Metabolism is the functional activity of all living cells, and thus this reflects the health status of an organism. Therefore the ability to measure global metabolism in quantitative detail is of fundamental importance in all aspects of biology. Metabolomics is the technical means to carry out global analyses of metabolism, via the ability to identify and quantify a large fraction of all of the metabolites present in a cell, and how they change in response to perturbations. This therefore requires high-end analytical instrumentation, of which mass spectrometry and NMR together are the most appropriate technologies ). Issues of sample preparation and data analysis/informatics are similarly critically important and are specifically addressed in the Center. Furthermore, at RC-SIRM, a particular emphasis is on measurements of metabolic rates and changes by tracing individual atoms though metabolic pathways, via the agency of SIRM, an approach pioneered by the directors of RC-SIRM . Environmental factors come into play both at the extrinsic level (macroenvironment) as represented by diet and pollutants for example, and local environments (microenvironment) as represented by the prevailing tissue conditions outside cells.
What is Stable Isotope-Resolved Metabolomics (SIRM)?
Global metabolomics, the quantification of a large number of metabolites in tissue or biofluids can identify disease states or response to therapeutics by reference to the normal condition. However, determining specific mechanisms, such as detecting which pathways are impacted in particular cell types within a tissue by measuring metabolic fluxes, requires additional information as many metabolites are present in different amounts in different cell types or within compartments of cells, as well as participating in several pathways simultaneously. To identify the precursor-product relationships, it is necessary to distinguish different sources of carbon, nitrogen etc. which necessitates some means of “labeling” individual atoms so that their fate can be traced through metabolic pathways. Traditionally this was achieved using radioisotopes. However, stable isotopes have several advantages, including being wholly biocompatible, and also individual atoms within a metabolite are easily distinguishable by NMR and mass spectrometry.
The general approach we have developed, which we call Stable Isotope Resolved Metabolomics or SIRM, combines the power of global (untargeted) metabolic profiling with atom-resolved tracking of metabolites during metabolic transformations within cells, tissue or whole organisms. The cell culture, tissue, or organism is provided with a source metabolite that is enriched at any or all of the atoms with a stable isotope (like 13C or 15N with natural abundances 1.1 % and 0.37%, respectively), and the products are analyzed by NMR and MS at different times after treatment. The specific isotopomer and isotopologue distributions in the various product metabolites are determined, along with the total amounts of the metabolites, which together provide detailed information about the relative importance of intersecting and parallel pathways. For example, lactate can be produced directly from glucose by lactic fermentation, as well as by glutaminolysis; the relative contributions from these independent pathways is readily determined from the isotope distributions in the lactate using either 13C-enriched glucose or glutamine as labeled sources. At the same time such labeling schemes provide simultaneous information about the flow of carbon through the pentose phosphate pathway, glycolysis, hexosamine pathway, the Krebs cycle and lipid biosynthesis among others.