Validation of lucigenin (bis-N-methylacridinium) as a chemilumigenic probe for detecting superoxide anion radical production by enzymatic and cellular systems.
Lucigenin is most noted for its wide use as a chemiluminescent detector of superoxide anion radical (O2-.) production by biological systems. However, its validity as a O2-.-detecting probe has recently been questioned in view of its ability to undergo redox cycling in several in vitro enzymatic systems, which produce little or no O2-.. Whether and to what extent lucigenin redox cycling occurs in systems that produce significant amounts of O2-. has not been carefully investigated. We examined and correlated three end points, including sensitive measurement of lucigenin-derived chemiluminescence (LDCL), O2 consumption by oxygen polarography, and O2-. production by 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline-N-oxide spin trapping to characterize the potential of lucigenin to undergo redox cycling and as such to act as an additional source of O2-. in various enzymatic and cellular systems. Marked LDCL was elicited at lucigenin concentrations ranging from 1 to 5 microM in all of the O2-.-generating systems examined, including xanthine oxidase (XO)/xanthine, lipoamide dehydrogenase/ NADH, isolated mitochondria, mitochondria in intact cells, and phagocytic NADPH oxidase. These concentrations of lucigenin were far below those that stimulated additional O2 consumption or O2-. production in the above systems. Moreover, a significant linear correlation between LDCL and superoxide dismutase-inhibitable cytochrome c reduction was observed in the XO/ xanthine and phagocytic NADPH oxidase systems. In contrast to the above O2-.-generating systems, no LDCL was observed at non-redox cycling concentrations of lucigenin in the glucose oxidase/glucose and XO/NADH systems, which do not produce a significant amount of O2-.. Thus, LDCL still appears to be a valid probe for detecting O2-. production by enzymatic and cellular sources.
Li
Y
Y
Department of Environmental Health Sciences, Johns Hopkins, University School of Hygiene and Public Health, Baltimore, Maryland 21205, USA.
Zhu
H
H
Kuppusamy
P
P
Roubaud
V
V
Zweier
J L
JL
Trush
M A
MA
D000166:Acridines; D002454:Cell Differentiation; D002460:Cell Line; D003497:Cyclic N-Oxides; D006801:Humans; D007202:Indicators and Reagents; D008163:Luminescent Measurements; D008264:Macrophages / Q000378:metabolism / Q000648:ultrastructure; D008956:Models, Chemical; D009000:Monocytes / Q000378:metabolism / Q000648:ultrastructure; D009243:NAD / Q000378:metabolism; D019255:NADPH Oxidases / Q000378:metabolism; D010101:Oxygen Consumption; D011048:Polarography; D011190:Potassium Cyanide / Q000494:pharmacology; D019097:Spin Trapping; D013481:Superoxides / Q000032:analysis*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D000166:Acridines; D003497:Cyclic N-Oxides; D007202:Indicators and Reagents; D009243:NAD; D013481:Superoxides; C098505:5-diethoxyphosphoryl-5-methyl-1-pyrroline N-oxide; C033472:10,10'-dimethyl-9,9'-biacridinium; D019255:NADPH Oxidases; D011190:Potassium Cyanide; D013755:Tetradecanoylphorbol Acetate