Ueno I, Kohno M, Mitsuta K, Mizuta Y, Kanegasaki S
Institute of Medical Science, University of Tokyo.
J Biochem. 1989 Jun;105(6):905-10. doi: 10.1093/oxfordjournals.jbchem.a122777.
By employing EPR spectrometry with the aid of a spin-trapping agent, 5,5-dimethyl-1-pyrroline-1-oxide (DMPO), the generation of superoxide anion and hydroxyl radical was reevaluated during the respiratory burst of porcine and human neutrophils. Properly prepared resting neutrophils did not generate any spin-trapped radical, and, when the cells were stimulated with phorbol myristate acetate, only DMPO-OOH, the spin-trapped adduct of superoxide anion, was detected. No formation of DMPO-OH, the spin-trapped adduct of the hydroxyl radical, was observed. DMPO-OOH was also detected principally when the neutrophils were stimulated with opsonized zymosan, a particulate stimulus. In the latter case, however, the formation of DMPO-OOH ceased shortly after the addition of zymosan and subsequent production of DMPO-OH was observed. The production of DMPO-OH was found to be associated with cell injury. DMPO at the concentration usually used for the experiment (0.045-0.09 M) injured phagocytizing neutrophils, causing lysis of the cells. On the other hand, an addition of cell homogenate or glutathione-glutathione peroxidase system to the suspension of intact cells which were producing DMPO-OOH resulted in the formation of DMPO-OH. Thus, DMPO-OH was probably derived from DMPO-OOH by the action of enzymes and/or factor(s) which were released from the lysed cells.
借助自旋捕获剂5,5-二甲基-1-吡咯啉-1-氧化物（DMPO），采用电子顺磁共振波谱法，对猪和人中性粒细胞呼吸爆发过程中超氧阴离子和羟自由基的生成进行了重新评估。制备得当的静息中性粒细胞不会产生任何自旋捕获自由基，当用佛波酯刺激细胞时，仅检测到超氧阴离子的自旋捕获加合物DMPO-OOH。未观察到羟自由基的自旋捕获加合物DMPO-OH的形成。当用调理酵母聚糖（一种颗粒刺激物）刺激中性粒细胞时，也主要检测到DMPO-OOH。然而，在后一种情况下，添加酵母聚糖后不久DMPO-OOH的形成就停止了，并且观察到随后有DMPO-OH的产生。发现DMPO-OH的产生与细胞损伤有关。实验中通常使用的浓度（0.045 - 0.09 M）的DMPO会损伤正在吞噬的中性粒细胞，导致细胞裂解。另一方面，向正在产生DMPO-OOH的完整细胞悬液中添加细胞匀浆或谷胱甘肽 - 谷胱甘肽过氧化物酶系统会导致DMPO-OH的形成。因此，DMPO-OH可能是由DMPO-OOH通过裂解细胞释放的酶和/或因子的作用产生的。
