Isolated Stratum Corneum Research Articles

Taurine levels and localisation in the stratified squamous epithelia.

The content and distribution of the amino acid taurine in squamous epithelia were studied using high-performance liquid chromatography and immunohistochemical methods. Quantitative analysis demonstrated that taurine was highly concentrated in the epidermis (5.49 mumol/g fresh tissue in the hairless skin of the hind footpad of the rat), although the values in the isolated stratum corneum were extremely low (< 0.073 mumol/g in the horny layer of the same skin area). No other analysed amino acid (such as glutamate, glutamine, glycine or alanine) showed this specific pattern of distribution. The immunohistochemical study revealed that in the dog and rat epidermis, taurine was present in the keratinocytes of the granular and upper spinous layers. The basal layer, lower spinous layer and stratum corneum were immunonegative. A similar immunostaining pattern was found in the epithelia of the different organs studied: the mouth, tongue and oesophagus of the dog and rat, the rat forestomach and the rat corneal epithelium. Other cell types, such as sebaceous and muscle cells, were immunolabelled. The existence of a circulating pool of taurine in the epidermis (via taurine release from keratinocytes before they reach the horny layer and its uptake by nearby cells) and its possible roles in these cells are discussed.

Microfabrication of Individual 200μm Diameter Transdermal Microconduits Using High Voltage Pulsing in Salicylic Acid and Benzoic Acid

We describe an extension of semiconductor fabrication methods that creates individual approximately 200 microm diameter aqueous pathways through human stratum corneum at predetermined sites. Our hypothesis is that spatially localized electroporation of the multilamellar lipid bilayer membranes provides rapid delivery of salicylic acid to the keratin within corneocytes, leading to localized keratin disruption and then to a microconduit. A microconduit penetrating the isolated stratum corneum supports a volumetric flow of order 0.01 ml per s with a pressure difference of only 0.01 atm (about 10(2) Pa). This study provides a method for rapidly microengineering a pathway in the skin to interface future devices for transdermal drug delivery and sampling of biologically relevant fluids.

A New Computer-Based Evaporimeter System for Rapid and Precise Measurements of Water Diffusion Through Stratum Corneum In Vitro

It is important to have reliable methods for evaluation of skin barrier function when questions such as barrier perturbing effects of different agents and occlusive effects of different formulations are to be elucidated. A wealth of clinical work relates to measurements of transepidermal water loss in vivo, a method much affected by ambient air relative humidity, temperature, skin irritation processes, psychologic status of the subject, etc., factors that cause the method to suffer from low precision (i.e., high random error). Relating to these obstacles, we have developed a closed in vitro system for measurements of water diffusion rate through pieces of isolated stratum corneum at steady-state conditions, where the relative humidity and temperature is held constant and data can be collected continuously. Our evaporimeter-based in vitro system has a more than 3-fold higher precision (lower random error) ( approximately 10%) than measurements of transepidermal water loss in vivo ( approximately 35%). The results of our study show that: (i) the corneocyte envelopes contribute to the barrier capacity of stratum corneum; (ii) removal of the lipid intercellular matrix results in approximately a 3-fold increase in the water diffusion rate through the isolated stratum corneum (n = 20; p < 0.05), not a 100-fold as has previously been suggested; (iii) exposure to sodium dodecyl sulfate in water does neither alter the water diffusion rate (n = 10; p > 0.05) nor the water holding capacity (n = 10; p > 0.05) of stratum corneum; (iv) exposure to 1 M CaCl2 in water yields an increased water diffusion rate through stratum corneum (n = 10; p < 0.05); and (v) when applied to the stratum corneum in excess concentrations, the penetration enhancer Azone has occlusive effects on water diffusion through the stratum corneum (n = 6; p < 0.05).

Measuring depth depends on frequency in electrical skin impedance measurements

Background/aims: Multi‐frequency electrical measurements in vivo on stratum corneum are frequently reported in the literature. It is well known that the measured volume of skin is dependent on measuring frequency, and we wanted to investigate to what extent viable skin influences the measured impedance as a function of frequency.Methods: Finite element modelling (FEM) was used to investigate the current and potential distribution in the skin when measuring impedance with two concentric surface electrodes.Results: The study showed that the impedance measured reflects mainly the stratum corneum at low frequencies, in this case below 1 kHz, and that the viable skin dominates at higher frequencies.Conclusion: It is concluded that a multi‐frequency measurement on isolated stratum corneum is not possible in vivo with conventional techniques, except within a very narrow and low frequency range.

Recessive x-Linked Ichthyosis: Role of Cholesterol-Sulfate Accumulation in the Barrier Abnormality

Cholesterol sulfate is a multifunctional sterol metabolite, produced in large amounts in squamous keratinizing epithelia. Because patients with recessive x-linked ichthyosis display not only a 10-fold increase in cholesterol sulfate, but also a 50% reduction in cholesterol, we assessed here whether cholesterol sulfate accumulation and/or cholesterol deficiency produce abnormal barrier function in recessive x-linked ichthyosis. Patients with recessive x-linked ichthyosis display both an abnormal barrier under basal conditions, and a delay in barrier recovery after acute perturbation, which correlate with minor abnormalities in membrane structure and extensive lamellar-phase separation. Moreover, both the functional and the structural abnormalities were corrected by topical cholesterol. Yet, topical cholesterol sulfate produced both a barrier abnormality in intact skin and extracellular abnormalities in isolated stratum corneum, effects largely reversed by coapplications of cholesterol. Together, these results suggest that cholesterol sulfate accumulation rather than cholesterol deficiency is responsible for the barrier abnormality. Despite the apparent importance of cholesterol sulfate-to-cholesterol processing for normal barrier homeostasis, neither steroid sulfatase activity nor mRNA levels are upregulated following acute perturbations. These results demonstrate both a potential role for cholesterol sulfate-to-cholesterol processing in normal permeability barrier homeostasis, and that basal levels of steroid sulfatase are sufficient to accommodate acute insults to the permeability barrier.

Testosterone Skin Permeation Enhancement by Menthol through Formation of Eutectic with Drug and Interaction with Skin Lipids

□ The aim of this investigation was to elucidate the mechanism of skin permeation enhancement of the lipophilic drug, testosterone, by menthol. Menthol was found to form eutectic mixtures with testosterone, cholesteryl oleate, and ceramides. DSC measurements showed that menthol drastically lowers the Tm of testosterone, from 153.7 to 39.9°C. The decrease in Tm resulted in an increase in the solubility of testosterone in an aqueous ethanol vehicle by 2.8-fold, which caused a corresponding 2.8-fold increase in the flux of testosterone. A further increase in skin flux, to eight times the base line, could be attributed to the effect of menthol on the skin barrier properties. This assumption is supported by DSC results showing that menthol decreases the Tm of cholesteryl oleate and ceramides and modifies the thermogram profile of isolated stratum corneum. The results of this investigation indicate that menthol affects skin permeation by a dual mechanism: by forming a eutectic with the penetrating compound, thereby increasing its solubility, and by altering the barrier properties of the stratum corneum. Moreover, this study indicates that both types of interactions must be taken into consideration when using chemical enhancers and that decreasing the melting temperature of the permeant through formation of a eutectic could be one approach for increasing solubility and permeation rates.

Selective complexing agents designed after adenosine receptor as percutaneous permeation enhancers of drugs

The objective was to evaluate selective complexing agents, naphthalene diamide diimide (I) and naphthalene diester diimide (II), as transport facilitators of adenosine across skin. Molecular modeling was used to show favored binding, energetically and conformationally, between adenosine and its selective complexing agents. The energy-minimized conformational structure of the adenosine-diamide diimide (I) complex indicated favored binding conformationally and energetically by potential energy saving of 8.26 kcal/mol. Permeation studies of adenosine alone (control) and with the complexing agent were conducted across polydimethyl siloxane ( PDMS), whole thickness human skin and isolated human stratum corneum using vertical franz diffusion cells and infinite dose technique. Adenosine flux across PDMS was increased 10-fold by diester diimide (II) (0.28 μg/min per cm 2 versus 0.027 μg/min per cm 2 for the control). During permeation across whole thickness human skin, adenosine was primarily metabolized to inosine, which further metabolized to hypoxanthine, and adenine was formed in small amounts by a minor pathway. The V max and K m values for adenosine metabolism were 6.7 × 10 −4 mM/min and 6.4 × 10 −3 mM, respectively. Thus, extensive metabolism within the skin concurrent with diffusion prevented the estimation of mass transfer rates of adenosine alone or in the presence of the complexing agent. Adenosine permeation across isolated stratum corneum was not enhanced by diester diimide (II); however, increased amounts of hypoxanthine and inosine were found in the receptor phase, indicating enhanced uptake of adenosine. These results also suggested residual metabolic activity in the stratum corneum. A novel mechanism was demonstrated for permeation enhancement of drugs across lipophilic solid-phase and biological membranes, using lipophilic selective complexing agents as transport facilitators. However, the inherent metabolic activity of biological membranes profoundly influences drug transport and synthetic lipophilic membranes may therefore have limitations as models of biological barriers.

Optimization and validation of freeze-drying for light and electron microscopic autoradiography of percutaneous steroid transport

A freeze-drying/autoradiography procedure to visualize steroid transport across human stratum corneum was optimized and validated. A finite dose of [ 3H]estradiol ( 3H-E2) was applied to human abdominal skin, i.e. isolated stratum corneum and dermatomed epidermal sheets, in an in vitro permeation set-up. The visualization procedure included cryofixation, freeze-drying, osmium tetroxide ( OsO 4) vapor fixation, Spurr resin embedding and autoradiography. The different steps of the sample preparation were carefully examined for morphological quality, extraction and diffusion of 3 H- E2 from the skin. It appeared that dermatomed skin was, for several reasons, more suitable to process than isolated stratum corneum. While cross-sections of dermatomed skin displayed a well-preserved stratum corneum, the viable epidermis suffered from decreased structural preservation due to cryofixation artifacts. The apparent extraction of 3 H- E2 during the infiltration with Spurr resin was due to donor phase residing at the skin surface. In the autoradiographs, there was no indication that 3 H- E2 had diffused out of the skin. Silver grains were mainly localized over the superficial corneocytes. Only a few grains were present over the deeper layers of the stratum corneum, both over the inter- and intracellular domains. An equation was deduced to estimate the exposure time of autoradiographs from skin samples, with the advantage that no time was wasted with empirical range finding tests. In the equation a distribution factor was introduced which can be used to quantify the localization of the silver grains over the different tissue structures. In conclusion, it can be stated that the visualization method is suitable to study the distribution of 3 H- E2 in human stratum corneum following topical application.

Mechanisms of action of skin penetration enhancers/retarders: Azone and analogues

The modifying effect of Azone and five analogues on diffusion of metronidazole through isolated stratum corneum (SC) is reported. All enhance diffusion except N-0915, which is a retarder. Enhancement ratios (amount penetrating with modifier/amount from control) at 40 min are Azone 6.7; N-0539 6.4; N-0253 3.4; N-0721 1.4; N-0131 1.1; N-0915 0.2. The sulphur analogue of Azone (N-0721) is much less effective than Azone itself, and the short hydrocarbon chain in N-1031 renders it ineffective. Similar results using diethyl m-toluamide as penetrant suggest that this effect of N-0195 is non-specific. Azone expands while N-0915 condenses monolayers of dipalmitoyl phosphatidylcholine (DPPC). The phase transition temperature ( Tm) at ∼40°C of multilamellar DPPC liposomes is lowered by the enhancers in rank order of their enhancing abilities, while N-0915 increases T m. Thus, modifier activity might be related to fluidising effect on lipid lamellae. A mechanism of modifier action based on alteration of the lateral bonding within SC lipid lamellae is proposed, based on molecular modelling of Azone and N-0915 and their hydrogen bonding capacities to cerebrosides.

Effects of β-cyclodextrins on skin: implications for the transdermal delivery of piribedil and a novel cognition enhancing-drug, S-9977

The effects of β-cyclodextrin (β-CD), randomly methylated β-cyclodextrin (RAMEB) and 2-hydroxypropyl β-cyclodextrin (HPβ-CD) on skin were investigated. The three cyclodextrins (CDs) were able to destabilize model liposomes and to extract significant amounts of cholesterol from isolated stratum corneum (SC). However, only RAMEB extracted all the major lipid classes from isolated SC, as shown by thin layer chromatography. Both RAMEB and HPβ-CD could release 5–10% of the extractable cholesterol as well as proteins from hairless rat skin. Nevertheless, CDs did not induce any major modification of the differential scanning calorimetry (DSC) profile or the Fourier-transformed infrared (FTIR) spectrum of SC. This was explained by the low percutaneous penetration of CDs. Furthermore, the influence of RAMEB on the transdermal diffusion through hairless rat skin of piribedil, a central dopaminergic agonist and of S-9977, a novel cognition enhancing drug, was studied. RAMEB was found to decrease the transdermal flux of piribedil, with which it forms an inclusion complex, as shown by NMR. Conversely, RAMEB increased by 2-fold the percutaneous absorption of the S-9977 hydrochloride, which does not interact with CD. Finally, a combination of oleic acid and RAMEB greatly increased by about 30-fold the flux of S-9977 hydrochloride.

The effect of glycerol and humidity on desmosome degradation in stratum corneum.

Moisturizers are known to have occlusive, emollient and humectant properties, all of which help to alleviate the symptoms of skin xerosis. Although the biological mode of action of moisturizers is poorly understood, the recent observation that skin xerosis is associated with incomplete desmosome digestion suggests that moisturizers improve the desquamation process in such conditions. To examine the possibility that certain moisturizers act by facilitating desmosomal digestion, we investigated the ability of glycerol, a common humectant, to influence this process in stratum corneum in vitro. Examining desmosome morphology in isolated stratum corneum by electron microscopy, it was observed that the desmosomes were in more advanced stages of degradation in glycerol-treated tissue compared with control tissue. This enhanced desmosomal degradation in glycerol-treated tissue was confirmed by significant decreases in the levels of immunoreactive desmoglein 1, a marker of desmosome integrity. Desmosomal degradation was also shown to be a humidity-dependent event, being significantly reduced at low relative humidity. The effect of glycerol on desmosome digestion was emphasized further in two in vitro model systems. Firstly, glycerol increased the rate of corneocyte loss from the superficial surface of human skin biopsies in a simple desquamation assay. Secondly, measurement of the mechanical strength of sheets of stratum corneum, using an extensiometer, indicated a dramatic reduction in the intercorneocyte forces following glycerol treatment. These studies demonstrated the ability of glycerol to facilitate desmosome digestion in vitro. Extrapolating from these results, we believe that one of the major actions of moisturizers in vivo is to aid the digestion of desmosomes which are abnormally retained in the superficial layers of xerotic stratum corneum.

Analysis of all stratum corneum lipids by automated multiple development high-performance thin-layer chromatography.

An optimized gradient enabling the separation of all stratum corneum lipids by automated multiple development on HPTLC plates is presented. An initial isocratic step separates sebum lipids. This is followed by a 25-step development using a gradient with a polarity range of methanol-water to hexane. Application to in-vivo extracted and isolated stratum corneum lipids demonstrates the possible quantification of the lipid classes with a "one-experiment" separation.

A METHOD FOR ASSESSING PERCUTANEOUS PERMEABILITY BY USING MODEL LIPIDS OF STRATUM CORNEUM

The maximum fluxes (Jmax) of six β-blockers across keratinized membranes were determined in vitro and compared with their melting points (mp). Rat abdominal skin and hamster cheek pouch mucosa were used as the model membranes. Linear relations of Jmax with mp were observed in both membrane. Permeation studies using the delipidized membranes and the isolated stratum corneum (SC) sheet of hamster cheek pouch clarified that the primary rate-limiting barrier function exists in the SC especially in the intercellular lipid. A lipid mixture system (ceramide : cholesterol : palmitic acid : cholesterol-3-sulfate = 40 : 25 : 25 : 10 by weight, a mimic of intercellular lipids of SC) was used as a lipid-coated membrane and liposomes to evaluate drug permeation through the SC. The Jmax values obtained in the lipid-coated membrane, which showed linear relations with those determined in rat skin and in hamster cheek pouch, correlated with the degree of drug partition to the liposomes prepared with the same model lipid mixture, suggesting the validity of this model lipid mixture system as an in vitro system substituted for animal skin in percutaneous drug permeation studies. The usefulness of the system in assessing the penetration enhancers which act on the lipids in the SC, such as oleic acid (OA), was also clarified.

&lt;b&gt;THE EFFECT OF ULTRAVIOLET B IRRADIATION ON THE ISOLATED PORCINE STRATUM CORNEUM: COLORIMETRIC AND ATR/FT-IR SPECTROSCOPIC &lt;/b&gt;&lt;b&gt;INVESTIGATIONS &lt;/b&gt;

To investigate the effect of ultraviolet B (UVB) on the isolated stratum corneum (SC), attenuated total reflectance/Fourier transform infrared spectroscopy and tristimulus colorimeter were used to evaluate the changes in secondary structure ofproteins and in skin color following UVB irradiation. After UVB radiation, the color of porcine SC became blanching with the increase of exposure time, because melanin pigmentation did not occur in the isolated porcine SC. The peaks assigned to lipid structure disappeared, and some peaks split into additional peaks and/or shifted to lower frequency, possibly because of the unsaturation of protein and lipid in the porcine SC after UVB irradiation

Electrochemical determination of oxygen permeability of isolated stratum corneum membranes

AbstractThe mammalian stratum corneum is a thin, highly organized polymeric membrane which is relatively impermeable to water but permeable to some gases including oxygen. Isolated stratum corneum membranes from perinatal Sprague‐Dawley rats were mounted under electrodes used to determine the rate of diffusion of oxygen through the membrane. These membranes were consistently found to be permeable to oxygen, which could be quantitatively determined at a gold electrode at −0.6 V (vs. a Ag/AgCl electrode). The stratum corneum membrane was found to be anisotropic, with greater oxygen permeability when the gas diffused toward the electrolyte from the side of the membrane which would be oriented toward the exterior of the animal than when it diffused in the opposite direction. There is also evidence of anatomic site heterogeneity in the permeability of the membranes.

Surfactant-Induced Stratum Corneum Hydration In Vivo: Prediction of the Irritation Potential of Anionic Surfactants

After repeated contact, many surfactants will cause skin irritation and, especially, dryness and scaliness. Earlier in vitro investigations suggested that the irritation potential of anionic surfactants was related to the induction of hydration of isolated stratum corneum membranes. We have now investigated early surfactant-stratum corneum interaction in vivo. Sodium salts of n-alkyl sulfates with variable carbon chain length (n = 8-14) were tested for the promotion of stratum corneum hydration by measurements of skin surface water loss and electrical capacitance measurements in healthy adult human volunteers. The surfactant-induced increase in water uptake was confirmed in vitro by means of isolated stratum corneum samples and surfactant solutions labeled with tritiated water. In a parallel experiment the irritation potential of these compounds was investigated by 24-h patch testing in human volunteers. The irritant responses were quantified non-invasively by erythema (skin color reflectance measurements) and transepidermal water loss measurements. Hydration of stratum corneum exposed for 5 min to surfactant solutions significantly exceeded that of controls (phosphate-buffered saline). It increased with application time and was concentration dependent, saturable with increasing concentration, and rapidly reversible. Baseline hydration was re-established only 10-15 min after treatment termination. Induction of hydration was closely correlated with the irritation potential of the investigated compounds. It initially increased with increasing carbon chain length. The maximum response was obtained for the C12 analogue (sodium lauryl sulfate). With further increases in molecular size induction of stratum corneum hydration subsequently decreased. We have demonstrated that anionic surfactants increase stratum corneum hydration in vivo. The present results suggest that the mechanisms responsible for the hydration are related to the irritation properties of these compounds.

Ontogeny of the epidermal barrier to water loss in the rat: correlation of function with stratum corneum structure and lipid content.

The mammalian epidermal permeability barrier is provided by highly hydrophobic lipids forming multiple membrane bilayers within the extracellular domains of the outer, cornified cell layers. To characterize the critical events associated with barrier maturation, we correlated the emergence of a competent barrier to transepidermal water loss with development of the lamellar body secretory system, the organization of stratum corneum membrane bilayers, and the lipid composition of these membranes in the perinatal rat. Whereas pups of 19 d estimated gestational age had no measurable barrier (transepidermal water loss greater than 10 mg/cm2/h), by 21 d the barrier was well established (mean transepidermal water loss 0.41 mg/cm2/h). Development of a functional barrier correlated with increasing thickness of the stratum corneum, as well as with development of a membrane pattern of lipid deposition, visualized with the hydrophobic fluorescent probe nile red. At 19 d estimated gestational age, the stratum corneum intercellular domains exhibited an abundance of secreted lamellar body contents, but they were not organized into basic bilayer unit structures. Lamellar unit structures became evident by 20 d and extended throughout the stratum corneum interstices by 22 d (term). The quantity of lipid in isolated stratum corneum increased significantly between 19 and 20 d (34.08 versus 50.08 mean micrograms lipid/cm2, respectively; p less than 0.02) and still further between 20 and 21 d estimated gestational age (74.49 micrograms lipid/cm2; p less than 0.001). This increase was due to progressive accumulation of neutral lipids, particularly cholesterol, as well as nonpolar ceramides, as shown by thin-layer chromatography/scanning densitometry. These studies imply that in the development of cutaneous barrier function in the fetal rat both the generation of sufficient quantities of hydrophobic lipids and the organization of these lipids into bilayer unit structures are required.

Keratinized epithelial transport of beta-blocking agents. I. Relationship between physicochemical properties of drugs and the flux across rat skin and hamster cheek pouch.

The maximum fluxes (Jmax) of beta-blockers through keratinized membranes were determined in vitro and compared with their physiochemical parameters such as lipophilicity (log k'0) and melting point (mp). Rat abdominal skin and hamster cheek pouch mucosa were used as the model membranes. Propranolol, metoprolol, timolol, pindolol, nadolol and agenolol were used as beta-blockers with a variety of physicochemical characters. Linear relations of Jmax with either log k'0 or mp were observed both in intact rat skin and in intact hamster cheek pouch, suggesting that the lipophilicity and thermodynamic activity of a drug in the crystal state primarily affect the drug's permeation through these membranes. However, the slope, dJmax/d(log k'0), for cheek pouch mucosa was greater than that for rat skin, corresponding to the lack of appendigeal shunt pathways in cheek pouch. Penetration studies using the delipidized membranes and the isolated stratum corneum sheet of hamster cheek pouch mucosa clarified that the primary rate-limiting barrier function might exist in the lipid layer of the stratum corneum. Jmax values for the tape-stripped and delipidized skins correlated with both the solubilities of drugs in the vehicle and with the mp, suggesting the polar porous characteristics of both model membranes. However, a theoretical approach confirmed that the contribution of an intracellular or aqueous pore route in the intact membrane to the permeation of drugs with positive lipophilic indexes is negligible.

The skin as a barrier membrane: relating in vitro measurements to in vivo behavior

Human skin serves as a semipermeable barrier that controls the exchange of various substances between the body and its environment. In vivo, its behavior is controlled by a variety of physiological conditions, especially skin blood flow. Previous, detailed analyses of permeation through human skin indicate a complex dependence of transcutaneous gas flux on the resistance of the stratum corneum, diffusivity in the bulk of the skin, total skin perfusion, and distribution of blood flow [1]. In vitro measurements can be used to isolate the principal barrier, the resistance of the stratum corneum. When these measurements are made with isolated stratum corneum samples, the effects of small defects in the stratum corneum may subtly influence the net resistance through the sample. This influence can be reduced by coating the sample with an adhesive film. To properly determine stratum corneum resistances from measurements on such composites, one must consider the two-dimensional nature of diffusion in the coating. We adopt methods from the study of synthetic membranes to suggest optimal choices for coating resistances and to determine the extent to which such coatings mask stratum corneum artifacts.

Localization of Lipid Microdomains and Thermal Phenomena in Murine Stratum Corneum and Isolated Membrane Complexes: An Electron Spin Resonance Study

The intercellular region of the stratum corneum can be isolated in the form of membrane complexes (intercellular lipids and adjacent cornified envelopes) which are devoid of the cytoplasmic components. In this study the temperature induced phase transitions and corresponding lipid domain reorganization in isolated stratum corneum (SC) sheets and SC membrane complexes (SCM) were determined using the electron spin resonance (ESR) spin probe technique. The spin probe perdeuterated di-tert-butyl nitroxide (pdDTBN) in SC and SCM revealed less well-defined physiologic phase transitions in the SCM and a more polar lipid domain in SC. However, the ESR results show the coexistence of highly ordered domains (immobilized spin probe) and less viscous domains in intact SC, which persist in SCM. Above approximately 20 degrees C the spin probe is dissolved in a highly disordered (isotropic) lipid domain in both SC and SCM. In both solvent extracted SC and SCM, the spin probe is dissolved in a highly ordered lipophilic domain associated with the lipids complexed to the corneocyte envelope and exhibiting no thermal transitions between -23 degrees to 60 degrees C. Further, the amount of mobile spin probe is related to the amount of residual lipid. An unexpected finding was the apparent reduction of the spin probe in solvent extracted SCM, suggesting the presence of a previously unrecognized free radical reducing mechanism in these sites. The mobilities of the spin probe when dissolved in model lipids, non-hydroxy, and hydroxy containing ceramides and cholesteryl oleate, differed significantly from those observed in SC or SCM. These studies demonstrate the usefulness of ESR for the localization and characterization of lipid microenvironments in the stratum corneum.