A Reappraisal of the Utility of L-012 to Measure Superoxide from Biologically Relevant Sources

The detection of superoxide anion (O ) in biological tissues remains challenging. Barriers to convenient and reproducible measurements include expensive equipment, custom probes, and the need for high sensitivity and specificity. The luminol derivative, L-012, has been used to measure O since 1993 w...

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Bibliographic Details
Published in:Antioxidants Vol. 12; no. 9; p. 1689
Main Authors: Haigh, Stephen, Brown, Zach L, Shivers, Mitch A, Sellers, Hunter G, West, Madison A, Barman, Scott A, Stepp, David W, Csanyi, Gabor, Fulton, David J R
Format: Journal Article
Language:English
Published: Switzerland MDPI AG 30-08-2023
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Summary:The detection of superoxide anion (O ) in biological tissues remains challenging. Barriers to convenient and reproducible measurements include expensive equipment, custom probes, and the need for high sensitivity and specificity. The luminol derivative, L-012, has been used to measure O since 1993 with mixed results and concerns over specificity. The goal of this study was to better define the conditions for use and their specificity. We found that L-012 coupled with depolymerized orthovanadate, a relatively impermeable tyrosine phosphatase inhibitor, yielded a highly sensitive approach to detect extracellular O . In O producing HEK-NOX5 cells, orthovanadate increased L-012 luminescence 100-fold. The combination of L-012 and orthovanadate was highly sensitive, stable, scalable, completely reversed by superoxide dismutase, and selective for O generating NOXes versus NOX4, which produces H O . Moreover, there was no signal from cells transfected with NOS3 (NO ) and NOS2(ONOO ). To exclude the effects of altered tyrosine phosphorylation, O was detected using non-enzymatic synthesis with phenazine methosulfate and via novel coupling of L-012 with niobium oxalate, which was less active in inducing tyrosine phosphorylation. Overall, our data shows that L-012 coupled with orthovanadate or other periodic group 5 salts yields a reliable, sensitive, and specific approach to measuring extracellular O in biological systems.
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ISSN:2076-3921
2076-3921
DOI:10.3390/antiox12091689