Studies on cholesterol 7alpha-hydroxylase.

The conversion of cholesterol into bile acids in the liver forms a major pathway of cholesterol catabolism and elimination in mammals. The main rate-limiting enzyme of this pathway, cholesterol 7alpha-hydroxylase, is a microsomal enzyme which displays some of the characteristics of a cytochrome P-450-dependent mixed-function oxidase. It can be hypothesised that a low activity of this hydroxylase may be causal in producing hypercholesteraemia and the associated occurrence of coronary heart disease. In the present study this hypothesis was tested using two animal models of hypercholesteraemia - the STR/lN mouse, and the vitamin D and cholesterol-fed rat. In both cases no consistent evidence for this proposal could be established. The nature of cholesterol 7alpha-hydroxylase was examined in rat liver microsomal preparations where typical mixed-function oxidase requirements for NADPH and atmospheric oxygen were demonstrated; evidence for a cytochrome P-450 dependency was derived from carbon monoxide inhibition and the additive effect on enzyme activity produced by NADPH and NADH in combination. Pretreatment of rats with various cytochrome P-450-dependent monooxygenase inducers such as phenobarbitone and 3-methylcholanthrene did not increase cholesterol 7alpha-hydroxylase activity, although administration of compounds such as SKF-525A, cholesterol and cholestyramine were associated with increases in both cholesterol 7alpha-hydroxylase activity and microsomal cytochrome P-450 content. Compounds such as a-naphthoflavone and SKF-525A were found to inhibit both cholesterol 7alpha-hydroxylation and biphenyl 4-hydroxylation in microsomal preparations. A cytochrome P-450 fraction and highly purified NADPH-cytochrome c (P-450) reductase, isolated from liver microsomal preparations of cholestyramine-pretreated rats, were used to reconstitute cholesterol 7alpha-hydroxylase activity in the presence of phospholipid. Cholesterol 7alpha-hydroxylase in the reconstituted system showed an absolute requirement for cytochrome P-450 and NADPH-cytochrome c reductase, and a partial requirement for phospholipid. It is concluded that cholesterol 7alpha-hydroxylase is a cytochrome P-450-dependent mixed-function oxidase