Simultaneous detection of superoxide anion radicals and determination of the superoxide scavenging activity of antioxidants using a N,N-dimethyl-p-phenylene diamine/Nafion colorimetric sensor

The widely used nitroblue tetrazolium (NBT) colorimetric method for superoxide anion radical (O-2(center dot-))detection suffers from limitations such as formazan solubility and interference from similar electron donors. This work reports the development of a novel assay for superoxides, where O-2(center dot-) generated by a NADH/PMS/O-2 system oxidizes the N, N-dimethyl-p-phenylene diamine (DMPD) reagent to pink-colored DMPD-semiquinone (DMPDQ) cationic radicals retained by a cation exchange Nafion (R) membrane and measured colorimetrically. Antioxidants exhibiting superoxide radical scavenging activity (SRSA) cause less DMPDQ radical production, resulting in an attenuated color intensity on the Nafion (R) membrane, the absorbance difference (Delta A) at 514 nm being proportional to the antioxidant concentration. The developed method was applied to 14 antioxidant compounds including trolox, phenolic and hydroxycinnamic acids, flavonoids, and thiols, and the linear concentration ranges, calibration equations (as Delta A versus concentration), and trolox-equivalent antioxidant capacities of these antioxidants were established. The results were discussed in the light of structure-activity relationships. This method was used for measuring the SRSA of antioxidant mixtures and a green tea extract. The Nafion (R) membrane-contacted green tea extract was transferred to an online-HPLC post-column CUPRAC system to measure unconsumed catechins. The percentage inhibitions for green tea found with the developed method were compared to those measured by the reference NBT method

Use of modified CUPRAC and dinitrophenylhydrazine colorimetric methods for simultaneous measurement of oxidative protein damage and antioxidant defense against oxidation

A modified CUPRAC (cupric reducing antioxidant capacity) method was developed for the simultaneous estimation of protein oxidation and counteracting antioxidant defense, and the results were compared with those of a modified 2,4-dinitrophenylhydrazine (DNPH) carbonyl assay. The alkaline carbonyl method was cleared off interferences by solvent extraction using a cationic surfactant. Both solution and Nafion membrane sensor CUPRAC methods were used to measure the oxidative hazard in protein solutions. Bovine serum albumin, fetal bovine serum and egg white were used as protein probes, exposed to oxidation by Fe(II)-induced Fenton reaction in the absence and presence of selected antioxidants (ascorbic acid, cysteine, gallic acid, glutathione, and N acetyl cysteine). Protein probes were initially unreactive toward the CUPRAC and DNPH reagents, but produced colored products upon Fenton oxidation which were bleached by antioxidants, enabling an indirect measurement of antioxidant activity (AOA) by difference. Spearman's rank test for antioxidants demonstrated that there was a strong correlation (+ 0.7 to + 0.9) between the modified CUPRAC and carbonyl assays. There was also a strong correlation between the results of the solution phase and optical sensing CUPRAC methods (R-2 > 0.95). As opposed to conventional antioxidant assays not using biologically relevant probes, this work utilizes protein probes for AOA assessment

Indirect colorimetric determination of trace hydrogen peroxide by its oxidizing power on chromium(III) oxide nanoparticles

Although enzymes are excellent catalysts in vivo, they are difficult to use in vitro. Therefore, enzyme-mimetic nanoparticles (NPs) or nanozymes, having the properties of certain enzymes but being much more resistant to chemicals and atmospheric conditions, offer a good alternative to biological enzymes. In the present study, Cr2O3 NPs were used as redox catalysts for determination of H2O2, a key compound for biochemistry, food industry and environmental engineering. This method proved to be more selective for H2O2 than other similar nanosensing methods based on Au/Ag NPs formation. This simple method had high selectivity toward H2O2 due to the high redox potential of Cr(VI)/Cr(III) couple, which could not be overcome by mild oxidants. Unlike most studies in the literature using peroxidase-like nanozymes for colorimetric H2O2 determination, a peroxidase substrate is not required in this study. Here, when H2O2 in the real samples (or in case of triacetone triperoxide (TATP) samples, H2O2 released by hydrolysis) were contacted with Cr2O3 NPs at pH 10, Cr(III) was oxidized to Cr(VI). At optimal temperature and time, there was a clear reaction with a net stoichiometry between the probe and analyte. Finally, the generated Cr(VI) was reacted by diphenylcarbazide (DPC) in acidic medium to form a pink colored compound, the color intensity of which was directly related to H2O2 concentration. This method yielded a linear calibration range of 4.0-58 mu M for H2O2. For the tested real samples and TATP hydrolyzate, the results obtained by the proposed procedure were compatible with those of the standard TiOSO4 colorimetric method. Finally, H2O2 scavenging effects of certain antioxidants (AOX) were successfully measured by the presented spectro-photometric Cr2O3 NPs method

Novel Colorimetric Assay of 2,3-Dihydroxybenzoate among Other Isomers as a Selective Indicator of Hydroxyl Radical Damage and Related Antioxidant Activity

Although reactive oxygen species can regulate intracellular signaling pathways, excessive amounts under oxidative stress conditions may cause damage to biomolecules. Hydroxyl radical ((OH)-O-center dot) is a most reactive oxidant that can harm DNA, lipids, and proteins. As the direct determination of (OH)-O-center dot by highly specialized electron paramagnetic resonance techniques is costly, indirect colorimetric determinations have attracted attention. Salicylic acid has been used both as an in vitro and in vivo probes to detect (OH)-O-center dot, itself being converted to 2,3-,2,4-, and 2,5-dihydroxybenzoic acids (DHBA) and catechol, but since 2,5-DHBA may also be generated enzymatically in the cyctochrome P-450 metabolism, 2,3-DHBA is the real marker of oxidative salicylate damage. This work is focused on the development of a selective hydroxyl radical detection assay by modifying a colorimetric nitrite-molybdate method concerning vic-diol determination of 2,3-DHBA among other DHBA isomers. Salicylic acid was hydroxylated to DHBAs and catechol upon the attack of (OH)-O-center dot produced in a Fenton system. An aliquot from the mixture was oxidized with a nitrite-molybdate(VI) reagent to give an intense red product in alkaline medium with maximal absorbance at 510 nm. The assay was selective to only 2,3-DHBA and catechol among all DHBA isomers. The residual salicylic acid, DHBAs, and catechol were measured using high-performance liquid chromatography (HPLC); the spectrophotometrically measured contents of 2,3-DHBA and catechol were compared with HPLC results. Antioxidants, when present, caused a reduction in the hydroxylation of salicylate probe producing less 2,3-DHBA and catechol, thereby enabling the development of an indirect antioxidant activity assay for colorimetrically measuring (OH)-O-center dot scavenging ability

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. (c) 2021 Elsevier B.V. All rights reserved