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Current issues in antioxidant measurement Re#at Apak J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b03657 • Publication Date (Web): 01 Jul 2019 Downloaded from pubs.acs.org on July 21, 2019

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Journal of Agricultural and Food Chemistry

Current Issues In Antioxidant Measurement Reşat Apak Istanbul University-Cerrahpasa, Faculty of Engineering, Department of Chemistry, Avcilar 34320 Istanbul-Turkey Turkish Academy of Sciences (TUBA), Piyade St. 27, Cankaya 06690, Ankara-Turkey E-mail: [email protected]

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Abstract

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The rationale and scope of the main issues of antioxidant measurement are presented,

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with basic definitions and terms in antioxidant research (such as reactive species and related

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antioxidative defenses, oxidative stress, antioxidant activity and capacity) in a historical

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background. An overwiew of technical problems and expectations are given in terms of

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interpretation of results, precision and comparability of methods, capability of simulating

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physical reality, and analytical performance (sensitivity, selectivity, etc.). Current analytical

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methods for measuring antioxidant and antiradical activity are classified from various

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viewpoints. Reaction kinetics and thermodynamics of current analytical methods are

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discussed, describing physico-chemical aspects of antioxidant action and measurement.

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Controversies and limitations of the widely used antioxidant assays are elaborated in detail.

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Emerging techniques in antioxidant testing (e.g., nanotechnology, sensors, electrochemistry,

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chemometry and hyphenated methods) are broadly introduced. Finally, hints for the selection

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of suitable assays (i.e., preferable for a specific purpose) and future prospects are given.

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Key words: Antioxidant activity, total antioxidant capacity, reactive species, antioxidant

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measurement, physical chemistry of antioxidants, current issues.

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1. Introduction (Background, scope and definitions)

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Antioxidants should be defined in a simple and understandable way familiar to all

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relevant communities (scientists, industrialists, engineers, clinicians, dieticians, technicians,

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customers, patients, consumers and stakeholders, etc.). In simple terms, an antioxidant is a

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substance inhibiting or delaying undesired oxidation reactions. This definition is closer to the

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most recent definition of Halliwell and Gutteridge1 because the relative magnitudes of

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concentrations of the antioxidant and its protected substrate is excluded from the original

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definition which had stated that antioxidant is “any substance that when present at low

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concentrations compared with those of an oxidizable substrate (every organic molecule found

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in vivo) significantly delays or prevents oxidation of that substrate”. Of course, the substrate

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undergoing oxidation, whatever its relative concentration, may be defined to fit our needs; it

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may be a food substrate (and the antioxidant counteracts its deterioration) or a biological

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macromolecule (lipid, protein, DNA, etc.), and the antioxidant protects the irreversible hazard

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on it by preventing its oxidative conversion via reactive species (reactive oxygen or nitrogen

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species, ROS or RNS, or collectively abbreviated as RONS). Shahidi2 defines antioxidants as

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substances that, when present in food, delay, control, or inhibit oxidation and deterioration of

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food quality (i.e. without imposing a limitation on the relative concentration of antioxidant).

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The use of antioxidants for preserving biological materials or food dates back to a long

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history. As molecular oxygen uptakes electrons in a stepwise manner to produce ROS in the

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respirative cycle, the capability to utilize ROS for cell signalling and regulation may have

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been the initial breakthrough in the evolution of complex aerobic life forms3, and the first

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cells should have developed antioxidant skills to protect themselves against oxidative damage.

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Ancient Egyptians used various antioxidant, antibacterial

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mummification, such as coniferous resin, mastic, myrrh, bitumen, beeswax, cassia, onions,

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lichen, and henna, to keep the moisture and bacteria away; of course these substances also 3 ACS Paragon Plus Environment

and antifungal substances in


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contained a great variety of polyphenols, organosulfur compounds, and terpenes as

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antioxidants to prevent oxidative deterioration of the corpse. Another historical example is the

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‘herby cheese’ produced for more than 200 years in the town of Van and nearby (East

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Anatolia, Turkey), known as ‘otlu peynir’ very famous in the region, in which the herbs

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mainly consisting of wild garlic (allium) species are incorporated in cheese as

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antioxidant/antimicrobial preservatives to be eaten under severe winter conditions when fresh

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vegetables are no longer available to the mountain villagers, raising the resistance of the

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population to diseases; the herbal ingredients of cheese also aid prevent the decomposition of

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proteins over a long period. Another miraculous soup base meal, ‘tarhana’, is a fermented,

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dried and powdered mixture of wheat flour, yoghurt, different vegetables (onion/garlic,

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paprika, tomato, etc.), yeast and spices. It was originally designed to safely store yoghurt for a

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long time. It was allegedly discovered in Central Asia, and later brought to Anatolia. The

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common theme behind all these durability examples is to keep the moisture away by salt and

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oil and to add special vegetables and spices (rich in phenolic antioxidants) to scavenge free

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radicals (responsible for deterioration) during the initial phase of storage. As early as 1901,

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the first preserving chemical for unvulcanized rubber, pyrogallol as a reducing agent (now

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recognized as an antioxidant) was found; this was followed by the discovery of various

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organo-sulfur and amine compounds between 1930-1950 used in the vulcanization of rubber

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to produce a durable cross-linked polymer to resist deterioration (due to ageing or

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atmospheric exposition).

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Even though ‘oxidative stress’, classified with respect to an intensity scale, i.e. from

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low to high levels,4 and defined as the imbalance in the redox status of the cell between

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physiological antioxidants and oxidants/prooxidants in favour of the latter or as a ‘disruption

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in redox signaling and control’5, is held responsible for the oxidative hazard on

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biomacromolecules (i.e., protein, lipid, DNA, etc.) and subsequent progression of certain 4 ACS Paragon Plus Environment

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diseases (e.g., cardiovascular and neurodegenerative diseases and some types of cancer),

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antioxidant measurement has not yet been a standard protocol parameter of clinical analysis

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regarding medical diagnosis and treatment. It has been a frequently asked question whether

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antioxidant measurements give useful hints for human health, good nutrition and well-being.

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Naturally the society is no longer at the expectation that antioxidants would be an essential

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remedy for all free radical oxidation-originated degenerative diseases, a thought that prevailed

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throughout the 1980s-90s, but still “a compelling case can be made for a role of radical-

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trapping antioxidants in the maintenance of good health and longevity”.6 The U.S.

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Department of Agriculture (USDA) removed its ORAC (oxygen radical absorbance capacity)

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database from the web in 2012 “due to mounting evidence that the values indicating

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antioxidant capacity have no relevance to the effects of specific bioactive compounds,

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including polyphenols on human health”. As the reason for withdrawal, USDA mentioned

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that in the course of preventing or ameliorating various oxidative stress−related chronic

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diseases, “the associated metabolic pathways are not completely understood and ORAC

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values are routinely misused by food and dietary supplement manufacturing companies”.7

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Moreover, a permanent categorization of antioxidants and oxidants as ‘good’ or ‘bad’,

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respectively, is not reasonable, because indiscriminate antioxidant supplementation may do

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more harm than good, while RONS perform such vital metabolic functions that removal of

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too many reactive species may distort cell signaling pathways and actually increase chronic

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disease risks8, further emphasizing the necessity of preservation of the miraculous balance of

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oxidants and antioxidants donated by mother nature to a healthy organism. Nevertheless, there

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is still a wide belief among health and nutrition scientists that antioxidants can prevent (if not

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cure) certain diseases originating from oxidative stress, and that they do it not singly but

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cooperatively, thereby giving credit for a ‘total antioxidant capacity’ (TAC) parameter. TAC

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is indicative of the collaborative (and usually additive) behaviour of all antioxidants present in



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a complex sample, enabling it to be recognized by leading researchers as a more useful

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parameter to assess the concerted action of food/plasma antioxidants.9 Antioxidants do not

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behave alone in vivo and act cooperatively in a magnificent network, e.g., glutathione can

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reduce dehydroascorbate to regenerate ascorbate (vitamin C) and ascorbate can in turn reduce

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α-tocopheroxyl radical to α-tocopherol (vitamin E) depending on their conditional redox

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potentials in biological media. In monitoring food quality and nutrition, antioxidants are

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analyzed for the meaningful comparison of foods in regard to their antioxidant content and for

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controlling variations within or between products. Because of the lack of a widely accepted

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total antioxidant activity/capacity parameter as a reference nutritional index for food and

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biological fluid labeling, researchers in the field are still in an attempt to develop and

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standardize novel methodologies to measure and compare the antioxidant abilities of food

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extracts and biological samples.

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One needs to distinguish ‘antioxidant capacity’ from ‘antioxidant activity’ (AOA).

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Antioxidant capacity has a thermodynamic definition concerning the oxidative conversion

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efficiency (linked to stoichiometry) of an antioxidant by an oxidizing agent, and is related to

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the equilibrium constant of this conversion. Antioxidant activity is mainly concerned with

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reaction kinetics, in that it reflects the rate of chemical oxidation of the tested antioxidant or

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the rate of quenching of a given reactive species by the antioxidant. Most ‘efficient’ assays

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are ‘fast’ at the same time, because the equilibrium constant of a reaction is defined as the

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ratio of the forward to backward rate constants, and a high rate constant of the forward

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reaction frequently implies a high equilibrium constant. Most electron transfer-based TAC

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assays rely on rapidly reacting reagents with antioxidant analytes, as redox reactions are

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generally known for their high equilibrium constants. As an example, p-coumaric acid is

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slowly oxidized by the CUPRAC reagent, i.e. Cu(Nc)22+, because of the small difference

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between the potentials of the oxidant and reductant, and at the same time, the TEAC (trolox 6 ACS Paragon Plus Environment

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equivalent antioxidant capacity) of p-coumaric acid as a measure of the oxidative conversion

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efficiency of this antioxidant is quite low. The units of antioxidant activity are usually lag

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times, percentage inhibition or scavenging relative to a reference compound, and finally

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reaction rate constants. On the other hand, antioxidant capacity corresponds to the

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thermodynamic conversion yield of reactive species by antioxidants, such as the number of

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moles of reactive species scavenged by one mole of antioxidant during a fixed time period.9 If

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trolox or gallic acid is taken as reference to express the antioxidant capacity of a test

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compound, the reducing ability of that antioxidant may be reported as its reference compound

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equivalents under the selected experimental conditions.

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In the investigation of antioxidative defense systems against reactive species, one

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needs to differentiate between RONS. The less toxic primary “reactive oxygen and nitrogen

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species” (RONS) are mainly composed of superoxide anion radical (O2•‒), hydrogen peroxide

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(H2O2) and nitrogen monoxide (NO•) which reversibly react with target biomolecules, while

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the more toxic secondary RONS, e.g., hydroxyl radical (•OH), peroxynitrite (ONOO‒) and

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hypochlorous acid (HOCl), are derived from the reaction of primary RONS with other species

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(such as the Fenton reaction giving rise to •OH from Fe(II) and H2O2, the Haber-Weiss

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reaction involving the conversion of O2•‒ into H2O2 and further to •OH, the formation of

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ONOO‒ from O2•‒ and NO•, or the formation of HOCl with an enzymatic reaction from H2O2

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and Cl‒) and may result in irreversible tissue damage. The primary RONS are quite

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controllable by superoxide dismutase (SOD), catalase and NO synthases (i.e., their levels in

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cells may be kept low by enzymes to minimize cytotoxicity), while the more toxic secondary

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species are much less controllable by antioxidative enzymatic defenses.10 The oxidation

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reactions realized by these primary and secondary RONS may give rise to different species,

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e.g., redox signaling by primary reactive species involves reversible oxidative conversions on

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biothiols (such as –SH oxidation in peroxiredoxins)11, whereas essentially irreversible 7 ACS Paragon Plus Environment


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oxidations by secondary species (such as peroxynitrite) on protein thiols may produce sulfinic

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(RSO2H) and sulfonic (RSO3H) acids.

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2. Overview of technical problems and expectations

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How should we interpret the results of antioxidant assays? Naturally, most antioxidant

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researchers, either from health/nutrition or food sciences, intend to extract maximal

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information from minimal investment of time and experimental work spent for antioxidant or

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radical scavenging assays. Actually, AOA assays are not ‘black box’ assays, as most people

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looking at analytical chemistry from a distance think, because one always needs to know what

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is being measured and how. Unfortunately, most AOA/TAC assays lack a detailed description

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of underlying initiators, targets, interaction of antioxidants among themselves or with oxidant

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species, reaction rates, pH, concentration and solvent effects, etc. Blind use of reagent kits

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without a clear idea on their outputs and mechanisms will often produce unscientific

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interpretation of results. If the protective action of antioxidants toward a biologically relevant

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substrate is measured, it is important to know which biomolecule (e.g., lipid, protein, DNA,

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nucleic acid, etc.) is protected from which reactive species.12 Certainly, there are approaches

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relating AOA testing essentially to the measurement of reaction kinetics of radical-trapping

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antioxidants with peroxyl radicals against lipid peroxidation;13 however, measuring the

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defense against lipid peroxidation does not stand as the only authentic assay of AOA, and

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there are other (equally valid) biomarkers. If total antioxidant capacity (TAC) is measured

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with the aid of a simulated assay in which antioxidants transfer hydrogen atoms or electrons

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to an oxidizing reagent, there are concerns that it may not reflect the biologically meaningful

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capacity to retard or suppress oxidation.14

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As opposed to the analytical determination of single analytes (i.e., specific elements or

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compounds) in different laboratories which should always yield the same result (with an 8 ACS Paragon Plus Environment

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acceptable error margin), no two antioxidant assays may give the same result for a test

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compound or sample because each assay has its own thermodynamics and kinetics, meaning

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that the same antioxidant would reduce or quench the oxidizing reagents or radicals of

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different TAC assays to different extents.15 Since the results of no two assays (e.g, even of the

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same radical reagent such as ABTS, DDPH or DMPD, prepared or reacted in different ways)

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may be identical, it is more meaningful to get a close rank of antioxidant effectiveness with

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different assays of wide use. Experimental efficacy rankings of antioxidants found with a

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selected antioxidant assay should also comply with theoretical structure-activity relationships

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of antioxidants. Consequently, in spite of the fact that the expert review panel on strategic

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food analytical methods has defined various analytical parameters for the evaluation of total

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antioxidant activity (such as limits of detection and quantification, repeatability precision,

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reproducibility and recovery factor), one should be aware of the fact that a statistical Student’s

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t-test comparison of the mean TAC values of two different assays carried out on the same

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sample is not possible. There are other ways of comparing results, e.g., the increase in

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oxidative status of a food or biological matrix subjected to RONS was correlated with the

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decrease in the antioxidant stock of the test medium.16 In addition, TAC/AOA may be

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correlated to the electrochemical properties (such as redox potential) of antioxidants. On the

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other hand, some researchers find the statistical comparisons among similar mechanism

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assays reduntant as they consider all electron transfer (ET)−based assays in an inherent

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correlatability,17 though one may find exceptions to this argument.

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How good do the assays simulate physical reality? In measuring AOA as the defense

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against reactive species, a frequently asked question is whether the reactant radicals truly

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represent biologically relevant radicals. Unfortunately, the most widely used reagents for the

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estimation of ‘free radical scavenging capacity’ (such as ABTS, DPPH and DMPD) do not

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meet these requirements. In addition, the biologically important radicals (such as •OH, O2•9 ACS Paragon Plus Environment


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and ROO•) that are used for AOA estimation are generated in excessive quantities and rates

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for such assays, which do not truly reflect the more temperate physiological conditions of

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oxidative conversion of biomacromolecules. Another requirement is that the concentration of

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antioxidant should be significantly low relative to that of the substrate it protects, as

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encountered under in vivo. As physical reality cannot be duplicated but reasonably simulated,

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it may be recommended that in vitro assays (especially those measuring antioxidative

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protection against lipid peroxidation) rely on the principle of ‘multifunctionality’. Since

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natural antioxidants behave in a multifunctional manner (e.g., due to the variability in system

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composition, types of oxidizable substrates, media of initiation and promotion of oxidation), a

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reliable antioxidant protocol necessitates the simultaneous measurement of a number of

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properties (e.g., radical scavenging, electron- and proton-transfer, phase distribution

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equilibria, etc.) pertaining to food and biological systems.18 Multifunctional studies may also

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involve the evaluation of several other factors in addition to the reactivity toward RONS, such

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as concentration, distribution, localization, fate of antioxidant-derived radical, interaction with

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other antioxidants, and metabolism. Versatility is another requirement to be considered

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together with multifunctionality. For example, the CUPRAC assay, originally developed to

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measure the reducing ability of antioxidants, later evolved into a ‘train of assays’ capable of

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measuring various reactive species together with the ability of antioxidants to scavenge those

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species (i.e., with the aid of carefully selected probes which respond to CUPRAC colorimetry

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initially or after reaction with antioxidants).9 Authenticity of simulating physiological

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antioxidant

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potential and pH values. Although it may seem at first glance that the influence of pH on a

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simulated AOA assay would not be more decisive than the actual differences of the selected

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assay conditions from physiological ones, it should be kept in mind that the efficacy of an

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antioxidant depends on the nature of the oxidant,19 which is not always a peroxyl or lipid

action in indirect assays obviously requires the selection of relevant redox


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radical, and each antioxidant shows different antioxidant activity against different radical

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forms.20 For example, except Folin−Ciocalteau phenolics assay having an indefinite redox

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potential, the widely used ET reagents have standard redox potentials in the useful range of

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0.6−0.7 V relevant for food and biological antioxidants.9 Among ET assays, the CUPRAC

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test uses an ammonium acetate buffer (at pH 7) closest to the physiological pH. If the

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TAC/AOA assay is carried out at an unrealistic pH (i.e., either too alkaline as in Folin-

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Ciocalteau where phenolics are proton-dissociated and become more vulnerable to oxidation,

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or too acidic as in FRAP not enabling full oxidation of free phenolics within the fixed

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protocol time of the assay), one may not get a clear idea of antioxidant action extrapolated to

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in vivo conditions.

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In accordance with the principle of multifunctionality, assays combining the

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measurement of oxidative status and AOA may serve the purpose better than pure antioxidant

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assays, since these techniques measure the oxidative hazard on a biologically relevant probe

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brought about by RONS, and the capacity of antioxidant defenses preventing or relieving this

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hazard. For example, when DNA was subjected to Fenton oxidation, the degradation products

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of DNA but not the original macromolecule was responsive to CUPRAC colorimetry;

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antioxidants, when present, scavenged hydroxyl radicals and mitigated both the DNA damage

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and subsequent CUPRAC absorbance of DNA degradation products. Moreover, the DNA

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damage could be electrochemically detected with differential pulse voltammetry (DPV) as

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reflected in the changes of the guanine oxidation signal in the absence or presence of

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antioxidants.21

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The analytical performance (e.g., sensitivity, linearity, selectivity) of the assay is

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important for precise and accurate measurement. Antioxidant researchers would normally

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prefer a perfectly linear response against concentration in each AOA/TAC; unfortunately to

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get a linear response (signal vs antioxidant concentration) is rarely possible. If a single 11 ACS Paragon Plus Environment


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chromophore emerges at the end of the ET reaction of a TAC reagent with the antioxidant,

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and this chromophore does not exhibit association, dissociation or further interactions with

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solvent molecules, then we may expect strict obedience to Beer’s law within a reasonable

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concentration range to get linear absorbance signals. The actual rate of inhibited autoxidation

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and lag time may linearly correlate with antioxidant concentration. In this regard, Pinchuk et

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al.22 proposed a linear correlation of lag time with antioxidant concentration, but their

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experimental data for ranking antioxidants based on their retardation effect on the

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peroxidation of human serum lipids23 did not exhibit full linearity. If several reactive species

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attack an absorptimetric or fluorometric probe, then there will emerge more than one product

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which will not obey Beer’s law. If TAC measurement is made at the end of an insufficient

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period of a fixed-time assay (i.e., most slow-reacting antioxidants are not fully oxidized

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within the protocol time of the assays under non-equilibrium conditions), then one may not

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get a linear relationship either. The ORAC assay, calculating antioxidant concentration via the

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difference between ‘area-under-curve’ (AUC) values of time-dependent fluorescence decay

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curves in the absence and presence of antioxidants, may yield nonlinear (polynomial)

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relationships with concentration.24 Electrochemical AOA assays operate under non-

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equilibrium conditions (because a current is passed while the measurement is made), so it is

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even harder to get linear responses. Electrochemical assays making single-potential

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measurements have a better chance of linearity than those measuring current within a

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predefined potential range (i.e., as in the AUC measurements of voltammetric curves), but in

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the former case, a universal TAC assay cannot be developed because each antioxidant has a

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separate redox potential.

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In general, absorptimetric probes give more linear responses to antioxidants than

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fluorometric or chemiluminometric ones, although better sensitivities may be achieved with

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the latter probes. Fluorescence quenching techniques generally yield a lower selectivity for 12 ACS Paragon Plus Environment

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antioxidants than fluorescence producing ones. Also, methods based on new absorbance or

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fluorescence formation are generally more sensitive than those relying on existing

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chromophore-bleaching or fluorophore-quenching, respectively, because of the high initial

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background color or fluorescence of the latter probes (which would eventually yield higher

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standard deviations for reagent blanks). Selectivity toward antioxidants is closely related to a

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thorough analysis of possible adverse effects (interferences), which should be properly stated

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(e.g., in free radical scavenging techniques, the used probes may be partially inaccessible to

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the antioxidants due to steric hindrance or there may be species such as activators or

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inhibitors, other than the target antioxidants, which aid to generate or quench these radicals,

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and their effects to the test system should also be considered; in enzymatic assays, there may

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be numerous species affecting the activity of enzymes, etc.). Possible synergistic-antagonistic

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interaction of antioxidant constituents among themselves, or prooxidant action of antioxidants

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usually in the presence of free or weakly-complexed transition metal ions should also be

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considered. If a TAC assay reagent, when reduced by the antioxidant, can undergo redox

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cycling with hydrogen peroxide or dissolved oxygen (i.e., a situation seen in Fe(III)-Fe(II)

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reduction based assays), this means that new reactive species may be generated in the assay

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causing negative errors.

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As for the selectivity of oxidative stress biomarkers, RONS in the organism can either

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be trapped (and the trapped molecules are measured) or the amount of oxidative damage

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performed by RONS is measured. Antioxidants can only be measured indirectly in such

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systems through their attenuation effect on oxidative damage of selected biomarkers. While

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testing in vivo antioxidant capacity, e.g., in antioxidant supplementation trials to improve

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clinical outcomes, it was recommended to use appropriate biomarkers that have been

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standardized by comparison of data collected in different laboratories.25 In making

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measurements with the widely used biomarker probes of oxidative stress, the most important 13 ACS Paragon Plus Environment


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interference comes from artifactual sources such as food or other metabolic processes which

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may cause confusion in the interpretation of results, as summarized in a comprehensive

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review by Halliwell & Whiteman,26 e.g., (i) among the dihydroxybenzoate isomers produced

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under •OH attack from a salicylate probe, 2,5-dihydroxybenzoate may derive from the

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catalytic activity of liver microsomes; (ii) urate as probe nonspecifically reacts with many

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RONS and its rise in blood plasma may be associated with kidney disease (hyperuricemia);

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(iii) malondialdehyde (MDA) in urine may be confounded by diet and may not indicate whole

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body lipid peroxidation

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(iv) while using a DNA probe, 8-hydroxy-2’-deoxyguanosine (8OHdG) levels may rise not

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only from oxidative damage to DNA but from other operations such as sample isolation and

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preparation for analysis; (v) the carbonyl assay presumably indicating oxidative protein

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damage may also respond to the binding of sugars or aldehydes to proteins.26

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3. Classification of measurement methods

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In order to prevent/retard undesired oxidation reactions and relieve oxidative stress, an

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antioxidant may act directly as a scavenger of reactive species (RONS) or as an inhibitor of

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their generation. Primary antioxidants sacrificially act by breaking the chain reactions

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involved in lipid peroxidation through inhibition of radical initiation −upon reaction with a

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lipid radical (L•)− or of radical propagation upon reaction with a lipid peroxyl (LOO•) or

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alkoxyl (LO•) radical. There are two chain-propagating steps, namely the fast LOO• formation

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from L• and O2, followed by the relatively slow hydroperoxide (LOOH) formation from LOO•

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and LH to generate new lipid radicals (L•). Radical trapping antioxidants (AH) can terminate

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the chain reaction by reacting with LOO• to convert it to LOOH and an antioxidant radical

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(A•), which may further degrade LOO• to non-radical products. Secondary antioxidants

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prevent Fenton-type oxidation reactions of biomacromolecules by either decomposing 14 ACS Paragon Plus Environment

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peroxides and/or hydroperoxides or by chelating free or weakly-complexed transition metal

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ions, where a Lewis base (such as a phenolic antioxidant) neutralizes a Lewis acid (metal ion)

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by covalently donating an electron-pair to the metal. There is also the possibility of its indirect

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action, e.g., via up-regulation of endogenous antioxidative defenses, control of gene

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expression of oxidative enzymes,12 and change of cell signaling.27 This perspective look is

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mainly aimed at methods for measuring chain-breaking non-enzymatic antioxidants which

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operate by deactivating reactive species through electron or H-atom transfer. The most widely

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used antioxidant assays, measuring the capacity or activity of antioxidants in deactivating

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reactive species, may be broadly classified as in vitro and in vivo, enzymatic and

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nonenzymatic, electron transfer (ET)− and hydrogen atom transfer (HAT)−based,17 direct and

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indirect assays, with thin borders between classes and subgroups. The mechanisms of direct

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and indirect assays are schematically shown in Figure 1. In a direct (competitive) assay

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(Figure 1-I), both the probe and the antioxidant are oxidized by the simultaneously generated

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reactive species, whereas in an indirect (noncompetitive) assay (Figure 1-II), the probe is

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reduced by the antioxidant in a simulated reaction. ET may be coupled to proton transfer

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reactions that may occur in a stepwise manner. Aside from the excellent papers of Prior et

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al.28 and Huang et al.,24 some recent reviews on the chemistry and mechanisms of antioxidant

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assays comprise the works of Shahidi & Zhong, Amorati & Valgimigli, Apak et al.2,9, 29–31

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Niki14 recommended that the antioxidant capacity of test compounds and their

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complex mixtures should be evaluated from their effect on the levels of plasma lipid

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peroxidation in vitro and of biomarkers of oxidative stress in vivo. Likewise, Pinchuk et al.22

342

considered the kinetic study of ex vivo peroxidation of lipids as the most relevant

343

characteristic of oxidative stress in the biological context, and proposed to quantify

344

antioxidant efficiency in terms of double-extension of the ‘lag time’ preceding lipid

345

peroxidation (i.e., observed in the absence of antioxidants). It has been established that lipid 15 ACS Paragon Plus Environment


Page 16 of 57

346

radicals, formed from RONS upon extraction of a H-atom from lipid side-chains, can react

347

with oxygen to form a peroxyl radical, which in turn extract further H-atoms from

348

surrounding molecules, thereby triggering a chain reaction of lipid peroxidation. Oxidized

349

polyunsaturated fatty acids (PUFA) are post-facto biomarkers of oxidative damage; in

350

addition to the radical chain reaction, PUFAs are susceptible to the attack of singlet oxygen

351

constituting a direct reaction of minor lipid oxidation pathway. However, there are

352

biomacromolecular probes other than lipids that can serve as oxidative stress biomarkers; for

353

example, proteins may undergo cross-links, peptide cleavage and oxidative modification of

354

their amino acids, DNA/RNA may show base-free sites, deletions, base modifications, frame

355

shifts,

356

carbohydrates may convert to aldehydes and ketones (e.g., glycolaldehyde and α- and β-

357

dicarbonyls), upon exposure to reactive species.32 As a widely used biomarker of oxidative

358

stress, the thiobarbituric acid (TBA) assay (basically measuring the lipid peroxidation end-

359

product MDA as a marker of aldehydes and ketones via formation of a colored compound

360

with TBA) was later found to be unacceptable in modern research by Halliwell &

361

Whiteman,26 because most TBA-reactive material in human body fluids is not related to lipid

362

peroxidation. MDA is one of the major aldehydes stemming from the breakdown of lipid

363

hydroperoxides, however it can also arise from free radical attack on sialic acid and

364

deoxyribose.1 MDA is basically formed from polyunsaturated fatty acids (PUFAs) with three

365

or more double bonds, e.g., MDA formation from linoleic acid has a very low yield whereas a

366

high yield from docosahexaenoic acid.33 TBA reacts not only with MDA but also with many

367

other compounds (e.g., carbohydrates, pigments, amino acids, pyridins, etc.), and its

368

selectivity for lipid peroxidation can be improved by isolating the TBA-MDA chromogenic

369

adduct with the use of reversed phase-HPLC before analysis. It should be emphasized that the

370

adoption of peroxidated lipids as the unique relevant oxidative biomarker does not seem

strand

breaks,

DNA–protein

cross-links,

and


chromosomal

arrangements,

Page 17 of 57


371

justified, and there are quite a number of possibilities in this regard with a wide spectrum of

372

oxidative conversion products, depending on the purpose (e.g., diagnosis and treatment of

373

certain oxidative stress−originated diseases). Needless to say, no one single conversion

374

species as oxidative stress biomarker (e.g., 8-OHdG, 8-hydroxyadenine, 5-hydroxyuracil,

375

MDA, thiobarbituric acid reactive substances (TBARS), acrolein, 4-hydroxy-2-nonenal

376

(HNE), conjugated dienes, F2 isoprostanes, lipid hydroperoxides (LOOH), protein carbonyls

377

and nitrotyrosine, etc.) can be the focal parameter of oxidant/antioxidant activity research,

378

serving all purposes.

379

In summary, HAT−based AOA assays generally rely on the scavenging reaction of

380

peroxyl radicals (ROO•) by a relevant biomolecular substrate (AH) in competition with an

381

antioxidant (such as a phenolic: ArOH) in accordance with the H-atom transfer reaction:

382

ROO• + AH/ArOH → ROOH + A•/ArO•

383

where the antioxidant should react faster with ROO• than the biomolecule protected. The

384

leading HAT-based assays comprise ORAC: oxygen radical absorbance capacity,34 TRAP:

385

total peroxyl radical-trapping antioxidant parameter,35 crocin bleaching, and β-carotene

386

bleaching assays.24 On the other hand, ET−based assays make use of simulated reactions in

387

which reactive species attacking antioxidants are replaced by oxidizing probes. The radical

388

scavenging reactions of antioxidants are assumed to take place by electron transfer coupled to

389

proton transfer:9

390

ROO• + AH/ArOH → ROO− + AH•+/ArOH•+

391

AH•+/ArOH•+ + H2O → A•/ArO• + H3O+

392

In ET−based spectroscopic assays, the chemically reduced probe is spectrally distinguishable

393

from its initial form; either the probe to which an antioxidant transfers an electron is 17 ACS Paragon Plus Environment


to

a

colored/fluorescent/chemiluminescent

species

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394

converted

or

the

initial

395

absorbance/fluorescence is quenched after reaction with the antioxidant. The most widely

396

used ET−based spectrophotometric TAC methods are Folin−Ciocalteau,36 ABTS/TEAC (2,2′-

397

azinobis(3-ethylbenzothiazoline-6-sulfonic acid/Trolox equivalent antioxidant capacity),37

398

DPPH (2,2-diphenyl-1-picrylhydrazyl),38 CUPRAC (cupric reducing antioxidant capacity),39

399

FRAP (ferric reducing antioxidant power),40 ferricyanide, and Ce(IV) reduction assays. Direct

400

assays are competitive in which the generated reactive species simultaneously attack the

401

probe and antioxidant, whereas indirect asssays are noncompetitive, because biological redox

402

reactions are simulated on an artificial probe, the structural changes of which are monitored

403

spectroscopically.

404

4. Physico-chemical aspects of antioxidant action and measurement

405

Certain thermodynamic parameters such as bond dissociation enthalpy (BDE) and

406

ionization potential (IP) of an antioxidant are very important in defining a potent antioxidant.

407

For example, the IP of an antioxidant (AH) should be high enough to prevent a proton-

408

coupled electron transfer to molecular oxygen (i.e., AH + O2 → [AH•+ + O2•-]), while in order

409

to be effective in chain termination, the BDE of AH should be low (i.e., ROO• + AH →

410

ROOH + A•).41 In fact, the relative magnitudes of BDE and IP may determine whether the

411

HAT or ET mechanism is predominant for a given phenol (PhOH).9 Kinetic solvent effects

412

should also be considered. For example, for common antioxidants (such as phenols) acting on

413

a HAT basis, the antioxidative ability (in terms H-atom abstraction rate from AH by a radical)

414

is strongly attenuated in hydrogen-bonding solvents42 because of steric inaccessibility of H-

415

bonded (PhOH…solvent) complexes.43 In investigating phenols oxidation with DPPH radical

416

in MeOH, the rate constants exceeded expected values, and were further increased or

417

decreased by the addition of methoxide or acetic acid, respectively, indicating a SPLET

418

(sequential proton-loss electron-transfer) mechanism, in which phenol deprotonation to 18 ACS Paragon Plus Environment

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419

phenoxide anion is followed by fast electron-transfer from phenoxide to the electron-deficient

420

radical; SPLET is enhanced in alkaline pH and favored by moderately acidic phenols in polar

421

solvents (e.g., methanol, ethanol, water) that support phenol ionization.43 SPLET is believed

422

to play a major role in biological media supporting ionization. SPLET was shown to be

423

effective –in addition to HAT mechanism– even for low-acidity tocopherols and chromanols

424

in a heterogeneous system of dispersed lipid micelles, because the ionization of even a very

425

small fraction of the phenolics increased the observed inhibition rate constant of methyl

426

linoleate peroxidation.44 Different types of proton-coupled electron transfer (PCET)

427

mechanisms were shown to operate in aqueous medium for less and more electron-rich

428

phenols; a SPLET-like mechanism was shown to be effective for trolox and 4-

429

methoxyphenol, which consists of the acidic dissociation of phenols before their trapping of

430

peroxyl radicals during inhibited autoxidations in water-THF mixtures.45

431

In electron transfer (ET) (or PCET: proton coupled electron transfer) assays of

432

antioxidant measurement, one needs to differentiate between inner- and outer-sphere ET

433

assays, as certain inner-sphere assays are strongly affected by the size and geometry of

434

reagents and antioxidants, and bulky antioxidants may not diffuse into the electron transfer

435

sites of the reagents giving rise to slow kinetics (i.e., the redox reaction may not go to

436

completion within a prespecified time). On the other hand, hexacyanoferrate(III) and

437

bis(neocuproine)copper(II) chelate type oxidizing reagents mainly act as outer-sphere electron

438

transfer agents which exhibit faster kinetics simply because the central metal ion of the

439

oxidant chelate (i.e., Fe(III) or Cu(II)) is coordinatively saturated and does not have to make

440

new bonds with the antioxidant substrate during the uptake of its electron. The mechanistic

441

difference in the degree of bonding in the precursor complexes immediately preceding the

442

transition states may greatly affect the ET rate constant and its dependence on the driving

443

force, temperature, and solvent.46 In order to have a fast electron transfer, the oxidized and 19 ACS Paragon Plus Environment


444

reduced forms of the coordination complex used as oxidizing agent for antioxidants should

445

not have a strong requirement for geometric rearrangement of the transition state (e.g., as is

446

valid for bis(neocuproine)copper(II,I) and hexacyanoferrate(III,II) redox couples). It should

447

be noted here that bulky radical reagents (such as ABTS and DPPH) may also encounter

448

accessibility difficulties during their reaction with multiphenolic antioxidants, which result in

449

incomplete electron transfer during the protocol time of the assay.47,48

450

Localization of antioxidants at phase boundaries is also important for antioxidant

451

action, a phenomenon known as the ‘polar paradox’.49 According to this hypothesis, polar

452

(hydrophilic) antioxidants in bulk oils (as the continuous phase) basically concentrate at the

453

oil-air interface and become more active than nonpolar antioxidants; on the other hand, in oil-

454

in-water emulsions (as dispersed system), nonpolar antioxidants better concentrate at the

455

oil/water interface and become more potent inhibitors of peroxidation than polar analogs. It

456

was shown over the years that lipophilized antioxidants designed in accordance with this

457

hypothesis were not always superior over their nonlipophilized analogues in emulsified and

458

liposomial systems because of the complexity of real systems not fully taken into account by

459

the hypothesis.50 The polar paradox phenomenon may be important in the antioxidative

460

preservation of food emulsions, and should be taken into account in AOA assays carried out

461

in emulsions. Surfactants and polysaccharides may partly solve solubility problems

462

encountered in AOA assays, but they also interfere with the redox reaction on which the assay

463

is focused (e.g., methylated cyclodextrins enhance the solubility of nonpolar antioxidants in

464

aqueous media, but they also form inclusion complexes with antioxidants such that the test

465

compound should first be released from this complex to undergo oxidation). Alternatively,

466

microemulsion systems were proposed as ideal solutions to the mentioned problem, especially

467

in studying the defense against lipid peroxidation, as they can accommodate both lipophilic

468

and hydrophilic antioxidants.51 Solubility problems may sometimes be relieved by incubating 20 ACS Paragon Plus Environment

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469

insoluble matrix constituents with redox reagents (i.e., known as “QUENCHER” methods) to

470

aid solubility equilibria with the predominant redox equilibria to facilitate “redox-coupled

471

dissolution” reactions. With the aid of this approach, one may be able to get a higher

472

antioxidant response from a sparingly soluble polyphenol in the selected solvent medium

473

containing the oxidant and simultaneously to skip certain tedious pre-separations and

474

extractions prior to AOA measurement.20 This approach is expected to be useful in the

475

foreseeable future, as security concerns of world nations may arouse interest in the analysis of

476

ammunition preserving antioxidants used in plastic bonded explosive formulations, where

477

AOA determination should be performed without fully solubilizing the matrix.

478

Synergism and antagonism may be defined as significant positive and negative

479

deviations, respectively, from the expected responses (calculated by assuming additivity of

480

responses) of mixtures to a given test. A good example of synergism was observed in the

481

inhibition of lipid oxidation by butylated hydroxyanisole (BHA) and butylated

482

hydroxytoluene (BHT) mixtures. Kurechi & Kato52 identified the mechanism of this

483

synergism between the two synthetic antioxidants by performing a DPPH assay: the rate of

484

decrease of DPPH concentration by BHA was ≈ 450 times that by BHT individually.

485

However in a binary mixture, DPPH oxidation of BHT was greatly accelerated, because BHA

486

was rapidly oxidized to its phenoxy radical first, and then this radical reacted with BHT to

487

regenerate BHA and form the BHT phenoxy radical (then transforming to a quinone-methide

488

intermediate of BHT), enabling enhanced oxidation of BHT in admixture compared to that of

489

lone BHT.52 Thus, in a fixed-time DPPH assay, both BHA and BHT exhibited 1-trolox

490

equivalent (or 2 H-atom donating) antioxidant capacities. Çelik et al.53 observed a similar

491

synergy with the CUPRAC method in (BHT + BHA) or (BHT + TBHQ) mixtures. Actually, a

492

hierarchic order (i.e., pecking order) of antioxidant consumption related to their radical-

493

scavenging capability can be predicted, on the basis of comparing one-electron reduction 21 ACS Paragon Plus Environment


494

potentials of the scavenged reactive species with the corresponding reduction potentials of

495

aryloxy/phenol redox couples. Despite their different distributions between lipid membrane

496

and aqueous phases, the fact that vitamin C (ascorbic acid) can physiologically regenerate

497

vitamin E (α-tocopherol) can be interpreted with the aid of reduction potentials of α-

498

tocopheroxyl (0.50 V) and ascorbate (0.282 V) radicals, because α-tocopheroxyl radical can

499

oxidize ascorbate and is itself converted back to α-tocopherol.54 By similar reasoning,

500

quercetin and catechin derivatives found in tea, showing reduction potentials of the

501

corresponding aryloxy radical/phenol couple less than 0.4 V, can thermodynamically

502

regenerate α-tocopherol from α-tocopheroxyl radical (aside from kinetic considerations).

503

Antioxidants may form synergistic pairs as reflected in their inhibition periods of lipid

504

peroxidation; this synergism can be quantified by the number of radical chains terminated, but

505

is complicated by phase distribution equilibria because some antioxidants do not display

506

distinctly measurable inhibition periods due to their non-uniform distribution between phases.

507

For example, ascorbate is an excellent peroxidation inhibitor in the aqueous phase but a poor

508

inhibitor in the lipid phase; however, ascorbate is a synergistic inhibitor of lipid peroxidation

509

toward both α-tocopherol and trolox.55 The collaborative action between ascorbate and α-

510

tocopherol in preventing lipid oxidation is perhaps the best-known example of synergism

511

donated by the design of nature, since the regeneration of α-tocopherol (α-TOC) by reduction

512

of α-tocopheroxyl radical (α-TOC•) by ascorbate prevents tocopherol-mediated peroxidation,

513

and effectively converts a water-soluble reductant to a lipid-soluble one,56 because ascorbate

514

(and also ubiquinol) can reduce α-TOC• to generate α-TOC before α-TOC• attacks lipids to

515

induce peroxidation. On the other hand, uric acid, another potent reducing agent and radical

516

scavenger in the aqueous phase, cannot reduce α-TOC• and spare α-TOC in the lipid phase,

517

showing the importance of the site of free radical generation on the expected synergistic

518

antioxidant action.14 In this regard, Valgimigli et al.56 developed the kinetic and 22 ACS Paragon Plus Environment

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Page 23 of 57


519

thermodynamic rationale for the design of highly reactive 3-pyridinols and 5-pyrimidinols as

520

N-atom bearing phenolics (ArOH) to act in synergistic pairs with less reactive phenols,

521

because these tailor-made compounds rapidly reacted with peroxyl radicals (ROO•) to be

522

converted to the corresponding aryloxyl radicals (ArO•), while less reactive phenols (such as

523

BHT) regenerated ArOH by acting as co-antioxidants to reduce ArO• back to ArOH before

524

ArO• reacted with a second ROO•.

525

The moderation of the electron-richness of phenols via inclusion of one or more

526

heteroatoms in the aromatic ring proved to be a good strategy for enhanced radical-trapping

527

activity.57 Thiol-phenol synergism was visible in peroxidations carried out in biphasic

528

systems. In regard to antioxidant activity against azo-initiated peroxidation of linoleic acid

529

both in homogeneous solution and in a biphasic system, the tellurium analogue of BHA

530

showed an extraordinary reactivity toward peroxyl radicals which was attributed to the octyl-

531

Te group located in ortho-position to the phenolic OH, and additionally, this BHA analogue

532

was regenerable in chlorobenzene-water in the presence of stoichiometric amounts of N-

533

acetylcysteine as a synergistic co-antioxidant in the aqueous phase.58 Likewise, phenolic 2,3-

534

dihydrobenzo[b]selenophene antioxidants bearing an OH-group in ortho-position to the Se-

535

atom quenched peroxyl radicals more efficiently than α-tocopherol by providing much longer-

536

lasting antioxidant protection, especially in the presence of thiol co-antioxidants (such as N-

537

acetylcysteine, glutathione and dithiothreitol) with which inhibition periods increased more

538

than five-fold.59

539

In thiol oxidations and similar reactions of thiol-disulfide interchange, various factors

540

affect the results such as acidity (pKa) of the thiol groups, nucleophilicity of the attacking

541

thiol, and stability of the leaving thiol group; as side reactions, 2-electron thiol oxidations to

542

disulfides through sulfenic acid (RSOH) intermediates, or thiyl (RS•) radical oxidations with

543

molecular oxygen to yield thiyl peroxyl radicals (RSOO•) are also possible, giving rise to 23 ACS Paragon Plus Environment


544

higher oxidation states. Ion-pairing, dipole-dipole interactions and hydrogen bonding can also

545

contribute to the nucleophilicity of the reacting thiol/thiolate, e.g., intermolecular H-bonding

546

can alter the macroscopic acidity constants (pKa) of the concerned thiols in mixtures, giving

547

rise to a greater relative abundance of thiolate responsible for fast reaction. Electron-donating

548

functional groups (such as those of phenolic antioxidants) may increase the nucleophilicity or

549

stabilize the transient state in thiol oxidations, thereby greatly increasing the reaction rates.60

550

All these factors, and even thiol-quinone adduct formation,61 may be responsible for the

551

observed overoxidation of thiols (i.e., though regarded as synergism, thiols may be

552

irreversibly oxidized up to the level of biologically irrelevant products) in complex mixtures

553

subjected to conventional antioxidant assays of ET nature. Since maintenance of

554

thiol/disulfide homeostasis in cellular systems is considered as a reliable wellness indicator

555

(e.g., GSH/GSSG ratio is an oxidative stress parameter), the lack of distinct spectroscopic

556

signatures of various thiol derivatives as well as the diversity of chemical factors that affect

557

the reaction rates pose challenges for future kinetic investigations60 that may enable more

558

precise determination of antioxidant activity of biological fluids and complex thiol-phenol

559

mixtures (including protein thiols).

560

Sulfenic acids are important biological antioxidants taking part in cellular signaling

561

related to redox homeostasis, via the formation of protein-disulfide linkages. Direct

562

measurements of the reaction kinetics of sulfenic acids showed difficulties (excluding a

563

persistent derivative, 9-triptycenesulfenic acid) owing to their high reactivity.62 Furthermore,

564

the high radical-trapping activity of alkyl sulfoxides and thiosulfinates can be attributed to the

565

formation of potent but transient sulfenic acids which may not be isolated in certain cases due

566

to rapid self-condensation.63 In the kinetic investigation of sulfenic acid radical-trapping

567

agents (e.g., taking part in the oxidation of organosulfur compounds from garlic and other

568

allium species), the H-atom transfer between a sulfenic acid and a peroxyl radical via proton24 ACS Paragon Plus Environment

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569

coupled electron transfer (PCET) was predicted to involve the initial formation of a hydrogen-

570

bonded complex, RSOH···•OOR’ resulting in a slightly lower-energy transition state and a

571

diffusion-controlled reaction with a very high rate constant, rendering sulfenic acids probably

572

the most potent of all peroxyl-radical trapping antioxidants.64 A lipophilic sulfenic acid acting

573

as a potent radical-trapper in lipid biyalers may synergistically interact with a water-soluble

574

thiol that may be hypothesized to play an important role in the in vivo control of oxidative

575

stress via regeneration of lipid-soluble/protein-bound sulfenic acids.63

576

Since antioxidants may function as prooxidants at certain conditions and

577

concentrations, prooxidant properties (i.e., enhancing lipid peroxidation or causing an

578

increase in certain oxidative stress biomarkers) should be studied along with antioxidant

579

activity of complex samples so as to assess possible health beneficial effects. As prooxidants

580

are more related to oxidative stress than antioxidants, biological and medicinal chemists have

581

a stronger tendency to investigate prooxidant activity rather than AOA; for example,

582

regarding flavonoids, their antioxidant efficacy is less documented than their prooxidant

583

properties in vivo. Especially ascorbic acid and α-tocopherol, and to a lesser extent

584

carotenoids and catechin group flavonoids, may act as prooxidants by reducing transition

585

metal ions (especially of iron and copper) to lower oxidation states, thereby inducing Fenton-

586

type oxidation reactions that may result in tissue damage. Prooxidant activity has been

587

assumed to be proportional to the total number of hydroxyl groups (preferably in the B-ring)

588

in a flavonoid molecule; evidence also exists in favour of the 2,3-double bond and 4-oxo

589

arrangement of flavones in promoting ROS formation in the presence of Cu(II)+O2.65 In

590

general, the more readily oxidizable catechol-group flavonoids were the most effective

591

prooxidants (e.g., in regard to their semi-quinone radicals that could generate ROS

592

responsible for DNA damage), because the lower redox potential catechols could be more

593

easily oxidized by peroxidase/H2O2.66 As the Cu(II)-reducing abilities of phenolics could be 25 ACS Paragon Plus Environment


594

correlated to their prooxidant properties (via ROS generating ability of the protein-bound

595

Cu(I) reduction product), a recent colorimetric nano-sensor using this theme was prepared by

596

Akyüz et al.67 using chicken egg white protein–protected gold nanoclusters (CEW-AuNCs) to

597

measure the Cu(II)-induced prooxidant activity of antioxidants, on which Cu(II) was reduced

598

to Cu(I), and the CEW-thiol bound Cu(I) was released with neocuproine, enabling an single-

599

pot prooxidant activity assay without tedious separations.

600

5. Controversies and limitations of widely used assays

601

Many well-established AOA assays of wide use may show unexpected limitations in

602

the course of analyzing complex samples, especially in respect to reaction conditions

603

pertaining to food and biological systems.68 For example, the ABTS/persulfate assay may be

604

influenced from activators (such as divalent iron) of persulfate to highly reactive

605

peroxysulfate radicals,69 because persulfate is one of the strongest oxidants known in aqueous

606

solution and can oxidize antioxidants (once activated) as well as ABTS to the colored ABTS•+

607

cation radical. The same assay may encounter kinetic difficulties when bulky antioxidants

608

having multiphenolic –OH groups are measured, since they are less able to diffuse to the

609

active sites of the sterically-hindered ABTS radical, limiting the accessibility of this radical

610

reagent during electron transfer (ET) and invalidate reporting antioxidant activity as trolox

611

equivalents.47 The ET rate constants of unhindered donor/acceptor couples are faster than

612

those of the hindered analogues. ET interactions between hindered donor-acceptor pairs (with

613

bulky substituents) may lead to substantial changes of the ET rate constants due to solvent

614

polarity, causing a large solvent-dependency of such reactions.46 Furthermore, the ABTS

615

assay shows TEAC coefficients greater than unity toward most thiol antioxidants, although

616

the physiologically relevant reversible oxidation of thiols to the corresponding disulfides

617

would necessitate a TEAC coefficient close to 0.5 (i.e., meaning 1-e oxidation), as in the

618

CUPRAC assay. In relation to cellular GSH and thiols metabolism, 2 molecules of GSH react 26 ACS Paragon Plus Environment

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619

with H2O2 or hydroperoxides through an enzymatic oxidation with glutathione peroxidase to

620

form 1 molecule of glutathione disulfide (GSSG), where GSH acts as a 1-e reductant.70 If

621

thiols are oxidized by ABTS•+ to sulfinic and sulfonic acids, this would not be a reversible

622

oxidation relevant for biological antioxidant action and would more likely correspond to

623

metal-catalyzed reactions of H2O2 or peroxynitrite with a thiol. Walker & Everette71

624

established that certain thiol antioxidants, especially aminothiols and amidothiols, are

625

oxidized by ABTS•+ in a biphasic kinetic pattern, consisting of a rapid conversion to the

626

corresponding disulfide followed by a slow step to higher oxidation products; thus, the

627

authors recommended that kinetic profiles for oxidation should first be established, then end-

628

point measurements should be made at a time when the reaction has reached near the end-

629

point (which is not guaranteed within the assay time). The equilibration times for tyrosine,

630

tryptophan and their related peptides were usually not sufficient within the protocol time of

631

the ABTS assay, and were strongly affected by pH.72

632

A kinetic investigation on DPPH radical scavenging carried out by Xie & Schaich48

633

demonstrated that none of the tested antioxidants exhibited a perfectly linear response range

634

with concentration and that all exhibited some saturation, showing that factors other than

635

antioxidant concentration also have important effects on reactivity. Contrary to expectations,

636

reaction stoichiometry (together with initial rate) showed negligible correlation to the number

637

of phenolic −OH groups per antioxidant molecule or to redox potential. Steric accessibility to

638

the hindered DPPH radical site may control the reaction rate to a stronger extent than specific

639

chemical properties of antioxidants (i.e., initial rates decrease with the bulkiness of multiple-

640

ring phenols). As DPPH scavenging is a mixed-mode (both ET− and HAT−based) assay,

641

antioxidants mainly acting with the HAT mechanism are strongly influenced by kinetic

642

solvent effects in that differences in the strength of hydrogen bonding of the solvent to

643

phenolic −OH groups and/or to DPPH interfere with the release of H-atoms (e.g., phenols 27 ACS Paragon Plus Environment


644

reacted fastest in methanol and slowed dramatically in ethanol or acetone). Steric accessibility

645

of the radical reagent, when combined with kinetic solvent effects, raise serious doubts

646

regarding the applicability of the DPPH assay for ranking antioxidants and natural extracts

647

and the quantitative evaluation of their antioxidant capacities, because there may be diverse

648

species in complex samples reacting with the DPPH radical at different rates, stoichiometries,

649

solvent and pH dependencies.48

650

Although the FRAP (ferric reducing antioxidant power) method is easy and robust, it

651

experiences kinetic difficulties in oxidizing antioxidants. Looking at the reactivity of thiols

652

with superoxide radicals (at a pH of 7.8), the order of second-order rate constants of

653

homocysteine (–SH pKa: 10.86) < gluthathione (–SH pKa: 9.65) < dithiothreitol (pKa of –SH

654

groups: 9.2 and 10.1) < cysteine (pKa: 8.18) was just the opposite of that of the corresponding

655

acidity constants (as pKa), meaning that thiols (in spite of their very favourable redox

656

potentials of oxidizibility to disulfides) may only be oxidized through the intermediary

657

formation of thiolate anions (RS‒) followed by thiyl radicals (RS•) with the overall reaction:

658

RSH + O2•‒ + H+ → RS• + H2O2.73 By similar reasoning, it was reported that the reactivities of

659

glutathione, cysteine, cysteamine, penicillamine, N-acetylcysteine, dithiothreitol and captopril

660

with both superoxide anion radicals and hydrogen peroxide (at pH 7.4) were inversely related

661

to the pKa of the thiol group.74 Likewise, seven low molecular-weight –SH compounds were

662

tested to reveal that thiols with the lower pKa reacted faster with peroxynitrite.75 Oxidation of

663

undissociated thiols (RSH) was proven to be slower in solutions at pH < pKa (–SH). Because

664

of the much stronger nucleophilicity of thiolate compared to thiol, the thiol-disulfide

665

exchange reaction basically proceeds through the thiolate anion even when its relative

666

abundance is low (i.e., at lower pH than the pKa).60 These examples show that the FRAP

667

method carried out at a pH of 3.6 (rather unfavorable for the formation of thiyl radicals)

668

cannot fully oxidize –SH compounds, constituting a major disadvantage for not accurately 28 ACS Paragon Plus Environment

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669

measuring thiol-type antioxidants. Moreover, the chemical inertness of high-spin Fe(III)

670

having half-filled d-orbitals (d5) –that presumably exists as the coordination center in the

671

FRAP reagent– may also be held responsible for the slow kinetics of this reagent toward

672

certain phenolic antioxidants that gives rise to incomplete oxidation of some phenolic acids

673

and flavonoids within the protocol time of the assay. While physiological pH enables partial

674

ionization of certain polyphenols to estimate a realistic antioxidant capacity, completely

675

unionized phenols at the acidic pH of the FRAP assay cannot exhibit their true antioxidant

676

power, yielding underrated results. As fast phenol oxidations performed with radicalic TAC

677

reagents in polar solvents (water or alcohol-water combinations) significantly proceed

678

through ionized phenols (i.e., phenoxide anions), this kinetic restriction arising from low pH

679

may also be important for FRAP and other ferric-based assays (excluding the ferricyanide-

680

Prussian blue assay which can be carried out at neutral pH).

681

Oxygen radical absorbance capacity (ORAC) is one of the most widely used AOA

682

assays (especially in the USA). ORAC was first established with the use of a β-phycoerythrin

683

probe, which was later replaced by the current fluorescein probe due to reproducibility

684

problems of the former.76 However, fluorescein also has a low reactivity toward peroxyl

685

radicals.43 Hydrophilic and lipophilic antioxidants should be assayed in different ORAC tests,

686

and in case when both types of antioxidants are tested in a 1:1 water-acetone solution

687

containing 7% randomly methylated β-cyclodextrin (Me-β-CD) to solubilize antioxidants,

688

there is the possibility for Me-β-CD‒antioxidant inclusion complex not to completely release

689

the antioxidant. The fluorescence (FL) decay of the probe is followed as a function of time in

690

the absence and presence of antioxidants, where FL quenching was delayed with antioxidants.

691

The difference between the AUC values under the fluorescence decay curve (∆AUC) in the

692

presence and absence of the antioxidant is not a linear but a polynomial function of

693

antioxidant concentration.24 The ORAC assay claims to measure both the rate and extent (i.e., 29 ACS Paragon Plus Environment


Page 30 of 57

694

thermodynamic efficiency and kinetic conversion) of antioxidant action in a single parameter

695

(AUC), but actually all fixed time assays implicitly measure the same two quantities because

696

the protocol time of most fixed time assays is not usually sufficient for a full oxidation of the

697

antioxidant (under nonequilibrium conditions), and the conversion achieved within a limited

698

time is also indicative of reaction kinetics. ORAC was reported to be highly influenced by the

699

presence of nonpolar solvents in the aqueous test medium, and among the tested amino acids,

700

tyrosine, tryptophan and histidine gave exceptionally high ORAC values while cysteine gave

701

a negative value. Possible polyphenol interactions with other food constituents had the largest

702

deviations (as found versus expected) in the ORAC assay with the overall order of

703

interference among four common assays being: ORAC > ABTS > DPPH > FRAP.77 Another

704

recently discovered drawback of ORAC lies in the fact that the ORAC values are determined

705

by the reactivity of antioxidants not only towards peroxyl but also alkoxyl radicals, which

706

may make the interpretation of ORAC values quite difficult for complex samples.78

707

6.

708

electrochemistry, chemometry and hyphenated methods)

Emerging

techniques

in

antioxidant

testing

(nanotechnology,

sensors,

709

Antioxidant scientists tend to increasingly use the basic structures of nature, which has

710

exploited energy-efficient self-assembly principles to create nanoscale structures during

711

evolution. Nanomaterials having at least one dimension between 1-100 nm have a high

712

specific surface area and a favourable hydrophilic-lipophilic interface, rendering their

713

effective use in imaging, encapsulation, processing into functional foods, biocompatible

714

delivery and activity determination of antioxidants. When the reducing power of antioxidant

715

compounds such as polyphenols were used to reduce Au(III) to elemental Au(0), the surface

716

plasmon resonance (SPR) band intensity of the resulting AuNPs could be used to measure

717

AOA, estimated from the generation (formation) and growth (enlargement) of nanoparticles.79

718

The localized SPR (LSPR) absorption band of noble metal (Au, Ag, Pt, Pd, Rh, etc.) 30 ACS Paragon Plus Environment

Page 31 of 57


719

nanoparticles is attributed to the collective excitation of electron gas in the particles, with a

720

periodic change in electron density at the surface; such bands may (in principle) give rise to

721

very sensitive antioxidant determinations. Unfortunately, chemical reduction–based assays of

722

noble metal nanoparticles yield molar absorptivities for antioxidants not more than the levels

723

observed in the widely used ET–based colorimetric assays. In order to reach extraordinarily

724

high sensitivities, one should aim at specific interactions or aggregation and disaggregation

725

behaviour of nanoparticles rather than simple redox reactions exploited by routine methods.80

726

For example, thiol-type antioxidants having a high affinity toward AuNPs (via the Au-thiol

727

covalent bond) may show specific interactions with nanoparticles81 and give rise to

728

aggregation enabling nM detection sensitivities for thiol analytes (instead of μM levels

729

achieved by conventional TAC assays). On the other hand, unlike noble metal nanoparticles

730

having very intense LSPR bands, metal oxide nanoparticles (such as nano-sized ceria, titania

731

and iron oxides) have not given rise to sensitive AOA determinations when merely measured

732

by their intrinsic color change upon contact with H2O282 or RONS, because of the weakness

733

of their charge-transfer bands (such as those in nanoceria, involving partial reduction of

734

Ce(IV) to Ce(III) by antioxidants forming a mixed-valent oxide, because either O (2p) →

735

Ce (4f) CT-transitions or electronic fluctuations between 4f → [4f + (5d6s)] and 4f → 4f

736

may not give rise to strong absorption bands). Another drawback with the use of transition

737

and lanthanide metal oxide nanoparticles is the possible interference of metal-polyphenol

738

complexation to the redox reactions upon which the AOA assay is based, due to the change of

739

redox potentials with complexation.

2-

4+

1

0

n

n-1

740

Most of the existing noble metal nanoparticle-based antioxidant sensors have exploited

741

the formation (seeding) rather than the enlargement (coating) of NPs upon reaction with

742

antioxidant compounds, and may not exhibit concentration-dependent linear responses due to

743

kinetic factors (i.e., NP size is dependent upon the reducing power of reductant; polyphenols 31 ACS Paragon Plus Environment


744

and flavonoids display a wide range of redox potentials, and may thus show slow, medium or

745

fast kinetics for reducing noble metal salts, giving rise to a wide range of sizes for noble metal

746

NPs with maximal wavelength shifts (λmax) for LSPR absorption which are not favourable for

747

analytical determinations). In this regard, our group made use of the LSPR absorption of

748

silver nanoparticles as a result of enlargement of citrate-reduced seed particles by antioxidant

749

addition subsequently enabling a linear response versus antioxidant concentration, because the

750

enhancement in LSPR band intensity at a stabilized λmax was a function of the enlargement

751

(coating) of nanoparticles rather than their initial formation (nucleation).83 In this respect,

752

Choleva et al.84 developed a paper-based assay of AOA determination, in which antioxidant

753

compounds directly produced AuNPs on the tetrachloaurate(III)‒impregnated paper, on which

754

the color intensity was analyzed by using IMAGE J software. Electrostatic, hydrogen bonding

755

or van der Waals interactions combined with chemical coordination–oriented donor-acceptor

756

bonding may be exploited in the design of creative colorimetric assays with noble metal

757

nanoparticles for detecting RONS-damaged DNA, protein, and other organic molecules

758

important in food chemistry or biochemistry.85 As biomacromolecules can easily be adsorbed

759

on noble metal nanoparticles, colorimetric assays can be designed utilizing the aggregation,

760

disaggregation, and displacement reactions of DNA- and protein-loaded Au/Ag nanoparticles

761

upon generation or quenching of reactive species in conjunction with RONS scavenging of

762

antioxidants.

763

Most of the colorimetric TAC sensors applicable to diverse food extracts yielding a

764

precisely linear response of absorbance versus concentration have been developed in our

765

laboratory, based on the principle of adsorption of positively-charged chromophores on the

766

negatively-charged perfluorosulfonate groups of Nafion membranes. The CUPRAC

767

colorimetric sensor –working like a pH indicator strip immersed in a test solution– relied on

768

Cu(II)→Cu(I) reduction by electron-donating antioxidants in the presence of neocuproine 32 ACS Paragon Plus Environment

Page 32 of 57

Page 33 of 57


769

(Nc) ligand; as there was not a drastic change in the coordination geometry of the copper-

770

chelate during electron-transfer, both the initial reagent: Cu(Nc)22+ and the emerging

771

chromophore: Cu(Nc)2+ were easily retained on the Nafion membrane, on which the measured

772

absorbance or reflectance of the cuprous-complex linearly correlated with antioxidant

773

concentration86 over a wide range of micromolar trolox-equivalent (TE) values. Similar

774

colorimetric TAC sensors were prepared by the immobilization of the cupric-neocuproine

775

cationic chelate onto a biopolymer (carrageenan)-incorporated membrane sensor having

776

cation-exchanger sulfate groups87 and of the Fe(III)-o-phenanthroline chelate onto Nafion.88

777

Since these coordination complexes acting as electron-transfer reagents were forced to a

778

planar configuration on the membrane surfaces, the geometry, size and charge (at the working

779

pH) of the approaching antioxidants affected the results, but altogether, the TAC values

780

measured with the aid of these membrane sensors were not significantly different from those

781

found in solution. These colorimetric sensors gave perfectly linear and additive responses

782

toward antioxidants because of the singularity of the resulting chromophores in accordance

783

with Beer’s law. A colorimetric membrane sensor capable of simultaneously measuring

784

oxidative status and AOA was prepared by immobilizing the pink-colored N,N-dimethyl-p-

785

phenylenediamine (DMPD) quinonic cation radicals on a Nafion membrane; hydroxyl or

786

superoxide radicals oxidized the DMPD probe to the corresponding cation radicals retained

787

on the membrane, and antioxidants, when present, bleached this color, where the absorbance

788

difference was indicative of their AOA.89

789

The general strategy with electrochemical techniques is the measurement of oxidative

790

damage produced on lipids, proteins or DNA, and restoration of their original electrochemical

791

signals in the presence of protective antioxidants.90 A significant number of electrochemical

792

sensing approaches target at cytochrome c, superoxide dismutase and DNA probes. These

793

bioelectrochemical sensors operated with the mechanism of following the changes of the 33 ACS Paragon Plus Environment


794

intrinsic anodic response (by the electrochemical technique of square wave voltammetry:

795

SWV) of the surface-confined guanine and adenine nucleic bases, resulting from their

796

interaction with ROS −derived from Fenton-type reactions− in the absence and presence of

797

measured antioxidants. The chemical reduction by antioxidants of the higher valencies of

798

transition metal ions (such as those of iron and copper) in coordination complexes can also be

799

exploited for indirect TAC measurement by monitoring the voltammetric peak and

800

chronoamperometric diffusional current; in this case, the electrochemical reduction current of

801

the remaining (intact) coordination complex may be measured after reaction with the

802

antioxidant. Electrodes coated with nanomaterials (such as noble metal nanoparticles) may

803

facilitate measurements by significantly increasing the surface area (thereby better catalyzing

804

electrochemical reactions) and conductivity, giving rise to more sensitive determinations.

805

High resolution screening that can combine chromatographic separation (such as

806

HPLC) with rapid post-column detection can both identify and quantify the active compounds

807

of complex mixtures. Chromatographic separation of constituents may be combined in

808

hyphenated techniques with on-line DAD, MS and NMR detection for fast detection of active

809

constituents. In setting up a well-controlled hyphenated assay, methods based on RONS

810

scavenging by antioxidants are not very practical and therefore have basically found limited

811

application; instead, methods based on a single chromogen or stable radical have been

812

popular.91 By similar reasoning, electrochemical detection (ED) exploiting the electroactivity

813

of antioxidant compounds have been used in conjunction with online-HPLC (HPLC-ED) or

814

flow injection analysis (FIA-ED). As a compensation for TAC assays giving an overall

815

nonspecific information for antioxidants rather than measuring them individually, online-

816

HPLC post-column spectrophotometric methods of TAC determination were originally

817

developed for DPPH and ABTS methods92 in which the HPLC-separated analytes reacted in a

818

post-column reactor with the colored radical solution, and the induced bleaching was detected 34 ACS Paragon Plus Environment

Page 34 of 57

Page 35 of 57


819

as a negative peak (corresponding to bleaching) by an absorbance detector at suitable

820

wavelengths. These methods combine chromatographic separation, constituent analysis, and

821

post-column identification of antioxidants in plant extracts. In the online-HPLC detection of

822

polyphenols by the CUPRAC method, antioxidant polyphenols in complex samples could be

823

separated on a C-18 HPLC column and further reacted with the Cu(II)-Nc reagent in a post-

824

column reactor to yield the Cu(I)-Nc chromophore detected at 450 nm. Thus, twice as much

825

information could be extracted from the same sample using these two consecutive

826

chromatograms, while a high selectivity was achieved because non-antioxidants did not yield

827

a peak in the post-column chromatogram.93 Flow-based methods in food and environmental

828

analysis using chemiluminescence (CL) have been excellently reviewed by Christodouleas et

829

al.;94 the CL produced by (luminol + oxidant) systems where the oxidant is hexacyanoferrate,

830

H2O2, iodine or permanganate are attenuated by phenolic antioxidants, whereas CL generated

831

by (permanganate + reductant) systems in which the reductant is H2O2 or formaldehyde are

832

enhanced by antioxidants, enabling an indirect determination of AOA. However, it is worthy

833

of mention that the mechanism of luminescence assays is not crystal-clear.43 CL detectors

834

could also be combined with LC in post-column detection systems to determine the free

835

radical scavenging activity of real samples.

836

The use of chemometric methods in antioxidant research has been reviewed by Apak

837

et al.31 When these methods are applied in conjunction with spectroscopic and

838

chromatographic assays to complex food matrices, under- or over-estimation of TAC values

839

may occur due to the presence of other food components (e.g., sugars, aminoacids, proteins,

840

etc.). Chemometric methods such as partial least-squares (PLS) or principal component

841

analysis (PCA) have been applied to food matrices to analyze complex spectra and

842

chromatograms with limited success. On the other hand, chemometry in chromatographic data

843

analysis was successful in eliminating/overcoming the adverse effects of background, 35 ACS Paragon Plus Environment


844

coelution/overlapping, retention time shifts and baseline drifts. Future trends for the use of

845

chemometry in antioxidant activity/capacity estimations may comprise the reliable prediction

846

of individual food components (such as impurities, authenticity, etc.) rather than of

847

cumulative parameters such as TAC values, which are themselves a complex function of the

848

efficacy and concentration of numerous food antioxidants rather than a selected few number

849

of compounds. Naturally when dealing with chemometry integrated to antioxidant analysis,

850

one should be aware of the chemistry and principles behind the method and samples so as to

851

better regulate input parameters and to interpret results.31

852

7. Hints for suitable assay selection and future perspectives

853

It may be argued that a carefully standardized TAC method should utilize a

854

biologically relevant or simulated oxidizing agent, be simple, reasonably rapid (enabling high-

855

throughput analysis), reproducible and versatile, have a clearly defined chemistry and

856

equivalence point, have readily available reagents and equipment, and not involve redox

857

cycling of end products (derived either from the antioxidant or reagent) in order to give

858

meaningful results.24,28 Quite similar principles apply for RONS biomarkers in that they

859

should be stable, give reproducible results for the same sample, and reliably indicate all or

860

most of the measured oxidative damage. Robustness (i.e., stability toward environmental

861

variations) is another important factor in assay selection. For example, DPPH• reduction

862

mechanisms and yields can be changed by various environmental factors of the reaction

863

medium (such as daylight, air oxygen, pH, solvent polarity, steric effects, temperature, etc.);

864

MeOH as solvent strongly inhibits the HAT mechanism and favours the SPLET (sequential

865

proton-loss electron-transfer) mechanism in DPPH• reduction by phenolic acids.95 A TAC

866

assay should preferably be robust with minimal influence from solvent variations, as was the

867

case with the CUPRAC method applied to edible oils.96


Page 36 of 57

Page 37 of 57


868

Some additional requirements for a preferable antioxidant assay, as summarized by

869

Apak et al.,9 can be listed as: (i) a working pH appreciably close to the physiological pH; (ii)

870

use of stable and reproducible probes; (iii) affinity of the assay reagent toward both aqueous

871

and organic phases; (iv) preferential absorption of the spectrophotometric assay chromophore

872

distinctly in the visible region so as to discriminate antioxidant analytes from other UV-

873

absorber organic molecules; (v) optimal redox potential of assay reagent high enough to

874

oxidize most biological and food antioxidants but low enough to leave out non-antioxidants

875

like sugars, citrate and certain amino acids; (vi) exclusion of strong chelators and reductants

876

when using transition metal ion coordination complexes as TAC reagents; (vii) absence of

877

redox cycling of transition metal coordination complexes (used as TAC reagents) with H2O2

878

or O2; (viii) avoid ‘short-circuit’ reactions in competitive assays (such as direct thiol-NBT

879

reaction in superoxide scavenging or peroxide-ABTS reaction in H2O2 scavenging tests); (ix)

880

selection of colorimetric assays that produce a single chromophore to avoid chemical

881

deviations from Beer’s law and comply with linearity of responses; (x) take measures to

882

distinguish between scavenging of reactive species and inhibition of oxidative enzymes by

883

antioxidants whenever RONS are generated enzymatically.

884

Although Huang & Tocmo51 evaluate that kinetics is more important than

885

thermodynamics when it comes to scavenging RONS, most of kinetic (i.e., reaction rate

886

measuring) and thermodynamic (i.e., measuring equilibrium conversion) TAC assays may be

887

collected under the common denominator of ‘fixed-time’ assays, in which a reproducible

888

conversion efficiency of the measurement probe is achieved within a predetermined time. In

889

this case, the end-point of the assay should be clearly defined to assure precision and

890

accuracy. It should be borne in mind that some researchers criticise end-point assays as

891

single-point titrations (usually carried out in alcohol-water mixtures far from lipid bilayers)

892

not reflecting the complex kinetics of lipid peroxidation.43 The controversies on this subject 37 ACS Paragon Plus Environment


893

have not yet been resolved, because while one literature source evaluates antioxidant activity

894

assays problematic with little evidence to suggest an in vivo role,97 others find colorimetric in

895

vitro screening tests useful for giving an idea on potential bioactivities and health benefits of

896

antioxidants.98,99 Granato et al.98 attribute the deserved importance to the versatility of

897

antioxidant assays operating in the HAT or ET and free radical scavenging modes making a

898

cumulative evaluation of integrated antioxidant action, as well as to the chromatographic

899

quantification of individual constituents in complex food and biological mixtures, and to the

900

antioxidative effect of bioactive compounds measured against various cell lines subjected to

901

oxidative stress.

902

How should one select an assay to fit a specific purpose? Antioxidant activity tests

903

give the most meaningful results when they are chosen as purpose-oriented. Antioxidants

904

extend the shelf-life of both oils and ammunition, prevent rancidity in nutritional fats, but the

905

AOA assay directed at measuring the exhaustion level of heated frying oils (e.g., monitoring

906

the extent of lipid peroxidation) cannot be identical to that of poorly-stored ammunition (e.g.,

907

measuring the generation or scavenging of reactive nitrogen species) or of partly degraded

908

petroleum products such as lubricating oils, fuels and plastics. Finley et al.8 have emphasized

909

that the term ‘antioxidant’ may appeal in varying modes to different audiences, such as the

910

ability of deactivating metabolically generated RONS (to biochemists and nutritionists), the

911

power to retard food oxidation (to food scientists), or the yield of high TAC values in ET- and

912

HAT-based in vitro assays (to a wider community of food science, commerce, and industry).

913

Cultured cells are being increasingly used in oxidative stress and antioxidant analyses,

914

because tests with experimental animals are controlled with tougher regulations and different

915

stressors and cell types can be applied in modeling certain diseases. Moreover, in vitro AOA

916

tests rarely consider the influences of absorption/adsorption, bioavailability and metabolism14

917

and the results of these tests will become more meaningful when all aspects of 38 ACS Paragon Plus Environment

Page 38 of 57

Page 39 of 57


918

bioaccessibility and bioavailability of phenolic antioxidants are further clarified and

919

elaborated.100 Another expectation is that, when the cells are subjected to oxidative stress, the

920

chosen AOA test should reveal the decrease in the cellular antioxidant reserve during combat

921

against RONS; when an oxidative stress was induced in vivo (e.g., via rabbit urinary bladder

922

outlet partial obstruction–mediated cyclic ischemia and reperfusion resulting in the generation

923

of both RONS), the CUPRAC assay –but not the FRAP assay– could detect the decrease in

924

the reactivity of antioxidants found within the obstructed bladder tissue as compared to the

925

control bladder tissue in both the muscle and mucosa.101

926

Cellular antioxidant activity (CAA) assays, performed within the cell medium, are

927

claimed to better reflect the uptake, distribution, and metabolism of antioxidants within cells.

928

Wolfe & Liu102 made use of peroxyl radical oxidation of the nonfluorescent probe 2’,7’-

929

dichlorofluorescin (DCFH) entrapped in human hepatocarcinoma HepG2 cells, where the

930

presence of antioxidants weakened the fluorescence signal of the oxidation product,

931

dichlorofluorescein (DCF), thereby enabling AOA determination. The probe may exert

932

photochemical instability and entrapping deficiency. As DCFH and DCF can diffuse out and

933

undergo extracellular reactions, care should be exercised when using a plate reader not to

934

confuse the light emission from the medium with that from the cells.26 López-Alarcón &

935

Denicola5 recommended the use of CAAs as genuine AOA assays against classical ones, but

936

the antioxidative efficacies of several compounds did not correlate well with those found by

937

ORAC. It was recently discovered by Kellett et al.27 that different models of cell lines may

938

give varying orders of antioxidant potency in CAA assays, e.g., both quercetin and catechin

939

were active against Caco-2 while only quercetin (but not catechin) was active against HepG2,

940

possibly due to differences in active membrane transport between cell lines.

941

It may be recommended to use several purpose-oriented assays of different

942

mechanisms to reveal the full capability of a real antioxidant sample. The usage of a variety of 39 ACS Paragon Plus Environment


943

methods and conditions was recommended so as to correctly describe the in vitro antioxidant

944

capacity of a sample supported by in vivo assay data.17 By similar reasoning, future

945

antioxidant tests are envisaged to aim at the determination of multiple antioxidants which are

946

expected to prevent diseases induced by multiple oxidants.19 Surprisingly, the results of tests

947

with different mechanisms (HAT, ET, and mixed-mode assays) may correlate well, as was the

948

case in DPPH, β-carotene bleaching, and NO inhibition radical scavenging tests carried out on

949

apple and pear peel and pulp samples.103 Recently, Huang & Tocmo51 suggested a novel

950

comprehensive evaluation of RONS scavenging capacity of an antioxidant in a chemical

951

system, relying on the proposition of Prior104 for a multiple radical approach of the ORAC test

952

(ORACMR) to provide a simple and visual way of presenting scavenging capacity of

953

antioxidants against several RONS, defined as the ‘spider web’ panel of antioxidative

954

defenses as the cumulative antioxidant capacity against six predominant reactive (O, N, and

955

Cl) species found in the human body. It should be borne in mind that ET−based assays are

956

complementary to HAT−based ones in fully disclosing the antioxidant ability of complex

957

samples, and that good work can be performed at relatively low cost. Li & Pratt13 state that

958

measurement of radical trapping antioxidants (i.e., basically focusing on peroxyl radical

959

quenching kinetics) usually require commonly available equipment such as HPLC, GC, or

960

fluorescent microplate readers rather than the much more expensive EPR or laser flash

961

spectrometers. The same reasoning is valid for most ET-based assays measuring the reducing

962

ability of antioxidants with simple absorption/emission spectrometers. As a concluding

963

remark, it may be added that whichever method is used for measuring antioxidant activity or

964

capacity, one should know what is being measured and how, with its limits (e.g., analytical

965

performance parameters) and weaknesses (such as interferences giving rise to deviations from

966

real values).

967


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968

References

969

1. Halliwell, B.; Gutteridge, J. M. C. Free Radicals in Biology and Medicine, 4; Clarendon,

970

Oxford, 2007.

971

2. Shahidi, F.; Zhong, Y. Measurement of antioxidant activity. J. Funct. Foods 2015, 18, 757–

972

781.

973

3. Gutteridge, J. M. C.; Halliwell, B. Mini-Review: Oxidative stress, redox stress or redox

974

success?. Biochem. Biophys. Res. Commun. 2018, 502, 183–186.

975

4. Sies, H. On the history of oxidative stress: Concept and some aspects of current

976

development. Curr. Opin. Toxicol. 2018, 7, 122–126.

977

5. Lopez‐Alarcon, C.; Denicola, A., Evaluating the antioxidant capacity of natural products: a

978

review on chemical and cellular‐based assays. Anal. Chim. Acta 2013, 763, 1–10.

979

6. Ingold, K. U.; Pratt, D. A. Advances in radical-trapping antioxidant chemistry in the 21st

980

Century: a kinetics and mechanisms perspective. Chem. Rev. 2014, 114, 9022–9046.

981

7. USDA, U.S. Department of Agriculture, Agriculture Research Service, 2010. Oxygen

982

Radical

983

www.ars.usda.gov/news/docs.htm?docid=15866 (Access date: 16 February 2019).

984

8. Finley, J. W.; Kong, A. N.; Hintze, K. J.; Jeffery, E. H.; Ji, L. L.; Lei, X. G. Antioxidants in

985

foods: state of the science important to the food industry. J. Agric. Food Chem. 2011, 59,

986

6837–6846.

Absorbance

Capacity

(ORAC)

of

Selected


Foods,

Release

2

Web:


987

9. Apak, R.; Özyürek, M.; Güçlü, K.; Çapanoğlu, E. Antioxidant Activity/Capacity

988

Measurement. 1. Classification, Physicochemical Principles, Mechanisms, and Electron

989

Transfer (ET)-Based Assays. J. Agric. Food. Chem. 2016, 64, 997–1027.

990

10. Weidinger, A.; Kozlov, A. V. Biological activities of reactive oxygen and nitrogen

991

species: oxidative stress versus signal transduction. Biomolecules 2015, 5, 472–484.

992

11. Radi, R. Oxygen radicals, nitric oxide, and peroxynitrite: Redox pathways in molecular

993

medicine. Proc. Natl. Acad. Sci. USA (PNAS) 2018, 115, 5839–5848.

994

12. Halliwell, B. Antioxidant characterization. Methodology and mechanism. Biochem.

995

Pharmacol. 1995, 49, 1341–1348.

996

13. Li, B.; Pratt, D. A. Methods for determining the efficacy of radical-trapping antioxidants.

997

Free Radic. Biol. Med. 2015, 82, 187–202.

998

14. Niki, E. Assessment of Antioxidant Capacity in vitro and in vivo. Free Radic. Biol. Med.

999

2010, 49, 503–515.

1000

15. Apak, R.; Gorinstein, S.; Böhm, V.; Schaich, K. M.; Özyürek, M.; Güçlü, K. IUPAC

1001

Technical Report, Methods of measurement and evaluation of natural antioxidant

1002

capacity/activity. Pure Appl. Chem. 2013, 85, 957–998.

1003

16. Demirci Çekiç, S.; Çetinkaya, A.; Avan, A. N.; Apak, R. Correlation of total antioxidant

1004

capacity with reactive oxygen species (ROS) consumption measured by oxidative conversion.

1005

J. Agric. Food Chem. 2013, 61, 5260–5270.

1006

17. MacDonald-Wicks, L. K.; Wood, L. G.; Garg, M. L. Methodology for the determination

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of biological antioxidant capacity in vitro: a review. J. Sci. Food Agric. 2006, 86, 2046–2056.


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1008

18. Frankel, E. N.; Meyer, A. S. The problems of using one dimensional methods to evaluate

1009

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Figure Captions

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Figure 1. (I) Direct (competitive) antioxidant assay, involving a fluorogenic or chromogenic

1263

probe and biologically relevant ROS/RNS; (II) Indirect (noncompetitive) antioxidant assay, in

1264

which physiological redox reactions (i.e., oxidant-antioxidant interactions) are simulated on

1265

an artificial probe without biologically relevant ROS/RNS.

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Figure Graphics

Figure 1


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