An enzymatic method to distinguish tetrahydrobiopterin from oxidized biopterins using UDP–glucose:tetrahydrobiopterin glucosyltransferase Hye-Lim Kim a,1 , Do Hyung Kim a,1 , Yeol Kun Lee a , Sun Ok Park a , Yong-Woo Lee b , O-Seob Kwon c , Young Shik Park a, * a Frontier Inje Research for Science and Technology Research Group, School of Biological Sciences, Inje University, Kimhae 621-749, Republic of Korea b Department of Biomedical Laboratory Science, Inje University, Kimhae 621-749, Republic of Korea c School of Environmental Science and Engineering, Inje University, Kimhae 621-749, Republic of Korea article info Article history: Received 25 August 2009 Available online 9 October 2009 Keywords: Tetrahydrobiopterin Pteridine glycosyltransferase HPLC abstract The quantitative determination of tetrahydrobiopterin (BH4) and its oxidized forms (dihydrobiopterin and biopterin) is important in searching for possible markers of neuropsychiatric and cardiovascular dis- orders as well as in diagnosing BH4 deficiencies. Currently, two high-performance liquid chromatography (HPLC) methods are available, although both have some limitations. We developed an enzymatic method to distinguish BH4 from the oxidized forms by employing BH4:UDP–glucose a-glucosyltransferase (BGluT), which catalyzes glucosyl transfer from UDP–glucose to BH4. The recombinant BGluT isolated from Escherichia coli converted essentially all of the BH4 in a mixture containing oxidized biopterins to the glucoside while leaving the oxidized forms intact. Therefore, acidic iodine oxidation of the reaction mixture followed by single fluorescence HPLC permitted the determination of biopterin and biopterin– glucoside, which represent oxidized biopterins and BH4, respectively. The validity of the method was evaluated using authentic biopterins and animal samples such as human urine, rat plasma, and rat liver. The BGluT-catalyzed reaction not only would reduce the burden of chromatographic separation but also would promise non-HPLC analysis of BH4. Ó 2009 Elsevier Inc. All rights reserved. As a cofactor for aromatic amino acid hydroxylases, tetrahydro- biopterin (BH4) 2 is essential for hepatic phenylalanine metabolism and neurotransmitter synthesis in humans [1]. It is well known that an inherent error in BH4 synthesis can cause hyperphenylalanine- mia, neurotransmitter deficiencies, and DOPA-responsive dystonia [1,2]. BH4 has also been studied for a possible association with sev- eral neuropsychiatric disorders such as Parkinson’s disease, Alzhei- mer’s disease, depression, autism, and schizophrenia [1,3–6]. In addition, BH4 plays a crucial role in endothelial dysfunction as a cofactor and regulator of nitric oxide synthase (NOS) [7]. Although BH4 is functional in the fully reduced form, it can be partially oxi- dized to dihydrobiopterin (BH2) and fully oxidized to biopterin in vivo, especially under oxidative stress conditions. Insufficient availability of BH4 in endothelial cells results in NOS-uncoupled pro- duction of superoxide anions instead of nitric oxide [8]. The situation is further ameliorated by the competitive binding of BH2 to NOS [9]. The resulting endothelial dysfunction is a well-known prognostic marker of cardiovascular diseases. Therefore, both BH4 and its oxi- dized forms have been measured extensively in biological samples for clinical diagnostics and biomedical research. Two high-performance liquid chromatography (HPLC) methods are currently available to quantify BH4 and its oxidized forms. A chemical method originally developed by Fukushima and Nixon [10] requires differential oxidation of specimens before HPLC. Total biopterin (BH4 + BH2 + biopterin) is simply determined by acidic iodine oxidation. Alkaline iodine oxidation converts BH2 to biop- terin and converts BH4 to pterin. The subtracted amount of biop- terin represents the amount of BH4. Another method depends on HPLC in anaerobic conditions to separate BH4 from its oxidized forms, which are measured directly by electrochemical detection or by fluorescence after postcolumn oxidation [11]. We previously isolated a novel enzyme named UDP–glu- cose:BH4 glucosyltransferase (BGluT) from the cyanobacterium Synechococcus sp. PCC 7942 [12–14]. The enzyme catalyzes the glu- cosyl transfer from UDP–glucose to BH4, generating BH4-a-gluco- side. While studying BGluT, we hypothesized that the enzyme may be useful in a BH4 assay. If BGluT catalysis is specific and irrevers- ible, essentially all of the BH4 in a mixture containing oxidized biopterins would be converted to BH4–glucoside while the oxi- dized biopterins would remain intact. An acidic iodine oxidation 0003-2697/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2009.10.007 * Corresponding author. Fax: +82 55 336 7706. E-mail address: mbyspark@inje.ac.kr (Y.S. Park). 1 These authors contributed equally to this article. 2 Abbreviations used: BH4, tetrahydrobiopterin; NOS, nitric oxide synthase; BH2, dihydrobiopterin; HPLC, high-performance liquid chromatography; BGluT, UDP–glucose:BH4 glucosyltransferase; IPTG, isopropyl b-D-1-thiogalactopyranoside; Ni–NTA, nickel–nitrilotriacetic acid; DTT, dithiothreitol; %RSD, percentage relative standard deviation; ELISA, enzyme-linked immunosorbent assay. Analytical Biochemistry 397 (2010) 79–83 Contents lists available at ScienceDirect Analytical Biochemistry journal homepage: www.elsevier.com/locate/yabio