title: Measurement of tissue acyl-CoAs using flow-injection tandem mass spectrometry: acyl-CoA profiles in short-chain fatty acid oxidation defects.
abstract: The primary accumulating metabolites in fatty acid oxidation defects are intramitochondrial acyl-CoAs. Typically, secondary metabolites such as acylcarnitines, acylglycines and dicarboxylic acids are measured to study these disorders. Methods have not been adapted for tissue acyl-CoA measurement in defects with primarily acyl-CoA accumulation. Our objective was to develop a method to measure fatty acyl-CoA species that are present in tissues of mice with fatty acid oxidation defects using flow-injection tandem mass spectrometry. Following the addition of internal standards of [(13)C(2)] acetyl-CoA, [(13)C(8)] octanoyl-CoA, and [C(17)] heptadecanoic CoA, acyl-CoA's are extracted from tissue samples and are injected directly into the mass spectrometer. Data is acquired using a 506.9 neutral loss scan and multiple reaction-monitoring (MRM). This method can identify all long, medium and short-chain acyl-CoA species in wild type mouse liver including predicted 3-hydroxyacyl-CoA species. We validated the method using liver of the short-chain-acyl-CoA dehydrogenase (SCAD) knock-out mice. As expected, there is a significant increase in [C(4)] butyryl-CoA species in the SCAD -/- mouse liver compared to wild type. We then tested the assay in liver from the short-chain 3-hydroxyacyl-CoA dehydrogenase (SCHAD) deficient mice to determine the profile of acyl-CoA accumulation in this less predictable model. There was more modest accumulation of medium chain species including 3-hydroxyacyl-CoA's consistent with the known chain-length specificity of the SCHAD enzyme.
authors.affiliation: Division of Endocrinology, Department of Pediatrics, Children's Hospital of Philadelphia and Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA.
authors.forename: Andrew A
authors.initials: AA
authors.lastname: Palladino
authors.forename: Jie
authors.initials: J
authors.lastname: Chen
authors.forename: Staci
authors.initials: S
authors.lastname: Kallish
authors.forename: Charles A
authors.initials: CA
authors.lastname: Stanley
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Bennett
mesh_terms: D000214:Acyl Coenzyme A / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D044944:Acyl-CoA Dehydrogenases / Q000235:genetics / Q000378:metabolism; D000818:Animals; D005232:Fatty Acids, Volatile / Q000737:chemistry / Q000378:metabolism; D008099:Liver / Q000737:chemistry / Q000201:enzymology; D051379:Mice; D018345:Mice, Knockout; D010084:Oxidation-Reduction; D015203:Reproducibility of Results; D053719:Tandem Mass Spectrometry
chemical_list: D000214:Acyl Coenzyme A; D005232:Fatty Acids, Volatile; D044944:Acyl-CoA Dehydrogenases