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Analytical strategies for the screening and determination of tryptophan metabolites in glioma human cell cultures

Grant number: 12/05198-0
Support Opportunities:Regular Research Grants
Duration: June 01, 2012 - November 30, 2014
Field of knowledge:Physical Sciences and Mathematics - Chemistry - Analytical Chemistry
Principal Investigator:Ernani Pinto Junior
Grantee:Ernani Pinto Junior
Host Institution: Faculdade de Ciências Farmacêuticas (FCF). Universidade de São Paulo (USP). São Paulo , SP, Brazil
Associated researchers:Ana Campa ; Ariane Rivellis Júlio ; Fabyana Maria dos Anjos


The aim of this study is the development of bioanalytical methods by on-line solid phase extraction coupled with two-dimensional HPLC for the simultaneous quantitative determination of tryptophan (TRP) metabolites, including those of kynurenine (KYN), serotonin (SER), tryptamine and dimethyltryptamine (DMT) pathways. The following detectors will be used in this study: (i) diode array (DAD), (ii) fluorescence (FD) and (iii) mass spectrometry (MS). Methods will be validated according to ANVISA 899. The biological matrix is glioma human cell cultures - strain A172. Extraction methods will also be optimized for the concentration of metabolites present in the supernatants and cell homogenates. The investigation of the catabolism of TRP and formation of its metabolites are of great importance, because several of the compounds present in these pathways act on the processes of tumor immune escape, tolerance and immunomodulation. The confirmation of some metabolites and determination of their concentrations in biological fluids can be of great value for the prognosis of various diseases. Additionally, recent results from our group showed that there is a cross-talking between the metabolic pathways of TRP and several other metabolites that affect the growth of tumors. Thus, there is a great interest in developing selective and sensitive analytical methodologies to monitor and demonstrate the formation of metabolites from TRP metabolism. Labled TRP with stable isotopes (13C and deuterium - D) at different positions in its structure and their metabolites will be monitored by LC-MS/MS (ion trap and Q-TOF). The choice of glioma A172 cultures is that all pathways involving TRP biotransformation are active in these cells, making it an excellent matrix for the method development. In addition, the KYN pathway, led by the enzyme indoleamine 2,3 dioxygenase (IDO), is strongly increased in the presence of interferon gamma (IFN-gama). This cell culture provides appropriate conditions for the development of new bioanalytical methods. (AU)

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Scientific publications
(References retrieved automatically from Web of Science and SciELO through information on FAPESP grants and their corresponding numbers as mentioned in the publications by the authors)
FARIAS, F. F.; MARTINS, V. A. P.; DOERR, F. A.; TRUJILLO, L. M.; PINTO, E.. Forced degradation study and characterization of main impurities of ibuprofen soft gelatin capsules by LC-MS-QTOF. Pharmazie, v. 76, n. 4, p. 138-144, . (12/05198-0)

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