Continuous-flow automated assay of steroids with nylon-tube-immobilized hydroxysteroid dehydrogenases

Several NAD(P)+-dependent hydroxysteroid dehydrogenases, namely 3 alpha-hydroxysteroid dehydrogenase, beta-hydroxysteroid dehydrogenase, 7 alpha-hydroxysteroid dehydrogenase, and 12 alpha-hydroxysteroid dehydrogenase were separately immobilized on nylon tubes for the continuous-flow automated assay of hydroxysteroids. 3 alpha-Hydroxysteroid dehydrogenase was also immobilized on pore glass. Spectrophotometric monitoring in the visible region, where blank values were markedly reduced, was achieved through the Meldola blue catalyzed transfer of hydrogen from NAD(P)H to a tetrazolium salt. Nylon-tube-immobilized enzymes maintained 45-55% of the original activity after 1 month of intermittent use. The operational range, using the "end point" approach, was 1-25 nmol of steroid and the assay speed 10-15 samples/h. Reliable results were obtained in the determination of 3 alpha-hydroxysteroids and 3 beta, 17 beta-hydroxysteroids in urine and total bile acids in serum.