title: Noninvasive diagnostic tool for inflammation-induced oxidative stress using electron spin resonance spectroscopy and an extracellular cyclic hydroxylamine.
abstract: Inflammation is one of the leading causes of the many pathological states associated with oxidative stress. A crucial role in the development of inflammation-induced oxidative stress is played by reactive oxidant species (ROS), which are very difficult to detect in vivo. One of the most sensitive and definitive methods in the detection of ROS is electron spin resonance, especially as used in conjunction with spin trapping. Unfortunately, the commonly used nitrone spin traps have a very low efficacy for trapping superoxide radicals, and their radical adducts are not stable. To address this deficiency, we have developed negatively charged cyclic hydroxylamines such as 1-hydroxy-4-phosphonooxy-2,2,6,6-tetramethylpiperidine (PP-H) for the detection of reactive oxidant species as a diagnostic tool for extracellular inflammation-induced oxidative stress. We used inflammation induced by a bacterial endotoxin lipopolysaccharide (LPS) as a model. ROS formation was tested in cultured macrophages, in blood and in vivo. PP-H reacts with reactive oxidant species generating the stable nitroxide radical 4-phosphonooxy-TEMPO. It was shown that a 5-h treatment of macrophages with LPS (1 microg/ml) leads to a threefold increase in superoxide formation as demonstrated using superoxide dismutase. Formation of reactive oxidant species 5 h after LPS (1 mg/kg) treatment of Fischer rats was analyzed in arterial blood; formation of reactive oxidant species in LPS-treated animals increased by a factor of 2.2 and was dependent upon the LPS dose. Diphenyleneiodonium (0.1 mM) inhibited formation of LPS-stimulated reactive oxidant species by 80%. We suggest that this test could be used as a noninvasive diagnostic tool for inflammation-induced oxidative stress.
authors.affiliation: Laboratory of Pharmacology and Chemistry, National Institute of Environmental Health Sciences, National Institutes of Health, 111 Alexander Drive, Research Triangle Park, NC 27709, USA.
authors.forename: Sergey I
authors.initials: SI
authors.lastname: Dikalov
authors.forename: Anna E
authors.initials: AE
authors.lastname: Dikalova
authors.forename: Ronald P
authors.initials: RP
authors.lastname: Mason
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D003497:Cyclic N-Oxides; D004578:Electron Spin Resonance Spectroscopy; D005110:Extracellular Space / Q000378:metabolism; D016166:Free Radical Scavengers; D007249:Inflammation / Q000139:chemically induced / Q000175:diagnosis* / Q000378:metabolism; D008070:Lipopolysaccharides; D008264:Macrophages / Q000378:metabolism; D008297:Male; D010755:Organophosphates; D018384:Oxidative Stress; D010880:Piperidines; D051381:Rats; D011916:Rats, Inbred F344; D017382:Reactive Oxygen Species / Q000097:blood*; D013113:Spin Labels; D013481:Superoxides / Q000378:metabolism
chemical_list: C113868:1-hydroxy-4-phosphonooxy-2,2,6,6-tetramethylpiperidine; D003497:Cyclic N-Oxides; D016166:Free Radical Scavengers; D008070:Lipopolysaccharides; D010755:Organophosphates; D010880:Piperidines; D017382:Reactive Oxygen Species; D013113:Spin Labels; D013481:Superoxides; C098505:5-diethoxyphosphoryl-5-methyl-1-pyrroline N-oxide