IMMUNOCHEMICAL STUDY ON THE CONTRIBUTION OF HYPOLIPIDEMIC-INDUCED CYTOCHROME-P-452 TO THE METABOLISM OF LAURIC ACID AND ARACHIDONIC-ACID

The influence of four hypolipidaemic drugs (clofibrate, WY-14,634, clobuzarit and benzafibrate) on hepatic cytochrome P-450 and fatty acid metaboism in male rate liver microsomes has been investigated. All of the hypolipidaemic drugs tested significantly induced the hydroxylation of lauric acid and, furthermore, this was accompanied by a concomitant 3-fold induction of a specific isoenzyme of cytochrome P-450 (termed cytochrome P-452) as determined by a single radial immuunodiffusion technique. In addition, immunochemical quantitation of cytochrome P-452 in control, uninduced rat liver microsomes revealed that this particular isoenzyme constituted 22% of the total carbon monoxide-discernible cytochrome P-450 population. This has led us to conclusion that cytochrome P-452 is a constitutive cytochrome P-450 isoenzyme and therefore that hypolipidaenic agents function as inducers of constitutive haemoprotein isoenzymes. Cytochrome P-452 plays a significant role in the hydroxylation of lauric acid as evidenced by inhibition of hydroxylase activity in the presence of an anti-P-452 IgG fraction. In addition, this antibody preferentially inhibits the 12-hydroxylation of lauric acid in rat liver microsomes by comparison to the 11-hydroxylase activity. Our studies have also shown that arachidonic acid serves as an excellent substrate for hypolipidaemic-induced cytochrome P-452, resulting in the formation of several metabolites that have been separated by reverse phase HPLC. Furthermore, a specific metabolite (or group of metabolites) of arachidonic acid is induced by clofibrate pretreatment and that the formation of this metabolite(s) is inhibited by an antibody of cytochrome P-452. By comparison, other metabolites of arachidonic acid remain refractory to induction by clofibrate and are not inhibited by the presence of anti-P-452 IgG. In addition, a reconstituted enzyme system containing highly purified cytochrome P-452 actively catalyses the above specific oxidation of arachidonic acid, a reaction that is significantly stimulated by the presence of cytochrome b5. Taken collectively, our data provide compelling evidence that hypolipidaemic agents induce a specific isoenzyme of hepatic microsomal P-450 that readily oxidizes fatty acids and that arachidonic acid may serve as an excellent endogenous substrate for this novel haemoprotein.