title: Measurement of NAD(P)H oxidase-derived superoxide with the luminol analogue L-012.
abstract: In the present study we sought to determine the ability of the chemiluminescence dye 8-amino-5-chloro-7-phenylpyridol[3,4-d]pyridazine-1,4-(2H,3H)dione sodium salt (L-012) to detect superoxide in different biological systems. In human whole blood or isolated leukocytes, the sensitivity of the luminol analogue L-012 to detect superoxide was higher as compared with luminol, lucigenin, coelenterazine, and the fluorescence dye dihydroethidine. In isolated leukocytes as well as aortic rings from control (New Zealand White) and hyperlipidemic (Watanabe heritable hyperlipidemic) rabbits, L-012-enhanced chemiluminescence was successful in detecting differences in superoxide formation under basal conditions and on stimulation with the direct activator of protein kinase C, phorbol 12,13-dibutyrate (PDBu). The effects of PDBu were abrogated by gliotoxin and inhibitors of protein kinase C such as chelerythrine, identifying NAD(P)H oxidase as the significant superoxide source. Experiments using electron paramagnetic resonance and the spin trap 5-diethoxyphosphoryl-5-methyl-1-pyrroline-N-oxide revealed that in contrast to lucigenin, L-012 is not subject to redox cycling. These findings indicate that L-012-enhanced chemiluminescence represents a sensitive and reliable probe to detect superoxide in whole blood, inflammatory cells, and vascular tissue.
authors.affiliation: Universitätsklinikum Hamburg-Eppendorf, Medizinische Klinik III, Angiologie und Kardiologie, Hamburg, Germany. daiber@uke.uni-hamburg.de
authors.forename: Andreas
authors.initials: A
authors.lastname: Daiber
authors.forename: Michael
authors.initials: M
authors.lastname: August
authors.forename: Stephan
authors.initials: S
authors.lastname: Baldus
authors.forename: Maria
authors.initials: M
authors.lastname: Wendt
authors.forename: Matthias
authors.initials: M
authors.lastname: Oelze
authors.forename: Karsten
authors.initials: K
authors.lastname: Sydow
authors.forename: Andrei L
authors.initials: AL
authors.lastname: Kleschyov
authors.forename: Thomas
authors.initials: T
authors.lastname: Munzel
mesh_terms: D000818:Animals; D000975:Antioxidants / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005453:Fluorescence; D006801:Humans; D007700:Kinetics; D007962:Leukocytes / Q000187:drug effects / Q000378:metabolism; D008163:Luminescent Measurements; D008165:Luminol / Q000031:analogs & derivatives* / Q000737:chemistry*; D019255:NADPH Oxidases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism; D011817:Rabbits; D017382:Reactive Oxygen Species / Q000378:metabolism; D015203:Reproducibility of Results; D013050:Spectrometry, Fluorescence; D013481:Superoxides / Q000032:analysis* / Q000097:blood
chemical_list: D000975:Antioxidants; D004791:Enzyme Inhibitors; D017382:Reactive Oxygen Species; D013481:Superoxides; C081614:L 012; D008165:Luminol; D019255:NADPH Oxidases; D011493:Protein Kinase C