Stoyanovsky D A, Melnikov Z, Cederbaum A I
Department of Biochemistry, Mount Sinai School of Medicine, New York, New York 10029, USA.
Anal Chem. 1999 Feb 1;71(3):715-21. doi: 10.1021/ac980657r.
The low stability of hydroxyl radical (OH.)-derived nitroxides is a limiting factor for direct spin-trapping of OH. in biological systems. The latter experimental difficulty is partly solved with the introduction of dimethyl sulfoxide (DMSO) into the studied systems. Hydroxyl radical oxidizes DMSO to methyl radical, which forms relatively stable nitroxides. The results of the present work provide evidence that in alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone (POBN) and alpha-phenyl-N-tert-butylnitrone (PBN) spin-trapping experiments aimed to detect methyl radical in biological systems, the nitroxides formed can be reduced to their ESR-"silent" hydroxylamine derivatives. The nitroxides and their hydroxylamine derivatives were successfully analyzed by HPLC with electrochemical (EC) and UV detection. The lowest limits of UV and EC detection of POBN/CH3 hydroxylamine was evaluated to be in the micro- and nanomolar range, respectively. In parallel ESR and HPLC-EC analysis of the metabolism of menadione by either HepG2 cells or isolated rat hepatocytes in the presence of DMSO, the HPLC-EC method has proven to be more sensitive in detecting the production of methyl radical. The use of the HPLC-EC detection of POBN/CH3 and PBN/CH3 is expected to be advantageous in detection of hydroxyl radical in biological systems in the presence of DMSO.
羟基自由基（OH·）衍生的氮氧化物稳定性较低，这是在生物系统中直接自旋捕获OH·的一个限制因素。通过将二甲基亚砜（DMSO）引入所研究的系统，部分解决了后一个实验难题。羟基自由基将DMSO氧化为甲基自由基，后者形成相对稳定的氮氧化物。本研究结果表明，在旨在检测生物系统中甲基自由基的α-（4-吡啶基-1-氧化物）-N-叔丁基硝酮（POBN）和α-苯基-N-叔丁基硝酮（PBN）自旋捕获实验中，形成的氮氧化物可被还原为其电子自旋共振（ESR）“沉默”的羟胺衍生物。通过高效液相色谱（HPLC）结合电化学（EC）和紫外检测成功分析了氮氧化物及其羟胺衍生物。POBN/CH3羟胺的紫外和EC检测最低限分别评估为微摩尔和纳摩尔范围。在DMSO存在下，对HepG2细胞或分离的大鼠肝细胞中维生素K3代谢进行的平行ESR和HPLC-EC分析表明，HPLC-EC方法在检测甲基自由基生成方面更灵敏。在DMSO存在的情况下，使用HPLC-EC检测POBN/CH3和PBN/CH3有望在生物系统中检测羟基自由基方面具有优势。
