Medicina

Omics strategies have triggered a revolution in the understanding of the microorganisms that reside in our body, and their implications in health and disease. For diagnosis and therapeutics, metabolomic fingerprinting is the most powerful approach, since the metabolites represent the actual interplay between humans and microbes. Studying the metabolome requires several new high-throughput analytical techniques and innovative computational methodologies. Herein, we will focus on the metabolomics workflow for gut microbiota analysis, including sampling, laboratory procedures, and available analytical techniques, paying special attention to microbiota isolation and multiplatform complementarity. Finally, we will summarize some applications and implications of gut microbiota metabolites in biomarkers discovery and several therapeutic strategies, such as fecal microbiota transplantation and the usage of prebiotics and probiotics.

GC-MS for untargeted metabolomics is a well-established technique. Small molecules and molecules made volatile by derivatization can be measured and those compounds are key players in main biological pathways. This tutorial provides ready-to-use protocols for GC-MS-based metabolomics, using either the well-known low-resolution approach (GC-Q-MS) with nominal mass or the more recent high-resolution approach (GC-QTOF-MS) with accurate mass, discussing their corresponding strengths and limitations. Analytical procedures are covered for different types of biofluids (plasma/serum, bronchoalveolar lavage, urine, amniotic fluid) tissue samples (brain/hippocampus, optic nerve, lung, kidney, liver, pancreas) and samples obtained from cell cultures (adipocytes, macrophages, Leishmania promastigotes, mitochondria, culture media). Together with the sample preparation and data acquisition, data processing strategies are described specially focused on Agilent equipments, including deconvolution software and database annotation using spectral libraries. Manual curation strategies and quality control are also deemed. Finally, considerations to obtain a semiquantitative value for the metabolites are also described. As a case study, an illustrative example from one of our experiments at CEMBIO Research Centre, is described and findings discussed.