Testosterone, Free, Direct

The concentration of free testosterone is very low, typically <2% of the total testosterone concentration. In most men and women, >50% of total circulating testosterone is bound to sex hormone-binding globulin, SHBG, and most of the rest is bound to albumin.^1,2 Routinely available assay methods used to measure total testosterone are not sensitive enough to accurately quantitate the free testosterone fraction directly. Free testosterone is estimated in this test by a direct, analogue immunoassay method. This assay uses a labeled testosterone analogue that has a low binding affinity for both SHBG and albumin but is bound by antitestosterone antibody used in the assay. Since the analogue is unbound in the plasma, it competes with free testosterone for binding sites on an antitestosterone antibody that is immobilized on the surface of the microtiter plate well.

Several authors have found that the analogue method has good correlation with equilibrium dialysis,^1-4 but have found that the analogue results were only about one-fourth as high. Another group found that the analogue method produced results directly comparable to equilibrium dialysis without multiplication by a factor.⁵ More recently, Winters and coworkers have found the analogue method to correlates better with total testosterone levels than with bioavailable testosterone determined by the ammonium sulfate precipitation method.⁶ They suggested that the analogue-free testosterone results might be misleading in men with low SHBG concentration.⁶ Ooi suggested that the problems observed by Winters⁶ might, in large part, be resolved by simply using a more appropriate population-based reference interval.⁷ Vermeulen and coworkers found that the analogue-free testosterone method correlated well with free testosterone by equilibrium dialysis, but did not agree well with a free testosterone calculated from total testosterone and SHBG.³