Assay of the Concentration and 13 C Isotopic Enrichment of Propionyl-CoA, Methylmalonyl-CoA, and Succinyl-CoA by Gas Chromatography–Mass Spectrometry Takhar Kasumov,* Wenjun Z. Martini,* Aneta E. Reszko,* Fang Bian,* Bradley A. Pierce,* France David,* Charles R. Roe,† and Henri Brunengraber* ,1 *Department and Nutrition, Case Western Reserve University, Cleveland, Ohio 44106-7139; and †Institute of Metabolic Diseases, Baylor University Medical College, Dallas, Texas 77030 Received November 19, 2001; published online April 25, 2002 We developed gas chromatography–mass spectrom- etry assays for the concentration and mass isotopomer distribution of propionyl-CoA, methylmalonyl-CoA, and succinyl-CoA in tissues. The assays involves per- chloric acid extraction of the tissue, spiking the ex- tract 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 car- tridge. Propionyl-CoA is reacted with sarcosine and the formed N-propionylsarcosine is assayed as its pen- tafluorobenzyl 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, re- flecting 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, threo- nine, and valine. © 2002 Elsevier Science (USA) Propionyl-CoA, methylmalonyl-CoA, and succinyl- CoA are intermediates in the catabolism of some amino acids (methionine, isoleucine, valine, and threonine), cholesterol (from the degradation of its side-chain), and odd-chain fatty acids (1). Propionyl-CoA is also formed in the liver from propionate derived from intestinal fermentations (2). Through propionyl-CoA, the above substrates contribute catalytic intermediates of the cit- ric acid cycle (anaplerosis) in all mammalian cells as well as gluconeogenic carbon to liver and kidney cortex. In inborn or dietary-acquired disorders in the metabo- lism of propionyl-CoA and methylmalonyl-CoA, there is formation of large amounts of methylcitrate, propio- nylcarnitine, hydroxypropionate, and methylmalonate (1). The concentration of the three CoA esters can be assayed by HPLC (3–5). However, there are no meth- ods for measuring their 13 C-enrichment and mass iso- topomer distribution (MID). 2 Such techniques could provide information on the regulation of propionyl-CoA metabolism. Over the past 10 years, mass isotopomer analysis has been applied to analytical procedures, studies of pathway control, and the synthesis of biopolymers (for a review, see (6)). As part of a study of anaplerosis from odd-chain fatty acids, we developed gas chromatography–mass spec- trometry (GC-MS) assays for the concentration and MID of the three CoA esters. The present report de- scribes these techniques and illustrates them by a study of the metabolism of [U- 13 C 3 ]propionate in per- fused rat livers. METHODS Materials. Chemicals and biochemicals were obtained from Sigma-Aldrich. [U- 13 C 3 ]Propionate, [ 2 H 5 ]propionate, 1 To whom correspondence should be addressed: Department of Nutrition, Room 280, Case Western Reserve University, 11000 Ce- dar Road, Cleveland, OH 44106-7139. Fax: (216) 368-6560. E-mail: hxb8@po.cwru.edu. 2 Abbreviations used: GC-MS, gas chromatography–mass spec- trometry; MID, mass isotopomer distribution; MPE, molar percent enrichment; PFB, pentafluorobenzyl; TBDMS, tert-butyl dimethyl- silyl. 90 0003-2697/02 $35.00 © 2002 Elsevier Science (USA) All rights reserved. Analytical Biochemistry 305, 90 –96 (2002) doi:10.1006/abio.2002.5639, available online at http://www.idealibrary.com on