Fig. 4. Authentication of oxysterol 7α-hydroxylase product.

Cholest-5-ene-3β,7α,25-triol, an anticipated product of the oxysterol 7α-hydroxylase enzyme activity in mouse liver, was chemically synthesized as described under “Experimental Procedures.” In lane 1, an aliquot of this compound was subjected to thin-layer chromatography in a solvent system containing toluene/ethyl acetate (2:3), and the lane was stained with phosphomolybdic acid (PMA). In lane 2, an aliquot of the authentic standard was mixed with the sterol products arising from incubation of mouse liver microsomes with [³H]cholest-5-ene-3β,25-diol (25-hydroxycholesterol), and the mixture was subjected to thin-layer chromatography and staining. In addition to steroids, dithiothreitol and Triton X-100, two components of the enzyme assay, were also stained by phosphomolybdic acid. In lane 3, an autoradiogram of lane 2 is presented. Comparison of the results obtained by autoradiography (lane 3) with those obtained by staining (lanes 1 and 2) reveals comigration of the authentic standard with the radiolabeled product of the enzyme.