The inner stratum corneum is likely to represent the location of the intact skin barrier, unperturbed by degradation processes. In our studies of the physical skin barrier a new high-performance liquid chromatography (HPLC)-based method was developed for the quantitative analysis of lipids of the inner stratum corneum. All main lipid classes were separated and quantitated by HPLC/light scattering detection (LSD) and the free fatty acid fraction was further analysed by gas-liquid chromatography (GLC). Mass spectrometry (MS) was used for peak identification and flame ionization detection (FID) for quantitation. Special attention was paid to the free fatty acid fraction since unsaturated free fatty acids may exert a key function in the regulation of the skin barrier properties by shifting the physical equilibrium of the multilamellar lipid bilayer system towards a noncrystalline state. Our results indicated that the endogenous free fatty acid fraction of the stratum corneum barrier lipids in essence exclusively consisted of saturated long-chain free fatty acids. This fraction was characterized as a very stable population (low interindividual peak variation) dominated by saturated lignoceric acid (C24:0, 39 molar%) and hexacosanoic acid (C26:0, 23 molar%). In addition, trace amounts of very long-chain (C32-C36) saturated and monounsaturated free fatty acids were detected in human forearm inner stratum corneum. Our analysis method gives highly accurate and precise quantitative information on the relative composition of all major lipid species present in the skin barrier. Such data will eventually permit skin barrier model systems to be created which will allow a more detailed analysis of the physical nature of the human skin barrier.

For a full understanding of the properties of the human skin barrier, physical macroscopic parameters of barrier function must be correlated to the structural organization of the barrier on a molecular level. This study was undertaken to relate differences in the relative composition of the three main lipid classes of human stratum corneum, i.e., free fatty acids, cholesterol, and ceramides, to differences in transepidermal water loss, stratum corneum electrical impedance, and corneometer value. A new high performance liquid chromatography/light scattering detection-based analysis method recently developed was used for collection of quantitative lipid data in conjunction with gas chromatography/mass spectrometry/flame ionization detection measurements on the free fatty acid fraction. After subtraction of contaminating lipid fractions we have estimated the molar ratio of the human skin barrier lipid composition to be, respectively, 15% cholesterol esters, 16% saturated long chain free fatty acids, 32% cholesterol, and 37% ceramides. The inter-individual difference in the relative amount of free fatty acids, cholesterol, and ceramides, respectively, can be >100% in the individual case. It was found that the relative amount of ceramides to cholesterol is larger in the wrist area, paralleled by a higher transepidermal water loss and corneometer value as well as different skin electrical impedance values as compared with the upper forearm area. We conclude that the site-dependent differences in the stratum corneum lipid composition are small compared with the large inter-individual variation. Interestingly, in the individual case, no correlation was registered between relative ceramide content and barrier properties.

Calcium plays an important role in the regulation of cellular differentiation and desquamation of epidermal keratinocytes. In this study, we examined the calcium distribution in reconstructed epidermis in an attempt to understand the physiology of keratinocyte differentiation and desquamation in vitro. Ion capture cytochemistry (the potassium oxalate-pyroantimonate method) was employed to localize ionic calcium in reconstructed epidermis generated under three different culture conditions (in serum-containing medium, serum-free medium, and serum-free medium supplemented with retinoic acid), allowing a comparison of the physiology of incompletely and well-differentiated keratinocytes. The reconstructed epidermis generated in serum-containing medium showed features of incomplete differentiation, and compared with the native skin, a high calcium content within incompletely differentiated cells in the stratum corneum. Use of serum-free medium containing vitamin and lipid supplements led to a marked improvement of the stratum corneum ultrastructure and penetration pathway across the stratum corneum, indicating improved barrier formation of the reconstructed epidermis. In parallel, the calcium distribution pattern was normalized showing the highest levels of calcium in the stratum granulosum and low levels in the inner stratum corneum. Addition of retinoic acid to the serum-free medium resulted in an altered keratinocyte differentiation and re-appearance of large quantities of calcium precipitates in the stratum corneum. Proton probe X-ray microanalysis was applied to investigate the calcium distribution quantitatively in native and reconstructed epidermis generated in serum-free medium, and verified the calcium distribution demonstrated by the precipitation technique. Regardless of the presence or absence of calcium in the stratum corneum, all examined culture systems exhibited insufficient desquamation, which correlates with the finding that stratum corneum chymotryptic enzyme was present predominantly as an inactive precursor. This study demonstrates that improvement of the stratum corneum barrier properties in vitro is concurrent with the normalization of the epidermal calcium gradient, whereas deregulation of terminal differentiation correlates with an accumulation of calcium ions within incompletely differentiated corneocytes.

The cellular part of the skin, the epidermis, is a very thin structure, approximately 120 μm thick, a fact which has hindered the exploration of the physiology of the epidermis in normal and pathological conditions. An additional complication is the fact that the epidermis contains layers of cells at different stages of differentiation. Therefore, conventional physiological capillary probes cannot, with any satisfactory precision, be located within a specified cell of a specified layer of the skin in vivo. Hence, alternative ways for the exploration of skin physiology have been sought for. In the past, analysis of the elemental content of skin was done as bulk measurements, and surprisingly wide ranges of elemental content were recorded. The width of these ranges was most certainly due to the sampling methods used rather than the sensitivity of the chosen method of analysis. Also, these older measurements did not discriminate between the different strata, and therefore the information provided little if any substance for a functional analysis of processes involved in normal and pathological differentiation of the epidermis. Particle probes, however, have been able to overcome such methodological problems. Over a period of 15 years we have studied normal human skin, normal‐looking, paralesional skin from psoriatics, and skin from persons suffering from atopic dermatitis using PIXE analysis. In recent years, trace elements have been shown to work as secondary messengers or regulatory substances. As an example, calcium (Ca2+) has proven to be a very important signalling substance in a great variety of cellular systems. Studies with the transmission electron microscope (TEM) as well as histochemical methods have allowed an understanding of the role of Ca2+ in the differentiation process of the epidermis. Ca2+ has also been shown to play an important role in apoptosis (programmed cell death), which is currently a hot subject for the obvious reason that the final differentiation step between the stratum granulosum level and the stratum corneum represents a particular aspect of programmed cell death. The importance of the balance between calcium and zinc in apoptosis has been clearly demonstrated in a number of cellular systems, but we have still to clarify the validity of topical treatment with Zn ointments in different skin conditions. Substantial iron (Fe) losses via psoriatic lesions were demonstrated more than two decades ago, and these data were given new meaning when we found that a more discrete loss occurs in clinically normal‐looking psoriatic skin. Obviously, such findings stress the importance of understanding the relation between the elemental content and normal and abnormal physiology. The ultimate goal of particle probe studies is to provide an understanding of the formation of a mature stratum corneum with a functional barrier reflected in physiological/biochemical mechanisms behind the properties of changed skin in patients afflicted with skin disorders of genetic or constitutional origin. This...

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