MMPC :: Catalog Item

13C-Labeling pattern of acetyl moiety of citrate (substrate oxidation)

Center	Case Western Reserve University
Center Core	Analytical Core
Test No.	CA2022
Test Price	$110/sample
Description	A number of investigators, who use 13C-labeled precursors of acetyl-CoA -in vivo- in isolated organs or in cell incubations, have attempted to estimate the labeling of mitochondrial acetyl-CoA to calculate the contribution of the substrate to the acetyl-CoA oxidized in the citric acid cycle (CAC). The best proxy for mitochondrial acetyl-CoA is the acetyl moiety of citrate. We developed an assay of the labeling of the acetyl moiety of citrate which involves (i) tissue extraction, (ii) alkaline hydrolysis of extant acetyl-CoA, (iii) after pH adjustment, cleavage of citrate with CoA + ATP-citrate lyase which we isolated from rat liver. The acetyl-CoA formed is either assayed as such by LC-MS, or reacted with thiophenol, followed by GC-MS assay of acetylthiophenol. This assay allows one to calculate the contribution of two or three substrates to mitochondrial acetyl-CoA in the same experiment. For example, consider a mouse heart perfused with unlabeled glucose + [1-13C]palmitate + [U-13C4]acetoacetate. These substrates yield acetyl-CoA that is unlabeled (M), singly labeled (M1), or doubly labeled (M2), respectively. So, the percent abundances of the M, M1, and M2 mass isotopomers of the acetyl moiety of citrate yield the contribution of each of the substrates to mitochondrial energy production.
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Energetics

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