Abstract
Using gas chromatography mass spectrometry (GC-MS) to analyze the citric acid cycle (CAC) and related intermediates (such as glutamate, glutamine, GABA, and aspartate) is an analytical approach to identify unexpected correlations between apparently related and unrelated pathways of energy metabolism. Intermediates can be as expressed as their absolute concentrations or relative ratios by using known amounts of added reference standards to the sample. GC-MS can also distinguish between heavy labeled molecules (2H- or 13C-labeled) and the naturally occurring most abundant molecules. Applications using tracers can also assess the turnover of specific metabolic pools under various physiological and pathological conditions as well as for pathway discovery. The following protocol is a relatively simple method that is not only sensitive for small concentrations of metabolic intermediates but can also be used in vivo or in vitro to determine the integrity of various metabolic pathways, such as flux changes within specific metabolite pools. We used this protocol to determine the role of phosphoenolpyruvate carboxykinase 1 (Pck1) gene in mouse macrophage cells to determine the percent contribution from a precursor of 13C labeled glucose into specific CAC metabolite pools.
Keywords:
Bone marrow-derived macrophages; Chromatography mass spectrometry methods; GC-MS; Immunology; Macrophage; Macrophage polarization; Metabolism; Stable isotopes.