PhD

dissertationA variety of pathologic processes including ischemia and reperfusion-associated injury, Adult Respiratory Distress Syndrome, and chronic inflammation are associated with the sequestration and inappropriate activation of neutrophils (PMN). PMN contribute to tissue injury by degranulating to release of lytic components and by generating massive amounts of free radicals upon activation of the neutrophil respiratory burst. In addition to the activation of PMN, these pathologic processes are associated with the generation of reactive oxygen species, molecules that are efficient at modifying the structure of macromolecules, incuding cell membranes, proteins, and nucleic acids. Due to their abundance and susceptibility to oxidation, the polyunsaturated fatty acids present in the cell membrane phospholipids are primary targets for free radical-mediated oxidation, with subsequent peroxidation, bond rearrangement, degradation, and hydroxy lation. This dissertation presents data supporting the hypothesis that oxidative modification of membrane phospholipids may alter their structures so they are able to bind to and activate receptors present on the neutrophil surface for a potent phospholipid autocoid, platelet-activating factor (PAF). PAF mediates a variety of effects, including profound hypotension, increased vascular permeability, and activation of inflammatory cells. The model oxidatively-fragmented phospholipid, l-palmitoyl-2(-5-oxovaleroyl)-phosphatidylcholine was shown to stimulate neutrophil function through the PAF receptor. Additionally, oxidatively-fragmented phospholipids formed during unregulated oxidation of the phospholipid hydroperoxide, l-palmitoyl-2-(15-hydroperoxyeicosatetraenoyO-phosphatidylcholine were shown to exhibit PAF-like activity. One of these bioactive products was identified by gas chromatography/mass spectrometry as, l-paimitoyl-2-propionoyl-GPC, an oxidized phospholipid with an exceedingly short sn-2 residue. Additionally, bioactive compounds generated upon unregulated oxidation were substrates for the plasma PAF acetylhydrolase, an enzyme that circulates in whole blood and degrades phospholipids with short sn-2 residues, such as PAF. This enzyme may help limit the injury mediated by these phospholipids by ablating their activity should they occur in vivo. Finally, the cell membrane phospholipids of the neutrophils themselves may be targets for oxidative modification. Oxidized phosphatidylcholines with PAF-like bioactivity were generated upon peroxidation of the neutrophil cell membrane phospholipids. This activity was mediated by species with sn-\ ether linkages, although there actually apprears to be selective oxidation for PMN membrane phosphatidylcholines with sn-1 ester-linked residues