Assessment of Nitric Oxide Production by Measurement of [15N]Citrulline Enrichment in Human Plasma Using High-Performance Liquid Chromatography–Mass Spectrometry

✍ Scribed by Fija M. Lagerwerf; Robert M.F. Wever; Herman J.M. van Rijn; Cornelis Versluis; Wigger Heerma; Johan Haverkamp; Hein A. Koomans; Ton J. Rabelink; Peter Boer

• Publisher: Elsevier Science
• Year: 1998
• Tongue: English
• Weight: 202 KB
• Volume: 257
• Category: Article
• ISSN: 0003-2697
• DOI: 10.1006/abio.1997.2515

✦ Synopsis

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.