ESR and HPLC-EC Analysis of the Interaction of Hydroxyl Radical with DMSO: Rapid Reduction and Quantification of POBN and PBN Nitroxides
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- 作者：Detcho A. Stoyanovsky ; Zoya Melnikov ; and Arthur I. Cederbaum
- 刊名：Analytical Chemistry
- 出版年：1999
- 出版时间：February 1, 1999
- 年：1999
- 卷：71
- 期：3
- 页码：715 - 721
- 全文大小：111K
- 年卷期：v.71,no.3(February 1, 1999)
- ISSN：1520-6882
文摘
The low stability of hydroxyl radical (OH
)-derived nitroxides is a limiting factor for direct spin-trapping of OH inbiological systems. The latter experimental difficulty ispartly solved with the introduction of dimethyl sulfoxide(DMSO) into the studied systems. Hydroxyl radical oxidizes DMSO to methyl radical, which forms relativelystable nitroxides. The results of the present work provideevidence that in 
-(4-pyridyl-1-oxide)-N-tert-butylnitrone(POBN) and -phenyl-N-tert-butylnitrone (PBN) spin-trapping experiments aimed to detect methyl radical inbiological systems, the nitroxides formed can be reducedto their ESR-"silent" hydroxylamine derivatives. The nitroxides and their hydroxylamine derivatives were successfully analyzed by HPLC with electrochemical (EC) andUV detection. The lowest limits of UV and EC detectionof POBN/CH3 hydroxylamine was evaluated to be in themicro- and nanomolar range, respectively. In parallel ESRand HPLC-EC analysis of the metabolism of menadioneby either HepG2 cells or isolated rat hepatocytes in thepresence of DMSO, the HPLC-EC method has proven tobe more sensitive in detecting the production of methylradical. The use of the HPLC-EC detection of POBN/CH3and PBN/CH3 is expected to be advantageous in detectionof hydroxyl radical in biological systems in the presenceof DMSO.
)-derived nitroxides is a limiting factor for direct spin-trapping of OH inbiological systems. The latter experimental difficulty ispartly solved with the introduction of dimethyl sulfoxide(DMSO) into the studied systems. Hydroxyl radical oxidizes DMSO to methyl radical, which forms relativelystable nitroxides. The results of the present work provideevidence that in -(4-pyridyl-1-oxide)-N-tert-butylnitrone(POBN) and -phenyl-N-tert-butylnitrone (PBN) spin-trapping experiments aimed to detect methyl radical inbiological systems, the nitroxides formed can be reducedto their ESR-"silent" hydroxylamine derivatives. The nitroxides and their hydroxylamine derivatives were successfully analyzed by HPLC with electrochemical (EC) andUV detection. The lowest limits of UV and EC detectionof POBN/CH3 hydroxylamine was evaluated to be in themicro- and nanomolar range, respectively. In parallel ESRand HPLC-EC analysis of the metabolism of menadioneby either HepG2 cells or isolated rat hepatocytes in thepresence of DMSO, the HPLC-EC method has proven tobe more sensitive in detecting the production of methylradical. The use of the HPLC-EC detection of POBN/CH3and PBN/CH3 is expected to be advantageous in detectionof hydroxyl radical in biological systems in the presenceof DMSO.