Estimation of nitric oxide synthase activity via liquid chromatography/tandem mass spectrometric assay determination of 15N3 -citrulline in biological samples.
BACKGROUND
We showed that the metabolite peaks of (15)N(3) -citrulline ((15)N(3) -CIT) and (15)N(3) -arginine ((15)N(3) -ARG) could be detected when (15) N(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 tandem mass spectrometric assay (LC/MS/MS) was utilized for the simultaneous analysis of (15)N(4) -ARG, ARG, CIT, (15)N(3) -CIT and (15)N(3) -ARG. (15)N(3) -CIT and (15)N(3) -ARG from impurities of (15)N(4) -ARG were determined and corrected for the calculation of their concentration. (15)N(4) -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)N(4) -ARG for 2 hours, the peak intensities of (15)N(3) -CIT and (15)N(3) -ARG significantly increased with (15)N(4) -ARG concentration and positively correlated with (15)N-nitrite production. The estimated Km values were independent of the metabolite (i.e., (15)N(3) -CIT, (15)N(3) -CIT+(15)N(3) -ARG or (15) N-nitrite) used for calculation. However, after correction for its presence as a chemical contaminant of (15)N(4) -ARG, (15)N(3) -ARG was only a marginal contributor for the estimation of NOS activity.


CONCLUSIONS
These data suggest that the formation of (15)N(3) -CIT can be used as an indicator of NOS activity when (15)N(4) -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.
College of Pharmacy, Catholic University of Daegu, Gyeongsan-si, Gyeongbuk, 712-702, Korea.
Beom Soo
BS
Shin
Department of Pharmaceutical Sciences, University at Buffalo, The State University of New York, Buffalo, NY, 14260, USA.
Ho-Leung
HL
Fung
BK21 Plus Team, Professional Graduate School of Oriental Medicine, Wonkwang University, Iksan, Jeonbuk, 570-749, Korea.
Mahesh
M
Upadhyay
Department of Pharmacy, Institute of Pharmaceutical Research and Development, College of Pharmacy, Wonkwang University, Iksan, Jeonbuk, 570-749, Korea.
Soyoung
S
Shin
D000818:Animals; D002460:Cell Line; D002853:Chromatography, Liquid / Q000379:methods*; D002956:Citrulline / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D008297:Male; D008930:Mitochondria, Liver; D019001:Nitric Oxide Synthase / Q000032:analysis* / Q000037:antagonists & inhibitors / Q000378:metabolism*; D009587:Nitrogen Isotopes / Q000737:chemistry / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D053719:Tandem Mass Spectrometry / Q000379:methods*
D009587:Nitrogen Isotopes; D002956:Citrulline; D019001:Nitric Oxide Synthase