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Novel hydroxyl radical scavenging antioxidant activity assay for water-soluble antioxidants using a modified CUPRAC method.
Biochem Biophys Res Commun. 2006 Jul 07; 345(3):1194-200.BB

Abstract

Reactive oxygen species (ROS) such as superoxide anion, hydroxyl ((*)OH), peroxyl, and alkoxyl radicals may attack biological macromolecules giving rise to oxidative stress-originated diseases. Since (*)OH is very short-lived, secondary products resulting from (*)OH attack to various probes are measured. Although the measurement of aromatic hydroxylation with HPLC/electrochemical detection is more specific than the low-yield TBARS test, it requires sophisticated instrumentation. As a more convenient and less costly alternative, we used p-aminobenzoate, 2,4- and 3,5-dimethoxybenzoate probes for detecting hydroxyl radicals generated from an equivalent mixture of Fe(II)+EDTA with hydrogen peroxide. The produced hydroxyl radicals attacked both the probe and the water-soluble antioxidants in 37 degrees C-incubated solutions for 2h. The CUPRAC (i.e., our original method for total antioxidant capacity assay) absorbance of the ethylacetate extract due to the reduction of Cu(II)-neocuproine reagent by the hydroxylated probe decreased in the presence of (*)OH scavengers, the difference being proportional to the scavenging ability of the tested compound. A rate constant for the reaction of the scavenger with hydroxyl radical can be deduced from the inhibition of color formation. The second-order rate constants of the scavengers were determined with competition kinetics by means of a linear plot of A(0)/A as a function of C(scavenger)/C(probe), where A(0) and A are the CUPRAC absorbances of the system in the absence and presence of scavenger, respectively, and C is the molar concentration of relevant species. The 2,4- and 3,5-dimethoxybenzoates were the best probes in terms of linearity and sensitivity. Iodide, metabisulfite, hexacyanoferrate(II), thiourea, formate, and dimethyl sulfoxide were shown by the modified CUPRAC assay to be more effective scavengers than mannitol, glucose, lysine, and simple alcohols, as in the TBARS assay. The developed method is less lengthy, more specific, and of a higher yield than the classical TBARS assay. The hydroxyl radical scavenging rate constants of ascorbic acid, formate, and hexacyanoferrate(II) that caused interference in other assays could be easily found with the proposed procedure.

Authors+Show Affiliations

Department of Chemistry, Faculty of Engineering, Istanbul University, Avcilar, 34320 Istanbul, Turkey.No affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

16716257

Citation

Bektaşoğlu, Burcu, et al. "Novel Hydroxyl Radical Scavenging Antioxidant Activity Assay for Water-soluble Antioxidants Using a Modified CUPRAC Method." Biochemical and Biophysical Research Communications, vol. 345, no. 3, 2006, pp. 1194-200.
Bektaşoğlu B, Esin Celik S, Ozyürek M, et al. Novel hydroxyl radical scavenging antioxidant activity assay for water-soluble antioxidants using a modified CUPRAC method. Biochem Biophys Res Commun. 2006;345(3):1194-200.
Bektaşoğlu, B., Esin Celik, S., Ozyürek, M., Güçlü, K., & Apak, R. (2006). Novel hydroxyl radical scavenging antioxidant activity assay for water-soluble antioxidants using a modified CUPRAC method. Biochemical and Biophysical Research Communications, 345(3), 1194-200.
Bektaşoğlu B, et al. Novel Hydroxyl Radical Scavenging Antioxidant Activity Assay for Water-soluble Antioxidants Using a Modified CUPRAC Method. Biochem Biophys Res Commun. 2006 Jul 7;345(3):1194-200. PubMed PMID: 16716257.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Novel hydroxyl radical scavenging antioxidant activity assay for water-soluble antioxidants using a modified CUPRAC method. AU - Bektaşoğlu,Burcu, AU - Esin Celik,Saliha, AU - Ozyürek,Mustafa, AU - Güçlü,Kubilay, AU - Apak,Reşat, Y1 - 2006/05/15/ PY - 2006/04/25/received PY - 2006/05/08/accepted PY - 2006/5/24/pubmed PY - 2006/8/2/medline PY - 2006/5/24/entrez SP - 1194 EP - 200 JF - Biochemical and biophysical research communications JO - Biochem. Biophys. Res. Commun. VL - 345 IS - 3 N2 - Reactive oxygen species (ROS) such as superoxide anion, hydroxyl ((*)OH), peroxyl, and alkoxyl radicals may attack biological macromolecules giving rise to oxidative stress-originated diseases. Since (*)OH is very short-lived, secondary products resulting from (*)OH attack to various probes are measured. Although the measurement of aromatic hydroxylation with HPLC/electrochemical detection is more specific than the low-yield TBARS test, it requires sophisticated instrumentation. As a more convenient and less costly alternative, we used p-aminobenzoate, 2,4- and 3,5-dimethoxybenzoate probes for detecting hydroxyl radicals generated from an equivalent mixture of Fe(II)+EDTA with hydrogen peroxide. The produced hydroxyl radicals attacked both the probe and the water-soluble antioxidants in 37 degrees C-incubated solutions for 2h. The CUPRAC (i.e., our original method for total antioxidant capacity assay) absorbance of the ethylacetate extract due to the reduction of Cu(II)-neocuproine reagent by the hydroxylated probe decreased in the presence of (*)OH scavengers, the difference being proportional to the scavenging ability of the tested compound. A rate constant for the reaction of the scavenger with hydroxyl radical can be deduced from the inhibition of color formation. The second-order rate constants of the scavengers were determined with competition kinetics by means of a linear plot of A(0)/A as a function of C(scavenger)/C(probe), where A(0) and A are the CUPRAC absorbances of the system in the absence and presence of scavenger, respectively, and C is the molar concentration of relevant species. The 2,4- and 3,5-dimethoxybenzoates were the best probes in terms of linearity and sensitivity. Iodide, metabisulfite, hexacyanoferrate(II), thiourea, formate, and dimethyl sulfoxide were shown by the modified CUPRAC assay to be more effective scavengers than mannitol, glucose, lysine, and simple alcohols, as in the TBARS assay. The developed method is less lengthy, more specific, and of a higher yield than the classical TBARS assay. The hydroxyl radical scavenging rate constants of ascorbic acid, formate, and hexacyanoferrate(II) that caused interference in other assays could be easily found with the proposed procedure. SN - 0006-291X UR - https://www.unboundmedicine.com/medline/citation/16716257/Novel_hydroxyl_radical_scavenging_antioxidant_activity_assay_for_water_soluble_antioxidants_using_a_modified_CUPRAC_method_ L2 - https://linkinghub.elsevier.com/retrieve/pii/S0006-291X(06)01085-0 DB - PRIME DP - Unbound Medicine ER -