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.
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.
Law
Lap-Kay
LK
Department of Chemical Pathology, The Chinese University of Hong Kong, Prince of Wales Hospital, Shatin, NT Hong Kong SAR.
Tang
Nelson Leung-Sang
NL
Hui
Joannie
J
Ho
Chung-Shun
CS
Ruiter
Jos
J
Fok
Tai-Fai
TF
Wanders
Ronald J A
RJ
Lam
Christopher Wai-Kei
CW
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*
D005227:Fatty Acids; D009097:Multienzyme Complexes; D010168:Palmitates; C116917:acylcarnitine; D003903:Deuterium; D002331:Carnitine