Quantitation of protein-bound 3-nitrotyrosine by high-performance liquid chromatography with electrochemical detection.

HPLC with electrochemical detection of the N-acetylated, dithionite-reduced derivative of NTyr provides a highly sensitive and selective means of measuring this nitrated residue in biological samples. The detection of protein-bound NTyr at baseline levels of approximately < or = 1 mumol per mole Tyr indicates that in plasma or total cellular extracts, endogenous nitration of tyrosine residues is low. This baseline level of NTyr and the marked increases that are observed during inflammatory conditions opens up the opportunity to observe more subtle changes in tyrosine nitration, thus broadening the range of studies that can be performed using this biomarker. This analytical approach may allow one to estimate protein nitration in an animal or individual exposed to elevated levels of peroxynitrite or other reactive nitrogen oxides, and it may assist in the evaluation of factors that contribute to this potentially important amino acid modification. Furthermore, this assay may allow one to assess the potential benefits of interventions that may limit nitration reactions in vivo.