Measurement of nitric oxide metabolites in brain microdialysates by a sensitive fluorometric high-performance liquid chromatography assay

Nitric oxide (NO), formed from arginine by a specific neuronal NO synthase, is an important neurotransmitter in various regions of the central nervous system. While intracerebral microdialysis is an elegant technique to study local extracellular neurotransmitter concentrations in vivo, NO metabolites (nitrate, nitrite (NO(x))) are difficult to study at high temporal resolution because of low tissue concentrations and small sample volumes. We developed a sensitive fluorometric high-performance liquid chromatography (HPLC)-coupled NO(x) assay adapted for the use in brain microdialysate samples. The assay includes an initial enzymatic step in which nitrate is reduced to nitrite. Nitrite is acidified to N2O3, which reacts with 2,3-diaminonaphthalene to form 1-(H)-naphthotriazole. This reaction product can be readily isolated and quantitated by HPLC with fluorometric detection. The theoretical assay sensitivity is less than 1 nM, but numerous sources of contamination must be eliminated in the sampling and assaying process to reliably monitor brain NO(x) outflow by microdialysis.