TY - JOUR
T1 - ESR and HPLC-EC analysis of the interaction of hydroxyl radical with DMSO
T2 - Rapid reduction and quantification of POBN and PBN nitroxides
AU - Stoyanovsky, Detcho A.
AU - Melnikov, Zoya
AU - Cederbaum, Arthur I.
PY - 1999/2/1
Y1 - 1999/2/1
N2 - 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 α-(4-pyridyl-1-oxide)-N-tert- butylnitrone (POBN) and α-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.
AB - 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 α-(4-pyridyl-1-oxide)-N-tert- butylnitrone (POBN) and α-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.
UR - https://www.scopus.com/pages/publications/0033083478
U2 - 10.1021/ac980657r
DO - 10.1021/ac980657r
M3 - Article
C2 - 9989388
AN - SCOPUS:0033083478
SN - 0003-2700
VL - 71
SP - 715
EP - 721
JO - Analytical Chemistry
JF - Analytical Chemistry
IS - 3
ER -