A combined adsorption-gel filtration technique for the determination of the cortisol-binding capacity of transcortin.

Abstract

A combined adsorption-gel filtration technique has been developed for the quantitation of the cortisol-binding capacity of transcortin: Endogenous steroids are removed from plasma by adsorption on uncoated charcoal. Saturation of the "stripped" binding sites of transcortin is accomplished by equilibrating the sample with a definite amount of labeled cortisol of low specific activity (0.1 muCi/mug). Transcortin-bound [4-14C]cortisol is isolated by gel filtration over Sephadex G-50 at 4 degrees C and measured by liquid scintillation counting. The cortisol-binding capacity of transcortin is calculated directly on the basis of the known specific activity of cortisol. The modification described eliminates methodological disadvantages associated with the original gel filtration procedures, i.e. the possible interference of various endogenous steroids with cortisol binding to transcortin, and the necessity of fluorometric or colorimetric determination of protein-bound cortisol. The values of the cortisol-binding capacity of transcortin in plasma obteined by this simplified assay are in close agreement with results reported in the literature (mean +/- S.D.): healthy males 261 +/- 23 mug/l) of transcortin-bound cortisol (n = 13), healthy nonpregnant females 255 +/- 31 mug/l (n = 15), and pregnant females prior to delivery 560 +/- 82 mug/l (n = 12).