THE COMBINED ESTIMATION OF CORTISOL AND 11-DESOXYCORTISOL IN PLASMA AS PORTER-SILBER CHROMOGENS

Abstract

Current methods of measuring the concentration of plasma 17-hydroxycorticosteroids, although adequate for cortisol, may vary in the recovery of 11-desoxycortisol. The present study demonstrates that procedures which employ solvent partition to transfer 11-desoxycortisol from the extracting solvent, dichloromethane, into the phenylhydrazine-sulfuric acid-ethanol color reagent lose almost 50 per cent of the steroid in this step. A modification of the Silber-Porter procedure has been developed, which overcomes this difficulty by evaporating the extracting solvent before addition of the color reagent. By the joint quantitation of both cortisol and 11-desoxycortisol this procedure provides a more accurate estimation of total free 17,21-dihydroxy-20-ketosteroids in plasma. The quantitative recovery of 11-desoxycortisol is of considerable practical value, since it is a biosynthetic precursor of cortisol and its presence is being recognized with greater frequency in various spontaneous and induced abnormal adrenal states. CURRENT methods for quantitating plasma levels of 17-hydroxycorticosteroids (17-OH-CS) in general utilize the color reaction first described by Porter and Silber (1). In this reaction, corticoids with 17, 21- dihydroxy-20-keto groups form phenylhydrazones which are yellow chromophores absorbing ultraviolet light maximally at 410 mμ (2). Although other nonsteroidal compounds may absorb similarly (3), the color reaction is presumed to measure principally cortisol,¹ the most abundant 17-OH-CS in the peripheral circulation of normal man (4). Recent interest in disorders of adrenocortical steroidogenesis, both congenital (5) and druginduced (6–8), has directed attention toward the quantitative measure ment of 11-desoxycortisol² as well. This compound, since it is structurally identical with cortisol except for the absence of an 11β-hydroxyl group, is also estimated by the Porter-Silber reaction (9). In initial experiments with the Peterson modification (10) of the method developed by Silber and Porter (11) it was found that excessive amounts of 11-desoxycortisol were lost. This observation prompted the present evaluation of the method and led to certain alterations in technique by which such losses may be avoided.