Development of radioimmunoassay methods for plasma T, DHT, A and DHEA and their application to the study of congenital defficiency of critical 21 - OH'ase

Radioimmunoassay methods have been developed for plasma T, DHT, A and DHEA. Plasma volumes usually employed were: 0,2 ml and 0,5 ml for men and women, respectively for T: 1,0 ml from DHT and 0,5 ml to A and DHEA. After preliminary extraction with diethyl ether, the extracts were applied to Sephadex LH-20 microcolumns to separate T from DHT and to Al2O3 microcolumns purification of DHEA. Following elution, the dried, purified extracts were incubated with specific antisera at temperature 40C overnight, the separation of antibody bound and free fractions being done on activated charcoal-dextrana T-70. The counting was done in liquid scintillations. Specificity studies have shown that the assays for all steroids were specific. The sensitivity was 5 pg for A and DHEA, 6 pg for T and DHT. The interassay coeficient of variation was 5-10% and the interessay precision was 10-15%. The accuracy was satisfactory with a - 8,74% for DHT, 104,82 +- 9,41% for A and 103,66 +- 5,71% for DHEA. The mean +- SD of plasma T, DHT, A and DHEA concentrations in normal men and women determined by these methods were as folows: in men, T:5,2 ng/ml +- 1,3, DHT:0,26 +- 0,18, A:0,9 +- 0,4 and DHEA:4,6 +- 17 and in females: T:0,5 ng/ml +- 0,2, DHT:0,4 +- 0,2, A:0,8 +- 0,5 and DHEA:4,8 +- 1,7. The methodology applied for blood collected simultaneously from peripheral and left and right adrenal vein effluents in basal conditions, after dexamethasone suppression and ACTH stimulation clearly demonstrated direct secretion (presence of a steroid gradient Peripheral/Adrenal vein) of the androgens studied in congenital defficiency of cortical 21-OH'ase. (Author)