Requirement for v and ( v – 1 ) - hydroxylations of fatty acids by human cytochromes P 450 2 E 1 and 4 A 11

Human liver microsomes and recombinant human P450 have been used as enzyme source in order to better understand the requirement for the optimal rate of v and ( v –1)-hydroxylations of fatty acids by cytochromes P450 2E1 and 4A. Three parameters were studied: alkyl chain length, presence and configuration of double bond(s) in the alkyl chain, and involvement of carboxylic function in the fatty acid binding inside the access channel of P450 active site. The total rate of metabolite formation decreased when increasing the alkyl chain length of saturated fatty acids (from C12 to C16), while no hydroxylated metabolite was detected when liver microsomes were incubated with stearic acid. However, unsaturated fatty acids, such as oleic, elaidic and linoleic acids, were v and ( v –1)-hydroxylated with an efficiency at least similar to palmitic acid. The ( v –1)/ v ratio decreased from 2.8 to 1 with lauric, myristic and palmitic acids as substrates, while the reverse was observed for unsaturated C18 fatty acids which are mainly v -hydroxylated, except for elaidic acid showing a metabolic profile quite similar to those of saturated fatty acids. The double bond configuration did not significantly modify the ability of hydroxylation of fatty acid, while the negatively charged carboxylic group allowed a configuration energetically favourable for v and ( v –1)-hydroxylation inside the access channel of active site. —Adas, F., J.P. Salaün, F. Berthou, D. Picart, B. Simon, and Y. Amet. Requirement for v and ( v – 1)-hydroxylations of fatty acids by human cytochromes P450 2E1 and 4A11. J. Lipid Res. 1999. 40: 1990–1997. Supplementary key words CYP4A11 • CYP2E1 • v and ( v –1)-hydroxylation • cytochrome b5 • human liver microsomes The ubiquitous cytochrome P450 (P450) enzymes comprise a superfamily of monooxygenases present in both eukaryote and procaryote organisms (1). Cytochromes P450 from mammals are involved in the oxidation of a large number of exogenous and endogenous compounds including fatty acids (2). The physiological role of cytochromes P450 catalyzing the hydroxylation of fatty acids remains to be clarified. The CYP4A family appears to be mainly involved in oxidation of fatty acids and derivatives. The physiological role of certain bioactive metabolites generated from eicosanoids by this P450 family is now increasingly documented, and several data show their involvement in various cell functions. In addition, a significant regulatory role of CYP4A induction in the overall balance of fatty acid degradation by b -oxidation system is becoming increasingly evident (3). The peroxisomal b oxidation system is particularly well suited for b -oxidation of fatty acids which are poor substrates for mitochondrial b -oxidation system (i.e., long-chain fatty acids). Unbalance for fatty acid degradation between a very efficient peroxisomal b -oxidation and the mitochondrial system should result in a rapid accumulation of toxic shortand medium-chain free fatty acids (i.e., lauric acid) generated by peroxisomes. Consequently, induction of members of CYP2E and CYP4A families should be associated with a detoxification process needed to reduce accumulation by cells of free fatty acids with the goal to maintain the membrane integrity. Study of the substrate specificity and the structure of metabolites generated by the major catalysts of fatty acid oxidation that belong to the CYP2E and CYP4A families are essential to demonstrate the individual role of P450s in the catabolic process. Several P450 isozymes are effective catalysts of hydroxylation of mediumand long-chain saturated and unsaturated fatty acids, although they show very different substrate selectivity and regiospecificity of the oxygene attack. The ethanol-inducible CYP2E1 isoform catalyzes not only the bioactivation of a large number of lipophilic compounds with low molecular weight, including aromatic and halogenated hydrocarbons, alcohols, ketones and nitrosamines (4–6), but also the hydroxylation of fatty acids. Recently, it was demonstrated that CYP2E1 from rat (7, 8), rabbit (9) and human (8, 10) livers was involved in the microsomal ( v –1)-hydroxyAbbreviations: P450, cytochrome P450 (E.C. 1.14.14.1) or CYP; RPHPLC, reverse phase-high performance liquid chromatography; APCI, atmospheric pressure chemical ionization; LC–MS, liquid chromatography– mass spectrometry; MNNG, 1-methyl-3-nitro-1-nitrosoganidine; PPAR, peroxisome proliferator-activated receptor. 1 To whom correspondence should be addressed. by gest, on S etem er 8, 2017 w w w .j.org D ow nladed fom