Submitted to: American Society for Mass Spectrometry
Publication Type: Abstract Only
Publication Acceptance Date: 6/1/2005
Publication Date: N/A
Citation: N/A Interpretive Summary:
Technical Abstract: Nitric oxide (NO) plays a critical role in several physiologic processes, including modulation of peripheral vascular resistance, gastrointestinal peristalsis, inflammation and neuronal function. NO is synthesized in tissues by three classes of nitric oxide synthases (NOS) and rapidly degraded to nitrite and nitrate before excretion. Direct measurement of NO production in humans by isotopic tracer measurements is difficult due to the innate toxicity and rapid local metabolism of the molecule. We describe here an indirect measure of whole-body NO production by measuring the conversion of 15N-arginine (Arg) to 15N-citrulline (Cit) using tandem LC/MS. The low fractional conversion of Arg to Cit by NOS requires a method with high sensitivity and precision in order to measure the low enrichment expected. Amino acids were converted to the N,N-dimethylaminonaphthalene sulfonamide (DANS) derivative by treatment of plasma (100 'L) with 15 mM DANSyl chloride at pH 9 to enhance ionization. Proteins were precipitated with acetonitrile and removed by centrifugation. The supernatant was evaporated and the DANS-AA were dissolved in HPLC mobile phase. The DANS-AA were separated on a C18 HPLC column using an acetonitrilewater gradient containing 0.4% formic acid. Isotopic enrichment measurements were performed on a ThermoElectron Quantum Discovery triple quadrupole instrument using electrospray ionization, collision induced dissociation and selected reaction monitoring. Specific reaction conditions are:(Arg) 408->170 @ 34 eV and (Cit) 409->392 @ 12 eV and 409->170 @ 28 eV (parent isotopic masses). Due to poor ionization efficiency and chromatography, plasma AA were converted to the DANSyl derivative using a facile single-step procedure. High proton affinity of the dimethylamino moiety in the DANSyl group contributes to the enhanced sensitivity. The primary CID fragment arises from scission of the naphthalene--|--sulfonamido bond resulting in loss of the sulfonamido-AA as a neutral species. Isotopic information is retained by selecting a specific precursor mass for Arg and Cit and monitoring a haracteristic product mass (m/z 170). A second reaction was monitored for citrulline in order to enhance the quantitation of 15N derived from arginine due to the expected low enrichment (~0.3 – 0.5 mole % excess). At low collision energy (10-12 eV), the ureido group of citrulline selectively loses NH3, which contains the 15N tracer from arginine. This fragment proved useful in quantifying low enrichments above a rather low threshold natural isotopic background of NH3. Patients (n = 12) were given a primed constant infusion of guanidino-labeled 15N2-arginine and -13C-4,4,5,5-2H4-citrulline. Timed blood samples were collected over 8 hours. The study is a randomized, placebo-controlled, double blind protocol. This study was done to evaluate the effects of soy isoflavones on blood pressure and NO production. Precision of the enrichment measurements in the baseline blood samples was +/- 0.01 – 0.03% for an individual isotopomer. Absolute accuracy of the natural abundance baseline samples is typically +/- 0.1%. Plateau enrichment in the primary amino acid compartment during the infusion study was typically 13-18 mole % excess (Arg) and 6-10 mole % excess (Cit). Enrichment in the secondary amino acid was 0.2 – 0.6 MPE (citrulline derived from arginine) and 0.4 – 1.3 MPE (arginine derived from citrulline).