Zhang J R, Scherch H M, Hall E D
CNS Diseases Research, Upjohn Company, Kalamazoo, Michigan 49001, USA.
J Neurochem. 1996 Jan;66(1):355-61. doi: 10.1046/j.1471-4159.1996.66010355.x.
The relationship between iron-dependent fetal mouse spinal cord neuron injury and the generation of endogenous lipid hydroperoxides (LOOHs) has been investigated. Cultured spinal cord neurons were incubated with ferrous iron (3-200 microM). Cell viability was measured in terms of the uptake of alpha-[methyl-3H]aminoisobutyric acid ([3H]AIB). Both endogenously and iron-generated LOOH, i.e., free fatty acid hydroperoxide (FFAOOH), phosphatidylethanolamine hydroperoxide (PEOOH), and phosphatidylcholine hydroperoxide (PCOOH), were measured directly by an HPLC-chemiluminescence (HPLC-CL) assay. The FFAOOH, PEOOH, and PCOOH levels in neurons incubated with 200 microM Fe2+ for 40 min were, respectively, 22-, 158-, and sevenfold higher than those in non-iron-exposed cultures, demonstrating that phosphatidylethanolamine (PE) was most sensitive to peroxidation. The dose-response and time course of Fe(2+)-induced generation of these LOOHs were also established. In both experiments, the LOOH levels were correlated directly with loss of neuronal viability, suggesting strongly a direct relationship between lipid peroxidation and cell injury. On examination of the time course of the LOOH generation, an immediate increase in PEOOH and PCOOH levels with only 30 s of Fe2+ incubation was observed. In contrast, a lag phase in the increase in FFAOOH level (2 min after Fe2+ addition) suggested a delay in the activation of phospholipase A2 (PLA2) required for the hydrolysis and generation of FFAOOH. This culture system provides an excellent model for screening antioxidant neuroprotective compounds with regard to their ability to protect against iron-dependent peroxidative injury and the relationship of the neuroprotection to inhibition of lipid peroxidation and/or PLA2.
已对铁依赖性胎鼠脊髓神经元损伤与内源性脂质氢过氧化物（LOOHs）生成之间的关系进行了研究。将培养的脊髓神经元与亚铁（3 - 200微摩尔）一起孵育。通过α-[甲基-³H]氨基异丁酸（[³H]AIB）摄取来测量细胞活力。内源性和铁生成的LOOH，即游离脂肪酸氢过氧化物（FFAOOH）、磷脂酰乙醇胺氢过氧化物（PEOOH）和磷脂酰胆碱氢过氧化物（PCOOH），通过高效液相色谱 - 化学发光（HPLC - CL）测定法直接测量。用200微摩尔Fe²⁺孵育40分钟的神经元中，FFAOOH、PEOOH和PCOOH水平分别比未暴露于铁的培养物中高22倍、158倍和7倍，表明磷脂酰乙醇胺（PE）对过氧化最敏感。还确定了Fe(2+)诱导这些LOOH生成的剂量反应和时间进程。在这两个实验中，LOOH水平与神经元活力丧失直接相关，强烈表明脂质过氧化与细胞损伤之间存在直接关系。在检查LOOH生成的时间进程时，观察到仅孵育30秒的Fe²⁺后，PEOOH和PCOOH水平立即增加。相比之下，FFAOOH水平增加的延迟期（添加Fe²⁺后2分钟）表明水解和生成FFAOOH所需的磷脂酶A2（PLA2）激活存在延迟。该培养系统为筛选抗氧化神经保护化合物提供了一个极好的模型，以研究它们对抗铁依赖性过氧化损伤的能力以及神经保护与脂质过氧化抑制和/或PLA2抑制之间的关系。
