Electrochemical detection of free 3-nitrotyrosine: application to microdialysis studies.
3-Nitrotyrosine (3-NT) is formed by the reaction of peroxynitrite with either free or protein-bound tyrosine residues and has been proposed as a biomarker of oxidative stress caused by reactive nitrogen species. This study describes the development of an HPLC electrochemical detection assay for free 3-NT capable of measuring this metabolite at the very low (nanomolar) levels encountered physiologically. We employed a dual-cell coulometric approach in which 3-NT is first reduced at an upstream cell to 3-aminotyrosine, which itself is then oxidized at the downstream cell. The method was shown to be linear over the range of 1-500 nM (r = 0.999), with a detection limit (signal/noise ratio of 3) of 0.5 nM (25 fmol on column). Ten consecutive injections of 2 and 20 nM 3-NT standards produced coefficients of variation of 5.88 and 1.87%, respectively. Validation of the identity of the 3-NT peak was confirmed by coelution with authentic standards and by the in vitro production of 3-NT by incubation of 3-morpholinylsydnoneimine (SIN-1, 100 microM), a molecule releasing nitric oxide and superoxide in solution at a pH of 7.0 or higher with tyrosine (10 microM). Using this method, 3-NT was detected in human liver microdialysate (levels up to 2.6 nM), although levels in rat spinal cord dialysate were below the limit of detection.
Richards
Douglas A
DA
Department of Pharmacology, Division of Neuroscience, Medical School, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK. d.a.richards@bham.ac.uk
Silva
Michael A
MA
Devall
Adam J
AJ
D000818:Animals; D002851:Chromatography, High Pressure Liquid; D004563:Electrochemistry / Q000379:methods*; D017551:Microdialysis; D010084:Oxidation-Reduction; D051381:Rats; D015203:Reproducibility of Results; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000032:analysis
C002744:3-nitrotyrosine; D014443:Tyrosine