The demonstration of DMPO superoxide adduct upon reperfusion using a low non-toxic concentration

Experiments were conducted in an attempt to circumvent the problem associated with the use of a high concentration of the spin trap, 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), which has been suggested to have serious toxic effects on tissue. A low concentration of DMPO (10 mM) was used to detect the free radical generation in hearts with ischemia/reperfusion insult. In the effluent immediately after reperfusion, DMPO-OOH, a superoxide spin adduct of DMPO, was obtained. DMPO in that concentration range did not interfere at all with the left ventricular (LV) function during the control perfusion period. Even after reperfusion, LV function was not depressed any more than that occurring in hearts without DMPO, whereas DMPO of the conventional concentration (100 mM) markedly depressed the ventricular function. Enzyme leakage from hearts also supported non-toxicity finding of DMPO at 10 mM; confirming that the DMPO superoxide adduct is real evidence of the generation of superoxide upon reperfusion and is not attributed to an artificial generation due to the cytotoxicity of DMPO.