CUPRAC colorimetric and electroanalytical methods determining antioxidant activity based on prevention of oxidative DNA damage.

An unbalanced excess of oxygen/nitrogen species (ROS/RNS) can give oxidative hazard to DNA and other biomacromolecules under oxidative stress conditions. While the 'comet' assay for measuring DNA damage is neither specific nor practical, monitoring oxidative changes on individual DNA bases and other oxidation products needs highly specialized equipment and operators. Thus, we developed a modified CUPRAC (cupric ion reducing antioxidant capacity) colorimetric method to determine the average total damage on DNA produced by Fenton oxidation, taking advantage of the fact that the degradation products of DNA but not the original macromolecule is CUPRAC-responsive. The DNA-protective effects of water-soluble antioxidants were used to devise a novel antioxidant activity assay, considered to be physiologically more realistic than those using artificial probes. Our method, based on the measurement of DNA oxidative products with CUPRAC colorimetry proved to be 2 orders-of-magnitude more sensitive than the widely used TBARS (thiobarbituric acid-reactive substances) colorimetric assay used as reference. Additionally, the DNA damage was electrochemically investigated using pencil graphite electrodes (PGEs) as DNA sensor platform in combination with differential pulse voltammetry (DPV). The interaction of the radical species with DNA in the absence/presence of antioxidants was detected according to the changes in guanine oxidation signal.

Spectrophotometric Determination of Phenolic Antioxidants in the Presence of Thiols and Proteins.

Development of easy, practical, and low-cost spectrophotometric methods is required for the selective determination of phenolic antioxidants in the presence of other similar substances. As electron transfer (ET)-based total antioxidant capacity (TAC) assays generally measure the reducing ability of antioxidant compounds, thiols and phenols cannot be differentiated since they are both responsive to the probe reagent. In this study, three of the most common TAC determination methods, namely cupric ion reducing antioxidant capacity (CUPRAC), 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt/trolox equivalent antioxidant capacity (ABTS/TEAC), and ferric reducing antioxidant power (FRAP), were tested for the assay of phenolics in the presence of selected thiol and protein compounds. Although the FRAP method is almost non-responsive to thiol compounds individually, surprising overoxidations with large positive deviations from additivity were observed when using this method for (phenols + thiols) mixtures. Among the tested TAC methods, CUPRAC gave the most additive results for all studied (phenol + thiol) and (phenol + protein) mixtures with minimal relative error. As ABTS/TEAC and FRAP methods gave small and large deviations, respectively, from additivity of absorbances arising from these components in mixtures, mercury(II) compounds were added to stabilize the thiol components in the form of Hg(II)-thiol complexes so as to enable selective spectrophotometric determination of phenolic components. This error compensation was most efficient for the FRAP method in testing (thiols + phenols) mixtures.

Colorimetric Determination of Sulfoxy Radicals and Sulfoxy Radical Scavenging-Based Antioxidant Activity.

Sulfoxy radicals (SORs) are oxygen- and sulfur-containing species such as SO3•-, SO4•-, and SO5•-. They can be physiologically generated by S(IV) autoxidation with transition metal catalysis. Due to their harmful effects, the detection of both SORs and their scavengers are important. Here, a simple and cost-effective method for the determination of SORs and the scavenging activity of different antioxidant compounds was proposed. A SOR was selectively generated by combining CoSO4·7H2O with Na2SO3. To detect SOR species as a whole, 3,3',5,5'-tetramethylbenzidine (TMB) was used as the chromogenic reagent, where SOR generated in the medium caused the formation of a blue-colored diimine from TMB. Additionally, the SOR scavenging effects of a number of antioxidant compounds (AOx) belonging to different classes were investigated, among which catechin derivatives were the most effective scavengers. The obtained results were compared with those of a reference rhodamine B decolorization assay. The radical scavenging effects of the tested AOx were ranked by both assays and then compared using the Spearman statistical test to yield a very strong correlation between the two rankings. The method was applied to real samples such as catechin-rich tea, that is, white, black, and green tea, among which white tea was determined as the most effective SOR scavenger.

Biomarkers of Oxidative Stress and Antioxidant Defense.

A number of reactive oxygen and nitrogen species are produced during normal metabolism in human body. These species can be both radical and non-radical and have varying degrees of reactivity. Although they have some important functions in the human body, such as contributing to signal transmission and the immune system, their presence must be balanced by the antioxidant defense system. The human body has an excellent intrinsic enzymatic antioxidant system in addition to different non-enzymatic antioxidants having small molecular masses. An extrinsic source of antioxidants are foodstuffs such as fruits, vegetables, herbs and spices, mostly rich in polyphenols. When the delicate biochemical balance between oxidants and antioxidants is disturbed in favor of oxidants, "oxidative stress" conditions emerge, under which reactive species can cause oxidative damage to biomacromolecules such as proteins, carbohydrates, lipids and DNA. This oxidative damage is often associated with cancer, aging, and neurodegenerative disorders. Because reactive species are extremely short-lived, it is almost impossible to measure their concentrations directly. Although there are certain methods such as ESR / EPR that serve this purpose, they have some disadvantages and are quite costly systems. Therefore, products generated from oxidative damage of proteins, lipids and DNA are often used to quantify the extent of oxidative damage rather than direct measurement of reactive species. These oxidative damage products are usually known as biomarkers. Determination of the concentrations of these biomarkers and changes in the concentration of protective antioxidants can provide useful information for avoiding certain diseases and keep healthy conditions.

Sulfate radical formation by Cr(III) activation of peroxydisulfate - Diphenylcarbazide spectrophotometric determination of sulfate radical and its scavenging activity.

Even though sulfate anion radical (SO4-) is a very reactive oxidant used in advanced oxidation processes, a reliably selective and simple colorimetric method for determining this radical can hardly be found. Peroxydisulfate (S2O82-) or peroxymonosulfate (HSO5-) can be activated with transition metal ions to produce SO4-. We have discovered that Cr(III) can be an activator for persulfate, generating Cr(VI) along with SO4-. By measuring the emerging chromate with diphenyl carbazide (DPC) spectrophotometry at 542 nm, we could measure both the formation of SO4- and its scavenging with antioxidant compounds. We could also investigate a number of UV-absorbing SO4- scavengers which could not be measured with other UV spectrometric methods. In addition to conventional antioxidants (phenolics such as quercetin, catechin, epicatechin, caffeic acid, thiols like cysteine and N-acetyl cysteine, and ascorbid acid), nitro-aromatics (represented by 2,4,6-trinitrophenol and 2,4-dinitrophenol) used in ammunition formulations could also be measured as scavengers. The presence of scavengers caused a reduction in the amount of Cr(VI) generated, where the difference in absorbance (ΔA) of chromate - with respect to the DPC method - in the absence and presence of scavengers was a linear function of SO4- scavenging capacity. Ethanol and tert-butanol were tested as solvents to show the selectivity of the method for SO4-. The method was statistically compared to a suitably modified ABTS/persulfate assay. The efficiency order of sulfate radical scavengers was determined and ranked (Spearman's test) using both the proposed method and modified ABTS/persulfate method to reveal a moderate correlation.

A colourimetric sensor for the simultaneous determination of oxidative status and antioxidant activity on the same membrane: N,N-dimethyl-p-phenylene diamine (DMPD) on Nafion.

A colourimetric sensor capable of simultaneously measuring oxidative status (OS) in terms of the hazard produced by reactive oxygen species (ROS) and antioxidant activity (AOA) in regard to ROS-scavenging ability of antioxidant compounds was developed. The coloured cationic semi-quinone derivatives, caused by ROS oxidative degradation of N,N-dimethyl-p-phenylene diamine hydrochloride (DMPD) in pH 5.7 acetate-buffered medium, were formed in solution and immobilized on a perfluorosulfonate-based Nafion membrane. ROS, namely hydroxyl (·OH) and superoxide (O2(·-)) radicals, were produced by Fenton/UV and xanthine/xanthine oxidase methods, respectively. The pink-coloured, (+)-charged chromophore (referred to as DMPD-quinone or DMPDQ), resulting from the reaction between DMPD and ROS, could be completely retained on the solid membrane sensor by electrostatic interaction with the anionic sulfonate groups of Nafion. After equilibration, the Nafion membrane surface was homogeneously coloured enabling an absorbance measurement at 514 nm, while the aqueous phase completely lacked colour. Antioxidants, when present, caused an absorbance decrease on the membrane due to their ROS scavenging action, giving rise to less DMPDQ production. The absorbance decrease on the sensor was linearly dependent on antioxidant concentration over a reasonable concentration range, enabling the simultaneous determination of OS and AOA-against ROS. The proposed antioxidant sensing method was tested in synthetic and real antioxidant mixtures, and validated against standard antioxidant capacity assays (i.e. ABTS and CUPRAC) for a variety of polyphenolic and antioxidant compounds. The dynamic linear ranges of antioxidants with the DMPD sensor in protection against hydroxyl and superoxide radicals generally varied within the micromolar to a few tens of micromolar concentration interval over one order-of-magnitude. Choosing three representative compounds in the high (epigallocatechin gallate), medium (quercetin) and low (p-coumaric acid) molar absorptivity range, the detection limits ranged within the concentration intervals of 0.2-0.9 µM, 0.3-0.8 µM, and 4-14 µM, respectively, depending on the radical scavenged.