{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,7,22]],"date-time":"2026-07-22T03:24:17Z","timestamp":1784690657334,"version":"3.55.0"},"reference-count":26,"publisher":"MDPI AG","issue":"1","license":[{"start":{"date-parts":[[2021,12,24]],"date-time":"2021-12-24T00:00:00Z","timestamp":1640304000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Antioxidants"],"abstract":"<jats:p>There is a considerable need for methods that allow quantitative determination in vitro and in vivo of transient oxidative species such as peroxynitrite (ONOOH\/ONOO\u2212) and superoxide (HO2\u2022\/O2\u2022\u2212). Cyclic hydroxylamines, which upon oxidation yield their respective stable nitroxide radicals, have been suggested as spin probes of peroxynitrite and superoxide. The present study investigated this approach by following the kinetics of peroxynitrite decay in the absence and presence of various 5-membered and 6-membered ring hydroxylamines, and comparing the yield of their respective nitroxides using electron paramagnetic spectroscopy. The results demonstrate that hydroxylamines do not react directly with peroxynitrite, but are oxidized to their respective nitroxides by the radicals formed during peroxynitrite self-decomposition, namely \u2022OH and \u2022NO2. The accumulated nitroxides are far below their expected yield, had the hydroxylamines fully scavenged all these radicals, due to multiple competing reactions of the oxidized forms of the hydroxylamines with \u2022NO2 and ONOO\u2212. Therefore, cyclic hydroxylamines cannot be used for quantitative assay of peroxynitrite in vitro. The situation is even more complex in vivo where \u2022OH and \u2022NO2 are formed also via other oxidizing reactions systems. The present study also compared the yield of accumulated nitroxides under constant flux of superoxide in the presence of various cyclic hydroxylamines. It is demonstrated that certain 5-membered ring hydroxylamines, which their respective nitroxides are poor SOD-mimics, might be considered as stoichiometric monitors of superoxide in vitro at highest possible concentrations and pH.<\/jats:p>","DOI":"10.3390\/antiox11010040","type":"journal-article","created":{"date-parts":[[2021,12,27]],"date-time":"2021-12-27T01:00:54Z","timestamp":1640566854000},"page":"40","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":10,"title":["Cyclic Hydroxylamines as Monitors of Peroxynitrite and Superoxide-Revisited"],"prefix":"10.3390","volume":"11","author":[{"given":"Uri","family":"Samuni","sequence":"first","affiliation":[{"name":"Department of Chemistry & Biochemistry, Queens College, City University of New York, Flushing, NY 11367, USA"},{"name":"The PhD Programs in Biochemistry and Chemistry, The Graduate Center of the City University of New York, New York, NY 10016, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Amram","family":"Samuni","sequence":"additional","affiliation":[{"name":"Institute of Medical Research, Israel-Canada Medical School, The Hebrew University of Jerusalem, Jerusalem 91120, Israel"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sara","family":"Goldstein","sequence":"additional","affiliation":[{"name":"Institute of Chemistry, The Hebrew University of Jerusalem, Jerusalem 91904, Israel"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2021,12,24]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"7","DOI":"10.1007\/s40828-019-0101-8","article-title":"Biological chemistry of superoxide radicals","volume":"6","author":"Winterbourn","year":"2020","journal-title":"ChemTexts"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"1041","DOI":"10.1063\/1.555739","article-title":"Reactivity of HO2\/O2\u2212 radicals in aqueous solution","volume":"14","author":"Bielski","year":"1985","journal-title":"J. Phys. Chem. Ref. Data"},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"662","DOI":"10.1038\/nrd2222","article-title":"Peroxynitrite: Biochemistry, pathophysiology and development of therapeutics","volume":"6","author":"Ischiropoulos","year":"2007","journal-title":"Nat. Rev. Drug Discov."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"1338","DOI":"10.1021\/acs.chemrev.7b00568","article-title":"Biochemistry of peroxynitrite and protein tyrosine nitration","volume":"118","author":"Campolo","year":"2018","journal-title":"Chem. Rev."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"4625","DOI":"10.1039\/C8AY01339J","article-title":"Methods for the detection of reactive oxygen species","volume":"10","author":"Zhang","year":"2018","journal-title":"Anal. Methods"},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"1433","DOI":"10.1089\/ars.2017.7396","article-title":"Electron paramagnetic resonance measurements of reactive oxygen species by cyclic hydroxylamine spin probes","volume":"28","author":"Dikalov","year":"2018","journal-title":"Antioxid. Redox Signal."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"2457","DOI":"10.1021\/cr0307087","article-title":"Chemistry of peroxynitrites as compared to peroxynitrates","volume":"105","author":"Goldstein","year":"2005","journal-title":"Chem. Rev."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"207","DOI":"10.1006\/abbi.1998.0772","article-title":"Myeloperoxidase and horseradish peroxidase catalyze tyrosine nitration in proteins from nitrite and hydrogen peroxide","volume":"356","author":"Sampson","year":"1998","journal-title":"Arch. Biochem. Biophys."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"54","DOI":"10.1006\/bbrc.1996.5880","article-title":"Quantification of peroxynitrite, superoxide, and peroxyl radicals by a new spin trap hydroxylamine 1-hydroxy-2,2,6,6-tetramethyl-4-oxo-piperidine","volume":"230","author":"Dikalov","year":"1997","journal-title":"Biochem. Biophys. Res. Commun."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"701","DOI":"10.1006\/bbrc.1997.6174","article-title":"Spin trapping of superoxide radicals and peroxynitrite by 1-hydroxy-3-carboxy-pyrrolidine and 1-hydroxy-2,2,6,6-tetramethyl-4-oxo-piperidine and the stability of corresponding nitroxyl radicals towards biological reductants","volume":"231","author":"Dikalov","year":"1997","journal-title":"Biochem. Biophys. Res. Commun."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"1245","DOI":"10.1016\/S0891-5849(98)00328-1","article-title":"Kinetics of superoxide-induced exchange among nitroxide antioxidants and their oxidized and reduced forms","volume":"26","author":"Zhang","year":"1999","journal-title":"Free Radic. Biol. Med."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"211","DOI":"10.1006\/bbrc.1998.8936","article-title":"Detection of superoxide radicals and peroxynitrite by 1-hydroxy-4-phosphonooxy-2,2,6,6-tetramethylpiperidine: Quantification of extracellular superoxide radicals formation","volume":"248","author":"Dikalov","year":"1998","journal-title":"Biochem. Biophys. Res. Commun."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"837","DOI":"10.1016\/j.freeradbiomed.2020.08.022","article-title":"Hydroxylamines inhibit tyrosine oxidation and nitration: The role of their respective nitroxide radicals","volume":"160","author":"Samuni","year":"2020","journal-title":"Free Radic. Biol. Med."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"789","DOI":"10.1021\/ja028190w","article-title":"The role of oxoammonium cation in the SOD-mimic activity of cyclic nitroxides","volume":"125","author":"Goldstein","year":"2003","journal-title":"J. Am. Chem. Soc."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"3679","DOI":"10.1021\/jp056869r","article-title":"Structure-activity relationship of cyclic nitroxides as SOD mimics and scavengers of nitrogen dioxide and carbonate radicals","volume":"110","author":"Goldstein","year":"2006","journal-title":"J. Phys. Chem. A"},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"653","DOI":"10.1016\/S0891-5849(97)00365-1","article-title":"Determination of optimal conditions for synthesis of peroxynitrite by mixing acidified hydrogen peroxide with nitrite","volume":"24","author":"Saha","year":"1998","journal-title":"Free Radic. Biol. Med."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"295","DOI":"10.1016\/0891-5849(88)90050-0","article-title":"A critical reevaluation of some assay methods for superoxide dismutase activity","volume":"4","author":"Goldstein","year":"1988","journal-title":"Free Radic. Biol. Med."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"937","DOI":"10.1016\/j.freeradbiomed.2006.05.026","article-title":"Kinetic properties of Cu,Zn-superoxide dismutase as a function of metal content-Order restored","volume":"41","author":"Goldstein","year":"2006","journal-title":"Free Radic. Biol. Med."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"1620","DOI":"10.1073\/pnas.87.4.1620","article-title":"Apparent hydroxyl radical production by peroxynitrite: Implications for endothelial injury from nitric oxide and superoxide","volume":"87","author":"Beckman","year":"1990","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"6664","DOI":"10.1021\/j100127a016","article-title":"Formation and decay of peroxynitrous acid: A pulse radiolysis study","volume":"97","author":"Loegager","year":"1993","journal-title":"J. Phys. Chem."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"1285","DOI":"10.1021\/tx970160x","article-title":"Formation and properties of peroxynitrite as studied by laser flash photolysis, high-pressure stopped-flow technique, and pulse radiolysis","volume":"10","author":"Kissner","year":"1997","journal-title":"Chem. Res. Toxicol."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"2799","DOI":"10.1016\/0013-4686(95)00261-C","article-title":"Reversible half-wave potentials of reduction processes on nitroxide radicals","volume":"40","author":"Kato","year":"1995","journal-title":"Electrochim. Acta"},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"317","DOI":"10.1016\/j.freeradbiomed.2004.09.037","article-title":"Kinetics and mechanism of the comproportionation reaction between oxoammonium cation and hydroxylamine derived from cyclic nitroxides","volume":"38","author":"Israeli","year":"2005","journal-title":"Free Radic. Biol. Med."},{"key":"ref_24","unstructured":"Mallard, W.G., Ross, A.B., and Helman, W.P. (1998). NIST Standard Reference Database. 40, Version 3.0."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"8719","DOI":"10.1021\/ja017587h","article-title":"Kinetics and mechanism of hydroxyl radical and OH-adduct radical reactions with nitroxides and with their hydroxylamines","volume":"124","author":"Samuni","year":"2002","journal-title":"J. Am. Chem. Soc."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"8364","DOI":"10.1021\/ja035286x","article-title":"Reaction of cyclic nitroxides with nitrogen dioxide:\u2009 The intermediacy of the oxoammonium cations","volume":"125","author":"Goldstein","year":"2003","journal-title":"J. Am. Chem. Soc."}],"container-title":["Antioxidants"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2076-3921\/11\/1\/40\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T07:53:10Z","timestamp":1760169190000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2076-3921\/11\/1\/40"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2021,12,24]]},"references-count":26,"journal-issue":{"issue":"1","published-online":{"date-parts":[[2022,1]]}},"alternative-id":["antiox11010040"],"URL":"https:\/\/doi.org\/10.3390\/antiox11010040","relation":{},"ISSN":["2076-3921"],"issn-type":[{"value":"2076-3921","type":"electronic"}],"subject":[],"published":{"date-parts":[[2021,12,24]]}}}