Autor(es): Pena, Izabella Agostinho, 1989- ; Marques, Lygia de Azevedo ; Laranjeira, Angelo Brunelli Albertoni, 1981- ; Yunes, José Andrés, 1967- ; Eberlin, Marcos Nogueira, 1959- ; Arruda, Paulo, 1952-
Data: 2016
Identificador Persistente: https://hdl.handle.net/20.500.12733/1059
Origem: Oasisbr
Assunto(s): Sacaropina; Espectrometria de massa; Saccharopine; Mass spectrometry; Lysine catabolism; Amino acid; Pipecolic acid; Artigo de pesquisa
Agradecimentos: This study was funded by FAPESP-10/50114-4. Izabella Agostinho Pena received Ph.D. fellowship from FAPESP-12/00235-5. Lygia A. Marques received a postdoctoral fellowship from FAPESP-13/23920-8. Paulo Arruda is a CNPq productivity research fellow. The funders had no role in study design, data collection, and analysis, decision to publish or preparation of the manuscript
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
CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQ
FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESP
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