Thermodynamic and Brewster Angle Microscopy ... - ACS Publications

J. Phys. Chem. B 2000, 104, 7735-7744

7735

Thermodynamic and Brewster Angle Microscopy Studies of Fatty Acid/Cholesterol Mixtures at the Air/Water Interface R. Seoane, J. Min˜ ones,* O. Conde, J. Min˜ ones, Jr., M. Casas, and E. Iribarnegaray Departamento de Quı´mica Fı´sica, Facultad de Farmacia, Campus Sur, UniVersidad de Santiago de Compostela, E-15706 Santiago de Compostela, Spain ReceiVed: March 27, 2000; In Final Form: June 6, 2000

Mixtures of cholesterol with stearic (STA), oleic (OA), and linoleic (LA) acids spread as monolayers at the air/water interface were used as model systems to examine the hypocholesterolemic effect of fatty acids. Miscibility and interactions between the components of the cholesterol/fatty acid systems were studied basing on the analysis of surface pressure/area isotherms completed with Brewster angle microscopy images. In monolayers, STA and cholesterol were found to be immiscible. In contrast, OA and LA were found to form miscible, but nonideal mixed monolayers with cholesterol. They exhibit negative deviations from ideality in the surface pressure/area plots. This reflects close-packing arrangements between bulky cholesterol molecule and the hydrocarbon chains of unsaturated fatty acids. The analysis of the excess free energies of mixing shows that the maximum negative value of ∆Gexc appears at about Xchol ) 0.5-0.7. Thus, the formation of the most stable 1:1 and 2:1 complexes between cholesterol and an unsaturated fatty acid molecule may account for the hypocholesterolemic effect of the acids in human organism by complexing free cholesterol, thereby hindering its deposition on artery walls.

Introduction In contrast to people who preferentially use unsaturated fatty acids, the human population whose diet is rich in saturated lipids exhibit increased blood cholesterol level, and hence has a higher risk of heart disease.1 Indeed, saturated fatty acids have been found to increase the levels of cholesterol in blood, especially cholesterol bound to low-density lipoproteins (LDL).2 Interestingly, monounsaturated acids, such as oleic acid (OA), have the opposite effect; i.e., they decrease the levels of LDL-bound cholesterol without affecting the fraction bound to high-density lipoproteins (HDL).3 On the other hand, ω-6-polyunsaturated fatty acids, such as linoleic acid (LA), reduce arteriosclerosis by diverting cholesterol to other body compartments (possibly to the cytoplasm of liver cells), thereby reducing substantially its levels in plasma.3 Therefore, a diet rich in olive oil and some fish meat, which contain unsaturated fatty acids and may thus help to reduce the incidence of vascular accidents such as myocardial infarction, has strongly been advised by nutritionists. Also, recent studies have revealed that ingesting olive oil normalizes the asymmetric distribution of cholesterol on the inner and outer walls of erythrocyte membranes as well as their motion inside, thereby significantly reducing blood pressure in hypertensive individuals.4-6 In order to get an insight into the mechanism of action of olive oil on cholesterol, we undertook studies herein on the surface behavior of its unsaturated fatty acids (OA and LA) in the presence of cholesterol. To this end, we used the monolayer technique which provides an excellent in vitro model for cell membranes. The results are compared with those obtained with a saturated fatty acid (viz., stearic acid, STA). Material and Methods Cholesterol (98% pure), STA (octadecanoic acid, 99% pure), OA (cis-octadecenoic acid, 99% pure), and LA (cis-9-cis-12-

octadecadienoic acid, 99% pure) were purchased from Sigma and used as received. Spreading solutions were prepared by using a 4:1 v/v mixture of chloroform and ethanol (both Merck p.a. chemicals). A fixed total number of molecules of the two components in mixture (2.3 × 1016) was always deposited on the subphase using a Microman-Gilson microsyringe precise to (0.2 µL. In order to study chol/fatty acids interactions in similar conditions to that of the living cells, water of pH 6.2 was used as subphase. The water was obtained by reversed osmosis, using a Milli RO-Milli Q system (Millipore); its pH and resistivity were 6.2 and 18 MΩ cm, respectively. Π-A curves were obtained with Lauda FW-1 balance furnished with a Teflon trough (total area ) 561 cm2). The compression rate applied was 43 Å2/molecule min. This rate ensured reproducible results, in spite of the fact that fatty acids tend to be dissolved in the subphase. Experiments were conducted at various temperatures ranging from 5 to 30 °C. The subphase temperature was controlled by circulating water through a coil in the bottom of the trough. Brewster angle microscope (mini BAM) from NTF (Go¨ttingen, Germany) was used for direct visualization of the monolayers. The microscope was mounted on the NIMA film balance (Coventry, England) and was equipped with a laser diode of 30 mW emitting at 688 nm as light source. Results and Discussion Stearic Acid/Cholesterol System. At 20 °C, the monolayer of STA exhibits typical of this compound Π-A isotherm (curve 1 in Figure 1) with a characteristic change of slope at 17 Å2/ molecule and π ) 23.5 mN/m, which reflects the film transition from the liquid condensed state (compressional modulus, Cs-1 ) 23.5 mN/m) to the solid state (Cs-1 ) 266 mN/m). These values are consistent with those previously reported.7-9 When the linear part of the isotherm, corresponding to the solid-

10.1021/jp001133+ CCC: $19.00 © 2000 American Chemical Society Published on Web 07/26/2000

7736 J. Phys. Chem. B, Vol. 104, No. 32, 2000

Figure 1. Surface pressure-area isotherms for STA/chol mixtures spread on water at 20 °C with different mole fractions of cholesterol (Xchol): (1) 0.0, (2) 0.1, (3) 0.3, (4) 0.5, (5) 0.7, (6) 0.9, (7) 1.0. Inset A: Plot of the mean molecular area of the mixed STA/chol monolayers as a function of the mole fraction of cholesterol at different surface pressures. Inset B: Phase diagram for the STA/chol mixed monolayer.

condensed phase, is extrapolated to zero pressure, the value of A0 is found to be 22.2 Å2/molecule, which is quite consistent with already reported values.10,11 The collapse pressure (at ca. 59.5 mN/m) appears when the compression isotherm exhibits a sharp spike followed by a drop in surface pressure. This was ascribed to the formation of a trilayer.12 The value of the collapse pressure is higher than those previously reported12,13 for solid surfactants, such as stearic acid. It should be pointed out here, however, that the collapse pressure strongly depends on the compression rate14 and in some papers the values of collapse pressure were found to be higher than 60 mN/m15. The BAM images, shown in Figure 2, provide valuable morphological information about STA monolayer. The images were taken at different points (A, B, C, D) of the Π-A isotherm (isotherm 1, Figure 1). Picture A reveals the presence of islands, shown as blight domains, surrounded by dark zones where no light is reflected (i.e., where no monolayer is present). Upon monolayer compression, the dark zones gradually disappear as the domains fuse to give an uniform image (B) at 21.5 Å2/molecule where the liquid condensed phase commences. This homogeneous image remains throughout the B-C region of the isotherm, even though the above-described transition from the liquid condensed to the solid phase occurs at 23.5 mN/m. At surface pressure of about 51 mN/m, the corresponding image (C) is dotted with bright spots, indicating the formation of small condensation nuclei or “embryos” (the germs of monolayer collapse). These spots cluster as the surface pressure is raised and, after the collapse, fuse (see image D in Figure 2, where the monolayer is in a three-dimensional state). These images are virtually identical with those obtained by Tanaka et al.16

Seoane et al. for monolayers of stearic acid containing 1.0 mol % of a fluorescent probe (rhodamine B octadecyl ester perchloride). Cholesterol gives a typical condensed monolayer (curve 7 in Figure 1) with a limiting area of 39 Å2/molecule (estimated by extrapolation of the steep, high-pressure, linear part of the Π-A curve to zero surface pressure) which is consistent with previously reported values.17-20 Under the dynamic compression conditions applied here, the collapse pressure was ca. 52.5 mN/ m, which is higher than the values obtained under quasi-static or near-equilibrium conditions.21,22 Figure 3 shows BAM images for the cholesterol monolayer. The images were taken at the points E, F, G, H, and I marked on the isotherm 7 in Figure 1. Immediately after spreading of the cholesterol solution, circular and ovoid domains with round borders are observed. They appear to arise from self-arrangement of the molecules which occurs even at large molecular areas, corresponding to picture E, where the monolayer is still in the gaseous state. As the film is further compressed, the domains gather in such a way that, at point F (where surface pressure begins to rise), the image exhibits fused macroscopic domains; however, some open spaces (dark zones in the image) are still present between them. This structure is consistent with the gasliquid-phase coexistence, where low molecular density states coexist with more compact ones. At a surface pressure of 35 mN/m, the monolayer is solid and hence uniform in appearance (Figure 3G). Upon collapse, a number of bright stripe domains are observed. (Figure 3H). Following the initial drop in the collapse pressure, upon further compression of the monolayer, the domains lose their brightness and rearrange in such a way that the space between them diminishes (Figure 3I). As noted by Snik et al.,23 it is quite possible that, at the collapse point, the collapsed 3D phase is formed, which is not stable and is rapidly transformed into another collapsed phase that is in equilibrium with the monolayer at a lower surface pressure. As a result, the BAM images H and I in Figure 3 may correspond to the two 3D phases involved in the collapse. The compression isotherms for all the investigated mixed monolayers (curves 2-6 in Figure 1) lie between those for the pure components. In the mixture with Xchol ) 0.1, the phase transition from liquid condensed to the solid state occurs at surface pressure of 27 mN/m. The other mixtures exhibit condensed monolayers, similar to those of pure cholesterol, and they collapse at the same surface pressure (ca. 53 mN/m). Further compression of those monolayers containing high proportion of STA (Xchol < 0.5) after the first collapse causes a new rise in the surface pressure and, eventually, a second collapse at roughly the same surface pressure as for STA. These results reveal that, during the first collapse, the more collapsible component (cholesterol) is “squeezed out” from the monolayer and the remaining component (STA) eventually collapses at surface pressure close to 60 mN/m. As can be seen from the plot of the mean molecular areas of mixed monolayers vs the mole fraction of cholesterol (inset A, Figure 1), the system obeys the additivity rule, whatever the surface pressure is at which the molecular areas were measured. Linear plots of this kind are typical of either immiscible films or monolayers containing miscible components of ideal behavior.14 In our case, the application of Crisp’s phase rule24 to the collapse region confirms the presence of three different surface phases. In fact, because the number of components C of the mixed system is 2 (cholesterol and STA), the degrees of freedom is f ) 3 - p, where p is the number of phases in equilibrium. As the collapse pressure does not change with the composition (line AB in inset B of Figure 1), the system is invariant (f ) 0)

Fatty Acid/Cholesterol Mixtures at the Air/Water Interface

J. Phys. Chem. B, Vol. 104, No. 32, 2000 7737

Figure 2. BAM images of pure STA monolayers at 20 °C corresponding to the points designated A, B, C, D in the Π-A isotherm (curve 1 of Figure 1).

and therefore there are three phases (p ) 3) in equilibrium, namely two surface phases corresponding to the immiscible components (cholesterol and STA) and that one consisting of collapsed cholesterol, (chol(c)) in 3D state. Similarly, line CD in the inset B of Figure 1 is consistent with the presence of three phases in equilibrium, namely two three-dimensional phases corresponding to the collapse of cholesterol and STA, and one consisting of the STA monolayer. Consequently, cholesterol and STA are immiscible in the condensed state, whatever the composition is, because they are so markedly different in chemical structure that they cannot form an ideal mixed monolayer.17 The two BAM images in Figure 4 for the mixed monolayer of Xchol ) 0.1, compressed at the surface pressures indicated as J and K in curve 2 in Figure 1, reveal the presence of channels with a collapsed structure, corresponding to cholesterol (J), and a series of clustered spots (K), corresponding to STA collapse, which confirms the separate expulsion of the two monolayer components at their own collapse pressures. This behavior is typical of immiscible systems. The results obtained over the temperature range 5-30 °C are virtually identical with those at 20 °C. Therefore, the temperature has no effect on the properties of monolayers of the pure components as well as on their immiscibility in mixed films.

Cholesterol/Unsaturated Fatty Acid Systems. The compression isotherm for oleic acid (OA) is much expanded than that for stearic acid; the monolayer properties are affected by the presence of the double bond in the nonpolar residue of the molecule. As can be seen from curve 1 in Figure 5, the surface pressure increases steadily as the monolayer is compressed, thereby suggesting the presence of an liquid expanded state. The molecular area corresponding to the initial rise in surface pressure is 54.5 Å2, which is consistent with reported values.8 The limiting molecular area obtained by extrapolation of the steep linear segment of the Π-A curve to Π ) 0 is 41.7 Å2, which is close to the 41 Å2 reported by Tomoaia-Cotisel et al.7 for subphases of pH 2. The consistency between these values confirms that OA is undissociated at pH 6. Because this acid is a liquid surfactant, the monolayer collapse occurs at surface pressures below its equilibrium spreading pressure (ESP).7 The value obtained, denoted by an arrow in curve 1 of Figure 5, was 31.6 mN/m (molecular area 27.5 Å2 ). If the monolayer is compressed after the collapse, the surface pressure continues to rise (up to 33 mN/m, which is similar to the 32.4 mN/m reported by Gericke et al.25 and similar to its ESP7,8,26). BAM images for this substance reveal that the film is quite uniform throughout the liquid expanded region, with no isolated domains within the observation field (Figure 6A). The image is similar to that of stearic acid in the solid phase, prior to its


Seoane et al.

Figure 3. BAM images of a pure cholesterol monolayer at 20 °C corresponding to the points designated E, F, G, H, I in the Π-A isotherm (curve 7 of Figure 1).

collapse (Figure 2B). After OA collapses, small condensation nuclei that cluster in stripes are formed and are separated by dark regions (Figure 6-B). The Π-A isotherms for the OA/cholesterol mixtures of Xchol < 0.5 (curves 2 and 3 of Figure 5) are similar to those for pure

OA, especially in the region of low surface pressures. In this situation, the addition of cholesterol to the OA film gradually shifts the isotherms to the y-axis. At Xchol > 0.5, the mixed films behave similarly to pure cholesterol monolayer and are incompressible (curves 5 and 6 of Figure 5). The mixture with



Figure 5. Surface pressure-area isotherms for OA/chol mixtures spread on water at 20 °C with different mole fractions of cholesterol (Xchol): (1) 0.0, (2) 0.1, (3) 0.3, (4) 0.5, (5) 0.7, (6) 0.9, (7) 1.0. Inset: Plot of the collapse surface pressure as a function of the mole fraction of cholesterol at 20 °C.

Figure 4. BAM images of STA/chol mixed monolayer (Xchol ) 0.9) corresponding to points J and I in the Π-A isotherm (curve 2 of Figure 1) after the collapse.

Xchol ) 0.5 behaves between the two pure. Thus, cholesterol reduces the area occupied by the mixed film as a result of not only its decreased molecular cross section caused by its incorporation into the OA monolayer, but also because of well-known condensing effect of cholesterol, which is discussed later on. OA/cholesterol mixed films exhibit collapse pressures between those for the pure components that increase gradually with increasing proportion of cholesterol in the mixture (see the arrows in Figure 5 and inset of the same figure). This shows that cholesterol molecules increase the stability of mixed monolayers, which, according to Crisp,24 is indicative of absolute miscibility between the two components throughout the composition range. BAM images of mixed monolayers in various surface states can be highly useful to confirm miscibility among the film components. However, the images of pure components of the OA/chol system in their liquid expanded state (OA) and solid state (cholesterol), respectively, are absolutely uniform and virtually indistinguishable (see Figures 6A and 3G). As a result, the different mixtures of these components also give uniform BAM images at surface pressures below that of collapse, thereby making it impossible to distinguish whether the components are miscible or immiscible. However, the images corresponding to the film collapse region may be of assistance in establishing

Figure 6. BAM images of pure OA monolayers corresponding to the liquid expanded phase (A) and collapse (B).


Seoane et al.

Figure 7. BAM images of OA/chol mixed monolayers at 20 °C corresponding to the collapse points denoted by arrows in the curves 2-6 of Figure 5: (A) Xchol ) 0.1; (B) Xchl ) 0.3; (C) Xchol ) 0.5; (D) Xchol ) 0.7; (E) Xcho l) 0.9.

the miscibility. Indeed, if the components are immiscible they are “squeezed out” from the monolayer at the surface pressure which coincides with the collapse pressure for the pure components. In this case, the BAM image for the first collapsed component differs from that for the second most stable one, as has been previously shown for the STA/chol system (Figure

4). On the other hand, if the monolayer components are miscible at the interface, they both collapse together (at different surface pressure other than that for pure components). Thus, the BAM images should not only differ from those for pure components, but also change with the mixture composition as they correspond to different structures.


Figure 8. Surface pressure-area isotherms for pure LA monolayers spread on water at different temperatures

Micrographs A-E in Figure 7 show the BAM images corresponding to the points a-e marked by arrows in the Figure 5. The images of the mixed monolayers with a high OA content (Xchol ) 0.1 and 0.3) are somewhat similar to that for OA at collapse (Figure 6B), although they differ from them. On the other hand, the BAM images for the mixtures with a high proportion of cholesterol (Xchol ) 0.7 and 0.9) are rather different from the previous one and resemble those for pure cholesterol (Figure 3) The equimolar mixture gives the image C with many small bright domains but exhibits no sign of a monolayer breakage after its collapse. Clearly, these images change as the proportion of the mixture components is altered. The different images can also be associated with different structures that arise from interactions between the components. This confirms their miscibility. The introduction of an additional double bond in the nonpolar hydrocarbon chain of OA to give linoleic acid (LA), substantially increases its solubility in the aqueous subphase at pH 6. This is clearly seen when LA monolayers are spread at different temperatures: it is evident from Figure 8 that the monolayers have less molecular areas as the temperature is increased, i.e., the isotherms shift to the y-axis as the temperature rises. Simultaneously, the collapse pressure decreases. These results contradict the expectations: an increase in temperature is known to cause fatty acid films to occupy longer areas14 through weakening the van der Waals forces between nonpolar chains. Consequently, the diminution of molecular areas observed should be ascribed to the solubility of the ionized film at pH 6, which increases with increasing temperature. For this reason, and in order to minimize the loss of molecules by dissolution, the isotherms for the LA/chol mixtures were obtained at 5 °C, at which the characteristic values for the LA monolayer are similar to those found at pH 27 (where the film is un-ionized


Figure 9. Surface pressure-area isotherms of LA/chol mixtures spread on water at 5 °C with different mole fractions of cholesterol (Xchol): (1) 0.0, (2) 0.1, (3) 0.3, (4) 0.5, (5) 0.7, (6) 0.9, (7) 1.0. Inset: Plot of the collapse surface pressure of LA/chol mixed monolayers as a function of the mole fraction of cholesterol at 5 °C.

and hence insoluble at the A/W interface). Under these conditions, the limiting area of the monolayer was 46 Å2/ molecule and its collapse pressure 30.6 mN/m, somewhat lower than that for OA. The behavior of LA/chol mixed monolayers (Figure 9) is very similar to that of the OA/chol system discussed above: at high LA contents, the mixed monolayers were of liquid expanded type (curves 2 and 3), similar to those for pure fatty acid; but at Xchol > 0.5 (curves 5 and 6), the mixed monolayers were of solid type, similar to that for pure cholesterol. Collapse of the mixed films occurs at surface pressures between those for the pure components and increases with increasing proportion of cholesterol in the mixture (see inset of Figure 9), which indicates that the two components are miscible throughout the mixture composition range. The BAM images corresponding to the monolayer collapse region confirm that their components are miscible. At surface pressures below that of collapse, the BAM images of LA are uniform (as those for OA). Following the collapse, small bright domains of collapsed phase appear (Figure 10 A) that increase gradually in size with increasing mole fraction of cholesterol in the mixture (Figure 10, B and C). Above Xchol ) 0.5, the number of bright domains increases markedly (Figure 10, D and E); the domains gradually cluster until nucleation is complete (Figure 10F). In summary, the addition of increasing amounts of cholesterol to the LA film causes its gradual condensation, which alters the collapse behavior of its mixed monolayers. This change with the composition of the mixed film reflects the miscible nature of their components. The condensing effect of cholesterol on monolayers of the two fatty acids studied can be readily inferred from a plot of the excess areas of mixing, Aexc, as a function of the mole


Seoane et al.

Figure 10. BAM images of pure LA (A) and LA/chol mixed monolayers after collapse. (B) Xchol ) 0.1; (C) Xchol ) 0.3; (D) Xchol ) 0.5; (E) Xchol ) 0.7; (F) Xchol ) 0.9.

fraction of cholesterol under isobaric conditions (i.e., Π ) const). Aexc, which is a measure of nonideality,27,28 is given by

Aexc ) A1,2 - (X1A1 + X2A2) where A1,2 is the area per molecule of the mixed monolayer; A1 and A2 are the molecular areas of the pure components at a given surface pressure; and X1 and X2 are their corresponding mole fractions. With mixtures where both components exhibit ideal miscibility or complete immiscibility, Aexc ) 0. Deviations

from the additivity rule, whether positive or negative, indicate miscibility and nonideality, with positive excess areas meaning greater cohesion between like molecules than the unlike components, and negative excess areas indicating greater cohesive forces between the unlike ones. Figure 11 shows the variation of Aexc with Xchol at surface pressures of 5, 20, and 30 mN/m for the two systems studied: OA/chol (solid lines) and LA/chol (broken lines). As can be seen, Aexc is negative for all mole fractions and surface pressures studied. These negative excess areas signify miscibility as well


Figure 11. Excess areas of mixing, ∆Aexc, as a function of the mole fraction of cholesterol in OA/chol (9-9) and LA/chol (O-O) mixtures at the surface pressures stated.

as favorable intermolecular interactions between cholesterol and OA or LA. The greatest deviations from ideality were observed over the Xchol range from 0.3 to 0.5 for both systems decreasing with increasing surface pressure. The condensing effect of cholesterol can be ascribed to straightening of the hydrocarbon chains of these unsaturated fatty acids in their expanded state. As a result, the mean molecular areas of the mixed films are decreased relative to those for the two pure components. This property of cholesterol, frequently referred to as a “stabilizing effect”, has been observed in most of the studies performed on systems containing phospholipids29,30 or fatty acid mixtures.31 The increased rigidity of the alkyl chains of stearic acid in relation to the unsaturated chains of OA and LA must be the origin of the virtual absence of a condensing effect of cholesterol on the STA/chol system, which reflects in linearity in the plots of molecular areas (see inset A in Figure 1). In summary, van der Waals attractions between cholesterol rings and tilted alkyl chains of unsaturated acids could be responsible for the deviations from ideality observed in these systems. On the other hand, the polar groups of the two compounds play no significant role in the interaction. The above-mentioned interaction can be analyzed in more quantitative terms by evaluating the excess free energy of mixing, ∆Gexc, at a given surface pressure, as determined by Goodrich,32 assuming that the components of the mixed film behave ideally at the limit of zero surface pressure:33

∆Gexc )

∫0π(A1,2 - X1A1 - X2A2) dπ


Figure 12. Plot of the excess free energy of mixing as a function of the mole fraction of cholesterol in OA/chol (20 °C) and LA/chol (5 °C) mixed monolayers at different surface pressures.

Figure 12 shows the excess free energies for mixed OA/chol and LA/chol monolayers, respectively, at different surface pressures on pure water. As can be seen, the excess free energies of both systems are all negative at any mole fraction of cholesterol and surface pressures, which is consistent with the thermodynamic conditions for miscibility and strong interactions in binary monolayers.32 At all compositions, these values become more negative with increasing surface pressure, thus indicating that the interaction between the two components is greater at increased Π values. The lowest value of ∆Gexc corresponds to the mixtures with Xchol ) 0.5-0.7 in the OA/ chol system and that with Xchol ) 0.7 in the LA/chol system. Therefore, the highest stability of the mixed film corresponds to the formation of 1:1 and 2:1 complexes between cholesterol and unsaturated fatty acids. This could be the responsible for the reduction of the free cholesterol in the blood and thereby preventing its deposition on artery walls. Conclusions On the basis of the results of this study, we have shown that in mixed monolayers cholesterol and STA are immiscible, but cholesterol and either OA or LA have strong interactions and good miscibility at all molar ratios and surface pressures studied. This reflects good matching between the bulky cholesterol molecule and the shorter hydrocarbon chains of unsaturated fatty acids. The BAM images obtained in the collapse region for the mixed films confirm the results of the Π-A isotherms. The formation of the most stable OA/chol and LA/chol complexes, of 1:1 and 2:1 stoichiometry, may be the origin of the

7744 J. Phys. Chem. B, Vol. 104, No. 32, 2000 hypocholesterolemic effect of these unsaturated fatty acids; they may act by “sequestering” free cholesterol, thereby preventing its deposition on artery walls. Acknowledgment. This work was supported for Counsellery for Education of Xunta de Galicia (Spain) under Projet XUGA 20313B96. References and Notes (1) Krummel, D. In Krause’s Food, Nutrition and Diet Therapy; Mahan, K., Escott-Stump, S., Eds. Saunders Co.: Philadelphia, PA, 1996; pp 526-568. (2) Grundy, S. M. Am. J. Clin. Nutr. 1994, 60, 9865. (3) Segura, R. In Nutricio´ n y Diete´ tica; Consejo General de Colegios Farmace´uticos: Madrid, 1993; Vol. 2, Chapter 17, pp 583-611. (4) Schroeder, F.; Jefferson, J. R.; Kier, A. B. Proc. Soc. Exp. Biol. Med. 1991, 196, 235. (5) Schroeder, F.; Memecz, G. In AdVances in Cholesterol Research; Teldford Press: London, 1990; p 49. (6) Muriana, F. J. G.; Ruiz Gutierrez, V. Grasas y aceites 1998, 49 (2), 139. (7) Tomoaia-Cotisel, M.; Zsako, J.; Mocanu, A.; Lupea, M.; Chifu, E. J. Colloid Interface Sci. 1987, 117, 464. (8) Linde´n, M.; Rosenholm, J. B. Langmuir 1995, 11, 4499. (9) Vogel, C.; Corset, V.; Billoudet, F.; Vincent, M.; Dupeyrat, M. J. Chim. Phys. 1980, 77, 947. (10) Datta, A.; Sanyal, M. K.; Dhanabalan, A.; Major, S. S. J. Phys. Chem. B 1997, 101, 9280. (11) Hwang, M. J.; Kim, K. Langmuir 1999, 15, 3563. (12) McFate, C.; Ward, D.; Olmsted III, J. Langmuir 1993, 9, 1036. (13) Ries, H. E. Jr.; Swift, H. J. Colloid Interface Sci. 1978, 64, 111.

Seoane et al. (14) Gaines, G. L. Jr. In Insoluble Monolayers at Liquid-Gas Interfaces; Wiley-Interscience: New York, 1996. (15) Joos, P. Bull. Soc. Chim. Belg. 1971, 80, 277. (16) Tanaka, H.; Akatsuka, T.; Murakami, T.; Ogoma, Y.; Abe, K.; Kondo, Y. J. Biochem. 1997, 121, 206. (17) Motomura, K.; Terazono, T.; Matuo, H.; Matuura, R. J. Colloid Interface Sci. 1976, 57, 52. (18) Mu¨ller-Landau, F.; Cadenhead, D. A. Chem. Phys. Lipids 1979, 25, 299. (19) Takano, E.; Ishida, Y.; Iwahashi, M. Langmuir 1997, 13, 5782. (20) Taneva, S.; Keough, K. M. W. Biochemistry 1997, 36, 912. (21) Galvez Ruiz, M. J.; Cabrerizo, M. A. Colloid Polym. Sci. 1991, 269, 77. (22) Demel, R. A.; Bruckdorfer, K. R.; Van Deenen, L. L. M. Biochim. Biopyhs. Acta 1972, 225, 311. (23) Snik, A. F. M.; Kruger, A. J.; Joos, P. J. Colloid Interface Sci. 1978, 66, 435. (24) Crisp, D. J. In Surface Chemistry Suppl. Research; Butterworth: London, 1949; pp 17-23. (25) Gericke, A.; Hu¨hnerfuss, H. Langmuir 1995, 11, 225. (26) Smith, R. D.; Berg, J. C. J. Colloid Interface Sci. 1980, 74, 273. (27) Dorfler, H. D. AdV. Colloid Interface Sci. 1990, 31. (28) Barnes, G. T. J. Colloid Interface Sci. 1991, 144, 299. (29) Pasenkiewicz-Gierula, M.; Subczynski, W. K.; Kusumi, A. Biochemistry 1990, 29, 4059. (30) Tanaka, K.; Manning, P. A.; Lau, V. K.; Yu, H. Langmuir 1999, 15, 600. (31) Sparr, E.; Ekelund, K.; Engblom, J.; Engstro¨m, S.; Wennerstro¨m, H. Langmuir 1999, 15, 6950. (32) Goodrich, F. C. Proc. 2nd Int. Congr. Surf. ActiVity, London1957, 1, 85. (33) Gershfeld, N. L. In Techniques of Surface and Colloid Chemistry and Physics; Good, R. J., Stromberg, R. R., Patrick, R. L., Eds.; Marcel Dekker: New York, 1972; Vol. 1, p 1.

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