Cells can synthesize fatty acids by ligating multiple acetyl units from acetyl-CoA. This is followed by desaturation and elongation reactions to produce a variety of fatty acids required for proper cellular functioning. Alternatively, exogenous lipid sources can contribute to cellular fatty acid pools. Here, we present a method based on incorporation of (13)C-carbon from labeled substrates into fatty acids and subsequent mass spectrometry analysis. The resulting labeling patterns can be used to determine (1) (13)C-enrichment of lipogenic acetyl-CoA, (2) the relative contributions of synthesis and uptake, and (3) absolute fatty acid fluxes. We begin by providing a background and general principles regarding the use of stable isotopes to study fatty acid metabolism. We then proceed with detailing procedures for sample preparation and both GC-MS and LC-MS analysis of isotope incorporation. Finally, we discuss the interpretation of the resulting fatty acid-labeling patterns.
Keywords: Acetyl-CoA; De novo lipogenesis; Fatty acid metabolism; Mass spectrometry; Stable isotopes; Tracing.
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