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

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. 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). 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 (