Impact of SOD-Mimetic Manganoporphyrins on Spin Trapping of Superoxide and Related Artifacts.

The superoxide dismutase (SOD)-mimetic effectiveness of [meso-tetrakis (R)porphyrinato]manganese with R = 1,3-di-N-ethylimidazolium-2-yl (Mn-TDEIP), 1,3-di-N-methylimidazolium-2-yl (Mn-TDMIP), 1,3-di-N-propylimidazolium-2-yl (Mn-TDPIP), N-ethyl-2-pyridyl (Mn-T2EPyP), 4-sulphonatophenyl (Mn-TSP), 1-methyl-4-pyridyl (Mn-T4PyP), 4-carboxyphenyl (Mn-TBAP), and β-octabromomeso-tetrakis(4-carboxyphenyl porphyrinato)manganese (MnBr8TBAP) was compared with Cu, Zn SOD. Superoxide generated by reaction of xanthine oxidase with hypoxanthine was trapped with 5-tert-butoxycarbonyl-5-methyl-1-pyrroline N-oxide (BMPO), forming BMPO-OOH, which was monitored by electron paramagnetic resonance. Manganoporphyrins with redox potentials ranging from -0.190 to 0.346 V relative to the standard hydrogen electrode were selected for this study. With 0.1 µM manganoporphyrins and 20 mM BMPO, the effectiveness of the manganoporphyrins in inhibiting formation of BMPO-OOH increases in the order Mn-TSP < Mn-TBAP < MnBr8TBAP < Mn-T4PyP < Mn-T2EPyP < Mn-TDEIP ~ Mn-TDMIP ~ Mn-TDPIP ~ Cu, Zn SOD. However, at higher concentrations of manganoporphyrin and BMPO, a BMPO-OH signal was observed. The formation of BMPO-OH was not inhibited by catalase or dimethylsulfoxide, which demonstrated that it was not produced from hydroxyl radical. The artifactual formation of BMPO-OH is attributed to oxidation of the water adduct of BMPO by the manganoporphyrins or decomposition of BMPO-OOH. Although spin trapping is an effective method for evaluating SOD-mimetic efficacy, caution must be exercised to ensure that artifact signals are not interpreted improperly.