Assay of the Concentration and 13C-Isotopic Enrichment of Malonyl–Coenzyme A by Gas Chromatography–Mass Spectrometry

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.