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

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.