Methods for evaluating the pote

ncy and efficacy of antioxidantsMickael Laguerrea, Eric A. Deckera, Jerome Lecomteb and Pierre Villeneuveb

aDepartment of Food Science, University ofMassachusetts, Chenoweth Laboratory, Amherst,Massachusetts, USA and bCIRAD, UMR IATE,Montpellier, France

Correspondence to Pierre Villeneuve, CIRAD, UMRIATE, Montpellier F-34398, FranceTel: +33 04 67 61 55 18; fax: +33 04 67 61 55 15;e-mail: villeneuve@cirad.fr

Current Opinion in Clinical Nutrition and

Metabolic Care 2010, 13:518–525

Purpose of review

The aim of this article is to present a brief panorama of the most widely used methods

and of new analytical approaches for evaluating antioxidant capacity and to discuss

them in terms of advantages and drawbacks.

Recent findings

To date, many in-vitro tests are available from the chemical assay performed in a

homogenous solution such as oxygen radical antioxidant capacity assay to more

complex cell-based methods using exogenic probes to detect oxidation. In complement

to these existing methods, novel approaches have recently been developed such as the

conjugated autoxidizable triene assay implemented in emulsions and using tung oil as

ultraviolet probe.

Summary

The complexity and diverse range of research topics investigated have led to the

development of a multitude of tests, but unfortunately none of them are universal. Thus,

one of the major challenges is to know which method is best suited for a particular

application.

Keywords

antioxidant capacity, conjugated autoxidizable triene assay, cell-based methods,

fluorescent probes, fluorogenic probes, oxygen radical absorbance capacity assay

Curr Opin Clin Nutr Metab Care 13:518–525� 2010 Wolters Kluwer Health | Lippincott Williams & Wilkins1363-1950

‘Progress in science depends on new techniques, new

discoveries, and new ideas, probably in that order.’

Sydney Brenner, 2002 Nobel Prize Winner in Physiology

and Medicine.

IntroductionFirst of all, it is important not to just consider antioxidant

capacity as being merely the reactivity of a compound to

reduce free radicals (and related reactive species) or

chelate transition metals. More importantly, the very

concept of antioxidant involves the notion of protecting

‘something’ from oxidation. But, what is that something?

Krinsky [1] has defined antioxidants as ‘compounds that

protect biological systems against the potentially harmful

effects of processes or reactions that cause extensive

oxidation’. Halliwell and Gutteridge [2] proposed that

an antioxidant is a ‘substance that, when present at low

concentrations compared to those of an oxidizable sub-

strate, significantly delays or prevents oxidation of that

substrate’. These two definitions are complementary, as

an oxidizable substrate is usually regarded as a biologi-

cally important molecule such as lipids, proteins, DNA,

and so on, whose oxidation leads to harmful effect. To

complicate matters further, many oxidation products of

antioxidants may still exhibit residual antioxidant

1363-1950 � 2010 Wolters Kluwer Health | Lippincott Williams & Wilkins

activity. In this way, a biological antioxidant could be

defined as a ‘substance that, when present at low con-

centrations compared to an oxidizable substrate, protects

(by itself and through its oxidation products) that sub-

strate from oxidation, and ultimately protects the organ-

ism from harmful effects of oxidative stress’.

Keeping this definition in mind, the question arises of

how to accurately evaluate the efficacy of antioxidants.

Many rapid in-vitro assays in test tubes or in cultured cells

are now available. The experimental models involve

various systems, from the simplest such as chemical

assays in homogeneous solution to more complex ones,

including heterogeneous systems such as emulsion, lipo-

some, or lipoprotein. Then, increasing the complexity

leads to living systems, including cells, animal models of

oxidative stress, and ultimately clinical tests in humans

(Fig. 1). The aim of this article is to present a brief

panorama of widespread methods and new analytical

approaches.

Test tube-based methodsAmong the multitude of test tube methods (Fig. 1),

we have focused on the widespread ones, especially

fluorescein-based oxygen radical absorbance capacity

DOI:10.1097/MCO.0b013e32833aff12

Methods for evaluating antioxidants Laguerre et al. 519

Figure 1 Methods classification as a function of the complexity of the system

aDPPH: 2,2-diphenyl-1-picrylhydrazyl radical. bFRAP: ferric-reducing antioxidant power. cCUPRAC: cupric reducing antioxidant capacity. dABTS: 2,20-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid). eORACbPE: b-phycoerythrin-based oxygen radical absorbance capacity. fORACFL: fluorescein-basedoxygen radical absorbance capacity. gTRAP: total radical-trapping antioxidant parameter. hCAT: conjugated autoxidizable triene. iSPME-GC: solid phasemicroextraction coupled to gas chromatography. jTBA: thiobarbituric acid. k4-HNE: 4-hydroxynonenal. lIn this context, in vitromeans that the animal tissuesare collected fromanimal and thenundergooxidation in test tube,whereas in vivomeans that the animal is ‘submitted’ tooxidativestressand thensacrificedto collect tissues. In one case, the test tube is the ‘medium’ (in vitro) and in the other one, the organism is the ‘medium’ (in vivo).

Figure 2 Chemical structure of disodium salt of fluorescein

COONa

OONaO

(ORACFL) assay and lipid oxidation measurement as well

as the newly developed conjugated autoxidizable triene

(CAT) assay.

Fluorescein-based oxygen radical absorbance capacity

method

The ORACFL method using fluorescein (Fig. 2) as oxi-

dizable substrate [3] results from a modification of the

ORACb-PE method [4] using b-phycoerithrin, which also

results from the original Glazer’s method [5,6]. The

ORACFL method is based on the loss of fluorescence

emission of fluorescein (lex/em: 490/515 nm) when oxi-

dized by peroxyl radicals derived from 20,20-azobis(ami-

dinopropane) dihydrochloride (AAPH) in a phosphate

buffer (pH 7.4); this fluorescence decay being monitored

using a fluorimeter with 1-cm cuvette or using a micro-

plate reader. The presence of antioxidants results in the

reduction of some of the peroxyl radicals, rendering them

unavailable to oxidize fluorescein, what leads to a

decrease in loss of fluorescence. The information on

the fluorescence decay is extracted through the calcu-

lation of the area under the curve and expressed as Trolox

equivalent. Trolox is a water-soluble a-tocopherol deriva-

tive that acts as a useful standard in this and several

other assays.

520 Assessment of nutritional status and analytical methods

As the method allows only the assessment of water-

soluble compounds, a new protocol was implemented

to solubilize lipophilic molecules in aqueous buffer using

randomly methylated b-cyclodextrins and acetone [7].

To date, two distinct protocols exist: one dedicated to

hydrophilic antioxidants (H-ORACFL) and another to

lipophilic molecules (L-ORACFL). In the case of blood

plasma, Prior et al. [8] assumed that H-ORACFL and

L-ORACFL values can be summated to give a total value

(T-ORACFL), estimating the whole antioxidant capacity

of blood plasma. This is, however, questionable, as

lipophilic and hydrophilic antioxidants are assessed in

different test tubes. Thus, the possible synergistic (or

antagonistic) effect(s) occurring from the interaction in

the bloodstream between antioxidants of different

polarity is not taken into account. In addition, antioxidant

(0.5–5mmol/l) is tested at concentrations much in excess

to fluorescein (63 nmol/l); thus, the conditions do not

meet the antioxidant definition of Halliwell and Gutter-

idge [2]. Despite these criticisms and because it is easy-

to-use, rapid, and high-throughput, this widespread

method has been used in various purposes, mainly for

assessing plant extracts or foodstuffs [9,10,11�] and a

comprehensive database of ORAC values is now available

at ORACValues.com (�300 foods so far).

Conjugated autoxidizable triene method

In 2008, we developed a new method called CAT, using

tung oil as oxidizable substrate [12]. Triacylglycerols

(TAGs) from this commercially available oil contain

85% eleostearic acid, encompassing a conjugated triene

(Fig. 3) [13�], which exhibits a strong ultraviolet (UV)

absorption at 273 nm. In emulsion and under oxidizing

conditions, the degradation of conjugated triene TAGs is

accompanied by a bleaching at 273 nm (Fig. 4a) [12,13�].

Addition of antioxidants results in a delay of oxidative

process (Fig. 4b) and enables the quantification of the

antioxidant capacity through the area under the curve

(Fig. 4c). This method has been applied to flavonoids

[12], phenolic acids [12], chlorogenate [14�] and rosmar-

inate esters [15�] as well as olive leaf extracts [16�].

Although efforts must be brought to enhance its repro-

ducibility, the CAT method already presents several

Figure 3 Chemical structure of trieleostearin

sn1

O

C

O

(CH2)7

(

C

O

O

sn3O

sn2

CO (

For simplicity, eleostearic acid is depicted in its alpha configuration [9(c), 1

advantages. First, as the ORACFL method, it is high-

throughput being performed in 96-well microplate

reader. In addition, as it uses TAG constituting the major

lipids found in biological and food systems (with phos-

pholipids), the CAT assay could provide more relevant

results than the other tests using nonrepresentative sub-

strates such as ORACFL method. Regarding this matter,

we recently reported a surprising correlation (R2¼ 0.87)

between CAT values measured in emulsion [14�] and the

antioxidant capacity of chlorogenate esters analyzed on

reactive oxygen/nitrogen species (RONS) overexpressing

fibroblasts (Laguerre M, et al., in preparation). This study

used 20,70-dichlorodihydrofluorescein as the fluorogenic

probe to measure the modulation of the oxidative stress

in fibroblasts induced by a homologous series of chlor-

ogenate esters and showed for the first time that the

relationship between hydrophobicity and antioxidant

capacity follows a nonlinear trend already observed in

emulsion.). Indeed, both assays showed dodecyl chlor-

ogenate as the more active compound. Work is in progress

to determine whether this result is a coincidence or,

more interestingly, whether the simple CAT assay allows

gaining reliable insight into the antioxidant properties

in biological systems such as cultured cells. Finally, the

possibility to assess both hydrophilic and lipophilic com-

pounds using methanolic predissolution of antioxidants

[14�] constitutes another interesting advantage and makes

it a promising supplement to currently used methods.

Measurement of lipid oxidation in emulsions,

liposomes, lipoproteins, and animal tissues

In contrast to ORACFL and CAT method whose kinetic

approach is based on the monitoring of the substrate loss,

the measurement of oxidation, especially lipid oxidation,

can employ a reverse kinetic strategy based upon the

monitoring of the appearance of oxidation products.

Although methods based on the assessment of oxidation

product formation are very sensitive and suitable for most

cases [micelles, emulsion, liposomes, low-density lipo-

protein (LDL), and animal tissues], they nevertheless

suffer from the lack of any universal oxidation marker

and, therefore, can have limited applicability. Indeed, in

the particular case of polyunsaturated lipids, oxidation

109

CH2)7

11 13 Conjugated triene

CH2)7

1(t), 13(t)]. Reproduced with permission from [13�].

Methods for evaluating antioxidants Laguerre et al. 521

Figure 4 Evolution of the ultraviolet spectrum of a stripped tung oil-in-water emulsion

(a) Example of a three-dimensional depiction of the evolution of the ultraviolet spectrum of a stripped tung oil-in-water emulsion (pH 7.2) duringaccelerated oxidation by 2,2-azobis(2-amidinopropane) dihydrochloride (AAPH) at 378C. (b) Example of kinetics of relative absorbance bleaching at273nm in the absence and presence of various concentrations of Trolox in stripped tung oil-in-water emulsion during AAPH-mediated oxidation. Thereaction mixture contained 115mmol/l tung oil, 17mmol/l Brij 35, 1mmol/l AAPH, and 0.25–2mmol/l Trolox in phosphate-buffered saline (PBS), pH7.2, at 378C. (c) Example of the net protection area under the curve (AUC Sample – AUCBlank) versus Trolox concentration. Adapted from [12] and [13�].

leads to the formation of primary oxidation products

(hydroperoxides and conjugated dienes), which are then

oxidized to secondary oxidation products [malondialde-

hyde (MDA), 4-hydroxy-2-nonenal, hexanal] that can

react with free amino groups of phospholipids, proteins,

DNA, RNA, and so on to form tertiary oxidation products

what leads to a complex network of reactions. It is,

therefore, essential to evaluate numerous oxidation com-

pounds to obtain a reliable overall picture of oxidation

status, which is tedious.

Regarding primary oxidation products, two main meth-

odologies can be implemented depending on the chemi-

cal group that is evaluated: the hydroperoxide group

(–C–OOH) or the conjugated diene system (–C=C–

C=C–C). First, hydroperoxides can be estimated by

iodometric, thiocyanate [17–19], orange xylenol [20],

gluthatione peroxidase [21], or diphenyl-1-pyrenylpho-

sphine (DPPP) [22�] assays. Concerning conjugated

dienes, their presence in oxidized natural fats (contain-

ing polyunsaturated fats) has been known for almost

80 years [23] and can be monitored spectrophotometri-

cally at 234 nm in both classical 1-cm cuvettes and

96-well microplates.

Secondary oxidation products such as aldehydes (propa-

nal, hexanal,. . .) can be collected and quantified by

coupling an extraction procedure (solid phase microex-

traction, dynamic headspace), a separation step by gas

chromatography, and finally detection by flame ionization

detector, mass spectrometer, or more sensitive detectors.

Although this methodology is reliable because most of

the oxidation products are identified, it is, nevertheless,

both time and sample consuming and can lead to a poor

reproducibility. An alternative is the well known thio-

barbituric acid (TBA) based on the condensation of MDA

with TBA to form a chromogen (lmax: 532 nm; Fig. 5)

[13�]. This method has, however, been criticized as

MDAs are not always specific to lipid oxidation. Indeed,

it can result from the oxidation of glucides, amino acids,

DNA, ascorbate, and deoxyglucose [13�]. In addition,

TBA can react with other compounds than MDA. So,

the initial specific evaluation of MDA is progressively

substituted by the less-specific notion of ‘TBA-reactive

substances, TBARS’; these latter limitations can be over-

looked after a preliminary purification of TBA/MDA

complex by high-performance liquid chromatography

techniques [24,25]. Moreover, according to the model

by Dahle et al. [26], MDA can only result from the

oxidation of fatty acids containing at least three double

bonds. This means that comparison of influence of v-3-

rich diet (those containing three double bonds) in com-

parison to v-6-rich diet (those containing less than two

double bonds) on lipid oxidation through the TBA assay

is totally wrong [13�].

Finally, these aldehydes can further react in vitro and

in vivo with amino groups of phospholipids, proteins,

DNA, and so on leading to lipofuscins (sometimes coined

as tertiary oxidation products) that exhibit fluorescent

522 Assessment of nutritional status and analytical methods

Figure 5 Formation of the chromogen product (lmax : 532nm) between malondialdehyde and 2-thiobarbituric acid

MDA, malondialdehyde; TBA, 2-thiobarbituric acid. Reproduced with permission from [13�].

properties (lex: 340–390; lem: 430–490 nm) measurable

by fluorimetry [27].

Cell-based methodsThe aim of the cell-based method is to monitor oxidation

of a given substrate with or without antioxidants. How-

ever, in such a complex system, it is almost impossible to

distinguish the signal of any endogenous oxidizable sub-

strate from the ‘cellular background noise’. This has led

to the use of exogenous artificial probes exhibiting

specific fluorescence properties.

Incorporation of fluorescent probes

Some fluorescent oxidizable substrates whose disappear-

ance can be monitored are cis-parinaric acid (cPnA), C11-

BODIPY581/591, BODIPY665/676, and alkylamide or phos-

pholipidic derivatives of fluorescein (Fig. 6) [13�]. These

probes can be used in aqueous dispersion and in liposo-

mal systems, but they can also be incorporated into

biological particles (LDL) or into living cells (via a

metabolic pathway in this latter case). The resulting

fluorescence can be observed by microscopy or measured

by spectrofluorimetry or flow cytometry. Overall, spectro-

fluorimetry is better suited to measuring adherent cells

such as fibroblasts, whereas flow cytometry is a better

choice for determining cells growing in suspension, such

as lymphocytes.

For instance, cPnA (Fig. 6a) has been extensively used to

measure lipid peroxidation in erythrocytes [28,29], sub-

mitochondrial particles [30], sarcoplasmic reticulum cells

[31], rat aortic smooth muscle cells [32], macrophages

[33], neonatal rat cardiomyocytes [34], and rapidly divid-

ing cell lines such as human leukemia cells [35]. More

recently, it has been incorporated to mitochondria iso-

lated from renal proximal tubular cells [36], acute

myeloid leukemia cell lines [37�], and lymphocytes

[38�]. Among other advantages, cPnA is a bioanalogue,

does not disturb the lipid bilayer, and has a broad fluor-

escence Stokes shift (�100 nm). However, cPnA acid is

air sensitive, is photolabile, and dimerizes under light,

which can make it difficult to use [39].

C11-BODIPY581/591 is a fatty acid analogue (Fig. 6b)

displaying red fluorescence. Once oxidized, the fluor-

escence shifts from red (�590 nm) to green (�520 nm;

Fig. 7) [40]. This probe has been used to monitor lipid

oxidation in boar [41,42�], equine [43], and human [44,45]

spermatozoa, rat fibroblasts [46], human dermal fibro-

blasts [47], human liver carcinoma cell (HepG2) [47],

neuronal human astrocytes [47], and Jurkat cells [48��].

Note that C11-BODIPY581/591 is not as biomimetic as

cPnA, but is in turn, less prone to photobleaching [49] and

about twice as sensitive to oxidation compared to cPnA

[50]. Other BODIPY derivatives can also be used such as

BODIPY665/676 (Fig. 6c), which is more oxidizable and

presents a slightly broader fluorescence Stokes shift than

that of C11-BODIPY581/591 [51].

Regarding alkylamide (Fig. 6d) or phospholipidic

(Fig. 6e) derivatives of fluorescein, their excitation wave-

length (488 nm) seems to be especially well suited to

routine use in flow cytometry, for which a laser emitting

visible blue light (488 nm) is used. In a large cell popu-

lation, flow cytometry allows the study of lipid peroxi-

dation cell by cell [52,53], which is a useful tool. How-

ever, these fluorescent probes are not yet widely used for

this purpose.

Incorporation of fluorogenic probe

In contrast to fluorescent probes losing their fluorescence

when oxidized, fluorogenic probes exhibit fluorescence

when oxidized. The basic principle consists of monitor-

ing the level of fluorescence as an estimation of the level

of oxidizing species in cultured cells. One can cite dihy-

dro-derivatives of 20,70-dichlorofluorescein (Fig. 8), fluor-

escein, and ethidium constituting the most widely used

probes. Depending on their charge, their chemical struc-

ture, and the cells involved, these probes can move into a

specific cell compartment: mitochondrion for dihydro-

fluorescein [54], nucleus for dihydroethidium [55], and

cytoplasm for 20,70-dihydrodichlorofluorescein [55]. By

Methods for evaluating antioxidants Laguerre et al. 523

Figure 6 Chemical structures of some lipid-soluble fluorescent probes

Chemical structure of (a) cis-parinaric acid, (b) C11-BODIPY581/591, (c) BODIPY665/676, (d) alkylated derivatives of fluorescein, and (e) phospholipidicderivative of fluorescein. Reproduced with permission from [13�].

example, this latter has been recently incorporated to

RONS (reactive oxygen/nitrogen species) overexpres-

sing fibroblasts to study antioxidant capacity of chloro-

genate ester (Laguerre M et al., in preparation) and

Figure 7 Emission spectra of 0.05mol% C11-BODIPY581/591 in

250mmol/l egg-phosphatidylcholine vesicles

Oxidation was induced with 250mmol/l CumOOH/100nmol/l heminand spectra were recorded every 20min. The spectra show a decreasein fluorescence at 595nm, with a concomitant increase in fluorescenceat 520nm and a shoulder at 545nm. Insert: molecular structure of C11-BODIPY581/591. At arrows: the diene interconnection is most likely thetarget for free radical-induced oxidation. Reproduced with permissionfrom [40].

showed for the first time that increasing hydrophobicity

does not necessarily lead to a more efficient anti-

oxidant drug.

In terms of advantages, these probes, generally dissolved

in dimethylsulfoxide and stored at �808C, are easy-to-use

(as much as fluorescent probes). The major concern is the

ambiguity of the response, whether these probes detect a

general oxidative stress or apoptosis. Indeed, cytochrome

c, whose release by mitochondria in cytosol is a key event

in apoptosis [56], strongly oxidizes 20,70-dichlorodihydro-

fluorescein [57]. Regarding this matter, it has been shown

that the antiapoptotic proto-oncoprotein bcl-2 prevents

20,70-dichlorodihydrofluorescein oxidation [58], which

suggested that bcl-2 prevents apoptosis through an anti-

oxidant pathway. In contrast, Burkitt and Wardman [57]

refute any antioxidant pathway and assumed that the

prevention of 20,70-dichlorodihydrofluorescein oxidation

is an artefact resulting from an apoptotic pathway leading

to the prevention of mitochondrial cytochrome c release,

which is thus unable to oxidize the probe located in the

cytosol. The use of fluorogenic probes has also been the

focus of criticism regarding their absence of specificity

toward RONS. Indeed, it is unclear what kind of species

react with these probes and what makes the latter the

‘markers’ of a general oxidative stress rather than of a

precise oxidative pathway.

524 Assessment of nutritional status and analytical methods

Figure 8 Bi-electronic oxidation of 20,70-dichlorodihydrofluorescein to 20,70-dichlorofluorescein

Reproduced with permission from [13�].

ConclusionWe have seen that the complexity and diversity of

investigation systems have led to the development of

a broad range of tests, but unfortunately, none of them

has the universal scope we are seeking and none of them

is free of criticism. In this context, it is preferable to use

several methods to assess multifaceted antioxidants.

Ultimately, the answer to the question, ‘what is the best

antioxidant?’ is dependent on the system in which this

activity is measured, especially pro-oxidants, cellular

compartment, and conditions. For instance, vitamin E

reportedly is the most important [59] and one of the least

important [60] plasma antioxidants depending on the

different assay conditions used. In other words, a ‘good’

antioxidant in a given system can be a ‘bad’ one in

another system, which means that it clearly does not

make sense to just consider the magnitudes of the anti-

oxidant capacities as being a value universally applicable

for all media, cells, oxidation conditions, or substrates

[39].

References and recommended readingPapers of particular interest, published within the annual period of review, havebeen highlighted as:� of special interest�� of outstanding interest

Additional references related to this topic can also be found in the CurrentWorld Literature section in this issue (pp. 595–596).

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13

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14

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15

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16

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33 McGuire SO, James-Kracke MR, Sun GY, Fritsche KL. An esterificationprotocol for cis-parinaric acid-determined lipid peroxidation in immune cells.Lipids 1997; 32:219–226.

34 Steenbergen RGH, Drummen GPC, Op den Kamp JAF, Post JA. The use ofcis-parinaric acid to measure lipid peroxidation in cardiomyocytes duringischemia and reperfusion. Biochim Biophys Acta 1997; 1330:127–137.

35 Ritov VB, Banni S, Yalowich JC, et al. Nonrandom peroxidation of differentclasses of membrane phospholipids in live cells detected by metabolicallyintegrated cis-parinaric acid. Biochim Biophys Acta 1996; 1283:127–140.

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37

�Khanim FL, Hayden RE, Birtwistle J, et al. Combined bezafibrateand medroxyprogesterone acetate: potential novel therapy for acutemyeloid leukaemia. PLos One 2009; 4:e8147. doi: 1371/journal.pone.0008147.

This work demonstrates that a combination of bezofibrate and medroxyprogester-one acetate elevated reactive oxygen species (ROS) in acute myeloid leukemiaand delivered the antineoplastic actions of 15d-PGJ2. This finding partly relies onthe use of cPnA to assess lipid peroxidation.

38

�Geetha S, Sai Ram M, Sharma SK, et al. Cytoprotective and antioxidantactivity of seabuckthorn (Hippophae rhamnoides L.) flavones against tert-butylhydroperoxide-induced cytotoxicity in lymphocytes. J Med Food 2009;12:151–158.

Example of the use of cPnA to measure lipid peroxidation in lymphocyte.

39 Laguerre M, Lecomte J, Villeneuve P. Evaluation of the ability of antioxidants tocounteract lipid oxidation: existing methods, new trends and challenges. ProgLipid Res 2007; 46:244–282.

40 Drummen GPC, van Liebergen LCM, op den Kamp JAF, Post JA. C11-BODIPY581/591, an oxidation-sensitive fluorescent lipid peroxidation probe:(micro)spectroscopic characterization and validation of methodology. FreeRadic Biol Med 2002; 33:473–490.

41 Guthrie HD, Welch GR. Use of fluorescence-activated flow cytometry todetermine membrane lipid peroxidation during hypothermic liquid storage andfreeze-thawing of viable boar sperm loaded with 4, 4-difluoro-5-(4-phenyl-1,3-butadienyl)-4-bora-3a, 4a-diaza-s-indaceundecanoic acid. J Anim Sci 2007;85:1402–1411.

42

�Awda BJ, Mackenzie-Bell M, Buhr MM. Reactive oxygen species and boarsperm function. Biol Reprod 2009; 81:553–561.

Example of the use of C11-BODIPY581/591 to measure lipid peroxidation in boarspermatozoa.

43 Ball BA, Vo A. Detection of lipid peroxidation in equine spermatozoa basedupon the lipophilic fluorescent dye C11-BODIPY581/591. J Androl 2002;23:259–269.

44 Aitken RJ, Wingate JK, De Iuliis GN, McLaughlin EA. Analysis of lipidperoxidation in human spermatozoa using BODIPY C11. Mol Hum Reprod2007; 13:203–211.

45 Cosci I, Moretti E, Collodel G. Lipid peroxidation in human spermatozoafrom men with genitourinary infections. Syst Biol Reprod Med 2008; 54:75–83.

46 Drummen GPC, Gadella BM, Post JA, Brouwers JF. Mass spectrometriccharacterization of the oxidation of the fluorescent lipid peroxidation reportermolecule C11-BODIPY581/591. Free Radic Biol Med 2004; 36:1635–1644.

47 Sayes CM, Gobin AM, Ausman KD, et al.Nano-C60 cytotoxicity is due to lipidperoxidation. Biomaterials 2005; 26:7587–7595.

48

��Vernier PT, Levine ZA, Wu YH, et al. Electroporating fields target oxidativelydamaged areas in the cell membrane. PLOS One 2009; 4:e7966;DOI:10.1371/journal.pone.0007966.

This article showed that oxidation of cell membrane enhances the susceptibility ofthe membrane to electropermeabilization, and so, that manipulation of the level ofoxidative stress may lead to efficient electrochemotherapy and electrotransfection-mediated gene therapy. The measurement of oxidative stress at membrane levelwas done using C11-BODIPY581/591.

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51 Naguib YMA. Antioxidant activities of astaxanthin and related carotenoids.J Agric Food Chem 2000; 48:1150–1154.

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