Moisturizing mechanism of glycerol and diglycerol on human stratum corneum studied by synchrotron X-ray diffraction.

OBJECTIVE: Polyols are moisturizers used in cosmetics. Using X-ray diffraction, we studied the moisturizingmechanism of polyol solutions in the stratum corneum (SC). We examined whether glycerol disrupts the ordered hydrocarbon chain packing structure in isolated SC, as previously proposed in an SC lipid model. METHODS: The SC samples were prepared by treatment with water and aqueous solutions of glycerol, diglycerol and glycerol/diglycerol. To examine the differences in the water-retention efficiency of polyols, using a solution cell, we performed dynamic X-ray diffraction to analyse the structural changes that occurred during water removal from the hydrated samples by a stream of dry nitrogen gas. We focused on two structures, the orthorhombic hydrocarbon chain packing structure in the intercellular lipids and the soft keratin in the corneocytes where most of the water is stored. RESULTS: The spacing formed by the soft keratin in the corneocytes immersed in a solution of water and diglycerol solution decreased rapidly by water removal. In contrast, it decreased gradually in the corneocytes immersed in a solution of glycerol and glycerol/diglycerol, indicating that the glycerol-containing solutions maintained the hydrated state of the corneocytes for longer. Furthermore, the characteristic change of the spacing in the orthorhombic hydrocarbon chain packing structure over time was observed during the drying process. The hydrated state was maintained longer, in water, followed by glycerol, diglycerol and glycerol/diglycerol, in increasing order. This is the first study to report such characteristic properties that could be indicators of the capacity of the SC to regulate water. CONCLUSION: The dynamic X-ray diffraction experiment on the structure of the soft keratin and the orthorhombic hydrocarbon chain packing structure during the drying of the hydrated SC provides an insight into the moisturizing mechanism of the polyol solutions in the SC. The results show that the glycerol/diglycerol solution functions as an effective SC moisturizer at the molecular level. Further, it was confirmed that the behaviour of glycerol in the isolated SC varies from that proposed in the SC lipid model, wherein glycerol was proposed to prevent the formation of a regular hydrocarbon chain packing structure.

OBJECTIF: les polyols sont des hydratants utilisés dans les cosmétiques. À l'aide de la diffractions des rayons X, nous avons étudié le mécanisme d'hydratation des solutions de polyols dans la couche cornée (stratum corneum, SC).Notre étude avait pour but de déterminer si le glycérol perturbe la structure d'empilement de la chaîne d'hydrocarbures ordonnée dans les SC isolés, comme proposé précédemment dans un modèle lipidique de SC. MÉTHODES: les échantillons de SC ont été préparés avec de l'eau et des solutions aqueuses de glycérol, de diglycérol et de glycérol/diglycérol. Pour examiner les différences d'efficacité de rétention d'eau des polyols à l'aide d'une cellule de solution, nous avons effectué une diffraction dynamique des rayons X pour analyser les changements structurels qui se sont produits pendant l'élimination de l'eau des échantillons hydratés par un flux d'azote sec. Nous avons mis l'accent sur deux structures, la structure d'empilement des chaînes d'hydrocarbures orthorhombiques dans les lipides intercellulaires et la kératine molle dans les cornéocytes où la majeure partie de l'eau est stockée. RÉSULTATS: l'espacement causé par la kératine molle dans les cornéocytes immergés dans une solution d'eau et de diglycérol a diminué rapidement en raison de l'élimination de l'eau. En revanche, il a diminué progressivement dans les cornéocytes immergés dans une solution de glycérol et de glycérol/diglycérol, ce qui indique que les solutions contenant du glycérol ont maintenu l'état hydraté des cornéocytes plus longtemps. De plus, la modification caractéristique de l'espacement dans la structure d'empilement de la chaîne d'hydrocarbures orthorhombique au fil du temps a été observée au cours du processus de séchage. L'état hydraté a été maintenu plus longtemps, dans l'eau, suivi par le glycérol, le diglycérol et le glycérol/diglycérol, dans un ordre croissant. C'est la première étude à faire état de telles propriétés caractéristiques qui pourraient être des indicateurs de la capacité du SC à réguler l'eau. CONCLUSION: l'expérience de diffraction dynamique des rayons X sur la structure de la kératine molle et la structure d'empilement de la chaîne d'hydrocarbures orthorhombique pendant le séchage du SC hydraté donne un aperçu du mécanisme hydratant des solutions de polyols dans le SC. Les résultats montrent que la solution de glycérol/diglycérol agit comme un hydratant de SC efficace au niveau moléculaire. De plus, il a été confirmé que le comportement du glycérol dans le SC isolé varie par rapport à celui proposé dans le modèle lipidique SC, où le glycérol a été proposé pour prévenir.

Evaluation of the molecular lipid organization in millimeter-sized stratum corneum by synchrotron X-ray diffraction.

BACKGROUND: The aim of this study was to investigate whether the lamellar and lateral structure of intercellular lipid of stratum corneum (SC) can be evaluated from millimeter-sized SC (MSC) by X-ray diffraction. MATERIALS AND METHODS: A 12 mm × 12 mm SC sheet from hairless mouse was divided into 16 pieces measuring 3 mm × 3 mm square. From another sheet, 4 pieces of ultramillimeter-sized SC (USC:1.5 mm × 1.5 mm square) were prepared. Small and wide-angle X-ray diffraction (SAXD and WAXD) measurements were performed on each piece. For MSC and USC, changes in the lamellar and lateral structure after the application of d-limonene were measured. RESULTS: The intensity of SAXD peaks due to the lamellar phase of long periodicity phase (LPP) and WAXD peaks due to the lateral hydrocarbon chain-packing structures varied in MSC and USC pieces, although over the 12 mm × 12 mm SC sheet. These results indicated that the intercellular lipid components and their proportion appeared nearly uniform. Application of d-limonene on MSC and USC piece with strong peaks in SAXD and the WAXD resulted in the disappearance of peaks due to the lamellar phase of LPP and decrease in peak intensity for the lateral hydrocarbon chain-packing structures. These changes are consistent with normal-sized sample results. CONCLUSION: We found that the selection of a sample piece with strong diffraction peaks due to the lamellar and lateral structure enabled evaluation of the SC structure in small-sized samples by X-ray diffraction.

Disruption of human stratum corneum lipid structure by sodium dodecyl sulphate.

OBJECTIVE: Surfactants are major ingredients of body soaps and cleansers, and such harsh ones have been demonstrated to damage the skin. Stratum corneum (SC), the outermost barrier of the skin layer, is rich in intercellular lipids. This lipid structure can be disrupted by surfactants, impairing the barrier function of the skin. Thus, we investigated the surfactant-induced disruption of the intercellular lipid structure of human SC at the molecular level using synchrotron X-ray diffraction. METHODS: SC samples from the breast of female Caucasians were treated with sodium dodecyl sulphate (SDS) and analysed by small-angle and wide-angle X-ray diffraction. RESULTS: We found that an aqueous SDS solution affected the long lamellar structure, which became disorganized. The final disordered lipid state was reached through two or more types of structural change. We propose that the disordered lipid state results from incorporation of SDS into the long lamellar structure. In contrast, the lattice constants in the short lamellar and the hydrocarbon-chain packing structures remained almost unchanged after SDS treatment. CONCLUSION: We conclude that the disruption of the long lamellar structure plays a key role in the damage to the SC caused by detergents. To our knowledge, this is the first report to clarify the details of the disorganization of the intercellular lipid structure upon surfactant application. The knowledge obtained herein may allow the development of skin restoration methods and cleanser products that do not affect skin barrier functions.

X-ray diffraction study on interdigitated structure of phosphatidylcholines in glycerol.

X-ray diffraction was used to study the interdigitated structure of phosphatidylcholines (PCs) in glycerol. In this study, we investigated five different saturated diacyl PCs with carbon number from 14 to 18 in their acyl chains. It was found that lamellar spacings increase linearly as increasing the carbon number in the chains and that the increment is 0.10+/-0.01 nm per one carbon atom. The lamellar diffraction intensity data were analyzed, by applying a method proposed by Adachi [Chem. Phys. Lipids 107 (2000) 93]. The results indicate that the moiety around polar headgroup regions is almost unchanged, being independent of the carbon number.

Effect of microsome-liposome fusion on the rotational mobility of cytochrome P450IIB4 in rabbit liver microsomes.

Membrane fusion of microsomes with soybean phospholipid vesicles was performed at pH 6.5 to investigate the effect of lipid-enrichment in the membrane on the rotational mobility of cytochrome P450. Rotational diffusion of cytochrome P450 in the microsomal membrane of phenobarbital-induced rabbit liver was measured by detecting the decay of absorption anisotropy after photolysis of the heme CO complex by a vertically polarized laser flash. The fusion procedures yielded three separate fractions upon sucrose density gradient centrifugation with lipid-to-protein ratio in weight (L/P) as follows: 1.5 in the bottom fraction, 2.2 in the middle fraction, and 3.9 in the top fraction. In each fraction, co-existence of mobile and immobile cytochrome P450 was observed. The percentage of rotationally mobile P450 (with the mean rotational relaxation time of phi=505-828 micros) in each of the different bands was found to be 59% in the bottom fraction, 61% in the middle fraction, and 68% in the top fraction. This increase in mobile population of P450 due to lipid-enrichment indicates that aggregated proteins in microsomal membranes dissociate with increasing L/P which is inversely proportional to the protein concentration in the membrane. With freeze-fracture electron microscopy, it was shown that the average distance increased between intramembrane particles by lipid-enrichment. Thus, the significant immobile population (32%) of P450 in microsomal membranes can be explained by nonspecific protein aggregation which is a consequence of the low L/P of 0.8. The decrease in the mobile population in the bottom fraction compared with intact microsomes was shown to be due to the pH 6.5 incubation used for fusion.

X-ray diffraction study on ordered, disordered and reconstituted intercellular lipid lamellar structure in stratum corneum.

From small angle X-ray diffraction for the stratum corneum of hairless mouse, it was obtained that in the normal stratum corneum, the 1st, 2nd and 3rd order diffraction peaks for the intercellular lipid lamellar structure appear at 13.8, 6.87 and 4.59 nm, respectively and also a broad hump for the 4th order reflection appears as observed by the previous researchers. In the damaged stratum corneum prepared by the treatment of sodium dodecyl sulfate, these small-angle diffraction peaks disappear and only the broad maxima remain around the 1st, 2nd and 3rd order diffraction peaks. These facts indicate that in the normal stratum the lamellar structure is ordered and in the damaged stratum corneum the lamellar structure is disordered. Furthermore, in the reconstituted lamellar structure obtained by immersing into the dilute suspension of the mixture of ceramide 3, cholesterol and stearic acid, the 1st, 2nd and 3rd order diffraction peaks reappear at 13.3, 6.67 and 4.44 nm, respectively. This fact indicates that the reorganization of the ordered lamellar structure takes place by adding the mixture to the damaged stratum corneum.

Conditions required for heat-capacity measurements using modulated-temperature calorimetry.

Modulated-temperature calorimetry, in which the same apparatus as a standard differential scanning calorimeter is used, is a powerful method for measuring heat capacities. Precise heat capacity measurements are useful, for example, to analyze anomalous behavior at the glass transition. However, owing to the periodically-modulated temperature we have to pay attention to: (1) the thickness of the sample; (2) the thermal contact resistance between the sample and the sample pan; and (3) the thermal contact resistance between the sample pan and the base plate of the apparatus. This article will illustrate the following. The thickness of a sample should be chosen so as to satisfy uniformly-modulated temperature conditions throughout a sample, and subsequently both thermal contact resistances should be reduced so that the relaxation times caused by the thermal contact resistances are adequately small in comparison with the period of modulated temperature.

Simultaneous X-ray diffraction and calorimetric study of metastable- to-stable solid phase transformation of 1,2-dipalmitoyl-sn-glycerol.

The thermal behaviour and structural changes associated with the phase transformation of 1,2-dipalmitoyl-sn-glycerol (DPG) were studied by means of simultaneous X-ray diffraction and differential scanning calorimetry. Metastable DPG solid phases are crystallized from the melted sample by thermal quenching. The metastable phase (alpha-phase) formed initially is converted into a stable phase (beta' phase) at approximately 50 degrees C on heating. It was found that the behaviour of the alpha- to beta'-phase transformation depends on the thermal history. DPG solid samples incubated at approximately 3 degrees C for more than 10 h after cooling transformed directly into the beta'-phase with heat release. On the other hand, in the solid samples without incubation, the alpha-phase once melted and then the crystallization of the beta'-phase occurred successively from the melted state.

An X-ray diffraction study of the effect of alpha-tocopherol on the structure and phase behaviour of bilayers of dimyristoylphosphatidylethanolamine.

The effect of alpha-tocopherol on the thermotropic phase transition behaviour of aqueous dispersions of dimyristoylphosphatidylethanolamine was examined using synchrotron X-ray diffraction methods. The temperature of gel to liquid-crystalline (Lbeta-->Lalpha) phase transition decreases from 49.5 to 44.5 degrees C and temperature range where gel and liquid-crystalline phases coexist increases from 4 to 8 degrees C with increasing concentration of alpha-tocopherol up to 20 mol%. Codispersion of dimyristoylphosphatidylethanolamine containing 2.5 mol% alpha-tocopherol gives similar lamellar diffraction patterns as those of the pure phospholipid both in heating and cooling scans. With 5 mol% alpha-tocopherol in the phospholipid, however, an inverted hexagonal phase is induced which coexists with the lamellar gel phase at temperatures just before transition to liquid-crystalline lamellar phase. The presence of 10 mol% alpha-tocopherol shows a more pronounced inverted hexagonal phase in the lamellar gel phase but, in addition, another non-lamellar phase appears with the lamellar liquid-crystalline phase at higher temperature. This non-lamellar phase coexists with the lamellar liquid-crystalline phase of the pure phospholipid and can be indexed by six diffraction orders to a cubic phase of Pn3m or Pn3 space groups and with a lattice constant of 12.52+/-0.01 nm at 84 degrees C. In mixed aqueous dispersions containing 20 mol% alpha-tocopherol, only inverted hexagonal phase and lamellar phase were observed. The only change seen in the wide-angle scattering region was a transition from sharp symmetrical diffraction peak at 0.43 nm, typical of gel phases, to broad peaks centred at 0.47 nm signifying disordered hydrocarbon chains in all the mixtures examined. Electron density calculations through the lamellar repeat of the gel phase using six orders of reflection indicated no difference in bilayer thickness due to the presence of 10 mol% alpha-tocopherol. The results were interpreted to indicate that alpha-tocopherol is not randomly distributed throughout the phospholipid molecules oriented in bilayer configuration, but it exists either as domains coexisting with gel phase bilayers of pure phospholipid at temperatures lower than Tm or, at higher temperatures, as inverted hexagonal phase consisting of a defined stoichiometry of phospholipid and alpha-tocopherol molecules.

Effects of diacylglycerol on the structure and phase behaviour of non-bilayer forming phospholipid.

The phase behaviour of mixed aqueous dispersions of the monomethyl derivative of dioleoylphosphatidylethanolamine and dipalmitoylglycerol has been characterised by X-ray diffraction, differential scanning calorimetry and freeze-fracture electron microscopy for mixtures containing dipalmitoylglycerol in the concentration range 0-20 mol%. Dispersions prepared at temperatures where the phospholipid exhibits a liquid-crystalline lamellar phase show that dipalmitoylglycerol is completely phase separated into aggregates of stable crystal phase (beta'-phase). Heating mixed dispersions results in transformation of lamellar into hexagonal-II structure commencing at approximately 45 degrees C. This temperature coincides with a disappearance of beta'-phase of DPG which becomes incorporated into hexagonal-II phase. The pure phospholipid is transformed upon cooling from hexagonal-II into characteristic cubic phases; the formation of cubic phase is prevented by the presence of dipalmitoylglycerol and mixed dispersions initially form a lamellar liquid-crystalline phase in which the lipids are phase separated. The X-ray and thermal data suggest that relatively small domains of metastable crystal phase (alpha-phase) of DPG form initially on cooling and these subsequently coalesce and transform to beta'-phase.

Trehalose-induced destabilization of interdigitated gel phase in dihexadecylphosphatidylcholine.

Trehalose is believed to have the ability to protect some organisms against low temperatures. To clarify the cryoprotective mechanism of trehalose, the structure and the phase behavior of fully hydrated dihexadecylphosphatidylcholine (DHPC) membranes in the presence of various concentrations of trehalose were studied by means of differential scanning calorimetry (DSC), static x-ray diffraction, and simultaneous x-ray diffraction and DSC measurements. The temperature of the interdigitated gel (Lbeta(i))-to-ripple (Pbeta') phase transition of DHPC decreases with a rise in trehalose concentration up to approximately 1.0 M. Above a trehalose concentration of approximately 1.0 M, no Lbeta(i) phase is observed. In this connection, the electron density profile calculated from the lamellar diffraction data in the presence of 1.6 M trehalose indicates that DHPC forms noninterdigitated bilayers below the P beta' phase. It was concluded that trehalose destabilizes the Lbeta(i) phase of DHPC bilayers. This suggests that trehalose reduces the area at the interface between the lipid and water. The relation between this effect of trehalose and a low temperature tolerance was discussed from the viewpoint of cold-induced denaturation of proteins.

Interlamellar waters in dimyristoylphosphatidylethanolamine-water system as studied by calorimetry and X-ray diffraction.

The number of water molecules incorporated into the interlamellar region in a gel phase of dimyristoylphosphatidylethanolamine (DMPE)-water system containing up to about 40 g% water was estimated by techniques of calorimetry and X-ray diffraction. The calorimetric estimation based upon enthalpy changes of deconvoluted ice-melting peaks revealed that bulk water existing outside lipid bilayers begins to appear although the gel phase is not fully hydrated. The gel phase showed a linear depression of its transition temperature proportional to the amount of freezable waters interposed between bilayers. For a fully hydrated gel phase, the numbers of non-freezable and freezable interlamellar waters estimated by calorimetric analysis were about 2.3 and 3.7 molecules per lipid, respectively. The limiting, total number of interlamellar waters, 6 H2O/lipid, agreed with that estimated from both the X-ray diffraction data and the absolute specific volume for a DMPE molecule. Furthermore, the analysis for the lamellar intensity data is also consistent with the result of calorimetric analysis.

Structure and phase behaviour of dimyristoylphosphatidic acid/poly(L-lysine) systems.

Differential scanning calorimetry (DSC) and X-ray diffraction studies on (DMPA)/poly(L-lysine) systems are reported. DSC studies revealed that addition of poly(L-lysine) to DMPA bilayers raises the gel to liquid-crystalline phase transition of the systems, and that this effect depends on the molecular weight of the poly(L-lysine). Small-angle X-ray diffraction measurements showed that, in the liquid-crystalline phase, the lamellar spacing of a DMPA/short-poly(L-lysine) (approximately 4000 mol. wt.) system is shorter than that of a DMPA/long-poly(L-lysine) (approximately 22000 mol. wt.). In this connection wide-angle X-ray diffraction measurements indicate that the long-poly(L-lysine) adopts a beta-sheet conformation on the DMPA bilayers in both the gel and the liquid-crystalline phases, but the short-poly(L-lysine) adopts this conformation only on gel phase DMPA bilayers. We found that the spacings of the hydrocarbon chain packing in a DMPA bilayer in the gel phase increases with temperature, while the spacing between neighbouring polypeptide chains in long-poly(L-lysine) in the beta-sheet conformation remains almost constant. These observations indicate that the positively charged lysine residues are structurally independent of the negatively charged head groups of the phospholipid. On the basis of the present results we propose a model to explain the elementary behaviour of extrinsic membrane proteins in biomembranes.

Cubic phases in hydrated 1:1 and 1:2 dipalmitoylphosphatidylcholine-dipalmitoylglycerol mixtures.

The structures of fully hydrated 1:1 and 1:2 (mol/mol) dipalmitoylphosphatidylcholine (DPPC)-dipalmitoylglycerol (DPG) mixtures were studied by means of small-angle x-ray diffraction. The x-ray diffraction pattern of the 1:1 (mol/mol) DPPC-DPG mixture at 65 degrees C contains three reflections with spacings in the ratio of 1:1/ square root of 2:1/ square root of 3 in addition to reflections of an inverted hexagonal (H11) phase. A possible interpretation of this result is that a cubic phase of the body-centered space group lm3m, with a lattice constant of 23.1 +/- 0.6 nm, is formed. This cubic phase appears at intermediate temperatures between the lamellar and the H11 phases. The 1:2 (mol/mol) DPPC-DPG mixture gives an x-ray diffraction pattern at temperatures higher than the lamellar-to-H11 transition containing a number of reflections that index a cubic phase structure. The space group of the cubic phase was assigned a face-centered group Fd3m with a lattice constant of 16.3 +/- 0.1 nm at 82 degrees C. The possible role of cubic phases in membrane phenomena such as transmembrane signal transduction and fusion is discussed.

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

Effects of cholesterol on the lamellar and the inverted hexagonal (HII ) phases of dielaidoylphosphatidylethanolamine (DEPE) were studied by means of not only differential scanning calorimetry (DSC) but also simultaneous 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 (L beta) to lamellar liquid-crystalline (L alpha) phase. In fact, in the broad transition region, a coexistence of two lamellar X-ray diffraction peaks of the L beta and L alpha phases take place. In samples containing above 30 mol% cholesterol, no peak at the L beta-L alpha phase transition was observed in the DSC thermogram. On the other hand, cholesterol causes biphasic effects on the L alpha-HII phase transition: At low cholesterol concentrations below 20 mol%, the incorporation of cholesterol reduces the transition temperature and at high cholesterol concentrations about 30 mol%, 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 HII phase. From the above results it is suggested that cholesterol in cell membranes works in keeping the bilayer membrane nature notwithstanding the change of external conditions.

Structural and thermotropic properties of calcium-dimyristoylphosphatidic acid complexes at acidic and neutral pH conditions.

Two kinds of calcium-dimyristoylphosphatidic acid (DMPA) complexes at acidic and neutral pH conditions were prepared in the following ways. The complex at pH 4 was obtained by adding Ca2+ to DMPA dispersion in pure water. On the other hand, the complex at pH 7.4 was obtained by adding Ca2+ to DMPA dispersion in the presence of NaOH. The stoichiometries of Ca2+ ion to DMPA molecule are 0.5-0.67 and approximately 1 for the complexes at pH 4 and 7.4, respectively. Static x-ray diffraction shows that the hydrocarbon chains of the Ca(2+)-DMPA complex at pH 4 at 20 degrees C are more tightly packed than those of the complex at pH 7.4 at 20 degrees C. Furthermore, the complex at pH 4 at 20 degrees C gives rise to several reflections that might be related to the ordered arrangement of the Ca2+ ions. These results indicate that the structure of the complex at pH 4 is crystalline-like. In the differential scanning calorimetry (DSC) thermogram, the complex at pH 7.4 undergoes no phase transition in a temperature range between 30 and 80 degrees C. On the other hand, in the DSC thermogram for the complex at pH 4, a peak appears at 65.8 degrees C in the first heating scan. In the successive second heating scan, a transition peak appears at 63.5 degrees C. In connection with the DSC results, the structural changes associated with these phase transitions were studied with temperature-scan x-ray diffraction. In the first heating scan, although a peak appears at 65.80C in the DSC thermogram, the hydrocarbon chain packing gradually converts from an orthorhombic lattice to a hexagonal lattice near 52 degree C, and successively the chain melting phase transition occurs near 670C. In the second heating scan, the hydrocarbon chains are packed in a hexagonal lattice over the whole temperature range and the chain melting phase transition occurs near 63.5 degree C. Therefore,the Ca2+-DMPA complex at pH 4 has a metastable state. The metastable state transforms to a stable state by maintaining the complex at pH 4 for about 90 h at 200C.

Interdigitated structure of phospholipid-alcohol systems studied by x-ray diffraction.

In the interdigitated structure of phosphatidylcholine/alcohol systems, the one-dimensional electron density profile in the direction normal to the membrane surface is generated from the x-ray diffraction pattern. The membrane thickness for these systems is expressed by the sum of the hydrocarbon chain lengths of phosphatidylcholine and alcohol molecules. For this study, various sets of phosphatidylcholines and 1-alcohols were used; a phosphatidylcholine has a carbon number from 14 to 18 in a hydrocarbon chain, and an alcohol has a carbon number from 1 (methanol) to 4 (1-butanol). Based upon the results, we propose a model for the interdigitated structure in which 1) two alcohol molecules occupy a volume whose surface is surrounded interstitially by the headgroups of phosphatidylcholine molecules, and 2) the methyl ends of both hydrocarbon chains in alcohol and phosphatidylcholine molecules face each other at the bottom of the volume.

Characterization of complexes formed in fully hydrated dispersions of dipalmitoyl derivatives of phosphatidylcholine and diacylglycerol.

The phase diagram of fully hydrated binary mixtures of dipalmitoylphosphatidylcholine (DPPC) with 1,2-dipalmitoylglycerol (DPG) published recently by López-García et al. identifies regions where stoichiometric complexes of 1:1 and 1:2 DPPC:DPG, respectively, are formed. In this study, the structural parameters of the 1:1 complex in the presence of pure DPPC was characterized by synchrotron low angle and static x-ray diffraction methods. Structural changes upon transitions through phase boundaries were correlated with enthalpy changes observed by differential scanning calorimetry in mixtures of DPPC with 5, 7.5, 10, and 20 mol% DPG dispersed in excess water. Phase separation of a complex in gel phase could be detected by calorimetry in the mixture containing 5 mol% DPG but was not detectable by synchrotron low angle x-ray diffraction. Static x-ray measurements show evidence of phase separation, particularly in the reflections indexing chain packing. In the mixture containing 7.5 mol% DPG, two distinct lamellar repeat spacings could be seen in the temperature range from 25 to 34 degrees C. The lamellar spacing of about 6.6 nm was assigned to pure gel phase DPPC because the change in the spacing corresponds with thermal transition of the pure phospholipid, and a longer repeat spacing of about 7.2 nm was assigned to domains of the 1:1 complex of DPPC-DPG. In the temperature range from 34 to 420C, i.e., in the region of coexistence of the ripple phase of DPPC and the gel phase of the complex, a single, rather broad lamellar reflection appears because of superposition of two reflections of DPPC and the complex; the lamellar spacing of DPPC in the ripple phase is similar to that of the gel phase of complex. In the coexistence region of the liquid-crystalline phase of DPPC and the gel phase of complex (-42-480C), the lamellar reflections of the both phases are present. The fluidus boundary lies between the coexistence region and the fluid region.In the fluid region (-48-550C), the gel state of complex persists up to the fluidus boundary, whereupon the liquid-crystalline state of complex replaces the gel state of the complex. This indicates that the complex is also immiscible with DPPC even above the fluidus boundary at least in the temperature range close to the phase boundary. For mixtures comprising 10 and 20 mol%DPG in DPPC, complex formation is clearly detectable in both the gel region and the coexistence region by x-ray diffraction.Synchrotron x-ray measurements indicate phase separation between pure DPPC and liquid-crystalline complex just above thefluidus boundary. Static, wide angle x-ray measurements also suggest phase separations of the 1:1 complex not only from the gel phase but also the liquid-crystalline phase of pure DPPC. Two distinct diffraction peaks were detected for the mixture of DPPC with 5, 10, and 20 mol% DPG. One is due to the chain spacing of the complex, and the other is due to that of the pure DPPC. In the coexistence region of the liquid-crystalline phase of DPPC and the gel phase of complex, two kinds of diffraction peaks of the hydrocarbon chain of the gel phase complex and the broad scattering profile for the chain melting of DPPC were observed in the wide angle region. Electron density reconstructed from the lamellar reflections indicates that the thicknesses of both the bilayer and the water layer of the gel phase complex are greater than those of the respective thicknesses of gel phase DPPC.

Temperature change of the ripple structure in fully hydrated dimyristoylphosphatidylcholine/cholesterol multibilayers.

The ripple structure was studied as a function of temperature in fully hydrated dimyristoylphosphatidylcholine (DMPC)/cholesterol multibilayers using synchrotron x-ray small-angle diffraction and freeze-fracture electron microscopy. In the presence of cholesterol, the ripple structure appears below the pretransition temperature of pure DMPC multibilayers. In this temperature range the ripple periodicity is relatively large (25-30 nm) and rapidly decreases with increasing temperature. In this region, defined as region I, we observed coexistence of the P beta' phase and the L beta' phase. The large ripple periodicity is caused by the formation of the P beta' phase region in which cholesterol is concentrated and the L beta' phase region from which cholesterol is excluded. An increase in ripple periodicity also takes place in the narrow temperature range just below the main transition temperature. We define this temperature region as region III, where the ripple periodicity increases dramatically toward the main transition temperature. In region II, between regions I and III, the ripple periodicity decreases gradually with temperature. This behavior is quite similar to that of pure DMPC. Temperature-versus-ripple periodicity curves are parallel among pure DMPC and DMPCs with various cholesterol contents. We explain this behavior in terms of a model proposed by other workers.