Isotope derivative assay of microsomal cholesterol 7 alpha-hydroxylase.
A rapid method was developed to measure cholesterol 7 alpha-hydroxylase activity of hepatic microsomes by the direct determination of the mass of 7 alpha-hydroxycholesterol formed. The method is based on the quantitative acetylation of the incubation mixture with [-3H]acetic anhydride and the separation of the biosynthetic 7 alpha-hydroxycholesterol as its diacetate by thin-layer chromatography on alumina. Amounts of 7 alpha-hydroxycholesterol as low as 0.1 nmole could be measured. A comparison of the proposed isotope derivative method with the previously used isotope incorporation method showed that the latter underestimated the enzyme activity by about 20 percent.
D000107:Acetylation; D000818:Animals; D002250:Carbon Radioisotopes; D002790:Cholesterol 7-alpha-Hydroxylase / Q000032:analysis*; D002855:Chromatography, Thin Layer; D005069:Evaluation Studies as Topic; D008297:Male; D008722:Methods; D008862:Microsomes, Liver / Q000201:enzymology*; D051381:Rats; D013250:Steroid Hydroxylases / Q000032:analysis*; D014316:Tritium
D002250:Carbon Radioisotopes; D014316:Tritium; D013250:Steroid Hydroxylases; D002790:Cholesterol 7-alpha-Hydroxylase