Measurement of Nitric Oxide Synthase Activity In Vivo and In Vitro by Gas Chromatography-Mass Spectrometry

Measurement of the nitric oxide (NO) metabolites nitrite and nitrate in biological matrices is a reliable method to assess NO synthase (NOS) activity. Unlike the “L -citrulline assay,” the “nitrite/nitrate assay” is generally applicable and permits acquisition of maximum information about what NOS did actually produce. From the various analytical approaches and methods available so far for the quantitation of nitrite and nitrate, mass-spectrometry-based methods, notably gas chromatography-mass spectrometry (GC-MS), offer the highest reliability in terms of accuracy, precision, specificity, minimum of interferences, sensitivity, and no limitation regarding biological matrix. Among the GC-MS methods, that one utilizing the derivatization reagent pentafluorobenzyl bromide is the single assay permitting measurement of nitrite and nitrate. This method possesses the unique opportunity to measure simultaneously stable-isotope-labeled nitrite and nitrate (i.e. [¹⁵ N]nitrite and [¹⁵ N]nitrate), which are exclusively produced by NOS-catalyzed oxidation of L -[guanidino -¹⁵ N₂ ]-arginine, the stable-isotope-labeled L -arginine analog. ^• NOS may concomitantly produce ^• NO and O₂ ^•− , thus forming peroxynitrite (ONOO⁻ ), which also degrades to nitrite and nitrate. Thus, the GC-MS nitrite/nitrate assay provides information about biologically relevant NOS activity. This chapter describes GC-MS protocols for the measurement of NOS activity in vitro (e.g., of isolated NOS preparations and in cells) and in vivo (in animals and humans).