Estimation of nitric oxide synthase activity via liquid chromatography/tandem mass spectrometric assay determination of ¹⁵N₃-citrulline in biological samples

Rationale We showed that the metabolite peaks of ¹⁵N₃-citrulline (¹⁵N₃-CIT) and ¹⁵N₃-arginine (¹⁵N₃-ARG) could be detected when ¹⁵N₄-ARG was metabolized by nitric oxide synthase (NOS) in endothelial cells. The usefulness of these metabolites as potential surrogate indices of nitric oxide (NO) generation is evaluated. Methods A hydrophilic-interaction liquid chromatography/electrospray tandem mass spectrometric assay (LC/MS/MS) was utilized for the simultaneous analysis of ¹⁵N₄-ARG, ARG, CIT, ¹⁵N₃-CIT and ¹⁵N₃-ARG. ¹⁵N₃-CIT and ¹⁵N₃-ARG from impurities of ¹⁵N₄-ARG were determined and corrected for the calculation of their concentration. ¹⁵N₄-ARG-derived NO, i.e., ¹⁵NO formation was determined by analyzing ¹⁵N-nitrite accumulation by another LC/MS/MS assay. Results After EA.hy926 human endothelial cells were challenged with ¹⁵N₄-ARG for 2 hours, the peak intensities of ¹⁵N₃-CIT and ¹⁵N₃-ARG significantly increased with ¹⁵N₄-ARG concentration and positively correlated with ¹⁵N-nitrite production. The estimated Km values were independent of the metabolite (i.e., ¹⁵N₃-CIT, ¹⁵N₃-CIT+¹⁵N₃-ARG or ¹⁵N-nitrite) used for calculation. However, after correction for its presence as a chemical contaminant of ¹⁵N₄-ARG, ¹⁵N₃-ARG was only a marginal contributor for the estimation of NOS activity. Conclusions These data suggest that the formation of ¹⁵N₃-CIT can be used as an indicator of NOS activity when ¹⁵N₄-ARG is used as a substrate. This approach may be superior to the radioactive ¹⁴C-CIT method which can be contaminated by ¹⁴C-urea, and to the ¹⁴N-nitrite method which lacks sensitivity.