title: Simultaneous detection of lysine metabolites by a single LC-MS/MS method: monitoring lysine degradation in mouse plasma.
abstract: Detection and quantification of lysine degradation metabolites in plasma is necessary for the diagnosis and follow-up of diseases such as pyridoxine-dependent epilepsy. The principal metabolites involved in the disease are related to the first steps of lysine oxidation, either through the saccharopine or the pipecolate pathways. Currently, there are three different analytical methods used to assess the content of these metabolites in urine and plasma, but they require different sample preparations and analytical equipment. Here, we describe a protocol that calls for a simple sample preparation and uses liquid chromatography tandem mass spectrometry (LC-MS/MS) that allows simultaneous detection and quantification of underivatized l-saccharopine, l-aminoadipic acid, l-pipecolic acid, piperideine-6-carboxylate, l-glutamic acid, and pyridoxal-5-phosphate in plasma samples. To validate the method we analyzed the time course degradation after intraperitoneal injection of l-lysine in C57BL/6/J mice. We observed that the degradation of lysine through the saccharopine pathway reached a maximum within the first 2 h. At this time point there was an increase in the levels of the metabolites saccharopine, aminoadipic acid, and pipecolic acid by 3-, 24- and 3.4-fold, respectively, compared to time zero levels. These metabolites returned to basal levels after 4-6 h. In conclusion, we have developed a LC-MS/MS approach, which allows simultaneous analysis of lysine degradation metabolites without the need for derivatization.
authors.affiliation: Centro de Biologia Molecular e Engenharia Genética, Universidade Estadual de Campinas (UNICAMP), 13083-875 Campinas, SP Brazil.
authors.forename: Izabella A
authors.initials: IA
authors.lastname: Pena
authors.affiliation: Thomson Mass Spectrometry Laboratory, Universidade Estadual de Campinas (UNICAMP), Campinas, SP 13083-861 Brazil.
authors.forename: Lygia A
authors.initials: LA
authors.lastname: Marques
authors.affiliation: Centro Infantil Boldrini, Universidade Estadual de Campinas (UNICAMP), Campinas, SP 13083-210 Brazil.
authors.forename: Angelo B A
authors.initials: AB
authors.lastname: Laranjeira
authors.affiliation: Centro Infantil Boldrini, Universidade Estadual de Campinas (UNICAMP), Campinas, SP 13083-210 Brazil ; Departamento de Genética Médica, Faculdade de Ciências Médicas, Universidade Estadual de Campinas (UNICAMP), Campinas, SP 13083-887 Brazil.
authors.forename: José A
authors.initials: JA
authors.lastname: Yunes
authors.affiliation: Thomson Mass Spectrometry Laboratory, Universidade Estadual de Campinas (UNICAMP), Campinas, SP 13083-861 Brazil.
authors.forename: Marcos N
authors.initials: MN
authors.lastname: Eberlin
authors.affiliation: Centro de Biologia Molecular e Engenharia Genética, Universidade Estadual de Campinas (UNICAMP), 13083-875 Campinas, SP Brazil ; Departamento de Genética e Evolução, Instituto de Biologia, Universidade Estadual de Campinas (UNICAMP), Campinas, SP 13083-970 Brazil.
authors.forename: Paulo
authors.initials: P
authors.lastname: Arruda
keywords: Amino acid; Lysine catabolism; Mass spectrometry; Pipecolic acid; Saccharopine