Human Stratum Corneum Lipids Research Articles

Individual variation of human plantar stratum corneum lipids, determined by automated multiple development of high-performance thin-layer chromatography plates.

The stratum corneum lipids are unique in composition and have been used frequently as a model system of the skin's lipid barrier. Automated multiple development (AMD) of high-performance thin-layer chromatography plates in combination with a 25-step gradient, based on methanol, diethyl ether and n-hexane separated the six major human plantar stratum corneum lipids. Post-chromatographic staining of these lipids with a solution of MnCl2-H2SO4 at 130 degrees C or a solution of CuSO4-H3PO4 at 140 degrees C allowed visualization of the lipids and quantification. The MnCl2-H2SO4 solution stained saturated fatty acids less intensely. Therefore, the CuSO4-H3PO4 solution was used for quantification and we found, on average, 2.06% (w/w) cholesterol 3-sulphate, 20.16% (w/w) free fatty acids, 20.25% (w/w) ceramides, 43.53% (w/w) non-esterified sterols, 4.56% (w/w) triacylglycerols and 9.4% (w/w) sterolesters in the human plantar stratum corneum extracts. The concentration of phospholipids was less than 1% (w/w). In addition, the lipid composition of twenty different human plantar stratum corneum extracts was determined. Statistics revealed a correlation between the ratio of free fatty acids and non-esterified sterols (r=0.832, p<0.01, n=20). Several control experiments proved that this correlation is not due to the extraction method, the post-chromatographic staining procedure or bacterial contamination of the stratum corneum.

Lateral diffusion of small compounds in human stratum corneum and model lipid bilayer systems

An image-based technique of fluorescence recovery after photobleaching (video-FRAP) was used to measure the lateral diffusion coefficients of a series of nine fluorescent probes in two model lipid bilayer systems, dimyristoylphosphatidylcholine (DMPC) and DMPC/cholesterol (40 mol%), as well as in human stratum corneum-extracted lipids. The probes were all lipophilic, varied in molecular weight from 223 to 854 Da, and were chosen to characterize the lateral diffusion of small compounds in these bilayer systems. A clear molecular weight dependence of the lateral diffusion coefficients in DMPC bilayers was observed. Values ranged from 6.72 x 10(-8) to 16.2 x 10(-8) cm2/s, with the smaller probes diffusing faster than the larger ones. Measurements in DMPC/cholesterol bilayers, which represent the most thorough characterization of small-solute diffusion in this system, exhibited a similar molecular weight dependence, although the diffusion coefficients were lower, ranging from 1.62 x 10(-8) to 5.60 x 10(-8) cm2/s. Lateral diffusion measurements in stratum corneum-extracted lipids, which represent a novel examination of diffusion in this unique lipid system, also exhibited a molecular weight dependence, with values ranging from 0.306 x 10(-8) to 2.34 x 10(-8) cm2/s. Literature data showed that these strong molecular weight dependencies extend to even smaller compounds than those examined in this study. A two-parameter empirical expression is presented that describes the lateral diffusion coefficient in terms of the solute's molecular weight and captures the size dependence over the range examined. This study illustrates the degree to which small-molecule lateral diffusion in stratum corneum-extracted lipids can be represented by diffusion in DMPC and DMPC/cholesterol bilayer systems, and may lead to a better understanding of small-solute transport across human stratum corneum.

Azone® induced fluidity in human stratum corneum. A fourier transform infrared spectroscopy investigation using the perdeuterated analogue

The effect of Azone® on human stratum corneum (SC) lipid fluidity was investigated by the use of FT-IR spectroscopy and its perdeuterated analogue (D-azone). SC lipid fluidity was monitored in terms of the degree of organisation of the lipid acyl chains (reflected by changes in the frequency of the C-H 2 symmetric stretching frequency) and in terms of the rate of motion of the lipid chains within the ordered environment (reflected in changes in the bandwidth of the C-H 2 symmetric stretching frequency at half maximum height). Two levels of D-azone treatment were used. At the lower level results indicated that D-azone did induce disordering of the lipid acyl chains by the introduction of gauche conformers above the SC lipid gel to liquid crystalline phase transition midpoint (T m). The rate of motion of the chains was increased at lower temperatures indicating that D-azone did affect one aspect of SC lipid fluidity below T m. At the higher level of D-azone treatment the SC lipid fluidity was increased with respect to both aspects of fluidity examined, both above and below the SC lipid phase transition. This action could suggest how the penetration of molecules through the skin is enhanced by Azone. The data also seem to suggest a homogeneous distribution of D-azone through the SC lipid array.

Quantitative screening of human stratum corneum lipids by sequential one‐dimensional thin layer chromatography

There is a need in many studies on the epidermal permeability barrier for practical and reliable separation of all major fractions of human stratum corneum lipids. Various methods have been described including thin layer chromatography, high performance thin layer chromatography and iatroscan. However, none of these methods seems to be applicable for an inexpensive, rapid and reliable analysis of a large number of samples. Here, such a method for the separation and quantification of all major stratum corneum lipid fractions is presented. This method employs the one-dimensional separation of stratum corneum lipids using thin layer chromatography. The systems used herein are based upon various modifications of the solvents, solvent ratio and developing distance of each system. For quantification of chromatographed and charred lipids a Desaga densitometer is used. The system described here had been successfully applied to 550 samples of human stratum corneum lipids.

Apparent pKa of the fatty acids within ordered mixtures of model human stratum corneum lipids.

The apparent pKa of the fatty acids within hydrated (30% w/w) model human stratum corneum (SC) lipid mixtures should be measured. The degree of ionisation of the fatty acids was calculated as a function of pH using Fourier transform infra-red spectroscopy. The relative intensity of the stretching bands of the unionized and ionized carboxylic groups was determined and fitted to the relevant expression for ionic equilibrium of a monoprotic acid. The pKa was then calculated for increasing proportion of unsaturated fatty acid in the lipid mixture. Values for pKa in the range 6.2-7.3 were found, increasing with greater proportion of oleic acid. These are some 1.5-3 pH units higher than the pKas of fatty acids in molecular solution. As there exists a pH-gradient across the SC, the degree of ionisation will also vary. In the innermost SC layers, a pH of 7 will produce 90% ionization of the fatty acids and head-group repulsion will be great. At the SC surface, the pH of 5 will cause almost minimal head-group repulsion, tending to increase crystallinity and promote a bilayer structure.

Thermoelectrical analysis of the human skin barrier

The influence of temperature on the resistive and capacitative properties of human stratum corneum in vitro was studied to determine where within substructures of stratum corneum, the electrical resistance R and capacitance C components reside. Heating-cooling cycles were designed in accordance with earlier calorimetric and spectroscopic studies of thermal transitions of human stratum corneum lipids and/or proteins. Two different protocols were used. (A) Heat treatment and electrical analysis were carried out simultaneously in pH 7.4 phosphate buffered saline, starting with prehydrated stratum corneum (70% w/w) of pH 7.4. (B) Heat treatment was performed before electrical analysis, using dried stratum corneum (< 10% w/w), followed by prehydration and measurement of the electrical properties in phosphate buffered saline at 20°C. Square-wave alternating current pulses of 13 μA cm −2 were applied every 60 s. Analysis of the resulting voltage waveform across stratum corneum yielded an equivalent electrical model of stratum corneum composed of a series connection of two RC circuits ( R 1 ∥ C 1 and R 2 ∥ C 2 ). Below 60°C a constant activation energy of 5.4 ± 0.7 kcal mol −1 was measured, which was close to the activation energy of K + diffusion in a fluid aqueous medium. The total resistance of stratum corneum was less than 100 kΩ cm 2 , which is very low compared to the resistance of black lipid membranes (1–10 MΩ cm 2 ). Both the low activation energy and resistance of human stratum corneum suggest the presence of highly conductive pathways through the membrane. Between 60 and 75°C an abrupt decline of the resistances R 1 and R 2 and a rapid rise of the capacitances C 1 and C 2 was observed. This temperature interval corresponded to the temperature interval of the second thermal transition observed in human stratum corneum, which is a lipid phase transition. Beyond 75°C, the resistances were fairly constant, while the capacitances continued to increase. The changes in the resistances and capacitances brought about by heating to 75 and 95°C were completely irreversible. This is in agreement with X-ray diffraction studies, which have shown that the originally predominant lamellar structure with a repeat distance of 6.4 nm does not reappear after recrystallization from 75 and 90°C. The final resistances after heating desiccated stratum corneum (⩽ 10% w/w hydration) to temperatures of 75 and 95°C and subsequent cooling were less different from the original values than those of hydrated samples, suggesting that the impact of heating depends on the level of stratum corneum hydration. After chloroform-methanol lipid extraction, stratum corneum was characterized by a single RC circuit. The resistance of extracted stratum corneum was less than 1% of the original pre-extraction value, which indicates that the resistance of stratum corneum is mainly determined by the intercellular lipids. Taken together, the correlation between the temperature dependence of the resistances and capacitances between 20 and 75°C suggests that the electrical barrier and the charge storage capacity of stratum corneum are determined by the same substructures. Most probably these structures are the intercellular lipid lamellae, because the changes of the electrical properties mainly take place within the interval 60 to 75°C, which corresponds to the phase transition of free intercellular lipids, and also because these changes are irreversible. This conclusion is also confirmed by chloroform-methanol extraction. However, the continued increases of the capacitances between 75 and 95°C, indicates that the charge storage capacity of stratum corneum also depends on lipids attached to protein. The low resistance and activation energy at temperatures between 20 and 60°C indicate that stratum corneum possesses highly conductive pathways.

6-Hydroxy-4-sphingenine in human epidermal ceramides.

The solvent-extractable lipids of human epidermal stratum corneum consist predominantly of ceramides. In addition two non-extractable ceramides are chemically bound to the stratum corneum protein. One of the bound ceramides, constituting 50% of the bound lipids, was previously shown to consist of very long chan omega-hydroxyacids in amide linkage with sphingosine. The second bound caramide, which forms 25% of the bound lipids, was shown to contain the same hydroxyacids, but the sphingoid base was neither sphingosine nor phytosphingosine. In the present study, the undefined bound ceramide was shown by NMR and chemical procedures to be the omega-hydroxyacid derivative of a new base, 6-hydroxy-4-sphingenine. In addition, a ceramide previously known to constitute 25% of the extractable human stratum corneum ceramides has been found to contain the same novel sphingoid base, amide-linked to long-chain alpha-hydroxyacids. Finally, a new acylceramide has been isolated and identified that consists of very long chain omega-hydroxyacids in amide linkage with the novel sphingolipid, with fatty acids esterified wit the terminal hydroxyl group of the hydroxyacid.

Diffusivity and structural polymorphism in some model stratum corneum lipid systems

Mixtures of model stratum corneum lipids were prepared in water from cholesterol, six fatty acids and ceramides. The influence of composition on the polymorphism of these mixtures and also on the diffusivity of a model drug within them, D lip , was determined. The former was obtained from X-ray diffraction and Fourier transform infrared spectrometry, and the latter from a diffusional release model. An Lβ structure was formed for the composition approximating that of the extracellular lipids in intact human abdominal stratum corneum. D lip was independent of water content in the range 20–40% w/w, with the bilayers showing one dimensional swelling without lateral expansion. Although removal of the ceramides did not result in a significant alteration in D lip , crystalline cholesterol now appeared. The ceramides were, therefore, necessary for solubilization within the fatty acid bilayers of the large proportion of cholesterol present in the lipid fraction of intact SC. They were also responsible for a thermal L α-H II transition observed at approx. 68°. At the concentration in which it exists in intact SC, cholesterol also had only a minimal effect on D lip , but was necessary to suppress H II phase formation within the fatty acids and ensure an Lβ structure. All lipid mixtures that had an L β structure presented a diffusional barrier approx. 1 order of magnitude greater than that of an unstructured, isotropic lipid mixture. H II structures formed at cholesterol/fatty acid proportions less than approx 8:92 mol% and appeared more permeable than L β ones. All the results indicate that the diffusional barrier within the model lipid mixtures is guaranteed essentially by the presence of an L β phase. Although the ceramides and cholesterol exert no intrinsic influence on the magnitude of D lip , their presence in necessary for the existence of an L β phase at 33° that is free of both crystalline cholesterol and H II character.

Transdermal absorption of steroids

The transport mechanisms of selected steroids in the presence of a series of aqueous ethanol solutions have been probed in vitro using hairless mouse and human tissue, as well as in vivo using the unique human skin sandwich flap model. Permeation and biophysical studies have also been performed under similar conditions in order to elucidate the biophysical alterations in hairless mouse and human stratum corneum induced by the vehicle solutions that contribute to the percutaneous transport mechanisms of steroids. Progesterone, estradiol and hydrocortisone transport across hairless mouse skin under the same conditions was examined to explore the effects of permeant properties. Steroid uptake and in vitro permeability coefficients across both skin species decreased with decreasing steroid lipophilicity. The polar character of the steroids influences not only partitioning, but also the effective diffusivity in the stratum corneum, as suggested from permeability coefficients calculated with a mathematical model. As steroids become more polar, the effective diffusivities decrease and the degree to which the stratum corneum controls overall diffusion increases. Estradiol permeability coefficients in vivo with the human skin sandwich flap were 10-fold higher than with human epidermis in vitro. Estradiol uptake and permeability coefficients decreased with the presence of increasing ethanol concentrations. Similarities in estradiol permeation in vitro through hairless mouse skin and human epidermis were further reflected in similarities of the human and hairless mouse stratum corneum lipid domain biophysical structures.

Influence of UV Irradiation on the Composition of Human Stratum Corneum Lipids

Irradiation with suberythemal doses of either UV-A or UV-B yielded an increase in the amount of stratum corneum lipids extracted from the lumbar skin area of 20 volunteers. These lipids were quantified after separation by high-performance thin-layer chromatography. Ten subfractions in the ceramide region were separated; two of them (fractions 7a and 7b) were only detectable after UV-A or UV-B irradiation. Improvement of barrier function after UV irradiation of human skin with suberythemal doses may be related to an increase in the stratum corneum ceramides.

Microanalytical Screening of All Major Stratum Corneum Lipids by Sequential High-performance Thin-layer Chromatography

For rapid and sensitive screening of lipid biochemical abnormalities of scaling skin disorders a sequential, one-dimensional high-performance thin-layer chromatographic method (HPTLC) has been developed. All major human stratum corneum lipid classes, i.e., cholesterol sulfate, glucosylceramides, six major ceramide fractions, free sterols, free fatty acids, triglycerides, sterol esters, squalene, and n-alkanes, are separated and quantitated after a stepwise development of a single silica gel 60 HPTLC-plate using three consecutive solvent systems. Reproducible results have been obtained by degradative charring as well as fluorescence detection. By fluorescence detection the method is particularly suitable for the determination of minor amounts of cholesterol sulfate and other sterols.

Covalently Bound Lipids of Human Stratum Corneum

In the present study, we demonstrate that human stratum corneum contains covalently bound lipids accounting for 1.4% of the dry weight of the tissue. The major component (53.3% of the total by weight) is a ceramide (CER-A) consisting of 30 through 34-carbon omega-hydroxyacids amide-linked to sphingosine. The other bound lipids in human stratum corneum include fatty acids (12.7%), omega-hydroxy acids (9.4%), and a second, more polar, omega-hydroxyacid-containing ceramide (CER-B, 24.8%). The predominant omega-hydroxyacids in both ceramides, as well as the free hydroxyacid fraction, are the 30-carbon saturated and 32- and 34-carbon monoenoic species. The bound fatty acids consist largely of 14 through 22-carbon saturated species, but significant proportions of monoenoic species and linoleic acid are also present. Psoriatic scale contains a similar total concentration of the same covalently bound lipids, but the proportions of the individual bound lipids are different from those found in normal stratum corneum. It is suggested that the principal function of the covalently bound lipids in human stratum corneum is the formation of a lipid envelope on the outer surface of the keratinized cells.

In Vitro and In Vivo Antistaphylococcal Activity of Human Stratum Corneum Lipids

Despite the assumption that sebum-derived fatty acids are responsible for cutaneous antimicrobial defense, no studies have assessed the contribution of epidermis-derived lipids. Herein, we tested the antistaphylococcal effects of human stratum corneum lipids, enriched in endogenous, keratinocyte-derived species obtained by lipid extraction and thin-layer chromatography, for antimicrobial activity in both in vitro and in vivo systems. Whereas the most potent species in vitro were the free fatty acids, polar lipids and glycosphingolipids also demonstrated antistaphylococcal activity in vitro, while other neutral lipids displayed virtually none, results that were confirmed with authentic standards in vitro. In a pilot study on delipidized forearm test sites in human volunteers, naturally occurring free fatty acids, polar lipids, and glycosphingolipids exhibited significantly more antistaphylococcal activity than other stratum corneum lipids or vehicle controls. Finally, biopsy specimens of incubated skin sites demonstrated penetration of staphylococci through lipid-enriched intercellular domains. These results provide the first evidence that endogenous, epidermis-derived skin lipids may contribute to cutaneous antimicrobial resistance.

In vitro and in vivo antistaphylococcal activity of human stratum corneum lipids

In vitro and in vivo antistaphylococcal activity of human stratum corneum lipids

Human stratum corneum lipids: characterization and regional variations

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