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Modified cupric reducing antioxidant capacity (CUPRAC) assay for measuring the antioxidant capacities of thiol-containing proteins in admixture with polyphenols

Proteins are not considered as true antioxidants but are known to protect antioxidants from oxidation in various antioxidant activity assays. This study aims to investigate the contribution of proteins, especially thiol-containing proteins, to the observed overall antioxidant capacity measured by known methods. To determine the antioxidant properties of thiol-containing proteins, the CUPRAC method of antioxidant assay using the oxidizing reagent Cu(II)-neocuproine previously used for simultaneous analysis of cystine and cysteine was adopted. While the CUPRAC method is capable of determining all antioxidant compounds including thiols in complex sample matrices, the Ellman method of thiol quantitation basically does not respond to other antioxidants. The antioxidant quantities in the selected samples were assayed with the ABTS and FRAP methods as well as with the CUPRAC method. In all applied methods, the dilutions were made with a standard pH 8 buffer used in the Ellman method by substituting the Na(2)EDTA component of the buffer with sodium citrate. On the other hand, the standard CUPRAC protocol was modified by substituting the pH 7 ammonium acetate buffer (at 1M concentration) with 8M urea buffer adjusted to pH 7 by neutralizing with 6M HCl. Urea helps to partly solubilize and denaturate proteins so that their buried thiols be oxidized more easily. All methods used in the estimation of antioxidant properties of proteins (i.e., CUPRAC, Ellman, ABTS, and FRAP) were first standardized with a simple thiol compound, cysteine, by constructing the calibration curves. The molar absorptivities of these methods for cysteine were: epsilon(CUPRAC)=7.71x10(3), epsilon(Ellman)=1.37x10(4), epsilon(ABTS)=2.06x10(4), and epsilon(FRAP)=2.98x10(3)L mol(-1)cm(-1). Then these methods were applied to various samples containing thiols, such as glutathione (reduced form:GSH), egg white, whey proteins, and gelatin. Additionally, known quantities of selected antioxidants were added to these samples to show the additivity of responses.

Spectrophotometric total protein assay with copper(II)-neocuproine reagent in alkaline medium.

Total protein assay was made using copper(II)-neocuproine (Nc) reagent in alkaline medium (with the help of a hydroxide-carbonate-tartarate solution) after 30min incubation at 40 degrees C. The absorbance of the reduction product, Cu(I)-Nc complex, was recorded at 450nm against a reagent blank. The absorptivity of the developed method for bovine serum albumin (BSA) was 0.023lmg(-1)cm(-1), greater than that of Lowry assay (0.0098), and much greater than that of Cu(II)-bicinchoninic acid (BCA) assay (0.00077). The linear range of the developed method (8-100mgl(-1) BSA) was as wide as that of Lowry, and much wider than that of BCA (200-1000mgl(-1) BSA) assay. The sensitivity of the method was greater than those of Cu-based assays (biuret, Lowry, and BCA) with a LOD of 1mgl(-1) BSA. The within-run and between-run precisions as RSD were 0.73 and 1.01%, respectively. The selectivity of the proposed method for protein was much higher than those of dye-binding and Lowry assays: Most common interferents to other protein assays such as tris, ethanolamine, deoxycholate, CsCl, citrate, and triton X-100 were tolerated at 100-fold concentrations in the analysis of 10mgl(-1) BSA, while the tolerance limits for other interferents, e.g., (NH(4))(2)SO(4) and acetylsalicylic acid (50-fold), SDS (25-fold), and glycerol (20-fold) were at acceptable levels. The redox reaction of Cu(II)-Nc as an outer-sphere electron transfer agent with the peptide bond and with four amino acid residues (cystine, cysteine, tryptophan, and tyrosine) was kinetically more favourable than that of Cu(II) alone in the biuret assay. Since the reduction product of Cu(II) with protein, i.e., Cu(I), was coordinatively saturated with Nc in the stable Cu(Nc)(2)(+) chelate, re-oxidation of the formed Cu(I) with Fenton-like reactions was not possible, thereby preventing a loss of chromophore. After conventional protein extraction, precipitation, and redissolution procedures, the protein contents of the minced meat (veal and turkey), sardine, various milk products, and egg white were analyzed with the proposed and Lowry methods, and the results correlated appreciably (r=0.98). The method was validated by Kjeldahl analyses of the tested samples; the data sets of complex samples assayed by Cu(II)-Nc and Lowry correlated to the findings of Kjeldahl yielded correlation coefficients r=0.96 and 0.97, respectively, with slopes being close to 1. Interferences of glucose and thiol compounds at relatively low concentrations could be compensated for by selecting a lower alkaline pH (i.e., pH 10) at a cost of slightly reduced sensitivity and adding an identical amount of interferent to the reagent blank, respectively, since the absorbances due to BSA and interferent were additive. Thus a novel spectrophotometric method for total protein assay using a stable reagent and chromophore, which was simple, rapid, sensitive, flexible, and relatively selective, was developed, and applied to a variety of food products.

Protein–Incorporated Serum Total Antioxidant Capacity Measurement by a Modified CUPRAC (CUPRIC Reducing Antioxidant Capacity) Method

Due to inadequacies in analytical methodology of total antioxidant capacity (TAC) measurement, proteins are initially separated from the human serum matrix by precipitation and are left unmeasured, thereby causing an important “antioxidant gap.” The aim of this work is to measure the TAC of serum with the modified CUPric Reducing Antioxidant Capacity (CUPRAC) method, and to identify the contribution of serum proteins, especially thiol-containing proteins, to TAC. CUPRAC results were statistically compared to those found by reference methods, namely ABTS (2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid), FRAP (ferric reducing antioxidant power), and Ellman thiols assay. The curves of absorbance vs thiol concentration, as well as of absorbance vs diluted serum (whole serum, trichloroacetic acid (TCA)−precipitated and redissolved serum protein solution, and TCA supernatant fractions) volume, of three distinct serum samples showed excellent linearity and low intercept values only with the modified CUPRAC method. The proposed method will help characterize the “antioxidant gap” of serum TAC originating from protein components which should not be neglected in future antioxidant measurements.

Correlation of total antioxidant capacity with reactive oxygen species (ROS) consumption measured by oxidative conversion.

Although both antioxidant capacity and oxidative conversion (hazard) are important in food and bioanalytical chemistry, there is considerable confusion in the literature between the results of these two types of assays. After the generation of ROS in the medium via Fe(III)-H₂O₂ reaction, attenuation of total oxidative conversion (TOC; as measured by thiobarbituric acid-reactive substances (TBARS) and N,N-dimethyl-p-phenylenediamine (DMPD) assays) was tested for possible correlation with the total antioxidant capacity (TAC; as measured by cupric reducing antioxidant capacity (CUPRAC) and trolox equivalent antioxidant capacity (ABTS/TEAC) assays) of the introduced antioxidant sample. The inverse relationship between oxidative conversion and antioxidant capacity was processed to establish a curvilinear relationship between the absolute values of TAC increments and TOC decrements as a function of added antioxidant concentration. This simple relationship may form a bridge between the two diverse disciplines of medical biochemistry and food analytical chemistry mainly using TOC and TAC results, respectively.

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.

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.

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.

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

ABSTRACT Although reactive oxygen species can regulate intracellular signaling pathways, excessive amounts under oxidative stress conditions may cause damage to biomolecules. Hydroxyl radical (•OH) is a most reactive oxidant that can harm DNA, lipids, and proteins. As the direct determination of •OH 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, 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 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 scavenging ability.

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.