Title: Requirement for ω and (ω–1)-hydroxylations of fatty acids by human cytochromes P450 2E1 and 4A11
Abstract: 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 ω and (ω–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 ω and (ω–1)-hydroxylated with an efficiency at least similar to palmitic acid. The (ω–1)/ω 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 ω-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 ω and (ω–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 ω and (ω–1)-hydroxylations of fatty acids by human cytochromes P450 2E1 and 4A11. J. Lipid Res. 1999. 40: 1990–1997. 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 ω and (ω–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 ω and (ω–1)-hydroxylated with an efficiency at least similar to palmitic acid. The (ω–1)/ω 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 ω-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 ω and (ω–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 ω and (ω–1)-hydroxylations of fatty acids by human cytochromes P450 2E1 and 4A11. J. Lipid Res. 1999. 40: 1990–1997. The ubiquitous cytochrome P450 (P450) enzymes comprise a superfamily of monooxygenases present in both eukaryote and procaryote organisms (1Nelson D.R. Koymans L. Kamataki T. Stegeman J.J. Feyereisen R. Waxman D.J. Watesman M.R. Gotoh O. Coon D.J. Estabrook R.W. Gunsalus I.C. Nebert D.W. P450 superfamily: update on new sequences, gene mapping, accession numbers and nomenclature.Pharmacogenetics. 1996; 6: 1-42Google Scholar). Cytochromes P450 from mammals are involved in the oxidation of a large number of exogenous and endogenous compounds including fatty acids (2Guengerich F.P. Characterization of human cytochrome P-450 enzymes.Annu. Rev. Pharmacol. Toxicol. 1989; 29: 241-264Google Scholar). 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 β-oxidation system is becoming increasingly evident (3Kroetz D.L. Yook P. Costet P. Bianchi P. Pineau T. Peroxisome proliferator-activated receptor alpha controls the hepatic CYP4A induction adaptive response to starvation and diabetes.J. Biol. Chem. 1998; 273: 31581-31589Google Scholar). The peroxisomal β-oxidation system is particularly well suited for β-oxidation of fatty acids which are poor substrates for mitochondrial β-oxidation system (i.e., long-chain fatty acids). Unbalance for fatty acid degradation between a very efficient peroxisomal β-oxidation and the mitochondrial system should result in a rapid accumulation of toxic short- and 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 medium- and 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 (4Guengerich F.P. Kim D.H. Iwasaki M. Role of human cytochrome P450 IIE1 in the oxidation of many low molecular weight cancer suspects.Chem. Res. Toxicol. 1991; 4: 168-179Google Scholar, 5Koop D.P. Tierney D.J. Multiple mechanisms in the regulation of ethanol inductible cytochrome P450IIE1.Bioessays. 1990; 12: 429-435Google Scholar, 6Raucy J.L. Kramer J.C. Lasker J.M. Bioactivation of halogenated hydrocarbons by cytochrome P450 2E1.Crit. Res. Toxicol. 1993; 23: 1-20Google Scholar), but also the hydroxylation of fatty acids. Recently, it was demonstrated that CYP2E1 from rat (7Amet Y. Berthou F. Goasduff T Salaün J.P. le Breton L. Ménez J.F. Evidence that cytochrome P450 2E1 is involved in the (ω–1)-hydroxylation of lauric acid in rat liver microsomes.Biochem. Biophys. Res. Commun. 1994; 203: 1168-1174Google Scholar, 8Clarke S.E. Baldwin S.J. Bloomer J.C. Ayrton A.D. Sozio R.S. Chenery R.J. Lauric acid as a model substrate for the simultaneous determination of cytochrome P450 2E1 and 4A in hepatic microsomes.Chem. Res. Toxicol. 1994; 7: 836-842Google Scholar), rabbit (9Fukuda T. Imai Y. Komori M. Nakamura M. Kusunose E. Satouchi K. Kusunose M. Different mechanisms of regioselection of fatty acid hydroxylation by laurate (omega–1)-hydroxylating P450s, P450 2C2 and P450 2E1.J. Biochem. 1994; 115: 338-344Google Scholar) and human (8Clarke S.E. Baldwin S.J. Bloomer J.C. Ayrton A.D. Sozio R.S. Chenery R.J. Lauric acid as a model substrate for the simultaneous determination of cytochrome P450 2E1 and 4A in hepatic microsomes.Chem. Res. Toxicol. 1994; 7: 836-842Google Scholar, 10Amet Y. Berthou F. Baird S. Dréano Y. Bail J.P. Ménez J.F. Validation of the (ω–1)-hydroxylation of lauric acid as an in vitro substrate probe for human liver CYP2E1.Biochem. Pharmacol. 1995; 50: 1775-1782Google Scholar) livers was involved in the microsomal (ω–1)-hydroxylation of lauric acid and of several other fatty acids. In addition, the hydroxylation of two isomeric C18 unsaturated fatty acids, i.e., oleic (11Adas F. Berthou F. Picart D. Lozac'h P. Beaugé F. Amet Y. Involvement of cytochrome P450 2E1 in the (ω–1)-hydroxylation of oleic acid in human and rat liver microsomes.J. Lipid Res. 1998; 39: 1210-1219Google Scholar) and elaidic (12Adas F. Picart D. Berthou F. Simon B. Amet Y. Liquid chromatography-mass spectrometry and gas chromatography-mass spectrometry of ω and (ω–1)-hydroxylated metabolites of elaidic and oleic acids in human and rat liver microsomes.J. Chromatogr. 1998; 714: 133-144Google Scholar) acids, was reported to be also catalyzed by the microsomal CYP2E1 from rat and human livers. The mammalian CYP4A family encodes several cytochrome P450 isozymes which are involved in the oxidation of the terminal carbon, and to a lesser extent, of the (ω–1)-carbon of both saturated and unsaturated fatty acids (13Simpson A.E.C.M. The cytochrome 4 (CYPA) family.Gen. Pharmacol. 1997; 28: 351-359Google Scholar). Interestingly, several members of the CYP4A subfamily also show the capability to catalyze mainly the ω-hydroxylation of potent chemotactic agents, such as prostaglandins and leukotrienes (14Bains S.K. Gardiner S.M. Mannweiler K. Gillett D. Gibson G.G. Immunochemical study on the contribution of hypolipidaemic-induced cytochrome P-452 to the metabolism of lauric acid and arachidonic acid.Biochem. Pharmacol. 1985; 34: 3221-3229Google Scholar, 15Sharma D.K. Doig M.V. Lewis D.F.W. Gibson G. Role of hepatic and renal cytochrome P450 IVA1 in the metabolism of lipid substrats.Biochem. Pharmacol. 1989; 38: 3621-3629Google Scholar, 16Aoyama T. Hardwick J.P. Imaoka S. Funae Y. Gelboin H.V. Gonzalez F.J. Clofibrate-inductible rat hepatic P450s IVA1 and IVA3 catalyze the ω- and (ω–1)-hydroxylation of fatty acids and the ω-hydroxylation of prostaglandins E1 and F2α.J. Lipid Res. 1990; 31: 1477-1482Google Scholar). CYP4A is induced in rat liver by hypolipidemic agents, such as clofibrate, and other peroxisomal proliferators (17Sharma R. Lake B.G. Foster J. Gibson G.G. Microsomal cytochrome P-452 and peroxisome proliferation by lipidaemic agents in rat liver. A mechanistic interrelationship.Biochem. Pharmacol. 1988; 37: 1193-1201Google Scholar). It has been recently demonstrated that CYP4A11 was the major lauric acid ω-hydroxylase in human liver (18Powell K.C. Wolf I. Lasker J.M. Identification of CYP4A11 as the major lauric acid ω-hydroxylase in human liver microsomes.Arch. Biochem. Biophys. 1996; 335: 219-226Google Scholar), while the ethanol-inducible CYP2E1 was the major catalyst of (ω-1)-hydroxylauric acid in rat and human liver microsomes (7Amet Y. Berthou F. Goasduff T Salaün J.P. le Breton L. Ménez J.F. Evidence that cytochrome P450 2E1 is involved in the (ω–1)-hydroxylation of lauric acid in rat liver microsomes.Biochem. Biophys. Res. Commun. 1994; 203: 1168-1174Google Scholar, 8Clarke S.E. Baldwin S.J. Bloomer J.C. Ayrton A.D. Sozio R.S. Chenery R.J. Lauric acid as a model substrate for the simultaneous determination of cytochrome P450 2E1 and 4A in hepatic microsomes.Chem. Res. Toxicol. 1994; 7: 836-842Google Scholar, 10Amet Y. Berthou F. Baird S. Dréano Y. Bail J.P. Ménez J.F. Validation of the (ω–1)-hydroxylation of lauric acid as an in vitro substrate probe for human liver CYP2E1.Biochem. Pharmacol. 1995; 50: 1775-1782Google Scholar). The role and significance of P450-dependent fatty acid hydroxylases are not well established. In rat and human, the induction of fatty acid hydroxylases by clofibrate and chemically related compounds involves transcriptional gene activation mediated by nuclear peroxisome proliferator-activated receptors (PPARs) (19Dreyer C. Keller H. Mahfoudi A. Laudet V. Krey G. Wahli W. Positive regulation of the peroxisomal beta-oxidation pathway acids through activation of peroxisome proliferator-activated receptors (PPAR).Biol. Cell. 1993; 77: 67-76Google Scholar, 20Keller H. Dreyer C. Medin J. Mahfoudi A. Ozato K. Wahli W. Fatty acids and retinoids control lipids metabolism through activation of peroxisome proliferator-activated receptor-retinoid X receptor heterodimers.Proc. Natl. Acad. Sci. USA. 1993; 90: 2160-2164Google Scholar). It is noteworthy that in mammalian systems, clofibrate selectively induces fatty acid ω-hydroxylases from the CYP4A family (17Sharma R. Lake B.G. Foster J. Gibson G.G. Microsomal cytochrome P-452 and peroxisome proliferation by lipidaemic agents in rat liver. A mechanistic interrelationship.Biochem. Pharmacol. 1988; 37: 1193-1201Google Scholar), while ethanol enhances (ω–1)-hydroxylation of lauric and oleic (11Adas F. Berthou F. Picart D. Lozac'h P. Beaugé F. Amet Y. Involvement of cytochrome P450 2E1 in the (ω–1)-hydroxylation of oleic acid in human and rat liver microsomes.J. Lipid Res. 1998; 39: 1210-1219Google Scholar) acids, mainly involving CYP2E1. The mechanism by which these P450 isoforms select the subterminal or the terminal carbon position for fatty acid hydroxylations remains unknown. It probably originates from substrate binding interactions with the active site of the enzyme. The CYP4A subfamilies are more selective for the terminal primary C–H bonds of the fatty acid, in preference to the more easily hydroxylated secondary C–H bonds at internal position such as (ω–1). Although P450-dependent fatty acid ω-hydroxylation in mammals was once thought to act as a catabolic pathway for eicosanoids, there are several lines of evidence that oxidized metabolites such as epoxy- and hydroxy-arachidonic acids are biologically active compounds (21Falck J.R. Lumin S. Blair I. Dishman E. Martin M.V. Waxman D.J. Guengerich F.P. Capdevila J.H. Cytochrome P-450-dependent oxidation of arachidonic acid to 16-, 17-, and 18-hydroxyeicosatetraenoic acids.J. Biol. Chem. 1990; 265: 10244-10249Google Scholar, 22Capdevila J.H. Falck J.R. Estabrook R.W. Cytochrome P450 and the arachidonate cascade.FASEB J. 1992; 6: 731-736Google Scholar, 23Zeldin D.C. Dubois R.N. Falck J.R. Capdevila J.H. Molecular cloning, expression and characterization of an endogenous human cytochromes P450 arachidonic acid epoxygenase isoform.Arch. Biochem. Biophys. 1995; 322: 76-86Google Scholar). In order to better understand the requirement for the optimal rate of ω and (ω–1)-hydroxylation of fatty acids by P450, three parameters were studied: i) the alkyl chain length, ii) the presence and configuration of double bond in the alkyl chain, and iii) the role of the carboxylic function. Human liver microsomes and recombinant human P450 cells were used as enzyme source. Lauric, myristic, palmitic, stearic, oleic, elaidic and linoleic acids were purchased from Fluka (Buchs, Switzerland), while radiolabeled [1-14C]lauric (58 mCi/mmol), [1-14C]myristic (50 mCi/mmol), [1-14C]palmitic (55 mCi/mmol), [1-14C]stearic (57 mCi/mmol), [1-14C]oleic (55 mCi/mmol), and [1-14C]linoleic acids (55 mCi/mmol) were from Amersham (Amersham, UK). NADPH and 1-methyl-3-nitro-1-nitrosoguanidine (MNNG) were purchased from Fluka-Sigma (St. Quentin Fallavier). Modified cell microsomes containing human P450s were obtained from Gentest (Woburn, MA), while cytochrome b5 was from Oxford Biomedical Research (Oxford, MI). All chemicals and solvents were of the highest purity obtainable and were from Merck (Darmstadt, Germany) or Fluka-Sigma. Human liver samples were obtained from subjects who died after traffic accidents. In accordance with French law, local ethical committee (CHU, Brest, France) approval was obtained prior to this study. Human liver samples were frozen immediately after removal and the microsomal fraction was prepared according to a previously described method (24Berthou F. Ratanasavanh D. Riché C. Picart D. Voisin T. Guillouzo A. Comparison of caffeine metabolism by slices, microsomes, and hepatocyte cultures from adult human liver.Xenobiotica. 1989; 19: 401-417Google Scholar), and stored at –80°C until use. Microsomal protein content was determined using the Bio-Rad protein assay (Bio-Rad, Munich, Germany) based on the Bradford dye-binding procedure (25Bradford M.M. A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of proteins-dye binding.Anal. Biochem. 1976; 72: 248-254Google Scholar), using bovine serum albumin as standard. Human microsomal contents, in terms of specific P450 and various monooxygenase enzymatic activities, have been reported previously (26Berthou F. Dréano Y. Belloc C. Kangas L. Gautier J.C. Beaune P. Involvement of cytochrome P450 3A enzyme family in the major metabolic pathways of toremifene in human liver microsomes.Biochem. Pharmacol. 1994; 47: 1883-1895Google Scholar). The hydroxylations at ω and (ω–1)-positions of a series of fatty acids were measured by adding microsomal protein (0.3 mg) to a reaction mixture containing substrate (0.1 mm; specific activity 2.5 mCi/mmol), 0.12 m potassium phosphate buffer, pH 7.4, and 5 mm MgCl2. The enzymatic reaction was initiated by the addition of 1 mm NADPH and stopped after 10–30 min by 0.8 mL of a 10% H2SO4 aqueous solution. The metabolites and residual substrate were extracted twice with 5 mL of diethylether. The organic phase was dried under a stream of nitrogen, the residue was then dissolved in 100 μL acetonitrile, and 20 μL were injected for RP-HPLC analysis. Enzyme kinetic parameters were determined by adding a series of fatty acids to the reaction mixture in the range 12.5–200 μm. The ω and (ω–1)-hydroxylated metabolites and residual substrates (lauric, myristic, palmitic, oleic, and linoleic acids) were separated by RP-HPLC using a 5-μm Ultrasphere C18 column 150 × 4.6 mm (Beckman, Gagny, France). The mobile phase (containing 0.2% acetic acid in a mixture of water–acetonitrile) program began isocratically for 30 min at a flow rate of 2 mL/min with mixtures of water–acetonitrile 75:25, 65:35, 60:40, 55:45, 60:40 (v/v) for C12, C14, C16, C18, C18:1, and C18:2, respectively. The isocratic phase was followed by a 5-min linear gradient of 95% acetonitrile in water for 15 min, in order to elute the residual substrate. Radioactivity of RP-HPLC effluents was monitored with a computerized on-line scintillation counter (Flo-One Beta radiometric detector, Packard, Meriden, CT). The rate of radiolabeled metabolites generated was calculated from peak surfaces and expressed as nmol/min per mg of protein. Hydroxylated metabolites were analyzed by HPLC/APCI–mass spectrometry on a Navigator LC/MS mass spectrometer (Thermo Quest, Manchester, UK), equipped with an ionization source at atmospheric pressure running on negative ion mode (11Adas F. Berthou F. Picart D. Lozac'h P. Beaugé F. Amet Y. Involvement of cytochrome P450 2E1 in the (ω–1)-hydroxylation of oleic acid in human and rat liver microsomes.J. Lipid Res. 1998; 39: 1210-1219Google Scholar, 12Adas F. Picart D. Berthou F. Simon B. Amet Y. Liquid chromatography-mass spectrometry and gas chromatography-mass spectrometry of ω and (ω–1)-hydroxylated metabolites of elaidic and oleic acids in human and rat liver microsomes.J. Chromatogr. 1998; 714: 133-144Google Scholar). The RP-HPLC chromatographic conditions were the same as described above. Human P450 isoforms CYP2E1 and CYP4A11 (Gentest) were obtained from human B-lymphoblastoid cell lines transfected separately with human P450 cDNAs. Cytochrome P450 2E1 (Gentest M106k) and 4A11 (Gentest M121a) preparations did not contain cytochrome b5. It was added to the incubation mixture in the molar ratio of 1:2 for P450/b5. The incubation method was the same as described above for human liver microsomes, except for the incubation time which was increased to 60 min. In a first step, diazomethane was prepared in a diethylether phase, following the procedure described by Sigma from 1-methyl-3-nitro-1-nitrosoguanidine (MNNG). Esterification was performed by adding [14C]palmitic acid (0.1 mm, specific activity 2.5 mCi/mmol) to 3 mL of ether containing diazomethane at room temperature for 2–3 h. The control of the complete esterification of the palmitate was checked by HPLC with radiometric detection. Methylated palmitic acid (0.1 mm) was then incubated with either human microsomes (FH3) at 37°C for 15 min, or human recombinant P450s (CYP2E1 and CYP4A11) at 37°C for 60 min. As methylpalmitate was not efficiently soluble in buffer, acetonitrile (1% final concentration) was added in the incubation mixture. Such a concentration of acetonitrile accompanied with a strong mixing allowed a total solubilization of the substrate in the buffer, and furthermore did not inhibit the catalytic activity of cytochromes P450. The RP-HPLC profiles of fatty acids incubated with human liver microsomes showed the presence of two major metabolites with retention times corresponding to (ω–1) and ω-hydroxylated metabolites. Furthermore, negative ion APCI–mass spectrometry analysis of these metabolites confirmed their elemental composition. They were characterized by selected ion monitoring as deprotonated molecule [M–H]+, with ions at m/z 215, 243, 271, 297, and 295 for the hydroxylated metabolites generated from lauric, myristic, palmitic, oleic or elaidic, and linoleic acids, respectively. These results were consistent with the expected mass fragmentation pattern of subterminal and terminal hydroxylated fatty acids. Mass spectra analysis of metabolites also showed that epoxide derivatives from oleic and elaidic acids were not generated to a detectable level by microsomes. Metabolites from linoleic acid were resolved by HPLC/APCI–MS analysis as a complex mixture containing epoxides, diols, and hydroxylated derivatives. Only the 17-hydroxy and 18-hydroxy-octadecadienoic derivatives characterized by their retention times and GC–MS (11Adas F. Berthou F. Picart D. Lozac'h P. Beaugé F. Amet Y. Involvement of cytochrome P450 2E1 in the (ω–1)-hydroxylation of oleic acid in human and rat liver microsomes.J. Lipid Res. 1998; 39: 1210-1219Google Scholar) were studied in this report. Human liver microsomal preparations hydroxylated fatty acids at the ω and (ω–1) positions (Fig. 1). The rate of total metabolites formation decreased when increasing the alkyl chain length from C12 to C16. The formation of both ω and (ω–1)-hydroxylated metabolites decreases from 2.7 ± 0.54 to 0.22 ± 0.04 and from 1.4 ± 0.27 nmol/min per mg to 0.24 ± 0.07 nmol/min per mg, respectively, when increasing the chain length from lauric to palmitic acid. Interestingly, no hydroxylated metabolite was detected when human liver microsomes were incubated with stearic acid. However, unsaturated analogs such as oleic (Z C18:1), elaidic (E C18:1), and linoleic (C18:2) acids were ω and (ω–1)-hydroxylated with an efficiency at least similar to palmitic acid. The (ω–1)-hydroxylation values were 0.20 ± 0.06, 0.60 ± 0.35, and 0.16 ± 0.09 nmol/min per mg, for oleic, elaidic, and linoleic acids, respectively, while the ω-hydroxylation activities were 0.70 ± 0.12, 0.50 ± 0.28, and 0.33 ± 0.03 nmol/min per mg, respectively. Enzyme properties were determined following classic Michaelis-Menten kinetics and are reported in Table 1. Kinetic parameters of fatty acid ω and (ω–1)-hydroxylations in human liver microsomes indicated the involvement of a single enzyme in the range of substrate concentrations used. Accordingly, the low affinity Km of lauric ω-hydroxylation higher than 500 μm (8Clarke S.E. Baldwin S.J. Bloomer J.C. Ayrton A.D. Sozio R.S. Chenery R.J. Lauric acid as a model substrate for the simultaneous determination of cytochrome P450 2E1 and 4A in hepatic microsomes.Chem. Res. Toxicol. 1994; 7: 836-842Google Scholar) was not detected in this range of substrate concentrations. High apparent velocities for (ω–1)-hydroxylated fatty acids were observed for lauric, myristic, oleic, palmitic and linoleic acids. Based on apparent Vmax, values of microsomal (ω–1)-oxidation were in decreasing order lauric > myristic > oleic > elaidic > palmitic > linoleic acids. Similarly, high apparent velocities for ω-hydroxylated fatty acids were observed for oleic, lauric, myristic, linoleic, and palmitic acids. Based on apparent Vmax (ω) values, catalytic efficiency of human liver microsomes was highest for lauric > oleic > linoleic > myristic > palmitic acids. The apparent Km values were in a same order of magnitude for all the fatty acids, but were 6 times higher for myristate when compared to laurate.TABLE 1.Kinetic parameters of fatty acid metabolism by human liver microsomes(ω-1)-Hydroxylated Metaboliteω-Hydroxlyated Metabolite(ω–1)ω RatioFatty AcidVmaxKmVmax/KmVmaxKmVmax/KmVmaxnmol/min·mgμmμL/min·mgnmol/min·mgμmμL/min·mgC12:0 (n = 5)7.2 ± 4.184 ± 4590 ± 302.1 ± 1.213 ± 6.6230 ± 2103.4 ± 0.2C14:0 (n = 2)1.17 ± 0.552.5 ± 17.720 ± 30.6 ± 0.382.5 ± 14.87 ± 32.8 ± 0.3C16:0 (n = 2)0.52 ± 0.3102 ± 13.45 ± 10.51 ± 0.2447 ± 9.98 ± 21 ± 0.2C18:0 (n = 2)NDND—NDND——C18:1 Z (n = 3)0.93 ± 0.0581 ± 3910 ± 22.6 ± 1.746.6 ± 2850 ± 60.35 ± 0.08C18:1 E (n = 1)1.428.549.10.4853.59.02.9C18:2 (n = 1)0.248.54.10.5552.610.50.36The ω and (ω–1)-hydroxylase activities were measured using purified radiolabeled substrates, as described in Materials and Methods section. The ratio Vmax/Km expressed as μL/min·mg represent the intrinsic clearance; ND, not detectable. Open table in a new tab The ω and (ω–1)-hydroxylase activities were measured using purified radiolabeled substrates, as described in Materials and Methods section. The ratio Vmax/Km expressed as μL/min·mg represent the intrinsic clearance; ND, not detectable. The intrinsic clearance Vmax/Km (μL/min·mg protein) allowing us to predict first-pass elimination in the liver showed a high ω-hydroxylation of lauric acid. Furthermore, the change of configuration of the double bond from cis (Z) (oleic acid) to trans (E) (elaidic acid) increased the intrinsic clearance of monounsaturated C18 fatty acid 5-fold. The Vmax (ω–1)/ω ratio decreased from 3.4 to 1 with lauric (C12), myristic (C14), and palmitic (C16) acids as substrates. Interestingly, the reverse was observed for unsaturated C18 fatty acids which are mainly ω-hydroxylated, except for elaidic acid which shows a metabolite profile more similar to those of saturated fatty acids. Saturated (from C12 to C18), and unsaturated (C18:1 and C18:2) fatty acids were incubated in the presence of genetically engineered human P450 2E1 and 4A11 at 37°C for 60 min. Figure 2A shows that the CYP2E1 enzyme was able of hydroxylating saturated and unsaturated fatty acids at the subterminal (ω–1) position, while CYP4A11 (Fig. 2B) shows mainly hydroxylated fatty acids at the terminal ω position, and to a lesser extent at the (ω–1) position. The turnover numbers of heterologously expressed CYP2E1 for (ω–1)-hydroxylation decreased from 3.6 to 0.13 min-1 when the alkyl chain length increased from lauric to stearic acids. The turnover of oleic and linoleic acids was 0.5 min-1. These values increased when cytochrome b5 was added in the incubation medium. Concerning the ω-hydroxylation of fatty acids, the turnover numbers of heterologously expressed CYP4A11 decreased from 7.2 to 0.85 min-1 when the alkyl chain length increased from lauric to palmitic acids. Although stearic acid was not a substrate of CYP4A11, the turover values for unsaturated C18 fatty acids (1.6 and 1.2 min-1 for oleic and linoleic acids, respectively) were higher than for palmitic acid. These values increased by 1.65 ± 0.17-fold when cytochrome b5 was added in the incubation medium. The CYP4A11 enzyme slightly metabolized lauric and myristic acids at the (ω–1) positions, with turnover values of 0.56 and 0.6 min-1, respectively. Methylated palmitic acid was incubated with human liver microsomes (FH3) or genetically engineered human P450s (2E1 and 4A11) in order to study the role of the free carboxylic group in the substrate access channel of the cytochrome P450 active site. As shown in Fig. 3B, no hydroxylated metabolite was detected when incubating methylated palmitic acid with human liver microsomes. Figure 3B shows only two peaks, corresponding to palmitic acid (peak 3) resulting from hydrolysis of ester group by esterases largely present in microsomal preparations, and the residual methylated palmitic acid (peak 4). In the same way, the incubation of methylester palmitate with genetically engineered cells P450 2E1 and 4A11 did not allow the production of hydroxylated metabolites (data not shown). The interplay of multiple enzymes involved in the oxidative metabolism of fatty acids makes difficult the understanding of the physiological role and effects of individual metabolites generated during the oxidative cascade of saturated and unsaturated fatty acids. The liver is mainly involved in detoxification processes because it contains several inductible P450 isoforms which are able to oxidize a large number of very lipophilic endogenous and exogenous compounds. The catalytic capability and the substrate specificity of individual P450 capable of oxidizing fatty acids might provide information as to their role in the catabolism of fatty acids, and in the synthesis of bioactive oxidized molecules. In this study, two P450 isoforms have been investigated in the hydroxylations of fatty acids, namely CYP2E1 and CYP4A that are shown to be highly regioselective for catalyzing (ω–1) and ω-hydroxylations, respectively. Two different criteria determine the preferred site of hydroxylation of fatty acids. One is thermodynamic, i.e., the ease of hydrogen atom abstraction by the ferryl oxygen complex of P450 that reflects the strength of the C–H bond. The other criterion is steric, i.e., the proximity of the H atom to the ferryl oxygen. The thermodynamic considerations favor oxidation of in-chain carbons over oxidation at the terminam methyl. Both experimental (this study) and computational studies (27Chang Y-T. Loew G.H. Homology modeling and substrate binding study of human CY