Validation of lucigenin (bis-N-methylacridinium) as a chemilumigenic probe for detecting superoxide anion radical production by enzymatic and cellular systems

J Biol Chem. 1998 Jan 23;273(4):2015-23. doi: 10.1074/jbc.273.4.2015.


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.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Acridines*
  • Cell Differentiation
  • Cell Line
  • Cyclic N-Oxides
  • Humans
  • Indicators and Reagents*
  • Luminescent Measurements
  • Macrophages / metabolism
  • Macrophages / ultrastructure
  • Models, Chemical
  • Monocytes / metabolism
  • Monocytes / ultrastructure
  • NAD / metabolism
  • NADPH Oxidases / metabolism
  • Oxygen Consumption
  • Polarography
  • Potassium Cyanide / pharmacology
  • Spin Trapping
  • Superoxides / analysis*
  • Tetradecanoylphorbol Acetate / pharmacology


  • Acridines
  • Cyclic N-Oxides
  • Indicators and Reagents
  • NAD
  • Superoxides
  • 5-diethoxyphosphoryl-5-methyl-1-pyrroline N-oxide
  • 10,10'-dimethyl-9,9'-biacridinium
  • NADPH Oxidases
  • Potassium Cyanide
  • Tetradecanoylphorbol Acetate