Beom Soo Shin1, Ho-Leung Fung2, Mahesh Upadhyay3, Soyoung Shin4. 1. College of Pharmacy, Catholic University of Daegu, Gyeongsan-si, Gyeongbuk, 712-702, Korea. 2. Department of Pharmaceutical Sciences, University at Buffalo, The State University of New York, Buffalo, NY, 14260, USA. 3. BK21 Plus Team, Professional Graduate School of Oriental Medicine, Wonkwang University, Iksan, Jeonbuk, 570-749, Korea. 4. Department of Pharmacy, Institute of Pharmaceutical Research and Development, College of Pharmacy, Wonkwang University, Iksan, Jeonbuk, 570-749, Korea.
Abstract
RATIONALE: 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.
RATIONALE: 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.hy926human 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.