We found a match
Your institution may have access to this item. Find your institution then sign in to continue.
- Title
Estimation of nitric oxide synthase activity via liquid chromatography/tandem mass spectrometric assay determination of <sup>15</sup>N<sub>3</sub>-citrulline in biological samples.
- Authors
Shin, Beom Soo; Fung, Ho‐Leung; Upadhyay, Mahesh; Shin, Soyoung
- 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 tandemmass 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 15Nnitrite) 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.
- Subjects
NITRIC oxide synthesis; LIQUID chromatography-mass spectrometry; METABOLITE synthesis; HYDROPHILIC interaction liquid chromatography; ENDOTHELIAL cells
- Publication
Rapid Communications in Mass Spectrometry: RCM, 2015, Vol 29, Issue 5, p447
- ISSN
0951-4198
- Publication type
Article
- DOI
10.1002/rcm.7124