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• W2110976514 abstract "Lipid lamellae present in the outermost layer of the skin protect the body from uncontrolled water loss. In human stratum corneum (SC), two crystalline lamellar phases are present, which contain mostly cholesterol, free fatty acids, and nine types of free ceramides. Previous studies have demonstrated that the SC lipid organization can be mimicked with model mixtures based on isolated SC lipids. However, those studies are hampered by low availability and high interindividual variability of the native tissue. To elucidate the role of each lipid class in the formation of a competent skin barrier, the use of synthetic lipids would offer an alternative. The small- and wide-angle X-ray diffraction results of the present study show for the first time that synthetic lipid mixtures, containing only three synthetic ceramides, reflect to a high extent the SC lipid organization. Both an appropriately chosen preparation method and lipid composition promote the formation of two characteristic lamellar phases with repeat distances similar to those found in native SC.From all synthetic lipid mixtures examined, equimolar mixtures of cholesterol, ceramides, and free fatty acids equilibrated at 80°C resemble to the highest extent the lamellar and lateral SC lipid organization, both at room and increased temperatures. Lipid lamellae present in the outermost layer of the skin protect the body from uncontrolled water loss. In human stratum corneum (SC), two crystalline lamellar phases are present, which contain mostly cholesterol, free fatty acids, and nine types of free ceramides. Previous studies have demonstrated that the SC lipid organization can be mimicked with model mixtures based on isolated SC lipids. However, those studies are hampered by low availability and high interindividual variability of the native tissue. To elucidate the role of each lipid class in the formation of a competent skin barrier, the use of synthetic lipids would offer an alternative. The small- and wide-angle X-ray diffraction results of the present study show for the first time that synthetic lipid mixtures, containing only three synthetic ceramides, reflect to a high extent the SC lipid organization. Both an appropriately chosen preparation method and lipid composition promote the formation of two characteristic lamellar phases with repeat distances similar to those found in native SC. From all synthetic lipid mixtures examined, equimolar mixtures of cholesterol, ceramides, and free fatty acids equilibrated at 80°C resemble to the highest extent the lamellar and lateral SC lipid organization, both at room and increased temperatures. One of the most important functions of the skin is to serve as a barrier to protect the body against uncontrolled water loss and to prevent the penetration of harmful agents. The protective function of the skin is provided primarily by the stratum corneum (SC), the outermost layer of the skin. SC has a unique morphology, in which keratin-filled corneocytes are surrounded by multilamellar lipid regions (1Elias P.M. Epidermal lipids, barrier function, and desquamation.J. Invest. Dermatol. 1983; 80: 44-49Google Scholar). The highly ordered intercellular lipid matrix is considered to play a crucial role in the maintenance of the barrier properties of the skin. Therefore, knowledge of the composition and organization of the SC lipids is essential to increase our insight into the skin barrier function. The composition of the SC lipids differs from that of biological membranes, because phospholipids are nearly absent. Ceramides (CER) belong to the major lipid species in the SC. Together with cholesterol (CHOL) and long-chain free fatty acids (FFA), they form the highly ordered intercellular lipid lamellae. At least nine different free CER types have been identified in human SC (2Ponec M. Weerheim A. Lankhorst P. Wertz P. New acylceramide in native and reconstructed epidermis.J. Invest. Dermatol. 2003; 120: 581-588Google Scholar, 3Robson K.J. Stewart M.E. Michelsen S. Lazo N.D. Downing D.T. 6-Hydroxy-4-sphingenine in human epidermal ceramides.J. Lipid Res. 1994; 35: 2060-2068Google Scholar, 4Stewart M.E. Downing D.T. A new 6-hydroxy-4-sphingenine-containing ceramide in human skin.J. Lipid Res. 1999; 4: 1434-1439Google Scholar, 5Wertz P.W. Miethke M.C. Long S.A. Strauss J.S. Downing D.T. The composition of the ceramides from human stratum corneum and from comedones.J. Invest. Dermatol. 1985; 84: 410-412Google Scholar), which are classified as CER1 to CER9. The CER are composed of a sphingosine (S), a phytosphingosine (P), or a 6-hydroxysphingosine (H) base to which a nonhydroxy (N) or α-hydroxy (A) fatty acid is chemically linked. The molecular structures of the CER, together with the two nomenclatures, are illustrated in Fig. 1A. CER1 (EOS), CER4 (EOH), and CER9 (EOP) have a unique molecular structure in that they contain linoleic acid bound to an ω-hydroxy fatty acid (EO) with a chain length of ∼30–32 carbon atoms. The lipids in the SC are organized in two coexisting crystalline lamellar phases: the short periodicity phase (SPP), with a repeat distance of ∼6 nm, and the long periodicity phase (LPP), with a periodicity of ∼13 nm (6Bouwstra J.A. Gooris G.S. van der Spek J.A. Bras W. The structure of human stratum corneum as determined by small angle X-ray scattering.J. Invest. Dermatol. 1991; 96: 1006-1014Google Scholar, 7Bouwstra J.A. Gooris G.S. van der Spek J.A. Bras W. Structural investigations of human stratum corneum by small angle X-ray scattering.J. Invest. Dermatol. 1991; 97: 1005-1012Abstract Full Text PDF Google Scholar, 8Bouwstra J.A. Gooris G.S. Bras W. Downing D.T. Lipid organization in pig stratum corneum.J. Lipid Res. 1995; 36: 685-695Google Scholar). Both the molecular organization of the LPP and the predominantly crystalline nature of its lipid packing in the presence of substantial amounts of CHOL are unique and are, therefore, suggested to be crucial for the barrier function of the skin. To elucidate the role of each lipid class in the formation of a competent skin barrier, the phase behavior of the SC lipids has been studied extensively. Using small- and wide-angle X-ray diffraction, it has been demonstrated that mixtures of CHOL, FFA, and CER isolated from either pig or human SC (natCER) closely mimic the lipid organization found in the SC. The results further reveal that in particular, CHOL and CER play a key role in the formation of the LPP, whereas FFA are required to induce the orthorhombic packing of the lipids (9Bouwstra J.A. Gooris G.S. Cheng K. Weerheim A. Bras W. Ponec M. Phase behavior of isolated skin lipids.J. Lipid Res. 1996; 37: 999-1011Google Scholar, 10Bouwstra J.A. Gooris G.S. Dubbelaar F.E.R. Weerheim A.M. Ponec M. pH, cholesterol sulfate, and fatty acids affect the stratum corneum lipid organization.J. Invest. Dermatol. 1998; 3: 69-73Google Scholar, 11Bouwstra J.A. Gooris G.S. Dubbelaar F.E.R. Ponec M. Phase behavior of stratum corneum lipid mixtures based on human ceramides: the role of natural and synthetic ceramide 1.J. Invest. Dermatol. 2002; 118: 606-617Google Scholar). Extensive studies with lipids isolated from the SC are hampered by the low availability and interindividual variability of the native tissue. In addition, the isolation and separation of the CER from the SC is very labor intensive. Therefore, the use of synthetic CER (synthCER) can offer an attractive alternative. Moreover, each subclass of natCER shows a variation in acyl chain length (2Ponec M. Weerheim A. Lankhorst P. Wertz P. New acylceramide in native and reconstructed epidermis.J. Invest. Dermatol. 2003; 120: 581-588Google Scholar), whereas synthCER have a well-defined acyl chain length. Thus, synthCER also enable us to study in detail the influence of acyl chain length on the SC lipid phase behavior. In recent years, several studies have been performed with model mixtures based on synthCER. The most frequently studied mixtures contain CHOL and/or palmitic acid (PA) and the commercially available bovine brain CER type III or type IV (12Fenske D.B. Thewalt J.L. Bloom M. Kitson N. Models of stratum corneum intercellular membranes: 2H NMR of microscopically oriented multilayers.Biophys. J. 1994; 67: 1562-1573Google Scholar, 13Kitson N. Thewalt J. Lafleur M. Bloom M. A model membrane approach to the epidermal permeability barrier.Biochemistry. 1994; 33: 6707-6715Google Scholar, 14Moore D.J. Rerek M.E. Mendelsohn R. Lipid domains and orthorhombic phases in model stratum corneum: evidence from Fourier transform infrared spectroscopy studies.Biochem. Biophys. Res. Commun. 1997; 231: 797-801Google Scholar, 15Neubert R. Rettig W. Wartewig S. Wegener M. Wienhold A. Structure of stratum corneum lipids characterized by FT-Raman spectroscopy and DSC. II. Mixtures of ceramides and saturated fatty acids.Chem. Phys. Lipids. 1997; 89: 3-14Google Scholar, 16Percot A. Lafleur M. Direct observations of domains in model stratum corneum lipid mixtures by Raman microspectroscopy.Biophys. J. 2001; 81: 2144-2153Google Scholar, 17Wegener M. Neubert R. Rettig W. Wartewig S. Structure of stratum corneum lipids characterized by FT-Raman spectroscopy and DSC. III. Mixtures of ceramides and cholesterol.Chem. Phys. Lipids. 1997; 88: 73-82Google Scholar). Other studies focused on mixtures prepared, for instance, with synthetic CER2, CER3, or CER5 (18Chen H. Mendelsohn R. Rerek M.E. Moore D.J. Fourier transform infrared spectroscopy and differential scanning calorimetry studies of fatty acid homogeneous ceramide 2.Biochim. Biophys. Acta. 2000; 1468: 293-303Google Scholar, 19Dahlen B. Pascher I. Molecular arrangements in sphingolipids. Thermotropic phase behaviour of tetracosanoylphytosphingosine.Chem. Phys. Lipids. 1979; 24: 119-133Google Scholar, 20Moore D.J. Rerek M.E. Insights into the molecular organisation of lipids in the skin barrier from infrared spectroscopy studies of stratum corneum lipid models.Acta Derm. Venereol. Suppl. 2000; 208: 16-22Google Scholar, 21Ohta N. Hatta I. Interaction among molecules in mixtures of ceramide/stearic acid, ceramide/cholesterol and ceramide/stearic acid/cholesterol.Chem. Phys. Lipids. 2002; 115: 93-105Google Scholar, 22Raudenkolb S. Hübner W. Rettig W. Wartewig S. Neubert R.H.H. Polymorphism of ceramide 3. Part 1. An investigation focused on the head group of N-octadecanoylphytosphingosine.Chem. Phys. Lipids. 2003; 123: 9-17Google Scholar). Using a variety of techniques, it has been determined that the packing of the lipids is mainly orthorhombic. However, the results demonstrate that the lipids are not properly mixed in one lattice but coexist in various phases, enriched in one of the components of the lipid mixture. Moreover, small-angle X-ray diffraction studies reveal that the characteristic LPP is not present in mixtures consisting of bovine brain CER type III, CHOL, and PA (23Bouwstra J.A. Thewalt J. Gooris G.S. Kitson N. A model membrane approach to the epidermal permeability barrier: an X-ray diffraction study.Biochemistry. 1997; 36: 7717-7725Google Scholar, 24McIntosh T.J. Stewart M.E. Downing D.T. X-ray diffraction analysis of isolated skin lipids: reconstitution of intercellular lipid domains.Biochemistry. 1996; 35: 3649-3653Google Scholar). In a recent study, we (25de Jager M.W. Gooris G.S. Dolbnya I.P. Bras W. Ponec M. Bouwstra J.A. The phase behaviour of lipid mixtures based on synthetic ceramides.Chem. Phys. Lipids. 2003; 124: 123-134Google Scholar) demonstrated that lamellar phases are formed in mixtures prepared with bovine brain CER type III or type IV. However, no LPP could be detected. In mixtures prepared with synthetic CER3 with an acyl chain length of 24 or 16 carbon atoms, several coexisting phases are present, including crystalline V-shaped CER structures. These V-shaped structures are different from the lipid organization observed in SC and therefore cannot be considered as representative for SC. In that study, we also studied the effect of synthetic CER1 on the phase behavior of synthetic skin lipid mixtures. From all of the mixtures examined, only one mixture, containing synthetic CER1 and CER3, CHOL, and FFA, showed phase behavior similar to that of SC. However, the repeat distance of the LPP was slightly shorter than that observed in SC. In the absence of CER1, no LPP was formed. This behavior is similar to that observed with mixtures prepared with isolated CER (11Bouwstra J.A. Gooris G.S. Dubbelaar F.E.R. Ponec M. Phase behavior of stratum corneum lipid mixtures based on human ceramides: the role of natural and synthetic ceramide 1.J. Invest. Dermatol. 2002; 118: 606-617Google Scholar, 24McIntosh T.J. Stewart M.E. Downing D.T. X-ray diffraction analysis of isolated skin lipids: reconstitution of intercellular lipid domains.Biochemistry. 1996; 35: 3649-3653Google Scholar, 26Bouwstra J.A. Gooris G.S. Dubbelaar F.E.R. Weerheim A.M. Ijzerman A.P. Ponec M. Role of ceramide 1 in the molecular organization of the stratum corneum lipids.J. Lipid Res. 1998; 39: 186-196Google Scholar). The objective of the present study is to generate a lipid mixture containing synthCER that closely mimics the natural SC lipid phase behavior. In a previous study performed with natCER, it became evident that a certain degree of fluidity of the lipid mixture is required for the formation of the LPP (11Bouwstra J.A. Gooris G.S. Dubbelaar F.E.R. Ponec M. Phase behavior of stratum corneum lipid mixtures based on human ceramides: the role of natural and synthetic ceramide 1.J. Invest. Dermatol. 2002; 118: 606-617Google Scholar). Because synthCER with uniform chain lengths form highly crystalline phases, it is reasonable to assume that increased lipid mobility and thus a possible enhancement of the formation of the LPP can be achieved by introducing variations in either acyl chain length or head group architecture. Therefore, in the present study, bovine brain CER type IV (referred to as ΣCERIV) was included in the lipid mixture. ΣCERIV consists of a sphingosine base linked to an α-hydroxy fatty acid with varying acyl chain lengths (Fig. 1B), in which C18 and C24 are the most abundantly present (M. W. de Jager, G. S. Gooris, M. Ponec et al., unpublished results). In addition, the preparation method was optimized to increase the degree of fluidity of the lipids to accomplish LPP formation. The present study shows that both a proper choice of the lipid composition and an optimal equilibration temperature during sample preparation are crucial for the formation of the LPP in mixtures based on synthCER. Palmitic acid, stearic acid, arachidic acid, behenic acid, docosatrienic acid, lignoceric acid, cerotic acid, CHOL, and ΣCERIV were purchased from Sigma-Aldrich Chemie GmbH (Schnelldorf, Germany). N-(30-Linoleoyloxy-triacontanoyl)-sphingosine [synthetic CER1 (C30)-linoleate] was a gift from Beiersdorf AG (Hamburg, Germany). N-Tetracosanoyl-phytosphingosine [synthetic CER3 (C24)] was generously provided by Cosmoferm B.V. (Delft, The Netherlands). Figure 1B shows the CER used in this study. All organic solvents used were of analytical grade and manufactured by Labscan Ltd. (Dublin, Ireland). All samples were prepared with a CER mixture consisting of CER1, CER3, and ΣCERIV at a molar ratio of 1:7:2. The CER mixture was mixed with CHOL in equimolar ratio. For the preparation of the CHOL:CER:FFA mixtures, the fatty acids C16:0, C18:0, C20:0, C22:0, C22:3, C24:0, and C26:0 were mixed at molar ratios of 1.3, 3.3, 6.7, 41.7, 5.4, 36.8, and 4.7%, respectively. This composition is similar to that found in the native SC. Appropriate amounts of individual lipids dissolved in chloroform-methanol (2:1) were combined to yield mixtures of ∼1.5 mg total dry weight at the desired composition with a total lipid concentration of 7 mg/ml. A Camag Linomat IV was used to apply the lipid mixtures onto mica. This was done at a rate of 4.3 μl/min under a continuous nitrogen stream. The samples were equilibrated for 10 min at appropriate temperatures that varied between 60°C and 100°C and subsequently hydrated with an acetate buffer of pH 5.0. Finally, the samples were homogenized by 10 successive freeze-thaw cycles between −20°C and room temperature, during which the samples were stored under gaseous argon. All measurements were performed at the European Synchrotron Radiation Facility (Grenoble, France) using station BM26B (27Bras W. A SAXS/WAXS beamline at the ESRF and future experiments.J. Macromol. Sci. Phys. B. 1998; 37: 557-566Google Scholar). The X-ray wavelength and the sample-to-detector distance were 1.24 Å and 1.7 m, respectively. Diffraction data were collected on a two-dimensional multiwire gas-filled area detector. The spatial calibration of this detector was performed using silver behenate. The samples were mounted in a temperature-controlled sample holder with mica windows. Static diffraction patterns of the lipid mixtures were obtained at room temperature for a period of 10 min. The temperature-induced phase changes were investigated by collecting diffraction patterns while increasing the temperature of the sample from 25°C to 95°C at a rate of 2°C/min. Each successive diffraction curve was collected for a period of 1 min. All measurements were performed at least in duplicate. Small-angle X-ray diffraction (SAXD) provides information about the larger structural units in the sample, namely the repeat distance of a lamellar phase. The scattering intensity I (in arbitrary units) was measured as a function of the scattering vector q (in reciprocal nanometers). The latter is defined as q = (4πsinθ)/λ, where θ is the scattering angle and λ is the wavelength. From the positions of a series of equidistant peaks (qn), the periodicity, or d-spacing, of a lamellar phase was calculated using the equation qn = 2nπ/d, where n is the order number of the diffraction peak. Wide-angle X-ray diffraction (WAXD) provides information about the lateral packing of the lipids within the lamellae. WAXD data were collected on a microstrip gas chamber detector with an opening angle of 60° (28Dolbnya I.P. Alberda H. Hartjes F.G. Udo F. Bakker R.E. Konijnenburg M. Homan E. Cerjak I. Goedtkindt P. Bras W. A fast position sensitive MSGC detector at high count rate operation.Rev. Sci. Instrum. 2002; 73: 3754-3758Google Scholar). The sample-to-detector distance was 36 cm and the X-ray wavelength was 1.24 Å. The spatial calibration of the detector was performed with a silicon/CHOL mixture. The SAXD and WAXD data were collected simultaneously. Mixtures consisting of CHOL:[CER1:CER3:ΣCERIV] at a molar ratio of 1:[0.1:0.7:0.2] were equilibrated at different temperatures ranging from 60°C to 100°C. The effect of the equilibration temperature on the lipid phase behavior is summarized in Table 1. The corresponding diffraction patterns are described below.TABLE 1The effect of equilibration temperature on the formation of the phases in equimolar cholesterol:synthCER (CHOL:synthCER) mixturesTemperatureLPPSPPCER3CHOL°Cnm60—5.7 (1, 2)3.7 (1, 2)—3.35 (1, 2)7012.8 (2)5.7 (1)3.7 (1, 2)—3.35 (1, 2)8012.5 (1, 2, 3)a12.5 (1, 2, 3) means that the periodicity of the phase is 12.5 nm and interpretation is based on the first-, second-, and third-order reflections.5.7 (1)3.7 (1, 2)4.3 (1)3.35 (1, 2)9012.6 (1, 2, 3*The peak is present as a shoulder.)5.5 (1*The peak is present as a shoulder., 3*The peak is present as a shoulder.)3.7 (1, 2)4.3 (1, 2)3.35 (1, 2)9512.3 (1, 2, 3*The peak is present as a shoulder., 4, 7)5.3 (1, 2)3.7 (1, 2)4.3 (1, 2)3.35 (1, 2)10012.2 (1, 2, 3*The peak is present as a shoulder., 4, 6, 7)5.3 (1, 3)—4.3 (1, 2)3.35 (1, 2)CER, ceramide; LPP, long periodicity phase; SPP, short periodicity phase; synthCER, synthetic ceramides.* The peak is present as a shoulder.a 12.5 (1, 2, 3) means that the periodicity of the phase is 12.5 nm and interpretation is based on the first-, second-, and third-order reflections. Open table in a new tab CER, ceramide; LPP, long periodicity phase; SPP, short periodicity phase; synthCER, synthetic ceramides. Figure 2Ashows the diffraction pattern of a lipid mixture equilibrated at 100°C. A lamellar structure with a repeat distance of 12.2 nm (LPP) is indicated by the presence of six diffraction peaks (q = 0.52, 1.03, 1.54, 2.06, 3.08, and 3.58 nm−1). The reflections at 1.17 and 3.51 nm−1 correspond to the first- and third-order maxima of a lamellar phase with a periodicity of 5.3 nm (SPP). The two sharp peaks at 1.46 and 2.91 nm−1 indicate the presence of a 4.3 nm phase, ascribed to crystalline CER3 in a V-shaped morphology (19Dahlen B. Pascher I. Molecular arrangements in sphingolipids. Thermotropic phase behaviour of tetracosanoylphytosphingosine.Chem. Phys. Lipids. 1979; 24: 119-133Google Scholar, 22Raudenkolb S. Hübner W. Rettig W. Wartewig S. Neubert R.H.H. Polymorphism of ceramide 3. Part 1. An investigation focused on the head group of N-octadecanoylphytosphingosine.Chem. Phys. Lipids. 2003; 123: 9-17Google Scholar, 25de Jager M.W. Gooris G.S. Dolbnya I.P. Bras W. Ponec M. Bouwstra J.A. The phase behaviour of lipid mixtures based on synthetic ceramides.Chem. Phys. Lipids. 2003; 124: 123-134Google Scholar). The presence of crystalline CHOL in separate domains can be deduced from the reflections at 1.87 and 3.74 nm−1.Fig. 2The effect of equilibration temperature on the phase behavior of equimolar cholesterol:synthetic ceramides (CHOL:synthCER) mixtures. The inset shows a magnification of the reflections in the q range between 2 and 4 nm−1. The Arabic numbers indicate the diffraction orders of the long periodicity phase (LPP), whereas the Roman numbers indicate the diffraction orders of the short periodicity phase (SPP). The letter C refers to the two crystalline phases of CER3. The asterisks indicate the reflections of crystalline CHOL located at 1.87 and 3.74 nm−1. A: Diffraction patterns of mixtures equilibrated at 100°C and 90°C. The various orders of the LPP are located at q = 0.52 nm−1 (1Elias P.M. Epidermal lipids, barrier function, and desquamation.J. Invest. Dermatol. 1983; 80: 44-49Google Scholar), 1.03 nm−1 (2Ponec M. Weerheim A. Lankhorst P. Wertz P. New acylceramide in native and reconstructed epidermis.J. Invest. Dermatol. 2003; 120: 581-588Google Scholar), 1.54 nm−1 (3Robson K.J. Stewart M.E. Michelsen S. Lazo N.D. Downing D.T. 6-Hydroxy-4-sphingenine in human epidermal ceramides.J. Lipid Res. 1994; 35: 2060-2068Google Scholar), 2.06 nm−1 (4Stewart M.E. Downing D.T. A new 6-hydroxy-4-sphingenine-containing ceramide in human skin.J. Lipid Res. 1999; 4: 1434-1439Google Scholar), 3.08 nm−1 (6Bouwstra J.A. Gooris G.S. van der Spek J.A. Bras W. The structure of human stratum corneum as determined by small angle X-ray scattering.J. Invest. Dermatol. 1991; 96: 1006-1014Google Scholar), and 3.58 nm−1 (7Bouwstra J.A. Gooris G.S. van der Spek J.A. Bras W. Structural investigations of human stratum corneum by small angle X-ray scattering.J. Invest. Dermatol. 1991; 97: 1005-1012Abstract Full Text PDF Google Scholar). The various orders of the SPP are located at q = 1.17 nm−1 (I) and 3.51 nm−1 (III). The reflections at 1.46 and 2.91 nm−1 are attributed to crystalline CER3 (4.3 nm phase). B: Diffraction patterns of mixtures equilibrated at 80°C and 70°C. The various orders of the LPP are located at q = 0.49 nm−1 (1Elias P.M. Epidermal lipids, barrier function, and desquamation.J. Invest. Dermatol. 1983; 80: 44-49Google Scholar), 0.99 nm−1 (2Ponec M. Weerheim A. Lankhorst P. Wertz P. New acylceramide in native and reconstructed epidermis.J. Invest. Dermatol. 2003; 120: 581-588Google Scholar), and 1.48 nm−1 (3Robson K.J. Stewart M.E. Michelsen S. Lazo N.D. Downing D.T. 6-Hydroxy-4-sphingenine in human epidermal ceramides.J. Lipid Res. 1994; 35: 2060-2068Google Scholar). The only reflection attributed to the SPP is located at q = 1.09 nm−1 (I). The reflection at 1.46 nm−1 is attributed to crystalline CER3 (4.3 nm phase), and the reflections at 1.71 and 3.41 nm−1 are attributed to crystalline CER3 (3.7 nm phase).View Large Image Figure ViewerDownload (PPT) A reduction in equilibration temperature from 100°C to 95°C does not affect the formation of the LPP and SPP (data not shown). However, X-ray diffraction patterns of lipid mixtures equilibrated at 95°C reveal the presence of a new structure with a repeat distance of 3.7 nm, as suggested by two reflections at 1.69 and 3.40 nm−1. This phase can be assigned to another crystalline V-shaped structure of CER3 (19Dahlen B. Pascher I. Molecular arrangements in sphingolipids. Thermotropic phase behaviour of tetracosanoylphytosphingosine.Chem. Phys. Lipids. 1979; 24: 119-133Google Scholar, 22Raudenkolb S. Hübner W. Rettig W. Wartewig S. Neubert R.H.H. Polymorphism of ceramide 3. Part 1. An investigation focused on the head group of N-octadecanoylphytosphingosine.Chem. Phys. Lipids. 2003; 123: 9-17Google Scholar, 25de Jager M.W. Gooris G.S. Dolbnya I.P. Bras W. Ponec M. Bouwstra J.A. The phase behaviour of lipid mixtures based on synthetic ceramides.Chem. Phys. Lipids. 2003; 124: 123-134Google Scholar). The intensities of the peaks attributed to the 4.3 nm phase are slightly reduced compared with those observed in the samples that were equilibrated at 100°C. The repeat distance of the LPP increases to 12.6 nm when an equilibration temperature of 90°C is used during sample preparation (Fig. 2A). However, only three reflections can be observed that are attributed to this phase. The periodicity of the SPP has increased slightly to 5.5 nm, as deduced from the first- and third-order reflections. Both reflections show partial overlap with other peaks in the diffraction pattern. Crystalline CHOL and the two coexisting crystalline CER3 phases are also present in the lipid mixture. The diffraction pattern of the lipid mixture prepared using an equilibration temperature of 80°C is illustrated in Fig. 2B. It is evident that decreasing the equilibration temperature reduces the intensities of the three equidistant reflections attributed to the LPP compared with the intensities of the peaks attributed to the SPP (and to crystalline CER3 and CHOL). The repeat distance of the SPP has changed slightly to 5.7 nm. Additionally, crystalline CHOL and crystalline CER3 are present in the lipid mixture. The latter forms predominantly a 3.7 nm phase, although a weak reflection at 1.46 nm−1 indicates that a small fraction of CER3 might still be present as a 4.3 nm phase. A further reduction in the equilibration temperature to 70°C (Fig. 2B) results in a diffraction pattern at which the diffraction peaks attributed to the LPP almost disappear. Only a weak reflection at 0.99 nm−1 (second order) reveals that a small fraction of lipids might form a LPP with a periodicity of ∼12.8 nm. Compared with the diffraction pattern of the lipid mixture equilibrated at 80°C, no significant shift is observed in the positions of the reflections attributed to the SPP and crystalline CHOL. All phase-separated CER3 is present as a 3.7 nm phase, because the only two additional reflections observed in the diffraction pattern are located at 1.69 and 3.40 nm−1. Equilibration of the lipid mixture at 60°C (data not shown) does not result in the formation of the LPP. Instead, broad reflections (1.13 and 2.26 nm−1) indicate the presence of a SPP with a repeat distance of 5.7 nm. In addition, crystalline CHOL and crystalline CER3 (3.7 nm phase) can be detected. To investigate the effect of FFA on the formation of the lamellar phases, FFA were added to the above-mentioned CHOL:[CER1:CER3:ΣCERIV] mixture prepared at a molar ratio of 1:[0.1:0.7:0.2]. Based on the results obtained with the CHOL:synthCER mixtures, the initially chosen equilibration temperature was 95°C. However, equilibration of an equimolar CHOL:synthCER:FFA mixture at this temperature did not result in the formation of the LPP but in melting of the lipids (data not shown). Therefore, the equilibration temperature was reduced to 80°C. The effect of FFA on the phase behavior was examined with CHOL:synthCER:FFA mixtures in which the amount of FFA gradually increased to achieve molar ratios ranging from 1:1:0 to 1:1:1.8. The results are summarized in Table 2. The corresponding diffraction patterns are described below. First, the diffraction curve of the CHOL:synthCER:FFA mixture at a molar ratio of 1:1:1 is described (Fig. 3A). Subsequently, the effect of decreased and increased FFA content on the lipid phase behavior is presented. The equimolar CHOL:synthCER:FFA mixture shows the presence of a LPP with a repeat distance of 12.2 nm, of which the first five reflections can be detected. The SPP with a periodicity of 5.5 nm is characterized by the presence of the first three diffraction peaks. Crystalline CHOL and the two coexisting crystalline phases of CER3 with periodicities of 3.7 and 4.3 nm can also be detected in the lipid mixture.TABLE 2The effect of FFA content on the formation of the phases in the lipid mixturesCHOL:CER:FFALPPSPPCER3 (C24)CHOLnm1:1:012.5 (1*The peak is present as a shoulder., 2, 3)5.5 (1*The peak is present as a shoulder.)3.7 (1, 2)—3.35 (1, 2)1:1:0.2512.5 (1*The peak is present as a shoulder., 2, 3)5.5 (1*The peak is present as a shoulder.)3.7 (1, 2)—3.35 (1, 2)1:1:0.512.5 (1, 2, 4)a12.5 (1, 2, 4) means that the periodicity of the phase is 12.5 nm and interpretation is based on the first-, second-, and fourth-order reflections.5.5 (1*The peak is present as a shoulder., 2)3.7 (1, 2)4.3 (1, 2)3.35 (1, 2)1:1:0.7512.3 (1, 2, 3*The peak is present as a shoulder., 4)5.5 (1, 2, 3)3.7 (1, 2)4.3 (1, 2)3.35 (1, 2)1:1:112.2 (1, 2, 3*The peak is present as a shoulder., 4, 5)5.5 (1, 2, 3)3.7 (1, 2)4.3 (1, 2)3.35 (1, 2)1:1:1.412.0 (1, 2, 3*The peak is present as a shoulder., 4)5.4 (1, 2)3.7 (1, 2)4.3 (1, 2)3.35 (1, 2)1:1:1.812.0 (1*The peak is prese" @default.
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• W2110976514 date "2004-05-01" @default.
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• W2110976514 title "Novel lipid mixtures based on synthetic ceramides reproduce the unique stratum corneum lipid organization" @default.
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