An improved method for assay of cholesterol 7 alpha-hydroxylase activity.
An improved assay method for cholesterol 7 alpha-hydroxylase which is accurate, sensitive, and yet still simple is described. The method consists of two parts: the first part is cholesterol 7 alpha-hydroxylation in liver microsomes utilizing cholesterol in situ as the substrate, and the second part is conversion of the product, 7 alpha-hydroxycholesterol, into 7 alpha-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.
D000818:Animals; D002783:Cholestenones / Q000032:analysis; D002790:Cholesterol 7-alpha-Hydroxylase / Q000032:analysis*; D002789:Cholesterol Oxidase; D002851:Chromatography, High Pressure Liquid; D008297:Male; D008862:Microsomes, Liver / Q000201:enzymology; D051381:Rats; D013056:Spectrophotometry, Ultraviolet; D013250:Steroid Hydroxylases / Q000032:analysis*
D002783:Cholestenones; C002656:7 alpha-hydroxy-4-cholesten-3-one; D002789:Cholesterol Oxidase; D013250:Steroid Hydroxylases; D002790:Cholesterol 7-alpha-Hydroxylase