Comparison of superoxide detection abilities of newly developed spin traps in the living cells.
This study compared the superoxide detection abilities of four spin traps, 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline N-oxide (DEPMPO), 5-(diphenylphosphinoyl)-5-methyl-1pyrroline N-oxide (DPPMPO) and 5-(2,2-dimethyl-1,3-propoxy cyclophosphoryl)-5-methyl-1-pyrroline N-oxide (CYPMPO) in living cells. Electron spin resonance (ESR) signals of the superoxide adducts were observed when spin traps were added to a suspension of human oral polymorphonuclear leukocytes (OPMNs) stimulated by phorbol 12-myristate 13-acetate. The ESR signal of the CYPMPO-superoxide adduct (CYPMPO-OOH) increased for 24 min after the initiation of the reaction, whereas the signals from DMPO-OOH and DPPMPO-OOH peaked at 6 and 10 min, respectively. The maximum concentrations of DMPO-OOH, DPPMPO-OOH and CYPMPO-OOH in OPMNs were 1.9, 6.0 and 10.7 microM, respectively. Furthermore, CYPMPO could more efficiently trap superoxide in blood PMNs compared with DEPMPO. From these results, it was concluded that CYPMPO performs better than DMPO, DPPMPO and DEPMPO for superoxide measurements in living cell systems because it has lower cytotoxicity and its superoxide adduct has a longer lifetime.
Saito
Keita
K
New Industry Creation Hatchery Center, Tohoku University, Sendai, Japan.
Takahashi
Miho
M
Kamibayashi
Masato
M
Ozawa
Toshihiko
T
Kohno
Masahiro
M
D002273:Carcinogens / Q000494:pharmacology; D002478:Cells, Cultured; D003497:Cyclic N-Oxides / Q000737:chemistry*; D004578:Electron Spin Resonance Spectroscopy; D005609:Free Radicals / Q000737:chemistry; D006801:Humans; D009504:Neutrophils / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D017382:Reactive Oxygen Species / Q000378:metabolism; D013113:Spin Labels; D013481:Superoxides / Q000737:chemistry*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
C516729:5-(2,2-dimethyl-1,3-propoxy cyclophosphoryl)-5-methyl-1-pyrroline N-oxide; D002273:Carcinogens; D003497:Cyclic N-Oxides; D005609:Free Radicals; D017382:Reactive Oxygen Species; D013113:Spin Labels; D013481:Superoxides; C017245:5,5-dimethyl-1-pyrroline-1-oxide; C055956:5,5-dimethyl-5-hydroperoxy-1-pyrrolidinyloxy; D013755:Tetradecanoylphorbol Acetate