Ste-Marie L, Boismenu D, Vachon L, Montgomery J
Department of Nutrition, Université de Montréal, Quebec, Canada.
Anal Biochem. 1996 Oct 1;241(1):67-74. doi: 10.1006/abio.1996.0379.
Molecular and tissue damage induced by reactive oxygen species is a serious consequence of the production of free radicals in biological systems. Biological markers produced by reactions with hydroxyl radicals are useful indices of free radical processes in vivo. In this respect, hydroxylation of aromatic compounds such as salicylate (2-hydroxybenzoate) has been used extensively as a measure of hydroxyl radical formation. 4-Hydroxybenzoate will also trap hydroxyl radicals with fewer of the complications for which salicylate has been criticized. We describe two sensitive and specific methods using gas chromatography-ion trap mass spectrometry and high-performance liquid chromatography with electrochemical detection for a number of these aromatic marker compounds in biological fluids. The use of an ion trap mass spectrometer provides enhanced sensitivity along with full mass spectral identification of the compounds of interest. 4-Hydroxybenzoate and salicylate were compared as hydroxyl radical traps (i) by determining relative hydroxyl radical trapping efficiencies in vitro, (ii) by measuring individual dihydroxybenzoate isomers in rat serum following intraperitoneal injection of either 2- or 4-hydroxybenzoate, and (iii) by comparing in vivo hydroxyl radical trapping using intrastriatal microdialysis in the rat. The techniques described have broad applications in the area of free radical biomedical research.
活性氧引起的分子和组织损伤是生物系统中自由基产生的严重后果。与羟基自由基反应产生的生物标志物是体内自由基过程的有用指标。在这方面，芳香族化合物如水杨酸盐（2-羟基苯甲酸盐）的羟基化已被广泛用作羟基自由基形成的度量。4-羟基苯甲酸盐也能捕获羟基自由基，且不存在水杨酸所遭受批评的诸多复杂问题。我们描述了两种灵敏且特异的方法，分别使用气相色谱 - 离子阱质谱法和高效液相色谱 - 电化学检测法来测定生物流体中的多种此类芳香族标志物化合物。离子阱质谱仪的使用提高了灵敏度，并能对目标化合物进行全质谱鉴定。通过（i）在体外测定相对羟基自由基捕获效率，（ii）在大鼠腹腔注射2 - 羟基苯甲酸盐或4 - 羟基苯甲酸盐后测量大鼠血清中的二羟基苯甲酸盐异构体，以及（iii）在大鼠中使用纹状体内微透析比较体内羟基自由基捕获情况，对4 - 羟基苯甲酸盐和水杨酸作为羟基自由基捕获剂进行了比较。所描述的技术在自由基生物医学研究领域具有广泛应用。
