LUMINOL-AMPLIFIED AND LUCIGENIN-AMPLIFIED CHEMILUMINESCENCE WITH RAT-LIVER MICROSOMES - KINETICS AND INFLUENCE OF ASCORBIC-ACID, GLUTATHIONE, DIMETHYLSULFOXIDE, N-T-BUTYL-A-PHENYLNITRONE, COPPER-IONS AND A COPPER COMPLEX, CATALASE, SUPEROXIDE-DISMUTASE, HEXOBARBITAL AND ANILINE

For the investigation of luminol (LM)-and lucigenin (LC)-amplified che miluminescence (CL) in rat liver microsomes using both a liquid-scinti llation counter (LKB/Wallac 1219 Rackbeta) and a Berthold luminometer (AutoLumat LB 953) optimal incubation mixtures and conditions and basi c 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 w ith 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 concentr ation, time course, temperature etc., but with differences. LM-CL does not work without addition of Fe2+, whereas LC-CL does. Both copper io ns and copper bound in a complex abolish CL, LC-CL being much more sen sitive. Isolated cytochrome P-450 (P450) and NADPH P450 reductase from liver of phenobarbital treated rats alone proved to be inactive in LM -and LC-CL production, whereas te combination 1:1 without and with add ition of lipid was highly active in both LM-and LC-CL. Ascorbic acid a nd glutathione as scavengers diminish both LM- and LC-CL in concentrat ions 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 conce ntrations 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 L C-CL more effectively than LM-CL. Clearcut differences were revealed b y the addition of catalase and superoxide dismutase: both enzymes dimi nished LM-CL only, without any influence on LC-CL. Hexobarbital, a pot ent uncoupler of P450, enhances LM-CL fivefold, whereas LC-CL is barel y influenced. Aniline (without uncoupling capability) decreased both L M-and LC-CL increasingly with increasing concentrations. Therefore the conclusion is drawn that LM-CL measures in liver microsomes predomina ntly 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 le tter being much more effective and more sensitive.