Stable Isotope Tracers for Metabolic Pathway Analysis
Stable isotope tracing allows a metabolic substrate to be followed through downstream biochemical reactions, thereby providing unparalleled insights into the metabolic wiring of cells. This approach stops short of modeling absolute fluxes but is relativel
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duction Interpretation of metabolomics data is often complicated by a lack of dynamic information; this is because significant changes in flux through a pathway can occur without corresponding changes in metabolite abundances (pool sizes). Stable isotope tracing, also referred to as qualitative flux analysis, can provide clarity in these situations by allowing visualization of the flow of a metabolic tracer through pathways of interest. Typically, the tracer contains 13C, 15 N, or 2H atoms (less commonly 18O and 34S have also been used). These heavy atoms can either be uniformly enriched throughout the tracer molecule or enriched at specific positions. The choice of tracer depends on the pathway being investigated, and examples of common isotopic tracers and their uses have recently been comprehensively reviewed [1]. In these experiments, a stable isotope tracer is introduced into the biological system as a metabolic substrate. Uptake and incorporation
Angelo D’Alessandro (ed.), High-Throughput Metabolomics: Methods and Protocols, Methods in Molecular Biology, vol. 1978, https://doi.org/10.1007/978-1-4939-9236-2_17, © Springer Science+Business Media, LLC, part of Springer Nature 2019
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of the tracer results in altered isotopic patterns (mass isotopologue distributions) in downstream metabolites. This approach is particularly useful when studying cell culture models which are inherently amenable to the introduction of stable isotope tracers through exchange of the culture medium. However, stable isotope tracing has also been successfully implemented in vivo, for example, by supplementing drinking water with D2O in animal models [2], and in human studies using preoperative tracer infusions followed by analysis of labeling patterns in resected tumor tissue [3, 4]. Suitable analytical platforms for these experiments include gas chromatography (GC) or liquid chromatography (LC) coupled to mass spectrometry (MS) and nuclear magnetic resonance (NMR) spectroscopy has also been used extensively [5, 6]. While NMR and MS/MS fragmentation can unambiguously determine the position of label incorporations, LC-MS analysis provides the highest throughput approach, particularly when considering a limited number of tracers and time points. When LC-MS is used, the chromatography should be robust and well-annotated, and a high-resolution detector is recommended to distinguish potentially overlapping mass isotopologue envelopes. To illustrate these and other key considerations involved in performing successful stable isotope tracing experiments we describe an in vitro tracing experiment using a macrophage cell line. Macrophages are frontline sentinels of the innate immune system responsible for the early detection of pathogens and coordinating the adaptive immune response. Macrophage polarization refers to their activation status and has been grouped into two major phenotypic states, termed M1 and M2 activation. M1 macrophages are associated with immunity to intracellular pathogens and bacteria and arise
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