Effects of cholesterol on the lamellar and the inverted hexagonal phases of dielaidoylphosphatidylethanolamine

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ct Biophysica A~ta ELSEVIER

Biochimica et Biophy,~i':aActa 1280 ( toq6l 2,t~tt-216

Effects of cholesterol on the lamellar and the inverted hexagonal phases of dielaidoylphosphatidylethanolamine Hiroshi Takahashi, Katsuki Sinoda. Ichiro Hatta

"

Department of Applied Ph.v.~ic,~,Nagoya Unirersitv. Nagoya 464.01. Japan Received II July 1095: revi~d 19 October 1995: accepted 15 November 1995

Abstract

Effects of cholesterol on the lamellar and the inverted hexagonal ( H,) phase,~ of dielaidoylphosphatidylethanolamine (DEPE) were studied by means of not only differential scanning calorimetry (DSC) but also ~imuhaneous X-ray diffraction and DSC (XDDSC). XDDSC shows that structural changes are related to thermotropic events of the mixtures. Addition of cholesterol to DEPE induces to broaden the transition from the lamellar gel (Lt~) to lamellar liquid-crystalline (L,,) pha.~, in fact, in the broad transition region, a coexistence of two lamellar X-ray diffraction peaks of the L~ and the L, phases take place. In sample:; containing above 30 tool% cholesterol, no peak at the L~-L,, phase transition was observed in the DSC thermogram. On the other hand. cholesterol c a u l s biphasic effects on the L~,-Hu phase transition: At low cholesterol concentrations below 20 tool%, the incorporation of cholesterol reduces the transition temperature and at high cholesterol concentrations above 30 tool%, the transition temperature increases by addition of cholesterol. Based upon the results of X-ray diffraction, the thermal expansion coefficients of lattice spacings, i.e.. the temperature dependence of lattice spacings, were calculated in each phase. Addition of cholesterol reduces the thermal expansion coefficients of the lamellar phases and, in contrast, increases that of the H n pha~. From the above r~,~ults it is suggested that cholesterol in cell membranes works in keeping the bilayer membrane nature notwithstanding the change of exte~ml ,-onditions. Keywords: Cholesterol; Phosphatidylethanolamine; Lamellar phase; Inverted hexagonal phase: DSC: X-ray diffraction

~. I n t r o d u c t i o n

Cholesterol is a major constituent of eucaryotic plasma membranes in animal cells, it is believed that the modulation of physical properties of lipid membrane induced by incorporation of cholesterol is relevant to the function of biomembranes. The interaction between cholesterol and phospholipids has therefore been a subject of considerable study. Phosphatidylcholine (PC)-cholesteroi mixtures have been studied extensively [1-3]. Recently, it has been revealed that PC-cholesterol mixtures form a presumably homogeneous phase above 20 tool% cholesterol concentra-

Abbreviations: PC. phosphatidylcholine. PE, phosphalidylethanolamine; DEPE. dielaidoylphosphatidylethanolamine" Hepes. hydroxyethylpiperazine-ethanesulfonie acid: EDTA, ethylenediaminetetraacetic acid: DSC. differential scanning calorimetry: XDDSC. simultaneous X-ray diffraction and differential ~anning calorimetry: L/j, lamellar gel phase: L,,. lamellar liquid-crystalline phase; HEn, inverted hexagonal phase " Corresponding author. Fax: +81 52 7893724; e-maih [email protected],ac.jp. 0304-4165/96/$15.00 © 1996 Elsevier Science [I.V. All rights reserved 5SDI 0304-4 165(95 )001 70-0

tions. This phase is called ~phase [4] or liquid-ordered phase [5]. This phase has a feature of both liquid-crystalline and gel pha~s. Phosphatidylethanolamines (PEs) are the second major component of membrane phospholipids in animal cells. Unlike common PCs, one of features of PEs i,~ to undergo a lamellar-inverted hexagonal ( H . ) phase transition, it has been pointed out that non-bilayer phase (H,n, cubic phase, etc.) plays an important role in membrane fusion process [6]. From this viewpoint, therefore, the effects of cholesterol on the non-bilayer phase of PEs are interesting ,,ubjects. There are many studies of the effects of choleste,ol on the iamellar phase [7-12]. However. there are relatively a few papers [13-16] which deal with the interaction between PEs and cholesterol in non-hilayer phase. Gallay and De Kruijff [14] have studied the effects of a variety of sterols on the lamellar-H n p h a ~ transition temperature of dielaidoylphosphatidylethanotamine (DEPE). Recently, the detailed differential scanning calorimetry (DSC) study has revealed a biphasic effect of cholesterol on the lamellar liquid-crystalline (L,,)-Ht, phase transition temperature for DEPE-

2 Ill

H. Takahmhi e! aL / llim'himi<'a el Biophy.vira A<'t
cholesterot mixtures [15]. Furthermore, Cheetham c t a l , [16] have shown that addition of cholesterol reduces the lattice constant of the Huj phase of DEPE. DEPE is lipid which is convenient for the study of cholesterol effects on the both lamellar and H . phases, because DEPE undergoes the lamellar gel (Lt3)-L,, phase transition and the L,,-H~+ phase transition at about 38°C and about 63°C, re:.,pectively [I 7.18] and therefore, DSC was easily applied to the measurements in the temperature range of O-iO0°C. However. the phase behavior of DEPE has the following characteristic features. The kinetics of the H u phase stabilization depends on lipid concentrations [19]. At the Lo to L , phase transition, two transition peaks appear in the cooling scan DSC thermogram [20]. Thus, in this paper. paying attention to these characteristic features of DEPE. we investigated the effects of cholesterol not only on the lamellar phase but also on the H , phase of DEPE. The phase behavior of DEPE-cholesterol mixtures was reexamined by means of differential scanning calorimetry (DSC) in the both heating and cooling scans. Furthermore. simultaneous X-ray diffraction and DSC (XDDSC) measurements were used to study the relationship between the structural changes and the phase transitions of the DEPEcholesterol mixtures. All experiments were performed for the samples with 40 wtCb lipid concentration. The effects of cholesterol on the thermal expansion coefficients (temperature dependence of lattice spacings) of each phase were also discussed.

2. Materials and methods 1.2-D ie laidoy I-sn-glyce ro- 3-phosphoethanolam ine (DEPE) was purchased from Avanti Polar Lipids (Alabaster. AL) and cholest-5-en-3/3-ol (cholesterol) was obtained from Sigma (St. Louis. MO). Hydroxyethylpiperazineethanesulfonic acid (Hepes) and ethylenediaminetetraacetic acid (EDTA) were purchased from Katayama (Osaka. Japan). No chromatographic analyses were performed but narrow transition width ( < 0.8°C in FWHM at I.O~C/min DSC scan) of DEPE was consistent with the supplier's claim of 99~ authentic compound. The purity of anhydrous cholesterol was checked using DSC and then. we observed a standard transition at 149°C [21]. DEPE and cholesterol were dissolved in chloroform and mixed in proportion to achieve the desired molar fractions. The solvent was evaporated under a stream of oxygen-free dry nitrogen and stored for 16 h in vacuum to remove remaining traces of solvent. The DEPE-cholesterol mixtures were hydrated by vortexing in !00 mM Hepes/I mM EDTA buffer (pH 7.37. The kinetics of the H , phase stabilization of DEPE depends on lipid concentrations [19]. Thus. the concentration of samples used in all measurements was 40 wt+;~ ((lipid + cholesterol)/water). The amount of cholesterol to phospholipid was expressed by mol~ as tool cholesteroi/(mol cholesterol + tool phospholipid). The

lipid-cholesterol mixtures were stored at 0-4°C until the measurements. Differential scanning calorimetry (DSC) was performed using a DSCI0 with SSC580 (SEIKO I and E. Tokyo, Japan) with the scan rate of 1.0°C/min. Lipid dispersions (about 20 /11) were placed in 40 p.I aluminum pans (Mettler, Highstown, NJ) and hermetically sealed. DSC data acquisition and analyses were performed on a microcomputer (PC-9801. NEC. Tokyo, JaFan) using a software provided by SEIKO I and E. Transition enthalpies were determined by the integration of the area under the transition curve and the transition temperatures were simply defined by the position of the peak maximum. Simultaneous X-ray diffraction and DSC (Xr~DSC) measurements were undcrtaken at station 15A of the Photon Factory (Tsukuba, Japan). The optical system in this station was described elsewhere [22]. In this measurements, time-resolved X-ray diffractograms were collected during heating or cooling of the samples, while at the same time the endo- or exothermic heat flow was recorded. Therefore. the relationship between the structural change and the thermal event at a phase transition is easily examined with this XDDSC measurements. In this XDDSC measurements. Mettler DSC apparatus (FP84)+ which was originally designed for simultaneous DSC and polarized light microscopy, was utilized. The first experiment of XDDSC was performed by Russell and Koberstein [23]. The details of our modifications were described elsewhere [24.25]. In each experiment, successive 70 diffraction patterns were recorded using a one-dimensional position-sensitive proportional counter with 512 channels (PSPC+ Rigaku, Tokyo, Japan). The exposure time for each frame was 30 s. The sample-to-detector distance was 1340 mm.

3. Results 3. I. D S C

We performed DSC measurements for DEPE-cholesterol mixtures with the range tYom 0 to 50 mol% cholesterol. Fig I shows the heating DSC thermograms of these mixtures and pure DEPE. The transition temperatures and the transition enthalpies are plotted as a function of cholesterol concentrations in Figs. 2 and 3, respectively. Addition of cholesterol to DEPE induces continuous decrease of the temperature of the lamellar gel (L~) to lamellar liquidcrystalline ( L , ) phase transition. In addition, broadening of the thermogram at the transition also was observed. The transition enthalpies also decrease as cholesterol concentration rises (Fig. 3). At cholesterol concentrations above 30 tool% no transition peak of the L~-L, phase transition was observed in the DSC thermograms. These results are in good agreement with that reported by Epand and Bottega [15]. In contrast to the L/j-L,, phase transition, another efl'ect of cholesterol on the L,, to inverted hexagonal (H in)

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width of the L,,-H, phase transition increase as cholesterol concentration rises (.see also Fig. 2). Addition of cholesterol to DEPE hardly affects the enthalpy of the L.-H,j phase transition (Fig. 3). These DSC results demonstrate that the effects of cholesterol on the La-L,, phase transition are more remarkable than that on the L , - H , pha~ transition and that cholesterol makes a biphasic effect on the transition temperature of the L,-H, phase transition. These results are consistent with that reported by Epand and Bottega [ i 5]. The therrnograms in a cooling scan are shown in Fig. lB. In the cooling scan. the peak temperature is lower than 12

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phase transition was observed. As displayed in Fig. I A. at cholesterol concentrations below 20 tool%, addition of cholesterol to DEPE reduces the transition temperature. and sightly affects the width of the L,.-H t, phase transition. On the other hand. at cholesterol concentrations above 30 mol%, both of the peak temperature and the

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that in the heating scan. The most marked feature of the thermograms is that two peaks were observed in the Lv-L,, phase transition regions for pure DEPE and the mixture containing I0 molCb cholesterol. In the mixtures containing 5 and 15 molC/E cholesterol, two transition peaks were also observed (thermograms not shown in Fig lB, see Fig, 2). This fact was already reported for pure DEPE by Epand and Epand [20]. in addition, for pure dipalmitoyI-PE, multi peak-pattern was observed in the DSC thermogram in the cooling scan by Yao et al, [26] and Lewis and McEihaney [27]. Yao et ai. [26] also performed the time resolved X-ray measurements. They observed the coexistence of two reflections corresponding to the Ltj and the L,, phases in the temperature range in which multi peak-pattern was observed in the thermogram, it is worthwhile to point out that such two transition peaks were observed even in the DEPE-cholestetoi mixtu0e: trc!e'" !5 mrs!% cholesterol concentration.

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normalized by the intensity after I min exposure to t'e I. The integrated intensity and the full width at half maximum were estimated by fitting Lorentzians to the observed diffraction patterns.

3.2. XDDSC In this XDDSC measurements, we used a high intensity X-ray beam afforded by a synchrotron storage ring. In experiments using X-ray beams with high intensity, the effects of radiation damage are necessary to be taken into consideration. In the beginning, we estimated the radiation aamage of the sample. The radiation damage by high intensity X-ray beam can be monitored by observing the small-angle diffraction profile. Recently, a phase transition arising as a consequence of radiation damage w.a~ ;'eported for PEs samples [28,29]. Cheng et al. [29] reported that for the fully hydrated dihexadecyI-PE, after irradiation of a high intensity X-ray beam. the diffraction peak,,, corresponding to the Ht~ phase appears below the usual L,,-H u phase transition temperature. In order to check the radiation damage, we observed the sequential diffraction profiles in I rain continuous exposure for the pure DEPE at 61°(2. about 3°(2 below the L,,-Htl phase transition temperature. Even after 90 rain exposure, the diffraction peaks of H u phase were not observed (the pattern not shown). As shown in Fig. 4, the intensity of the lamellar diffraction decreases about 8% after 90 min exposure. However, the full width at half maximum of the diffraction peak is almost constant. This result implies that the radiation damage might occur but the contribution is not serious in this study. Because, no sample was exposed to the X-ray beam for > 35 rain. The discrepancy between our result and that of Cheng et al. might be due to the difference of the intensity of the incident beams. Tne measured X-ray beam intensily was ! - 2 . I0 x photons/s in a focused spot of 1.0 mm X 2.0 mm in this study. On the other hand, Cheng et al. reported that the intensity was 2 . 1 0 I" photons/s in a 0.3 mm diameter collimator size. In addition, in a sample cell, an aluminium plate with I00 /zm tidck absorbs about 80oh X-ray beam. XDDSC measurements were performed for the mixtures

containing 0, I0, 20, 30 and 40 mol% cholesterol in the temperature range from 20°C to 85°(:. Fig. 5 shows DSC thermograms obtained using XDDSC. The results of DSC obtained with simultaneous measurements are essentially identical to that of DSC measured independently (Fig, I A). However, there are a few different points. Comparing Fig. 5 with Fig. I A, the transition peaks exhibit rather broader due to the faster scan rate (2.0°C/rain). In the thermogram of 30 tool% cholesterol mixture, broad transition peaks are seen in the range from 25°C to 40°C (see Fig. 5), however,

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lamellar rcflcctions coexist. One reflection with shorter spacing (about 5.~-6.0 nm) is due to the L,, pha~ and the other reflection with longer ~pacing (about 6.5 nm) originates from the region of the LIj phase, in about 35-55°C, only lamellar reflection originating from the L,, pha~ was observed. The diffraction pattern above 63°C indicates the formation of the Hn,.~ha~ which gives the Bragg spacings with the ratio I: I / ¢3 : I / 2 . In the I.,.-H ~t pha~ transition region, the coexistence of two pha~s was ob~rved. The coexistence of two phases was also ob~rved for the mixture containing 20 tool% cholesterol (patterns not shown). Furthermore, for the sample with 20 molr/i cholesterol, an additional reflection with about 5.6 nm spacing due to an uncharactedzed structure was detected together with the reflections of the L,, and HII phases in the temperature range of 62-65°C. In the presence of 30 mol% and 40 mol% cholesterol, only one lamellar reflection originating from the L,. phase appears below the L,,-H,t phase transition (diffraction patterns not shown), i.e.. no transition from the Lt~ to L,, phase appears in the temperature range 20-60°C. It was confirmed from the data of XDDSC measurements that there is a wide coexistence region of the Ltj and the L,, phases for the sample containing 10-20 tool% cholesterol below the peak temperature of the L:j-L,, phase transition as obtained by DSC.

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as will be described later, no change of the X-ray diffraction profile was observed in the corresponding temperature range. In connection with this fact. we have no explanation for two peaks observed in the range from 25°C to 40°C for 30 tool%. In the present XDDSC measurements, small-angle X-ray diffraction patterns were recorded in the range of S = 0-0.5 nm -~. S is defined as 2sin0/,4 (20= scattering angle. A = wavelength of X-ray). But, the patterns in 0-0.05 nm -l are out of observation as shadowed by a beam stopper. For the all mixtures, in a high temperature region {above 70°C). three reflections were observed in the range of S = 0 . 0 5 - 0 . 5 nm -I (see Fig. 6). These reflections correspond to the (10). (11) and (20,~ planes of the hexagonal lattice of the H it phase in order. For the L/j or the t,,, phase, only the first order of lamellar reflections was observed. The second order of lamellar reflections could not be detected due to its weak intensity. In the static X-ray diffraction measurements with a longer exposure time. the second order reflection couht he detected. For pure DEPE. the changes of diffraction patterns were observed at each phase transition temperature (diffraction pattern not shown). Fig. 6 shows small-angle X-ray diffraction patterns in the range of S = 0.1-0.4 nm -I obtained by XDDSC for the sample of DEPE containing I0 tool% cholesterol. Below about 35°C, two kinds of

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H. Takahu.~'hi el aL /Bhu'him!ca el IJkJphysit'a Acre 1289 f1996~ 209-2 Ili

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The lattice spacings obtained by XDDSC are plotted in Fig. 7 as a function of temperature. In this paper, the lattice spacing means the laltice basis vector length in the unit cell. i.e.. the lattice spacings are the lamellar repeat distance and the center-to-center distance between the water core for the lamellar and the hexagonal phases, respectively. The spacings for pure DEPE are in good agreement with published data [30]. Addition of cholesterol reduces the lattice spacing of the Lt~ and the H , phase and. m cnntra,~t, it lengthens the lattice spacing of the L,, phase. These tendency are almost in agreement with that reported by Cheetham et al. [16], The temperature dependence of the lattice spacing was observed for the all mixtures. The thermal expansion coefficients of the lattice spacings calculated from the data of Fig. 7 are summarized in Table I. In contrast with the La or the L,, phase, the H , phase has a strong temperature dependence of the lattice spacing. This is one of common feature for Hit phase [31-35]. The order of the thermal expansion coefficients for pure DEPE in each phase is in good agreement with that of dihexadecyI-PE [31]. Cholesterol reduces the thermal expansion coeMcients of the Lt~ and L , phase. In contrast, the thermal expansion coefficients of the H , phase increase as cholesterol concentrations rise.

4. D i ~ u ~ i o n DSC and XDDSC measurements in this study show that a distinct transition from the L~ to L,, phase,,, disappears at cholester,.~l concentratiens above 30 mol'/r. However, Blume [10] reported that a very broad transition is ob.,,erred at cholesterol concentrations above 30 molC~ using high-sen,,itivity DSC for the dimyristoyI-PE- or dipalmitoyI-PE-cholesteroi mixtures. Furthermore. it is well known that for dipalmitoyI-PC-cholesterol mixtures, at cholesterol

concentrations from 0 to about 20 sol%, the endothermic DSC peak consists of two components [36,37]. One is sharp and the other is very broad. The sharp component disappears in the DSC thermogram at cholesterol concentrations above about 20 mo1%. On the other hand, the broad component persists in the thermogram until cholesterol concentralions reach 50 sol%. McMullen et al, [37] pointed out that such less energetic and less cooperative phase transitions can not be detected using a low-sensitivity DSC. Therefore. there is a possibility that a very broad transition of the DEPE-cholesterol mixtures remains at cholesterol concentrations above 30 sol%. The our results and those of previous papers [15,16] by Epand's group exhibited a biphasic effect of cholesterol on the L,,-H. transition temperature. At cholesterol concentrations below 25 mol%. cholesterols stabilize the H u phase of DEPE. On the other hand, cholesterol with concentration above 30 s o l % destabilizes the H , phase. In phosphatidylcholin~-cholesterol mixtures, it has been known that a liquid-ordered phase is formed instead of a L,, phase at cholesterol concentration above 20 s o l % [3-5]. Such a liquid-ordered phase might be also formed in DEPE-cholesteroi mixtures above 30 s o l % cholesterol. The biphasic effect of cholesterol seems to be associated with the formation of a liquid ordered phase. However, to determine the formation of ordered-liquid phase a further study is required. It is worthwhile to point out that the two lamellar reflections of the L a and the L,, phases coexist in the presence of 10-20 molC/~ cholesterol. In the DSC thermogram, a broad transition peak was observed in the temperalure range of 25-35°C. These facts indicate that a phase separation occurs for the mixtures containing less than 30 s o l % cholesterol. One region might be pure DEPE-rich domain and the other might be cholesterol-rich domain. It is well known that addition of cholesterol increases the fluidity of bilayers. Therefore, the cholesterol-rich domain gives the reflection with shorter spacing, such as the reflection of the L,, phase. From the results of carbon-13 and deuterium nuclear magnetic resonance [I 1,12], it was reported that there are two conformationally and dynamically inequivalent dipalmitoyl-PE molecules in dipalmitoyI-PE-cholesterol mixtures over the wide ranges of temperature and composition in its phase diagram. The coexistence of the two reflections was also observed in the L,-H I, phase transition region. The coexistence temperature range for pure DEPE is wide (about 10°C) in particular. Recently. Epand and Lemay [39] reported that the L,, phase of DEPE is metastable in the temperature range of 63-65°C. In addition. Tate et al. [30] reported that there exist the long time kinetics of the L,-Hi, phase transition of dioleoyi-PE lot a small temperature jump. where the L,,-H. phase transition temperature for dioleoyI-PE is about 3°C. In the case of the temperature jump from 2°C to 4°C, the reflection of the L , phase remained after a week. For pure DEPE. the coexistence of

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the Lhermal e x p a n s i o n c o e f f i c i e n t in t h e l a m e l l a r p h a ~ . Thi,, fi=cl m i g h t i m p l y that the physical properties o f t h e hilayer,,, s u c h as the f l u i d i t y or the r o t a l i o n a l m o t i o n o f the lipid m o l e c u l e , , , are kept u n c h a n g i n g a g a i n s t l e m p e r a t u r e . " ~,, * ... ... .. . . .r ... ., - c . . ' m e (~f f u n c t i o n s ¢~f c h o l e s t e r o l in b i o l o g i c a l m e m b r a n e , , m i g h ! c o n t r i b u t e the c o n , , l a n c y o f t h e m e m b r a n - n a t u r e a[~ainst t h e c h a n g e o f e x t e r n a l ~:onditions.

Acknowledgements DEPE-cholesterol

DEPE

0 chelesterol

water-core

Fig. 8. Schemalic view of the Hi1 pha.,,e for pure I)EPE (uppcrl and DEPE-cholesterol mixture (hott-)m) in cms.~ ~,ection. The ,,hope nf DI'PE molecule in the H uu phase has a truncated wedge lypc. ('holcstcroi molecules arc siluatcd between the neighboring hydr~:arhon chain.,, of DEPE.

two phase in the L,,-Hun phase transition region might he due to kinetic effects. Let us consider cholesterol effects cm the structure of the H n phase. Structures of lipid assembly in water depend on the shape of the lipid molecule [38.39]. The lipid molecules with a truncated wedge type shape prefer to stabilize the H n phase (see Fig. 8). Addition of small hydrophobic molecules, for example, alkane [34.40] or squalene [40,41] etc., to PE-water systems increases the lattice spacing of the Hll phase. It is supposed that alkane and squalene enter into the perimeter and the corner of the hexagons of PE in the H ll phase. On the other hand. addition of diacylglycerol to the lipid membranes containing PE reduces the Hun phase lattice constant [42]. It is copcluded that diacylglycerol molecules do not pack into the perimeter or the corner of the hexagons, owing to the large molecule size as compared with alkanes and the hydrophilicity of the glycerol part. i.e.. the hydrocarbon chain and glycerol moiety of diacylglycerol might t~e situated near the hydrocarbon chains and glycerol moiety of phospholipids, respectively [42]. The situation of cholesterol seems to be similar to diacylglycerol. Therefore. the effective area at the end of hydrocarbon chains hecomes large (Fig. 8). As a result, the lattice spacings of the DEPE-cholesteroi mixtures in the H , phase are smaller than that of pure DEPE. Therefi)re, addition of cholesterol reduces the diameter of water core of DEPE in the H H phase. Usually,

biomembranes

form a bilayer structure. The

present study revealed that addition of c h o l e s t e r o l r e d u c e s

The author,, are grateful to Dr. Y. Amemiya fi)r inv:.luable advice in the instrumentation of synchrotron X-ray diffraction experiments. A part of this work has been perfi~rmcd under approval of the Photon Factory Program Advisory Committee (Proposal No. 93G078). This work is ~upported in pa,'l by Grant-in-Aid fi~r General Scientific Research from Ministry of Education. Science and Culture. Japan.

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