Lipid composition of cohesive and desquamated corneocytes from mouse ear skin

An organ culture system has been used to examine differences in the lipid compositions of materials derived from cohesive and desquamated mouse ear stratum corneum. Within this culture system, skin explants display rates of cell replication and differentiation comparable to those observed in vivo for up to 2 weeks and, during this period, loosened or dishesive material accumulates at the surface. Lipid compositions were determined for both intact and loosened stratum corneum derived from cultured skin and also for freshly prepared stratum corneum. In all 3 cases, the profiles of the nonpolar lipids and the ceramides were essentially the same; some of the nonpolar lipids appeared to be of sebaceous origin. The only changes detected upon desquamation were reductions of cholesteryl sulfate and a second unidentified lipid of similar polarity. Cholesteryl sulfate constitutes 4-5% of the polar lipid in fresh stratum corneum or stratum corneum from organ culture. This is reduced to 0.4% in the desquamated material which accumulates in the culture system. The unidentified lipid decreases from 1-2% of the polar lipid in intact fresh or cultured stratum corneum to 0.1% in the desquamated material. The possible function of cholesteryl sulfate in corneocyte cohesion is discussed.     

Composition and morphology of epidermal cyst lipids

The contents of epidermal cysts were used as a source of desquamated human keratinocytes uncontaminated by sebaceous, subcutaneous, or bacterial lipids. Lipids extracted with chloroform:methanol mixtures included six series of ceramides (41% of the total extractable lipid), cholesterol (27%), cholesteryl esters (10%), fatty acids (9%), cholesteryl sulfate (1.9%), a novel class of ceramide esters (3.8%), and a sterol diester (0.9%). Electron microscopy revealed that the lipids in the cyst contents existed as multiple intercellular lamellae, as in stratum corneum. One lamella, adjacent to the horny cell protein envelope, was resistant to lipid extraction and is thought to represent covalently bound lipid on the outer surface of the keratinocyte. The results indicate that the degradation of intercellular lipid lamellae is not required for desquamation.     

Preparation of model membranes for skin permeability studies using stratum corneum lipids

Liposomes were prepared from stratum corneum lipids consisting of epidermal ceramides (55% by weight), cholesterol (25%), free fatty acids (15%), and cholesteryl sulfate (5%). Multiple lamellae were formed by air-drying the liposomal suspensions on hydrophilic filter disks, and water permeabilities through these filter-supported lamellae were measured using a diffusion cell. Ultrastructure of the lipid lamellae was characterized by scanning and thin-section electron microscopy. Water flux data and ultrastructure of the model membranes are discussed in relation to stratum corneum, the horny layer of the epidermis that constitutes the epidermal barrier.     

Fusion patterns of liposomes formed from stratum corneum lipids

Stratum corneum lipid liposomes formed from epidermal ceramides (40%), cholesterol (25%), palmitic acid (25%), and cholesteryl sulfate (10%), when exposed to hypertonic medium, form flattened liposomes. Epidermal acylglucosyl-ceramides (AGCs) and acylceramides (ACs) cause aggregation and fusion of these flattened vesicles. This could serve as a model to study (a) the fusion of membranous disks in the intercellular space of the stratum corneum and (b) the roles of AGCs and ACs in the assembly of lamellar structures in the epidermis.     

Decreased level of ceramides in stratum corneum of atopic dermatitis: an etiologic factor in atopic dry skin?

Stratum corneum lipids are an important determinant for both water-retention function and permeability-barrier function in the stratum corneum. However, their major constituent, ceramides, have not been analyzed in detail in skin diseases such as atopic dermatitis that show defective water-retention and permeability-barrier function. In an attempt to assess the quantity of ceramides per unit mass of the stratum corneum in atopic dermatitis, stratum corneum sheet was removed from the forearm skin by stripping with cyanoacrylate resin and placed in hexane/ethanol extraction to yield stratum corneum lipids. The stratum corneum was dispersed by solubilization of cyanoacrylate resin with dimethylformamide, and after membrane filtration, the weight of the stratum corneum mass was measured. The ceramides were quantified by thin-layer chromatography and evaluated as microgram/mg stratum corneum. In the forearm skin of healthy individuals (n = 65), the total ceramide content significantly declined with increasing age. In atopic dermatitis (n = 32-35), there was a marked reduction in the amount of ceramides in the lesional forearm skin compared with those of healthy individuals of the same age. Interestingly, the non-lesional skin also exhibited a similar and significant decrease of ceramides. Among six ceramide fractions, ceramide 1 was most significantly reduced in both lesional and non-lesional skin. These findings suggest that an insufficiency of ceramides in the stratum corneum is an etiologic factor in atopic dry skin.     

Relationship between cholesterol sulfate and intercellular cohesion of the stratum corneum: demonstration using a push-pull meter and an improved high-performance thin-layer chromatographic separation system of all major stratum corneum lipids.

To investigate the role of cholesterol sulfate (CS) as an intercellular glue or cement in the stratum corneum, we compared the relationship between CS levels and magnitude of the intercellular cohesion of the stratum corneum between the palm and the upper arm. Using a push-pull meter, the palm displayed approximately seven times the magnitude of cohesion of the stratum corneum as the upper arm (n = 11). CS and other stratum corneum lipids were extracted from the palm and the upper arm (n = 22) by a cup method and determined by our improved high-performance thin-layer chromatography (HPTLC). Despite a great difference in the magnitude of cohesion (p less than 0.01), CS levels and ratios of CS to ceramides and CS to cholesterol in the stratum corneum showed no significant differences between the palm and the upper arm. Our results suggest that differences in CS cannot account for the differences in cohesion between palm and upper arm.     

Quantitative study of stratum corneum ceramides contents in patients with sensitive skin

People with sensitive skin (SS) are those who state their skin is more sensitive than that of average persons. The stratum corneum is responsible for maintaining skin barrier function. Ceramides, major constituents of stratum corneum lipids, have been shown to predominantly contribute to the role. It has been suggested that barrier function in SS is decreased. However, we could find very few reports about stratum corneum ceramides in SS. This study was done to find out differences in stratum corneum ceramides between SS and non-SS groups. Fifty individuals (20 with SS and 30 with non-SS) were recruited. Lactic acid sting test (LAST) was performed on the left cheek. On six sites including the right cheek, arm, thigh, leg, back and palm, transepidermal water loss (TEWL) and erythema index (EI) were measured. On the above six sites, stratum corneum sheets were obtained by stripping with cyanoacrylate resin and stratum corneum lipids were extracted, then, analyzed by high-performance liquid chromatography electrospray ionization mass spectrometry. LAST scores were higher in the SS group, but not statistically significant. There were no differences in TEWL and EI values between the two groups. The mean value of the quantity of stratum corneum ceramides on the face was significantly lower in the SS group. On other sites, mean values were also lower in the SS group, but not statistically significant. The quantity of ceramides was significantly decreased in the face of the SS group compared to that of the non-SS group. These results suggest that the decrease in stratum corneum ceramides on facial skin could be related to SS development.     

Human epidermal glucosylceramides are major precursors of stratum corneum ceramides

Ceramides are the major component of the stratum corneum, accounting for 30%-40% of stratum corneum lipids by weight, and are composed of at least seven molecular groups (designated ceramides 1-7). Stratum corneum ceramides, together with cholesterol and fatty acids, form extracellular lamellae that are responsible for the epidermal permeability barrier. Previous studies indicated that beta-glucocerebrosidase- and sphingomyelinase-dependent ceramide production from glucosylceramides and sphingomyelins, respectively, is important for epidermal permeability barrier homeostasis. A recent study indicated that sphingomyelins are precursors of two stratum corneum ceramide molecular groups (ceramides 2 and 5). In this study, we have examined the role of glucosylceramides in the generation of each of the seven stratum corneum ceramide molecular groups. First, the structures of various glucosylceramide species in human epidermis were determined by gas chromatography-mass spectrometry, fast atom bombardment-mass spectrometry, and nuclear magnetic resonance. The results indicate that total epidermal glucosylceramides are composed of six distinct molecular groups, glucosylceramides 1-6. Glucosylceramide 1 contains sphingenine and nonhydroxy fatty acids, glucosylceramide 2, phytosphingosine and nonhydroxy fatty acids, glucosylceramide 3, phytosphingosine with one double bond and nonhydroxy fatty acids, glucosylceramide 4, sphingenine and alpha-hydroxy fatty acids, glucosylceramide 5, phytosphingosine and alpha-hydroxy fatty acids, and glucosylceramide 6, phytosphingosine with one double bond and alpha-hydroxy fatty acids. The nonhydroxy fatty acids typically have 16-24-carbon-length chains, whereas alpha-hydroxy fatty acids are limited to 24-, 25-, and 26-carbon chains. The sphingosine bases are C18 or C20 chains. Next, acylglucosylceramides and glucosylceramides were treated with beta-glucocerebrosidase and the ceramides released were compared with stratum corneum ceramides. Ceramide moieties of acylglucosylceramides and glucosylceramides 1, 2, 4-6 correspond to stratum corneum ceramides 1-7. These results, together with those of our previous reports characterizing epidermal sphingomyelins, indicate that all ceramide species, including omega-hydroxy fatty-acid-containing ceramides, are derived from glucosylceramides, and fractions of ceramides 2 and 5 are from sphingomyelins. Furthermore, structural analysis of glucosylceramides revealed that human epidermal glycosphingolipids display a unique lipid profile that is rich in very long chain hydroxylated (alpha- and omega-hydroxy) fatty acids and phytosphingosine.     

Variations in lipids in different layers of porcine epidermis

Frozen cryosections, 8 microns thick, were cut parallel to the surface of porcine skin so as to provide separate samples representing various epidermal layers. These samples were dried, extracted with chloroform-methanol mixtures, and the lipids chromatographed on silica gel plates in different solvent systems. After spraying with sulfuric acid and charring, lipids were quantified using a scanning densitometer. It was thus possible to determine lipid concentrations in 12 consecutive epidermal layers, extending 96 microns into the skin. The phospholipids that were characterized all decreased in concentration toward the surface, whereas the neutral lipids and ceramides all increased. Glucosylceramide and acylglucosylceramide reached a peak concentration in the stratum granulosum and then decreased in the surface layers. Cholesterol sulfate reached a maximum concentration in the deeper stratum corneum and then abruptly decreased in the surface layer. These changes in patterns of lipid concentration are consistent with current theories regarding the formation of a water barrier in the stratum corneum that is composed mainly of neutral lipids, and with a possible function of cholesterol sulfate in cellular adhesion in the stratum corneum.     

Stratum corneum lipids in skin xerosis

Lipids of the stratum corneum are implicated in cohesion and desquamation of the stratum corneum as well as in the maintenance of normal barrier function. Evidence linking the intercellular lipids to such processes has mainly been derived from studies on acquired or inherited diseases of lipid metabolism manifesting abnormalities in the structure and the function of the stratum corneum. We have studied the composition of stratum corneum lipids in clinically normal individuals with typical xerosis or 'winter dry skin' in order to establish if the lipid composition differs from that of normal individuals, showing no signs of xerosis. The amount of total stratum corneum lipids was not related to xerosis (22.0 +/- 1.8 micrograms/cm2 for normal skin, and 26.3 +/- 2.9 micrograms/cm2 for severe xerosis), and no correlation was evident between polar lipids, cholesterol sulfate (2.8 +/- 0.5% for normal skin, and 1.6 +/- 0.2% for severe xerosis), or ceramides types I-VI, and dry skin. It therefore appears that dramatic changes in stratum corneum lipids are not detectable in normal 'winter dry' skin. However, a decreased proportion of neutral lipids (sterol esters, triglycerides), coupled to increased amounts of free fatty acids, were found associated to the severity of dry skin. Apart from a decline in the sebaceous function and in esterases activity, winter dry skin does not appear to be associated to dramatic changes in polar stratum corneum lipids.     

Lipid composition and acid hydrolase content of lamellar granules of fetal rat epidermis

Lipids and acid hydrolases have been characterized in a subcellular fraction, enriched with lamellar granules (LG), derived from fetal rat epidermis. This fraction contains 23% glycosyl ceramides and ceramides, 15% free sterols, and 34% phospholipids. The lipid/protein ratio is 2.0. The sterols and sphingolipids were present in proportions similar to those previously reported in stratum corneum. These findings provide direct biochemical evidence for the widely accepted hypothesis that stratum corneum lipids are derived from exocytosis of lamellar granules into the intercellular space. The LG fraction was enriched in certain acid hydrolases including glucosidase, acid phosphatase, phospholipases A, and sphingomyelinase; other acid hydrolases, i.e., amino-glycosidases, glactosidase and aryl sulfatase (pH 5.5), and steroid sulfatase were not preferentially localized in this fraction. By modulation of phospholipids, glycolipids, and proteins in the membrane regions of stratum corneum, the acid hydrolases of LG may play a role relevant to the function and desquamation of stratum corneum.     

Stratum corneum lipid liposomes: calcium-induced transformation into lamellar sheets

The epidermal water barrier in mammalian stratum corneum is formed of broad lamellar sheets of lipids consisting principally of ceramides (40%), cholesterol (25%), cholesteryl sulfate (10%), and free fatty acids (25%). Such lipid mixtures have been shown to form lipid bilayers in the form of small, unilamellar liposomes when sonicated at 80 degrees C in water containing Tris buffer and 100 mM NaCl. In the present study it is shown that such liposomes are slowly transformed into large unilamellar liposomes and then into broad lamellar sheets after the addition of stoichiometric amounts of calcium chloride. The presence of free fatty acids was a necessary condition for this calcium-induced fusion. These observations may provide a useful analogy for the transformation of flattened liposomes into broad lamellar sheets that occurs during transition of epidermal granular cells into corneocytes.     

Relationship between covalently bound ceramides and transepidermal water loss (TEWL)

The stratum corneum, which is the outermost layer of the skin, functions as an important barrier to maintain biological homeostasis. The multilamellar structures formed by intercellular lipids present in the stratum corneum are considered to play an important role in barrier function. Most intercellular lipids are unbound and can be extracted by organic solvents, but some intercellular lipids are covalently bound to cornified envelope proteins. Decreases in unbound lipid levels reduce the barrier function of the stratum corneum, but the relationship between bound lipid and the barrier function of the stratum corneum is not well understood. In this study, we examined the relationship between the amount of covalently bound ceramide, the main bound lipid, and the barrier function of the stratum corneum. A single dose of UVB irradiation (2 x MED), or continuous UVB irradiation (0.5 x MED/day for 14 days) to the back, or feeding with an essential fatty acid-deficient (EFAD) diet for 8 weeks caused a significant elevation of TEWL and a significant reduction in covalently bound ceramides in hairless rats. Transmission electron microscopy revealed that the intercellular multilamellar structures in the stratum corneum of treated rats were incomplete (folding, defects, unclear images) compared to the structures seen in the stratum corneum of non-UVB-irradiated and non-EFAD rats. These results suggest that the amount of covalently bound ceramides is highly correlated with the barrier function of the skin, and that covalently bound ceramides play an important role in the formation of lamellar structures, and are involved in the maintenance of the barrier function of the skin.     

Ceramide profiles of the uninvolved skin in atopic dermatitis and psoriasis are comparable to those of healthy skin

Ceramides are sphingolipids consisting of sphingoidbases, which are amide-linked to fatty acids. In the stratum corneum, they represent the major constituent of the free extractable intercellular lipids and play a significant role in maintaining and structuring the water permeability barrier of the skin. Using thin layer chromatography, which represents the method of the first choice in analyzing the stratum corneum ceramides, at least seven classes can be distinguished. Each ceramide class contains various species, which have the same head group and different chain lengths. As in many other skin disorders, atopic dermatitis and psoriasis show derangements in content and profile of the ceramides. Such derangements were reported for both the lesional involved as well as for the normal-appearing uninvolved skin. In this study, we focused on investigating the stratum corneum ceramides of the uninvolved skin in atopic dermatitis and psoriasis patients compared to healthy skin. The aim of the investigations was to explore possible significant and specific differences which can be accomplished for purposes of early diagnostics. The skin lipids were collected by means of an in vivo topical extraction procedure using an extraction mixture consisting of n-hexane and ethanol, (2:1). An automated multiple development-high performance thin layer chromatography (AMD-HPTLC) method with photodensitometric detection were applied to separate the ceramides and to estimate their contents. For studying their molecular profile within each ceramide class, a new method of normal phase HPLC with atmospheric pressure chemical ionization mass spectrometry were used. The results obtained by AMD-HPTLC exposed no significant alterations regarding the relative composition of the major stratum corneum lipids and primarily the ceramides. In addition, the mass spectrometric profiles within each ceramide class were similar in the patients and the healthy control subjects. In conclusion, this study revealed that the normal-appearing uninvolved skin of atopic dermatitis and psoriasis patients does not prove significant or specific deficiencies with respect to the free extractable major stratum corneum lipids and mainly the ceramides, when compared to healthy skin. Thus, they cannot be used for diagnostic purposes. Furthermore, our data are not consistent with the concept that impairments in the ceramide composition represent an obligate etiologic factor for both diseases.     

Sodium lauryl sulfate (SLS) induced irritant contact dermatitis: a correlation study between ceramides and in vivo parameters of irritation

Sodium lauryl sulfate (SLS), a surfactant frequently used in the induction of experimental irritant contact dermatitis in animals and in humans, characterstically induces a dose-related increase in TEWL (transepidermal water loss). Ceramides are considered to be important in the regulation of the skin barrier. We therefore examined the relationship between initial ceramide content of stratum corneum and induced changes in skin color (erythema) and barrier function, after SLS application under occlusion (1% and 3% in water) to the forearm of 14 volunteers. Stratum corneum sheets were removed, stratum corneum lipids extracted, and ceramide composition determined from chromatograms (TLC) using densitometry. After determining baseline skin color and TEWL at each area. 2 samples of stratum corneum were obtained from each volunteer. Clinical and instrumental controls of the SLS-induced irritation were performed at 24, 48, 72 and 96 h. Erythema was evaluated by colorimetry; barrier impairment by changes in TEWL. We found inverse correlations between baseline ceramide 61 (weight) and the 24 h erythema score for SLS: between ceramide I and 24 h TEWL, and between ceramide 611 and 72 h TEWL for SLS 3%. Our findings suggest that low levels of these ceramides may determine a proclivity to SLS-induced irritant contact dermatitis.     

The water-holding capacity of the stratum corneum measured by 1H-NMR

The amount of bound water in the stratum corneum of the hairless rat was measured by using proton nuclear magnetic resonance (1H-NMR) spectrometry in the temperature range of -5 degrees C to -30 degrees C. A decrease in the bound water content was observed when the stratum corneum was extracted using water, a mixed solvent of chloroform:methanol (2:1), or a 1% sodium dodecyl sulfate (SDS) aqueous solution. However, extraction using a mixed solvent of acetone:ether (1:1) did not change the bound water content at temperatures above -20 degrees C. The lipids extracted by acetone:ether-extraction method consisted of sebaceous gland lipids, cholesterol, free fatty acids, and ceramides. In contrast, the lipids extracted by chloroform:methanol-extraction method contained more polar lipids such as sphingomyelin, which had the same amount of bound water in itself as that of the water-extracted stratum corneum. These results suggest that the polar lipids soluble in the chloroform and methanol mixture may contribute to enhance the bound water content. Therefore, it is hypothesized that the water-holding capacity of the stratum corneum is dependent on both the hygroscopic components such as the natural moisturizing factor (NMF) and the polar lipids, such as sphingolipids, existing in the intercellular spaces of the stratum corneum.     

Effect of lactic acid isomers on keratinocyte ceramide synthesis,stratum corneum lipid levels and stratum corneum barrier function

Alpha-hydroxy acids are effective agents for the treatment of skin xerosis and it is known that, following treatment with lotions containingd,l-lactic acid, the stratum corneum prevents xerosis more effectively. To date, the relative efficacy of the different isomers of lactic acid has not been evaluated and the mode of action of lactic acid in improving stratum corneum resilience is not known. The objective of the present studies was to determine the effects of lactic acid isomers on keratinocyte ceramide biosynthesis, stratum corneum barrier function and the resistance of the straum corneum to the appearance of skin xerosis. In vitro, lactic acid enhanced the production of ceramides by keratinocytes.l-Lactic acid was more effective than thed isomer (300% increase vs 100% increase). Carbon label from lactic acid was incorporated into all keratinocyte lipid species and a greater incorporation of label into ceramides was achieved withl-lactate than withd-lactate. In vivo, lactic acid increased the levels of stratum corneum ceramides. Whereas, lotions containingl-lactic acid resulted in the greatest increase (48% increase) followed byd,l-lactic acid (25% increase),d-lactic acid had no effect on stratum corneum ceramide levels. The increases in stratum corneum ceramide levels following lactic acid treatment also led to improvements in stratum corneum barrier function, measured by transepidermal water loss following a challenge to the skin with SLS and in the regression phase of a moisturization efficacy study. Significant improvements in barrier function and resistance to the appearance of skin xerosis were observed followingl-lactic acid andd,l-lactic acid, but not followingd-lactic acid treatment. From these results we believe that lactic acid, particularly thel isomer, stimulates ceramide biosynthesis leading to increased stratum corneum ceramide levels which results in superior lipid barrier and a more effective resistance against xerosis.     

Evidence that the corneocyte has a chemically bound lipid envelope

The stratum corneum of mammalian epidermis contains a mixture of ceramides, free fatty acids, cholesterol, and cholesteryl sulfate, amounting to 14% of the dry weight of the tissue, that can be removed by exhaustive extraction with chloroform/methanol. Subsequent mild alkaline hydrolysis liberates additional lipid, consisting almost exclusively of C30-C34 omega-hydroxyacids in amide linkage with sphingosine, equal to 2% of the tissue mass. In the present study, transmission electron microscopy was used to demonstrate that the initial extraction removes the intercellular lamellae that constitute the epidermal water barrier but leaves the lucent band that has been termed the corneocyte plasma membrane. The subsequent alkaline hydrolysis and lipid extraction remove the lucent band, which must therefore contain the omega-hydroxyacylsphingosines. From the results of in situ derivatization of these lipids and the construction of molecular models, it is inferred that the bound lipids exist in ester linkage with protein on the surface of the corneocyte envelope. The tightly packed hydroxyacylsphingosine molecules thus form a lipid envelope for each corneocyte.     

Impaired cutaneous permeability barrier function, skin hydration, and sphingomyelinase activity in keratin 10 deficient mice

Point mutations in the suprabasal cytokeratins 1 (K1) or 10 (K10) in humans have been shown to be the cause of the congenital ichthyosis epidermolytic hyperkeratosis. Recently, a K10 deficient mouse model was established serving as a model for epidermolytic hyperkeratosis. Homozygotes suffered from severe skin fragility and died shortly after birth. Heterozygotes developed hyperkeratosis with age. To see whether phenotypic abnormalities in the mouse model were associated with changes in skin barrier function and skin water content we studied basal transepidermal water loss and capacity for barrier repair after experimental barrier disruption as well as stratum corneum hydration. Also, we determined the activities of acid and neutral sphingomyelinase key enzymes of the tumor necrosis factor and interleukin-1 signal transduction pathways generating the ceramides most important for epidermal permeability barrier homeostasis. Neonatal homozygotes showed an 8-fold increase in basal transepidermal water loss compared with wild type controls. Adult heterozygotes exhibited delayed barrier repair after experimental barrier disruption. Stratum corneum hydration was reduced in homozygous and heterozygous mice. Acid sphingomyelinase activity, which is localized in the epidermal lamellar bodies and generates ceramides for extracellular lipid lamellae in the stratum corneum permeability barrier, was reduced in homozygous as well as heterozygous animals. Neutral sphingomyelinase activity, which has a different location and generates ceramides involved in cell signaling, was increased. The reduction in acid sphingomyelinase activity may explain the recently described decreased ratio of ceramides to total lipids in K10 deficient mice. In summary, our results demonstrate the crucial role of the keratin filament for permeability barrier function and stratum corneum hydration.