Stratum Corneum Ceramides Research Articles

A reconstructed human epidermal keratinization culture model to characterize ceramide metabolism in the stratum corneum

To examine factors that regulate ceramide production during keratinization of the human stratum corneum (SC), we developed a reconstructed human epidermal keratinization model in which a fresh layer of SC is newly formed within 1 week. Addition of the UDP-glucose: ceramide glucosyltransferase inhibitor 1-phenyl-2-decanoylamino-3-morpholino-1-propanol significantly diminished SC ceramide levels (expressed as µg/mg protein) with decreased glucosylceramide levels. Desipramine hydrochloride, an inhibitor of sphingomyelinase, also significantly reduced SC ceramide levels. Similarly, conduritol B epoxide, an inhibitor of β-glucocerebrosidase, significantly down-regulated SC ceramide levels and significantly increased glucosylceramide levels. These results indicate the reliability of this model to elucidate ceramide synthesis regulating factors. Using this model, we assessed the effects of the inflammatory cytokine interleukin-1α (IL-1α), several bioactive sphingolipids and all-trans retinoic acid (RA) on ceramide levels in the SC. Whereas treatment with IL-1α (at 10 nM) significantly down-regulated ceramide levels, treatment with sphingosylphosphorylcholine (at 50 µM) or sphingosine-1-phosphate (at 10 or 20 µM) distinctly up-regulated ceramide levels. Interestingly, RA (at low as 10 nM) significantly up-regulated ceramide levels without affecting the formation of the SC or levels of keratinization-related proteins in the epidermis. The increased levels of ceramide were accompanied by a significantly increased secretion of granulocyte-macrophage colony-stimulating factor as well as by a significantly down-regulated expression of acid-ceramidase at both the gene and protein levels. Taken together, our results underscore the superiority of this reconstructed human epidermal keratinization model to analyze factors that regulate ceramide synthesis, especially in human SC.

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.

Alteration of stratum corneum ceramide profiles in spontaneous canine model of atopic dermatitis

Ceramides (CERs) in the stratum corneum (SC) are thought to play a key role in cutaneous barrier function. It has been reported that human SC contains 11 free CER classes and that their profiles are altered in humans with atopic dermatitis (AD). Although decreased proportions of free CERs or quantities of protein-bound CERs in the SC have been reported in dogs with AD, the overall profile of CERs in the canine SC has not been fully elucidated. The aim of this study was thus to investigate the profile of free CERs in the canine SC and to identify alterations in the CER profiles in dogs with AD. Normal-phase liquid chromatography-electrospray ionization-mass spectrometry indicated 11 clusters of peaks for free CER classes, similar to those recognized in the human SC. The fractions of free SC CER in dogs with AD and in breed- and age-matched healthy dogs were quantitatively compared using high-performance thin-layer chromatography. CER[EOS], CER[EOP] and CER[NP], which are known to be decreased in the skin of humans with AD, were also decreased in the skin of dogs with AD. These findings highlight canine AD as a spontaneous animal model for investigating the disruption of CER-associated cutaneous barrier functions in the corresponding human disease.

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

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

Investigation of the Molecular Structure of the Human Stratum Corneum Ceramides [NP] and [EOS] by Mass Spectrometry

The aim of this study was to characterize the two ceramide (CER) subclasses CER[NP] and CER[EOS] of human stratum corneum and to identify the chemical structures of their subspecies. High-performance thin-layer chromatography and normal-phase high-performance liquid chromatography were used for the separation of CER fractions, whereas nanoelectrospray ionization tandem mass spectrometry was applied to investigate the chemical structures in detail. Thus, CER[EOS] fragmentation revealed that in addition to linoleic acid other esterified fatty acids occur in the ω-position. Of particular interest is the identification of a 17:2 fatty acid located in this part of the molecule. Several subspecies of CER[NP], including subspecies with odd numbers of carbon atoms in both chains, the non-α-hydroxylated fatty acid moiety (part N) and the phytosphingosine (part P), were identified. Furthermore, it was found that 12% of CER[NP] subspecies have an odd number of carbon atoms in both chains of the molecule. Similar results were obtained for CER[EOS]. Both the esterified fatty acid (part E) and the sphingosine base (part S) were found to contain odd-numbered chain lengths. These results underline the heterogeneity of the CER composition in the stratum corneum lipid bilayer.

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 (8–24C) 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.

Structural and functional differences in barrier properties of African American, Caucasian and East Asian skin

Differences in structural and functional skin characteristics have been linked with ethnical background. But racial differences in skin have not been thoroughly investigated by objective methods and the data are often contradictory. This study was undertaken to compare skin barrier-related parameters of the stratum corneum on African American, Caucasian and East Asian skin by objective measurements. Baseline values of trans epidermal water loss were collected on the face. Consecutive stratum corneum D-squame tape strippings were collected on the panelist's ventral forearm and face to evaluate skin barrier strength and cohesion. Stratum corneum ceramides, maturation, measured as the transglutaminase-mediated cross-linking of stratum corneum proteins, and stratum corneum trypsin like enzyme activity were measured on the D-squame tape strippings. East Asian and to some extent Caucasian skin was characterized by low maturation and relatively weak skin barrier. African American skin was characterized by low ceramide levels and high protein cohesion in the uppermost layers of the stratum corneum. These data can be interpreted in terms of the high prevalence of xerosis in black skin and increased skin sensitivity in East Asian skin. These results demonstrate that skin properties at the level of the stratum corneum vary considerably among these ethnic groups. This contributes to an improved understanding of physiological differences between these study populations.

Changes in skin barrier during treatment with systemic alitretinoin: focus on skin susceptibility and stratum corneum ceramides

Alitretinoin is a new drug for systemic treatment of chronic hand eczema. Previous functional tests of skin topically treated with retinoids have indicated impaired skin barrier function, but no data are available on barrier parameters after systemic alitretinoin treatment. To investigate the effect of systemic alitretinoin on skin barrier function and response to irritants, a secondary objective was to determine if changes occur in the lipid profile of stratum corneum after treatment with systemic alitretinoin. We conducted an open clinical intervention study on eight people ascribed to systemic alitretinoin treatment. The criteria for being ascribed to alitretinoin were chronic hand eczema and insufficient therapeutic response to potent topical corticosteroids. Before initiation and after 2 months of systemic treatment with 30 mg alitretinoin, a challenge with sodium lauryl sulphate (SLS) was performed on the volar forearm and evaluated by trans-epidermal water loss (TEWL), erythema, and a cyanoacrylate skin sample was obtained for lipid analysis. We found no significant changes in response to SLS irritation as evaluated by TEWL and erythema, after treatment with alitretinoin for 2 months. No significant changes in stratum corneum lipids were found after 2 months of treatment. In conclusion, systemic alitretinoin does not influence skin susceptibility to irritants or the ceramide profile of stratum corneum.

Analysis of free and protein-bound ceramides by tape stripping of stratum corneum from dogs

The free and protein-bound ceramides of dog stratum corneum (SC) were analyzed by thin-layer chromatography after tape stripping of the abdomen of five dogs. The sphingoid bases were identified by gas-liquid chromatography as sphingosine, phytosphingosine, and 6-hydroxysphingosine. Electrospray ionization-ion trap mass spectrometry was used to characterize the protein-bound ceramides containing sphingosine and omega-hydroxy long-chain fatty acids. Although the molecular species were the same ones in all dogs, wide quantitative variations in the patterns of SC ceramides were observed in different breeds of dogs. The free ceramide concentration changed with the depth of SC, with a higher concentration in the deep layers, whereas the concentration of protein-bound ceramides remained constant. These results show that canine SC is close to that of humans with respect to ceramides.

Investigation of the Detailed Molecular Structure of the Human Stratum Corneum Ceramides [NP] and [EOS] by APCI and Nano-ESI Mass Spectrometry

Investigation of the Detailed Molecular Structure of the Human Stratum Corneum Ceramides [NP] and [EOS] by APCI and Nano-ESI Mass Spectrometry

Percutaneous penetration modifiers and formulation effects

The enhancement/retardation of percutaneous permeation of diethyl- m-toluamide (DEET) in the presence of five percutaneous penetration modifiers (laurocapram, 3-dodecanoyloxazolidin-2-one (N-0915), S, S-dimethyl- N-(4-bromobenzoyl) iminosulfurane (DMBIS), S, S-dimethyl- N-(2-methoxycarbonylbenzenesulfonyl) iminosulfurane (DMMCBI) and tert-butyl 1-dodecyl-2-oxoazepan-3-yl-carbamate (TBDOC)) was investigated. These permeation modifiers were formulated in either water, propylene glycol (PG), ethanol or polyethylene glycol 400 (PEG 400). The permeation studies indicated that laurocapram enhanced DEET permeation in PG, but retarded in PEG 400. Likewise, N-0915 acted as a retardant with ethanol and PEG 400, but not with water. DMBIS decreased the permeation with ethanol as compared to permeation with water, PEG 400 or PG. Similarly, DMMCB acted as a retardant with ethanol and PEG 400, but not with water or PG. TBDOC formulations revealed its activity as a retardant with ethanol, but behaved as enhancer with water, PG and PEG 400. In addition, penetration modifier interactions with stratum corneum ceramide were investigated using chemical modeling. This investigation is significant since it confirms the role of pharmaceutical formulations and shows for the first time that an enhancer can become a retardant or vice versa depending upon the vehicle in which it is applied to the skin. Hence, we should be using the term “penetration modifiers” for all such compounds.

Comprehensive quantification of ceramide species in human stratum corneum

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

Short-Chain Ceramides Decrease Skin Barrier Properties

Stratum corneum ceramides are major determinants of skin barrier function. Although their physiological and pathological role has been widely investigated, to date no structure-activity relationships have been established. In this study, a series of short-chain ceramide analogues with polar head structure identical to ceramide NS, a sphingosine length of 12 carbons and an acyl chain length of 2–12 carbons was synthesized. Their effect on skin permeability was evaluated using porcine skin and two model drugs, theophylline and indomethacin, and compared to that of a physiological ceramide NS. The results showed that the ceramide chain length was crucial for their barrier properties. Ceramides with a 4- to 8-carbon acyl chain were able to increase skin permeability for both drugs up to 10.8 times with maximum effect at a 6-carbon acyl chain. No increase in permeability was found for ceramide analogues with 2- and 12-carbon acyl chains and ceramide NS. The same relationships were obtained for skin concentrations of the model drugs. The relationship between ceramide acyl chain length and its ability to perturb skin barrier showed striking similarity to the behavior of short-chain ceramides in sphingomyelin/phospholipid membranes and confirmed that short-chain ceramides do not act as natural ceramides and their use as experimental tools should be cautious.

Atopic dermatitis: Therapeutic concepts evolving from new pathophysiologic insights

Recent insights into the relevance of the epidermal barrier function and its interaction with components of the innate and adaptive immune responses in patients with atopic dermatitis (AD) give rise to a number of novel potential treatment options. In particular, the identification of loss-of-function mutations in the barrier protein filaggrin and of a diminished expression of certain antimicrobial peptides in AD skin stimulates new concepts to think beyond the T(H)1/T(H)2 paradigm. This review will focus on these most recent discoveries and will discuss new and corresponding proof-of-concept trials in patients with AD. It will further speculate on novel ways to restore the homeostasis among the 3 major components in AD skin suspected to be clinically relevant.

Dietary Phospholipid Concentrate from Bovine Milk Improves Epidermal Function in Hairless Mice

We investigated the effect of dietary phospholipid (PL) concentrate from bovine milk on the epidermis. Thirteen-week-old hairless male and female mice (Hos:HR-1) were separated into two experimental groups, each fed two experimental diets: the control group and the PL group. The mice were given the experimental diets for 6 weeks. Stratum corneum hydration and transepidermal water loss (TEWL) were measured using Corneometer CM825 and Tewameter TM300 (Courage and Khazaka Electronics, Cologne, Germany) at 3 weeks and 6 weeks. After the feeding period, ceramides in stratum corneum were analyzed. We found that stratum corneum hydration and ceramides in the PL group were significantly higher than those in the control group and that TEWL in the PL group tended to decrease. These results indicate that dietary PL concentrate improves epidermal function by increasing the amount of ceramides, resulting in higher hydration.

Characterization of overall ceramide species in human stratum corneum

Ceramides (CERs) in human stratum corneum (SC) play physicochemical roles in determining barrier and water-holding functions of the skin, and specific species might be closely related to the regulation of keratinization, together with other CER-related lipids. Structures of those diverse CER species, however, have not been comprehensively revealed. The aim of this study was to characterize overall CER species in the SC. First, we constructed 3D multi-mass chromatograms of the overall CER species, based on normal-phase liquid chromatography (NPLC) connected to electrospray ionization-mass spectrometry (ESI-MS) using a gradient elution system and a postcolumn addition of a volatile salt-containing polar solvent. The CERs targeted from the 3D chromatograms were structurally analyzed using NPLC-ESI-tandem mass spectrometry (MS/MS), which resulted in the identification of 342 CER species in the inner forearm SC. This led to the discovery of a new CER class consisting of alpha-hydroxy fatty acid and dihydrosphingosine moieties, in addition to the 10 classes generally known. The results also revealed that those CERs contain long-chain (more than C(18))-containing sphingoids and a great number of isobaric species. These novel results will contribute not only to physiochemical research on CERs in the SC but also to lipidomics approaches to CERs in the skin.

The Effect of Gromwell (Lithospermum erythrorhizon) Extract on the Stratum Corneum Hydration and Ceramides Content in Atopic Dermatitis Patients.

A disruption of the balance between the water content of the stratum corneum (SC) and skin surface lipids may lead to the clinical manifestation of dryness of skin in patients with atopic dermatitis (AD). To determine whether supplementation of gromwell (Lithospermum erythrorhizon), one of herbs used in East Asia in remedies for various abnormal skin conditions, may improve the SC level of hydration and ceramides, major lipid in SC in patients with AD. A total of 28 subjects with AD were randomly assigned into two groups: either gromwell group received dextrose contained capsules with 1.5 g of gromwell extracts or placebo group received only dextrose contained capsules for 10 weeks. In contrast to no alteration of SC hydration and ceramides in placebo group, the SC hydration in gromwell group was significantly increased in parallel with an increase of SC ceramides. Furthermore, % increase of SC hydration in gromwell group bore a positive correlation with the clinical severity, which suggests that the increase of SC hydration in gromwell group was more effective as AD was more severe. Supplementation of gromwell improves SC hydration in parallel with an increase of ceramides in part.

Characterization of a Hapten-Induced, Murine Model with Multiple Features of Atopic Dermatitis: Structural, Immunologic, and Biochemical Changes following Single Versus Multiple Oxazolone Challenges

Atopic dermatitis (AD) is a chronic dermatosis bearing clinical, histological, and immunologic similarities to chronic allergic contact dermatitis (ACD). AD shows a Th2 cell-dominant inflammatory infiltrate, elevated serum IgE levels, a permeability barrier abnormality, and Staphylococcus aureus colonization. Repeated hapten challenges reportedly produce a Th2-like hypersensitivity reaction (Th2-like HR). Here, 9-10 challenges with oxazolone (Ox) to hairless mice also produced a chronic Th2-like HR. Permeability barrier function and expression of differentiation proteins, filaggrin, loricrin, and involucrin, became abnormal. CRTH-positive Th2-dominant inflammatory infiltrate, with increased IL-4 expression, and a large increase in serum IgE levels were observed. The barrier abnormality was associated with decreased stratum corneum (SC) ceramide content and impaired lamellar body secretion, resulting in abnormal lamellar membranes, as in human AD. Furthermore, as in human AD, epidermal serine protease activity in SC increased and expression of two lamellar body-derived antimicrobial peptides, CRAMP and mBD3, declined after Ox challenges, paralleling the decrease of their human homologues in AD. Thus, multiple Ox challenges to normal murine skin produce a chronic Th2-like HR, with multiple features of human AD. Because of its reproducibility, predictability, and low cost, this model could prove useful for evaluating both pathogenic mechanisms and potential therapies for AD.

Increase of Skin-Ceramide Levels in Aged Subjects following a Short-Term Topical Application of Bacterial Sphingomyelinase from Streptococcus Thermophilus

Several studies have demonstrated that ceramides play an essential role in both the barrier and water-holding functions of healthy stratum corneum, suggesting that the dysfunction of the stratum corneum associated with ageing as well that observed in patients with several skin diseases could result from a ceramide deficiency. In a previous study our group reported a significant increase in skin ceramide levels in healthy subjects after treatment in vivo with a cream containing a preparation of Streptococcus thermophilus. The presence of high levels of neutral sphingomyelinase activity in this organism was responsible for the observed increase of stratum corneum ceramide levels, thus leading to an improvement in barrier function and maintenance of stratum corneum flexibility. The aim of the present work is to investigate the effects of the topical treatment of a Streptococcus thermophilus-containing cream on ceramide levels of stratum corneum of healthy elderly women. The ceramide levels, transepidermal water loss and capacitance were evaluated on stratum corneum sheets from the forearms of 20 healthy female subjects treated with a base cream or the same cream containing a sonicated preparation of the lactic acid bacterium Streptococcus thermophilus. A 2-week topical application of a sonicated Streptococcus thermophilus preparation led to significant and relevant increase of stratum corneum ceramide levels. Moreover, the hydration values of the treated forearm of each subject was significantly higher than control sites. These results suggest that the experimental cream was able to improve the lipid barrier and to increase a resistance against ageing-associated xerosis.

β‐Glucocerebrosidase Activity in the Stratum Corneum of House Sparrows following Acclimation to High and Low Humidity

Skin is an important avenue of water loss in terrestrial birds, so environmental conditions that necessitate water conservation should favor physiological mechanisms that reduce cutaneous water loss (CWL). Skin resistance to CWL is conferred by a barrier of lipid molecules located in the stratum corneum (SC), the outer layer of the epidermis. In mammals, SC barrier function depends on the conversion of cerebrosides to ceramides by the enzyme beta -glucocerebrosidase ( beta -GlcCer'ase). Avian SC contains both cerebrosides and ceramides, suggesting that observed plasticity in CWL may be mediated by changes in beta -GlcCer'ase activity and resultant SC lipid composition. We tested the hypothesis that changes in ambient humidity would alter beta -GlcCer'ase activity by acclimating house sparrows (Passer domesticus) to either dry (6.5 g H(2)O m(-3) absolute humidity) or humid (31 g H(2)O m(-3)) conditions for 5 and 21 d at 30 degrees C and then measuring beta -GlcCer'ase activity from SC homogenates. Our results provide the first characterization of beta -GlcCer'ase activity in any nonmammalian vertebrate. Relative to nonacclimated controls, both dry- and humid-acclimated sparrows had significantly elevated beta -GlcCer'ase activity at 21 d postacclimation. Across individuals, we observed negative correlations between beta -GlcCer'ase activity and both CWL and SC ceramide content. Although dry- and humid-acclimated sparrows did not differ in beta -GlcCer'ase activity, these results are consistent with our findings that both humidity treatments caused a reduction in CWL and similar changes in SC lipid composition. Our results demonstrate physiological plasticity in CWL and provide tentative support for a role of beta -GlcCer'ase in mediating this response.