Kramer J H, Dickens B F, Misík V, Weglicki W B
Department of Medicine, George Washington University Medical Center, Washington, DC 20037, USA.
J Mol Cell Cardiol. 1995 Jan;27(1):371-81. doi: 10.1016/s0022-2828(08)80034-x.
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.
内皮细胞已被证明在缺氧后复氧过程中会产生以氧为中心的初级自由基（羟基、超氧阴离子），但在该模型中，关于随后引发脂质过氧化损伤的证据很少。采用电子自旋共振（ESR）光谱结合α-苯基-N-叔丁基硝酮（PBN）自旋捕获技术，监测培养的牛主动脉内皮细胞悬液在37℃下暴露于缺氧（A，45分钟，氮气）和复氧（R，15分钟，95%氧气/5%二氧化碳）时形成的脂质过氧化（LPO）衍生自由基。在一些研究中，在复氧前立即加入超氧化物歧化酶（SOD，10微克/毫升），以评估这种初级自由基清除剂对LPO衍生自由基产生的影响。在不同时间，取出等分试样，在甲苯萃取和ESR光谱分析之前，将PBN加入细胞悬液等分试样（最终浓度8 mM PBN，1分钟）或相应的无细胞滤液（最终浓度60 mM PBN）中。在复氧过程中，使用两种捕获方法均观察到PBN的LPO衍生烷氧基自由基加合物（PBN/RO·，超精细分裂αN = 13.63 G，αH = 1.94 - 1.98 G），在4 - 5分钟时产生量最大，在10分钟时出现第二个较小峰值。SOD有效降低了PBN/RO·的产生，并提高了缺氧/复氧细胞的活力。在平行研究中，通过高效液相色谱法评估缺氧/复氧细胞脂质提取物中的脂质过氧化氢产生情况。它们的分离图谱显示，在复氧4 - 5分钟和10分钟时采集的样品中，氧化脂质峰出现在磷脂酰乙醇胺（PE）和磷脂酰胆碱（PC）之间。将细胞脂质提取物重新溶解在含有乙酰丙酮钴（II）和PBN的无氧苯中会导致烷氧基自由基产生，但仅在氧化脂质样品中出现，这证实了过氧化氢的存在。这些结果表明，缺氧/复氧会导致初级自由基诱导的内皮细胞脂质过氧化损伤，如源自氧化膜磷脂的烷氧基自由基产生所示。
