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

Background: 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. Methods: Incubations were optimized with L-carnitine and [16-H-2(3), 15-H-2(2)]-palmitate at 37 degrees 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. Results: 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 degrees C does little or not alter capacities of whole-blood samples to generate labeled acylcarnitines over a period of 48 h. Conclusion: 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 degrees C make it promising for the diagnostic exploration of FAOD. (C) 2009 Elsevier B.V. All rights reserved.