Structural identification of a novel pro-inflammatory epoxyisoprostane phospholipid in mildly oxidized low density lipoprotein

One of the earliest steps in the development of the atherosclerotic lesion is the accumulation of monocyte/macrophages within:the vessel wall. Oxidize d lipids present in minimally modified-low density lipoproteins (RIM-LDL) c ontribute to this process by activating endothelial cells to express; monoc yte-specific adhesion molecules and chemoattractant factors. A major focus of our group has been the isolation and characterization of the biologicall y active oxidized lipids in MRI-LDL. We have previously characterized three oxidized phospholipids present in MM-LDL, atherosclerotic lesions of fat f ed rabbits, and autoxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phospho choline (Ox-PAPC) that induced human aortic endothelial cells to adhere hum an monocytes in vitro. We have used sequential normal and reverse phase-hig h performance liquid chromatography to isolate various isomers of an oxidiz ed phospholipid from autoxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-ph osphocholine. The fatty acid in the sn-2 position of this biologically acti ve isomer and its dehydration product was released by phospholipase A(2) an d characterized. Hydrogenation with platinum(IV) oxide/hydrogen suggested a cyclic moiety, and reduction with sodium borohydride suggested two reducib le oxygen-containing groups in the molecule. The fragmentation pattern prod uced by:electrospray ionization-collision induced dissociation-tandem mass spectrometry was consistent with a molecule resembling an E-ring prostaglan din with an epoxide at the 5,6 position. The structure of this lipid was co nfirmed by proton nuclear magnetic resonance spectroscopy analysis of the f ree fatty acid isolated from the dehydration product of m/z 828.5, Based on these studies, we arrived at the structure of the biologically active oxid ized phospholipids as 1-palmitoyl-2-(5,6-epoxyisoprostane E-2)-sn-glycero-3 -phosphocholine. The identification of this molecule adds epoxyisoprostanes to the growing list of biologically active isoprostanes.