Ae. Reszko et al., Assay of the concentration and C-13-isotopic enrichment of malonyl-coenzyme A by gas chromatography-mass spectrometry, ANALYT BIOC, 298(1), 2001, pp. 69-75
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.