Reestimation of Cypridina luciferin analogs (MCLA) as a chemiluminescence probe to detect active oxygen species--cautionary note for use of MCLA.

Cypridina luciferin analogs have been widely used as a specific chemiluminescence probe for the detection of superoxide anion (O2-) and singlet oxygen (1O2). However, light emission during the reaction of Cypridina luciferin analogs and other active oxygen species (AOS) has not been reported in detail. Therefore, we re-evaluated 2-methyl-6-(p-methoxyphenyl)-3,7-dihydroimidazo[1,2-a]pyrazine-3-one (MCLA), one of the Cypridina luciferin analogs, as a chemiluminescence probe to detect various AOS. MCLA-dependent chemiluminescence was observed when MCLA was incubated with the following systems; 1) hypoxanthine plus xanthine oxidase (O2-), 2) thermolysis of endoperoxide (1O2), 3) hydrogen peroxide plus ferrous ion (hydroxyl radical), 4) ferrous ion, 5) thermolysis of azo compound (alkyl peroxyl radical) and 6) hydrogen peroxide. Superoxide dismutase inhibited MCLA-dependent chemiluminescence observed during ferrous ion-induced decomposition of hydrogen peroxide. Alkyl peroxyl radical reacted with MCLA, but light was not emitted when the concentration of MCLA was high. These results suggest that radicals, except O2-, appeared not to be direct inducers of MCLA-dependent light emission. In summary, MCLA-dependent chemiluminescence was induced by various AOS in addition to O2- and 1O2, but active species must be O2- and 1O2 in many cases. These points should be appreciated when Cypridina luciferin analogs, such as MCLA, are used for the detection of AOS.