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

Authors :
Stephen Haigh
Zach L. Brown
Mitch A. Shivers
Hunter G. Sellers
Madison A. West
Scott A. Barman
David W. Stepp
Gabor Csanyi
David J. R. Fulton
Source :
Antioxidants, Vol 12, Iss 9, p 1689 (2023)
Publication Year :
2023
Publisher :
MDPI AG, 2023.

Abstract

The detection of superoxide anion (O2●−) 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 O2●− 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 O2●−. In O2●− 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 O2●− generating NOXes versus NOX4, which produces H2O2. Moreover, there was no signal from cells transfected with NOS3 (NO●) and NOS2(ONOO−). To exclude the effects of altered tyrosine phosphorylation, O2●− 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 O2●− in biological systems.

Details

Language :
English
ISSN :
20763921
Volume :
12
Issue :
9
Database :
Directory of Open Access Journals
Journal :
Antioxidants
Publication Type :
Academic Journal
Accession number :
edsdoj.0a9fab2f08ad48e9b3da209f3c6b8be7
Document Type :
article
Full Text :
https://doi.org/10.3390/antiox12091689