Biochemical characterization of lucigenin (Bis-N-methylacridinium) as a chemiluminescent probe for detecting intramitochondrial superoxide anion radical production

Direct detection of intramitochondrial superoxide anion radical (O₂^-1) production is of critical importance for investigating the pathophysiological consequences resulting from altered cellular reactive oxygen homeostasis. The purpose of this study with isolated mitochondria was to characterize the biochemical basis for lucigenin as a chemiluminescent probe to detect intramitochondrial O₂^-1 production. Incubation of isolated mitochondria with lucigenin at non-redox cycling concentration produced lucigenin-derived chemiluminescence (LDCL), which was increased markedly by mitochondrial substrates, pyruvate/malate or succinate. The LDCL was reduced greatly by the membrane permeable superoxide dismutase (SOD) mimetics, 2,2,6,6-tetra-methylpiperidine-N-oxyl and Mn(III)tetrakis(1-methyl-4-pyridyl)porphyrin, but not by Cu,Zn-SOD. With an ion-pair HPLC method, a concentration-dependent accumulation of lucigenin was detected within mitochondria. The accumulation of lucigenin by mitochondria was reduced markedly in the presence of carbonyl cyanide p-(trifluoromethoxy)phenyhyldrazone, an uncoupler known to dissipate the mitochondrial membrane potential. With submitochondrial particles, we observed that both complexes I and III of the mitochondrial electron transport chain appear to be able to catalyze the one electron reduction of lucigenin, a critical step involved in LDCL. After incubation of mitochondria with lucigenin at non-redox cycling concentrations, formation of N-methylacridone, the proposed end product of the reaction pathway leading to LDCL, within the mitochondrial fraction was also detected. In addition, a significant linear correlation was observed between the LDCL and either the lucigenin accumulation or the N-methylacridone formation within the mitochondria. Taken together, our results conclusively demonstrate that when properly used LDCL can reliably detect intramitochondrial O₂^-1 production.