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Antioxidant activity/capacity measurement: II. Hydrogen atom transfer (HAT)-based, mixed mode (ET/HAT) and lipid peroxidation assays Re#at Apak, Mustafa Özyürek, Kubilay Guclu, and Esra Capanoglu J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.5b04743 • Publication Date (Web): 25 Jan 2016 Downloaded from http://pubs.acs.org on January 25, 2016

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Journal of Agricultural and Food Chemistry

Antioxidant activity/capacity measurement: II. Hydrogen atom transfer (HAT)-based, mixed mode (ET/HAT) and lipid peroxidation assays

Reşat Apak1, Mustafa Özyürek1, Kubilay Güçlü1, Esra Çapanoğlu2

1

Department of Chemistry, Faculty of Engineering, Istanbul University, Avcilar 34320,

Istanbul-Turkey

2

Department of Food Engineering, Faculty of Chemical and Metallurgical Engineering,

Istanbul Technical University, Maslak 34469, Istanbul-Turkey

* Corresponding Author. Tel.: +90 212 4737070 Fax: +90 212 473 7180 E-mail: [email protected]

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ABSTRACT

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Measuring the antioxidant activity/capacity levels of food extracts and biological fluids is

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useful for determining the nutritional value of foodstuffs and for the diagnose, treatment and

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follow-up of numerous oxidative stress-related diseases. Biologically, antioxidants play their

6

health-beneficial roles via transfering a hydrogen (H) atom or an electron (e-) to reactive

7

species, thereby deactivating them. Antioxidant activity assays imitate this action, i.e.

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antioxidants are measured by their H-atom transfer (HAT) or e--transfer (ET) to probe

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molecules. Antioxidant activity/capacity can be monitored by a wide variety of assays with

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different mechanisms, including HAT, ET, and mixed-mode (ET/HAT) assays, generally

11

without distinct boundaries between them. Understanding the principle mechanisms,

12

advantages and disadvantages of the measurement assays is important for proper selection of

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method for valid evaluation of antioxidant properties in desired applications. This work

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provides a general and up-to-date overview of HAT based, mixed mode (ET/HAT) and lipid

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peroxidation assays available for measuring antioxidant activity/capacity and the chemistry

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behind them, including a critical evaluation of their advantages and drawbacks.

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Keywords: Hydrogen atom transfer assays, mixed-mode assays; lipid peroxidation assays;

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antioxidant mechanisms; food analytical methods.

20 21 22 23 24


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1. INTRODUCTION

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Various methods are used to investigate the antioxidant property of individual compounds and

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complex samples (food extracts, beverages and biological fluids). Antioxidant activity (AOA)

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assays: total peroxyl radical trapping antioxidant parameter (TRAP),1,2 crocin bleaching,3,4

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oxygen radical absorbance capacity (ORAC),5,6 total oxyradical scavenging capacity

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(TOSC),7,8 etc. are usually competitive and work on a HAT mechanism (monitor competitive

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reaction kinetics), whereas total antioxidant capacity (TAC) measurement methods are

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usually non-competitive ET and mixed-mode (ET/HAT) assays (generally involving a redox

34

reaction with the probe (oxidant)). Although the 2,2-diphenyl-1-picrylhydrazyl (DPPH) and

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2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS) assays are usually classified as

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ET reactions, these two radicals in fact may be deactivated either by radical quenching via

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HAT or by direct reduction through ET mechanisms.9 Mechanisms and reactivity patterns are

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thus difficult to interpret without detailed structural information about the tested antioxidant

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compounds. In competitive assays, the oxidant reacts with target species, called probes,

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leading to changes in its spectroscopic properties (i.e., absorbance, fluorescence,

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luminescence), or any other measurable property, where antioxidants compete against the

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probe for the related oxidant.10 Due to the competition between the probe and antioxidants for

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oxidant, the probe undergoes less oxidative conversion by reactive oxygen species (ROS) or

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reactive nitrogen species (RNS) in the presence of antioxidant compounds. In other respects,

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in ET−based methods, the probe undergoing reduction with the antioxidant is either converted

46

to a colored, fluorescent, or chemiluminescent species, or the initial absorbance/fluorescence

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of the probe is attenuated as a consequence of the antioxidation reaction.

48



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Lipid peroxidation is the oxidation of lipids, especially unsaturated fatty acids in

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cellular membranes mediated by oxidative stress in cells. In recent years, different

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measurement strategies for estimating lipid peroxidation can be used to directly assess the

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AOA of a compound toward a lipid substrate (i.e., β-carotene bleaching assay, iodometric

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hydroperoxide measurement, thiobarbituric acid-reactive substances (TBARS) assay,

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ultraviolet (UV) spectroscopic measurement of conjugated dienes, ferric-thiocyanate and

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ferric-xylenol orange assays and mass spectrometry (MS) techniques).11

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In this review, the relevance, advantages, and drawbacks of HAT-based, mixed mode

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(ET/HAT) and lipid peroxidation assays are critically discussed, with respect to their

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chemistry and to the mechanisms of antioxidant activity/capacity.

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2. HAT−BASED METHODS

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These methods measure the capability of an antioxidant compound to scavenge ROS (e.g.,

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peroxyl radical: ROO•) by hydrogen donation as in Eq.s 1&2. ROO• are generally chosen as

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the reactive species in these assays because of their higher biological relevance and longer

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half-life (compared to hydroxyl: •OH and superoxide anion radicals: O2•−). The HAT reaction

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mechanism include the transferring of hydrogen atom (H•) of antioxidants (AH/Ar-OH) to a

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ROO• to give more stable free radicals (A• and ArO•), summarized by the reaction scheme

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(Eq.s 1-3):

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ROO• + AH

→ ROOH + A•

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ROO• +Ar-OH → ROOH + ArO•

… (Eq. 2)

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2 ROO•→ Non Radical Products

… (Eq. 3)

… (Eq. 1)

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where the radical of the antioxidant (A•) and aryloxyl radical (ArO•) is usually stabilized by

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resonance. A potent phenolic antioxidant (Ar-OH) need to react faster than the target to be

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protected with the oxidant (ROS), and A• must be rapidly converted to less reactive species.12

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In these methods using a fluorometric probe, both the probe and antioxidant compound react

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with ROO• simultaneously and the AOA can be calculated from competition kinetics by

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measuring the fluorescence decay curve of the probe in the presence and absence of

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antioxidant, and integrating the Area Under the Curves (AUC approach). The AUC difference

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between reagent blank and sample is then related to tested antioxidant concentration in the

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sample.13,14 HAT−based assays include ORAC assay, TRAP assay using either β-

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phycoerythrin (β-PE) or fluorescein (3ˈ,6ˈ-dihydroxyspiro[isobenzofuran-1[3H],9ˈ[9H]-

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xanthen]-3-one) (FL) as the fluorogenic probe, TOSC assay, crocin bleaching test.13,14

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2.1. ORAC Assay

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The original ORAC assay5,15 has been widely used in measuring the content of antioxidants in

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food samples. Initially, β-PE fluorescent protein isolated from Porphyridium cruentum, was

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used as the target/probe (λexcitation=540 nm; λemission=565 nm) and 2,2’azobis (2-

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methylpropionamidine) dihydrochloride (AAPH) was used as a ROO• generator in this assay

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with the thermolysis reaction: (AAPH + O2 → 2 ROO• + N2), where R: HN=C(NH2)-

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C(CH3)2-.16 The ROO• preferably abstracts a H• from antioxidant molecule. A chain-breaking

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antioxidant competes (AH) with the probe for quenching ROO•, and the reaction between

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ROO• and the probe is retarded or inhibited (Eq.s 5-7). The ORAC assay measures the AOA

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against ROO• based on the time-dependent decrease in the fluorescence intensity of the β-PE

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probe, a fluorescent hydrosoluble protein. β-PE absorbs the visible light and possesses a high

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fluorescent yield, proving highly sensitive to ROS.5 Basically, the ROO• reacts with a 5 ACS Paragon Plus Environment


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fluorogenic probe to form a nonfluorescent product, and the conversion of initial probe can be

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quantitated by a fluorescence decay (Figure 1). ORAC assay was also automated in a

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microplate with a COBAS FARA II analyzer to improve the throughput.

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Figure 1

104 probe Probe + ROO•  → Probe•

....(Eq. 5)

k AH AH + ROO• → A•

....(Eq. 6)

k → Probe + A • Probe• +AH ←

....(Eq. 7)

k

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106 107

Eq.s 5&6 represent the one-electron oxidation reactions of the fluorogenic probe and

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antioxidant molecule with ROO•, respectively. The computed time-course of probe oxidation

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displays the characteristic lag phase during which antioxidant compound is consumed linearly

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with time (e.g., an efficient antioxidant like Trolox, a water-soluble vitamin E analog,

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produces a well defined lag phase). The equilibrium constant of Eq. 7 (K) is related to the

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relative difference in reduction potential (∆E°) between Probe• and A • :

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∆ E° = (RT / nF) ln K

....Eq. 8

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A potent antioxidant is expected to have a low intrinsic value of E° and hence a larger

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value for K pertaining to (Eq. 8). Therefore, AOA of an antioxidant compound is dependent

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on the reactivity of the selected probe with ROO• (kprobe), and thermodynamics of the

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equilibrium reaction represented by Eq. 8.


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The β-PE probe reacts with ROO• more slowly than biologically important compounds

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(i.e., thiols, uric acid, bilirubin, and ascorbic acid), but this probe is more reactive than other

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non-antioxidant molecules. Thus, all of the antioxidant compounds are fully oxidized before

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this probe starts to become oxidized, and it facilitates the measurement of the TAC of the

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studied sample. Using β-PE and AAPH, the decay in the intensity of fluorescence of β-PE

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followed zero-order reaction kinetics, and the AOA of an antioxidant, synthetic mixture or

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complex analyte was related to the decrease in the rate constant and/or to the increase

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observed in the lag phase. Cao and Prior16 quantified the level of antioxidant protection by

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measuring the AUC of the sample being tested as compared to that of the blank (Figure 2).

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Thus, this method combines both inhibition percentage and time of the ROO• action by

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antioxidant compound into a single quantity. In this assay, the incubation reaction is

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monitored for extended periods (> 30 min) in the absence and presence of antioxidants, and

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results were expressed as µM Trolox (TR) equivalents (TE). The net integrated response

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curves (net AUC) was obtained by subtracting the AUC of the blank from that of the sample

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( AUC

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Eq.s 1 & 2 as follows (Eq. 9):

sample

− AUCblank ) . The relative ORAC value of a sample (µM TE) was calculated from

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Figure 2

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ORAC value =

( AUC

sample

− AUCblank )

( AUCTR − AUCblank )

x

[ TR ]

[sample]

…(Eq. 9)

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The basic disadvantage of this assay is the use of β-PE as the probe, because it varies

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from one production lot to another (decreasing reproducibility), is not photostable, can be

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photobleached after an exposure to excitation light, can interact with polyphenolic 7 ACS Paragon Plus Environment


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antioxidants (especially with proanthocyanidins) by non-specific protein binding. Considering

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these disadvantages, ORAC assay was significantly improved by Ou et al. (ORAC-FL

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method).6 FL was used as a more stable fluorogenic probe for oxidation by ROO•. FL probe

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in comparison with β-PE is less reactive, does not interact with antioxidant compounds, and

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shows excellent photostability.6 However, ORAC with a FL probe also has some

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shortcomings such as not differentiating between rate and efficiency of radical scavenging,

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overestimating the activity of slow-reacting antioxidants and giving rise to unusually high

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TEAC coefficients for certain antioxidants, and poorly correlating with the activity for

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inhibition of ROS-mediated oxidation.17-19

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The ORAC-FL method was initially developed by Ou et al.6 to evaluate the protective

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effect of hydrophilic chain-breaking antioxidant compounds, and Huang et al.20 extended its

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usage to lipophilic antioxidants using an acetone/water solution containing 7% of methyl-β-

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cyclodextrin (M-β-CD) as a solubility enhancer, which allows one to measure the AOA of

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both lipophilic and hydrophilic antioxidant compounds in a sample being tested using the

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same ROO• source (ORACFL-LIPO).20 However, high concentrations of M-β-CD may hinder

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antioxidative action, due to the relative stability of the inclusion complex with antioxidant.

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The ORAC method is superior to other AOA/TAC spectroscopic methods because it provides

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a unique evaluation in which the inhibition time and degree of ROS are measured as the

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reaction goes to completion.13 AOA of water- and lipid-soluble antioxidants in the complex

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matrix are measured by the hydrophilic ORAC (H-ORAC) and lipophilic ORAC (L-ORAC)

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methods, respectively.21 Wu et al.22 demonstrated that the ORAC values for the hydrophilic

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extracts of fruits and vegetables were much higher than those of the lipophilic extracts.

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Rautiainen et al.23 reported that vegetables and fruits in Sweden were the major contributors

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to food-frequency questionnaire- based ORAC (≥50%). H-ORAC values of vegetables and

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fruits are usually observed much higher than L-ORAC values22 with improved precision24.


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Originally promoted in a machine-based assay (COBAS FARA II), the ORAC assay

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was also coupled to a microplate reader with injection systems,25 and to a microplate robotics

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system.20 Automation indeed reduces analysis time and improves the efficiency of the assay

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in multiple analyses. Another method was developed for microplate-based ORAC using

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pyrogallol red (PGR) as probe with good linearity, precision, and accuracy.26 However there

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are no published data on a manual ORAC assay. The BIOOXYTECH® AOP-490™

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microplate assay based on the reduction of Cu(II) to Cu(I) by the antioxidants in the

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oxygenated sample resulting in DNA cleavage was developed,27 and has not been directly

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compared to the original ORAC method.

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Some advantages of the ORAC assay over other AOA-TAC assays include the use of

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physiologically relevant and relatively long-lived peroxyl radicals (ROO•) as oxidants, and

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utilization of physiological pH so that the antioxidant compounds react with an overall charge

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and protonation state similar to that in the human body.28 This assay also takes into account

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both thermodynamic and kinetic properties of radical-antioxidant reactions.

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Dorta et al.29 demonstrated that ORAC results do not correlate with the activity of

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antioxidants to trap ROO•, suggesting a dominant role of RO• in the assay. The ORAC index

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is not related to the total free radical scavenging capacity of the complex mixtures due to the

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concentration, chemical characteristics, and possible interaction between the antioxidant

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compounds present in the tested sample.30 The ORAC index of a specific sample does not

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reflect the capacity of the antioxidants to trap ROO• or RO• generated from AAPH.31

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Recently, the ORAC-index (database) for approximately 300 selected fruits, vegetables, nuts

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was also removed from the website by US Department of Agriculture - Nutrient Data

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Laboratory due to the fact that the ORAC values indicating AOA have no relevance to the

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effects of specific bioactive compounds (e.g., polyphenols) on human health.32



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Kohri et al.33 proposed an electron paramagnetic resonance (EPR) spin trapping

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method employing in situ photolysis of AAPH to generate RO• for the direct measurement of

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RO• scavenging capacity. This method determines the level of RO• in the absence and

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presence of antioxidants. In addition, there was no correlation between ORAC-EPR values

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and those measured by conventional ORAC method. The proposed method was also claimed

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to be superior to conventional ORAC assay in terms of analyzing thermally labile biological

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specimens.

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2.2. TRAP Assay

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Secondary antioxidants (e.g., vitamin E, superoxide dismutase) trap ROO• directly, thereby

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preventing the oxidation chain reactions and thus limiting the amplification of ROS damage.

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TRAP method was introduced by Wayner et al.1 for determination of the secondary

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antioxidant status of plasma (as target) and results (TRAP value) were expressed as the

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number (µmoles) of ROO• trapped by one liter of plasma.1,2 This test was based on the

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measurement of O2 (as probe) uptake during a controlled peroxidation reaction induced by

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ABAP thermolysis, which yields effective quantities of ROO• at a constant rate, Ri (Figure

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3).

211 212

Figure 3

213 214

After addition of ABAP to the human plasma, the measured parameter was the “lag

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time” that O2 uptake by oxidizable biological sample (e.g., plasma) is inhibited by antioxidant

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compounds. The “lag time” is the induction period, rplasma, and is measured with the aid of an

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O2 electrode by diluting plasma in aqueous buffer. These induction periods provide a 10 ACS Paragon Plus Environment

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quantitative measurement of the TAC of plasma as the TRAP value. This value can be

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calculated from rplasma by Eq. 10,

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TRAP= Ri rplasma … (Eq. 10)

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The Ri value is obtained by adding a known quantity of TR to the plasma after the tested

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antioxidants have been fully consumed, i.e., well after rplasma when peroxidation is proceeding

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rapidly. In this test, TR produces a second induction period, rTR, which yields Ri via Eq. 11:

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Ri= n [TR]/ rTR … (Eq. 11)

227 228

where n is the stoichiometric factor (the number of ROO• trapped per molecule of TR).

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This method generally measure antioxidant capability to interfere with the reaction

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between ROO• and probe. This method are also variants of ORAC assays in principle, but

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they use a broader range of probes, initiators, and end-point measurements (e.g., Trolox-

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equivalent antioxidant capacity (TEAC) assays). Initiators ROO• have been produced

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selectively by azides (e.g., AAPH),1,2 enzymes (e.g., horseradish peroxidase),34 or by H2O2-

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hemin,35 •NO,36 and singlet oxygen (1O2)37. Some of the probes used in TRAP assays include

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FL,37 dichlorofluorescein diacetate (DCFH-DA),38 phycoerythrin,39,40 luminol,34 and ABTS41.

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As a more efficient, and less costly alternative to the conventional techniques, a

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DCFH-DA probe was used for detecting ROO• generated by thermal decomposition of

238

AAPH.38 Its oxidation by ROO• converts this probe to highly fluorescent dichlorofluorescein

239

(DCF) at room temperature. High TRAP values were obtained in the case of addition of some

240

antioxidant compounds to the assay medium in vitro (e.g., vitamin E).38

241



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Plasma antioxidants (i.e., vitamins C and E, uric acid) react more rapidly with ROO•

243

−generated by thermolysis of ABAP− than β-PE, while the other compounds provide partial

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concentration-dependent protection from ROS attack.39 A change in the intensity of time-

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resolved fluorescence provides a measure of the rate of ROO• damage by exploiting the

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unusual reactivity (over 100-fold slower than that of ascorbate or vitamin E analogs) of β-PE

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toward peroxyl radicals. Delange and Glazer39 described the estimation of ROO• scavenging

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activity of human plasma in comparison with proteins, DNA, vitamins, catecholamine

249

neurotransmitters, and other low molecular weight biological compounds.

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In another modified TRAP assay, the rate of peroxidation induced by ABAP was

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determined through the loss of fluorescence of β-PE (probe).40 The lag-phase induced by

252

plasma sample was compared to that induced by Trolox as the reference compound. Proteins

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without their sulphydryl groups interfere with this assay. These molecules provides partially

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protection of the probe when all of the plasma antioxidants are consumed. Therefore, either

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other compounds present in plasma are likely to exert AOA, or a synergistic activity between

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antioxidants should be postulated to exist. Bartosz et al.41 found that the simultaneous

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inactivation of ascorbate and thiol groups produces a greater loss in TAC of plasma.

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The major drawback of TRAP assay is the possible error in end-point detection caused

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by instability of the O2 electrode, as reaching the end-point is too lengthy (≈ 2 h) for the AOA

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measurement of multiple samples. As a more convenient, chemiluminescence has been used

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to determine the reaction end-point precisely. An enhanced chemiluminescence assay using

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luminol and horseradish peroxidase (HRP) was developed by Bastos et al.35 for the highly

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senstivite measurement of TAC in biological sample. The addition of antioxidant solutions

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(i.e., vitamin E, ascorbate, urate) or biological samples to a glowing chemiluminescent

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reaction temporarily interrupts light output. The light emission is restored after a time interval


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which is linearly related to the molar concentration of added antioxidant, enabling TAC

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quantitation of various biological fluids.

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A spectrophotometric method for the detection of ROO•-trapping capacity of food

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products and biological fluids was developed, involving the decomposition of ABAP as the

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source of ROO• which oxidizes ABTS to ABTS•+.41 Antioxidants present in a sample inhibit

271

the reaction and the induction period of the oxidation reaction provides measurement of AOA

272

of several types of beverages.

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2.3. TOSC Assay

275 276

TOSC method described by Winston et al.7 is based on the evaluation of different

277

antioxidants specifically toward three potent oxidants (i.e., •OH, ROO•, and ONOO-).42 The

278

oxidation of α-keto-γ-methiolbutyric acid (KMBA) to ethylene is realized by ROS; ethylene

279

formation relative to a control reaction is monitored by a head space-gas chromatography

280

(HS-GC). TOSC assay is based on the inhibition of ethylene formation in the presence of

281

antioxidant compounds that compete with KMBA for ROS. The reaction between ROO• and

282

KMBA7 represented by Eq. 12:

283 284

CH3S-CH2-CH-CO-COOH / •OOH(R) → ½ (CH3S)2 + (R)HOO- + CO2↑ + CH2=CH2↑. (Eq.

285

12)

286 287

The method uses an AUC of ethylene concentration versus the reaction time, which

288

can be up to 300 min. The AUC kinetic linear dose-response curves for antioxidant

289

compounds can be obtained by method of competitive reactions. TOSC value is then



∫ sample

290

quantified by the following equation (Eq. 13), where

291

integrated AUC of the sample and control reactions, respectively.

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and

∫ control

are the

292

293

 sample  ∫ • 100  TOSC = 100 −   ∫ control   

.....(Eq.13)

294 295

A modified version of the TOSC assay in combined with ion flow tube- mass

296

spectrometry (SIFT-MS) was used for measuring the AOA of antioxidant compounds and real

297

samples (e.g., plant extracts, biological fluids). This modified assay utilizing the

298

quantification of ethylene produced from the antioxidant sample overcomes the unsolved

299

scattering and cloudiness interference problems in spectroscopic aqueous-phase AOA/TAC

300

assays. This method is reproducible, sensitive and has also a well-defined end-point.43

301 302

The main limitation of this method is the long reaction time and the necessity of multiple chromatographic analyses for each experiment.44

303

This assay is not suitable for high-throughput analyses because of the requirements of

304

multiple injections of each sample in order to measure the production of ethylene.13 The

305

reaction kinetic of this method does not permit a linear relationship between the percentage

306

inhibition of the probe and antioxidant concentration.45 There is absolutely no relationship

307

between the different multiple end-points, so it is difficult to make a comparison between

308

foods.13

309 310

2.4. Crocin Bleaching Assay

311 312

This assay described by Bors et al.3 is based on the competitive kinetic reaction of an

313

antioxidant and crocin as a naturally occurring carotenoid derivative (probe) with ROO• 14 ACS Paragon Plus Environment

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formed through thermolysis of AAPH in the presence of O2. Inhibition of crocin bleaching by

315

an antioxidant compound, as retarding the decrease in absorbance of the probe caused by

316

ROO•, was monitored at a wavelength of 443 nm (Ɛ=1.3x105 M-1cm-1) for 10 min. The crocin

317

bleaching rate constants and AOA of some phenolic antioxidants obtained by different

318

methods were well correlated each other. In this assay, abstraction of hydrogen atoms to the

319

polyene structure of crocin results in crocin bleaching.

320

Tubaro et al.4 later modified this method for the analysis of plasma antioxidant

321

capacity. Blanks without crocin were also monitored under the same wavelength to eliminate

322

sample-based interferences.

323

Initial crocin bleaching rate constants are calculated from the Eq. 7 where [AH] and

324

[crocin] are the concentrations of the antioxidant compound and probe (crocin), respectively,

325

and kAH and kcrocin are the second order rate constants for the reaction of the ROO• with

326

antioxidant and probe, respectively. The ratio of the rate constants (kAH/kC) could then

327

calculated by measuring the V0/V value at a known ratio of [AH] to [C] using the Stern–

328

Volmer-like relation (Eq. 14):46

329 330

V0 [ AH ] k = 1 + AH V kcrocin [ crocin ]

....(Eq. 14)

331 332

A rapid HPLC-crocin bleaching assay system for on-line detection of antioxidants was

333

developed using crocin as a substrate and AAPH as ROO• generator. This method is based on

334

detection of antioxidants by their inhibitory effect on the bleaching of crocin. Advantages of

335

this method are: usage of ROO• and polyunsaturated substrate as well as simplicity and

336

detection of positive peaks in the chromatogram. On the other hand, disadvantages of this



337

method are: reduced sensitivity, requirement of baseline correction, and high temperature of

338

reaction coil for significant oxidation of crocin.47

339

The AOA of ascorbic acid in the crocin assay (as TE)48 is unusually high as 7.7 (for

340

comparison, the cupric reducing antioxidant capacity (CUPRAC) value of vitamin C is 0.95,

341

in accordance with its 2-e oxidation to dehydroascorbic acid). Some phytochemicals compete

342

with crocin for ROO•, and the degree of inhibition of crocin oxidation may vary greatly,

343

depending on the AOA of tested sample. As a consequence, the reaction rates are very small

344

and are not sensitive to the changes of tested sample concentration. In addition, this test

345

suffers from the initial color of crocin in the presence of other carotenoids and colored

346

compounds. Therefore, the interpretation of the results can be complicated in analysis of

347

different food samples.49 Finally, crocin is subject to lot-to-lot variability, which limits its

348

application for reproducible TAC determination in a quantitative procedure.

349 350

2.5. Kinetics of HAT-Based Assays

351 352

The HAT mechanism involves the concerted transfer of H• from a donor (XH) to an acceptor

353

(Y) according to the reaction: (X-H + Y → X + H-Y). In HAT, the proton and electron of the

354

donated H-atom are transferred to the same atomic orbital (i.e. the transferring electron and

355

proton start and end in the same bond) whereas proton-coupled electron transfer (PCET)

356

involves several molecular orbitals.50 In fact, HAT may be visualized as a special case of

357

concerted PCET involving electronically adiabatic proton transfer.51 HAT and PCET have

358

one common point in that H+ and e- are transferred in one kinetic step from one group to

359

another.

360


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361

The distinction between HAT and PCET can be demonstrated by a comparison

362

between the phenoxyl/phenol and benzyl/toluene self-exchange reactions. The PCET

363

mechanism, responsible for phenoxyl/phenol exchange, requires the formation of a hydrogen

364

bond, and therefore is not valid for benzyl/toluene exchange.52 The phenoxyl/phenol reaction,

365

which involves nonadiabatic transitions between the reactant and product electron-proton

366

vibronic states (i.e. between different sets of orbitals), corresponds to PCET, while the

367

benzyl/toluene reaction, which involves electronically adiabatic proton transfer between the

368

same sets of orbitals and instantaneous electron response to the proton motion, corresponds to

369

HAT.53

370

Although Leopoldini et al.54 stated that the relative magnitudes of bond dissociation

371

enthalpy (BDE) and ionization potential (IP) determine whether the HAT- or ET-mechanism

372

is predominant for a given phenolic compound (i.e. a low BDE is required for a strong

373

phenolic antioxidant essentially acting by H-atom donation), Mayer showed that HAT rate

374

constants correlated better with R-H free energies rather than with their BDEs.55 Mayer also

375

demonstrated that the intrinsic barrier for H• transfer is also a rate-determining parameter, and

376

this barrier can be partly interpreted in terms of Marcus-type inner-sphere reorganization

377

energy, i.e. the energy required to reorganize the reactants and their surrounding solvent to the

378

structure of the product without the electron transferring.55 O-H bonds are more amenable to

379

HAT than C-H bonds of equal strength, and consequently, tert-butyl peroxy radicals (t-

380

BuOO•) abstract H• approximately 105 times faster from phenol than from toluene.50

381

However, the radical character at the abstracting atom is not a primary determinant of HAT

382

reactivity (e.g., metal-oxo abstracting groups in Ru(IV), V(V) and Mn(VII) complexes are not

383

radicalic).55

384



385

Leopoldini et al.54 calculated the BDEs and IPs of various phenolics with the help of

386

density functional theory (DFT), and hypothesized that α-tocopherol, hydroxytyrosol, gallic

387

acid, caffeic acid and epicatechin should predominantly oxidize by HAT-, while kaempferol

388

and resveratrol essentially by ET-mechanism. Warren et al.50 summarized the main preference

389

modes of oxidation for polyphenols, and proposed the primary mechanisms as: HAT for

390

simple phenols (with the exception of acidic phenols and under basic conditions, where

391

sequential proton loss electron transfer (SPLET) is predominant), HAT for α-tocopherol and

392

related phenols (such as the water-soluble analogue, trolox), PCET for butylated phenols (oil-

393

soluble synthetic antioxidants such as BHT, BHA, 2,4,6-Bu3PhOH), and PCET for catechols,

394

hydroquinones, and ascorbate. It should be emphasized that in ionizing solvents and under

395

basic conditions, ET may predominate over HAT for phenolic compounds, and in non-

396

hydrogen bond accepting solvents, catechols can be easily oxidized to ortho-quinones by H•

397

donation due to strong stabilization of the intermediary (semi-quinone) radicals by

398

intramolecular H-bonding.

399 400

3. MIXED-MODE (ET‒ AND HAT‒BASED) METHODS

401 402

3.1. ABTS/TEAC Assay

403 404

ABTS/TEAC assays use intensely-colored radical cation of ABTS as useful colorimetric

405

probes (Figure 4) accepting hydrogen atoms or electrons supplied by antioxidant compounds.

406

Antioxidant ability is measured as the ability of the test compound (e.g., Ph-OH) to decrease

407

ABTS•+ color by intercepting initial oxidation and preventing ABTS•+ production, or reacting

408

directly with the preformed radical cation (Eq.s 15&16).

409 18 ACS Paragon Plus Environment

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410


Figure 4

411 412

S2O82− +ABTS → SO42− +SO4•− + ABTS•+

… (Eq. 15) (λmax=734 nm)

413

ABTS•+ + PhOH → ABTS + PhO• + H+

… (Eq. 16)

414 415

Thus, aryloxyl radicals emerge as the first oxidation products from phenolics in the

416

ABTS assay. Thermodynamically, compounds with 0.68 V > Eo can reduce ABTS•+. The

417

high Eo of ABTS•+ increases the likelihood of interferences by side reactions with oxidizable

418

molecules, particularly alcohols, mono-phenols, and amino acids in natural extracts.56

419

Essentially, two strategies may be used in the design of ABTS assays; either the ABTS•+

420

radical is enzymatically generated and its formation retarded by antioxidants (lag time assays)

421

or a stable radical is first produced, and then its decolorization monitored after the addition of

422

antioxidants (fixed time assays). The original assay developed by Miller and Rice-Evans57,58

423

(original TEAC) utilized metmyoglobin-H2O2 to generate ABTS•+, which then reacted with

424

antioxidants. In fact, original TEAC and modified TEAC with potassium persulfate as oxidant

425

were totally different from one other, were applicable to different solvent media, and their

426

results for a tested antioxidant could vary significantly.59 The original TEAC assay was based

427

on the activation of metmyoglobin with H2O2/peroxidase to produce the ferryl myoglobin

428

radical, which in turn oxidized ABTS to the blue-green colored ABTS•+ in the presence or

429

absence of antioxidant compounds. In this assay, antioxidants are added before ABTS•+

430

formation is initiated by H2O2, resulting in a delay in radical formation (‘lag-time’) as an

431

indication of AOA. However, this reaction was criticized because faster reacting antioxidants

432

might also contribute to the reduction of the ferryl myoglobin radical as well as reacting with

433

ABTS•+, causing an overestimation of antioxidant activity.60 In addition, some antioxidants

434

may not show a distinct lag-time. The assay was modified by firstly generating a stable form 19 ACS Paragon Plus Environment


435

of the radical cation, ABTS•+, using oxidizing agents such as potassium persulfate,61-63 solid

436

manganese dioxide (MnO2),64-66 in situ electrochemical oxidation,67 and then adding

437

antioxidants and following direct reaction with the colored radical resulting in decolorization,

438

caused by H-atom abstraction of the colored ABTS•+ from the antioxidant. The absorbance

439

diminution at 734 nm of ABTS•+ could be monitored in the presence of TR,62,63,66 chosen as

440

reference standard. Thus, the TEAC value for an individual antioxidant molecule is actually

441

the number of ABTS radical cations it scavenges, compared to that of TR. In variations of this

442

approach, laccase,68 Br2•-,69 H2O2+HRP,70,71 and ROO•56 have also been used as oxidants to

443

generate a stable radical. Of all these, persulfate oxidation is frequently preferred for its high

444

ABTS•+ yields and radical/antioxidant inertness,72 and therefore forms the basis of current

445

TEAC assays. ABTS•+ has absorption maxima at wavelengths 645, 734 and 815 nm, as

446

reported previously,57,73,74 as well as at the more commonly used wavelength of 415 nm.

447

The modified assay uses stock solutions of concentrated ABTS•+ stored in the

448

refrigerator (stable for several months). The stock solution is diluted with water or buffer

449

prior to reaction. As antioxidants cause an absorbance decrease to a non-zero value, the

450

starting solution should have an absorbance of A=0.70, as recommended by Re et al.61, or 1.0

451

(corresponding to an initial radical concentration of 67 µM ABTS•+) as recommended by

452

Apak et al.72 Reactions are followed optically at 415 or 734 nm. The molar extinction

453

coefficient is higher at 415 nm (3.60x104 M-1cm-1)75 than at 734 nm (1.5x104 M-1cm-1 in water

454

and 1.6x104 M-1cm-1 in ethanol).61 However, some carotenoids, polyphenols or their oxidation

455

products may have overlapping absorbance at 400-450 nm, while 734 nm is out of the range

456

of possible interference, making the latter wavelength a suitable choice for TAC quantitation.

457

Initial absorbance of the ABTS•+ at 734 nm is monitored, the antioxidant compound is added,

458

and the decrease in absorbance is measured after reaction periods ranging from 4 minutes to a

459

few hours. ABTS•+ absorbance is monitored over a specified time with antioxidant, in parallel 20 ACS Paragon Plus Environment

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460

with a TR standard and with solvent (blank). TEAC is calculated by finding the ratio of the

461

extent of test compound reaction to that of TR reaction76 (Eq.17):

462

463

( A 0 − A f ) − ( A 0 − A f )blank  sample  TEAC (unitless) =  ( A 0 − A f )TR − ( A 0 − A f ) blank 

.....Eq.17

464 465

Practically, the TEAC value can be found from the ratio of the slope of (A0 − Af)sample

466

versus test compound concentration to that of a standard curve of (A0 − Af)TR plotted versus

467

TR concentration.61 TEAC values (unitless) reflect the ratio of the ABTS•+ scavenging action

468

of test compounds to that of TR. TEAC is the numerical value of mM TR-equivalent ABTS•+

469

scavenging capacity of 1.0 mM solution of the compound under investigation. For example, if

470

the TEAC coefficient of gallic acid is reported as 3.0 in the ABTS/persulfate method, this

471

means that 1.0 mM gallic acid solution behaves as 3.0 mM TR solution in decolorizing a

472

reference ABTS•+ solution.

473

In general, the ABTS/TEAC assay offers significant advantages contributing to its

474

widespread popularity in screening AOAs of a wide range of complex matrixes covering both

475

food and biological fluids. TEAC is operationally simple, reactions are rapid (some methods

476

use 30 minutes or less) and run over a wide range of pH. ABTS•+ is soluble in both aqueous

477

and organic solvents, enabling the TAC determination of both hydrophilic and lipophilic

478

antioxidants.72 Reactions can be automated and adapted to microplates,76,77 flow injections,78

479

and stopped flow methods. Nevertheless, to achive more reproducible results, many

480

parameters must be controlled, such as the method used for ABTS•+ generation with a suitable

481

oxidant, pH, temperature, time of ABTS•+ quenching (especially for slow reacting

482

antioxidants), ageing and storage conditions of the radical reagent, initial concentrations of



483

both reagent and analyte (antioxidant), choice of solvent, and calculation method, since the

484

results are strongly dependent upon these parameters.

485 486

Several criticisms have been directed to the ABTS/TEAC assay, which are summarized below:

487

(i) The tests do not measure ‘biologically relevant’ radical scavenging activity, and

488

ABTS•+ is N-centered rather than O-centered, so it may not truly represent the radical

489

reactions occurring with antioxidants in foods and biological systems.

490

(ii) As molecular size and steric accessibility are important parameters in the TEAC

491

test, small molecules and reducing agents give more reasonable results. The responsiveness of

492

the radical cation of ABTS to polyphenolics with bulky substituents is sterically limited.

493

Although reaction stoichiometry (e.g., TEAC coefficients) correlated well with the number of

494

phenolic groups (r2>0.81), the Ph-OH groups of heterocyclic polymeric phenols (e.g.,

495

condensed tannins) may show hindered steric accessibility to the ABTS•+ site.79 In this case, a

496

stable end-point may not be reached within the protocol time of the decolorization assay.

497

While linear curves indicate free access to the ABTS•+ site, a decreasing curve with increasing

498

antioxidant concentrations may reflect impaired access.

499

(iii) The radical cation of ABTS non-selectively oxidizes phenolic –OH groups

500

irrespective of their antioxidant power in real systems.80 For example, ROO• -scavenging rate

501

constants of catechol and hydroquinone were higher than that of resorcinol;81 moreover, in a

502

microsomal model, the antioxidant protective effect of catechol and hydroquinone was much

503

stronger (by 1-2 orders-of-magnitude) than that of resorcinol.82 Yet, the TEAC coefficient of

504

resorcinol (2.49) was much higher than those of either catechol (1.43) or p-hydroquinone

505

(1.33), revealing the contradiction between in vivo TAC and in vitro ABTS/TEAC

506

measurements.80


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507

(iv) ABTS•+ is a mixed-mode assay reagent in the sense that it reacts by both ET and

508

HAT mechanisms (with reducing agents and H-atom donors, respectively), so its reaction

509

mechanism with individual antioxidants or extracts is not clear and can vary with reaction

510

conditions. Tian and Schaich79 have proposed that instantaneous reactions result from

511

dominant ET-mechanism by individual phenol groups with greatest steric access to the

512

ABTS•+ site, and that slow sustained reactions with no initial absorbance decrease result from

513

HAT-mechanism.

514

(v) In its present form, the end-point assay reports stoichiometric conversion (e.g.,

515

TEAC coefficients) rather than kinetic rates for phenols oxidation, which does not precisely

516

correspond to the in vivo action of antioxidants; antioxidant compounds having very fast

517

initial rates toward ABTS•+ may show low TEAC values.79 In a recent critical study on

518

ABTS•+ kinetics, Tian and Schaich79 were able to identify six distinguishable kinetic patterns

519

of phenols oxidation, including both immediate and extended (sometimes up to many hours)

520

reaction components, and found that rates were highly dependent upon antioxidant

521

concentration. Under the described conditions, the reaction between antioxidants and ABTS•+

522

may not reach completion within the time span of the assay protocol, leading to an

523

underestimation of the TEAC values of these antioxidants; thus different approaches to the

524

calculation of antioxidant activity were made.83 Consequently, Tian and Schaich79

525

recommended a different calculation of ABTS/TEAC test results for further research by

526

simultaneously considering reactivity as well as stoichiometric conversion efficiency. In

527

addition, due to strong dependency of reaction rates on antioxidant concentration, they

528

recommended the use of a wide range of concentrations to construct the concentration curves

529

from which second-order rate constants and response saturation could be determined.

530

(vi) Walker and Everette84 compared the ABTS•+ oxidation rates of various

531

antioxidants and found that the reactions followed first-order kinetics, chlorogenic acid and 23 ACS Paragon Plus Environment


532

caffeic acid showing the longest half-lives among the tested phenolic compounds. Most

533

phenolics showed a biphasic kinetic reaction pattern involving fast and slow steps, and

534

aminothiols followed a fast step up to disulfide formation, followed by a slow step leading to

535

further oxidation products. The authors concluded that before determining TEAC coefficients

536

in an ‘end-point’ assay, one has to observe the kinetic profiles of the tested antioxidants for

537

ABTS•+ oxidation so as to decide whether longer incubation times are needed to obtain

538

reliable data.84

539

(vii) Non-specific side reactions in ABTS•+ oxidations are common. For example,

540

thiols (-SH) are probably oxidized to higher oxidation products (e.g., sulfenic acids: -SOH)

541

with ABTS•+ rather than to the physiologically relevant disulfides (-SS-), because the TEAC

542

value of glutathione in ABTS/persulfate assay was reported as 1.3-1.5, much larger than the

543

CUPRAC-TEAC value of 0.5 corresponding to a neat one-electron oxidation to the

544

corresponding disulfide (2RSH → RSSR + 2H+ + 2e-).85 On the other hand, surprisingly poor

545

reactivity of protein thiols toward ABTS•+ was reported in other sources.72

546

(viii) The applicability of the assay to either the establishment of structure-activity

547

relationships of phenolic compounds or to proper ranking of antioxidants may not be relevant,

548

because in certain cases, the initial oxidation product formed with ABTS•+ may react faster

549

with the assay reagent than the parent phenolic such as chrysin.86 Likewise, the antioxidant

550

efficiency order of hydroxycinnamic acids in protection against lipid peroxidation and in

551

CUPRAC tests as: caffeic acid > ferulic acid > p-coumaric acid87 was completely reversed in

552

the ABTS/TEAC test. The unexpected high AOA (i.e. more than 2-electrons per –OH) of

553

ferulic acid was attributed to the generation of secondary products such as quinones that may

554

further react with ABTS•+.79,88

555


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556

ABTS•+ as a large nitrogen-centered and sterically-hindered radical may not be

557

suitable for simulating small highly-reactive radicals (e.g., •OH, NO•, O2•−) that are active in

558

biological tissues and foods.89 It may also miss the activity of certain bioactive compounds

559

(e.g., thiols and proteins). Even if the ABTS/TEAC assay may not be exactly suitable for

560

TAC ranking in food and biochemistry, our research group has found the test competitive to

561

CUPRAC in various cases, because it is simple, versatile, reproducible, and applicable to both

562

hydrophilic and lipophilic antioxidants, especially in biological fluids. Thus, this test can be

563

most fruitful in reflecting the TAC changes of a given sample or interrelated group of

564

samples, such as in monitoring the changes in TAC of human fluids of a given individual

565

under medical treatment, or of a given food sample subjected to processing (e.g., heat

566

treatment applications including drying, roasting, and packaging film extrusion). In such cases

567

of comparative TAC measurement, even though limitations to the assay continue to exist,

568

antioxidant components remain constant in the measured system and variations in ABTS•+

569

accessibility are not the prime determinant of reactivity, therefore meaningful results can be

570

obtained.79

571

Hyphenated techniques enable the rapid and selective detection of radical scavengers

572

in the tested samples. Kalili et al.90 investigated the AOA of individual phenolic antioxidants

573

in natural products (e.g., cocoa, red grape seed and green tea) by using an on-line two-

574

dimensional liquid chromatography (LC/LC) coupled to the ABTS assay, allowing detailed

575

characterization of phenolic compounds in complex matrixes. Polar phenolic antioxidants

576

were separated by hydrophilic interaction chromatography (HILIC) in the first dimension,

577

while nonpolar phenolics were separated by reversed-phase liquid chromatography (RP-LC)

578

in the second dimension. On-line HILIC/RP-LC–ABTS method offers high throughput, while

579

the off-line method as an alternative to conventional AOA/TAC assays offers higher

580

resolution. 25 ACS Paragon Plus Environment


581

The first flow injection analysis (FIA)-ABTS•+ assay consisting of a single-channel

582

flow manifold was developed by Pellegrini et al.78 for the evaluation of TAC of some food

583

extracts and beverages (i.e., cola, coffee, beer). The results of the proposed assay were

584

compared to those of the batch assay, and it was demonstrated that the TAC values obtained

585

by the two methods were not statistically different. On the other hand, LOQ was lower by at

586

least two orders of magnitude in comparison to the batch assay, attributed by the authors to

587

the geometry of the FIA system. However, TAC measurement in biological samples (e.g.,

588

plasma) using this FIA-ABTS•+ assay partly failed, possibly due to the presence of diverse

589

constituents that may produce masking features at high concentrations in plasma. In this

590

context, Bompadre et al.91 introduced temperature control of the reaction coil and proposed

591

minor changes in the flow manifold (e.g., reaction coil configuration and sample volume).

592

Using this modified system, a controlled temperature (35 °C) was demonstrated as a critical

593

point in the repeatable determination of TAC of plasma sample and of less complex biological

594

mixtures (white wines and mouthrinse). One of the limitations of single-channel flow systems

595

is the depletion of reagent in the central zone of the sample plug. This limitation was

596

overcome using an ABTS•+ assay adopted to a double-line FIA system,92 which allowed the

597

addition of reagent to sample plug with different channel. This double-line FIA system offers

598

a 4-times higher sample throughput in comparison to the single channel system. A stopped-

599

flow method was also developed by93 to examine the pH and timing effect on the ABTS•+

600

scavenging reactions of various antioxidants. The authors showed that TEACABTS values of all

601

studied antioxidant compounds (i.e., p-coumaric acid, albumin, BHT, glutathione, quercetin)

602

were dependent on reaction time and pH, because of the different reaction rates of the tested

603

antioxidants. Generally, FIA-ABTS•+ assays rely on the ABTS•+ preformed off-line by

604

chemical oxidation of ABTS with persulfate or H2O2. This radical generation technique is

605

time consuming because of the slow kinetics of ABTS oxidation. Ivekovic et al.94 introduced 26 ACS Paragon Plus Environment

Page 26 of 65

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606

an improved TEAC decolorization assay in conjunction with FIA for the evaluation of the

607

AOA of antioxidant compounds and some beverages in which ABTS•+ was generated on-line

608

by electrochemical oxidation in the flow-through electrolysis cell. This technique avoids the

609

time consuming step of ABTS•+ preparation by chemical oxidation, resulting in reduced

610

analysis time. In addition to the on-line electrochemical oxidation, an on-line enzymatic

611

generation of ABTS•+ with electrochemical determination of AOA was described by

612

Milardovic et al.95 This method is based on the continuous enzymatic production of ABTS•+

613

by HRP in a tubular flow-through reactor and biamperometric measurement (i.e.,

614

measurement of the current flowing between two identical working electrodes polarized at a

615

small potential difference and placed in a solution containing a reversible redox couple) of

616

residual reduced concentration of ABTS•+. In FIA-based methods, TEAC values of slow

617

reacting antioxidants such as catechins are strongly dependent on time, and the oxidation

618

reaction may not reach completion within the selected time interval resulting in

619

underestimated TEAC values. To overcome this limitation, a kinetic matching approach for

620

the fast determination of TAC using a sequential injection system with a miniaturized lab-on-

621

valve (LOV) was proposed by Ramos et al.96 The method was applied to the evaluation of the

622

TAC of red wines in less than 1 min using tannic acid as reference, and the results were

623

correlated with the TAC determined by microplate-based ABTS method.

624 625

3.2. DPPH Radical Scavenging Assay

626 627

The stable chromogen radical DPPH (Figure 5) was first proposed for quantitating

628

antioxidant content more than half a century ago, when Blois97 used the thiol-containing

629

amino acid cysteine as his model antioxidant. Later it was used as a phenol reagent.98 The

630

more recently introduced method of Brand-Williams et al.99 has been used as a reference 27 ACS Paragon Plus Environment


631

assay by various researchers.100,101 Reaction with DPPH was adapted for measuring radical

632

quenching kinetics,102,103 and since then numerous variations for following the reaction and

633

for calculating relative AOA by reaction stoichiometry have evolved.72,99 The reaction

634

equation can be formulated with respect to HAT-mechanism, although proton-coupled ET-

635

mechanism cannot be excluded, especially in phenol-ionizing solvents and at alkaline pH (Eq.

636

18):

637 638

DPPH• + PhOH → DPPH2 + PhO• … (Eq. 18)

639 640

where DPPH• is a stable chromogen radical (Figure 5) with λmax=515 nm.

641 642

The DPPH assay is low-cost and simple, and therefore has been widely used in

643

conventional laboratories for extensive applications. This method was citicized for lacking

644

standardization in sample preparation, analytical protocols, reaction conditions, and

645

expression of findings.13

646 647

Figure 5

648 649

DPPH crystals are generally dissolved in ethanol or methanol to give an initial

650

absorbance of ~1.0. Then an aliquot of the antioxidant is added, the mixture incubated for 30

651

min, and the final absorbance recorded. The original purple color of the solution fades to

652

yellow due to the reduction of DPPH• to DPPH2 by H-atom abstraction from antioxidants.

653

The extent of reaction is usually measured as (A0 − Af) and AOA is expressed either as IC50

654

(total antioxidant concentration necessary to reduce the initial DPPH• absorbance by 50%) or

655

mM TR-equivalent (TE) concentration of the tested sample solution in comparison to the 28 ACS Paragon Plus Environment

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656

decolorization provided by the reference compound, TR. IC50 is not a sufficiently objective

657

parameter for comparing results among different laboratories, because the IC50 values

658

reported in literature by different researchers for ascorbic acid and BHT vary over more than

659

one order-of-magnitude.104 As alternative to spectrophotometry, the loss in EPR signal of the

660

DPPH• can also be measured.80 Since these approaches report the extent of reaction (i.e.

661

reaction stoichiometry) rather than rate, fast and slow reacting antioxidants cannot be

662

distinguished from conventional reporting of results. Monitoring absorbance loss continually

663

during reaction of a range of antioxidant concentrations provides a series of curves from

664

which rates and rate constants can be calculated.

665

Kinetic and thermodynamic requirements of the method were met with two different

666

(dynamic and static) versions of the assay. In the dynamic version, the rate of DPPH• decay

667

was measured right after the addition of phenolic antioxidant,105,106 while in the dynamic

668

version, the consumption of the chromogen radical after a fixed time of reaction with the

669

antioxidant sample was measured107,108 to reveal stoichiometric conversion. However, fixed-

670

time assays have the disadvantage of underestimating the radical scavenging activities of slow

671

reacting antioxidants whose steady-state decolorization of DPPH• may take as long as several

672

hours. Sánchez-Moreno et al.109 tried to unify both (dynamic and static) approaches by

673

defining a new activity parameter, antiradical efficiency (AE), such that AE = 1/(EC50×t50),

674

where EC50 was the concentration of antioxidant compound necessary to decrease the initial

675

DPPH• concentration by 50%, and t50 was the time required to reach a steady-state

676

concentration corresponding to EC50. Although AE was claimed to be a more discriminatory

677

parameter than either the reaction rate or stoichiometric conversion of DPPH• decay, its

678

physical meaning was vague and antioxidants were listed in an uncommon order of DPPH•

679

scavenging power (e.g., ascorbic acid > caffeic acid ≥ gallic acid > tannic acid ≥ α-tocopherol

680

> rutin ≥ quercetin > resveratrol) using this parameter.80,109 29 ACS Paragon Plus Environment


681

Several criticisms have been directed to the DPPH• assay together with some

682

recommendations for further research, and these have been well summarized by Mishra et

683

al.,104 Apak et al.72 and Xie and Schaich110 to focus on the following points:

684

(i) DPPH• is a long-lived N-radical, whereas most physiological antioxidants act

685

against ROS; therefore it is difficult to model in vivo antioxidant activity with in vitro DPPH

686

assays. Additionally, DPPH• decay kinetics and thermodynamics are rather different from

687

those of other assays, revealed in the low correlations with other assay results in comparative

688

TAC evaluation. The assay is known to work well with lipophilic (rather than hydrophilic)

689

antioxidants in alcohol solvents.111

690 691

(ii) Steric accessibility to the DPPH• radical site may be more rate-controlling than the chemical structural features of antioxidants.110

692

(iii) There is strong solvent- and pH-dependency in the mixed-mode (HAT and ET)

693

DPPH• reactions. Solvent effects on fast reactions of DPPH• decay and phenols oxidizability

694

have been well documented.112,113 In phenol-ionizing solvents, the ET-rate is primarily

695

controlled by the formation of phenolate anion with concomitant increase in pH. In hydrogen-

696

bond accepting (HBA) solvents, the HAT-mechanism is essentially hindered because of the

697

difficulty in H-atom donation of an inter-molecularly bonded phenol (i.e. Ph-OH…S), where

698

(S) denotes a solvent molecule.113 Strong hydrogen bonding solvents impede HAT.114 As a

699

result, ET-reactions with DPPH• are very fast and not diffusion-controlled, whereas HAT-

700

mechanism is slower and essentially diffusion-controlled.110 Concerning the rates of cinnamic

701

acids oxidation with DPPH•, Foti et al.115 reached the conclusion that in (DPPH• + ArOH)

702

reactions performed in methanol and ethanol, the rate-determining step consists of a fast ET-

703

process from the phenoxide anions to DPPH•, leaving the very slow H• abstraction from

704

neutral ArOH by DPPH• a marginal reaction path because of the strong HBA character of

705

alcohol solvents. Adventitious acids and bases (that may enter the medium from various 30 ACS Paragon Plus Environment

Page 30 of 65

Page 31 of 65


706

sources) may also affect the rates, because a rise in pH dramatically increases the reaction

707

rates by supporting phenols ionization, while Brønsted acids significantly retard the reaction.

708

In this regard, DPPH• reactions may be recommended to be carried out in methanol or

709

aqueous mixtures of methanol, because methanol is the alcohol that best supports phenols

710

ionization (and subsequently the ET-mechanism of phenols oxidation).

711

(iv) There are fast (e.g., ascorbic acid, in seconds), medium (e.g., most phenolics, with

712

5-min end points) and slow (e.g., curcumin, 3 h) reacting antioxidants in the system, with

713

some known antioxidants (e.g., melatonin, α-lipoic acid and pentoxifylline) almost not

714

reacting at all.104,110 The oxidations of compounds known to react with basically HAT-

715

mechanism (e.g., uric acid and thiols) are generally slow. Both eugenol and BHT oxidations

716

with DPPH• are second-order (with fractional dependency on phenol and DPPH•

717

concentrations) and reversible, reaching steady state within 2 h and 5 h, respectively.116

718

Mishra et al.104 found the steady-state absorbance decrease time of gallic acid and ferulic acid

719

up to 3 h, and of BHT and curcumin around 6 h. The reversibility of the reactions of o-

720

methoxyphenols structurally similar to eugenol may give rise to falsely low readings in TAC

721

measurement with the DPPH method.14

722

(v) The assay should be run over a full range of antioxidant concentrations (e.g., 3

723

orders-of-magnitude) to reach reliable results. Tests carried out at a great excessive

724

concentration of antioxidant over DPPH• may yield questionable results due to the changes in

725

reaction kinetics, especially when large polymeric phenolics and extracts of unknown

726

composition are to be analyzed.110

727

(vi) The evaluation of AOA by the change in DPPH• absorbance should be carefully

728

interpreted since the absorbance of DPPH• at 517 nm after reaction with a tested antioxidant

729

may be decreased by some other parameters (i.e., pH, O2, light, and type of solvent).117 While

730

neither O2 nor its first reduction product, O2•−, is known to directly affect DPPH• within the 31 ACS Paragon Plus Environment


731

protocol time, each antioxidant should be evaluated for possible O2 effects (for example,

732

higher results of DPPH• scavenging for ascorbic acid were obtained under argon or nitrogen

733

atmosphere).72

734

(vii) Fixed-time assays may underestimate the radical scavenging activities of slow

735

reacting antioxidants.104 As with the initial rate of reaction, stoichiometry showed negligible

736

correlation to either redox potential or to the number of phenolic hydroxyl groups.110 Since

737

the ionization of phenols and subsequently the reaction rates are greatly affected by pH and

738

solvent composition, the DPPH test results may be questionable for ranking antioxidant

739

compounds and natural extracts,110 chemically unsound and should always be comparatively

740

evaluated with those of other (in vitro or in vivo) assays of AOA.14

741

Recently, Foti118 has concluded that EC50 is not considered as a kinetic parameter in a

742

standard DPPH end-point assay run for a prespecified arbitrary time. The EC50 value is

743

essentially related to the stoichiometric factor of Eq.19 (n = ∆[DPPH]/∆[AH]) which is an

744

important complementary parameter for antioxidant compounds (for a potent antioxidant, n ≥

745

2).

746

747

AH + nDPPH • → A • +DPPH-H

....(Eq.19)

748

EC50, as being the effective concentration of antioxidant scavenging 50% of initial

749

[DPPH], is inversely proportional to n when the reaction (Eq.19) is complete and thus

750

phytochemicals with large n can be classified by this test as potent antioxidants with low EC50

751

values. When decreasing concentrations of DPPH are plotted against increasing

752

concentrations of antioxidant: [AH], ideally a linear curve is obtained with the Eq. 20:

753 754

[DPPH] = -n [AH] + [DPPH]o

…. Eq. 20


Page 32 of 65

Page 33 of 65


755

Since at 50% scavenging of the initial radical concentration: [DPPH]o, the instantaneous value

756

of [DPPH] will be equal to [DPPH]o/2, we derive (Eq. 21):

757 758

EC50 = [DPPH]o/2n

… Eq. 21

759 760

As can be seen from this equation, EC50 is rather a subjective parameter, and is

761

dependent on both the initial concentration of DPPH and the stoichiometric factor of the

762

antioxidant for DPPH scavenging under the chosen experimental conditions (e.g., solvent, pH,

763

temperature, time, etc.). Moreover, n (and therefore EC50) may depend on [AH] used in the

764

experiments. It should also be noted that the products formed in reactions ( A • + DPPH • ) and

765

(A

•

+ A • ) may also react with DPPH•, resulting in an increase in the value of n.118

766

Andrei et al.119 developed a DPPH•-based electrochemical method, based on the

767

reduction of DPPH at a screen-printed gold electrode, for measuring the TAC of wine. Since

768

DPPH was detected on this selected electrode after preincubation of (DPPH+sample) mixture

769

and injection in a continuous buffer flow, TAC values of samples, especially slow reacting

770

samples with DPPH, were correctly evaluated by this method. As an alternative to

771

conventional DPPH assay, an amperometric method for colored plant samples was introduced

772

by Tyurin et al.,120 which is based on the monitoring of the signal from the reduction of

773

DPPH at a carbon nanotube‒modified glassy carbon electrode.

774

Nuengchamnong and Ingkaninan adapted a hyphenated technique using a combination

775

of RP-HPLC separation coupled to electrospray ionization (ESI)-MS detection with DPPH

776

assay for screening multiple antioxidant molecules in Antidesma thwaitesianum Muell. fruit

777

wine.121 The phenolic antioxidant molecules with different chemical structures could be

778

successfully determined in one run by using the information of hyphenated chromatographic

779

technique. These molecules were simultaneously monitored and identified (i.e., gallic acid, 33 ACS Paragon Plus Environment


780

catechin, caffeic acid, cyanidin-3-sophoroside, delphinidin-3-sambubioside, monogalloyl

781

glucoside, and pelargonidin-3-malonyl glucoside). This assay might be useful for the rapid

782

characterization of antioxidant molecules in a large number of plant extracts.121 Antioxidants

783

in the aqueous extract of Houttuynia cordata, mainly consisting of chlorogenic acid and its

784

derivatives, catechin and procyanidin B, were also characterized using the on-line DPPH

785

assay coupled with LC-ESI-MS.122

786

Ukeda et al.123 reported a FIA-DPPH system using ESR spectrometer for the rapid

787

estimation of AOA of various substances and food samples via DPPH• scavenging. In a

788

double line flow system, DPPH was fed into a flat cell, providing the largest change in the

789

ESR signal at a fixed magnetic field (335.3 mT). When the antioxidant compound was

790

injected into the carrier system, a negative peak was obtained in proportion to the

791

concentration of antioxidant. This assay, having a standard reaction time of 16-30 min for a

792

single plant extract, was claimed to be superior to the conventional DPPH method for

793

screening large series of food samples. The automation of DPPH assay was described by

794

Polasek et al.124 using a sequential injection analysis (SIA) manifold for fast screening AOA

795

of biological samples. A multi-syringe FIA (MSFIA)-DPPH• automatic system was further

796

developed for the assessment of TAC by monitoring the absorbance decrease along time.125 A

797

stopped-flow approach was chosen because of the different reaction kinetics of the tested

798

antioxidants. The TAC of samples containing fast antioxidants (e.g., ascorbic acid) can be

799

determined by using a single absorbance measurement after flow stop, while a combination of

800

FIA system and mathematical modeling can be applied to estimate the TAC of slow reacting

801

antioxidant samples. Bukman et al.126 proposed an FIA-DPPH system in which flow

802

parameters were optimized with the response surface methodology employing central

803

composite rotatable design. The proposed method was applied to wine samples, and the

804

results showed good correlation with the conventional DPPH assay.


Page 34 of 65

Page 35 of 65

805


3.3. DMPD Radical Scavenging Assay

806 807

N,N-Dimethyl-p-phenylenediamine dihydrochloride (DMPD) is a compound that is normally

808

used to measure the antioxidant capacity of fruit juices and other natural products. It is

809

converted to the quinonic DMPD•+ in the presence of oxidants such as ROS (•OH, O2•-) or

810

ferric iron, which is scavenged by antioxidants present in studied matrixes. Thus, the DMPD

811

method is capable of measuring both the oxidative status and antioxidant capacity of

812

unknown samples. This method is based on the conversion of the DMPD to colored DMPD•+

813

in the presence of a suitable oxidant at weakly acidic pH.127 DMPD•+ shows maximum

814

absorbance at 505 nm. Antioxidant molecules which are able to transfer a H-atom (or an

815

electron, depending on the polarity of solvent and ionization potential of donor) to DMPD•+

816

quench the radical and cause decolorization of the solution. Alternatively, antioxidants may

817

react with ROS and hinder the formation of DMPD•+ from DMPD. The decolorization

818

reaction is rapid, and the very stable end-point is taken as a measure of the antioxidant

819

potency. This is very important when large-scale screening is required. The use of this colored

820

radical cation has been widely extended to evaluate the TAC of different food products (i.e.,

821

fruits, vegetables) and wine.127-129 This method focuses on the ability of the antioxidant

822

molecules to transfer a hydrogen atom to the colored DMPD•+, turning it into an uncolored

823

DMPD.127 The basic drawback of this method was indicated as the loss in sensitivity and

824

reproducibility when hydrophobic antioxidant molecules (e.g., BHT or tocopherol) were

825

studied.130 Corral-Aguayo et al.129 examined the correlation between some nutritional

826

parameters and the TAC values measured with six different assays (i.e., DPPH, DMPD,

827

FRAP, ORAC, TEAC, and TOSC assays) in eight horticultural crops. They found that from

828

hydrophilic extracts, vitamin C and total soluble contents were highly correlated with TAC



829

values for all assays, while from lipophilic extracts, total carotenoids and β-carotene contents

830

possessed a high correlation with TAC only in the DMPD assay.

831

On the other hand, Gil et al.131 examined the TAC of pomegranate juices by different

832

spectrophotometric TAC methods (i.e., DPPH, ABTS, FRAP, and DMPD) in comparison

833

with green tea infusion and red wine. Since some water-soluble constituents of pomegranate

834

juice reacted with DMPD•+, high TAC values were observed for these fruit juices with respect

835

to the DMPD method. On the other hand, these water soluble compounds did not present free-

836

radical scavenging activity with the other two methods. According to the results, it was

837

reported that DMPD method should be used with caution for evaluating the TAC, especially

838

in those food products which are rich in organic acids (e.g., citric acid).

839

Mehdi and Rizvi132 modified the DMPD assay for the detection of plasma oxidative

840

capacity during human aging by measuring the oxidant potential of plasma using DMPD

841

reagent. As mentioned above, in the presence of Fe3+, DMPD•+ is formed which is scavenged

842

by antioxidants present in the studied analyte. This modified method is fast and

843

reproducible132, but it cannot be applied to the determination of AOA of plasma in the

844

presence of iron. The researchers indicated that the method is fast and reproducible.132

845

A colorimetric DMPD sensor was developed in our laboratory by immobilizing

846

cationic DMPD semi-quinone derivatives (DPDMQ), formed from the reaction between ROS

847

and DMPD, on a persulfonate-based Nafion membrane for simultaneous determination of

848

oxidative status and AOA.133 The decrease in absorbance of the sensor in the presence of

849

antioxidants enabled the determination of TAC of a complex sample. This sensor can

850

succesfully detect oxidants along with antioxidant compounds and basically yield higher

851

molar absorptivities for antioxidants in comparison to the CUPRAC sensor.

852


Page 36 of 65

Page 37 of 65


853

4. KINETICS AND MEASUREMENT OF LIPID PEROXIDATION AND

854

ITS INHIBITION

855 856

Lipid peroxidation is a radical chain reaction process including the initial formation of lipid

857

radical (L•), hydrogen atom abstraction by lipid alkoxyl radical (LO•) or lipid peroxyl radical

858

(LOO•), O2 addition to carbon centered radical, LOO• addition to carbon-carbon double bonds

859

and finaly LOO•-LOO• termination steps resulting in the oxidative destruction of

860

polyunsaturated fatty acids (PUFAs) containing lipids.134 The overall process of lipid

861

peroxidation mainly consists of three steps:

862

(i) initiation: formation of L• ;

863

(ii) propagation: oxygen uptake by lipid radicals to produce lipid peroxyl radicals, which in

864

turn generate new lipid radicals through radical chain reactions;

865

(iii) inhibition and termination: formation of non-radical products, where chain-breaking

866

antioxidants, when present, are consumed by reacting with these lipid-oxidizing radicals to

867

produce the more stable antioxidant radicals.

868

The overall process may be symbolized by reaction Eq.s (22-27):

869 870

Initiation:

kd In-In  → In • + In • (initiator)

(Eq. 22)

kiLH In • + LH → InH (reduced form) + L•

(Eq. 23)

perox L• + O 2  → LOO•

k

871

872

Propagation:

Termination:

•

kp

(Eq. 24) •

LOO + LH → LOOH + L

(Eq. 25)

kt 2LOO•  → [ LOO-OOL]

(Eq. 26)

[ LOO-OOL]

(Eq. 27)

→ Non radical products + O 2

873



874

The initiation step involves the formation of a lipid radical ( L• ) which may later

875

maintain the chain reaction of lipid oxidation. Various methods have been used to initiate the

876

in vitro oxidation of lipids such as usage of enzymes (e.g., lipoxygenase), copper and iron

877

salts, •OH, and cultured cells that can generate ROS/RNS.135 Some azo compounds (i.e.,

878

AAPH, AMVN) as free radical initiators have also been used.135 Thermolysis of these azo

879

compounds by first order-kinetics result in the production of free radicals. For a first order

880

decomposition reaction of azo compounds, kd is strongly affected by structure. Two carbon

881

centered alkyl radicals (R•) are produced from AAPH (kd= 1.5x10-6 s-1; 37 °C, methanol

882

phase), while two RO• generated from DTBN (di-tert-butylhyponitrite) (kd= 8x10-6 s-1; 37 °C,

883

iso-octane phase) that can initiate free radical chain process.135 The rate of lipid peroxidation

884

is not affected by lipid chain length, but by the number of bis-allylic carbons (e.g., PUFA) in

885

lipids and by the dissociation energies of carbon-hydrogen (C-H) bonds in lipid chain.136

886

The propagation step involves the addition of molecular O2 to L• to generate LOO•

887

(Eq. 24) and hydrogen atom transfer from the organic substrate to LOO• (Eq. 25). The rate of

888

the first reaction, near the diffusion controlled rate,137 is dependent on O2 concentration (Eq.

889

28). As compared to the other reactions of the free radical chain oxidation, the propagation

890

rate of hydrogen atom transfer reaction is generally slow.138

891

892

−

d [O2 ] d [t]

=

kp

( 2kt )

1/2

[ LH ] Ri1/2

....(Eq.28)

893 894

In Eq. (26), Ri is the radical generation rate, and kp and kt are the rate constants of

895

propagation and termination reactions, respectively. The propagation rate constants of lipid

896

samples were generally lower than those of other radical reactions, because the slow step in

897

radical chain oxidation is usually the H-atom transfer from LH to LOO• in Eq. (25).139


Page 38 of 65

Page 39 of 65


898

Arachidonic acid, a polyunsaturated fatty acid that is normally thought of as being particularly

899

prone to peroxidation, was over an order of magnitude less reactive in chain propagation than

900

7-dehydrocholesterol, known as the immediate biosynthetic precursor of cholesterol.139

901

Hydrogen atom abstraction from a bis-allylic site on the lipid chain to LOO• is

902

thermodinamically favorable with a Gibbs free energy change of ∆G=-9 kcal mol-1.140

903

Moreover, LOO• with high standard reduction potential (+1.0 V) can oxidize PUFA. The

904

termination step consists of the reaction of LOO•s with each other and self-destruction to

905

form non-radical products.

906

Antioxidants (AH), when present, quench lipid peroxyl radicals and subsequently

907

retard lipid (LH) autoxidation with the inhibition reaction (i.e., LOO• + AH → LOOH + A•)

908

having a rate constant: ki. The antioxidant is sacrificed in this reaction, and its derived radical

909

(A•) is rapidly converted into stable products without damaging lipids (with the assumption

910

that the tested antioxidant does not exhibit prooxidative behavior). Under under steady-state

911

conditions, the rate of uninhibited (runh) and inhibited (rinh) oxidations can be expressed by the

912

equations (Eq.s 29 & 30):

913 914

runh = kp [LH] {Ri /(2kt)}1/2

915

rinh = kp [LH] Ri / (n ki [AH]) … (Eq. 30)

… (Eq. 29)

916 917

where Ri is the constant flux rate of peroxyl radicals thermally generated from an azo-

918

compound and n is the number of radicals scavenged per molecule of AH.14 The antioxidant

919

activity of the tested antioxidant can be evaluated by the relative magnitude of the rate of

920

inhibited reaction (rinh) with respect to that of uninhibited (runh). Antioxidants efficient in

921

inhibiting lipid peroxidation should have a large ratio of (ki/kp). Roginsky and Lissi80 39 ACS Paragon Plus Environment


922

extensively reviewed the quantitation methods of chain-breaking antioxidant activity by the

923

kinetic approach. By equalizing (rinh/runh) to 0.5, the 50% inhibitive concentration of the

924

tested antioxidant, IC50, can be found for n=2 antioxidants using the Eq. (31):

925 926

IC50 = (2kt Ri)1/2/ ki

... (Eq. 31)

927 928

which is a good way of expressing AOA, because effective antioxidants should show low IC50

929

values in the test system.80

930

In recent years, various indirect methods have been developed to evaluate the relative

931

AOA of chain-breaking antioxidants for protection against lipid peroxidation. The most

932

widely used and practical methods involve measuring11:

933

(i) substrate loss: β-Carotene (βC) bleaching assay

934

(ii) primary oxidation products: Determination of hydroperoxides by iodometry and

935

colorimetric ferric-thiocyanate or ferric-xylenol orange assays; ultraviolet (UV) spectroscopic

936

measurement of conjugated dienes

937

(iii) secondary oxidation products: TBARS assay

938 939

4.1. β-Carotene Bleaching Assay

940 941

One of the most common techniques for assessing lipid peroxidation described by Marco141,

942

βC bleaching assay, is based on the measurement of βC absorbance decay at 470 nm,

943

resulting from βC-linoleic acid degradation. Radicals generated from the autoxidation of

944

linoleic acid (used in a lipid model system) cause βC bleaching, which is retarded by

945

antioxidants. The reaction is performed in an aqueous emulsion of βC and linoleic acid using

946

tween surfactant (e.g., Tween 40). Radicals generated by the spontaneous oxidation of linoleic


Page 40 of 65

Page 41 of 65


947

acid are promoted by thermal induction at 50°C. The addition of an antioxidant compound

948

retards βC bleaching. The low reproducibility, incorrect quantification, complexity of the

949

reagent preparation, and certain interference parameters (e.g., pH, temperature, and solvents)

950

are the main limitations of this test.142 This nonspecific method involves uncontrolled

951

thermally-induced oxidation, which often induces data variability. This method is also

952

hampered by interferent absorbent compounds in the βC spectrum.11 It is not easy to interpret

953

the AOA findings because βC itself is an O2-sensitive antioxidant. Additionally, the lack of

954

reproducibility of initiation, the crude kinetic treatment and the complexity of the oxidation

955

reaction involving carotenes under O2 are the other limitations of this assay.44

956 957

4.2. Iodometric Hydroperoxide Measurement

958 959

Iodometric method is one of the oldest colorimetric methods used for total hydroperoxides

960

determination and measurement of the ‘peroxide number’ (i.e., oxidative rancidity parameter

961

of fats or oils, defined as the quantity of –O-O– groups per unit mass).143 This UV-based

962

method involves the oxidation of iodide (I-) to iodine (I2) by lipid hydroperoxides, and

963

monitoring the 360 nm-absorption of the triiodide comple x ion (I3-) formed from I2 and

964

excess I-.144 At higher peroxide values, the titrimetric method is more precise than

965

colorimetry, as the liberated iodine may be titrated with standard thiosulfate solution using

966

starch as indicator. UV measurement of lipid (hydro)peroxides at 365 nm in biological

967

samples with cholesterol has been used to evaluate lipid peroxidation.145 Protein peroxides,

968

O2, and H2O2 with the possible ability to oxidize I- have been also reported to interfere with

969

the method. This method has two main limitations: (i) I2 can be absorbed at unsaturated

970

bonds (e.g., iodine addition to –C=C–) in the lipid sample, to result in unfavorably lower lipid



971

peroxidation measurement; (ii) I2 can be liberated from potassium iodide in the presence of O2

972

(especially in acidic medium), resulting in erroneous findings.146

973 974

4.3. Ferric-Thiocyanate and Ferric-Xylenol Orange Assays

975 976

Lipid hydroperoxides may oxidize ferrous iron to the ferric state, which in turn forms a red-

977

colored complex with thiocyanate (SCN-)147 or xylenol orange (XO) in acidic medium148 to

978

enable a colorimetric assay of the primary oxidation products of lipids. The ferric-xylenol

979

orange assay is abbreviated as the ‘FOX’ assay, and was applied to biological fluids.149 Free

980

hydrogen peroxide as well as lipid hydroperoxides can oxidize Fe(II) to Fe(III), however,

981

both assays are nonspecific. Fe(III)-SCN- and Fe(III)-XO complexes absorb in the visible

982

range, with absorption maxima around 500 nm and 560 nm, respectively, and antioxidants,

983

when present, attenuate the resulting color intensity, forming the basis of a lipid peroxidation

984

inhibition assay.

985 986

4.4. Ultraviolet Measurement of Conjugated Dienes

987 988

Lipid peroxidation produces diene conjugates, e.g., the formation of the PUFA• free radical is

989

accompanied by bond rearrangement that results in a diene-bond character. The conjugated

990

diene structures of alternating single and double bonds between carbon atoms (-C=C-C=C-)

991

absorb UV light in the wavelengths of 230-235 nm. UV absorption spectrometry150 is used

992

for the detection of non-specific lipid peroxidation caused by ROS in isolated lipoprotein

993

fractions (LDL lipoproteins).11 This technique is not suitable for the direct analysis of plasma

994

because of the UV-absorbing interferents (e.g., purines, pyrimidines, heme proteins).151 In

995

relation to the extraction of lipids into organic solvents, sensitivity of the method can be


Page 42 of 65

Page 43 of 65


996

improved by combining HPLC with UV detection.11 However, this coupled method yields

997

surprising results for lipid extracts of human body fluids, because the conjugated isomer of

998

linoleic acid ( cis-9, trans-11-octadecadienoic acid) also makes a major contribution to the

999

measured absorption at wavelengths typical for conjugated dienes.152

1000

An important limitation of this assay is that UV method has enough sensitivity for

1001

following the early stages of the oxidation process, whereas at later stages, the absorption

1002

bands of secondary oxidation products overlap in the UV region of interest.153 Since there is a

1003

strong dependence on fatty acid composition in the tested oil sample, the method cannot be

1004

applied to oils with different composition of PUFA. Oils with high amounts of PUFAs will

1005

have a dramatic increase in conjugated dienes compared to oils with less PUFAs.

1006 1007

4.5. TBARS Assay

1008 1009

TBARS spectrophotometric assay has been commonly employed to screen and monitor lipid

1010

peroxidation, for the ease of operation and low cost. In this method, malondialdehyde

1011

(MDA) emerging as an advanced product of unsaturated lipid degradation reacts with

1012

thiobarbituric acid (TBA) under acidic conditions to give a characteristic chromogenic adduct

1013

(MDA-(TBA)2) (Figure 6). The amount of MDA-(TBA)2 produced at high temperature

1014

(100°C) is measured spectrophotometrically at 532 nm.154 The MDA equivalents (µmol) of

1015

the samples being tested are calculated with the use of the molar extinction coefficient of the

1016

adduct, i.e., 1.56x105 M-1cm-1. TBARS assay measures the amount of MDA formed from

1017

lipid peroxidation, but other aldehydes simultaneously generated during lipid peroxidation

1018

may react with TBA and also absorb at 532 nm.155

1019 1020

Figure 6 43 ACS Paragon Plus Environment


1021

TBARS assay is not a specific method for lipid peroxidation products.156 On the other

1022

hand, in the application of TBARS method to biological fluids, some substances (e.g.,

1023

glycoproteins) interfere with the method by their ability of reacting with TBA.157 TBA

1024

reacts with different forms of aldehydes, not just those formed as a result of lipid

1025

peroxidation, and also ascorbic acid, homocysteine, deoxyribose, glucose, and certain amino

1026

acids (i.e., proline, arginine and glutamate). The sensitivity of the TBARS assay can be

1027

increased by combining it with HPLC techniques158, but some other aldehydes originating

1028

from lipid peroxidation can form an adduct with TBA, as demonstrated in this assay.159

1029

TBARS method cannot distinguish between the kinetics and the stoichiometry of the

1030

oxidation reaction. It also suffers from limitations due to oxidation reaction of TBA with other

1031

substances not associated with lipid peroxidation, and to the formation of Schiff bases

1032

between MDA and amines, leading to misestimation of the antioxidant protection.160

1033 1034

4.6. MS Techniques for Identifying Lipid Peroxidation Products

1035 1036

MS techniques have the advantage of more accurate identification and quantification of lipid

1037

peroxidation products. GC-MS has been seen as a traditional approach for studying the

1038

products of lipid peroxidation with high sensitivity. The lack of thermal stability of the

1039

peroxide bond formed during lipid peroxidation makes this assay difficult to implement in the

1040

direct analysis of hydroperoxides.161 To overcome this drawback, aldehydes generated from

1041

lipid peroxidation reactions were converted into the corresponding oximes using

1042

pentafluorobenzyl-hydroxylamine and further derivatized into the trimethylsilyl ethers, and

1043

finally formed derivatized lipid peroxidation products which give characteristic fragment ions

1044

were measured by GC-electron ionization (EI)-MS with selected ion monitoring (SIM)

1045

technique.162 On the other hand, coordination ionspray (CIS)-MS technique is nowadays 44 ACS Paragon Plus Environment

Page 44 of 65

Page 45 of 65


1046

considered a routine technique for the detection and identification of intact phospholipid

1047

hydroperoxides and cholesterol ester, without any prior derivatization in comparison to other

1048

MS methods.161 Silver(I) ion has been used in CIS-MS for quantitation of lipid peroxidation

1049

products because of its ability to coordinate with double bonds or aromatics (Ag+ CIS-MS).

1050

CIS-MS method is advantageous for the analysis of intact cholesterol esters and

1051

phospholipids, but the introduction of a coordinating Ag+ provides additional complications

1052

because its binding efficiency is expected to be related to the degree of unsaturation in the

1053

molecule. LC-electrospray ionization (ESI)-MS has been employed to analyze lipid

1054

peroxidation products (i.e., 5-hydroperoxyeicosatetraenoic acid,163 F2-isoprostane (F2-IsoPs)

1055

regioisomers as a specific in vivo marker of oxidative stress status or lipid peroxidation164).

1056

The main drawback of this method is that the phospholipid hydroperoxides must be

1057

hydrolyzed to the free acids prior to quantification. The quantitation of F2-IsoPs, produced

1058

from the peroxidation of the esterified arachidonic acid, by electron capture (EC)-atmospheric

1059

pressure chemical ionization (APCI)-MS has also been studied.135 EC-APCI-MS method is

1060

based on the ionization by EC reactions at atmospheric pressure (105 Pa). The combination of

1061

APCI-MS and the EC negative ionization mode provides approximately two orders of

1062

magnitude greater sensitivity than conventional APCI methods, due to the high efficiency of

1063

capture of a thermal or near thermal electron and formation of a dominant anion product.

1064

As an alternative to the common spectrophotometric techniques, a combination of

1065

LC/MS and LC/electron spin resonance (ESR) technique to investigate the carbon-centered

1066

spin adducts generated from soybean lipoxygenase-catalysed eicosapentaenoic acid (a major

1067

dietary ω-3 PUFA) peroxidation using a nitrone spin trap: α-[4-pyridyl 1-oxide]-N-tert-butyl

1068

nitrone was performed.165 The combination of spin-trapping with LC/ESR/MS for exploring

1069

cyclooxygenase/lipoxygenase-catalyzed lipid peroxidation offers a high resolution and

1070

selective platform alternative to conventional lipid peroxidation assays. This method also



1071

greatly improves the reliability of radical detection. As a result, this advanced technique

1072

becomes a potent tool for not only radical-mediated lipid peroxidation but also for PUFA’s

1073

bioactivities.166

1074

Oxidative damage in clinical trials can be evaluated by measurement of depletion of

1075

antioxidant compounds or of formation of lipid peroxidation products. However, more

1076

experimental evidence is needed to pinpoint the mechanisms of lipid peroxidation biomarkers

1077

at steady-state levels and/or at increased levels. Regarding a whole bunch of assays available

1078

for the detection of lipid peroxidation and its inhibition, it is recommended that more than one

1079

technique (i.e., a combination of suitable techniques) be used to provide a better estimate of

1080

lipid peroxidation in vivo. The hyphenated techniques for the assessment of ROS/RNS

1081

damage will also help us to evaluate the potential role of antioxidants in the prevention of

1082

oxidative stress-originated diseases in future studies.

1083 1084

ACKNOWLEDGMENTS

1085 1086

The authors wish to thank the Istanbul University-Application & Research Center for the

1087

Measurement of Food Antioxidants (Istanbul Universitesi Gida Antioksidanlari Olcumu

1088

Uygulama ve Arastirma Merkezi).

1089 1090 1091 1092 1093 1094 1095 1096 1097 1098 1099 1100 46 ACS Paragon Plus Environment

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LIST OF ABBREVIATIONS AAPH = 2,2’azobis (2-methylpropionamidine) dihydrochloride ABAP = 2,2'-azobis-(2-amidinopropane) dihydrochloride ABTS = 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid AE = antiradical efficiency AOA = antioxidant activity Ar-OH = phenol AUC = area under curve BDE = bond dissociation enthalpy β-PE = β-phycoerythrin CUPRAC = cupric reducing antioxidant capacity DCF = dichlorofluorescein DFT = density functional theory DMPD = N,N-dimethyl-p-phenylenediamine dihydrochloride DPPH = 2,2-di(4-tert-octylphenyl)-1-picrylhydrazyl EC50 = effective antioxidant concentration scavenging 50% of initial radical concentration EPR = electron paramagnetic resonance ESR = electron spin resonance ET = electron transfer FIA = flow injection analysis FRAP = ferric reducing antioxidant power HAT = hydrogen atom transfer HILIC = hydrophilic interaction chromatography HRP = horseradish peroxidase H2O2 = hydrogen peroxide IP = ionization potential KMBA = α-keto-γ-methiolbutyric acid • NO = nitric oxide radical 1 O2 = singlet oxygen O2•− = superoxide anion radical • OH = hydroxyl radical ONOO− = peroxynitrite anion ORAC = oxygen radical absorbance capacity PCET = proton-coupled electron transfer PUFAs = polyunsaturated fatty acids RO• = alkoxyl radical ROO• = peroxyl radical ROS = reactive oxygen species RNS = reactive nitrogen species RP-LC = reversed-phase liquid chromatography SIFT-MS = selected ion flow tube- mass spectrometry SPLET = sequential proton loss electron transfer TAC = total antioxidant capacity TBARS = thiobarbituric acid-reactive substances TE = Trolox-equivalent TEAC = Trolox-equivalent antioxidant capacity TOSC = total oxyradical scavenging capacity TR = Trolox 47 ACS Paragon Plus Environment


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Figure Captions

Figure 1. Reaction mechanism of ORAC assay.

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Figure 2. ORAC AOA of tested antioxidant expressed as the net AUC.

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Figure 3. Generation of ROO• by ABAP. Figure 4. Reaction of ABTS radical cation (intensely green) with a polyphenolic antioxidant (ArOH). Figure 5. Reaction of DPPH radical (deep purple color) with a polyphenolic antioxidant (ArOH).

Figure 6. Condensation reaction between TBA and MDA to form the (TBA)2-MDA chromogen adduct.


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Figure 1. Reaction mechanism of ORAC assay.

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1725 1726 1727 1728 1729 1730 1731 1732 1733 1734 1735 1736 1737 1738 1739 1740 1741 1742 1743 1744 1745 1746 1747 1748 1749 1750 1751 1752 1753

Figure 2. ORAC AOA of tested antioxidant expressed as the net AUC.


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1754 1755

2R RN=NR

[R N2 R]

-N2

(ABAP)

H3C

Stable products NH

R= H3C

NH2

1756 1757 1758

2O2 Ri

Figure 3. Generation of ROO• by ABAP.

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2ROO


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1790 1791 HO3S

S

S N

N

N

1792 1793 1794

1795 1796 1797 1798 1799 1800 1801 1802 1803 1804 1805 1806 1807 1808 1809 1810 1811 1812 1813 1814 1815 1816 1817 1818 1819 1820 1821 1822 1823 1824 1825 1826 1827

SO3H

N Et

Et

ABTS•+

decreased color

Figure 4. Reaction of ABTS radical cation (intensely green) with a polyphenolic antioxidant (ArOH). .


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1828 1829

N

N O

N O

N

O

+

O

-

-

+

ArOH O

PCET/HAT

N

N

H N

N

-

+

ArO

.

+

O +

+

N O

1830 1831 1832 1833 1834 1835 1836 1837 1838 1839 1840 1841 1842 1843 1844 1845 1846 1847 1848 1849 1850 1851 1852 1853 1854 1855 1856 1857 1858 1859 1860 1861 1862 1863 1864 1865 1866

O

-

+

O

+

O

N O

-

O

DPPH•

-

DPPH-H

Figure 5. Reaction of DPPH radical (deep purple color) with a polyphenolic antioxidant (ArOH).



1867 1868

1869 1870 1871 1872 1873 1874 1875 1876 1877 1878 1879 1880 1881 1882 1883 1884 1885 1886 1887 1888 1889 1890 1891 1892 1893 1894 1895 1896 1897 1898 1899 1900 1901 1902 1903 1904 1905 1906 1907 1908 1909

Figure 6. Condensation reaction between TBA and MDA to form the (TBA)2-MDA chromogen adduct.


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1910 1911 1912 1913


TOC Graphic

1914


Capacity Measurement. 2 ... - ACS Publications

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