Evaluation of a new X-ray fluorescent analysis technique for the creation of a Nordic hair database: elemental distributions within the root and the virgin segment of hair fibers.

A new, non-destructive X-ray fluorescence technique for quantitative estimation of elemental content in biological tissues has been developed. Technical and instrumental characteristics of the ITRAX X-ray spectrometer have been evaluated in relation to the properties of biological samples, i.e., human hair fibers. Thus, attenuation variations of the fluorescent X-rays in the hair bulk mass were demonstrated by analysis of sulfur, calcium, and zinc in a virgin part near the root of one hair fiber with elliptical cross section. By rotation of the hair fiber and successive analyses made of the same part of the hair fiber, the results showed that concentrations of elements varied as functions of the diameter of the analyzed hair volume. Other sources of errors are also discussed. The ITRAX instrument allows for precise, fast, non-destructive, simultaneous, quantitative recording of the detected elements and trace elements down to levels of 1 ppm (microg/g). It was used for assessment of normal values of physiologically important elements present in hair in a cohort of normal, healthy Swedish, Caucasian individuals. The database constructed from data retrieved from a conceivably homogeneous ethnic set of individuals represents, to our knowledge, the first of its kind.

The skin barrier: analysis of physiologically important elements and trace elements.

Changes in the properties of the skin barrier should have correlates in the physiological status of the differentiating epidermal cells. However, the quantitative distributions of physiologically important elements and trace elements of the skin has been a neglected area of research for lack of tools to investigate this highly differentiated tissue. With the event of the particle probes, the electron microprobe and the scanning proton microprobe, it has become possible to investigate different aspects of normal skin physiology as well as pathophysiological processes. In addition penetration profiles of allergenic metals can be demonstrated with the trace element sensitive proton probe. Future approaches to the study of skin physiology in normal and pathological conditions should incorporate other techniques including immunological and biochemical tagging of particular cells to achieve a broad basis for interpretations of data.

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).

Elemental analysis mirrors epidermal differentiation.

Using a scanning nuclear microprobe, the distribution of elements and trace elements of skin cross sections of normal skin, non-lesional psoriatic skin and in dry atopic skin have been mapped. In non-lesional psoriatic skin and in dry atopic skin the epidermal Ca-gradient is higher than that of normal skin. In addition, abnormally high Fe and Zn levels were recorded in the stratum granulosum and corneum regions in the pathological skin. It is suggested that these findings correlate to an increased cell turnover in the basal cell layer of the psoriatic and atopic skins. The ratio of Ca/Zn in stratum corneum of paralesional psoriatic skin is approximately 8:1 compared to 12: 1 in normal skin and 15: 1 in atopic skin. This suggests that the differentiation process in paralesional psoriatic skin may actually be an example of disturbed programmed cell death.

Inter- and intra-individual differences in human stratum corneum lipid content related to physical parameters of skin barrier function in vivo.

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.

A new HPLC-based method for the quantitative analysis of inner stratum corneum lipids with special reference to the free fatty acid fraction.

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.

Normalization of epidermal calcium distribution profile in reconstructed human epidermis is related to improvement of terminal differentiation and stratum corneum barrier formation.

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.

Electrical impedance and other physical parameters as related to lipid content of human stratum corneum.

BACKGROUND/AIMS: In previous studies we have shown that variations in the properties of the stratum corneum are reflected by alterations in electrical impedance. The aim of this study was to explore the ability of the electrical impedance technique to detect changes in the lipid content of the stratum corneum, and to compare It with the other non-invasive methods, measurement of transepidermal water loss and of skin moisture. METHODS: Twenty-two healthy test subjects were recruited. Transepidermal water loss was measured at standard sites on the forearms and wrists, followed by skin moisture estimation by electrical capacitance, and finally by the recording of electrical impedance spectra in the frequency range 1 kHz to 1 MHz. Readings by all three methods were taken before the start of each series of test procedures, as well as after cyclohexane swabbing, a skin stripping procedure and lipid extraction, and also during the recovery process. A mixture of hexane:isopro-panol was used for lipid extraction of the skin, and the extracts were evaluated using HPLC/LSD and GC/MS/FID analysis. Biopsy samples for light and electron microscopy were obtained after lipid extraction. RESULTS: Electrical impedance results showed greater changes after lipid extraction than either transepidermal water loss or skin moisture content. Baseline values varied from the cubital fossa to the wrist, both for the non-invasive methods and for lipid composition. CONCLUSIONS: The electrial impedance is dependent on the lipid content of the stratum corneum, as studied by lipid extraction experiments.

Stratum corneum swelling. Biophysical and computer assisted quantitative assessments.

The aim of this study was to characterize the swelling behaviour of the stratum corneum. Stratum corneum pieces isolated from the breast region of 20 different females were incubated in distilled water at two different temperatures (20 degrees C and 45 degrees C) for 90 min and 24 h, respectively. Half of the stratum corneum pieces were previously extracted with chloroform-methanol (2:1). The area-enlargement was photographically recorded. The thickness enlargement was determined using a confocal laser scanning microscope. The average swelling (99% confidence interval) in the area dimension at 20 degrees C was 8.4% +/- 1.4% (n = 20), which corresponded to an average swelling in the length (lateral) dimension of approximately 4.1%. The swelling in the thickness dimension was 26.3% +/- 16.3% (n = 8). The swelling was most pronounced in the thickness dimension and was complete after 90 min of water immersion (P < 0.01, n = 5). In addition, the removal of the intercellular lipids with chloroform/methanol (2:1) induced a decreased swelling in the samples (P < 0.01, n = 20). An increase in temperature of the water from 20 degrees C to 45 degrees C resulted in an increase in swelling (P < 0.01, n = 20). Taken together our results support the idea that the mechanism of stratum corneum swelling is linked to the intercellular lipid structure and hence to skin barrier function.

Aspects on the physiology of human skin: studies using particle probe analysis.

The cellular part of the skin, the epidermis, is a very thin structure, approximately 120 microns 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 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 paper aims to give an overview of the state of the art in skin physiology made possible through the use of particle probes.

A novel approach to the understanding of human skin barrier function.

The basis for externally caused skin disorders is penetration of the skin barrier. A recent model for the skin barrier, the domain mosaic model, based on current knowledge of the physics of lipid bilayer organization gave tentative explanations for several aspects of function. It is demonstrated here that a development of the model explains how the requirements are met for a water-tight structure that will still allow a controlled, minute loss of water, the perspiratio insensibilis, necessary for maintaining plasticity of the keratin. A major advantage of the extended model is that it allows an interpretation of the changes imposed on the structure when in contact with detergents and/or penetration enhancers.

Pixe analysis of pathological skin with special reference to psoriasis and atopic dry skin.

The nuclear microprobe is used for element analysis of human skin cross sections, providing new insight into the physiology of normal and pathological conditions. Special interest is focused on trace elements as they work as secondary messengers or regulatory substances. The distribution of ions in normal tissues serves as reference for pathological changes. Calcium (Ca2+) is assumed to take an important role in the differentiation process of the epidermis. This paper presents new data on elemental and trace elemental distributions in skin. Samples are prepared from skin biopsies obtained from patients with skin disorder and from individuals with no records of skin disorder serving as controls. Using the nuclear microprobe, both elemental maps and quantitative depth profiles are obtained. Previous findings of abnormal Fe distribution in psoriatic skin are confirmed, and new observations of altered Zn and Ca profiles in atopic skin are reported. The relation to possible physiological/biochemical mechanisms and apoptosis ("programmed cell death") is discussed. The study is a part in a larger survey aiming at an understanding of the formation of a mature stratum corneum with a functional barrier, and its changed properties in cases of skin disorder.

Distribution of calcium and sulphur in the blue-light-exposed rat retina.

BACKGROUND: Blue-light exposure inhibits cytochrome oxidase and may therefore inhibit retinal metabolism. The reduced metabolism decreases the extrusion of calcium from the photoreceptor cell. Overload of calcium is proposed as one of the factors that lead to photoreceptor degeneration after light exposure. The light-induced photoreceptor degeneration can be ameliorated by calcium overload blocker. In the present study the calcium concentration was measured in the inner and outer segment layer of the rat retina. METHODS: Six eyes were exposed to blue (404 nm) light at a retinal dose of 380 kJ/m2. Five eyes served as the control group. The calcium and sulphur distributions were measured with a nuclear microprobe in the freeze-dried rat retina. The proton beam size was 12 x 12 microns and the energy of the protons was 2.55 MeV. The calcium concentration was calculated using sulphur as a reference. RESULTS: The level of calcium per milligram sulphur was 21 micrograms (range 17-23 micrograms) in the inner segment of the control retina. It increased to 62 micrograms/mg sulphur (range 57-67 micrograms) and 61 micrograms/mg sulphur (range 58-66 micrograms) 1 h and 12 h after blue-light exposure, respectively. CONCLUSION: The findings of the present study support the idea that accumulation of calcium in the inner segment layer is one of the factors that cause photoreceptor degeneration.

Human skin physiology studied by particle probe microanalysis.

Particle probe methods (electron probe and proton probe X-ray microanalysis) have been applied to investigate the distribution of elements and water over the different layers of the epidermis. For major elements, electron probe X-ray microanalysis (XRMA) provides the advantage of superior spatial resolution, but for trace element analysis the more sensitive proton probe (particle induced X-ray emission, PIXE) analysis has to be used. On a dry weight basis, the concentration of S is rather constant across the epidermis, whereas the concentrations of P, K, Cl and Na show gradients with high levels in stratum germinativum (basale) and stratum spinosum but low levels in the stratum granulosum and stratum corneum. Essentially, Fe and Zn are confined to the basal region in normal skin. The concentration of Ca, however, increased steadily from the basal region to the stratum corneum. The probe technique allows quantitative analysis of stratum-specific changes in elemental content in a variety of pathological conditions, e.g., changes induced by nickel, detergents and other chemicals, or in psoriatic skin. Of particular interest are findings of increased Fe and Zn in non-involved psoriatic skin. Since the different layers of the skin have different elemental concentrations and react differently under pathological conditions, the probe techniques are far superior to bulk chemical analysis in elucidating physiological and pathological processes in the skin.

A domain mosaic model of the skin barrier.

The skin barrier primarily protects the body against uncontrolled loss of water and in addition prevents water and matter of the environment from indiscriminately entering the living system. The current concept of the skin barrier suggests that permeability is governed by a hydrophilic and a hydrophobic "channel". To account both for the barrier function and the hydrophilic and hydrophobic pathways through this barrier, we propose a new model, "the domain mosaic model of the skin barrier", which depicts the bulk of the lipids as segregated into crystalline/gel domains bordered by "grain borders" where lipids are in the fluid crystalline state. Such an arrangement provides for an effective "water-tight" barrier that allows a minute and controlled loss of water to keep the corneocytes moistened. In addition the model provides for the necessary mechanical properties permitting bending and stress imposed on the skin surface. Furthermore, the fluid character of the "grain borders" represents areas where lipid and hydrophobic molecules may diffuse through the system on down-hill gradients. It is suggested that in the border areas between the crystalline domains, structural transformations of the lipid organization due to permeation promoters may take place without structural changes in the bulk organization of lipids in the crystalline or gel phase.

Quantitative and 3-dimensional analysis of Langerhans' cells following occlusion with patch tests using confocal laser scanning microscopy.

Quantitative and detailed 3-dimensional (3-D) morphological information can be obtained from the same tissue volume using a confocal laser scanning microscope (CLSM). In the present study, we used CLSM for evaluation of Langerhans' cells (LC) in human skin at 0 h, 24 h and 48 h following occlusion with patch tests for 48 h. The relative volume of epidermal CD1a reactivity was quantified with CLSM on 25 microns thick sections stained with indirect immunofluorescence. No statistically significant difference was found when comparing the values obtained on the biopsy specimens from occluded skin (n = 36) with those from non-occluded skin (n = 9). Nor were any statistically significant changes detected in the number of epidermal CD1a+ cells as determined with immunoperoxidase staining between occluded and non-occluded skin. The occlusion produced a transient mild inflammatory reaction with an induced expression of intercellular adhesion molecule-1 (ICAM-1) on keratinocytes and an increased number of CD3+ epidermal lymphocytes. In addition, 3-D reconstructions revealed spatial information on the distribution of LC dendrites towards the skin surface.

Water and ion distribution profiles in human skin.

Bulk sample x-ray microanalysis (XRMA) of human gluteal skin was performed to provide data on water and ion profiles over the epidermal cross section. All samples were analyzed both in the frozen hydrated state and in the frozen dried state. This allows, for the first time, a quantitative determination of the local water content in different strata of the skin. A steep water gradient was found in the stratum corneum towards the stratum granulosum, while the water content was constant in all deeper layers of the skin, including the papillary dermis. Previously demonstrated distributions over the epidermis of the monovalent ions sodium (Na), potassium (K) and chlorine (Cl) given in concentrations per unit dry weight were confirmed, as were the distributions of phosphorus (P) and sulfur (S). Combining the water and ion distributions, our data indicate the major monovalent ions Na, K and Cl to be in equilibrium between the stratum corneum and the dermis if the assumption is that all monovalent ions are in solution and that all the water of the tissue is available as solvent for the ions. This result does not disprove the existence of an ion transport gradient in the epidermis but sets significant bounds to it.

The temperature effect on in vitro penetration of sodium lauryl sulfate and nickel chloride through human skin.

Irritant contact dermatitis is a major problem in dermatology. One important group of substances causing irritant dermatitis is detergents. Exposure of the skin to detergents is frequent in both work and domestic environments. In the present paper we have studied how the penetration through the skin, and thus the effect, of the detergent sodium lauryl sulfate (SLS) is altered when the temperature is raised from 22 degrees C to 40 degrees C or 60 degrees C. We found that the penetration of sodium lauryl sulfate increased with increasing temperature. When comparing the increased penetration of sodium lauryl sulfate with the change in NiCl penetration at the same temperatures, we found that the increase in penetration was more pronounced for the detergent. This implies that the detergent also had a different effect on the structure and function of the epidermal barrier itself. The results underline the importance of choosing the right (low) temperature when working with detergent solutions to reduce the risk of developing irritant contact reactions.