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.     

Neutron diffraction studies of oral stratum corneum model lipid membranes

In this work we have investigated model lipid mixtures simulating a lipid component of oral stratum corneum (OSC). Neutron diffraction experiments on oriented samples have revealed that SM (bovine brain)/dipalmitoylphosphatidylethanolamine/dipalmitoylphosphatidylcholine (DPPE/DPPC) mixtures at molar ratios of 1/2/1 and 1/1/1 are one-phase membranes. The incorporation of low concentrations of ceramide 6 and cholesterol into SM/DPPC/DPPE bilayers does not result in a phase separation, affecting membrane hydration. The model OSC membrane composed of ceramide 6/cholesterol/fatty acids/cholesterol sulfate/SM (bovine brain)/DPPE/DPPC is characterized by coexistence of several lamellar phases, that behave differently during their hydration in water excess. The phase with lamellar repeat distance of about 45 Å is likely a ceramide-rich phase and shows a restricted swelling in water, while another phase with repeat distance of 50 Å swells very quickly on 15 Å and then disappears. Our results indicate that phospholipid-rich and ceramide-rich domains could possibly coexist in the intercellular space of oral epithelium.     

Effects of sphingomyelin/ceramide ratio on the permeability and microstructure of model stratum corneum lipid membranes

The conversion of sphingomyelin (SM) to a ceramide (Cer) by acid sphingomyelinase (aSMase) is an important event in skin barrier development. A deficiency in aSMase in diseases such as Niemann–Pick disease and atopic dermatitis coincides with impaired skin barrier recovery after disruption. We studied how an increased SM/Cer ratio influences the barrier function and microstructure of model stratum corneum (SC) lipid membranes. In the membranes composed of isolated human SC Cer (hCer)/cholesterol/free fatty acids/cholesteryl sulfate, partial or full replacement of hCer by SM increased water loss. Partial replacement of 25% and 50% of hCer by SM also increased the membrane permeability to theophylline and alternating electric current, while a higher SM content either did not alter or even decreased the membrane permeability. In contrast, in a simple membrane model with only one type of Cer (nonhydroxyacyl sphingosine, CerNS), an increased SM/Cer ratio provided a similar or better barrier against the permeation of various markers. X-ray powder diffraction revealed that the replacement of hCer by SM interferes with the formation of the long periodicity lamellar phase with a repeat distance of d = 12.7 nm. Our results suggest that SM-to-Cer processing in the human epidermis is essential for preventing excessive water loss, while the permeability barrier to exogenous compounds is less sensitive to the presence of sphingomyelin.     

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].     

Effect of galactosylceramide on stratum corneum intercellular lipid synthesis in a three-dimensional cultured human epidermis model

Intercellular lipids in the stratum corneum (SC), such as ceramide (CER), free fatty acid (FFA), and cholesterol (CHOL), contribute to the formation of stable lamellar structures in the SC, making them important for skin barrier function. β-Galactosylceramide (GalCer) is a glycosphingolipid that is used in some cosmetics and quasi-drugs in anticipation of a moisturizing effect. GalCer promotes keratinocyte differentiation and increases CER production by increasing β-glucocerebrosidase (β-GCase) activity. However, few reports have described the mechanism of these effects, and detailed studies on the role of GalCer in intercellular lipid production in the SC have not been conducted. This study investigated the effect of GalCer on the metabolism and production of intercellular lipids in the SC in a three-dimensional cultured epidermis model. After reacting GalCer with a homogenate solution of three-dimensional cultured epidermis, GalCer was hardly metabolized. Treatment of the three-dimensional cultured epidermis with GalCer increased the expression of genes involved in the β-GCase metabolic pathway and promoted CER production. In addition, GalCer treatment reduced the expression of FFA metabolism-related genes as well as palmitic acid levels. In addition, transepidermal water loss, which is a barrier index, was reduced by GalCer treatment. These findings suggested that GalCer, which is hardly metabolized, affects the production of intercellular lipids in the SC and improves skin barrier function.     

Effect of dimethyl sulfoxide on the phase behavior of model stratum corneum lipid mixtures

Dimethyl sulfoxide (DMSO), an efficient transdermal enhancer, is proposed to alter the skin barrier by, at least partially, disturbing the lipid phase of the stratum corneum (SC). We have investigated, using differential scanning calorimetry and vibrational microspectroscopy, the effect of DMSO on the phase behavior of a lipid mixture formed by N-palmitoyl-d-erythro-sphingosine, deuterated palmitic acid, and cholesterol, mimicking the SC lipid phase. Our results reveal that DMSO favors the disordering of the lipid acyl chains. Moreover, the effect of DMSO is strongly concentration dependent and this dependence is reminiscent of that describing the DMSO transdermal enhancement. DMSO-induced fluidification affects primarily the fatty acid in the mixture. Therefore, it is proposed that the molecular mechanism of the transdermal transport enhancement caused by DMSO is associated with its H-bonding properties; its presence alters the interfacial H-bond network involving the fatty acid molecules and consequently the cohesive lipid packing.     

Evidence of free fatty acid interdigitation in stratum corneum model membranes based on ceramide [AP] by deuterium labelling

This research paper provides direct evidence concerning the localisation of free fatty acids in stratum corneum lipid model membranes. We employed partially deuterated free fatty acids to gain further information about the assembly of a stratum corneum lipid model membrane based on a ceramide of the phytosphingosine-type (ceramide [AP]) with particular respect to the position of the deuterated groups of the free fatty acids. The application of behenic-22,22,22-d₃-acid and cerotic-12,12,13,13-d₄-acid confirmed that the short-chain ceramide [AP] forces the longer-chained free fatty acids to incorporate into the bilayer created by ceramide [AP]. The ceramide [AP] molecules determine the structural assembly of this model membrane and obligate the long-chain free fatty acids to either arrange inside this formation or to separate as a fatty acid rich phase.     

The relationship between water loss,mechanical stress,and molecular structure of human stratum corneum ex vivo

Proper hydration of the stratum corneum (SC) is important for maintaining skin's vital functions. Water loss causes development of drying stresses, which can be perceived as ‘tightness’, and plays an important role in dry skin damage processes. However, molecular structure modifications arising from water loss and the subsequent development of stress has not been established. We investigated the drying stress mechanism by studying, ex vivo, the behaviors of the SC components during water desorption from initially fully hydrated samples using Raman spectroscopy. Simultaneously, we measure the SC mechanical stress with a substrate curvature instrument. Very good correlations of water loss to the mechanical stress of the stratum corneum were obtained, and the latter was found to depend mainly on the unbound water fraction. In addition to that, the water loss is accompanied with an increase of lipids matrix compactness characterized by lower chain freedom, while protein structure showed an increase in amount of α‐helices, a decline in α‐sheets, and an increase in folding in the tertiary structure of keratin. The drying process of SC involves a complex interplay of water binding, molecular modifications, and mechanical stress. This article provides a better understanding of the molecular mechanism associated to SC mechanics. (© 2015 WILEY‐VCH Verlag GmbH &Co. KGaA, Weinheim)     

Transition processes in stratum corneum model lipid membranes with a mixture of free fatty acids

The hydration of model membranes based on ceramide 6 with a mixture of free fatty acids most commonly encountered in the native lipid matrix of stratum corneum, the outermost layer of the mammalian skin, has been studied by neutron diffraction. Membrane hydration with water vapor at a temperature of 25°C is characterized by a small increase in the repeat distance Δd ₀ = 1.0 Å, which is comparable with membrane swelling in the presence of excess water. The kinetics of changes in the repeat distance, connected with an increase of the water layer between bilayers during hydration, and water exchange during the processes of hydration and H-D isotopic substitution, consists of a fast initial and a subsequent slow stage and is well described by exponentials with two characteristic times lying in the range from a few tens of minutes to several hundreds of minutes. During hydration at a temperature of 57°C, the repeat distance increases by Δd ₀ = 1.6 Å, after which the membrane irreversibly separates into two phases. One of the phases is formed mainly by long-chain free fatty acids and is characterized by a large decrease in the repeat distance Δd _ph = 8.3 Å on dehydration. The investigation of the structure of model membranes in the temperature range 20–72°C indicated that the system with 20% (w) of cholesterol in the range of 63–67°C undergoes a structural phase transition caused by the melting of hydrocarbon chains of lipids. In the system with a smaller content of cholesterol, no phase transition was observed up to a temperature of 72°C.     

LC/MS analysis of stratum corneum lipids: ceramide profiling and discovery

Ceramides (CERs) in the upper layer of the skin, the stratum corneum (SC), play a key role in the skin barrier function. In human SC, the literature currently reports 11 CER subclasses that have been identified. In this paper, a novel quick and robust LC/MS method is presented that allows the separation and analysis of all known human SC CER subclasses using only limited sample preparation. Besides all 11 known and identified subclasses, a 3D multi-mass chromatogram shows the presence of other lipid subclasses. Using LC/MS/MS with an ion trap (IT) system, a Fourier transform-ion cyclotron resonance system, and a triple quadrupole system, we were able to identify one of these lipid subclasses as a new CER subclass: the ester-linked ω-hydroxy fatty acid with a dihydrosphingosine base (CER [EOdS]). Besides the identification of a new CER subclass, this paper also describes the applicability and robustness of the developed LC/MS method by analyzing three (biological) SC samples: SC from human dermatomed skin, human SC obtained by tape stripping, and SC from full-thickness skin explants. All three biological samples showed all known CER subclasses and slight differences were observed in CER profile.     

Micron-scale assessment of molecular lipid organization in human stratum corneum using microprobe X-ray diffraction

Lipid and protein components of the stratum corneum (SC) are organized in complex supramolecular arrangements. Exploring spatial relations between various possible substructures is important for understanding the barrier function of this uppermost layer of epidermis. Here, we report the first study where micro-focus X-ray scattering was used for assessing fine structural variations of the human skin barrier with micrometer resolution. We found that the scattering profiles were unchanged when scanning in the direction parallel to the SC surface. Furthermore, small-angle scattering profiles did not change as a function of depth in the SC, confirming that the lipid lamellar spacings remained the same throughout the SC. However, the wide-angle scattering data showed that the orthorhombic phase was more abundant in the middle layers of the SC, whereas the hexagonal phase dominated in the surface layers both at the external and the lowest part of the SC; i.e., the lipids were most tightly packed in the middle region of the SC. Taken together, our results demonstrate that microprobe X-ray diffraction provides abundant information about spatial variations of the SC lipid structure and thus may be a promising tool for assessing the effects of topical formulations on the barrier function of skin.     

Investigation of stratum corneum lipid model membranes with free fatty acid composition by neutron diffraction

The outermost epidermal layer, the stratum corneum (SC), is the main skin barrier. Studies of SC model systems enable characterization of the influence of individual lipids on the organization of the SC lipid matrix, which is the main pathway of water through the skin. This work presents a neutron diffraction study of the SC model membranes based on short-chain ceramide 6 with nearly realistic composition of free fatty acids (FFA) at physiological temperature of the SC. The influence of FFA and the effect of cholesterol–cholesterol sulfate substitution on the structure and hydration of the SC model membranes are described. The structure of the SC membrane with FFA is close to the structure of the earlier studied SC membrane based on short-chain palmitic acid (PA) and does not vary significantly under changes of the ratio of the main membrane components. FFA accelerates membrane swelling at the same low level of hydration of both PA- and FFA-containing membranes. The substitution of cholesterol sulfate by cholesterol in the membrane composition decreases membrane swelling and leads to phase separation in the model system.     

Comprehensive quantification of ceramide species in human stratum corneum

One of the key challenges in lipidomics is to quantify lipidomes of interest, as it is practically impossible to collect all authentic materials covering the targeted lipidomes. For diverse ceramides (CER) in human stratum corneum (SC) that play important physicochemical roles in the skin, we developed a novel method for quantification of the overall CER species by improving our previously reported profiling technique using normal-phase liquid chromatog­raphy-electrospray ionization-mass spectrometry (NPLC-ESI-MS). The use of simultaneous selected ion monitoring measurement of as many as 182 kinds of molecular-related ions enables the highly sensitive detection of the overall CER species, as they can be analyzed in only one SC-stripped tape as small as 5 mm × 10 mm. To comprehensively quantify CERs, including those not available as authentic species, we designed a procedure to estimate their levels using relative responses of representative authentic species covering the species targeted, considering the systematic error based on intra-/inter-day analyses. The CER levels obtained by this method were comparable to those determined by conventional thin-layer chromatography (TLC), which guarantees the validity of this method. This method opens lipidomics approaches for CERs in the SC.     

The phase behavior of lipids in photosynthetic membranes

The phase behavior of the main classes of polar lipids found in the photosynthetic membranes of higher plants and algae is reviewed and compared to that of binary lipid mixtures and total lipid extracts of such membranes. Particular interest is paid to the way in which factors such as temperature and acyl chain saturation influence the phase behavior of these lipids and the implications this has in terms of the ability of photosynthetic membranes to resist environmental stress.     

Study of human stratum corneum and extracted lipids by thermomicroscopy and DSC

A study on the thermal behavior of human stratum corneum and lipids is described. The use of high scanning rate DSC for both SC and extracted lipids allows the consistent determination of transition temperatures, including those of lower energy. Changes are found both at physiological and higher temperatures. There is a clear correspondence between the thermotropic behavior of these two systems. However, one of the transitions found in human SC (approximately 55 degrees C) is absent in extracted lipids and may be ascribed to those covalently-linked to corneocytes. Lipidic thermotropic behavior is clearly found above 100 degrees C, in which proteins do not play an exclusive role. Changes related to most transitions are observed directly by polarized light thermal microscopy in extracted lipids. This technique also allowed for the observation of large segregated domains in the extracted lipids. A drastic change is observed at approximately 60 degrees C, corresponding to the disruption of the lamellar structure.     

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.     

Arrangement of ceramide [EOS] in a stratum corneum lipid model matrix: new aspects revealed by neutron diffraction studies

The lipid matrix in stratum corneum (SC) plays a key role in the barrier function of the mammalian skin. The major lipids are ceramides (CER), cholesterol (CHOL) and free fatty acids (FFA). Especially the unique-structured omega-acylceramide CER[EOS] is regarded to be essential for skin barrier properties by inducing the formation of a long-periodicity phase of 130 angstroms (LPP). In the present study, the arrangement of CER[EOS], either mixed with CER[AP] and CHOL or with CER[AP], CHOL and palmitic acid (PA), inside a SC lipid model membrane has been studied for the first time by neutron diffraction. For a mixed CER[EOS]/CER[AP]/CHOL membrane in a partly dehydrated state, the internal membrane nanostructure, i.e. the neutron scattering length density profile in the direction normal to the surface, was obtained by Fourier synthesis from the experimental diffraction patterns. The membrane repeat distance is equal to that of the formerly used SC lipid model system composed of CER[AP]/CHOL/PA/ChS. By comparing both the neutron scattering length density profiles, a possible arrangement of synthetic long-chain CER[EOS] molecules inside a SC lipid model matrix is suggested. The analysis of the internal membrane nanostructure implies that one CER[EOS] molecule penetrates from one membrane layer into an adjacent layer. A 130 angstroms periodicity phase could not be observed under experimental conditions, either in CER/CHOL mixtures or in CER/CHOL/FFA mixture. CER[EOS] can be arranged inside a phase with a repeat unit of 45.2 angstroms which is predominately formed by short-chain CER[AP] with distinct polarity.