Thematic review series: skin lipids. The role of epidermal lipids in cutaneous permeability barrier homeostasis

The permeability barrier is required for terrestrial life and is localized to the stratum corneum, where extracellular lipid membranes inhibit water movement. The lipids that constitute the extracellular matrix have a unique composition and are 50% ceramides, 25% cholesterol, and 15% free fatty acids. Essential fatty acid deficiency results in abnormalities in stratum corneum structure function. The lipids are delivered to the extracellular space by the secretion of lamellar bodies, which contain phospholipids, glucosylceramides, sphingomyelin, cholesterol, and enzymes. In the extracellular space, the lamellar body lipids are metabolized by enzymes to the lipids that form the lamellar membranes. The lipids contained in the lamellar bodies are derived from both epidermal lipid synthesis and extracutaneous sources. Inhibition of cholesterol, fatty acid, ceramide, or glucosylceramide synthesis adversely affects lamellar body formation, thereby impairing barrier homeostasis. Studies have further shown that the elongation and desaturation of fatty acids is also required for barrier homeostasis. The mechanisms that mediate the uptake of extracutaneous lipids by the epidermis are unknown, but keratinocytes express LDL and scavenger receptor class B type 1, fatty acid transport proteins, and CD36. Topical application of physiologic lipids can improve permeability barrier homeostasis and has been useful in the treatment of cutaneous disorders.     

The role of sphingolipid metabolism in cutaneous permeabilitybarrier formation

The epidermal permeability barrier of mammalian skin is localized in the stratum corneum. Corneocytes are embedded in an extracellular, highly ordered lipid matrix of hydrophobic lipids consisting of about 50% ceramides, 25% cholesterol and 15% long and very long chain fatty acids. The most important lipids for the epidermal barrier are ceramides. The scaffold of the lipid matrix is built of acylceramides, containing ω-hydroxylated very long chain fatty acids, acylated at the ω-position with linoleic acid. After glucosylation of the acylceramides at Golgi membranes and secretion, the linoleic acid residues are replaced by glutamate residues originating from proteins exposed on the surface of corneocytes. Removal of their glucosyl residues generates a hydrophobic surface on the corneocytes used as a template for the formation of extracellular lipid layers of the water permeability barrier. Misregulation or defects in the formation of extracellular ceramide structures disturb barrier function. Important anabolic steps are the synthesis of ultra long chain fatty acids, their ω-hydroxylation, and formation of ultra long chain ceramides and glucosylceramides. The main probarrier precursor lipids, glucosylceramides and sphingomyelins, are packed in lamellar bodies together with hydrolytic enzymes such as glucosylceramide-β-glucosidase and acid sphingomyelinase and secreted into the intercelullar space between the stratum corneum and stratum granulosum. Inherited defects in the extracellular hydrolytic processing of the probarrier acylglucosylceramides impair epidermal barrier formation and cause fatal diseases: such as prosaposin deficiency resulting in lack of lysosomal lipid binding and transfer proteins, or the symptomatic clinical picture of the “collodion baby” in the absence of glucocerebrosidase. 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.     

Integrity and barrier function of the epidermis critically depend on glucosylceramide synthesis

Ceramides are vital components of the water barrier in mammalian skin. Epidermis-specific, a major ceramide portion contains omega-hydroxy very long chain fatty acids (C30-C36). These omega-hydroxy ceramides (Cers) are found in the extracellular lamellae of the stratum corneum either as linoleic acyl esters or protein bound. Glucosylceramide is the major glycosphingolipid of the epidermis. Synthesized from ceramide and UDP-glucose, it is thought to be itself an intracellular precursor and carrier for extracellular omega-hydroxy ceramides. To investigate whether GlcCer is an obligatory intermediate in ceramide metabolism to maintain epidermal barrier function, a mouse with an epidermis-specific glucosylceramide synthase (Ugcg) deficiency has been generated. Four days after birth animals devoid of GlcCer synthesis in keratinocytes showed a pronounced desquamation of the stratum corneum and extreme transepidermal water loss leading to death. The stratum corneum appeared as a thick unstructured mass. Lamellar bodies of the stratum granulosum did not display the usual ordered inner structure and were often irregularly arranged. Although the total amount of epidermal protein-bound ceramides remained unchanged, epidermal-free omega-hydroxy ceramides increased 4-fold and omega-hydroxy sphingomyelins, almost not detectable in wild type epidermis, emerged in quantities comparable with lost GlcCer. We conclude that the transient formation of GlcCer is vital for a regular arrangement of lipids and proteins in lamellar bodies and for the maintenance of the epidermal barrier.     

Effect of ceramide acyl chain length on skin permeability and thermotropic phase behavior of model stratum corneum lipid membranes

Stratum corneum ceramides play an essential role in the barrier properties of skin. However, their structure-activity relationships are poorly understood. We investigated the effects of acyl chain length in the non-hydroxy acyl sphingosine type (NS) ceramides on the skin permeability and their thermotropic phase behavior. Neither the long- to medium-chain ceramides (8-24 C) nor free sphingosine produced any changes of the skin barrier function. In contrast, the short-chain ceramides decreased skin electrical impedance and increased skin permeability for two marker drugs, theophylline and indomethacin, with maxima in the 4-6C acyl ceramides. The thermotropic phase behavior of pure ceramides and model stratum corneum lipid membranes composed of ceramide/lignoceric acid/cholesterol/cholesterol sulfate was studied by differential scanning calorimetry and infrared spectroscopy. Differences in thermotropic phase behavior of these lipids were found: those ceramides that had the greatest impact on the skin barrier properties displayed the lowest phase transitions and formed the least dense model stratum corneum lipid membranes at 32°C. In conclusion, the long hydrophobic chains in the NS-type ceramides are essential for maintaining the skin barrier function. However, this ability is not shared by their short-chain counterparts despite their having the same polar head structure and hydrogen bonding ability.     

Optimization of submerged keratinocyte cultures for the synthesis of barrier ceramides

Epidermal differentiation results in the formation of the extracellular lipid barrier in the stratum corneum, which mainly consists of ceramides, free fatty acids, and cholesterol. Differentiating keratinocytes of the stratum granulosum synthesize a series of complex long-chain ceramides and glucosylceramides with different chain lengths and hydroxylation patterns at intracellular membranes of the secretory pathway. Formation of complex extracellular ceramides parallels the transition of keratinocytes from the stratum granulosum to the stratum corneum, where their precursors, complex glucosylceramides and sphingomyelin, are secreted and exposed to extracellular lysosomal lipid hydrolases. Submerged cultures used so far showed a reduced ceramide content compared to the native epidermis or the air-exposed, organotypic culture system. In order to investigate the sphingolipid metabolism during keratinocyte differentiation, we optimized a simple cell culture system to generate the major barrier sphingolipids. This optimized model is based on the chemically well-defined serum-free MCDB medium. At low calcium ion concentrations (0.1mM), keratinocytes proliferate and synthesize mainly Cer(NS) and a small amount of Cer(NP). Supplementation of the MCDB cell culture medium with calcium ions (1.1mM) and 10 microM linoleic acid triggered differentiation of keratinocytes and synthesis of a complex pattern of free and covalently bound ceramides as found in native epidermis or air-exposed organotypic cultures, though at a reduced level. The mRNA levels of the differentiation markers keratin 10 and profilaggrin increased, as well as those of ceramide glucosyltransferase and glucosylceramide-beta-glucosidase. The described culture system was thus suitable for biochemical studies of the sphingolipid metabolism during keratinocyte differentiation. The addition of serum or vitamin A to the medium resulted in a decrease in ceramide and glucosylceramide content. Lowering the medium pH to 6, while maintained cell viability, led to an increase in the processing of probarrier lipids glucosylceramide and sphingomyelin to free ceramides and protein-bound ceramide Cer(OS).     

Avian sebokeratocytes and marine mammal lipokeratinocytes: structural, lipid biochemical, and functional considerations

In terrestrial mammals, stratum corneum lipids derive from two sources: deposition of lamellar body lipids in stratum corneum interstices and excretion of sebaceous lipids onto the skin surface, resulting in a two-compartment ("bricks and mortar") system of lipid-depleted cells surrounded by lipid-enriched intercellular spaces. In contrast, intracellular lipid droplets, normally not present in the epidermis of terrestrial mammals, are prominent in avian and marine mammal epidermis (cetaceans, manatees). We compared the transepidermal water loss, ultrastructure, and lipid biochemistry of the viable epidermis and stratum corneum of pigeon apterium, fledgling (featherless) zebra finches, painted storks, cetaceans, and manatees to those of humans and mice. Marine mammals possess an even more extensive lamellar-body secretory system than do terrestrial mammals; and lamellar-body contents, as in terrestrials, are secreted into the stratum corneum interstices. In cetaceans, however, glycolipids, but not ceramides, persist into the stratum corneum; whereas in manatees, glycolipids are replaced by ceramides, as in terrestrial mammals. Acylglucosylceramides, thought to be critical for lamellar-body deposition and barrier function in terrestrial mammals, are present in manatees but virtually absent in cetaceans, a finding that indicates that they are not obligate constituents of lamellar-body-derived membrane structures. Moreover, cetaceans do not elaborate the very long-chain, saturated N-acyl fatty acids that abound in terrestrial mammalian acylglucosylceramides. Furthermore, cold-water marine mammals generate large, intracellular neutral lipid droplets not found in terrestrial and warm-water marine mammals; these lipid droplets persist into the stratum corneum, suggesting thermogenesis, flotation, and/or cryoprotectant functions. Avians generate distinctive multigranular bodies that may be secreted into the intercellular spaces under xerotic conditions, as in zebra fledglings; ordinarily, however, the internal lamellae and limiting membranes deteriorate, generating intracellular neutral lipid droplets. The sphingolipid composition of avian stratum corneum is intermediate between terrestrials and cetaceans (approximately equal to 50% glycolipids), with triglycerides present in abundance. In the midstratum corneum of avians, neutral lipid droplets are released into the interstices, forming a large extracellular, lipid-enriched compartment, surrounding wafer-thin corneocytes, with a paucity of both lipid and keratin ("plates-and-mortar" rather than the "bricks-and-mortar" of mammals).(ABSTRACT TRUNCATED AT 400 WORDS)     

The skin barrier in healthy and diseased state

The primary function of the skin is to protect the body for unwanted influences from the environment. The main barrier of the skin is located in the outermost layer of the skin, the stratum corneum. The stratum corneum consists of corneocytes surrounded by lipid regions. As most drugs applied onto the skin permeate along the lipid domains, the lipid organization is considered to be very important for the skin barrier function. It is for this reason that the lipid organization has been investigated quite extensively. Due to the exceptional stratum corneum lipid composition, with long chain ceramides, free fatty acids and cholesterol as main lipid classes, the lipid organization is different from that of other biological membranes. In stratum corneum, two lamellar phases are present with repeat distances of approximately 6 and 13 nm. Moreover the lipids in the lamellar phases form predominantly crystalline lateral phases, but most probably a subpopulation of lipids forms a liquid phase. Diseased skin is often characterized by a reduced barrier function and an altered lipid composition and organization. In order to understand the aberrant lipid organization in diseased skin, information on the relation between lipid composition and organization is crucial. However, due to its complexity and inter-individual variability, the use of native stratum corneum does not allow detailed systematic studies. To circumvent this problem, mixtures prepared with stratum corneum lipids can be used. In this paper first the lipid organization in stratum corneum of normal and diseased skin is described. Then the role the various lipid classes play in stratum corneum lipid organization and barrier function has been discussed. Finally, the information on the role various lipid classes play in lipid phase behavior has been used to interpret the changes in lipid organization and barrier properties of diseased skin.     

The skin barrier in healthy and diseased state

The primary function of the skin is to protect the body for unwanted influences from the environment. The main barrier of the skin is located in the outermost layer of the skin, the stratum corneum. The stratum corneum consists of corneocytes surrounded by lipid regions. As most drugs applied onto the skin permeate along the lipid domains, the lipid organization is considered to be very important for the skin barrier function. It is for this reason that the lipid organization has been investigated quite extensively. Due to the exceptional stratum corneum lipid composition, with long chain ceramides, free fatty acids and cholesterol as main lipid classes, the lipid organization is different from that of other biological membranes. In stratum corneum, two lamellar phases are present with repeat distances of approximately 6 and 13 nm. Moreover the lipids in the lamellar phases form predominantly crystalline lateral phases, but most probably a subpopulation of lipids forms a liquid phase. Diseased skin is often characterized by a reduced barrier function and an altered lipid composition and organization. In order to understand the aberrant lipid organization in diseased skin, information on the relation between lipid composition and organization is crucial. However, due to its complexity and inter-individual variability, the use of native stratum corneum does not allow detailed systematic studies. To circumvent this problem, mixtures prepared with stratum corneum lipids can be used. In this paper first the lipid organization in stratum corneum of normal and diseased skin is described. Then the role the various lipid classes play in stratum corneum lipid organization and barrier function has been discussed. Finally, the information on the role various lipid classes play in lipid phase behavior has been used to interpret the changes in lipid organization and barrier properties of diseased skin.     

Structure of the skin barrier and its modulation by vesicular formulations

The natural function of the skin is to protect the body from unwanted influences from the environment. The main barrier of the skin is located in the outermost layer of the skin, the stratum corneum. Since the lipids regions in the stratum corneum form the only continuous structure, substances applied onto the skin always have to pass these regions. For this reason the organization in the lipid domains is considered to be very important for the skin barrier function. Due to the exceptional stratum corneum lipid composition, with long chain ceramides, free fatty acids and cholesterol as main lipid classes, the lipid phase behavior is different from that of other biological membranes. In stratum corneum crystalline phases are predominantly present, but most probably a subpopulation of lipids forms a liquid phase. Both the crystalline nature and the presence of a 13 nm lamellar phase are considered to be crucial for the skin barrier function. Since it is impossible to selectively extract individual lipid classes from the stratum corneum, the lipid organization has been studied in vitro using isolated lipid mixtures. These studies revealed that mixtures prepared with isolated stratum corneum lipids mimic to a high extent stratum corneum lipid phase behavior. This indicates that proteins do not play an important role in the stratum corneum lipid phase behavior. Furthermore, it was noticed that mixtures prepared only with ceramides and cholesterol already form the 13 nm lamellar phase. In the presence of free fatty acids the lattice density of the structure increases. In stratum corneum the ceramide fraction consists of various ceramide subclasses and the formation of the 13 nm lamellar phase is also affected by the ceramide composition. Particularly the presence of ceramide 1 is crucial. Based on these findings a molecular model has recently been proposed for the organization of the 13 nm lamellar phase, referred to as "the sandwich model", in which crystalline and liquid domains coexist. The major problem for topical drug delivery is the low diffusion rate of drugs across the stratum corneum. Therefore, several methods have been assessed to increase the permeation rate of drugs temporarily and locally. One of the approaches is the application of drugs in formulations containing vesicles. In order to unravel the mechanisms involved in increasing the drug transport across the skin, information on the effect of vesicles on drug permeation rate, the permeation pathway and perturbations of the skin ultrastructure is of importance. In the second part of this paper the possible interactions between vesicles and skin are described, focusing on differences between the effects of gel-state vesicles, liquid-state vesicles and elastic vesicles.     

The important role of epidermal triacylglycerol metabolism for maintenance of the skin permeability barrier function

Survival in a terrestrial, dry environment necessitates a permeability barrier for regulated permeation of water and electrolytes in the cornified layer of the skin (the stratum corneum) to minimize desiccation of the body. This barrier is formed during cornification and involves a cross-linking of corneocyte proteins as well as an extensive remodeling of lipids. The cleavage of precursor lipids from lamellar bodies by various hydrolytic enzymes generates ceramides, cholesterol, and non-esterified fatty acids for the extracellular lipid lamellae in the stratum corneum. However, the important role of epidermal triacylglycerol (TAG) metabolism during formation of a functional permeability barrier in the skin was only recently discovered. Humans with mutations in the ABHD5/CGI-58 (α/β hydrolase domain containing protein 5, also known as comparative gene identification-58, CGI-58) gene suffer from a defect in TAG catabolism that causes neutral lipid storage disease with ichthyosis. In addition, mice with deficiencies in genes involved in TAG catabolism (Abhd5/Cgi-58 knock-out mice) or TAG synthesis (acyl-CoA:diacylglycerol acyltransferase-2, Dgat2 knock-out mice) also develop severe skin permeability barrier dysfunctions and die soon after birth due to increased dehydration. As a result of these defects in epidermal TAG metabolism, humans and mice lack ω-(O)-acylceramides, which leads to malformation of the cornified lipid envelope of the skin. In healthy skin, this epidermal structure provides an interface for the linkage of lamellar membranes with corneocyte proteins to maintain permeability barrier homeostasis. This review focuses on recent advances in the understanding of biochemical mechanisms involved in epidermal neutral lipid metabolism and the generation of a functional skin permeability 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.     

The roles of ABCA12 in epidermal lipid barrier formation and keratinocyte differentiation

ATP-binding cassette (ABC) transporters form a large superfamily of transporters that bind and hydrolyze ATP to transport various molecules across limiting membranes or into vesicles. The ABCA subfamily members are thought to transport lipid materials. ABCA12 is a keratinocyte transmembrane lipid transporter protein associated with the transport of lipids via lamellar granules. ABCA12 is considered to transport lipids including ceramides to form extracellular lipid layers in the stratum corneum of the epidermis, which is essential for skin barrier function. ABCA12 mutations are known to underlie the three major types of autosomal recessive congenital ichthyoses: harlequin ichthyosis, lamellar ichthyosis and congenital ichthyosiform erythroderma. ABCA12 mutations result in defective lipid transport via lamellar granules in the keratinocytes, leading to ichthyosis phenotypes from malformation of the stratum corneum lipid barrier. Studies on ABCA12-deficient bioengineered models have revealed that lipid transport by ABCA12 is required for keratinocyte differentiation and epidermal morphogenesis. Defective lipid transport due to loss of ABCA12 function leads to the accumulation of intracellular lipids, including glucosylceramides and gangliosides, in the epidermal keratinocytes. The accumulation of gangliosides seems to result in the apoptosis of Abca12^−/− keratinocytes. It was reported that AKT activation occurs in Abca12^−/− granular-layer keratinocytes, which suggests that AKT activation serves to prevent the cell death of Abca12^−/− keratinocytes. 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.     

The Lipid Organisation of the Skin Barrier: Liquid and Crystalline Domains Coexist in Lamellar Phases

The superficial layer of the skin, the stratum corneum, is the main barrier for diffusion of substances across the skin. The stratum corneum is composed of corneocytes embedded in lipid lamellae. In previous studies two lamellar phases have been identified with periodicities of 6.4 and 13.4 nm of which the 13.4 nm phase (long periodicity phase = LPP) is considered to be very important for the skin banier function. The main lipid classes in stratumcorneum are ceramides, free fatty acids and cholesterol. Until now 8 subclassesof ceramides are identified in human stratum corneum referred to as ceramide 1 to 8. Studies with mixtures prepared with isolated human ceramides revealed that cholesterol and ceramides are very important for the formation of the lamellar phases. After addition of free fatty acids the lipids are organised in an orthorhombic packing with a small proportion of lipids in a liquid phase. Our most recent results show that the presence of ceramide 1 and the formation of a liquid phase are crucial elements for the formation of the LPP. These observations and the broad-narrowbroad sequence of lipid layers in the LPP led us to propose a molecular model for this phase. This consists of one narrow central lipid layer with fluid domains with on both sides a broad layer with a crystalline structure. This model is referred to as `the sandwich model'.     

Avian epidermal differentiation: role of lipids in permeability barrier formation

Though avian skin is known to possess a highly lipogenic epidermis, little is known about its permeability barrier function. We correlated epidermal barrier function, fine structure and lipid biochemistry in the pigeon, Columbia livia, and compared these features with terrestrial mammalian systems. Whereas barrier function, as assessed by transepidermal water loss was not as efficient as in mammals, both groups shared certain morphological features including substantial compartmentalization of lipids in stratum corneum intercellular domains. Avian intercellular lipids derive from extrusion of intracellular non-membrane-bound droplets from lowermost corneocytes, rather than by secretion of lamellar discs from multigranular bodies, as previously reported in some avians, and in mammals. Instead, both the internal lamellae and the limiting membranes of multigranular bodies appear to degenerate, leading to the formation of non-membrane-bound droplets. The lipid content of avian epidermis and stratum corneum demonstrates important similarities to terrestrial mammals, i.e. abundant sphingolipids, a paucity of phospholipids, and abundant neutral lipids, but also certain striking differences, i.e. persistence of glycosphingolipids and triglycerides into the stratum corneum. Thus, avian stratum corneum forms a two-compartment system of lipid-depleted cells embedded in non-polar-lipid enriched intercellular domains, analogous to mammals. But, in contrast to mammals, the highly attenuated corneocytes of avians, which results from a paucity of keratin filaments, produce a 'straws-and-mortar' tissue, rather than the 'bricks-and-mortar' tissue of mammals.     

Sphingolipid activator proteins are required for epidermal permeability barrier formation

The epidermal permeability barrier is maintained by extracellular lipid membranes within the interstices of the stratum corneum. Ceramides, the major components of these multilayered membranes, derive in large part from hydrolysis of glucosylceramides mediated by stratum corneum beta-glucocerebrosidase (beta-GlcCerase). Prosaposin (pSAP) is a large precursor protein that is proteolytically cleaved to form four distinct sphingolipid activator proteins, which stimulate enzymatic hydrolysis of sphingolipids, including glucosylceramide. Recently, pSAP has been eliminated in a mouse model using targeted deletion and homologous recombination. In addition to the extracutaneous findings noted previously, our present data indicate that pSAP deficiency in the epidermis has significant consequences including: 1) an accumulation of epidermal glucosylceramides together with below normal levels of ceramides; 2) alterations in lipids that are bound by ester linkages to proteins of the cornified cell envelope; 3) a thickened stratum lucidum with evidence of scaling; and 4) a striking abnormality in lamellar membrane maturation within the interstices of the stratum corneum. Together, these results demonstrate that the production of pSAP, and presumably mature sphingolipid activator protein generation, is required for normal epidermal barrier formation and function. Moreover, detection of significant amounts of covalently bound omega-OH-GlcCer in pSAP-deficient epidermis suggests that deglucosylation to omega-OH-Cer is not a requisite step prior to covalent attachment of lipid to cornified envelope proteins.     

Direct visualization of lipid domains in human skin stratum corneum's lipid membranes: effect of pH and temperature

The main function of skin is to serve as a physical barrier between the body and the environment. This barrier capacity is in turn a function of the physical state and structural organization of the stratum corneum extracellular lipid matrix. This lipid matrix is essentially composed of very long chain saturated ceramides, cholesterol, and free fatty acids. Three unsolved key questions are i), whether the stratum corneum extracellular lipid matrix is constituted by a single gel phase or by coexisting crystalline (solid) domains; ii), whether a separate liquid crystalline phase is present; and iii), whether pH has a direct effect on the lipid matrix phase behavior. In this work the lateral structure of membranes composed of lipids extracted from human skin stratum corneum was studied in a broad temperature range (10 degrees C-90 degrees C) using different techniques such as differential scanning calorimetry, fluorescence spectroscopy, and two-photon excitation and laser scanning confocal fluorescence microscopy. Here we show that hydrated bilayers of human skin stratum corneum lipids express a giant sponge-like morphology with dimensions corresponding to the global three-dimensional morphology of the stratum corneum extracellular space. These structures can be directly visualized using the aforementioned fluorescence microscopy techniques. At skin physiological temperatures (28 degrees C-32 degrees C), the phase state of these hydrated bilayers correspond microscopically (radial resolution limit 300 nm) to a single gel phase at pH 7, coexistence of different gel phases between pH 5 and 6, and no fluid phase at any pH. This observation suggests that the local pH in the stratum corneum may control the physical properties of the extracellular lipid matrix by regulating membrane lateral structure and stability.     

Pathogenesis of permeability barrier abnormalities in the ichthyoses: inherited disorders of lipid metabolism

Many of the ichthyoses are associated with inherited disorders of lipid metabolism. These disorders have provided unique models to dissect physiologic processes in normal epidermis and the pathophysiology of more common scaling conditions. In most of these disorders, a permeability barrier abnormality "drives" pathophysiology through stimulation of epidermal hyperplasia. Among primary abnormalities of nonpolar lipid metabolism, triglyceride accumulation in neutral lipid storage disease as a result of a lipase mutation provokes a barrier abnormality via lamellar/nonlamellar phase separation within the extracellular matrix of the stratum corneum (SC). Similar mechanisms account for the barrier abnormalities (and subsequent ichthyosis) in inherited disorders of polar lipid metabolism. For example, in recessive X-linked ichthyosis (RXLI), cholesterol sulfate (CSO(4)) accumulation also produces a permeability barrier defect through lamellar/nonlamellar phase separation. However, in RXLI, the desquamation abnormality is in part attributable to the plurifunctional roles of CSO(4) as a regulator of both epidermal differentiation and corneodesmosome degradation. Phase separation also occurs in type II Gaucher disease (GD; from accumulation of glucosylceramides as a result of to beta-glucocerebrosidase deficiency). Finally, failure to assemble both lipids and desquamatory enzymes into nascent epidermal lamellar bodies (LBs) accounts for both the permeability barrier and desquamation abnormalities in Harlequin ichthyosis (HI). The barrier abnormality provokes the clinical phenotype in these disorders not only by stimulating epidermal proliferation, but also by inducing inflammation.     

Human stratum corneum lipid organization as observed by atomic force microscopy on Langmuir-Blodgett films

The barrier function of skin ultimately depends on the physical state and structural organisation of the stratum corneum extracellular lipid matrix. Ceramides, cholesterol and a broad distribution of saturated long-chain free fatty acids dominate the stratum corneum lipid composition. Additionally, smaller amounts of cholesterol sulfate and cholesteryl oleate may be present. A key feature determining skin barrier capacity is thought to be whether or not different lipid domains coexist laterally in the stratum corneum extracellular lipid matrix. In this study, the overall tendency for lipid domain formation in different mixtures of extracted human stratum corneum ceramides, cholesterol, free fatty acids, cholesterol sulfate and cholesteryl oleate were studied using atomic force microscopy (AFM) on Langmuir-Blodgett (LB) films on mica. It is shown that the saturated long-chain free fatty acid distribution of human stratum corneum prevents hydrocarbon chain segregation. Further, LB-films of human stratum corneum ceramides express a pattern of connected elongated domains with a granular domain interface. The dominating effect of both cholesterol and cholesterol sulfate is that of increased ceramide domain dispersion. This effect is counteracted by the presence of free fatty acids, which preferentially mix with ceramides and not with cholesterol. Cholesteryl oleate does not mix with other skin lipid components, supporting the hypothesis of an extra-endogenous origin. In the system composed of endogenous human ceramides and cholesterol plus 15 wt% stratum corneum distributed free fatty acids, i.e., the system mimicking most closely the lipid composition of the stratum corneum extracellular space, LB-films on mica express lateral domain formation.     

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.     

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.