title: Ceruloplasmin (ferroxidase) oxidizes hydroxylamine probes: deceptive implications for free radical detection.
abstract: Ceruloplasmin (ferroxidase) is a copper-binding protein known to promote Fe(2+) oxidation in plasma of mammals. In addition to its classical ferroxidase activity, ceruloplasmin is known to catalyze the oxidation of various substrates, such as amines and catechols. Assays based on cyclic hydroxylamine oxidation are used to quantify and detect free radicals in biological samples ex vivo and in vitro. We show here that human ceruloplasmin promotes the oxidation of the cyclic hydroxylamine 1-hydroxy-3-carboxy-2,2,5,5-tetramethylpyrrolidine hydrochloride (CPH) and related probes in Chelex-treated phosphate buffer and rat serum. The reaction is suppressed by the metal chelators DTPA, EDTA, and desferal, whereas heparin and bathocuproine have no effect. Catalase or superoxide dismutase additions do not interfere with the CPH-oxidation yield, demonstrating that oxygen-derived free radicals are not involved in the CPH oxidation mediated by ceruloplasmin. Plasma samples immunodepleted of ceruloplasmin have lower levels of CPH oxidation, which confirms the role of ceruloplasmin (ferroxidase) as a biological oxidizing agent of cyclic hydroxylamines. In conclusion, we show that the ferroxidase activity of ceruloplasmin is a possible biological source of artifacts in the cyclic hydroxylamine-oxidation assay used for reactive oxygen species detection and quantification.
authors: Free Radical Metabolism Section, Laboratory of Toxicology and Pharmacology, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC 27709, USA.
authors: Douglas
authors: D
authors: Ganini
authors: Donatella
authors: D
authors: Canistro
authors: JinJie
authors: J
authors: Jiang
authors: JinJie
authors: J
authors: Jang
authors: Krisztian
authors: K
authors: Stadler
authors: Ronald P
authors: RP
authors: Mason
authors: Maria B
authors: MB
authors: Kadiiska
mesh_terms: D000818:Animals; D016477:Artifacts; D001681:Biological Assay; D002374:Catalase / Q000737:chemistry; D002570:Ceruloplasmin / Q000737:chemistry*; D002614:Chelating Agents / Q000737:chemistry; D003676:Deferoxamine / Q000737:chemistry; D004492:Edetic Acid / Q000737:chemistry; D005609:Free Radicals / Q000097:blood*; D006493:Heparin / Q000737:chemistry; D006801:Humans; D006898:Hydroxylamines / Q000737:chemistry*; D016877:Oxidants / Q000737:chemistry*; D010084:Oxidation-Reduction; D004369:Pentetic Acid / Q000737:chemistry; D010618:Phenanthrolines / Q000737:chemistry; D051381:Rats; D013482:Superoxide Dismutase / Q000737:chemistry
chemical_list: D002614:Chelating Agents; D005609:Free Radicals; D006898:Hydroxylamines; D016877:Oxidants; D010618:Phenanthrolines; D004369:Pentetic Acid; D006493:Heparin; D004492:Edetic Acid; C002478:bathocuproine; D002374:Catalase; D013482:Superoxide Dismutase; D002570:Ceruloplasmin; D003676:Deferoxamine