2-Ethylhydracrylic Aciduria in Short/Branched-Chain Acyl-CoA Dehydrogenase Deficiency: Application to Diagnosis and Implications for the R-Pathway of Isoleucine Oxidation

Background: Isolated excretion of 2-methylbutyrylglycine (2-MBG) is the hallmark of short/branched-chain acyl-CoA dehydrogenase deficiency (SBCADD), a recently identified defect in the proximal pathway of l-isoleucine oxidation. SBCADD might be underdiagnosed because detection and recognition of urine acylglycines is problematic. Excretion of 2-ethylhydracrylic acid (2-EHA), an intermediate formed in the normally minor R -pathway of l-isoleucine oxidation, has not previously been described in SBCADD. Methods: Samples from four patients with 2-MBG excretion were analyzed by gas chromatography–mass spectrometry for urine organic acids, quantification of 2-MBG, and chiral determination of 2-methylbutyric acid. Blood-spot acylcarnitines were measured by electrospray–tandem mass spectrometry. Mutations in the ACADSB gene encoding SBCAD were identified by direct sequencing. Results: SBCADD was confirmed in each patient by demonstration of different ACADSB gene mutations. In multiple urine samples, organic acid analysis revealed a prominent 2-EHA peak usually exceeding the size of the 2-MBG peak. Approximately 40–46% of total 2-methylbutyric acid conjugates were in the form of the R -isomer, indicating significant metabolism via the R -pathway. Conclusions: If, as generally believed, SBCAD is responsible for R -2-MBG dehydrogenation in the R -pathway, 2-EHA would not be produced in SBCADD. Our observation of 2-ethylhydracrylic aciduria in SBCADD implies that a different or alternative enzyme serves this function. Increased flux through the R -pathway may act as a safety valve for overflow of accumulating S -pathway metabolites and thereby mitigate the severity of SBCADD. Awareness of 2-ethylhydracrylic aciduria as a diagnostic marker could lead to increased detection of SBCADD and improved definition of its clinical phenotype.