title: An enzymatic method to distinguish tetrahydrobiopterin from oxidized biopterins using UDP-glucose:tetrahydrobiopterin glucosyltransferase.
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 disorders 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 alpha-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.
authors.affiliation: Frontier Inje Research for Science and Technology Research Group, School of Biological Sciences, Inje University, Kimhae 621-749, Republic of Korea.
authors.forename: Hye-Lim
authors.initials: HL
authors.lastname: Kim
authors.forename: Do Hyung
authors.initials: DH
authors.lastname: Kim
authors.forename: Yeol Kun
authors.initials: YK
authors.lastname: Lee
authors.forename: Sun Ok
authors.initials: SO
authors.lastname: Park
authors.forename: Yong-Woo
authors.initials: YW
authors.lastname: Lee
authors.forename: O-Seob
authors.initials: OS
authors.lastname: Kwon
authors.forename: Young Shik
authors.initials: YS
authors.lastname: Park
mesh_terms: D000818:Animals; D001708:Biopterins / Q000031:analogs & derivatives* / Q000097:blood / Q000378:metabolism / Q000652:urine; D016695:Glycosyltransferases / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D010084:Oxidation-Reduction; D051381:Rats; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D014532:Uridine Diphosphate Glucose / Q000378:metabolism*
chemical_list: D011994:Recombinant Proteins; D001708:Biopterins; C017226:7,8-dihydrobiopterin; D016695:Glycosyltransferases; C003402:sapropterin; D014532:Uridine Diphosphate Glucose