Britigan B E, Hamill D R
Department of Internal Medicine, Veterans Administration Medical Center, Iowa City, Iowa.
Arch Biochem Biophys. 1989 Nov 15;275(1):72-81. doi: 10.1016/0003-9861(89)90351-2.
Activation of human neutrophils leads to secretion of myeloperoxidase (MPO) with resulting generation of several oxidant species including OCl-. Spin trapping techniques employing 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) are being applied increasingly to the investigation of free radical production by in vitro and in vivo experimental systems which contain neutrophils. Because such knowledge is critical to the interpretation of these data, we examined the impact of MPO and MPO-derived oxidants on DMPO spin adduct formation and stability. Addition of increasing concentrations of OCl- to DMPO yielded a number of EPR-detectable products including DMPO-OH. However, the concentration of OCl- required was in excess of that expected under physiologic conditions. Addition of purified human MPO and H2O2 to DMPO yielded EPR spectra consisting of small DMPO-OH peaks. The addition of MPO and H2O2 to preformed DMPO-OH and DMPO-CH3 resulted in rapid destruction of these spin adducts. Thus MPO/H2O2 appeared to both generate and destroy DMPO spin adducts. Neutrophils stimulated with phorbol myristate acetate or opsonized zymosan generated large DMPO-OOH and DMPO-OH peaks as well as small DMPO-CH3 peaks. Addition of the MPO inhibitor azide to the reaction mixture had no effecting on resulting DMPO-OH or DMPO-CH3 peak amplitudes but increased that of DMPO-OOH. These data suggest that MPO-derived oxidants likely have little impact on the nature of EPR spectra resulting from DMPO spin trapping of free radical species following neutrophil stimulation. Because MPO oxidants did appear to react with DMPO the ability of DMPO to protect a biologic target from in vitro MPO injury was examined. DMPO (greater than 10 mM) significantly decreased MPO/H2O2/Cl- -mediated erythrocyte hemolysis as assessed by 51Cr release. The experimental and/or pharmacologic implications of this observation require further study.
人类中性粒细胞的激活会导致髓过氧化物酶（MPO）的分泌，进而产生包括次氯酸根离子（OCl-）在内的多种氧化剂。采用5,5-二甲基-1-吡咯啉-N-氧化物（DMPO）的自旋捕获技术越来越多地应用于对含有中性粒细胞的体外和体内实验系统中自由基产生的研究。由于此类知识对于解读这些数据至关重要，我们研究了MPO及其衍生的氧化剂对DMPO自旋加合物形成和稳定性的影响。向DMPO中添加浓度不断增加的OCl-会产生许多可通过电子顺磁共振（EPR）检测到的产物，包括DMPO-OH。然而，所需的OCl-浓度超过了生理条件下预期的浓度。向DMPO中添加纯化的人MPO和过氧化氢（H2O2）会产生由小的DMPO-OH峰组成的EPR光谱。向预先形成的DMPO-OH和DMPO-CH3中添加MPO和H2O2会导致这些自旋加合物迅速被破坏。因此，MPO/H2O2似乎既能产生也能破坏DMPO自旋加合物。用佛波酯肉豆蔻酸酯或调理酵母聚糖刺激的中性粒细胞会产生大的DMPO-OOH和DMPO-OH峰以及小的DMPO-CH3峰。向反应混合物中添加MPO抑制剂叠氮化物对产生的DMPO-OH或DMPO-CH3峰幅度没有影响，但增加了DMPO-OOH的峰幅度。这些数据表明，MPO衍生的氧化剂可能对中性粒细胞刺激后DMPO捕获自由基产生的EPR光谱性质影响很小。由于MPO氧化剂似乎确实与DMPO发生反应，因此研究了DMPO保护生物靶标免受体外MPO损伤的能力。通过51Cr释放评估，DMPO（大于10 mM）显著降低了MPO/H2O2/Cl-介导的红细胞溶血。这一观察结果的实验和/或药理学意义需要进一步研究。
