Abstract
Supplementation of tetrahydrobiopterin (BH_4), an essential cofactor in nitric oxide and monoamine production, is used in treating a systemic deficiency of BH_4 despite of high cost medication due to short retention of supplied BH_4. Although it also potentially targets a local deficiency such as in some cases of cardiovascular dysfunction, understanding of transport mechanisms underlying the rapid BH_4-excretion is required to improve the therapeutic efficiency. Application of a membrane-protein expression system using Xenopus oocytes followed by a radioisotope assay has widely been employed in transporter research. The assay, however, is made difficult by the labile character of BH_4 while undergoing aerobic oxidation and enzymic conversion. We report here an application of a Xenopus-oocyte expression system to hENT1 and hENT2 for characterization of putative BH_4 transporter. The method allows for the efficient extraction of reduced pterins from a small number of oocytes followed by HPLC and highly sensitive fluorescence detection.
