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-PUFA act as lipid mediators. In biological samples the major part occurs esterified in glycerophospholipids or other lipids. In this work, the incorporation into glycerophospholipids of 15(S)-HETE, 15(S)-HEPE, 17(S)-HDHA, and 13(S)-HODE was investigated in oxylipin-supplemented HEK293T 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 while > 75% are bound to distinct glycerophospholipid classes, depending on the hydroxy-PUFA. 15-HETE and 15-HEPE were found in PI/PS, while 17-HDHA was in PE and 13-HODE in 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 glycerophospholipids 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 16:0/18;13OH. These results indicate that hydroxy-PUFA are bound to glycerophospholipids in a specific manner. The distinct incorporation of 15-LOX-2 products from different PUFA into glycerophospholipids might contribute to the biological effect of these oxylipins and their precursor fatty acids.