Arachidonic acid is oxidatively metabolized by rat liver microsomes at a rate of approximately 5 nmol per min per mg of protein at 25C. This reaction is dependent on the presence of NADPH and oxygen. Studies with various inhibitors indicate a role for membrane-bound cytochrome P-450 in the transformation of arachidonic acid to a mixture of hydroxy acid derivatives. The stoichiometry of the reaction conforms to that of a monooxygenase reaction-i.e., one mole of NADPH is oxidized per mole of oxygen utilized-suggesting a reaction mechanism different from that proposed for lipid peroxidation reactions. No evidence for the formation of prostaglandin-like metabolites was obtained. The diene character of some of the metabolites formed suggests another role for cytochrome P-450-i.e., participation in hydrogen abstraction reactions for the activation of various substrates.The oxidative metabolism ofarachidonic acid can lead to a wide variety of metabolites. Interest in the physiological function of prostaglandins, thromboxanes, leukotrienes, and other oxidative metabolites of arachidonic acid, as well as the reactions of lipid peroxidation, have established the need to better understand the role of the various enzymes responsible for oxygen incorporation during the process of arachidonic acid metabolism-e.g., cyclooxygenase, lipoxygenase, etc. (1, 2).The microsomal fraction from rat liver, like that from many other tissues, is rich in phospholipids containing arachidonic acid (3). During the NADPH-and oxygen-dependent function of the liver microsomal cytochrome P-450-containing electron transport system, a significant portion of electrons is diverted to either the formation of hydrogen peroxide or the oxidative transformation of "endogenous substrates" (4, 5). The latter reaction conforms to the stoichiometry of a monooxygenase reaction in that one mole of NADPH is oxidized for each mole of oxygen consumed. The nature of the endogenous substrate(s) of liver microsomes has eluded characterization, although Schenkman et al. (6) have provided evidence that unsaturated fatty acids, in particular oleic acid, might serve in this capacity. In addition, it has been reported that unsaturated fatty acids appear to be released from liver microsomal phospholipids during the course of NADPH oxidation (7,8). In the presence of an iron chelate, such as the ADP-Fe3+ complex, and an electron donor, such as NADPH or ascorbate, malonaldehyde is rapidly formed concomitant with a rapid rate of oxygen utilization (9). It is generally assumed that one source of malonaldehyde is from the oxidation of arachidonic acid (1, 10). Thus, it was of interest to evaluate the oxidative metabolism ofarachidonic acid by the electron transport system associated with rat liver microsomes.It is the purpose of this communication to describe the role of liver microsomal cytochrome P-450 in the oxidation of arachidonic acid. It is suggested that free arachidonic acid, as well as that derived from microsomal phospholipids, may contribute, in part, to the...

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Arachidonic acid is oxidized by a NADPH-dependent oxygenase of rat liver microsomes to a number ofoxygencontaining products, which can be resolved by HPLC. Several of these products have been purified and characterized. They exhibit an absorbance in the UV region of the spectrum that has a maximum at =235 nm, indicative ofthe presence ofa conjugated diene function. Mass spectral analysis of the trimethylsilyl ether derivatives of the methyl esters of the hydrogenated and nonhydrogenated metabolites shows that they are the 9-, 11-, 12-, and 15-monohydroxy derivatives of arachidonic acid, the hydroxyicosatetraenoic acids (HETEs). Their UV absorbance and chromatographic properties suggest that these products possess cis,transdiene geometry characteristic ofHETEs isolated from other mammalian sources. The isolation of these isomeric HETEs suggests that cytochrome P-450 may play a role in the oxidative metabolism of arachidonic acid to physiologically and pharmacologically important hydroxylated unsaturated fatty acids.The oxygenation of arachidonic acid to hydroxyicosatetraenoic acids (HETEs), which contain a conjugated diene function, is catalyzed by enzyme(s) present in several mammalian tissues and has important pathophysiological consequences (1, 2). The isolation of each of the isomeric HETEs has been reported, although significant tissue variation in the composition of the products formed has been observed (3-6). HETEs and their hydroperoxy acid precursors (HPETEs) exhibit diverse biological activities and may play an important role in leukocyte chemotaxis and the inflammatory response (1).HETEs are produced by an oxygenation reaction involving either lipoxygenases (3), singlet molecular oxygen (7), or autooxidation (8). Animal lipoxygenases have not been extensively purified and the nature and properties of this class of mammalian enzymes have scarcely been characterized in most tissues that biosynthesize HETEs. We have recently reported a novel route for the oxidative metabolism ofarachidonic acid (9). The reaction is catalyzed by liver microsomes and requires NADPH and molecular oxygen. The reaction is inhibited by carbon monoxide and metyrapone, suggesting a role for microsomal cytochrome P450 (9). The metabolism ofarachidonic acid can also be demonstrated in reconstitution experiments using purified cytochromes P450 and NADPH-cytochrome P-450 reductase (10).The present paper describes studies to characterize some of the metabolites formed during the rat liver microsomal cytochrome P450-dependent oxidation of arachidonic acid. We report here that the major components of that group of products are the 9-, 11-, 12-, and 15-hydroxy derivatives of arachidonic acid-the HETEs. MATERIALS AND METHODSMicrosomal fractions were prepared from homogenates of rat livers as described (11). Male Sprague-Dawley rats (150-200 g body weight) were treated by four daily intraperitoneal injections ofphenobarbital (75 mg/kg ofbody weight) and starved overnight prior to sacrifice.The oxygenation products of arachidonic m...