A novel functional assay for simultaneous determination of total fatty acid β-oxidation flux and acylcarnitine profiling in human skin fibroblasts using 2H31-palmitate by isotope ratio mass spectrometry and electrospray tandem mass spectrometry
| Autor: | Joannie Hui, Ronald J.A. Wanders, Chung Shun Ho, Jos P.N. Ruiter, L. K. Law, Nelson L.S. Tang, Christopher W.K. Lam, Tai Fai Fok |
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| Přispěvatelé: | AGEM - Amsterdam Gastroenterology Endocrinology Metabolism, Laboratory Genetic Metabolic Diseases |
| Rok vydání: | 2007 |
| Předmět: |
Spectrometry
Mass Electrospray Ionization Electrospray Clinical Biochemistry Palmitates Human skin Dehydrogenase Tandem mass spectrometry Biochemistry Lipid Metabolism Inborn Errors Cell Line Multienzyme Complexes Tandem Mass Spectrometry Carnitine medicine Humans Isotope-ratio mass spectrometry Skin chemistry.chemical_classification Chromatography Fatty Acids Biochemistry (medical) Fatty acid General Medicine Fibroblasts Deuterium Mitochondria chemistry Cell culture Oxidation-Reduction medicine.drug |
| Zdroj: | Clinica chimica acta; international journal of clinical chemistry, 382(1-2), 25-30. Elsevier |
| ISSN: | 0009-8981 |
| DOI: | 10.1016/j.cca.2007.03.011 |
| Popis: | BACKGROUND: Two separate and complementary assays, total mitochondrial fatty acid beta-oxidation (FAO) flux rate and acylcarnitine profiling, have been used to establish a definitive diagnosis of FAO defects (FAOD) in cultured cells. We developed a novel functional assay for total FAO rate assay by measurement of deuterated water enrichment and to combine it with the conventional acylcarnitine profiling method into a single tracer incubation experiment. METHODS: Skin fibroblasts were incubated in a medium containing universal deuterium-labeled palmitate ((2)H(31)-palmitate) and l-carnitine without glucose supplementation for 96 h. The culture medium was assayed for deuterated water enrichment using isotope ratio mass spectrometry (IRMS) and acylcarnitine profiling by electrospray-ionization tandem mass spectrometry (ESI/MS/MS). RESULTS: The medians of (2)H(2)O enrichment after 96 h of incubation of (2)H(31)-palmitate of the control, other inherited metabolic diseases and FAOD cell lines were 109.9, 102 and 23.1 ppm/mg protein/96 h, respectively. All fibroblasts with FAOD except carnitine uptake defective, multiple acyl-CoA dehydrogenase and short-chain 3-hydroxyacyl-CoA dehydrogenase deficient cells were well separated from the control ( |
| Databáze: | OpenAIRE |
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