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-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.
Reszko
A E
AE
Department of Biochemistry, Case Western Reserve University, Cleveland, Ohio 44106, USA.
Kasumov
T
T
Comte
B
B
Pierce
B A
BA
David
F
F
Bederman
I R
IR
Deutsch
J
J
Des Rosiers
C
C
Brunengraber
H
H
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
D000085:Acetates; D001639:Bicarbonates; D002247:Carbon Isotopes; D011773:Pyruvates; D008316:Malonyl Coenzyme A