Synthesis of 27-hydroxycholesterol in rat liver mitochondria: HPLC assay and marked activation by exogenous cholesterol.
Sterol 27-hydroxylase, the mitochondrial enzyme that catalyzes the first step in oxidation of the sterol side chain in hepatic bile acid synthesis, also catalyzes the synthesis of 27-hydroxycholesterol from cholesterol. We have developed a high performance liquid chromatography (HPLC) assay for this enzyme, using either endogenous or exogenous cholesterol as substrate and cholesterol oxidase to convert 27-hydroxycholesterol to 4-cholesten-27-hydroxy-3-one. The alpha,beta-unsaturated ketone product was separated by normal phase HPLC and quantitated via absorption at 240 nm. Addition of cholesterol dissolved in 2-hydroxypropyl-beta-cyclodextrin to the assay mixture raised the enzyme activity of rat liver mitochondria more than 10-fold. 2-Hydroxypropyl-beta-cyclodextrin itself was partially effective, apparently by making more endogenous cholesterol accessible to the enzyme. Availability of cholesterol to the enzyme limits synthesis of 27-hydroxycholesterol in rat liver. Using our assay to simultaneously determine the activities of cholesterol 7 alpha-hydroxylase and cholesterol 27-hydroxylase in rat liver homogenates, we demonstrated that the two enzymes are separately regulated.
Petrack
B
B
Research Department, CIBA-GEIGY Corporation, Summit, NJ 07901.
Latario
B J
BJ
D000818:Animals; D002784:Cholesterol / Q000378:metabolism / Q000494:pharmacology; D002851:Chromatography, High Pressure Liquid; D004789:Enzyme Activation; D006888:Hydroxycholesterols / Q000378:metabolism*; D008297:Male; D008930:Mitochondria, Liver / Q000201:enzymology / Q000378:metabolism*; D006899:Mixed Function Oxygenases / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley
D006888:Hydroxycholesterols; C076996:27-hydroxycholesterol; D002784:Cholesterol; D006899:Mixed Function Oxygenases