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Biochemical and Biophysical Research Communications 288, 225–232 (2001)

doi:10.1006/bbrc.2001.5765, available online at http://www.idealibrary.com on

staxanthin and Peridinin Inhibit Oxidative Damagen Fe21-Loaded Liposomes: Scavenging Oxyradicalsr Changing Membrane Permeability?

arcelo P. Barros,*,1 Ernani Pinto,*,† Pio Colepicolo,† and Marianne Pedersen*Department of Botany, Stockholm University, SE-10691 Stockholm, Sweden; andDepartamento de Bioquimica, IQUSP, C.P. 26077, 05599-970, Sao Paulo, Brazil

eceived August 30, 2001

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Astaxanthin and peridinin, two typical carotenoidsf marine microalgae, and lycopene were incorpo-ated in phosphatidylcholine multilamellar liposomesnd tested as inhibitors of lipid oxidation. Contrarilyo peridinin results, astaxanthin strongly reducedipid damage when the lipoperoxidation promoters—

2O2, tert-butyl hydroperoxide (t-ButOOH) or ascor-ate—and Fe21:EDTA were added simultaneously tohe liposomes. In order to check if the antioxidantctivity of carotenoids was also related to their effectn membrane permeability, the peroxidation pro-esses were initiated by adding the promoters to Fe21-oaded liposomes (encapsulated in the inner aqueousolution). Despite that the rigidifying effect of carote-oids in membranes was not directly measured here,eridinin probably has decreased membrane perme-bility to initiators (t-ButOOH > ascorbate > H2O2)ince its incorporation limited oxidative damage onron-liposomes. On the other hand, the antioxidantctivity of astaxanthin in iron-containing vesiclesight be derived from its known rigidifying effect and

he inherent scavenging ability. © 2001 Academic Press

Key Words: astaxanthin; peridinin; antioxidant; lipo-ome; lipoperoxidation.

Peridinin is an unusual C37 carbon skeleton carot-noid with epoxy, hydroxy, and acetate groups on-rings, an allene moiety and a lactone group conju-ated to the p-electron system (Fig. 1) (1). In addition

Abbreviations used: BHT, butylated hydroxytoluene; EDTA, eth-lenediaminotetraacetic acid; Iron-PCL, Fe21:EDTA-loaded egg-yolkhosphatidylcholine liposomes; MDA, malondialdehyde; PCL, egg-olk phosphatidylcholine liposomes; PUFA, polyunsaturated fattycids; ROS, reactive oxygen species; TBARS, thiobarbituric acideactive substances; t-ButOOH, tert-butyl hydroperoxide; Trolox,1)-6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid.

1 To whom correspondence should be addressed at Departamentoe Bioquımica, IQUSP, Bloco 9 superior, C.P. 26077, 05599-970, Saoaulo, Brazil. Fax: 155-11-38182170. E-mail: mpbarros@botan.su.se.

225

hotosystem II (PSII), dinoflagellates also contain aater-soluble external antenna complex, the peridinin-

hlorophyll-protein (PCP). Peridinins in PCP and inodel antenna systems effectively transfers electronic

xcitation to chlorophyll a (88 to 95%) which is able toass this excitation energy to membrane-bound light-arvesting complexes on PSII (1–4). Recently, Pinto etl. (5) have demonstrated that peridinin is the majoringlet molecular oxygen [O2(

1Dg)] quencher in Lingu-odinium polyedra, despite being less efficient than-carotene. However, it has not been clearly shown ifinoflagellates contain peridinin molecules on antennaomplexes of the photosystems within thylakoid mem-ranes (6).The ketocarotenoid astaxanthin (Fig. 1) is a red pig-ent common to several aquatic organisms including

lgae, salmon, troute, and shrimp (7–9). Several re-orts indicate that astaxanthin is one of the most ef-ective antioxidant against lipid peroxidation and oxi-ative stress in many in vitro and in vivo systems10–15). It has also been shown that simultaneousepletion of astaxanthin and a-tocopherol influencesutoxidative defense, fatty acid metabolism and syn-hesis of coenzyme thiamine-pyrophosphate in Balticea salmon affected by the M74 syndrome (16–20).Another relevant property of carotenoids is how

hese compounds affect fluidity and permeability ofatural and artificial membranes. Carotenoids witheto and hydroxy groups on both ends of the moleculee.g., zeaxanthin, astaxanthin, and canthaxanthin)trongly decrease water and small molecules perme-bility across the lipid bilayer (21). Thus, in addition todirect scavenging ability against reactive oxygen spe-

ies (ROS), some polar carotenoids also inhibit theenetration of oxidative substances and, consequently,he initiation of a lipid peroxidation process.

The aim of this work is to study the antioxidantctivity of astaxanthin and peridinin, two of the most

0006-291X/01 $35.00Copyright © 2001 by Academic PressAll rights of reproduction in any form reserved.

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Vol. 288, No. 1, 2001 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

bundant carotenoids among marine microalgal spe-ies. For that purpose, the carotenoids were incorpo-ated into egg-yolk phosphatidylcholine multilamellariposomes (PCL) and challenged by different ROShich were generated by classical lipoperoxidation ini-

iators. In order to check if the carotenoid antioxidantctivity is exclusively or partially derived from its ri-idifying effect on membranes, the liposomes were pre-iously loaded with Fe21:EDTA complexes (Iron-PCL).hus, to initiate ROS generation in Iron-PCL, the li-operoxidation agents—H2O2, tert-butyl hydroperox-de (t-ButOOH) and ascorbate—must cross the lipidilayers and react with the metal ion present inside theesicles. These experiments were also performed withycopene and butylated hydroxytoluene (BHT), classi-al antioxidants, as controls.

ATERIALS AND METHODS

Materials. All chemicals were obtained from Sigma–Aldrich Swe-en AB, except FeSO4.7H2O and liquid chromatography grade sol-ents n-hexane, chloroform, methanol, and ethanol from Merck Co.Darmstadt, Germany); ascorbic acid and Perdrogen (H2O2 30%)rom Riedel-deHaen (Seelze, Germany); and (1)-6-hydroxy-2,5,7,8-etramethylchromane-2-carboxylic acid (Trolox) from Fluka ChemikaBuchs, Switzerland). Peridinin was isolated from Lingulodiniumolyedra as described by Pinto et al. (5). The dialysis membranesere Spectra/Por MWCO 2000 from Spectrum Medical Industries

Los Angeles, CA).

Carotenoid stock solutions. All carotenoids were solubilized inrganic solvents previously to their incorporation into egg-yolk phos-hatidylcholine liposomes (PCL) and the absorbances of these stockolutions were measured to evaluate their effective concentrations.eridinin («469 5 85.8 3 103 M21 cm21) was solubilized in chromatog-aphy grade methanol while astaxanthin («468 5 125 3 103 M21 cm21)nd lycopene («472 5 186 3 103 M21 cm21) were dissolved in purified-hexane (22). The stock solutions were stored at 280°C freezer androtected from light to avoid oxidation.

Preparation of multilamellar liposomes (PCL). In order to pre-ent aggregate formation and loss of material during the procedure,he carotenoids were isolated from stock solution by flushing theespective organic solvent with a N2 stream until dryness. After that,

FIG. 1. Chemical structures of peridinin and astaxanthin.

226

hosphatidylcholine solution in CHCl3 was mixed for a final carote-oid:lecithin proportion of 0.5% (25 mM and 5 mM, respectively). Eggolk phosphatidylcholine was selected for its unsaturated fatty acidontent which offers suitable oxidation targets for ROS (23, 24). Afterrief mixing, chloroform was evaporated by flushing N2 in a round-ottom flask adapted to a rotavapor apparatus working at a lowpeed to allow the formation of a homogeneous dried film. Theipid-carotenoid film was stored overnight in the dark under vacuumo eliminate traces of chloroform. The PCL vesicles were prepared byixing 100 mM phosphate buffer (pH 7.4) to the lipid film followed

y strong vortexing for 5 min. The formation of carotenoid aggre-ates was avoided by preparing the PCL at 40°C, which is high abovehe transition temperature of 30°C for egg-yolk phosphatidylcholine25). The suspension was centrifuged at 15,000 rpm for 20 min toliminate eventual formed aggregates.

Preparation of Fe21-incorporated multilamellar liposomes (Iron-CL). The first method tested for Iron-PCL preparation envolvedonication of the lipid-carotenoid film with 100 mM phosphate bufferpH 7.4) on ice until the dispersion becomes clean (26). However, thislassic method of liposome preparation proved to be unefficient forur purposes since it caused a 8.5-fold higher level of lipid oxidationdata not shown). Thus, the Iron-PCL was prepared as PCL: mixinghe lipid-carotenoid film with 5 mL of 100 mM phosphate buffer (pH.4) plus 5 mM Fe21:EDTA solution (to a final concentration of 0.1M) and strong vortexation. A dialysis procedure was used to elim-

nate external and loosely bound iron complexes from the liposomes.bout 5 mL of uncleaned Iron-PCL were dialysed in Spectra/Porolecularporous membrane (MWCO 2000) at room temperature

gainst 2 L of destilled water for 2 h with smooth agitation by aagnetic stirrer. In the beginning, the liposome suspensions were

ialysed against 2 L of 100 mM phosphate buffer (pH 7.4) but thisrocedure did not efficiently remove the metal ions supposed to belaced outside the liposomes (data not shown). The iron content inhe PCL was checked before and after every dialysis process tostimate loss of iron complexes during the procedure.

Induction of lipid peroxidation. Either PCL or Iron-PCL, contain-ng significant concentrations of unsaturated lipids (27), were oxi-ized by incubation for 45 min at 30°C with 1 mM solution of threeifferent initiators: H2O2, t-ButOOH or ascorbic acid. To stimulateipid oxidation in PCL, 0.1 mM Fe21:EDTA was simultaneouslydded. Trolox (0.5 mM in 0.1 M phosphate buffer pH 7.4) and 5 mMutylated hydroxytoluene (BHT) were used as controls. Trolox, aater-soluble derivative of a-tocopherol with similar scavenging ac-

ivity (28), was used as a probe for checking the sites of ROS gener-tion in multilamellar vesicles since it is not supposed to permeateiposome lipid bilayers (Fig. 2).

Measurement of lipoperoxidation extent (TBARS test). After thencubation period, the oxidative reaction was stopped by adding 20L of 0.2 M BHT (ethanol solution). To produce the coloured adduct,50 mL of sample were incubated with 700 mL of 0.375% thiobarbi-uric acid (TBA) in 0.25 M HCl and 1% Triton X-100 at 100°C for 15in. After reaching the room temperature, the absorbance of the

olutions were measured at 535 nm using malondialdehyde (MDA)s standard (29). Controls for residual absorption of carotenoids at35 nm were made using 0.25 M HCl plus 1% Triton X-100 solutionithout TBA.

Iron determination. The iron incorporation in the liposomes washecked before and after the dialysis procedure by a modification ofhe method described by Bralet et al. (30). Aliquotes of 300 mL wereaken from the liposome suspensions and 3 mL of Triton X-100 wasdded to disrupt the vesicles. The samples were added to 50 mMlycine hydrochloride buffer (pH 2.5) with 20 mg/mL ascorbate, 10g/mL pepsin and 5 mM 2,29-bipyridine. After incubation for 2 h at

7°C, the absorbance was measured at 520 nm and results comparedo FeSO4.7H2O standard curve.

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Vol. 288, No. 1, 2001 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Statistics. Data are presented as means 6 SD (standard devia-ion) and statistical analysis performed with the Student’s t test atignificance level of 5%.

ESULTS AND DISCUSSION

onloaded Liposomes (PCL)

The TBARS concentration after PCL preparationsere (0.169 6 0.043 nmol MDA/mmol PC) and (0.209 6.031 nmol MDA/mmol PC), respectively for PCL andron-PCL. As expected, the encapsulation of Fe21:DTA complexes in PCL resulted in higher lipid oxi-ation level (c.a. 25%). The coordination of Fe21 withDTA does not prevent it to react with ROS and,ypothetically, it would be easier to eliminate (by di-lysis) a water-soluble Fe21:EDTA complex than aembrane-associated Fe21:phosphatidylcholine che-

ate (31, 32).Probably, the osmotic pressure must have led to re-

rganization of PCL membranes and coalescence ofipid vesicles during the dialysis performed againstistilled water (33). Even with distinguished polarityroperties, lycopene and astaxanthin induced Fe21:DTA elimination from liposomes at the same extent

c.a. 30%). When peridinin was associated, the effectas less intense (23%). On the other hand, a higher

oss of iron chelate was measured in carotenoid-freeiposomes (53%) (Fig. 3).

Ascorbic acid can behave as a prooxidant since it caneduce Fe31 to Fe21, a well-known strong promoter ofipoperoxidation (33). However, at millimolar concen-rations the ability of ascorbate to scavenge HO• be-omes more significant. Ascorbate is also able to reduceocopheryl radicals, generated by hydrogen abstrac-ion from a-tocopherol, back to its active antioxidantorm. Trolox, with similar scavenging mechanism as-tocopherol, is supposed to be constantly regenerated

FIG. 2. Trolox scavenging activity against ROS produced by2O2, t-ButOOH and ascorbate in PCL and Iron-PCL.

227

y ascorbate from the Trolox radical form in the aque-us solution (28). Ascorbate is also supposed to beharged at pH 7.4 (ascorbic acid pKa1 and pKa2, 4.17nd 11.57, respectively) thus with low permeabilityhroughout membranes.

Butylated hydroxytoluene (BHT) was very efficientn scavenging free radicals generated by all lipoperoxi-ation agents in PCL even at micromolar range (Fig.). This effect was probably due to its higher diffusibil-ty into membranes (34) which would allow this anti-xidant to scavenge oxyradicals at several spotshrough out the lipid bilayer. Trolox, mostly present inhe aqueous solution, required millimolar concentra-ions to inhibit lipid oxidation to the same extention as

FIG. 3. Iron concentrations in Iron-PCL in the presence or ab-ence of lycopene (Iron-PCL/LYC), astaxanthin (Iron-PCL/AST) oreridinin (Iron-PCL/PER), during a dialysis process (nmols Fe21/mol PC). Shown are the means 6 SD of 3 experiments; *P , 0.05.

FIG. 4. Effects of BHT and Trolox in lipoperoxidation of PCL inhe absence of carotenoids induced by mixing lipoperoxidationromoters—H2O2, t-ButOOH, or ascorbate—to chelated Fe21 ionsnmols MDA/mmol PC). Shown are the means 6 SD of 4 experiments;P , 0.05.

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Vol. 288, No. 1, 2001 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

HT in H2O2/Fe21 system. Higher levels of TBARSere produced by addition of Fe21 and t-ButOOH toCL: 1.9-fold higher compared to 94% obtained with2O2. When lipoperoxidation process is initiated by

-ButOOH most of the free radicals detected in the lipidilayer is peroxyl radical (ROO•) (35). A significantroportion of alkoxyl radical (RO•) and singlet oxygenO2(

1Dg)] have also to be considered (23, 26, 35–38).rolox was not able to efficiently protect the PCL mem-ranes in t-ButOOH-induced peroxidation. Usually,rolox is more reactive with ROS than BHT, especiallyoncerning peroxyl radicals (39), but the higher perme-bility of the phenolic compound may have compen-ated for its lower reactivity.To evaluate the single effect of iron addition to PCL

with or without carotenoids), the TBARS measure-ents were also performed in the absence of peroxida-

ion agents. As an extra control, PCL was also pre-ared containing 25 mM a-tocopherol as described byalozza & Krinsky (40). As could be observed in Fig. 5,staxanthin and peridinin were able to inhibit lipoper-xidation before the addition of iron complexes. Theddition Fe21:EDTA to PCL, in the absence of carote-oids, did not change TBARS production although an

ncrease of 25% in MDA content was previously ob-erved after Iron-PCL preparation. The lipid oxidationn astaxanthin- (PCL/AST) and peridinin-incorporatediposomes (PCL/PER) were both, approximately, 25%ower than in PCL although only PCL/AST was insen-itive to iron addition. Lycopene was the only carot-noid which reduced (25%) the level of lipoperoxidationfter Fe21:EDTA addition (Fig. 5).The carotenoid-incorporated liposomes were chal-

enged by ROS produced outside, when initiator andron complexes were added simultaneously. These re-ults are presented in Fig. 6. The simultaneous addi-ion of ferrous salt and ascorbate resulted in a moreoderate increase of TBARS concentration (21%) than

FIG. 5. Levels of TBARS promoted by addition of Fe21:EDTA inCL containing a-tocopherol (PCL/TOC), lycopene (PCL/LYC), peri-inin (PCL/PER) and astaxanthin (PCL/AST). Shown are theeans 6 SD of 4 experiments; *P , 0.05.

228

esting the previously described dual effect of ascor-ate concerning its action against free radicals. It isoteworthy that, usually, iron ions are contaminatingscorbic acid by 0.02% which would allow the initia-ion of lipid oxidation even without adding Fe21 solu-ion (31).

Astaxanthin proved to be the best antioxidant in allxperiments performed with both peroxidation initia-or and iron chelate placed outside the PCL, as ex-ected from other authors (10, 17). The ketocarotenoidas the only tested compound to avoid extreme high

evels of lipid damage caused by concomitant additionf ferrous ions and H2O2, t-ButOOH or ascorbate: re-pectively, 45, 45, and 33% lower lipid oxidation thanCL added with iron (II). As observed with the exper-

ments without peroxidation agents (Fig. 5), astaxan-hin also induced the lowest enhancement of MDAroduction upon iron ions addition.No antioxidant activity was found for peridinin when

ncorporated into PCL and challenged by free radicalsroduced outside. Actually, peridinin led to intenseugmentation of oxidated lipid levels after ferrous ionsere added to H2O2- and t-ButOOH-treated liposomes:.9-fold and 3.5-fold higher, respectively. The TBARSevel obtained after incubation of PCL/PER with ascor-ate 1 mM in the absence of Fe21 (0.65 6 0.14 nmolDA/mmol PC) was one of the highest of all experi-ents performed.Lycopene, under the reaction conditions described

ere, could not inhibit the lipoperoxidation process inCL. The effect of chelated iron (II) inclusion toscorbate-treated PCL/LYC was lower than with otheripid peroxidation agents despite being the highestalue measured (0.73 6 0.03 nmol MDA/mmol PC).polar carotenoids, e.g., b-carotene and lycopene, have

FIG. 6. TBARS levels induced by H2O2, t-ButOOH or ascorbatend Fe21:EDTA in carotenoid-associated PCL. Shown are theeans 6 SD of 4 experiments; *P , 0.05.

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Vol. 288, No. 1, 2001 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

een reported to perturb the acyl chain packing and toncrease bilayer permeability (41, 42). In some circum-tances, efficient in vivo antioxidants like b-carotenend lycopene could also act, or partially offer, a prooxi-ative effect in lipid peroxidation process masking itsntioxidant activity.

ron-Loaded Liposomes (Iron-PCL)

After the dialysis, an insignificant concentration ofe21:EDTA was present outside the PCL. As it ishown in Fig. 7, no significant variation was observedn H2O2-generating system when 25 mM, 50 mM, 0.25

M, or 0.5 mM Trolox were added. This aspect sug-ests that these oxyradicals were generated in thenternal aqueous solution, triggered by the permeationf the easily diffusible molecule, H2O2. When both ironII) and H2O2 were added to the external aqueous so-ution, 0.5 mM Trolox and 5 mM BHT inhibited lipoper-xidation by 40 and 55%, respectively (Fig. 4).A constant (13%), but not significant, inhibition ofDA production in t-ButOOH-treated Iron-PCL was

aused by Trolox in the concentration range from 25M to 0.25 mM (Fig. 7). However, 0.5 mM Troloxignificantly suppressed lipid peroxidation: 23.6%.ven also being a small and uncharged molecule,

-ButOOH was expected to permeate membranes in aess extention than H2O2. Paradoxically, higher lipidxidation products were measured after incubation ofron-PCL with t-ButOOH than with H2O2. BHT wasot able to prevent lipid oxidation in this system al-hough, when peroxyl and alkoxyl were generated out-

FIG. 7. Effects of 5 mM BHT and 25 mM, 50 mM, 0.25 mM and 0.5M Trolox in lipoperoxidation of Iron-PCL (in the absence of caro-

enoids) induced by adding 1 mM ROS promoters—H2O2, t-ButOOH,nd ascorbate (nmols MDA/mmol PC). Shown are the means 6 SD ofexperiments; *P , 0.05.

229

as obtained.Ascorbate addition to Iron-PCL also resulted in a

igher lipid oxidation despite being negatively chargedt pH 7.4 and not assumed to penetrate intensely theipid bilayers. A possible explanation is the 0.02%sual iron contamination of commercial ascorbic acid31). The effect of Trolox on lipoperoxidation is anotherndication that the oxidation process was initiated athe outer moiety. In fact, the addition of increasingoncentrations of Trolox led to gradual higher protec-ion of the membranes against oxidative damage. An-ther indication of external action of free radicals is the3% inhibition of lipoperoxidation in Iron-PCL inducedy 5 mM BHT.As shown in Fig. 8, lipoperoxidation in Iron-PCL was

ntensely stimulated by the addition of peroxidationromoters—H2O2, t-ButOOH and ascorbate, respec-ively—2.4-, 4-, and 3.8-fold higher than MDA concen-rations obtained without promoters (dotted line;.12 6 0.02 nmol MDA/mmol PC). The MDA concentra-ions found when H2O2 and t-ButOOH were added toron-PCL were significantly lower than those mea-ured when iron ions and promoter were added simul-aneously to the vesicles (Fig. 4). When ascorbate/Fe21:DTA was used as lipoperoxidation initiator system,n equivalent MDA concentration was obtained foroth types of vesicles: (0.45 6 0.06) and (0.45 6 0.07)mol MDA/mmol PC for, respectively, Iron-PCL and PCL.Astaxanthin was the more efficient antioxidant since

t suppressed the H2O2-induced lipoperoxidation inron-PCL by 26% (Fig. 8). Peridinin showed a moreodest inhibition of lipid oxidation process (17.7%),

uggesting that, due to its incorporation into lipid bi-ayer, it could have limited the permeation of the per-xidation agent, H2O2 in these experiments. On

FIG. 8. TBARS levels induced by addition of H2O2, t-ButOOH orscorbate to Iron-PCL in the presence of lycopene (Iron-PCL/LYC),eridinin (Iron-PCL/PER) or astaxanthin (Iron-PCL/AST). Shownre the means 6 SD of 4 experiments; *P , 0.05.

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Vol. 288, No. 1, 2001 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

as enhanced membrane permeability to H2O2 and-ButOOH, since increases of c.a. 21% in MDA concen-rations (not significant) were observed in both sys-ems. When 1 mM ascorbate was added to Iron-PCL,eridinin significantly limited lipoperoxidation whichas comparable to the values obtained with astaxan-

hin: respectively, 33.5% and 46.4%. Lycopene wasnly able to protect liposome membranes when ascor-ate was used as a promoter of lipid oxidation (17.4%ower than control).

ONCLUSIONS

Carotenoids, especially astaxanthin and zeaxanthin,how high rate constants for reactions with peroxyladicals (ROO•) and as a [O2(

1Dg)] quencher (24, 37).himidzu et al. (43) developed in vitro assays to studyhe quenching efficiency of several carotenoids fromarine organisms against [O2(

1Dg)] and has evidencedstaxanthin as one of the most efficient. Recently,into et al. (5) have demonstrated that peridinin, de-pite being less efficient than b-carotene, is the majorO2(

1Dg)] quencher in Linulodinium polyedra, mainlyue its elevated concentration in this organism. Thentioxidant effect of carotenoids is probably also de-ived from its rigidifying effect on membranes whichould lead to a limitation in metal ions or oxidativeompound penetration into lipid bilayers (44, 45). Onhe other hand, hydrophobic carotenoids (e.g., lycopenend b-carotene) make the membranes more fluid and,nder some circumstances, more fluid and, under someircumstances, more susceptible to oxidative damage41). The data reported here suggest that lycopene,nder the described reaction conditions, was not ableo protect membrane lipids against iron-induced oxida-ion process. This fact has also been recently pointedut as a possible explanation for the ambiguous actionf b-carotene challenged by oxyradicals in differentipid systems (42).

Astaxanthin, as previously demonstrated in vitrond in vivo (10, 17, 46–50), was able to strongly inhibithe propagation step of lipoperoxidation in all testedystems. It is possible that two combined properties ofstaxanthin were responsible for this fact: (i) the rigid-fying effect on membrane, which could have limitedhe penetration of lipoperoxidation promoters—H2O2,-ButOOH and ascorbate—into the liposome mem-ranes (21, 51, 52); and (ii) the inherent antioxidantctivity of this ketocarotenoid (53). However, the samexplanation is not valid for antioxidant action of peri-inin in Iron-PCL assays. Peridinin did not show anyntioxidant property when the ROS were producedutside the liposomes. However, when the peridinin-ssociated vesicles were pre-loaded with Fe21:EDTAomplexes, a significant inhibition of lipoperoxidationas observed (in all tested systems).

230

ipid bilayers was available in the literature, it isempting to suggest, by a preliminary analysis of itshemical structure, that this carotenoid might have aertical or more angular orientation in egg-yolk leci-hin liposomes. Thus, it is possible, despite being spec-lative, that peridinin also shows polar-carotenoid ri-idifying effect on membranes and, consequently, itsetected inhibitory action on lipid oxidation might beelated to a peridinin-induced decrease in the perme-tion of lipoperoxidation promoters in membranes.

CKNOWLEDGMENTS

This work was supported by the Swedish Foundation for Interna-ional Cooperation in Research and Higher Education (STINT Pro-ram), Sweden, and Fundacao de Amparo a Pesquisa do Estado deao Paulo (FAPESP), Brazil. We are grateful to Universidaderuzeiro do Sul (Brazil) and University of Uppsala (Sweden) forllowing, respectively, Marcelo Barros, Ph.D., and Ernani Pinto totay at Stockholm University to perform the experiments and pre-are this article.

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