[ureido-15N]Citrulline UPLC–MS/MS nitric oxide synthase (NOS) activity assay: Development, validation, and applications to assess NOS uncoupling and human platelets NOS activity

In healthy human subjects, less than 0.2% of l-arginine is converted to l-citrulline and nitric oxide (NO) by NO synthases (NOS), a metabolic pathway present in all cell types. Assessment of NOS activity in vitro and in vivo by measuring l-citrulline or NO is difficult. l-citrulline is formed from l-arginine to a much higher extent by other pathways including the urea cycle. Furthermore, NO is a very short-lived gaseous molecule and is oxidized to nitrite and nitrate which are ubiquitous. In fact, nitrite and nitrate are also derived from food and air and are major laboratory contaminants. Further, NOS (in the uncoupled state) are also able to produce superoxide in addition and/or instead of l-citrulline and NO. The difficulties of NOS assays based on l-citrulline and NO measurement can only in part be overcome by sophisticated techniques including use of radio-labeled (3H or 14C) and stable-isotope labeled (15N2 at the guanidine group) l-arginine analogs as substrates for NOS and measurement of radio-labeled l-citrulline and 15N-labeled nitrite and nitrate, respectively. In the present work, we report on the development, validation and application of an UPLC–MS/MS method for the assessment of the activity of recombinant NOS enzymes by using [guanidino-15N2]-l-arginine (20 μM for recombinant NOS, 5 mM in cell systems) as the substrate and by measuring [ureido-15N]-l-citrulline as the reaction product (usually formed at concentrations below 1 μM) using 2H7-l-citrulline as the internal standard. The lower limit of detection of the method is about 80 fmol 2H7-l-citrulline. In cell systems, exceeding [guanidino-15N2]-l-arginine is removed by strong cation exchanger solid-phase extraction. The method was cross-validated by a GC–MS assay that measures simultaneously 15N-nitrite and 15N-nitrate as pentafluorobenzyl derivatives, with unlabeled nitrite and nitrate serving as the internal standards. By means of this UPLC–MS/MS 15N-citrulline assay, NG-nitro-arginine (100 μM) was found to inhibit recombinant inducible NOS (iNOS) activity (by 38%), whereas nitrite and GSSG (each at 500 μM) did not affect iNOS activity at all. Nitrite and GSSG at pathophysiological concentrations are unlikely to uncouple NOS. NOS activity was not detectable in platelets of healthy humans by the UPLC–MS/MS and GC–MS assays.