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

Tetrahydrobiopterin (BH4) is an essential cofactor of endothelial nitric oxide (NO) synthase and when depleted, endothelial dysfunction results with decreased production of NO. BH4 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 BH4 oxidation and subsequent decrease in NO production and bioavailability. In order to define the process of ROS-mediated BH4 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 BH4 and its related pteridine metabolites. To study, in detail, the BH4 oxidative pathways, a rapid direct sensitive HPLC assay of BH4 and its pteridine derivatives was adapted using sequential electrochemical and fluorimetric detection. We examined BH4 autoxidation, hydrogen peroxide- and superoxide-driven oxidation, and Fenton reaction hydroxyl radical-driven BH4 transformation. We demonstrate that the formation of the primary two-electron oxidation product, dihydrobiopterin (BH2), predominates with oxygen-induced BH4 autoxidation and superoxide-catalyzed oxidation, while the irreversible metabolites, pterin and dihydroxanthopterin (XH2), are largely produced during hydroxyl radical-driven BH4 oxidation.