Deuterated palmitate-driven acylcarnitine formation by whole-blood samples for a rapid diagnostic exploration of mitochondrial fatty acid oxidation disorders

The biochemical diagnosis of mitochondrial fatty acid oxidation defects (FAOD) currently rests on enzyme assays. A dynamic ex vivo exploration consisting of incubations of whole-blood samples with stable-labeled palmitate and determining leukocyte capacities to produce deuterated acylcarnitines was developed on healthy controls ( n = 52) and patients with very-long- (VLCADD) ( n = 2), medium- (MCADD) ( n = 6), or short- (SCADD) ( n = 1) chain acyl-CoA dehydrogenase deficiencies. Incubations were optimized with l-carnitine and [16- 2H 3, 15- 2H 2]-palmitate at 37 °C for various time periods on MCADD and control whole-blood samples. Labeled acylcarnitines were quantified by electrospray-ionization tandem mass spectrometry after thawing, extraction and derivatization to their butyl esters and the method was applied to patients with defects mentioned above. The production of acylcarnitines was linear until 6 h of incubation and optimal on 50 to 200 nmol deuterated substrate. A good discrimination between MCADD patient and control data was found, with median C8/C4 acylcarnitine production rate ratios of 81.0 (5th–95th percentile range: 16.6–209.9) and 0.21 (5th–95th percentile range: 0.06–0.79), respectively. The method also discriminated from controls the VLCADD and SCADD patients. Preliminary studies on a healthy control indicated that the storage at 4 °C does little or not alter capacities of whole-blood samples to generate labeled acylcarnitines over a period of 48 h. The rapid management afforded by the method, its abilities to characterize patients and to work on whole-blood samples after a stay of 24–48 h at 4 °C make it promising for the diagnostic exploration of FAOD.