Principle Of Method: Confluent layers of cells are incubated with 1-[14C]-leucine in a microtitre plate. Endogenous transaminase converts the leucine to a-ketoisocaproic acid which is subsequently oxidatively decarboxylated to 14CO2 and isovaleryl-CoA. The released 14CO2 is trapped in a small glass fibre disc soaked in sodium hydroxide and is determined by scintillation counting. Use of labelled leucine rather than labelled a-ketoisocaproic acid as substrate leads to lower blanks in this assay. The leucine is also more stable and less expensive.

At the end of the incubation the cells are digested with alkali and their protein content is determined by the Lowry method.

Uses And Limitations Of The Method: The assay is used to diagnose maple syrup urine disease (MSUD; branched chain ketoaciduria), both the severe neonatal type and later onset variants. These disorders are characterised by accumulation of the branched chain amino acids leucine, isoleucine and valine, and also of the corresponding branched-chain a-keto acids. The presence of alloisoleucine in urine is diagnostic of MSUD. In this assay classical cases of the disease have virtually undetectable activity, but variant forms have some residual activity, severity of the disease corresponding to some extent with extent of the deficiency.

Branched-chain ketoacid dehydrogenase is a multi-enzyme complex comprised of three catalytic components and two regulatory enzymes. The catalytic enzymes are a thiamine pyrophosphate-dependent decarboxylase (E1) with a a2b2 structure, a transacylase (E2) and a homodimeric flavoprotein dehydrogenase (E3). The regulatory enzymes are a kinase and a phosphatase. In total 6 loci are involved with coding for these proteins. Mutations in E1a, E1b, E2 and E3 have been described. Those in E3 result in a combined deficiency of branched chain ketoacid decarboxylase, pyruvate dehydrogenase and a-ketoglutarate dehydrogenase because E3 is a common component of the other dehydrogenase complexes. Patients with E3 deficiency have severe lactic acidosis in addition to the biochemical abnormalities of MSUD and should be detected by the assay.

Prenatal diagnosis of MSUD is possible by assay of the enzyme in cultured amniotic cells grown in a microtitre plate, or of cultured chorionic villus cells. Direct assay of chorionic villi is not offered by the laboratory, and heterozygote detection is not considered reliable by this method.

Specimen Requirements: Fibroblasts cultured from a skin biopsy are needed. Biopsy material should be collected aseptically into a sterile bottle containing tissue culture medium (available from the laboratory), and sent at room temperature to arrive at the laboratory within 24 hours.

Biopsies for tissue culture should not be frozen.

Fibroblast cultures established in other laboratories should be sent in plastic 25 cm2 flasks filled with medium.

The Laboratory Recommends Use Of A Courier Service Or Royal Mail Special Delivery For Sending All Specimens To The Laboratory.

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