The Interaction of Abietic Acid with Phospholipid Membranes Francisco J

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The interaction of abietic acid with phospholipid membranes Francisco J. Aranda ), JoseÂÂ Villalaõn Departamento de BioquõmicaÂÂ y Biologõa Molecular() A , Edificio de Veterinaria, UniÕersidad de Murcia, Apdo. 4021, E-30080 Murcia, Spain Received 1 November 1996; revised 19 February 1997; accepted 27 February 1997

Abstract

Abietic acid is a major component of the oleoresin synthesized by many conifers and constitutes a major class of environmental toxic compounds with potential health hazard to animal, including human, and plant life. Being an amphipathic molecule, the study of the influence of abietic acid on the structure of membranes would be important to get insight into the mechanism of toxic action of the molecule. The interaction of abietic acid with model membranes of dipalmitoylphosphatidylcholineŽ. DPPC and dielaidoylphosphatidylethanolamine Ž. DEPE has been studied by differential scanning calorimetry and 31 P-nuclear magnetic resonance spectroscopy. It has been found that abietic acid greatly affects the phase transition of DPPC, shifting the transition temperature to lower values, giving rise to the appearance of two peaks in the thermogram and to the presence of fluid immiscible phases. In a similar way, the phase transition of DEPE, in the presence of abietic acid, was shifted to lower temperatures, and two peaks appeared in the thermograms. The temperature of

the lamellar to hexagonal HII phase transition was also decreased by the presence of abietic acid, but phase immiscibilities were not detected. The possible implications of these effects on the action of abietic acid on biological membranes are discussed.

Keywords: Abietic acid; Model membrane; DSC; NMR, 31P-; Lipid polymorphism

1. Introduction hazard to animal and plant life. Abietic acidŽ Scheme 1. is a major component of the rosin fraction of The amount and variety of aromatic hydrocarbons oleoresin synthesized by grand fir Ž.Abies grandis , and their derivatives used in industrial and commer- lodgepole pine Ž.Pinus contorta , and many other cial activities has been increasing over the years. The conifer specieswx 1 . The production of oleoresins by release of these compounds into the environment is conifer species is an important component of the of a great concern because of their potential health defense response against insect attack and fungal pathogen infectionwx 2 . Resin acids are a major class of toxicants discharged in the effluent of pulp and paper mills to aquatic environmentswx 3 and they have Abbreviations: DEPE, dielaidoylphosphatidylethanolamine; been shown to be lethal to fish affecting respiration DPPC, dipalmitoylphosphatidylcholine; DSC, differential scan- 31 31 and energy metabolismwx 4 and causing liver dysfunc- ning calorimetry; P-NMR, P-nuclear magnetic resonance; Tc, main gel to liquid-crystalline phase transition temperature tionwx 5 . It is known that the dehydro derivative of ) Corresponding author. Fax: q34 68 364147. abietic acid is toxic to the rat, impairing co-ordination

phism'. These non-bilayer structures include the mi-

cellar phase, the hexagonal HII phase and lipidic particleswx 16 , which can greatly affect the functional behavior of the membranewx 17 , being intermediates in vesicle fusion and involved in lipid flip-flop. Non- bilayer structures may also modulate the energetics and kinetics of transport across membraneswx 18 . There Scheme 1. Structure of abietic acidŽ 13-isopropylpodocarpa- is also strong evidence that the function of membrane 7,13-dien-15-oic acid. . proteins can be affected by the modification of the properties of the lipid matrix in which they are embeddedwx 19 . and producing paralysiswx 6 . It has been suggested Since lipid polymorphism has such potential bio- that abietic acid might be the specific etiologic agent logical importance, in this investigation we have in the development of acute and chronic lung disease checked whether abietic acid might modulate it. We in workers exposed to resin derived from pine wood have studied the interaction between abietic acid and wx7. phosphatidylethanolamine because this phospholipid, The mechanism whereby abietic acid causes the being a major component of eukaryotic membranes, toxic effects is still unknown. Since many amphi- spontaneously adopts hexagonal HII phases in the pathic compounds affect the function and integrity of presence of excess aqueous buffer at physiological cell membranes, it seems possible that resin acids, temperatures. At the same time, we have investigated also amphipathic compounds, might act in a similar the interaction of abietic acid with phosphatidyl- manner. In this respect, it has been shown that the choline, the most important class of membrane phos- dehydro derivative of abietic acid affects the physical pholipids. We have used differential scanning state of cytoskeletal proteins and the lipid bilayer of calorimetryŽ. DSC and31 P-nuclear magnetic reso- erythrocyte membraneswx 8 . Abietic acid and deriva- nanceŽ 31 P-NMR. to assess the influence of abietic tives have been reported to act as non-specific in- acid on the phase behavior of the phospholipids. The hibitors of platelet aggregationwx 9 , they affect the modulation of DPPC thermotropic properties and permeability of synaptosomal membraneswx 10 and DEPE polymorphic behavior by abietic acid is dis- have a direct lytic activity to alveolar epithelial cells cussed in the light of its possible mechanism of toxic producing profound lung epithelial injurywx 11 . The action. above facts, together with the marked amphipathicity of resin acids, points to biological membranes as one of their target sites of toxic action. From this point of 2. Materials and methods view, the knowledge of the interaction between abietic acid and membrane lipids may help to get insight 2.1. Materials into the mechanism of action of the amphipathic toxin. Dipalmitoylphosphatidylcholine and dielaidoyl- The purpose of this study was to investigate in phosphatidylethanolamine were obtained from Avanti detail the interaction of abietic acid with membranes Polar LipidsŽ. Birmingham, AL, USA . Abietic acid using lipid vesicles as model systems. Membrane was obtained from Aldrich ChemieŽ Steinheim, Ger- model systems are widely used to study the mecha- many. . Organic solvents were obtained from Merck nism of interaction of membrane active molecules Ž.Darmstadt, Germany . Deionized and twice distilled with lipidswx 12±15 . It is known that dispersion of water was used. individual or mixtures of phospholipids of biological origin or synthetic ones can adopt several non-bilayer 2.2. Differential scanning calorimetry structures, in addition to the familiar bilayer organization. The ability of certain lipids to adopt these The lipid mixtures for calorimetry measurements different structures is known as `lipid polymor- were prepared by combination of chloroformr F.J. Aranda, J. VillalaõnÂ rBiochimica et Biophysica Acta 1327() 1997 171±180 173 methanolŽ. 1:1 solutions containing 4 mmol of the tion decays were obtained from up to 1600 scans. A phospholipid and the appropriate amount of abietic spectral width of 25 000 Hz, a memory of 32 K data acid as indicated. The organic solvents were evapo- points, a 2 s interpulse time and a 808 radio frequency rated under a stream of dry N22 , free of O , and the pulse were used. Prior to Fourier transformation, an last traces of solvents were removed by a further 3 h exponential multiplication was applied resulting in a evaporation under high vacuum. After the addition of 100 Hz line broadening. 1 ml of 0.1 mM EDTA, 100 mM NaCl, 10 mM HepesŽ. pH 7.4 buffer, multilamellar liposomes were formed by mixing, using a bench-vibrator, always 3. Results keeping the samples at a temperature above the lamellar gel to lamellar liquid-crystalline phase tran- The DSC profiles for pure DPPC and mixtures of sition temperature of the lipid. The suspensions were DPPC with abietic acid are shown in Fig. 1. The pure centrifuged at 13 000 rpm in a bench microfuge and phospholipid showed its main lamellar gel to lamellar the pellets were collected and placed into small alu- liquid-crystalline phase transition temperature Ž.Tc at minum pans. Pans were sealed and scanned in a 40.3"0.28C, in agreement with previous reportswx 21 . Perkin±Elmer DSC-4 calorimeter, using a reference The thermotropic pretransition of DPPC was greatly pan containing buffer. The heating rate was 4 C8rmin affected by the presence of a very low concentration in all the experiments. The DSC instrument was set at of abietic acid, being already abolished at an abietic a sensitivity of 1 mcalrs full scale. For the determi- acid mol fraction of 0.03. Increasing the concentra- nation of the total phospholipid contained in a pan, tion of abietic acid in the membrane produced the this was carefully opened, the lipid was dissolved broadening and shifting of the transition peak to with chloroformrmethanolŽ. 1:1 and the phosphorus lower temperatures. At an abietic acid mol fraction of content were determined using the method of BotcherÈ et al.wx 20 . The instrument was calibrated using in- dium as standard. The points used to construct the boundary lines in the phase diagrams where obtained from the beginning and the end of the transition peaks in the heating thermograms.

2.3. 31P-Nuclear magnetic resonance

The samples for 31P-NMR were prepared by combination of chloroformrmethanolŽ. 1:1 solutions containing 50 mg of phospholipid and the appropriate amount of abietic acid. Evaporation of the solvents and multilamellar vesicles were formed as described above. The suspensions were centrifuged at 13 000 rpm in a bench microfuge and pellets were placed into conventional 5 mm NMR tubes and 31P-NMR spectra were obtained in the Fourier Transform mode in a Varian Unity 300 spectrometer. All chemical shift values are quoted in parts per millionŽ. ppm with reference to pure lysophosphatidylcholine mi- cellesŽ. 0 ppm , positive values referring to low-field Fig. 1. DSC thermograms for mixtures of DPPCrabietic acid. The concentration of abietic acid in the membraneŽ. mol fraction shifts. All spectra were obtained in the presence of a is expressed on the curves. The profiles correspond to heating gated-broad band decouplingŽ 10 W input power scans. Arrows indicate the peak chosen to determine the begin- during acquisition time. and accumulated free induc- ning of the transition. 174 F.J. Aranda, J. VillalaõnÂ rBiochimica et Biophysica Acta 1327() 1997 171±180

Table 1 phase transition of DPPC. A direct relationship be- The enthalpy changes Ž.DH, kcalrmol for the lamellar gel to tween DH and the abietic acid concentration was not lamellar liquid-crystalline phase transition of mixtures of observed. DPPCrabietic acid and DEPErabietic acid at different abietic acid mol fractionsŽ values are means standard deviation of four In order to check whether abietic acid affects the " 31 different experiments. phase behavior of DPPC, P-NMR measurements at different temperatures were carried out. As shown in Abietic acid mol fraction DH Ž.kcalrmol Fig. 2, the presence of abietic acid did not change the DPPC DEPE phospholipid phase organization, which remained in 0 8.5"0.6 8.3"0.3 the lamellar phase over the whole range of tempera- 0.01 7.8"0.9 8.0"0.4 0.03 8.5"0.5 6.4"0.6 tures under study. These data indicated that the dif- 0.05 7.9"0.2 6.7"0.6 ferent endotherms presented in the various thermo- 0.07 7.6"0.6 6.5"0.1 grams corresponded solely to lamellar gel to lamellar 0.10 7.4"0.1 5.8"0.5 liquid-crystalline phase transitions. However, the 0.15 6.3"0.8 6.2"0.6 spectra obtained at high concentrations of abietic acid 0.20 7.0"0.2 5.8"0.2 Ž 0.30 8.9"0.9 6.5"1.0 showed a reduced chemical shift anisotropy Fig. 0.50 7.6"0.6 6.7"0.3 2D. , suggesting that the presence of the toxicant increased the mobility of the DPPC molecules. From the beginning and the end temperatures of 0.15 two different peaks were present in the thermo- the heating thermograms we constructed the partial gram. The incorporation of higher concentrations of phase diagram for DPPCrabietic acid systemŽ shown abietic acid produced the shifting of both peaks to in Fig. 3. . This system is a mixture of a phospholipid lower temperatures, this effect being greater on the Ž.DPPC and a molecule with a very different struc- low temperature transition peak than on the high tureŽ. abietic acid which does not undergo any phase temperature one. As shown in Table 1, the presence transition in the range of temperature under study, of abietic acid did not produce a drastic decrease of and therefore, all the observed thermotropic transi- DH of the lamellar gel to lamellar liquid-crystalline tions arose from the phospholipid component in the

31 Fig. 2. P-NMR spectra at different temperatures corresponding to pure DPPCŽ. A ; DPPCrabietic acid, 0.05 mol fraction Ž. B ; DPPCrabietic acid, 0.10 mol fractionŽ. C and DPPCrabietic acid, 0.30 mol fraction Ž. D . F.J. Aranda, J. VillalaõnÂ rBiochimica et Biophysica Acta 1327() 1997 171±180 175

and the lamellar to hexagonal HII structural phase transition occurs around 648C in agreement with previous datawx 22 . The latter has a much smaller transition enthalpy due to the fluid character of both the

lamellar and the hexagonal HII phasewx 23 . As shown in Fig. 4, the presence of increasing concentrations of abietic acid produced a broadening and shifting of the lamellar gel to lamellar liquid-crystalline phase tran- Fig. 3. Partial phase diagram for DPPC in mixtures of sition peak to lower temperatures. At 0.07 and 0.10 DPPCrabietic acid. Black Ž.v and open circles Ž.` were ob- abietic acid mol fraction a shoulder at the lower tained from the begining and the end temperatures of the transi- temperature side of the endotherm is clearly ob- tions, respectivelyŽ. see text for details . G indicates a lamellar gel served. The effect of abietic acid on the lamellar to X phase, F and F indicate different lamellar liquid-crystalline hexagonal H phase transition was even more dras- Ž II phases. Points represent the average of four experiments error tic, it shifted the transition to lower temperatures such bars are shown when larger than the symbols. . that at an abietic acid mol fraction of 0.15 the transition could no longer be observedŽ. Fig. 4 . The presence of abietic acid only produced a slight de- mixture. Because of that, this phase diagram corre- crease in the enthalpy change of the lamellar gel to sponds to a partial phase diagram for the DPPC lamellar liquid crystalline phase transition of DEPE component in mixtures of the phospholipid and abi- Ž.Table 1 . etic acid. To simplify the partial phase diagram, we did not consider the pretransition of DPPC. The thermotropic analysis of the pretransition is difficult as it is abolished at very low abietic acid contentŽ see Fig. 1. . The beginning and the end temperatures of the transitions gave us the points to obtain the solid and fluid lines, respectively. The temperature of the solid line decreased upon increasing the concentration of abietic acid. The fluid line kept horizontal, i.e., constant temperature, up to 0.2 mol fraction of abietic acid, whereas higher concentrations of abietic acid produced a decline of the fluid line, i.e., a decrease of temperature. The phospholipid component evolved from a lamellar gel phaseŽ. G phase to a lamellar liquid-crystalline phaseŽ. F phase through a broad region of phase coexistenceŽ. GqF , but in a concentration range of abietic acid from 0 to 0.2 mol fraction, a fluid phase immiscibility was observed X ŽFqF.. The effect of abietic acid on the thermotropic phase transitions of DEPE is shown in Fig. 4. Aque- ous dispersion of DEPE can undergo a gel to liquid- crystalline phase transition in the lamellar phase and in addition a lamellar to hexagonal HII structural phase transitionwx 22 . This is shown in the thermo- Fig. 4. DSC thermograms for the mixture DEPErabietic acid. gram corresponding to DEPE dispersed in buffer The concentration of abietic acid in the membraneŽ. mol fraction Ž.Fig. 4, upper part . The lamellar gel to lamellar is expressed on the curves. The profiles correspond to heating liquid-crystalline phase transition occurs around 368C scans. 176 F.J. Aranda, J. VillalaõnÂ rBiochimica et Biophysica Acta 1327() 1997 171±180

of a small signal at 608CŽ. Fig. 5B , superimposed to the bilayer lineshape. The resonance position of this signal coincides with the chemical shift of the low-

field peak of the hexagonal HII phase lineshape observed at higher temperatures. This is in agreement with the shift to lower temperatures of the lamellar to

hexagonal HII phase observed by DSCŽ. Fig. 4 . At an abietic acid 0.10 mol fraction the characteristic spec-

trum corresponding to the hexagonal HII phase appeared at 548CŽ. Fig. 5C and at an abietic acid 0.30

mol fraction the hexagonal HII is already present at 408CŽ. Fig. 5D . Using the DSC data and the information of phospholipid structural organization obtained from 31P- NMR, we constructed a partial phase diagram for DEPE in mixtures of DEPErabietic acidŽ. Fig. 6 . In a concentration range of abietic acid from 0 to 0.1 mol fraction, both the main lamellar gel to lamellar liquid-crystalline phase transition and the structural

lamellar to hexagonal HII phase transition of DEPE Fig. 5. 31P-NMR spectra at different temperatures corresponding were clearly distinguishableŽ. Fig. 4 . We used the to pure DEPEŽ. A ; DEPErabietic acid, 0.05 mol fraction Ž. B ; temperature of the beginning and the end of the DEPErabietic acid, 0.10 mol fractionŽ. C and DEPErabietic lamellar gel to lamellar liquid-crystalline phase tran- acid, 0.30 mol fractionŽ. D . sition to obtain the solid and fluid lines, respectively, whereas the temperature of the beginning and the end

The effect of abietic acid on the thermotropic of the lamellar to hexagonal HII phase transition phase transitions of DEPE was further investigated were used to obtain the lamellar and hexagonal lines by31 P-NMRŽ. Fig. 5 . DEPE when organized in respectively. At concentrations of abietic acid higher bilayer structures gives rise to a characteristic asym- than 0.1 mol fraction, the lamellar to hexagonal H II metrical 31P-NMR line-shape, with a high-field peak phase transition peak is not observed by DSCŽ Fig. and a low-field shoulderwx 24 . The chemical shift 4. . In these cases we used the temperature of the anisotropyŽ measured as 3 times the chemical shift difference between the 90 degrees edge and the position of isotropically-moving lipid molecules. is ap- proximately 40 ppm in the liquid-crystalline state, in agreement with previous datawx 24 , characteristically of an axially symmetrical tensorŽ. Fig. 5A . In the lamellar gel state, the lineshape is broadened com- pared with the lamellar liquid-crystalline state where the lineshape is considerably narrower. For DEPE organized in hexagonal HII phaseŽ. Fig. 5A, 758C additional motional averaging is experienced due to diffusion of the phospholipids around the cylinders of Fig. 6. Partial phase diagram for DEPE in mixtures of which this phase is composed. This results in a 2-fold DEPErabietic acid. Black and open symbols were obtained from reduction in effective chemical shift anisotropy and a the beginning and the end temperatures of the transitionsŽ see text . Ž for details . G indicates a lamellar gel phase, F indicates a reversed asymmetry i.e., a high-field shoulder and a lamellar liquid-crystalline phases and H indicates an hexagonal low-field peak. 16,25 . The incorporation of abietic II wx HII phase. Points represent the average of four experimentsŽ error acid at a 0.05 mol fraction produces the appearance bars are shown when larger than the symbols. . F.J. Aranda, J. VillalaõnÂ rBiochimica et Biophysica Acta 1327() 1997 171±180 177 beginning and the end of the observed transition to ones. These observations are compatible with the obtain the solid and fluid lines. According to 31P- abietic acid molecule aligning itself with the prevail- NMR data, the boundaries defined by these solid and ing directions of the phospholipid acyl chains. The fluid lines are different from those defined at lower polar carboxyl group of abietic acid will prevent the abietic acid content. The temperature of all the molecule to be located in the terminal methylene boundary lines decreased as the concentration of region of the fatty acyl chains of the phospholipids, abietic acid increased. Up to a concentration of abi- which otherwise would be the preferred location for a etic acid of 0.1 mol fraction the system evolved from completely hydrophobic molecule. The carboxyl a lamellar gel phaseŽ. G phase to a lamellar liquid- group tends to locate itself near the polar group of the crystalline phaseŽ. F phase through a very narrow phospholipids, i.e., near the water interface, where it coexistence regionŽ. GqF , and then to a hexagonal could form hydrogen bonding with water and might HII phaseŽ. H II phase through a coexistence region also establish other types of interactions with the Ž. FqHII . At concentrations of abietic acid higher polar part of the phospholipids. This would restrict than 0.1 mol fraction, the system evolved from a the location of the hydrophobic rings of the abietic lamellar gel phaseŽ. G phase to a hexagonal H II acid molecule to the upper part of the phospholipid Ž. Ž phase HII phase through a coexistence region Gq palisade. Due to the length of the abietic acid HII. , apparently without an intervening lamellar liq- molecule, this region would probably be that com- uid-crystalline phase. prised between methylene C1 and methylene C7 of the fatty acyl chain of the phospholipid. Such a location would perturb the phospholipid acyl chains, 4. Discussion and consequently we found that abietic acid drastically decreased the onset temperature for the lamellar Abietic acid is a tricyclic diterpenoid carboxylic gel to lamellar liquid-crystalline phase transition. At acid with an isopropyl substituent. The amphipathic concentrations higher than 0.10 mol fraction of abi- nature of this toxic molecule makes biological mem- etic acid, the thermograms are more complex and branes one of its most likely sites of action. Using several components are observed. This might be due DSC and 31P-NMR, we have carried out the study of to a lateral phase separation of abietic acid rich the interaction between abietic acid and lipid vesicles domains. Nevertheless, enough abietic acid seemed to formed by two different classes of phospholipid of remain in the bulk phase so that there was a further major relevance, phosphatidylcholine and phos- decrease of Tc of the components of the thermo- phatidylethanolamine. We used DSC in order to char- grams. It seems that the presence of abietic acid acterize the influence of abietic acid on the ther- perturbs the packing of some phospholipids but these motropic properties of the phospholipids. The profile perturbed molecules undergo a transition with a DH of a DSC thermogram of a phospholipid phase transi- similar to that of the unperturbed phospholipidsŽ Ta- tion is determined by the transition temperature and ble 1. . Similar effects on the lamellar gel to lamellar the enthalpy change. Determining the temperatures of liquid-crystalline phase transition of DPPC, i.e., a the transitions allows the construction of phase dia- decrease of Tc with no change in DH, have been grams, which provide information regarding the equi- previously reported for C510 ±C alcohols and C 710 ±C librium between different phases. fatty acidswx 26 . For DPPCrabietic acid systems we observed a Adding a foreign moleculeŽ. abietic acid to a progressive broadening of the transition peak and a phospholipid systemŽ. DPPC would normally be ex- shift of the Tc to lower temperaturesŽ. Fig. 1 . Abietic pected to change the transition temperature of the acid perturbs the cooperative behavior of the phos- phospholipid, if both molecules are miscible. We pholipid. This could be explained by the establish- observed that the temperature of the solid line de- ment of a molecular interaction between the phospho- creased as more abietic acid is present in the system lipid acyl chain and the abietic acid molecule. This Ž.Fig. 3 . This indicates that DPPC and abietic acid interaction would be the consequence of the intercala- are miscible in the lamellar gel phase, and the interca- tion of the abietic acid molecules between the DPPC lation of the abietic acid molecule into the phospho- 178 F.J. Aranda, J. VillalaõnÂ rBiochimica et Biophysica Acta 1327() 1997 171±180 lipid palisade will perturb the thermotropic properties to hexagonal HII phase transition which has a low of the phospholipid. We found that the fluid line kept heat content due to the liquid-crystalline state of the horizontal in a concentration range of abietic acid acyl chains in both phases. Our 31P-NMR experi- from 0 to 0.2 mol fraction, at the temperature of the ments confirmed the above interpretations, showing end of the transition of pure DPPC. The fact that no that abietic acid is able to promote hexagonal H II perturbation in the temperature is observed indicates structures at temperatures lower than for pure DEPE. that a fluid immiscibility occurs, suggesting the for- At an abietic acid concentration of 0.05 mol fraction Ž. mation of an abietic acidrDPPC domain F phase the transition to the hexagonal HII phase is shifted immiscible with free DPPCŽ FX phase. in the lamellar down by about 4 C8 Ž.Fig. 5B . The effect on this liquid-crystalline phase. This is an interesting obser- transition is greater at higher abietic acid concentra- vation which has been reported for unsaturated fatty tionsŽ. Fig. 5, C and D where shifts of 10 and 25 C8 acidswx 26 , diglycerides wx 27 and retinol wx 28 . This were observed. type of immiscibility was predicted on the basis of The location of abietic acid in the upper part of the theoretical calculations for mixtures of DPPC and phospholipid palisade would enable the polar part of anaestheticswx 29 , where a relatively strong interaction the molecule to establish hydrogen bonding with the between the anaesthetic molecules was supposed, so polar head of DEPE molecules, and this could lead to that clusters were formed. Above 0.2 mol fraction of an effective reduction of the headgroup area of the abietic acid, the temperature of the fluid line de- phospholipid, explaining the facilitating effect of abi- creased indicating a miscibility in the lamellar etic acid on the formation of hexagonal HII struc- liquid-crystalline phase at these concentrations of the tures. We found the presence of a minor isotropic toxicant. signal in the spectra corresponding to 0.1 and 0.3 mol Abietic acid produces a broadening and a shifting fraction of abietic acid in the gel phaseŽ.Ž 258C Fig. to lower temperature of the main lamellar gel to 5, C and D. . The presence of an isotropic signal in lamellar liquid-crystalline phase transition of DEPE. phosphatidylethanolamine systems have been previ- However this effect is less severe than that observed ously reportedwx 30±32 . From the phase diagramŽ Fig. in DPPCŽ. compare Fig. 1 with Fig. 4 . The effect of 6. , it can be concluded that the system does not abietic acid on the lamellar to hexagonal HII phase present immiscibilities in any of the described phases transition of DEPE is more pronounced. Incorpora- as the temperature of all boundary lines decreased as tion of increasing concentrations of the toxicant re- more abietic acid is present. Interestingly, at concen- sults in a progressive decrease of enthalpy and tem- trations of abietic acid higher than 0.10 mol fraction perature of this transition. At a 0.15 molar fraction of the system evolved directly from the lamellar gel abietic acid this transition cannot be observed any phase to the hexagonal HII phase. more. This can be interpreted that upon abietic acid The main result of this study is that abietic acid is incorporation, the DEPE molecules interacting with able to be incorporated in phospholipid membranes, the toxicant give rise to a broad lamellar liquid-crys- probably aligning itself with the phospholipid acyl talline to hexagonal HII phase transition which is chains, perturbing the packing of the lipids and af- shifted to lower temperatures. When the content of fecting their thermotropic properties. When consider- abietic acid is higher than 0.10 molar fraction a very ing the possible action of abietic acid in biological broad lamellar to hexagonal HII phase transition is membranes, it is interesting to note that, as shown present. In the presence of these high amounts of above, the system may present immiscibilities in the abietic acid, the structural lamellar to hexagonal H II fluid phase, a remarkable effect, given the usual fluid phase transition occurs at such a low temperature that condition of biological membranes. This fluid-phase it takes place concomitantly with the chain melting immiscibilities of abietic acid could give rise to the Ž.lamellar gel to lamellar liquid-crystalline transition formation of domains, where the concentration of the of the phospholipid. The endothermic transitions ob- toxic molecule could be specially high. On the other served in the thermograms corresponding to high mol hand abietic acid is able to affect lipid polymorphism fraction of abietic acid originate from the melting of on phosphatidylethanolamine systems, promoting the the acyl chains. These endotherms mask the lamellar hexagonal HII phase. F.J. Aranda, J. VillalaõnÂ rBiochimica et Biophysica Acta 1327() 1997 171±180 179

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