A novel functional assay for simultaneous determination of total fatty acid β-oxidation flux and acylcarnitine profiling in human skin fibroblasts using 2H 31-palmitate by isotope ratio mass spectrometry and electrospray tandem mass spectrometry

Abstract

Background Two separate and complementary assays, total mitochondrial fatty acid β-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 ( 2H 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 2H 2O enrichment after 96 h of incubation of 2H 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.