A comprehensive review of CUPRAC methodology

Measuring the antioxidant activity/capacity levels of food and biological fluids is carried out for the meaningful comparison of the antioxidant content of foodstuffs and for the diagnosis and treatment of oxidative stress-associated diseases in clinical biochemistry. Current literature clearly states that there is no widely adopted/accepted “total antioxidant parameter” as a nutritional index available for the labeling of food and biological fluids due to the lack of standardized quantitation methods. The “parent” CUPRAC (CUPric Reducing Antioxidant Capacity) method of antioxidant measurement, introduced by our research group to world literature, is based on the absorbance measurement of Cu(I)-neocuproine (Nc) chelate formed as a result of the redox reaction of chain-breaking antioxidants with the CUPRAC reagent, Cu(II)-Nc, where absorbance is recorded at the maximal light absorption wavelength of 450 nm; thus this is an electron-transfer (ET)-based method. From the parent CUPRAC method initially applied to food (apricot, herbal teas, wild edible plants, herby cheese etc.) and biological fluids (as hydrophilic and lipophilic antioxidants together or in separate fractions), a number of “daughter” methods have evolved, such as the simultaneous assay of both lipophilic and hydrophilic antioxidants in acetone-water as methyl-β-cyclodextrin inclusion complexes, determination of ascorbic acid alone in the presence of flavonoids (with preliminary extraction of flavonoids as their La(III)-complexes), determination of hydroxyl radical scavenging activity of both water-soluble antioxidants (using benzoate derivatives and salicylate as hydroxylation probes) and of polyphenols using catalase to stop the Fenton reaction so as to prevent redox cycling of antioxidants, measurement of Cu(II)-catalyzed hydrogen peroxide scavenging activity and of xanthine oxidase inhibition activity of polyphenols, TAC measurement of protein thiols in urea buffer, development of a CUPRAC-based antioxidant sensor on a Nafion cation-exchanger membrane, the off-line HPLC-CUPRAC assay and finally the on-line HPLC-CUPRAC assay of antioxidants with post-column detection. The current direction of CUPRAC methodology can be best described as a self-sufficient and integrated train of measurements providing a useful “antioxidant and antiradical assay package”. This review attempts to unify and summarize various methodologies of main and modified CUPRAC procedures that can normally be extracted from quite different literature sources.