ASSESSMENT OF NITRIC-OXIDE PRODUCTION BY MEASUREMENT OF [N-15]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 co nvert arginine into citrulline. A decreased in vivo NO availability ca n be the result of an increased NO inactivation or a decreased NO prod uction. The latter can be assessed by measurement of isotopic enrichme nt of plasma citrulline during infusion of isotopically labeled argini ne. The potential of high-performance liquid chromatography (HPLC) cou pled to mass spectrometry (MS) to determine enrichments of [N-15(2)]ar ginine and [N-15]citrulline in plasma during infusion of [N-15(2)]argi nine in humans was investigated. Two types of MS instruments were eval uated: a sector-type mass spectrometer equipped with a frit fast-atom bombardment (FAB) interface and a quadrupole instrument with electrosp ray ionization (ESI). FAB-MS appeared to be unsuitable for determinati on 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 te chnique. However, the amount of material that is introduced in the mas s spectrometer is critical and should be carefully controlled. During infusion of [N-15(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 t he 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 determinatio n of arginine and citrulline enrichments in plasma, thus providing a u seful tool for assessment of in vivo NO production. (C) 1998 Academic Press.