Production of OH-Radical-Type Oxidant by Lucigenin

Abstract

In the presence of NADH- reductases (dihydrolipoamide: NAD oxidoreductase E. C.l.8.1.4 from pig heart or from Clostridium kluyveri; frequently also addressed as “diaphorases”) and NADH lucigenin strongly increases ethylene production from a-ketomethylthiobutyrate (KMB) as an indicator for strong oxidants of the OH-radical type. These reactions are further stimulated in the presence of Fe3+ ions. With these NADH- “diaphorases”, the structurally similar poison, paraquat, in the absence or presence of Fe3+ has no effect. With ferredoxin-NADP reductase (E. C.1.18.1.2.), however, paraquat reacts quasi identical to lucigenin. Superoxide dismutase, catalase, free radical- or OH - scavengers such as mannitol, propylgallate, DABCO, and desferal inhibit the reaction whereas EDTA (in the presence or absence of added Fe3+) is stimulatory. From these data we conclude that the superoxide - indicator LUC is redox-active after unspecific coupling to several almost ubiquitory NAD(P)H- reductases catalyzing monovalent oxygen reduction. Lucigenin thus should no longer be used as a “specific” superoxide indicator. This report is in agreement with very recent results by Liochev and Fridovich (Arch. Biochem. Biophys. 337, 115 [1997]) and Vasquez-Vivar et al. (FEBS Lett. 403, 127 (1997).

Use of spectroscopic probes for detection of reactive oxygen species

“…Although the intensity of luminescence measured under these conditions may be too low for typical luminometers, a scintillation counter switched to the ''outof-coincidence'' mode may be used. Lucigenin cation radical can also reduce substances such as cytochrome c [111] and, in the presence of NADH reductases, can enhance ethylene production from a-keto-methylthiobutyrate (KMB), indicating formation of a strong oxidant similar to the hydroxyl radical [112].…”

Chemiluminescent Detection of Oxidants in Vascular Tissue

“…This will enhance nonrespiratory mitochondrial oxygen consumption 32 because of lucigenin-mediated redirection of reducing equivalents to O 2 • and H 2 O 2 formation. 30 Thus, it would be expected that a significant fraction of mitochondrial lucigenin-dependent chemiluminescence is due to direct respiratory chain reduction of lucigenin, extramitochondrial diffusion of the mono-cation radical, and subsequent autooxidation to O 2…”

Antimicrobial peptides and endotoxin inhibit cytokine and nitric oxide release but amplify respiratory burst response in human and murine macrophages

“…To confirm enhancement of ROS release by AMP and endotoxin, an inhibitable cytochrome c reduction assay was employed as an alternative approach for the chemiluminescent detection method with lucigenin or luminol (Han et al ., 1998). High concentrations of lucigenin have been proposed to effect ROS generation (Heiser et al ., 1998; Liochev and Fridovich, 1998). THP‐1 cells were primed with 5.6 pmole ml −1 of N. meningitidis LOS overnight in presence or absence of AMP (2 µg ml −1 ).…”