TY - JOUR
T1 - Reevaluation of the 2-deoxyribose assay for determination of free radical formation
AU - Genaro-Mattos, Thiago C.
AU - Dalvi, Luana T.
AU - Oliveira, Ricardo G.
AU - Ginani, Janini S.
AU - Hermes-Lima, Marcelo
N1 - Funding Information:
The authors would like to thank Fernanda Rosa for the help with HPLC experiments and Rafaella Franco for the technical support. T. Genaro-Mattos, J. Ginani and L. Dalvi received studentships from CAPES or CNPq (Brazil). CNPq-Universal, Milênio-Redoxoma (CNPq) and INCT-CNPq (Brazil) supported this study.
PY - 2009/12
Y1 - 2009/12
N2 - 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.
AB - 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.
KW - 2-Deoxyribose
KW - Fenton
KW - Hydroxyl radical
KW - Iron autoxidation
KW - TBARS
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U2 - 10.1016/j.bbagen.2009.09.003
DO - 10.1016/j.bbagen.2009.09.003
M3 - Article
C2 - 19747523
AN - SCOPUS:71549114848
SN - 0304-4165
VL - 1790
SP - 1636
EP - 1642
JO - Biochimica et Biophysica Acta - General Subjects
JF - Biochimica et Biophysica Acta - General Subjects
IS - 12
ER -