PHOSPHOLIPID HYDROPEROXIDES ARE PRECURSORS OF LIPID ALKOXYL RADICALS PRODUCED FROM ANOXIA REOXYGENATED ENDOTHELIAL-CELLS

Endothelial cells have been shown to generate primary oxygen-centered free radicals (hydroxyl, superoxide anion) during post-anoxic reoxygen ation, but little evidence is available concerning subsequent initiati on of lipid peroxidative injury in this model. Electron spin resonance (ESR) spectroscopy with alpha-phenyl-N-tert-butylnitrone (PBN) spin t rapping was used to monitor lipid peroxidation (LPO)-derived free radi cals formed by cultured bovine aortic endothelial cell suspensions exp osed (37 degrees C) to anoxia (A, 45 min, N-2 gas) and reoxygenation ( R, 15 min, 95% O-2/5% CO2). In some studies, superoxide dismutase (SOD , 10 mu g/ml) was introduced just prior to R to assess the effects of this primary free radical scavenger on LPD-derived free radical produc tion. At various times, aliquots were removed and PEN was introduced t o either the cell suspension aliquot (8 mM PBN final, 1 min), or to th e corresponding cell-free filtrate (60 mM PBN final), prior to extract ion with toluene and ESR spectroscopy. A LPO-derived alkoxyl radical a dduct of PBN (PBN/RO ., hyperfine splitting alpha(N)=13.63 G and alpha (H)=1.94-1.98 G) was observed during R using both trapping procedures, with maximal production at 4-5 min and a second minor peak at 10 min. SOD effectively reduced PBN/RO . production and improved viability of A/R cells, In parallel studies, lipid hydroperoxide production was as sessed in lipid extracts of A/R cells by high-performance liquid chrom atography. Their separation profiles revealed a peak of oxidized lipid occurring between phosphatidylethanolamine (PE) and phosphatidylcholi ne (PC) in samples taken at 4-5 min and 10 min of R. Resolubilizing ce ll lipid extracts in oxygen-free benzene containing cobalt(II) acetyla cetonate and PEN led to alkoxyl radical production, but only in the ox idized lipid samples, confirming the presence of hydroperoxides. These results suggest that A/R leads to primary free radical induced-lipid peroxidative injury to endothelial cells, as indicated by alkoxyl radi cal production originating from oxidized membrane phospholipids.