Using ESR spectroscopy, it was showed that manganous could substitute for iron or copper ion in Fenton system to evolve hydroxyl radical. In the presence of 5,5-dimethyl-1-pyrroline-1-oxide (DMPO), four hyperfine splitting line signal of DMPO/•OH adduct was observed. HPLC with fluorescence detection (excitation 277 nm and emission 306 nm) was also used to detect the generation of hydroxyl radical in this system. L-Phenylalanine, which could be converted to highly fluorescent L-tyrosine in the presence of •OH, was used as fluorescence probe. The variety of fluorescence intensity of L-tyrosine reflected the generation of •OH. ESR spectrum and HPLC result proved that hydroxyl radical was generated in this system. The effect of several in vivo environment factors, such as Cu-Zn superoxide dismutase (Cu-Zn SOD), chelators (phosphate, pyrophosphate, EDTA and desferrioxamine) were also considered. ESR spectrum showed that Cu-Zn SOD restrained DMPO/•OH adduct signal markedly, which could not be restrained by denatured enzyme. Chelators partly restricted the generation of •OH, but could not eliminate the radical. It is suggested that hydroxyl radical initiated by manganous complex and hydrogen peroxide might be an important reason why manganous could induce oxidative stress in vivo.

Key words: manganous, hydroxyl radical, Fenton like reaction, ESR, HPLC, oxidative stress