{"doi":"10.3390/antiox12091689","title":"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.","journal":"Antioxidants","year":2023,"id":377487,"datarank":0.0,"base_score":0.0,"endowment":0.0,"self_citation_contribution":0.0,"citation_network_contribution":0.0,"self_endowment_contribution":0.0,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":3,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9568,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2023-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":1044464,"name":"Zachary Brown","orcid":"0000-0002-1590-4534","position":1,"is_corresponding":false},{"id":1141560,"name":"Mitch Shivers","orcid":null,"position":2,"is_corresponding":false},{"id":1002419,"name":"Hunter Sellers","orcid":"0000-0001-8994-9404","position":3,"is_corresponding":false},{"id":1141179,"name":"Madison A. West","orcid":"0000-0002-9338-5872","position":4,"is_corresponding":false},{"id":330831,"name":"Scott A. Barman","orcid":"0000-0001-9085-5122","position":5,"is_corresponding":false},{"id":719660,"name":"David W. Stepp","orcid":"0000-0003-0184-8321","position":6,"is_corresponding":false},{"id":355756,"name":"Gábor Csányi","orcid":"0000-0002-4429-3031","position":7,"is_corresponding":false},{"id":250316,"name":"David Fulton","orcid":"0000-0002-1528-1517","position":8,"is_corresponding":false},{"id":665654,"name":"Stephen Haigh","orcid":null,"position":0,"is_corresponding":true}],"reference_count":40,"raw_metadata":null,"created_at":"2026-07-19T01:16:40.346877Z","pmid":"37759992","pmcid":null,"fwci":null,"citation_percentile":null,"influential_citations":0,"oa_status":null,"license":null,"views":0,"total_file_size_bytes":0,"version_count":0,"fair_f":null,"fair_a":null,"fair_i":null,"fair_r":null,"fair_zscore":null,"fair_rationale":null,"fair_model":null,"fair_agent_version":null,"fair_fulltext_source":null,"fair_has_llm":null,"fair_computed_at":null,"clinical_trials":[],"software_tools":[],"db_accessions":[],"linked_datasets":[],"topics":[]}