Simultaneous Measurement of [15N]Nitrate and [15N]Nitrite Enrichment and Concentration in Urine by Gas Chromatography Mass Spectrometry as Pentafluorobenzyl Derivatives

Abstract

Quantification of (15)N-labeled nitrate and determination of the (15)N-enrichment in urine upon administration of (15)N-labeled precursors such as L-[guanidino-(15)N(2)]arginine is a suitable approach to study formation and metabolism of nitric oxide (NO) and its metabolites in vivo. Previously, we have reported on the simultaneous derivatization and accurate quantification of nitrite and nitrate in various biological fluids using pentafluorobenzyl bromide and GC/MS. We report here on a modification of this method that allows for the simultaneous determination of (15)N-enrichment of [(15)N]nitrate and [(15)N]nitrite and the simultaneous quantification of [(15)N]nitrate, [(14)N]nitrate, [(15)N]nitrite, and [(14)N]nitrite in human urine. In a pilot study, using the carbonic anhydrase inhibitor drug acetazolamide at therapeutical oral doses (5.4 and 5 mg per kg bodyweight) and by oral intake of [(15)N]nitrite (0.31 and 0.5 micromol per kg bodyweight) by two healthy volunteers, we demonstrate for the first time that renal carbonic anhydrase activity is mainly responsible for the reabsorption of nitrite from the primary urine and confirm previous findings on nitrate.