Electrochemical detection of 3-nitrotyrosine: optimisation for 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 I 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 n M (25 fmol column). Tell consecutive injections or 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 mu M), a molecule releasing nitric oxide and superoxide in solution at a pH of 7.0 or higher with tyrosine (10 mu M). Using this method, 3-NT was detected in human liver microdialysate (levels up to 2.6 nM), although levels ill rat spinal cord dialysate were below the limit of detection. (c) 2006 Elsevier Inc. All rights reserved.