title: Assessment of nitric oxide production by measurement of [15N]citrulline enrichment in human plasma using high-performance liquid chromatography-mass spectrometry.
abstract: 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 [15N2]arginine and [15N]-citrulline in plasma during infusion of [15N2]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 [15N2]arginine in 14 healthy subjects, a mean arginine-to-citrulline conversion rate of 0.22 +/- 0.07 (SD) mumol.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 mumol.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.
authors.affiliation: Department of Mass Spectrometry, Utrecht University, The Netherlands.
authors.forename: F M
authors.initials: FM
authors.lastname: Lagerwerf
authors.forename: R M
authors.initials: RM
authors.lastname: Wever
authors.forename: H J
authors.initials: HJ
authors.lastname: van Rijn
authors.forename: C
authors.initials: C
authors.lastname: Versluis
authors.forename: W
authors.initials: W
authors.lastname: Heerma
authors.forename: J
authors.initials: J
authors.lastname: Haverkamp
authors.forename: H A
authors.initials: HA
authors.lastname: Koomans
authors.forename: T J
authors.initials: TJ
authors.lastname: Rabelink
authors.forename: P
authors.initials: P
authors.lastname: Boer
mesh_terms: D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D002956:Citrulline / Q000097:blood*; D004791:Enzyme Inhibitors / Q000008:administration & dosage; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods*; D009569:Nitric Oxide / Q000096:biosynthesis* / Q000097:blood; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors; D009587:Nitrogen Isotopes; D019323:omega-N-Methylarginine / Q000008:administration & dosage
chemical_list: D004791:Enzyme Inhibitors; D009587:Nitrogen Isotopes; D019323:omega-N-Methylarginine; D002956:Citrulline; D009569:Nitric Oxide; D019001:Nitric Oxide Synthase