Dual-cell electrochemical detection OF 3-nitrotyrosine following reversed-phase HPLC separation

Douglas A Richards & Adam J Devall, Department of Pharmacology, Division of Neuroscience, Medical School, University of Birmingham, B15 2TT, UK.

Nitric oxide (NO) is a highly diffusible, reactive and unstable free radical, produced in vivo by both constitutive and inducible isoforms of the enzyme, nitric oxide synthase. NO has been associated with both physiological and pathological roles, and amongst mechanisms by which it may be detrimental to tissue is by its reaction with superoxide radicals to form peroxynitrite. There is considerable evidence associating damaging peroxynitrite radicals in the pathology of a number of conditions, including Parkinson 's and Alzheimer 's disease. Whilst peroxynitrite is itself unstable, it can form a stable by-product, 3-nitrotyrosine (3-NT), by reaction with either free or protein-bound tyrosine residues. As a result 3-NT has become a recognized molecular marker for disrupted NO metabolism. The aim of this study was to develop an HPLC electrochemical detection assay for 3-NT that would be capable of measuring this metabolite at the very low (nanomolar) levels encountered physiologically ( Duncan, 2003), and which are presently most accurately measured by mass-spectrometric methods.

Although 3-NT can be directly oxidized, the potential required to achieve this is over 800mV, resulting in highly complex chromatograms containing numerous peaks at concentrations several orders of magnitude greater than 3-NT when analyzing biological fluids. We therefore adopted a dual-cell approach in which 3-NT is first reduced at an upstream cell to 3-aminotyrosine, which is itself then oxidized at the downstream cell at a potential far lower than that required to oxidize 3-NT directly. The final optimized method consisted of a 3 μm reversed-phase (C18) column, with mobile phase consisting of 20mM sodium dihydrogen phosphate buffer, pH 3.2, containing 20% v/v methanol, delivered at 0.3 ml/min, and detection via a Coulochem II coupled to a 5021A conditioning cell (set at +550mV), and a 5014b microdialysis cell (cell 1, -1200mV; cell2, -100mV) in series. Under these conditions, 3-NT was eluted in around 7 min.

This study describes (a) the optimisation of the three electrode potentials that were varied in order to achieve an assay that was both sensitive and selective, and then (b) the characterization of the final method. Injecting 50μ l aliquots of authentic 3-NT standards, the method was shown to be linear over the range 1-500 nM (r=0.999), with a detection limit (signal/noise ratio of 3) of 0.5nM (25 femtomoles on column). Ten consecutive injections of 2nM and 20nM 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 co-elution with authentic standards, and by the in vitro production of 3-NT by incubation of the peroxynitrite donor, 3-morpholinylsydnoneimine (SIN-1; 100 μM), with tyrosine (10 μM). Finally, a peak corresponding to 3-NT in an authentic microdialysis sample (approx 2nM) was shown to disappear on decreasing the potential of the reduction cell so that 3-NT could not be reduced to 3-aminotyrosine.

The described dual-cell electrochemical detection method is capable of the sensitive and specific measurement of 3-NT, using equipment more widely available than mass spectrometry, and its applicability to microdialysis studies has been demonstrated.

Duncan , M.W. (2003) Amino Acids. 25, 351-361.