Enzymatic estimation of steroids in subpicomole quantities by hydroxysteroid dehydrogenases and nicotinamide nucleotide cycling

Extremely sensitive methods are described for the measurements of 3α- and 3β-hydroxysteroids, as well as 3-ketosteroids, based on their reaction with highly purified bacterial hydroxysteroid dehydrogenases and the amplification of the accompanying changes in nicotinamide nucleotides by enzymatic cycling procedures. Conditions have been devised under which the steroid oxidation and reduction reactions lead to the formation of stoichiometric quantities of NADH or NAD⁺, respectively, even in the presence of large excesses of reaction products. The scope of these methods is illustrated by application to the analysis of minute volumes of human pregnancy urine, high pressure liquid chromatography fractions derived from such urine samples, and human serum. The steroid contents of milligram quantities of rat prostate have been determined. The methods have been applied also to the measurement of the activities of steroid-transforming enzymes, such as the 3α-hydroxysteroid dehydrogenase of prostate microsomes. At the present time the sensitivity of the described methods allows the accurate determination of 0.2-0.4 pmol of steroids.