Localisation of partially deuterated cholesterol in quaternary SC lipid model membranes: a neutron diffraction study

This letter presents our first results in using the benefit of selective deuteration in neutron diffraction studies on stratum corneum (SC) lipid model systems. The SC represents the outermost layer of the mammalian skin and exhibits the main skin barrier. It is essential for studying drug penetration through the SC to know the internal structure and hydration behaviour on the molecular level. The SC intercellular matrix is mainly formed by ceramides (CER), cholesterol (CHOL) and long- chain free fatty acids (FFA). Among them, CHOL is the most abundant individual lipid, but a detailed knowledge about its localisation in the SC lipid matrix is still lacking. The structure of the quaternary SC lipid model membranes composed of either CER[AP]/CHOL-D6/palmitic acid (PA)/cholesterol sulphate (ChS) or CER[AP]/CHOL-D7/PA/ChS is characterized by neutron diffraction. Neutron diffraction patterns from the oriented samples are collected at the V1 diffractometer of the Hahn-Meitner-Institute, Berlin, measured at 32°C, 60% humidity and at different D₂O contents. The neutron scattering length density profile in the direction normal to the surface is restored by Fourier synthesis from the experimental diffraction patterns. The analysis of scattering length density profile is a suitable tool for investigating the internal structure of the SC lipid model membranes. The major finding is the experimental proof of the CHOL localisation in SC model membrane by deuterium labelling at prominent positions in the CHOL molecules.     

Fatty acid interdigitation in stratum corneum model membranes: a neutron diffraction study

The influence of the chain length of the free fatty acid (FFA) in a stratum corneum (SC) lipid model membrane composed of N-(alpha-hydroxyoctadecanoyl)-phytosphingosine (CER [AP]), cholesterol (Ch), FFA and cholesterol sulphate (ChS) was investigated by neutron diffraction. The internal nanostructure of the SC lipid membrane in addition to the water distribution function was determined via calculation of the neutron scattering length density profile (Fourier profile). The Fourier profiles of the studied SC model membranes revealed that such membranes have a repeat distance approximately equal to the membrane thickness. Increasing the chain length of the FFA in the CER[AP] based model membrane did not cause an alteration of the internal nanostructure but led to a decrease in the membrane repeat distance from 45.6 A (palmitic acid, C16:0) to 43.7 A (cerotic acid, C26:0) due to a partial interdigitation of the FFA chains. Ceramide [AP] forces the long chain fatty acids to incorporate into the unchanged spacing of the bilayer, thereby obligating the FFA protrude partly through opposing leaflet. Furthermore, the longer chained free fatty acids tend to form a new separate so-called "fatty acid rich phase". Therefore, the elongation of the chain length of the FFA decreases the solubility of the FFA in the SC model membrane based on CER[AP].     

Basic Nanostructure of Stratum Corneum Lipid Matrices Based on Ceramides [EOS] and [AP]: A Neutron Diffraction Study

The goal of this study was to investigate the nanostructure of SC lipid model membranes comprising the most relevant SC lipids such as the unique-structured ω-acylceramide [EOS] in a near natural ratio with neutron diffraction. In models proposed recently the presence of ceramide [EOS] and FFA are necessary for the formation of one of the two existent crystalline lamellar phases of the SC lipids, the long-periodicity phase as well as for the normal barrier function of the SC. The focus of this study was placed on the influence of the FFA BA on the membrane structure and its localization within the membrane based on the ceramides [EOS] and [AP]. The internal nanostructure of such membranes was obtained by Fourier synthesis from the experimental diffraction patterns. The resulting neutron scattering length density profiles showed that the exceptionally long ceramide [EOS] is arranged in a short-periodicity phase created by ceramide [AP] by spanning through the whole bilayer and extending even further into the adjacent bilayer. Specifically deuterated BA allowed us to determine the exact position of this FFA inside this SC lipid model membrane. Furthermore, hydration experiments showed that the presented SC mimic system shows an extremely small intermembrane hydration of ∼1 Å, consequently the headgroups of the neighboring leaflets are positioned close to each other.     

The phase behaviour of skin lipid mixtures based on synthetic ceramides

The lipid lamellae present in the outermost layer of the skin, the stratum corneum (SC), form the main barrier for diffusion of molecules across the skin. The main lipid classes in SC are cholesterol (CHOL), free fatty acids (FFA) and at least nine classes of ceramides (CER), referred to as CER1 to CER9. In the present study the phase behaviour of four synthetic CER, either single or mixed with CHOL or CHOL and FFA, has been studied using small and wide angle X-ray diffraction. The lipid mixtures showed complex phase behaviour with coexistence of several phases. The results further revealed that the presence of synthetic CER1 as well as a proper composition of the other CER in the mixture were crucial for the formation of a phase with a long periodicity, characteristic for SC lipid phase behaviour. Only a mixture containing synthetic CER1 and CER3, CHOL and FFA showed similar phase behaviour to that of SC.     

The role of ceramide composition in the lipid organisation of the skin barrier.

The lipid lamellae in the stratum corneum (SC) play a key role in the barrier function of the skin. The major lipids are ceramides (CER), cholesterol (CHOL) and free fatty acids (FFA). In pig SC at least six subclasses of ceramides (referred to as CER 1, 2-6) are present. Recently it was shown that in mixtures of isolated pig SC ceramides (referred to as CER(1-6)) and CHOL two lamellar phases are formed, which mimic SC lipid organisation very closely [J.A. Bouwstra et al., 1996, J. Lipid Res. 37, 999-1011] [1]. Since the CER composition in SC originating from different sources/donors often varies, information on the effect of variations in CER composition on the SC lipid organisation is important. The results of the present study with mixtures of CHOL including two different CER mixtures that lack CER 6 (CER(1-5) mixtures) revealed that at an equimolar molar ratio their lipid organisation was similar to that of the equimolar CHOL:CER(1-6) and CHOL:CER(1,2) mixtures, described previously. These observations suggest that at an equimolar CHOL:CER ratio the lipid organisation is remarkably insensitive toward a change in the CER composition. Similar observations have been made with equimolar CHOL:CER:FFA mixtures. The situation is different when the CHOL:CER molar ratio varies. While in the CHOL:CER(1-6) mixture the lamellar organisation hardly changed with varying molar ratio from 0.4 to 2, the lamellar organisation in the CHOL:CER(1-5) mixtures appeared to be more sensitive to a change in the relative CHOL content, especially concerning the changes in the periodicities of the lamellar phases. In summary, these findings clearly indicate that at an equimolar CHOL:CER molar ratio the lamellar organisation is least sensitive to a variation in CER composition, while at a reduced CHOL:CER molar ratio the CER composition plays a more prominent role in the lamellar phases. This observation may have an implication for the in vivo situation when both the CER composition and the CHOL:CER molar ratio change simultaneously.     

The effect of the chain length distribution of free fatty acids on the mixing properties of stratum corneum model membranes

The stratum corneum (SC) plays a fundamental role in the barrier function of the skin. The SC consists of corneocytes embedded in a lipid matrix. The main lipid classes in the lipid matrix are ceramides (CERs), cholesterol (CHOL) and free fatty acids (FFAs). The aim of this study was to examine the effect of the chain length of FFAs on the thermotropic phase behavior and mixing properties of SC lipids. Fourier transform infrared spectroscopy and Raman imaging spectroscopy were used to study the mixing properties using either protonated or deuterated FFAs. We selected SC model lipid mixtures containing only a single CER, CHOL and either a single FFA or a mixture of FFAs mimicking the FFA SC composition. The single CER consists of a sphingoid base with 18 carbon atoms and an acyl chain with a chain length of 24 carbon atoms. When using lignoceric acid (24 carbon atoms) or a mixture of FFAs, the CER and FFAs participated in mixed crystals, but hydration of the mixtures induced a slight phase separation between CER and FFA. The mixed crystalline structures did not phase separate during storage even up to a time period of 3 months. When using palmitic acid (16 carbon atoms), a slight phase separation was observed between FFA and CER. This phase separation was clearly enhanced during hydration and storage. In conclusion, the thermotropic phase behavior and the mixing properties of the SC lipid mixtures were shown to strongly depend on the chain length and chain length distribution of FFAs, while hydration enhanced the phase separation.     

Lipid organization in human and porcine stratum corneum differs widely, while lipid mixtures with porcine ceramides model human stratum corneum lipid organization very closely

The conformational disordering and lateral packing of lipids in porcine and human isolated stratum corneum (SC) was compared using Fourier transform infrared spectroscopy (FTIR). It was shown that SC of both species differ markedly, porcine SC lipids being arranged predominantly in a hexagonal lattice while lipids in human SC are predominantly packed in the denser orthorhombic lattice. However, the lipid organization of equimolar ceramide:cholesterol:free fatty acid (CER:CHOL:FFA) mixtures prepared with isolated porcine CER or human CER is very similar, only the transition temperatures differed being slightly lower in mixtures with porcine CER. Therefore, the difference in lateral packing between human and porcine stratum corneum is not due to the difference in CER composition. Furthermore, it is possible to use more readily available porcine CER in model lipid mixtures to mimic lipid organization in human SC. As the equimolar porcine CER:CHOL:FFA mixtures closely mimic the lipid organization in human SC, both human SC and this mixture were selected to examine the effect of glycerol on the lipid phase behaviour. It was found that high concentrations of glycerol change the lamellar organization slightly, while domains with an orthorhombic lateral packing are still observed.     

Lipid organization in human and porcine stratum corneum differs widely, while lipid mixtures with porcine ceramides model human stratum corneum lipid organization very closely

The conformational disordering and lateral packing of lipids in porcine and human isolated stratum corneum (SC) was compared using Fourier transform infrared spectroscopy (FTIR). It was shown that SC of both species differ markedly, porcine SC lipids being arranged predominantly in a hexagonal lattice while lipids in human SC are predominantly packed in the denser orthorhombic lattice. However, the lipid organization of equimolar ceramide:cholesterol:free fatty acid (CER:CHOL:FFA) mixtures prepared with isolated porcine CER or human CER is very similar, only the transition temperatures differed being slightly lower in mixtures with porcine CER. Therefore, the difference in lateral packing between human and porcine stratum corneum is not due to the difference in CER composition. Furthermore, it is possible to use more readily available porcine CER in model lipid mixtures to mimic lipid organization in human SC. As the equimolar porcine CER:CHOL:FFA mixtures closely mimic the lipid organization in human SC, both human SC and this mixture were selected to examine the effect of glycerol on the lipid phase behaviour. It was found that high concentrations of glycerol change the lamellar organization slightly, while domains with an orthorhombic lateral packing are still observed.     

Models of stratum corneum intercellular membranes: 2H NMR of macroscopically oriented multilayers.

Deuterium NMR was used to characterize model membrane systems approximating the composition of the intercellular lipid lamellae of mammalian stratum corneum (SC). The SC models, equimolar mixtures of ceramide:cholesterol:palmitic acid (CER:CHOL:PA) at pH 5.2, were contrasted with the sphingomyelin:CHOL:PA (SPM:CHOL:PA) system, where the SPM differs from the CER only in the presence of a phosphocholine headgroup. The lipids were prepared both as oriented samples and as multilamellar dispersions, and contained either perdeuterated palmitic acid (PA-d31) or [2,2,3,4,6-2H5]CHOL (CHOL-d5). SPM:CHOL:PA-d31 formed liquid-ordered membranes over a wide range of temperatures, with a maximum order parameter of approximately 0.4 at 50 degrees C for positions C3-C10 (the plateau region). The quadrupolar splitting at C2 was significantly smaller, suggesting an orientational change at this position, possibly because of hydrogen bonding with water and/or other surface components. A comparison of the longitudinal relaxation times obtained at theta = 0 degrees and 90 degrees (where theta is the angle between the normal to the glass plates and the magnetic field) revealed a significant T1Z anisotropy for all positions. In contrast to the behavior observed with the SPM system, lipid mixtures containing CER exhibited a complex polymorphism. Between 20 and 50 degrees C, a significant portion of the entire membrane (as monitored by both PA-d31 and CHOL-d5) was found to exist as a solid phase, with the remainder either a gel or liquid-ordered phase. The proportion of solid decreased as the temperature was increased and disappeared entirely above 50 degrees C. Between 50 and 70 degrees C, the membrane underwent a liquid-ordered to isotropic phase transition. These transitions were reversible but displayed considerable hysteresis, especially the conversion from a fluid phase to solid. The order profiles, relaxation behavior, and angular dependence of these parameters suggest strongly that both the liquid-ordered CER- and SPM-membranes are bilayers. The unusual phase behavior observed for the CER-system, particularly the observation of solid-phase lipid at physiological temperatures, may provide insight into the functioning of the permeability barrier of stratum corneum.     

Modelling the stratum corneum lipid organisation with synthetic lipid mixtures: the importance of synthetic ceramide composition

Cholesterol (CHOL), free fatty acids (FFA) and nine classes of ceramides (CER1-CER9) form the main constituents of the intercellular lipid lamellae in stratum corneum (SC), which regulate the skin barrier function. Both the presence of a unique 13-nm lamellar phase, of which the formation depends on the presence of CER1, and its dense lateral packing are characteristic for the SC lipid organisation. The present study focuses on the lipid organisation in mixtures prepared with CHOL, FFA and a limited number of synthetic CER, namely CER1, CER3 and bovine brain CER type IV (SigmaCERIV). The main objective is to determine the optimal molar ratio of CER3 to SigmaCERIV for the formation of the 13-nm lamellar phase. CER3 contains a uniform acyl chain length, whereas SigmaCERIV contains fatty acids with varying chain lengths. Using small angle X-ray diffraction (SAXD), it is demonstrated that the CER3 to SigmaCERIV ratio affects the formation of the 13-nm lamellar phase and that the optimal ratio depends on the presence of FFA. Furthermore, the formation of the 13-nm lamellar phase is not very sensitive to variations in the total CER level, which is similar to the in vivo situation.     

Modelling the stratum corneum lipid organisation with synthetic lipid mixtures: the importance of synthetic ceramide composition

Cholesterol (CHOL), free fatty acids (FFA) and nine classes of ceramides (CER1-CER9) form the main constituents of the intercellular lipid lamellae in stratum corneum (SC), which regulate the skin barrier function. Both the presence of a unique 13-nm lamellar phase, of which the formation depends on the presence of CER1, and its dense lateral packing are characteristic for the SC lipid organisation. The present study focuses on the lipid organisation in mixtures prepared with CHOL, FFA and a limited number of synthetic CER, namely CER1, CER3 and bovine brain CER type IV (∑CERIV). The main objective is to determine the optimal molar ratio of CER3 to ∑CERIV for the formation of the 13-nm lamellar phase. CER3 contains a uniform acyl chain length, whereas ∑CERIV contains fatty acids with varying chain lengths. Using small angle X-ray diffraction (SAXD), it is demonstrated that the CER3 to ∑CERIV ratio affects the formation of the 13-nm lamellar phase and that the optimal ratio depends on the presence of FFA. Furthermore, the formation of the 13-nm lamellar phase is not very sensitive to variations in the total CER level, which is similar to the in vivo situation.     

New insights into the structure and hydration of a stratum corneum lipid model membrane by neutron diffraction

The structure and hydration of a stratum corneum (SC) lipid model membrane composed of N-(-hydroxyoctadecanoyl)-phytosphingosine (CER6)/cholesterol (Ch)/palmitic acid (PA)/cholesterol sulfate (ChS) were characterized by neutron diffraction. The neutron scattering length density across the SC lipid model membrane was calculated from measured diffraction peak intensities. The internal membrane structure and water distribution function across the bilayer were determined. The low hydration of the intermembrane space is a major feature of the SC lipid model membrane. The thickness of the water layer in the SC lipid model membrane is about 1 Å at full hydration. For the composition 55% CER6/25% Ch/15% PA/5% ChS, in a partly dehydrated state (60% humidity) and at 32°C, the lamellar repeat distance and the membrane thickness have the same value of 45.6 Å . The hydrophobic region of the membrane has a thickness of 31.2 Å . A decrease of the Ch content increases the membrane thickness. The water diffusion through the SC lipid model multilamellar membrane is a considerably slow process relative to that through phospholipid membranes. In excess water, the membrane hydration follows an exponential law with two characteristic times of 93 and 44 min. At 81°C and 97% humidity, the membrane separates into two phases with repeat distances of 45.8 and 40.5 Å . Possible conformations of CER6 molecules in the dry and hydrated multilayers are discussed.     

Impact of the long chain ω-acylceramides on the stratum corneum lipid nanostructure. Part 1: Thermotropic phase behaviour of CER[EOS] and CER[EOP] studied using X-ray powder diffraction and FT-Raman spectroscopy

The stratum corneum (SC), the outermost layer of the mammalian skin, is the main skin barrier. Ceramides (CERs) as the major constituent of the SC lipid matrix are of particular interest. At the moment, 11 classes of CERs are identified, but the effect of each single ceramide species is still not known.Therefore in this article, the thermotropic behaviour of the long chain ω-acylceramides CER[EOS] and CER[EOP] was studied using X-ray powder diffraction and FT-Raman spectroscopy.It was found that the ω-acylceramides CER[EOS] and CER[EOP] do not show a pronounced polymorphism which is observed for shorter chain ceramides as a significant feature. The phase behaviour of both ceramides is strongly influenced by the extremely long acyl-chain residue. The latter has a much stronger influence compared with the structure of the polar head group, which is discussed as extremely important for the appearance of a rich polymorphism. Despite the strong influence of the long chain, the additional OH-group of the phyto-sphingosine type CER[EOP] influences the lamellar repeat distance and the chain packing. The less polar sphingosine type CER[EOS] is stronger influenced by the long acyl-chain residue. Hydration is necessary for the formation of an extended hydrogen-bonding network between the polar head groups leading to the appearance of a long-periodicity phase (LPP). In contrast, the more polar CER[EOP] forms the LPP with densely packed alkyl chains already in the dry state.     

FTIR studies show lipophilic moisturizers to interact with stratum corneum lipids, rendering the more densely packed

Lipophilic moisturizers are widely used to treat dry skin. However, their interaction with the lipids in the upper layer of the skin, the stratum corneum (SC), is largely unknown. In the present study this interaction of three moisturizers, isostearyl isostearate (ISIS), isopropyl isostearate (IPIS) and glycerol monoisostearate (GMIS), has been elucidated using lipid mixtures containing isolated ceramides (CER), cholesterol (CHOL) and free fatty acids (FFA), mimicking the lipid composition and organization in SC. The conformational ordering and the lateral packing of the lipid mixtures were examined by Fourier transformed infrared spectroscopy. Equimolar CER:CHOL:FFA mixtures show an orthorhombic to hexagonal phase transition between 22 and 30 degrees C and an ordered-disordered phase transition between 46 and 64 degrees C. Addition of 20% m/m ISIS or IPIS increased the thermotropic stability of the orthorhombic lateral packing, while GMIS had no influence. Furthermore, small amounts of all three moisturizers are incorporated into the CER:CHOL:FFA lattice, while the majority of the moisturizer exists in separate domains. Especially the thermotropic stabilization of the orthorhombic lateral packing, which might reduce water loss from the skin, is considered to contribute to the moisturizing effect of IPIS and ISIS in stratum corneum.     

Cholesterol sulfate and calcium affect stratum corneum lipid organization over a wide temperature range

The main diffusion barrier for drugs penetrating through the skin is located in the intercellular lipid matrix in the upper layer of the skin, the stratum corneum (SC). The main lipid classes in the SC are ceramides (CER), free fatty acids (FFA) and cholesterol (CHOL). The lipids in SC are organized into two lamellar phases with periodicities of approximately 13 and 6 nm, respectively. Similar lipid organization has been found with equimolar CHOL:CER:FFA mixtures in SAXD studies performed at room temperature. However, one may conclude that the phase behavior of the mixtures is similar to that in SC only when the lipid organization of the lipid mixtures resembles that in SC over a wide temperature range. Therefore, in the present study, the organization of the lipid mixtures has been studied in a temperature range between 20 degrees and 95 degrees C. From these experiments it appeared that at elevated temperatures in equimolar CHOL:CER:FFA mixtures a new prominent 4.3 nm phase is formed between 35;-55 degrees C, which is absent or only weakly formed in intact human and pig SC, respectively. As it has been suggested that gradients of pH and cholesterol sulfate exist in the SC and that Ca(2+) is present only in the lowest SC layers, the effect of pH, cholesterol sulfate, and Ca(2+) on the lipid phase behavior has been investigated with lipid mixtures. Both an increase in pH from 5 (pH at the skin surface) to 7.4 (pH at the SC;-stratum granulosum interface) and the presence of cholesterol sulfate promote the formation of the 13 nm lamellar phase. Furthermore, cholesterol sulfate reduces the amount of CHOL that is present in crystalline domains, causes a shift in the formation of the 4.3 nm phase to higher temperatures, and makes this phase less prominent at higher temperatures. The finding that Ca(2+) counteracts the effects of cholesterol sulfate indicates the importance of a proper balance of minor SC components for appropriate SC lipid organization. In addition, when the findings are extrapolated to the in vivo situation, it seems that cholesterol sulfate is required to dissolve cholesterol in the lamellar phases and to stabilize SC lipid organization. Therefore, a drop in cholesterol sulfate content in the superficial layers of the SC is expected to destabilize the lipid lamellar phases, which might facilitate the desquamation process.     

Infrared spectroscopy studies of mixtures prepared with synthetic ceramides varying in head group architecture: Coexistence of liquid and crystalline phases

The barrier function of the skin is provided by the stratum corneum (SC), the outermost layer of the skin. Ceramides (CERs), cholesterol (CHOL) and free fatty acids (FFAs) are present in SC and form highly ordered crystalline lipid lamellae. These lamellae are crucial for a proper skin barrier function. In the present study, Fourier transform infrared spectroscopy was used to examine the lipid organization of mixtures prepared from synthetic CERs with CHOL and FFAs. The conformational ordering and lateral packing of these mixtures showed great similarities to the lipid organization in SC and lipid mixtures prepared with native CERs. Therefore, mixtures with synthetic CERs serve as an excellent tool for studying the effect of molecular architecture of CER subclasses on the lipid phase behavior. In SC the number of OH-groups in the head groups of CER subclasses varies. Furthermore, acylCERs with a linoleic acid chemically bound to a long acyl chain are also identified. The present study revealed that CER head group architecture affects the lateral packing and conformational ordering of the CER:CHOL:FFA mixtures. Furthermore, while the majority of the lipids form a crystalline packing, the linoleate moiety of the acylCERs participates in a “pseudo fluid” phase.     

FTIR studies show lipophilic moisturizers to interact with stratum corneum lipids, rendering the more densely packed

Lipophilic moisturizers are widely used to treat dry skin. However, their interaction with the lipids in the upper layer of the skin, the stratum corneum (SC), is largely unknown. In the present study this interaction of three moisturizers, isostearyl isostearate (ISIS), isopropyl isostearate (IPIS) and glycerol monoisostearate (GMIS), has been elucidated using lipid mixtures containing isolated ceramides (CER), cholesterol (CHOL) and free fatty acids (FFA), mimicking the lipid composition and organization in SC. The conformational ordering and the lateral packing of the lipid mixtures were examined by Fourier transformed infrared spectroscopy. Equimolar CER:CHOL:FFA mixtures show an orthorhombic to hexagonal phase transition between 22 and 30 °C and an ordered-disordered phase transition between 46 and 64 °C. Addition of 20% m/m ISIS or IPIS increased the thermotropic stability of the orthorhombic lateral packing, while GMIS had no influence. Furthermore, small amounts of all three moisturizers are incorporated into the CER:CHOL:FFA lattice, while the majority of the moisturizer exists in separate domains. Especially the thermotropic stabilization of the orthorhombic lateral packing, which might reduce water loss from the skin, is considered to contribute to the moisturizing effect of IPIS and ISIS in stratum corneum.     

The long periodicity phase (LPP) controversy part I: The influence of a natural-like ratio of the CER[EOS] analogue [EOS]-br in a CER[NP]/[AP] based stratum corneum modelling system: A neutron diffraction study

This study used neutron diffraction to investigate a ceramide-[NP] C24/[AP] C24 /[EOS]-br C30/cholesterol/lignoceric acid (0.6: 0.3: 0.1: 0.7: 1) based stratum corneum modelling system. By adding specifically deuterated ceramides-[NP]-D₃, [AP]-D₃, and [EOS]-br-D₃, detailed information on the lamellar and the nanostructure of the system was obtained. For the short periodicity phase a natural-like lamellar repeat distance of 5.47?±?0.02?nm was observed, similar to the [NP]/[AP] base system without the [EOS]-br. Unlike in this system the ceramides here were slightly tilted, hinting towards a slightly less natural arrangement. Due to the deuteration it was possible to observe that the long ceramide chains were overlapping in the lamellar mid-plane. This is considered to be an important feature for the natural stratum corneum. Despite the presence of a ceramide [EOS] analogue – able to form a long phase arrangement – no distinct long periodicity phase was formed, despite a slightly higher than natural ω-acyl ceramide ratio of 10?mol%. The deuterated variant of this ceramide determined that the very long ceramide was integrated into the short periodicity phase, spanning multiple layers instead. The – compared to the base system – unchanged repeat distance highlights the stability of this structure. Furthermore, the localisation of the very long ceramide in the short periodicity phase indicates the possibility of a crosslinking effect and thus a multilayer stabilizing role for the ceramide [EOS]. It can be concluded, that additionally to the mere presence of ceramide-[EOS] more complex conditions have to be met in order to form this long phase. This has to be further investigated in the future.     

Effect of the ω-acylceramides on the lipid organization of stratum corneum model membranes evaluated by X-ray diffraction and FTIR studies (Part I)

The lipid organization in the outermost layer of the skin, the stratum corneum, is important for the skin barrier function. The stratum corneum lipids are composed of ceramides (CER), free fatty acids (FFA) and cholesterol (CHOL). In the present study Fourier transform infrared (FTIR) and small-angle X-ray scattering (SAXS) techniques were utilized to evaluate the effect of three C18 fatty acid esterified ω-acylceramides (CER EOS) on the lipid organization of stratum corneum model membranes. FTIR spectra (scissoring and rocking bands) showed as a function of temperature significant line-shape changes for both components assigned to the orthorhombic phase. Second-derivative analyzes revealed a significant decrease in the interchain coupling strength (Δν values) for the samples formed by CER EOS with the linoleate (CER EOS-L) and oleate (CER EOS-O) moiety around 28.5 °C. However, only a gradual decrease in the Δν values was noticed for the mixture formed with CER EOS with the stearate moiety (CER EOS-S) over the whole temperature range. In the absence of CER EOS the decrease started already at 25.5 °C, demonstrating that CER EOS stabilized the orthorhombic lattice. This stabilization was most pronounced for the CER EOS-S. Spectral fittings allowed to evaluate the orientation changes of the skeletal plane within the orthorhombic unit cell (θ values) for a given temperature range. From the best-fit parameters (peak area values), a decrease in the orthorhombic phase contribution to the scissoring band was also monitored as a function of the temperature. SAXS studies showed the coexistence of two lamellar phases with a periodicity of ∼5.5 nm (short periodicity phase, SPP) and ∼12 nm (LPP) in the presence of the CER EOS-L and CER EOS-O. However, no diffraction peaks associated to the LPP were detected for CER EOS-S. While CER EOS-S most efficiently stabilized the orthorhombic phase, CER EOS-L and CER EOS-O promoted the presence of the LPP. Therefore, the presence of all three CER EOS as observed in human stratum corneum may contribute to a proper skin barrier function.     

Hydration properties of N-(alpha-hydroxyacyl)-sphingosine: X-ray powder diffraction and FT-Raman spectroscopic studies

The thermotropic properties of N-(alpha-hydroxyacyl)-sphingosine (CER[AS]) in dry and hydrated state were studied by means of X-ray powder diffraction and FT-Raman spectroscopy. The polymorphic states of the CER[AS]/water mixture (lamellar crystalline, lamellar hexagonal gel, liquid crystalline) depend on the thermal pre-treatment of the sample. Only by heating the CER[AS]/water mixture above the melting chain transition can the system be hydrated. At room temperature, both dry and hydrated states form lamellar structures, which differ in their repeat distance and packing of hydrocarbon chains. Above the melting chain transition, hydrated CER[AS] forms a liquid crystalline hexagonal phase, whereas anhydrous CER[AS] forms an isotropic liquid phase. The various phases of hydrated CER[AS] are distinguished on the basis of the corresponding Raman spectra.