Metabolism

Metabolism is the functional activity of all living cells, and thus 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. 

Technology Solutions

Metabolomics requires high-end analytical instrumentation, of which mass spectrometry and NMR together are the most appropriate technologies. The Center also can quantify protein levels by Reverse Phase Protein Arrays (RPPI) in a highly multiplexed manner. Elemental analyses can be carried out using ICP-MS, which when coupled with laser ablation, permits tissue slice imaging. Issues of sample preparation  and data analysis/informatics  are similarly critically important and are specifically addressed in the Center for Environmental and Systems Biochemistry.

Furthermore, at CESB a particular emphasis is on determination of metabolic pathway and nutrient utilization and reprogramming by tracing individual atoms through metabolic pathways, via the agency of stable isotope-resolved metabolomics (SIRM), an approach pioneered by the directors of CESB. Environmental factors come into play both at the extrinsic level (macroenvironment) as represented by diet, pollutants and drugs for example, and local environments (microenvironment) as represented by the prevailing tissue conditions outside cells.

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.  More recently, we have extended the technique to multiplexing stable isotopes to maximize the information retrieval from small quantities of samples such as biopsies.