Estimation of nitric oxide synthase activity via liquid chromatography/tandem mass spectrometric assay determination of 15N3‐citrulline in biological samples. (28th January 2015)

• Record Type: Journal Article
• Authors: Shin, Beom Soo; Fung, Ho‐Leung; Upadhyay, Mahesh; Shin, Soyoung
• Is Part Of: Rapid communications in mass spectrometry. Volume 29:Number 5(2015)
• Page Start: 447
• Page End: 455
• Publication Date: 2015-01-28
• Subjects: Mass spectrometry -- Periodicals; 543.65
• DOI: 10.1002/rcm.7124
• Languages: English
• ISSNs: 0951-4198

Abstract

Abstract : Rationale: We showed that the metabolite peaks of 15 N3 ‐citrulline ( 15 N3 ‐CIT) and 15 N3 ‐arginine ( 15 N3 ‐ARG) could be detected when 15 N4 ‐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 15 N4 ‐ARG, ARG, CIT, 15 N3 ‐CIT and 15 N3 ‐ARG. 15 N3 ‐CIT and 15 N3 ‐ARG from impurities of 15 N4 ‐ARG were determined and corrected for the calculation of their concentration. 15 N4 ‐ARG‐derived NO, i.e., 15 NO formation was determined by analyzing 15 N‐nitrite accumulation by another LC/MS/MS assay. Results: After EA.hy926 human endothelial cells were challenged with 15 N4 ‐ARG for 2 hours, the peak intensities of 15 N3 ‐CIT and 15 N3 ‐ARG significantly increased with 15 N4 ‐ARG concentration and positively correlated with 15 N‐nitrite production. The estimated Km values were independent of the metabolite (i.e., 15 N3 ‐CIT, 15 N3 ‐CIT+ 15 N3 ‐ARG or 15 N‐nitrite) used for calculation. However, after correction for its presence as a chemical contaminant of 15 N4 ‐ARG, 15 N3 ‐ARG was only a marginal contributor for the estimation of NOS activity. Conclusions: These data suggest that the formation of 15 N3 ‐CIT can be used as an indicator of NOS activity when 15 N4 ‐ARG is used as a substrate. This approach may be superior to the radioactive 14 C‐CIT method which can be contaminated by 14 C‐urea, and to the 14 N‐nitrite method which lacks sensitivity. Copyright © 2015 John Wiley & Sons, Ltd.