Abstract
Coenzyme A (CoA) is an essential cofactor for dozens of reactions in intermediary metabolism. Dysregulation of CoA synthesis or acyl CoA metabolism can result in metabolic or neurodegenerative disease. Although several methods use liquid chromatography coupled with mass spectrometry/mass spectrometry (LC-MS/MS) to quantify acyl CoA levels in biological samples, few allow for simultaneous measurement of intermediates in the CoA biosynthetic pathway. Here we describe a simple sample preparation and LC-MS/MS method that can measure both short-chain acyl CoAs and biosynthetic precursors of CoA. The method does not require use of a solid phase extraction column during sample preparation and exhibits high sensitivity, precision, and accuracy. It reproduces expected changes from known effectors of cellular CoA homeostasis and helps clarify the mechanism by which excess concentrations of etomoxir reduce intracellular CoA levels.
Highlights
IntroductionPublisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations
We detail an LC-MS/MS method that allows for the sensitive detection of shortchain acyl Coenzyme A (CoA) and metabolites in the CoA biosynthetic pathway
Use of SSA circumvents the need for solid phase extraction (SPE) [17,31] as this single solvent can be used as both a deproteinizing agent and solvent for the reconstitution of acyl CoAs
Summary
Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Coenzyme A (CoA) is an obligatory co-factor in all organisms [1]. It is involved in several aspects of mammalian cellular metabolism including the Krebs cycle, oxidation of fatty acids and branched chain amino acids, as well as synthesis of fatty acids and sterols. CoA acts as an acyl group carrier forming thioester linkages with organic acids to yield acyl CoAs (e.g., acetyl CoA or palmitoyl CoA) [1]. The formation of a CoA thioester serves multiple functions. The large free energy of hydrolysis of the thioester bond serves as a means to ‘charge’ or ‘activate’ the adjoining acyl group for further metabolism
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