PRINCIPLES OF THE ENZYMATIC MEASUREMENT OF STEROIDS

Abstract

SUMMARY The sensitive and specific microestimation of steroids by means of LY- and fl-hydroxysteroid dehydrogenases has been described. Factors in- fluencing the equilibria of the reactions catalyzed by these enzymes have been examined. Conditions for obtaining complete oxidations of hydroxy- steroids or reductions of ketosteroids have been established. Quantitative oxidations of steroids have been obtained by using a high pH and adding a ketone-binding reagent. Complete reductions of ketosteroids have been achieved by the destruction with DPNase of DPNf formed in the reac- tion. The enzymes have been employed for the estimation of SOL-, 3&, and 17P-hydroxysteroids as well as 3- and 17-ketosteroids, singly as well as in mixtures. The sensitivity of the method is about 1 mpmole of ster- oid. The methods have been illustrated by the measurement of single steroids, steroid mixtures, and chromatographic fractions. These pro- cedures are also applicable to the determination of steroid purity and con- tamination by isomers. Some of the steroids used in these studies were generously donated by Dr. A. Zaffaroni, Syntex, S. A., Mexico, and Dr. E. W. Meyer, The Glid- den Company, Chicago. The authors are grateful to Mr R. C. Leek for aid in developing the system for silicic acid chromatography of steroids.