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 (O-2(-.)) p roduction is of critical importance for investigating the pathophysiologica l consequences resulting from altered cellular reactive oxygen homeostasis. The purpose of this study with isolated mitochondria was to characterize t he biochemical basis for lucigenin as a chemiluminescent probe to detect in tramitochondrial O-2(-.) production. Incubation of isolated mitochondria wi th lucigenin at non-redox cycling concentration produced lucigenin-derived chemiluminescence (LDCL), which was increased markedly by mitochondrial sub strates, pyruvate/malate or succinate. The LDCL was reduced greatly by the membrane permeable superoxide dismutase (SOD) mimetics, 2,2,6,6-tetramethyl piperidine-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 accu mulation 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 dissipa te the mitochondrial membrane potential. With submitochondrial particles, w e observed that both complexes I and III of the mitochondrial electron tran sport chain appear to be able to catalyze the one electron reduction of luc igenin, a critical step involved in LDCL. After incubation of mitochondria with lucigenin at non-redox cycling concentrations, formation of N-methylac ridone, the proposed end product of the reaction pathway leading to LDCL, w ithin the mitochondrial fraction was also detected. In addition, a signific ant linear correlation was observed between the LDCL and either the lucigen in accumulation or the N-methylacridone formation within the mitochondria. Taken together, our results conclusively demonstrate that when properly use d LDCL can reliably detect intramitochondrial O-2(-.) production. (C) 1999 Academic Press.