Enzymic color development of urinary 3 alpha-hydroxysteroids on thin-layer chromatograms.

Abstract

I describe the use of 3 alpha-hydroxysteroid dehydrogenase (EC 1.1.1.50) for enzymic color development of 3 alpha-hydroxysteroids. Urinary 3 alpha-hydroxysteroids are converted to 3-ketosteroids by the enzyme, and the NADH formed is reacted with 2-p-iodophenyl-3-p-nitrophenyl-5-phenyl-tetrazolium chloride and "diaphorase" to form the colored formazan. The dye so formed on a thin layer chromatographic plate is measured by densitometoric scanning. I illustrate use of this method by showing results for urinary excretion of 3 alpha-hydroxysteroids by some various sorts of patients.