Testosterone, Free, Equilibrium Ultrafiltration With Total Testosterone

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.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 profile by an indirect method, equilibrium ultrafiltration. Tritiated testosterone is added to the sample and allowed to come to equilibrium with testosterone in the serum at physiological temperature.^3,4 The amount of the added radiolabeled testosterone must be low enough to ensure that the addition will not significantly increase the total testosterone concentration. Once equilibrium is achieved, the free testosterone is separated from the bound by filtration through a membrane by centrifugal ultrafiltration.⁵ The radioactivity of the protein-free ultrafiltrate is measured and used to calculate the percent free testosterone. The concentration of free testosterone is then calculated by multiplying the percent free testosterone by the total testosterone concentration.