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.
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.
Authors: Sebastian M Fica-Contreras; Sydney O Shuster; Nathaniel D Durfee; Gregory J K Bowe; Nathaniel J Henning; Staci A Hill; Geoffrey D Vrla; David R Stillman; Kelly M Suralik; Roger K Sandwick; Sunhee Choi Journal: J Biol Inorg Chem Date: 2017-10-16 Impact factor: 3.358
Authors: Rok Martinčič; Janez Mravljak; Urban Švajger; Andrej Perdih; Marko Anderluh; Marjana Novič Journal: PLoS One Date: 2015-10-16 Impact factor: 3.240