Stratum Corneum Proteins Research Articles

New arrangement of proteins and lipids in the stratum corneum cornified envelope

A new arrangement of proteins and lipids of stratum corneum (SC) cornified envelope (CE) is proposed. The chemical analysis of CE revealed the presence of free fatty acids (FFA), ceramides (Cer), and important percentages of glutamic acid/glutamine (Glx) and serine (Ser) residues. The molecular structure of these components suggests the existence of covalent links not only between Cer and Glx but also between FFA and Ser. The protein distribution of extracellular surface of CE, i.e., the proteins that could be involved in the bonds with lipids, was studied using post- and pre-embedding immunolabeling electron microscopy. Some loricrin (protein rich in Ser) was detected in the outermost part of the CE protein layer. The external arrangement of some domains of this protein may give rise to form linkages with FFA, yielding further insight into the CE arrangement in which Cer–Glx bonds and FFA–Ser bonds would be involved. Although the importance of fatty acids in the cohesion and barrier function of SC has been widely demonstrated, their role could be associated not only to the presence of these lipids in the intercellular lamellae but also in the CE, in the same way that Cer.

Heavy Water Labeling of Keratin as a Non-Invasive Biomarker of Skin Turnover In Vivo in Rodents and Humans

Measurement of skin turnover has been problematic in humans. Heavy water (2H2O) labeling has recently been developed as a safe, simple method to study in vivo kinetics of many biosynthetic processes, including DNA and protein synthesis. Here, we apply this approach to the measurement of 2H incorporation into skin keratin and show close agreement between keratin and keratinocyte turnover data in the epidermis of rodents. Elevated turnover rates of both keratin and keratinocytes were observed in the epidermis of the flaky skin mouse, although topical treatments effective in human psoriasis had no effect on either turnover rate in these mice. In humans, keratin turnover was monitored non-invasively by serial tape stripping during and after 2H2O labeling. Kinetic data were consistent with previous estimates of epidermal turnover, with a lag time of 18 days before label appeared at the skin surface and a transit time of 4-5 weeks. Variability in skin keratin turnover rates was present among healthy individuals. In summary, 2H2O labeling of skin keratin represents a non-invasive approach for assessing skin turnover dynamics in pre-clinical models and in human subjects.

Thermotropic and Spectroscopic Behavior of Skin: Relationship with Percutaneous Permeation Enhancement

Stratum corneum (SC) is comprised of lipids, protein and low molecular weight water-soluble components. Changes in these skin micro constituents can be understood by instrumental methods like differential scanning calorimetry (DSC) and attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy. The former provides information about changes in thermotropic behavior of SC lipids and proteins, whereas the latter provides data about alterations at molecular and conformational level. Most of the DSC thermograms of intact mammalian SC show two reversible and two irreversible transitions in the temperature range of 25-125 degrees C. The reversible endotherms are ascribed to lipid melting transitions, whereas the irreversible endotherms are ascribed to protein denaturation. Similarly, the FTIR spectral bands of SC occurring between 2920-2850 cm-1 and between 1650-1550 cm-1 have been suggested to arise from lipid and protein molecular vibrations, respectively. Treatment of skin with solvents or permeation enhancers alters the composition of lipids or their molecular arrangement in the skin microenvironment, which leads to changes in permeability of drug molecules. Furthermore, inhibition of lipid synthesis in epidermis with concomitant decrease in enthalpy of lipid endothermic transitions and reduction in height and area of asymmetric and symmetric C-H stretching peaks have been found to be directly correlated with enhanced permeation of drugs. In addition, method of skin preparation, type of skin, types of enhancer etc. also influence both the nature and intensity of responses recorded in spectrographs and thermograms. Therefore, the modification in spectrographs and thermograms of skin samples treated with various enhancers, vehicles etc. are expected to provide better insight into their mechanism of action on the skin. This review article shall critically evaluate the thermotropic and infrared spectroscopic data of SC/epidermis after various treatments.

Late Cornified Envelope Family in Differentiating Epithelia—Response to Calcium and Ultraviolet Irradiation

The late cornified envelope (LCE) gene cluster within the epidermal differentiation complex on human chromosome one (mouse chromosome three) contains multiple conserved genes encoding stratum-corneum proteins. Within the LCE cluster, genes form "groups" based on chromosomal position and protein homology. We link a recently accepted nomenclature for the LCE cluster (formerly XP5, small proline-rich-like, late-envelope protein genes) to gene structure, groupings, and chromosomal organization, and carry out a pan-cluster quantitative expression analysis in a variety of tissues and environmental conditions. This analysis shows that (i) the cluster organizes into two "skin" expressing groups and a third group with low-level, tissue-specific expression patterns in all barrier-forming epithelia tested, including internal epithelia; (ii) LCE genes respond "group-wise" to environmental stimuli such as calcium levels and ultraviolet (UV) light, highlighting the functional significance of groups; (iii) in response to UV stimulation there is massive upregulation of a single, normally quiescent, non-skin LCE gene; and (iv) heterogeneity occurs between individuals with one individual lacking expression of an LCE skin gene without overt skin disease, suggesting LCE genes affect subtle attributes of skin function. This quantitative and pan-cluster expression analysis suggests that LCE groups have distinct functions and that within groups regulatory diversification permits specific responsiveness to environmental challenge.

Quantification of stratum corneum removal by adhesive tape stripping by total protein assay in 96‐well microplates

Examination of stratum corneum (SC) content with tape stripping and a colorimetric method is increasingly used. We examined the possible use of microplates in tandem with a colorimetric method to examine SC removed with tape stripping. As a corollary to this examination, the homogeneity of tape strips was examined. The commonly used Lowry assay was adapted for 96-well plates. Tapes were divided into four regions and sample disks of 5 mm diameter were taken from each and analyzed for SC mass using the adapted Lowry assay. Homogeneity of SC removal over different areas across a tape strip is limited. Quantification of SC by means of a 96-well microplate-based colorimetric method is feasible and shortens the time of analysis. However, when using D-Squame tape disks, SC removal on a limited area of the tape is not predictive for SC removal on the entire tape as removal is inhomogenous. Therefore, SC protein extraction should be performed on a large enough area, eventually on the entire tape when quantifying SC mass removed by tape stripping.

Design principles of chemical penetration enhancers for transdermal drug delivery.

Chemical penetration enhancers (CPEs) are present in a large number of transdermal, dermatological, and cosmetic products to aid dermal absorption of curatives and aesthetics. This wide spectrum of use is based on only a handful of molecules, the majority of which belong to three to four typical chemical functionalities, sporadically introduced as CPEs in the last 50 years. Using >100 CPEs representing several chemical functionalities, we report on the fundamental mechanisms that determine the barrier disruption potential of CPEs and skin safety in their presence. Fourier transform infrared spectroscopy studies revealed that regardless of their chemical make-up, CPEs perturb the skin barrier via extraction or fluidization of lipid bilayers. Irritation response of CPEs, on the other hand, correlated with the denaturation of stratum corneum proteins, making it feasible to use protein conformation changes to map CPE safety in vitro. Most interestingly, the understanding of underlying molecular forces responsible for CPE safety and potency reveals inherent constraints that limit CPE performance. Reengineering this knowledge back into molecular structure, we designed >300 potential CPEs. These molecules were screened in silico and subsequently tested in vitro for molecular delivery. These molecules significantly broaden the repertoire of CPEs that can aid the design of optimized transdermal, dermatological, and cosmetic formulations in the future.

Dynamics of proteins and lipids in the stratum corneum: Effects of percutaneous permeation enhancers

We have spin labeled the stratum corneum (SC) with a lysine specific reagent, succinimidyl-2,2,5,5-tetramethyl-3-pirroline-1-oxyl-carboxylate spin label (SSL), to assess the dynamics and hydration degree of SC proteins by electron paramagnetic resonance (EPR) spectroscopy taking measurements directly from the intact tissue. Treating the SC with two percutaneous penetration enhancers, 8 M urea or 20% (v/v) 1-methyl-2-pyrrolidone (1 MP), destabilizes the proteins thus promoting more mobile and solvent-exposed protein conformations. Upon SC lipid depletion the nitroxide side chain becomes more solvent exposed, suggesting that the removal of hygroscopic substances in the extraction process favors more hydrated protein conformations. On the other hand, the treatments with 8 M urea or 40% (v/v) 1 MP did not alter significantly the fluidity in the SC lipid domain as assessed by the probe 5-doxyl stearic acid; these permeation enhancers, specially 1 MP, seem to increase the probe solubility in the solvent leading to a considerable fraction of spin label to be removed from the lipid domain.

Interaction of 1-dodecyl-azacycloheptan-2-one with mouse stratum corneum

The interactions of 1-dodecyl-azacycloheptan-2-one (Azone®), a penetration enhancer, with mouse skin were analyzed by fluorescence microscopy, solid-state 13C-CP/MAS-NMR spectroscopy and Attenuated Total Reflectance Fourier-transform infrared (ATR–FT-IR) spectroscopy. Ferulic acid was employed as fluorescent probe to observe the delivery pathway in the stratum corneum (SC) after treatment with Azone®. Results suggested that the interaction between Azone® and the SC occurs in the lipid domains as well as the protein domains. FT-IR measurements show that treatment with Azone® results in significant shifts toward larger wavenumbers at v asCH2 and v sCH2, indicating an increased gauche conformational isomer content of lipid CH2. Further, a decrease of 13CT1 values and a shift of the SC protein amide-II band to a short wavenumber were found when the SC was pretreated with Azone®. It is concluded that Azone® can partially convert the SC protein from an α-helix conformation to a β-sheet conformation and loosen the aggregating SC keratins at room temperature.

Near-infrared Fourier transform Raman spectroscopic analysis of proteins, water and lipids in intact normal stratum corneum and psoriasis scales.

Previous biochemical studies demonstrated differences in the structure of lipids in normal skin and psoriatic lesions. Raman spectroscopy provides a unique possibility of studying the molecular structure of proteins, lipids and water in intact skin. Near-infrared Fourier transform Raman spectroscopy was used to study changes in molecular structure and conformation of proteins and lipids of stratum corneum in healthy persons and patients with psoriasis. In vitro Raman spectra were obtained from intact psoriatic scales in 11 patients and from normal stratum corneum of forearm, elbow and heel in a group of age-matched healthy persons. The spectra of stratum corneum differed between psoriasis and normal skin but not between investigated regions. No major changes of lipid band positions in Raman spectra were found, but the crystalline lipid structure was disrupted in psoriatic scales (assessed as the ratio of the symmetric methylene C-H stretching-mode intensities, S(lat)). Major spectral differences were seen in the molecular structure of the proteins. In the spectra of psoriatic scales, the peak position of the amide I band, in comparison with the normal skin, was shifted to higher wavenumbers, suggesting unfolding of proteins. Moreover, alterations in the disulfide stretch bonds of proteins were found in psoriasis scales, resulting in a less energetically favourable gauche-gauche-trans conformation (band around 520 cm(-1)). Psoriatic scales and normal stratum corneum did not statistically differ in their water content. The findings further define the molecular abnormalities in the stratum corneum in psoriasis.

Evidence that unrestricted legumain activity is involved in disturbed epidermal cornification in cystatin M/E deficient mice.

Homozygosity for Cst6 null alleles causes the phenotype of the ichq mouse, which is a model for human harlequin ichthyosis (OMIM 242500), a genetically heterogeneous group of keratinization disorders. Here we report evidence for the mechanism by which deficiency of the cysteine protease inhibitor cystatin M/E (the Cst6 gene product) leads to disturbed cornification, impaired barrier function and dehydration. Absence of cystatin M/E causes unrestricted activity of its target protease legumain in hair follicles and epidermis, which is the exact location where cystatin M/E is normally expressed. Analysis of stratum corneum proteins revealed a strong decrease of soluble loricrin monomers in skin extracts of ichq mice, although normal levels of loricrin were present in the stratum granulosum and stratum corneum of ichq mice, as shown by immunohistochemistry. This suggested a premature or enhanced crosslinking of loricrin monomers in ichq mice by transglutaminase 3 (TGase 3). In these mice, we indeed found strongly increased levels of TGase 3 that was processed into its activated 30 and 47 kDa subunits, compared to wild-type mice. This study shows that cystatin M/E and legumain form a functional dyad in epidermis in vivo. Disturbance of this protease-antiprotease balance causes increased enzyme activity of TGase 3 that could explain the observed abnormal cornification.

Passive and iontophoretic transport enhancement of insulin through porcine epidermis by depilatories: permeability and fourier transform infrared spectroscopy studies.

The effect of thioglycolate-based depilatory lotions was studied on the in vitro passive and iontophoretic permeability of insulin through porcine epidermis and biophysical changes in the stratum corneum (SC) lipids and proteins. The porcine epidermis and Franz diffusion cells modified for iontophoresis were used for the in vitro transport studies. Cathodal iontophoresis was performed at 0.2 mA/cm2 current density. Resistance of the control- and depilatory-lotion-treated epidermis was determined according to Ohm's law. Biophysical changes were studied on porcine SC before (control) and after treatment with the depilatory lotions using Fourier transform infrared (FT-IR) spectroscopy. Asymmetric (approximately 2915 cm(-1)) and symmetric approximately 2848 cm(-1)) Carbon-Hydrogen (C-H) stretching absorbances were studied to estimate the extent of lipid extraction. Fourier self-deconvolution and second derivative procedures were applied to amide I band (1700-1600 cm(-1)) in order to estimate quantitatively the changes in the secondary structure of the SC protein. The passive permeability of insulin was significantly (P <.05) increased through depilatory-lotion-treated (ie, Better Off, Marzena, and Sally Hansen) epidermis in comparison to control. Iontophoresis significantly enhanced (P <.05) the permeability of insulin through depilatory-pretreated epidermis in comparison with the control epidermis. Further, we were able to achieve the desired flux of insulin (5.25 U/cm2/d) through Better Off-treated epidermis using 0.2 mA/cm2 current density and 100 U/mL donor concentration of insulin. The SC treated with depilatory lotions showed a decrease in peak areas of C-H stretching absorbances in comparison with untreated SC. Depilatory lotion treatment also decreased (P <.05) the epidermal resistance in comparison with the control epidermis. The decrease in the alpha-helix conformation and the increase in the random and turn structures were observed in the SC proteins due to depilatory lotion treatment. The changes in the secondary structure of proteins and lipid extraction from the SC are suggested as the cause of the decrease in the epidermal resistance and the increase in the passive and iontophoretic permeability of insulin through depilatory-pretreated epidermis in comparison with the control epidermis.

Evaluation of the Pseudo-Absorption Method to Quantify Human Stratum corneum Removed by Tape Stripping Using Protein Absorption

Tape stripping is a well-known method to study the barrier function of the stratum corneum (SC) and penetration processes of topically applied substances into the horny layer. The quantification of the removed corneocytes for each tape strip is the prerequisite for these studies. The pseudo-absorption of the corneocytes was proposed as a measure for the quantification of the removed corneocyte aggregates. In this study, the pseudo-absorption of the corneocytes in the visible range (430 nm) is compared with the protein absorption in the UV range (278 nm) and an absorption at 652 nm obtained after staining of the SC proteins with Trypan blue. Both the protein absorption and the absorption measured after staining correlate well with the pseudo-absorption measured at 430 nm (R² = 0.92 ± 0.04 and R² = 0.95 ± 0.04, respectively).

PH‐induced alterations in stratum corneum properties

Skin-cleansing compositions based on alkyl carboxylates (soaps) have a higher irritation potential than those based on syndet surfactants such as alkyl isethionates or alkyl ether sulphates. Contributing factors include inherent differences in the irritation potential of soaps and syndet surfactants, pH-induced changes in surfactant solution chemistry, and the direct effects of pH on the physical properties of the stratum corneum (SC). Past work has not directly addressed the effect of solution pH on the SC itself and its potential role in cleanser-induced skin irritation. In the current work, alterations to SC properties induced by buffered pH solutions and two strongly ionizable surfactants, sodium dodecyl sulphate and sodium lauryl ether sulphate, at different pH values are measured. By utilizing optical coherence tomography (OCT) and infrared (IR) spectroscopy we have directly measured physical changes in SC proteins and lipids. Our results indicate that SC swelling, which reflects alterations to SC structural proteins, is increased significantly at pH 10, compared to pH 4 and 6.5. The transition temperature (T(m)) of SC lipids is found to increase at pH 10, compared to pH 4 and 6.5, suggesting a more rigid SC lipid matrix. Surfactants cause a further increase in swelling and lipid rigidity. Some aspects of what these results mean for SC physical properties as well as their implications to potential mechanisms of surfactant-induced skin irritation are discussed.

Percutaneous absorption, biophysical, and macroscopic barrier properties of porcine skin exposed to major components of JP-8 jet fuel

JP-8 has been associated with toxicity in animal models and humans. There is a great potential for human exposure to JP-8. Quantitation of percutaneous absorption of JP-8 is necessary for assessment of health hazards involved in its occupational exposure. In this study, we selected three aliphatic (dodecane, tridecane, and tetradecane) and two aromatic (naphthalene and 2-methylnaphthalene) chemicals, which are major components of JP-8. We investigated the changes in skin lipid and protein biophysics, and macroscopic barrier perturbation from dermal exposure of the above five chemicals. Fourier transform infrared (FTIR) spectroscopy was employed to investigate the biophysical changes in stratum corneum (SC) lipid and protein. FTIR results showed that all of the above five components of JP-8 significantly ( P<0.05) extracted SC lipid and protein. Macroscopic barrier perturbation was determined by measuring the rate of transepidermal water loss (TEWL). All of the five JP-8 components studied, caused significant ( P<0.05) increase in TEWL in comparison to control. We quantified the amount of chemicals absorbed assuming 0.25 m 2 body surface area exposed for 8 h. Our findings suggest that tridecane exhibits greater permeability through skin among aliphatic and naphthalene among aromatic JP-8 components. Amount of chemicals absorbed suggests that tridecane, naphthalene and its methyl derivatives should be monitored for their possible systemic toxicity.

Stratum Corneum Protein Dynamics as Evaluated by a Spin-Label Maleimide Derivative: Effect of Urea

The stratum corneum (SC) protein dynamics in the sulfhydryl group regions was studied by electron paramagnetic resonance (EPR) spectroscopy of a covalently attached maleimide derivative spin label. A two-state model for the nitroxide described the coexistence of two spectral components in the EPR spectra. The so-called strongly immobilized component arises from a spin-label fraction with the nitroxide moiety hydrogen-bonded to protein (rigid structure) and the weakly immobilized component is provided by the spin labels with higher mobility (∼10 times greater) exposed to the aqueous environment. The relative populations between these two states are in thermodynamic equilibrium. The apparent energetic gain for the nitroxide to form a hydrogen bond with the backbone rather than to be dissolved in the local environment was ∼10 kcal/mol in the temperature range of 2–30°C and ∼6 kcal/mol in the range of 30–70°C. Urea treatment caused a drastic increase in the segmental motion of the polypeptide chains that was completely reversible by its removal. Our analyses also indicated that the urea induced unfolding of the SC proteins opening the thiol group cavities. This work can also be useful to improve the spectral analysis of site-directed spin-labeling, especially for a more quantitative description of the nitroxide side chain mobility.

Effect of liposomes on delipidized stratum corneum structure: an ‘in vitro’ study based on high resolution low temperature scanning electron microscopy

The structural modifications induced in the delipidized stratum corneum (SC) by the addition of SC lipid liposomes were investigated ‘in vitro’ using double-layer for high resolution low-temperature scanning electron microscopy technique (HRLTSEM). Liposomes were prepared from a lipid mixture extracted from the SC with organic solvents and characterized by freeze-fracture electron microscopy. After delipidization and subsequent incubation with liposomes no structural modifications were detected in the SC protein domains. However, the lipid lamellar structure detected in the native SC was lacking after delipidization. The subsequent treatment with liposomes of this delipidized tissue led to the formation of new structures in the intercellular spaces that showed similar organization to the lipid lamellae structure of the native SC. Image analyses of the desmosomes found in the delipidized SC and of the structures formed after treatment with liposomes demonstrated that these new structures did not correspond to desmosomes in spite of the similar appearance of both structures. Hence, the new structures could be associated with a restoration of the damaged lipid lamellae structure of the delipidized SC by the SC lipid liposomes. This effect would involve the interaction between the lipids of the liposomes and the remaining material after delipidization.

Stratum corneum protein mobility as evaluated by a spin label maleimide derivative

The molecular dynamics in the vicinity of sulfhydryl groups of stratum corneum (SC) proteins has been studied by electron paramagnetic resonance (EPR) spectroscopy of maleimide spin labels covalently bound to the proteins. The total amount of bound maleimide was around 4 nmol per mg of SC. We have interpreted the coexistence of two spectral components in the EPR spectra by a two-state model with a fraction of label hydrogen bonded to proteins and another fraction exposed to the aqueous environment. We showed that the relative populations among these two states, determined by spectral simulation, are in thermodynamic equilibrium. The calculated energetic gain for the nitroxide to form hydrogen bond with SC proteins rather than to be dissolved in the buffer was ∼12 kcal/mol in the temperature range of 2–30°C and ∼5 kcal/mol in the range of 30–86°C. Temperature profiles of other EPR parameters related to the rotational diffusion of the probe also showed changes in the temperature interval of 26–42°C, suggesting alterations in the vibration modes of SC proteins which are sensitive to higher motional freedom above 26–42°C. We also compared samples of intact and lipid-depleted SC and we found that the delipidization process does not alter significantly the backbone mobility in the SH group regions, but the data suggest that the protein cavity is more open in the case of the delipidized samples. These results contribute to the understanding of the protein participation in the barrier function of SC, and can be useful to improve the spectral analysis of site-directed spin labeling, particularly for a more quantitative description of the dynamic modes of the nitroxide side chains.

A study on structural and diffusion properties of porcine stratum corneum based on very small angle neutron scattering data.

Generation of valuable information about the biphasic geometrical configuration of porcine stratum corneum from Very Small Angle Neutron Scattering (VSANS) data and investigation of its effect on the corresponding effective diffusivity. Spectra of porcine stratum corneum are mathematically transformed in order to obtain the corresponding auto-correlation function (ACF). Model stratum corneum structures, matching this experimentally determined ACF, are then produced based on the "brick-and-mortar" configuration. The effective diffusivity through these model domains is calculated using an appropriate numerical method. The most appropriate geometry of porcine stratum corneum's lipid and protein phases in a "brick-and-mortar" configuration is quantitatively determined and correlated with the barrier properties (diffusivity) of the stratum corneum model structures. The ACF analysis indicates the most appropriate values for the dimensions of the corneocyte thickness and the surrounding lipid gap, while the corneocyte length is estimated from the diffusion study.

Fetal Epidermal Differentiation and Barrier Development In Vivo is Accelerated by Nuclear Hormone Receptor Activators1

Nuclear receptors which interact with the retinoid X receptor are involved in the regulation of epidermal differentiation and development. We have recently shown that activators of the peroxisome proliferator-activated receptor and of the farnesoid X-activated receptor accelerate epidermal barrier maturation in fetal rat skin in vitro. In this study we asked whether cutaneous development in utero was affected by peroxisome proliferator-activated receptor or farnesoid X-activated receptor activators, or by an activator of another retinoid X receptor partner, liver X receptor. Activators of the peroxisome proliferator-activated receptor (clofibrate or linoleic acid), farnesoid X-activated receptor (farnesol or juvenile hormone III), or liver X receptor (22R-hydroxycholesterol), were injected into the amniotic fluid of fetal rats on gestational day 17. Fetal epidermal barrier function and morphology was assessed on day 19. Whereas vehicle-treated fetal rats displayed no measurable barrier (transepidermal water loss > 10 mg per cm2 per h), a measurable barrier was induced by the intra-amniotic administration of all activators tested (transepidermal water loss range 4.0-8.5 mg per cm2 per h). By light microscopy, control pups lacked a well-defined stratum corneum, whereas a distinct stratum corneum and a thickened stratum granulosum were present in treated pups. By electron microscopy, the extracellular spaces of the stratum corneum in control pups revealed a paucity of mature lamellar unit structures, whereas these structures filled the stratum corneum interstices in treated pups. Additionally, protein and mRNA levels of loricrin and filaggrin, two structural proteins of stratum corneum, were increased in treated epidermis, as were the activities of two lipid catabolic enzymes critical to stratum corneum function, beta-glucocerebrosidase and steroid sulfatase. Finally, peroxisome proliferator-activated receptor-alpha and -delta and liver X receptor-alpha and -beta mRNAs were detected in fetal epidermis by reverse transcriptase-polymerase chain reaction and northern analyses. The presence of these receptors and the ability of their activators to stimulate epidermal barrier and stratum corneum development suggest a physiologic role for peroxisome proliferator-activated receptor and liver X receptor and their endogenous ligands in the regulation of cutaneous development.

What's water got to do with it? A nuclear magnetic resonance study of molecular motion in pig stratum corneum.

The 1H and 2H magnetic resonance signals from pig stratum corneum were measured as a function of hydration. The 1H free induction decay contained two components, one motionally restricted and the other isotropically mobile on the timescale of 10(-5) s. From its T2 decay, the mobile signal was further subdivided into two components; one, which was 11% of the signal and 5.5% of the mass of the dehydrated stratum corneum, was assigned to nonaqueous tissue (likely hydrocarbons) and the other to water. As water content increased from 0 to 0.25 gH2O/gSC, the second moment of the motionally restricted signal decreased from 5.4 x 10(9) to 3.6 x 10(9) s(-2), whereas the water T2 time increased from less than 0.3 ms to 3.3 ms. The 2H quadrupolar echo from stratum corneum hydrated in 2H2O had a signal from motionally restricted deuterons, attributed to deuterons exchanged onto O-H and N-H groups, and a mobile signal from 2H2O. The amount of exchange, 9.5% of the hydrogen sites in the motionally restricted fraction, was close to the number of exchangeable sites on keratin, the most abundant protein in stratum corneum. Our results are consistent with a model in which the bulk of the water interacts closely with the corneocytes in stratum corneum.