A one-step enzymatic assay for the measurement of 17β-hydroxy- and 17-oxo-steroid profiles in biological samples

We describe a simple enzymatic method for the sensitive and specific detection and quantitation of families of hydroxy- and oxo-steroids in biological mixtures. Analysis of the profiles of individual steroids may be achieved following their Chromatographie separation. The objectives of this analytical system are, therefore, different from conventional methods which are designed to measure single steroids with a high degree of specificity. The method employs highly purified and active bacterial hydroxysteroid dehydrogenases (HSD) which promote stereospecific, nicotinamide nucleotide-dependent oxidations and reductions at specified positions of steroids. In the presence of catalytic quantities of steroids these enzymes promote the transfer of hydrogen (transhydrogenation) between NADH and NAD analogues. A recently purified 17β-HSD from an Alcaligenes species (D. W. Payne and P. Talalay, J. biol. Chem., 260, 13468-13655, 1985) shows almost complete specificity for the 17β-hydroxy- and 17-oxogroups of both C₁₈ and C₁₉ steroids. This enzyme catalyzes steroid-dependent transhydrogenation between NADH and the thionicotinamide analogue of NAD (S-NAD). When these components are incubated at pH 8.5 in the presence of minute quantities of steroid substrates, S-NADH (measured at 398 nm where NADH does not absorb) accumulates at a constant rate which is proportional to the concentrations of steroid and enzyme. The linear increase in absorbance with time is a measure of the total concentration of 17β-hydroxy- and 17-oxo-steroids, and can be used to detect subpicomol quantities of steroids. The method is illustrated by the detection and identification of free and conjugated androgens in human serum following their separation by high pressure liquid chromatography. The specificity of the transhydrogenase assay is completely dependent on the specificity of the enzyme and is thus applicable to the detection of other hydroxy- and oxo-steroids by making use of HSDs with appropriate specificities (e.g. 3α-HSD for the measurement of 3α-hydroxy- and 3-oxo-steroids). The simple one-step reaction lends itself to automation, needs no auxiliary detection systems, and requires only an inexpensive colorimeter.