title: 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.
authors.affiliation: Department of Biochemistry, Case Western Reserve University, Cleveland, Ohio 44106, USA.
authors.forename: A E
authors.initials: AE
authors.lastname: Reszko
authors.forename: T
authors.initials: T
authors.lastname: Kasumov
authors.forename: B
authors.initials: B
authors.lastname: Comte
authors.forename: B A
authors.initials: BA
authors.lastname: Pierce
authors.forename: F
authors.initials: F
authors.lastname: David
authors.forename: I R
authors.initials: IR
authors.lastname: Bederman
authors.forename: J
authors.initials: J
authors.lastname: Deutsch
authors.forename: C
authors.initials: C
authors.lastname: Des Rosiers
authors.forename: H
authors.initials: H
authors.lastname: Brunengraber
mesh_terms: D000085:Acetates / Q000378:metabolism; D000818:Animals; D001639:Bicarbonates / Q000378:metabolism; D002247:Carbon Isotopes / Q000737:chemistry*; D002849:Chromatography, Gas / Q000379:methods; D066298:In Vitro Techniques; D008099:Liver / Q000201:enzymology*; D008316:Malonyl Coenzyme A / Q000032:analysis* / Q000378:metabolism*; D013058:Mass Spectrometry / Q000379:methods; D009206:Myocardium / Q000201:enzymology*; D010477:Perfusion / Q000379:methods; D011773:Pyruvates / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D014018:Tissue Distribution
chemical_list: D000085:Acetates; D001639:Bicarbonates; D002247:Carbon Isotopes; D011773:Pyruvates; D008316:Malonyl Coenzyme A