Determination of l-methionine using methionine-specific dehydrogenase for diagnosis of homocystinuria due to cystathionine β-synthase deficiency

To determine the l-methionine (l-Met) concentration in an extract from dried blood spots (DBSs) for newborn mass screening for homocystinuria (HCU) due to cystathionine β-synthase (CBS) deficiency, a new fluorometric microplate assay using a methionine-specific dehydrogenase (MetDH) and the diaphorase/reazusrin system was established. We created by directed mutagenesis an NAD+-dependent MetDH from phenylalanine dehydrogenase (PheDH) showing higher substrate specificity toward l-Met than l-phenylalanine (l-Phe). However, it also exhibited notable activity for branched-chain amino acids (BCAAs). BCAAs in blood clearly interfered with the determination of l-Met in the DBS specimens using a single application of MetDH. To measure l-Met selectively, we used a branched-chain amino acid transaminase (BCAT) to eliminate the BCAAs in the specimens and screened for a BCAT with low activity toward l-Met. In microplate assays using MetDH, pretreatment of specimens with the BCAT from Lactobacillus delbrueckii subsp. bulgaricus coupled with l-glutamate oxidase minimized the effects of BCAAs, and l-Met concentrations were determined with high accuracy even at elevated BCAA concentrations. This enzymatic end-point assay is suitable for determining l-Met concentrations in DBSs for neonatal screening for HCU due to CBS deficiency.