Abstract
Summary: Maple syrup urine disease (MSUD) is caused by a congenital defect of the branched-chain α-ketoacid dehydrogenase complex (BCKADC), and is one of the target disorders in newborn screening. However, it is not always easy to confirm the diagnosis; conventional methods of enzyme assay require cell culture, isolation of mitochondria, or radioisotope-labelled reagents, and disease-causing mutations can exist in any of the genes encoding the three enzyme subunits. To realize a practical test for diagnostic confirmation, we developed a simple and rapid enzymatic assay for BCKADC. In this procedure, the production of isovaleryl-CoA from 2-ketoisocaproic acid was measured using high-performance liquid chromatography. Detection of the BCKADC product was significantly reproducible depending on concentration of the substrates. We applied the assay to two patients with MSUD and demonstrated pathologically low levels of residual activity in both subjects. These results indicate that our method is a practical and sensitive assay for BCKADC, and that it can be a useful adjunct in newborn screening for MSUD.
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REFERENCES
Chuang DT, Shih VE (1995) Disorders of branched-chain amino acid and keto acid metabolism. In Scriver CR, Beaudet AL, Sly WS, Valle D, eds. The Metabolic and Molecular Bases of Inherited Disease, 7th edn. New York: McGraw-Hill, 1239–1277.
Chuang DT, Ku LS, Kerr DS, et al (1982) Detection of heterozygotes in maple syrup urine disease: measurements of branched-chain alpha-ketoacid dehydrogenase and its components in cell cultures. Am J Hum Genet 34: 416–424.
Danner DJ, Lemmon SK, Besharse JC, et al (1979) Puri?cation and characterization of branched-chain alpha-ketoacid dehydrogenase from bovine liver mitochondria. J Biol Chem 254: 5522–5526.
Elsas LJ, Priest JH, Wheeler FB, et al (1974) Maple syrup urine disease: coenzyme function and prenatal monitoring. Metabolism 23: 569–579.
Fensom AH, Benson PF, Baker JE (1978) A rapid method for assay of branched-chain keto acid decarboxylation in cultured cells and its application to prenatal diagnosis of maple syrup urine disease. Clin Chim Acta 87: 169–174.
Pettit FH, Yeaman SJ, Reed LJ (1978) Puri?cation and characterization of branched-chain alpha-ketoacid dehydrogenase complex of bovine kidney. Proc Natl Acad Sci USA 75: 4881–4885.
Toshima K, Kuroda Y, Yokota I, et al (1985) Activation of branched-chain alpha-ketoacid dehydrogenase complex by alpha-chloroisocaproate in normal and enzyme-de?cient ?broblasts. Clin Chim Acta 147: 103–108.
Wendel U, Wentrup H, Rudiger W (1975) Maple syrup urine disease: analysis of branched-chain ketoacid decarboxylation in cultured ?broblasts. Pediatr Res 9: 709–717.
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Tajima, G., Yofune, H., Bahagia Febriani, A.D. et al. A simple and rapid enzymatic assay for the branched-chain α-ketoacid dehydrogenase complex using high-performance liquid chromatography. J Inherit Metab Dis 27, 633–639 (2004). https://doi.org/10.1023/B:BOLI.0000042988.31581.ed
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DOI: https://doi.org/10.1023/B:BOLI.0000042988.31581.ed