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.
Kasumov
Takhar
T
Department and Nutrition, Case Western Reserve University, 11000 Cedar Road, Cleveland, OH 44106-7139, USA.
Martini
Wenjun Z
WZ
Reszko
Aneta E
AE
Bian
Fang
F
Pierce
Bradley A
BA
David
France
F
Roe
Charles R
CR
Brunengraber
Henri
H
D000214:Acyl Coenzyme A / Q000032:analysis* / Q000097:blood / Q000378:metabolism; D000818:Animals; D002138:Calibration / Q000592:standards; D002250:Carbon Radioisotopes / Q000737:chemistry; D003903:Deuterium / Q000737:chemistry; D008401:Gas Chromatography-Mass Spectrometry / Q000379:methods; D007553:Isotope Labeling / Q000379:methods; D008099:Liver / Q000201:enzymology; D013058:Mass Spectrometry / Q000379:methods; D008970:Molecular Weight; D010477:Perfusion / Q000379:methods; D051381:Rats; D017207:Rats, Sprague-Dawley; D012521:Sarcosine / Q000737:chemistry
D000214:Acyl Coenzyme A; D002250:Carbon Radioisotopes; C015357:methylmalonyl-coenzyme A; C009061:propionyl-coenzyme A; D003903:Deuterium; C012046:succinyl-coenzyme A; D012521:Sarcosine