Resolvin D4 biosynthetic evidence by an epoxide hydrolase and identification in human brain

Resolvins play pivotal roles in inflammation resolution and tissue homeostasis. These active compounds are formed by enzymatic oxygenation and further transformation of omega-3 polyunsaturated fatty acids in mammalian cells. Here, we report analytical data for the identification of resolvin D4 (4S,5R,17S-trihydroxy-6E,8E,10Z,13Z,15E,19Z-docosahexaenoic acid) in human brain tissues and confirm the involvement of the soluble epoxide hydrolase (sEH) in the biosynthesis of this potent bioactive product. Using synthetic 4,5(S,S) epoxide intermediate (4S,5S-oxido-17S-hydroxy-6E,8E,10Z,13Z,15E,19Z-docosahexaenoic acid) prepared by total chemical synthesis, UV, and liquid chromatography–tandem mass spectrometry (LC-MS/MS)-based analyses including linear ion trap MS ³ fragmentation, our results demonstrated that this allylic epoxide is a substrate for human recombinant sEH. Incubation of the epoxide with sEH in H ₂ ¹⁸O-enriched buffer led to incorporation of ¹⁸O into resolvin D4 at C-5 position, as judged by MS ³ ion trap experiments. These results give direct evidence for the role of the 4,5(S,S) epoxide in the biosynthesis of resolvin D4 by epoxide hydrolases such as sEH.