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.

Enzymatic determination of hypoxanthine in fish samples as a freshness indicator using the CUPRAC colorimetric sensor.

Fish consumption is essential for a healthy diet. However, all seafood including fish are susceptible to deterioration unless properly preserved. Controlling the freshness of fresh or packaged fish is a challenging issue for the food industry in terms of human health and shelf life determination. One of the main indicators showing the freshness of fish is undoubtedly the amount of hypoxanthine (Hx). As soon as the organism dies, Hx begins to be released with the cessation of ATP synthesis and shows a gradual increase over time. Therefore, Hx determination is an important indicator in the control of fish freshness. Based on this fact, a colorimetric method for the enzymatic determination of Hx using the CUPRAC (Cupric ion Reducing Antioxidant Capacity) sensor was developed. Uric acid (UA) and H2O2 are enzymatically produced by xanthine oxidase (XOD) from Hx, and both products respond to the CUPRAC reagent to produce the cuprous neocuproine (Cu(I)-Nc) chromophore chelate formed in situ on a Nafion anionic membrane on which the cationic Cu(II)-Nc complex was fixed. Hx was measured at different time intervals in the meat samples taken from sea bass (Dicentrarchus labrax), which was left to stand at room temperature for a time period between 0 and 24 h; the level of spoilage was determined from the coloration of the CUPRAC membrane sensor (via absorbance measurement at 450 nm). It was observed that there was a linear increase in the amount of Hx during the measurement period. The method was optimized for Hx determination, verified with interference analysis and standard additions to real samples, and validated against HPLC. The linear detection range of the developed method for Hx was 2.0-32.0 µM with an LOD of 0.79 µM, and early stages of fish degradation could be detected at several nanomoles of Hx per gram of fish meat. The proposed method was demonstrated to have distinct superiority over many recent colorimetric sensors of fish freshness in regard to its lower LOD for Hx, wider linear range, capability to cope with interferents (including biologically important antioxidants, such as cysteine, reduced glutathione, ascorbic acid, UA and α-tocopherol) and applicability to real samples.

Colorimetric Nanobiosensor Design for Determining Oxidase Enzyme Substrates in Food and Biological Samples.

Biological enzymes have high catalytic activity and unique substrate selectivity; their immobilization may provide cost reduction and reusability. Using magnetic nanoparticles (MNPs) as support materials for enzymes ensures easy separation from reaction media by an external magnetic field. Thus, MNPs were prepared by the coprecipitation method, coated with silanol groups, then -NH2-functionalized, and finally activated with glutaraldehyde. Finally, three different oxidase enzymes, i.e., uricase, glucose oxidase, and choline oxidase, were separately immobilized on their surfaces by covalent attachment. Hence, colorimetric nanobiosensors for the determination of three biologically important substrates, i.e., uric acid (UA), glucose (Glu), and choline (Ch), were developed. Hydrogen peroxide liberated from enzyme-substrate reactions was determined by the cupric ion reducing antioxidant capacity (CUPRAC) reagent, bis-neocuproine copper(II) chelate. In addition, UA-free total antioxidant capacity could also be measured via the developed system. Kinetic investigations were carried out for these nanobiosensors to enable the calculation of their Michaelis constants (K m), revealing no affinity loss for the substrate as a result of immobilization. Enzyme-immobilized MNPs could be reused at least five times. The linear concentration ranges were 5.43-65.22 µM for UA, 11.1-111.1 µM for Glu, and 2.78-44.4 µM for Ch, and the limit of detection (LOD) values with the same order were 0.34, 0.59, and 0.20 µM, respectively. Besides phenolic and thiol-type antioxidants, UA could be determined with an error range of 0.18-4.87%. The method is based on a clear redox reaction with a definite stoichiometry for the enzymatically generated H2O2 (which minimizes chemical deviations from Beer's law of optical absorbances), and it was successfully applied to the determination of Glu and UA in fetal bovine serum and Ch in infant formula as real samples.

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.

Novel Spectroscopic and Electrochemical Sensors and Nanoprobes for the Characterization of Food and Biological Antioxidants.

Since an unbalanced excess of reactive oxygen/nitrogen species (ROS/RNS) causes various diseases, determination of antioxidants that can counter oxidative stress is important in food and biological analyses. Optical/electrochemical nanosensors have attracted attention in antioxidant activity (AOA) assessment because of their increased sensitivity and selectivity. Optical sensors offer advantages such as low cost, flexibility, remote control, speed, miniaturization and on-site/in situ analysis. Electrochemical sensors using noble metal nanoparticles on modified electrodes better catalyze bioelectrochemical reactions. We summarize the design principles of colorimetric sensors and nanoprobes for food antioxidants (including electron-transfer based and ROS/RNS scavenging assays) and important milestones contributed by our laboratory. We present novel sensors and nanoprobes together with their mechanisms and analytical performances. Our colorimetric sensors for AOA measurement made use of cupric-neocuproine and ferric-phenanthroline complexes immobilized on a Nafion membrane. We recently designed an optical oxidant/antioxidant sensor using N,N-dimethyl-p-phenylene diamine (DMPD) as probe, from which ROS produced colored DMPD-quinone cationic radicals electrostatically retained on a Nafion membrane. The attenuation of initial color by antioxidants enabled indirect AOA estimation. The surface plasmon resonance absorption of silver nanoparticles as a result of enlargement of citrate-reduced seed particles by antioxidant addition enabled a linear response of AOA. We determined biothiols with Ellman reagent-derivatized gold nanoparticles.

The assessment of total antioxidant capacity and superoxide dismutase levels, and the possible role of manganese superoxide dismutase polymorphism in acromegaly.

Oxidative status is attributed to endothelial dysfunction and might be one of the key mechanisms of endothelial dysfunction in acromegaly. In this study, we aimed to investigate the effect of acromegaly on superoxide dismutase (SOD) and total antioxidant capacity (TAC) levels, and the possible influence of human manganese superoxide dismutase (MnSOD) polymorphism on these levels. 51 acromegaly patients and 57 age and sex matched healthy subjects were recruited to the study in Bezmialem Vakif University Hospital between 2011 and 2014. The median SOD and TAC levels were 42.7 (33-60) pg/mL and 1,313.7 (155-1,902) µM in acromegaly; and 46.3 (38-95) pg/mL and 1,607.3 (195-1,981) µM in healthy subjects (p < 0.001, p < 0.001). SOD levels were decreased in controlled and uncontrolled patients compared to healthy subjects (p = 0.05 and p = 0.002, respectively). Controlled and uncontrolled acromegaly displayed significantly decreased levels of TAC compared to healthy subjects (p < 0.05 and p < 0.001, respectively). SOD levels were not associated with MnSOD polymorphisms in acromegaly. In conclusion, this study showed that acromegaly was associated with decreased levels of SOD and TAC, and controlling the disease activity could not adequately improve these levels.

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.

Selective determination of catechin among phenolic antioxidants with the use of a novel optical fiber reflectance sensor based on indophenol dye formation on nano-sized TiO2.

The optical sensor for "tea catechins" was built by immobilizing 2,2'-(1,4-phenylenedivinylene)bis-8-hydroxyquinoline (PBHQ) on TiO2 nanoparticles (NPs). The sensor worked by "indophenol blue" dye formation on PBHQ-immobilized TiO2 NPs as a result of p-aminophenol (PAP) autoxidation with dissolved O2 at pH 10. Among quercetin, rutin, naringenin, naringin, gallic acid, caffeic acid, ferulic acid, p-coumaric acid, catechin, epicatechin, epicatechin gallate, epigallocatechin, epigallocatechin gallate, and trolox, only catechin group antioxidants delayed the color formation on NPs, as measured by the reflectance signal at 710 nm. For quantitative analysis, reflectance signal versus time was recorded, and the difference between the areas under curve (ΔAUC) in the presence and absence of catechin was correlated (r = 0.98) to catechin concentration. The selectivity of the sensor for catechins was shown in tea infusions compared to other plant extracts and was ascribed to the nonplanar structure of catechin interfering with the formation of perfectly conjugated indophenol blue on TiO2 surface.