Quantification of 15N-Nitrate in Urine with Gas Chromatography Combustion Isotope Ratio Mass Spectrometry to Estimate Endogenous NO Production

The use of stable isotope labeled substrates and subsequent analysis of urinary nitrate, forms a noninvasive test for evaluation of the in vivo NO metabolism. The present paper describes a new method for simultaneous quantification of N-15-nitrate and total nitrate with gas chromatography combustion isotope ratio mass spectrometry (GC-C-IRMS). Nitrate, isolated from urine with a nitrate selective resin, was reduced to nitrite using copperized cadmium. Subsequently, Sudan I was formed by diazotation. Sudan II was added as internal standard, and both molecules were analyzed with GCC-IRMS as tert-butyldimethylsilyl derivatives. The accuracy was determined during a recovery study of two different known nitrate concentrations kind two N-15-enrichments. A recovery of 101.6%, and 103.9% for total nitrate and 107.6% kind 91.2% for N-15-nitrate was obtained, respectively. ne validated method was applied on complete 72 h urine collections after intravenous administration of N-15-nitrate and N-15-arginine in humans. On average, 51.8% (47.0-71.0%) of administered N-15-nitrate was excreted, while 0.68% (0.44-1.17%) of N-15-arginine was metabolized to nitrate. In conclusion, this method can be used for accurate simultimeous detemination of N-15-nitrate and total nitrate concentrations in urine and can be applied in clinical studies for noninvasive evaluation of NO metabolism in vivo.