Reevaluation of the 2-deoxyribose assay for determination of free radical formation

Thiago C. Genaro-Mattos, Luana T. Dalvi, Ricardo G. Oliveira, Janini S. Ginani, Marcelo Hermes-Lima

Research output: Contribution to journalArticle

21 Scopus citations

Abstract

Background: The 2-deoxyribose (2-DR) degradation assay is a widely used test for determining anti/pro-oxidant properties of molecules and plant extracts. Most reports use reaction blanks omitting 2-DR or thiobarbituric acid (TBA). However, when studying Fe(II)-mediated reactions, we verified that these blanks are not appropriate. Fe(III) - a product of these reactions - causes a relevant artifact in the assay, where 2-DR is oxidized by Fe(III). Method: 2-DR degradation was determined at 532 nm as TBA-reactive substances. Results and conclusion: HPLC determinations indicated that Fe(III) added after or before TBA generates considerable amounts of malondialdehyde (2-DR degradation product) in comparison with assays employing Fenton reagents or Fe(II) autoxidation. Addition of catalase and thiourea has no effect on Fe(III)-induced 2-DR degradation indicating lack of ROS involvement. This Fe(III)-mediated 2-DR damage is dependent on iron and 2-DR concentrations, but not on H2O2, buffer composition or iron-chelators. Depending on the assay conditions Fe(III)-interference accounts for 20% to 90% of 2-DR degradation mediated by Fe(II). Significance: A new reaction blank is proposed herein-based on the use of Fe(III)-for the assay. The lack of such correction has caused the underestimation of antioxidant capacity of various compounds in many studies in the last 2 decades.

Original languageEnglish (US)
Pages (from-to)1636-1642
Number of pages7
JournalBiochimica et Biophysica Acta - General Subjects
Volume1790
Issue number12
DOIs
StatePublished - Dec 1 2009

Keywords

  • 2-Deoxyribose
  • Fenton
  • Hydroxyl radical
  • Iron autoxidation
  • TBARS

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology

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