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

Abstract

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.

Tracer-based metabolomics for profiling nitric oxide metabolites in a 3D microvessel-on-a-chip model

“…This approach utilizes stable isotope-labelled precursors to trace complex pathways by following the labeled atom(s) to downstream metabolites (Chokkathukalam et al, 2014; Paul Lee et al, 2010). This technique, applied to enzymatic reactions involved in NO production (Figueroa et al, 2020; Gambardella et al, 2020; Kucharzewska et al, 2010; Shatanawi et al, 2020; Shin et al, 2015; Siervo et al, 2011) enhances accuracy and sensitivity, overcoming the challenges associated with traditional NO measurement methods.…”