Luminol-and lucigenin-amplified chemiluminescence with rat liver microsomes: Kinetics and influence of ascorbic acid, glutathione, dimethylsulfoxide, N-t-butyl-a-phenyl-nitrone, copper-ions and a copper complex, catalase, superoxide dismutase, hexobarbital and aniline

Abstract

For the investigation of luminol (LM)-and lucigenin (LC)-amplified chemiluminescence (CL) in rat liver microsomes using both a liquid-scintillation counter (LKB/Wallac 1219 Rackbeta) and a Berthold luminometer (AutoLumat LB 953) optimal incubation mixtures and conditions and basic kinetics have been established. Whereas calibration curves for both LM- and LC-CL are performed with hydrogenperoxide (LC quantum yield is 6.25 fold higher as that of LM), distinct differences were revealed with microsomes, indicating that different reactive oxygen species (ROS) are determined: Both LM- and LC-CL follow the kinetics of enzymatic reactions in terms of dependence on protein and NADPH or NADH concentration, time course, temperature etc., but with differences. LM-CL does not work without addition of Fe2+, whereas LC-CL does. Both copper ions and copper bound in a complex abolish CL, LC-CL being much more sensitive. Isolated cytochrome P-450 (P450) and NADPH P450 reductase from liver of pheno-barbital treated rats alone proved to be inactive in LM-and LC-CL production, whereas te combination 1:1 without and with addition of lipid was highly active in both LM-and LC-CL. Ascorbic acid and glutathione as scavengers diminish both LM- and LC-CL in concentrations higher then 10(5). Dimethyl-sulfoxide (DMSO) was ineffective in LM-CL up to concentrations of 0.2 M, the very high concentration of 2 M diminished LM-CL only to 1/3. LC-CL was diminished starting at concentrations of 100 mM and at 2 M only 10% of maximum LC-CL was observed. The trap substance N-t-butyl-a-phenylnitrone (BNP) also diminished LC-CL more effectively than LM-CL. Clearcut differences were revealed by the addition of catalase and superoxide dismutase: both enzymes diminished LM-CL only, without any influence on LC-CL. Hexobarbital, a potent uncoupler of P450, enhances LM-CL fivefold, whereas LC-CL is barely influenced. Aniline (without uncoupling capability) decreased both LM-and LC-CL increasingly with increasing concentrations. Therefore the conclusion is drawn that LM-CL measures in liver microsomes predominantly superoxide anion radicals, whereas LC-CL is mainly a measure for microsomal hydroxyl radical formation or of reactive organic radicals. With microsomes of phenobarbital and beta-naphthoflavone treated rats CL was much higher but in principle the same kinetic characteristics could be shown. All results on microsomes were obtained uniformly with the liquid scintillation counter and the Berthold luminometer, the letter being much more effective and more sensitive.