Resumen de Aplicaciones de la metabolómica basada en lc-ms en el campo clínico y toxicológico
Metabolomics consists of the study and comparison of metabolomes, that is, the collection of low molecular weight metabolites (< 1 KDa) in biological samples. The principal aim of this emerging omic science is to provide an insight into the metabolic status of complex living systems. Unlike genes and proteins, the functions of which are subject to epigenetic regulation and post-translational modifications, respectively, metabolites serve as direct signatures of biochemical activity and are, therefore, easier to correlate with phenotype. Comparison of themetabolic profiles from different phenotypes can be used to identify specific metabolic changes, leading to the understanding of physiology, toxicology and disease progression.There are two metabolomic approaches, targeted and untargeted metabolomics. Targeted metabolomics refers to a method in which a specified list of metabolites is measured, typically focusing on one or more related pathways of interest. On the contrary, untargeted metabolomics aims to measure simultaneously as many metabolites as possible from a biological sample without bias. Nowadays the more widespread analytical platform in metabolomics is liquid chromatography coupled to mass spectrometry (LC-MS), closely followed by nuclear magnetic resonance spectroscopy (NMR). LC-MS is a very sensitive technique that enables to analyze simultaneously thousands of metabolites requiring a small sample volume for analysis. In this research work LC-MS based metabolomics has been applied to different clinical and toxicological issues:¿ Firstly 8 human plasma preparation protocols were evaluated for their suitability for metabolomic studies by ultra-high performance liquid chromatography coupled to time-of-flight mass spectrometry (UHPLC-TOF-MS). Different criteria were used to evaluate the different protocols: multivariate and univariate analysis, number of extracted features, reproducibility of a series of selected features and the amount of protein remaining in each reconstituted plasma extract. The best results were offered by an organic solvent protein precipitation with acetonitrile in a 2:1 organic solvent:plasma ratio.¿ Another study aimed to better understand the variability of galactosamine mediated hepatotoxicity. Firstly, the best sample treatment protocol for the study of global metabolic profiling of rat faecal samples by UHPLC-TOF-MS was selected and then, the optimal protocol was applied to the hepatotoxicity study. Classification of faecal metabolic profiles according to histopathological analysis was observed.¿ The third project was based on the optimization of a UHPLC-TOF-MS method for obtaining lipid profiles of human plasma samples. This method was applied to the study of the effect of two nutritional challenges on the lipid profile of plasma samples from women of different age and body mass indexes. Significant differences were observed in the intensity obtained for some features in the comparisons performed.¿ Last project was based on a targeted liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) approach for the determination of metabolites involved in the central carbon metabolism. The proposed LC-MS/MS was firstly validated in stock solutions and then applied to human dried blood spots samples.
