Determination of L-methionine using methionine-specific dehydrogenase for diagnosis of homocystinuria due to cystathionine b-synthase deficiency Shino Yamasaki-Yashiki 1 , Shinjiro Tachibana 2 , Yasuhisa Asano ⇑ Biotechnology Research Center, Toyama Prefectural University, 5180 Kurokawa, Imizu, Toyama 939-0398, Japan article info Article history: Received 11 March 2012 Received in revised form 5 June 2012 Accepted 21 June 2012 Available online 29 June 2012 Keywords: Enzymatic determination Methionine-specific dehydrogenase Branched-chain amino acid transaminase Homocystinuria due to cystathionine b- synthase deficiency abstract 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 b-synthase (CBS) deficiency, a new fluorometric microplate assay using a methionine-specific dehydrogenase (MetDH) and the diaph- orase/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 trans- aminase (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 Lacto- bacillus 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. Ó 2012 Elsevier Inc. All rights reserved. The homocystinurias (HCU) 3 are autosomal recessively inher- ited disorders due to deficiencies of cystathionine b-synthase (CBS, EC 4.2.1.22), methylenetetrahydrofolate reductase (EC 1.5.1.20), and methionine synthase (EC 2.1.1.13) as well as several disorders of vitamin B 12 metabolism [1]. The most common cause of HCU is CBS deficiency in the transsulfuration pathway by which L-methionine (L-Met) is converted to cysteine. The deficiency causes the abnormal accumulation of homocystine in urine and of total homocysteine (tHcy, i.e., homocysteine and its disulfide derivatives) and L-Met in blood [2]. Newborn screening for HCU due to CBS deficiency is carried out worldwide by routine measurement of tHcy or L-Met levels in blood [3,4]. The most widely used method for newborn screening for HCU due to CBS deficiency is a semi- quantitative bacterial inhibition assay (BIA) for measuring L-Met concentration in dried blood spots (DBSs) [5]. Several methods for the determination of tHcy or L-Met levels have been reported, including immunoassay [6,7], enzymatic microplate assay [8], high-performance liquid chromatography (HPLC) [9,10], and tandem mass spectrometry (MS/MS) [11,12]. Because of their convenience and cost, enzymatic microplate assays are used in routine screening for several inborn diseases. One of the first enzymatic microplate fluorometric assays to be established was that for practical screening for phenylketonuria (PKU) using an NAD + -dependent phenylalanine dehydrogenase (PheDH, EC 1.4.1.20) from Bacillus sphaericus [13–15]. The discov- ery of an L-phenylalanine (L-Phe)-specific amino acid dehydroge- nase from Bacillus badius IAM 11059 [16] enabled the diagnosis of PKU with high accuracy and expedition [17,18]. This assay is car- ried out with PheDH, diaphorase (EC 1.6.99.2), and resazurin and has been used for the past decade in Japan and several other coun- tries [19]. A similar enzymatic assay system has also been used for mass screening for galactosemia (GAL) using galactose dehydroge- nase [20] and maple syrup urine disease (MSUD) using L-leucine dehydrogenase [21]. However, such an assay system has not been applied to HCU due to CBS deficiency because an L-Met-specific 0003-2697/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.ab.2012.06.019 ⇑ Corresponding author. Fax: +81 766 56 2498. E-mail address: asano@pu-toyama.ac.jp (Y. Asano). 1 Current address: Toyama Prefectural Institute for Pharmaceutical Research, 17-1 Nakataikoyama, Imizu, Toyama 939-0363, Japan. 2 Department of Bioscience and Biotechnology, Faculty of Agriculture, University of the Ryukyus, 1 Senbaru, Nishihara-cho, Okinawa 903-0213, Japan. 3 Abbreviations used: HCU, homocystinuria; CBS, cystathionine b-synthase; L-Met, L- methionine; tHcy, total homocysteine; BIA, bacterial inhibition assay; DBS, dried blood spot; HPLC, high-performance liquid chromatography; MS/MS, tandem mass spectrometry; PKU, phenylketonuria; PheDH, phenylalanine dehydrogenase; L-Phe, L- phenylalanine; GAL, galactosemia; MSUD, maple syrup urine disease; OPA, o- phthalaldehyde; 2ME, 2-mercaptoethanol; MetDH, methionine-specific dehydroge- nase; BCAA, branched-chain amino acid; BCAT, branched-chain amino acid transam- inase; UPLC, ultra-performance liquid chromatography; PCR, polymerase chain reaction; LB, Luria–Bertani; IPTG, isopropyL-b-D-thiogalactopyranoside; KPB, potas- sium phosphate buffer; PLP, pyridoxal phosphate; L-Leu, L-leucine; CV, coefficient of variation. Analytical Biochemistry 428 (2012) 143–149 Contents lists available at SciVerse ScienceDirect Analytical Biochemistry journal homepage: www.elsevier.com/locate/yabio