44 Chapter 2 Mass spectrometric assays The main analytical platform that is used to detect and quantify a wider range of metabolites is MS (111–113). Different kinds of separation methods can be coupled to MS to increase the depth of analysis as well as to cover a wider range of compounds. Gas chromatography (GC) and liquid chromatography (LC) are most widely used (114). LC is better suited to measure polar and charged molecules, whereas GC is preferred when investigating short-chain fatty acids, esters, hydrocarbons, and volatile and thermally stable molecules. Most metabolite measurements are done at a fixed time point, while it is of interest to rather assess turnover rates, as this will provide a more dynamic picture of the contribution of a given metabolic pathway in, for example, central carbon metabolism. Such measurements can be done using stable-isotope-labeled metabolic substrates (see paragraph “Mass spectrometric metabolic flux analysis”). Metabolite analyses by MS can be performed in a targeted or untargeted manner. With targeted MS methods it is possible to have accurate and precise quantitative information on known metabolites, such as those that are involved in metabolic reprogramming of macrophages. While many metabolites, that play a key role in macrophage metabolic reprogramming, are known, recent work shows that new metabolites are still being discovered using untargeted MS (115). One example is the discovery of the role of uridine diphosphate N-acetylglucosamine in macrophage polarization (116).Once such a metabolite has been discovered, it may be further investigated in greater detail with a targeted MS approach. Mass Spectrometric metabolic flux analysis Knowing the concentration and/or abundance of the metabolites at a given moment in time can help to understand which metabolite levels are altered at the moment of metabolite extraction. However, to understand the dynamics of metabolic reprogramming, it is also relevant to know which metabolic pathways are up- or down-regulated, which can be deduced from measuring production and consumption rates of metabolites by following the incorporation of stable isotopes into metabolites over time. This is accomplished by using stable isotope labelled metabolic tracer molecules to study the flux of stable isotopes through different pathways for a set of key metabolites (117). An untargeted LC-MS metabolomics flux analysis approach with stable isotope labeling of metabolites was used to get more insight into substrate use in different macrophage phenotypes after stimulation with LPS or IL4 (118). A change in phenotype due to a certain stimulation was first confirmed by flow cytometry and then different metabolic pathways were
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