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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Published in: | Antioxidants Vol. 12; no. 9; p. 1689 |
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Main Authors: | , , , , , , , , |
Format: | Journal Article |
Language: | English |
Published: |
Switzerland
MDPI AG
30-08-2023
MDPI |
Subjects: | |
Online Access: | Get full text |
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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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Bibliography: | ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23 |
ISSN: | 2076-3921 2076-3921 |
DOI: | 10.3390/antiox12091689 |