Astaxanthin and Peridinin Inhibit Oxidative Damage in Fe 2ϩ-Loaded Liposomes:Scavenging Oxyradicals or Changing Membrane Permeability?
Marcelo P.Barros,*,1Ernani Pinto,*,†Pio Colepicolo,†and Marianne Pederse ´n*
*Department of Botany,Stockholm University,SE-10691Stockholm,Sweden;and †Departamento de Bioquimica,IQUSP,C.P.26077,05599-970,Sa ˜o Paulo,Brazil
Received August 30,2001
Astaxanthin and peridinin,two typical carotenoids of marine microalgae,and lycopene were incorpo-rated in phosphatidylcholine multilamellar liposomes and tested as inhibitors of lipid oxidation.Contrarily to peridinin results,astaxanthin strongly reduced lipid damage when the lipoperoxidation promoters—H 2O 2,tert -butyl hydroperoxide (t -ButOOH)or ascor-bate—and Fe 2؉:EDTA were added simultaneously to the liposomes.In order to check if the antioxidant activity of carotenoids was also related to their effect on membrane permeability,the peroxidation pro-cesses were initiated by adding the promoters to Fe 2؉-loaded liposomes (encapsulated in the inner aqueous solution).Despite that the rigidifying effect of carote-noids in membranes was not directly measured here,peridinin probably has decreased membrane perme-ability to initiators (t -ButOOH >ascorbate >H
2O 2)since its incorporation limited oxidative damage on iron-liposomes.On the other hand,the antioxidant activity of astaxanthin in iron-containing vesicles might be derived from its known rigidifying effect and the inherent scavenging ability.©2001Academic Press
Key Words:astaxanthin;peridinin;antioxidant;lipo-some;lipoperoxidation.
Peridinin is an unusual C 37carbon skeleton carot-enoid with epoxy,hydroxy,and acetate groups on -rings,an allene moiety and a lactone group conju-gated to the -electron system (Fig.1)(1).In addition
to the membrane-bound light harvesting complex of Photosystem II (PSII),dinoflagellates also contain a water-soluble external antenna complex,the peridinin-chlorophyll-protein (PCP).Peridinins in PCP and in model antenna systems effectively transfers electronic excitation to chlorophyll a (88to 95%)which is able to pass this excitation energy to membrane-bound light-harvesting complexes on PSII (1–4).Recently,Pinto et al.(5)have demonstrated that peridinin is the major singlet molecular oxygen [O 2(1⌬g )]quencher in Lingu-lodinium polyedra,despite being less efficient than -carotene.However,it has not been clearly shown if dinoflagellates contain peridinin molecules on antenna complexes of the photosystems within thylakoid mem-branes (6).
The ketocarotenoid astaxanthin (Fig.1)is a red pig-ment common to several aquatic organisms includin
g algae,salmon,troute,and shrimp (7–9).Several re-ports indicate that astaxanthin is one of the most ef-fective antioxidant against lipid peroxidation and oxi-dative stress in many in vitro and in vivo systems (10–15).It has also been shown that simultaneous depletion of astaxanthin and ␣-tocopherol influences autoxidative defense,fatty acid metabolism and syn-thesis of coenzyme thiamine-pyrophosphate in Baltic Sea salmon affected by the M74syndrome (16–20).Another relevant property of carotenoids is how these compounds affect fluidity and permeability of natural and artificial membranes.Carotenoids with keto and hydroxy groups on both ends of the molecule (e.g.,zeaxanthin,astaxanthin,and canthaxanthin)strongly decrease water and small molecules perme-ability across the lipid bilayer (21).Thus,in addition to a direct scavenging ability against reactive oxygen spe-cies (ROS),some polar carotenoids also inhibit the penetration of oxidative substances and,consequently,the initiation of a lipid peroxidation process.
The aim of this work is to study the antioxidant activity of astaxanthin and peridinin,two of the most
Abbreviations used:BHT,butylated hydroxytoluene;EDTA,eth-ylenediaminotetraacetic acid;Iron-PCL,Fe 2ϩ:EDTA-loaded egg-yolk phosphatidylcholine liposomes;MDA,malondialdehyde;PCL,egg-yolk phosphatidylcholine liposomes;PUFA,polyunsaturated fatty acids;ROS,reactive oxygen species;TBARS,thiobarbituric acid reactive substances;t -ButOOH,tert -butyl hydroperoxide;Trolox,(ϩ)-
6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid.1
To whom correspondence should be addressed at Departamento de Bioquı´mica,IQUSP,Bloco 9superior,C.P.26077,05599-970,Sa ˜o Paulo,Brazil.Fax:ϩ55-11-38182170.E-mail:mpbarros@botan.su.se.
Biochemical and Biophysical Research Communications 288,225–232(2001)doi:10.1006/bbrc.2001.5765,available online at www.idealibrary
on
abundant carotenoids among marine microalgal spe-cies.For that purpose,the carotenoids were incorpo-rated into egg-yolk phosphatidylcholine multilamellar liposomes(PCL)and challenged by differe
nt ROS which were generated by classical lipoperoxidation ini-tiators.In order to check if the carotenoid antioxidant activity is exclusively or partially derived from its ri-gidifying effect on membranes,the liposomes were pre-viously loaded with Fe2ϩ:EDTA complexes(Iron-PCL). Thus,to initiate ROS generation in Iron-PCL,the li-
poperoxidation agents—H
2O
2
,tert-butyl hydroperox-
ide(t-ButOOH)and ascorbate—must cross the lipid bilayers and react with the metal ion present inside the vesicles.These experiments were also performed with lycopene and butylated hydroxytoluene(BHT),classi-cal antioxidants,as controls.
MATERIALS AND METHODS
Materials.All chemicals were obtained from Sigma–Aldrich Swe-den AB,except FeSO4.7H2O and liqui
d chromatography grade sol-vents n-hexane,chloroform,methanol,and ethanol from Merck Co. (Darmstadt,Germany);ascorbic acid and Perdrogen(H2O230%) from Riedel-deHae¨n(Seelze,Germany);and(ϩ)-6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid(Trolox)from Fluka Chemika (Buchs,Switzerland).Peridinin was isolated from Lingulodinium polyedra as described by Pinto et al.(5).The dialysis membranes were Spectra/Por MWCO2000from Spectrum Medical Industries (Los Angeles,CA).
Carotenoid stock solutions.All carotenoids were solubilized in organic solvents previously to their incorporation into egg-yolk phos-phatidylcholine liposomes(PCL)and the absorbances of these stock solutions were measured to evaluate their effective concentrations. Peridinin(469ϭ85.8ϫ103MϪ1cmϪ1)was solubilized in chromatog-raphy grade methanol while astaxanthin(468ϭ125ϫ103MϪ1cmϪ1)
and lycopene(472ϭ186ϫ103MϪ1cmϪ1)were dissolved in purified n-hexane(22).The stock solutions were stored atϪ80°C freezer and protected from light to avoid oxidation.
Preparation of multilamellar liposomes(PCL).In order to pre-vent aggregate formation and loss of material during the procedure, the carotenoids were isolated from stock solution byflushing the respect
ive organic solvent with a N2stream until dryness.After that,500L of chloroform were added to eachflask and the egg-yolk
phosphatidylcholine solution in CHCl3was mixed for afinal carote-noid:lecithin proportion of0.5%(25M and5mM,respectively).Egg
yolk phosphatidylcholine was selected for its unsaturated fatty acid content which offers suitable oxidation targets for ROS(23,24).After brief mixing,chloroform was evaporated byflushing N2in a round-bottomflask adapted to a rotavapor apparatus working at a low speed to allow the formation of a homogeneous driedfilm.The lipid-carotenoidfilm was stored overnight in the dark under vacuum to eliminate traces of chloroform.The PCL vesicles were prepared by mixing100mM phosphate buffer(pH7.4)to the lipidfilm followed by strong vortexing for5min.The formation of carotenoid aggre-gates was avoided by preparing the PCL at40°C,which is high above the transition temperature of30°C for egg-yolk phosphatidylcholine (25).The suspension was centrifuged at15,000rpm for20min to eliminate eventual formed aggregates.
Preparation of Fe2ϩ-incorporated multilamellar liposomes(Iron-PCL).Thefirst method tested for Iron-PCL preparation envolved sonication of the lipid-carotenoidfilm with100mM phosphate buffer (pH7.4)o
n ice until the dispersion becomes clean(26).However,this classic method of liposome preparation proved to be unefficient for our purposes since it caused a8.5-fold higher level of lipid oxidation (data not shown).Thus,the Iron-PCL was prepared as PCL:mixing the lipid-carotenoidfilm with5mL of100mM phosphate buffer(pH 7.4)plus5mM Fe2ϩ:EDTA solution(to afinal concentration of0.1 mM)and strong vortexation.A dialysis procedure was used to elim-inate external and loosely bound iron complexes from the liposomes. About5mL of uncleaned Iron-PCL were dialysed in Spectra/Por molecularporous membrane(MWCO2000)at room temperature against2L of destilled water for2h with smooth agitation by a magnetic stirrer.In the beginning,the liposome suspensions were dialysed against2L of100mM phosphate buffer(pH7.4)but this procedure did not efficiently remove the metal ions supposed to be placed outside the liposomes(data not shown).The iron content in the PCL was checked before and after every dialysis process to estimate loss of iron complexes during the procedure.
Induction of lipid peroxidation.Either PCL or Iron-PCL,contain-ing significant concentrations of unsaturated lipids(27),were oxi-dized by incubation for45min at30°C with1mM solution of three different initiators:H2O2,t-ButOOH or ascorbic acid.To stimulate lipid oxidation in PCL,0.1mM Fe2ϩ:EDTA was simultaneously added.Trolox(0.5mM in0.1M phosphate buffer pH7.4)and5M
butylated hydroxytoluene(BHT)were used as controls.Trolox,a water-soluble derivative of␣-tocopherol
with similar scavenging ac-
tivity(28),was used as a probe for checking the sites of ROS gener-ation in multilamellar vesicles since it is not supposed to permeate liposome lipid bilayers(Fig.2).
Measurement of lipoperoxidation extent(TBARS test).After the incubation period,the oxidative reaction was stopped by adding20L of0.2M BHT(ethanol solution).To produce the coloured adduct, 350L of sample were incubated with700L of0.375%thiobarbi-
turic acid(TBA)in0.25M HCl and1%Triton X-100at100°C for15 min.After reaching the room temperature,the absorbance of the solutions were measured at535nm using malondialdehyde(MDA) as standard(29).Controls for residual absorption of carotenoids at 535nm were made using0.25M HCl plus1%Triton X-100solution without TBA.
Iron determination.The iron incorporation in the liposomes was checked before and after the dialysis procedure by a modification of the method described by Bralet et al.(30).Aliquotes of300L were taken from the liposome suspensions and3L of Triton X-100was added to disrupt the vesicles.The samples were added to50mM glycine hydrochloride buffer(pH2.5)with20mg/mL ascorbate,10 mg/mL pepsin and5mM2,2Ј-bipyridine.After incubation for2h at 37°C,the absorbance was measured at520nm and re
sults compared to FeSO4.7H2O standard
curve. FIG.1.Chemical structures of peridinin and astaxanthin.
Statistics.Data are presented as means ϮSD (standard devia-tion)and statistical analysis performed with the Student’s t test at significance level of 5%.
RESULTS AND DISCUSSION Nonloaded Liposomes (PCL)
The TBARS concentration after PCL preparations were (0.169Ϯ0.043nmol MDA/mol PC)and (0.209Ϯ0.031nmol MDA/mol PC),respectively for PCL and Iron-PCL.As expected,the encapsulation of Fe 2ϩ:EDTA complexes in PCL resulted in higher lipid oxi-dation level (c.a.25%).The coordination of Fe 2ϩwith EDTA does not prevent it to react with ROS and,hypothetically,it would be easier to eliminate (by di-alysis)a water-soluble Fe 2ϩ:EDTA complex than a membrane-associated Fe 2ϩ:phosphatidylcholine che-late (31,32).
Probably,the osmotic pressure must have led to re-organization of PCL membranes and coalescence of lipid vesicles during the dialysis performed against distilled water (33).Even with distinguished polarity properties,lycopene and astaxanthin induced Fe 2ϩ:EDTA elimination from liposomes at the same extent (c.a.30%).When peridinin was associated,the effect was less intense (23%).On the other hand,a higher loss of iron chelate was measured in carotenoid-free liposomes (53%)(Fig.3).
Ascorbic acid can behave as a prooxidant since it can reduce Fe 3ϩto Fe 2ϩ,a well-known strong promoter of lipoperoxidation (33).However,at millimolar concen-trations the ability of ascorbate to scavenge HO •be-comes more significant.Ascorbate is also able to reduce tocopheryl radicals,generated by hydrogen abstrac-tion from ␣-tocopherol,back to its active antioxidant form.Trolox,with similar scavenging mechanism as ␣-tocopherol,is supposed to be constantly regenerated
by ascorbate from the Trolox radical form in the aque-ous solution (28).Ascorbate is also supposed to be charged at pH 7.4(ascorbic acid pKa 1and pKa 2,4.17and 11.57,respectively)thus with low permeability throughout membranes.
Butylated hydroxytoluene (BHT)was very efficient in scavenging free radicals generated by all lipoperoxi-dation agents in PCL even at micromolar range (Fig.4).This effect was probably due to its higher diffusibil-ity into membranes (34)which would allow this anti-oxidant to scavenge oxyradicals at several spots through out the lipid bilayer.Trolox,mostly present in the aqueous solution,required millimolar concentra-tions to inhibit lipid oxidation to the same extention
as
FIG. 2.Trolox scavenging activity against ROS produced by H 2O 2,t -ButOOH and ascorbate in PCL and
Iron-PCL.
FIG.3.Iron concentrations in Iron-PCL in the presence or ab-sence of lycopene (Iron-PCL/LYC),astaxa
nthin (Iron-PCL/AST)or peridinin (Iron-PCL/PER),during a dialysis process (nmols Fe 2ϩ/mol PC).Shown are the means ϮSD of 3experiments;*P Ͻ
0.05.
FIG.4.Effects of BHT and Trolox in lipoperoxidation of PCL in the absence of carotenoids induced by mixing lipoperoxidation promoters—H 2O 2,t -ButOOH,or ascorbate—to chelated Fe 2ϩions (nmols MDA/mol PC).Shown are the means ϮSD of 4experiments;*P Ͻ0.05.
BHT in H 2O 2/Fe 2ϩsystem.Higher levels of TBARS were produced by addition of Fe 2ϩand t -ButOOH to PCL:1.9-fold higher compared to 94%obtained with H 2O 2.When lipoperoxidation process is initiated by t -ButOOH most of the free radicals detected in the lipid bilayer is peroxyl radical (ROO •)(35).A significant proportion of alkoxyl radical (RO •)and singlet oxygen [O 2(1⌬g )]have also to be considered (23,26,35–38).Trolox was not able to efficiently protect the PCL mem-branes in t -ButOOH-induced peroxidation.Usually,Trolox is more reactive with ROS than BHT,especially concerning peroxyl radicals (39),but the higher perme-ability of the phenolic compound may have compen-sated for its lower reactivity.
To evaluate the single effect of iron addition to PCL (with or without carotenoids),the TBARS measure-ments were also performed in the absence of peroxida-tion agents.As an extra control,PCL was also pre-pared containing 25M ␣-tocopherol as described by Palozza &Krinsky (40).As could be observed in Fig.5,astaxanthin and peridinin were able to inhibit lipoper-oxidation before the addition of iron complexes.The addition Fe 2ϩ:EDTA to PCL,in the absence of carote-noids,did not change TBARS pr
oduction although an increase of 25%in MDA content was previously ob-served after Iron-PCL preparation.The lipid oxidation in astaxanthin-(PCL/AST)and peridinin-incorporated liposomes (PCL/PER)were both,approximately,25%lower than in PCL although only PCL/AST was insen-sitive to iron addition.Lycopene was the only carot-enoid which reduced (25%)the level of lipoperoxidation after Fe 2ϩ:EDTA addition (Fig.5).
The carotenoid-incorporated liposomes were chal-lenged by ROS produced outside,when initiator and iron complexes were added simultaneously.These re-sults are presented in Fig.6.The simultaneous addi-tion of ferrous salt and ascorbate resulted in a more moderate increase of TBARS concentration (21%)than
those observed for H 2O 2and t -ButOOH systems sug-gesting the previously described dual effect of ascor-bate concerning its action against free radicals.It is noteworthy that,usually,iron ions are contaminating ascorbic acid by 0.02%which would allow the initia-tion of lipid oxidation even without adding Fe 2ϩsolu-tion (31).
Astaxanthin proved to be the best antioxidant in all experiments performed with both peroxidation initia-tor and iron chelate placed outside the PCL,as ex-pected from other authors (10,17).The ketocar
otenoid was the only tested compound to avoid extreme high levels of lipid damage caused by concomitant addition of ferrous ions and H 2O 2,t -ButOOH or ascorbate:re-spectively,45,45,and 33%lower lipid oxidation than PCL added with iron (II).As observed with the exper-iments without peroxidation agents (Fig.5),astaxan-thin also induced the lowest enhancement of MDA production upon iron ions addition.
No antioxidant activity was found for peridinin when incorporated into PCL and challenged by free radicals produced outside.Actually,peridinin led to intense augmentation of oxidated lipid levels after ferrous ions were added to H 2O 2-and t -ButOOH-treated liposomes:2.9-fold and 3.5-fold higher,respectively.The TBARS level obtained after incubation of PCL/PER with ascor-bate 1mM in the absence of Fe 2ϩ(0.65Ϯ0.14nmol MDA/mol PC)was one of the highest of all experi-ments performed.
Lycopene,under the reaction conditions described here,could not inhibit the lipoperoxidation process in PCL.The effect of chelated iron (II)inclusion to ascorbate-treated PCL/LYC was lower than with other lipid peroxidation agents despite being the highest value measured (0.73Ϯ0.03nmol MDA/mol PC).Apolar ,-carotene and lycopene,
have
FIG.6.TBARS levels induced by H 2O 2,t -ButOOH or ascorbate and Fe 2ϩ:EDTA in carotenoid-associated PCL.Shown are the means ϮSD of 4experiments;*P Ͻ
0.05.
FIG.5.Levels of TBARS promoted by addition of Fe 2ϩ:EDTA in PCL containing ␣-tocopherol (PCL/TOC),lycopene (PCL/LYC),peri-dinin (PCL/PER)and astaxanthin (PCL/AST).Shown are the means ϮSD of 4experiments;*P Ͻ0.05.
been reported to perturb the acyl chain packing and to increase bilayer permeability (41,42).In some circum-stances,efficient in vivo antioxidants like -carotene and lycopene could also act,or partially offer,a prooxi-dative effect in lipid peroxidation process masking its antioxidant activity.
Iron-Loaded Liposomes (Iron-PCL)
After the dialysis,an insignificant concentration of Fe 2ϩ:EDTA was present outside the PCL.As it is shown in Fig.7,no significant variation was observed in H 2O 2-generating system when 25M,50M,0.25mM,or 0.5mM Trolox were added.This aspect sug-gests that these oxyradicals were generated in the internal aqueous solution,triggered by the permeation of the easily diffusible molecule,H 2O 2.When both iron (II)and H 2O 2were added to the external aqueous so-lution,0.5mM Trolox and 5M BHT inhibited lipoper-oxidation by 40and 55%,respectively (Fig.4).
A constant (13%),but not significant,inhibition of MDA production in t -ButOOH-treated Iron-PCL was caused by Trolox in the concentration range from 25M to 0.25mM (Fig.7).However,0.5mM Trolox significantly suppressed lipid peroxidation:23.6%.Even also being a small and uncharged molecule,t -ButOOH was expected to permeate membranes in a less extention than H 2O 2.Paradoxically,higher lipid oxidation products were measured after incubation of Iron-PCL with t -ButOOH than with H 2O 2.BHT was not able to prevent lipid oxidation in this system al-though,when peroxyl and alkoxyl were generated out-
side the liposomes (Fig.4)a 55%lowed MDA content was obtained.
Ascorbate addition to Iron-PCL also resulted in a higher lipid oxidation despite being negatively charged at pH 7.4and not assumed to penetrate intensely the lipid bilayers.A possible explanation is the 0.02%usual iron contamination of commercial ascorbic acid (31).The effect of Trolox on lipoperoxidation is another indication that the oxidation process was initiated at the outer moiety.In fact,the addition of increasing concentrations of Trolox led to gradual higher protec-tion of the membranes against oxidative damage.An-other indication of external action of free radicals is the 53%inhibition of lipoperoxidation in Iron-PCL induced by 5M BHT.
As shown in Fig.8,lipoperoxidation in Iron-PCL was intensely stimulated by the addition of peroxidation promoters—H 2O 2,t -ButOOH and ascorbate,respec-tively—2.4-,4-,and 3.8-fold higher than MDA concen-trations obtained without promoters (dotted line;0.12Ϯ0.02nmol MDA/mol PC).The MDA concentra-tions found when H 2O 2and t -ButOOH were added to Iron-PCL were significantly lower than those mea-sured when iron ions and promoter were added simul-taneously to the vesicles (Fig.4).When ascorbate/Fe 2ϩ:EDTA was used as lipoperoxidation initiator system,an equivalent MDA concentration was obtained for both types of vesicles:(0.45Ϯ0.06)and (0.45Ϯ0.07)nmol MDA/mol PC for,respectively,Iron-PCL and PCL.Astaxanthin was the more efficient antioxidant since it suppressed the H 2O 2-induced lipoperoxidation in Iron-PCL by 26%(Fig.8).Peridinin showed a more modest inhibition of lipid oxidation process (17.7%),suggesting that,due to its incorporation into lipid bi-layer,it could have limited the permeation of the per-oxidation agent,H 2O 2in these experiments.
On
FIG.8.TBARS levels induced by addition of H 2O 2,t -ButOOH or ascorbate to Iron-PCL in the presence of lycopene (Iron-PCL/LYC),peridinin (Iron-PCL/PER)or astaxanthin (Iron-PCL/AST).Shown are the means ϮSD of 4experiments;*P Ͻ
0.05.
FIG.7.Effects of 5M BHT and 25M,50M,0.25mM and 0.5mM Trolox in lipoperoxidation of Iron-PCL (in the absence of caro-tenoids)induced by adding 1mM ROS promoters—H 2O 2,t -ButOOH,and ascorbate (nmols MDA/mol PC).Shown are the means ϮSD of 4experiments;*P Ͻ0.05.
the other hand,lycopene showed evidences that it
has enhanced membrane permeability to H
2O
2
and
t-ButOOH,since increases of c.a.21%in MDA concen-
trations(not significant)were observed in both sys-tems.When1mM ascorbate was added to Iron-PCL, peridinin significantly limited lipoperoxidation which was comparable to the values obtained with astaxa
n-thin:respectively,33.5%and46.4%.Lycopene was only able to protect liposome membranes when ascor-bate was used as a promoter of lipid oxidation(17.4% lower than control).
CONCLUSIONS
Carotenoids,especially astaxanthin and zeaxanthin, show high rate constants for reactions with peroxyl radicals(ROO•)and as a[O
2
(1⌬g)]quencher(24,37). Shimidzu et al.(43)developed in vitro assays to study the quenching efficiency of several carotenoids from
marine organisms against[O
2
(1⌬g)]and has evidenced astaxanthin as one of the most efficient.Recently, Pinto et al.(5)have demonstrated that peridinin,de-spite being less efficient than-carotene,is the major
[O
2
(1⌬g)]quencher in Linulodinium polyedra,mainly due its elevated concentration in this organism.The antioxidant effect of carotenoids is probably also de-rived from its rigidifying effect on membranes which could lead to a limitation in metal ions or oxidative compound penetration into lipid bilayers(44,45).On the other hand,hydrophobic ,lycopene and-carotene)make the membranes morefluid and, under some circumstances,morefluid and,under some circumstances,more susceptible to oxidative damage (41).The data reported here suggest that lycopene, under the described reaction conditions,was not able to protect membrane lipids against iron-induced oxida-tion process.This fact has also been recently pointed out as a possible explanation for the ambiguous action of-carotene challenged by oxyradicals in different lipid systems(42).
Astaxanthin,as previously demonstrated in vitro and in vivo(10,17,46–50),was able to strongly inhibit the propagation step of lipoperoxidation in all tested systems.It is possible that two combined properties of astaxanthin were responsible for this fact:(i)the rigid-ifying effect on membrane,which could have limited
the penetration of lipoperoxidation promoters—H
2O
reactive oxygen species (ros)2
,
t-ButOOH and ascorbate—into the liposome mem-branes(21,51,52);and(ii)the inherent antioxidant activity of this ketocarotenoid(53).However,the same explanation is not valid for antioxidant action of peri-dinin in Iron-PCL assays.Peridinin did not show any antioxidant property when the ROS were produced outside the liposomes.However,when the peridinin-associated vesicles were pre-loaded with Fe2ϩ:EDTA complexes,a significant inhibition of lipoperoxidation was observed(in all tested systems).
Despite that no report about peridinin orientation on lipid bilayers was available in the literature,it is tempting to suggest,by a preliminary analysis of its chemical structure,that this carotenoid might have a vertical or more angular orientation in egg-yolk leci-thin liposomes.Thus,it is possible,despite being spec-ulative,that peridinin also shows polar-carotenoid ri-gidifying effect on membranes and,consequently,its detected inhibitory action on lipid oxidation might be related to a peridinin-induced decrease in the perme-ation of lipoperoxidation promoters in membranes. ACKNOWLEDGMENTS
This work was supported by the Swedish Foundation for Interna-tional Cooperation in Research and Higher Education(STINT Pro-gram),Sweden,and Fundac¸a˜o de Amparo a`Pesquisa do Estado de Sa˜o Paulo(FAPESP),Brazil.We are grateful to Universidade Cruzeiro do Sul(Brazil)and University of Uppsala(Sweden)for allowing,respectively,Marcelo Barros,Ph.D.,and Ernani Pinto to stay at Stockholm University to perform the experiments and pre-pare this article.
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