Detection of superoxide and peroxynitrite in model systems and mitochondria by the luminol analogue L-012.
In the present study we investigated the specificity and sensitivity of the chemiluminescence (CL) dye and luminol analogue 8-amino-5-chloro-7-phenylpyrido[3,4-d]pyridazine-1,4-(2H,3H) dione (L-012) to detect reactive oxygen species (ROS) such as superoxide, peroxynitrite and hydrogen peroxide in cell free systems as well as in isolated mitochondria. The results obtained by L-012 were compared with other CL substances such as luminol, lucigenin, coelenterazine and the fluorescence dye dihydroethidine. The results indicate that the L-012-derived chemiluminescence induced by superoxide from hypoxanthine/xanthine oxidase (HX/XO) or by 3-morpholino sydnonimine (SIN-1)-derived peroxynitrite largely depends on the incubation time. Irrespective of the experimental conditions, L-012-derived CL in response to HX/XO and SIN-1 was 10-100 fold higher than with other CL dyes tested. In a cell-free system, authentic peroxynitrite yielded a higher L-012-enhanced CL signal than authentic superoxide and the superoxide-induced signal in cell-free as well as isolated mitochondria increased in the presence of equimolar concentrations of nitrogen monoxide (NO). The superoxide signal/background ratio detected by L-012-enhanced CL in isolated mitochondria with blocked respiration was 7 fold higher than that obtained by the superoxide sensitive fluorescence dye dihydroethidine. We conclude that L-012-derived CL may provide a sensitive and reliable tool to detect superoxide and peroxynitrite formation in mitochondrial suspensions.
Daiber
Andreas
A
Universitätsklinikum Hamburg-Eppendorf, Medizinische Klinik III, Angiologie und Kardiologie, Martinistr. 52, 20246 Hamburg, Germany. daiber@uke.uni-hamburg.de
Oelze
Matthias
M
August
Michael
M
Wendt
Maria
M
Sydow
Karsten
K
Wieboldt
Hartwig
H
Kleschyov
Andrei L
AL
Munzel
Thomas
T
D000818:Animals; D002474:Cell-Free System / Q000737:chemistry; D005456:Fluorescent Dyes / Q000737:chemistry; D008163:Luminescent Measurements; D008165:Luminol / Q000031:analogs & derivatives* / Q000737:chemistry*; D008928:Mitochondria / Q000737:chemistry / Q000378:metabolism*; D009566:Nitrates / Q000032:analysis* / Q000378:metabolism; D009589:Nitrogen Oxides / Q000378:metabolism; D010084:Oxidation-Reduction; D051381:Rats; D017382:Reactive Oxygen Species / Q000032:analysis / Q000378:metabolism; D013481:Superoxides / Q000032:analysis* / Q000378:metabolism
D005456:Fluorescent Dyes; D009566:Nitrates; D009589:Nitrogen Oxides; D017382:Reactive Oxygen Species; D013481:Superoxides; C081614:L 012; C039642:peroxynitric acid; D008165:Luminol