HPLC Analysis of Tetrahydrobiopterin and Its Pteridine Derivatives Using Sequential Electrochemical and Fluorimetric Detection: Application to Tetrahydrobiopterin Autoxidation and Chemical Oxidation

Overview

Journal Arch Biochem Biophys

Publisher Elsevier

Specialties Biochemistry
Biophysics

Date 2012 Jan 31

PMID 22286026

Citations 9

Authors

Roberto Biondi

Giuseppe Ambrosio

Francesco De Pascali

Isabella Tritto

Enrico Capodicasa

Lawrence J Druhan

Craig Hemann

Jay L Zweier

Affiliations

Soon will be listed here.

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.

Citing Articles

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