A Novel Tandem Mass Spectrometry Method for Rapid Confirmation of Medium- and Very Long-Chain acyl-CoA Dehydrogenase Deficiency in Newborns

BACKGROUND: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. 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. METHODOLOGY:LC-MS/MS was used to measure MCAD- or VLCAD-catalyzed production of enoyl-CoA and hydroxyacyl-CoA, in human lymphocytes. PRINCIPAL FINDINGS:VLCAD 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%. CONCLUSIONS:Our 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

Corresponding increase in long-chain acyl-CoA and acylcarnitine after exercise in muscle from VLCAD mice

Long-chain acylcarnitines accumulate in long-chain fatty acid oxidation defects, especially during periods of increased energy demand from fat. To test whether this increase in long-chain acylcarnitines in very long-chain acyl-CoA dehydrogenase (VLCAD−/−) knock-out mice correlates with acyl-CoA content, we subjected wild-type (WT) and VLCAD−/− mice to forced treadmill running and analyzed muscle long-chain acyl-CoA and acylcarnitine with tandem mass spectrometry (MS/MS) in the same tissues. After exercise, long-chain acyl-CoA displayed a significant increase in muscle from VLCAD−/− mice [C16:0-CoA, C18:2-CoA and C18:1-CoA in sedentary VLCAD−/−: 5.95 ± 0.33, 4.48 ± 0.51, and 7.70 ± 0.30 nmol · g−1 wet weight, respectively; in exercised VLCAD−/−: 8.71 ± 0.42, 9.03 ± 0.93, and 14.82 ± 1.20 nmol · g−1 wet weight, respectively (P < 0.05)]. Increase in acyl-CoA in VLCAD-deficient muscle was paralleled by a significant increase in the corresponding chain length acylcarnitine. Exercise resulted in significant lowering of the free carnitine pool in VLCAD−/− muscle. This is the first study demonstrating that acylcarnitines and acyl-CoA directly correlate and concomitantly increase after exercise in VLCAD-deficient muscle

Octanoyl-CoA oxidation, plasma medium-chain acylcarnitine levels and gene analysis of both <i>ACADM</i> alleles in subjects with suspected MCAD-deficiency.

<p>Case 1 to 18 were identified in NBS, with the exception of 4 and 8.</p>1<p>Relative residual MCAD enzyme activities are presented as a percentage of the mean of lymphocytes from healthy control (2.38 nmol · min⁻¹ · mg⁻¹, n = 6).</p>2<p>First octanoylcarnitine specimen (C8:0 I) obtained on day 2–5 of life and subsequent repeat specimen (C8:0 II) are shown in µmol · L⁻¹, cut-off was set at 0.30.</p>3<p>Parents of 12.</p>4<p>Parents of 16.</p>5<p>Intervening sequences: IVS2-32C>G, IVS3+10T>C, IVS5+32C>G, IVS7-22C>A.</p>6<p>Intervening sequences: IVS2-32C>G, IVS3+10T>C, IVS5+32C>G, IVS6-14A>G and IVS7-22C>A.</p>7<p>subjects with clinical suspicion of MCADD.</p

Plasma octanoyl-carnitine (C8:0) concentrations determined in dried blood spots during initial newborn screening and subsequent follow-up from patients carrying two confirmed <i>ACADM</i> mutations.

<p>Plasma octanoyl-carnitine (C8:0) concentrations determined in dried blood spots during initial newborn screening and subsequent follow-up from patients carrying two confirmed <i>ACADM</i> mutations.</p

Multiple Reactant Monitoring (MRM) chromatograms of quenched VLCAD assay samples.

<p>C16:OH-CoA, m/z 1022.4 → 515.2 (A and B) and C16:1-CoA, m/z 1004.4 → 497.2 (C and D) after 0 (A and C) and 5 (B and D) min. of incubation.</p