In Vivo Detection of Free Radical Generation

自旋捕集技术已经成为检测体内高活性自由基分子的最成功的方法。该技术涉及直接检测初级自由基，由于稳态浓度低或弛豫时间非常短，导致非常宽的线，因此无法通过常规ESR直接观察到初级自由基。所有报道的体内自旋捕获研究都使用了硝酮自旋捕获剂（PBN，POBN，DMPO）。虽然自旋捕获技术在体内的主要困难是仅仅检测自由基加合物，但当自旋捕获器在体内施用时，必须考虑其他因素，例如吸收、分布、代谢和排泄。此外，自由基加合物结构归属中的人为因素和模糊性必须认真考虑。该小组率先将ESR自旋捕获技术应用于生物化学、药理学和毒理学问题，采用体内自旋捕获和离体ESR检测自由基的两种主要方法：组织中产生的自由基加合物的脂质提取和检测生物液体中的自由基加合物。 I.金属介导的自由基的产生：急性镉和/或柴油机废气颗粒中毒的ESR自旋捕获研究表明，在体内形成的羟基自由基在肝脏或脂质衍生的自由基在肺部。 二.通过HPLC/ESR/MS表征内源性自由基加合物：使用在线高效液相色谱（HPLC）/电子自旋共振（ESR）系统与质谱分析（MS）的组合来鉴定体外和体内脂质过氧化形成的各种脂质衍生自由基。首次发现并鉴定了亚油酸碳中心自由基的POBN加合物。 三.新的甲醇和乙二醇自由基代谢：乙醇自由基代谢的研究已经使用基因敲除小鼠，化学类似物，如乙二醇和甲醇，及其代谢产物，如甲酸。在急性甲酸钠中毒期间，通过ESR自旋捕获来自甲酸盐代谢的自由基提供了体内甲酸盐衍生自由基代谢物的证据，这些自由基在胆汁和尿液中作为自由基加合物被检测到。

Spin Trapping has been the most successful method for the detection of highly reactive free radical molecules in vivo. This technique involves the direct detection of primary free radicals that cannot be directly observed by conventional ESR due to low steady-state concentrations or to very short relaxation times, which lead to very broad lines. All the reported in vivo spin-trapping investigations have used the nitrone spin traps (PBN, POBN, DMPO). Although the major difficulty of the spin-trapping technique in vivo is the mere detection of a radical adduct, other factors such as absorption, distribution, metabolism and excretion must be considered when spin traps are administered in vivo. In addition, artifacts and ambiguities in the assignment of radical adduct structure must be taken into serious consideration. The group has pioneered the application of the ESR spin-trapping technique to biochemical, pharmacological and toxicological problems by using two major approaches for in vivo spin-trapping and ex vivo ESR detection of free radicals: lipid extraction of radical adducts generated in tissues and detection of radical adducts in biological fluids. i. Metal-mediated Free Radical Production: ESR spin-trapping investigations of acute cadmium- and/or diesel exhaust particle poisoning have shown the in vivo formation of either the hydroxyl radical in the liver or lipid-derived radicals in the lungs. ii. Characterization of Endogenous Radical Adducts by HPLC/ESR/MS: The combination of an on-line high performance liquid chromatography (HPLC)/electron spin resonance (ESR) system with mass spectrometric analysis (MS) was used to identify a variety of lipid-derived radicals formed from lipid peroxidation both in vitro and in vivo. For the first time, POBN adducts of linoleic acid carbon-centered pentadienyl radicals were detected and identified. iii. Novel Methanol and Ethylene Glycol Free Radical Metabolites: The investigations of ethanols free radical metabolism have been extended using knockout mice, chemical analogues such as ethylene glycol and methanol, and their metabolites such as formic acid. The evidence for formate-derived radical metabolites in vivo, during acute sodium formate poisoning is provided by ESR spin trapping of the radicals from the metabolism of formate, which are detected in bile and urine as radical adducts.