title: A novel functional assay for simultaneous determination of total fatty acid beta-oxidation flux and acylcarnitine profiling in human skin fibroblasts using (2)H(31)-palmitate by isotope ratio mass spectrometry and electrospray tandem mass spectrometry.
abstract: 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 (<60% control median, p<0.05) and could be identified by IRMS assay. Accumulations of disease-specific acylcarnitines due to blockage in the carnitine cycle and FAO spiral were also demonstrated by acylcarnitine profiling.
  
  
  CONCLUSIONS
  This novel functional assay is less time consuming and relatively simple by comparison to other published methods and can be used to investigate patients suspected to have FAO defects.
authors: Department of Chemical Pathology, The Chinese University of Hong Kong, Prince of Wales Hospital, Shatin, NT Hong Kong SAR.
authors: Lap-Kay
authors: LK
authors: Law
authors: Nelson Leung-Sang
authors: NL
authors: Tang
authors: Joannie
authors: J
authors: Hui
authors: Chung-Shun
authors: CS
authors: Ho
authors: Jos
authors: J
authors: Ruiter
authors: Tai-Fai
authors: TF
authors: Fok
authors: Ronald J A
authors: RJ
authors: Wanders
authors: Christopher Wai-Kei
authors: CW
authors: Lam
mesh_terms: D002331:Carnitine / Q000031:analogs & derivatives* / Q000032:analysis / Q000378:metabolism; D002460:Cell Line; D003903:Deuterium / Q000032:analysis / Q000378:metabolism; D005227:Fatty Acids / Q000378:metabolism*; D005347:Fibroblasts / Q000378:metabolism; D006801:Humans; D008052:Lipid Metabolism, Inborn Errors / Q000175:diagnosis* / Q000378:metabolism; D008928:Mitochondria / Q000378:metabolism*; D009097:Multienzyme Complexes / Q000378:metabolism; D010084:Oxidation-Reduction; D010168:Palmitates / Q000032:analysis / Q000378:metabolism; D012867:Skin / Q000166:cytology / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D005227:Fatty Acids; D009097:Multienzyme Complexes; D010168:Palmitates; C116917:acylcarnitine; D003903:Deuterium; D002331:Carnitine