Simultaneous measurement of cholesterol 7 alpha-hydroxylase activity by reverse-phase high-performance liquid chromatography using both endogenous and exogenous [4-14C]cholesterol as substrate.

The HPLC-spectrophotometric method (T. Ogishima and K. Okuda (1986) Anal. Biochem. 158, 228-232) for measuring cholesterol 7 alpha-hydroxylase activity was modified by using a C-18 reverse-phase column to separate 7 alpha-hydroxy-4-cholesten-3-one and 4-cholesten-3-one and by adding 7 beta-hydroxycholesterol to each reaction mixture as an internal recovery standard. With this method, we were able to simultaneously measure cholesterol 7 alpha-hydroxylase activity using endogenous cholesterol and exogenous [4-14C]cholesterol as substrate. Rat liver cytosol differentially stimulated (286%) the 7 alpha-hydroxylation of exogenous [4-14C]-cholesterol. In contrast, total cholesterol 7 alpha-hydroxylase activity was stimulated only 35% by cytosol. This method should prove useful for studying mechanisms of cholesterol delivery to cholesterol 7 alpha-hydroxylase.