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

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-C-13(3)]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 [C-13]substrates in perfused rat livers and hearts. In livers perfused with [1,2-C-13(2)]acetate, malonyl-CoA is doubly labeled from [1,2-C-13(2)]acetate and singly labeled from (CO2)-C-13. In livers perfused with either (NaHCO3)-C-13 or [3-C-13]lactate + [3-C-13]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 (NaHCO3)-C-13, was about 1.25 min. Thus, our assay allows us to measure the turnover of tissue malonyl-CoA, the contribution of various [C-13]substrates to its production in lipogenic and nonlipogenic organs, and the cycling between acetyl-CoA and malonyl-CoA in nonlipogenic organs. (C) 2001 Academic Press.