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

✍ Scribed by Aneta E. Reszko; Takhar Kasumov; Blandine Comte; Bradley A. Pierce; France David; Ilya R. Bederman; Joseph Deutsch; Christine Des Rosiers; Henri Brunengraber

• Publisher: Elsevier Science
• Year: 2001
• Tongue: English
• Weight: 90 KB
• Volume: 298
• Category: Article
• ISSN: 0003-2697
• DOI: 10.1006/abio.2001.5349

✦ Synopsis

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