The isolation from P. testosteroni of adequate quantities of purified and highly active hydroxysteroid dehydrogenases of known specificity pointed the way for the development of precise and specific methods for the enzymatic microanalysis of certain common steroid hormones and their metabolites (70, 71, 73, 168). Knowledge of the equdibrium constants and of factors affecting the equdibria of various hydroxy- and ketosteroid in-terconversions permitted the definition of rigorous experimental conditions under which microgram quantities of steroids could be quantitatively and specifically oxidized or reduced. The changes in the ultraviolet light absorption of reduced DPN which accompanied these reactions could then be used as stoichiometric measures of the quantities of steroid oxidized or reduced. The high degree of specificity of these methods for steroids generally, and for specific groups of steroids in particular, is due to the relatively restricted substrate specificities of the hydroxysteroid dehydrogenases. The ultimate limits of sensitivity of these methods have not yet been explored, but accurate measurement of 1-10 millimicromoles (ca. 0.3-3.0 Mg.) of individual steroids have been achieved without special equipment or precautions.

The measurement of steroid concentrations in tissues and in body fluids was one of the major objectives for which the enzymatic microanalysis of steroids was developed. Some progress toward this goal has been achieved (70, 73)- Simple methods have been devised for the extraction and hydrolysis of neutral (non-phenolic) hydroxy- and ketosteroids from small aliquots of human urine. Adequate quantities of steroids for analysis are present in a few millditers of normal urine. By sequential reaction of these crude extracts with the α- and β-hydroxysteroid dehydrogenases of P. testosteroni, measurements may be obtained of the following groups of compounds: 3α-hydroxysteroids, 3 βhydroxysteroids, 17β-hydroxyster-oids, 3-ketosteroids, and 17-ketosteroids. Values have been established for the quantities of a-and β-hydroxysteroids and 17-ketosteroids excreted by normal adults of both sexes and by some patients with abnormalities of the endocrine system (70,73). Measurements of various groups of steroid alcohols and ketones have provided information on the patterns of excretion of these substances in man, without the necessity of carrying out elaborate preliminary separations. It is not yet possible to assess the clinical importance of such measurements, although the value of a simple method of measuring the 3β-hydroxysteroid fraction has been demonstrated in the diagnosis and treatment of adrenal cortical hyperfunction (73). If crude urine extracts are fractionated, or individual compounds isolated by chromatographic methods, enzymatic measurements may then be carried out on these compounds or fractions. By enzymatic means, certain steroids can be measured for which no adequate analytical methods have been previously available.

The application of enzymatic microanalysis of steroids is not confined to the measurement of individual steroids or of steroid mixtures. The procedures may be used for the determination of the purity of steroids, and especially for the detection of the presence of contaminating isomers (168). In addition, 17β-hydroxysteroid dehydrogenase has been used in the resolution of totally synthetic racemic testosterone (168). The enzymatic methods provide powerful tools for the determination and correlation of steric configuration (174). They have also proved useful in carrying out quantitative oxidations of very small quantities of radioactive steroids (p. 81).