Nitrolipids are a new class of signal molecules, whose biological effects have not been analysed in detail to-date, but which seem to possess anti-inflammatory properties. As several of the nitro-lipids identified in vivo are polyunsaturated fatty acids (PUFA) and PUFAs are excellent substrates for vascular cytochrome P450 (CYP) enzymes we speculated that nitro-lipids are CYP substrates and that CYP derivatives generated from nitro-lipids are bioactive and involved in cell signalling.

The nitrolipids (9-, 10-nitrooleate and nitrolinoleate) could be converted to distinct products; w/w-1-hydroxy-nitrooleate, w-hydroxy-nitrolinoleate and unidentified epoxy-derivates, by mouse liver microsomes and the products were analysed by HPLC and LC-MS/MS. The formation of the products was NADPH-dependent and could be inhibited by the CYP inhibitor miconazole, but not by the cyclooxygenase inhibitor diclofenac. Furthermore, we could generate distinct products using different human vascular CYP enzymes including; CYP2C9, CYP2J2, CYP4F2 and CYP4A11. The turnover rates ranged from 2 to 16% of the substrate and MS/MS-analysis clearly identified the products as hydroxy- and epoxy-derivates of nitrooleate and nitrolinoleate. While nitrolinoleate was able to elicit protein tyrosine phosphorylation in human umbilical vein endothelial cells and attenuate thrombin-induced platelet aggregation, w-hydroxy-nitrooleate was able to activate peroxisome proliferator-activated receptor (PPARg) in the nano molar range, as assessed in a PPRE/PPARg reporter gene assay. Taken together, these data indicate that nitro-lipids are excellent substrates for CYP hydroxylases. It is therefore tempting to speculate that the resulting CYP-derived nitro-metabolites represent a novel class of cellular mediators.