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Hydroxyl radical detection with a salicylate probe using modified CUPRAC spectrophotometry and HPLC.
Talanta. 2008 Oct 19; 77(1):90-7.T

Abstract

Reactive oxygen species (ROS) may attack biological macromolecules giving rise to oxidative stress-originated diseases, so it is important to establish efficient methods to screen hydroxyl radical scavengers for antioxidant therapy. Since *OH is very short-lived, secondary products resulting from *OH attack to various probes are measured. As a low-cost measurement technique, we used a salicylate probe 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 2 h. The CUPRAC (cupric ion reducing 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. Attack by *OH radicals upon salicylate produced 2,3-dihydroxybenzoate, 2,4-dihydroxybenzoate, and 2,5-dihydroxybenzoate as major products. HPLC separation combined with CUPRAC spectrophotometry was used to identify and quantify hydroxylated salicylate derivatives in the presence of synthetic water-soluble antioxidants and green tea infusion. The developed spectrophotometric method for *OH detection was validated with HPLC, i.e., the concentrations of dihydroxybenzoates produced by radical attack from the probe were determined by HPLC, and the sum of (concentrationxabsorptivity) products of these components approximately agreed with the experimentally found CUPRAC absorbances, confirming the validity of Beer's law for the selected system. Statistical comparison of the results found with the proposed methodology and HPLC was made with two-way ANOVA (analysis of variance) test. Under optimal conditions, about 53% of the probe (salicylate) was converted into dihydroxybenzoate isomers in the absence of *OH scavengers, and these isomers were more specific markers of hydroxyl radicals than the non-specific malondialdehyde end-product of the TBARS test. Thus, the more costly and less speedy HPLC method could advantageously be substituted with the proposed spectrophotometric assay of *OH detection, which was also of much higher yield than the TBARS colorimetric assay.

Authors+Show Affiliations

Istanbul University, Faculty of Engineering, Department of Chemistry, Avcilar 34320, Istanbul, Turkey.No 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

18804604

Citation

Bektaşoğlu, Burcu, et al. "Hydroxyl Radical Detection With a Salicylate Probe Using Modified CUPRAC Spectrophotometry and HPLC." Talanta, vol. 77, no. 1, 2008, pp. 90-7.
Bektaşoğlu B, Ozyürek M, Güçlü K, et al. Hydroxyl radical detection with a salicylate probe using modified CUPRAC spectrophotometry and HPLC. Talanta. 2008;77(1):90-7.
Bektaşoğlu, B., Ozyürek, M., Güçlü, K., & Apak, R. (2008). Hydroxyl radical detection with a salicylate probe using modified CUPRAC spectrophotometry and HPLC. Talanta, 77(1), 90-7. https://doi.org/10.1016/j.talanta.2008.05.043
Bektaşoğlu B, et al. Hydroxyl Radical Detection With a Salicylate Probe Using Modified CUPRAC Spectrophotometry and HPLC. Talanta. 2008 Oct 19;77(1):90-7. PubMed PMID: 18804604.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Hydroxyl radical detection with a salicylate probe using modified CUPRAC spectrophotometry and HPLC. AU - Bektaşoğlu,Burcu, AU - Ozyürek,Mustafa, AU - Güçlü,Kubilay, AU - Apak,Reşat, Y1 - 2008/06/05/ PY - 2008/01/29/received PY - 2008/05/20/revised PY - 2008/05/29/accepted PY - 2008/9/23/pubmed PY - 2008/11/4/medline PY - 2008/9/23/entrez SP - 90 EP - 7 JF - Talanta JO - Talanta VL - 77 IS - 1 N2 - Reactive oxygen species (ROS) may attack biological macromolecules giving rise to oxidative stress-originated diseases, so it is important to establish efficient methods to screen hydroxyl radical scavengers for antioxidant therapy. Since *OH is very short-lived, secondary products resulting from *OH attack to various probes are measured. As a low-cost measurement technique, we used a salicylate probe 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 2 h. The CUPRAC (cupric ion reducing 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. Attack by *OH radicals upon salicylate produced 2,3-dihydroxybenzoate, 2,4-dihydroxybenzoate, and 2,5-dihydroxybenzoate as major products. HPLC separation combined with CUPRAC spectrophotometry was used to identify and quantify hydroxylated salicylate derivatives in the presence of synthetic water-soluble antioxidants and green tea infusion. The developed spectrophotometric method for *OH detection was validated with HPLC, i.e., the concentrations of dihydroxybenzoates produced by radical attack from the probe were determined by HPLC, and the sum of (concentrationxabsorptivity) products of these components approximately agreed with the experimentally found CUPRAC absorbances, confirming the validity of Beer's law for the selected system. Statistical comparison of the results found with the proposed methodology and HPLC was made with two-way ANOVA (analysis of variance) test. Under optimal conditions, about 53% of the probe (salicylate) was converted into dihydroxybenzoate isomers in the absence of *OH scavengers, and these isomers were more specific markers of hydroxyl radicals than the non-specific malondialdehyde end-product of the TBARS test. Thus, the more costly and less speedy HPLC method could advantageously be substituted with the proposed spectrophotometric assay of *OH detection, which was also of much higher yield than the TBARS colorimetric assay. SN - 1873-3573 UR - https://www.unboundmedicine.com/medline/citation/18804604/Hydroxyl_radical_detection_with_a_salicylate_probe_using_modified_CUPRAC_spectrophotometry_and_HPLC_ L2 - https://linkinghub.elsevier.com/retrieve/pii/S0039-9140(08)00443-8 DB - PRIME DP - Unbound Medicine ER -