title: In situ assay of fatty acid β-oxidation by metabolite profiling following permeabilization of cell membranes.
abstract: Quantitative analysis of mitochondrial FA β-oxidation (FAO) has drawn increasing interest for defining lipid-induced metabolic dysfunctions, such as in obesity-induced insulin resistance, and evaluating pharmacologic strategies to improve β-oxidation function. The aim was to develop a new assay to quantify β-oxidation function in intact mitochondria and with a low amount of cell material. Cell membranes of primary human fibroblasts were permeabilized with digitonin prior to a load with FFA substrate. Following 120 min of incubation, the various generated acylcarnitines were extracted from both cells and incubation medium by protein precipitation/desalting and subjected to solid-phase extraction. A panel of 30 acylcarnitines per well was quantified by MS/MS and normalized to citrate synthase activity to analyze mitochondrial metabolite flux. Pretreatment with bezafibrate and etomoxir revealed stimulating and inhibiting regulatory effects on β-oxidation function, respectively. In addition to the advantage of a much shorter assay time due to in situ permeabilization compared with whole-cell incubation systems, the method allows the detection of multiple acylcarnitines from an only limited amount of intact cells, particularly relevant to the use of primary cells. This novel approach facilitates highly sensitive, simple, and fast monitoring of pharmacological effects on FAO.
authors.affiliation: Research Center, Dr. von Hauner Children's Hospital, Ludwig-Maximilians-Universität München, 80337 Munich, Germany. Electronic address: Regina.Ensenauer@med.uni-muenchen.de.
authors.forename: Regina
authors.initials: R
authors.lastname: Ensenauer
authors.affiliation: Laboratory Becker, Olgemöller and Colleagues, 81671 Munich, Germany.
authors.forename: Ralph
authors.initials: R
authors.lastname: Fingerhut
authors.affiliation: Research Center, Dr. von Hauner Children's Hospital, Ludwig-Maximilians-Universität München, 80337 Munich, Germany.
authors.forename: Sonja C
authors.initials: SC
authors.lastname: Schriever
authors.affiliation: Research Center, Dr. von Hauner Children's Hospital, Ludwig-Maximilians-Universität München, 80337 Munich, Germany.
authors.forename: Barbara
authors.initials: B
authors.lastname: Fink
authors.affiliation: Research Center, Dr. von Hauner Children's Hospital, Ludwig-Maximilians-Universität München, 80337 Munich, Germany.
authors.forename: Marc
authors.initials: M
authors.lastname: Becker
authors.affiliation: Research Center, Dr. von Hauner Children's Hospital, Ludwig-Maximilians-Universität München, 80337 Munich, Germany.
authors.forename: Nina C
authors.initials: NC
authors.lastname: Sellerer
authors.affiliation: Lehrstuhl für Genomorientierte Bioinformatik, Technische Universität München, 85350 Freising, Germany.
authors.forename: Philipp
authors.initials: P
authors.lastname: Pagel
authors.affiliation: Lehrstuhl für Genomorientierte Bioinformatik, Technische Universität München, 85350 Freising, Germany.
authors.forename: Andreas
authors.initials: A
authors.lastname: Kirschner
authors.affiliation: Laboratory Becker, Olgemöller and Colleagues, 81671 Munich, Germany.
authors.forename: Torsten
authors.initials: T
authors.lastname: Dame
authors.affiliation: Laboratory Becker, Olgemöller and Colleagues, 81671 Munich, Germany.
authors.forename: Bernhard
authors.initials: B
authors.lastname: Olgemöller
authors.affiliation: Laboratory Becker, Olgemöller and Colleagues, 81671 Munich, Germany.
authors.forename: Wulf
authors.initials: W
authors.lastname: Röschinger
authors.affiliation: Research Center, Dr. von Hauner Children's Hospital, Ludwig-Maximilians-Universität München, 80337 Munich, Germany.
authors.forename: Adelbert A
authors.initials: AA
authors.lastname: Roscher
mesh_terms: D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D002463:Cell Membrane Permeability; D002648:Child; D005227:Fatty Acids / Q000378:metabolism*; D005347:Fibroblasts / Q000166:cytology; D006801:Humans; D007231:Infant, Newborn; D055432:Metabolomics / Q000191:economics / Q000379:methods*; D008928:Mitochondria / Q000378:metabolism; D010084:Oxidation-Reduction; D015203:Reproducibility of Results; D021241:Spectrometry, Mass, Electrospray Ionization; D053719:Tandem Mass Spectrometry; D013997:Time Factors
chemical_list: D005227:Fatty Acids