Novel lipid mixtures based on synthetic ceramides reproduce the unique stratum corneum lipid organization

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 degrees C resemble to the highest extent the lamellar and lateral SC lipid organization, both at room and increased temperatures.     

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.     

Whole picture of human stratum corneum ceramides,including the chain-length diversity of long-chain bases

Ceramides are essential lipids for skin permeability barrier function, and a wide variety of ceramide species exist in the stratum corneum (SC). Although ceramides with long-chain bases (LCBs) of various lengths have been identified in the human SC, a quantitative analysis that distinguishes ceramide species with different LCB chain lengths has not been yet published. Therefore, the whole picture of human SC ceramides remains unclear. Here, we conducted LC/MS/MS analyses to detect individual ceramide species differing in both the LCB and FA chain lengths and quantified 1,327 unbound ceramides and 254 protein-bound ceramides: the largest number of ceramide species reported to date. Ceramides containing an LCB whose chain length was C16–26 were present in the human SC. Of these, C18 (28.6%) was the most abundant, followed by C20 (24.8%) and C22 (12.8%). Each ceramide class had a characteristic distribution of LCB chain lengths and was divided into five groups according to this distribution. There was almost no difference in FA composition between the ceramide species containing LCBs of different chain lengths. Furthermore, we demonstrated that one of the serine palmitoyltransferase (SPT) complexes, SPTLC1/SPTLC3/SPTSSB, was able to produce C16–24 LCBs. The expression levels of all subunits constituting the SPT complexes increased during keratinocyte differentiation, resulting in the observed chain-length diversity of LCBs in the human SC. This study provides a molecular basis for elucidating human SC ceramide diversity and the pathogenesis of skin disorders.     

Lipid mixtures prepared with well-defined synthetic ceramides closely mimic the unique stratum corneum lipid phase behavior

Lipid lamellae present in the outermost layer of the skin, the stratum corneum, form the main barrier for the diffusion of molecules through the skin. The presence of a unique 13 nm lamellar phase and its high crystallinity are characteristic for the stratum corneum lipid phase behavior. In the present study, small-angle and wide-angle X-ray diffraction were used to examine the organization in lipid mixtures prepared with a unique set of well-defined synthetic ceramides, varying from each other in head group architecture and acyl chain length. The results show that equimolar mixtures of cholesterol, free fatty acids, and synthetic ceramides (resembling the composition of pig ceramides) closely resemble the lamellar and lateral stratum corneum lipid organization, both at room and higher temperatures. Exclusion of several ceramide classes from the mixture does not affect the lipid organization. However, complete substitution of ceramide 1 (acylceramide with a sphingosine base) with ceramide 9 (acylceramide with a phytosphingosine base) reduces the formation of the long periodicity lamellar phase. This indicates that the head group architecture of acylceramides affects the lipid organization. In conclusion, lipid mixtures prepared with well-defined synthetic ceramides offer an attractive tool with which to unravel the importance of the molecular structure of individual ceramides for proper lipid organization.     

Hydration of stratum corneum

A model for the hydration behavior of human stratum corneum has been developed from measurements on in vitro samples isolated in a manner which minimized tissue treatment and trauma. Water sorption/desorption rate measurements as a function of water activity, temperature, and tissue integrity are reported. These data, together with thermodynamic data (infrared and nmr results reported earlier) are consistent with a model in which rapidly sorbed/desorbed water (ca. 0.5 mg water/mg stratum corneum) is associated with (“bound” by) the tissue, while slowly sorbed/desorbed “free” water (up to 12 mg water/mg stratum corneum) is kinetically restricted and probably intracellular in location. Both equilibrium water binding and desorption kinetic data suggest structural changes of this cellular water barrier upon hydration. Evidence for hysteresis in water sorption isotherms (reported by others) could not be reproduced.     

Separation and identification of ceramides derived from human plasma sphingomyelins

Sphingomyelins from human blood plasma have been converted into ceramides by enzymatic hydrolysis with phospholipase C. After acetylation the ceramides were fractionated by thin-layer chromatography on silica gel containing silver nitrate. Four main fractions obtained by this method were subsequently converted to di-O-trimethylsilyl ether derivatives and separated by gas-liquid chromatography on 1% OV-1. 2-11 components could be distinguished in each of the four fractions. The major fractions emerging from the gas chromatograph were analyzed by mass spectrometry and their main molecular species were identified. Two of the gas chromatographic fractions contained essentially pure molecular species, namely N-tetracosenoyl sphingosine and N-tetracosenoylsphinga-4, 14-dienine.     

Infrared spectroscopic study of stratum corneum model membranes prepared from human ceramides, cholesterol, and fatty acids

The outermost layer of the skin, the stratum corneum, consists of corneocytes surrounded by lipid domains. The main lipid classes in stratum corneum are cholesterol, ceramides (CER), and free fatty acids forming two crystalline lamellar phases. However, only limited information is available on whether the various lipid classes participate in the same crystalline lattices or if separate domains are formed within the lipid lamellae. In this article infrared spectroscopic studies are reported of hydrated mixtures prepared from cholesterol, human CER, and free fatty acids. Evaluation of the methylene stretching vibrations revealed a conformational disordering starting at approximately 60 degrees C for all mixtures. Examination of the rotational ordering (scissoring and rocking vibrations) of mixtures prepared from equimolar cholesterol and CER with a variation in the level of free fatty acids showed that at lower free fatty acid content orthorhombic and hexagonal domains coexist in the lipid lamellae. Increasing the fatty acid level to an equimolar cholesterol/CER/fatty acid mixture reveals the dominant presence of an orthorhombic lattice, confirming x-ray diffraction studies. Replacing the protonated free fatty acid chains by their perdeuterated counterparts demonstrates that free fatty acids and CER participate in the same orthorhombic lattice up to a level of slightly less than 1:1:0.75 cholesterol/CER/free fatty acids molar ratio but that free fatty acids also form separate domains within the lipid lamellae at equimolar ratios at room temperature. However, no evidence for this has been observed at 32 degrees C. Extrapolating these findings to the situation in stratum corneum led us conclude that in stratum corneum, fatty acids and CER participate in the orthorhombic lattice at 32 degrees C, the skin temperature.     

Influence of different ceramides on the structure of in vitro model lipid systems of the stratum corneum lipid matrix

Human stratum corneum (SC) consists of several layers of keratinized corneocytes embedded in a lipid matrix of ordered lamellar structure which is considered to constitute the major barrier to percutaneous penetration. Artificial mixtures of SC lipids are often used as model systems to mimic the skin barrier or to investigate the effects of substances on the phase behaviour of the models. In the present study a SC lipid model composed of cholesterol, fatty acids and ceramides was used to investigate the effect of three different commercially available ceramide types on the microstructure and the physicochemical behaviour of the lipids. Polarized light microscopy, transmission electron microscopy, small-angle X-ray diffraction, wide-angle X-ray diffraction and differential scanning calorimetry (DSC) were used for physicochemical characterization. The results revealed a lamellar structure for all models but showed differences with regard to the thermal and optical behaviour depending obviously on the composition of the ceramide mixtures. A model containing a mixture of Cer[AS] was comparable to human SC lipids.     

Modeling of composite sorption isotherm for stratum corneum

Equilibrium water sorption in stratum corneum (SC) is considered by treating it as a biocomposite with two main phases, namely, corneocytes and lipids. To validate the rule of mixtures for the individual phase sorption isotherms, a new flexible fitting model is introduced by accounting for characteristic features observed in the variations of the thermodynamic correction factors corresponding to the individual sorption isotherms. The comparison of the model fitting performance with that of the five-parameter Park's model shows a remarkably good ability to fit experimental data for different types of sorption isotherms. The effect of the lipids content on the variance of the composite sorption isotherm of stratum corneum is highlighted. The sensitivity analysis reveals that for the typical water content 20–30 wt%, which corresponds to the SC in a stable condition, the sensitivity of the composite sorption isotherm to the variation of the lipids content on dry basis is predominantly positive and sufficiently small. The good agreement observed between the experimental sorption isotherm for SC and the composite isotherm, which is based on the rule of mixtures for the individual phase sorption isotherms, yields a plausible conclusion (hypothesis) that the corneocytes–lipids mechanical interaction during unconstrained swelling of the SC membrane in the in vitro laboratory experiment is negligible.     

Heterogeneous drying stresses in stratum corneum

We study the drying of stratum corneum, the skin's outermost layer and an essential barrier to mechanical and chemical stresses from the environment. Even though stratum corneum exhibits structural features across multiple length-scales, contemporary understanding of the mechanical properties of stratum corneum is based on the assumption that its thickness and composition are homogeneous. We quantify spatially resolved in-plane traction stress and deformation at the interface between a macroscopic sample of porcine stratum corneum and an adherent deformable elastomer substrate. At length-scales greater than a millimeter, the skin behaves as a homogeneous elastic material. At this scale, a linear elastic model captures the spatial distribution of traction stresses and the dependence of drying behavior on the elastic modulus of the substrate. At smaller scales, the traction stresses are strikingly heterogeneous and dominated by the heterogeneous structure of the stratum corneum.     

Beta-glucocerebrosidase activity in mammalian stratum corneum

Although previous studies have demonstrated a crucial role for the enzyme beta-glucocerebrosidase (GlcCer'ase) in the final steps of membrane structural maturation in mammalian stratum cornuem (SC) and epidermal homeostasis, the precise in vivo localization of GlcCer'ase activity and protein is not known. Here, we developed a fluorogenic in situ assay on histologic sections (zymography) to elucidate the in vivo distribution of GlcCer'ase activity, and further characterized and localized the SC GlcCer'ase activity in vitro. The zymographic technique revealed higher GlcCer'ase activity in upper stratum granulosum and SC, both in murine and human SC; activity that was both inhibited by conduritol B epoxide, a specific GlcCer'ase inhibitor, and pH-dependent; i.e., present at pH 5.2, and absent or significantly reduced at neutral pH (7.4), consistent with the known pH optimum for epidermal GlcCer'ase in vitro. Immunohistochemical staining for GlcCer'ase protein showed enhanced fluorescent signal in the outer layers of human epidermis, concentrated at the apex and margins of stratum granulosum and lower SC. Moreover, in extracts from individual epidermal layers, GlcCer'ase activity was present throughout murine epidermis, with the highest activity in the SC, peaking in the lower-to-mid-SC. The SC activity was stimulated >10-fold by sodium taurocholate, and inhibited by bromoconduritol B epoxide. Finally, isolated membrane couplets, prepared from SC sheets, also demonstrated significant GlcCer'ase activity. These data localize GlcCer'ase activity to the outer epidermis by three different techniques, and support the role of this enzyme in extracellular processing of glucosylceramides to ceramides, required for permeability barrier maturation and function.     

Very long-chain polyunsaturated fatty acids are the major acyl groups of sphingomyelins and ceramides in the head of mammalian spermatozoa

Very long-chain (C24 to C34) polyunsaturated fatty acids (VLCPUFA) are important constituents of sphingomyelin (SM) and ceramide (Cer) in testicular germ cells. In the present paper we focused on the SM and Cer and their fatty acids in spermatozoa and their main regions, heads and tails. In bull and ram spermatozoa, SM was the third most abundant phospholipid and VLCPUFA were the major acyl groups ( approximately 70%) of SM and Cer. In rat epididymal spermatozoa the SM/Cer ratio was low in the absence of and could be maintained high in the presence of the cation chelator EDTA, added to the medium used for sperm isolation. This fact points to the occurrence of an active divalent cation-dependent sphingomyelinase. Bull and rat sperm had an uneven head-tail distribution of phospholipid, with virtually all the VLCPUFA-rich SM located at the head, the lower SM content in the rat being determined by the lower sperm head/tail size ratio. Most of the SM from bull sperm heads was readily solubilized with 1% Triton X-100 at 4 degrees C. The detergent-soluble SM fraction was richer in VLCPUFA than the nonsoluble fraction and richer in saturated fatty acids. Cer was produced at the expense of SM, thus decreasing severalfold the SM/Cer ratio in rat spermatozoa incubated for 2 h in presence of the sperm-capacitating agents, calcium, bicarbonate, and albumin. The generation of Cer from SM in the sperm head surface may be an early step among the biochemical and biophysical changes known to take place in the spermatozoon in the physiological events preceding fertilization.     

Restoration of stratum corneum with nacre lipids

To discover potential new products for the atopic dermatitis treatment, lipids extracted from nacre from the oyster Pinctada margaritifera were tested on artificially dehydrated skin explants. Expression of filaggrin and transglutaminase 1 was investigated after treatment of dehydrated skin with P. margaritifera lipid extracts according to light microscopy after labelling with specific monoclonal antibodies. The lipids were extracted from the nacre with methanol/chloroform mixture at room temperature and the extract composition was determined according to TLC and densitometry measures. Relative to the dry nacre material, a yield of extraction in lipids of 0.54% (w/w) was determined. Fatty acids, triglycerides, cholesterol and ceramides were in low abundance. Then, application of lipid formulations on skin explants previously dehydrated gave after 3 h an overexpression of filaggrin and a decrease of transglutaminase expression as shown by light microscopy. Using immunofluorescence labelling, we showed that lipids extracted from the mother of pearl of P. margaritifera induced a reconstitution of the intercellular cement of the stratum corneum. The signaling properties of the nacre lipids could be used for a development of new active product treatment against the symptoms of the dermatitis.     

Covalently bound omega-hydroxyacylsphingosine in the stratum corneum

Pig epidermis was heat separated, and the stratum corneum was isolated after trypsinization. Exhaustive extraction of the stratum corneum fraction with chloroform/methanol mixtures yielded 14.7% lipid on a dry weight basis. After mild saponification of the extracted residue, additional lipid could be extracted which accounted for 2.1% of the stratum corneum weight. This bound lipid proved to consist mainly (91.9%) of N-(omega-hydroxyacyl)sphingosines in which the amide-linked omega-hydroxyacids were 28 to 34 carbon atoms in length. The release of this lipid by mild alkaline hydrolysis indicates that it is bound through an ester linkage. Half of the hydroxyceramide molecules reacted in situ with acidic acetone, suggesting that half of these molecules are attached to the stratum corneum through the omega-hydroxyl function, while the other half may be linked through one of the hydroxyl groups of the sphingosine.     

Two new methods for preparing a unique stratum corneum substitute

Stratum corneum lipids play an important role in the barrier function of the skin. An in vitro permeation model consisting of synthetic lipids has previously been developed to replace human stratum corneum (SC) in permeation studies. This model is referred to as the stratum corneum substitute (SCS). In order to improve its reproducibility and to increase the efficiency in preparing the SCS, two new preparation methods are developed. Subsequently the properties of the SCS prepared by the various methods, i.e. the manual airbrush method, the rotor airbrush method and the linomat method, are investigated. The results show that the SCS prepared with the various methods share the properties of a uniform lipid composition and lipid distribution. Furthermore, irrespective of the preparation method, the lipids form crystalline lamellar phases, mimicking the lipid organization and orientation in human SC. As a result, permeation profiles of benzoic acid through SCS are very similar to human SC. The rotor method increases the efficiency and reproducibility of the manual airbrush method, while the linomat method reduces the lipid loss during preparation and results in SCS with a more uniform membrane thickness. In conclusion, the linomat method was chosen as the preferred method for preparing the substitute.     

The stratum corneum lipid thermotropic phase behavior.

The stratum corneum, the outermost layer of mammalian skin, is considered the least permeable skin layer to the diffusion of water and other solutes. It is generally accepted that the intercellular lipid multilayer domain is the diffusional pathway for most lipophilic solutes. Fluidization of the lipid multilayers is believed to result in the loss of barrier properties of the stratum corneum. Current investigations address the lipid thermotropic phase behavior in terms of lipid alkyl chain packing, mobility and conformational order as measured by Fourier transform infrared (FTIR) spectroscopy. A solid-solid phase transition is observed with increased alkyl chain mobility followed by a gel to liquid-crystalline phase transition near 65 degrees C. These results further elucidate the role of lipid fluidity that may contribute to the transport properties of the stratum corneum.