Glycosylated proteins of stratum corneum, nail and hair in diabetes mellitus: correlation with cutaneous manifestations

Nonenzymatic glycosylation of protein may play some role in the development of diabetic complications. To study the association of nonenzymatically glycosylated protein in keratinized tissues with the prevalence of cutaneous manifestations frequently observed in diabetics, we measured furosine values of stratum corneum, nail and hair from 61 diabetics and assessed their cutaneous manifestations. The manifestation most frequently found in this study was 10 cases of pigmented pretibial patches. We did not detect significant correlations between the prevalence of any cutaneous manifestations and furosine values of any keratinized tissues. However, 11 of the 17 patients with yellow nail showed high nail furosine values. Our data suggest that nonenzymatically glycosylated protein levels in the keratinized tissues do not correlate with the prevalence of cutaneous manifestations in diabetics, but do not exclude a role in the formation of yellow nail.     

Non-enzymatic glycosylation of keratin from the stratum corneum of the diabetic foot

Non-enzymatic glycosylation of keratin from the stratum corneum of the sole of the foot was measured by the thiobarbituric acid technique in thirty diabetic and thirty control subjects. A significant increase in the level of glycosylation was demonstrated in the diabetic subjects (P less than 0.001). HbA1 levels were measured in a further eighteen subjects at the same time as keratin was removed, and in this group a significant association between non-enzymatic glycosylation of that protein and diabetic control was demonstrated (P less than 0.01). In vitro incubation of keratin in the presence of free glucose produced increased non-enzymatic glycosylation (P less than 0.01) and this effect was blocked by incubation in the presence of increasing concentrations of aspirin (P less than 0.01). Measurement of non-enzymatic glycosylation of keratin in a further group of twenty diabetics with neuropathic ulceration showed a significant increase in levels when compared with a group of diabetics without ulcers (P less than 0.05). As keratin is the principle structural protein of the stratum corneum of the sole of the foot, it is possible that changes in this protein associated with non-enzymatic glycosylation may contribute to abnormalities seen in the skin of the feet of diabetics.     

Glycosylated proteins of skin, nail and hair: application as an index for long-term control of diabetes mellitus

The purpose of the present study was to compare the degrees of nonenzymatically glycosylated proteins in the skin (stratum corneum), the nail, the hair, and hemoglobin obtained simultaneously from the same subject and to evaluate the most useful sample for management of diabetic complications. Fifty-one diabetic patients and 20 control patients were examined, utilizing furosine determination. Furosine value of the skin in diabetics was 2.14 +/- 1.70%, whereas that in controls was 1.65 +/- 0.47%. Furosine value of the nail in diabetics was 6.67 +/- 3.30%, whereas that in controls was 4.16 +/- 1.62%. Furosine value of the hair in diabetics was 1.30 +/- 1.11%, whereas that in controls was 1.29 +/- 1.71%. Close correlations were detected between HbA1 (glycosylated hemoglobin) and furosine of the nail (r = 0.58, p less than 0.001), HbA1 and furosine of the skin (r = 0.48, p less than 0.001), and HbA1 and furosine of the hair (r = 0.43, p less than 0.01); however, poor correlations were found between furosine of the hair and the skin (r = 0.35, p less than 0.05) and furosine of the nail and the hair (r = 0.33, p less than 0.05). Furosine of the nail was significantly correlated with the FBS (fasting blood sugar) of the same time, previous 6, and previous 12 months. Furosine value of the nail, we believe, is the most useful indicator for evaluating long term control of diabetics and may provide useful information for management of diabetic complications.     

Electron diffraction provides new information on human stratum corneum lipid organization studied in relation to depth and temperature.

The outermost layer of mammalian skin, the stratum corneum, provides the body with a barrier against transepidermal water loss and penetration of agents from outside. The lipid-rich extracellular matrix surrounding the corneocytes in the stratum corneum is mainly responsible for this barrier function. In this study (cryo-) electron diffraction was applied to obtain information about the local lateral lipid organization in the extracellular matrix in relation to depth in human stratum corneum. For this purpose, stratum corneum grid-strips were prepared from native skin in vivo and ex vivo. It was found that the lipid packing in samples prepared at room temperature is predominantly orthorhombic. In samples prepared at 32 degrees C the presence of a hexagonal packing is more pronounced in the outer layers of the stratum corneum. Gradually increasing the specimen temperature from 30 to 40 degrees C induced a further transition from an orthorhombic to a hexagonal sublattice. At 90 degrees C all lipids were present in a fluid phase. These results are in good agreement with previously reported wide angle X-ray diffraction and Fourier transformed infrared spectroscopy studies. We conclude that the lipids in human stratum corneum are highly ordered throughout the stratum corneum and that electron diffraction allows monitoring of the local lipid organization, which contributes to the understanding of stratum corneum barrier function.     

Plantar skin in type II diabetes: an investigation of protein glycation and biomechanical properties of plantar epidermis

The generation of thickened plantar stratum corneum (SC) in response to elevated pressures, places individuals with diabetes at risk of ulceration. Such a response may culminate from altered biochemical and physical states of the epidermis as a result of non-enzymatic glycation (NEG). The objective of this study was to quantify specific glycation products generated in plantar epidermal proteins in individuals with Type 2 Diabetes Mellitus (T2DM) and age-matched controls (n = 103 and n = 87, respectively) and to compare these data with the viscoelastic properties (in vivo) of the epidermis. Plantar SC and venous blood samples were collected from all participants for the quantification of furosine and pentosidine using high performance liquid chromatography (HPLC). The viscoelastic properties of plantar epidermis were measured by the application of negative pressure on the surface of the skin. Plantar epidermal thickness was measured using high frequency (20 MHz) ultrasonography. There was a significantly greater concentration of pentosidine in the SC samples from people with T2DM (p = 0.001). There was no correlation between the concentration of glycated proteins in the epidermal proteins and serum proteins (furosine r = - 0.115, pentosidine r = - 0.023). The plasticity of the epidermis was significantly lower in the T2DM group than the control group (p = 0.007). The results suggest that alterations in the glycation of plantar epidermal proteins may constitute additional aggravators of ulceration in people with T2DM.     

Lipid thermotropic transitions in human stratum corneum

The techniques of differential scanning calorimetry (DSC) and thermal perturbation infrared (IR) spectrometry were used to investigate thermal transitions in intact, fractionated, and lipid-extracted human stratum corneum. The DSC results show 3 major and one minor thermal transition in the range of 30-120 degrees C. Of particular interest to this study are 2 transitions seen near 65 degrees and 75 degrees C in intact stratum corneum and a stratum corneum membrane preparation, but absent from lipid-extracted samples. Results of IR spectrometry show that thermally induced spectral changes related to enhanced motion of the lipid acyl chains also occur in the region of about 60-80 degrees C. The combined DSC and IR results show that the thermal transitions near 65 degrees and 75 degrees C depend on water concentration in a manner identical to that seen for a variety of lipid-water systems. Taken together, these results suggest that thermal transitions occurring near 65 degrees and 85 degrees C involve increased acyl chain motion of stratum corneum lipids.     

Isolation and characterization of proteinase from Candida albicans: substrate specificity

Candida albicans was able to produce a keratinolytic proteinase (KPase) when cultivated in a medium containing human stratum corneum as a nitrogen source. The KPase was purified to 108.5-fold by ion-exchange chromatography and gel filtration. The molecular weight of the enzyme was estimated to be 42,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and gel filtration through Sephacryl S-200, while the isoelectric point was determined to be at pH 4.5. The enzyme had an optimum pH of 4.0 and was "inactive" below pH 2.5 and above pH 6.0. The activity of KPase after preincubation at various temperatures was stable up to 50 degrees C. The keratinolytic activity was not affected by the addition of nonionic detergents and divalent cations. The enzyme was a glycoprotein and contained a high content of aspartic acid residues (172/1000). Pepstatin and chymostatin inhibited the activity in a dose-dependent manner; however, neither the other group specific inhibitors tested nor the pepsin specific inhibitors, DAN or EPNP, showed any effect on the enzyme. From these inhibitory profiles, this enzyme was determined to be a carboxyl proteinase such as cathepsin D. Among the various substrates for proteolytic enzymes, KPase digested human stratum corneum as much as albumin and hemoglobin. In the three fractions (water soluble, keratin filamentous, and membranous) prepared from human stratum corneum, the keratin filamentous fraction was more susceptible to degradation by KPase than the other two fractions were. KPase also digested much less human fingernail (13%) than human stratum corneum, but did not show any signs of there being any digestion of human scalp hair. These studies suggest that KPase from C. albicans may play an important role in superficial infection by affecting the human stratum corneum of the skin and nail.     

Drug permeation through human skin: I. Effect of storage conditions of skin

The purpose of this study was to establish conditions for the preparation and storage of excised human skin such that it most closely retained the permeation characteristics seen with skin samples studied immediately after having been removed from cadavers. Using skin taken from the inner thighs of cadavers within 48 hr post mortem, we have compared drug permeation through samples that were used immediately following removal with samples that had been stored in either the frozen or dried state. With the chromone acid under investigation, it was found that samples of stratum corneum attached to the underlying epidermis used without prior storage consistently showed less permeation than samples taken from the same individual that had been frozen at -17 degrees C upon removal and then thawed before permeation studies were started. However, drug permeation through samples of stratum corneum plus epidermis that had been allowed to dry under controlled humidity conditions at room temperature were found to be similar to those obtained with fresh skin. These results indicate that dried skin, when appropriately rehydrated before use, exhibits rates of drug permeation similar to those found with fresh skin. With the chromone acids under investigation, the use of frozen samples of excised skin should be avoided since the extent of permeation exceeds that obtained with fresh skin samples.     

The water-holding capacity of the stratum corneum measured by 1H-NMR

The amount of bound water in the stratum corneum of the hairless rat was measured by using proton nuclear magnetic resonance (1H-NMR) spectrometry in the temperature range of -5 degrees C to -30 degrees C. A decrease in the bound water content was observed when the stratum corneum was extracted using water, a mixed solvent of chloroform:methanol (2:1), or a 1% sodium dodecyl sulfate (SDS) aqueous solution. However, extraction using a mixed solvent of acetone:ether (1:1) did not change the bound water content at temperatures above -20 degrees C. The lipids extracted by acetone:ether-extraction method consisted of sebaceous gland lipids, cholesterol, free fatty acids, and ceramides. In contrast, the lipids extracted by chloroform:methanol-extraction method contained more polar lipids such as sphingomyelin, which had the same amount of bound water in itself as that of the water-extracted stratum corneum. These results suggest that the polar lipids soluble in the chloroform and methanol mixture may contribute to enhance the bound water content. Therefore, it is hypothesized that the water-holding capacity of the stratum corneum is dependent on both the hygroscopic components such as the natural moisturizing factor (NMF) and the polar lipids, such as sphingolipids, existing in the intercellular spaces of the stratum corneum.     

Properties of acid phosphatase in human stratum corneum

Sheets of stratum corneum were prepared by a trypsinization procedure from human skin samples, homogenized with a freeze press and then fractionated into a soluble fraction and a sediment by centrifugation at 50,000 g. Acid phosphatase (AcP) activity was found in both fractions but the bulk of the activity was detected in the supernatant. Highest activities were observed after treatment with Triton X-100. The bulk of the AcP activity remained bound to the pellet, if suspension and fractionation of the homogenized stratum corneum were performed in acetate buffer in the range between pH 4.0-5.0, probably due to ionic or hydrophobic interactions. AcP activity was totally lost if homogenates or fractions were stored frozen at -20 degrees C in buffers with pH values lower than 4.0. Triton X-100 extracts from whole skin, epidermis, stratum corneum, cultured skin fibroblasts and leukocytes were compared by isoelectric focusing. Extracts from whole skin, epidermis and stratum corneum yielded almost identical patterns with one main AcP activity band at pI of 5.65, whereas a second pronounced band from whole skin behaved similarly to one band from cultured skin fibroblasts and leukocytes (pI 6.1). The prominent band from extracts of stratum corneum and epidermis was not observed in extracts of skin fibroblasts and leukocytes. Hence, we conclude that stratum corneum and epidermis contain a tissue-specific AcP.     

There is no influence of a temperature rise on in vivo adsorption of UV filters into the stratum corneum

Temperature influences the stratum corneum adsorption of several topically applied compounds. This study was designed to evaluate the influence of the temperature on the stratum corneum adsorption of 3 UV filters. The UV filters were solubilized in two vehicles, an emulsion gel and petroleum jelly and applied at respectively, 31 and 40 degrees C during 30 min. In vivo stratum corneum UV filter content was measured using the tape stripping method. Similar amounts of UV filter were detected in the stratum corneum when comparing applications at the different temperatures. Application of the UV filters in the emulsion gel resulted in higher stratum corneum UV filter concentrations compared with application in the petroleum jelly. The application temperature did not influence the stratum corneum adsorption of the tested UV filters while the nature of the vehicle significantly influenced the amount of UV filters recovered from the stratum corneum.     

Structural investigations of human stratum corneum by small-angle X-ray scattering.

The structure of human stratum corneum was investigated with small-angle X-ray scattering (SAXS). At room temperature the scattering curve was characterized by a strong intensity at low scattering vector (Q less than 0.8 nm-1) and two complicated diffraction peaks originating from a lamellar structure of the lipids. The lamellar lipid structure in the stratum corneum transformed to a disordered structure between 65 degrees C and 75 degrees C, the same temperature region at which a thermal lipid transition occurred. After cooling down to room temperature a recrystallization of at least a part of the lipids took place, after which only one unit cell with a repeat distance of 13.4 nm could be detected. Comparison of the scattering curve of the stratum corneum after crystallization with the scattering curve of the stratum corneum before recrystallization leads to the conclusion that in the original curve the lipids are arranged in two unit cells with repeat distances of 6.4 nm and 13.4 nm. From model calculations it appears that the latter unit cell consists of more than one bilayer. The scattering curves of stratum corneum hydrated to various levels were measured. A change in the water content of stratum corneum between 6% w/w and 60% w/w (fully hydrated) did not result in swelling of the bilayers, but the scattering curve obtained with stratum corneum hydrated to 60% w/w differed from those at lower hydration levels: the scattering curve at 60% w/w showed only the diffraction peaks corresponding to a unit cell with a repeat distance of 6.4 nm. This observation implies that the ordering of a part of the lipids is reduced at very high water contents, which may explain the strong penetration-enhancing effects of water in the stratum corneum.     

Direct comparison of skin physiology in children and adults with bioengineering methods

The aim of the study was to investigate whether the physiologic skin parameters of transepidermal water loss (TEWL), stratum corneum hydration (capacitance and conductance), dynamic stratum corneum hydration parameters (hygroscopicity and water-holding capacity), skin color (a* and L* axes; chromameter), cutaneous blood perfusion [laser Doppler flowmetry (LDF)], and pH value differ between a sample of 44 children [C] (average age 3.5 years) and a directly comparable sample of 44 adults (their parents) [P] (average age 34.6 years). The results can be described as follows: TEWL C: 6.2 g/m2/h, P: 5.4 g/m2/h; stratum corneum hydration, capacitance C: 75.4 AU, P: 76.1 AU; conductance C: 27.1 microS, P: 19.2 microS; hygroscopicity C: 129.0 AU, P: 132.7 AU; water-holding capacity: C: 127.7 AU, P: 127.6 AU; redness (a*) C: 7.31, P: 8.21; lightness (L*) C: 67.63, P: 66.36; LDF (%) C: 24.6, P: 18.7; pH value C: 4.91, P: 5.07. In comparison to the skin of the adult sample we investigated (the parents of the 44 children), the skin of the small child can be characterized in the following way: it has a significantly lower hygroscopicity, a lighter (higher L* values) and less red color (lower a* values), and an increased cutaneous blood perfusion (LDF).     

Stratum corneum hydration and flexibility are useful parameters to indicate clinical severity of congenital ichthyosis

To determine any correlation between the stratum corneum barrier function and the phenotypic severity of congenital ichthyosis, we studied stratum corneum hydration, flexibility, thickness and transepidermal water loss (TEWL) in patients with congenital ichthyosis. Seven patients with congenital ichthyosis aged 2-46 years and age-matched controls were included in the present study. We divided seven patients into two groups; patients with non-bullous type (non-bullous congenital ichthyosiform erythroderma patients) and patients with the bullous type of congenital ichthyosis (bullous congenital ichthyosiform erythroderma and ichthyosis bullosa of Siemens). Stratum corneum hydration, thickness and flexibility were measured using a Corneometer ASA-M2. The stratum corneum thickness was also examined using a skin biopsy technique. TEWL was measured using Evaporimeter AS-TW1. The clinical severity of ichthyosis phenotype was evaluated using a visual analogue scale (VAS). Stratum corneum hydration and flexibility were significantly reduced in both congenital ichthyosis patient groups. Stratum corneum thickness was significantly increased in both groups. In the patient group with non-bullous congenital ichthyosis, significant negative correlations were confirmed between the VAS score and stratum corneum hydration and between the VAS score and flexibility. A significant, positive correlation was also observed between the VAS score and stratum corneum thickness. There was a positive correlation between the VAS score and TEWL on both the extensor and flexor sides of the forearm and back. We conclude that stratum corneum hydration, flexibility and thickness measured by the corneometer, and TEWL on the arm may be a useful indicator of the severity of ichthyosis phenotype.     

Hyaluronan exists in the normal stratum corneum

Hyaluronan is well known to exist as a water-sorbed macromolecule in the extracellular matrix. We here examined whether hyaluronan exists in the normal stratum corneum. High performance liquid chromatography was used to quantify hyaluronan content in the stratum corneum, epidermis (including stratum corneum), and dermis of mice, with the resulting dry weights being 22.3 +/- 2.9, 15.1 +/- 1.5, and 738.6 +/- 31.6 microg per g, respectively. Normal mouse skin was then labeled with [3H]-glucosamine in an organ culture, and accumulation of [3H]-labeled hyaluronan and its molecular mass were determined separately for the stratum corneum, epidermis, and dermis. In the stratum corneum, [3H]-labeled hyaluronan was accumulated linearly over the 3-d culture period. After the 3-d culture period, the epidermis synthesized twice the amount (expressed as dpm per mg dry weight) of [3H]-labeled hyaluronan as the dermis, whereas the stratum corneum and dermis showed nearly the same content of [3H]-labeled hyaluronan. The molecular mass of [3H]-labeled hyaluronan was highest (>1.0 x 106) in the dermis and clearly lower (<6.0 x 104) in the stratum corneum. Based on these results, we here confirm that hyaluronan is supplied from keratinocytes beneath the stratum corneum layer, and is present in the normal stratum corneum. We speculate that hyaluronan may play a role in moisturizing the stratum corneum and/or regulating its mechanical properties.     

The dependence of detergent-induced cell dissociation in non-palmo-plantar stratum corneum on endogenous proteolysis.

We have recently shown that cell cohesion in plantar stratum corneum is mediated to a significant extent by protein structures, and that endogenous proteolysis plays an important role in desquamation in this tissue. This paper is a report of our investigations into whether similar mechanisms for cell cohesion and desquamation can be found in non-palmo-plantar stratum corneum. Biopsies of non-palmo-plantar human skin were incubated at 37 degrees C, pH 8, in a buffer with and without additions of detergents (a mixture of N,N-dimethyldodecylamine oxide and sodium dodecyl sulphate), ethylene diamine tetraacetate (EDTA), and the proteinase inhibitor aprotinin. Released cells were examined by phase contrast microscopy and counted. The incubated biopsies were examined by light microscopy. As has been previously shown by others, we found that in the presence of detergents there was a dissociation of stratum corneum cells. This dissociation was stimulated by EDTA and inhibited by aprotinin. After 36 h of incubation the entire stratum corneum and, on some parts of the biopsies, the stratum granulosum had dissociated. There was no evidence of cell dissociation in the spinous or basal epidermal layers. We conclude that the detergent-induced cell dissociation in non-palmo-plantar human stratum corneum is dependent on the action of proteinases present in the tissue on protein structures. These structures may be of significant importance for non-palmo-plantar stratum corneum cell cohesion.     

Stratum corneum and nail lipids in patients with atopic dermatitis. Decrease in ceramides--a pathogenetic factor in atopic xerosis?

Dry skin is seen in many patients with atopic dermatitis and correlates with a disturbed epidermal barrier function demonstrated by such features as increased transepidermal water loss and diminished stratum corneum hydration. With regard to the importance of stratum corneum lipids for the permeability barrier, we have analysed plantar (n = 8) and lumbar (n = 20) stratum corneum and nail lipids (n = 15) of atopic subjects by high-performance thin-layer chromatography (HPTLC). Compared with controls our investigations show a decrease in the ceramide fraction as a percentage of total lipid and a diminished ratio of ceramides and free sterols in atopic subjects. This implies that impaired ceramide synthesis may be a factor in the pathogenesis of atopic xerosis.     

Factors affecting human percutaneous penetration of methotrexate and its analogues in vitro.

Factors affecting the percutaneous penetration of methotrexate (MTX) and its analogues through excised human skin were studied. With application of 0.05% MTX solution to the epidermis, 0.07% of the applied dose was recovered in the epidermis and less than 0.005% penetrated completely through the skin. The percent of drug which penetrated remained constant from 0.05% to 2.0% MTX and was also unchanged by increasing incubation temperature from 28 degrees C to 37 degrees C. Cellophane tape stripping of stratum corneum following drug incubation removed essentially all the MTX that had been measured in whole epidermis. Binding of MTX was localized to the outermost layers of the stratum corneum. With removal of the stratum corneum prior to drug incubation, 25% of the applied MTX penetrated through the skin. Various vehicles were tested to determine their effect on MTX percutaneous absorption. Dimethylsulfoxide (80%), dimethylacetamide (25%), or retinoic acid (0.1%) had no effect on penetration. Maximum enhancement of penetration by 25-fold and 143-fold was obtained with retinoic acid (saturated solution in aqueous ethanol) and C-10-methylsulfoxide (2.5%), respectively. The penetration of lipid-soluble derivatives of MTX was also tested. The compound that penetrated best was the dimethyl ester of dichloro-MTX with a 3-fold increase in penetration over MTX.     

Biophysical changes after mechanical injury of the stratum corneum in normal skin

Scrubbing off the stratum corneum with a rough towel after soaking in warm water is a bathing custom unique to Korea. However, Korean dermatologists have advised against this practice due to the potential harm that it may cause, though there is little data to support this advice. This study aimed to evaluate the changes in biophysical characteristics after such mechanical injury of stratum corneum, as reflected by water-holding capacity, stratum corneum barrier function, and stratum corneum turnover time (SCTT). 10 Korean female volunteers (aged 19-34 years) were enrolled in this study to investigate the acute effects and 32 volunteers (aged 19-49 years) the chronic effects. After soaking in warm water (36 degrees C) for 5 min, the flexor surfaces of the forearms were scrubbed with a rough towel. The volunteers' forearms were divided into 4 compartments, and each compartment was scrubbed 0, 5, 10, and 15x, respectively. After a single injury of the stratum corneum, there was an initial decrease in water-holding capacity until 6 h. Thereafter, it increased until day (D) 3, when it began to descend to the normal range. The transepidermal water loss (TEWL) values also peaked at D2 and declined thereafter. However, there were no changes in either water-holding capacity or skin barrier function, and only the SCTT was significantly shortened, after chronic injury of the stratum corneum.     

Stratum corneum chymotryptic enzyme: a proteinase which may be generally present in the stratum corneum and with a possible involvement in desquamation.

A chymotrypsin-like proteinase that may be involved in the desquamation process in plantar stratum corneum has recently been partially characterized. The aim of the present study was to elucidate whether a similar proteinase is also present in non-palmo-plantar stratum corneum. Stratum corneum was obtained by tape stripping of volar forearm skin after the skin surface had been painted with colourless nail varnish. The adherent tissue was released from the tape strips by acetone treatment, then extracted with diethyl ether and dried. Extracts of this acetone-ether powder were analyzed with respect to proteolytic activity by means of electrophoresis under non-reducing conditions in polyacrylamide gels containing sodium dodecyl sulphate and casein. The extracts were found to contain one major chymotrypsin-like proteinase with an apparent molecular weight of around 25 kDa, and several minor proteinases with trypsin-like activity. The 25 kDa proteinase was active at pH 5.5-8, and could be inhibited by aprotinin, chymostatin and zinc ion, but not by leupeptin. No difference could be found between the 25 kDa enzyme in forearm stratum corneum and the recently described chymotrypsin-like enzyme in dissociated plantar stratum corneum cells as regards electrophoretic mobility, pH dependency, and inhibitor profile. The fact that the enzyme could degrade casein at pH 5.5 and that it appears to be present in stratum corneum in general suggests that it may play a role in the desquamation process under in vivo conditions. The tentative name "stratum corneum chymotryptic enzyme" is proposed for this newly discovered proteinase.