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


METHODS
2-DR degradation was determined at 532 nm as TBA-reactive substances.


CONCLUSIONS
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).


CONCLUSIONS
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.
Genaro-Mattos
Thiago C
TC
Oxyradical Research Group, Departamento de Biologia Celular, Universidade de Brasília, 70910-900 Brasília, DF, Brazil.
Dalvi
Luana T
LT
Oliveira
Ricardo G
RG
Ginani
Janini S
JS
Hermes-Lima
Marcelo
M
D000818:Animals; D001681:Biological Assay / Q000379:methods*; D002021:Buffers; D002417:Cattle; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D003855:Deoxyribose / Q000032:analysis* / Q000378:metabolism; D005609:Free Radicals / Q000032:analysis* / Q000378:metabolism*; D006861:Hydrogen Peroxide / Q000737:chemistry / Q000494:pharmacology; D007501:Iron / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D009994:Osmolar Concentration; D010084:Oxidation-Reduction
D002021:Buffers; C045076:Fenton's reagent; D005609:Free Radicals; D003855:Deoxyribose; D006861:Hydrogen Peroxide; D007501:Iron