Principles of the enzymatic measurement of steroids.

Information on steroid concentrations in tissues is important for an understanding of the metabolism of these substances and also for diagnostic purposes in certain clinical conditions. Existing procedures for determining steroids, either singly or in mixtures, are based upon calorimetric methods or tedious bioassays. The merits and limitations of these procedures have been critically appraised (l-3), their principal drawbacks being lack of specificity and sensitivity. This paper concerns the microestimation of steroid hormones and their metabolites by enzymatic methods suitable for the measurement of these compounds in biological systems, and which appear to offer the promise of a high degree of sensitivity and specificity. Enzymatic steroid assays depend upon the quantitative interconversion of certain hydroxyand ketosteroids by highly purified DPNl-linked hydroxysteroid dehydrogenases prepared from Pseudomonas testosteroni (4-6). The accompanying changes in DPNH concentration are determined spectrophotometrically at 340 rnp (7) and constitute a stoichiometric measure of the amount of steroid oxidized or reduced. The enzymatic microestimation of steroids was demonstrated in 1953 (4) and its applications extended when more highly purified hydroxysteroid dehydrogenases became available (6). The enzymatic estimation of urinary steroids has been reported briefly (8). The present paper contains a detailed examination of the principles of the enzymatic measurements of steroids and considers their specificity, sensitivity, and accuracy. At this time, two hydroxysteroid dehydrogenases are suitable for analytical purposes by virtue of their purity and high activity and a knowledge of their substrate specificities. The enzymes are highly active because of their inductive (adaptive) nature, and increases in specific activity of several 100-fold have been easily achieved by adding various steroids to the growth medium (6). The substrate specificities of these hydroxysteroid dehydrogenases have been described in detail (9, 10). In brief,