Stratum corneum extraction

The third class of lipids found in stratum corneum extracts is represented by cholesterol and cholesteryl esters. The actual role of cholesterol remains enigmatic, and no clear reason for its role in the barrier function has been proposed so far. However, it is possible that contrary to what is the role in cell membranes where cholesterol increases close packing of phospholipids, it can act as kind of a detergent in lipid bilayers of long-chain, saturated lipids.30,31 This would allow some fraction of the barrier to be in a liquid crystalline state, hence water permeable in spite of the fact that not only ceramides, but also fatty acids found in the barrier are saturated, long-chain species.28,32... 

Differential Scanning Calorimetry (DSC) Studies. Hairless mouse abdomen stratum corneum, extracted lipids and protein residues were studied with a Perkin Elmer 4 differential scanning calorimeter (DSC) equipped with a thermal analysis data system (TADS). Scanning rates were 10°C per minute over the temperature region -10 to 237°C. Stratum corneum, extracted lipid and protein residue samples obtained from the abdomen of the hairless mice (average 10 mg/sample) were studied in the desiccated state following evaporation of any residue water or solvents by vacuum drying at 10 4 Torr. 

Differential scanning calorimetric and infrared spectroscopic investigations of intact stratum corneum, extracted lipids and keratinized protein residue sheets suggested the thermal transitions occurring within the 30 to 70°C region were associated with increased molecular mobility of the lipids. The permeability coefficients of lipophilic molecules through hairless mouse skin increased by several orders of magnitude over the same temperature... 

Altered partitioning into stratum corneum Extraction of intercellular lipids... 

Drug levels within the stratum corneum can be assessed by sampling single corneocyte layers with adhesive film. The drug is then extracted from the tape-strips and quantified by a suitable analytical method. Usually, scintillation counting (for radioactive compounds) or high performance liquid... 

Substituting hx = 3.6 cm and K ip/w = K - into Eq. 28 Johnson et al. calculated solute lateral diffusion coefficients in stratum corneum bilayers from macroscopic permeability coefficients. Measurements with highly ionized or very hydrophilic compounds were not performed because of the possible transport along a nonlipoidal pathway. Comparison of the computed Aat values with experimentally determined data for fluorescent probes in extracted stratum corneum lipids [47] showed a highly similar curve shape. The diffusion coefficient for the lateral transport showed a bifunctional size dependence with a weaker size dependence for larger, lipophilic compounds (> 350 Da), than... 

Compounds that penetrate the stratum corneum via the transepidermal route may follow a transcellular (or intracellular) or intercellular pathway (see Figure 11.1). Because of the highly impermeable character of the cornified envelope (see previous section), the tortuous intercellular pathway has been suggested to be the route of preference for most drug molecules [32], This is confirmed by several microscopic transport studies, in which compounds have been visualized in the intercellular space of the stratum corneum [33-35]. Moreover, it has been demonstrated that drug permeation across stratum corneum increases many folds after lipid extraction [36], Hence, knowledge of the structure and physical properties of the intercellular lipids is crucial to broaden our insight into the skin barrier function. 

There is substantial history regarding the application of conventional vibrational spectroscopy methods to study the intact surface of skin, the extracted stratum corneum and the ceramide-cholesterol-fatty acid mixtures that constitute the primary lipid components of the barrier. The complexity of the barrier and the multiple phases formed by the interactions of the barrier components have begun to reveal the role of each of these substances in barrier structure and stability. The use of bulk phase IR to monitor lipid phase behavior and protein secondary structures in the epidermis, as well as in stratum corneum models, is also well established 24-28 In addition, in vivo and ex vivo attenuated total reflectance (ATR) techniques have examined the outer layers of skin to probe hydration levels, drug delivery and percutaneous absorption at a macroscopic level.29-32 Both mid-IR and near-IR spectroscopy have been used to differentiate pathological skin samples.33,34 The above studies, and many others too numerous to mention, lend confidence to the fact that the extension to IR imaging will produce useful results. 

Special honors are also due to S. Jacobi, who like Irwin Blank was not a clinician but a Ph.D. scientist. He discovered the famous natural moisturizing factor, made up of low molecular weight, water-soluble substances, dominantly amino acids, which are chiefly responsible for the ability of the stratum corneum to absorb water and to hold onto it in the face of a hostile dry environment.8 He went on to show that an extract of the natural moisturizing factor could ameliorate dry skin. 

Most recently, Nakagawya et al.34 demonstrated that topical application of potassium lactate restored stratum corneum hydration after NMF extraction and exhibited a significantly higher restorative effect than sodium lactate. The authors speculate that this is due to the structure-destructive properties of the potassium ion and may influence hydrogen bonding in the keratin matrix. 

Hantschel, D. et al. Urea analysis of extracts from stratum corneum and the role of urea-supplemented... 

Urea is a physiological substance occurring in human tissues, blood, and urine. The amount in urine is of the order of 2%. The extraction of pure urea from urine was first accomplished by Proust in 1821, and it was first synthesized by Wohler in 1828.1 Urea is also a major constituent of the water-soluble fraction of the stratum corneum, as a component of the natural moisturizing factor (NMF).2 The level of urea in the stratum corenum is significantly reduced in patients with atopic dermatitis.3... 

Lipid Extracts. Lipids were extracted from the hairless mouse stratum corneum using a modified method based on methods developed by Bligh and Dyer (37) and Elias and coworkers (17.20.21). 

The abdomen stratum corneum samples were homogenized in 1 2 0.8 (v/v/v) chloroform methanol deionized water (1 ml solvent mixture/ 10 mg tissue). Solvents (Baker HPLC grade) were distilled prior to use. Homogenized tissue was left overnight and then filtered through a fine mesh fritted glass filter. Lipid extraction media were separated into two phases with the addition of chloroform and water (7.6 2 2 (v/v/v) extraction medium chloroform water)... 

Preparation of Protein Residue Sheets. Individual abdomen stratum corneum sheets were placed in 20 ml 1 2 0.8 chloroform methan-ol water and agitated lightly continually for 24 hours. The solution was then removed and replaced daily with methanol. Stratum corneum sheets were extracted for a total of two, four, seven or thirteen days. The extracted protein residue sheets were placed on Teflon sheets and dried in a vacuum oven (10- Torr, 25°C) for eight hours and then stored under vacuum. 

Fourier Transform Infrared Spectroscopy (FTIR) Studies. Infrared spectra of hairless mouse stratum corneum, lipid extract and protein residue are illustrated in Figures 3 and 4 for the 4000 to 2600 cm-2 and 1800 to 1360 cm-2 regions, respectively. 

Figure 3. Transmission FTIR spectra of desiccated, hairless mouse stratum corneum, protein residue and extracted lipids from 4000 to 2400 cm-. ...

Figure 13. Wavenumber versus temperature plot for CH2 asymmetric C-H stretching band during the heating cycle following 2, 4, 7 and 13 days extended extraction of iesiccated, hairless mouse stratum corneum in methanol.

Calorimetric results for the hairless mouse stratum corneum and isolated components confirmed thermal transitions associated with increased mobility occurred in the temperature region where large increases in permeability of lipophilic components also occurred. Thermal transitions for the extracted lipids were in the 27 to 67°C range, while the protein residue sheets (primarily... 

Figure 15. Wavenumber versus temperature plot for N-H stretching region during the heating-cooling cycle following 13 days extensive methanol extraction of desiccated, hairless mouse stratum corneum.

---