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The influence of antioxidants in the thiyl radicalinduced lipid peroxidation and geometricalisomerization in micelles of linoleic acid

Ivana Tartaro Bujak, Branka Mihaljević, Carla Ferreri & ChryssostomosChatgilialoglu

To cite this article: Ivana Tartaro Bujak, Branka Mihaljević, Carla Ferreri & ChryssostomosChatgilialoglu (2016) The influence of antioxidants in the thiyl radical induced lipid peroxidation andgeometrical isomerization in micelles of linoleic acid, Free Radical Research, 50:sup1, S18-S23,DOI: 10.1080/10715762.2016.1231401

To link to this article: http://dx.doi.org/10.1080/10715762.2016.1231401

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ORIGINAL ARTICLE

The influence of antioxidants in the thiyl radical induced lipid peroxidationand geometrical isomerization in micelles of linoleic acid

Ivana Tartaro Bujaka, Branka Mihaljevi�ca, Carla Ferrerib and Chryssostomos Chatgilialoglub,c

aDivision of Materials Chemistry, Radiation Chemistry and Dosimetry Laboratory, Rud-er Bo�skovi�c Institute, Zagreb, Croatia; bISOF,Consiglio Nazionale delle Ricerche, Bologna, Italy; cInstitute of Nanoscience and Nanotechnology, NCSR Demokritos, Athens, Greece

ABSTRACTThe biomimetic model of micelles of linoleic acid containing 2-mercaptoethanol and the antioxi-dant was examined under gamma irradiation up to 400Gy in aerobic or deoxygenated conditionswhere thiyl radicals are the main reactive species. Lipid peroxidation was retarded by ascorbicacid and a-tocopherol, whereas this process was strongly inhibited by resveratrol as effectively asthe ascorbic acid/a-tocopherol mixture. Furthermore, antioxidants have a much stronger inhibi-tory effect on the peroxidation in the presence of 2-mercaptoethanol, and at the same timeshow protective properties of the double bond, decreasing the cis–trans isomerization. Underanaerobic conditions, cis–trans isomerization occurred and antioxidants efficiency increased alongthe series: resveratrol < a-tocopherol< ascorbic acid. This result is explained taking into accountthe double bond localization in the hydrophobic core of the micelle and the need of co-localiza-tion of the antioxidant in order to get an anti-isomerizing activity and protection of the naturallipid geometry.

ARTICLE HISTORYReceived 27 June 2016Revised 23 August 2016Accepted 28 August 2016

KEYWORDSFree radicals; gamma-irradiation; geometricalisomerization; lipids;peroxidation; antioxidants

Introduction

Biomimetic lipid models are useful to explore lipid reac-tion outcomes and products in a simplified context thatclosely mimics the biological environment in livingorganism, thus allowing mechanistic basis of molecularprocesses to be assayed. There are two main classes offree radical reactions involving polyunsaturated fattyacids (PUFA). One of them is lipid peroxidation [1–3]and the second one is the process of geometrical iso-merization of naturally occurring cis unsaturated lipidswhich can be catalyzed by thiyl radicals (RS�) [4–6].It has been shown that one process can be parallelwith the occurrence of the other and both lipid hydro-peroxides and trans lipids are the resulting productsof oxidative free radical conditions [7] (Scheme 1).Investigations of peroxidation and isomerization proc-esses in liposome models were shown to occur bychemical agents, such as the antitumor drug bleomycin,known to form metal complexes, thus producing oxida-tive stress conditions [8]. Both processes induce pro-found changes of lipid structures, especially whenphospholipids, present in organized compartments

such as cell membranes, are involved. Indeed, the lossof PUFA from membrane phospholipids due to the oxi-dation processes is known to affect bilayer asset and itsfluidity, as well as the release of lipid mediators frommembranes and signaling cascades [9,10]. On the otherhand, the transformation of the natural cis geometryinto trans is a subtle change that influences membraneorganization and biochemical processes, with harmfuleffects on health [11].

Preventive or repairing strategies for the lipid integ-rity and good model systems for testing their effi-ciency are therefore necessary. In recent studies ofunilamellar liposomes, several aspects of the lipid iso-merization of monounsaturated fatty acids (MUFA) inorganized systems and the effectiveness of knownantioxidants was were shown [12,13]. In linoleic acid(LH) micelles, the effects of aromatic amines and phe-nols to the mechanisms of Scheme 1 have been alsorecently addressed [14].

Here, we wish to present the micelle model made ofLH (9cis,12cis-octadecadienoic acid, 9c,12c-18:2) as rep-resentative PUFA in membranes, to elucidate the

CONTACT Ivana Tartaro Bujak itartaro@irb.hr Division of Materials Chemistry, Radiation Chemistry and Dosimetry Laboratory, Rud-er Bo�skovi�cInstitute, Zagreb, Croatia; Chryssostomos Chatgilialoglu chrys@isof.cnr.it; c.chatgilialoglu@inn.demokritos.gr ISOF, Consiglio Nazionale delle Ricerche,Bologna, Italy; Institute of Nanoscience and Nanotechnology, NCSR Demokritos, Athens, Greece

Supplemental data for this article can be accessed here

� 2016 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis GroupThis is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial NoDerivatives License (http://creativecommons.org/Licenses/by-nc-nd/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited, and is not altered, transformed,or built upon in any way.

FREE RADICAL RESEARCH, 2016VOL. 50, NO. S1, S18–S23http://dx.doi.org/10.1080/10715762.2016.1231401

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influence of different naturally occurring antioxidantson both peroxidation and geometrical isomerizationprocesses. Interesting insights of the lipid protectionemerged in aerobic and anaerobic conditions, highlight-ing the role of molecular diversity of antioxidants in “oilin water” heterogeneous systems.

Materials and methods

Materials

LH (9cis,12cis-octadecadienoic acid, 9c,12c-18:2, >99%pure), non-ionic surfactant polyoxyethylene sorbitanmonolaureate (TweenVR -20), 2-mercaptoethanol (RSH),and resveratrol (ResOH) (>99%, pure) were purchasedfrom Sigma-Aldrich. DL-a-Tocopherol (a-TOH) (97%,pure) and L� (þ) ascorbic acid (AscH) were purchasedfrom “Alfa Aesar”. Sodium dihydrogen phosphate(�98%) obtained from Carlo Erba, ferrous sulfate(FeSO4�7H2O), and potassium thiocyanate (KSCN)from Merck. These products were used as received andall other used chemicals were of analytical reagentgrade purity. Water was triply distilled, first from potas-sium permanganate and sodium dichromate, then with-out additives. On the basis of UV absorption, allsolvents used were of analytical grade purity.

LH micelles

Model system containing mixed surfactant micelles andbuffer was prepared by slow solubilization of LH in non-ionic surfactant micelles previously formed by mixingTweenVR -20 and NaH2PO4, pH 6.5. The composition ofinvestigated model systems was typically 5.0� 10�4 MLH, 2.8� 10�4 M TweenVR -20, and 5.0� 10�3 M NaH2PO4

(pH �5). Different antioxidants of define concentrationswere added during preparation of model systems. Theaddition of the amphiphilic thiol RSH (2.8� 10�3 M) isadded to previously prepared micelles just beforeirradiation.

Continuous irradiation and product studies

Gamma radiolysis was performed at room temperatureusing panoramic 60Co source at different doses anddose rates. LH micelles were irradiated in air-equilibriumor after saturation with N2O. After irradiation, LH com-ponents were extracted with a solvent mixture ofCH2Cl2/MeOH (2:1, v/v) de-aerated by nitrogen, and analiquot of the sample was taken out from the lowerlayer for the quantitative determination of hydroperox-ide of LH (LOOH) by spectrophotometric method. Theconcentration of LOOH was determined by the spectro-photometric ferric thiocyanate method following theprocedure described by us earlier, using UV/VIS spectro-photometer Varian Cary 4000 [15]. The rest of the lipidextract was used for GC analysis of geometrical isomersusing known conditions for the separation of cis andtrans isomers [7,16]. In order to transform LH and itsgeometrical isomers in the corresponding methyl ester,the reaction solutions were treated with an etherealsolution of diazomethane [17]. We used Varian 450 gaschromatograph equipped with a flame ionizationdetector and an Rtx-2330 (90% biscyanopropyl/10%phenylcyanopropyl polysiloxane capillary column; 105m� 0.25mm). Temperature started from 180 �C held for35min, followed by increase in 10 �C/min up to 250 �Cand held for 5min. Methyl esters were identified bycomparison with the retention times of authentic sam-ples, which were commercially available.

Results and discussion

Formation of LOOH in irradiated LH micellesunder aerobic conditions

LH micelles were irradiated by 50, 100, 200, 300, and400Gy doses under air-equilibration. After irradiation,lipid components were extracted with CH2Cl2–MeOH(2/1, v/v) and analyzed spectrophotometrically for lipidhydroperoxide (LOOH) determination. Figure 1 shows

Scheme 1. Radical-based peroxidation and geometrical isomerization processes of linoleic acid (9cis,12cis-octadecadienoic acid,9c,12c-18:2).

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the dose profiles of LOOH formation in absence or pres-ence of various antioxidants. Vitamin E (a-TOH) is themajor lipophilic, radical-scavenging antioxidant in vivo[18]. Using concentration of 50 lM in this work, its effi-ciency was confirmed. Another phenolic antioxidant likeresveratrol (ResOH) showed an analogous behavior, forexample using 80 lM of ResOH the formation of LOOHis about half of that one using 50lM a-TOH [19]. Theeffect of 60 lM AscH to limit LOOH formation was alsoremarkable in our model (see below the section onAscH). In the presence of a-TOH/AscH mixture, oxida-tion of LH was inhibited better than with a-TOH orAscH alone. The synergistic or additive action of a-TOHand AscH to inhibit propagation of lipid peroxidation isknown also in liposomes [18,20] and in biological sys-tems, essentially connected either to a recycling of toc-opherol radical by ascorbate anion or to the typicaldistribution between lipophilic and hydrophilic com-partments of the two antioxidants [18,20]. Such effectwas confirmed in this model of LH micelles where thetwo antioxidants can act by chemical recycling and bytrapping of radicals distributed in the system.

Geometrical isomerization in irradiated LHmicelles under aerobic conditions

The same samples described above for the measure-ments of LOOH were used for the follow-up of the

geometrical isomerization of LH. Lipid componentswere extracted as methyl esters after treatment of thereaction mixture for esterification, as previouslydescribed [7]. GC analysis is the gold standard for thegeometrical isomer identification and quantification.Figure 2 shows the dose profiles of LH disappearanceand trans isomers appearance in the presence of antiox-idants, that is, AscH (60 lM), a-TOH (50 lM), and ResOH(80lM) as well as mixture of 50 lM a-TOH and 60 lMAscH. In all cases, the trans isomers increased with dose,indicating that the cis–trans isomerization parallels theperoxidation process even in the presence of variousantioxidants.

Geometrical isomerization in irradiated LHmicelles under anaerobic conditions

LH micelles were saturated by N2O prior to irradiation(anaerobic conditions). The samples were irradiated by50, 100, 200, 300, and 400Gy doses and treated as pre-viously explained to obtain the isomerization profiles.The disappearance of LH is replaced by its trans isomersas shown in Figure 3 in function of the dose and inabsence or presence of various antioxidants, that is,AscH (60 lM), a-TOH (50 lM), and ResOH (80lM). AscH

Figure 2. The geometrical isomer distribution of linoleic acidmethyl ester (symbols (�, �, *, �) represent 9cis,12cis-18:2and symbols (s, �, �, D) represent the sum of trans isomers)in the presence of natural occurring antioxidants under air-equilibration (dose rate: 274.8 Gy/min): (*, �) LH micelles inthe presence of 60lM AscH; (�, �) LH micelles in the pres-ence of 80lM ResOH; (�,D) LH micelles in the presence of50lM a-TOH; (�, s) LH micelles in the presence of mixtureof 50 lM a-TOH and 60 lM AscH; LH micelles formed by5.0� 10�4 M LH, 2.8� 10�4 M TweenVR -20, 5.0� 10�3 MNaH2PO4, and 2.8� 10�3 M RSH at pH 5. Reported values rep-resent the mean of three independent measurements.

Figure 1. The formation of LOOH in LH micelles as a functionof irradiation dose (dose rate 274.8 Gy/min) under air-equili-bration: (�) LH micelles without antioxidants; (*) LH micellesin the presence of 60lM AscH; (~) LH micelles in the pres-ence of 80lM ResOH; (j) LH micelles in the presence of50 lM a-TOH; (�) LH micelles in the presence of mixture of50 lM a-TOH and 60 lM AscH; LH micelles formed by5.0� 10�4 M LH, 2.8� 10�4 M TweenVR -20, 5.0� 10�3 MNaH2PO4, and 2.8� 10�3 M RSH at pH 5. Reported values rep-resent the mean of three independent measurements.

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in the presence of RSH is the most effective inhibitor ofradiation-induced trans-isomer formation. This resultconfirms the efficiency of this hydrophilic antioxidant tocounteract lipid isomerization, as already known forMUFA in liposome models [12,13]. Slightly weaker effectwas observed by the addition of an AscH/a-TOH mix-ture, while in the case of ResOH inhibition of trans-iso-mer formation did not significantly differ from thatprocess in the control micelles.

Thiyl radical generation and cis–transisomerization mechanism

Radiolysis of neutral water leads to reactive species,including solvated electrons (e�aq), hydrogen atoms (H�),and hydroxyl radicals (HO�) as shown in Reaction 1. Thevalues in parentheses represent the radiation chemicalyields (G) in units of lmol J�1 [21]. In a N2O-saturated solution (�0.02 M of N2O), e�aq are trans-formed into HO� (Reaction 2, k2¼9.1� 109 M

�1 s�1),affording a G(HO�)¼ 0.55 lmol J�1, that is, HO� radicalsand H� atoms account for 90% and 10%, respectively,of the reactive species. In the presence of thiols such asthe amphiphilic 2-mercaptoethanol (RSH), hydrogen

abstraction by HO�, and H� directly produce thiyl radi-cals (Reaction 3, k3¼6.8� 109 M

�1 s�1 for HO� andk3¼1.7� 109 M

�1 s�1 for H�) [21].

H2O e�aqð0:27Þ; HO�ð0:28Þ;H�ð0:06Þ (1)

e�aq þ N2Oþ H2O ! HO� þ N2 þ HO� (2)

HO�=H� þ RSH ! RS� þ H2O=H2 (3)

Scheme 2 shows the reaction mechanism of cis–transisomerization that consists of RS� radical reversible add-ition to the double bond. Indeed, the reconstitution ofthe double bond is obtained by b-elimination of RS�

and the result is in favor of trans geometry, the mostthermodynamically favorable disposition. The rate con-stants for RS� addition to the cis and trans isomers(oleic acid and elaidic acid, respectively) were foundto be rather similar (ka

cis¼ 1.6� 105 M�1 s�1 andka

trans¼ 2.9� 105 M�1 s�1), whereas the thiyl radicalelimination from the intermediate adduct radicalswere substantially different (kf

cis¼ 1.7� 107 s�1 andkftrans¼ 1.6� 108 s�1) [22,23]. It is worth noting that the

RS� radical acts as a catalyst for cis–trans isomerization.Considering LH, the isomerization mechanism occurs asa step-by-step process, that is, each isolated doublebond behaves independently [4,16].

The disappearance of the starting material (mol kg�1)divided by the absorbed dose (1 Gy ¼1 J kg�1) givesthe radiation chemical yield or G[-(9c,12c-18:2)]. Bytreating the data of Figure 3 in this way and by plottingthe G[-(9c,12c-18:2)] versus dose (see Figure S1 inSupporting Information), we obtained further informa-tion on the catalytic process. For example, the extrapo-lation to zero dose for LH micelles without antioxidantsgives G¼ 192 lmol J�1. Assuming that the G(RS�) is0.52 lmol J�1 (ca. 85% of the initial radical species), wecalculated the catalytic cycle to be 370 at the initialphase. For the analogous isomerization of LH micellesin the presence of AscH, a catalytic cycle of 96 was cal-culated, whereas the effect of ResOH was negligiblesince the catalytic cycle is close to the one without anti-oxidants (Figure S1).

The effect of ascorbic acid or resveratrolconcentration on the LOOH formation andgeometrical isomers distribution

Next, we considered the role of AscH in our model sys-tem in some details at fixed irradiation dose of 100Gy.

Figure 3. The disappearance of LH (9c,12c-18:2 isomer) versusdose in the presence of different natural occurring antioxi-dants under anaerobic conditions (dose rate: 274.8 Gy/min):(�) LH micelles without antioxidants; (*) LH micelles in thepresence of 60lM AscH; (�) LH micelles in the presence of80 lM ResOH; (~) LH micelles in the presence of 50lMa-TOH; (�) LH micelles in the presence of mixture of 50lMa-TOH and 60 lM AscH; LH micelles formed by 5.0� 10�4 MLH, 2.8� 10�4 M TweenVR -20, 5.0� 10�3 M NaH2PO4, and2.8� 10�3 M RSH at pH 5.

Scheme 2. Reaction mechanism for the cis–trans isomerization catalyzed by thiyl radicals.

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In particular, Figure 4(A) shows the increase in AscHconcentration range from zero to 8� 10�5 M in theabsence (w) or presence (�) of 2.8mM RSH under air-equilibration. At zero concentration of AscH, the LOOHyield is higher with thiol than in the absence of thiol,indicating that thiyl radicals activate peroxidation. Byincreasing the concentration of AscH to 2� 10�5 M,AscH alone has a small inhibiting effect, whereas in thepresence of both AscH and thiol complete inhibition ofperoxidation occurs, indicating an effective synergismbetween AscH and RSH. With further increase in AscHconcentration, a pro-oxidative effect was observedalthough the synergic effect of RSH remains. Figure 4(B)shows the geometrical isomers distribution for thesame experiments when the thiol is present. Therefore,the cis–trans isomerization process operates in parallelwith lipid peroxidation. Figure 4(C) shows the analo-gous experiments under anaerobic conditions, wherethe process of lipid peroxidation is suppressed. At zeroconcentration of AscH, 50% of LH is transformed inmono-trans (40%) and di-trans (10%) isomers. Byincreasing the concentration of AscH, the isomerizationprocess with trans-isomer formation decreased and at4� 10�5 M is almost completely inhibited.

It is worth mentioning that rate constants for thereactions of RS� (from cysteamine, cysteine, and gluta-thione) and ascorbate were reported to be �109 M�1

s�1 by pulse radiolysis determination [24]. It is alsoknown that the RS� radicals are moderate oxidants (e.g.E(HOCH2CH2S

�, Hþ/HOCH2CH2SH)¼ 1.3 V) and oxidizeascorbate to ascorbyl radicals which terminate due todisproportionation (k¼ 106 M�1 s�1). Based on theseresults, it can be assumed that in the model system of

LH, a rapid reaction between AscH and RS� radicals inthe hydrophilic part of the micelles takes place so thatquenching of the majority of RS� occurs, preventing thesulfur-center radical migration in the hydrophobic partof micelles to reach the double bond, thereby inhibitingthe isomerization process.

Similar experiments with ResOH are performed andreported in Figure S2 (Supporting Information). Theresults of the geometrical LH isomer distribution in LHmicelles in the presence of RSH obtained in anaerobicconditions at irradiation dose of 100Gy are summarizedin Table 1. It is evident that ResOH inhibits isomeriza-tion, but it is not so effective as compared with AscH.Using the mixture of the two antioxidants, the isomer-ization process shows intermediate reactivity.

Conclusion

The “oil in water” system of micelles was used as bio-mimetic model of the lipid isomerization and peroxida-tion reactions occurring under irradiation conditions

Table 1. The influence of AscH and ResOH on the formationof geometrical isomers of LH micelles after 100 Gy of irradi-ation at 274.8 Gy/min under anaerobic conditionsa.Sample % 9c,12c % 9c,12t þ 9t,12c % 9t,12t

LH 38.9 43.5 17.5LH þ100lM ResOH 47.8 39.9 12.3LH þ60 lM AscH 98.4 1.6 0LH þ100lM ResOH

þ60 lM AscH88.2 10.9 1.7

aLH micelles formed by 5.0� 10�4 M LH, 2.8� 10�4 M TweenVR -20,5.0� 10�3 M NaH2PO4, and 2.8� 10�3 M 2-mercaptoethanol at pH 5.Reported values represent the mean of three independentmeasurements.

Figure 4. (A) Effect of the AscH concentrations on LOOH formation after 100 Gy of irradiation (dose rate: 274.8 Gy/min) under air-equilibration: (w) LH micelles and AscH (0–8)� 10�5 M; (�) LH micelles, AscH (0–8)� 10�5 M and RSH 2.8mM; (B) Effect of theAscH concentrations on the distribution of linoleic acid methyl ester geometrical isomers after 100 Gy of irradiation of LH micelles,AscH (0–8)� 10�5 M and RSH 2.8mM under air-equilibration: (�) 9c,12c-18:2, (�) 9t,12c-18:2 and 9c,12t-18:2, (�) 9t,12t-18:2; (C)like in B under anaerobic conditions; LH micelles formed by 5.0� 10�4 M LH, 2.8� 10�4 M TweenVR -20, 5.0� 10�3 M NaH2PO4,and 2.8� 10�3 M RSH at pH 5. Reported values represent the mean of three independent measurements (errors ±5%).

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and inhibited by natural compounds known as antioxi-dants. Efficiency of the inhibition was correlated to thecompartment where radical initiation occurs and to thedouble bond location in organized systems, that is inthe lipophilic interior.

Under air-equilibrated conditions, the addition of dif-ferent natural occurring antioxidants retarded the pro-cess of lipid peroxidation along the series:a-TOH<AscH< ResOH<AscH/a-TOH mixture. At thesame time and conditions, isomerization was observedat low radical concentration in the presence of differentantioxidants as compared to control LH system. Underanaerobic conditions, isomerization is a process moreeffective than in aerobic conditions. The presence ofantioxidants in LH model systems decreased trans-isomer level after gamma irradiation at low dose (up to200Gy) along the series: ResOH < a-TOH<AscH/a-TOHmixture<AscH.

Acknowledgements

All authors gratefully acknowledge the support and network-ing opportunities they received from COST Action CM1201on “Biomimetic Radical Chemistry”.

Disclosure statement

The authors report no conflicts of interest. The authors aloneare responsible for the content and writing of this article.

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