Assay of the Concentration and 13C Isotopic Enrichment of Propionyl-CoA, Methylmalonyl-CoA, and Succinyl-CoA by Gas Chromatography–Mass Spectrometry

✍ Scribed by Takhar Kasumov; Wenjun Z. Martini; Aneta E. Reszko; Fang Bian; Bradley A. Pierce; France David; Charles R. Roe; Henri Brunengraber

• Publisher: Elsevier Science
• Year: 2002
• Tongue: English
• Weight: 85 KB
• Volume: 305
• Category: Article
• ISSN: 0003-2697
• DOI: 10.1006/abio.2002.5639

✦ Synopsis

We developed gas chromatography-mass spectrometry assays for the concentration and mass isotopomer distribution of propionyl-CoA, methylmalonyl-CoA, and succinyl-CoA in tissues. The assays involves perchloric acid extraction of the tissue, spiking the extract with [ 2 H 5 ]propionyl-CoA and [ 2 H 4 ]succinyl-CoA internal standards, and isolation of short-chain acyl-CoA fraction on an oligonucleotide purification cartridge. Propionyl-CoA is reacted with sarcosine and the formed N-propionylsarcosine is assayed as its pentafluorobenzyl derivative. Methylmalonyl-CoA and succinyl-CoA are hydrolyzed and the corresponding acids assayed as tert-butyl dimethylsilyl derivatives. The assay was applied to a study of [U-13 C 3 ]propionate metabolism in perfused rat livers. While propionyl-CoA is only M3 labeled, succinyl-CoA is M3, M2, and M1 labeled because of isotopic exchanges in the citric acid cycle. Methylmalonyl-CoA is M3 and M2 labeled, reflecting reversal of S-methylmalonyl-CoA mutase. Thus, our assays allow measuring the turnover of the coenzyme A derivatives involved in anaplerosis of the citric acid cycle via precursors of propionyl-CoA, i.e., propionate, odd-chain fatty acids, isoleucine, threonine, and valine.