An improved assay method for cholesterol 7α-hydroxylase which is accurate, sensitive and yet still simple is described. The method consists of two parts: the first part is cholesterol 7α-hydroxylation in liver microsomes utilizing cholesterol in situ as the substrate, and the second part is conversion of the product, 7α-hydroxycholesterol, into 7α-hydroxy-4-cholesten-3-one having an intense absorption at 240 nm by the action of cholesterol oxidase. The converted sterol is then analyzed by high-performance liquid chromatography. During the second enzyme reaction, the first enzyme reaction is halted and further metabolism of the product is prevented. In consequence, the method had more than 10-fold increase in the sensitivity than the previous one.
All Science Journal Classification (ASJC) codes
- Molecular Biology
- Cell Biology