Maulik D, Numagami Y, Ohnishi S T, Mishra O P, Delivoria-Papadopoulos M
Department of Obstetrics and Gynecology, Winthrop-University Hospital, 259 First Street, Mineola, NY 11501, USA.
Brain Res. 1998 Jul 6;798(1-2):166-72. doi: 10.1016/s0006-8993(98)00408-9.
The present study tested the hypothesis that maternal hypoxia induces oxygen free radical generation in the fetal guinea pig brain utilizing techniques of electron spin resonance spectroscopy and alpha-phenyl-tert-butyl nitrone (PBN) spin trapping. Pregnant guinea pigs of 60 days gestation were divided into normoxic and hypoxic groups and exposed to 21% or 7% oxygen for 60 min. Free radical generation was documented by measuring the signal of PBN spin adducts. Fluorescent compounds were determined as an index of lipid peroxidation and the activity of Na+,K+-ATPase was determined as an index of brain cell membrane function. Hypoxic fetal cerebral cortical tissue showed a significant increase in spin adducts (normoxic: 33.8+/-9.3 units/g tissue vs. hypoxic: 57.9+/-9.2 units/g tissue, p<0.01) and fluorescent compounds (normoxic: 0.639+/-0.054 microg quinine sulfate/g brain vs. 0.810+/-0.102 microg quinine sulfate/g brain, p<0.01) and a decrease in Na+,K+-ATPase activity (normoxic: 43.04+/-2.50 micromol Pi/mg protein/h vs. hypoxic: 33. 80+/-3.51 micromol Pi/mg protein/h, p<0.001). These results demonstrate an increased free radical generation during hypoxia in the fetal guinea pig brain. The spectral characteristics of the radicals were consistent with those of alkoxyl radicals. The increased level of fluorescent compounds and decreased activity of Na+,K+-ATPase indicated hypoxia induced brain cell membrane lipid peroxidation and dysfunction, respectively. These results directly demonstrate an increased oxygen free radical generation during hypoxia and suggest that hypoxia-induced increase in lipid peroxidation and decrease in membrane function, as indicated by a decrease in Na+,K+-ATPase activity, are consequences of increased free radicals. The nature of predominantly present alkoxyl radical indicates ongoing lipid peroxidation during hypoxia. The direct demonstration of oxygen free radical generation during hypoxia is the critical missing link in the mechanism of hypoxia-induced brain cell membrane dysfunction and damage.
本研究利用电子自旋共振光谱技术和α-苯基叔丁基硝酮（PBN）自旋捕捉技术，验证了母体缺氧会在胎儿豚鼠大脑中诱导氧自由基生成这一假设。将妊娠60天的豚鼠分为常氧组和缺氧组，分别暴露于21%或7%的氧气环境中60分钟。通过测量PBN自旋加合物的信号来记录自由基的生成情况。测定荧光化合物作为脂质过氧化的指标，并测定Na⁺，K⁺-ATP酶的活性作为脑细胞膜功能的指标。缺氧胎儿的大脑皮质组织中，自旋加合物显著增加（常氧组：33.8±9.3单位/克组织，缺氧组：57.9±9.2单位/克组织，p<0.01），荧光化合物也显著增加（常氧组：0.639±0.054微克硫酸奎宁/克脑，缺氧组：0.810±0.102微克硫酸奎宁/克脑，p<0.01），而Na⁺，K⁺-ATP酶活性降低（常氧组：43.04±2.50微摩尔无机磷/毫克蛋白质/小时，缺氧组：33.80±3.51微摩尔无机磷/毫克蛋白质/小时，p<0.001）。这些结果表明，胎儿豚鼠大脑在缺氧期间自由基生成增加。自由基的光谱特征与烷氧基自由基一致。荧光化合物水平升高和Na⁺，K⁺-ATP酶活性降低分别表明缺氧诱导了脑细胞膜脂质过氧化和功能障碍。这些结果直接证明了缺氧期间氧自由基生成增加，并表明缺氧诱导的脂质过氧化增加和膜功能降低（如Na⁺，K⁺-ATP酶活性降低所示）是自由基增加的结果。主要存在的烷氧基自由基的性质表明缺氧期间脂质过氧化持续进行。缺氧期间氧自由基生成的直接证明是缺氧诱导脑细胞膜功能障碍和损伤机制中关键的缺失环节。
