Accepted Manuscript Spectroscopic studies of alpha tocopherol interaction with a model liposome and its influence on oxidation dynamics Dubravka Krilov, Marin Kosović, Kristina Serec PII: DOI: Reference:

S1386-1425(14)00498-3 http://dx.doi.org/10.1016/j.saa.2014.03.087 SAA 11920

To appear in:

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy

Received Date: Revised Date: Accepted Date:

27 September 2013 2 March 2014 22 March 2014

Please cite this article as: D. Krilov, M. Kosović, K. Serec, Spectroscopic studies of alpha tocopherol interaction with a model liposome and its influence on oxidation dynamics, Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy (2014), doi: http://dx.doi.org/10.1016/j.saa.2014.03.087

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Spectroscopic studies of alpha tocopherol interaction with a model liposome and its influence on oxidation dynamics Dubravka Krilova, Marin Kosovića and Kristina Sereca a

University of Zagreb, School of Medicine, Šalata 3, 10001 Zagreb, Croatia [email protected], [email protected], [email protected]

Corresponding author: Dubravka Krilov, University of Zagreb, School of Medicine, Šalata 3, 10001 Zagreb, Croatia; tel. (+3851) 4566951; e-mail: [email protected]

Abstract The influence of – tocopherol on the surface conformation of liposome, as a model component of lipoproteins, and its role in oxidation process were studied. FT-IR spectra from suspensions of neat liposome, mixtures of liposome and  – tocopherol and liposome with incorporated  – tocopherol were analyzed. When  – tocopherol was incorporated into liposome, intensities of some bands were decreased or increased in comparison with the spectra of liposome and  – tocopherol mixture. These changes reflect the different localization of  – tocopherol in two types of liposome suspensions. The oxidation of liposome suspensions was initiated by addition of cupric ions. After prolonged oxidation, the differences in FT-IR spectra of oxidized samples were recorded. Differences were observed in comparison with spectra of native and oxidized liposomes were analyzed. The rate of oxidation was measured by EPR oximetry. Oxidation was generally very slow, but faster in liposome without  – tocopherol, indicating the protective role of  – tocopherol against liposome oxidation. On the other hand, liposome suspensions with EDTA in the buffer were not oxidized at all, while those with  – tocopherol and liposome mixture were only slightly oxidized. In this case the consumption of oxygen was the result of liposome oxidation supported by  – tocopherol. These results reflect the ambivalent role of  – tocopherol in liposome oxidation, similarly to findings in studies of lipoprotein oxidation. Keywords: Liposome,  – tocopherol, Lipid layer, Oxidation, FT-IR, EPR

1. Introduction Lipoprotein oxidation, mainly of low density lipoprotein (LDL), and its influence on atherogenesis was extensively studied in vitro from the basic chemistry to the biological effects of oxidized LDL on culture cells [1]. In vivo clinical studies were mostly focused on understanding the role of antioxidants, specifically  – tocopherol, in prevention of the development of atherosclerosis. Earlier studies, where strong oxidation of LDL initiated by transition metals was performed in vitro, confirmed the protective role of  – tocopherol acting as a trap for peroxyl radicals formed in oxidation chain. If the oxidation hypothesis of atherogenesis were to be correct, it was expected that the regular uptake of vitamin E supplements would prevent or slow down the progress of atherosclerosis. However, numerous clinical studies failed to confirm that the uptake of  – tocopherol had any influence on the development of atherosclerosis and cardiovascular disease [2]. Later studies of LDL oxidation in vitro demonstrated that under some conditions,  – tocopherol could act as prooxidative agent, increasing the rate of oxidation. This process was called tocopherol mediated peroxidation (TMP) and suggested at first by Stocker and his group [3]. Our group had investigated the slow oxidation of LDL in a closed system by EPR measurement of oxygen concentration using spin probe [4]. On the basis of these experiments we had built the theoretical probabilistic model of LDL oxidation in which  – tocopherol in interaction with transition metal was acting as a prooxidant on one site of the oxidation cycle and as an antioxidant on the other site [5]. Results from literature confirmed that oxidation process is not limited only to the lipid domain of the particle but free radicals were transferred to the protein domain, by reaction of phospholipids with lysine groups [6]. EPR spin trapping experiments of lipid and protein fractions of oxidized LDL also demonstrated that larger number of radicals was trapped in protein domain [7]. Fluorescent measurements of LDL oxidation demonstrated that it starts by decomposition of tryptophan residues in apolipoprotein B, immediately after addition of cupric ions, with subsequent reaction of tryptophan peroxides with lipids [8]. This process did not depend on  – tocopherol. In order to get more insight into interaction of  – tocopherol and the lipid domain of lipoprotein, in our experiment liposome was used as a model of LDL particle. Three types of liposome suspensions were prepared: neat liposome in suspension with EDTA and without EDTA, liposome suspension with added  – tocopherol to preformed vesicles, and liposome suspension into which  – tocopherol was added to the mix of lipids and cholesterol before the formation of vesicles. We applied FT-IR spectroscopy to inspect the possible conformational differences in the spectra of liposome induced by the proximity or incorporation of  – tocopherol into vesicles. The oxidation was initiated by addition of small amounts of CuCl2 to the suspension. The oxidation was followed by measuring oxygen consumption in aqueous domain using EPR oximetry [4]. FT-IR spectra of oxidized liposome samples were recorded and compared with the spectra native samples. Differences in spectra indicated the conformation changes due to oxidation.

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2. Experimental All chemicals were purchased from Sigma Aldrich and used without further purification. Preparation of liposome vesicles Stock solutions of phosphatidylcholine (PC), 20 mg / 2 mL, sphingomyelin (SM), 15 mg / 1 mL and cholesterol (C), 20 mg / 2 mL were prepared in chloroform-methanol (2:1, v/v). The aliquots were combined to obtain the molar ratio of PC:SM:C as 2:1:0.3. The solvent was slowly removed from lipid solution under a stream of nitrogen and additionally under vacuum for 1 hour. The lipid film was further dispersed in 2 mL of 0.1 M phosphate buffer saline (PBS) by shaking for 5 minutes in vortex. For some oxidation experiments, EDTA was added to the buffer solution. EDTA is widely used in biological systems as a chelator for the metal ions and therefore is considered as a protector against the oxidation. The final concentration of liposome in the buffer was 1.65 mmol/mL. The sonication of the liposomal solution led to formation of unilamellar vesicles. The formation of vesicles was tested in the images obtained by transmission electron microscope using facility at Ruđer Bošković Institute in Zagreb.  – tocopherol solutions   – tocopherol was dissolved in chloroform-methanol (2:1, v/v), 20 mg in 2 mL as a stock solution. The aliquots of tocopherol solution were added to liposome suspension in a ratio of one molecule of  – tocopherol to 7 molecules of lipids for IR measurements and one molecule of  – tocopherol to 56 molecules of lipids for EPR measurements. In the other experiment 0.66 mmol of  – tocopherol was added to lipid and cholesterol mixture to obtain vesicles with 20% embedded  – tocopherol. Copper (II) chloride dihydrate solutions Cupric ions were used as the initiators of lipid peroxidation. Stock solution of CuCl22H2O was prepared by dissolving 20.46 mg CuCl22H2O in 10 mL of PBS to obtain the concentration of 12 mmol/mL. This stock solution was further diluted to obtain the desired concentration in aliquots of 10 L which were added to the liposome suspensions. The ratios of cupric ions to lipids in various samples were 1:10, 1:20, 1:50 and 1:100. Oxidation measurements Spin probe 3-carbamoyl-2,2,5,5-tetramethyl-3-pyroline-1-yloxy (CTPO) was dissolved in PBS, 4.18 mg/100 mL. The concentration of solution was 0.22 mM. The final concentration of CTPO in the sample was 0.11 mM. Copper and CTPO solutions were added to the liposome suspension immediately before incubation. Samples were incubated in sealed quartz micropipettes at room temperature for two days before EPR measurements. EPR spectrum of CTPO consists of triplet of nitrogen lines with super hyperfine pattern of 10 lines from 4 methyl groups. Their resolution is sensitive to the concentration of oxygen in the aqueous phase of the sample. Oxygen concentration was determined from EPR spectral parameters of CTPO, using calibrated curves from the literature [9].

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FT-IR spectroscopy For the spectroscopic measurements, the liposome solution (10 L) was placed on ZnSe window and dried under nitrogen for 20 minutes to obtain thin film. The film was further dried under vacuum for another 15 minutes. The oxidized samples were obtained by prolonged incubation of liposome suspensions with added cupric ions (1 per 10 lipids). FT-IR spectra were recorded with PerkinElmer Spectrum GX spectrometer equipped with a DTGS detector. Each spectrum was obtained from 100 coadded scans in the region 4000 – 600 cm-1, with 4 cm-1 resolution, at room temperature. The spectrometer was continuously purged with nitrogen to remove the water vapor from the detector and the sample compartment.

3. Results FT-IR spectroscopy FT-IR spectra of the liposome film in high wavenumber region are presented in Fig. 1. Figure 1 In this and other figures, spectrum A is of neat liposomes, spectrum B of liposomes with incorporated  – tocopherol and spectrum C of liposomes with  – tocopherol added after vesicle formation. For the comparison, the spectrum of a pure  – tocopherol, D, is added at the bottom in Fig. 1 and Fig. 2. The asymmetric stretching band from methyl groups, marked by arrow, increases from spectrum A to spectrum C. This indicates the contribution of  – tocopherol, spectrum D, which has 8 methyl groups in the molecule. It is more expressed in spectrum C than in spectrum B which implies that  – tocopherol is only partially embedded into lipid layer or is located very close to the surface. Spectral region 1800-600 cm-1 is presented in Fig. 2. The assignments of some lipid bands were described in our earlier paper [10]. Figure 2 The stretching band from ester bond (C=O) at 1740 cm-1, marked by 1, is structurally different in three spectra. It is somewhat broader in spectra B and C, indicating the presence of two unresolved components. A group (2) of stretching bands from skeletal C=C vibrations of polyunsaturated fatty acid (PUFA) chains appears in the region 1700-1500 cm-1. There is a significant difference in the pattern of these bands among three spectra. In spectrum A, three well resolved bands at 1645, 1595 and 1548 cm-1 are present. In spectrum B there are two groups of vibrations around 1645 and 1550 cm-1 and in spectrum C these bands are hardly observable. That indicates different localization of  – tocopherol and therefore its influence on C=C vibrations. The bands 1 and 2 are not present in spectrum D. The band, marked 3, is a deformation scissoring band from CH2 group. It is rather narrow, at 1467 cm-1, in spectra A and B but broad one and relatively stronger, at 1464 cm-1, in spectrum C. This band is the strongest one in spectrum D due to the large number of CH2 groups in the molecule. Intensities of deformation bands (4) formed of symmetric deformation from CH3 group, at 1377 cm-1, and wagging deformation from CH2 group increase from spectrum A and B to 4

spectrum C. The position of latter band is also changed from 1340 cm-1 (A and B) to 1344 cm-1 (C). In spectrum D, this group of bands is sharper and relatively stronger in comparison with the band 3. Two bands, labelled with 5, belong to the pair of stretching vibrations from PO2- group. Asymmetric stretching band is at 1240 cm-1 in spectrum A and at 1235 cm-1 in spectrum B. In spectrum C it is at 1246 cm-1 and has a shoulder at about 1220 cm-1. It can reflect the contributionof – tocopherol which has a band in vicinity, at 1212 cm-1 as could be seen in spectrum D. Symmetric stretching band from PO2- group is at 1090 cm-1 in spectrum A and at 1087 cm-1 in spectra B and C. The intensity of both phosphate bands is lower in spectrum C. In spectrum D is a sharp band close to the position of the symmetric phosphate band. The band, marked by 6, is attributed to stretching vibrations of ester sn-1 and sn-2 CO-O single bonds in phospholipids. In spectrum A it is weak and at 1172 cm-1 with shoulder at 1143 cm-1. The shoulder is not observed in spectrum B and in spectrum C where the band, at 1165 cm-1, is much stronger. The band, marked by 7, is ester symmetric stretching vibration from C-O-C group at 1061 cm-1. It is weaker in spectrum C. The relatively strong band (8) at 970 cm-1 is asymmetric stretching vibration from choline group N+(CH3)3. It has much lower intensity in spectrum C than in spectrum B. On the other hand, the skeletal band, marked 9, a C–C stretching vibration from the region near the head of phospholipids, is therefore more expressed in spectrum C than in B. It is also present in spectrum D. That also indicates the difference in  – tocopherol localization in two systems. The broad band (10) around 850 cm-1 is known as a combination of symmetric stretching vibration from choline group and C–C stretching band from the region near the end of chain. It is much stronger in spectrum A. Two small rocking bands, marked 11, from CH group at 762 cm-1 and doublet, marked 12, at 721/702 cm-1 from CH2 group are broader and less resolved in spectrum C. These bands are not visible in spectrum D.

FT-IR spectroscopy of oxidized liposomes FT-IR spectra of native and oxidized samples from neat liposomes and those with embedded  – tocopherol were recorded. Differences were observed in fingerprint and low wavenumber regions and are indicated by arrows in the upper spectrum in following figures. Spectra of native (A) and oxidized (B) liposome samples in the region 1800-600 cm-1 are presented in Fig. 3. Figure 3 The relative intensity of C=O stretching band in comparison with C=C stretching band at 1650 cm-1 is slightly higher in spectrum B. The additional band from C=C vibration was observed at 1562 cm-1 in spectrum B which is probably poorly resolved in spectrum A. Two deformation bands, at 1377 cm-1 and 1340 cm-1, are better expressed in spectrum B. This spectral region is presented enlarged in the left islet above spectrum B. The relative intensity of symmetric over asymmetric stretching band from PO2- is higher in spectrum B. In the wavenumber region below 1000 cm-1 dominate two bands: asymmetric stretching band from choline group at 970 cm-1 and broad combination band around 850 cm-1, both with somewhat different profile in spectrum B. Rocking band from CH group at 768 cm-1 in spectrum A is split into doublet at 771/756 cm-1 in spectrum B. The other rocking band from CH2 group is stronger in spectrum B and its other component is also visible at 697 cm-1. The spectral region of rocking bands is presented enlarged in the right islet above spectrum B. FT-IR spectra of liposome samples with embedded  – tocopherol also show changes induced by oxidation. Contrary to the samples of neat liposome, the relative intensity of

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stretching C=O band in comparison with C=C band is significantly lower in spectrum B of oxidized sample, Fig. 4. Figure 4 In the group of C=C stretching bands there appears a new band in spectrum B, at 1606 cm , which was not observed previously. On the other side, C=C band at 1573 cm-1 in spectrum A cannot be observed in spectrum B. The deformation scissoring band of CH2 group is relatively weaker in spectrum B and the complex (CH3)s + w(CH2) has a different profile, as presented in the left islet above spectrum B. As in spectra of neat liposome, the relative intensity of symmetric over asymmetric stretching PO2- band is higher in spectrum B. The relative intensity of the complex band around 850 cm-1 in comparison with choline band at 970 cm-1 is higher in spectrum B. In low wavenumber region, the rocking band from CH group, at 766 cm-1, in spectrum A is shifted to 772 cm-1 in spectrum B. The other rocking band from CH2 group is stronger in spectrum A. This region is also presented enlarged in the right islet above spectrum B. -1

Oxygen consumption The oxygen concentration was measured in the suspensions with limited amount of dissolved oxygen as a function of incubation time. A small number of cupric ions in comparison with number of lipids, and at the same time exclusion of atmospheric oxygen assured the conditions for very slow oxidation [4, 7]. In the first experiment, we used PBS with added EDTA, 1g/L, as a solvent for all chemical compounds. Oxidation curves for suspensions of neat liposomes (○) and of liposomes and  – tocopherol mixture (□) are presented in Fig. 5, panel 1. In the suspensions, the ratio of cupric ions to lipids was 1:10. Figure 5 The samples without  – tocopherol did not consume oxygen even after 30 days, in spite of presence of cupric ions. The resistance to oxidation could be explained by the absence of preexisted lipid hydroperoxides in the liposome vesicles and/or protective role of EDTA. In samples with added  – tocopherol the oxygen concentration decreased with time, indicating that  – tocopherol did act as a prooxidant, as was earlier observed in slow oxidation of LDL [4]. In order to check how variation of copper concentration changes the oxidation dynamics of liposome with added  – tocopherol, different amounts of cupric ions were added into liposome suspension, with ratio of one ion to 10 (▲), 20 (▼), 50 () and 100 (■) lipid molecules. These results are presented in Figure 5, panel 2. As expected, the oxidation was faster when more metal ions were present in the sample, confirming its role as initiator of oxidation. In the second experiment, suspensions of liposome and liposome with 20% of embedded  – tocopherol were prepared in PBS without EDTA. The same buffer was used for the preparation of copper (II) chloride dihydrate and CTPO. The copper concentration was 1:10 lipids. Oxidation curves are presented in Fig. 6 for the suspension of neat liposomes () and those with embedded  – tocopherol (■).

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Figure 6 The sample of neat liposomes consumes oxygen faster, demonstrating a protective role of  – tocopherol when it is incorporated within the vesicle. The change of copper concentration from 1:10 to 1:20 did not have influence on the rate of oxidation.

4. Discussion FT-IR spectra of liposome films with  – tocopherol demonstrate the different localization of this molecule as a consequence of its interaction with liposome vesicle. When  – tocopherol was added to the mixture of lipids and cholesterol, it became the intrinsic component of lipid layer in the process of vesicle formation and therefore it is expected to be buried between fatty acid chains. This is in accordance with earlier studies of liposomes with embedded  – tocopherol, which had shown that the exposure of chromanol ring is low at the surface of liposome. This statement was based on the results of fluorescence measurements which showed low quenching of  – tocopherol fluorescence by a water soluble quencher [11]. Therefore,  – tocopherol must be located deeper in the layer, occupying the available space between two PUFA chains. If it is so, the presence of  – tocopherol would not affect much the ordering of the layer's surface as was observed earlier by spin label EPR spectroscopy [12]. Magnetic resonance measurements of spin-lattice relaxation time of 13C labeled  – tocopherol incorporated into liposome membrane indicated that the affinity of CH3 ring groups of  – tocopherol toward PUFA is larger then of its chain groups [13]. Therefore, the position of chromanol ring must be below polar region and closer to the lipid chains. These arguments are in agreement with our spectroscopic results which show that  – tocopherol added to already formed vesicles induces larger changes in vibration bands of liposome than incorporated  – tocopherol. The comparison of spectrum A and B in Fig. 2 shows that  – tocopherol has no influence on the asymmetric band from choline and phosphate groups which are on the surface of lipid layer. On the other hand, it changes the band pattern of stretching vibrations from C=C groups, which are below the surface. Deformation bands of side groups and skeletal stretching bands of fatty acid chain are only slightly affected indicating the alignment of phytyl chain with fatty acid chains. Spectrum C demonstrates that  – tocopherol, added after formation of liposome vesicle, remains in the interface close to the lipid layer or is partially embedded into the layer. It is confirmed by the increase of asymmetric stretching band from CH3 groups and deformation band from CH2 groups due to contribution of these vibrations from  – tocopherol. Such localization could have effect on the surface lipid packing and the orientation of head groups. Larger changes in skeletal and deformation bands show the change in lipid ordering. Stretching vibrations from C=C groups are significantly decreased, but there is an increase in deformation bands from side groups and of skeletal stretching bands from the chain region close to choline group. That increase reflects the contribution of the same bands from  – tocopherol. These observations confirm that  – tocopherol managed to enter partly the lipid layer affecting mostly vibrations from polar part but also from backbone and side groups of fatty acid chains in its vicinity. Differences in FT-IR spectrum of oxidized neat liposome reflect conformational changes within lipid chains but also in the polar region. This is the consequence of the formation of lipid hydroperoxides in PUFA. On the other hand, in the spectrum of oxidized

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liposome with incorporated  – tocopherol, changes in FT-IR spectrum due to oxidation are not the same. The relative decrease of C=O stretching band in comparison with the group of C=C stretching bands and appearance of the new band in this group are the most pronounced differences. In this system, the oxidation chain of reactions includes formation of  – tocopheroxyl radical and its reaction with lipids or its transformation to non radical products which can influence the conformation of lipid layer. EPR measurements showed that lipid oxidation was generally very slow, even when samples were not protected against oxidation with EDTA. The process of liposome oxidation was much slower than in lipoprotein suspension which emphasizes the important role of protein domain in the mechanism of LDL oxidation [5,7]. This could be also in agreement with the hypothesis about the initiation of oxidation in amino acid residues [8]. It was found that 80% of cupric ions are likely to be bound to the apo B in the vicinity of LDL lipids [14]. The consumption of oxygen in liposome solution with EDTA was practically negligible indicating that the presence of EDTA was sufficient to prevent oxidation of lipids by cupric ions. However, in the liposome solution with added  – tocopherol, the oxygen concentration dropped about 32% after 30 days. The absence of lipid hydroperoxides at the moment of initiation is not a prerequisite for further chain reactions if  – tocopherol is present in the system, in this case only 1 molecule per 56 lipid molecules. Earlier, in our theoretical model of lipid peroxidation it was shown that the initiation of oxidation process is possible even without preexisting lipid hydroperoxides starting by reaction of  – tocopherol with cupric ion and subsequent reaction of tocopheroxyl radical with the lipid molecule [15]. Our results demonstrate that the presence of  – tocopherol is essential for oxidation process in liposome suspension with EDTA and that it acts as a prooxidant in TMP. [3] Metal ions initiate lipid oxidation which was confirmed that for the same number of added  – tocopherol molecules, the oxidation was faster for higher number of cupric ions in the liposome. That suggests the interaction of metal ions with  – tocopherol and the action of  – tocopheroxyl radical in further promotion of lipid oxidation. In our experiments the concentration of copper was very low in comparison with other in vitro studies and closer to the LDL environment in the blood or the artery wall. In the studies on the role of vitamin E in atherosclerosis it was found that vitamin E is minimally oxidized in the atherosclerotic lesions and only in the combination with other antioxidant can act against development of the disease [2]. The other experiment was prepared without EDTA in buffer. Oxidation curves for neat liposome and liposome with incorporated  – tocopherol were quite different. Liposome without  – tocopherol was oxidized almost completely after 20 days. The slope of oxidation curve showed faster oxygen consumption in the first time interval and slower one in the second time interval. The reaction of PUFA with cupric ions leads to the formation of alkyl radicals which in the presence of oxygen transform to peroxyl radicals. They can further transform to lipid hydroperoxides which could be again decomposed to radicals by cuprous ions. In that way, the metal ion oscillates between the oxidized and the reduced state. These two ways of radical generation have different rate constants and that is reflected in the change of the slope of oxidation curve. The oxidation curve did not depend on the concentration of cupric ions. When  – tocopherol was incorporated into liposome vesicle, the oxidation curve was not the same. There was a long lag period before the onset of oxidation which indicates the role of  – tocopherol as antioxidant [16]. After about 15 days, the oxidation became faster and the concentration of oxygen reached the value close to that of the liposome without  – tocopherol, at same time interval. Again, the change of copper concentration did not change

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the slope of the curve. This result is different than in the first experiment where concentration of copper had influence on the rate of oxidation. In this case the oxidation process is not supported by  – tocopherol meaning that cupric ions, at least in the first phase, react more readily with lipids than with  – tocopherol, contrary to the mechanism that was suggested in the model of TMP. The molecule of  – tocopherol acts therefore as an antioxidant transforming peroxyl radicals into hydroperoxides. The rate constant for the reaction of  – tocopheroxyl radical with PUFA is rather low [17] and therefore the oxidation process is very slow. After depletion of  – tocopherol, the oxidation becomes faster than in neat liposome in that time interval. 5. Conclusions FT-IR spectroscopic studies demonstrate the different localization of  – tocopherol in its complex with liposome, depending on the way of vesicle preparation. EPR oxidation measurements suggest also the different mechanism of lipid oxidation and the role of  – tocopherol in these two systems due to its different position in the liposome. The significantly slower oxidation of liposome in comparison with lipoproteins, under same conditions, indicates that protein part of the particle is important in the oxidation process.

Acknowledgement This work was supported by Ministry of Science, Education and Sports of the Republic of Croatia (project No. 108-1080134-3105). We acknowledge Department of Biophysics, Faculty of Pharmacy and Biochemistry for the use of EPR spectrometer and Department of Chemistry and Biochemistry, University of Zagreb School of Medicine for the use of ultrasound bath.

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[14] M. Kuzuya, K. Yamada, T. Hayashi, C. Funaki, M. Naito, K. Asai, A. Kanichi, F. Kuzuya, "Role of lipoprotein-copper complex in copper catalyzed-peroxidation of lowdensity lipoprotein", Biochim. Biophys. Acta 1123 (1992) 334-341. [15] D. Krilov, J.N. Herak, "Probabilistic kinetic model of slow peroxidation of low-density lipoprotein: 3. Hydroperoxide-free initiation", J. Chem. Inf. Model. 45 (2005) 1616-1620. [16] M. Iwatsuki, E. Niki, D. Stone, V. Darley Usmar, " – tocopherol mediated peroxidation of copper (II) and met myoglobin induced oxidation of human low density lipoprotein: the influence of lipid hydroperoxides", FEBS Lett. 1995 (360) 271-276. [17] G.W. Burton, K.U. Ingold, " Vitamin E: application of the principles of physical organic chemistry to the exploration if its structure and function", Acc. Chem. Res. 19 (1986) 194201.

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Figure captions Fig.1. FT-IR spectra in 3200 - 2600 wavenumber region: (A) neat liposome, (B) liposome with incorporated - tocopherol, (C) liposome and - tocopherol mixture, (D) neat tocopherol; the arrow marks (C–H) band from methyl groups. Fig.2. FT-IR spectra in 1800 - 600 wavenumber region: (A) neat liposome, (B) liposome with incorporated - tocopherol, (C) liposome and - tocopherol mixture, (D) neat tocopherol; 1- (C=O) from ester bond, 2 - (C=C) skeletal vibrations, 3 - (CH2)sci, 4 (CH3)sym + w(CH2), 5 - (PO2-)as and (PO2-)sym, 6 - (CO–O) of ester sn-1 and sn-2 single bonds, 7 - (C–O–C), 8 - (N+(CH3)3)as, 9 - (C–C) skeletal vibrations near phospholipid head, 10 - (N+(CH3)3)sym + (C–C) skeletal vibrations near end of chain, 11 - r(CH), 12 r(CH2). Fig.3. FT-IR spectra in 1800 - 600 wavenumber region: (A) native liposome, (B) oxidized liposome; arrows above spectrum B indicate changes in vibration bands induced by oxidation; in islets are two enlarged parts of spectra presenting deformation bands from side chain groups. Fig.4. FT-IR spectra in 1800 - 600 wavenumber region: (A) native liposome with incorporated  - tocopherol, (B) oxidized liposome with incorporated  - tocopherol; arrows above spectrum B indicate changes in vibration bands induced by oxidation; in islets are two enlarged parts of spectra presenting deformation bands from side chain groups. Fig.5. Consumption of oxygen in liposome suspension with EDTA as a function of incubation time. Oxygen concentration was measured by EPR oximetry using CTPO as a spin probe. Oxidation was initiated by addition of cupric ions 1 to 10 lipids: panel 1 – neat liposomes (○) and of liposomes and  – tocopherol mixture (□); panel 2 – oxidation curves for liposomes and  – tocopherol mixture with different number of added cupric ions to one lipid: 10 (▲), 20 (▼), 50 () and 100 (■). Fig.6. Consumption of oxygen in liposome suspension without EDTA as a function of incubation time. Oxygen concentration was measured by EPR oximetry using CTPO as a spin probe. Oxidation was initiated by addition of cupric ions 1 to 10 lipids: neat liposome (), liposome with embedded  – tocopherol (■).

12

A

A

B

B

C

C D

3200

3100

3000

2900

2800

wavenumber / cm-1

2700

2600

5

7

3

1

8

2

10

11 A

A 9

4 B

B

6

12

C C

D 1800

1600

1400

1200

1000

wavenumber / cm-1

800

600

800 750 700 1400 1350 1300

B B

A

A 1800

1600

1400

1200

1000

wave number / cm-1

800

600

[O2] / µM

200 180 160

panel 1

140

[O2] / µM

200 180 160 140

panel 2 120 0

5

10

15

20

t / day

25

30

35

250

200

[O2] / µM

150

100

50

0 0

5

10

15 t / day

20

25

prooxidant

antioxidant

liposome

α-tocopherol

Highlights  Liposome was used as a model system for lipid monolayer of lipoprotein.  Interaction of -tocopherol with liposome was studied by FT-IR spectroscopy.  Localization of -tocopherol depended on a way of its import into lipid system.  Oxidation dynamics of liposome and that with -tocopherol was studied by EPR.  Depending on localization, -tocopherol acts as antioxidant or prooxidant.