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.