Cholesterol Mole Fraction Research Articles

A model membrane approach to the epidermal permeability barrier: an X-ray diffraction study.

The permeability of mammalian skin is determined in large part by lamellar lipid domains packed between cells of the upper layer of the epidermis, the stratum comeum. Although these lamellae have features in common with typical biological membranes, they differ in having a lipid population composed mainly of ceramides, cholesterol, and free fatty acids. In our initial studies of the relationship between lipid composition and phase behavior in this unusual system, we used deuterium NMR [Kitson et al. (1994) Biochemistry 33, 6707-6715] to examine aqueous dispersions of nonhydroxylated bovine brain ceramide, cholesterol, and perdeuterated palmitic acid, and found complex phase behavior as a function of temperature and pH, whereas analogous dispersions in which sphingomyelin replaced ceramide resulted in spectra consistent with a fluid lamellar phase under the same conditions. To extend these observations, we examined the same dispersions at pH 5.2 by means of X-ray diffraction. The significant findings are as follows: (1) the ceramide dispersions form complex crystalline phases between room temperature and about 40 degrees C; (2) the majority of the crystalline cholesterol is not in a separate phase; and (3) the analogous sphingomyelin dispersions form a fluid lamellar phase under the same conditions. We conclude that ceramides, even in the presence of considerable mole fractions of cholesterol, can form crystalline lamellar structures. We suggest that the existence of such structures in stratum corneum may be important in the function of the epidermal permeability barrier, and that the interaction between ceramide and cholesterol in other biological membranes may result in regions having unique physical properties.

Electrochemical studies of lipophilic ion transport through BLM. The influence of sterols on its transport

AbstractCyclic voltammetry was employed to study the influence of sterols on the lipophilic ion transport through the BLM. The mole fraction of the sterols (cholesterol, oxidized cholesterol), as referred to total lipid, was varied in a range of 0–0.8. Data demonstrate that a thin‐layer model is suitable to this BLM system, By this model, the number of charges transported per lipophilic ion, the concentration of the ion in the membrane hulk phase and the aqueous/membrane phase partition coefficient could be calculated. These parameters proved that sterols had an obvious influence on the lipophilic ion transport. Cholesterol had a stronger influence on the ion transport than oxidized cholesterol. Its incorporation into egg lecithin membranes increased the partition coefficientβ of the ion up to more than 3‐fold. Yet, oxidized cholesterol incorporated into egg lecithin membranes only increased the β up to less than 2‐fold, and the β had no great variation at different oxidized cholesterol mole fractions. The higher β obtained was partly due to the trace amount of solvent existing in the core of the lipid bilayers. At the different sterol mole fractions, combining the change of β with the change of peak current, we also concluded that sterols had somewhat inhibiting effect on the ion transport at the higher sterols mole fraction (> 0.4). These results are explained in terms of the possible change of dipole potential of the membrane produced by sterols and the decrease of the membrane fluidity caused by the condensation effect of sterols and the thinning effect caused by sterols. The substituting group (in the oxidized cholesterol) had some inhibiting effects on the ion transport at higher mole fractions (oxidized cholesterol mole fraction > 0.4).

Phospholipid surface density determines the partitioning and permeability of acetic acid in DMPC:cholesterol bilayers.

Relationships between the permeability coefficient (PHA) and partition coefficient (K m/w) of acetic acid and the surface density of DMPC:cholesterol bilayers have been investigated. Permeability coefficients were measured in large unilamellar vesicles by NMR line broadening. Bilayer surface density, sigma, was varied over a range of 0.5-0.9 by changing cholesterol concentration and temperature. The temperature dependence of PHA for acetic acid exhibits Arrhenius behavior with an average apparent activation energy (Ea) of 22 +/- 3 kcal/mole over a cholesterol mole fraction range of 0.00-0.40. This value is much greater than the enthalpy change for acetic acid partitioning between bulk decane and water (delta H degree = 4.8 +/- 0.8 kcal/mole) and the calculated Ea (= 8.0 kcal/mole) assuming a "bulk phase" permeability model which includes the enthalpy of transfer from water to decane and the temperature dependence of acetic acid's diffusion coefficient in decane. These results suggest that dehydration, previously considered to be a dominant component, is a minor factor in determining Ea. Values of 1n PHA decrease linearly with the normalized phospholipid surface density with a slope of kappa = -12.4 +/- 1.1 (r = 0.90). Correction of PHA for those temperature effects considered to be independent of lipid chain order (i.e., enthalpy of transfer from water to decane and activation energy for diffusion in bulk hydrocarbon) yielded an improved correlation (kappa = -11.7 +/- 0.5 (r = 0.96)). The temperature dependence of Km/w is substantially smaller than that for PHA and dependent on cholesterol composition. Values of 1n K m/w decrease linearly with the surface density with a slope of kappa = -4.6 +/- 0.3 (r = 0.95), which is 2.7-fold smaller than the slope of the plot of 1n PHA vs. sigma. Thus, chain ordering is a major determinant for molecular partitioning into and transport across lipid bilayers, regardless of whether it is varied by lipid composition or temperature.

Lateral Organization of Liquid-Crystalline Cholesterol-Dimyristoylphosphatidylcholine Bilayers. Evidence for Domains with Hexagonal and Centered Rectangular Cholesterol Superlattices

The lateral organization of fluid cholesterol-dimyristoylphosphatidylcholine (DMPC) bilayers was studied by measuring the response of fluorescent membrane probes, dipyrenylphosphatidylcholines (diPyrxPCs) or merocyanine 540, to the variation of cholesterol concentration. Parallel absorbance and light-scattering measurements were also carried out. The excimer-to-monomer ratio of diPyrxPCs displayed abrupt deviations at particular cholesterol mole fractions (CMFs). The most notable of these occurred at CMFs of 0.15, 0.33, and 0.67. Deviations were also frequently observed at CMFs of 0.12, 0.20, 0.25, and 0.40. Merocyanine 540 reproducibly reported deviations at CMFs of 0.15 and 0.33 and frequently reported values close to 0.12, 0.20, and 0.25. In absorbance (turbidity) and light scattering versus CMF plots, well-defined kinks were observed at CMFs of 0.16, 0.33, 0.52, and 0.67. The occurrence of kinks or other deviations at those particular CMFs is most readily explained in terms of a superlattice model previously developed to explain the lateral distribution of pyrenylphospholipids in bilayers [Somerharju, et al. (1985) Biochemistry 24, 2773-2781; Virtanen, J. A., et al. (1988) J. Mol. Electron. 4, 233-236]. This model is based on the assumptions that (i) each cholesterol molecule replaces a single acyl chain in a hexagonal lattice, (ii) cholesterol molecules, because of their larger size, perturb the lattice, (iii) this perturbation is minimized when the cholesterol molecules are maximally separated from each other, and (iv) the maximal separation is achieved when the cholesterol molecules form a hexagonal or centered rectangular superlattice. All detected critical CMFs, except that at CMF 0.67, are predicted by the model, thus strongly supporting its validity. The critical CMF at 0.67 is a limiting case, which can be accounted for by assuming that cholesterol and phospholipid molecules form alternating rows, i.e., formation of a cholesterol superlattice with rectangular symmetry. As predicted by the superlattice model, composition-driven order-to-disorder transitions occur between the critical CMFs, as indicated by increased data scatter and sample fluctuations in those regions. Another important prediction of the superlattice model is that domains with different cholesterol superlattices should coexist at most cholesterol concentrations. Such domains do not have to be extensive to account for the critical events observed here; rather, they are expected to be dynamic entities of limited size. It is very likely that such microscopic domains with distinct cholesterol superlattices also coexist in biological membranes. This is expected to have remarkable effects on both the structure and functions of these membranes.

The Low-Frequency Dielectric Response of Liposomes

The dielectric response of dipalmitoylphosphatidylcholine (DPPC) liposomes has been examined over a frequency range from 105 to 10-2 Hz under a variety of conditions in order to investigate the low-frequency dispersion noted in previous studies. The data have been interpreted in terms of the model proposed by Hill and Pickup (J. Mater. Sci. 20, 4431 (1985)), which predicts that the response over this frequency range will be a function of the presence of an electrode barrier layer in series with a bulk layer. The barrier response was found to be related to the nature of the liposomal bilayer, as a linear relationship was observed between the low-frequency capacitance and the mole fraction of cholesterol in the phospholipid bilayer. The size of the vesicles, the use of different lipid isomers, and changing the head group of the phospholipid were all found to alter the bilayer response. It is suggested that the low-frequency response is associated with the presence of a layer of liposomes absorbed onto the electrode surface, hence the low-frequency technique has potential as a means of characterizing liposomal suspensions.

Hydrophobic Barriers of Lipid Bilayer Membranes Formed by Reduction of Water Penetration by Alkyl Chain Unsaturation and Cholesterol

The hydrophobicity profiles across phosphatidylcholine (PC)-cholesterol bilayer membranes were estimated in both frozen liposome suspensions and fluid-phase membranes as a function of alkyl chain length, unsaturation, and cholesterol mole fraction. A series of stearic acid spin labels, with the probe attached to various positions along the alkyl chain, cholesterol-type spin labels (cholestane and androstane spin labels), and Tempo-PC were used to examine depth-dependent changes in local hydrophobicity, which is determined by the extent of water penetration into the membrane. Local hydrophobicity was monitored primarily by observing the z component of the hyperfine interaction tensor (Az) of the nitroxide spin probe in a frozen suspension of the membrane at -150 degrees C and was further confirmed in the fluid phase by observing the rate of collision of Fe(CN)6(3-) with the spin probe in the membrane using saturation recovery ESR. Saturated-PC membranes show low hydrophobicity (high polarity) across the membrane, comparable to 2-propanol and 1-octanol, even at the membrane center where hydrophobicity is highest. Longer alkyl chains only make the central hydrophobic regions wider without increasing the level of hydrophobicity. Introduction of a double bond at C9-C10 decreases the level of water penetration at all locations in the membrane, and this effect is considerably greater than the cis configuration than with the trans configuration. Incorporation of cholesterol (30 mol %) dramatically changes the profiles; it decreases hydrophobicity (increases water penetration) from the polar headgroup region to a depth of approximately C7 and C9 for saturated- and unsaturated-PC membranes, respectively, which is about where the bulky rigid steroid ring structure of cholesterol reaches in the membrane. Membrane hydrophobicity sharply increases at these positions from the level of methanol to the level of pure hexane, and hydrophobicity is constant in the inner region of the membrane. Thus, formation of effective hydrophobic barriers to permeation of small polar molecules requires alkyl chain unsaturation and/or cholesterol. The thickness of this rectangular hydrophobic barrier is less than 50% of the thickness of the hydrocarbon regions. Results obtained in dioleoyl-PC-cholesterol membranes in the fluid phase are similar to those obtained in frozen membranes. These results correlate well with permeability data for water and amino acids in the literature.

Sodium transport by an ionizable and a neutral mobile carrier: effects of membrane structure on the apparent activation energy

Temperature-jump relaxation experiments on Na + transport by (221)C 10-cryptand (ionizable mobile carrier) and nonactin (neutral mobile carrier) were carried out in order to study the effects of cholesterol and the degree of acyl chain unsaturation, and their temperature-dependence on ion transport through thin lipid membranes. The experiments were performed on large, negatively charged unilamellar vesicles (LUV) prepared from mixtures of phosphatidylcholine (egg phosphatidylcholine, dioleoylphosphatidylcholine and dilinoleolylphosphatidylcholine), phosphatidic acid and cholesterol (mole fraction 0–0.43), at various temperatures and carrier concentrations. The apparent rate constants of Na + translocation by (221)C 10 and nonactin increased with the carrier concentration, the degree of acyl chain unsaturation and the temperature. The incorporation of cholesterol into the membranes significantly reduced the carrier concentration-, acyl chain unsaturation- and temperature-dependence of this parameter. The apparent energy required to activate the transport decreased significantly with increasing (221)C 10 concentrations and remained constant with increasing those of nonactin at any given cholesterol molar fraction and degree of acyl chain unsaturation. It increased significantly with increasing the cholesterol molar fraction at any given carrier concentration to an extent depending on the degree of acyl chain unsaturation. Our interpretation of the action of cholesterol on these transport systems is based on the assumption that the adsorption plane of Na +-(221)C 10 and Na +-nonactin complexes is likely to be located towards the aqueous and the hydrocarbon side of the dipole layer, respectively. The results are discussed in terms of the structural, physico-chemical and electrical characteristics of carriers and complexes, and of the interactions occuring between an ionizable or a neutral mobile carrier and the membrane.

Temperature-dependent effects of cholesterol on sodium transport through lipid membranes by an ionizable mobile carrier

Temperature-jump relaxation experiments on Na + transport by (221)C 10-cryptand were carried out in order to study the influence of cholesterol and its temperature-dependence on ion transport through thin lipid membranes. The experiments were performed on large, negatively charged unilamellar vesicles (LUV) prepared from mixtures of dioleoylphosphatidylcholine, phosphatidic acid and cholesterol (mole fractions 0–0.43), at various temperatures and carrier concentrations. The initial rates of Na + transport and the apparent rate constants of its translocation by (221)C 10 increased with the carrier concentration and the temperature. The incorporation of cholesterol into the membranes significantly reduced the carrier concentration- and temperature-dependence of these two parameters. The apparent energy required to activate the transport decreased significantly with increasing carrier concentrations at any given cholesterol molar fraction, and increased significantly with the cholesterol molar fraction at any given carrier concentration. Our interpretation of the action of cholesterol on this transport system is based on the assumption that the binding cavity of cryptands is likely to be located towards the aqueous side of the dipole layer. The results are discussed in terms of the structural, physico-chemical and electrical characteristics of carriers and complexes, and of the interactions occurring between an ionizable mobile carrier and the membrane.

Cholesterol modulation of lipid intermixing in phospholipid and glycosphingolipid mixtures. Evaluation using fluorescent lipid probes and brominated lipid quenchers

Carbazole- and indole-labeled phospholipids have been used to monitor the homo- or heterogeneity of lipid mixing in several types of lipid bilayers combining a brominated and a nonbrominated lipid with varying amounts of cholesterol. Experimental quenching curves (relating the normalized probe fluorescence intensity to the mole fraction of brominated lipid) show a characteristic smooth, monophasic form for homogeneous liquid-crystalline lipid mixtures. However, for mixtures exhibiting lipid lateral segregation, such curves show marked perturbations in form over the region of composition where segregation occurs. Using this approach, it is found that high mole fractions of cholesterol (40-50 mol %) promote the formation of apparently homogeneous solutions in mixtures of disaturated and monounsaturated phosphatidylcholines (PCs) that exhibit extensive thermotropic phase separations in the absence of sterol. At only slightly lower levels of cholesterol, however, these systems exhibit inhomogeneous lipid mixing over a wide range of relative proportions of the two PC components. Mixtures of cerebroside and monounsaturated PCs, even at high bilayer cholesterol contents, exhibit significant inhomogeneity in lipid mixing over a wide range of cerebroside/PC ratios. Phase-separating PC/PC and PC/cerebroside mixtures can readily form long-lived metastable solutions when the level of the higher-melting component in the liquid-crystalline phase exceeds its equilibrium solubility by as much as 20-30 mol %; this tendency is significantly increased by cholesterol. Cholesterol shows no significant ability to enhance lipid intermixing in a third type of phase-separating lipid system, combining a monounsaturated PC with a monounsaturated phosphatidic acid--calcium complex. Experiments using cleavable phospholipid conjugates, linking a fluorescent lipid to a brominated lipid, suggest that each fluorescent molecule probes a local lipid domain comprising approximately less than 40-50 nearby acyl chains.

Membrane structural dynamics of plasma membranes of living human prostatic carcinoma cells differing in metastatic potential

Rigidity of the outer hemileaflet of the plasma membrane of two prostatic carcinoma cell lines with different metastatic potential, 1-LN and 1-LN-EMS-10, was assessed by steady-state anisotropy, using a battery of fluorescent probes. The “bulk” membrane rigidity sensed by diphenylhexatriene, trimethylammonio-DPH, 1-palmitoyl-2-[DPH-ethylcarbonyl]-phosphatidylcholine, and 10-pyrenedecanoic acid indicated slightly higher rigidity in the membrane of the highly metastatic line (1-LN). This was accompanied by 26% greater mole fraction of cholesterol and 9% lower phospholipid, resulting in 40% greater cholesterol/phospholipid ratio. Phosphatidylethanolamine was increased 12%, but corresponding decreases in phosphatidylserine and phosphatidylinositol resulted in no significant change in molar ratio of choline/noncholine phospholipids. Whereas unsaturation index was slightly higher in 1-LN, fatty acids of 1-LN plasma membranes contained 15% more 18:1, 43% more 20:4, 26% more 22:4, and 38% less 18:2. Anisotropy gradients were determined for the two cell lines using a series of n-(9-anthroyloxy) fatty acid probes with n = 2, 3, 6, 7, 9, 12, and 16. Gradients differed only in position of anisotropy maxima, which occurred with n = 6, in 1-LN, and n = 7, in 1-LN-EMS-10. Possible relationships between observed anisotropy gradients and differences in membrane cholesterol and fatty acid composition are discussed.

The effect of protons or calcium ions on the phase behavior of phosphatidylserine-cholesterol mixtures

The influence of protons or calcium ions on the miscibility of cholesterol in phosphatidylserine has been examined using differential scanning calorimetry and X-ray diffraction. At pH 2.6, where the car☐yl group of the serine moiety is protonated, two endothermic transitions are observed in cholesterol-phosphatidylserine mixtures. The midpoint of the first is at 35°C in the absence of cholesterol and decreases to approx. 15°C for molar fraction of cholesterol 0.5. The second transition is centered at approx. 44°C, almost independent of cholesterol content. The two lower temperature phases are lamellar and the high temperature phase has hexagonal symmetry. Cholesterol is more miscible in protonated phosphatidylserine than in the sodium form: cholesterol crystals are detected at a molar ratio of phosphatidylserine to cholesterol of about 1.7:1 as compared to about 2.3:1 at neutral pH. In the presence of calcium ions (1.3 Ca 2+ per phosphatidylserine), a lamellar phase is observed with layer spacing 53Åwhich is independent of temperature (25°C–65°C) and of cholesterol content. Calcium ions cause reduced cholesterol solubility: crystallites are detected already at a molar ratio of 4:1.

Microimmiscibility and three-dimensional dynamic structures of phosphatidylcholine-cholesterol membranes: translational diffusion of a copper complex in the membrane

Saturated and unsaturated phosphatidylcholine (PC)-cholesterol membranes have been studied, with a special attention paid to fluid-phase immiscibility in cis-unsaturated phosphatidylcholine (PC)-cholesterol membranes as previously proposed and to the three-dimensional structure of the membrane. The investigation was carried out with dual probes: a membrane-soluble, square-planar copper complex, (3-ethoxy-2-oxobutyraldehyde bis(N4,N4-dimethylthiosemicarbazonato]copper(II) (CuKTSM2), and one of several nitroxide radical lipid-type spin-labels. Bimolecular collision rates between metal ion and spin-label were determined by measuring the nitroxide spin-lattice relaxation times (T1's) in the presence and absence of CuKTSM2 by use of saturation-recovery ESR techniques, and from these measured rates, translational diffusion coefficients of CuKTSM2 were estimated. Profiles of the collision rate across the membrane bilayer were obtained with Tempocholine phosphatidic acid ester, 5-doxylstearic acid, 16-doxylstearic acid, and cholesterol-type spin-labels as a function of cholesterol mole fraction, length and unsaturation of acyl chains, and temperature. In the liquid-crystalline phase of saturated PC membranes, incorporation of cholesterol decreases the collision rate at all depths in the membrane, and the effect of cholesterol is smallest in the middle of the bilayer. In trans-unsaturated PC membranes, a cholesterol-induced decrease of the collision rate was also observed, except in the head-group regions. In cis-unsaturated PC membranes, virtually no effect of cholesterol was observed on the collision rate, either with phospholipid-type spin-labels or with cholesterol-type spin-labels. This result is in clear contrast with our previous observation, in which the effect of cholesterol in cis-unsaturated PC membranes is small on the alkyl-chain motion of phospholipid-type spin-labels but large on the wobbling rotational diffusion of cholesterol-type spin-labels [Pasenkiewicz-Gierula, M., Subczynski, W. K., & Kusumi, A. (1990) Biochemistry 29, 4059-4069]. A model is proposed to explain these results in which the fluid-phase immiscibility is prevalent in cis-unsaturated PC-cholesterol membranes, but where cholesterol-rich (cholesterol oligomeric) domains are small (several lipids) and/or of short lifetime (10(-9) s to less than 10(-7) s). It is suggested that this microimmiscibility arises from the structural nonconformability between the rigid cholesterol ring structure and the rigid bend at the cis double bonds in PC alkyl chains.(ABSTRACT TRUNCATED AT 400 WORDS)

Rotational diffusion of a steroid molecule in phosphatidylcholine-cholesterol membranes: fluid-phase microimmiscibility in unsaturated phosphatidylcholine-cholesterol membranes

Rotational diffusion of androstane spin-label (ASL), a sterol analogue, in various phosphatidylcholine (PC)-cholesterol membranes was systematically studied by computer simulation of steady-state ESR spectra as a function of the chain length and unsaturation of the alkyl chains, cholesterol mole fraction, and temperature for a better understanding of phospholipid-cholesterol and cholesterol-cholesterol interactions. Special attention was paid to the differences in the cholesterol effects on ASL motion between saturated and unsaturated PC membranes. ASL motion in the membrane was treated as Brownian rotational diffusion of a rigid rod within the confines of a cone imposed by the membrane environment. The wobbling rotational diffusion constant of the long axis, its activation energy, and the cone angle of the confines were obtained for various PC-cholesterol membranes in the liquid-crystalline phase. Cholesterol decreases both the cone angle and the wobbling rotational diffusion constant for ASL in all PC membranes studied in this work. The cholesterol effects are the largest in DMPC membranes. An increase of cholesterol mole fraction from 0 to 30% decreases the rotational diffusion constant by a factor of 9-15 (depending on temperature) and the cone angle by a factor of about 2. In dioleoyl-PC membranes, addition of 30 mol % cholesterol reduces both the rotational diffusion constant and the cone angle of ASL by factors of approximately 2.5 and approximately 1.3, respectively, while it was previously found to cause only modest effects on the motional freedom of phospholipid analogue spin probes [Kusumi, A., Subczynski, W. K., Pasenkiewicz-Gierula, M., Hyde, J. S., & Merkle, H. (1986) Biochim. Biophys. Acta 854, 307-317]. It is proposed that fluid-phase microimmiscibility takes place in dioleoyl-PC-cholesterol membranes at physiological temperatures, which induces cholesterol-rich domains in the membrane, partially due to the steric nonconformability between the rigid fused-ring structure of cholesterol and the 30 degrees bend at the C9-C10 cis double bond of the alkyl chains of dioleoyl-PC. The mechanism by which cholesterol influences the lipid dynamics in the membrane is different between saturated and unsaturated PC membranes.

Adsorption of Cholesterol at Hexane/Water Interface

Abstract The interfacial tension between hexane solution of cholesterol and water was measured as a function of temperature and the mole fraction of cholesterol in hexane under atmospheric pressure. The interfacial tension vs. mole fraction of cholesterol curve was observed to have two break points at a certain temperature; the concentration of the break point increases with increasing temperature. The experimental results were analyzed by applying the rigorous thermodynamics of adsorption at interfaces developed previously. The excess number of moles of cholesterol and the thermodynamic quantity changes associated with its adsorption were found to show the discontinuous changes at the break points. It was concluded that the first-order phase transition takes place from the gaseous to the expanded state and from the expanded to the condensed one in the adsorbed film of cholesterol at the hexane/water interface.

Thermodynamics of phosphatidylcholine-cholesterol mixed model membranes in the liquid crystalline state studied by the orientational order parameter

It is shown that good estimates of the activity of cholesterol in phosphatidylcholine-cholesterol mixed model membranes are obtained by examining the orientational order parameter S of cholestane spin probe (CSL) that is obtained from electron spin resonance by spectral simulation. By introducing thermodynamic stability conditions of liquid mixtures, the variation of activity (or S) as a function of cholesterol mole fraction is utilized to predict the concentration at which the phase separation occurs. These results for DMPC and cholesterol binary mixtures agree very well with those of Tempo-partitioning experiments. The comparison of activity coefficients and the phase boundary in DMPC/cholesterol mixtures with those of POPC/cholesterol mixtures suggests that acyl chain unsaturation leads to poorer mixing of cholesterol in phosphatidylcholine model membranes at higher temperatures (i.e., greater than 35 degrees C). In ternary solutions of DMPC, POPC, and cholesterol, it is found that cholesterol shows less deviation from ideality than in either of the two binary mixtures, and this implies that the phase separation occurs at higher cholesterol concentration than in either of the two binary mixtures. The present analysis suggests that there may not be a critical point in DMPC/cholesterol mixtures, even though phase separation does occur.

Role of the stereochemistry of the hydroxyl group of cholesterol and the formation of nonbilayer structures in phosphatidylethanolamines

The phase behavior of mixtures of cholesterol or epicholesterol with phosphatidylethanolamine was studied by differential scanning calorimetry and by X-ray diffraction. Discrete domains of cholesterol are detected by X-ray diffraction in the L alpha phase of phosphatidylethanolamine from egg yolk and synthetic dielaidoylphosphatidylethanolamine beginning at mole fractions of 0.35-0.4 cholesterol. Separate domains of crystalline epicholesterol can also be detected in the L alpha phase of dielaidoylphosphatidylethanolamine by X-ray diffraction at as little as 0.16 mole fraction of epicholesterol. This is a result of poor miscibility of the epicholesterol with dielaidoylphosphatidylethanolamine. Epicholesterol does not alter the L beta----L alpha transition or bilayer spacing. Epicholesterol also has little effect on the diameter of the cylinders in the hexagonal phase. Formation of the inverted hexagonal phase is facilitated by addition of small amounts of cholesterol (mole fraction less than 0.2) in both egg phosphatidylethanolamine and dielaidoylphosphatidylethanolamine. However, at higher mole fractions of cholesterol, the stability of the liquid-crystalline phase is found to increase markedly for dielaidoylphosphatidylethanolamine but not for egg phosphatidylethanolamine, indicating the importance of the structure of the acyl chains in controlling the relative stability of the lamellar and nonlamellar phases in these systems. In contrast to cholesterol, epicholesterol markedly lowers the L alpha----HII phase transition temperature at low mole fraction of sterol. This result demonstrates the importance of the orientation and motional properties of an additive in determining the L alpha----HII transition temperature.

Rotational diffusion of a steroid molecule in phosphatidylcholine membranes: effects of alkyl chain length, unsaturation, and cholesterol as studied by a spin-label method.

Rotational diffusion of cholestane spin-label (CSL), a sterol analogue, in various phosphatidylcholine (PC)-cholesterol membranes was systematically studied by computer simulation of steady-state ESR spectra as a function of chain length and unsaturation of alkyl chains, cholesterol mole fraction, and temperature for better understanding of phospholipid-cholesterol and cholesterol-cholesterol interactions. CSL motion in the membrane was treated as Brownian rotational diffusion of a rigid rod within the confines of a cone imposed by the membrane environment. The wobbling rotational diffusion constant of the long axis, its activation energy, and the cone angle of the confines are obtained for various membranes in the liquid-crystalline phase. The wobbling diffusion constant decreases in the order dilauroyl-PC greater than dimyristoyl-PC greater than dioleoyl-PC approximately dipalmitoyl-PC greater than distearoyl-PC greater than dioleoyl-PC/cholesterol = 3/1 greater than dioleoyl-PC/cholesterol = 1/1 membranes. Activation energy for the wobbling diffusion of the long axis of CSL is strongly dependent on alkyl chain length, unsaturation, and cholesterol mole fraction. It decreases with decrease in alkyl chain length and by introduction of unsaturation in the alkyl chains. In dioleoylphosphatidylcholine membranes, activation energy decreases by a factor of approximately 3 in the presence of 50 mol % cholesterol. Activation energy for wobbling diffusion of CSL in phosphatidylcholine membranes is smaller than the activation energy for translational diffusion of a phospholipid. The former is more dependent on alkyl chain length and unsaturation.(ABSTRACT TRUNCATED AT 250 WORDS)

A calorimetric study of the thermotropic behaviour of mixtures of brain cerebrosides with other brain lipids

We have used a computer-controlled differential scanning calorimeter to determine the phases present in mixtures of the brain galactocerebrosides with other representative brain lipids. There are two types of brain galactocerebroside, those which posses an α-hydroxy substituent on the acyl chain (HFA) and those that do not (NFA). In the liquid crystalline state both cerebrosides were miscible with all the lipids studied, but in the gel state they were immiscible with cholesterol and the brain phosphatidylcholines. However, cholesterol mixtures in which the cholesterol mole fraction exceeded one third formed homogeneous metastable gel states on cooling from above the melting point of the cerebroside. Relaxation to the stable two phase state took place slowly over several hours. The solubilities of the galactocerebrosides in the other main brain sphingolipid, sphingomyelin, were much higher. Only in the case of the NFA galactocerebroside and at low mole fractions of sphingomyelin was immiscibility detected. Ternary mixtures of the two cerebrosides with sphingomyelin/cholesterol and phosphatidylcholine/cholesterol (PC/Chol) showed different miscibility characteristics. On cooling from 80°C all mixtures formed homogeneous gel states. However, on standing the cerebrosides separated into discrete gel phases in all mixtures but one, that in which HFA galactocerebrosides were mixed with sphingomyelin and cholesterol. The cerebroside in the mixture with the composition closest to that of myelin, HFA/PC/Chol, melted at 38°C. On scanning guinea pig CNS myelin which had been equilibrated at 5°C a transition was detected with T max 33°C. On the basis of comparison with the HFA/PC/Chol mixture we propose that the transition in myelin at this temperature is due to the melting of a galactocerebroside gel phase.

Modulation of the phase transition behavior of phosphatidylethanolamine by cholesterol and oxysterols.

Cholesterol lowers the bilayer to hexagonal phase transition temperature of phosphatidylethanolamines up to a mole fraction of about 0.1. At cholesterol mole fractions above about 0.3, the effect of this sterol is to stabilize the bilayer phase. The relatively weak effects of cholesterol in altering the bilayer to hexagonal phase transition temperature can be explained on the basis of lateral phase separation. This is indicated by the horizontal liquidus line for the gel to liquid-crystalline transition in the phase diagram for mixtures of cholesterol with dielaidoylphosphatidylethanolamine (DEPE) as well as the fact that cholesterol does not greatly decrease the cooperativity of the bilayer to hexagonal phase transition. The enthalpy of this latter transition increased with increasing mole fractions of cholesterol. Two oxidation products of cholesterol are 5-cholesten-3 beta,7 alpha-diol and cholestan-3 beta,5 alpha,6 beta-triol. Compared with cholesterol, 5-cholesten-3 beta,7 alpha-diol had a greater effect in decreasing the bilayer to hexagonal phase transition temperature and broadening this transition. It is suggested that its effectiveness is due to its greater solubility in the DEPE. In contrast, cholestan-3 beta,5 alpha,6 beta-triol raises the bilayer to hexagonal phase transition temperature of DEPE. This is due to its larger and more hydrophilic head group. In addition, its length, being shorter than that of DEPE, would not allow it to pack efficiently in a hexagonal phase arrangement. We suggest that this same effect is responsible for cholesterol raising the bilayer to hexagonal phase transition temperature at higher mole fractions.

Cardiolipin-cholesterol interactions in the liquid-crystalline phase: a steady-state and time-resolved fluorescence anisotropy study with cis- and trans-parinaric acids as probes.

The potency of cholesterol to affect the acyl chain order and dynamics of cardiolipin membranes in the liquid-crystalline state was monitored by steady-state and time-resolved fluorescence anisotropy as well as excited-state lifetime measurements with cis- and trans-parinaric acids as probes. Up to a cholesterol mole fraction (mean chl) of congruent to 0.20, no measurable effect on any of the fluorescence parameters of either probe in cardiolipin bilayers was evidenced. This was in striking contrast to the situation in dioleoylphosphatidylcholine (DOPC), for which a cholesterol mole fraction of 0.20 corresponded to the half-maximal effect on the fluorescence parameters, reflecting the classical ordering effect of cholesterol observed in lecithin systems in the liquid-crystalline phase. Whereas in DOPC bilayers this order effect plateaued at mean chl = 0.50, in cardiolipins the increase in acyl chain order was observable up to a mole fraction as high as 0.80. This indicated that cardiolipins were able to incorporate about 4 mol of cholesterol/mol of cardiolipin (i.e., 1 mol of cholesterol per fatty acyl chain). Besides, 31P NMR spectra of multilamellar liposomes obtained from pure cardiolipins and cardiolipin--cholesterol mixtures evidenced a line shape characteristic of lamellar structures. These results clearly indicate that the presence of high levels of cardiolipins in inner mitochondrial membrane does not impede cholesterol uptake by these membranes. However, the absence of an effect of cholesterol in the physiological range of the cholesterol mole fraction (congruent to 0.20) would signify weaker sterol-cardiolipin interactions than with lecithins and in turn would explain the relative dearth of cholesterol in these membranes.