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

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.

SUBMITTER: Haigh S

PROVIDER: S-EPMC10525458 | biostudies-literature | 2023 Aug

REPOSITORIES: biostudies-literature

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

Haigh Stephen S Brown Zach L ZL Shivers Mitch A MA Sellers Hunter G HG West Madison A MA Barman Scott A SA Stepp David W DW Csanyi Gabor G Fulton David J R DJR

Antioxidants (Basel, Switzerland) 20230830 9

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 tha ...[more]

PMID: 37759992

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

Publications

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

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