title: Intracellular in vitro probe acylcarnitine assay for identifying deficiencies of carnitine transporter and carnitine palmitoyltransferase-1.
abstract: Mitochondrial fatty acid oxidation (FAO) disorders are caused by defects in one of the FAO enzymes that regulates cellular uptake of fatty acids and free carnitine. An in vitro probe acylcarnitine (IVP) assay using cultured cells and tandem mass spectrometry is a tool to diagnose enzyme defects linked to most FAO disorders. Extracellular acylcarnitine (AC) profiling detects carnitine palmitoyltransferase-2, carnitine acylcarnitine translocase, and other FAO deficiencies. However, the diagnosis of primary carnitine deficiency (PCD) or carnitine palmitoyltransferase-1 (CPT1) deficiency using the conventional IVP assay has been hampered by the presence of a large amount of free carnitine (C0), a key molecule deregulated by these deficiencies. In the present study, we developed a novel IVP assay for the diagnosis of PCD and CPT1 deficiency by analyzing intracellular ACs. When exogenous C0 was reduced, intracellular C0 and total AC in these deficiencies showed specific profiles clearly distinguishable from other FAO disorders and control cells. Also, the ratio of intracellular to extracellular C0 levels showed a significant difference in cells with these deficiencies compared with control. Hence, intracellular AC profiling using the IVP assay under reduced C0 conditions is a useful method for diagnosing PCD or CPT1 deficiency.
authors.affiliation: Department of Pediatrics, Shimane University School of Medicine, Izumo, Shimane, Japan.
authors.forename: Jamiyan
authors.initials: J
authors.lastname: Purevsuren
authors.forename: Hironori
authors.initials: H
authors.lastname: Kobayashi
authors.forename: Yuki
authors.initials: Y
authors.lastname: Hasegawa
authors.forename: Kenji
authors.initials: K
authors.lastname: Yamada
authors.forename: Tomoo
authors.initials: T
authors.lastname: Takahashi
authors.forename: Masaki
authors.initials: M
authors.lastname: Takayanagi
authors.forename: Toshiyuki
authors.initials: T
authors.lastname: Fukao
authors.forename: Seiji
authors.initials: S
authors.lastname: Fukuda
authors.forename: Seiji
authors.initials: S
authors.lastname: Yamaguchi
mesh_terms: D001692:Biological Transport; D009202:Cardiomyopathies / Q000175:diagnosis* / Q000201:enzymology / Q000378:metabolism; D002331:Carnitine / Q000031:analogs & derivatives* / Q000032:analysis / Q000172:deficiency / Q000378:metabolism; D002333:Carnitine Acyltransferases / Q000172:deficiency; D002334:Carnitine O-Palmitoyltransferase / Q000172:deficiency / Q000378:metabolism; D002478:Cells, Cultured; D005227:Fatty Acids / Q000378:metabolism; D005347:Fibroblasts / Q000737:chemistry / Q000201:enzymology / Q000378:metabolism; D006801:Humans; D022124:Hyperammonemia / Q000175:diagnosis* / Q000201:enzymology / Q000378:metabolism; D007003:Hypoglycemia / Q000175:diagnosis* / Q000201:enzymology / Q000378:metabolism; D008052:Lipid Metabolism, Inborn Errors / Q000175:diagnosis* / Q000201:enzymology / Q000378:metabolism; D008928:Mitochondria / Q000378:metabolism; D009135:Muscular Diseases / Q000175:diagnosis* / Q000201:enzymology / Q000378:metabolism; D010084:Oxidation-Reduction; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D005227:Fatty Acids; C116917:acylcarnitine; D002333:Carnitine Acyltransferases; D002334:Carnitine O-Palmitoyltransferase; D002331:Carnitine