Isolated Stratum Corneum Research Articles

Percutaneous absorption of benzoic acid across human skin. I. In vitro experiments and mathematical modeling.

The percutaneous absorption of benzoic acid across human skin in vitro was experimentally and mathematically modeled. Skin partition coefficients were measured over a range of benzoic acid concentrations in both saline and distilled water. The permeation of benzoic acid was measured across isolated stratum corneum, stratum corneum and epidermis, and split-thickness skin. These experiments demonstrated that the stratum corneum was the rate-limiting barrier and that the flux is proportional to the concentration of the undissociated species. The permeation data were analyzed with a comprehensive non-steady-state mathematical model of diffusion across skin. Two adjustable parameters, the effective skin thickness and diffusivity, were fit to the permeation data by nonlinear regression.

The Role of the Corneocyte Lipid Envelopes in Cohesion of the Stratum Corneum

Treatment of isolated stratum corneum with certain detergents results in complete disaggregation of the corneocytes within hours at 45 degrees C without agitation. This is prevented by prior heating of the tissue to 80 degrees C or by solvent extraction of the intercellular lipids. In the present study, electron microscopy revealed that the heated or solvent-extracted tissue was characterized by cell-to-cell contacts that appeared to involve the chemically bound hydroxyceramides which constitute the corneocyte lipid envelope. It is proposed that the irreversible bonding between corneocytes that results from heating or lipid extraction results from interdigitation of the sphingosine chains belonging to those hydroxyceramides that are bound to the corneocyte protein envelope by the omega-hydroxyl function of the 30- and 32-carbon hydroxyacid moieties. Similar interdigitation of adjacent envelopes might be involved in natural stratum corneum cohesion, limited mostly to the periphery of corneocytes where the absence of intercellular lamellae allows the appropriate cell-to-cell contact.

FUNCTIONS OF LIPIDS ON HUMAN SKIN

Abstract In order to clarify the roles of lipids in the water-holding property of stratum corneum, the forearm skin of healthy male volunteers was treated with acetone/ether (1/1) or sodium dodecyl sulfate (5%) for 1-30 min. A prolonged treatment period of 5-30 min produced a chapped and scaly appearance of the stratum corneum without any inflammatory reactions. Under these conditions, there was a marked decrease in the water-holding capacity of the stratum corneum accompanied by a selective loss of stratum corneum lipids such as cholesterol, cholesterol esters, and sphingolipids. Two daily applications of the isolated stratum corneum lipids to experimentally induced dry skins caused a significant increase of conductance, accompanied by a marked improvement in the level of scaling. Meanwhile, the isolated sebaceous lipids exhibited no significant recovery in both the conductance value and the scaling. Out of chroma-tographically separated fractions of the stratum corneum lipids, topical applications of ce...

Heterogeneity effects on permeability-partition coefficient relationships in human stratum corneum.

The relationship between the permeability of solutes undergoing transport via the lipid pathway of the stratum corneum and the degree to which the same solutes partition into the stratum corneum has been explored by measuring the permeability coefficients and stratum corneum/water partition coefficients of a series of hydrocortisone esters varying in lipophilicity. Isolated human stratum corneum, used in both the permeability and the uptake experiments, was shown to resemble full-thickness skin in its overall resistance and selectivity to solute structure. As with full-thickness skin, delipidization destroys the barrier properties of isolated stratum corneum. Although a linear relationship is frequently assumed to exist between permeability coefficients and membrane/water partition coefficients, a log-log plot of permeability coefficients versus the intrinsic stratum corneum/water partition coefficients for the series of hydrocortisone esters studied is distinctly nonlinear. This nonlinearity arises from the fact that the transport of these solutes is rate limited by a lipid pathway in the stratum corneum, while uptake reflects both lipid and protein domains. From the relative permeability coefficients of 21-esters of hydrocortisone varying in acyl-chain structure, group contributions to the free energy of transfer of solute into the rate-limiting barrier microenvironment of the stratum corneum lipid pathway are calculate for a variety of functional groups including the -CH2-, -CONH2, -CON(CH3)2, -COOCH3, -COOH, and -OH groups.(ABSTRACT TRUNCATED AT 250 WORDS)

Filaggrin breakdown to water binding compounds during development of the rat stratum corneum is controlled by the water activity of the environment

Filaggrin is a specific epidermal protein which is the precursor of the free amino acids, urocanic acid and pyrrolidone carboxylic acid which are largely responsible for the ability of the stratum corneum of the skin to remain hydrated at low environmental humidity. The distribution of filaggrin shown by immunofluorescence in the stratum corneum of the rat changed dramatically during the first hours of postnatal life. During late foetal development, filaggrin accumulated through the entire thickness of the stratum corneum, indicating that there was a block on the subsequent processing of the protein which normally would convert it to free amino acids. Immediately after birth this block was lifted and normal proteolysis of the filaggrin took place in the outer part of the stratum corneum, leaving the normal adult pattern of a thin zone of cells containing filaggrin at the bottom of the stratum corneum. This activation of filaggrin proteolysis was dependent on the drop in external water activity caused by the transition from an aqueous environment in utero to a dryer environment after birth and it could be blocked by maintaining a 100% humidity atmosphere around the newborn rat after birth. In isolated stratum corneum in vitro, filaggrin proteolysis took place only between 80 and 95% relative humidity, both higher and lower relative humidity blocked the proteolysis. Application of occlusive patches to adult rats prevented the normal proteolysis of filaggrin, indicating that this mechanism controls not only the massive filaggrin proteolysis occurring after birth but also the proteolysis occurring during normal stratum corneum maturation. The stratum corneum therefore has the ability to respond to changes in external humidity by altering the level of the stratum corneum where it converts its reserves of filaggrin into water binding amino acids, such that under humid conditions water binding components with be produced in only the most superficial stratum corneum, or even not produced at all.

Transmission of human epidermis and stratum corneum as a function of thickness in the ultraviolet and visible wavelengths.

Abstract The dependence of radiation transmission on sample thickness was studied in isolated samples of human stratum corneum and full‐thickness epidermis. The investigation also included samples of skin repeatedly exposed to UV‐B. Transmission was measured in the ultraviolet and in the visible from 248–546 nm. Two methods, one microscopic and the other mechanical, were used to measure thickness. There was a good correlation between the results.The dependence of transmission on thickness in these samples could be described satisfactorily by an exponential function, implying that the Lambert‐Beer law is approximately valid. Thus, a single parameter, such as the half‐value layer (d½), is sufficient to characterize absorption in the skin samples.Water content of the isolated stratum corneum was influenced by maintenance conditions: samples floating on water containing a small amount of NaCl were more hydrated than samples floating on a more concentrated salt solution, or stored in air. Changes in water content of the samples resulted in changes of thickness and, to a lesser extent, of transmission. Approximate in vivo values of d½ were computed after estimating the in vivo water content of stratum corneum.Differences found in the shape of the transmission spectra of stratum corneum and full‐thickness epidermis may reflect differences in chemical composition. The influence of wetting of the skin on its sensitivity to sunlight is explained in a new way.

Isolation and Lipid Biochemical Characterization of Stratum Corneum Membrane Complexes: Implications for the Cutaneous Permeability Barrier

Despite substantial histochemical and ultrastructural evidence suggesting that mammalian stratum corneum is a heterogenous tissue composed of lipid-depleted corneocytes embedded in a neutral lipid-enriched matrix, controversy remains about the distribution of lipids in this layer. In this study we attempted to resolve this issue by cell fractionation and lipid biochemistry of isolated stratum corneum membrane complexes. Intact homogeneous sheets of stratum corneum were obtained from neonatal mice by treatment first with the staphylococcal epidermolytic toxin and then by removal of residual granular cells with trypsin. The resultant sheets were pooled, pulverized at −196°C and homogenized at 20–30,000 psi in a Stansted cell disrupter. Treatment with 0.005% Subtilisin (protease-type VIII) in Tris-HCl buffer, pH 8.8, for 60min at 25°C stripped keratin filaments from beneath the plasma membrane. The resulting homogeneous population of apposed membrane complexes revealed trapped intercellular lipid bilayers when it was examined ultrastructurally. Whereas whole stratum corneum sheets contained 10% lipid/total lipid + protein weight, purified membrane complexes contained about 50% lipid, a 5-fold concentration of lipid in membrane fractions over whole cells. From available quantitative data stratum corneum cytoplasmic domains contained less than 3% lipid, while membrane complexes accounted for over 80% of the total stratum corneum lipid. Moreover, freeze-fracture replicas of isolated membrane complexes revealed intercellular membrane bilayers that were: ( a ) identical to those encountered in whole stratum corneum and ( b ) removed by lipid solvent extraction. Finally, thin-layer chromatography of lipids extracted from membrane complexes revealed a similar spectrum and weight distribution of lipids as encountered in extracts from whole sheets. These results provide direct evidence that stratum corneum cell membrane regions are preferentially enriched in lipids over whole tissue, confirming earlier morphological studies that lipids are progressively segregated in membrane domains during cornification. This heterogeneous structural/chemical organization has profound implications for skin barrier function, stratum corneum cohesion, and for avenues of percutaneous transport.

Factors controlling the expressed activity of histidine ammonia-lyase in the epidermis and the resulting accumulation of urocanic acid.

The synthesis of urocanic acid by histidine ammonia-lyase in guinea-pig epidermis was investigated in various ways. 1. In epidermal homogenates the enzyme obeys Michaelis-Menten kinetics and shows marked dependence of its activity of pH, such that below pH 6 it is inactive. 2. Part-thickness skin samples cultured with radioactive histidine do not accumulate detectable radioactive urocanic acid during 3 days in culture. 3. Very little histidine ammonia-lyase activity can be detected in the living cells of the epidermis. The enzyme is almost completely restricted to the dead cells of the stratum corneum. 4. Radioactive histidine injected into living animals does not result immediately in the accumulation of radioactive urocanic acid. By 3 days after the injection, however, both radioactive urocanic acid and histidine appear, apparently at the expense of radioactive proteins, 5. In isolated stratum corneum, the residual histidine can be converted into urocanic acid by the histidine ammonia-lyase in the tissue only if the natural acidity of the tissue is neutralized. It is concluded from these observations that the biosynthesis of urocanic acid occurs naturally only in the stratum corneum, which contains only dead cells. The amount of urocanic acid accumulated is limited by the availability of free histidine produced by proteolysis of residual protein in these cells and by the penetration into the stratum corneum of the 'acid mantle' of the skin.

Immunologic Cross-reaction of Stratum Corneum Basic Protein and a Keratohyalin Granule Protein

Antiserum to the stratum corneum basic protein (SCBP) of newborn rat epidermis was used to test for cross-reactive proteins in extracts of skin and in tissue sections. The antibody reacts strongly with epidermal extracts but very poor with dermal extracts. Buffer extracts of epidermis give a reaction of partial identity or identity with the antigen, SCBP. Urea extracts of isolated stratum corneum give a reaction of identity with SCBP. When the proteins of these extracts are separated by SDS polyacrylamide gel electrophoresis, the immunoreactive peak in the 4 M urea extract corresponds to the 49,000 MW SCBP. The immunoreactive peak in the 1 M potassium phosphate extract corresponds to a 52,000 MW protein. This protein is rapidly and transiently labeled after injection of 3H-histidine into newborn rats, in contrast to the SCBP which is labeled after a 5-hr lag. The 52,000 MW protein appears to be an immunologically related precursor of the SCBP. Immunoreactive proteins were localized in tissue sections by the indirect immunoperoxidase method. A strong positive reaction was seen in keratohyalin granules and in the stratum corneum. The reaction of keratohyalin granules corroborates the extraction of a cross-reactive protein by 1 M potassium phosphate, a method for extraction of keratohyalin granules from epidermis. These results are consistent with the hypothesis that a protein of 52,000 MW is present in keratohyalin and is converted to the SCBP (49,000 MW) concomitantly with the conconversion of a granular cell to a stratum corneum cell.

VISCOELASTIC PROPERTIES OF INTACT HUMAN SKIN: INSTRUMENTATION, HYDRATION EFFECTS, AND THE CONTRIBUTION OF THE STRATUM CORNEUM

Past attempts to relate mechanical properties of the stratum corneum to its state of hydration or disease condition have been hampered by the lack of adequate in vivo methodology. With the technique described herein, viscoelastic properties of the skin surface can be measured in vivo or on excised skin in vitro. This method is sensitive enough to detect changes in the properties of the stratum corneum induced by topically applied agents or by mechanical disruption of it. The viscoelastic data suggest that the outermost layers of the stratum corneum may become dry in vivo despite their proximity to the underlying wet tissue, and that the instrument is sensitive enough to measure the changes that occur. It also appears that the in vivo behavior of the stratum corneum is similar to that of isolated stratum corneum membranes which become brittle when they are dried and supple when they are hydrated.

Stratum corneum of frog skin: inferences for studies of Na entry and transport pool

Pieces of isolated stratum corneum from frog skins were studied. Preliminary data showed that approximately 56% of the total sodium of split skins was associated with the stratum corneum. In more detailed studies, the sodium space estimated with 22Na and the volume of tissue water were identical, averaging 0.95 mul/cm2. Thus, the stratum corneum contained approximately 0.11 mueq Na+/cm2. Similar determinations of the sucrose, mannitol, and inulin spaces of the stratum corneum gave mean values of 0.84, 2.21, and 1.84 mul/cm2, respectively. Surprisingly, the mannitol and inulin spaces were observed to be at least twice as large as the space occupied by the tissue water, and this can be interpreted to mean that mannitol and inulin bind in appreciable amounts and thus are unsuitable markers of the sodium space. Whereas the kinetics of sodium uptake and washout could be described simply, similar studies for sucrose, mannitol, and inulin were more complex and consistent with appreciable binding of the substances to the stratum corneum.

The effect of polyene antibiotics on the aldosterone induced changes in sodium transport across isolated frog skin

It is shown that: (1) The isolated stratum corneum has a very low electrical resistance. 1. (2) The passive sodium flux through the isolated s. corneum is 20–100 times higher than the active sodium transport. 2. (3) Polyene antibiotics (amphotericin B and filimarisin) abolish the aldosterone induced inhibition of the active sodium transport. 3. (4) Aldosterone induces a recovery in the electrical potential across skins where it had been reduced previously by the addition of a polyene antibiotic. 4. (5) An hypothesis is presented to explain these observations.

An Electron Microscopic Study of the Effects of Subtilisin and Detergents on Human Stratum Corneum

Human stratum corneum was treated with the protcolytic enzyme, subtilisin, in the presence and absence of surfactant (alkylbenzene sulfonates) and examined by electron microscopy to determine what effect the enzyme and surfactant had upon this tissue. Treatments were carried out both in vitro with isolated stratum corneum and in vivo with human volunteers. The attack of the enzyme is principally upon the most superficial cells of the tissue with little evidence of proteolysis elsewhere. Physical rupture of the intact sheet causes a marked increase in the number of digested cells. Where digestion is complete, the insoluble cytoplasmic constituents are degraded and only the resistant cell envelopes, desmosomes, and melanin granules are left as recognizable elements. Surfactant does not appear to increase the susceptibility of the stratum corneum to proteolysis. It is concluded that the outer cell envelope, which is resistant to digestion, protects the intracellular proteins from proteolysis and only through damage to the envelope do cells become susceptible.