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Catalog Item
13C-Labeling pattern of acetyl moiety of citrate (substrate oxidation)
Common Name(s): Metabolites of oxidative metabolism

CenterCase Western Reserve University
Center CoreAnalytical Core
Test No.CA2022
Test Price$175/sample
DescriptionA 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.
DataData will be collected for this catalog item
Keywordsenergy metabolism, metabolic flux, mitochondria, oxidation, partitioning of
substrates, TCA cycle
Tissues

No catalog groups / subjects found.





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