A symmetric dialysis method for the determination of free testosterone in human plasma

We describe a symmetric dialysis (SYD) method for the determination of the fraction (fT), and the concentration (FT) of non-protein-bound testosterone (T) in human plasma. In SYD fT is estimated from the rate at which radiolabelled testosterone redistributes between two identical (undiluted) plasma aliquots separated by a dialysis membrane. We compared the effect of tracer contamination and sample dilution on FT as measured by SYD and equilibrium dialysis (EQD) in five plasma pools differing in Sex Hormone Binding Globulin (SHBG) and total T concentration. In addition, FT was calculated by computer simulation of the interaction between binding proteins and steroid hormones. With SYD relatively small decreases in FT were observed following two- to tenfold dilution (14.3 +/- 4.4%). A comparable decrease was calculated by computer simulation (13.3 +/- 2.2%). The apparent fall in FT as estimated by EQD was much larger (44.7 +/- 2.2%) and, contrary to assessment by SYD, this fall depended on the radiochemical purity of the 3HT-tracer. Moreover, since SYD allows the assessment of fT in undiluted samples, problems relating to the interference of buffer components with T-binding are circumvented. We conclude that SYD is to be preferred over EQD for the accurate and precise measurement of fT and consequently FT.