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

We developed gas chromatography-mass spectrometry assays for the concentrat ion and mass isotopomer distribution of malonyl-CoA in tissues. The assay i nvolves 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 cartrid ge, alkaline hydrolysis to malonate, trimethylsilyl derivatization, and ana lysis of the mass isotopomer distribution of malonate. The assay was applie d 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-Co A 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-Co A, 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, a nd the cycling between acetyl-CoA and malonyl-CoA in nonlipogenic organs. ( C) 2001 Academic Press.