Article pubs.acs.org/Langmuir
Resveratrol Interaction with Lipid Bilayers: A Synchrotron X‑ray Scattering Study Ana Rute Neves,† Cláudia Nunes,*,† Heinz Amenitsch,‡ and Salette Reis† †
UCIBIO, REQUIMTE, Department of Chemical Sciences, Faculty of Pharmacy, University of Porto, Rua de Jorge Viterbo Ferreira, 228, 4050-313 Porto, Portugal ‡ Institute of Inorganic Chemistry, Graz University of Technology, Stremayergasse 6/V, 8010 Graz, Austria
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S Supporting Information *
ABSTRACT: Resveratrol belongs to the large group of biologically active polyphenol compounds, with several beneficial health effects including antioxidant activity, antiinflammatory action, cardiovascular protection, neuroprotection, and cancer chemoprevention. In the present study, the possibility that the effects of resveratrol described above are caused by resveratrol membrane interactions and structural modifications of lipid bilayers is evaluated. In this context, it is possible that resveratrol interacts selectively with lipid domains present in biological membranes, thereby modulating the localization of the anchored proteins and controlling their intracellular cascades. This study was conducted in a synchrotron particle accelerator, where the influence of resveratrol in the structural organization of lipid domains in bilayers was investigated using small- and wide-angle X-ray scattering (SAXS and WAXS) techniques. Membrane mimetic systems composed of egg L-α-phosphatidylcholine (EPC), cholesterol (CHOL), and sphingomyelin (SM), with different molar ratios, were used to access the effects of resveratrol on the order and structure of the membrane. The results revealed that resveratrol induces phase separation, promoting the formation of lipid domains in EPC, EPC:CHOL [4:1], and EPC:CHOL:SM [1:1:1] bilayers, which brings some structural organization to membranes. Therefore, resveratrol controls lipid packing of bilayers by inducing the organization of lipid rafts. Moreover, the formation of lipid domains is important for modulating the activity of many receptors, transmembrane proteins, and enzymes whose activity depends on the structural organization of the membrane and on the presence or absence of these organized domains. This evidence can thereby explain the therapeutic effects of resveratrol.
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INTRODUCTION Resveratrol can be found in a wide variety of plants and fruits, including blueberries, mulberries, cranberries, rhubarb, peanuts, pine nuts, coconuts, and cocoa; grapes and red wine are the most important sources of this compound.1 Resveratrol exhibits pleiotropic beneficial health effects, including antioxidant activity, anti-inflammatory action, cardiovascular protection, neuroprotection, and cancer chemoprevention.2,3 Some studies suggest that resveratrol can induce apoptosis, inhibit cell proliferation, scavenge free radicals, overexpress SIRT1, suppress cyclooxygenase (COX) activity, and inhibit phospholipase C (PLC) and protein kinase C (PKC).4−9 However, its mechanism of action is still uncertain.10 Meanwhile, it is possible that the effects of resveratrol described above are caused by resveratrol membrane interactions and structural modifications of lipid bilayers, similar to several drugs that act through a membrane-lipid therapy.11−13 Recent studies have revealed that resveratrol is able to incorporate in biomembranes, either fluidizing or stiffening the membrane depending on its initial fluidity state, and inducing the formation of ordered domains.14−16 In fact, cell membranes are non© 2016 American Chemical Society
homogeneous lipid mixtures composed of dynamic clusters referred to as microdomains or lipid rafts that assemble essential cell signaling proteins.13,17 In this context, it is possible that resveratrol interacts with these lipid domains in membranes, thereby modulating the localization of the anchored proteins and controlling their intracellular cascades. Therefore, the importance of membrane events in the myriad of effects of resveratrol is a fundamental question in this study, and the influence of resveratrol in the structural organization of lipid domains in biological membranes was investigated using X-ray scattering techniques. To the best of our knowledge, only one study of our group has been published so far regarding the effects of resveratrol on 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) model systems using X-ray diffraction techniques.16 To achieve this purpose, membrane mimetic systems composed of egg L-α-phosphatidylcholine (EPC), cholesterol (CHOL), and sphingomyelin (SM), with different molar ratios between the chosen lipids, were used to access the Received: September 30, 2016 Published: October 27, 2016 12914
DOI: 10.1021/acs.langmuir.6b03591 Langmuir 2016, 32, 12914−12922
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Figure 1. Chemical structures of the intervenient species in this study. (A) EPC unsaturated predominant species31% 18:1 PC; (B) sphingomyelin (SM) predominant species33%; (C) cholesterol; and (D) trans-resveratrol. EPC, EPC:CHOL [4:1], or EPC:CHOL:SM [1:1:1] in a chloroform/ methanol mixture (3:1, v/v). Lipid films were formed by drying the samples under a nitrogen stream and left overnight under reduced pressure to remove all traces of the organic solvents. The lipid films were then hydrated with phosphate buffer (pH 7.4) and vortexed, at 40 °C (well above the main phase transition temperature of the lipid mixtures). The lipid dispersions were transferred into glass capillaries of 1.5 mm diameter, and the flame-sealed capillaries were stored at 4 °C until the X-ray measurements. Small- and Wide-Angle X-ray Measurements. The evaluation of the long-range membrane order and the hydrocarbon chain packing by X-ray scattering measurements was carried out as described in previous studies.24 Small- and wide-angle X-ray scattering (SAXS and WAXS) measurements were recorded at the Austrian SAXS beamline in the electron storage ring Elettra (Trieste, Italy).25 Monochromatic radiation with a wavelength of 0.154 nm was used, and the SAXS and WAXS Pilatus detectors (Dectris, Baden Switzerland) were calibrated with silver behenate powder (d-spacing = 58.376 Å)26 and with pbromo benzoic acid with d-spacings taken from Ohkura et al.,27 respectively. Measurements were recorded at different temperatures (10, 37, and 45 °C) using a custom-made sample cell with a Unistat water bath (Huber, Offenburg, Germany) for cooling. Each diffraction pattern was presented as normalized scattering intensity in arbitrary units versus the reciprocal spacing in nm−1, s (s = (2 sin θ)/λ, where 2θ is the scattering angle and λ is the X-ray wavelength). The X-ray diffraction patterns obtained have been subjected to detailed analysis using a peak fitting tool in OriginPro 8.5.1 software (OriginLab Corporation). The Bragg peaks were fitted to a single Lorentzian peak function until converged. The single peak fit was then compared with the fit of two or three Lorentzian peak functions. The best fit to the data was selected according to the fitting coefficient (R2 greater than 0.99) and with the fewest deconvoluted peaks. The positions of maximum intensities (s) were determined and used to calculate the lipid bilayer + water layer distances, d (d = 1/s), whereas the widths at half maximum (w) were used to calculate the correlation lengths between the lipid bilayers, ξ (ξ = 2π/w).
effects of trans-resveratrol on the order and structure of the membrane (Figure 1). EPC is a naturally occurring mixture of phospholipids obtained from egg yolk, which have the same choline head group but different acyl chain lengths and degrees of unsaturation: C16:0, 32%; C18:1, 30%; C18:2, 15%; C18:0, 13%; C20:4, 4%; C22:6, 1%; C16:1, 1%; and others, 4%.18,19 Phosphatidylcholine is the major membrane phospholipid in eukaryotic cells.17 Lipid mixtures with cholesterol (EPC:CHOL, 4:1 molar ratio) and sphingomyelin (EPC:CHOL:SM, 1:1:1 molar ratio) were used to mimic biological membranes as they allow the characterization of lipid domains. In fact, cholesterol and sphingomyelin molecules co-exist in tightly packed liquid-ordered domains (lo) dispersed in liquiddisordered (ld) phospholipids,20−22 which provides an additional membrane environment for the assembly of different raft proteins.23 The present study contributed to a better knowledge of resveratrol interaction with biological membranes, particularly its effects on the order and structure of lipid domains in the membrane. The results showed the induction of phase separation in EPC model systems and also the promotion of lipid domain formation in EPC:CHOL and EPC:CHOL:SM bilayers. In this way, resveratrol seems to induce the organization of the lipid rafts, which may play an important role in the control of proteins and receptors and the activity of enzymes in cell membranes.
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EXPERIMENTAL SECTION
Materials. trans-Resveratrol (>99% purity) and EPC were obtained from Sigma Aldrich (St. Louis, MO, USA). Cholesterol and sphingomyelin were purchased from Avanti Polar Lipids, Inc. (Alabama, USA). Both cholesterol and sphingomyelin were naturally extracted from ovine wool and from bovine milk, respectively. Resveratrol and lipid suspensions were prepared with phosphate buffer (pH 7.4). For the preparation of phosphate buffer solutions, potassium phosphate monobasic was obtained from Sigma Aldrich and sodium hydroxide was obtained from Riedel-de Haën AG (Seelze, Germany). The buffer was prepared using double deionized water from arium water purification system (resistivity >18 MΩ cm, Sartorius, Goettingen, Germany), and the ionic strength was adjusted with NaCl (I = 0.1 M) to mimic physiological conditions. Lipid Dispersion Preparation. Different molar fractions of resveratrol (0, 5, and 10 mol %) were mixed with molar ratios of
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RESULTS AND DISCUSSION The structural effect of resveratrol on EPC, EPC:CHOL [4:1], and EPC:CHOL:SM [1:1:1] bilayers at pH 7.4 was studied using X-ray scattering techniques. SAXS and WAXS are appropriate tools to assess the phase coexistence in model membranes. SAXS permitted us to obtain the distances (d) of the bilayer thickness (db) plus the water layer (dw), and WAXS 12915
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Figure 2. (A) Temperature-dependent SAXS diffraction patterns of EPC at pH 7.4. (B) SAXS diffraction patterns of EPC in the absence and presence of 5 and 10 mol % of resveratrol, at 37 °C and pH 7.4. Note: inset boxes represent the magnified images of first-order Bragg peaks.
Table 1. Long-Range Distances (d) and Correlation Lengths (ξ) Determined from SAXS Diffraction Patterns for EPC in the Absence and Presence of 5 and 10 mol % of Resveratrol at 10, 37, and 45 °C and pH 7.4 long distances system EPC
EPC + 5 mol % RSV
EPC + 10 mol % RSV
T (°C) 10 37 45 10 37 45 10 37 45
d1 (Å) 64.0 64.9 65.5 63.8 64.6 65.0 63.8 64.6 65.1
± ± ± ± ± ± ± ± ±
d2 (Å)
0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5
60.9 60.7 61.1 59.7 59.4 61.7 60.6 59.5
± ± ± ± ± ± ± ±
0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5
ξ1 (Å) 614 614 528 896 656 581 846 707 585
± ± ± ± ± ± ± ± ±
10 10 10 10 10 10 10 10 10
ξ2 (Å) 347 310 534 350 343 546 344 334
± ± ± ± ± ± ± ±
10 10 10 10 10 10 10 10
results were found in the literature where X-ray diffraction patterns recorded from dispersions of pure EPC show evidence of two bilayer structures assigned to the fluid phase of EPC over the temperature range from 20 to 50 °C.33 The explanation for fluid−fluid immiscibility in EPC bilayers is not yet known but may be the result of phase separation of molecular species with different acyl chain lengths.34 In fact, EPC comprises a mixture of phosphatidylcholines with the same head groups and different acyl chain lengths and degrees of unsaturation.18,19 The WAXS diffraction patterns (Figure S1) confirm the presence of a liquid-crystalline structure of EPC because it presents a diffuse reflection characteristic of the disordered molten chains of the fluid phase.35,36 The addition of resveratrol leads to some changes in the SAXS diffraction patterns of EPC bilayers (Figure 2B and Table 1). The first consideration is that resveratrol induces phase separation by itself at 10 °C because at this temperature a second first-order peak appears (Table 1). This finding might be attributed to the formation of lipid domains composed of resveratrol. This phenomenon has already been described in the literature in the presence of cholesterol molecules.37,38 Wesolowska and colleagues have already suggested that resveratrol induces phase separation in DMPC bilayers by promoting the formation of resveratrol-rich and resveratrolpoor domains in the phospholipid bilayers.39 Indeed, resveratrol accumulates into liquid-ordered domains, binding preferentially to lipid rafts.40 The creation of ordered domains is probably caused by the favorable van der Waals interactions between the phenol rings of resveratrol and the acyl chains of
provides information about the short-range order of the system, that is, the packing of the lipid acyl chains.28,29 The measurements were recorded at 10, 37, and 45 °C because the order and packing of the lipids are temperature-dependent. Effect of Resveratrol on the Structure of EPC Bilayers. Figure 2A displays the temperature-dependent X-ray diffraction patterns obtained for SAXS of EPC at pH 7.4. The respective long-range distances (d) and correlation lengths (ξ) of the firstorder Bragg peaks are listed in Table 1. The results obtained indicate that the EPC bilayer selfassembles into a lamellar phase at all temperatures studied because two series of reflections can be detected with an S position ratio on the order of 1:2, which is in agreement with previous studies.19,30,31 Takeda et al. reported a d value for pure EPC bilayers of 65 Å at room temperature,19 and Kamo et al. found a d value of ca. 64 Å at 25 °C,31 which are consistent with the d1 values obtained in this study for EPC in the absence of resveratrol (Table 1). The increase in the temperature slightly increased the long-range distances of the EPC bilayer + water layer but considerably decreased the correlation length obtained. Indeed, it was already reported that the bilayer cooperativity decreases with increasing temperature mainly because the disordering effect of the temperature increases the spaces between the phospholipids.32 In fact, a broad and diffuse WAXS band of EPC is attributable to the liquid-crystalline state (Lα) typical of these lipid bilayers in the range of temperatures studied.19 From the interpretation of SAXS diffraction patterns of Figure 2A, it is possible to observe a phase separation phenomenon with the increase in the temperature. Similar 12916
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Figure 3. Schematic representation of the proposed effect of resveratrol in the structure of EPC membrane model system. Note: water molecules are not represented for simplification.
Figure 4. (A) Temperature-dependent SAXS diffraction patterns of EPC:CHOL [4:1] at pH 7.4. (B) SAXS diffraction patterns of EPC:CHOL [4:1] in the absence and presence of 5 and 10 mol % of resveratrol, at 37 °C and pH 7.4. Note: inset boxes represent the magnified images of first-order Bragg peaks.
Table 2. Long-Range Distances (d) and Correlation Lengths (ξ) Determined from SAXS Diffraction Patterns for EPC:CHOL [4:1] in the Absence and Presence of 5 and 10 mol % of Resveratrol at 10, 37, and 45 °C and pH 7.4 long distances system EPC:CHOL [4:1]
EPC:CHOL [4:1] + 5 mol % RSV
EPC:CHOL [4:1] + 10 mol % RSV
T (°C) 10 37 45 10 37 45 10 37 45
d1 (Å) 65.5 66.6 67.7 65.8 67.1 68.1 70.5 72.5 72.4
± ± ± ± ± ± ± ± ±
d2 (Å)
0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5
65.2 ± 0.5 65.7 ± 0.5 65.3 65.5 65.6 66.6 66.3
± ± ± ± ±
0.5 0.5 0.5 0.5 0.5
ξ1 (Å) 1532 1323 1304 1366 1072 994 565 370 367
± ± ± ± ± ± ± ± ±
10 10 10 10 10 10 10 10 10
ξ2 (Å) 593 ± 10 535 ± 10 446 374 483 276 228
± ± ± ± ±
10 10 10 10 10
correlation lengths (ξ) of the first-order Bragg peaks are listed in Table 2. The SAXS results for EPC:CHOL bilayers (Figure 4A) reveal the presence of lamellar structures. At 10 °C, there is an homogeneous and uniformly distributed mixture of lipids, where the d1 value corresponds to the thickness of the EPC:CHOL bilayer + water layer (Table 2). However, increasing the temperature to 37 and 45 °C induces the formation of lipid domains, with phase separation, which is demonstrated by the appearance of two distinct d values (Table 2), as already described in the literature.32,43,44 The first distance (d1 ≈ 67−68 Å) corresponds to the thickness of the EPC:CHOL bilayer + water layer (liquid-ordered phaselo), whereas the second distance (d2 ≈ 65 Å) corresponds to the width of the pure EPC bilayer + water layer without cholesterol (liquid-disordered phaseld), showing a redistribution of the
phospholipids.15,41,42 The presence of small liquid-ordered domains (lo) composed of resveratrol can consequently explain the increase in the correlation length of EPC bilayer (Table 1) for the first peak because it brings some structural organization and order to the membrane. The second peak, which should correspond to the liquid-disordered (ld) phase, remains almost unchanged as far as correlation length is concerned. Overall, EPC is still in a liquid-crystalline phase because there is no evidence of a peak in the WAXS diffraction patterns even in the presence of resveratrol (Figure S1). Figure 3 shows a schematic representation of the proposed effect of resveratrol in the structure of EPC bilayer. Effect of Resveratrol on the Structure of EPC:CHOL Bilayers. The temperature-dependent SAXS diffraction patterns of EPC:CHOL [4:1] at pH 7.4 are shown in Figure 4A, whereas the respective long-range distances (d) and 12917
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Figure 5. Schematic representation of the proposed effect of resveratrol in the structure of the EPC:CHOL membrane model system. Note: water molecules are not represented for simplification.
The WAXS diffraction patterns in both the absence and the presence of resveratrol (Figure S1) are characterized by broad bands typical of acyl chains in a disordered fluid state, confirming the coexistence of two liquid-crystalline phases (lo and ld) in this EPC:CHOL bilayer.36,47 Figure 5 shows a schematic illustration of the suggested effect of resveratrol in the structure of the EPC:CHOL bilayer. Effect of Resveratrol on the Structure of EPC:CHOL:SM Bilayers. SAXS temperature-dependent diffraction patterns of EPC:CHOL:SM [1:1:1] at pH 7.4 are shown in Figure 6, and Table 3 displays the respective long-range distances (d) and correlation lengths (ξ) of the first-order Bragg peaks.
cholesterol molecules that are now composed of the lipid raftslo. The distance obtained for the lipid rafts is greater than the distance obtained for the liquid-disordered phase because the presence of cholesterol leads to the stiffening of the bilayer and its compression, which increases d thickness. On the contrary, the EPC-rich area is more fluid and hence causes the relaxation of the acyl chains and the lateral spreading of the phospholipids, which reduces its thickness. Indeed, comparing the thickness d of a pure EPC bilayer, ca. 64−65 Å (Table 1), to the distance d obtained for the EPC:CHOL membrane, ca. 66−68 Å (Table 2), one can conclude that the incorporation of 20 mol % of cholesterol increases the d value at all studied temperatures because of a tendency of the upper portion of the lipid hydrocarbon chains to adopt a trans configuration, thereby becoming effectively longer.45,46 In fact, it is well documented that cholesterol molecules induce the reorientation of the acyl chains of the phospholipids into a fully extended and vertically orientated position that may order the system, increasing the d values.43 At the same time, when cholesterol is incorporated into a phospholipid bilayer, the head groups shield the nonpolar structure of cholesterol from exposure to water, the so-called umbrella model.20 Therefore, under the umbrella head groups, the acyl chains and cholesterol molecules become tightly packed, whereas the head groups become expanded, increasing the d value.21 In terms of correlation lengths, an increasing effect similar to that of resveratrol in the EPC bilayer was observed. In the presence of resveratrol (Figure 4B and Table 2), it is possible to notice a greater tendency for the formation of these domains or lipid rafts because they arise from lower temperatures. In fact, at 10 °C, the SAXS diffraction patterns already displayed phase separation. Therefore, we anticipate that resveratrol induces a phase separation in the membrane, promoting the formation of lipid rafts. At the same time, it also appears that the d value increases with increasing concentrations of resveratrol. However, only d1 increases but not d2, suggesting that resveratrol has a higher effect on the ordered lipid domains rich in cholesterol than on the disordered ones, favoring the formation of such lipid rafts. Concerning the cooperativity (Table 2), we found that both temperature and resveratrol tend to decrease the value of the correlation factor of EPC:CHOL bilayers, which may also be related with the phase separation phenomenon. In fact, with the formation of these lipid domains, the value of cooperativity seems to decrease as the system becomes more heterogeneous because of the presence of a mixture of cholesterol and resveratrol molecules disturbing the membrane which induce the appearance of various distances of thickness and, consequently, cause the broadening of the Bragg peaks.
Figure 6. Temperature-dependent SAXS diffraction patterns of EPC:CHOL:SM [1:1:1] at pH 7.4. Note: inset boxes represent the magnified images of first-order Bragg peaks.
In the SAXS diffraction patterns of EPC:CHOL:SM [1:1:1] bilayers (Figure 6), the two series of reflections indicate a lamellar structure for all studied temperatures. Moreover, at 37 and 45 °C, two lamellar phases are present within each order of Bragg reflection, where overlapping peaks are in agreement with the literature.32,47,48 Therefore, it is possible to assume a clear phase separation with increasing temperature that may be associated with the separation of liquid-ordered domains (lo) from a liquid-disordered phase (ld).47,49,50 This information is in agreement with the PC:CHOL:SM phase diagrams where regions of coexistence of phases have been described.51,52 When temperature increases from 10 to 37 °C, a phase separation occurs (lo → lo/ld) in POPC/SM/CHOL (1:1:1) dispersions.52 This phase separation is very likely to occur in cell membranes with 30−40% of cholesterol molecules, mainly at higher temperatures where the lo/ld coexistence region is much broader in PSM/POPC/CHOL (1:1:1) systems.51 12918
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Table 3. Long- and Short-Range Distances (d) and Correlation Lengths (ξ) Determined from SAXS and WAXS Diffraction Patterns, Respectively, for EPC:CHOL:SM [1:1:1] in the Absence and Presence of 5 and 10 mol % of Resveratrol at 10, 37, and 45 °C and pH 7.4 long distances system EPC:CHOL:SM [1:1:1]
EPC:CHOL:SM [1:1:1] + 5 mol % RSV
EPC:CHOL:SM [1:1:1] + 10 mol % RSV
T (°C) 10 37 45 10 37 45 10 37 45
d1 (Å) 67.7 67.5 67.5 67.6 66.9 67.0 68.1 67.0 67.1
± ± ± ± ± ± ± ± ±
0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5
d2 (Å) 65.2 ± 0.5 64.6 ± 0.5
short distances
ξ1 (Å) 741 542 407 1301 1174 1061 1081 986 909
± ± ± ± ± ± ± ± ±
10 10 10 10 10 10 10 10 10
ξ2 (Å)
d (Å)
ξ (Å)
1208 ± 10 1195 ± 10 4.64 4.65 4.65 4.64 4.65 4.65
398 206 142 479 427 369
± ± ± ± ± ±
10 10 10 10 10 10
Figure 7. SAXS and WAXS diffraction patterns of EPC:CHOL:SM [1:1:1] in the absence and presence of 5 and 10 mol % of resveratrol, at 37 °C and pH 7.4. Note: inset box represents the magnified image of the first-order Bragg peaks.
in an umbrella conformation with a ratio of two cholesterol molecules for each molecule of sphingomyelin.22 In this case, as we have a ratio of CHOL:SM [1:1], all cholesterol molecules will be involved in the formation of these liquid-ordered domains, with an excess of sphingomyelin molecules that will uniformly distribute along the liquid-disordered phase of EPC, thus causing phase separation. The insertion of resveratrol into the EPC:CHOL:SM bilayers (Figure 7 and Table 3) leads to a decrease in the phase separation, which can be explained if resveratrol molecules behave like cholesterol, also forming umbrella-shaped domains constituted by one molecule of sphingomyelin and two molecules of resveratrol. For this reason, we no longer have two completely separate phases with different distances but two similar ordered domains composed of two different types of umbrellas with similar distances, d1 (EPC:CHOL:SM ≈ EPC:RSV:SM). In what concerns cooperativity, the correlation lengths decreased with the temperature in the absence of resveratrol (Table 3), which is perfectly understandable when we think that there is a phase separation with temperature and, therefore, an increase in the half-width of the peak. Indeed, a more fluid system is itself a more disordered model consisting of several irregular phases and leading to low values of cooperativity. However, in the presence of resveratrol, the correlation length increases, thereby explaining the reversion of phase separation that has already been explained before. In fact, in the presence
Moreover, the tendency of sphingomyelin and phosphatidylcholines to phase separate seems to be important in this context. The lo phase state is rich in sphingomyelin (33%− 42%) and cholesterol (44%−48%) molecules with high acylchain order, whereas the ld domains are predominantly composed of phosphatidylcholines (57%−69%) and the lipids are loosely packed.50,51,53 A pseudo-ternary phase diagram at 37 °C has also been constructed by Quinn et al. from the analysis of X-ray data recorded from binary and ternary mixtures of EPC, cholesterol, and sphingomyelin (EPC:CHOL:SM).33 The coexisting of liquid-ordered and liquid-disordered (lo + ld) phases seems to dominate the phase diagram and may be assigned to the phase separation of a structure composed of a mixture of sphingomyelin and cholesterol (lo) and a singlephase composed mainly of EPC (ld).33 Tessier et al. suggested d values of 69.3 Å for lo domains and 65.4 Å for ld domains in EPC:CHOL:SM [1:1:1] bilayers at 37 °C,50 whereas Quinn et al. found d values of ca. 68 Å for lo domains and ca. 65 Å for ld domains in EPC:CHOL:SM [1:1:1] bilayers at 50 °C,47 which are consistent with the d1 value of ca. 68 Å and d2 value of ca. 65 Å obtained in the present study for EPC:CHOL:SM [1:1:1] in the absence of resveratrol (Table 3). The formation of ordered domains can be explained by the umbrella model,20,21 where cholesterol must rely on the head group of sphingomyelin to cover its large nonpolar structure from the aqueous environment.53 Indeed, it is well known that cholesterol and sphingomyelin molecules can form domains 12919
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Figure 8. Schematic representation of the proposed effect of resveratrol in the structure of the EPC:CHOL:SM membrane model system. Note: water molecules are not represented for simplification.
The results obtained revealed that resveratrol induces phase separation in EPC model systems and also seems to promote the formation of lipid domains in EPC:CHOL and EPC:CHOL:SM bilayers. This brings some structural organization and order to the membranes. In a recent study of our group, Förster resonance energy transfer and DPH fluorescence quenching assays have also been applied to reveal potential molecular interactions between resveratrol and lipid domains.15 In fact, resveratrol has been shown to induce phase separation in lipid bilayers, favoring the formation of lipid domains in EPC, EPC:CHOL, and EPC:CHOL:SM model systems.15 As a result, we conclude that resveratrol controls the lipid packing of bilayers by inducing the organization of lipid rafts. Moreover, the formation of lipid domains is important for modulating the activity of many receptors, transmembrane proteins, and enzymes whose activity depends on the structural organization of the membrane and on the presence or absence of these organized domains.55−59 Therefore, it is likely that the action of resveratrol on these targets is dependent on the interaction with lipid domains within the membranes where they are inserted because rafts are implicated in many biological processes such as signal transduction, membrane trafficking, and protein sorting.60 Consequently, the understanding of how resveratrol modulates lipid rafts and how these domains interfere in cell fate may lead to an important advance in the knowledge of resveratrol action and in the design of new strategies for the treatment of several diseases.
of resveratrol, the membrane becomes uniform because of the presence of similar umbrella conformations composed of EPC:CHOL:SM and EPC:RSV:SM, which have similar thicknesses, as we have seen in the previous analysis. Finally, the absence of a sharp WAXS peak in the EPC:CHOL:SM bilayer without resveratrol (Figure 7) indicates that no gel phases are present in this lipid membrane, as in the previous models.36,47 However, in the presence of resveratrol, a peak located at a d-spacing of 4.6 Å can be found from the WAXS profiles (Figure 7), which by itself indicates the higher organization state of the system. Nevertheless, the packing of the lipid acyl chains is still typical of disordered hydrocarbon chains in a fluid state.49,54 The schematic representation of the anticipated effect of resveratrol in the structure of an EPC:CHOL:SM bilayer is presented in Figure 8.
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CONCLUSIONS The current work tried to elucidate the way by which resveratrol affects membrane order and structure, particularly with regard to lipid domain formation, using SAXS and WAXS techniques. The concentration of 10 mol % corresponds to membrane effective concentrations, [RSV]m, of 0.1 M resveratrol for EPC bilayers, 0.05 M for EPC:CHOL [4:1], and 0.04 M for EPC:CHOL:SM [1:1:1], calculated according to the following equation [RSV]m =
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K p[RSV]T K pαm + (1 − αm)
ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.langmuir.6b03591. WAXS of EPC and EPC:CHOL [4:1] (PDF)
where Kp is the partition coefficient of resveratrol calculated for each membrane model system in a recent publication of our group,14 [RSV]T is the total concentration of resveratrol, and αm is the volume fraction of membrane phase (αm = VØ[L]; VØ is the lipid molar volume and [L] is the lipid molar concentration). To make sure that these concentrations can really occur biologically, here we sum up some relevant bioavailability data of resveratrol. Several studies have reported a peak plasma level of ≈2 μM after the oral administration of 25 mg of resveratrol by a person with 70 kg of weight.2 Additionally, 500 mg per day of resveratrol has been shown to be adequate, and no adverse effects have been found in vivo.10 Considering this, a plasma concentration of ca. 40 μM resveratrol can be achieved after the oral administration of 500 mg supplement. Considering the Kp values of 3400 for EPC bilayers, 1400 for EPC:CHOL [4:1], and 900 for EPC:CHOL:SM [1:1:1] determined in a recent study using liposomes/water systems,14 one can conclude that ca. 0.1, 0.05, and 0.04 M resveratrol concentrations, respectively, can be achieved in biological membranes, which is comparable to the concentrations used in this study.
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AUTHOR INFORMATION
Corresponding Author
*E-mail: cdnunes@ff.up.pt. Phone: (+351)220428672. Fax: (+351)226093390. Author Contributions
The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work received financial support from the European Union (FEDER funds) and National Funds (FCT/MEC, Fundaçaõ para a Ciência e a Tecnologia and Ministério da Educaçaõ e 12920
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Ciência) under the Partnership Agreement PT2020 UID/ MULTI/04378/2013POCI/01/0145/FEDER/007728. The authors thank Elettra Synchrotron beamline (Trieste, Italy) for beam time and support through the project 20135320. A.R.N. and C.N. also acknowledge their post-doc grants under the project NORTE-01-0145-FEDER-000011 and SFRH/BPD/ 81963/2011. To all financing sources, the authors are greatly indebted.
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