Determination of 17,21-Dihydroxy-20-Ketosteroids in Blood Plasma*

IN 1950 Porter and Silber (1) observed that a color reaction occurred if 17,21-dihydroxy-20-ketosteroids were exposed to a phenylhydrazinesulfuric acid solution. Utilizing this color reaction, Nelson and Samuels (2), Glenn and Nelson (3) and Reddy et al. (4) have published methods for the determination of adrenal steroids having a 17,21-dihydroxy-20-keto configuration. Since then, numerous publications have appeared dealing with adrenocortical physiology and pathology, using levels of 17-hydroxycorticosteroids as an index for these conditions. Marks and Leftin (5), however, were able to find butanol-extractable substances in human urine which gave rise to a positive Porter-Silber reaction without fulfilling the 17,21-dihydroxy-20-keto qualification. These findings raise an essential point of methodology: How much of the Porter-Silber chromogen in plasma or urine estimated by the simpler techniques may be ascribed to nonsteroidal material? Even with the Nelson and Samuels method as originally described, the Porter-Silber chromogen in blood plasma from normal subjects seldom exhibits a peak absorbancy at 410 mμ, whereas crystalline cortisol, even when extracted from water with chloroform and subjected to chromatographic treatment on Florisil columns, exhibits such a peak.