Zweier J L, Kuppusamy P, Williams R, Rayburn B K, Smith D, Weisfeldt M L, Flaherty J T
Department of Medicine, Johns Hopkins Medical Institutions, Baltimore, Maryland 21224.
J Biol Chem. 1989 Nov 15;264(32):18890-5.
Electron paramagnetic resonance spectroscopy has been applied to measure radical generation in the postischemic heart; however, there is controversy regarding the methods used and the conclusion as to whether radicals are generated. In order to resolve this controversy, direct and spin trapping measurements of the time course and mechanisms of radical generation were performed in isolated perfused rabbit hearts. In reperfused tissue, 3 prominent radical signals are observed: A, isotropic g = 2.004 suggestive of a semiquinone; B, anisotropic g parallel = 2.033 and g perpendicular = 2.005 suggestive of ROO.; and C, a triplet g = 2.000 and aN = 24 G suggestive of a nitrogen centered radical. B and C, however, are highly labile and disappear at temperatures probably encountered in some previous studies. In normally perfused hearts, A is observed with only small amounts of B and C. During ischemia, B and C increase reaching a maximum after 45 min while A decreases. On reflow with oxygenated perfusate all 3 signals increase. With varying duration of ischemia and reflow, peak signal intensities occurred after 15 s of reflow following 30 min of ischemia. Reperfusion with superoxide dismutase, deferoxamine, or mannitol abolished the reperfusion increase of B. Measurements performed with the spin trap 5,5'-dimethyl-1-pyrroline-N-oxide (DMPO) demonstrated a similar time course of radical generation with prominent DMPO-OH and DMPO-R signals peaking between 10 and 20 s of reflow. Superoxide dismutase and deferoxamine also quenched these signals. Thus, .O2- derived .OH, R., and ROO. radicals are generated in postischemic myocardium. While the experimental techniques used can result in loss of intrinsic radicals and generation of extraneous radicals, with proper care and controls valid measurements of free radicals in biological tissues can be performed.
电子顺磁共振波谱已被用于测量缺血后心脏中的自由基生成；然而，关于所使用的方法以及是否产生自由基的结论存在争议。为了解决这一争议，在离体灌注兔心脏中对自由基生成的时间进程和机制进行了直接和自旋捕获测量。在再灌注组织中，观察到3个显著的自由基信号：A，各向同性g = 2.004，提示为半醌；B，各向异性g平行 = 2.033且g垂直 = 2.005，提示为ROO.；以及C，三重态g = 2.000且aN = 24 G，提示为以氮为中心的自由基。然而，B和C非常不稳定，在一些先前研究可能遇到的温度下会消失。在正常灌注的心脏中，仅观察到少量B和C时出现A。在缺血期间，B和C增加，在45分钟后达到最大值，而A减少。用含氧灌注液再灌注时，所有3个信号均增加。随着缺血和再灌注持续时间的变化，在30分钟缺血后再灌注15秒时出现信号强度峰值。用超氧化物歧化酶、去铁胺或甘露醇再灌注可消除B的再灌注增加。用自旋捕获剂5,5'-二甲基-1-吡咯啉-N-氧化物（DMPO）进行的测量表明，自由基生成的时间进程相似，显著的DMPO-OH和DMPO-R信号在再灌注10至20秒之间达到峰值。超氧化物歧化酶和去铁胺也会淬灭这些信号。因此，缺血后心肌中会产生源自.O2-的.OH、R.和ROO.自由基。虽然所使用的实验技术可能导致内源性自由基的损失和外源性自由基的产生，但通过适当的小心操作和控制，可以对生物组织中的自由基进行有效的测量。
