Protein-enriched 'regular products' and their effect on protein intake in acute hospitalized older adults; a randomized controlled trial.

BACKGROUND & AIMS: Especially in older adults, maintaining muscle mass is essential to perform activities of daily living. This requires a sufficient protein intake. However, protein intake in hospitalized older adults is often insufficient. Thus far different nutrition intervention strategies have failed to show success in reaching sufficient protein intake in hospitalized older adults. The effect of recently developed protein-enriched bread and drinking yoghurt on protein intake is still unknown. Therefore, the objective of this study was to examine the effect of protein-enriched bread and drinking yoghurt on the protein intake of acute hospitalized older adults (≥55 years). METHODS: This study was performed as a single blind randomized controlled trial in 47 hospitalized elderly acutely admitted to a university hospital. During three consecutive days participants received either ad libitum protein-enriched bread and drinking yoghurt or normal, non-enriched products as part of their daily meals. The protein-enriched bread contained 6.9 g of protein per serving and the normal bread 3.8 g of protein. For drinking yoghurt this was 20.0 g and 7.5 g of protein per serving respectively. The products were almost isocaloric. Food intake of participants was measured and nutritional values were calculated according to the Dutch Food Composition Table. An independent sample t-test was used to compare protein intake between the intervention and control group. RESULTS: Analyses illustrate a protein intake in the intervention group of 75.0 ± 33.2 g per day versus 58.4 ± 14.5 g in the control group (p = 0.039). Intervention patients had a mean protein intake of 1.1 g/kg/day, with 36% of the patients reaching the minimum requirement of 1.2 g/kg/day; in control patients this was 0.9 g/kg/day (p = 0.041) and 8% (p = 0.030). Bread and drinking yoghurt contributed almost equally to the increased intake of protein in the intervention group. CONCLUSIONS: The use of protein-enriched bread and drinking yoghurt, consumed as part of regular meals, is a promising and feasible solution to increase the protein intake of acutely ill patients. It needs to be confirmed whether the use of these products will also result in a better clinical outcome. ClinicalTrials.gov ID number: NCT01907152.

Effects of the daily consumption of protein enriched bread and protein enriched drinking yoghurt on the total protein intake in older adults in a rehabilitation centre: a single blind randomised controlled trial.

OBJECTIVES: To investigate the effects of protein enriched bread and drinking yoghurt, substituting regular products, on the total protein intake and the distribution of protein intake over the day in older adults. DESIGN: A single blind randomised controlled trial. SETTING: Rehabilitation centre. PARTICIPANTS: Older adults (≥ 55 years) admitted to a rehabilitation centre after hospital discharge (n=34). INTERVENTION: Participants received a high protein diet (protein enriched bread and protein enriched drinking yoghurt; n=17) or a regular diet (regular bread and regular drinking yoghurt; n=17) for three consecutive weeks. MEASUREMENTS: Total protein intake and protein intake per meal, measured twice weekly over a three weeks period (six measurements per participant). RESULTS: Compared with controls, patients who received the protein enriched products had a significantly higher protein intake (115.3 g/d vs 72.5 g/d, P<0.001; 1.6 g/kg/d vs 1.1 g/kg/d, P<0.001). The intervention group consumed quantities over the recommended level (25-30 g/meal) during each of the three meals (32.5 g, 30.0 g, 34.8 g/meal), where the control group consumed quantities below the recommended level during breakfast (17.7 g) and lunch (18.4 g). CONCLUSIONS: The use of protein enriched products, replacing regular products, results in a significant increased daily protein intake in older adults. In addition, the daily consumption of protein enriched products improves protein distribution over the day.

Reconstruction of a human skin equivalent using a spontaneously transformed keratinocyte cell line (HaCaT).

Reconstruction of a skin equivalent using an immortalized human keratinocyte line, HaCaT, was investigated in an attempt to generate an in vitro system representative for human skin. Three different substrates were used to establish air-exposed cultures of HaCaT cells: de-epidermized dermis, collagen gels, and filter inserts. Effects of variations in culture conditions on tissue morphology, on the expression of proliferation-specific and differentiation-specific protein markers, and on lipid profiles were investigated. When grown at the air-liquid interface HaCaT cells initially developed a multilayered epithelium, but during the course of culture marked alterations in tissue architecture were observed. Ultrastructurally, a disordered tissue organization was evident as judged from the presence of rounded cells with abnormally shaped nuclei. Keratins K1 and K10 were irregularly expressed in all cell layers, including stratum basale. Staining of K6/K16 was evident in all cell layers. Locally, basal and suprabasal cells were positive for K4 and additionally expressed K13 and K19. The cornified envelope precursors were expressed only in older cultures (>2 wk after air exposure), except for transglutaminase and small proline rich protein 1, which were irregularly expressed in both early and older cultures. In addition, HaCaT cells showed an impaired capacity to synthesize lipids that are necessary for a proper barrier formation as indicated by the absence of free fatty acids and a very low content and incomplete profile of ceramides. Our data demonstrate that the ultimate steps of terminal differentiation in HaCaT cells do not occur irrespective of the type of substrate or the culture conditions.

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.

Nutritional skin care: health effects of micronutrients and fatty acids.

Human skin is continuously exposed to internal and external influences that may alter its condition and functioning. As a consequence, the skin may undergo alterations leading to photoaging, inflammation, immune dysfunction, imbalanced epidermal homeostasis, or other skin disorders. Modern nutritional science is developing new insights into the relation between food intake and health, and effects of food ingredients may prove to be biologically relevant for optimal skin condition. The objective of this review was to evaluate the present knowledge about the interrelation of nutrients and skin, particularly the photoprotective effects of nutrients, the influences of nutrients on cutaneous immune responses, and therapeutic actions of nutrients in skin disorders. The nutrients of focus were vitamins, carotenoids, and polyunsaturated fatty acids. Supplementation with these nutrients was shown to provide protection against ultraviolet light, although the sun-protection factor was relatively small compared with that of topical sunscreens. An increase in delayed-type hypersensitivity skin responses after supplementation with nutrients has proven beneficial, especially in elderly people, and may boost cell-mediated immunity. Dietary consumption of certain plants or fish oil is known to modulate the balance of lipid inflammatory mediators and, therefore, is valuable in the treatment of inflammatory skin disorders. It was concluded that nutritional factors exert promising actions on the skin, but information on the effects of low-to-moderate doses of nutrients consumed long term by healthy individuals is obviously lacking, as are data on direct effects on basal skin properties, including hydration, sebum production, and elasticity.

In vivo human skin permeability enhancement by oleic acid: a laser Doppler velocimetry study.

Topical application of a skin permeation enhancer such as oleic acid (OA) can result in: (i) higher skin permeability for many exogenous substances (ii) an irritation reaction. Laser Doppler velocimetry (LDV) is one of few techniques which can assess both effects using appropriate protocols. Direct LDV measurement, measuring cutaneous blood flow, has been preferred as a tool to evaluate skin irritation. By comparing the LDV value of an irritant-treated site with an untreated site, an irritation index for the irritant can be obtained. Occlusive application of 0.16 M OA in propylene glycol (PG) for either 3 or 24 h produced irritation in form of redness and slight swelling. Using LDV, we obtained an irritation index of 2 and 4, respectively. The vehicle, PG alone, produced an index of around 1, which corresponded well to the slight to almost no irritation observed visually. The duration of the high irritation index assessed by LDV after OA-PG application is comparable to the duration of the increase in transepidermal water loss following the same application. This indicates a correlation between skin irritation and barrier perturbation caused by OA. LDV can also be used to elucidate the effect of enhancers on skin using hexyl nicotinate (HN) as a model drug, since its vasodilative effect can be clearly assessed by LDV. Pre-treatment of PG for 3 h significantly reduced the t0 and tmax of HN. No further reduction could be observed when OA was added into PG, suggesting that OA-PG is not more effective than PG alone in enhancing the permeation of HN.

Tape stripping of human stratum corneum yields cell layers that originate from various depths because of furrows in the skin.

Tape stripping of human stratum corneum is widely used as a method for studying the kinetics and penetration depth of drugs. Several factors can influence the quantity of stratum corneum that is removed by a piece of tape, such as the manner of tape stripping, the hydration of the skin, cohesion between cells, body site and interindividual differences. However, few data are available about the influence of furrows in the human epidermis on the tape-stripping technique. In this study, we investigated the efficacy of tape stripping in removing complete cell layers from the superficial part of the human stratum corneum. A histological section of skin that was tape-stripped 20 times clearly showed nonstripped skin in the furrows, indicating persistent incomplete tape stripping. Replicas of tape-stripped skin surface demonstrated that even after removing 40 tape strips the furrows were still present. We validated the tape-stripping method further with X-ray microanalysis in the mapping mode by scanning electron microscopy, using a TiO2-containing compound as a marker. TiO2 applied to the skin before the tape-stripping procedures was still present after the tenth tape strip, and was specifically located on the rims of the furrows. We emphasize that results from studies using the tape-stripping method have to be viewed from the perspective that cells on one tape strip of the stratum corneum may be derived from different layers, depending on the position of the tape strip in relation to the slope of the furrow, and such results should be interpreted with considerable caution.

Determinants of skin sensitivity to solar irradiation.

BACKGROUND: Acute effects of UV irradiation include UV-induced erythema. Sunlight plays an important role in the development of skin cancer. Several predictive factors of UV-induced erythema could also be predictive for skin cancer. OBJECTIVE: Our objective was to quantitatively assess phenotypical and nutritional determinants of sensitivity to UV irradiation, as assessed by the minimal erythema dose (MED). DESIGN: We conducted a cross-sectional study among 335 volunteers. Sensitivity to UV irradiation was established through assessment of the MED. Phenotypical determinants, including skin melanin content, hair color and iris color were determined by skin reflectance spectrometry, a subjective questionnaire and an objective classification system, respectively. Furthermore, dietary exposure was measured by carotenoids, vitamin C, retinol and alpha-tocopherol in serum. RESULTS: Male subjects were found to be more sensitive to UV irradiation; that is, the MED was significantly lower compared to female subjects. Skin melanin content, which was positively associated with iris color in both sexes and with hair color in men, was the main phenotypical determinant of sensitivity to UV irradiation. No associations were found between serum carotenoids and MED in the total study group. Vitamin C was inversely associated with MED. However, associations between carotenoids concentrations and MED showed a positive trend in subjects with melanin values above and a negative trend in subjects below the median after adjustment for gender and total cholesterol. CONCLUSIONS: Skin melanin content and gender are important determinants of sensitivity to UV irradiation. No relation was found between serum carotenoids and MED in the total study group. The inverse association between vitamin C and MED was against our hypothesis. For the modifying effect of melanin on the association between carotenoids and MED, we do not have a clear biological explanation.

Assessment of the potential irritancy of oleic acid on human skin: Evaluation in vitro and in vivo.

As skin barrier modulating compounds, fatty acids are frequently used in formulations for transdermal or topical delivery. In this study the effects of oleic acid on keratinocytes in vitro was compared with its in vivo skin irritancy in humans. Dose- and time-dependent effects of oleic acid were examined in submerged human keratinocyte cultures, in reconstructed human epidermis (RE-DED), and in excised human skin, using alterations in morphology and changes in interleukin-1alpha mRNA levels as endpoints. In vitro results were compared with responses of living human skin after topical application of oleic acid, using non-invasive bioengineering methods. Direct interaction of oleic acid and submerged keratinocyte cultures resulted in cell toxicity at very low concentrations of the fatty acid. By contrast, when oleic acid was applied topically on RE-DED or on excised skin, no alterations in morphology were observed. Modulation of stratum corneum thickness indicated a key role of the stratum corneum barrier in the control of oleic acid-induced toxicity. In agreement with these findings, no epidermal tissue damage was seen in vivo, whereas oleic acid induced a mild but clearly visible skin irritation and inflammatory cells were present in the upper dermal blood vessels. Small amounts of oleic acid induced IL-1alpha mRNA expression in submerged keratinocyte cultures, whereas in RE-DED and in excised skin, IL-1alpha mRNA levels were increased only when the concentration applied topically was at least two orders of magnitude higher. It is concluded that minute amounts of oleic acid are sufficient to cause local (i.e. inside the viable epidermis) modulation of cytokine production. These concentrations do not affect morphology but induce skin irritation in vivo. To achieve comparable effects in the skin, much higher topical doses are needed than expected according to the locally required levels, owing to the rate-limiting transport of the fatty acid across the stratum corneum barrier.

An in vivo-In vitro study of the use of a human skin equivalent for irritancy screening of fatty acids.

A human skin equivalent (HSE) consisting of reconstructed epidermis on a fibroblast-populated collagen gel was evaluated as a model for irritancy screening. The irritancy potential of a series of saturated and unsaturated fatty acids was investigated in vivo under short-term exposure conditions using transepidermal water loss (TEWL), laser Doppler velocimetry (LDV) and the penetration of hexyl nicotinate as parameters. The effects of the fatty acids in vitro were studied after topical application on HSE using changes in epidermal morphology, changes in interleukin (IL)-1alpha and interleukin-8 mRNA expression and protein levels, and alterations in activity of plasminogen activators as endpoints. The unsaturated fatty acids increased both TEWL and LDV and elevated IL-1alpha and IL-8 mRNA levels, whereas their effects on protein levels were minimal. In contrast, the saturated fatty acids were not very effective in vivo but induced an increase in IL-1alpha protein levels. The type of fatty acid determines not only the way and the extent of skin barrier modulation, but also the pattern of cell mediator production and release. This study stresses the neccessity of investigating multiple endpoints for the characterization of a test compound, in particular when studying mild and moderate irritants.

Lipid and ultrastructural characterization of reconstructed skin models.

The study aimed at evaluating tissue architecture and quality of the permeability barrier in commercially available reconstructed human skin models; EpiDerm, SkinEthic and Episkin in comparison to native tissue. For this purpose, tissue architecture was examined by electron microscopy and epidermal lipid composition was analyzed by HPTLC. Stratum corneum lipid organization was investigated by electron microscopy in combination with RuO(4) post-fixation and by SAXD. Ultrastructurally, the overall tissue architecture showed high similarities with native epidermis. In the stratum corneum extracellular space, lipid lamellae consisting of multiple alternating electron-dense and electron-lucent bands were present. This regular pattern was not seen throughout the whole stratum corneum probably due to the observed irregular lamellar body extrusion in some areas. Lipid analyses revealed the presence of all major epidermal lipid classes. Compared with native epidermis the content of polar ceramides 5 and 6 was lower, ceramide 7 was absent, and the content of free fatty acids was very low. These differences in lipid composition may account for differences observed in SAXD pattern of Episkin and EpiDerm penetration models. In the latter only the long-distance periodicity unit of about 12 nm was observed and the short periodicity unit was missing. In conclusion, all three skin models provide a promising means for studying the effects of topically applied chemicals, although the observed deviations in tissue homeostasis and barrier properties need to be optimized.

Effects of calcitriol on fibroblasts derived from skin of scleroderma patients.

BACKGROUND: Scleroderma is a fibrotic disorder of unknown etiology that is characterized by excessive collagen synthesis and its deposition in the skin and various internal organs. OBJECTIVE: To examine whether an overproduction of extracellular matrix molecules is a result of either increased fibroblast proliferation or increased collagen synthesis. As results of clinical trials with 1,25-dihydroxyvitamin D3 (calcitriol) have suggested beneficial effect in the treatment of scleroderma patients, the effects of calcitriol on fibroblasts derived from scleroderma and normal skin has been examined as well. METHODS: Cultures of fibroblasts were established from biopsies from involved and uninvolved skin of scleroderma patients and from skin of healthy subjects, and compared with respect to proliferation, collagen synthesis and collagen lattice contraction. RESULTS: No significant differences in cell proliferation and in the extent of fibroblast-induced collagen lattice contraction have been found between scleroderma patients exhibited a disorganized growth pattern in a monolayer culture in contrast to normal fibroblasts. Collagen synthesis tends to be higher in scleroderma fibroblasts as compared with controls. Calcitriol exerted an antiproliferative and antisynthetic effect on fibroblasts, which, however, did not discriminate healthy fibroblasts from fibroblasts derived from involved or uninvolved scleroderma plaques. CONCLUSIONS: Our findings suggest that collagen accumulation may not result from increased proliferation or altered dynamic properties of fibroblasts in a scleroderma lesion but from increased collagen biosynthesis. We additionally found that calcitriol does not selectively affect scleroderma fibroblasts.

Expression of skin-derived antileukoproteinase (SKALP) in reconstructed human epidermis and its value as a marker for skin irritation.

For the investigation of the skin irritancy potential of chemicals in an in vitro model, it is necessary to have sensitive end-points that predict the effects on native human skin. Our aim was to investigate whether the induction of the proteinase inhibitor SKALP in reconstructed epidermis can be used as a marker. The influence of culture conditions and the effect of topical application of sodium lauryl sulfate and oleic acid on SKALP expression were evaluated using immunohistochemistry and Northern blotting. SKALP expression was induced by serum, epidermal growth factor and fibroblasts. In the presence of retinoic acid and 1,25-dihydroxyvitamin D3 SKALP expression was inhibited, whereas supplementation with ascorbic acid and a-tocopherol had no effect. Tape-stripping of excised skin and topical treatment with sodium lauryl sulfate induced SKALP protein expression. Application of sodium lauryl sulfate and oleic acid on reconstructed epidermis also induced SKALP at the protein level but no significant effects could be demonstrated at mRNA levels. In conclusion, SKALP expression, which was increased upon application of sodium lauryl sulfate and oleic acid, can be used as an in vitro end-point for skin irritancy, irrespective of the modifying effects of culture conditions.

Covalently bound lipids in reconstructed human epithelia.

The composition of free and covalently bound lipids in reconstructed epithelia generated with normal human keratinocytes, HaCaT cells and squamous carcinoma cells was investigated and compared with native skin. Stratum corneum isolated from native human and reconstructed epidermis was subjected to extensive extraction with chloroform-methanol mixtures followed by alkaline hydrolysis to release covalently bound lipids. High-performance thin layer chromatography was used for analysis of solvent-extractable and non-extractable lipids and gas liquid chromatography was performed to assess the fatty acid profile in extractable lipids. In both native and reconstructed tissue covalently bound lipids consisted of omega-hydroxyceramides, omega-hydroxyacids and free fatty acids. Small amounts of omega-hydroxyacids could already be detected in solvent-extractable fractions. omega-Hydroxyceramides consisted of Ceramide A, Ceramide B and a small fraction of unknown ceramides with intermediate polarity. The relative proportions of individual omega-hydroxyceramides were similar in both native and reconstructed stratum corneum. In contrast, differences were found in profiles of both solvent-extractable and non-extractable lipids isolated from epithelia reconstructed with transformed cell lines (HaCaT, SCC-12F2 and SCC-13 cells). Compared with native or reconstructed epidermis, in epithelia reconstructed with transformed cell lines the ceramide content was low, the most polar ceramides were missing and the content of free fatty acids was low. The same holds true for covalently bound lipids that were virtually absent in these epithelia. Marked similarities were demonstrated in the overall lipid composition of free and bound stratum corneum lipids in native epidermis and in epidermis reconstructed with normal human keratinocytes. The observed imbalance in fatty acid profile may account for differences in phase behaviour of stratum corneum lipids.

Temperature-sensitive regulation of epidermal morphogenesis and the expression of cornified envelope precursors by EGF and TGF alpha.

Epidermis reconstructed on de-epidermized dermis was used to investigate the effects of growth factors and culture temperature on epidermal morphogenesis and the expression of cornified envelope precursors. Cultures grown at 33 degreesC or 37 degreesC in the absence or presence of transforming growth factor alpha (TGFalpha), keratinocyte growth factor (KGF), basic fibroblast growth factor (bFGF), or insulin-like growth factor (IGF) show a similar morphology to that of native epidermis. Loricrin and SPRR2 are expressed in the stratum granulosum and SPRR3 is absent. Cultures grown in epidermal growth factor (EGF)-supplemented medium at 37 degrees C have a normal morphology, whereas cultures grown at 33 degrees C have a disorganized basal layer, no stratum granulosum, and nuclei are present in the stratum corneum. Loricrin is absent, and SPRR2 and SPRR3 expression extend into the spinous layers. Irrespective of the culture condition used, involucrin is aberrantly expressed in all suprabasal layers. EGF stimulated keratinocyte proliferation and migration to a greater degree than TGFalpha. Epidermis reconstructed on fibroblast-populated collagen gels at 33 degrees C led to the same disturbances in keratinocyte differentiation as seen in cultures grown on de-epidermized dermis at 33 degrees C in the presence of EGF, whereas parallel cultures grown at 37 degrees C have a similar morphology to that of native epidermis.

Microdialysis technique as a method to study the percutaneous penetration of methyl nicotinate through excised human skin, reconstructed epidermis, and human skin in vivo.

PURPOSE: The aim was to assess the feasibility of cutaneous microdialysis as a method to study percutaneous penetration of methyl nicotinate through human skin in vitro and in vivo. METHODS: Microdialysis was applied in vitro in excised human skin, in isolated dermis, in reconstructed human epidermis and in vivo in the volar forearm skin of volunteers using methyl nicotinate (MN) as a model compound. After topical application of MN, aliquots of the perfusate were collected and analyzed for the presence of MN spectrophotometrically and by HPLC. In vivo, visual scoring and laser Doppler perfusion imaging (LDPI) were used to monitor the effects on skin blood flow. RESULTS: In vitro, MN was detected in the dialysate after a 1 min exposure of excised skin to concentrations as low as 25 mM. Higher concentrations up to 500 mM showed increased levels. Prolongation of the application time to 60 min resulted in increased levels of MN in the perfusate as the duration of application increased. Reconstructed epidermis and isolated dermis showed an almost 2- and 20-fold higher penetration compared to excised skin, respectively. In vivo, LDPI measurements showed a rapid increase in skin blood flow after application of 25 to 100 mM MN for 1 min. MN was only detectable in the microdialysate after application of 100 mM for 10 min (two of three subjects). CONCLUSIONS: Cutaneous microdialysis may be a tool for comparative studies linking responses in human skin in vivo to in vitro data using the same technique and endpoint.

Characterization and comparison of reconstructed skin models: morphological and immunohistochemical evaluation.

Reconstructed human skin equivalents are currently being investigated as in vitro models for the prediction of human skin toxicity and irritation responses. Three different industrial reconstructed skin models (EpiDerm, Episkin and SkinEthic) and one in-house equivalent were characterized and compared using light microscopy, immunohistochemistry and reduction of (3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyl tetrazolium bromide) (MTT). Their inter- and intra-batch variation was evaluated. Histological examination showed a completely stratified epithelium in all skin models, which closely resembled normal human epidermis. Low intra-batch variation in tissue architecture was observed in all skin models, but moderate to considerable inter-batch variation was noticed. Evaluation of the expression and localization of a number of differentiation-specific protein markers revealed that all skin models showed an aberrant expression of keratin 6, skin-derived antileukoproteinase, small proline rich proteins, involucrin and transglutaminase. Although variation within batches was low, in particular keratin 6, involucrin and skin-derived antileukoproteinase expression demonstrated some inter-batch variation. Reduction of MTT in vehicle-treated cultures showed high similarities between skin models, but marked differences were observed when 1.0% sodium lauryl sulfate was applied topically for 3 or 16 h. Most pronounced effects were noticed in SkinEthic cultures. Intra-batch variations were low and moderate variations were observed between batches. All skin models tested reproduced many of the characteristics of normal human epidermis and therefore provide a morphologically relevant in vitro means to assess skin irritation and other skin-related studies.

Retinoic acid metabolites in plasma are higher after intake of liver paste compared with a vitamin A supplement in women.

The objectives of the present study were to compare the bioavailability of vitamin A from liver paste and from a vitamin A supplement at three nutritionally relevant levels of intake, and to estimate levels of "safe" intake based on concentrations of retinoic acid and its metabolites in plasma after a single dose of vitamin A from liver paste. Women (n = 35; 19-47 y of age) consumed 3.0, 7.5 or 15 mg vitamin A as liver paste or as a vitamin A supplement with a test meal in a randomized design, with a combined crossover (two sources) and parallel approach (three dosages). Retinyl esters and retinoic acid (RA) metabolites were quantified in blood samples at 2-24 h after dosing. The areas under the time-response curves (AUC) were calculated to evaluate responses in plasma vitamin A after intake of liver paste and the vitamin A supplements. For retinyl esters, the AUC was significantly affected by the dosage, but not by the source. The formation of 13-cis-RA, 13-cis-4-oxo-RA, and to a lesser extent all-trans-RA was significantly higher after consumption of liver paste compared with the supplement, especially at higher dosages. Long-term baseline concentrations of retinol were not affected by a single intake of vitamin A. In conclusion, the bioavailability of vitamin A from single doses of liver paste and a vitamin A supplement does not differ, but the plasma concentrations of RA metabolites are higher after intake of liver paste. Thus, pregnant women should indeed limit the intake of vitamin A from liver products.

In vivo human skin barrier modulation by topical application of fatty acids.

The in vivo effects of fatty acids on skin barrier function were assessed by measuring: (i) transepidermal water loss (TEWL), (ii) diffusion lag times for hexyl nicotinate (HN), and (iii) irritant skin response using laser Doppler velocimetry (LDV) in combination with visual scoring. Two classes of fatty acids have been investigated: straight-chain saturated fatty acids (SFA), having 6-12 carbon atoms, and unsaturated fatty acids (UFA): oleic, linoleic, alpha-linolenic and arachidonic acids. It has been reported that these acids can enhance the permeation of various compounds across the skin. After topical and occlusive application as a solution in propylene glycol (PG) for 3 h on the volar arm of human subjects, SFA only caused a slight irritation and increase in TEWL. The diffusion lag times of HN were reduced by the application SFA to the same extent as and not more than by the application of the pure solvent PG. In contrast, the application of UFA caused a significant increase in TEWL and LDV (irritation) responses. The TEWL values after oleic acid application were higher than those observed for the other three acids, while the irritation potential of arachidonic acid was the highest among UFA. As with SFA, sites treated with UFA did not show significantly different lag times of HN diffusion from PC-treated sites. The data suggest that the degree of irritation and the degree of barrier modulation for fatty acids are not necessarily correlated.

Barrier function in reconstructed epidermis and its resemblance to native human skin.

One of the prerequisites for the use of human skin equivalents for scientific and screening purposes is that their barrier function is similar to that of native skin. Using human epidermis reconstructed on de-epidermized dermis we demonstrated that the formation of the stratum corneum (SC) barrier in vitro proceeds similarly as in vivo as judged from the extensive production of lamellar bodies, their complete extrusion at the stratum granulosum/SC interface, and the formation of multiple broad lamellar structures in the intercorneocyte space. The presence of well-ordered lipid lamellar phases was confirmed by small-angle X-ray diffraction. Although the long periodicity lamellar phase was present in both the native and the reconstructed epidermis, the short periodicity lamellar phase was present only in native tissue. In addition, the SC lipids predominantly formed the hexagonal sublattice. Analysis of lipid composition revealed that all SC lipids are synthesized in vitro. Differences in SC lipid organization in reconstructed epidermis may be ascribed to the differences in fatty acid content and profile indicating that further improvement in culture conditions is required for generation of in vitro reconstructed epidermis with stratum barrier properties of the native tissue.