Interaction of methanol, ethanol, propanol and butanol with human plasma low density lipoproteins (LDL) was studied in this work. The surface lipid monolayer of LDL was spin labeled and the electron spin resonance (ESR) spectra were measured in the presence and absence of alcohols. The decomposition of the complex ESR spectra was performed via theoretical simulations of experimental data. The results gained from this study indicate that the influence of alcohol could be observed through the changes of lipid ordering in the surface of LDL monolayer. This observation supports the hypothesis on the mechanism of alcohol action through its interference with lipid-protein interactions at the level of macromolecular surface

Greta Pifat

Marina Kveder

Milan Schara

Slavko Pečar

English

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ISSN-0011-1643 Original Scientific PaperCCA-2488The Influence of Lower Alcohols on the Surface LipidMonolayer in LDLMarina Kveder,a Greta Pifat,a Slavko Pe~ar,b,c and Milan ScharabaRu|er Bo{kovi} Institute, P.O.B. 1016, 10001 Zagreb, Croatia(e-mail: kveder@rudjer.irb.hr)bJ. Stefan Institute, 61000 Ljubljana, SloveniacFaculty of Pharmacy, University of Ljubljana, 61000 Ljubljana, SloveniaReceived July 2, 1997; revised October 3, 1997; accepted October 27, 1997Interaction of methanol, ethanol, propanol and butanol with hu-man plasma low density lipoproteins (LDL) was studied in thiswork. The surface lipid monolayer of LDL was spin labeled and theelectron spin resonance (ESR) spectra were measured in the pre-sence and absence of alcohols. The decomposition of the complexESR spectra was performed via theoretical simulations of experi-mental data. The results gained from this study indicate that theinfluence of alcohol could be observed through the changes of lipidordering in the surface of LDL monolayer. This observation sup-ports the hypothesis on the mechanism of alcohol action throughits interference with lipid-protein interactions at the level of ma-cromolecular surface.INTRODUCTIONVarious species circulating in the bloodstream can undergo mutual in-teractions. This is especially important when the influence of alcohol on thelipid-protein assemblies is considered. So far, there is still a disagreementas to whether the primary site to be affected by alcohol is the lipid or pro-tein phase of the biosystem.1Reported data2 claim that chronic abusers can survive bloodstreamethanol levels in excess of 0.3 M. On the other hand, it is known3 that thelevel of low density lipoproteins (LDL) in human serum is about 1.2 mM.Considering this LDL concentration range it could be expected that, besidesCROATICA CHEMICA ACTA CCACAA 71 (1) 189¿198 (1998)other molecules circulating in the blood, also the interaction of the blood-stream alcohol with LDL particles is possible. Therefore, it is important tounderstand the molecular mechanism of their interaction. In our previouswork,4 the interaction of lower alcohols with human plasma LDL was ad-dressed. Using the electron spin resonance (ESR) spectroscopic method, theexperimental evidence of alcohol-LDL interaction was offered. The LDL par-ticles were spin labeled at the level of apoB and the ESR data were explai-ned following the arguments of the phospholipid mediated mechanism of al-cohol action, which results in the protein conformational change. In order tocontinue along the same line of investigation, in this work the surface lipidmonolayer of LDL was spin labeled and the ESR spectra analyzed in thepresence of different alcohols. The aim of the study was to detect whetherthe changes in the ordering of the surface lipid phase in LDL are inducedupon the interaction with alcohol.MATERIALS AND METHODSMaterialsThe spin label 1-oxyl-2,2,-dimethyl-4-undecyl-4-methoxy-carbonylpropyl-oxazolidine (MeFASL(10,3)) was synthesized by one of the authors (S.P.) ac-cording to the published procedure.5Alcohols were of p.a. purity and purchased from Kemika, Zagreb. In or-der to get the desired final alcohol concentration in the lipoprotein solution,a double alcohol concentration of the stock solution was prepared and thenmixed with the sample in 1:1 ratio.All the experiments were performed in the 0.1 M Tris-HCl buffer (1 g/LEDTA; pH = 7.4).Isolation of LDLHuman plasma was obtained from two female normolipidemic donorsand lipoproteins were isolated according to the published procedures.6,7 Toavoid oxidation of lipoproteins, EDTA was present in all preparation stepsand all buffers were flushed with nitrogen.Purity of the lipoprotein samples was checked by electrophoresis.The LDL concentration was determined gravimetrically by dry weightestimation including correction for the salt content of the buffer.Spin Labeling of LipoproteinsThe spin label dissolved in ethanol was deposited as a dry thin film onthe wall of the glass tubing into which the LDL sample was added. The190 M. KVEDER ET AL.maximal expected molar ratio of nitroxide dissolved in LDL versus the esti-mated surface lipid content was always kept less than 1:100. Incubation un-der moderate shaking lasted for 5 h at 30 °C. After exhaustive dialysis inTris-HCl buffer, the LDL samples were filtered through 450 nm sterile fil-ters and the ESR measurements were started.ESR Spectroscopy and AnalysisExperiments were performed in glass capillaries (1 mm inner diameter)on an X-band Varian E-109 ESR spectrometer. The following settings wereused: microwave power, 10 mW; modulation amplitude, 0.1 mT; modulationfrequency, 100 kHz and scan range 20 mT. The acquisition and the analysisof the ESR data were performed using the EW Scientific Software Servicesprogram.8 All ESR spectra were measured at t = 37 °C.For a detailed analysis of the experimental data, computer simulation ofthe ESR spectra was performed assuming a fast nitroxide rotation about thenormal on the lipoprotein surface.9 This framework allows the use of the or-der parameter, S, defined as:  S  A AA A||||where Aand A are the crystal values of the components of hyperfinetensor while the values in brackets are obtained from the best fit. Theparameter  takes into account the polarity correction for the A hyperfinetensor.RESULTSThe molecule of MeFASL(10,3) and the assumed model of its incorpora-tion into the surface lipid monolayer of LDL is presented in Figure 1. Thisspin label probes the phospholipid acyl chain region of LDL and the orienta-tion of the nitrogen 2p orbital in the paramagnetic group of nitroxide ispreferentially parallel to the long molecular axis which coincides with thenormal to the lipoprotein surface. The ESR spectrum of the spin labeled na-tive LDL is shown in Figure 2 along with simulation of the experimentaldata. This approach revealed the superimposition of two components in theESR spectrum, denoted as A and B, with different order parameters, i.e. SA= 0.15 and SB = 0.5 and the corresponding weight factors of WA = 0.55 andWB = 0.45 respectively (insert to Figure 2). In the presence of 0.3 M metha-nol, ethanol and propanol, no substantial changes of the spin labeled LDLspectra could be observed (Figure 3) so that the same fitting parameters asINTERACTION OF LOWER ALCOHOLS WITH LDL 191in the case of the native samples are applicable (Table I). However, in theexperiments with 0.3 M butanol (Figure 3), the ESR spectrum indicates anincrease in disorder which is reflected in an increase of the weight factor forthe component with the smaller order parameter (Table I). This phenome-non can be observed for methanol and ethanol if the concentration of alcoholis increased by one order of magnitude (Table I). In the presence of 3 M pro-panol, the ESR spectrum shows denaturation of the LDL particle (Figure 4),where both components indicate an extremely low level of ordering.In order to check whether the empirical rotational correlation time, ,deduced from the ESR spectra10 for MeFASL(10,3) incorporated in LDL issensitive to the presence of alcohol, the following equation was used: = K 	H(0) [(Io/I¿1)1/2 ¿ 1] (1)where K is the proportionality constant, Io and I¿1 are the first derivativeamplitudes of the central and the high field hyperfine components in the192 M. KVEDER ET AL.Figure 1. The molecule of MeFASL(10,3) and the assumed model for its incorpora-tion into the surface lipid monolayer of LDL.ESR spectrum, respectively, and 	H(0) is the corresponding linewidth.These results are presented in Table II. Calculated  values suggest that therotational correlation time extracted directly from the spectral amplitudesand linewidth can reliably discriminate only between the native and highlydenatured particles.DISCUSSIONIn this work, the influence of alcohols, with short alkyl chains, on LDLparticles was studied at the level of the lipoprotein surface monolayer (Fig-ure 1). Methanol, ethanol, propanol and butanol were applied at two concen-trations. i.e. 0.3 M, which represents the physiological upper limit, and 3 M,INTERACTION OF LOWER ALCOHOLS WITH LDL 193Figure 2. The ESR spectrum of native LDL spin labeled with MeFASL(10,3).The concentration of LDL was 12.8 M. The experimental data (dotted) were fitted(line) with two superimposed components A and B (Insert to Figure 2) which contri-bute to the total spectrum with weight factors WA = 0.55 and WB = 0.45 respectively.The following parameters were used for the components: order parameter, SA = 0.15,SB = 0.5; rotational correlation time, tA = tB = 1.5 ns; half width at half maximum ofthe Lorentzian line shape for m = 0, GA = GB = 0.2 mT; polarity correction in the Ahyperfine tensor, kA = 1.07, kB = 1.05.194 M. KVEDER ET AL.TABLE ITheoretical simulation of the ESR spectra of spin labeled LDL exposed todifferent alcohol concentrationsType of alcoholalog(part.coeff.) ¿Methanol¿0.73Ethanol¿0.32Propanol0.34Butanol0.88Concentration,mol dm3 0 0.3 3 0.3 3 0.3 3 0.3WA0.55 0.55 0.65 0.55 0.65 0.55 0.65 0.65WB0.45 0.45 0.35 0.45 0.35 0.45 0.35 0.35SA0.15 0.15 0.15 0.15 0.15 0.15 0.01 0.15SB0.5 0.5 0.5 0.5 0.5 0.5 0.05 0.5A, ns 1.5 1.5 1.5 1.5 1.5 1.5 0.3 1.5B, ns 1.5 1.5 1.5 1.5 1.5 1.5 0.5 1.5A1.07 1.07 1.07 1.07 1.07 1.07 1.0 1.07B1.05 1.05 1.05 1.05 1.05 1.05 1.07 1.05bGA, mT 0.2 0.2 0.2 0.2 0.2 0.2 0.1 0.2GB, mT 0.2 0.2 0.2 0.2 0.2 0.2 0.15 0.2athe experimentally determined partition coefficients for each alcohol in the octanol/watersystem quoted by W. D. Stein and W. R. Lieb, Transport and Diffusion across Cell Membranes,Academic Press, 1986, p. 88.bG denotes the half width at half maximum of the Lorentzian line shape for m = 0.TABLE IIThe empirical rotational correlation time, t, for MeFASL(10,3) incorporated inLDL in the presence of alcohol deduced from the ESR spectraType of alcohol Concentration of alcohol t, ns*¿ 0 2.9methanol 0.3 M 3.0ethanol 0.3 M 2.8propanol 0.3 M 2.9butanol 0.3 M 2.5methanol 3 M 3.0ethanol 3 M 2.7propanol 3 M 0.7*The reproducibility of t determination from equation (1) is within the precision of determin-ation 	H(0), Io, I¿1 i.e.  0.2 ns.which provides the extreme denaturing condition. The interaction of alco-hols with LDL was followed by analyzing the induced changes in the ESRspectra via theoretical simulation of the experimental data.In the native LDL samples (Figure 2), at least two domains of differentordering could be assigned to populate the monolayer, which is in accor-dance with previous findings.9 The order parameter, S = 0.5, indicates arather ordered domain, whereas S = 0.15 reveals the domain with a less mo-tional restriction which slightly predominates (weight factor 0.55). The exis-tence of domains could be understood in terms of the complex chemical com-position of constituent molecules in the particle.11 Namely, the length anddegree of saturation of fatty acid building blocks in the monolayer governthe packing properties of phospholipids with cholesterol.12 In the presenceof 0.3 M methanol, ethanol and propanol (Figure 3), the ESR spectra do notindicate any visible changes of the lipid state. However, in the presence of0.3 M butanol (Table I), the increase of the weight factor to 0.65 for the com-ponent with the smaller order parameter could be observed. Thus, the lipidmonolayer became less ordered in the presence of butanol. Since differentalcohols were used at the very same molar concentrations, the difference inINTERACTION OF LOWER ALCOHOLS WITH LDL 195Figure 3. Influence of different lower alcohols on the ESR spectra of spin labeled LDL.In all experiments, the concentration of LDL was 12.8 mM and the concentration ofalcohol was 0.3 M. The numbering on each spectrum indicates the presence of a cer-tain alcohol in the experiment according to: »0« (control sample), »1« (methanol), »2«(ethanol), »3« (propanol) and »4« (butanol).the length of the respective alkyl chains is responsible for the induced per-turbation in the LDL monolayer. This observation is consistent with the re-sults of similar experiments at the apoB level.4 Therefore, the explanationfor these findings is searched in the involvement of alcohols in the lipid-protein interactions. Namely, the incorporation of alcohol at the lipid-waterinterface displaces the water molecules bound at the macromolecular sur-face.13 This event provokes a modulation of the lipid packing free volumeand induces a change in the protein conformation. Thus, the difference inthe lipophilicity of alcohols, which governs their solubility in the monolayerof the LDL particle, is the principal determinant of the alcohol-LDL interac-tions. To induce the change in the lipid chain order, the concentration ofmethanol and ethanol should be increased by one order of magnitude (Table I).Furthermore, in the presence of 3 M propanol, the ESR spectrum reflects196 M. KVEDER ET AL.Figure 4. The ESR spectra in the presence of 3 M propanol.The concentration of LDL was 12.8 mM. The experimental data (dotted) were fitted(line) with two superimposed components A and B (Insert to Figure 4) whichcontribute to the total spectrum with weight factors 0.65 and 0.35, respectively. Thefollowing parameters were used for the components: order parameter, SA = 0.01, SB =0.05; rotational correlation time, tA = 0.3 ns, tB = 0.5 ns; half width at half maximumof the Lorentzian line shape for m = 0, GA = 0.1 mT, GB = 0.15 mT; polarity correctionin the A hyperfine tensor, kA = 1.0, kB = 1.07.LDL particle denaturation (Figure 4). Both spectral components indicatethe presence of extremely disordered domains (SA = 0.01 with weight factorWA = 0.65 and SB = 0.05 with weight factor WB = 0.35). Hence, these obser-vations show that the influence of alcohol could be observed through thechanges of lipid ordering in the surface of the LDL monolayer.It should be stressed that the conclusions gained from this study could bederived only through theoretical simulations of the experimental data, allow-ing decomposition of the complex spectra. For comparison, the empirical rota-tional correlation time extracted directly from the amplitudes and linewidthsin the ESR spectra (Table II) should be taken only as a rough estimation ofthe order of magnitude for the time scale describing molecular motions.In conclusion, together with the results of our previous study,4 the ESRmethod provided the experimental evidence of LDL interaction with alco-hols. Furthermore, the explanation of the molecular mechanism of this pro-cess is proposed as the disruption of the lipid-protein interaction when alco-hol competes with water molecules for hydrogen bonding at the LDLsurface. Consequently, changes in the lipid packing are induced and propa-gated in the perturbation of the native protein conformation of apoB.Acknowledgments. ¿ This work was partly supported by the Ministry of Scienceof the Republic of Croatia, by the Ministry of Science and Technology of the Republicof Slovenia and partly by the joint Slovenia-Croatia project: »Structure anddynamics of synthetic and biological species«.REFERENCES1. R. W. Peoples, C. Y. Li, and F. F. Weght, Annual Review of Pharmacology andToxicology 36 (1996)185¿201.2. I. Diamond, Cecil Textbook of Medicine, W. B. Saunders (Ed.), Philadelphia, 1992,pp. 44¿47.3. H. Esterbauer, J. Gebicki, H. Puhl, and G. Jürgens, Free Rad. Biol. Med. 13 (1992)341¿390.4. M. Kveder, G. Pifat, S. Pe~ar, and M. Schara, Chem. Phys. Lipids 87 (1997) 125¿135.5. W. L. Hubbell and H. M. McConnell, J. Am. Chem. Soc. 93 (1971) 314¿326.6. G. Jürgens, H. F. Hoff, G. M. Chisolm and H. Esterbauer, Chem. Phys. Lipids 45(1987) 315¿336.7. H. Puhl, G. Waeg, and H. Esterbauer, Methods in Enzymology, L. Packer (Ed.),Academic Press, 1994, pp. 425¿441.8. P. D. Morse II, Biophys. J. 51 (1987) 440a.9. M. Kveder, G. Pifat, S. Pe~ar, and M. Schara, Chem. Phys. Lipids 70 (1994) 101¿ 108.10. D. Kivelson, J. Chem. Phys. 33 (1960) 1094¿1106.11. M. Kveder, G. Pifat, S. Pe~ar, and M. Schara, Chem. Phys. Lipids, 85 (1997) 1¿12.12. J. F. Tocanne, L. Cezanne, A. Lopez, B. Piknowa, V. Chram, J. F. Tournier, and M.Welby, Chem. Phys. Lipids 73 (1994) 139¿158.13. J. A. Barry and K. Gawrisch, Biochemistry 33 (1994) 8082¿8088.INTERACTION OF LOWER ALCOHOLS WITH LDL 197SA@ETAKUtjecaj ni`ih alkohola na povr{inski lipidni monosloj u LDLMarina Kveder, Greta Pifat, Slavko Pe~ar i Milan ScharaProu~avano je me|udjelovanje metanola, etanola, propanola i butanola s lipopro-teinima niske gusto}e (LDL) izoliranima iz ljudske plazme. Povr{inski monosloj LDLspinski je ozna~en, a spektri elektronske spinske rezonancije (ESR) mjereni su u pri-sutnosti i odsutnosti alkohola. Rastav slo`enih spektara ESR proveden je teorijskomsimulacijom eksperimentalnih podataka. Rezultati dobiveni u ovom istra`ivanju po-kazuju da se utjecaj alkohola mo`e opaziti kroz promjenu ure|enosti lipida povr-{inskog monosloja LDL. To opa`anje podr`ava hipotezu o mehanizmu djelovanjaalkohola kroz njegovu interferenciju s me|udjelovanjem lipid/protein na makromole-kulskoj povr{ini.198 M. KVEDER ET AL.

The Influence of Lower Alcohols on the Surface Lipid Monolayer in LDL

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