Ultrastructural organization of avian stratum corneum lipids as the basis for facultative cutaneous waterproofing

The ultrastructure of naked neck epidermis from the ostrich (Struthio camelus) and ventral apterium from watered, and water-deprived, Zebra finches (Taeniopygia [Poephila] guttata castanotis) is presented. The form and distribution of the fully differentiated products of the lipid-enriched multigranular bodies are compared in biopsies post-fixed with osmium tetroxide or ruthenium tetroxide. The fine structure of ostrich epidermis suggests it is a relatively poor barrier to cutaneous water loss (CWL). The fine structure from watered, and 16-hr water-deprived Zebra finches, considered in conjunction with measurements of CWL, confirms previous reports of “facultative waterproofing,” and emphasizes the rapidity of tissue response to dehydration. The seemingly counterintuitive facts that one xerophilic avian species, the ostrich, lacks a “good barrier” to CWL, whereas another, the Zebra finch, is capable of forming a good barrier, but does not always express this capability, are discussed. An explanation of these data in comparison to mammals centers on the dual roles of the integument of homeotherms in thermoregulation and conserving body water. It is concluded that birds, whose homeothermic control depends so much on CWL, cannot possess a permanent “good barrier,” as such would compromise the heat loss mechanism. Facultative waterproofing (also documented in lizards) protects the organism against sudden reductions in water availability. In birds, and probably in snakes and lizards, facultative waterproofing involves qualititative changes in epidermal cell differentiation. Possible control mechanisms are discussed. © 1996 Wiley-Liss, Inc.     

Stratum corneum hydration: Phase transformations and mobility in stratum corneum, extracted lipids and isolated corneocytes

The outermost layer of skin, stratum corneum (SC), functions as the major barrier to diffusion. SC has the architecture of dead keratin filled cells embedded in a lipid matrix. This work presents a detailed study of the hydration process in extracted SC lipids, isolated corneocytes and intact SC. Using isothermal sorption microcalorimetry and relaxation and wideline ¹H NMR, we study these systems at varying degrees of hydration/relative humidities (RH) at 25 °C. The basic findings are (i) there is a substantial swelling both of SC lipids, the corneocytes and the intact SC at high RH. At low RHs corneocytes take up more water than SC lipids do, while at high RHs swelling of SC lipids is more pronounced than that of corneocytes. (ii) Lipids in a fluid state are present in both extracted SC lipids and in the intact SC. (iii) The fraction of fluid lipids is lower at 1.4% water content than at 15% but remains virtually constant as the water content is further increased. (iv) Three exothermic phase transitions are detected in the SC lipids at RH = 91-94%, and we speculate that the lipid re-organization is responsible for the hydration-induced variations in SC permeability. (v) The hydration causes swelling in the corneocytes, while it does not affect the mobility of solid components (keratin filaments).     

Stratum corneum hydration: phase transformations and mobility in stratum corneum, extracted lipids and isolated corneocytes

The outermost layer of skin, stratum corneum (SC), functions as the major barrier to diffusion. SC has the architecture of dead keratin filled cells embedded in a lipid matrix. This work presents a detailed study of the hydration process in extracted SC lipids, isolated corneocytes and intact SC. Using isothermal sorption microcalorimetry and relaxation and wideline (1)H NMR, we study these systems at varying degrees of hydration/relative humidities (RH) at 25 degrees C. The basic findings are (i) there is a substantial swelling both of SC lipids, the corneocytes and the intact SC at high RH. At low RHs corneocytes take up more water than SC lipids do, while at high RHs swelling of SC lipids is more pronounced than that of corneocytes. (ii) Lipids in a fluid state are present in both extracted SC lipids and in the intact SC. (iii) The fraction of fluid lipids is lower at 1.4% water content than at 15% but remains virtually constant as the water content is further increased. (iv) Three exothermic phase transitions are detected in the SC lipids at RH=91-94%, and we speculate that the lipid re-organization is responsible for the hydration-induced variations in SC permeability. (v) The hydration causes swelling in the corneocytes, while it does not affect the mobility of solid components (keratin filaments).     

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.     

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.     

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.     

Evidence for the hydrolysis of topical applied liposomal lipids in human stratum corneum

Liposomes were incubated with an extract of human plantar stratum corneum. The liposomal lipids were hydrolysed, if composed of soy-bean phosphatidylcholine or phosphatidylglycerol. Rigid lipids were not degraded. The temperature optimum of the hydrolysis was between 30-35degC. CaCl enhanced, while EDTA reduced the rate of hydrolysis, indicating that the hydrolysis is due to a phospholipase A(2).     

Cutaneous water loss and lipids of the stratum corneum in two syntopic species of bats

The lipid matrix of the stratum corneum (SC), the outer layer of the epidermis of mammals and birds, constitutes the barrier to diffusion of water vapor through the skin. The lipids of the SC are structured in the intercellular spaces of the mammalian epidermis in ordered layers, called lamellae, which have been postulated to prevent water loss. Lipids in the mammalian SC are mainly cholesterol, free fatty acids and ceramides, the latter forming the structural support for the lamellae. However, knowledge on how the lipid composition of the SC alters cutaneous water loss (CWL) in mammals is rudimentary, and is largely derived from studies on laboratory animals and humans. We measured CWL of individuals of two species of syntopic bats, Tadarida brasiliensis and Myotis velifer. In the first study of its kind on wild mammals, we correlated CWL with the lipid composition of the SC, measured using thin layer chromatography and high performance liquid chromatography coupled with atmospheric pressure photoionization mass spectrometry. Surface-specific CWL was 20.6% higher in M. velifer than in T. brasiliensis, although differences were not significant. Compared with individuals of M. velifer, individuals of T. brasiliensis had more classes, and a higher proportion, of polar ceramides in the SC, a feature associated with lower CWL. Individuals of T. brasiliensis also had a class of non-polar ceramides that presumably spans the lamellae and gives more cohesiveness to the lipid matrix of the SC. We conclude that qualitative and quantitative modifications of the lipid composition of the SC contribute to regulate CWL of these two species of bats.     

The important role of stratum corneum lipids for the cutaneous barrier function

The skin protects the body from unwanted influences from the environment as well as excessive water loss. The barrier function of the skin is located in the stratum corneum (SC). The SC consists of corneocytes embedded in a lipid matrix. This lipid matrix is crucial for the lipid skin barrier function. This paper provides an overview of the reported SC lipid composition and organization mainly focusing on healthy and diseased human skin. In addition, an overview is provided on the data describing the relation between lipid modulations and the impaired skin barrier function. Finally, the use of in vitro lipid models for a better understanding of the relation between the lipid composition, lipid organization and skin lipid barrier is discussed. This article is part of a Special Issue entitled The Important Role of Lipids in the Epidermis and their Role in the Formation and Maintenance of the Cutaneous Barrier. This article is part of a Special Issue entitled The Important Role of Lipids in the Epidermis and their Role in the Formation and Maintenance of the Cutaneous Barrier. Guest Editors: Kenneth R. Feingold and Peter Elias.     

Human stratum corneum lipids: characterization and regional variations

The lipids of mammalian stratum corneum are known to be important regulators of skin permeability. Since the human stratum corneum displays remarkable regional variations in skin permeability, we assessed the total lipid concentration, the distribution of all major lipid species, and the fatty acid composition in Bligh-Dyer extracts from four skin sites (abdomen, leg, face, and sole) that are known to display widely disparate permeability. Statistically significant differences in lipid weight were found at the four sites that were inversely proportional to their known permeability. In all four sites, among the polar lipids, the stratum corneum contained negligible phospholipids, but substantially more cholesterol sulfate (1-7%) than previously appreciated. As in the stratum corneum from other mammals, the bulk of the lipids consisted of neutral (60-80%) and sphingolipids (15-35%). Of the neutral lipids, free sterols (4- to 5-times greater than esterified sterols), free fatty acids, triglycerides, and highly nonpolar species (n-alkanes and squalene) predominated. n-Alkanes, which were present in greater quantities than previously appreciated, comprised a homologous series of odd- and even-chained compounds ranging from C19 to C34. The sphingolipids comprised over 80% ceramides vs. lesser quantities of glycosphingolipids. In all four sites, the sphingolipids were the major repository of long-chain, saturated fatty acids. The neutral lipid:sphingolipid ratio generally was proportional to the known permeability of each site: higher neutral lipids and lower sphingolipids generally were associated with superior barrier properties. These studies provide: 1) the first detailed, quantitative analysis of human stratum corneum lipids and 2) information about the variability in lipid composition at four skin sites with known differences in permeability. The latter results suggest that variations in neutral lipids, rather than sphingolipids, may underlie local variations in skin permeability.     

Deuterium NMR investigation of polymorphism in stratum corneum lipids

The intercellular lipid lamellae of stratum corneum constitute the major barrier to percutaneous penetration. Deuterium magnetic resonance and freeze-fracture electron microscopic investigation of hydrated lipid mixtures consisting of ceramides, cholesterol, palmitic acid and cholesteryl sulfate and approximating the stratum corneum intercellular lipid composition, revealed thermally induced polymorphism. The transition temperature of bilayer to hexagonal transition decreased as the ratio of cholesterol to ceramides in these mixtures was lowered. Lipid mixtures in which the stratum corneum ceramides were replaced by synthetic dipalmitoylphosphatidylcholine did not show any polymorphism throughout the temperature range used in the present study. The ability of the ceramide-containing samples to form hexagonal structures establishes a plausible mechanism for the assembly of the stratum corneum intercellular lamellae during the final stages of epidermal differentiation. Also, the bilayer to hexagonal phase transition of these nonpolar lipid mixtures could be used to enhance the penetration of drugs through skin.     

Effect of epidermal acylglucosylceramides and acylceramides on the morphology of liposomes prepared from stratum corneum lipids

Epidermal acylglucosylceramides (AGC) and acylceramides (AC) cause aggregation and stacking of stratum corneum lipid liposomes formed from a lipid mixture containing epidermal ceramides (40%), cholesterol (25%), palmitic acid (25%), and cholesteryl sulfate (10%). This demonstrates the ability of these sphingolipids to hold adjacent bilayers in close apposition and their roles in the assembly of lamellar structures in the epidermis. However, AGC and AC in their hydrogenated form also caused aggregation and stacking of the stratum corneum lipid liposomes. This throws into doubt the proposed structural specificity of linoleate in the function of AGC and AC as molecular rivets in the assembly of the epidermal lamellar granules and the stratum corneum intercellular lamellae, respectively.     

Permeability parameters of the toad isolated stratum corneum.

A technique for isolating the stratum corneum from the subjacent layers of the epithelium was developed which permits studying the stratum corneum as an isolated membrane mounted between half-chambers. The method basically consists of an osmotic shock induced by immersing a piece of skin in distilled water at 50 degrees C for 2 min. When the membrane is bathed on each surface by NaCl-Ringer's solution, its electrical resistance is 14.1 +/- 1.3 omega cm2 (n=10). This value is about 1/100 of the whole skin resistance in the presence of the same solution. The hydraulic filtration coefficient (Lp) measured by a hydrostatic pressure method, with identical solutions on each side of the membrane, is 8.8 X 10(-5) +/- 1.5 X 10(-5) cm sec-1 atm-1 (n=10) in distilled water and 9.2 X 10(-5) +/- 1.4 X 10(-5) cm sec-1 atm-1 (n=10) in NaCl-Ringer's solution. These values are not statistically different and are within the range of 1/80 to 1/120 of the whole skin Lp. The stratum corneum shows an amphoteric character when studied by KCl diffusion potentials at different pH'S. The membrane presents an isoelectric pH of 4.6 +/- 0.3 (n=10). Above the isoelectric pH the potassium transport number is higher than the chloride transport number; below it, the reverse situation is valid. Divalent cations (Ca++ or Cu++) reduce membrane ionic discrimination when the membrane is negatively charged and are ineffective when the membrane fixed charges are protonated at low pH.     

New arrangement of proteins and lipids in the stratum corneum cornified envelope

A new arrangement of proteins and lipids of stratum corneum (SC) cornified envelope (CE) is proposed. The chemical analysis of CE revealed the presence of free fatty acids (FFA), ceramides (Cer), and important percentages of glutamic acid/glutamine (Glx) and serine (Ser) residues. The molecular structure of these components suggests the existence of covalent links not only between Cer and Glx but also between FFA and Ser. The protein distribution of extracellular surface of CE, i.e., the proteins that could be involved in the bonds with lipids, was studied using post- and pre-embedding immunolabeling electron microscopy. Some loricrin (protein rich in Ser) was detected in the outermost part of the CE protein layer. The external arrangement of some domains of this protein may give rise to form linkages with FFA, yielding further insight into the CE arrangement in which Cer-Glx bonds and FFA-Ser bonds would be involved. Although the importance of fatty acids in the cohesion and barrier function of SC has been widely demonstrated, their role could be associated not only to the presence of these lipids in the intercellular lamellae but also in the CE, in the same way that Cer.     

Physical and chemical perturbations of the supramolecular organization of the stratum corneum lipids: In vitro to ex vivo study

Background: FTIR spectroscopy is classically used to study the supramolecular organization of the stratum corneum lipids. Exposure to UV_A is responsible for a small decrease in packing observed on cutaneous lipid films. Methods: Lipid films and human skin biopsies were either exposed to UV_A irradiation of 120 J/cm², UV_B irradiation of 0.15 J/cm² or put in contact with ethanol. Using FTIR in vitro and IR microspectroscopy ex vivo provided information on: i) the precise localisation of the stratum corneum in the skin, ii) its thickness, and iii) the organization of its constituted lipids. Results: Different action modes were observed for UV irradiation and the contact with ethanol with a certain destabilisation of the lipidic layer. Ethanol was also found to be responsible for the creation of pores. The destabilisation of the lipid cement was mainly observed ex vivo. Conclusion: The barrier function of the skin is affected by the action of physical and chemical external agents at the molecular level. The increased laxity of the lipid packing could enable the percutaneous penetration velocity of actives.     

New arrangement of proteins and lipids in the stratum corneum cornified envelope

A new arrangement of proteins and lipids of stratum corneum (SC) cornified envelope (CE) is proposed. The chemical analysis of CE revealed the presence of free fatty acids (FFA), ceramides (Cer), and important percentages of glutamic acid/glutamine (Glx) and serine (Ser) residues. The molecular structure of these components suggests the existence of covalent links not only between Cer and Glx but also between FFA and Ser. The protein distribution of extracellular surface of CE, i.e., the proteins that could be involved in the bonds with lipids, was studied using post- and pre-embedding immunolabeling electron microscopy. Some loricrin (protein rich in Ser) was detected in the outermost part of the CE protein layer. The external arrangement of some domains of this protein may give rise to form linkages with FFA, yielding further insight into the CE arrangement in which Cer-Glx bonds and FFA-Ser bonds would be involved. Although the importance of fatty acids in the cohesion and barrier function of SC has been widely demonstrated, their role could be associated not only to the presence of these lipids in the intercellular lamellae but also in the CE, in the same way that Cer.