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

초록

RATIONALE: We showed that the metabolite peaks of N-15(3)-citrulline (N-15(3)-CIT) and N-15(3)-arginine (N-15(3)-ARG) could be detected when N-15(4)-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 tandemmass spectrometric assay (LC/MS/MS) was utilized for the simultaneous analysis of N-15(4)-ARG, ARG, CIT, N-15(3)-CIT and N-15(3)-ARG. N-15(3)-CIT and N-15(3)-ARG from impurities of N-15(4)-ARG were determined and corrected for the calculation of their concentration. N-15(4)-ARG-derived NO, i.e., (NO)-N-15 formation was determined by analyzing N-15-nitrite accumulation by another LC/MS/MS assay. RESULTS: After EA. hy926 human endothelial cells were challenged with N-15(4)-ARG for 2 hours, the peak intensities of N-15(3)-CIT and N-15(3)-ARG significantly increased with N-15(4)-ARG concentration and positively correlated with N-15-nitrite production. The estimated Km values were independent of the metabolite (i.e., N-15(3)-CIT, N-15(3)-CIT+ N-15(3)-ARG or N-15-nitrite) used for calculation. However, after correction for its presence as a chemical contaminant of N-15(4)-ARG, N-15(3)-ARG was only a marginal contributor for the estimation of NOS activity. CONCLUSIONS: These data suggest that the formation of N-15(3)-CIT can be used as an indicator of NOS activity when N-15(4)-ARG is used as a substrate. This approach may be superior to the radioactive C-14-CIT method which can be contaminated by C-14-urea, and to the N-14-nitrite method which lacks sensitivity. Copyright (C) 2015 John Wiley & Sons, Ltd.

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