title: Simultaneous detection of stable isotope-labeled and unlabeled L-tryptophan and of its main metabolites, L-kynurenine, serotonin and quinolinic acid, by gas chromatography/negative ion chemical ionization mass spectrometry.
abstract: A method for the detection of unlabeled and (15)N2 -labeled L-tryptophan (L-Trp), L-kynurenine (L-Kyn), serotonin (5-HT) and quinolinic acid (QA) in human and rat plasma by GC/MS is described. Labeled and unlabeled versions of these four products were analyzed as their acyl substitution derivatives using pentafluoropropionic anhydride and 2,2,3,3,3-pentafluoro-1-propanol. Products were then separated by GC and analyzed by selected ion monitoring using negative ion chemical ionization mass spectrometry. L-[(13)C11, (15)N2]-Trp, methyl-serotonin and 3,5-pyridinedicarboxylic acid were used as internal standards for this method. The coefficients of variation for inter-assay repeatability were found to be approximately 5.2% for L-Trp and (15)N2-Trp, 17.1% for L-Kyn, 16.9% for 5-HT and 5.8% for QA (n = 2). We used this method to determine isotope enrichments in plasma L-Trp over the course of a continuous, intravenous infusion of L-[(15) N2 ]Trp in pregnant rat in the fasting state. Plasma (15)N2-Trp enrichment reached a plateau at 120 min. The free Trp appearance rate (Ra) into plasma was 49.5 ± 3.35 µmol/kg/h. The GC/MS method was applied to determine the enrichment of (15)N-labeled L-Trp, L-Kyn, 5-HT and QA concurrently with the concentration of non-labeled L-Trp, L-Kyn, 5-HT and QA in plasma. This method may help improve our understanding on L-Trp metabolism in vivo in animals and humans and potentially reveal the relative contribution of the four pathways of L-Trp metabolism.
authors.affiliation: UMR 1280 Physiologie des Adaptations Nutritionnelles, INRA, Université de Nantes, Nantes, France; Laboratories of Food Science and Nutrition, School of Human Culture, The University of Shiga Prefecture, Shiga, Japan.
authors.forename: Mitsue
authors.initials: M
authors.lastname: Sano
authors.forename: Véronique
authors.initials: V
authors.lastname: Ferchaud-Roucher
authors.forename: Charlotte
authors.initials: C
authors.lastname: Nael
authors.forename: Audrey
authors.initials: A
authors.lastname: Aguesse
authors.forename: Guillaume
authors.initials: G
authors.lastname: Poupeau
authors.forename: Blandine
authors.initials: B
authors.lastname: Castellano
authors.forename: Dominique
authors.initials: D
authors.lastname: Darmaun
mesh_terms: D000328:Adult; D000818:Animals; D005260:Female; D008401:Gas Chromatography-Mass Spectrometry / Q000379:methods*; D006801:Humans; D007553:Isotope Labeling; D007737:Kynurenine / Q000097:blood* / Q000737:chemistry; D016014:Linear Models; D008297:Male; D011247:Pregnancy; D017378:Quinolinic Acid / Q000097:blood* / Q000737:chemistry; D051381:Rats; D017207:Rats, Sprague-Dawley; D017208:Rats, Wistar; D015203:Reproducibility of Results; D012701:Serotonin / Q000097:blood* / Q000737:chemistry; D014364:Tryptophan / Q000097:blood* / Q000737:chemistry; D055815:Young Adult
chemical_list: D012701:Serotonin; D007737:Kynurenine; D014364:Tryptophan; D017378:Quinolinic Acid
keywords: GC/MS; l-Kynurenine; l-Tryptophan; serotonin; stable isotope