title: Phospholipid hydroperoxides are precursors of lipid alkoxyl radicals produced from anoxia/reoxygenated endothelial cells.
abstract: Endothelial cells have been shown to generate primary oxygen-centered free radicals (hydroxyl, superoxide anion) during post-anoxic reoxygenation, but little evidence is available concerning subsequent initiation of lipid peroxidative injury in this model. Electron spin resonance (ESR) spectroscopy with alpha-phenyl-N-tert-butylnitrone (PBN) spin trapping was used to monitor lipid peroxidation (LPO)-derived free radicals formed by cultured bovine aortic endothelial cell suspensions exposed (37 degrees C) to anoxia (A, 45 min, N2 gas) and reoxygenation (R, 15 min, 95% O2/5% CO2). In some studies, superoxide dismutase (SOD, 10 micrograms/ml) was introduced just prior to R to assess the effects of this primary free radical scavenger on LPO-derived free radical production. At various times, aliquots were removed and PBN was introduced to either the cell suspension aliquot (8 mM PBN final, 1 min), or to the corresponding cell-free filtrate (60 mM PBN final), prior to extraction with toluene and ESR spectroscopy. A LPO-derived alkoxyl radical adduct 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 assessed in lipid extracts of A/R cells by high-performance liquid chromatography. Their separation profiles revealed a peak of oxidized lipid occurring between phosphatidylethanolamine (PE) and phosphatidylcholine (PC) in samples taken at 4-5 min and 10 min of R. Resolubilizing cell lipid extracts in oxygen-free benzene containing cobalt (II) acetylacetonate and PBN led to alkoxyl radical production, but only in the oxidized 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 radical production originating from oxidized membrane phospholipids.
authors.affiliation: Department of Medicine, George Washington University Medical Center, Washington, DC 20037, USA.
authors.forename: J H
authors.initials: JH
authors.lastname: Kramer
authors.forename: B F
authors.initials: BF
authors.lastname: Dickens
authors.forename: V
authors.initials: V
authors.lastname: Misík
authors.forename: W B
authors.initials: WB
authors.lastname: Weglicki
mesh_terms: D000332:Aerobiosis; D000818:Animals; D001011:Aorta; D002417:Cattle; D015687:Cell Hypoxia; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D003497:Cyclic N-Oxides; D004578:Electron Spin Resonance Spectroscopy; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D005609:Free Radicals / Q000032:analysis / Q000378:metabolism*; D007700:Kinetics; D015227:Lipid Peroxidation; D008054:Lipid Peroxides / Q000378:metabolism*; D009589:Nitrogen Oxides; D010743:Phospholipids / Q000378:metabolism*; D013113:Spin Labels; D013482:Superoxide Dismutase / Q000494:pharmacology; D013997:Time Factors
chemical_list: D003497:Cyclic N-Oxides; D005609:Free Radicals; D008054:Lipid Peroxides; D009589:Nitrogen Oxides; D010743:Phospholipids; D013113:Spin Labels; C029217:phenyl-N-tert-butylnitrone; D013482:Superoxide Dismutase