Interaction of cholesterol with galactocerebroside and galactocerebroside-phosphatidylcholine bilayer membranes

The interaction of the galactocerebroside, N-palmitoylgalactosylsphingosine (NPGS), with cholesterol has been studied by differential scanning calorimetry (DSC) and x-ray diffraction. Thermal and structural studies demonstrate complex behavior characterized by two endothermic transitions: transition I (TI approximately equal to 50-60 degrees C) corresponding to an NPGS-cholesterol bilayer gel----bilayer liquid crystal transition II (TII where TI less than TII less than TNPGS) corresponding to an NPGS bilayer crystal (stable E form)----bilayer liquid crystal transition. For mixtures containing from 6 to 80 mol % cholesterol, x-ray diffraction studies at 22 degrees C (T less than TI) indicate two separate lamellar phases; an NPGS crystal bilayer phase and a cholesterol monohydrate phase. For cholesterol concentrations less than 50 mol % at TI less than T less than TII, NPGS-cholesterol liquid crystal bilayer and excess NPGS crystal bilayer phases are observed. For greater than 50 mol % cholesterol concentrations at these temperatures, an excess cholesterol monohydrate phase coexists with the NPGS-cholesterol liquid crystal bilayers. At T greater than TII, complete NPGS-cholesterol miscibility is only observed for less than 50 mol % cholesterol concentrations, whereas at greater than 50 mol % cholesterol an excess cholesterol phase is present. The solid phase immiscibility of cerebroside and cholesterol at low temperatures is suggested to result from preferential NPGS-NPGS associations via hydrogen bonding. The unique thermal and structural behavior of NPGS-cholesterol dispersions is contrasted with the behavior of cholesterol-phosphatidycholine and cholesterol-sphingomyelin bilayers. Thermal and structural studies of NPGS in dipalmitoylphosphatidylcholine (DPPC)/cholesterol (1:1, molar ratio) bilayers have been performed. For dispersions containing less than 20 mol % NPGS at 22 degrees C there are no observable calorimetric transitions and x-ray diffraction studies indicate complete lipid miscibility. At greater than 20 mol % NPGS, a high temperature transition is observed that is shown by x-ray diffraction studies to be due to an excess NPGS crystal bilayer----liquid crystal bilayer transition. Complete miscibility of NPGS in DPPC/cholesterol bilayers is observed at T greater than TNPGS. The properties of NPGS/DPPC/cholesterol bilayers are discussed in terms of the lipid composition of the myelin sheath.     

Phase diagram of stigmasterol-dipalmitoylphosphatidylcholine mixtures dispersed in excess water

As a simple model of rafts in plant cells, the effect of stigmasterol, one of the predominant sterols in plant plasma membranes, on the phase behavior of dipalmitoylphosphatidylcholine (DPPC) multilayers has been studied by X-ray diffraction (XRD), differential scanning calorimetry (DSC), and freeze-fracture electron microscopy (FFEM) techniques. A partial phase diagram of the binary system has been constructed. Particularly, the stigmasterol concentrations of the “left endpoint” and “right endpoint” of the three-phase line have been determined using the newly developed linear and nonlinear fitting method. They are 6.2 and 23.7 mol%, respectively. Furthermore, the resemblance and difference of phase diagrams of DPPC/stigmasterol, DPPC/cholesterol, and DPPC/ergosterol have been compared and the efficiency of these sterols in promoting the formation of the liquid-ordered domains (rafts) have also been discussed.     

Phase stability of phosphatidylcholines in dimethylsulfoxide solutions.

The temperature dependence of the phase stability of dispersions of dimyristoyl, dipalmitoyl, and distearoyl derivatives of phosphatidylcholines in excess aqueous dimethylsulfoxide has been examined by differential scanning calorimetry and synchrotron x-ray diffraction methods. There was a close correlation between the enthalpic transitions and the structural changes associated with the pre- and main transitions of the phospholipids in the range of concentrations up to mole fractions of dimethylsulfoxide in water of 0.1333. The temperature of the pre- and main transitions of the three phospholipids were found to increase linearly with increasing mole fraction of dimethylsulfoxide. The difference in phase stability between the lamellar gel and ripple phases induced by increasing dimethylsulfoxide concentration resulted in disappearance of the ripple phase and direct transition between lamellar gel and lamellar liquid-crystal phases. The effect of changing the properties of the solvent by the addition of dimethylsulfoxide on the dimensions of dipalmitoylphosphatidylcholine and solvent layers of the bilayer repeat structure has been determined from electron density distribution calculations. The lamellar repeat spacing recorded at 25 degrees C decreased from 6.36 nm in aqueous dispersion to 6.04 nm in a dispersion containing a mole fraction of 0.1105 dimethylsulfoxide. The results indicate that dipole interactions between solvent and phospholipid and dielectric properties of the solvent are important factors in the determination of the structure of saturated phosphatidylcholines.     

Antioxidant activity of probucol and its effects on phase transitions in phosphatidylcholine liposomes

The effect of probucol on the phase behavior of dimyristoylphosphatidylcholine (DMPC) was examined by fluorescence polarization and differential scanning calorimetry (DSC). Probucol broadens and shifts the temperature of the main phase transition of DMPC liposomes as measured by fluorescence polarization with diphenylhexatriene and trimethyl-ammonium-diphenylhexatrine at concentrations as low as 5 mole%. As measured by DSC, probucol reduces the transition temperature of the gel----liquid-crystalline phase transition of DMPC by approx. 2 C degrees at all concentrations above about 5 mole% probucol and eliminates the pretransition at less than 1 mole%. In addition, the phase transition of DMPC is broadened and the enthalpy of the transition reduced by approx. 50%. Even at high concentrations of probucol, the gel----liquid-crystalline phase transition of DMPC is not eliminated. Similar effects are observed with dipalmitoylphosphatidylcholine liposomes. Based on these DSC measurements, measurements of the melting of probucol in dry mixtures with DMPC and observations of probucol mixtures with DMPC under polarizing optics, the maximum solubility of probucol in DMPC is approx. 10 mole%. This concentration exceeds that required (approx. 0.5 mole%) to prevent peroxidation of 10 mole% arachidonic acid in DMPC liposomes for 30 min in the presence of 0.05 mM Fe(NH4)(SO4)2 at 4 degrees C. Thus, probucol has a limited solubility in saturated phosphatidylcholine bilayers, but is an effective antioxidant at concentrations lower than its maximum solubility.     

Phase diagram of stigmasterol-dipalmitoylphosphatidylcholine mixtures dispersed in excess water

As a simple model of rafts in plant cells, the effect of stigmasterol, one of the predominant sterols in plant plasma membranes, on the phase behavior of dipalmitoylphosphatidylcholine (DPPC) multilayers has been studied by X-ray diffraction (XRD), differential scanning calorimetry (DSC), and freeze-fracture electron microscopy (FFEM) techniques. A partial phase diagram of the binary system has been constructed. Particularly, the stigmasterol concentrations of the "left endpoint" and "right endpoint" of the three-phase line have been determined using the newly developed linear and nonlinear fitting method. They are 6.2 and 23.7 mol%, respectively. Furthermore, the resemblance and difference of phase diagrams of DPPC/stigmasterol, DPPC/cholesterol, and DPPC/ergosterol have been compared and the efficiency of these sterols in promoting the formation of the liquid-ordered domains (rafts) have also been discussed.     

Kinetics of the lamellar gel–fluid transition in phosphatidylcholine membranes in the presence of sugars

Phase diagrams are presented for dipalmitoylphosphatidylcholine (DPPC) in the presence of sugars (sucrose) over a wide range of relative humidities (RHs). The phase information presented here, determined by small angle X-ray scattering (SAXS), is shown to be consistent with previous results achieved by differential scanning calorimetry (DSC). Both techniques show a significant effect of sucrose concentration on the phase behaviour of this phospholipid bilayer. An experimental investigation into the effect of sugars on the kinetic behaviour of the gel to fluid transition is also presented showing that increasing the sugar content appears to slightly increase the rate at which the transition occurs.     

A comparative calorimetric study of the effects of cholesterol and the plant sterols campesterol and brassicasterol on the thermotropic phase behavior of dipalmitoylphosphatidylcholine bilayer membranes

We present a comparative differential scanning calorimetric study of the effects of the animal sterol cholesterol (Chol) and the plant sterols campesterol (Camp) and brassicasterol (Bras) on the thermotropic phase behavior of dipalmitoylphosphatidylcholine (DPPC) bilayers. Camp and Bras differ from Chol in having a C24 methyl group and, additionally for Bras, a C22 trans-double bond. Camp and especially Bras decrease the temperature, cooperativity and enthalpy of the DPPC pretransition more than Chol, although these effects are attenuated at higher sterol levels. This indicates that they destabilize gel-state DPPC bilayers to a greater extent, but are less soluble, than Chol. Not surprisingly, all three sterols have similar effects on the sterol-poor sharp component of the DPPC main phase transition. However, Camp and especially Bras less effectively increase the temperature and decrease the cooperativity and enthalpy of the broad component of the main transition than Chol. This indicates that at higher sterol concentrations, Camp and Bras are less miscible and less effective than Chol at ordering the hydrocarbon chains of the sterol-enriched fluid DPPC bilayers. Overall, these alkyl side chain modifications generally reduce the ability of Chol to produce its characteristic effects on DPPC bilayer physical properties. These differences are likely due to the less extended and more bent conformations of the alkyl side chains of Camp and Bras, producing sterols with a greater effective cross-sectional area and reduced length than Chol. Hence, the structure of Chol is likely optimized for maximum solubility in, as opposed to maximum ordering of, phospholipid bilayers.     

Alterations in the organization of phosphatidylcholine/cholesterol bilayers by tetrahydrocannabinol

The interactions of delta 9-tetrahydrocannabinol (THC) with various phosphatidylcholines (PCs) was studied in model membranes by differential scanning calorimetry. THC present in PC bilayers above a certain concentration complexed stoichiometrically with phospholipids containing both saturated and unsaturated fatty acids. When the bilayer PCs were sufficiently dissimilar for phase separation to occur, THC preferentially associated with the lower melting point lipid. The presence of cholesterol below 20 mol% in dipalmitoylphosphatidylcholine bilayers enhanced THC X PC complex formation. Above 20 mol% cholesterol, there was no indication of THC X dipalmitoylphosphatidylcholine complex formation. This is in agreement with a phase rearrangement occurring in PC bilayers at concentrations of cholesterol of approximately 20 mol%. These studies suggest several possible mechanisms for the modulation of membrane activities by hydrophobic drugs such as THC.     

胆固醇对二棕榈酰磷脂酰胆碱（DPPC）多层膜结构的影响

用小角Ｘ射线衍射（ＳＡＸＤ）和差示扫描量热（ＤＳＣ）方法研究胆固醇对二棕榈酸磷脂酰胆碱多层膜结构的影响。在不同胆因醇浓度及不同温度下的衍射实验和热吸收曲线实验显示纯磷脂的尖锐的凝胶－液晶相变随胆固醇含量增加而逐渐消失。当胆固醇含量约为４０（ｍｏｌ）％或更多时,衍射曲线和热吸收曲线均显示存在分相现象。     

Galactocerebroside-phospholipid interactions in bilayer membranes.

Differential scanning calorimetry (DSC) and x-ray diffraction have been used to study the interaction of hydrated N-palmitoylgalactosylsphingosine (NPGS) and dipalmitoylphosphatidylcholine (DPPC). For mixtures containing less than 23 mol% NPGS, complete miscibility of NPGS into hydrated DPPC bilayers is observed in both the bilayer gel and liquid-crystal phases. X-ray diffraction data demonstrate insignificant differences in the DPPC-bilayer gel-phase parameters on incorporation of up to 23 mol% NPGS. At greater than 23 mol% NPGS, additional high-temperature transitions occur, indicating phase separation of cerebroside. For these cerebroside concentrations, at 20 degrees C, x-ray diffraction shows two lamellar phases, hydrated DPPC-NPGS gel bilayers (d = 64 A) containing 23 mol% NPGS, and NPGS "crystal" bilayers (d = 55 A). On heating to temperatures greater than 45 degrees C, the mixed DPPC-NPGS bilayer phase undergoes chain melting, and on further increasing the temperature progressively more NPGS is incorporated into the liquid-crystal DPPC-NPGS bilayer phase. At temperatures greater than 82 degrees C (the transition temperature of hydrated NPGS), complete lipid miscibility is observed at all DPPC/NPGS molar ratios.     

Interactions of N-stearoyl sphingomyelin with cholesterol and dipalmitoylphosphatidylcholine in bilayer membranes.

Differential scanning calorimetry and x-ray diffraction have been utilized to investigate the interaction of N-stearoylsphingomyelin (C18:0-SM) with cholesterol and dipalmitoylphosphatidylcholine (DPPC). Fully hydrated C18:0-SM forms bilayers that undergo a chain-melting (gel -->liquid-crystalline) transition at 45 degrees C, delta H = 6.7 kcal/mol. Addition of cholesterol results in a progressive decrease in the enthalpy of the transition at 45 degrees C and the appearance of a broad transition centered at 46.3 degrees C; this latter transition progressively broadens and is not detectable at cholesterol contents of >40 mol%. X-ray diffraction and electron density profiles indicate that bilayers of C18:0-SM/cholesterol (50 mol%) are essentially identical at 22 degrees C and 58 degrees C in terms of bilayer periodicity (d = 63-64 A), bilayer thickness (d rho-p = 46-47 A), and lateral molecular packing (wide-angle reflection, 1/4.8 A-(1)). These data show that cholesterol inserts into C18:0-SM bilayers, progressively removing the chain-melting transition and altering the bilayer structural characteristics. In contrast, DPPC has relatively minor effects on the structure and thermotropic properties of C18:0-SM. DPPC and C18:0-SM exhibit complete miscibility in both the gel and liquid-crystalline bilayer phases, but the pre-transition exhibited by DPPC is eliminated at >30 mol% C18:0-SM. The bilayer periodicity in both the gel and liquid-crystalline phases decreases significantly at high DPPC contents, probably reflecting differences in hydration and/or chain tilt (gel phase) of C18:0-SM and DPPC.     

Effects of replacement of the hydroxyl group of cholesterol and tocopherol on the thermotropic behavior of phospholipid membranes

The role of the hydroxyl groups of cholesterol and tocopherol in mediating their interaction with phospholipid bilayers has been a subject of considerable interest. We have examined this question by using derivatives of cholesterol and tocopherol in which the hydroxyl group is esterified to succinate. The hemisuccinate esters of cholesterol and alpha-tocopherol can be readily incorporated into phospholipid membranes and in fact can by themselves form closed membrane vesicles as demonstrated by the encapsulation of [3H]sucrose. The thermotropic behavior of mixtures containing each succinate ester and phospholipid was studied by differential scanning calorimetry. The effect of cholesteryl hemisuccinate on the thermotropic properties of dipalmitoylphosphatidylcholine and dimyristoylphosphatidylethanolamine is very similar to that of cholesterol. This indicates that the 3 beta-OH is not required for the formation of a cholesterol-phospholipid complex. In mixtures of tocopherol acid succinate and phospholipids the peak transition temperature is progressively shifted to lower temperatures as the mole fraction of alpha-tocopherol succinate is increased, while the enthalpy of the transition is only slightly affected. At a tocopherol succinate/phospholipid molar ratio of 9/1 a phase transition is still detectable. A comparison between tocopherol succinate and tocopherol indicates that the substitution of the hydroxyl group reduces the interaction of tocopherol with phospholipids to a small but measurable extent. Thus, the hydroxyl group of tocopherol is more important than the hydroxyl group of cholesterol in influencing their interactions with phospholipids.     

A comparative study of the effects of cholesterol and sclareol, a bioactive labdane type diterpene, on phospholipid bilayers

Sclareol (labd-14-ene-8,13-diol) is a highly water-insoluble molecule that belongs to the labdane type diterpenes and is characterized as a biologically active molecule, due to its cytotoxic and cytostatic effects against human leukemic cell lines. A superimposition study between sclareol and cholesterol, based on their corresponding hydrophobic and polar molecular segments calculated from their lipophilic profiles, revealed their spatial similarities. This structural similarity between the two molecules prompted us to compare their effects on the structure and stability of phospholipid dipalmitoylphosphatidylcholine (DPPC) membranes. Differential scanning calorimetry (DSC) was applied to compare the thermal changes caused by either cholesterol or sclareol when are incorporated in DPPC bilayers. The results showed that sclareol is incorporated into phospholipid model membranes and mimics the thermal effects of cholesterol especially at concentrations up to X(sclareol)=9.1 mol%. These effects can be summarized as the abolition of pre-transition, lowering of the main phase transition and reduction of the enthalpy change (DeltaH) of the gel to liquid-crystalline phase transition of DPPC bilayers. At concentrations X> or =16.7 mol%, sclareol and cholesterol caused different heterogeneity in lipid bilayers or a reversible transition from a vesicular suspension to an extended peak bilayer network. This different fluidization, exerted by the two molecules at high concentration, may be related to their different stability and the z-average mean diameter of the liposomes they form. Small unilamellar vesicles, prepared by the thin film hydration method showed that DPPC bilayers containing a high concentration of sclareol in equimolar ratio sclareol:cholesterol were unstable, in contrast to the ones containing only cholesterol.     

Interaction of dolichol and dolichyl phosphate with phospholipid bilayers

The thermotropic phase transition of dipalmitoylphosphatidylcholine vesicles reconstituted with dolichol or dolichyl phosphate was investigated as a function of the lipid-to-polyisoprenoid ratio by means of differential scanning calorimetry and fluorescence depolarization of the embedded probe 1,6-diphenyl-1,3,5-hexatriene. At the concentrations studied, dolichol and dolichyl phosphate lowered and broadened the transition temperature of dipalmitoylphosphatidylcholine bilayers. Dolichol was found to increase the motional freedom of the bilayer both below and above the transition temperature as determined by fluorescence depolarization. In contrast, low concentrations of dolichyl phosphate decreased the bilayer motional freedom below the transition temperature while high concentrations increased the motional freedom. Above the transition temperature, dolichyl phosphate decreased bilayer 'fluidity' at all concentrations. The data suggest that these polyisoprenoids perturb the bilayer lattice, with the neutral species dolichol increasing membrane 'fluidity', while dolichyl phosphate acts to 'stiffen' the membrane.     

The effect of the sterol oxygen function on the interaction with phospholipids

The effect of cholesteryl ethers (namely cholesteryl methyl ether, cholesteryl ethyl ether, cholesteryl n-propyl ether, cholesteryl isopropyl ether, cholesteryl butyl ether, cholesteryl methoxymethyl ether, cholesteryl (2'-hydroxy)-3-ethyl ether) and cholesteryl ester (namely cholesteryl acetate) is tested on the interaction with phosphatidylcholines in liquid-crystalline and crystalline state. The interfacial properties of sterols are tested at the air-water interface. The cholesteryl ethers show a reduced interfacial stability with increasing hydrophobicity of the ether-linked moiety. The interaction between the sterol derivatives and phospholipids in mixed monolayers is indicated by measuring the deviation from the simple addivity rule (condensing effect). An interaction is found only for cholesteryl (2'-hydroxy)-3-ethyl ether, cholesteryl methyl ether and cholesteryl ethyl ether. These sterols also reduce the glucose permeability of liposomal membranes in this order. In this respect cholesteryl (2'-hydroxy)-3-ethyl ether is as effective as cholesterol. Cholesteryl methyl ether and cholesteryl ethyl ether show 62 and 33 percent of the effect observed with cholesterol. The effect of the sterol derivatives on the gel-to-liquid-crystalline phase transition of dipalmitoylphosphatidylcholine is measured by differential scanning calorimetry. Cholesteryl methyl ether, cholesteryl ethyl ether, and cholesteryl (2'-hydroxy)-3-ethyl ether reduce the energy content of the phase transition nearly as effective as cholesterol, cholesteryl n-propyl ether has only a small effect. Although cholesteryl acetate, and cholesteryl methoxymethyl ether have no condensing or permeability-reducing effect, they have a considerable effect on the gel-to-liquid-crystalline phase transition. Cholesteryl isopropyl ether and cholesteryl butyl ether have no effect. It is concluded that a free 3 beta-hydroxy group is not a prerequisite to observe a sterol-like effect in membranes. However, the interfacial stability and the orientation of the sterol and oxygen moiety at the sterol 3-position are important.     

Lipid phase separations induced by the association of cholera toxin to phospholipid membranes containing ganglioside GM1

The interactions of cholera toxin and their isolated binding and active subunits with phospholipid bilayers containing the toxin receptor ganglioside GM1 have been studied by using high-sensitivity differential scanning calorimetry and steady-state and time-resolved fluorescence and phosphorescence spectroscopy. The results of this investigation indicate that cholera toxin associates with phospholipid bilayers containing ganglioside GM1, independent of the physical state of the membrane. In the absence of Ca2+, calorimetric scans of intact cholera toxin bound to dipalmitoylphosphatidylcholine (DPPC) large unilamellar vesicles containing ganglioside GM1 result in a broadening of the lipid phase transition peak and a slight decrease (less than 5%) in the transition enthalpy. In the presence of Ca2+ concentrations sufficient to cause ganglioside phase separation, the association of the intact toxin to the membrane results in a significant decrease of enthalpy change for the lipid transition, indicating that under these conditions the toxin molecule perturbs the hydrophobic core of the bilayer. Calorimetric scans using isolated binding subunits lacking the hydrophobic toxic subunit did not exhibit a decrease in the phospholipid transition enthalpy even in the presence of Ca2+, indicating that the binding subunits per se do not perturb the hydrophobic core of the bilayer. On the other hand, the hydrophobic A1 subunit by itself was able to reduce the phospholipid transition enthalpy when reconstituted into DPPC vesicles. These calorimetric observations were confirmed by fluorescence experiments using pyrene phospholipids.(ABSTRACT TRUNCATED AT 250 WORDS)     

Structures and mechanisms of lipid phase transitions in nonaqueous media. Dipalmitoylphosphatidylethanolamine in fused salt.

The phase transitions for dipalmitoylphosphatidylethanolamine (DPPE) dispersed in water and in N-ethylammonium nitrate (EAN) were examined using differential scanning calorimetry and time-resolved x-ray diffraction. Subgel, pre-, and main-phase transitions were observed for DPPE in water, whereas only the pre- and main transitions were observed for DPPE in EAN. Hysteresis was observed for both dispersions upon cooling. In addition, the lamellar (L alpha) to hexagonal (H alpha) phase transition was observed for DPPE dispersed in EAN when using time-resolved x-ray diffraction but not when using calorimetry. This low enthalpy process occurred at 73-77 degrees C, which is significantly lower than that observed for DPPE in water. The presence of EAN stabilizes the existence of the H alpha phase in DPPE by its influence on the bilayer interfacial properties, primarily on the area per lipid head group.     

Phospholipid studies of marine organisms: 26. Interactions of some marine sterols with 1-stearoyl-2-oleoyl phosphatidylcholine (SOPC) in model membranes.

The thermotropic behavior of multilamellar vesicles (MLV) composed of different mole fractions of various marine sterols and 1-stearoyl-2-oleoyl phosphatidylcholine (SOPC) was examined by differential scanning calorimetry (DSC), and was compared to pure SOPC as well as their mixtures with cholesterol. The marine sterols investigated were capable of interacting with the phospholipid bilayers. Upon addition of marine sterols, the apparent transition temperature (Tm) of SOPC decreased significantly. Desmosterol (cholesta-5,24-dien-3 beta-ol) had the least interaction with SOPC, as reflected by the larger delta H values of its mixtures with the phospholipid. Fucosterol (24-ethylcholesta-5,24(28)-dien-3 beta-ol) showed a non-linear trend as the mole percent of the sterol increased. Mixtures of sutinasterol (24R-24-ethyl-26,26-dimethylcholesta-7,25(27)-dien-3 beta-ol) with SOPC had similar enthalpy values to cholesterol. The shape of the SOPC/marine sterol endotherm and their delta H values were not identical when liposomes prepared by dialysis were compared to MLV.     

Cholesterol-phospholipid interaction in membranes. 1. Cholestane spin-label studies of phase behavior of cholesterol-phospholipid liposomes

The effect of cholesterol concentration on the thermotropic phase behavior of aqueous phospholipid multi-bilayers was monitored by means of electron spin resonance spectroscopy (ESR) of a cholestane spin-label (CSL). The spin-label itself induces an additional transition in several different phospholipids, which is attributed to local melting around the spin probe. In contrast, cholesterol prevents its neighboring phospholipids from undergoing fluidization. Small additions of cholesterol affect the position of the probe-induced lipid mobilization curve. The phospholipid main gel-liquid-crystal transition, which is also observed as a separate change in probe mobilization, is not affected by low concentrations of cholesterol. These observations indicate the presence of two phases, a cholesterol-rich phase and a pure phospholipid phase, and indicate that CSL preferentially enters the cholesterol-rich phase. Addition of more than 20 mol % cholesterol abolishes the bulk phospholipid phase. This is evidenced by the disappearance of the gel-liquid-crystal transition as observed by ESR. However, the CSL-induced transition is present at all concentrations of cholesterol and CSL. The behavioral differences between the two sterols caution against using this probe as a direct substitute for cholesterol. However, it remains a useful tool for monitoring the phase behavior of cholesterol-phospholipid bilayer systems.     

The phase behavior of hydrated cholesterol.

The thermotropic phase behavior of cholesterol monohydrate in water was investigated by differential scanning calorimetry, polarizing light microscopy, and x-ray diffraction. In contrast to anhydrous cholesterol which undergoes a polymorphic crystalline transition at 39 degrees C and a crystalline to liquid transition at 151 degrees C, the closed system of cholesterol monohydrate and water exhibited three reversible endothermic transitions at 86, 123, and 157 degrees C. At 86 degrees C, cholesterol monohydrate loses its water of hydration, forming the high temperature polymorph of anhydrous cholesterol. At least 24 hours were required for re-hydration of cholesterol and the rate of hydration was dependent on the polymorphic crystalline form of anhydrous cholesterol. At 123 degrees C, anhydrous crystalline cholesterol in the presence of excess water undergoes a sharp transition to a birefringent liquid crystalline phase of smectic texture. The x-ray diffraction pattern obtained from this phase contained two sharp low-angle reflections at 37.4 and 18.7 A and a diffuse wide-angle reflection centered at 5.7 A, indicating a layered smectic type of liquid crystalline structure with each layer being two cholesterol molecules thick. The liquid crystalline phase is stable over the temperature range of 123 to 157 degrees C before melting to a liquid dispersed in water. The observation of a smectic liquid crystalline phase for hydrated cholesterol correlates with its high surface activity and helps to explain its ability to exist in high concentrations in biological membranes.