van den Berg J J, Op den Kamp J A, Lubin B H, Roelofsen B, Kuypers F A
Children's Hospital Oakland Research Institute, CA 94609.
Free Radic Biol Med. 1992;12(6):487-98. doi: 10.1016/0891-5849(92)90102-m.
To provide a detailed description of the time course and the site specificity of hydroperoxide-induced oxidative stress in red blood cells (RBCs), we have characterized the action of a membrane-soluble (cumene hydroperoxide [cumOOH]) and a water-soluble (hydrogen peroxide [H2O2]) oxidant. The fluorescent polyunsaturated fatty acid (PUFA) parinaric acid (PnA) was used to probe peroxidation processes in the membrane, and oxidation of hemoglobin (Hb) was measured spectrophotometrically as an indicator of cytosolic oxidative stress. The observed degradation patterns of PnA and Hb were clearly distinct for each oxidant. At comparable oxidant concentrations, the cumulative oxidative stress on the RBC membrane was always much higher with cumOOH, whereas much more Hb oxidation was measured with H2O2. The kinetics of Hb oxidation as well as the nature of the products formed were different for each oxidant. The main Hb oxidation product generated gradually by cumOOH was metHb, whereas H2O2 caused the rapid formation of ferrylHb. CumOOH caused more oxidation of endogenous PUFAs and of vitamin E, while the degradation pattern of vitamin E closely resembled that of PnA. At high oxidant concentrations, extensive cell lysis was observed after prolonged incubation. Butylated hydroxytoluene (BHT) completely prevented oxidation of endogenous PUFAs but did not completely prevent hemolysis, indicating that factors other than lipid peroxidation are also important in causing lysis of RBCs. The action of cumOOH is characterized by a gradual reaction with Hb, generating radicals that produce an oxidative stress primarily directed at the membrane, which increases in time to a maximum and then gradually decreases. In contrast, H2O2 crosses the RBC membrane and reacts rapidly with Hb, generating a very reactive radical species that has Hb, not the membrane, as a prime target. H2O2-induced oxidative stress is at a maximum immediately after addition of this oxidant and decreases rapidly to zero in a short time. These findings provide further insight into the mode of action of hydroperoxides and the mechanism of compartmentalization of RBC oxidative damage.
为了详细描述过氧化氢诱导的红细胞（RBC）氧化应激的时间进程和位点特异性，我们对一种膜溶性氧化剂（异丙苯过氧化氢[cumOOH]）和一种水溶性氧化剂（过氧化氢[H2O2]）的作用进行了表征。荧光多不饱和脂肪酸（PUFA）十八碳四烯酸（PnA）用于探测膜中的过氧化过程，血红蛋白（Hb）的氧化通过分光光度法进行测量，作为胞质氧化应激的指标。每种氧化剂对PnA和Hb的观察到的降解模式明显不同。在相当的氧化剂浓度下，cumOOH对RBC膜的累积氧化应激总是高得多，而H2O2导致的Hb氧化则更多。每种氧化剂的Hb氧化动力学以及形成的产物性质都不同。cumOOH逐渐产生的主要Hb氧化产物是高铁血红蛋白（metHb），而H2O2导致高铁血红蛋白（ferrylHb）的快速形成。cumOOH对内源性PUFAs和维生素E的氧化作用更强，而维生素E的降解模式与PnA非常相似。在高氧化剂浓度下，长时间孵育后观察到广泛的细胞裂解。丁基羟基甲苯（BHT）完全阻止了内源性PUFAs的氧化，但没有完全阻止溶血，这表明除脂质过氧化外的其他因素在导致RBC裂解中也很重要。cumOOH的作用特点是与Hb逐渐反应，产生自由基，主要针对膜产生氧化应激，这种应激随时间增加到最大值，然后逐渐降低。相比之下，H2O2穿过RBC膜并与Hb迅速反应，产生一种非常活泼的自由基物种，其主要靶标是Hb而非膜。添加这种氧化剂后，H2O2诱导的氧化应激立即达到最大值，并在短时间内迅速降至零。这些发现为过氧化氢的作用模式和RBC氧化损伤的区室化机制提供了进一步的见解。
