Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton, Alberta T6G 2E1, Canada.
Anal Chem. 2012 Aug 7;84(15):6739-46. doi: 10.1021/ac301142c. Epub 2012 Jul 12.
Free radicals are conventionally detected by electron paramagnetic resonance (EPR) spectroscopy after being trapped as spin adducts. Albeit this technique has demonstrated utmost efficacy in studying free radicals, its application to biological settings is intrinsically hampered by the inevitable bioreduction of radical-derived paramagnetic adducts. Herein, we describe a reliable technique to detect and quantify free radical metabolites, wherein reduced alkyl- and phenyl-5,5-dimethyl-1-pyrroline N-oxide (DMPO) adducts are converted into ultrastable N-naphthoate esters. To mimic the ubiquitous in vivo microenvironment, bioreductants, exogenous thiols, and sodium borohydride were studied. Nitroxyl reduction was confirmed using EPR and triphenyltetrazolium chloride. The formation of the N-naphthoyloxy derivatives was established by liquid chromatography/mass spectrometry (LC/MS). The derivatives were chromatographed using a binary eluent. HPLC and internal standards were synthesized using Grignard addition. The labeled DMPO adduct is (1) fluorescent, (2) stable as opposed to nitroxyl radical adducts, (3) biologically relevant, and (4) excellently chromatographed. Applications encompassed chemical, biochemical, and biological model systems generating C-centered radicals. Different levels of phenyl radicals produced in situ from whole blood were successfully determined. The method is readily applicable to the detection of hydroxyl radical. Analogously, DMPO, the spin trap, could be detected with extreme sensitivity suitable for in vivo applications. The developed method proved to be a viable alternative to EPR, where for the first time the reductive loss of paramagnetic signals of DMPO-trapped free radicals is transformed into fluorescence emission. We believe the proposed methodology could represent a valuable tool to probe free radical metabolites in vivo using DMPO, the least toxic spin trap.
自由基通常通过电子顺磁共振（EPR）光谱法检测，方法是将其作为自旋加合物捕获。尽管该技术在研究自由基方面已证明具有极高的功效，但由于自由基衍生的顺磁加合物不可避免的生物还原，其在生物环境中的应用受到固有阻碍。在此，我们描述了一种可靠的技术，用于检测和定量自由基代谢物，其中还原的烷基和苯基-5,5-二甲基-1-吡咯啉 N-氧化物（DMPO）加合物被转化为超稳定的 N-萘酸酯。为了模拟无处不在的体内微环境，研究了生物还原剂、外源性巯基化合物和硼氢化钠。使用 EPR 和三苯基氯化四唑证实了氮氧自由基的还原。通过液相色谱/质谱（LC/MS）证实了 N-萘酰氧基衍生物的形成。使用二元洗脱剂对衍生物进行色谱分离。使用格氏加成合成 HPLC 和内标。标记的 DMPO 加合物（1）具有荧光性，（2）与氮氧自由基加合物相比稳定，（3）具有生物学相关性，（4）出色的色谱分离。该方法适用于产生 C 中心自由基的化学、生化和生物学模型系统。成功地测定了来自全血原位产生的不同水平的苯基自由基。该方法易于应用于羟基自由基的检测。类似地，DMPO（自旋捕集剂）可以以非常高的灵敏度检测到，适用于体内应用。所开发的方法被证明是 EPR 的可行替代方法，其中首次将 DMPO 捕获的自由基的顺磁信号的还原损失转化为荧光发射。我们相信，该方法可以代表一种使用 DMPO（毒性最低的自旋捕集剂）在体内探测自由基代谢物的有价值的工具。
