testosterone, and lower, but still reasonable (r=0.88) concordance between Vermeulen and other methods. Fundamentally, the Vermeulen and Sodergard equations were derived from experimentally derived association constants from the law of mass action, as opposed to the other algorithms, which rely on experimentally derived free and bioavailable testosterone measurements that was modeled by regression equations, and thus depends on the accuracy of these measurements. Though the experimental basis underlying the Vermeulen and Sodergard equations is stronger, it is known that supraphysiologic concentrations of other steroid hormones (estradiol or dihydrotestosterone), in competition for binding sites to SHBG, can significantly underestimate free testosterone by any of these methods. Of course, inaccuracies in the measurement of total testosterone or SHBG can significantly affect results, as well as significant perturbations in total serum protein concentrations (as mentioned above).

Since the publication of the above work, additional calculations for free testosterone accounting for other modes of interaction of SHBG such as allostery and dimerization have been published that may further improve concordance with AFTC;7,8 however, further study is needed to determine if these methods actually result in superior calculated FT measurement for clinical decision making, as well as changes in sensitivity to interference.

Why do accurate free testosterone measurements matter?

Testosterone bound to serum albumin is essentially inactive; therefore, the only testosterone that is biologically relevant is free (and to a lesser extent, bound to SHBG). Current consensus guidelines still support the use of total testosterone for defining hypogonadism in men,9,10 although emerging studies and newer task-force consensus groups11,12 highlight an emerging role for both calculated and free testosterone measurements in addition to total testosterone. The role of direct free testosterone measurement is still hotly debated; a recent analysis of CAP proficiency data indicates considerable heterogeneity among laboratories using the reference methods described above, and suggests considerable cost savings without significant loss of reliability can be achieved by using calculated or FT bioavailable T over direct FT measurement.13 Further standardization of these assays is needed to better understand the tradeoffs here.


  1. Ajayi A a. L, Halushka PV. Castration reduces platelet thromboxane A2 receptor density and aggregability. QJM. 2005;98(5):349-356. doi:10.1093/qjmed/hci054
  2. Shea JL, Wong P-Y, Chen Y. Free testosterone: clinical utility and important analytical aspects of measurement. Adv Clin Chem. 2014;63:59-84.
  3. Botelho JC, Shacklady C, Cooper HC, et al. Isotope-Dilution Liquid Chromatography–Tandem Mass Spectrometry Candidate Reference Method for Total Testosterone in Human Serum. Clinical Chemistry. 2013;59(2):372-380. doi:10.1373/clinchem.2012.190934
  4. Dittadi R, Fabricio ASC, Michilin S, Gion M. Evaluation of a sex hormone-binding globulin automated chemiluminescent assay. Scand J Clin Lab Invest. 2013;73(6):480-484. doi:10.3109/00365513.2013.805807
  5. Ronde W de, Schouw YT van der, Pols HAP, et al. Calculation of Bioavailable and Free Testosterone in Men: A Comparison of 5 Published Algorithms. Clinical Chemistry. 2006;52(9):1777-1784. doi:10.1373/clinchem.2005.063354
  6. Vermeulen A, Verdonck L, Kaufman JM. A Critical Evaluation of Simple Methods for the Estimation of Free Testosterone in Serum. None. 1999;84(10):3666-3672. doi:10.1210/jcem.84.10.6079
  7. Heinrich-Balard L, Zeinyeh W, Déchaud H, et al. Inverse relationship between hSHBG affinity for testosterone and hSHBG concentration revealed by surface plasmon resonance. Molecular and Cellular Endocrinology. 2015;399:201-207. doi:10.1016/j.mce.2014.10.002
  8. Zakharov MN, Bhasin S, Travison TG, et al. A multi-step, dynamic allosteric model of testosterone’s binding to sex hormone binding globulin. Mol Cell Endocrinol. 2015;399:190-200. doi:10.1016/j.mce.2014.09.001
  9. Margo KL, Winn R. Testosterone Treatments: Why, When, and How? AFP. 2006;73(9):1591-1598.
  10. American Association of Clinical Endocrinologists Medical Guidelines for Clinical Practice for the Evaluation and Treatment of Hypogonadism in Adult Male Patients—2002 Update. Endocrine Practice. 2002;8(6):439-456. doi:10.4158/EP.8.6.439
  11. Bhasin S, Cunningham GR, Hayes FJ, et al. Testosterone therapy in men with androgen deficiency syndromes: an Endocrine Society clinical practice guideline. J Clin Endocrinol Metab. 2010;95(6):2536-2559. doi:10.1210/jc.2009-2354
  12. Liu Z, Liu J, Shi X, et al. Comparing calculated free testosterone with total testosterone for screening and diagnosing late-onset hypogonadism in aged males: A cross-sectional study. J Clin Lab Anal. 2017;31(5). doi:10.1002/jcla.22073
  13. Morales A, Collier CP, Clark AF. A critical appraisal of accuracy and cost of laboratory methodologies for the diagnosis of hypogonadism:  the role of free testosterone assays. Can J Urol. 2012;19(3):6314-6318.

-Dr. Jim Hsu is a 2nd year pathology resident currently in training at Houston Methodist Hospital. After completing a M.D./Ph.D at the University of Texas Medical Branch in Galveston, he realized his passions remained in the lab, but wanted to bring that passion into patient care, and soon realized that pathology was the key to achieving both. His love for all things data drew him to pathology informatics, and with the suggestion of his mentor Dr. Wesley Long, to API. In particular, he is interested in the transformative power of data analysis in improving best practices, reducing error, and combating bias. Outside of the lab, he is interested in financial markets, algorithms, neuroscience, reading, and traveling (for the food, of course).

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