Synthesis of 27-hydroxycholesterol in rat liver mitochondria: HPLC assay and marked activation by exogenous cholesterol.

Abstract:

: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.

journal_name

J Lipid Res

authors

Petrack B,Latario BJ

subject

Has Abstract

pub_date

1993-04-01 00:00:00

pages

643-9

issue

4

eissn

0022-2275

issn

1539-7262

journal_volume

34

pub_type

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