Abstract
BackgroundNewborn screening for medium- and very long-chain acyl-CoA dehydrogenase (MCAD and VLCAD, respectively) deficiency, using acylcarnitine profiling with tandem mass spectrometry, has increased the number of patients with fatty acid oxidation disorders due to the identification of additional milder, and so far silent, phenotypes. However, especially for VLCADD, the acylcarnitine profile can not constitute the sole parameter in order to reliably confirm disease. Therefore, we developed a new liquid chromatography tandem mass spectrometry (LC-MS/MS) method to rapidly determine both MCAD- and/or VLCAD-activity in human lymphocytes in order to confirm diagnosis.MethodologyLC-MS/MS was used to measure MCAD- or VLCAD-catalyzed production of enoyl-CoA and hydroxyacyl-CoA, in human lymphocytes.Principal FindingsVLCAD activity in controls was 6.95±0.42 mU/mg (range 1.95 to 11.91 mU/mg). Residual VLCAD activity of 4 patients with confirmed VLCAD-deficiency was between 0.3 and 1.1%. Heterozygous ACADVL mutation carriers showed residual VLCAD activities of 23.7 to 54.2%. MCAD activity in controls was 2.38±0.18 mU/mg. In total, 28 patients with suspected MCAD-deficiency were assayed. Nearly all patients with residual MCAD activities below 2.5% were homozygous 985A>G carriers. MCAD-deficient patients with one other than the 985A>G mutation had higher MCAD residual activities, ranging from 5.7 to 13.9%. All patients with the 199T>C mutation had residual activities above 10%.ConclusionsOur newly developed LC-MS/MS method is able to provide ample sensitivity to correctly and rapidly determine MCAD and VLCAD residual activity in human lymphocytes. Importantly, based on measured MCAD residual activities in correlation with genotype, new insights were obtained on the expected clinical phenotype.
Highlights
If undiagnosed and untreated, fatty acid oxidation defects (FAOD), such as medium- and very long-chain acyl-CoA dehydrogenase deficiency (MCADD and VLCADD, respectively), are associated with high morbidity and mortality [1]
Based on measured MCAD residual activities in correlation with genotype, new insights were obtained on the expected clinical phenotype
As enoyl-CoA products are rapidly converted into hydroxyacylCoA by matrix 2-enoyl-hydratases, in order to avoid product inhibition in-vivo [12,13], it was critical to include hydroxyacylCoA as acyl-CoA dehydrogenase product to avoid underestimation of catalytic turnover
Summary
Fatty acid oxidation defects (FAOD), such as medium- and very long-chain acyl-CoA dehydrogenase deficiency (MCADD and VLCADD, respectively), are associated with high morbidity and mortality [1]. NBS for MCADD and VLCADD using tandem mass spectrometry screening identifies false-positive cases and the outcome of the expanded NBS program in Germany, including MCADD and VLCADD, was recently assessed by Schulze et al. Genotyping does, not always constitute a swift and cost-effective confirmatory diagnostic technique for a suspected FAOD in all cases. Newborn screening for medium- and very long-chain acyl-CoA dehydrogenase (MCAD and VLCAD, respectively) deficiency, using acylcarnitine profiling with tandem mass spectrometry, has increased the number of patients with fatty acid oxidation disorders due to the identification of additional milder, and so far silent, phenotypes. We developed a new liquid chromatography tandem mass spectrometry (LC-MS/MS) method to rapidly determine both MCAD- and/or VLCAD-activity in human lymphocytes in order to confirm diagnosis
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