Samouilov Alexandre, Roubaud Valerie, Kuppusamy Periannan, Zweier Jay L
Center for Biomedical EPR Spectroscopy Imaging, Davis Heart and Lung Research Institute, Division of Cardiovascular Medicine, Department of Internal Medicine, Ohio State University College of Medicine, Columbus, OH 43210, USA.
Anal Biochem. 2004 Nov 1;334(1):145-54. doi: 10.1016/j.ab.2004.07.026.
Because short-lived reactive oxygen radicals such as superoxide have been implicated in a variety of disease processes, methods to measure their production quantitatively in biological systems are critical for understanding disease pathophysiology. Electron paramagnetic resonance (EPR) spin trapping is a direct and sensitive technique that has been used to study radical formation in biological systems. Short-lived oxygen free radicals react with the spin trap and produce paramagnetic adducts with much higher stability than that of the free radicals. In many cases, the quantity of the measured adduct is considered to be an adequate measure of the amount of the free radical generated. Although the intensity of the EPR signal reflects the magnitude of free radical generation, the actual quantity of radicals produced may be different due to modulation of the spin adduct kinetics caused by a variety of factors. Because the kinetics of spin trapping in biochemical and cellular systems is a complex process that is altered by the biochemical and cellular environment, it is not always possible to define all of the reactions that occur and the related kinetic parameters of the spin-trapping process. We present a method based on a combination of measured kinetic data for the formation and decay of the spin adduct alone with the parameters that control the kinetics of spin trapping and radical generation. The method is applied to quantitate superoxide trapping with 5-diethoxyphosphoryl-5-methyl-1-pyrroline N-oxide (DEPMPO). In principle, this method is broadly applicable to enable spin trapping-based quantitative determination of free radical generation in complex biological systems.
由于诸如超氧化物等短寿命活性氧自由基与多种疾病过程有关，因此在生物系统中定量测量其生成量的方法对于理解疾病病理生理学至关重要。电子顺磁共振（EPR）自旋捕获是一种直接且灵敏的技术，已被用于研究生物系统中的自由基形成。短寿命的氧自由基与自旋捕获剂反应，生成稳定性远高于自由基的顺磁性加合物。在许多情况下，所测加合物的量被认为是对生成的自由基量的一种充分衡量。尽管EPR信号强度反映了自由基生成的程度，但由于多种因素对自旋加合物动力学的调制，实际产生的自由基量可能会有所不同。由于生化和细胞系统中自旋捕获的动力学是一个复杂的过程，会受到生化和细胞环境的影响，因此并不总是能够确定所有发生的反应以及自旋捕获过程的相关动力学参数。我们提出了一种方法，该方法基于仅对自旋加合物形成和衰变的测量动力学数据与控制自旋捕获和自由基生成动力学的参数相结合。该方法用于定量用5 - 二乙氧基磷酰基 - 5 - 甲基 - 1 - 吡咯啉N - 氧化物（DEPMPO）捕获超氧化物。原则上，该方法广泛适用于基于自旋捕获的复杂生物系统中自由基生成的定量测定。
