Assessment of nitric oxide production by measurement of [15N]citrulline enrichment in human plasma using high-performance liquid chromatography mass spectrometry

Nitric oxide (NO) is formed by a class of NO synthases (NOS), which convert arginine into citrulline. A decreased in vivo NO availability can be the result of an increased NO inactivation or a decreased NO production. The latter can be assessed by measurement of isotopic enrichment of plasma citrulline during infusion of isotopically labeled arginine. The potential of high-performance liquid chromatography (HPLC) coupled to mass spectrometry (MS) to determine enrichments of [(15)N(2)]arginine and [(15)N]citrulline in plasma during infusion of [(15)N(2)]arginine in humans was investigated. Two types of MS instruments were evaluated: a sector-type mass spectrometer equipped with a frit fast-atom bombardment (FAB) interface and a quadrupole instrument with electrospray ionization (ESI). FAB-MS appeared to be unsuitable for determination of isotope ratios, because background ions influenced the observed isotope ratio in an unpredictable way. In combination with either off-or on-line reversed-phase HPLC, ESI-MS proved to be a more reliable technique. However, the amount of material that is introduced in the mass spectrometer is critical and should be carefully controlled. During infusion of [(15)N(2)]arginine in 14 healthy subjects, a mean arginine-to-citrulline conversion rate of 0.22 +/- 0.07 (SD) mu mol.kg(-1).h(-1) was found. In 4 subjects who received an intravenous infusion with the NOS antagonist L-NMMA, the conversion rate decreased from 0.30 +/- 0.14 to 0.10 +/- 0.06 mu mol.kg(-1).h(-1). It is concluded that ESI-MS in combination with HPLC can be successfully applied for determination of arginine and citrulline enrichments in plasma, thus providing a useful tool for assessment of in vivo NO production. (C) 1998 Academic Press.