Investigation of rat liver microsomal 6 beta-hydroxylation of 4-androstene-3,17-dione and 4-pregnene-3,20-dione using methodology which excludes steroid-3-imine induced introduction of the 6 beta-hydroxyl group.

To avoid artefactual 6 beta-hydroxylation of 3-oxo-4-ene steroids due to steroid-3-imine formation and rearrangement a combined extraction and liquid chromatography purification procedure for incubated rat liver microsomes has been worked out. With this procedure no nonenzymatic 6 beta-hydroxylation could be observed. Conventional termination of incubations with male rat liver microsomes (105,000 g sediments) and 4-14 C-labelled 4-androstene-3,17-dione (or progesterone) by solvent extraction and evaporation might lead to a 30% overestimation of the formation of 6 beta-hydroxy-derivatives at substrate saturation. Furthermore this work-up procedure produces 6-oxo-derivatives which were not observed when the new procedure was used. By elimination of the artefactual 6-oxygenation some properties of the male rat liver microsomal 4-androstene-3,17-dione 6 beta-hydroxylase have been studied, and the activity has been compared to the artefact produced by solvent extraction and evaporation. Using the combined extraction-liquid chromatography purification it was demonstrated that the microsomal 6 beta-hydroxylase active on 4-androstene-3,17-dione and progesterone was inhibited to 95% by carbon monoxide. This makes previous suggestions regarding participation of a non cytochrome P450-dependent 4-androstene-3,17-dione 6 beta-hydroxylase less likely.