Puntarulo S, Cederbaum A I
Department of Biochemistry, Mount Sinai School of Medicine, New York, New York 10029.
Arch Biochem Biophys. 1988 Aug 1;264(2):482-91. doi: 10.1016/0003-9861(88)90313-x.
The interaction of microsomes with iron and NADPH to generate active oxygen radicals was determined by assaying for low level chemiluminescence. The ability of several ferric complexes to catalyze light emission was compared to their effect on microsomal lipid peroxidation or hydroxyl radical generation. In the absence of added iron, microsomal light emission was very low; chemiluminescence could be enhanced by several cycles of freeze-thawing of the microsomes. The addition of ferric ammonium sulfate, ferric-citrate, or ferric-ADP produced an increase in chemiluminescence, whereas ferric-EDTA or -diethylenetriaminepentaacetic acid (detapac) were inhibitory. The same response to these ferric complexes was found when assaying for malondialdehyde as an index of microsomal lipid peroxidation. In contrast, hydroxyl radical generation, assessed as oxidation of chemical scavengers, was significantly enhanced in the presence of ferric-EDTA and -detapac and only weakly elevated by the other ferric complexes. Ferric-desferrioxamine was essentially inert in catalyzing any of these reactions. Chemiluminescence and lipid peroxidation were not affected by superoxide dismutase, catalase, or competitive hydroxyl radical scavengers whereas hydroxyl radical production was decreased by the latter two but not by superoxide dismutase. Chemiluminescence was decreased by the antioxidants propylgallate or glutathione and by inhibiting NADPH-cytochrome P-450 reductase with copper, but was not inhibited by metyrapone or carbon monoxide. The similar pattern exhibited by ferric complexes on microsomal light emission and lipid peroxidation, and the same response of both processes to radical scavenging agents, suggests a close association between chemiluminescence and lipid peroxidation, whereas both processes can be readily dissociated from free hydroxyl radical generation by microsomes.
通过检测低水平化学发光来确定微粒体与铁和NADPH相互作用以产生活性氧自由基的情况。将几种铁络合物催化发光的能力与其对微粒体脂质过氧化或羟基自由基生成的影响进行比较。在不添加铁的情况下，微粒体发光非常低；微粒体经过几个冻融循环可增强化学发光。添加硫酸铁铵、柠檬酸铁或铁 - ADP会使化学发光增加，而铁 - EDTA或二乙烯三胺五乙酸（detapac）具有抑制作用。以丙二醛作为微粒体脂质过氧化指标进行检测时，发现对这些铁络合物有相同的反应。相比之下，以化学清除剂氧化来评估的羟基自由基生成，在铁 - EDTA和detapac存在时显著增强，而其他铁络合物仅使其微弱升高。铁 - 去铁胺在催化这些反应中基本无活性。化学发光和脂质过氧化不受超氧化物歧化酶、过氧化氢酶或竞争性羟基自由基清除剂的影响，而羟基自由基生成会被后两者降低，但不受超氧化物歧化酶影响。化学发光会被抗氧化剂没食子酸丙酯或谷胱甘肽以及用铜抑制NADPH - 细胞色素P - 450还原酶所降低，但不受甲吡酮或一氧化碳抑制。铁络合物在微粒体发光和脂质过氧化上表现出的相似模式，以及这两个过程对自由基清除剂的相同反应，表明化学发光和脂质过氧化之间存在密切关联，而这两个过程都可以很容易地与微粒体产生的游离羟基自由基生成相分离。
