Heart disease and stroke are the most common causes of death in Westernized societies. Mounting evidence over the past decade suggests that oxidation of low density lipoprotein (LDL) plays an important role in the initiation and promotion of atherosclerosis. Platelet-activating factor (PAF) is a phospholipid with potent, diverse physiological actions, particularly as a mediator of inflammation. Some PAF-like phospholipids, formed during the oxidative modification of LDL, i.e., oxPAF, may act via the PAF receptor.
In this work, a diverse family of oxidatively truncated ether phospholipids was prepared to facilitate the identification and biological testing of these compounds that are generated through oxidative cleavage of polyunsaturated phospholipids. Using HPLC with on-line tandem mass spectrometry, the formation of these phospholipids in oxidation reactions of docosahexaenoate, arachidonate and linoleate esters of 1-alkyl-2-hydroxy-sn-glycero-3-phosphocholine(lyso-PAF) was demonstrated. The oxidation of LDL by myeloperoxidase (MPO) also produced oxidized alkyl acyl phosphocholines (oxPAFs). We discovered a profound influence of membrane environment on the stability of γ-hydroxyalkenal oxPAFs. Under autoxidative conditionsthe aldehyde group of γ-hydroxyalkenal oxPAFs is stable in small unilamellar vesicles (SUVs) composed of saturated diacyl-PCs, but is rapidly oxidized in SUVs that contain 1 equivalent of the linoleic acid ester of 2-lysophosphatidylcholine (LA-PC).
Various oxPAFs are potent platelet-activating factor receptor (PAFR) agonists, such as OB-PAF, S-PAF, KHdiA-PAF, KOdiA-PAF and HOHA-PAF. Several of these oxPAFs, at micromolar concentrations, can initiate calcium accumulation and these responses were increased in a concentration-dependent way. The oxidatively truncated alkyl acyl phosphocholines tested were all biologically active as mitochondrial depolarizing agents. Apoptosis was initiated upon exposure of HL 60 cells to these oxPAFs. The potencies of these oxPAFs apparently show structural dependences.
With the recent development of soft ionization methods, electrospray ionization (ESI)has become an informative method for detecting oxidized phospholipids. In the present study, the fragmentation pattern of a series of oxidatively truncated PAF analogs, formed during oxidation of alkyl phosphocholines, was systematically investigated using negative mode ESI-MS/MS. In comparison with the positive ion mode, the negative mode provides more structural information, obviating the need for derivatization. Negative ion MS fragmentation readily distinguishes structurally similar acyl phosphocholine (PC), phosphatidylethanolamine (PE) and phosphatidylserine (PS) oxidative products without the need for phospholipase A1 treatment, that is commonly used to distinguish alkyl from acyl phosphatidylcholines.