Donald C. Swartzendruber

Regional variation in content, composition and organization of porcine epithelial barrier lipids revealed by thin-layer chromatography and transmission electron microscopy

Epidermis and oral epithelia provide permeability barriers that limit penetration of potentially harmful agents. Barrier function is determined by lipids in the superficial epithelial layers and varies regionally by more than 10-fold. The purpose of this study was to determine whether differences in lipid content, composition or organization could account for this variation in barrier function. Stratum corneum from skin, gingiva and palate and superficial layers from buccal regions and the floor of the mouth were isolated, and lipids were extracted and analysed by thin-layer chromatography. Tissue from each region was examined by electron microscopy. There was an inverse correlation between permeability and ceramide content and a direct correlation with triglyceride content. Electron microscopy revealed that the intercellular space in epidermal stratum corneum contained multiple lipid lamellae displaying an alternating broad-narrow-broad spacing. In palatal and gingival stratum corneum, uniformly spaced lamellae were present at the periphery of dilations of the intercellular space, but the interiors of the dilations contained disorganized lamellae and electron-dense material. In the non-keratinized barriers, there was a single, broad lamella at the cell periphery and occasional short stacks of lamellae traversing the intercellular space. These intercellular lamellae may be derived from a population of membrane-coating granules that contain internal lamellae. The results suggest that ceramides may be important barrier components, even in non-keratinizing epithelia where they are very minor components. Regional differences in the physical organization of barrier lipids may also contribute to differences in barrier function.

Regional variation in the structure and permeability of oral mucosa and skin

Abstract Molecules of low to moderate size are capable of passage through the skin and oral mucosa by simple diffusion. The diffusional resistance of the tissue is determined primarily by lipids in the intercellular spaces of the outer layers of the epithelium. In the skin, palate, and gingiva, this barrier function is provided by a stratum corneum, the intercellular spaces of which contain mainly ceramides, cholesterol and fatty acids. The more flexible regions of the oral mucosa are lined by a non-keratinizing epithelium, the outer half to one-third of which provides a permeability barrier. The barrier region of the non-keratinized epithelia contains phospholipids, cholesterol and monohexosylceramides as major constituents. Differences in the composition and organization of lipids in the epithelial barriers result in regional variation in permeability.

Effects of essential fatty acid deficiency on epidermal O-acylsphingolipids and transepidermal water loss in young pigs

Linoleate-rich O-acylglucosylceramides and acylceramides are thought to be of major significance for the physical structure and function of the epidermal permeability barrier. In the present investigation, the effects of a linoleate-free diet on O-acylsphingolipids and their associated functions were investigated. Starting at 5 days of age, male pigs were fed diets containing 12% of either lard or hydrogenated coconut oil. Transepidermal water loss was measured with an electrolytic water analyzer at weekly intervals. Pigs were killed at intervals, and epidermal lipids were isolated and analyzed. Fatty acid compositions were determined by gas-liquid chromatography (GLC). Within 2-3 weeks, pigs on the diet containing coconut oil began to display biochemical and physiological symptoms of essential fatty acid deficiency. Within 2 months, this group had extremely scaly skin and transepidermal water loss was elevated to five times that of controls. The progressive increase in transepidermal water loss correlated with replacement of linoleate by oleate in both acylceramide and acylglucosylceramide. The formation of lamellar granules and intercellular lipid sheets in the stratum corneum was not impaired in essential fatty acid deficiency as judged by electron microscopy. These results suggest that the linoleic acid normally found in the O-acylsphingolipids is not essential for formation of the epidermal membrane system. Rather, it appears that the nature of the ester-linked fatty acid in the O-acylsphingolipids regulates the permeability of this membrane system.

In vitro reconstitution of stratum corneum lipid lamellae

In the final stages of differentiation in the epidermis of terrestrial mammals, lipids are extruded into the intercellular spaces. The initially extruded lipid becomes transformed into broad, multilamellar sheets that are found in the intercellular spaces throughout the stratum corneum. These lamellae display an unusual alternating broad-narrow-broad pattern of lucent bands as revealed by transmission electron microscopy (TEM). This arrangement results in two periodicities that can be measured from electron micrographs and are also evident in X-ray diffraction-5 nm (broad) and 13 nm (broad-narrow-broad). The goal of the present study was to reconstitute these lamellae in vitro. Porcine stratum corneum lipids were applied to Millipore filters. The disks were placed in water and heated to 80 degrees C for 1 h. After cooling, the disks were stored over desiccant. At each stage, the disks were prepared for TEM. TEM revealed that the application of the lipid solutions onto the disks resulted to deposition of mostly amorphous material. Heating in water resulted in the formation of many lamellae. The width of the lamellae was uniform and in the range of 5 to 6 nm with no broad-narrow-broad pattern; however, after storage under desiccating conditions, the broad-narrow-broad pattern was reproduced.

Organization of the intercellular spaces of porcine epidermal and palatal stratum corneum: a quantitative study employing ruthenium tetroxide.

Previous studies have demonstrated that the intercellular spaces of the stratum corneum contain multilamellar lipid sheets with variable ultrastructure in addition to desmosomes or desmosomal remnants. The intercellular lamellae are thought to provide a permeability barrier whereas the desmosomes are responsible for cell-cell cohesion. In this study, transmission electron microscopy of RuO4-fixed tissue was used to compare the proportions of the intercellular spaces in epidermal and palatal stratum corneum occupied by desmosomes and by different patterns of lamellae. Desmosomes are more abundant in palatal than in epidermal stratum corneum (46.9 vs 15.0% length of intercellular space). In epidermis the most frequent lamellar arrangements involve 3 (23.5%) or 6 (24.2%) lucent bands with an alternating broad-narrow-broad pattern, whereas the most frequent lamellar arrangements in palatal tissue are 2 (17.2%) or 4 (10.5%) lucent bands of uniform width. Most of the nondesmosomal portion of the intercellular space in palatal stratum corneum was dilated and had elongated lamellae at the periphery and short disorganized lamellae and amorphous electron-dense material in the interior. It is concluded that the multilamellar lipid sheets are less extensive in palatal than in epidermal stratum corneum, which could explain the greater permeability of the palate.

Isolation of corneocyte envelopes from porcine epidermis

SummarySheets of porcine stratum corneum were dispersed into individual corneocytes after 4 h in a solution consisting of 8 mM N,N-dimethyldodecylamine oxide and 2 mM sodium dodecylsulfate in phosphate-buffered isotonic saline, at 45°C. With continued detergent treatment and moderate sonication, most of the cells lost their keratin contents and were then separated from the remaining intact cells by centrifugation in cesium chloride solution of density 1.280. Electron microscopy showed that the cell envelopes retained both the cross-linked protein envelope and its attached lipid envelope. The dry weight of envelopes was approximately 7% of the estimated dry weight of the original stratum corneum, while the corneocytes surviving intact also amounted to 7% of the starting weight. Mild alkaline hydrolysis of the corneocyte envelopes allowed the extraction of hydroxyceramides amounting to 10% of the dry weight of the envelopes. The procedure therefore provides isolated corneocyte envelopes suitable for studying both the protein and lipid components of this compound sheath.

Covalently bound lipids in keratinizing epithelia

Covalently bound lipids have been identified and compared in keratinizing porcine epithelia including epidermis and oral epithelium from palate and gingiva. Stratum corneum was isolated by tryptic digestion, and after extensive extraction of lipids using a series of chloroform-methanol mixtures, the residual tissue was subjected to alkaline hydrolysis to release covalently bound lipids. The lipids so released were analyzed by quantitative thin-layer chromatography. Stratum corneum from each of the three anatomical sites contained omega-hydroxyceramides, omega-hydroxyacids and fatty acids. In epidermal stratum corneum the total covalently bound lipids represented 2.4% of the dry weight of the tissue, but in the oral epithelia this figure was consistently lower: 0.24% in palatal stratum corneum and 0.20% in gingival stratum corneum. Transmission electron microscopy before and after lipid extraction confirms the presence of a lipid envelope in epidermal stratum corneum and demonstrates the absence of this structure in oral stratum corneum.

Lipids of hamster cheek pouch epithelium

The hamster cheek pouch is a much used but incompletely understood experimental model. In particular, the cheek pouch epithelial lipids, which are important for permeability barrier function as well as other aspects of epithelial biology, have not been completely characterized. In the present study, the complete lipid class composition has been determined by thin-layer chromatography in conjunction with photodensitometry. The major lipid classes were phospholipids, free sterols, and ceramides. Minor amounts of monohexosylceramides, sterol esters, fatty acids, and triglycerides were also present. Significant amounts of covalently bound ω-hydroxyceramide was also detected. Transmission electron micrographs reveal extensive, largely paired, lipid bilayers in the intercellular spaces of the stratum corneum.