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

Roberto Biondi, Giuseppe Ambrosio, Francesco De Pascali, Isabella Tritto, Enrico Capodicasa, Lawrence J. Druhan, Craig Hemann, Jay L. Zweier

Research output: Contribution to journalArticle

Abstract

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.

Original languageEnglish (US)
Pages (from-to)7-16
Number of pages10
JournalArchives of Biochemistry and Biophysics
Volume520
Issue number1
DOIs
StatePublished - Apr 1 2012
Externally publishedYes

Keywords

  • Fenton reaction
  • Hydroxyl radical
  • Nitric oxide
  • Nitric oxide synthase
  • Reactive oxygen species
  • Superoxide

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Molecular Biology

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