An enzymatic method to distinguish tetrahydrobiopterin from oxidized biopterins using UDP-glucose:tetrahydrobiopterin glucosyltransferase.
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.
Kim
Hye-Lim
HL
Frontier Inje Research for Science and Technology Research Group, School of Biological Sciences, Inje University, Kimhae 621-749, Republic of Korea.
Kim
Do Hyung
DH
Lee
Yeol Kun
YK
Park
Sun Ok
SO
Lee
Yong-Woo
YW
Kwon
O-Seob
OS
Park
Young Shik
YS
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*
D011994:Recombinant Proteins; D001708:Biopterins; C017226:7,8-dihydrobiopterin; D016695:Glycosyltransferases; C003402:sapropterin; D014532:Uridine Diphosphate Glucose