In Vivo Detection of Free Radical Generation

自旋捕捉法是目前检测体内高活性自由基分子最成功的方法。这项技术涉及直接检测初级自由基，而传统的ESR由于稳态浓度低或弛豫时间很短而无法直接观察到这些自由基，这导致了非常宽的谱线。所有报道的体内自旋捕获研究都使用了硝酮自旋陷阱(PBN，POBN，DMPO)。虽然体内自旋捕获技术的主要困难是仅仅检测到自由基加合物，但在体内应用自旋捕获时必须考虑其他因素，如吸收、分布、代谢和排泄。此外，必须认真考虑词根加合物结构赋值中的伪像和歧义。该小组率先将ESR自旋捕获技术应用于生化、药理学和毒理学问题，使用了体内自旋捕获和体外ESR检测自由基的两种主要方法：对组织中生成的自由基加合物进行脂质提取，以及检测生物液中的自由基加合物。 I.金属介导的自由基产生：对急性镉和/或柴油废气颗粒中毒的ESR自旋捕获研究表明，肝脏中的羟基自由基或肺中的脂类自由基在体内形成。 二、内源性自由基加合物的高效液相色谱/电子自旋共振/质谱联用表征：采用在线高效液相色谱/电子自旋共振与质谱分析相结合的方法，对体内和体外多种脂质过氧化生成的自由基进行鉴定。首次检测和鉴定了亚油酸碳中心戊二烯自由基的POBN加合物。 三、新型甲醇和乙二醇自由基代谢物：乙醇自由基代谢的研究已扩展到使用基因敲除小鼠、化学类似物(如乙二醇、甲醇)及其代谢物(如甲酸)。在急性甲酸钠中毒期间，体内存在甲酸盐衍生的自由基代谢物的证据是通过ESR自旋捕捉甲酸盐代谢产生的自由基，这些自由基在胆汁和尿液中以自由基加合物的形式被检测到。

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 particles poisoning have shown the in vivo formation of either the hydroxyl radical in the liver or a lipid derived radicals in the lungs. ii. Characterization of Endogenous Radical Adducts by HPLC/ESR/MS: The combination of 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 ethanol's free radical metabolism have been extended using both knockout mice, chemical analogues such as ethylene glycol, 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.