Progress in Lipid Research

ARTICLE IN PRESS

Progress in Lipid Research Progress in Lipid Research 46 (2007) 244–282 www.elsevier.com/locate/plipres

Review

Evaluation of the ability of antioxidants to counteract lipid oxidation: Existing methods, new trends and challenges M. Laguerre, J. Lecomte, P. Villeneuve

*

UMR 1208 Ingeńierie des Agropolyme`res et Technologies Emergentes, CIRAD, INRA, Montpellier SupAgro, Universite´ Montpellier 2, F-34000 Montpellier, France Received 30 March 2007; received in revised form 9 May 2007; accepted 11 May 2007

Abstract Oxidative degradation of lipids, especially that induced by reactive oxygen species (ROS), leads to quality deterioration of foods and cosmetics and could have harmful effects on health. Currently, a very promising way to overcome this is to use vegetable antioxidants for nutritional, therapeutic or food quality preservation purposes. A major challenge is to develop tools to assess the antioxidant capacity and real efficacy of these molecules. Many rapid in vitro tests are now available, but they are often performed in dissimilar conditions and different properties are thus frequently measured. The so-called ‘direct’ methods, which use oxidizable substrates, seem to be the only ones capable of measuring real antioxidant power. Some oxidizable substrates correspond to molecules or natural extracts exhibiting biological activity, such as lipids, proteins or nucleic acids, while others are model substrates that are not encountered in biological systems or foods. Only lipid oxidation and direct methods using lipid-like substrates will be discussed in this review. The main mechanisms of autoxidation and antioxidation are recapitulated, then the four components of a standard test (oxidizable substrate, medium, oxidation conditions and antioxidant) applied to a single antioxidant or complex mixtures are dealt with successively. The study is focused particularly on model lipids, but also on dietary and biological lipids isolated from their natural environment, including lipoproteins and phospholipidic membranes. Then the advantages and drawbacks of existing methods and new approaches are compared according to the context.

Abbreviations: ROS, reactive oxygen species; LDL, low-density lipoproteins; 3O2, triplet oxygen; 1O2, singlet oxygen; OH, hydroxyl radical; LO, alkoxyl radical; LOO, peroxyradical; O 2 , superoxyde radical; SOD, superoxide dismutase; GSH-Px, glutathion peroxidase; GSH, glutathion; CAT, catalase; BDE, bond dissociation energy; PLPC, palmitoyl linoleoyl phosphatidylcholine; BSA, bovine serum albumin; EGCG, epigallocatechin gallate; AAPH, 2,2 0 -azobis(2-amidinopropane) dihydrochloride; ORAC, oxygen radical absorbance capacity; TAG, triacylglycerols; C11-BODIPY581/591, 4,4-difluoro-5-(4-phenyl-1,3-butadienyl)-4-bora-3a,4a-diaza-s-indacene-3-undecanoic acid; AIBN, 2,2-azobisisobutyronitrile; AIPH, 2,2 0 -azobis[2-(2-imidazolin-2-yl)propane] dihydrochloride; AMVN, 2,2 0 -azobis(2,4dimethylvaleronitrile); MeO-AMVN, 2,2 0 -azobis(4-methoxy-2,4-dimethylvaleronitrile); EDTA, ethylenediaminetetracetic acid; C11-fluor, 5-(N-dodecanoyl)aminofluorescein; C16-fluor, 5-hexadecanoylaminofluorescein; C18-fluor, 5- octadecanoylaminofluorescein; fluor-DHPE, fluoresceinated dihexadecanoylglycerophosphoethanolamine; BODIPY665/676, 4,4-difluoro-3,5-bis(4-phenyl-1,3-butadienyl)-4-bora3a,4a-diaza-s-indacene; PV, peroxide value; TBA, thiobarbituric acid; TBARS, thiobarbituric acid-reactive substances; HPLC, high performance liquid chromatography; GC-FID, gas chromatography-flame ionization detector; PAV, p-anisidine value; GC–MS, gas chromatography–mass spectrometry; SDE, simultaneous steam distillation; SHS, static headspace; DHS, dynamic headspace; HS-SPME, headspace-solid phase microextraction. * Corresponding author. Tel.: +33 (0) 467 615 518; fax: +33 (0) 467 615 515. E-mail address: [email protected] (P. Villeneuve). 0163-7827/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.plipres.2007.05.002

Please cite this article in press as: Laguerre M et al., Evaluation of the ability of antioxidants to counteract lipid ..., Prog Lipid Res (2007), doi:10.1016/j.plipres.2007.05.002

ARTICLE IN PRESS M. Laguerre et al. / Progress in Lipid Research 46 (2007) 244–282

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Finally, recent trends based on the chemometric strategy are introduced as a highly promising prospect for harmonizing in vitro methods. Ó 2007 Elsevier Ltd. All rights reserved. Keywords: Antioxidants; Antioxidant capacity; Lipid oxidation; Lipidic oxidizable substrate; Reactive oxygen species

Contents 1. 2.

3.

4.

5.

6.

7.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Theory of lipidic oxidation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Initiation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Propagation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3. Termination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Antioxidant mechanisms of action. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Preventive antioxidants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1.1. Transient metal chelators . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1.2. Singlet oxygen quenchers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1.3. ROS detoxification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Chain-breaking antioxidants. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3. Proxidant effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Essential test system components . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1. Antioxidant substances . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1.1. Synergistic and antagonistic effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1.2. Interfering substances present in complex mixtures . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2. Oxidizable substrates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2.1. Potential interference sources . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2.2. Substrate representativeness . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3. Medium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3.1. Homogeneous medium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3.2. Heterogeneous medium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.4. Oxidation conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.4.1. Heat . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.4.2. ROO generation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.4.3. HO generation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Adopted measurement strategies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.1. Oxygen depletion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2. Substrate loss . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2.1. b-Carotene bleaching assay. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2.2. Fluorescence decay of cis-parinaric acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2.3. Lipophilic fluorescein-based flow cytometry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2.4. Fluorescence decay of BODIPY probes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.3. Formation of primary oxidation products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.3.1. Iodometric hydroperoxide measurement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.3.2. Ultraviolet measurement of conjugated dienes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.4. Formation of secondary oxidation products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.4.1. Thiobarbituric acid (TBA) test . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.4.2. Aldehyde measurement by the anisidine test . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.4.3. Chromatographic measurement of volatile compounds . . . . . . . . . . . . . . . . . . . . . . . . . Final considerations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.1. Test system parametering. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.2. Measurement strategy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.3. Information processing and data presentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Future prospects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

246 249 249 250 250 250 251 251 251 252 252 254 254 255 255 256 256 256 257 258 259 259 261 261 261 262 263 263 264 265 266 266 267 269 269 270 270 270 271 272 273 273 274 275 275 276


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1. Introduction Reactive oxygen species (ROS) and their likely involvement in some human physiopathologies have attracted growing interest from the health sector over the last few decades. Oxidative stress, caused by an imbalance between antioxidant systems and the production of oxidants, including ROS, seems to be associated with many multifactorial diseases, especially cancers [1], cardiovascular diseases [2] and inflammatory disorders [3]. The mechanisms by which these pathologies develop generally involve oxidative alteration of physiologically critical molecules, including proteins, lipids, carbohydrates and nucleic acids, along with modulation of gene expression and the inflammatory response. The human organism has developed defense systems to deal with this oxidative stress. These include enzymatic systems, especially superoxide dismutases, catalases, gluthation peroxidases and thioredoxin systems, which are recognized as being highly efficient in ROS detoxification. The main nonenzymatic antioxidants present in the human organism are gluthation, bilirubin, estrogenic sex hormones, uric acid, coenzyme Q, melanin, melatonin, a-tocopherol and lipoic acid. Moreover, many studies have now confirmed that exogenic antioxidants, especially supplied by foods, are essential for counteracting oxidative stress. These antioxidants mainly come from plants in the form of phenolic compounds (flavonoids, phenolic acids and alcohols, stilbenes, tocopherols, tocotrienols) (Fig. 1–3), ascorbic acid and carotenoids (Fig. 4). The quest for natural antioxidants for dietary, cosmetic and pharmaceutical uses has become a major industrial and scientific research challenge over the last 20 years. Extracts of aromatic herbs [4], tea, grapes and derivative products, citrus fruit peel and seeds are amongst the most studied natural antioxidants. Despite major research, extensive knowledge has not yet been gained into the power of antioxidants derived from plants, nor has their potential been substantially tapped. Reliable in vitro methods to screen for antioxidant activity are therefore needed, as indicated by the number of review papers on this topic [5–17]. The complexity and diverse range of research topics investigated has nevertheless led to the development of a multitude of tests, but unfortunately none of them are universal. Databases on the antioxidant power of molecules, plant extracts and foodstuffs could be enhanced through better control, improvement and harmonization of current methods. The first problem is that antioxidant activity can be conveyed via many different pathways such as proxidant enzyme inhibition, singlet oxygen deactivation, UV filtration, enzymatic detoxification of

Hydroxybenzoic acids

R2

Hydroxycinnamic acids

O

R1

R1 O

OH

R2

R3 OH

R3

R4 R1=R2=R3=R4=H R1=R4=H, R2=R3=OH R1=H, R2=R3=R4=OH R1=OH, R2=R3=R4=H R1=R4=OH, R2=R3=H

Benzoic acid (non phenolic) Protocatechic acid Gallic acid Salicylic acid Gentisic acid

R1=R2=R3=H R1=R3=H, R2=OH R1=R2=OH, R3=H R1=OCH3, R2=OH, R3=H R1=R3=OCH3, R2=OH

Cinnamic acid (non phenolic) p-Coumaric acid Caffeic acid Ferulic acid Sinapic acid

Hydroxycinnamates (Chlorogenic acids)

O R1=R2=OH, R3 R1=R3=H, R2=OH R1=OCH3, R2=OH,R3=H R1=R3=OCH3, R2=OH

Caffeoyl quinic acid p-Coumaroyl quinic acid Feruloyl quinic acid Sinapoyl quinic acid

R1 O HOOC

5

HO

R2

4

1 2

OH OH

R3

3

Fig. 1. Main phenolic acids and esters found in plant kingdom.


ARTICLE IN PRESS M. Laguerre et al. / Progress in Lipid Research 46 (2007) 244–282 Flavones

8

4’

6

8 7

2’

6

5

O

O

Daidzein : 7=4’=OH Genistein : 5=7=4’=OH

7

6

6’ 6

4 5

5’ 6’

5’

2*

3

4’

O

7

O

4

3’

3’ 8

5’

Naringenin : 5=7=4’=OH Hesperitin : 5=7=3’=OH, 4’=OCH3 Naringin : Naringenin-7-neohesperidoside Hesperidin : Hesperitin-7-rutinoside

2’ 4’

O

O

Flavonols

2’

4’

5

4’

6’ 5’

Flavanols

8

6

5

Apigenin : 5=7=4’=OH Luteolin : 5=7=3’=4’=OH Diosmetin : 5=7=3’=OH, 4’=OCH3 Isovitexin : 5=7=4’=OH, 6=Glucose

3’

6’ 3’

3

4

O

7

6’

Flavanones 2’

8

5’

O

7

Isoflavones

3’ 2’

247

4 5

3*

3

OH

O

OH

Kaempferol : 5=7=4’=OH Quercetin : 5=7=3’=4’=OH Morin : 5=7=2’=4’=OH Fisetin : 7=3’=4’=OH Myricetin : 5=7=3’=4’=5’=OH

Catechin (2*R, 3*S) : 5=7=3’=4’=OH Epicatechin (2*R, 3*R) : 5=7=3’=4’=OH Epigallocatechin (2*R, 3*R) : 5=7=3’=4’=5’=OH Epicatechin gallate (2*R, 3*R) : 5=7=3’=4’=OH, 3-gallic acid ester Epigallocatechin gallate (2*R, 3*R) : 5=7=3’=4’=5’=OH, 3-gallic acid ester

Anthocyanidins

3’ 4’

2’ 8 7

+ O

Pelargonidin : 5=7=4’=OH Cyanidin : 5=7=3’=4’=OH Delphinidin : 5=7=3’=4’=5’=OH Malvidin : 5=7=4’=OH, 3’=5’=OCH3

5’ 6’

6

4 5

3

OH

Anthocyanins Cyanidin 3-glucoside Cyanidin 3-galactoside Cyanidin 3-rutinoside Malvidin 3-glucoside

Fig. 2. Main flavonoid antioxidants found in plant kingdom.

ROS, chelation of transition metals, as well as ROS stabilization through hydrogen radical transfer. Different strategies have thus been adopted to gain insight into antioxidation. According to Halliwell and Gutteridge [18], the term antioxidant refers to ‘‘a substance that, when present at low concentrations compared to those of an oxidizable substrate, significantly delays or prevents oxidation of that substrate’’. Logically, by this definition, any method that does not involve such a substrate could not measure antioxidant activity. These are called indirect methods, which are generally used to measure the capacity of a molecule to reduce a stable artificial free radical (by hydrogen or electron transfer), or a transition metal (simply by electron transfer). 2,2-Diphenyl-1-picrylhydrazyl [19,20] and ferric-reducing antioxidant power assays [21], as well as cyclic voltammetry [22,23], are used in some of these indirect antioxidant determination methods. Conversely, direct evaluation methods involve an oxidizable substrate. They are based on assessing the inhibitory effect of a potentially antioxidant substance on the oxidative degradation of a substrate in a test system subjected to natural or accelerated oxidation conditions. The oxidizable substrate Please cite this article in press as: Laguerre M et al., Evaluation of the ability of antioxidants to counteract lipid ..., Prog Lipid Res (2007), doi:10.1016/j.plipres.2007.05.002


M. Laguerre et al. / Progress in Lipid Research 46 (2007) 244–282 Tocopherols

R1 HO

CH3

R2

O R3

CH3

CH3 CH3

CH3

R1=R2=R3=CH3 R1=R3=CH3, R2=H R1=H, R2=R3=CH3 R1=R2=H, R3=CH3

Tocotrienols

R1 HO

CH3

R2

O R3

CH3

α β γ δ

CH3 CH3

CH3

CH3

Trolox

HO H 3C

O CH3

O

CH 3 OH

Fig. 3. Chemical structure of tocopherols, tocotrienols and Trolox (a-tocopherol without a phytyl chain).

β -carotene

Lycopene

OH

Lutein

HO

O OH

Astaxanthin

HO O

Fig. 4. Chemical structure of main carotenoid antioxidants.

usually consists of individual or mixed lipids, plant proteins, fluorophores, chromophores, DNA, or fluids containing biologically active chemical species such as low-density lipoproteins (LDLs) and biological membranes. Please cite this article in press as: Laguerre M et al., Evaluation of the ability of antioxidants to counteract lipid ..., Prog Lipid Res (2007), doi:10.1016/j.plipres.2007.05.002


249

Unsaturated lipid substrates are prime targets of oxidation in foods, cosmetics and biological environments. In vitro lipid oxidation is a major concern for agrifood and cosmetics industries, since they utilize unsaturated fatty acids to an increasing extent, such as those derived from the highly oxidation-sensitive n 3 (x 3) family. Besides altering the taste (rancidification) and nutritional quality (loss of vitamins and essential fatty acids) of foodstuffs, the ultimate oxidation of lipids into highly reactive and toxic compounds is a real danger for consumers. Moreover, the in vivo involvement of lipid oxidation products in the etiology of atherosclerosis [24–26] is clearly established, along with their role in many other human pathologies such as Alzheimer’s disease [27], cancers [28], inflammation, aging, etc. In this review, we first briefly outline the theories on the main autoxidation and antioxidation mechanisms, and then sequentially cover the principle components of a test system for direct in vitro measurement of the antioxidant capacity of plant molecules and extracts against lipid oxidation. The study focuses specifically on model lipids, in addition to dietary and biological lipids isolated from their natural environment. Finally, a comparative analysis of the main advantages and drawbacks of currently available methods is presented. 2. Theory of lipidic oxidation Lipid oxidation is a complex phenomenon induced by oxygen in the presence of initiators such as heat, free radicals, light, photosensitizing pigments and metal ions. It occurs via three reaction pathways: (i) nonenzymatic chain autoxidation mediated by free radicals, (ii) nonenzymatic and nonradical photooxidation, and (iii) enzymatic oxidation. The first two types of oxidation consist of a combination of reactions involving triplet oxygen 3O2, which could be considered as a ground-state biradical OO, and the singlet oxygen 1O2, which corresponds to an excited state of the molecule. There are many sources of 1O2 but its presence is often coupled with UV photonic impact in presence of photosensitizers. According to Frankel [29], there are two types of photosensitizer: type I for riboflavin, and type II for chlorophyll and erythrosine. Briefly, the photooxidation mechanism is dependent on the type of photosensitizer. In type II reactions, a triplet photosensitizer (3sens, reaction (1)) absorbs photons and becomes a singlet, thereafter transmitting its energy to molecular oxygen, which in turn becomes excited and forms singlet oxygen – around 1500-fold more reactive than triplet oxygen: 3

hm

sens ! 1 sens þ 3 O2 ! 1 O2

ð1Þ

The singlet oxygen formed through reaction (1) is electrophilic and can thus bind directly to C@C double bonds, leading to hydroperoxide formation. However, this so-called nonradical photooxidation seems to be a minor reaction in comparison to 3O2-induced radical chain autoxidation. It has thus been suggested that photooxidation mainly generates hydroperoxides that break down into free radicals that could initiate autoxidation reactions [30]. Autoxidation therefore seems to be a key mechanism in lipid oxidation. It mainly generates hydroperoxides and volatile compounds, generally through a three-phase process (initiation, propagation and termination). 2.1. Initiation From a mechanistic standpoint, the initiation phase involves homolytic breakdown of hydrogen in a position relative to the LH fatty acid chain double bond. It is unlikely that reaction (2) occurs spontaneously with 3 O2 because of the very high activation energy [31] arising from the spin barrier between lipids and 3O2. LH þ 3 O2 ! LOOH

ð2Þ

The reaction can, however, be initiated via external physical agents such as heat, ionizing radiation or a photonic impact in the ultraviolet spectrum, and also by chemical agents such as metal ions, free radicals and metalloproteins (reaction (3)), through a still controversial mechanism. Indeed, the initiation step of autoxidation is difficult to define because of the low concentration of radicals and the likelihood of there being more than one process. initiator

L1 H ! L1 þ H

ð3Þ




This results in the formation of L1 free radicals, i.e. chemical species with an unpaired electron. These are highly unstable, short-lived intermediates that stabilize by abstracting an hydrogen from another chemical species. The oxidation process remains slow during this phase. At the end of the initiation period, oxidation suddenly accelerates, oxygen consumption becomes high and the peroxide content increases substantially. Lipid oxidation is primarily initiated by hydroxyl (OH) and hydroperoxyl (HOO) radicals, as well as lipid alkoxyl (LO) and peroxyl (LOO)1 radicals. Hydroxyl radicals have by far the highest reaction rate with lipids. 2.2. Propagation In aerobic environments, the L1 radical centered on the carbon molecule and formed during the initiation phase reacts very quickly with triplet oxygen to generate different radical species, including L1 OO peroxyradicals. L1 þ 3 O2 ! L1 OO

ð4Þ

Reaction (4) has very low activation energy and a high rate constant, so the L1 OO concentration becomes much higher than the L1 content in all oxygen-bearing systems [31]. The peroxyradical then abstracts a hydrogen atom from another unsaturated lipid molecule to form hydroperoxide (primary oxidation compound) and another L2 radical (reaction (5)), thus replenishing reaction (4). L1 OO þ L2 H ! L1 OOH þ L2

ð5Þ

This so-called ‘self-sustained’ radical chain reaction thus propagates at a high rate, as clearly shown by a marked increase in the hydroperoxide formation kinetics. This is a rapidly peaking irreversible reaction. It has been calculated that around 25 fatty acid molecules are oxidized during the propagation phase after radical-initiated oxidation [32]. Many hydroperoxide isomers are thus formed during this phase. Hydroperoxide formation from unsaturated fatty acids is generally accompanied by stabilization of the radical state via double-bond rearrangement (electron delocalization), which gives rise to conjugated dienes and trienes. Frankel [29] summarized the potential autoxidation mechanisms of oleate, linoleate, linolenate, triacylglycerol and phospholipid models. Maximum peroxide formation marks the onset of the termination phase. 2.3. Termination The oxidation process then continues with the transformation of hydroperoxides into secondary nonradical oxidation compounds. The main hydroperoxide decomposition mechanism involves scission of the double bond adjacent to the hydroperoxyl group, leading to the formation of hydrocarbons, aldehydes, alcohols and volatile ketones. Other nonvolatile secondary compounds are also formed, including nonvolatile aldehydes, oxidized triacylglycerols and their polymers. Decomposition of primary oxidation compounds is a complex mechanism in which a single hydroperoxide can generate several types of volatile or nonvolatile molecules. The type of by-products obtained after fatty acid oxidation is determined by the hydroperoxide composition and by the type of scission of double bonds in the fatty acid chain. The reaction can then also terminate after polymer formation. Moreover, many antioxidants can facilitate termination of radical chain oxidation. 3. Antioxidant mechanisms of action Antioxidants counteract oxidation in two different ways, i.e. by protecting target lipids from oxidation initiators or by stalling the propagation phase. In the first case, the so-called preventive antioxidants hinder 1 ROS formation or scavenge species responsible for oxidation initiation (O 2 , O2, etc.). In the second case, the so-called ‘chain breaking’ antioxidants intercept radical oxidation propagators (LOO) or indirectly participate in stopping radical chain propagation. The mechanisms of action are sequentially reviewed here, but 1

For clarity, and wherever possible, peroxyradicals derived from lipid substrates are represented by LOO, and those derived from azoinitiators by ROO.



251

it should be kept in mind that antioxidants often act via mixed mechanisms that combine different types of antioxidation. 3.1. Preventive antioxidants There are many different ‘preventive’ antioxidation pathways because of the diverse range of available oxidation initiators. These pathways include chelation of transition metals, singlet oxygen deactivation, enzymatic ROS detoxification, UV filtration, inhibition of proxidant enzymes, antioxidant enzyme cofactors, etc. Here, we will only describe the most renowned ones. 3.1.1. Transient metal chelators Chelators of transition metals such as copper and iron can prevent oxidation by forming complexes or coordination compounds with the metals. These are proteins such as transferrin, ferritin and lactalbumin that sequester iron, or ceruloplasmin and albumin that sequester copper. Polyphosphates, ethylenediaminetetracetic acid (EDTA), citric acid, phenolic acids and flavonoids are also known for their transition metal chelation capacity. In flavonoids, the two points of attachment of transition metal ions are o-diphenolic groups in 3 0 ,4 0 dihydroxy positions in the B ring (Fig. 5a), and ketol structures 4-keto, 3-hydroxy (Fig. 5b) or 4-keto and 5hydroxy (Fig. 5c) in the C ring of flavonols [33]. Hence, glycosylation in these hydroxyl positions reduces the chelating power of flavonoids. The extent of this type of antioxidation seems, however, to depend directly on the oxidizable target being protected. This mechanism of action is minor for lipid peroxidation inhibition as compared to anti-radical activity via ROS scavenging [34], but paramount in the inhibition of DNA strand breakage [35]. 3.1.2. Singlet oxygen quenchers To our current knowledge, carotenoids are the most efficient molecules for 1O2 quenching. Around 600 carotenoids occur naturally in the environment, most of which have 40 carbon atoms. They can be pure hydrocarbons, i.e. carotenes (lycopene, b-carotene, etc.), or include an oxygenated functional group, i.e. xanthophylls (astaxanthin, lutein, etc.) [36] (Fig. 4). As discussed later, carotenoids, like many antioxidants, have antioxidant activity through several different but highly complementary mechanisms, i.e. chain breaking antioxidants and 1O2 quenchers. This latter mechanism of action occurs through deactivation of 1O2 into 3O2. 1

O2 þ b-carotene ! 3 O2 þ b-carotene

ð6Þ

Through the long conjugated polyenic system of these molecules, the excess energy generated in their excited state (b-carotene*) is dissipated via vibrational and rotational interactions with the solvent or the environment [36]. b-carotene ! b-carotene þ heat

ð7Þ 1

Regenerated b-carotene can begin a new O2 quenching cycle through this energy (heat) dissipation mechanism and thus become a nonstochiometric quencher. It is estimated that each carotenoid molecule could quench around 1000 1O2 molecules prior to chemical reaction and product formation.

(n-2)+ O M

O 3’

O

O

4’

B

C 4

O

0

O A

3

C

5

4

O M (n-1)+

O

O M

(n-1)+

0

Fig. 5. Metallic ion complexation by flavonoids via the 3 -4 -o-diphenolic group in the B ring (a) and ketol structures 4-keto, 3-hydroxy in the C ring (b) or 4-keto, 5-hydroxy in the C and A rings (c).




In addition to carotenoids, there are other 1O2 quenchers, especially tocopherols and thiols. Di Mascio et al. [37] investigated the ability of some antioxidants to quench 1O2 pregenerated by thermodissociation of the endoperoxide of 3,3 0 -(1,4-naphthylidene) dipropionate. Their efficacy was found to decrease in the following order: lycopene, c-carotene, bixin, zeaxanthin, lutein, bilirubin, biliverdin, tocopherols and thiols. However, these authors point out that the least active quenchers occur in the highest concentrations in biological tissues. Carotenoids and tocopherols therefore equally contribute to the protection of tissues against the deleterious effects of 1O2, since tocopherols in vivo are estimated to be around 30-fold more abundant than b-carotene. 3.1.3. ROS detoxification ROS detoxification is a crucial oxidation prevention pathway, mainly mediated by endogenous enzymatic antioxidant systems. First, superoxide dismutase (SOD), a metalloenzyme that is omnipresent in eukaryotic organisms, catalyzes superoxide anion dismutation into hydrogen peroxide and oxygen. SOD

þ 2O 2 þ 2H ! H2 O2 þ O2

ð8Þ

Glutathion peroxidase (GSH-Px), another enzyme, has detoxification activity concerning three reactive species: hydrogen peroxide, lipid hydroperoxides and peroxynitrite, which is a highly reactive nitrogen species. GSH-Px is a selenodependant enzyme containing four selenium atoms located, in the form of selenocystein, at the active core of the enzyme. In particular, this enzyme accelerates glutathion (peptidic thiol, symbolized here by GSH) oxidation by hydrogen peroxide, which is then reduced to water. GSH-Px

H2 O2 þ 2GSH ! 2H2 O þ GSSG

ð9Þ

A third enzyme, i.e. catalase (CAT), only has hydrogen peroxide as substrate, which it reduces to water and molecular oxygen (reaction (10)). This heminic enzyme mainly occurs in peroxisomes and erythrocytes. CAT

2H2 O2 ! 2H2 O þ O2

ð10Þ

It is usually assumed that there is direct cooperation between these different enzymes in vivo. SOD activity, which dismutates superoxide anions, leads to the formation of hydrogen peroxide, which in turn is detoxified by the catalase and/or GSH-Px systems. 3.2. Chain-breaking antioxidants In lipid peroxidation, chain-breaking antioxidants usually lose a hydrogen radical to LOO (reaction (11)), thus halting radical oxidation propagation. A-H þ LOO ! A þ LOO-H

ð11Þ

This primarily involves mono- or poly-hydroxylated phenol compounds (tocopherols, tocotrienols, flavonoids, phenolic acids and alcohols, stilbenes, etc.) with different substituents on one or several aromatic rings. Theoretically, the capacity of a phenol to dispose of an H atom could be quantified by the homolytic dissociation energy of the O–H bond, i.e. bond dissociation energy (BDE). The donor capacity of an H atom increases as the phenol BDE decreases. This is not the only factor that governs the chain-breaking trait – it is under multifactorial control. A suitable position and good mobility towards LOO production sites are key attributes which ensure that an antioxidant will be a good chain breaker. Finally, the reactivity of antioxidant-derived radicals with unsaturated lipids should also be taken into account. This fate is generally dictated by the capacity of the antioxidant to stabilize unpaired electrons by delocalization. From this standpoint, the aromatic structure and the potential presence of bulky groups able to extend this delocalization, increases the stability of phenol radicals. Note that in vivo regeneration systems also control the reduction of residual radical forms, e.g. tocopherols. From a kinetic standpoint, chain-breaking antioxidants induce a lag phase during which the substrate is not substantially oxidized. This phase continues until the antioxidant is completely consumed (Fig. 6). Once the antioxidant has disappeared, the peroxidation rate rises sharply until it reaches the same rate as during Please cite this article in press as: Laguerre M et al., Evaluation of the ability of antioxidants to counteract lipid ..., Prog Lipid Res (2007), doi:10.1016/j.plipres.2007.05.002


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Lag phase

Substrate quantity

Chain-breaking antioxidant (α -tocopherol) Retarder antioxidant

Uninhibited oxidation of substrate

Time

Fig. 6. Theoretical time-course curves of antioxidation by chain-breaker or retarder.

uninhibited oxidation. Conversely, retarder antioxidants reduce the peroxidation rate without inducing a distinct lag phase. The most powerful natural chain breakers currently known are definitely tocopherols (Fig. 3), which exhibit a very distinct lag phase. Many studies have shown that these chain breakers scavenge two peroxyradical molecules [38,39]. Vitamin C and polyphenols also seem to be able to directly reduce peroxyradicals [40], but their hydrophilic nature and remoteness from lipophilic radicals seem to hinder all direct contact reactions. It is now clearly established that vitamin C is involved mainly during regeneration (reduction) of tocopheroxyl obtained through the antiradical activity of tocopherol (Fig. 7). Tappel [41] was the first to suggest that these two molecules could act synergistically. This regeneration, which underlies the antiradical protection of biological membranes and LDLs, has been widely documented in vitro on methyl linoleate [39], membrane systems [42] and LDLs [43], as well as in vivo [44]. This regeneration through ascorbate to a-tocopherol hydrogen transfer is hypothetically possible due to the low reducing potential of ascorbic acid (E0 0.28 V) relative to that of a-tocopherol (E0 0.5 V) [13]. Concerning polyphenols, many in vitro studies [45–51] have shown that a 1,2-dihydroxy substitution on the B ring (catecholic structure) is a key factor in determining the antioxidant activity of phenolic compounds, in agreement with the low BDE of the corresponding OH groups. The higher antioxidant efficacy of ortho-diphenols is usually explained by stabilization of the phenoxy AO radical through formation of an intramolecular hydrogen bond. In vivo, it is quite likely that flavonoids with a catechol-like B cycle act in the same way as ascorbic acid [52]. This pathway seems to involve regeneration of the chromanoxyl radical into chromanol through polyphenol oxidation into phenoxyl radicals (Fig. 7). Contrary to ascorbyl radicals, there are no known phenoxyl radical regeneration systems in the animal kingdom [53]. Finally, transfer of hydrogen atoms to propagator free radicals such as LOO is not the only chain-breaker mechanism of action. In this respect, b-carotene hindrance of chain lipoperoxidation occurs via other

Membrane LOO•

Water-phase

α -TOH

Fl• AscO-•

AscO LOOH

α -TO

•

Fl

Tocopheroxyl radical

Fig. 7. Potential recycling mechanism between a-tocopherol (a-TOH), ascorbate (AscO) and flavonoid (Fl) in membrane systems.




pathways, such as adduct formation. Although H transfer from b-carotene to ROO is thermodynamically possible [54], since the BDE of the most labile hydrogen in b-carotene (309 kJ/mol) is lower than those of LOO–H (370–380 kJ/mol) [55], the addition mechanism yielding a nonradical product terminating the chain reaction (reactions (12 and 13)) has been widely hypothesized as the chain breaking antioxidant mechanism of b-carotene on lipid oxidation [56–59]. ROO þ CAR ! ROO–CAR

ð12Þ

ROO–CAR þ ROO ! ROO–CAR–ROO

ð13Þ

Black [60] recently proposed a mechanism by which b-carotene would regenerate the tocopheroxyl radical, with b-carotene radical cations being reduced by ascorbic acid. However, this still relatively unknown mechanism of action is refuted by some experimental data which indicate that b-carotene does not interact with the tocopheroxyl radical [61]. These crucial studies are aimed at gaining insight into the role of b-carotene in vivo and could considerably clarify the scheme of antioxidant interdependance. Note finally that the sites of interaction between antioxidants are not solely on biological membranes and lipoproteins. They occur especially in the digestive tract lumen and should now be investigated in this environment, while taking the broad range of different food matrices into account [53]. 3.3. Proxidant effects It is extremely important to understand the antioxidant and proxidant behaviors of bioactive substances according to their structure, chemical environment and test experimental conditions involved (transition metals, azo-initiators, etc.). It is now well established that flavonoids can have a proxidant effect [62] in the presence of Cu2+. For a class of flavonoids with the same basic structural motif, their proxidant activity towards one plant protein (b-phycoerythricin) in aqueous solution at 37 °C was found to increase with the number of aromatic hydroxyls. However, the most oxidant flavonoids in the presence of Cu2+ were more antioxidant in the presence of: (i) a peroxyradical initiator (absence of Cu2+) or (ii) a Fenton-like system (Cu2+/H2O2) (reaction (14)) generating hydroxyl radicals. Mn þ H2 O2 ! Mðnþ1Þ þ OH þ OH

ðM ¼ Cu; Fe; Co; and n ¼ 2Þ

ð14Þ

These results should be considered in relation with those obtained in other studies which highlighted that some flavonoids (quercetin, myricetin and kaempferol) may induce lipoperoxidation and nuclear DNA damage in the presence of transition metals [63–66]. Similarly, Maiorino et al. [67] demonstrated that, during peroxidation of palmitoyl linoleoyl phosphatidylcholine (PLPC) micelles induced by Cu2+, the addition of a-tocopherol led to a drastic increase in PLPC hydroperoxide accumulation. According to these authors, the proxidant effect of a-tocopherol could largely come from its capacity to reduce Cu2+ into Cu+. Indeed, the reaction between phospholipid and Cu2+ hydroperoxides generates peroxyradicals, while that with Cu+ generates alcoxyl radicals [68]. However, it is known that alkoxyl radicals are far more reactive than peroxyl radicals in Habstraction reactions [69]. As regards ascorbic acid, it is well known that it acts as a pro- rather than an antioxidant in the presence of transition metal ions. This is because ascorbic acid reduces transition metal ions (Me(n+1)+ ! Men+) and generates hydrogen peroxide through autoxidation, which drives production of hydroxyl radicals via the Fenton reaction [70]. Finally, carotenoids can also have oxygen pressure dependant proxidant activity. According to Burton and Ingold [56], b-carotene behaves like a radical-trapping antioxidant, only when the oxygen partial pressure is below 150 torr (normal ambient oxygen pressure). However, when the pressure increases, b-carotene loses its antioxidant activity and has an autocatalytic proxidant effect which increases with its concentration. 4. Essential test system components Methods for direct in vitro assessment of antioxidant activity generally focus on four components: (i) an oxidizable substrate, whose oxidative degradation can be monitored by physicochemical or sensorial analysis Please cite this article in press as: Laguerre M et al., Evaluation of the ability of antioxidants to counteract lipid ..., Prog Lipid Res (2007), doi:10.1016/j.plipres.2007.05.002


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methods, (ii) a medium, in which the different components come in contact, (iii) the oxidation conditions, in which the substrate is oxidized, and (iv) antioxidant substances, that are to be evaluated in terms of their capacity to protect the oxidizable substrate. Two experiments could thus be conducted. The first experiment involves testing the in situ ability of an antioxidant to protect a food or biological tissue (natural substrate) removed from its native environment and subjected to natural oxidative aging. However, to be representative, this test requires an analysis time that is not compatible with routine mass-sample screening. The most common alternative is to assess the efficacy of an antioxidant with a natural substrate whose oxidation has been artificially accelerated. This type of partially modeled approach, as discussed hereafter, is often combined with a measurement strategy based on the detection of oxidation products. The second experiment involves evaluating the antioxidant activity of a given molecule in an oxidizable substrate that is artificial or labeled with a fluorescent probe, and subjected to accelerated oxidation. We will see that this completely modeled approach is often combined with a measurement strategy based on substrate loss. It may be hard to extrapolate modeling test results to a real situation under natural conditions, but the analyst must try to correlate the results and adopt the most optimal parametering, while rejecting all other options. It is currently well known that the antioxidant efficacy (or antioxidation mechanism) is dependant on most of the real or modeled parameters involved: medium polarity, temperature, type of substrate, oxidation conditions and physical state (solid, liquid or emulsion). This explains the marked differences sometimes noted between many reported results, e.g. differences in the antioxidant power classification for the same molecule evaluated in different tests (Table 2 in [71]). It should therefore be kept in mind that the magnitudes of the antioxidant activities that we present are only comparable for given process conditions, i.e. antioxidant efficacy values cannot be considered as universally applicable for all media, oxidation conditions or substrates. In this chapter, we will cover the test system parametering issue through a detailed description of each essential component, including the antioxidant substances, oxidizable substrate, medium and oxidation conditions. 4.1. Antioxidant substances Antioxidant efficacy measurement tests must involve antioxidant substances, but some points could lead to ambiguities in measurements and interpreting the results. The first concerns nonlinear synergistic and antagonistic effects that could arise when the substances are mixed. The second concerns the bias that these substances may induce when their efficacy is analyzed. 4.1.1. Synergistic and antagonistic effects As pointed out by Becker et al. [13], the concepts underlying nonlinear synergistic and antagonistic effects are commonly used by scientists but seldom defined. According to an initial definition [72], ‘‘synergism is, in general, the phenomenon in which a number of compounds, when present together in the same system, have a more pronounced effect than that which would be derived from a simple additivity concept’’. Antagonism may be defined likewise by substituting ‘more’ with ‘less’. There are mainly four types of synergy. (i) Firstly, the regeneration of highly active antioxidants by less active forms could explain the synergies noted between a-tocopherol and some phenolic compounds (()-epicatechin, (+)-catechin and quercetin) in a methyl linoleate solution in organic medium [73]. (ii) Secondly, synergies can also be created through the interaction of antioxidants with different mechanisms of action (singlet oxygen quenchers and chain-breaking antioxidants). The presence of a transition metal chelator can also reduce the need for chain breakers, which are often considered as the most powerful antioxidants. (iii) In multiphase media, the interaction of antioxidants with different polarities, which are thus distributed in different phases or solvents, could induce synergy, as already discussed with respect to ascorbate-induced regeneration of a-tocopherol. (iv) There can also be synergies between antioxidants and substances without any antioxidant activity, such as bovine serum albumin (BSA). Hence, Almajano and Gordon [74] clearly showed that the antioxidant activity of caffeic acid, epigallocatechin gallate (EGCG) and Trolox (a-tocopherol without a phytyl chain, Fig. 3) increased very significantly when BSA was present in the emulsion medium. It was also noted that the efficacy order was partially reversed in the absence (caffeic > ECGC > Trolox) or presence Please cite this article in press as: Laguerre M et al., Evaluation of the ability of antioxidants to counteract lipid ..., Prog Lipid Res (2007), doi:10.1016/j.plipres.2007.05.002



(ECGC > caffeic > Trolox) of BSA. According to these authors, the antioxidant position within the emulsion system is likely the mechanism underlying this synergy. It is actually considered that these polar phenolic compounds, which usually occur in the aqueous phase, could bind to BSA and then be transported to the oil–water interface where they become active. This hypothesis is based on the fact that BSA can stabilize emulsions [75] and also that oxidation is initiated at the system interfaces. Contrary to the results reported by Pedrielli and Skibsted [73], Peyrat-Maillard et al. [76] observed antagonistic effects between a-tocopherol and certain phenolic acids (rosmarinic and caffeic acids) during oxidation of an aqueous dispersion of linoleic acid induced by the azo-initiator 2,2 0 -azobis(2-amidinopropane) dihydrochloride (AAPH). The authors explained this antagonism by the fact that a fraction of these acids (highly active) would regenerate the less active a-tocopherol. Although these test systems (especially the antioxidants) were not identical, it is still surprising that Pedrielli and Skibsted consider that a-tocopherol has greater antioxidant activity than polyphenols, whereas the reverse phenomenon was noted by Peyrat-Maillard et al. This paradox is likely due to the fact that Pedrielli and Skibsted used a liposoluble azo-initiator that generates ROO at the interface, thus promoting a-tocopherol, whereas Peyrat-Maillard et al. used a hydrosoluble azo-initiator that generates ROO in the aqueous phase, where phenolic compounds occur. This indicates that the type of azo-initiator can have a considerable impact on the results, so it is always essential to assess the data relative to the process conditions – a crucial point that we stress throughout this paper. Finally, the presence of these nonlinear phenomena clearly highlights the need to assess the situation on several global (mixed substances) and individual (isolated substances) levels. Measurements on a global level will provide very little information on the antioxidant power of each molecule, but potential nonlinear effects are taken into account, while individual measurements of the antioxidant capacity of each component of a mixture requires preliminary separation, assay and identification. Nonlinear effects are neglected in this case. We have, however, seen that it is not always possible to determine the global antioxidant activity of the raw extract on the basis of the cumulated effects of each component. The best tradeoff is obviously to strike a balance between these two approaches by measuring the antioxidant capacity of an extract globally along with that of each elementary component. 4.1.2. Interfering substances present in complex mixtures When measuring the antioxidant properties of a complex mixture, it is essential to know what nonantioxidant components could interfere in the reaction, i.e. which could lead to overestimation of the antioxidant power and consequently to a false positive result. Pe´rez-Jimeńez and Saura-Calixto [77] recently showed that tyrosine and tryptophane, which are not antioxidants, have high oxygen radical absorbance capacity (ORAC) values at very low concentration, thus confirming previous results obtained by Yilmaz and Toledo [78]. This indicates that proteins and amino acids present at high concentration in some plants can generate considerable interference. Carbohydrates (glucose, pectin and galacturonic acid) do not seem to generate interference, at least in ORAC, ferric-reducing antioxidant power, 2,2 0 -azinobis(3-ethylbenzothiazoline-6-sulphonate) (ABTS+) and 2,2-diphenyl-1-picrylhydrazyl tests. The second type of interference can be associated with the measurement technique, e.g. absorption spectrophotometry. Indeed, antioxidant substances may absorb in the same spectral window as both oxidizable substrate or oxidation products. Note, in this case, that this problem can be avoided through proper use of a blank containing the antioxidant substances. 4.2. Oxidizable substrates Note first that all chemical or biological structures consisting of unsaturated lipid compounds such as triacylglycerols, carotenoid pigments, cholesterol, lipoproteins and biological membranes are prone to oxidation. 4.2.1. Potential interference sources When selecting an oxidizable substrate, commercially available compounds with a clearly defined composition should be selected whenever possible. However, it is sometimes necessary, or more advantageous, to use noncommercial lipid compounds or mixtures such as vegetable oils or biological fluids. Noncommercial vegetable oils should always be obtained from the same source in order to minimize potential variability due to Please cite this article in press as: Laguerre M et al., Evaluation of the ability of antioxidants to counteract lipid ..., Prog Lipid Res (2007), doi:10.1016/j.plipres.2007.05.002


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soil–climate conditions and plant variety. The different chemical species in these mixtures should be clearly identified in order to be able to gain insight into the mechanisms involved in oxidation and antioxidation reactions, and to adopt the most suitable measurement strategy. Note also that crude and refined vegetable oils and some biological particles, such as LDLs or microsomes, contain endogenous antioxidants (tocopherols, tocotrienols and carotenoids) that may compete with the tested antioxidants, which could markedly bias the results. These contaminants must be eliminated in oils, especially through purification on an alumina column. It is also important to make sure that the lipid oxidizable substrate does not initially contain oxidation compounds or proxidant substances like transition metals. Some authors have suggested that free fatty acids [79] and some secondary oxidation products such as hexanal and 2,4-decadienal [80] could intervene as proxidant agents. A high initial concentration of these components could result in premature antioxidant consumption or acceleration of the oxidation kinetics, thus biasing assessment of their efficacy. Even trace amounts of transition metals can accelerate oxidation. Finally, the substrate signal should remain completely stable when there is no oxidation. There can be two pitfalls: (i) photobleaching (concerning fluorimetry) and (ii) nonspecific interactions with antioxidants. Some fluorescent substrates like cis-parinaric acid were found to be photosensitive to the excitation wavelength [81], which leads to gradual fluorescence extinction, even when no oxidation initiators are present. This is generally very problematic – even though a mathematical correction might be possible if the deviation is low and regular – because it leads to overestimation of the extent of oxidation. Concerning nonspecific interactions, it is essential to choose a substrate that is chemically inert with respect to the antioxidant molecule, thus ensuring that its signal will not change in the absence of oxidant agents. These two parameters can be checked by monitoring the signal of the substrate mixed with other test components, except oxidant agents. 4.2.2. Substrate representativeness The assessment procedure, especially for choosing an oxidizable substrate, will differ depending on whether the antioxidant from plant sources is to be utilized for nutritional, therapeutic or preservation purposes. Firstly, it is essential to select a substrate in which oxidative degradation can be monitored. Secondly, it should have high representativeness relative to in situ conditions in which the antioxidant has a protective effect. Finally, the best tradeoff should be found between the substrate representativeness and certain process constraints, such as the analysis time, cost, feasibility, specificity of the substrate reaction, signal stability and obtaining a high signal-to-noise ratio. (i) When the aim is to assess the antioxidant efficacy of a substance in preserving a foods, it is thus best to use a substrate derived from the foods. However, a specific test should be developed for each food. This approach is suitable if the analyst focuses the investigation just on a few foods, but not if he/she wants to gain global insight into the antioxidant activity of any substance in a large panel of foodstuffs. Tests using model substrates can provide a satisfactory compromise solution in such cases. Triacylglycerols (TAGs) and phospholipids are therefore the best sources of oxidizable substrate as they are the most representative of dietary lipids [29]. Although free fatty acids should be avoided because the micelles that are formed will behave differently from those obtained with real lipids [29], linoleic acid and its methyl ester in aqueous dispersion are the substrates most commonly used in this field – they are easy to use, the oxidation kinetics are rapid and the mechanisms involved are relatively simple (with or without antioxidant inhibition) compared to more realistic substrates. Few tests currently use TAGs as model substrates [82–84]. This is probably related to the fact that the triglyceride environment markedly slows down the oxidation kinetics and leads to high analysis times, or the analyses have to be performed under harsher oxidation conditions. Finally, the most generic tests, such as the b-carotene and linoleic acid cooxidation test [85,86], are often used for this type of assessment. (ii) When the aim is to assess the ability of a substance to protect biologically important lipids in the organism, LDLs and natural membrane structures (extracts of biological media) like microsomes, or model (artificial) structures like liposomes, are the most commonly used substrates. Moreover, over the last 15 years or so, in vitro studies performed on cell cultures biosynthetically incorporated with fluorescent probe such as cisparinaric acid [87] have helped to enhance the detection sensitivity of cellular oxidative processes. First, LDL has four potential oxidation targets (Fig. 8): free or esterified cholesterol, polyunsaturated fatty acids bound to surface phospholipids, triacylglycerols containing unsaturated fatty acids, and apolipoprotein B. Please cite this article in press as: Laguerre M et al., Evaluation of the ability of antioxidants to counteract lipid ..., Prog Lipid Res (2007), doi:10.1016/j.plipres.2007.05.002


M. Laguerre et al. / Progress in Lipid Research 46 (2007) 244–282 Monolayer shell of phospholipids (~ 700 molecules a) Apolipoprotein B

Unesterified cholesterols (~ 600 molecules a)

C A

D

B

HO

Cholesteryl esters (~ 1600 molecules a) Triacylglycerols (~ 170 molecules a) + Non enzymatic antioxidants (~ 9 molecules a) including : α -tocopherol (~ 7 molecules), β -tocopherol, β - and α -carotene, lycopene, cryptoxanthin and ubiquinol-10

Fig. 8. Simplified structure of LDL particle. (aFrom reference [26].)

The mechanism of LDL oxidation in vivo is largely a matter of speculation. According to one of the most likely scenarios lipid peroxidation would begin in polyunsaturated fatty acids of surface phospholipids, then propagate to the nucleus and end with oxidation of all lipids of the particle, followed by degradation of free lysine groups of apolipoprotein B. Aldehyde-modified apolipoprotein B has altered receptor affinity, causing it to be scavenged by macrophages in an uncontrolled manner with the development of foam cells and the initiation of atherosclerotic lesions [26]. Note that cholesterol, which is an unsaturated lipid, oxidizes in the same way as an unsaturated fatty acid. This oxidation mainly concerns unsaturation affecting the B steroid cycle, while promoting attack in the allylic position. Hydroperoxides are first formed, which then degrade into epoxides, primary, secondary and tertiary alcohols and ketones. Some fluorescent lipid or lipid-like probes such as cis-parinaric acid have also been incorporated in LDLs for fluorimetric analysis of oxidation and antioxidation [88,89]. Liposomes, which were first described by Bangham [90], are closed spherical structures with one or several phospholipid bilayers organized between two aqueous compartments. A distinction is made between small, large and giant multilamellar and unilamellar liposomes. To our knowledge, Porter et al. [91] were the first to demonstrate free radical oxidation of membrane phospholipids, thus opening a new avenue of highly seminal research. Many researchers have tried to model membrane structures from animal tissues in order to study the oxidation response of phospholipids in biological media or meat products and the capacity of endo- and exogenous antioxidants to preserve this oxidizable substrate. Some authors have reconstructed mono- or multi-lamellar liposome systems [92–94], while others have studied microsomes extracted from rabbit liver [95] or liposomes from brain [96]. Finally, as discussed later, very many studies have highlighted the advantages of incorporating hydrophobic fluorescent probes (cis-parinaric acid, C11-BODIPY581/591 probe and lipophilic derivative of fluorescein) in liposomal structures. 4.3. Medium There are three general types of medium: homogeneous solutions, emulsions (oil-in-water, water-in-oil, etc.) and aqueous suspensions of liposomes or LDLs. It is now well established that antioxidants behave differently in media with different polarities and phase states. Note that no universal medium is available that can solubilize all antioxidants in a neutral way, so no single method can be implemented to extract and/or thoroughly test antioxidant compounds. In any case, such a hypothetical medium would not be useful since antioxidants Please cite this article in press as: Laguerre M et al., Evaluation of the ability of antioxidants to counteract lipid ..., Prog Lipid Res (2007), doi:10.1016/j.plipres.2007.05.002


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are not targeted for universal applications. With these limitations in mind, the analyst should study the antioxidant activity of a fraction of molecules belonging to the same class and extracted by a specific type of solvent. Moreover, the polarities of the medium used for the in vitro test and the plant extract should be compatible. It would thus be interesting to assess the same oxidizable substrate/oxidation condition combination in solvents with different polarities. Access to such a battery of tests would facilitate investigation of a diverse range of molecules and plant extracts, while also enabling studies on the impact of the medium and its polarity on the antioxidant behavior – a model to predict the antioxidant power according to this parameter should be developed. This would be a first step towards understanding the behavior of antioxidant substances in complex matrices, such as foods or certain biological tissues, where antioxidants undergo many interactions with the matrix components. 4.3.1. Homogeneous medium Homogeneous media (solutions) can be obtained with low concentrations of lipids dissolved in solvent, or just with vegetable oils. Many studies concerning methods for measuring antioxidant activity in organic media have shown that hydrogen bonding of solvents can induce a sharp change in the antioxidant capacity of phenol compounds [77,97] and especially in their ability to shed an H atom [98]. These solvent effects are mainly explained in terms of hydrogen bonds between the phenol compound (hydrogen donor) and the solvent (hydrogen acceptor). Barclay et al. [99] suggested that the antioxidant activity of catechols is mainly controlled by the stabilization, via intramolecular hydrogen bonds, of the aroxyl radical formed after inactivation of the first peroxyradical. This should be considered in the light of the fact that the antioxidant capacity of quercetin and epicatechin is greater in apolar solvents such as chlorobenzene than in more polar solvents like tert-butyl alcohol [100]. The rate of reaction of quercetin or epicatechin with the peroxyradical generated by 2,2-azobisisobutyronitrile (AIBN, Table 1) substantially decreases when switching from chlorobenzene to tert-butyl alcohol. Hydrogen bonding in the solvent also affects the antioxidant mechanism. Hence, quercetin does not function like chain-breaking antioxidant in tert-butyl alcohol, which is a protic medium, but rather as retarder, while it acts like chain breakers in chlorobenzene, which is an aprotic solvent [100]. This suggests that the type of solvent could influence the mechanism of flavonoid antioxidant action, i.e. from a chain breaker to an oxidation retarder role. This could be even more important when evaluating their efficacy if the analyst focuses on the lag phase. Finally, it would be senseless to discuss antioxidant efficacy without mentioning the solvent in which this property is measured. 4.3.2. Heterogeneous medium There are two main types of heterogeneous media: (i) oil-in-water emulsions, and (ii) aqueous suspensions of liposomes or LDLs. In such systems, oxidation is highly influenced by the type of interface and its microviscosity, the size and distribution of oil and liposome droplets, the partitioning and diffusion of oxygen towards reaction centers, and the antioxidant location. Moreover, lipid oxidation in these systems is generally faster than in bulk oil media, especially due to an increase in the surface contact area. The antioxidant location in the emulsion is critical for the chemical reactivity since these compounds are obviously more efficient when located close to the oxidation site. This is clearly illustrated by Porter’s polar paradox [101] in which polar antioxidants are more often active in lipid solutions than apolar antioxidants, whereas apolar antioxidants are more efficient in emulsion media than their polar homologues. This paradox is based on the interface properties of antioxidants, on their partitioning in multiphase media [102] and on the fact that lipid oxidation is initiated at the system interfaces. Hence, in bulk oil media, oxidation occurs at the air–oil interface where hydrophilic antioxidants are concentrated, whereas in emulsion media it occurs at the oil–water interface where lipophilic antioxidants are located. The interface membrane, and especially the type of surface active agent used (neutral, anionic or cationic), also have essential roles. Indeed, the repulsion or electrostatic attraction exerted by the membrane environment affects the oxidation and antioxidation efficacy. The anionic emulsion droplet charge can, for instance, attract transition metals, and consequently increase metal–lipid type interactions, leading to an increase in the oxidation rate [103]. Note, finally, that lipid oxidation is dependant on the pH in oil-in-water emulsions, and in liposomes. Frankel [29] reported that lipid oxidation is generally lower at high pH, and consequently the oxidation rate increases with the pH decrease. This author proposes, Please cite this article in press as: Laguerre M et al., Evaluation of the ability of antioxidants to counteract lipid ..., Prog Lipid Res (2007), doi:10.1016/j.plipres.2007.05.002

260

Water soluble

Solvent soluble

Formula

Comments Me

N

Me

Me

N

2HCl

N N Me

N

Me

H

H

N

Me

N

Me

Me

N

Me

N

N N H

H

HN

Me

Me

2HCl

NH 2HCl

N N H 2N

a b c

Me

Me

NH 2

Me

0

2,2 -Azobis[2-(5-methyl-2-imidazolin-2yl)propane]dihydrochloridea (41 °C)b MW: 351.32 Soluble in water: 30 wt% Insoluble in toluene, hexane

2,2 0 -Azobis[2-(2-imidazolin-2yl)propane]dihydrochloride (44 °C)b AIPH-MW: 323.33 Soluble in water >3 wt% Slightly methanol soluble: 0.5–3 wt% Insoluble in toluene, hexane 2,2 0 -Azobis(2methylpropionamide)dihydrochloride (56 °C)b AAPH-MW: 271.19 Soluble in water, methanol >3 wt% Slighty soluble in toluene

Not commercially available. 10 h half-life decomposition temperature in water. 10 h half-life decomposition temperature in toluene.

Comments

OMe Me

Me

CH 2

Me N N

CN

Me

Me N N

CH 2 Me

CN

Me Me

Me Me CN

Me

Me

CH 2 CN

N N CN

Me

CH 2 CN

Me

Me

OMe

Me

2,2 0 -Azobis(4-methoxy-2,4-dimethyl valeronitrile) (30 °C)c MeO-AMVN-MW: 308.42 Soluble in toluene, acetone > 3 wt% Insoluble in water

2,2 0 -Azobis(2,4dimethylvaleronitrile) (51 °C)c AMVN-MW: 248.37 Soluble in toluene, acetone, methanol Insoluble in water 2,2 0 -Azobisisobutyronitrile (65 °C)c AIBN-MW: 164.21 Soluble in methanol, ethanol Insoluble in water

ARTICLE IN PRESS

N

Formula



Table 1 Chemical structure of main azo-initiators commercially available


261

among other hypotheses, that this oxidative behavior could be linked with the solubilization of metal catalysts following a decline in pH. 4.4. Oxidation conditions According to Frankel [29], there is no ideal accelerated aging system and test conditions should be as close as possible to those in which the antioxidant has a protective effect. Here, we will present three oxidation systems: heat, ROO initiators and the different OH generation systems. 4.4.1. Heat Temperature is likely the most influential factor when assessing the extent of oxidation since the mechanism involved, especially hydroperoxide decomposition, is closely dependent on this parameter. Antioxidant efficacy also seems to be inversely proportional to the heat [104], which could be due to the volatization or thermal degradation of labile antioxidants at high temperatures. An analyst conducting a test at an excessively high temperature relative to real or biological conditions would therefore not be sure whether the antioxidant activity of the substance or its thermostability was being measured. Excess heat can also prompt polymerization and cyclization of the lipid substrate – reactions that would not necessarily occur under natural conditions. Finally, an excessively high temperature can result in oxygen depletion since oxygen solubility decreases with the heat. These five points underline that it is clearly essential to carry out tests at temperatures close to natural conditions, e.g. ambient (25 °C) or physiological (37 °C) temperatures. 4.4.2. ROO generation ROO peroxyradicals are not as reactive as hydroxyl radicals, thus facilitating their elimination by certain antioxidants, which means that they are a prime target for assessing antiradical activity. Azo-initiators are generally used for ROO generation, but other systems are also available (ethanol irradiation). With a generic formula of R–N@N–R, these initiators can generate R free radicals through spontaneous low temperature decomposition. D

R–N@N–R ! 2R þ N2

ð15Þ

In aerobic medium, free radicals R react immediately with oxygen (reaction (16)) to form ROO peroxyradicals, 2R þ 2O2 ! 2ROO

ð16Þ

which then abstract an allylic H atom from the L1H lipid substrate (reaction (17)), thus propagating the oxidative process (reactions 18,19). ROO þ L1 H ! ROOH þ L1 L1 þ O2 ! L1 OO

ð17Þ ð18Þ

L1 OO þ L2 H ! L1 OOH þ L2

ð19Þ

The increasing interest of using azo-initiators in in vitro tests is explained by the fact that they are easy to handle and generate ROO at a constant reproducible rate at moderate temperatures. They can be classified (Table 1) according to their hydrophilic, lipophilic or amphiphilic nature. 2,2 0 -Azobis(2-amidinopropane) dihydrochloride (AAPH, also abbreviated in the literature as ABAP) and 2,2 0 -azobis[2-(2-imidazolin-2-yl)propane] dihydrochloride (AIPH, also abbreviated in the literature as ABIP) are the most hydrosoluble commercial azo-initiators, and AIPH generates more peroxyradicals than AAPH in aqueous medium. 2,2 0 -Azobis(2,4dimethylvaleronitrile) (AMVN) and its methoxyl derivative 2,2 0 -azobis(4-methoxy-2,4-dimethylvaleronitrile) (MeO-AMVN) are the most widely used organic solvent-soluble azo-initiators. AMVN has the advantage of being highly soluble (in decreasing order) in toluene, methanol, ethanol and hexane. MeO-AMVN has a lower decomposition temperature, so it has a 15-fold higher decomposition rate in acetonitrile, at 37 °C, than AMVN [105], but it is less soluble in the above-mentioned solvents. Please cite this article in press as: Laguerre M et al., Evaluation of the ability of antioxidants to counteract lipid ..., Prog Lipid Res (2007), doi:10.1016/j.plipres.2007.05.002



Myriad studies have been carried out with azo-initiators, so we will simply focus on a few examples. These initiators have been used to investigate the oxidation response of linoleic acid micelles and multilamellar liposomes of dilinoleoylphosphatidylcholine [93], as well as LDLs and multilamellar liposomes from soybean oil [105]. Lie´geois et al. [106] measured the antioxidant capacity of wort, malt and hops in an aqueous dispersion of linoleic acid, with oxidation induced by AAPH. Finally, Van Dyck et al. [107] studied oxidation and antioxidation in complex food matrices such as minced pork and mayonnaise. It is essential to control the physicochemical factors that dictate the behavior of azo-initiators. Their efficacy is highly influenced by temperature, pH [108], the type of medium and its (micro)viscosity, as well as solvent cage effects. Concerning the (micro)viscosity of the medium, Barclay et al. [93] demonstrated that the decomposition rate of di-tert-butylhyponitrite (lipid-soluble) was substantially lower in liposome systems than in micelles, but also lower in micelles than in chlorobenzene. This difference was attributed to the decrease in the bilayer (micro)viscosity, to the micelles and to chlorobenzene, which is likely associated with the impact of this parameter on ROO diffusion. Prior et al. [109] also showed that preheating the solvent to the temperature at which the azo-initiator is subsequently diluted will reduce the variation coefficient (standard deviation/ mean) by around 50%. Concerning the drawbacks, Frankel and Meyer [8] reported some relevant examples where the use of azo-initiators in emulsion systems may give unclear results. For instance, a mixture of a-tocopherol and ascorbic acid has an additive antioxidant effect (the sum of the activities of each antioxidant is equal to the overall activity of the mixture) on liposomes oxidized in the presence of AAPH, thus generating peroxyradicals in the aqueous phase. However, the same mixture was found to have a synergistic antioxidant effect with AMVN, thus generating ROO in the lipid phase. This differential behavior could be explained by the fact that a-tocopherol traps free radicals in the lipid phase to be subsequently regenerated by ascorbic acid at the interface (second example), which does not occur during ROO generation in the aqueous phase (first example). Frankel and Meyer also reported that, contrary to proxidant metals, azo-initiators do not promote hydroperoxide decomposition into secondary oxidation compounds. This can lead to artificial hydroperoxide accumulation to the detriment of secondary oxidation compounds, which is an oversimplification of the natural autoxidation phenomenon. Moreover, it is hard to determine whether antioxidation involves direct trapping of azo-compound-derived peroxyradicals or of (more biologicallyimportant) lipoperoxides from the oxidizable substrate [5]. According to Niki [110], ascorbic acid, uric acid and other water-soluble antioxidants clearly quench radicals generated from AAPH but not lipid-derived radicals. Finally, Culbertson and Porter [111] synthesized lipophilic derivatives of AIPH upon which a fat moiety (C8, C12 and C16) was grafted on one side, resulting in a nonsymmetric amphiphilic azo-initiator. In aqueous dispersions of methyl linoleate (Triton X100), LDLs and phosphatidylcholine multilamellar liposomes, AIPH lipophilization was found to significantly increase total ROO production (ROO accessible to the aqueous compartment + ROO accessible to the lipid compartment). In LDLs and liposomes, there were more ROOs accessible to the lipid compartment for nonsymmetric azo-initiators as compared to AIPH and MeO-AMVN. These new initiators could be effective tools for future studies to assess lipid peroxidation and its antioxidant inhibition. 4.4.3. HO generation Hydroxyl radicals are clearly the most destructive species in oxidative stress because of their high oxidizing power and extremely high oxidation rate of very many bioorganic substrates. It would therefore be almost impossible to develop an in vivo chemical system to curb attacks from these radicals. Their rate constant is limited by diffusion, i.e. just by the molecule movement but not by an energy barrier. Consequently, the lifespan of OH radicals is extremely short (< a microsecond) and the distances that they can travel are also very short (< around 10 nm). Diffusion of these radicals is therefore very low and they generally react at the site of their production. Five main hydroxyl radical generating systems are found in the scientific literature [112], including: (i) the Haber–Weiss reaction (reaction (20)) which involves the hypoxanthine/xanthine oxidase system and its alternative, Please cite this article in press as: Laguerre M et al., Evaluation of the ability of antioxidants to counteract lipid ..., Prog Lipid Res (2007), doi:10.1016/j.plipres.2007.05.002

ARTICLE IN PRESS M. Laguerre et al. / Progress in Lipid Research 46 (2007) 244–282 3þ O ! OHþ OH þ O2 þ Fe2þ 2 þ H2 O2 þ Fe

263

ð20Þ

(ii) the standard Fenton reaction (reaction (14)) in buffered medium (pH 7.4) which involves the Fe3+/ EDTA/ascorbic acid/H2O2 system and its alternatives, (iii) use of a ‘Fenton-like’ reagent such as Co2+/H2O2 or Cu2+/H2O2 systems, (iv) by pulse radiolysis of water, (v) by the ‘photo-Fenton’ system using photosensitizers. According to Moore et al. [112], the hypoxanthine/xanthine oxidase system generates superoxide radicals in addition to OH radicals and may have interference from enzyme inhibitors, which could result in overestimation of the actual capacity of the OH scavenger. Radiolysis of water, which involves submitting an aqueous dispersion of lipids to ionizing radiation (X, c and electron beam), closely simulates oxidative stress conditions by generating hydroxyl and superoxide radicals. However, this type of equipment is not very widespread, which limits the use of this technique. The ascorbic acid/Fe3+ system was criticized by Niki and Noguchi [10] because ascorbic acid acts like a proxidant when the ascorbic acid/Fe3+ ratio is low, whereas it acts as an antioxidant at higher ratios. On the other hand, it is harder to distinguish between the transition metal chelation activity of antioxidants and the scavenging activity of OH radicals. In addition, transition metals fully participate in the radical autoxidation mechanism via OH generation, but they are also involved in hydroperoxide decomposition, which can mask their actual contribution, especially if a tailored measurement strategy is not used. We have also noted that transition metals can give rise to proxidant effects that, despite the fact that they have been extensively monitored, can also complicate interpretation of in vitro results. Finally, OH radicals have very high and almost identical rate constants against oxidizable substrates and antioxidant agents. The antioxidant concentration must therefore be grossly increased relative to concentrations that actually occur in biological media or foodstuffs, so antioxidant measurements will not reflect the real situation. 5. Adopted measurement strategies Tests used to measure antioxidant power require assessment of the extent of oxidation of the lipid substrate in the presence or absence of a potential antioxidant molecule or plant extract. A rapid and reliable evaluation of the best oxidation markers in terms of the specificity and representativeness of the oxidative process would be a good measurement strategy. However, the problem of implementing such a method is related to the actual chemical mechanism of the oxidation reaction. In practical terms, four different measurement strategies can be used to directly assess the antioxidant capacity of a molecule toward a lipid substrate, these involve measuring: (i) oxygen depletion, (ii) substrate loss, and formation of (iii) primary and/or (iv) secondary oxidation products. In this chapter, we will discuss problems that may arise in such evaluations and roughly review the most common tests, which we will classify according to the adopted measurement strategy. The respective advantages and drawbacks of each method will also be discussed. 5.1. Oxygen depletion The duration of the initiation phase and its extension in the presence of antioxidant agents can be measured by assessing the oxygen consumption patterns. The measurement methods can be manometric [113], gravimetric via measurement of weight increases following oxygen fixation on fatty acids [114], or polarographic [115] using a Clark electrode [116]. Roginsky and Barsukova [115] used this latter method to investigate oxygen consumption induced by chain peroxidation of methyl linoleate in Triton X-100 aqueous micelles induced by AAPH. This procedure was used to determine the antioxidant capacity of nine red wines and single samples of green and black teas, white wine, beer and soluble coffee. Headspace chromatography can also be implemented, whereby oxygen absorption is measured using a thermoconductimetry detector [117]. The main shortcoming of all of these methods is that oxygen-consuming interference reactions may occur in complex media. Note also that the assay equipment must be completely sealed to avoid artifacts. Please cite this article in press as: Laguerre M et al., Evaluation of the ability of antioxidants to counteract lipid ..., Prog Lipid Res (2007), doi:10.1016/j.plipres.2007.05.002



5.2. Substrate loss Monitoring oxidative degradation in a real system such as a food or biological sample using a substrate loss measurement strategy is quite complicated. There are many hard to identify potentially oxidizable biomolecules in these complex systems. Moreover, a ‘matrix effect’ due to the presence of interfering substances can considerably confound the analysis. Considering these few limitations, this measurement strategy seems more adapted for determining the extent of oxidation in a model system (i.e. single fully characterized substrate, simple medium) or labeled system (fluorophore-labeled biological particles or living cells). Methods that use this strategy – apart from a few chromatographic methods designed for assaying residual nonoxidized substrates – generally involve a spectral measurement system (Table 2). Most oxidizable substrates used in Table 2 Main methods based on measurement of initial substrate loss Substrate

Detection

Oxidizing conditions/media/temperature a

Reference

Tween-emulsified linoleic acid with b-carotene

Spectrophotometry (470 nm) Spectrophotometry (460 nm)

AAPH /emulsified aqueous system/32 °C Soybean lipoxygenase type 1-S/buffer (pH 7)/25 °C

[123] [124]

cPnAb labeled liposomes

Fluorimetry (324/413 nm)

[126]

cPnA labeled LDLs


cPnA labeled erythrocytes


cPnA labeled cardiac myocytes


C11-fluorc labeled erythrocytes

Fluorimetry (500/520 nm) and flow cytometry (kex: 488 nm) Fluorimetry (488/520 nm) and flow cytometry (kex: 488 nm) Fluorimetry (570/600 nm) Fluorimetry (540/600 nm)

CuSO4 + H2O2/(150 mM NaCl,10 mM Tris–HCl buffer (pH 7.4))/25 °C AAPH/110 mM NaCl, 20 mM phosphate buffer (pH 7.4)/37 °C Cumene hydroperoxide + cofactors (Fe3+, Cu2+and hemin-Fe3+)/150 mM NaCl, 10 mM Tris–HCl (pH 7.4)/25 °C Cumene hydroperoxide/buffer (pH 7.4)/37 °C CuSO4 + H2O2/Buffer (pH 7.4)/37 °C AAPH/buffer (pH 7.4)/37 °C Cumene hydroperoxide

Fluor-DHPEd labeled erythrocytes C11-BODIPY581/591e DOPCg liposomesincorporated C11BODIPY581/591 C11-BODIPY581/591

Fluorimetry (488/500–700 nm) and ESI-MSh (negative mode)

C11-BODIPY581/591 labeled rat fibroblasts

Fluorimetry (488/500–700 nm) and ESI-MSh (negative mode)

BODIPY665/676m

Fluorimetry (620/675 nm) Fluorimetry (600/700 nm)

[89] [127]

[134]

[140]

Benzoyl peroxide or Cumene hydroperoxide

[141]

AMVNf/Octane:butyronitrile (9:1, v:v)/41 °C AMVN/20 mM Tris–HCl buffer (pH 7.4)/42 °C

[139]

MeO-AMVNi or AAPH/ethanol/37 °C CuSO4 + cumene hydroperoxide/ethanol/37 °C CuSO4 + H2O2/ethanol/37 °C Peroxynitrite/ethanol/37 °C Cellular depletion of GSHj by BSOk and serum-free DMEMl/37 °C Peroxynitrite/37 °C AMVN/octane:butyronitrile (9:1, v:v)/39 °C AMVN/20 mM Tris–HCl buffer (pH 7.4)/39–41 °C

[146]

[146]

[152]

Abbreviations: a AAPH: 2,2 0 -azobis(2-amidinopropane) dihydrochloride. b cPnA: cis-parinaric acid. c C11-fluor: 5-(N-dodecanoyl)aminofluorescein. d Fluor-DHPE: fluoresceinated dihexadecanoylglycerophosphoethanolamine. e C11-BODIPY581/591: 4,4-dichloro-5-(4-phenyl-1,3-butadienyl)-4-bora-3a,4a-diaza-s-indacene-3-undecanoic acid. f AMVN: 2,2 0 -azobis(2,4-dimethylvaleronitrile). g DOPC: dioleoylphosphatidylcholine. h ESI-MS: electrospray ionization mass spectrometry. i MeO-AMVN: 2,2 0 -azobis(4-methoxy-2,4-dimethylvaleronitrile). j GSH: glutathion. k BSO: L-buthionine-(S,R)-sulfoximine. l DMEM: Dulbecco’s modified Eagle’s medium. m BODIPY665/676: 4,4-difluoro-3,5-bis(4-phenyl-1,3-butadienyl)-4-bora-3a,4a-diaza-s-indacene.



265

these tests have a conjugated polyenic structure, which means they have absorption and/or fluorescence properties in the UV–VIS spectrum so their oxidative degradation can be readily monitored. Here, we cover such methods as a complement to the review of Gomes et al. [17]. 5.2.1. b-Carotene bleaching assay This UV-spectrophotometric technique initially developed by Marco [85] and modified by Miller [86] involves measuring b-carotene bleaching at 470 nm resulting from b-carotene oxidation by linoleic acid degradation products. Tween is used for dispersion of linoleic acid and b-carotene (Fig. 9a) in the aqueous phase. Linoleic acid oxidation is nonspecifically catalyzed with heat (50 °C). The addition of an antioxidant-containing sample, individual antioxidants [118], or plant extracts [119,120], results in retarding b-carotene bleaching. A close correlation (R2: 0.935) was obtained between the quantity of polyphenols in fruit extracts measured by the Folin–Ciocalteu method and the antioxidant capacity results obtained in this test [121].

β -Carotene

a O HO

+ (CH2) 7

(CH2)4-CH3 Linoleic acid

c

C O OH

Lipophilic derivatives of fluorescein n = 10 (C11-fluor) n = 14 (C16-fluor) n = 16 (C18-fluor)

cis-Parinaric acid

d

Phospholipidic derivatives of fluorescein Fluor-DHPE

HN

C N

O

F

f F

(CH2) 14CH3

HC O C

(CH2) 14CH3

(CH2)2 O P O CH2

O

O

N

F

H2C O C

O

+ B

(CH 2) n CH3

O

S

N

HN CO

COOH H

N

COOH

O

O

HO

e

O

O

HO

b

(CH2)10

OH

C11-BODIPY581/591

+ B

N F

BODIPY665/676

Fig. 9. Chemical structure of some lipidic or lipid-like oxidizable substrates.




This spectrophotometric measurement method is sensitive, relatively rapid (2 h), simple and generates results in the visible spectral range. Another advantage is that it can be combined with thin layer chromatography [122]. After chromatographic separation, a mixture of b-carotene and linoleic acid is pulverized on the plate and then exposed to daylight or UV for several hours, until the yellow background fades. Antioxidant substances are indicated by bands that have remained yellow. However, this thermically-induced oxidation is not controlled and is thus quite unspecific, which often induces data variability. Some authors have attempted to overcome this problem by using oxidants rather than heat, with AAPH [123] and soybean lipoxygenase [124] being the most reproducible of these agents. This method is also hampered by interference from absorbent compounds in the b-carotene spectral window. Frankel [125] also criticized the use of free fatty acids, which are not realistic lipid models. Finally, it is not easy to interpret the results because b-carotene itself is an oxygen-sensitive antioxidant. 5.2.2. Fluorescence decay of cis-parinaric acid In 1987, Kuypers et al. [126] demonstrated the advantages of using cis-parinaric acid as fluorescent probe to evaluate the extent of lipid peroxidation in membrane systems. cis-Parinaric acid, or 9-cis,11-trans,13-trans,15cis,octadecatetraenoic acid, is a polyunsaturated fatty acid with four conjugated double bonds (Fig. 9b). This linear conjugated tetraene gives the molecule high fluorescence properties (kex/em: 320/432 nm), as well as high sensitivity to free radical attacks as compared to most polyunsaturated fatty acids and their derivatives. The key point is that the fluorescence of cis-parinaric acid is irreversibly lost during its oxidation, so many tests have been developed using this substrate. Moreover, cis-parinaric acid can be metabolically integrated in the membranes of various cell types, which has led to enhancement of the detection sensitivity of oxidative processes in these highly organized structures. Consequently, cis-parinaric acid has been used extensively to measure lipid peroxidation in erythrocytes [127,128], sub-mitochondrial particles [129], sarcoplasmic reticulum [130], rat aortic smooth muscle [87], lens membrane [131], macrophage [132], neonatal rat cardiomyocytes [133], cultured cardiac myocytes [134] and rapid dividing cell lines such as human leukemia cells [135]. This method has also been used for measuring the antioxidant capacity of various substances toward cis-parinaric incorporated in LDL particles [88] or liposomes [136]. For example, Osaka et al. [137] assessed the ability of amphotericin B to overcome peroxidation of cis-parinaric acid complexed with human serum albumin or incorporated in liposomes – AAPH was used to initiate oxidation in the complex, while AMVN was the initiator in the liposomal system. Laranjinha et al. [89] studied, in terms of structure–activity relationships, the antioxidant activity of various dietary phenolic compounds toward AAPH-induced oxidation of cis-parinaric incorporated into LDL. Amongst other advantages, cis-parinaric acid is a bioanalog and does not disturb the lipid bilayer. It also has a very broad fluorescence Stockes shift (110 nm). Finally, the fact that it can be metabolically incorporated into membrane phospholipids of cultured cells makes cis-parinaric acid a suitable and straightforward membrane probe for detecting the initial stages of lipid peroxidation [133–135]. Concerning the drawbacks, cis-parinaric acid is air sensitive and photolabile and undergoes photodimerization under illumination, which results in loss of fluorescence and overestimation of the extent of lipid peroxidation [81,138,139]. Moreover, cis-parinaric absorbs in the UV region at 320 nm where most test compounds also absorb, especially flavonoids which have an absorption peak at 320 nm. In practical terms, the results can be biased if the interfering molecule absorbs all or part of the excitation and/or emission photons. 5.2.3. Lipophilic fluorescein-based flow cytometry Based on the principle that fluorimetry can only be used to study lipid peroxidation in a large cell population, but not in single cells, Makrigiorgos et al. [140] developed a flow cytometry-based analysis method. Hence, 5-(N-dodecanoyl)aminofluorescein (C11-fluor, Fig. 9c), a commercially available lipophilic fluorescein, was incorporated into the cellular membranes of red blood cells. C11-fluor oxidation induced by cumene hydroperoxide was accompanied by gradual fluorescence extinction. In this test, Trolox and vitamin E clearly inhibit lipid peroxidation when incubated with the cells, as shown by the steady slowdown in the fluorescence extinction kinetics. However, a major prerequisite for the routine use of this method for the detection of lipid peroxidation in specific cell subpopulations is that the fluorescent probe is not exchangeable between cells. Please cite this article in press as: Laguerre M et al., Evaluation of the ability of antioxidants to counteract lipid ..., Prog Lipid Res (2007), doi:10.1016/j.plipres.2007.05.002


267

Any between-cell differences in peroxidation could not be detected if this exchange were to occur. Maulik et al. [141] thus examined and compared four lipophilic fluoresceins, i.e. C11-fluor, 5-hexadecanoylaminofluorescein (C16-fluor), 5-octadecanoylaminofluorescein (C18-fluor) and fluoresceinated dihexadecanoylglycerophosphoethanolamine (fluor-DHPE) (Fig. 9c and d). Of these, fluor-DHPE was found to be the only nonexchangeable flow-cytometric probe. This method was then used for studying the antioxidant efficacy of a-tocopherol, ascorbic acid and uric acid toward erythrocyte membranes labeled with C11-fluor and oxidized by cumene hydroperoxide [142]. The results indicated that a-tocopherol was the most efficient antioxidant and that ascorbate halted chain propagation, probably through a mechanism involving redox recycling of a-tocopherol. In 2004, Amer et al. [143] used fluor-DHPE to label phospholipidic membranes of erythrocytes from normal donors and thalassemia patients (genetic disease in which hemoglobin synthesis is impaired). The extent of oxidation induced by H2O2 was measured by flow cytometry, and the results showed that cells from the thalassemia patients were the most H2O2-sensitive. Concerning the advantages, the excitation wavelength of these probes (488 nm) seems to be especially well adapted to cells and to routine use in flow cytometry, for which a laser emitting visible blue light (488 nm) is generally used. Moreover, fluor-DHPE mimics membrane lipids relatively well. Overall, the method, which can be used on an individual cell or a subpopulation scale, despite its cost, is an especially valuable tool for scientists. The only drawback is that, to our knowledge, this method has only been used to assess erythrocytes. As pointed out by Gomes et al. [17], it should now be confirmed that fluor-DHPE is a sufficiently sensitive probe to be used with other cell types. 5.2.4. Fluorescence decay of BODIPY probes This fluorimetric analysis method was initially developed using the fatty 4,4-difluoro-5-(4-phenyl-1,3butadienyl)-4-bora-3a,4a-diaza-s-indacene-3-undecanoic acid (C11-BODIPY581/591) as oxidizable substrate [139]. This fluorescent fatty acid analog (Fig. 9e) in which the BODIPY core is connected to a phenyl moiety via a conjugated diene (that acts as a resonance bridge), displays bright red fluorescence, with an emission peak in the 591–595 nm range, depending on the solvent used. This substrate is highly oxidizable by ROO because of the number of conjugated double bonds. It was found that the rate of reaction between C11-BODIPY and ROO is around twofold higher than that between polyunsaturated lipids and ROO [144]. Oxidation of C11-BODIPY581/591 leads to gradual extinction of the fluorescent signal. The principle of the method, as set out by Naguib [139], is to assess the capacity of antioxidant molecules to protect the C11-BODIPY581/591 probe from attack by ROO generated by AMVN at around 40 °C. The antioxidant capacity is then shown by the relatively marked lag phase, depending on the quantity of chain-breaking antioxidants used. Naguib thus used two complementary approaches: (i) C11-BODIPY581/591 is directly solubilized in an apolar organic medium consisting of octane and butyronitrile (9:1, v:v), and (ii) this fluorescent probe can also be incorporated in liposomes of dioleoylphosphatidylcholine suspended in Tris-HCl buffer. In both cases, Trolox was used as reference antioxidant molecule and the information was derived from analytical data by calculating the area under the curve. Aldini et al. [145] then adapted the method for determination of the oxidizability of aqueous and lipidic compartments in plasma. To monitor this oxidation in the plasma lipid compartment, MeO-AMVN and C11-BODIPY581/591 were used, whereas for aqueous plasma the extent of oxidation was determined by using AAPH and 2 0 ,7 0 -dichlorodihydrofluorescein. One of the advantages of the method is that relatively consistent documentation is available on the oxidation of this probe by various oxidant agents, and the type of products obtained according to their fluorescence spectra. The diene link between the phenyl ring and the BODIPY core was determined as the prime target of C11-BODIPY581/591 oxidation [146]. These authors showed that, in ethanol solution at 37 °C, oxidation of C11-BODIPY581/591 by various oxidants (AAPH, MeO-AMVN, cumene hydroperoxides/CuSO4 and H2O2/ CuSO4) led to the formation of two stable end products with 419 and 445 amu (pathway 1, Fig. 10), leading to a shift in fluorescence emission (from 595 to 520 nm). This should be considered in the light of the fact that the emission spectrum of oxidized C11-BODIPY581/591 contains two maxima [147,148], in contrast to the single emission peak in nonoxidized C11-BODIPY581/591, suggesting the formation of multiple oxidation products. However, when oxidation is induced by peroxynitrite in ethanol solution (pathway 2, Fig. 10), the two stable end products (419 and 445 amu) noted above are only detected in trace amounts. Instead, a single Please cite this article in press as: Laguerre M et al., Evaluation of the ability of antioxidants to counteract lipid ..., Prog Lipid Res (2007), doi:10.1016/j.plipres.2007.05.002


M. Laguerre et al. / Progress in Lipid Research 46 (2007) 244–282 m/z : 445 amu N

HO

N F

F

O

+

O

+ B

(CH2)10

OH

Stable end products λ ex/em : 488/520 nm

m/z : 419 amu

N

HO O

+ B

N F

F

Fluorescence shift from red to green

O

(CH2)10

OH

a

Pathway 1 : ROS attack toward ethanolic 581/591 solution of C11-BODIPY 581/591

Non-oxidized C11-BODIPY Diene interconnection : Resonance bridge

N F

b

B

O

+ N F

(CH2)10

λex/em : 488/595 nm

OH

Pathway 3 : Peroxynitrite attack c toward rat 581/591 fibroblasts labelled with C11-BODIPY

Pathway 2 : Peroxynitrite attack toward ethanolic solution of C11-

m/z = 519 amu

m/z : 445 amu HO

N

N (CH2)10

F

F

O

Trace amounts

B

OH

O

+

N

OH

F

F 581/591

C11-BODIPY

O

F

-OH λ ex/em : 498/539 nm

(CH )10

m/z : 445 amu

OH

2

Major oxidation product (CH )10 2

OH

N

HO O

581/591

C11-BODIPY

(CH )10

OH

2

m/z : 419 amu

N F

F

N F

O

+ B

B

F

O

m/z : 519 amu

+

O

+

N

HO

OH

OH

2

N

B

F

N

(CH )10

+

O

+

N

Small amounts

N

B

m/z : 419 amu HO

O

+

F

O

+ B

N F

(CH )10 2

OH

-OH

λ ex/em : 498/539 nm

Fig. 10. Oxidative degradation of C11-BODIPY581/591 probe by different oxidants (adapted from reference [146]) (a After 48 h incubation at 37 °C in ethanol with either 20 mM AAPH, 20 mM MeO-AMVN, 20 mM cumene hydroperoxide/250 lM CuSO4or 20 mM H2O2/ 250 lM CuSO4, bAfter exposure to 10 mM peroxynitrite for 1 h at 37 °C, cExogenously oxidative stress induced by exposure C11BODIPY581/591 labelled rat fibroblasts to 100 lM peroxynitrite).

hydroxyl oxidation product (C11-BODIPY581/591-OH) is detected with a single emission peak at 539 nm. These authors then conducted a more in-depth study on cell cultures of rat fibroblasts labeled with C11-BODIPY581/591. It was found that peroxynitrite-induced oxidation (pathway 3, Fig. 10) mainly led to the formation of two stable end products (419 and 445 amu), and just small amounts of the hydroxyl product were detected. This fluorescent probe thus generated a uniform oxidation profile in rat fibroblasts in all oxidation conditions tested. However, in ethanol solution, the pattern differed and a distinction could be made between ROS and peroxynitrite. Concerning the advantages, this method can be applied using microplate readers for highthroughput analysis, and also using flow cytometry for assessment of cell subpopulations [149]. Concerning the drawbacks, Huang et al. [150] showed that the C11-BODIPY581/591 probe could lose 30% of its fluorescence in the absence of AMVN. This suggests that, like cis-parinaric acid, C11-BODIPY581/591 is photosensitive. According to these authors, this photobleaching is likely caused by cis/trans isomerization of olefinic double bonds, under the impact of an excitation beam at around 545 nm. This is a disconcerting result because Naguib [139], using the original method, confirmed that C11-BODIPY581/591 is thermo- and photostable on the basis of his results obtained under identical conditions (except for the type of reader, i.e. microplate reader vs. 1-cm quartz cuvette spectrofluorometer) and those reported by Haugland [138]. This was then further confirmed especially by Beretta et al. [151], who showed that C11-BODIPY581/591 fluorescence was stable at 37 °C in the absence of oxidant agents in either phosphatidylcholine intermediate unilamellar vesicle suspension or Please cite this article in press as: Laguerre M et al., Evaluation of the ability of antioxidants to counteract lipid ..., Prog Lipid Res (2007), doi:10.1016/j.plipres.2007.05.002


269

human plasma. Drummen et al. [147] noted that photobleaching was virtually absent when measurements were obtained with a fluorimeter, but under high-intensity illumination conditions using confocal laser scanning microscopy, photobleaching of C11-BODIPY581/591 becomes the main factor limiting fluorescence detectability and should thus be taken into account. Note that other BODIPY derivatives have also been used. Naguib [152] tested the feasibility of the method by substituting C11-BODIPY581/591 by BODIPY665/676 (Fig. 9f). Amongst other advantages, BODIPY665/676 is more readily oxidizable than C11-BODIPY581/591 because of extension of the conjugated polyene system, so less AMVN is required. Moreover, its Stockes shift (15 nm) is slightly broader than that of C11BODIPY581/591 (10 nm) [138], resulting in less interference of scatter to its emission. 5.3. Formation of primary oxidation products Contrary to the measurement strategy based on substrate loss, that based on the formation of oxidation products (primary or secondary) seems relatively well adapted for studying all types of system, including model systems, foods or biological samples isolated from their environment. Although no universal markers are available, oxidation of complex systems generate certain oxidation products that can be detected by physicochemical methods. Hydroperoxides are considered as primary oxidation products that generate the most relevant information on lipoperoxidation intensity. Hydroperoxide measurement is likely the approach that has been the focus of the most development. The main techniques for assaying these products will be covered in this chapter. 5.3.1. Iodometric hydroperoxide measurement A conventional standardized method for quantifying total hydroperoxides and hydrogen peroxide involves iodometric assay [153]. This is one of the oldest and most commonly used methods for assessing lipid substrate oxidation. In acidic medium, hydroperoxides (reaction (21)) and ROORs (reaction (22)) react with the iodide ion to generate iodine, which is titered using a sodium thiosulfate solution in the presence of starch solution: ROOH þ 2Hþ þ 2I ! I2 þ ROH þ H2 O þ

ROOR þ 2H þ 2I ! I2 þ 2ROH I2 þ

2S2 O2 3

!

S4 O2 6

þ 2I

ð21Þ ð22Þ ð23Þ

The peroxide value (PV) is considered to represent the quantity of active oxygen (in mg) contained in 1 g of lipid and which could oxidize potassium iodide, followed by iodine release. PV peaks during the propagation phase and then decreases during the termination phase when the hydroperoxide decomposition kinetics are higher than the formation kinetics. Note, however, that this method has some drawbacks. PV measurements are only relevant for samples in which autoxidation is not too advanced and under temperature conditions that are mild enough to avoid hydroperoxide decomposition. Moreover, light exposure, iodine absorption by unsaturated fatty acids and iodine formation through the oxidation of iodide ions in the presence of ambient oxygen can interfere and lead to under- or overestimation, depending on the case [29]. Moreover, this method is relatively easy to use for anhydrous systems like bulk oil, but not with emulsions, foods or biological media where the presence of water is detrimental. In these three cases, a prerequisite quantitative, selective and nonaltering extraction of lipids is required, so some precautionary measures must be taken to avoid oxidative stress. For emulsions, two strategies may be implemented: (i) breaking the emulsion by increasing the ionic strength or by centrifugation, or (ii) performing liquid–liquid lipid extraction followed by an evaporation step to eliminate the solvent. Concerning the drawbacks, lipid extraction carried out in the presence of oxygen can generate hydroperoxides, sometimes in greater quantities than originally present. Cold extraction of lipids is also recommended using a methanol–chloroform mixture according to a method recommended for lipids derived from animal tissues [154]. High-temperature solvent removal may induce substantial peroxide decomposition. Vacuum evaporation solvent extraction is the most commonly used procedure. Most of these problems can be overcome, but PV measurement in emulsions, foods and biological samples is still markedly hampered by the discontinuous aspect of the process and potential interference. Alternative methods have thus been developed, including colorimetric assays using thiocyanate Please cite this article in press as: Laguerre M et al., Evaluation of the ability of antioxidants to counteract lipid ..., Prog Lipid Res (2007), doi:10.1016/j.plipres.2007.05.002



[155,156], orange xylenol [157], enzymatic assays using glutathion peroxidase [158] or Fourier transform infrared spectroscopy [159]. 5.3.2. Ultraviolet measurement of conjugated dienes In 1931, Gillam et al. [160] demonstrated that, during storage, natural fats had a peak absorption of around 230–235 nm [161]. Over 90% of hydroperoxides formed by lipoperoxidation have a conjugated dienic system resulting from stabilization of the radical state by double bond rearrangement. These relatively stable compounds absorb in the UV range (235 nm) forming a shoulder on the main absorption peak of nonconjugated double bonds (200–210 nm), so they can be measured in the UV spectrum by absorption spectrophotometry. All substances containing polyunsaturated fatty acids can be used as oxidizable substrates for this method since conjugated dienes can only be formed from fatty acids with at least two double bonds, e.g. linoleic acid. In 1993, Pryor et al. [162] assessed the antioxidant capacity of synthetic and natural compounds by measuring their impact on the kinetics of conjugated diene formation via AAPH-induced oxidation of linoleic acid micelles in aqueous medium. Amongst other advantages, these authors reported that this measurement strategy is more sensitive than that based on oxygen consumption. The antioxidant activities of 44 berry fruit wines and liquors were compared by this method, with methyl linoleate used as substrate [163]. Although this method can be adopted for continuous measurement of lipid peroxidation, the problem is that the absorption peak of conjugated dienes often appears as a shoulder of a broad band generated by nonoxidized lipids [164]. To overcome this poor resolution, several authors have used second derivative spectrometry for the analysis of vegetable oils, food lipids and biological samples [165–167]. In addition, Antolovich et al. [14] reported that diene conjugation measurements often cannot be performed directly on tissues and body fluids because many other interfering substances are present, such as haem proteins, chlorophylls, purines and pyrimidines that absorb strongly in this UV region. Compounds that could interfere should thus be identified in order to be able to correct the experimental bias as well as possible. Systematic extraction of lipids using an organic solvent prior to analysis could be done to overcome the interference problem. Moreover, like PV, it is essential to ensure that this reaction does not reach an advanced oxidation stage and that the process conditions are mild enough to limit hydroperoxide decomposition. As is the case for all measurement strategies based on the formation of primary oxidation products, we think that the use of azo-initiators could be beneficial as they would minimize the formation of secondary oxidation products [8]. 5.4. Formation of secondary oxidation products This strategy for measuring secondary oxidation products, like that based on the formation of primary oxidation products, seems to be suitable for studying model lipid systems, as well as real lipids isolated from their natural environment (microsomes, LDLs, etc.). We will now discuss the three tests most commonly implemented in research and industrial conditions. 5.4.1. Thiobarbituric acid (TBA) test This test, which was developed in the late 1940s, is very commonly used both in vitro and in vivo. It involves reacting thiobarbituric acid with malondialdehyde produced by lipid hydroperoxide decomposition to form a red chromophore with peak absorbance at 532 nm. This colored complex results in the condensation of 2 moles of TBA and 1 mole of malondialdehyde (Fig. 11), under the joint effect of the medium temperature and pH. TBA is defined as the quantity of malondialdehyde (in mg) present in 1 kg of sample. Many oxidizable substrates have been used in thiobarbituric acid-reactive substances (TBARS) determination, including free fatty acids, LDL and fluids (urine, serum) from cells or tissues. This method has, however, been the focus of much criticism. The first is that malondialdehyde only forms from fatty acid chains containing at least three double bonds, like linolenic acid, to the exclusion of linoleic and oleic acid peroxide decomposition products [168]. Secondly, TBA is not specific to malondialdehyde because it can react with other aldehydes, browning reaction products, protein and sugar degradation products, amino acids and nucleic acids [29,169]. There is also a risk of underestimating the response since malondialdehyde can, under in vivo conditions, form linear Please cite this article in press as: Laguerre M et al., Evaluation of the ability of antioxidants to counteract lipid ..., Prog Lipid Res (2007), doi:10.1016/j.plipres.2007.05.002

ARTICLE IN PRESS M. Laguerre et al. / Progress in Lipid Research 46 (2007) 244–282 O

271

O OH O

O

O

O Malondialdehyde formation pathway (a)

OH O

O

O

OH

O C

N

HS O

O

+

2

N

+

OH H

OH 2-thiobarbituric acid

O

O

HCl H2O

+

(b)

OH

N

S

OH HO

SH

N N

N OH

OH

TBA chromophore (λ max : 532 nm)

Fig. 11. Malondialdehyde formation pathway from peroxyl radical of triunsaturated C18 fatty acid (a) and formation of TBA chromophore from TBA and malondialdehyde (b).

or cyclical Schiff bases, or even crosslinked bonds, with lysine and arginine from proteins and then form advanced glycation end products (AGEs). Note finally that malondialdehyde does not occur in many oxidized lipids, and is often a minor secondary oxidation product [29], which is detrimental to the representativeness due to the assumed lipid oxidation marker role of this compound. Alternative techniques that are more specific to malondialdehyde have thus been developed, including the methods of Tatum et al. [170] and Wong et al. [171]. The first involves purifying the TBA/malondialdehyde complex by HPLC to eliminate all contaminants, and then quantifying it with a fluorimetric detector. Picomoles of malondialdehyde can thus be assayed in plasma and liver. This technique has also been used to study urine samples [172]. The plasma assessment method proposed by Wong et al. combines HPLC separation and spectrophotometric detection. Note also that malondialdehyde can be assayed without prior condensation via TBA. There are two key techniques in which malondialdehyde is analyzed by GC after derivatization: (i) in stable tetramethylacetal with GC-FID analysis [173], and (ii) in methylpyrazole with analysis using GC combined with a nitrogen/phosphorus specific detector [174]. 5.4.2. Aldehyde measurement by the anisidine test Calculating the p-anisidine value (PAV) is one of the oldest methods for evaluating secondary lipid oxidation. It is based on the reactiveness of the aldehyde carbonyl bond on the p-anisidine amine group, leading to the formation of a Schiff base that absorbs at 350 nm R–CH@O þ H2 N–U–OMe ! R–CH@N–U–OMe ðSchiff baseÞ þ H2 O

ð24Þ

This test is mainly used in the analysis of animal fats and vegetable oils targeted for dietary, cosmetic or industrial applications. It generates useful information on carbonyl compounds, especially a-unsaturated aldehydes. Other high molecular weight decomposition products, such as triglyceride dimers, can also be measured with this method. In addition, PAV accounts for nonvolatile aldehyde, or core aldehydes, that also form during lipid oxidation processes. These aldehydes could even explain some inconsistencies noted during the analysis Please cite this article in press as: Laguerre M et al., Evaluation of the ability of antioxidants to counteract lipid ..., Prog Lipid Res (2007), doi:10.1016/j.plipres.2007.05.002



of some products. An oil obtained by refining a highly oxidized raw material could thus have a high PAV, a low PV, and an undetectable rancid odor simply due to the presence of nonvolatile aldehydes, which are hard to remove by refining. The results, however, are interpreted in a relatively empirical manner since each of the many aldehydes produced during oxidation generates a specific signal. It is well known that the colorimetric response with panisidine varies according to the extent of aldehyde unsaturation. Hence, at identical concentrations, the response is more intense with di-unsaturated aldehydes than with mono-unsaturated aldehydes, which in turn are more sensitive than saturated aldehydes. Moreover, p-anisidine reacts with all aldehydes, irrespective of their origin. This is especially the case for some phenol compounds of virgin olive oil, such as decarboxymethyl oleuropeine dialdehyde, which could interfere in the assessment. Finally, studies on correlations between PAV, or the totox (PAV + PV), and the organoleptic quality highlighted the efficacy of this test for measuring oxidation in many different lipids. However, these correlations may vary markedly between lipids and also according to the prevailing oxidation conditions. Caution is thus required when interpreting this index. 5.4.3. Chromatographic measurement of volatile compounds Chromatography can be used to quantify volatile molecules derived mainly from hydroperoxide decomposition. The main molecules measured are aldehydes, ketones, alcohols, short carboxylic acids and hydrocarbons. Some of these volatile compounds are highly specific to the oxidative degradation of a particular polyunsaturated fatty acid family. Propanal is the main marker of oxidation of fatty acids of the n–3 family, while hexanal and pentanal are markers of oxidation of fatty acids of the n-6 family. Hexanal is the most frequently measured lipid oxidation end product. Snyder et al. [175] showed that hexanal formation is much greater than that of most secondary oxidation products, apart from a few exceptions. However, measuring the extent of oxidation with just one or two markers is a cyclopean-type approach [14]. Methods involving assessment of large set of compounds should be promoted, especially as recent advances and widespread adoption of GC–MS analysis techniques can considerably facilitate this task. Different methods may be used to recovered volatile oxidization markers, including: (i) extraction and (ii) headspace analysis (HS). (i) Concerning extraction, note especially simultaneous steam distillation (SDE), where the sample is dispersed in water and heated, with the extraction solvent contained in a separate flask. The sample is distilled for several hours. The system is then refrigerated and the solvent is removed from the flask, dried using a drying agent and concentrated by slow evaporation. The combined SDE-GC technique has been used especially to quantify hexanal, which is generated in ground turkey and ground pork during storage [176]. SDE has the advantage of being able to extract high quantities of target compounds since the volatile fractions generally have a high solubility in organic solvent. However, this method is long and laborious and requires a solvent evaporation step, which can lead to substantial volatile compound degradation. (ii) HS analysis can be performed by static headspace (SHS), dynamic purge-and-trap headspace (DHS) or headspace-solid phase microextraction (HS-SPME) techniques. Concerning SHS, the sample is placed in an airtight vial. Most compounds that are volatile at the analysis temperature evaporate from the liquid or solid fraction and pass into the overhead gas HS. At equilibrium (no variations in the HS composition), an HS aliquot is harvested and injected on the GC column. According to Frankel et al. [177] this method, combined with GC, is rapid (15 determinations/h), hexanal-specific and could be used for measuring lipoperoxidation in biological samples such as rat liver. SHS has also been used to measure the antioxidant effect of butylated hydroxytoluene on the oxidation of meat products such as cooked beef patties [178]. The main advantages of this method are that solvent extraction is not required, and it is relatively inexpensive, easy to use and can be automated. SHS can, however, only quantify a fraction of the target compounds. Since equilibrium is established between the volatile compounds in the HS and those remaining in the sample, only low quantities of compounds are actually recovered, which limits the sensitivity. The increase in the extraction temperature could increase the volatilization of the target compounds and thus increase the quantities recovered, but the temperature must be kept as low as possible in order to minimize generation of new oxidation products and/or thermal degradation of oxidation markers. Please cite this article in press as: Laguerre M et al., Evaluation of the ability of antioxidants to counteract lipid ..., Prog Lipid Res (2007), doi:10.1016/j.plipres.2007.05.002


273

Contrary to SHS, in which an equilibrium must be established between the volatile compounds in the HS and those remaining in the sample, the dynamic headspace (DHS) technique hampers establishment of this equilibrium. In DHS, the sample is thus continually purged by inert gas to extract volatile compounds. The gas effluent then passes through a porous polymer trap that collects volatile analytes. TenaxÒ can adsorb many volatiles and is the most widely used of all available traps. As volatiles contained in the sample are constantly released and trapped, a high concentration of compounds are injected on the GC column. Several studies have highlighted the efficacy of DHS-GC in assessing the oxidative status of different meats [179–181]. Despite its high sensitivity, one shortcoming of DHS is that the volatile compound concentration profile can vary according to the availability of the oxygen in the vial – the instrumentation is also complex and expensive, thus increasing the sources of error (trap drying, trap transfer, purging efficiency, etc.). DHS requires around 15 min more analysis time per sample as compared to SHS [182]. SPME is a solvent-free gas-solid extraction method which utilizes the adsorbant properties of a polymeric film that coats the inside of fibers placed in contact with the HS. This method consists of three steps: (i) the equilibrium phase in which the volatile compounds migrate from the sample to the HS until a balance is reached, (ii) the adsorption phase, when a balance is reached in the volatile compounds shared between the fiber and the HS, and (iii) the thermal desorption phase, when the fiber is introduced in a GC injector. The analytes are then quickly desorbed and penetrate into the column. HS-SPME has been used in many studies to evaluate lipid oxidation in the presence or absence of antioxidants in meat products [183–186] or in vegetable oil emulsions [74]. Like DHS, close correlations were obtained in HS-SPME between the hexanal concentration [187] or pentanal concentration [183] and the TBARS test. HS-SPME is clearly the most promising and easy-to-use method, especially since extraction can now be automated. However, the main drawback is fiber degradation occurs quite rapidly, thus necessitating long-term replicated studies. Note that these fibers can seldom be used for more than 50 extractions. HS contamination by alkylcyclosiloxanes has also been observed [188,189], which is likely due to fiber degradation. In conclusion, each extraction method has its shortcomings, but HS-SPME is being used to an increasing extent on account of its user friendliness. 6. Final considerations 6.1. Test system parametering Test system parametering involves many exploratory operations, but a few general points could be helpful for analysts. The first key parameter is the representativeness of the oxidizable substrate relative to the actual conditions in which antioxidant protection takes effect. TAGs, LDLs and liposomal membrane structures seem to be good candidates in this respect, along with lipid substrates directly derived from foods or biological tissues. It is also essential to strike a balance between this representativeness objective and certain process constraints such as the analysis time, cost, feasibility, the specificity and stability of the substrate reaction and obtainment of a high signal-noise ratio. Finally, the different chemical species in these mixtures have to be meticulously identified in order to gain full insight into the oxidative and antioxidative mechanisms involved, and to be able to determine the most suitable measurement strategy. Concerning the medium, the most suitable polarity with respect to the plant extract should be sought. It would thus be especially interesting to test the same oxidizable substrate in different media. A diverse range of molecules and plant extracts could be investigated with this available battery of tests. Concerning antioxidants, it is essential to assess their efficacy in a physiologically-relevant concentration range [5]. Tests using antioxidant concentrations higher than those occurring in foods or biological media do not measure the antioxidant activity as defined by Halliwell and Gutteridge [18] and discussed in Section 1. It is also important, if possible, to compare antioxidant substances on the basis of their molar concentration, not on their mass concentration [8], as this latter approach could bias the results. Indeed, in molecules with the same number of aromatic hydroxyls, a comparison based on the mass concentration would favor the smallest one (with all other factors being equal), since it would be present in the highest quantity. However, comparing antioxidants on the basis of the molar concentration requires quantification and identification of Please cite this article in press as: Laguerre M et al., Evaluation of the ability of antioxidants to counteract lipid ..., Prog Lipid Res (2007), doi:10.1016/j.plipres.2007.05.002



the different chemical species present in the extract, and liquid chromatography is generally used for this purpose. Considerable progress has fortunately been made in this area, e.g. Sakakibara et al. [190] achieved high separation quality after developing an HPLC-diode array detector method that can isolate most glycosylated flavonoids or aglycones present in plants. Concerning the oxidation system, the test conditions should be as realistic as possible. First, temperatures above 37–40 °C should not be used. Although a multitude of reactive species are involved in vivo, we focused this review on ROO and HO radicals, which are the most common in vitro procedures. ROO radicals are prime targets for testing antioxidant activity. However, despite the fact that these radicals are biologically active, ROO generation systems are not always completely suitable, e.g. the fact that the antioxidant capacity depends directly on the polarity of the azo-initiator. Through an extreme simplification, the analyst can almost predict the order of antioxidant activity of molecules with different polarities based just on the polarity of the initiator. The antioxidant capacity of polar substances is therefore often higher than that of apolar substances with a water-soluble azo-initiator, and the trend is reversed if a solvent-soluble azo-initiator is used. Unfortunately, this over-simplified in vitro behavior is the rule more than the exception and gives rise to problems without any miraculous solution, except possibly varying the azo-initiator and more generally the oxidation conditions as much as possible. In this setting, nonsymmetric and amphiphilic azo-initiators thus seem to be an especially interesting alternative, but their widespread adoption is dependant on their market availability. 6.2. Measurement strategy As already mentioned, measurement of substrate loss seems to be best adapted to determining the extent of oxidation in (i) model systems, or in (ii) biological or bio-mimetic systems (LDLs, liposomes or living cells) prelabeled with a fluorescent probe. These substrate loss measurement methods are generally based on spectral analysis, apart from some that involve oxygen consumption measurements or chromatographic analysis to assay residual nonoxidized substrates. Oxidizable substrates used in such tests mainly have a conjugated polyenic structure, which gives them absorption and/or fluorescence properties in the UV–VIS spectrum, thus facilitating assessment of their oxidative degradation. Fluorimetry is the preferred method for this because it is more sensitive and specific than absorption spectrophotometry and is not as vulnerable to any kind of interference. Note, however, that fluorimetry is not as widely used as absorption spectrophotometry, which most research laboratories are equipped to perform. Flow cytometry is a valuable tool for collecting information on individual cells. Finally, the main advantage of these methods is that they can be used to measure loss of a single substrate without interference from a multitude of oxidation products – so the oxidation status is thus evaluated directly without bias. Methods based on measurement of oxidation product formation seem to be well adapted for assessing oxidation in all systems, i.e. modeled or based on lipids isolated from their natural environment. These methods are nevertheless limited by the fact that no universal oxidation marker is currently available. Primary and secondary compounds are generally also highly unstable multiple reaction intermediaries, often with different molar masses and polarities. These factors considerably complicate evaluations on the extent of oxidation and antioxidant efficacy. It is therefore essential to evaluate both primary (hydroperoxides, conjugated dienes) and secondary (carbonyls, volatile compounds) oxidation products in order to obtain a reliable overall picture of the oxidation status. Decker et al. [191] reported two relevant examples on this topic. In some cases, the addition of antioxidants can boost the level of primary oxidation products through cession of a hydrogen atom to a peroxyradical to form a lipid hydroperoxide. The secondary oxidation product concentration consequently declines. If the analyst only measures the primary oxidation products, he/she will conclude that a proxidant effect is involved, despite the fact that the tested molecule has acted as an antioxidant. Similarly, some proxidant agents like transition metals can accelerate the kinetics of hydroperoxide decomposition into secondary oxidation products. If these hydroperoxides are the only oxidation compounds measured, then a biological or plant extract with a high iron content could appear as an antioxidant, whereas it will actually prompt an oxidation reaction at more advanced oxidation stages. This review highlights that neither of these two key assessment strategies (measurement of substrate loss or formation of oxidation products) is better overall than the other since they measure different but complementary aspects of oxidation. Please cite this article in press as: Laguerre M et al., Evaluation of the ability of antioxidants to counteract lipid ..., Prog Lipid Res (2007), doi:10.1016/j.plipres.2007.05.002


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6.3. Information processing and data presentation The way in which relevant information is extracted from acquired analytical data are extremely important. There are many possible alternatives. These include measurement of the area under the curve (AUC), the initial rate, inhibition time, reaction rate with free radicals, and the IC 50 (antioxidant concentration to achieve 50% inhibition). Then there are two different highly complementary objectives. Firstly, fundamental studies can be focused on mechanistic, kinetic and stoichiometric aspects and preferentially take the inhibition time and reaction rate into account. Note also that when mathematical expression is possible from the experimental data the inhibition time can be determined from the extremum of the second derivative of the function. Secondly, measurement of the area under the curve (if possible) seems especially suitable when the main aim is to compare the antioxidant capacity of various substances. The advantage of this method is that it takes both the inhibition time and inhibition degree into account. Hence, the AUC measurement provides a bidimensional view, whereas other approaches may extract more specific information but the results are unidimensional. Moreover, Cao et al. [62] found that glutathion does not induce a lag phase, so it is impossible to quantify the antioxidant power of his compound simply by measuring the lag phase. Finally, it clearly does not make sense from a chemical standpoint to just consider the antioxidant capacity without also taking the medium, substrate and oxidation conditions in which this property was measured into account. The chaotic results discussed by some authors seems to be mainly due to a lack of reference to the test conditions (temperature, type of substrate, etc.) rather than to differences in analysis methods used. The choice of all of these parameters will therefore determine the information and possible interpretations concerning the antioxidant properties of natural and synthetic substances. 7. Future prospects This review covers the issue of assessing the antioxidant capacity by direct methods, in relation to the setting, and autoxidation and antioxidation mechanisms. We have seen that the complexity and diversity of investigation systems has led to the development of a broad range of tests, but unfortunately none of them has the universal scope we are seeking. The main problem concerns both the choice of test system parametering and the measurement strategy to adopt. The improvement of current methods and the development of new evaluation tools are clearly prime objectives for the future only if efforts are seriously focused on harmonizing methods to ensure genuine progress. This latter objective is much harder to fulfill since the antioxidant capacity involves a multitude of different mechanisms and pathways. Holistic (or global) antioxidation studies are therefore impossible. This has naturally prompted researchers to adopt a reductionist approach, whereby the complexity of the antioxidant capacity is broken down into different parts which are then studied separately. We feel that these two approaches – which are far from being antinomic – could be dovetailed to a certain extent. As each measurement method only generates information on part of the overall antioxidant activity, it would be logical to consider each result generated by a test as a variable of a multivariate equation representing the global antioxidant capacity, thus accounting for the many mechanisms and pathways involved. The problem then is to determine the exact weight to be allocated to each variable to ensure the consistency and interpretation of this set of diverse data. Some authors have recommended adopting a multidimensional approach, while alluding to this crucial question. One very recent solution looks highly promising – it involves subjecting a set of samples to various analysis methods and then processing the results through a chemometric procedure [192]. In the simplest case of principal components analysis, the initial variables, which correspond to different test results, are linearly combined to generate a small number of new variables. This multivariate analysis enables the analyst at first to clarify the data by eliminating outliers, while still preserving much of the information contained in the initial variables. We also consider that these new artificial variables could be very good antioxidant capacity markers. This chemometric approach could give rise to a set of compatible methods, and then a classification of methods could be drawn up on the basis of the extent of homology in the results. In addition to this harmonization strategy, it would be useful to pool knowledge acquired on antioxidation against lipids with data obtained on other oxidizable substrates (nucleic acid, proteins, etc.). A much more global understanding of the topic could emerge from this integrated approach. It should be Please cite this article in press as: Laguerre M et al., Evaluation of the ability of antioxidants to counteract lipid ..., Prog Lipid Res (2007), doi:10.1016/j.plipres.2007.05.002



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