Comparative Studies on the Influence of β-Sitosterol and Stigmasterol

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Comparative Studies on the Influence of β-Sitosterol and Stigmasterol on Model Sphingomyelin Membranes: A Grazing-Incidence X-ray Diffraction Study Katarzyna Ha˛c-Wydro,*,† Michał Flasin´ski,† Marcin Broniatowski,† Patrycja Dynarowicz-Ła˛tka,† and Jarosław Majewski‡ Faculty of Chemistry, Jagiellonian UniVersity, Ingardena 3, 30-060 Krako´w, Poland, and Lujan Neutron Scattering Center, Los Alamos National Laboratory, Los Alamos, New Mexico 87545 ReceiVed: February 7, 2010; ReVised Manuscript ReceiVed: April 18, 2010

Sterols are essential constituents of membranes, both in the plant world and in human organisms. Therefore, their activity on model lipid systems has systematically been studied. Despite intensive investigations, differences in the effect induced by β-sitosterol (β-sito) and stigmasterol (stigma) (two major phytosterols) are very controversial and still under debate. To compare the influence of these compounds on model membranes, we have performed grazing incidence X-ray diffraction (GIXD) experiments on phytosterol/ sphingomyelin (Sph) monolayers. The analysis of the X-ray scattering and the resulting in-plane parameters provided information on the lateral organization of pure lipid films and the mixed systems. The obtained results prove a nonideal mixing between the investigated lipids in the monolayers and the existence of strong interactions between phytosterols and Sph. Both the plant sterols incorporated into sphingolipid film condense the monolayer and order Sph chains. The results of GIXD experiments, compared with those obtained previously from Langmuir monolayer studies allowed us to observe the comparable influence of β-sito and stigma on model membrane organization. Introduction Stigmasterol (stigma) and β-sitosterol (β-sito) are major representatives of vast array of sterols found in plants tissues, mainly in cellular plasma membranes. These compounds play an essential role as modulators of membrane parameters such as fluidity and permeability as well as participate in the regulation of various biological processes in plant organisms, e.g., plant growth, metabolic cycles, or modulation of the activity of membrane-bound enzymes.1-3 They are also required for the synthesis of a wide variety of secondary metabolites, such as glycoalkaloids or saponins.3 Although plant sterols cannot be synthesized by animals themselves, including humans, they can be assimilated from food.4,5 It is believed that both stigma and β-sito have beneficial influence on cardiovascular and immune systems in humans and show anticancer activity; however, they are mainly known for lowering of the blood cholesterol level.4-9 Similarly to cholesterol in animal membranes, stigma and β-sito alter membrane properties and induce condensing and ordering effects on phospho- and sphingolipids. The foregoing abilities, leading to the formation of a liquid-ordered (LO) phase, manifest in the decrease of the average area per lipid and the increase of order of lipid acyl chains in the mixed systems, respectively.10,11 Thus, by interacting with other lipids, these sterols modulate the fluidity of the cellular membrane and change its lateral organization by the formation of the ordered state and microdomains (‘rafts’).10,12-16 Phytosterols differ from cholesterol only in the structure of the side chain. Namely, both β-sito and stigma molecules have an additional ethyl group as compared to mammalian sterol. Moreover, stigma also possesses a double bond in the side chain. * Corresponding author. E-mail: [email protected]. Fax: +48 0-12634-05-15. Phone: +48 0-12 633-20-82. † Jagiellonian University. ‡ Los Alamos National Laboratory.

Figure 1. Structural formulas of β-sito and stigma with the numeration of carbon atoms.

Numerous studies have been done on differences in the effect of cholesterol and phytosterols on model lipid systems. It was proved that β-sito and stigma, because of having bulkier side chains, are markedly less effective regarding condensing and ordering effects than human sterol.2,15,17-21 Contradictions appear, however, when the properties of stigma and β-sito are compared. The factor distinguishing the foregoing lipids is a double bond in the stigma side chain between C22 and C23 (see Figure 1). As it was found in steady-state fluorescence polarization experiments on dipalmitoylphosphatidylcholine (DPPC)/sterol bilayers, the double bond in the stigma molecule

10.1021/jp101196e  2010 American Chemical Society Published on Web 04/30/2010

Influence of β-Sito and Stigma on Model Sph Membranes side chain decreases volume fluctuations in the steroid alkyl chain region, which manifests in its stronger condensing and ordering abilities as compared to β-sito.17 Similarly, the analysis of differential scanning calorimetry thermograms21 for DPPC/ sterol bilayers proved a stronger ability of stigma versus β-sito to order and stabilize these systems. On the other hand, in the Langmuir monolayer experiments by Su et al.,20 small-angle X-ray scattering, dilatometry, and ultrasound velocimetry studies on bilayers by Hodzic et al.22 as well as in permeability investigations on liposomes performed by Yamauchi et al.,23 a stronger condensing effect of β-sito as compared to stigma on DPPC was proved as well as higher efficiency of the former in modulating the thickness and elasticity of dimyristoylphosphatidylcholine (DMPC) bilayers. β-sito is also more effective than stigma in stabilization of LO phase, as it was found in the comprehensive studies on the phase behavior of a sphingomyelin (Sph) bilayer, performed using differential scanning calorimetry, synchrotron X-ray diffraction, freeze-fracture electron microscopy, and Fourier-transform infrared spectroscopy.24 Interestingly, there are also results indicating that both these plant sterols are of similar effect on saturated phosphatidylcholines (DMPC and DPPC). In the synchrotron X-ray diffraction and differential scanning calorimetry experiments25 and in the NMR investigations on sterol/saturated phosphatidylcholine bilayers (DMPC and DPPC),26 it was found that β-sito and stigma behave almost identically in the interactions with DPPC25 and that both plant sterols are of similar potency as far as their ordering and rigidifying effect on saturated phosphatidylcholines is concerned.26 It should be noticed that, when the influence of the plant sterols on unsaturated lipids is compared, the results seem to be more consistent, but only for the same phospholipid studied. In detergent solubilization experiments based on light scattering measurements, performed on palmitoyloleoylphosphatidylcholine (POPC) (16:0/18:1 PC) vesicles,21 β-sito was found to provoke a tighter packing in POPC bilayers than stigma. This is in agreement with the results for POPC bilyers obtained by Hodzic et al.22 as well as with the experiments by Schuler et al.27 In this work,27 the incorporation of sterols into soybean phosphatidylcholine bilayers was investigated, and it was found that β-sito orders phospholipid acyl chains more strongly than stigma. However, in the studies on 16:0/18:2-PC model membranes, the influence of both these plant sterols on the polarity and molecular mobility at the hydrophilic/ hydrophobic interface was similar.28 The above-analyzed controversies encouraged us to perform Langmuir monolayer experiments, in which the influence of both the phytosterols on Sph and DPPC films18 was verified. It was recognized that β-sito interacts with DPPC and Sph only slightly stronger than stigma, and induces only a slightly higher ordering effect on the investigated lipids. Thus, in fact, both phytosterols are of comparable influence on model biomembranes.18 Similar results were obtained when ternary DPPC/Sph/phytosterol monolayers were investigated: at a low content of sterols in a model system (up to 30%), β-sito and stigma were found to induce similar ordering effect on model membranes.19 Generally, the investigation of the effect of phytosterols on model membranes is really a challenging task, as minute changes in the sterol constitution can induce significant effect on the membrane organization. In this contribution, we present the results of systematic studies of the interactions of stigma and β-sito with Sph in Langmuir monolayers, using the grazing incidence X-ray diffraction (GIXD) method. The application of this modern technique to the analysis of lateral organization of the lipid films

J. Phys. Chem. B, Vol. 114, No. 20, 2010 6867 provides information that is not available from classical Langmuir monolayer experiments. To the best of our knowledge, phytosterols/Sph mixed films have not been studied before with GIXD. Although the GIXD probes only the ordered (or gel) part of the monolayer, the application of this method provides information regarding the in-plane organization of the molecules within the investigated films. Experimental Section Materials. The investigated phytosterols, β-sito and stigma (for chemical structures, see Figure 1) were synthetic compounds (>99%) purchased from Sigma, while egg Sph (g98%) was purchased from BioChemica. To prepare the spreading solutions, the respective compounds were dissolved in chloroform p.a. (POCh, Poland). Mixed solutions of various composition (the molar ratio of sterol/Sph ) 1:9 and 3:7) were prepared from the respective stock solutions. Spreading solutions were deposited onto the water subphase with a Hamilton microsyringe, precise to 2.0 µL. Methods. X-ray scattering experiments were performed at the BW1 (undulator) beamline at the HASYLAB synchrotron source (Hamburg, Germany) using a dedicated liquid surface diffractometer29 with an incident X-ray wavelength λ ≈ 1.3 Å. A thermostatted Langmuir trough made of one block of Teflon, equipped with a movable barrier for monolayer compression, was placed in a gastight container and mounted on the diffractometer. The surface pressure was monitored with a Wilhelmy balance equipped with a filter paper (Whatman ashless) as a surface pressure sensor. The subphase temperature was controlled to be 20.0 ( 0.1 °C by a circulating water bath. After spreading the solution onto the subphase, at least 40 min were allowed for the trough container to be flushed with helium to reduce the scattering background and to minimize beam damage during X-ray scans. Afterward, the films were compressed to the surface pressure of 30 mN/m (the surface pressure at which the properties of monolayer can be compared with those of bilayers),30 at which the X-ray experiments were performed. GIXD Experiments. Regarding the GIXD experiments, the X-ray scattering theory and the liquid diffractometer used here have been described previously.31-33 GIXD experiments were carried out to obtain lateral ordering information of the samples. The scattered intensity is measured by scanning over a range of horizontal scattering vectors Qxy:

Qxy ≈

4π sin(2θxy /2) λ

where 2θxy is the angle between the incident and diffracted beam projected on the liquid surface. The GIXD intensity resulting from a powder of two-dimensional (2D) crystallites can be represented as Bragg peaks, resolved in the Qxy direction, by integrating the scattered intensity over the Qz direction, which is measured by the position-sensitive detector placed perpendicular to the air-water interface. Conversely, the Bragg rod profiles were resolved in the Qz direction (Qz ) (2π/λ) sin Rf, where Rf is the X-ray exit angle) and obtained by integrating the scattered intensity over Qxy corresponding to the Bragg peak. The angular positions of the Bragg peaks determine the d-spacing, d ) 2π/Qxy (where the Qxy is the position of the maximum of the Bragg peak along Qxy) for the 2D lattice. From the line width of the peaks, it is possible to determine the 2D crystalline coherence length, Lxy, the average distance in the

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Figure 2. Background-subtracted GIXD diffraction data for the Langmuir monolayers of the investigated lipids, compressed to 30 mN/ m. Left column: Bragg peak profiles I(Qxy); right column: corresponding Bragg rod profiles I(Qz) for Sph (a,b), β-sito (c,d), and stigma (e,f). The solid lines were fitted to Bragg peaks using Voigt functions. The Bragg rods were fitted by approximating the scattering unit of the molecule by a cylinder of constant electron density.29

direction of the reciprocal lattice vector Qxy, over which the ordering extends. The intensity distribution along the Bragg rod can be analyzed to determine the magnitude and direction of the molecular tilt and the coherently scattering length (Lz) of the alkyl tail measured along the backbone. Results and Discussion The Bragg diffraction peaks together with corresponding Bragg rod profiles collected for one-component lipid monolayers are presented in Figure 2. The values of structural parameters obtained from Bragg profiles are listed in Table 1. Egg Sph forms a stable homogeneous monolayer at the air/ water interface.18 During compression, the surface pressure starts to increase at an area of ∼78 Å2/molecule, and the film collapses

at π ≈ 60 mN/m. In the course of the π-A curve appears a characteristic region, reflecting a phase transition between liquid expanded (LE) and liquid condensed (LC) states. The area per molecule value estimated from the isotherm at π ) 30 mN/m is 43.4 Å2.18 For a Sph monolayer, one Bragg peak and the corresponding Bragg rod were registered at π ) 30 mN/m (Figure 2a,b). This indicates a 2D ordering of the acyl chains and may suggest a hexagonal arrangement of the lipids molecules at the water/air interface. The small asymmetric shape of the Bragg peak can testify to some deviations from a hexagonal to a distorted hexagonal unit cell. In our GIXD measurements, no ordering of the Sph head groups was observed at the lower Qxy values. The peak reaches its maximum at Qxy ≈ 1.46 Å-1, which corresponds to a d spacing of ∼4.30 Å and consequently results in the hexagonal unit cell of the dimension aH ≈ 4.97 Å (see Table 1). The 2D crystalline coherence length Lxy (reflecting the approximate size of the crystalline domains) calculated according to the Scherer formula34-36 was estimated to be 51 Å. This means that the surface 2D domains are crystalline within a radius of ∼10 aH periods and consist of ca. 100 molecules. The Bragg rod is relatively wide as compared with the rods of the two investigated sterols, with the maximum intensity above the horizon (Qz ) 0 Å-1). This can be the result of the above discussed distortion of the hexagonal unit cell originating from a moderate collective tilt of the Sph alkyl chains toward the nearest neighbor (NN). In this situation, the Bragg peak and Bragg rod are the superposition of two overlapping peaks (1,-1) and (1,0)+(0,1). The fitting of the Bragg rod data resulted in a tilt angle of 12° and a length of the coherently scattering molecular unit (measured along the backbone of the molecule) of Lz ) 15.1 ((0.8) Å. For comparison, the length of the palmitic chain, assuming its all-trans configuration, is 19.1 Å. The two chains of Sph are not identical in length, which, together with the thermal movements of the termini of the alkyl chains, leads to the effective shortening of the coherently scattering unit of the molecule. The area per molecule (A) values for Sph calculated from GIXD data was found to be A ) 42.6 Å2, which agrees well with the value obtained from the π-A isotherm. The GIXD method has been widely applied to study the lateral ordering of cholesterol thin films (see, e.g., refs 37-40). The organization of mammalian sterol monolayers has been studied at several points in the course of the isotherm.37,39,40 At a temperature of 5 °C at larger areas (uncompressed state), one Bragg peak (d spacing ) 5.7 Å) was detected and related to a hexagonal unit cell (a ) b ) 6.6 Å). On the basis of the Bragg rod analysis (at Qz ) 0 Å-1,) it was found that the molecules are oriented normal to the plane of water. During further investigations performed according to molecular modeling, it was found that a hexagonal unit cell may not be the most

TABLE 1: In-Plane Structural Parametersa Obtained from GIXD Measurements for Phytosterols and Sphingomelin, and for Their 1:9 and 3:7 Mixturesb d (Å) ((0.003) aH (Å) ((0.003) Lz (Å) ((0.8) Lxy (Å) ((1) A (Å2) ((0.2)

Sph

β-sito

stigma

β-sito/Sph 1:9

β-sito/Sph 3:7

stigma/Sph 1:9

stigma/Sph 3:7

4.301 4.966 15.1 51 42.6

5.772 6.664 13.3 67 38.4

5.740 6.630 14.1 38 38.0

4.319 4.987 9.7 35 40.9

4.398 5.078 12.2 21 38.0

4.301 4.966 10.9 37 40.5

4.370 5.046 13.2 23 37.4

a d: d-spacing; aH: parameter of the 2D unit cell; Lz: coherently scattering part of the molecules; Lxy: in-plane coherence length; A: averaged area per molecule). b Measurements were performed at the surface pressure of 30 mN/m. The presented errors for the respective parameters represent maximum values estimated from the fitting procedure and calculated with the exact differential method.

Influence of β-Sito and Stigma on Model Sph Membranes favorable structure because of the too short intermolecular contact. Instead, a trigonal arrangement of a unit cell a′ ) b′ ) 3a was proposed. Beyond the collapse, several sharp Bragg peaks were recorded, corresponding to a rectangular unit cell in a crystalline bilayer. Further transformation of the bilayer to a crystalline trilayer phase was also detected. As it is also shown in Figure 2c-f for both investigated sterols, single Bragg peaks were obtained. This may suggest the hexagonal arrangement of the molecules within the film. The maximum of Bragg peaks for both sterols appears at nearly the same value (Qxy ≈ 1.09 Å-1), and the calculated d-spacings are 5.77 Å and 5.74 Å for β-sito and stigma, respectively. The value of the unit cell aH for β-sito is ca. 6.66 Å and is noticeably larger than that for Sph. However, it should be realized that the repeating unit is the bulky tetracyclic entity, characteristic of sterols, whereas in the case of Sph the repeating unit is a single alkyl chain. As it has been mentioned, the peak has a relatively small width at half-maximum, leading to the Lxy value of ca. 67 Å. The in-plane order extends over ca. 10 aH units, and an average ordered domain of β-sito consists of ca. 100 molecules. The Bragg rod has its maximum at 0 Å-1, which proves perpendicular arrangement of the molecules at the air/water interface. The length of the coherently scattering molecular moiety (Lz) was fitted to be 13.3 ((0.8) Å. We refer to the Cambridge Structural Database System (CSDS) crystallographic database41 to check how the Lz of 13.3 Å corresponds to the length of the alkyl chain of β-sito molecule known from the monocrystalline X-ray data. There is one record regarding β-sito. According to ref 42, the length of the alkyl counted from C3 to C27 is ca. 16 Å in the solid phase. The value of ca. 13.5 Å corresponds to the distance from C3 to C24, which is reasonable, taking into consideration the thermal movement of the bulky peripheries (the fragment from C24 to C29) of the terminal alkyl fragment. Because of the thermal movement of the molecules at the air/water interface, the terminal part of β-sito molecule can be very disordered, which reduces the coherently scattering moiety only to the fragment from C3 to C24. The value of aH calculated for stigma (6.63 Å) is nearly the same as that for β-sito (6.66 Å). The Bragg peak is, however, nearly twice as wide at half-maximum than in the case of the latter sterol molecule, leading to an Lxy of only ca. 38 Å. Therefore, the in-plane periodicity extends at only ca. 6 unit cells, and the statistical ordered domain contains only 35 molecules. The lowering of the size of the coherently scattering domains can result from the chemical structure of stigma. The molecule has a double bond in the side alkyl chain, making it less flexible and more difficult to order. The Bragg rod has its maximum at 0 Å-1 and a course very similar to that for β-sito, suggesting that the molecules pack in the undistorted 2D hexagonal lattice and the molecules stack perpendicularly at the air-water interface. The coherently scattering part of the molecule Lz was calculated from the best fit to be 14.1 Å. According to CSDS41 there are two publications43,44 concerning the crystal structure of stigma. The structure is very similar to that of the above-mentioned β-sito. The length of the whole hydrophobic chain is ca. 16 Å. Here the Lz of 14.1 Å is slightly larger than the 13.3 Å for β-sito, which can be caused by the presence of the double bond between C22 and C23. Even in the stigma monocrystal the terminal bulky peripheries of the alkyl chain are disordered, which was underlined by the cited authors.44 Therefore, similarly to β-sito, we can postulate that the coherently scattering moiety is the molecular fragment from C3 to C24. Interestingly, the film thickness estimated from

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Figure 3. Background-subtracted GIXD diffraction data for mixed films composed of 1:9 and 3:7 β-sito/Sph, compressed to 30 mN/m. Left column: Bragg peak profiles I(Qxy); right column: corresponding Bragg rod profiles I(Qz) for 1:9 (a,b) and 3:7 (c,d) β-sito/Sph monolayers. The solid lines were fitted to Bragg peaks using Voigt functions. The Bragg rods were fitted by approximating the scattering unit of the molecule by a cylinder of constant electron density.29

GIXD experiments for pure cholesterol monolayers on water was reported37 to be Lz )14.5 Å. This value is also lower than 16 Å (the full length of cholesterol), also suggesting significant disorder in the terminal alkyl chain. Moreover, the areas per molecule (A) calculated for the investigated sterols (A ) 38.4 Å2 for β-sito and 38.0 Å2 for stigma) do agree very well with those determined from π-A isotherms at the surface pressure π ) 30 mN/m (A ) 38.0 Å2 for β-sito and 38.1 Å2 for stigma).18 In Figures 3 and 4 the GIXD data collected for the mixtures of sterols and Sph are shown. For both investigated sterols, regardless of the mole fractions of a sterol molecule, one Bragg peak and one corresponding Bragg rod was observed. This may imply the arrangement of the molecules in the 2D hexagonal unit cell at the water/air interface. With an increase of the sterol content in the mixture, the Bragg peak maxima shift to slightly lower Qxy values; however, this effect is similar for both investigated phytosterols. Comparing the changes of d-spacing and aH values for the respective systems with the increase of sterol concentration, we find that the differences in the values of the foregoing parameters for 1:9 and 3:7 mixtures are similar for β-sito- and stigma-containing monolayers (e.g., the shift of d-spacing for β-sito/Sph monolayers is 0.08 Å, while that for stigma/Sph mixtures is 0.07 Å). The in-plane Lxy coherence lengths calculated from the Bragg peak widths at half-maximum are relatively low (see Table 1), and for both sterol/Sph systems they are considerably lower than those observed for pure components. This indicates that the mixed domains are of smaller dimensions and statistically contain ca. 50 (for 1/9 mixtures) and ca. 20 (for 3/7 mixtures) molecules. Additionally, with the increase of sterol proportion in the mixed system, the size of domains in the monolayer decreases: the lowering of the values of Lxy may indicate the increase in packing defects in the lattice. When the average areas per lipid values were calculated, a trend similar to that for Lxy values versus composi-

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Figure 4. Background-subtracted GIXD diffraction data for the mixed films composed of 1:9 and 3:7 stigma/Sph, compressed to 30 mN/m. Left column: Bragg peak profiles I(Qxy); right column: corresponding Bragg rod profiles I(Qz) for 1:9 (a,b) and 3:7 (c,d) stigma/Sph monolayers. The solid lines were fitted to Bragg peaks using Voigt functions. The Bragg rods were fitted by approximating the scattering unit of the molecule by a cylinder of constant electron density.29

Figure 5. aH in-plane lattice constants (open symbols) versus the molar fraction (Xsterol) of the sterol in the monolayers for β-sito/Sph (open squares) and stigma/Sph (open circles) films compressed to 30 mN/m. The solid symbols connected by the dashed lines represent the hypothetical values of the parameters obtained from linear combinations of the aH of pure components weighted by their molar fraction in the monolayer. The difference between the calculated and measured values demonstrates the condensing effect of sterols on the mixtures.

tion was observed: the A values for mixtures are not the linear combination of the areas of pure components (see Table 1) and are in good agreement with the values determined from the Langmuir isotherms.18 The foregoing dependence between the area per molecule and the sterol content is a consequence of the condensing effect of sterols and ordering of Sph acyl chains, and proves the existence of strong interactions between both components in a mixed system. It is of interest to plot the aH lattice constant versus the molar ratio of the sterols and compare them with linear combinations of aH of pure components (Figure 5). It can be seen in Figure 5 that the aH lattice constants of the hexagonal unit cells of the mixed monolayers are not just weighted by the molar fraction linear combinations of the lattice constants of the pure components (black symbols in Figure 5), but they are systematically

Ha˛c-Wydro et al. smaller than the weighted average. Such a behavior proves considerable attractive interactions between the monolayer components. From Figure 5 it is evident that the deviations from linearity are similar for both investigated plant sterols within the error range. Bragg rods for sterol/Sph mixtures possess their maxima close to the horizon, i.e., at Qz ≈ 0 Å-1, indicating that the scattering moieties are untilted at both mol ratios. The calculated values of the lengths of the coherently scattering moieties (Lz) for the mixtures are visibly lower than those for pure components (Table 1). It can be assumed that mainly the tetracyclic cyclopentaneperhydrophenantrene unit of the sterol molecule is aligned and well packed with the alkyl chains of the Sph molecule, whereas it is more difficult to align the side chains of the sterol molecules with the terminal parts of the Sph alkyl chains. Taking into consideration that the introduction of sterol molecules into the Sph monolayer enlarges the distances between alkyl side chains of Sph, it is obvious that the rotational freedom of their terminal parts will be larger, which, in consequence, leads to the shortening of Lz. The fact that Lz values for stigma/Sph mixtures are slightly larger than in the case of the mixtures containing β-sito may suggest that the interactions between the hydrophobic tail of stigma and alkyl chains of Sph are more effective as compared to that of β-sito. This can originate from the presence of the double bond in the side chain of stigma molecule, making this fragment less flexible and reducing its thermal movements, which, in consequence, leads to the greater length of the coherently scattering moiety. On the other hand, it should also be pointed out that the Lz for the respective sterols differ by only ∼1 Å, which may reflect in small differences in the values obtained for mixed systems. Moreover, taking into consideration errors of Lz, it is reasonable to conclude that both investigated plant sterols similarly interact with Sph. The GIXD method has been widely applied to study the mixed systems composed of membrane lipids and cholesterol (see, e.g., refs 45-47). Comparing the results of our experiments for plant sterols/Sph monolayers with those for dihydrocholesterol (DChol)/Sph mixtures,46,47 similarities in the behavior of the system upon sterol addition can be found. Namely, at π ) 25 mN/m and a Dchol content in the mixed system lower than 35%, one Bragg peak was recorded, which shifts only slightly with the increase of sterol content in the monolayer. In consequence, d-spacing values change only slightly with sterol addition. This was attributed to the ordered lipid-cholesterol entity with excess unordered lipid, which does not affect the position of the Bragg peak. Moreover, the coherence length for mixed systems was lower than those for pure components and indicates the existence of nanoscale domains (similar trend was also found in our experiments). It was concluded that favorable interactions between Sph and Dchol provoke the formation of a short-range unique ordered structure. Conclusions The results of GIXD experiments suggest a hexagonal arrangement of the ordered part of the investigated monolayers of the lipid molecules at the water/air interface. The comparison of the parameters calculated from Bragg profiles (Table 1) enables us to conclude that the plant sterols, incorporated into Sph monolayers, induce monolayer condensation and ordering of Sph alkyl tails. A nonlinear change of the aH lattice constant with monolayer composition suggests strong interactions between the monolayer components and a nonideal mixing between the investigated lipids. The reduction of area per lipid

Influence of β-Sito and Stigma on Model Sph Membranes and decrease of the values of the coherence length parameter (Lxy) for the mixed systems prove the condensing effect of sterols on Sph, found also in the Langmuir isotherm experiments. Although the results obtained from GIXD studies may suggest a slightly stronger ordering effect of stigma on the Sph model membrane, there is no prevailing effect of one of the investigated plant sterols on the lateral ordering of Sph film as compared to the other one. It should be stressed that the parameters obtained for both sterols from Bragg peaks are very similar, and moreover, their error ranges cannot be ignored. Therefore, the influence of β-sito and stigma on membrane organization is comparable. Acknowledgment. The authors are grateful to DESYHASYLAB, Hamburg (Germany), for granting synchrotron beam time for the realization of the project. LANSCE is funded by the DOE Office of Basic Energy Sciences under DOE Contract DE-AC52-06NA25396. K.H.-W. wishes to thank The Foundation for Polish Science for financial support. References and Notes (1) Hartmann, M.-A. Trends Plant Sci. 1998, 3, 170. (2) Dufourc, E. J. J. Chem. Biol. 2008, 1, 63. (3) Piironen, V.; Lindsay, D. G.; Miettinen, T. A.; Toivo, J.; Lampi, A. M. J. Sci. Food Agric. 2000, 80, 939. (4) de Jong, A.; Plat, J.; Mensink, R. P. J. Nutr. Biochem. 2003, 14, 362. (5) Kritchevsky, D.; Chen, S. C. Nutr. Res. (N.Y.) 2005, 25, 413. (6) Moreau, R. A.; Whitaker, B. D.; Hicks, K. B. Prog. Lipid Res. 2002, 41, 457. (7) Brufaua, G.; Canelab, M. A.; Rafecasa, M. Nutr. Res. (N.Y.) 2008, 28, 217. (8) Bouic, P. J. D.; Clark, A.; Lamprecht, J.; Freestone, M.; Pool, E. J.; Liebenberg, R. W.; Kotze, D.; van Jaarsveld, P. P. Int. J. Sports Med. 1999, 20, 258. (9) Berger, A.; Jones, P. J. H.; Abumweis, S. S. Lipids Health Dis. 2004, 3, 5. (10) Ohvo-Rekila, H.; Ramstedt, B.; Leppimaki, P.; Slotte, J. P. Prog. Lipid Res. 2002, 41, 66. (11) Ro´g, T.; Pasenkiewicz-Gierula, M.; Vattulainen, I.; Karttunen, M. Biochim. Biophys. Acta 2009, 1788, 97. (12) Martin, S. W.; Glover, B. J.; Davies, J. M. Trends Plant Sci. 2005, 10, 263. (13) Mongrand, S.; Morel, J.; Laroche, J.; Claverol, S.; Carde, J.-P.; Hartmann, M.-A.; Bonneu, M.; Simon-Plas, F.; Lessire, R.; Bessoule, J.-J. J. Biol. Chem. 2004, 279, 36277. (14) Bhat, R. A.; Panstruga, R. Planta 2005, 223, 5. (15) Xu, X.; Bittman, R.; Duportail, G.; Heissler, D.; Vilcheze, C.; London, E. J. Biol. Chem. 2001, 276, 33540. (16) Beck, J. G.; Mathieu, D.; Loudet, C.; Buchoux, S.; Dufourc, E. J. FASEB J. 2007, 21, 1714. (17) Bernsdorff, C.; Winter, R. J. Phys. Chem. B 2003, 107, 10658. (18) Ha˛c-Wydro, K.; Dynarowicz-Ła˛tka, P. J. Phys. Chem. B 2008, 112, 11324.

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