Assay of the concentration and (13)C isotopic enrichment of propionyl-CoA,methylmalonyl-CoA,and succinyl-CoA by gas chromatography-mass spectrometry

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.