title: An improved enzyme assay for carnitine palmitoyl transferase I in fibroblasts using tandem mass spectrometry.
abstract: Carnitine palmitoyl transferase I (CPTI), which converts acyl-CoA and carnitine into acyl-carnitine and free CoASH, is the rate limiting enzyme of hepatic mitochondrial beta-oxidation. CPTI-deficiency is a severe disorder characterized by Reye-like attacks with hypoketotic hypoglycemia, hepatomegaly, elevated liver enzymes and hyperammonemia. We developed a simple tandem-MS-based assay to measure CPTI activity in human fibroblasts. Surprisingly, a large part of the palmitoyl-carnitine formed in our assay by CPTI was degraded into C14- to C2-acyl-carnitines. Degradation of the product of CPTI leads to under estimation of the CPTI activity. When we used potassium cyanide to inhibit enzymes downstream of CPTI and thereby degradation of the product, we measured four times more CPTI activity than the previous methods. This inhibition is essential for correct calculation of CPTI activity. In fibroblasts of CPTI-deficient patients, CPTI activity was not detectable and this assay can be used for the diagnosis of CPTI-deficiency.
authors.affiliation: Department of Clinical Chemistry and Pediatrics, Laboratory Genetic Metabolic Diseases (F0-224), Academic Medical Center, University of Amsterdam, PO Box 22700, 1100 DE Amsterdam, The Netherlands.
authors.forename: Naomi
authors.initials: N
authors.lastname: van Vlies
authors.forename: Jos P N
authors.initials: JP
authors.lastname: Ruiter
authors.forename: Mirjam
authors.initials: M
authors.lastname: Doolaard
authors.forename: Ronald J A
authors.initials: RJ
authors.lastname: Wanders
authors.forename: Frédéric M
authors.initials: FM
authors.lastname: Vaz
mesh_terms: D002334:Carnitine O-Palmitoyltransferase / Q000032:analysis* / Q000172:deficiency / Q000235:genetics; D002478:Cells, Cultured; D005347:Fibroblasts / Q000201:enzymology* / Q000473:pathology; D006801:Humans; D013379:Substrate Specificity / Q000235:genetics; D053719:Tandem Mass Spectrometry
chemical_list: D002334:Carnitine O-Palmitoyltransferase