Tags

Type your tag names separated by a space and hit enter

Total antioxidant capacity assay of human serum using copper(II)-neocuproine as chromogenic oxidant: the CUPRAC method.
Free Radic Res. 2005 Sep; 39(9):949-61.FR

Abstract

BACKGROUND

Tests measuring the combined antioxidant effect of the nonenzymatic defenses in biological fluids may be useful in providing an index of the organism's capability to counteract reactive species known as prooxidants, resist oxidative damage and combat oxidative stress-related diseases. The selected chromogenic redox reagent for the assay of human serum should be easily accessible, stable, selective, respond to all types of biologically important antioxidants such as ascorbic acid, alpha-tocopherol, beta-carotene, reduced glutathione (GSH), uric acid and bilirubin, regardless of chemical type or hydrophilicity. Currently, there is no rapid method for total antioxidant assay of human serum meeting the above criteria.

METHODS

Our recently developed cupric reducing antioxidant capacity (CUPRAC) spectrophotometric method for a number of polyphenols and flavonoids using the copper(II)-neocuproine reagent in ammonium acetate buffer was now applied to a complete series of plasma antioxidants for the assay of total antioxidant capacity (TAC) of serum, and the resulting absorbance at 450 nm was recorded either directly (e.g. for ascorbic acid, alpha-tocopherol and glutathione) or after incubation at 50 degrees C for 20 min (e.g. for uric acid, bilirubin and albumin), quantitation being made by means of a calibration curve. The lipophilic antioxidants, alpha-tocopherol and beta-carotene, were assayed in dichloromethane (DCM). Lipophilic antioxidants of serum were extracted with n-hexane from an ethanolic solution of serum subjected to centrifugation. Hydrophilic antioxidants of serum were assayed after perchloric acid precipitation of proteins in the centrifugate.

RESULTS

The molar absorptivities, linear ranges and trolox equivalent antioxidant capacity (TEAC) coefficients of the serum antioxidants were established with respect to the CUPRAC spectrophotometric method, and the results (TEAC, or TEAC coefficients) were evaluated in comparison to the findings of the ABTS/TEAC reference method using persulfate as oxidant. As for hydrophilic phase, a linear correlation existed between the CUPRAC and ABTS findings (r=0.58), contrary to current literature reporting that either serum ORAC or serum ferric reducing antioxidant potency (FRAP) does not correlate at all with serum TEAC. The analytical responses of serum antioxidants were shown to be additive, enabling a TAC assay. The intra- and inter-assay CVs were 0.7 and 1.5%, respectively, for serum.

CONCLUSIONS

The CUPRAC assay proved to be efficient for glutathione and thiol-type antioxidants, for which the FRAP test was nonresponsive. The findings of CUPRAC completely agreed with those of ABTS-persulfate for lipophilic phase. The additivity of absorbances of all the tested antioxidants confirmed that antioxidants in the CUPRAC test did not chemically interact among each other so as to cause an intensification or quenching of the theoretically expected absorbance. As a distinct advantage over other electron-transfer based assays (e.g. Folin, FRAP, ABTS, DPPH), CUPRAC is superior in regard to its realistic pH close to the physiological pH, favourable redox potential, accessibility and stability of reagents and applicability to lipophilic antioxidants as well as hydrophilic ones.

Authors+Show Affiliations

Faculty of Engineering, Department of Chemistry, Istanbul University, Avcilar 34320, Instanbul, Turkey. rapak@istanbul.edu.trNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

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

Language

eng

PubMed ID

16087476

Citation

Apak, Reşat, et al. "Total Antioxidant Capacity Assay of Human Serum Using copper(II)-neocuproine as Chromogenic Oxidant: the CUPRAC Method." Free Radical Research, vol. 39, no. 9, 2005, pp. 949-61.
Apak R, Güçlü K, Ozyürek M, et al. Total antioxidant capacity assay of human serum using copper(II)-neocuproine as chromogenic oxidant: the CUPRAC method. Free Radic Res. 2005;39(9):949-61.
Apak, R., Güçlü, K., Ozyürek, M., Karademir, S. E., & Altun, M. (2005). Total antioxidant capacity assay of human serum using copper(II)-neocuproine as chromogenic oxidant: the CUPRAC method. Free Radical Research, 39(9), 949-61.
Apak R, et al. Total Antioxidant Capacity Assay of Human Serum Using copper(II)-neocuproine as Chromogenic Oxidant: the CUPRAC Method. Free Radic Res. 2005;39(9):949-61. PubMed PMID: 16087476.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Total antioxidant capacity assay of human serum using copper(II)-neocuproine as chromogenic oxidant: the CUPRAC method. AU - Apak,Reşat, AU - Güçlü,Kubilay, AU - Ozyürek,Mustafa, AU - Karademir,Saliha Esin, AU - Altun,Mehmet, PY - 2005/8/10/pubmed PY - 2006/9/7/medline PY - 2005/8/10/entrez SP - 949 EP - 61 JF - Free radical research JO - Free Radic. Res. VL - 39 IS - 9 N2 - BACKGROUND: Tests measuring the combined antioxidant effect of the nonenzymatic defenses in biological fluids may be useful in providing an index of the organism's capability to counteract reactive species known as prooxidants, resist oxidative damage and combat oxidative stress-related diseases. The selected chromogenic redox reagent for the assay of human serum should be easily accessible, stable, selective, respond to all types of biologically important antioxidants such as ascorbic acid, alpha-tocopherol, beta-carotene, reduced glutathione (GSH), uric acid and bilirubin, regardless of chemical type or hydrophilicity. Currently, there is no rapid method for total antioxidant assay of human serum meeting the above criteria. METHODS: Our recently developed cupric reducing antioxidant capacity (CUPRAC) spectrophotometric method for a number of polyphenols and flavonoids using the copper(II)-neocuproine reagent in ammonium acetate buffer was now applied to a complete series of plasma antioxidants for the assay of total antioxidant capacity (TAC) of serum, and the resulting absorbance at 450 nm was recorded either directly (e.g. for ascorbic acid, alpha-tocopherol and glutathione) or after incubation at 50 degrees C for 20 min (e.g. for uric acid, bilirubin and albumin), quantitation being made by means of a calibration curve. The lipophilic antioxidants, alpha-tocopherol and beta-carotene, were assayed in dichloromethane (DCM). Lipophilic antioxidants of serum were extracted with n-hexane from an ethanolic solution of serum subjected to centrifugation. Hydrophilic antioxidants of serum were assayed after perchloric acid precipitation of proteins in the centrifugate. RESULTS: The molar absorptivities, linear ranges and trolox equivalent antioxidant capacity (TEAC) coefficients of the serum antioxidants were established with respect to the CUPRAC spectrophotometric method, and the results (TEAC, or TEAC coefficients) were evaluated in comparison to the findings of the ABTS/TEAC reference method using persulfate as oxidant. As for hydrophilic phase, a linear correlation existed between the CUPRAC and ABTS findings (r=0.58), contrary to current literature reporting that either serum ORAC or serum ferric reducing antioxidant potency (FRAP) does not correlate at all with serum TEAC. The analytical responses of serum antioxidants were shown to be additive, enabling a TAC assay. The intra- and inter-assay CVs were 0.7 and 1.5%, respectively, for serum. CONCLUSIONS: The CUPRAC assay proved to be efficient for glutathione and thiol-type antioxidants, for which the FRAP test was nonresponsive. The findings of CUPRAC completely agreed with those of ABTS-persulfate for lipophilic phase. The additivity of absorbances of all the tested antioxidants confirmed that antioxidants in the CUPRAC test did not chemically interact among each other so as to cause an intensification or quenching of the theoretically expected absorbance. As a distinct advantage over other electron-transfer based assays (e.g. Folin, FRAP, ABTS, DPPH), CUPRAC is superior in regard to its realistic pH close to the physiological pH, favourable redox potential, accessibility and stability of reagents and applicability to lipophilic antioxidants as well as hydrophilic ones. SN - 1071-5762 UR - https://www.unboundmedicine.com/medline/citation/16087476/Total_antioxidant_capacity_assay_of_human_serum_using_copper_II__neocuproine_as_chromogenic_oxidant:_the_CUPRAC_method_ L2 - http://www.tandfonline.com/doi/full/10.1080/10715760500210145 DB - PRIME DP - Unbound Medicine ER -