HPLC analysis of tetrahydrobiopterin and its pteridine derivatives using sequential electrochemical and fluorimetric detection: Application to tetrahydrobiopterin autoxidation and chemical oxidation

摘要

Tetrahydrobiopterin (BH _4) is an essential cofactor of endothelial nitric oxide (NO) synthase and when depleted, endothelial dysfunction results with decreased production of NO. BH _4 is also an anti-oxidant being a good "scavenger" of oxidative species. NADPH oxidase, xanthine oxidase, and mitochondrial enzymes producing reactive oxygen species (ROS) can induce elevated oxidant stress and cause BH _4 oxidation and subsequent decrease in NO production and bioavailability. In order to define the process of ROS-mediated BH _4 degradation, a sensitive method for monitoring pteridine redox-state changes is required. Considering that the conventional fluorescence method is an indirect method requiring conversion of all pteridines to oxidized forms, it would be beneficial to use a rapid quantitative assay for the individual detection of BH _4 and its related pteridine metabolites. To study, in detail, the BH _4 oxidative pathways, a rapid direct sensitive HPLC assay of BH _4 and its pteridine derivatives was adapted using sequential electrochemical and fluorimetric detection. We examined BH _4 autoxidation, hydrogen peroxide- and superoxide-driven oxidation, and Fenton reaction hydroxyl radical-driven BH _4 transformation. We demonstrate that the formation of the primary two-electron oxidation product, dihydrobiopterin (BH _2), predominates with oxygen-induced BH _4 autoxidation and superoxide-catalyzed oxidation, while the irreversible metabolites, pterin and dihydroxanthopterin (XH _2), are largely produced during hydroxyl radical-driven BH _4 oxidation.