Chemistry and Physics of Lipids

114 (2002) 81–98

Copper-induced peroxidation of liposomalpalmitoyllinoleoylphosphatidylcholine (PLPC), effect ofantioxidants and its dependence on the oxidative stress

Orit Bittner a,1, Sigal Gal a,1, Ilya Pinchuk a, Dganit Danino b,Hadassah Shinar c, Dov Lichtenberg a,*

a Department of Physiology and Pharmacology, Sackler Faculty of Medicine, Sackler Medical School, Tel-A�i� Uni�ersity,Tel-A�i� 69978, Israel

b Department of Chemical Engineering, Technion, Israel Institute of Technology, Haifa 32000, Israelc School of Chemistry, Tel-A�i� Uni�ersity, Tel-A�i� 69987, Israel

Received 12 March 2001; received in revised form 21 November 2001; accepted 13 December 2001

Abstract

In an attempt to deepen our understanding of the mechanisms responsible for lipoprotein peroxidation, we havestudied the kinetics of copper-induced peroxidation of the polyunsaturated fatty acid residues in model membranes(small, unilamellar liposomes) composed of palmitoyllinoleoylphosphatidylcholine (PLPC). Liposomes were preparedby sonication and exposed to CuCl2 in the absence or presence of naturally occurring reductants (ascorbic acid (AA)and/or �-tocopherol (Toc)) and/or a Cu(I) chelator (bathocuproinedisulfonic acid (BC) or neocuproine (NC)). Theresultant oxidation process was monitored by recording the time-dependence of the absorbance at several wave-lengths. The observed results reveal that copper-induced peroxidation of PLPC is very slow even at relatively highcopper concentrations, but occurs rapidly in the presence of ascorbate, even at sub-micromolar copper concentra-tions. When added from an ethanolic solution, tocopherol had similar pro-oxidative effects, whereas when introducedinto the liposomes by co-sonication tocopherol exhibited a marked antioxidative effect. Under the latter conditions,ascorbate inhibited peroxidation of the tocopherol-containing bilayers possibly by regeneration of tocopherol.Similarly, both ascorbate and tocopherol exhibit antioxidative potency when the PLPC liposomes are exposed to thehigh oxidative stress imposed by chelated copper, which is more redox-active than free copper. The biologicalsignificance of these results has yet to be evaluated. © 2002 Elsevier Science Ireland Ltd. All rights reserved.

Keywords: PUFA; Peroxidation; Copper; Liposomes; Antioxidants; Cu(I) chelators

www.elsevier.com/locate/chemphyslip

Abbre�iations: AA, L-ascorbic acid, vitamin C; AAPH, 2,2�-azobis(2-amidinopropane) hydrochloride; BC, bathocuproinedisulfonicacid; L�, lipidic radicals; LDL, low-density lipoprotein; LOO�, lipid peroxyl radicals; LOOH, hydroperoxides; NC, neocuproine; OD,optical density; PLPC, palmitoyllinoleoylphosphatidylcholine; PUFA, LH, polyunsaturated fatty acids; QLS, quasielastic lightscattering; Toc, �-tocopherol, vitamin E.

* Corresponding author. Tel.: +972-3-640-7305; fax: +972-3-640-9113.E-mail address: physidov@post.tau.ac.il (D. Lichtenberg).1 Parts of this study constitute a part of the M.Sc. thesis of Orit Bittner, other parts are from the Ph.D. thesis of Sigal Gal. These

two authors contributed equally to the present work.

0009-3084/02/$ - see front matter © 2002 Elsevier Science Ireland Ltd. All rights reserved.

PII: S 0009 -3084 (01 )00208 -0

O. Bittner et al. / Chemistry and Physics of Lipids 114 (2002) 81–9882

1. Introduction

Oxidative modification of low-density lipo-protein (LDL) is believed to play a key role inatherogenesis (Esterbauer and Ramos, 1995), be-cause it interferes with the normal internalizationof LDL into cells through the down-regulatedmechanism mediated by the LDL receptor (Stein-brecher, 1999). In addition, peroxidation results inthe formation of several chemoattractants (Mc-Murray et al., 1993) and toxic products (Ester-bauer 1993; Chisolm, 1991) that are involved inatherogenesis. Much effort has, therefore, beendevoted to gain understanding of the variousmechanisms by which LDL lipids may becomeoxidized.

Several in-vitro peroxidation processes are com-monly used to model the very complex process ofLDL peroxidation in vivo. These include studieson the oxidation of fractionated LDL induced byorganic free radical generators (Noguchi et al.1998; Frei and Gaziano, 1993; Esterbauer andJurgens, 1993), hypochlorite (Panasenko et al.1997; Hazell et al. 1999), free radical enzymaticreactions (Esterbauer and Ramos, 1995; Hei-necke, 1997) and transition metals, mostly copperions (Esterbauer and Ramos, 1995; Frei andGaziano, 1993; Esterbauer and Jurgens, 1993).

The latter, most commonly used process is un-doubtedly an oversimplified model (Babiy andGebicki, 1999). Yet, even this process is a verycomplex function of many interrelated factors,including the size of the LDL particles, theirsurface charge, their composition with respect toproteins, cholesterol, cholesteryl-esters, phospho-lipids and lipid-soluble antioxidants, as well as thechain length and degree of saturation of both thefree and esterified polyunsaturated fatty acids(PUFA). Furthermore, LDL, fractionated by dif-ferent procedures, may have varying composition,which is also likely to alter their susceptibility tooxidation.

In view of this complexity, several authors haveattempted to gain understanding of the mecha-nisms responsible for oxidation in simple modelsystems, made by dispersing oxidizable lipids inthe form of liposomes, emulsions, microemulsionsor lipid-surfactant mixed micellar systems. In such

systems it is possible to monitor peroxidationunder varying conditions while changing the fac-tors that govern the reaction in a controllablefashion, one at a time. These studies yielded inter-esting results concerning the influence of variousfactors on the kinetics of copper-induced peroxi-dation. These include the surface charge of mi-celles (Yoshida and Niki, 1992), the fatty acidcomposition of the phospholipid chains (Ya-mamoto et al., 1984; Vossen et al., 1993), and thepresence and concentration of lipidic hydroperox-ides (LOOH) (Patel et al., 1997) and of variousantioxidants (Haase and Dunkley, 1969; Niki etal., 1985b; Barclay, 1993; Yoshida et al., 1994;Zhang et al., 1994; Maiorino et al., 1995;Kritharides, 1999).

The most intriguing results obtained in thesestudies relate to the effects of L-ascorbic acid,vitamin C (AA) and �-tocopherol, vitamin E(Toc) on PUFA peroxidation in different modelsystems. Both these naturally abundant vitaminsare potent antioxidants under conditions of highoxidative stress. Under such conditions, everymolecule of each of these antioxidants is capableof quenching up to two free radicals (Frankel,1998) and by that they can inhibit peroxidation(Niki et al., 1984, 1985b; Barclay, 1993). By con-trast, at low oxidative stress, Toc can promotecopper-induced peroxidation, probably by form-ing free radicals upon reducing Cu(II) to Cu(I).This was shown for lipoproteins (Kontush et al.,1996; Neuzil et al., 1997), and for micelles(Yoshida et al., 1994; Maiorino et al., 1995). Thepro-oxidative effect of AA on copper-induced per-oxidation was demonstrated in liposomes (Zhanget al., 1994).

In view of this pro-oxidative effect, as observedat micromolar copper concentrations (Yoshida etal., 1994; Zhang et al., 1994; Maiorino et al.,1995), we found it of interest to test whetherin the presence of AA and Toc, aggregated (lipo-somal) phospholipids can be oxidized bysub-micromolar copper concentrations. Such be-havior was expected in light of the relativelyrapid peroxidation of potassium linoleate in solu-tions containing AA, first observed by Haase andDunkley (1969), and recently interpreted in termsof AA-catalyzed, transition metal-induced peroxi-dation (Kritharides, 1999).

O. Bittner et al. / Chemistry and Physics of Lipids 114 (2002) 81–98 83

By contrast, at sufficiently high oxidative stressboth AA and Toc are expected to inhibit copper-induced peroxidation. Phospholipid liposomescan, in fact, be subjected to higher copper-inducedoxidative stress by introducing copper-chelatorsthat form copper-chelates of higher oxidative po-tency than free copper. Such copper-chelates areexpected to accelerate the peroxidation process, asearlier reported for LDL and linoleic acid peroxi-dation in the presence of bathocuproinedisulfonicacid (BC) and neocuproine (NC) (Perugini et al.,1997; Ueda et al., 1999, Pinchuk et al., 2001). Inthe present study we found that in the presence ofthese chelates, trace amounts of transition metalswere sufficient to induce rapid oxidation of theliposomal palmitoyllinoleoylphosphatidylcholine(PLPC). Similarly, rapid peroxidation was in-duced by traces of transition metals when thesolution contained AA. By contrast, under theconditions of high oxidative stress, imposed bymoderate concentrations of both BC and copper,both ascorbate and tocopherol can act asantioxidants.

2. Materials and methods

2.1. Chemicals

PLPC, 1-Palmitoyl-2-Linoleoyl-sn-Glycero-3-Phosphocholine was purchased from Avanti Po-lar-Lipids, Inc. (Alabaster, AL).

AAPH, 2,2�-azobis(2-amidinopropane) hydro-chloride was obtained from Poly Sciences (War-rington, PA).

Neocuproine, 2,9-Dimethyl-1,10-phenanthrolinehydrochloride (NC), Bathocuproinedisulfonic aciddisodium salt, 2,9-dimethyl-4,7-diphenyl-1,10-phenantrolinedisulfonic acid disodium salt (BC),L-absorbic acid(vitamin C), (� )-�-Tocopherol,(vitamin E), and polyoxyethylene(23)laurylether(Brij 35) were all purchased from Sigma (St.Louis, MO).

CuCl2, EDTA, NaCl, NaH2PO4, and Na2HPO4

were purchased from Merck (Darmstadt,Germany).

2.2. Preparation of PLPC liposomes for kineticstudies

A saline solution (146 mM NaCl) was added atroom temperature either to dry PLPC or to PLPClyophilized from chloroform. The dispersed PLPC(30 mM) was mixed to homogeneity using a vor-tex-mixer. Liposomes were prepared by sonication(10–13 min) under nitrogen and ice cooling(Huang, 1969), using a Heat Systems Inc. XL-2020 probe sonicator.

In several experiments, 0.12 mM EDTA wasadded to the saline solution before sonication tominimize peroxidation by residual transitionmetals during sonication; the figure legends indi-cate the addition. This affected only slightly thekinetics of oxidation in the diluted solutions, atany given final concentration of non-chelatedcopper.

After sonication, the liposomes underwent fur-ther dilution to the specified concentrations inPBS (pH 7.4, 146 mM NaCl, 3.3 mM sodiumphosphate).

AA, BC, NC and CuCl2 were added fromaqueous solutions to the diluted liposomal disper-sions just prior to the onset of the experiment.The reaction solutions were mixed with a pasteurpipette and the monitoring of absorbance wascommenced.

Toc was either added externally to the liposo-mal dispersions just before the addition of thewater-soluble additives in a constant volume (20�l) of ethanolic solutions to yield the indicatedconcentrations (control experiments were con-ducted in solutions containing 20 �l ethanol), orby co-lyophylization of a chloroformic solution ofPLPC and an ethanolic solution of the appropri-ate concentrations of Toc. This organic solutionwas mixed, evaporated under nitrogen and thenunderwent further overnight lyophilization. Theresulting film was dispersed in saline using a vor-tex mixer, and sonicated as described above.

Solutions of CuCl2, AA and Toc were freshlyprepared before each experiment. BC and NCsolutions were refrigerated and used within aweek of their preparation. Liposomes were storedunder nitrogen in a refrigerator until being used,typically within 2 weeks of their preparation.

O. Bittner et al. / Chemistry and Physics of Lipids 114 (2002) 81–9884

2.3. Characterization of liposomal dispersions

Prior to the addition of CuCl2 for peroxidationstudies, the reaction mixtures were characterizedwith respect to the physical properties of theliposomes, the concentration of residual copperpresent in the buffer solution, and in mixturescontaining Toc, the liposomes/water partitioningof Toc was also evaluated, as described below.Water-soluble additives were assumed to essen-tially reside in the aqueous solution.

2.3.1. Characterization of the physical propertiesof the liposomes

The mean size of the resultant liposomes wasevaluated by quasielastic light scattering measure-ments (QLS), either at 90°, using Malvern’s pho-ton correlation spectrometer Model 4700,equipped with an argon laser with a wavelengthof 488 nm, or at 173°, using ALV’s high perfor-mance particle sizer, model ALV-NIBS/HPPSequipped with a HeNe-laser at 632.8 nm. Themean diameter, as measured for different lipo-some preparations, varied within the range of30–50 nm. The addition of co-sonicated Toc tothe liposomes had little influence, if any, on themean diameter readings. In several cases up toabout 15% of the PLPC gave rise to larger vesi-cles, of diameters of about 110 nm, in agreementwith the observation of multilamellar vesicles bycryo-TEM of a vitrified specimen of the lipo-somes, using a Philips CM120 microscope, oper-ated at 120 kV (not shown).

This observation is consistent with the out/inratio of about 1.5 found for the choline peak (notshown) using nuclear magnetic resonance spec-troscopy in the presence of the shift reagent PrCl3(Eigenberg and Chan, 1980; Shaw and Thomp-son, 1982; Lichtenberg and Barenholz, 1988).NMR measurements were conducted on a BrukerARX-500 MHz NMR spectrometer at room tem-perature. Liposomes (30 mM) for these measure-ments were prepared in PBS made in D2O andfurther diluted in saline made in D2O to a finalconcentration of 2 mM. The final PrCl3 concen-tration was 4 mM.

Prior to dilution in PBS, the phospholipid con-centration in the liposome dispersions was deter-

mined by Bartlett’s inorganic phosphate assay(Bartlett, 1959).

Dilution of the liposomes to the final concen-trations used in the experiment had little effect, ifany, on the average size of the particles, as deter-mined by further QLS analysis.

The assessment of the concentration of pre-formed LOOH on the basis of their absorbance islimited because the liposomes scatter light. Thetypical initial optical density (OD) at 234 nm for0.25 mM PLPC was between 0.2 and 0.5 ODunits. Under the false assumption that this ODrepresents only absorbance of light by LOOH, itfollows that their maximal concentration is 8–20�M (�234=24 400 M−1 cm−1). This over-esti-mated range of values can be corrected for thecontribution of light scattering on the basis of theOD measured at 325 nm, where the scattering oflight is the sole contributor to the OD. AssumingRayleigh scattering, the contribution of light scat-tering to the OD observed at 234 nm is given by:(OD325nm)× (325/234)4= (OD325)×3.72 (Moore,1962). Therefore, the corrected values of ab-sorbance at 234 nm are given by:

A234 nm= (OD234 nm)corrected

= (OD234 nm)measured− (OD325)measured

×3.72 (1)

The computed values of absorbance were onlyup to 0.05 OD units, indicating that the ab-sorbance by LOOH made only a small contribu-tion to the OD observed at 234 nm. Thiscorresponds to 2 �M LOOH, which means thatless than 1% of the linoleate was pre-oxidized.

2.3.2. Determination of the residual copperconcentration

Under certain conditions peroxidation occurredwithout copper addition (see results), hence wehad to evaluate the concentration of residual cop-per in the PBS, which was below the limit ofspectroscopic detection. Therefore, we first con-centrated the buffer solution by a factor of tenand subsequently mixed it with a solution con-taining 50 �M BC (CRC, 1982), and 50 �M AA.This AA concentration is sufficient to reduce allthe copper in the system to Cu(I). The absorbance

O. Bittner et al. / Chemistry and Physics of Lipids 114 (2002) 81–98 85

of the (BC)2–Cu(I) complex in the concentratedbuffer solution was monitored at 483 nm andcompared with a calibration curve prepared fromknown Cu(II) concentration in the range of 0–10�M in the presence of 50 �M AA and 50 �M BC.

In several experiments we have tested howmuch EDTA is required to prevent peroxidationinduced by residual transition metals. The resultsof these experiments (not shown) correspondedwith the evaluation obtained as described above.

2.3.3. Partitioning of tocopherol between bilayersand aqueous solutions

Partitioning of tocopherol between the lipidicand aqueous phases was evaluated on the basis ofits fluorescence intensity at 334 nm following exci-tation at 298 nm. The fluorescence intensity wasmeasured using the ISS K2 multifrequency crosscorrelation phase and modulation fluorometer(ISS-Champaign Illinois), with excitation slits of0.1 nm and emission slits set to 0.2 nm. Allmeasurements were conducted in quartz cuvetteswith a final volume of 1.5 ml.

It is assumed that the non-soluble Toc does notcontribute to the overall apparent intensity due toquenching. On the basis of this assumption themeasured apparent fluorescence intensity (Fapp) isthe sum of any background fluorescence or lightscattering (Fcontrol), the fluorescence intensity ofaqueous Toc (Faq.), and that of Toc that resides inthe lipidic environment (Flip.), where its quantumyield is higher (Ramos-Lledo et al., 2001). Toestimate the partitioning of Toc between lipo-somes and the aqueous solution, we assumed thatthe quantum yield of the fluorescence of liposo-mal Toc is about equal to that of micellar Toc.

To evaluate the specific fluorescence intensity ofaqueous and lipid-associated Toc, we have con-ducted two control experiments:1. Fluorescence intensity measurements of

aqueous solutions of Toc (0.25–10 �M) inmedia containing 20 �l ethanol in 1.5 ml PBS.

2. Fluorescence intensity measurements of thesame concentrations of Toc and ethanol inmedia containing 10 mM Brij 35, in whichessentially all the Toc resides in the hydropho-bic core of the Brij micelles (Ramos-Lledo etal., 2001).

Both these control experiments yielded lineardependencies of the fluorescence intensity on theToc concentrations (r2=0.987 and 0.971, respec-tively). The specific fluorescence intensity for themicellar solutions was about seven fold higherthan for the aqueous Toc (110 000 arbitrary unitsper �M, as compared with about 15 000 arbitraryunits per �M).

Accordingly, we treated our results as follows:1. Background values of fluorescence, pre-

sumably due mostly to light scattered by theliposomes or micelles, (Fcontrol), have been sub-tracted from the apparent fluorescence reading(Fapp).

2. The resultant value (Fnet) is given by:

Fnet=Fapp−Fcontrol=Faq+Flip (2)

3. The fluorescence intensity of Toc in the PLPCliposomal dispersion (0.25 mM) was measuredin the absence and presence of Brij 35 (10mM). Under the above assumptions, the ratio(R) between Fnet observed in the presence ofBrij (Fnet)micellar, and that observed in its ab-sence, (Fnet)liposomes is given by Eq. (3):

R=(Fnet)micellar

(Fnet)liposomes

=(Flip)

Faq+ (Flip)liposomes

=7

1+6flip

(3)

where flip denotes the fraction of Toc presentin the lipidic phase.

This procedure enables evaluation of flip suchthat the ratios of fluorescent intensities are usedinstead of the absolute fluorescence intensity inarbitrary units.

In our experiments the partitioning of Toc be-tween the vesicles and the aqueous media exhib-ited a strong dependence on how the tocopherolwas introduced to the liposomal dispersions. Spe-cifically, when Toc was added from ethanol topre-formed liposomes Fnet was only slightly higherthan Faq and solubilization of the PLPC (0.25mM) by Brij 35 (10 mM) resulted in about a sevenfold increase of Fnet, indicating that prior to solu-bilization the Toc resided essentially in the waterphase and not in the liposomes.

O. Bittner et al. / Chemistry and Physics of Lipids 114 (2002) 81–9886

For the co-sonicated liposomes (5 �M Toc) weassume that the light scattered from these lipo-somes is equal to that of pure PLPC liposomes.Based upon this assumption, Fnet increased by afactor of only two upon solubilization. Accordingto Eq. (3), this means that the fraction of Toc inthe liposomes ( flip) is approximately 50%.

2.4. Peroxidation of PLPC liposomes

PLPC peroxidation was monitored at 37 °C bycontinuous recording of the absorbance at threewavelengths (234, 268, and 325 nm) using a Kon-tron (Uvikon 933) double-beam spectrophotometerequipped with a 12 position automated samplechanger. Measurements were carried out in quartzcuvettes containing a final volume of 1.5 ml. PLPCwas diluted in PBS to a final concentration of either1 or 0.25 mM.

Typically, 99 time points were recorded, withintervals of 10–15 min between measurements.Each reported kinetic profile is typical of two to

four identical experiments. Given the critical de-pendence of oxidation on the concentration ofpre-formed LOOH in the liposomes (and, there-fore, on the individual preparation and its fresh-ness), each kinetic profile was compared withcontrol experiments conducted with identical lipo-somal dispersions.

Several experiments were also conducted withsamples incubated at 37 °C out of the UV beamand the absorbance was measured prior to andfollowing the continuous recording in the UVspectrometer of identical samples that were contin-uously monitored. The close similarity of ab-sorbance indicates that the UV irradiation in thecourse of continuous monitoring had only slighteffects, if any, on the kinetics of peroxidation.

2.5. E�aluation of kinetic parameters from kineticprofiles

The major contribution to the time-dependentincrease of absorbance at 234 nm is that of theconjugated dienic lipid LOOH, whereas the majorcontributors to the absorbance at 268 nm are thedienals formed during the reaction (Esterbauer andRamos, 1995; Pinchuk and Lichtenberg, 1996). Theabsorbance at 325 nm was recorded in order tomonitor changes in turbidity.

Initial OD values were recorded immediatelybefore and after the addition of the peroxidation-inducing agent(s). The presented time dependenciesof absorbance were corrected by subtracting theOD value recorded after the addition, from thelater time points. Several kinetic profiles measuredat 234 nm were also corrected for the contributionof turbidity changes. This correction was per-formed using Eq. (1), the figure legends indicate thecorrection.

Fig. 1 depicts a typical time-course of ab-sorbance. Each time-dependence was characterizedby three factors, namely the maximal absorbance(ODmax), the maximal rate of increase of ab-sorbance (Vmax) and the time at which the rate wasmaximal (tmax). The latter factor has been previ-ously shown to correlate with the more commonlyused ‘lag time’ (Ramos et al., 1995).

Data analysis was performed by the standardprocedures provided by MICROSOFT EXCEL 2000

and MICROCAL ORIGIN 5.0 software.

Fig. 1. Characterization of the kinetics of peroxidation. Thekinetic parameters of copper-induced peroxidation of PLPC,as monitored by continuous recording of UV absorption ofPLPC oxidation products at 234 nm, are defined in this figure.The upper panel demonstrates a typical time course of ab-sorbance (i.e. of product accumulation). It was observed dur-ing peroxidation of PLPC (1 mM) by CuCl2 (20 �M). Thelower panel depicts the first derivative of this time course andis, therefore, the time-dependence of the rate of accumulationof the absorbing products.

O. Bittner et al. / Chemistry and Physics of Lipids 114 (2002) 81–98 87

Fig. 2. Copper-induced peroxidation of PLPC. The specified CuCl2 concentrations were added to PLPC (1 mM), the peroxidationwas monitored at 37 °C by recording the time dependence of absorption at 234 nm.

3. Results

Liposomal PLPC undergoes slow oxidationupon exposure to CuCl2 (Fig. 2). Accumulation ofthe resultant conjugated dienic LOOH, asrecorded by continuous monitoring of the ab-sorbance at 234 nm, is characterized by a ‘lagphase’ followed by a ‘propagation phase’ of fasteraccumulation, as previously observed byMaiorino et al. (1995). Both the lag time and themaximal rate depend on the concentration ofcopper, as exemplified for 1 mM PLPC vesicles inFig. 2. Increasing the copper concentration withinthe range of 0–10 �M shortened the apparent lag(and tmax) and enhanced the maximal rate (Vmax).A further increase in the copper concentration (20�M) had little effect on the kinetic profile.

In several other series of experiments, we used alower PLPC concentration (0.25 mM) and foundthat the dependence of the lag on the copperconcentration varied such that it was a functionof the copper/lipid ratio rather than of the copperconcentration itself (not shown).

In each of the experiments described in Fig. 2,the kinetics of accumulation of reaction productsabsorbing at 268 nm, where final products ofLOOH decomposition make the major contribu-

tion, was quite similar to the kinetics obtained at234 nm except that the lag observed at 268 nm(not shown) was somewhat longer than that ob-served at 234 nm.

3.1. Effect of ascorbic acid and tocopherol

AA promoted the copper-induced peroxidationof PLPC. Moreover, in the presence of AA, oxi-dation occurred even at sub-micromolar concen-trations of copper. Examples are given in Fig. 3.In the experiments depicted in this figure, PLPCvesicles (0.25 mM) were exposed to 50 nM CuCl2in the absence and presence of AA (0.2–2.0 �M).As evident from these experiments, low concen-trations of AA enhanced the peroxidation quitedramatically, but at somewhat higher concentra-tions (above 0.5 �M) the tendency was less pro-nounced (Fig. 3).

When externally added in ethanol, Toc had thesame pro-oxidative effects as AA on the copper-induced peroxidation of PLPC liposomes. Exam-ples are given in Fig. 4 for peroxidation of PLPC(1 mM) at two copper concentrations (5 and 10�M). As seen in this figure, increasing the concen-tration of Toc accelerated peroxidation, the effectbeing more pronounced at 5 �M than at 10 �Mcopper (Fig. 4).

O. Bittner et al. / Chemistry and Physics of Lipids 114 (2002) 81–9888

By contrast, when Toc was added via co-soni-cation with the PLPC, it had pronounced antioxi-dative effects as shown in Fig. 5. This figuredepicts the kinetics of copper-induced peroxida-tion of PLPC liposomes (0.25 mM), and ofPLPC-Toc liposomes made by co-sonication ofPLPC with Toc (final concentrations of 0.25 mMPLPC, and 5 �M Toc). In the latter, Toc-contain-ing vesicles, ascorbate, at concentrations between0–50 �M, exhibited antioxidative effects (Fig. 6),in contrast to the pro-oxidative effects of AA onthe peroxidation of pure PLPC vesicles (Figs. 3and 6). In Fig. 6, CuCl2 (5 �M) was added to purePLPC vesicles or PLPC vesicles containing 5 �MToc in the absence or presence of AA (0–50 �M).

3.2. Effects of Cu(I) chelators

Both BC and NC are strong chelators of Cu(I)(log �2�19). Both these chelators also bindCu(II), although with much lower affinity(log �2�11) (CRC, 1982; Lappin et al., 1980;

Fig. 4. Effect of externally added tocopherol on the peroxida-tion of PLPC. Peroxidation of PLPC (1 mM) at 37 °C,induced by 5 �M CuCl2 (solid lines) or 10 �M CuCl2 (dashedlines) in the absence or presence of the specified concentrationsof Toc added as an ethanolic solution (see Section 2), asrecorded by continuous monitoring of the absorption at 234nm.

Fig. 3. Effect of AA on the kinetics of peroxidation of PLPC.At time zero, 50 nM CuCl2 and various concentrations of AA(0–2 �M, as indicated in the figure) were added to PLPC (0.25mM) at 37 °C and the absorbance was continuously moni-tored at 234 nm.

Sayre, 1996). Both accelerated the copper-inducedperoxidation of liposomal PLPC (0.25 mM) to theextent that in the presence of 0.5 �M chelator,relatively rapid peroxidation occurred even whenthe added CuCl2 concentration was merely 50 nM(Fig. 7). Notably, under all the studied conditions,BC accelerated peroxidation more than NC (Fig.7).

In many of the experiments conducted in thepresence of copper chelators (e.g. Fig. 7 at 0.5 �MBC), the kinetic profile exhibited an apparentbiphasic shape, i.e. following the maximal accu-mulation of absorbing products, the absorbancedecreased and subsequently increased, sometimesto levels higher than the first maximum. In severalexperiments, we have corrected the absorbanceobserved at 234 nm for the contribution of lightscattering on the basis of the time dependence ofOD at 325 nm (see Section 2 Eq. (1)). Thiscorrection revealed that the second ‘phase’ of

O. Bittner et al. / Chemistry and Physics of Lipids 114 (2002) 81–98 89

Fig. 5. Effect of tocopherol co-sonicated with PLPC on the peroxidation of liposomes. CuCl2 (1 or 5 �M) was added to PLPCliposomes (0.25 mM, solid lines) or to PLPC liposomes (0.25 mM) co-sonicated with Toc (5 �M, dashed lines). The absorbance wasmonitored at 234 nm at 37 °C. The sonication of both the PLPC and the PLPC–Toc mixture was performed in the presence ofEDTA (final concentration 1 �M).

Fig. 6. Combined effects of AA and tocopherol (co-sonicated with PLPC) on the peroxidation of liposomes. CuCl2 (5 �M) and AA,at the indicated concentrations (0–50 �M), were added to PLPC liposomes (0.25 mM, solid lines) or to liposomes made byco-sonication of PLPC (0.25 mM) and Toc (5 �M, dashed lines). Peroxidation was monitored by recording the absorbance at 234nm. Temperature was maintained at 37 °C. Sonication was performed in the presence of EDTA (final concentration 1 �M).

O. Bittner et al. / Chemistry and Physics of Lipids 114 (2002) 81–9890

Fig. 7. Kinetic profiles of the peroxidation of PLPC in thepresence and absence of copper chelators. CuCl2 (50 nM) wasadded at time zero to PLPC liposomes (0.25 mM) in theabsence or presence of NC or BC, at the indicated concentra-tions. The absorbance at 234 nm was continuously monitoredat 37 °C.

rapid increase of OD relates to increased scatter-ing, probably due to peroxidation-induced aggre-gation and/or fusion (Gast et al., 1982; Barclay etal., 1987). More detailed studies of these processesare currently underway.

At BC concentrations above 0.05 �M, oxida-tion occurred even when no CuCl2 was added tothe system, as demonstrated in Fig. 8 for mixturesof BC (0–5 �M) and liposomal PLPC (0.25 mM).As seen in this figure, increasing the BC concen-tration up to 0.5 �M BC shortened tmax andaccelerated Vmax. Increasing the BC concentrationto higher levels (5 �M BC) further acceleratedVmax but prolonged the lag preceding rapid perox-idation (Fig. 8).

Addition of EDTA (1 �M), with no addedcopper, completely blocked peroxidation at all BCconcentrations (not shown), indicating that theoxidation observed when no copper was added tothe system was induced by contamination of thesolution with transition metals and not by the BCitself. Similar rationale has been recently raised inKritharides’s review (Kritharides, 1999) of theresults of Haase and Dunkley (1969). Such con-tamination most likely consists of a mixture oftransition metals. The concentration of copperions in the buffer used in our study, determined as

Fig. 8. Peroxidation of PLPC in the presence of BC with no added copper. BC, at the indicated concentrations, was added to PLPC(0.25 mM) at time zero, and the absorbance at 234 nm was continuously monitored at 37 °C.

O. Bittner et al. / Chemistry and Physics of Lipids 114 (2002) 81–98 91

Fig. 9. Effects of antioxidants on the peroxidation of PLPC in the presence of BC with no added copper. BC (0.1 �M) was addedto PLPC vesicles (0.25 mM) with no added copper, in the absence or presence of different concentrations of externally added Tocor AA, as indicated on the graph. Absorbance at 234 nm was continuously recorded at 37 °C.

described in the Section 2 was below 100 nM, inagreement with the maximal copper concentrationof up to 300 nM quoted by the manufacturer asthe maximal contamination by this ion.

3.3. Effect of Toc and of ascorbic acid on PLPCoxidation induced by copper in the presence ofchelators

The combined effects of bathocuproine andeither Toc or AA are interrelated and complex, asdemonstrated by the following examples:1. In the absence of BC and Toc, AA, within the

studied range, always exhibited pro-oxidativeeffects independent of the copper concentra-tion, (e.g. Fig. 3). By contrast, in the presenceof BC, the effect of AA depended on theconcentrations of both copper and BC. As anexample, in the presence of 0.1 �M BC and noadded copper, AA exhibited dose-dependentpro-oxidative effects (Fig. 9). Notably, the ODapparently did not decrease after attaining itsmaximal level, indicating that the decomposi-tion of LOOH stopped. To explain this find-ing, we note that the actual concentration of

LOOH in the system is a reflection of thebalance between their production and decom-position. Copper catalyses both these reac-tions. However, our preliminary resultsindicate that decomposition requires highercopper concentrations than initiation (Gal etal., in preparation).

The dose-dependencies of the antioxidativeeffect of AA, as measured at low oxidativestress and at high oxidative stress, are de-scribed in Fig. 10. Interestingly, the antioxida-tive potency of AA increased with theoxidative stress, as evident from the findingthat the lag obtained at 6 �M CuCl2 and 10�M BC was longer than that obtained at 2 �MCuCl2 and 2 �M BC (Fig. 10). Note that thereaction mixture contained a final EDTA con-centration of 1 �M, therefore, the net BC/cop-per ratio is 2:1.

2. As described above, in the absence of BC, theeffect of Toc depended on how it was added tothe liposomes. Specifically, when added exter-nally from ethanol, Toc exhibited pro-oxida-tive effects at all studied copper andtocopherol concentrations (e.g. Fig. 4), in con-

O. Bittner et al. / Chemistry and Physics of Lipids 114 (2002) 81–9892

trast to the antioxidative effects of Toc incor-porated into liposomes by co-sonication (e.g.Fig. 5). In the presence of BC, the effect ofexternally added tocopherol depended on itsconcentration as well as on the concentrationsof BC and copper. In the presence of BC,under all the studied conditions with no addedcopper, Toc added from ethanol was a morepotent antioxidant than AA at the same con-centration. This was particularly clear whenthe medium contained 0.1 �M BC (Fig. 9).Under these conditions, AA was a pro-oxi-dant, whereas Toc was a potent antioxidant(Fig. 9). However, the dose dependence of thelatter effect was complex, as demonstrated inFig. 11 for increasing tocopherol concentra-tions introduced from ethanol into liposomalsystems containing 0.25 mM PLPC, 0.25 �MBC and no added copper. As obvious fromthis figure, the strongest antioxidative effect(i.e. the longest lag and lowest Vmax) was ob-tained at 0.5 �M tocopherol whereas furtheraddition of Toc shortened the lag periodmarkedly.

3.4. Effects of antioxidants on PLPC oxidationinduced by AAPH

The influence of tocopherol and AA on thekinetics of AAPH-induced peroxidation of PLPCliposomes was very different from their influenceon copper-induced peroxidation of the same lipo-somes in the same range of concentrations of theantioxidants. As seen in Fig. 12, AA (5 �M)affected the oxidation of PLPC induced by AAPH(1 mM) only slightly (if at all). Toc added exter-nally from ethanol into the liposomes had some-what larger antioxidative effects, whereas Tocincorporated into the liposomes by co-sonicationexerted the largest antioxidative effects (Fig. 12).Similar results were obtained for an AAPH con-centration of 3 mM (not shown).

4. Discussion

Peroxidation induced by transition metals re-quires the occurrence of a redox cycle. Initiation

Fig. 10. The influence of oxidative stress on the effect of AA on tmax. Different concentrations of AA (0, 1, 2, 5 or 20 �M) wereadded to PLPC liposomes (0.25 mM) at two different concentrations of BC-chelated-copper. The solid line represents the results ofan experiment conducted at relatively low oxidative stress (the solution contained 2 �M CuCl2, 1 �M EDTA and 2 �M BC). Thedashed line depicts the results of an experiment conducted at higher oxidative stress (the solution contained 6 �M CuCl2, 1 �MEDTA and 10 �M BC). The tmax values were derived from the respective kinetic profiles, as described in Fig. 1.

O. Bittner et al. / Chemistry and Physics of Lipids 114 (2002) 81–98 93

Fig. 11. Effect of externally added Toc on the peroxidation of PLPC in the presence of BC with no added copper. Toc (0–5 �M,as indicated) was added from ethanol to PLPC liposomes (0.25 mM) with BC (0.25 �M). Peroxidation was monitored at 37 °C. Theabsorbance at 234 nm, as presented in the figure, was corrected for changes in light scattering, on the basis of the absorbance at 325nm, as described in the Section 2.

Fig. 12. Effects of antioxidants on the peroxidation of PLPC induced by AAPH. AAPH (1 mM) was added to PLPC vesicles (0.25mM) in the absence or presence of 5 �M AA or Toc that was either added externally in an ethanolic solution or incorporated intothe liposomes by co-sonication. The absorbance at 234 nm was continuously monitored at 37 °C.

O. Bittner et al. / Chemistry and Physics of Lipids 114 (2002) 81–9894

Cu(II)+RH�R�+H++Cu(I) (4)

where RH must be a strong reductant for thereaction to occur. RH may be an antioxidant(RH=AH=AA or Toc) or a hydroperoxide(RH=LOOH):

Cu(II)+AH�A�+H++Cu(I) (5a)

Cu(II)+LOOH�LOO�+H++Cu(I) (5b)

Propagation of the chain reaction requires oxi-dation of Cu(I) back to Cu(II), either by molecu-lar oxygen:

Cu(I)+O2�Cu(II)+O2−� (slow) (6)

or via a Fenton-type reaction:

Cu(I)+LOOH�Cu(II)+LO�+OH− (fast)(7)

The radicals formed in these reactions (X�=R�,A�, LOO�, LO�) may, under the appropriate con-ditions, contribute to the propagation of the chainreaction:

LH+X��L�+XH (8)

L�+O2�LOO� (fast) (9)

Subsequently, the peroxyl radicals may eitherpropagate the chain reaction according to Eq. (8)(X�=LOO�) or, undergo biradical quenching,which terminates the reaction:

X�+Y��NRP (non-reactive products)(10)

where Y�, X�=LOO�, LO�, A�, L�, R� or anyother radical formed in the system.

The entire lag phase observed during oxidationof naturally occurring lipids in lipoproteins ormembranes has been previously attributed to thescavenging of peroxyl radicals by lipid-associatedantioxidants (Esterbauer and Jurgens, 1993). Inthe context of this interpretation, the timepoint atwhich fast propagation begins has been previouslyidentified with complete consumption of the an-tioxidants (Esterbauer and Jurgens, 1993). Morerecently, it has been demonstrated that for cop-per-induced peroxidation only 20–50% of the lagis due to the protective effect of antioxidants(Abuja and Esterbauer, 1995) and that a major

part of the lag phase is due to continuous growthof the concentration of free radicals via the inter-related processes of formation and breakdown ofthe reactive intermediates—LOOH (Pinchuk etal., 1998).

Unlike in LDL, in PLPC liposomes containingno lipid-associated antioxidants, initiation mustdepend on the LOOH produced during prepara-tion of the liposomes (Eq. (5b)) and on the au-tooxidation process. Accordingly, the whole ‘lag’must be attributed to autoacceleration and theperoxidation is initially slow. Furthermore, sincethe binding of copper to the surface of the zwitte-rionic phospholipid liposomes is weak, it is notsurprising that oxidation occurs only at relativelyhigh copper/PLPC ratios.

The overall effect of both tocopherol and ascor-bate represents a sum of their pro-oxidative andantioxidative effects. The net effect of these reduc-ing agents depends on three major factors, namelyon their concentration, on where they reside andon the oxidative stress applied to the system.Under the mild oxidative stress induced by CuCl2alone, the water-soluble reducing agent AA, andto a lesser extent externally added Toc, exhibitedpro-oxidative effects (Figs. 3 and 4). Under theseconditions, biradical quenching (via Eq. (10)) islikely to be relatively slow and the reducing agentsprobably accelerate the reaction by ‘supplying’free radicals (via Eq. (5a)), which interact withLH (Eq. (8); Bowry and Stocker, 1993; Pinchukand Lichtenberg, 1999). Furthermore, oxidationof the Cu(I) formed via Eq. (5a) by pre-formedLOOH (Eq. (7)), further propagates the processby producing more free radicals.

Simultaneously, both AA and externally addedToc can act as antioxidants (Fukuzawa et al.,1981; Niki et al., 1985b; Barclay, 1993) byquenching up to two free radicals per molecule ofantioxidant via reactions Eqs. (11) and (12):

AH + X��A�+XH (11)

A�+X��NRP (12)

This effect is particularly pronounced underconditions of high oxidative stress (Figs. 9–11),when the free radicals formed during peroxida-tion, at a relatively high concentration, have a

O. Bittner et al. / Chemistry and Physics of Lipids 114 (2002) 81–98 95

high probability of undergoing biradical quenching(reaction Eq. (12)).

Accordingly, the actual effect of a given antiox-idant is a complex function of the external oxidativestress, and of its concentration in the bilayer andin the aqueous solution: when the antioxidantresides in the solution (e.g. when AA is added tothe system), under all the conditions studied here,the concentration of the radicals formed upon itsoxidation may be too low to allow for biradicalquenching, so the AA is pro-oxidative. Further-more, in agreement with previous studies (Yoshidaet al., 1994), our results indicate that the water-sol-uble antioxidant AA is a more potent reductant forCu(II) than Toc. This interpretation is also consis-tent with the finding that in the presence of AA (butnot Toc), oxidation occurs even at sub-micromolarconcentrations of copper (Fig. 3). By contrast,when the antioxidant resides mainly in the bilayers(e.g. in Toc containing vesicles), its local concentra-tion is high, the biradical quenching is likely to berapid, and its net effect is, therefore, likely to beantioxidative, as observed throughout the studiedrange of experimental conditions (Fig. 5), and inagreement with previous studies (Niki et al., 1985a;Barclay, 1993).

Notably, under high oxidative stress, Toc exertsprofound anti-oxidative effects even when it isexternally added (Figs. 9 and 11), although most ofit resides in the aqueous solution. We think that thenet antioxidative effect can be attributed to thatfraction of Toc that resides in the liposomes. Yet,at high tocopherol concentrations (e.g. above 0.5�M in Fig. 11), increasing the Toc concentrationresults in reduction of the antioxidative effect,which we attribute to the pro-oxidative effect of thewater soluble Toc via Eq. (5a).

Another finding of interest is that when theliposomes contained Toc, AA exhibited markedantioxidative effects. In previous studies, it hasbeen shown that AA enhances the antioxidativeeffects of Toc, presumably via recovery of Toc fromtocopheroxyl radicals by AA (Niki et al., 1984;Doba et al., 1985; Niki et al., 1985b; Bowry andStocker, 1993; Wang and Quinn, 1999). A similarreaction may occur in our tocopherol-containingPLPC liposomes when AA is added to the disper-sion. This reaction may affect peroxidation more

than the production of ascorbyl radicals, so that thenet effect of AA is antioxidative.

To explain the dramatic pro-oxidative effect ofthe copper chelating agents BC and NC, it isimportant to recall that the affinity of both thesechelators for Cu(I) is several orders of magnitudehigher than their affinity to Cu(II). Hence, thesechelators stabilize Cu(I) at the expense of Cu(II),which may explain the finding that the redoxpotential of chelated copper (0.62 V, Lappin et al.,1980) is higher than that reported for the equi-librium Cu(II)–Cu(I) (0.15 V, Skoog and West,1976).

Copper-catalyzed peroxidation proceeds via aCu(I)–Cu(II) redox cycle. Stabilization of copperin its reduced state may, therefore, either inhibitperoxidation, when oxidation of Cu(I) to Cu(II) israte limiting, or else, accelerate peroxidation whenthe chelated Cu(I) can still be oxidized back toCu(II) by LOOH. This explains the complex depen-dence of copper-induced peroxidation on the con-centrations of copper and chelating agents, asrecently described for LDL (Lynch and Frei, 1995;Pinchuk et al., 2001). It may also explain theinhibitory effect of BC on copper-induced LDLperoxidation, observed by Abuja et al. (1997) whenall the copper was in the form of (BC)2–Cu(I) andthe conditions were probably such that the Cu(I)could not have been efficiently oxidized to Cu(II).Under our experimental conditions, both BC andNC accelerated the peroxidation of liposomalPLPC even when the system contained BC at muchhigher concentrations (up to ten fold) than that ofcopper (Fig. 8), so that essentially all the copper(both Cu(I) and Cu(II)) was chelated. Hence, theobserved BC-induced acceleration of peroxidationis likely to be a consequence of the higher redoxpotential of chelated copper (Ueda et al., 1999;Pinchuk et al., 2001).

Interestingly, in all our experiments, BC was amore potent pro-oxidant than NC (Fig. 7). Thereason for this difference is not clear. Explaining itas being a result of the more hydrophobic natureof NC (and of its copper chelates) is not trivial. Thehigher hydrophobicity could have actually beenexpected to result in faster production of freeradicals at the interface when oxidation is inducedby NC-copper chelates than when it is induced bythe less hydrophobic BC-copper chelates. However,

O. Bittner et al. / Chemistry and Physics of Lipids 114 (2002) 81–9896

Cu(I) bound to the liposomes may inhibit peroxida-tion, possibly by quenching the free radicals formedby the copper chelates at the interface (Costanzo etal., 1995). As a consequence the higher hydropho-bicity of the NC chelates may result not only in theproduction of more free radicals, but also in moreefficient quenching, which may explain the lowerpro-oxidative potency of NC.

Notably, the dependence of the lag on theconcentration of BC is bell-shaped, the lag at 0.5�M BC being much shorter than the lag observedin the absence of BC, but also shorter than the lagobserved at 5 �M BC (Fig. 8). By contrast, themaximal rate increases monotonically with thechelator’s concentration (Fig. 8) throughout thewhole studied range of BC concentrations. Theprolongation of the lag observed upon increasingthe BC concentration can be explained in terms ofthe stabilization of Cu(I), to the extent that theoxidation of Cu(I) to Cu(II) becomes rate limiting(Perugini et al., 1997). Nonetheless, towards theend of the lag, the LOOH concentration is suffi-ciently high to rapidly oxidize Cu(I) back to Cu(II)and by that reinstate the redox cycle, so that thereaction rate will depend on the total concentrationof chelated copper. As a consequence, the maximalrate increases monotonically upon increasing theBC concentration.

In an attempt to evaluate the influence of oxida-tive stress on the effect of AA and Toc we usedchelated copper to induce high oxidative stress.Under these conditions, the radical R�, formed viaEq. (4) is quenched via Eq. (10) faster than it caninteract with LH (Eq. (8)). Consequently, evenexternally added Toc, most of which resides in theaqueous phase, is a potent antioxidant (Figs. 9 and11). Moreover, even AA exhibits antioxidativeeffects under such conditions, although, as ex-pected, Toc is a more potent antioxidant than AA.In fact, under certain conditions (e.g. Fig. 9),externally added Toc was a potent antioxidant,whereas AA accelerated the peroxidation. A rea-sonable explanation for this phenomenon is thattocopheroxyl radicals formed near the liposomesurface are likely to become concentrated in theliposomes and undergo biradical quenching fasterand more efficiently than the ascorbyl radicalsformed in the large aqueous volume.

Interpretation of the pronounced pro-oxidativeeffect of antioxidants as being a consequence of freeradical production upon reduction of Cu(II) im-plies that these antioxidants should not promoteAAPH-induced peroxidation. The inhibition ofAAPH-induced peroxidation by both AA and Toc(Fig. 12) is consistent with this interpretation, aswell as with the results of Niki et al. (1985b) andWaters et al. (1997) obtained with soybean PCliposomes. Notably, inhibition by co-sonicated Tocis much more pronounced than inhibition by exter-nally added Toc, whereas AA exhibits only a slightanti-oxidative effect.

In conclusion, liposomal PLPC is susceptible tooxidation induced by copper ions only at micromo-lar copper concentrations. Under these conditions,both AA and externally added Toc act as ‘pro-ox-idants’. By contrast, when Toc was incorporatedinto the liposomes, it acted as an antioxidant. In thepresence of either of the chelating agents BC or NC,which stabilize Cu(I) on the expense of Cu(II),sub-micromolar concentrations of the copperchelates induce rapid peroxidation of the lipids.Under these conditions, peroxidation induced bycopper chelates may either be accelerated or inhib-ited by AA and Toc, depending on the concentra-tions of copper, chelators and antioxidants. In viewof these results, we propose that under certainconditions peroxidation of PLPC vesicles may beinduced by biologically-relevant, sub-micromolarconcentrations of transition metals. The depen-dence of the ‘threshold peroxidizing concentration’of copper on the presence and concentration ofother chelates of transition metals, as well as on thelipid composition, is presently under investigation.Although the relevance of such studies to lipidoxidation in vivo is quite questionable, these studiesare important to gain basic understanding of thefactors affecting oxidation of aggregated lipidsbearing polyunsaturated fatty acid chains.

Acknowledgements

We thank the Israel Science Foundation,founded by the Israel Academy of Science andHumanities-Centers of Excellence Program, forfinancial support.

O. Bittner et al. / Chemistry and Physics of Lipids 114 (2002) 81–98 97

References

Abuja, P.M., Esterbauer, H., 1995. Simulation of lipid peroxi-dation in low-density lipoprotein by a basic ‘skeleton’ ofreactions. Chem. Res. Toxicol. 8, 753–763.

Abuja, P.M., Albertini, R., Esterbauer, H., 1997. Simulationof the induction of oxidation of low-density lipoprotein byhigh copper concentrations: evidence for a nonconstantrate of initiation. Chem. Res. Toxicol. 10, 644–651.

Babiy, A.V., Gebicki, J.M., 1999. Decomposition of lipidhydroperoxides enhances the uptake of low-density lipo-protein by macrophages. Acta Biochim. Polon. 46, 31–42.

Barclay, L.R.C., 1993. Model biomembranes: quantitativestudies of peroxidation, antioxidant action, partitioning,and oxidative stress. Can. J. Chem. 71, 1–16.

Barclay, L.R.C., Baskin, K.A., Kong, D., Locke, S.J., 1987.Autoxidation of model membranes. The kinetics andmechanism of autoxidation of mixed phospholipid bilay-ers. Can. J. Chem. 65, 2541–2550.

Bartlett, G.R., 1959. Phosphorus assay in column chromatog-raphy. J. Biol. Chem. 234, 466–468.

Bowry, V.W, Stocker, R., 1993. Tocopherol-mediated peroxi-dation. The pro-oxidant effect of Vitamin E on the radical-initiated oxidation of human low-density lipoprotein. J.Am. Chem. Soc. 115, 6029–6044.

Chisolm, G.M., 1991. Cytotoxicity of oxidized lipoproteins.Curr. Opin. Lipidol. 2, 311–316.

Costanzo, L.L., De Guidi, G., Giuffrida, S., Sortino, S.,Condorelli, G., 1995. Antioxidant effect of inorganic ionson UVC and UVB induced lipid peroxidation. J. Inorg.Biochem. 59, 1–13.

CRC, 1982. Handbook of organic analytical reagents. CRCPress, Inc, Florida pp. 331–334.

Doba, T., Burton, G.W., Ingold, K.U., 1985. Antioxidant andco-antioxidant activity of vitamin C. The effect of vitaminC, either alone or in the presence of vitamin E or a watersoluble vitamin E analogue, upon the peroxidation ofaqueous multilamellar phospholipid liposomes. Biochim.Biophys. Acta 835, 298–303.

Eigenberg, K.E., Chan, S.I., 1980. The effect of surface curva-ture on the head-group structure and phase transitionproperties of phospholipid bilayer vesicles. Biochim. Bio-phys. Acta 599, 330–335.

Esterbauer, H., 1993. Cytotoxicity and genotoxicity of lipid-oxidation products. Am. J. Clin. Nutr. 57, 779S–786S.

Esterbauer, H., Jurgens, G., 1993. Mechanistic and geneticaspects of susceptibility of LDL to oxidation. Curr. Opin.Lipidol. 4, 114–124.

Esterbauer, H., Ramos, P., 1995. Chemistry and pathophysiol-ogy of oxidation of LDL. Rev. Physiol. Biochem. Pharma-col. 127, 31–64.

Frankel, E.N., 1998. Lipid Oxidation. The Oily Press Ltd,Dundee, Scotland, pp. 128–160.

Frei, B., Gaziano, J.M., 1993. Content of antioxidants, pre-formed lipid hydroperoxides, and cholesterol as predictorsof the susceptibility of human LDL to metal ion-dependentand-independent oxidation. J. Lipid Res. 34, 2135–2145.

Fukuzawa, K., Chida, H., Tokumura, A., Tsukatani, H., 1981.Antioxidative effect of �-tocopherol incorporation intolecithin liposomes on ascorbic acid-Fe(II)-induced lipidperoxidation. Arch. Biochem. Biophys. 206, 173–180.

Gast, K., Zirwer, D., Ladhoff, A.M., Schreiber, J., Koelsch,R., Kretschmer, K., Lasch, J., 1982. Auto-oxidation-in-duced fusion of lipid vesicles. Biochim. Biophys. Acta 686,99–109.

Haase, G., Dunkley, W.L., 1969. Ascorbic acid and copper inlinoleate oxidation. II. Ascorbic acid and copper as oxida-tion catalysts. J. Lipid Res. 10, 561–567.

Hazell, L.J., Davies, M.J., Stocker, R., 1999. Secondary radi-cals derived from chloramines of apolipoprotein B-100contribute to HOCl-induced lipid peroxidation of low-den-sity lipoproteins. Biochem. J. 339, 489–495.

Heinecke, J.W., 1997. Mechanisms of oxidative damage oflow-density lipoprotein in human atherosclerosis. Curr.Opin. Lipidol. 8, 268–274.

Huang, C., 1969. Studies on phosphatidylcholine vesicles. For-mation and physical characteristics. Biochemistry 8, 344–352.

Kontush, A., Meyer, S., Finckh, B., Kohlschutter, A.,Beisiegel, U., 1996. �-Tocopherol as a reductant for Cu(II)in human lipoproteins. J. Biol. Chem. 271, 11106–11112.

Kritharides, L., 1999. Ascorbic acid and copper in linoleateoxidation—Dunkley revisited. Redox report 4, 259–262.

Lappin, A.G., Youngblood, M.P., Margerum, D.W., 1980.Electron transfer reactions of copper(I) and copper(III)complexes. Inorg. Chem. 19, 407–413.

Lichtenberg, D., Barenholz, Y., 1988. Liposomes: preparation,characterization, and preservation. In: David, G. (Ed.),Methods of Biochemical Analysis. Wiley, New York, pp.337–462.

Lynch, S.M., Frei, B., 1995. Reduction of copper, but notiron, by human low density lipoprotein (LDL). J. Biol.Chem. 270, 5158–5163.

Maiorino, M., Zamburlini, A., Roveri, A., Ursini, F., 1995.Copper-induced lipid peroxidation in liposomes, micelles,and LDL: which is the role of vitamin E. Free Radic. Biol.Med. 18, 67–74.

McMurray, H.F., Parthasarathy, S., Steinberg, D., 1993. Oxi-datively modified low-density lipoprotein is a chemoattrac-tant for human T lymphocytes. J. Clin. Invest. 92,1004–1008.

Moore, W.J., 1962. Physical Chemistry, fourth ed. LongmansGreen and Co Ltd, London, pp. 761–763.

Neuzil, J., Thomas, S.R., Stocker, R., 1997. Requirement for,promotion, or inhibition by �-tocopherol of radical-in-duced initiation of plasma lipoprotein lipid peroxidation.Free Radic. Biol. Med. 22, 57–71.

Niki, E., Saito, T., Kawakami, A., Kamiya, Y., 1984. Inhibi-tion of oxidation of methyl linoleate in solution by vitaminE and vitamin C. J. Biol. Chem. 259, 4177–4182.

Niki, E., Kawakami, A., Saito, M., Yamamoto, Y., Tsuchiya,J., Kamiya, Y., 1985a. Effect of phytyl side chain ofvitamin E on its antioxidant activity. J. Biol. Chem. 260,2191–2196.

O. Bittner et al. / Chemistry and Physics of Lipids 114 (2002) 81–9898

Niki, E., Kawakami, A., Yamamoto, Y., Kamiya, Y., 1985b.Oxidation of lipids. VIII. Synergistic inhibition of oxidationof phosphatidylcholine liposomes in aqueous dispersion byvitamin E and vitamin C. Bull. Chem. Soc. Jpn. 58,1971–1975.

Noguchi, N., Yamashita, H., Gotoh, N., Yamamoto, Y.,Numano, R., Niki, E., 1998. 2,2�-Azobis (4-Methoxy-2,4-Dimethylvaleronitrile), a new lipid-soluble azo initiator:application to oxidations of lipids and low-density lipo-protein in solution and in aqueous dispersions. Free Radic.Biol. Med. 24, 259–268.

Panasenko, O.M., Arnhold, J., Vladimirov, Y.A., Arnhold, K.,Sergienko, V.I., 1997. Hypochlorite-induced peroxidation ofegg yolk phosphatidylcholine is mediated by hydroperox-ides. Free Radic. Res. 27, 1–12.

Patel, R.P., Svistunenko, D., Wilson, M.T., Darley-Usmar,V.M., 1997. Reduction of Cu(II) by lipid hydroperoxides:implications for the copper-dependent oxidation low-densitylipoprotein. Biochem. J. 322, 425–433.

Perugini, C., Seccia, M., Albano, E., Bellomo, G., 1997. Thedynamic reduction of Cu(II) to Cu(I) and not Cu(I)availability is a sufficient trigger for low density lipoproteinoxidation. Biochim. Biophys. Acta 1347, 191–198.

Pinchuk, I., Lichtenberg, D., 1996. Continuous monitoring ofintermediates and final products of oxidation of low densitylipoprotein by means of UV-spectroscopy. Free Rad. Res.24, 351–360.

Pinchuk, I., Lichtenberg, D., 1999. Copper-induced LDL perox-idation: interrelated dependencies of the kinetics on theconcentrations of copper, hydroperoxides and tocopherol.FEBS Letts. 450, 186–190.

Pinchuk, I., Schnitzer, E., Lichtenberg, D., 1998. Kineticanalysis of copper-induced peroxidation of LDL. Biochim.Biophys. Acta 1389, 155–172.

Pinchuk, I., Gal, S., Lichtenberg, D., 2001. The dose-dependenteffect of copper-chelating agents on the kinetics of peroxida-tion of low-density lipoprotein (LDL). Free Radic. Res. 34,349–362.

Ramos, P., Gieseg, S.P., Schuster, B., Esterbauer, H., 1995.Effect of temperature and phase transition on oxidation

resistance of low density lipoprotein. J. Lipid Res. 36,2113–2128.

Ramos-Lledo, P., Vera, S., San Andres, M.P., 2001. Determina-tion of vitamins A and E in milk samples by fluorescencein micellar media. Fresenius J. Anal. Chem. 369, 91–95.

Sayre, L.M., 1996. Alzheimer’s precursor protein and the useof bathocuproine for determining reduction of copper(II).Science 274, 1933–1934.

Shaw, J.M., Thompson, T.E., 1982. Effect of phospholipidoxidation products on transbilayer movement of phospho-lipids in single lamellar vesicles. Biochemistry 21, 920–927.

Skoog, D.A., West, D.M, 1976. Fundamentals of AnalyticalChemistry, third ed. Holt, Rinehart and Winston, NewYork.

Steinbrecher, U.P., 1999. Receptors for oxidized low-densitylipoprotein. Biochim. Biophys. Acta 1436, 279–298.

Ueda, J., Anzai, K., Miura, Y., Ozawa, T., 1999. Oxidation oflinoleic acid by copper(II) complexes: effects of ligand. J.Inorg. Biochem. 76, 55–62.

Vossen, R.C.R.M., van Dam-Mieras, M.C.E, Hornstra, G.,Zwaal, R.F.A., 1993. Continuous monitoring of lipid perox-idation by measuring conjugated diene formation in anaqueous liposome suspension. Lipids 28, 857–861.

Wang, X., Quinn, P., 1999. Vitamin E and its function inmembranes. Prog. Lipid Res. 38, 309–336.

Waters II, R.E., White, L.L., May, J.M., 1997. Liposomescontaining �-tocopherol and ascorbate are protected froman external oxidant stress. Free Rad. Res. 26, 373–379.

Yamamoto, Y., Niki, E., Kamiya, Y., Shimasaki, H., 1984.Oxidation of lipids. 7. Oxidation of phosphatidylcholines inhomogeneous solution and in water dispersion. Biochim.Biophys. Acta 795, 332–340.

Yoshida, Y., Niki, E., 1992. Oxidation of methyl linoleate inaqueous dispersions induced by copper and iron. Arch.Biochem. Biophys. 295, 107–114.

Yoshida, Y., Tsuchiya, J., Niki, E., 1994. Interaction of �-toco-pherol with copper and its effect on lipid peroxidation.Biochim. Biophys. Acta 1200, 85–92.

Zhang, D., Yasuda, T., Yu, Y., Okada, S., 1994. Physicochem-ical damage to liposomal membrane induced by iron-orcopper-mediated lipid peroxidation. Acta Med. Okayama48, 131–136.