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Reevaluation of the 2-deoxyribose assay for determination of free radical formation
Authors:Thiago C. Genaro-Mattos  Luana T. Dalvi  Ricardo G. Oliveira  Janini S. Ginani  Marcelo Hermes-Lima
Affiliation:1. Oxyradical Research Group, Departamento de Biologia Celular, Universidade de Brasília, 70910-900 Brasília, DF, Brazil;2. Instituto de Química, Universidade de Brasília, 70910-900 Brasília, DF, Brazil;3. Programa de Pós-Graduação em Nutrição Humana, Universidade de Brasília, 70910-900 Brasília, DF, Brazil
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.
Keywords:Fenton   Iron autoxidation   Hydroxyl radical   TBARS   2-Deoxyribose
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