Free fatty acids and fatty acids of triacylglycerols in normal and hyperkeratotic human stratum corneum

The content of the free fatty acids and the fatty acids of triacylglycerols has been measured in human plantar stratum corneum from normal and hyperkeratotic subjects with palmoplantar keratoderma. Fatty acids of triacylglycerols in normal tissues showed a characteristic pattern with a relative abundance of short-chain length and unsaturated fatty acids. Free fatty acid fraction was characterized by the predominance of saturated compounds. The relative amount of short-chain and monoene fatty acids in the hyperkeratotic stratum corneum was increased. These results seem to show a defect in the maturation of fatty acids in the living epidermis and present new evidence that the abnormality of lipid metabolism can influence the process of desquamation in stratum corneum.     

pH directly regulates epidermal permeability barrier homeostasis,and stratum corneum integrity/cohesion

Both exposure of stratum corneum to neutral pH buffers and blockade of acidification mechanisms disturb cutaneous permeability barrier homeostasis and stratum corneum integrity/cohesion, but these approaches all introduce potentially confounding variables. To study the consequences of stratum corneum neutralization, independent of hydration, we applied two chemically unrelated superbases, 1,1,3,3-tetramethylguanidine or 1,8-diazabicyclo [5,4,0] undec-7-ene, in propylene glycol:ethanol (7:3) to hairless mouse skin and assessed whether discrete pH changes alone regulate cutaneous permeability barrier function and stratum corneum integrity/cohesion, as well as the responsible mechanisms. Both 1,1,3,3-tetramethylguanidine and 1,8-diazabicyclo [5,4,0] undec-7-ene applications increased skin surface pH in parallel with abnormalities in both barrier homeostasis and stratum corneum integrity/cohesion. The latter was attributable to rapid activation (<20 min) of serine proteases, assessed by in situ zymography, followed by serine-protease-mediated degradation of corneodesmosomes. Western blotting revealed degradation of desmoglein 1, a key corneodesmosome structural protein, in parallel with loss of corneodesmosomes. Coapplication of serine protease inhibitors with the superbase normalized stratum corneum integrity/cohesion. The superbases also delayed permeability barrier recovery, attributable to decreased beta-glucocerebrosidase activity, assessed zymographically, resulting in a lipid-processing defect on electron microscopy. These studies demonstrate unequivocally that stratum corneum neutralization alone provokes stratum corneum functional abnormalities, including aberrant permeability barrier homeostasis and decreased stratum corneum integrity/cohesion, as well as the mechanisms responsible for these abnormalities.     

The how,why and clinical importance of stratum corneum acidification

In this article, I review the multiple endogenous mechanisms that contribute to the highly acidic pH of normal stratum corneum (SC). Then, I describe how each mechanism potentially impacts specific defensive functions of the SC. Finally, I review the rapidly expanding, clinical implications and potential therapeutic applications of SC acidification.     

The content of free amino acids in the stratum corneum is increased in senile xerosis

Xerosis is one of the characteristics of aged skin. Xerosis may be caused by a decrease in the stratum corneum free amino acids which are natural moisturizing factors derived from filaggrin. In aged skin, filaggrin is immunohistochemically decreased compared with the levels in young skin. However, the differences in stratum corneum amino acids between aged and young skin have not been analyzed quantitatively. Therefore, in this study we determined the stratum corneum amino acids per 1000 stratum corneum cells in aged and young skin by high-performance liquid chromatography. The amount of filaggrin mRNA in the epidermis was also compared between aged and young skin using RT-PCR. The total amount of amino acids in the stratum corneum was larger in aged senile xerosis skin than in young skin. Only a few amino acids were found in the stratum corneum of ichthyosis vulgaris patients (control skin). The expression of filaggrin mRNA in aged skin was, however, similar to that in young skin. These findings suggest that the immunohistochemical decrease in filaggrin in aged skin may be caused by promotion of filaggrin proteolysis in the upper layers of the stratum spinulosum.     

Antimicrobial activity of stratum corneum lipids from normal and essential fatty acid-deficient mice

Among the cutaneous effects of an essential fatty acid deficient (EFAD) diet are hyperdesquamation, increased transepidermal water loss (TEWL), and altered lipid profiles, characteristics also common to inflammatory dermatoses. Because fatty acids are antimicrobial, we examined the indigenous skin flora of normal and EFAD hairless mice, and compared the antimicrobial efficacy of lipids extracted from their stratum corneum. EFAD mice supported 100-fold more bacteria than normal mice, and were the only group from which Staphylococcus aureus were routinely isolated. Despite this greater carriage, in vitro experiments demonstrated that EFAD lipids are more lethal than normal lipids against Streptococcus pyogenes, S. aureus, S. epidermidis, Micrococcus sp., and a coryneform. Skin fungi were equally susceptible to both extracts. After thin layer chromatography, the most active fractions were found to be glycosphingolipids and phospholipids. EFAD extracts had 35% more free fatty acids and 75% more glycosphingolipids; normal extracts had more triglycerides and phospholipids. S. aureus strain 502A survived equally well on EFAD as on normal mice. Normal lipids applied on EFAD mice had no additional effect, but EFAD lipids on normal mice brought about a 35% reduction of the inoculated bacteria. If the mice were pretreated with alcohol, carriage of strain 502A was reduced by 71%. If instead the mice were previously washed with acetone to increase TEWL, a 97% reduction of the staphylococcus occurred. The application of normal flora to such acetone-washed mice decreased the efficacy to 76%. EFAD and normal lipids on human subjects were equally ineffective in eliminating strain 502A.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Comparison of lipids from epidermal and palatal stratum corneum.

Stratum corneum has been isolated by tryptic digestion of porcine epidermis and palatal epithelium, and the lipid concentrations and compositions have been compared by thin-layer chromatography in conjunction with photodensitometry. Palatal stratum corneum contained 47 +/- 6 micrograms lipid/mg tissue or 115 +/- 16 micrograms lipid per cm2 of stratum corneum surface, whereas epidermal stratum corneum contained 105 +/- 17 micrograms lipid/mg tissue or 135 +/- 16 micrograms/cm2. The difference in total lipid content does not account for the tenfold higher permeability constant for the permeation of water through the former tissue compared to the latter; therefore, the difference in permeability must be based on differences in lipid composition. In this regard, palatal stratum corneum includes 12.1% phospholipids, although phospholipids were undetected in epidermal stratum corneum. Differences in the content and location of non-polar liquid-phase lipids may also be of significance for permeability. Other factors that may contribute to the greater permeability of the palatal horny layer relative to epidermal stratum corneum include generally lower proportions of cholesterol, fatty acids, and ceramides, a dramatically lower proportion of the linoleate-containing acylceramide, and a tenfold lower content of covalently bound lipids associated with the corneocyte envelope.     

Preparation of liposomes from stratum corneum lipids

Cutaneous and subcutaneous blood flow (CBF, SBF) were studied in non-lesional psoriatic skin (NLS) of 10 patients with only minimal psoriatic skin manifestations, using the local 133Xe washout method. Measurements of the CBF and SBF in the NLS of the patients and 10 normal individuals yielded no statistically significant differences. The results of the present study indicate that the activity of psoriasis can be monitored by the CBF measurements in the NLS, since previously published values for CBF of NLS have shown increasing values with increasing psoriatic activity. The significance of these findings may be more evidence of humoral factors playing a role in the pathogenesis of the disease. The tissue-to-blood partition coefficient for 133Xe was calculated on the basis of biochemical estimations of the relative content of lipids, proteins, and water in skin biopsies from non-lesional skin sites of 8 psoriatic patients. The relative content of lipids, proteins, and water was normal. Thus, the normal 133Xe partition coefficient of 0.7 ml/g should be used for measurements of the CBF in NLS.     

Topically applied fatty acids are elongated before incorporation in the stratum corneum lipid matrix in compromised skin

In several skin diseases, both the lipid composition and organization in the stratum corneum (SC) are altered which contributes to the impaired skin barrier function in patients. One of the approaches for skin barrier repair is treatment with topical formulations to normalize SC lipid composition and organization. Vernix caseosa (VC), a white cheesy cream on the skin during gestational delivery, has shown to enhance skin barrier repair. In this study, we examined how a fatty acid (FA) containing formulation mimicking the lipid composition of VC interacts with the lipid matrix in the SC. The formulation was applied on ex vivo human skin after SC removal. Subsequently, the ex vivo human skin generated SC during culture. The effect of FA containing formulations on the lipid organization and composition in the regenerated SC was analysed by Fourier transform infrared (FTIR) spectroscopy and liquid chromatography mass spectroscopy (LC/MS), respectively. FTIR results demonstrate that the FAs are intercalated in the lipid matrix of the regenerated SC and partition in the same lattice with the endogenous SC lipids, thereby enhancing the fraction of lipids forming an orthorhombic (very dense) packing in the SC. LC/MS data show that the topically applied FAs are elongated before intercalation in the lipid matrix and are thus involved in the lipid biosynthesis in the skin.     

Essential fatty acids and epidermal integrity

The intercellular spaces of the stratum corneum contain multilamellar lipid sheets derived from the extruded contents of lamellar granules. In the absence of linoleic acid, lamellar granules appear empty, and only fragmentary extracellular sheets are found. This defective differentiation is attributable to substitution of oleate for linoleate in O-acylsphingolipids. Normally, linoleate is ester-linked to 30- to 34-carbon omega-hydroxyacids, which, in turn, are amide-linked to sphingosine. Acylglucosylceramides, bearing a beta-D-glucosyl moiety on the sphingosine, may provide the driving force for lamellar granule assembly. The omega-hydroxyacyl chains are long enough to span a lipid bilayer, while the linoleate inserts into an adjacent bilayer. This interaction could promote assembly of lamellar granules. It has also been proposed that acylceramides may stabilize the extracellular sheets by a similar mechanism. In addition, the horny cell has been found to possess a covalently bound lipid envelope consisting principally of omega-hydroxyacylsphingosines derived from O-acylsphingolipids.     

Extraction and quantification of amino acids in human stratum corneum in vivo

Background Amino acid (AA) levels in stratum corneum (SC) are potential biomarkers of skin health while their systemic levels may be used to diagnose inherited metabolic diseases. Objectives To examine reverse iontophoresis, in human volunteers, as a minimally invasive tool to analyse AAs within the skin and subdermally. Methods In four volunteers, the amounts of iontophoretically extracted AAs were compared with those determined in the SC following repetitive tape stripping and with the plasma concentrations. Glucose levels, evaluated in the different compartments, were used as a control. Results SC concentrations of 13 essentially zwitterionic AAs were ∼100‐fold higher than the respective plasma levels. Passive and reverse iontophoretic extraction for 4 h did not deplete the SC depot of AAs, a fact reinforced by postextraction tape stripping, which revealed that AAs remained in the SC at this time. In contrast, glucose was much less abundant in the SC and was fully and relatively quickly extracted by reverse iontophoresis. Conclusions It follows that reverse iontophoresis is useful for quantifying AAs in the SC and these data are highly correlated with levels obtained by tape stripping. However, reverse iontophoresis is impractical for the routine monitoring of AA plasma concentrations (unlike the situation for glucose, the skin reservoir of which is much smaller).     

Thermal analysis of human stratum corneum.

A thermal analysis technique has been developed and used to detect the melting of lipids and denaturation of proteins in stratum corneum. Trasitions were observed at 40 degrees C, 75 degrees C, 85 degrees C, and 107 degrees C. The transitions at 40 degrees C were attributed to the melting of lipids. The transition at 85 degrees C was identified as due to the denaturation of alpha-keratin, and the transition at 107 degrees C to the denaturation of a nonfibrous protein. It was found that alteration of the conformation of the nonfibrous protein changed the state of water absorbed, and that water contributed to the ordering of the lipid and protein. The thermal analysis technique may prove to be of value in studying the interaction of materials with skin.     

Mechanisms by which psychologic stress alters cutaneous permeability barrier homeostasis and stratum corneum integrity

Although many skin disorders, including psoriasis and atopic dermatitis, are adversely affected by psychologic stress (PS), the pathophysiologic link between PS and disease expression remains unclear. Recent studies demonstrated PS-induced alterations in permeability barrier homeostasis, mediated by increased endogenous glucocorticoids. Here, we assessed the mechanisms by which PS alters stratum corneum (SC) function. Insomniac psychologic stress (IPS) altered both barrier homeostasis and SC integrity. IPS decreased epidermal cell proliferation, impaired epidermal differentiation, and decreased the density and size of corneodesmosomes (CD), which was linked to degradation of CD proteins (e.g., desmoglein1). Barrier compromise was linked to decreased production and secretion of lamellar bodies (LB), which in turn could be attributed to a decrease in de novo synthesis of epidermal lipids. Topical physiologic lipids (equimolar cholesterol, ceramides, and free fatty acids) normalized both barrier homeostasis and SC integrity in IPS mice, further evidence that lipid deficiency accounted for these functional abnormalities. Thus, PS inhibition of epidermal lipid synthesis results in decreased LB formation and secretion, as well as decreased CD, compromising both permeability barrier homeostasis and SC integrity. These studies suggest that topical treatment with epidermal physiologic lipids could be beneficial in stress-induced, barrier-associated dermatoses, such as psoriasis and atopic dermatitis.     

Characteristics of cytokeratins from stratum corneum of palms

We have examined cytokeratins from stratum corneum (s. corneum) of the palm and back of hands by biochemical techniques which characterize cytokeratin No. 9 (CK9). By two-dimensional gel electrophoresis, cytokeratins from s. corneum of the palm (PSC) were resolved into three spots (designated as CK-A, CK-B and CK-C by us), and cytokeratins from s. corneum of the back of hands (BSC) were resolved into two spots (CK-A and CK-C). The results of polypeptide mapping and the spot positions in two-dimensional gels suggest that CK-A, CK-B and CK-C are degraded products derived from CK1/2, CK9 and CK10/11 in living keratinocytes of epidermis, respectively. While the spots of CK-A and CK-B were prominent and the spot of CK-C was very weak in PSC, the spots of CK-A and CK-C were prominent and the spot of CK-B was not detected in BSC. In filament reconstitution in vitro, cytokeratins from PSC formed intermediate-sized filaments similar to those commonly observed using cytokeratins from whole epidermis. In contrast, cytokeratins from BSC formed only short filaments and small granules. In order to examine this difference in detail, polypeptides were eluted electrophoretically from gel slices containing spots. Addition of eluted CK-B to cytokeratins from BSC promoted the formation of intermediate-sized filaments similar to those from PSC. Furthermore, although the mixture of eluted CK-A and CK-C did not form intermediate-sized filaments, the combination of CK-A and CK-B alone did form them.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Stratum corneum acidification in neonatal skin: secretory phospholipase A2 and the sodium/hydrogen antiporter-1 acidify neonatal rat stratum corneum

At birth, human stratum corneum (SC) displays a near-neutral surface pH, which declines over several days to weeks to months to an acidic pH, comparable to that of adults. Recent studies suggest that an acidic pH is required for normal permeability barrier homeostasis and SC integrity/cohesion. We assessed here the basis for postnatal acidification in the neonatal rat, where SC pH, as measured with a flat surface electrode, declines progressively from near-neutral levels (pH 6.63) on postnatal days 0 to 1 to adult levels (pH 5.9) or even below over the subsequent 7 to 8 d. The postnatal decline in SC pH was paralleled by a progressive activation of a pH-dependent hydrolytic enzyme, beta-glucocerebrosidase. Because SC acidification could not be linked to commonly implicated exogenous factors, such as bacterial colonization, or the deposition of sebaceous gland products. We next assessed whether changes in one or more of three endogenous mechanisms demonstrate postnatal activity changes that contribute to the progressive development of an acidic SC pH. Although the histidine-to-urocanic acid pathway has been implicated in acidification of the adult SC, surface pH is completely normal in histidase-deficient (his/his, Peruvian) mice, ruling out a requirement for this mechanism. In contrast, when sodium/hydrogen antiporter-1 (NHE1), which predominantly acidifies membrane domains at the stratum granulosum-SC interface, is inhibited, postnatal acidification of the SC is partially blocked. Likewise, SC secretory phospholipase A2 (sPLA2) activity, measured with a fluorometric assay, is low at birth, but increases progressively (by 66%) over the first 5 d after birth, and inhibition of sPLA2 between days 0 to 1 and days 5 to 6 delays postnatal SC acidification. Together, these results describe a neonatal model, in which the development of an acidic surface pH can be ascribed, in part, to progressive SC acidification by two endogenous mechanisms, namely, sPLA2 and NHE1, which are known to be important for acidification of adult rodent SC. Conversely, the impaired acidification of neonatal SC, which has important functional and clinical consequences, can be explained by the relatively low activities of one or both of these mechanisms at birth.