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.