Analysis of 3-Nitrotyrosine in Biological Fluids and Protein Hydrolyzates by High-Performance Liquid Chromatography Using a Postseparation, On-line Reduction Column and Electrochemical Detection: Results with Various Nitrating Agents

Abstract

Nitric oxide reacts rapidly with superoxide to form the strong nitrating agent peroxynitrite, which is responsible for much of the tissue damage associated with diverse pathophysiological conditions such as inflammation. The occurrence of free or protein-bound nitrotyrosine (NTYR) has been considered as evidence forin vivoformation of peroxynitrite. However, various agents can nitrate tyrosine, and their relative significancein vivohas not been determined due to lack of a sensitive method to analyze NTYR in tissue proteins and biological fluids. We have developed a new HPLC-electrochemical detection method to analyze NTYR in protein hydrolyzates or biological fluids. The sample is injected directly into a reversed-phase HPLC column and NTYR is subsequently reduced by a platinum column to 3-aminotyrosine, which is quantified with an electrochemical detector. The method is simple, selective, and sensitive (detection limit, 0.1 pmol per 20-μl injection). We have applied this method to comparein vitrothe ability of various nitrating agents to form NTYR in bovine serum albumin and human plasma. Yields of NTYR formed in human plasma proteins incubated with 1 or 10 mM nitrating agent decreased in the following order: synthetic peroxynitrite > 3-morpholino-sydonimine, a generator of both NO and superoxide > Angeli's salt, which forms nitroxyl anion (NO−) > spermine-NONOate, which releases NO > sodium nitrite plus hypochlorite, which forms the nitrating agent nitryl chloride (NO2Cl). A simple purification method using a C18 Sep-Pak cartridge is also described for analysis of free NTYR in human plasma.