Substrate-dependent incorporation of 15-lipoxygenase products in glycerophospholipids: 15-HETE and 15-HEPE in PI, 17-HDHA in plasmalogen PE, and 13-HODE in PC.

Several oxylipins including hydroxy-PUFAs act as lipid mediators. In biological samples, the major part occurs esterified in glycero-phospholipids (PLs) or other lipids. In this work, the incorporation into glycero-PLs of 15-hydroxyeicosatetraenoic acid (15(S)-HETE), 15(S)-hydroxyeicosapentaenoic acid (15(S)-HEPE), 17(S)-hydroxydocosahexaenoic acid (17(S)-HDHA), and 13-(S)-hydroxyoctadecadienoic acid (13(S)-HODE) was investigated in oxylipin-supplemented human embryonic kidney 293T cells and cells overexpressing 15-lipoxgenase-2 (15-LOX-2, ALOX15B). Indirect quantification of esterified oxylipins in lipid fractions showed that >97% of each supplemented 15-LOX-2 product is esterified and that <25% are bound to neutral lipids, whereas >75% are bound to distinct glycero-PL classes, depending on the hydroxy-PUFA. 15-HETE and 15-HEPE were found in phosphatidylinositol (PI)/phosphatidylserine, whereas 17-HDHA was in phosphatidylethanolamine (PE) and 13-HODE in phosphatidylcholine (PC). The same pattern was found for oxylipins endogenously formed by overexpression of 15-LOX-2. A new targeted method for the analysis of oxidized glycero-PLs enabled to pinpoint the specific molecular species of the oxylipins. 15-HETE (20:4;15OH) and 15-HEPE (20:5;15OH) are dominantly found as PI 18:0/20:4;15OH (70%) and PI 18:0/20:5;15OH (80%), respectively. This preferential incorporation of 20:4;15OH and 20:5;15OH into PI may be biologically relevant for PI signaling pathways. In contrast, >50% of 17-HDHA (22:6;17OH) was found in PE P-16:0/22:6;17OH, PE P-18:0/22:6;17OH, and PE P-18:1/22:6;17OH. At least 40% of 13-HODE (18:2;13OH) was incorporated into PC 16:0/18:2;13OH, and relevant amounts were found in PI 18:0/18;13OH, PC 18:1/18;13OH, and PC-O (ether PC) 16:0/18;13OH. These results indicate that hydroxy-PUFAs are bound to glycero-PLs in a specific manner. The distinct incorporation of 15-LOX-2 products from different PUFAs into glycero-PLs might contribute to the biological effect of these oxylipins and their precursor FAs.