Development and validation of bioanalytical methods for monitoring the products of metabolic pathways of tryptophan in human glioma cell line

Abstract

The aim of this study is development of bioanalytical methods for High Performance Liquid Chromatography (HPLC) for the two-dimensional and simultaneous quantitative determination of metabolites of tryptophan (TRP), including those of airway quinureninas (via KYN), serotonin (via RSS) and tryptamine (including dimethyltryptamine, DMT). Will be used to detect different types: (i) by diode array (DAD); (ii) fluorescence (DF) and (iii) mass spectrometry (MS). The validation of the methods follow the specifications of Resolution RE n º 899 of May 29, 2003 ANVISA. As a biological matrix, will be used glioma cell cultures of strain A172. Besides the development of methods using two-dimensional chromatography for the simultaneous detection of the analytes, 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 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 the presence 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 of the collaborative group (Laboratory of Clinical Biochemistry FCF-USP) show 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 interest in developing selective and sensitive analytical methodologies to monitor and demonstrate the formation of the products of the metabolism of TRP. For roads to be shown will be used TRP marked with stable isotopes (13C and deuterium - D) at different positions in its structure and the products will be monitored by LC-MS/MS (ion trap and Q-TOF). The choice of the lineage of glioma A172 is due to the route of catabolism of TRP, and KYN pathway via tryptamines, 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 line provides appropriate conditions for the development of new bioanalytical methods selective, robust and sensitive for the study of metabolism of TRP. (AU)