Stereoselective epoxidation of the last double bond of polyunsaturated fatty acids by human cytochromes P450

Abstract

Cytochromes P450 (CYPs) metabolize polyunsaturated long-chain fatty acids (PUFA-LC) to several classes of oxygenated metabolites. Through use of human recombinant CYPs, we recently showed that CYP1A1, -2C19, -2D6, -2E1, and -3A4 are mainly hydroxylases, whereas CYP1A2, -2C8, -2C9, and -2J2 are mainly epoxygenases of arachidonic acid (AA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA), respectively. It is worth noting that the last double bond of these PUFAs, i.e., omega6 in AA or omega3 in EPA and DHA, respectively, was preferentially epoxidized. In this study, we have characterized the stereoselectivity of this epoxidation reaction by comparison with the PUFA-LC epoxide stereoisomers obtained from the enantioselective bacterial CYP102A1 F87V. The stereoselectivity of the epoxidation of the last olefin of AA (omega6), EPA (omega3), or DHA (omega3) differed between the CYP isoforms but was similar for EPA and DHA. These data give additional insight into the PUFA-LC epoxide enantiomers generated by the hepatic CYPs.

Fig. 1.

DHA metabolism by CYP102A1 F87V. 1A: RP-HPLC Chromatographic resolution of the products generated by CYP102A1 F87V during the metabolism of DHA; Peak 1: 19,20-EDP; Peak 2: 16,17-EDP. A total of 100 µM of [1-¹⁴C] DHA was incubated with10 µg of crude extract CYP102A1 F87V. The HPLC analysis was carried out as described in Experimental Procedures. 1B: RP-HPLC chromatographic resolution of the products generated by chemical epoxidation of DHA; Peak 1: 19,20-EDP; Peak 2: 16,17-EDP; Peak 3: 13,14-EDP; Peak 4: 10,11-EDP; Peak 5: 7,8-EDP. These peaks were previously identified by LC-MS (9). 1C: LC-MS of 16,17- and 19,20-EDP generated by incubation of DHA with CYP102A1 F87V as described in Experimental Procedures. Only significant fragments of the epoxide position in the alkyl chain were indicated.

Fig. 2.

Stereochemistry of epoxidation of the last double bond of AA and EPA by CYPs. Results are expressed as percentages of the two enantiomers generated by incubation of AA (A) and EPA (B) with CYP102A1 F87V and human recombinant CYPs. Enantiomers were identified by comparison with the S,R enantiomer generated by CYP102A1 F87V. * CYP2J2 data were previously reported (42).

Fig. 3.

HPLC chiral analysis of 19,20 EDP. Representative HPLC chiral analysis illustrating the stereoselectivity of epoxidation of CYP102A1 F87V and CYP1A2 during the 19,20-double bond epoxidation of DHA versus the racemisation of this olefin by chemical epoxidation.

Fig. 4.

Stereochemistry of epoxidation of the last double bond of DHA by CYPs. Results are expressed as percentages of the two enantiomers generated by incubation of DHA with CYP102A1 F87V and human recombinant CYPs. Enantiomers were identified by comparison with the S,R enantiomer generated by CYP102A1 F87V.