Assay of the concentration and 13C-isotopic enrichment of malonyl-coenzyme A by gas chromatography-mass spectrometry

Aneta E. Reszko, Takhar Kasumov, Blandine Comte, Bradley A. Pierce, France David, Ilya R. Bederman, Joseph Deutsch, Christine Des Rosiers, Henri Brunengraber*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

43 Scopus citations

Abstract

We developed gas chromatography-mass spectrometry assays for the concentration and mass isotopomer distribution of malonyl-CoA in tissues. The assay involves perchloric acid extraction of the tissue, spiking the extract with [U-13C3]malonyl-CoA or dimethylmalonyl-CoA internal standard, isolation of short-chain acyl-CoA fraction on an oligonucleotide purification cartridge, alkaline hydrolysis to malonate, trimethylsilyl derivatization, and analysis of the mass isotopomer distribution of malonate. The assay was applied to labeling of malonyl-CoA from various [13C]substrates in perfused rat livers and hearts. In livers perfused with [1,2-13C2]acetate, malonyl-CoA is doubly labeled from [1,2-13C2]acetate and singly labeled from 13CO2. In livers perfused with either NaH13CO3 or [3-13C]lactate + [3-13C]pyruvate, the half-lives of singly labeled malonyl-CoA were less than 20 s and 6.95 min, respectively. In rat heart, the half-life of malonyl-CoA, traced with NaH13CO3, was about 1.25 min. Thus, our assay allows us to measure the turnover of tissue malonyl-CoA, the contribution of various [13C]substrates to its production in lipogenic and nonlipogenic organs, and the cycling between acetyl-CoA and malonyl-CoA in nonlipogenic organs.

Original languageEnglish
Pages (from-to)69-75
Number of pages7
JournalAnalytical Biochemistry
Volume298
Issue number1
DOIs
StatePublished - 1 Nov 2001

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