Vol. 42, No. 1, pp.16-21, 2008Decrease in the skin transparency induced by protein carbonylation in the stratum corneum

It has been reported in recent years that the carbonyl modification of protein plays a part in various diseases. The existence of the protein carbonylation in the stratum corneum also came to be known in the last few years, but the effect on the properties of the stratum corneum including the skin appearance was not clarified. In this study, we examined the influence of protein carbonylation in the stratum corneum to provide helpful information for skin care products focusing on transparency of the stratum corneum. Firstly, we developed a method to assess the protein carbonylation level numerically by the image analysis of tape-stripped stratum corneum reacted with a fluorescent hydrazide. The level of stratum corneum carbonylation of the sun-exposed site (face) was higher than that of unexposed site (upper arm), and the surface part of the stratum corneum showed a higher level of carbonylation compared with the deeper layer. Stratum corneum carbonylation was induced by in vitro UV irradiation. In addition, it was shown that the skin transparency score was significantly low in the woman with high level of protein carbonylation in the stratum corneum of the cheek. An experimentally carbonylated stratum corneum sheet in vitro became opaque. Among the amino acids investigated, L-lysine was the most effective to prevent carbonylation of the stratum corneum ex vivo . Moreover, L-lysine inhibited the decrease in the skin transparency caused by experimental carbonylation of the stratum corneum in the human skin. These results suggest that preventing the carbonylation of the stratum corneum due to adverse effects from the environment by l-lysine could improve the skin transparency.
Keywords: stratum corneum, protein carbonylation, appearance, skin transparency, l-lysine, amino acids, fluorescent hydrazide, tape-stripping     

Quantification of skin penetration of antioxidants of varying lipophilicity

Antioxidants play a vital role in protecting the skin from environmental distress. As the skin is constantly exposed to harmful UV radiation, endogenous antioxidants present in the superficial layers of the skin neutralize reactive oxygen species. Over time, antioxidants become depleted and loss their protective effect on the skin. Therefore, supplementing skin with topical antioxidant can help replenish this loss and fight the oxidative stress. The objective of this study was to deliver antioxidants topically and quantify the amount permeated in the stratum corneum and underlying skin. Polyphenols (catechin, resveratrol and curcumin) and vitamin (retinol) with various lipophilic properties were delivered via porcine ear skin, using propylene glycol as a vehicle. The amount in the stratum corneum and underlying skin was quantified using tape stripping and skin extraction methods, respectively, and samples were analysed via HPLC. All four antioxidants permeated into the skin from the propylene glycol vehicle. The order of the amount of antioxidant in the stratum corneum was catechin > resveratrol~ retinol> curcumin, whereas that in the underlying skin was retinol > catechin~ resveratrol~ curcumin. Of the total amount of polyphenols in the skin, approximately 90% was retained in the stratum corneum whereas 10% was quantified in the underlying skin. In contrast, 10% of retinol was retained in the stratum corneum whereas 90% permeated in the underlying skin. Polyphenols (catechin, resveratrol and curcumin) showed high concentration in the stratum corneum whereas retinol showed high accumulation in the underlying layers of the skin.     

Efficacy of levulinic acid-sodium dodecyl sulfate against Encephalitozoon intestinalis, Escherichia coli O157:H7, and Cryptosporidium parvum

Foodborne parasites are characterized as being highly resistant to sanitizers used by the food industry. In 2009, a study reported the effectiveness of levulinic acid in combination with sodium dodecyl sulfate (SDS) in killing foodborne bacteria. Because of their innocuous properties, we studied the effects of levulinic acid and SDS at various concentrations appropriate for use in foods, on the viability of Cryptosporidium parvum and Encephalitozoon intestinalis. The viability of Cryptosporidium and E. intestinalis was determined by in vitro cultivation using the HCT-8 and RK-13 cell lines, respectively. Two Escherichia coli O157:H7 isolates were also used in the present study: strain 932 (a human isolate from a 1992 Oregon meat outbreak) and strain E 0018 (isolated from calf feces). Different concentrations and combinations of levulinic acid and SDS were tested for their ability to reduce infectivity of C. parvum oocysts (10(5)), E. intestinalis spores (10(6)), and E. coli O157:H7 (10(7)/ml) when in suspension. Microsporidian spores were treated for 30 and 60 min at 20 ± 2°C. None of the combinations of levulinic acid and SDS were effective at inactivating the spores or oocysts. When Cryptosporidium oocysts were treated with higher concentrations (3% levulinic acid-2% SDS and 2% levulinic acid-1% SDS) for 30, 60, and 120 min, viability was unaffected. E. coli O157:H7, used as a control, was highly sensitive to the various concentrations and exposure times tested. SDS and levulinic acid alone had very limited effect on E. coli O157:H7 viability, but in combination they were highly effective at 30 and 60 min of incubation. In conclusion, Cryptosporidium and microsporidia are not inactivated when treated for various periods of time with 2% levulinic acid-1% SDS or 3% levulinic acid-2% SDS at 20°C, suggesting that this novel sanitizer cannot be used to eliminate parasitic contaminants in foods.     

Stability study of lipoic acid in the presence of vitamins A and E in o/w emulsions for cosmetic application

The effectiveness of any cosmetic product containing a functional ingredient is determined by the skin delivery of the active molecule, which is influenced by the type of carrier and the molecule itself. Furthermore, the functional ingredient should be stable in the formulation. The purpose of this paper is to study the stability of lipoic acid in the presence of vitamins A (as palmitate) and E (as acetate) in semisolids for cosmetic use. The systems formulated were studied in regard to their aspect, pH, stability under centrifugation and rheological behavior. The chemical analyses of lipoic acid and vitamins A and E were carried out by HPLC after studying the specificity of the method employed in each case. The quantitation of the active principles was performed by HPLC with C18 (5 μm) columns. The mobile phase was methanol for the vitamins, with spectrophotometric detection at 325 nm for vitamin A and 230 nm for vitamin E. The mobile phase for lipoic acid was methanol : water (80 : 20) and phosphoric acid at pH 3.0, with spectrophotometric detection at 332 nm. All systems were stable to centrifugation, and no significant modification of rheological behavior was observed in relation to the base emulsion used as control. The chemical studies performed indicated that although lipoic acid is not very stable in these formulations, the presence of vitamin A favors its chemical stability.     

Investigation of interactions between silicones and stratum corneum lipids

Ingredients of topically applied skin care formulations have not only positive effects on the appearance of human skin but can also disturb the Stratum corneum (SC) lipid barrier. In the present study, the influence of silicones (PDMS), as often used cosmetic ingredients, on the microstructure of SC lipids was investigated. For this purpose the interactions of four different PDMS with excised human SC were examined first using differential scanning calorimetry (DSC) and wide angle X-ray diffraction for physical characterization. Because the physical properties of human stratum corneum strongly depend on the lipid composition, showing inter-and intra-individual differences, the interactions with an in vitro model lipid system containing SC fatty acids were also studied, using polarized light microscopy, transmission electron microscopy, small angle X-ray diffraction and DSC. The results revealed that the investigated PDMS do not change either the microstructure of excised human SC or the biphasic lamellar/inverse hexagonal structure of the in vitro model. We concluded that PDMS will not cause any side-effects when topically applied and that our simplified in vitro model could be helpful for estimating interactions between cosmetic ingredients and other topically applied substances and the skin barrier at an early moment of formulation development.     

Determination of lipoic acid in meat of commercial quality.

For the quantitative determination of lipoic acid in meat a sensitive GC/MS method in the chemical ionisation mode with methane as reactant gas has been developed. Firstly, the cleavage of protein-bound lipoic acid from the epsilon-amino group of lysine residues was optimized by hydrolysing the synthesized model compound epsilon-lipoyllysine with several organic and inorganic acids and proteolytic enzymes. The concentrations of lipoyllysine and lipoic acid during this test hydrolysis were monitored by HPLC. Optimum hydrolytic conditions were heating at 120 degrees C in 2 mol H2SO4 for seven hours. After tissue hydrolysis, the lipoic acid in the hydrolysate was separated by a diethylether/sodium bicarbonate/diethylether extraction and then derivatised for GC with MBDSTFA. The highest amounts of lipoic acid in meat of commercial quality were detected in liver, heart and kidney whereas in muscle tissues its content was lower.     

Preparation and structural properties of small‐particle cassava starch

Acid and enzyme hydrolyses followed by ball milling were applied to fracture cassava starch granules. Microscopic and chromatographic evidence suggested different mechanisms of the two hydrolyses. Using the enzyme process, granules with a sponge‐like structure and shells with the interior hydrolysed were produced. Amylose and amylopectin were subjected equally to multiple attacks by enzymes, with no significant change in granule crystallinity. The hydrolysed residues could not be effectively broken down by ball milling, although the crystallinity was destroyed. In contrast, the acid treatment caused superficial external corrosion, mainly at the amorphous lamellae, ie the branch points of amylopectin. Acid‐lintnerised starch granules were mostly of Degree of polymerization, DP 10–15 and exhibited increased crystallinity and brittleness, making them more susceptible to breakdown upon milling. Ball milling, although destroying some degree of crystallinity, could effectively reduce the size of acid‐hydrolysed starch, with no further degradation of amylodextrin molecules. By a combination of lintnerisation and ball milling, smaller particle starch (3–8 µm compared with 3–30 µm for native starch) could be produced. It is clear that removal of the amorphous phase prior to milling is critical for effective rupture of the granules. Copyright © 2003 Society of Chemical Industry     

Influence des differents constituents de la couche cornee sur la mesure de son élasticité

Un nouvel appareillage, permettant une détermination simultanée des modules d'élasticité statique de dynamique du Stratum Corneum, a été mis au point.
L'étude systématique de ces paramètres mécaniques pour différents échantillons de SC humain permet de mettre en évidence les avantages de la mesure du module d'élasticité dynamique pour quantifier l'effet des traitements.
L'effet de certains traitements 'kératolytiques'sur l'élasticité du SC peut ainsi être discuté. Ces résultats ainsi que ceux obtenus par microscopie électronique sur l'effet de ces produits, permettent de discuter les relations existant entre la structure et les paramètres élastiques.
The effect of various constituants of the corneous layer on measurements of its elasticity

Summary

A new instrument has been developed which enables simultaneous determinations of the static and dynamic moduli of elasticity of the stratum corneum to be made.
A systematic study of these mechanical parameters for various samples of human SC demonstrates the advantages of the dynamic measurement of the modulus of elasticity in evaluating the effect of treatments.
The effect of treatment of thioglycollic acid (TA) and salicylic acid (SA) on the stratum corneum was studied by means of this technique and electron microscopy.
TA markedly reduces the elastic modulus. Electron microscopy shows that horny cell structure is fully disorganized. On the contrary, SA treatment increases the elastic modulus without any noticeable changes on corneocytes. This last study confirms the preferential effect of SA on the intercorneocyte spaces.     

Guaraná, a supplement rich in caffeine and catechin,modulates cytokines: evidence from human in vitro and in vivo protocols

Guaraná powder is an antiobesogenic supplement; however, its effect on inflammatory biomarkers has not yet been determined. Therefore, this study analysed whether guaraná supplementation can differentially modulate the levels of proinflammatory cytokines [interleukin 6 (IL-6), tumour necrosis factor-alpha, interleukin 1 beta (IL-1β), interferon-gamma (Ig-γ)] and anti-inflammatory interleukin 10 (IL-10) from in vitro and in vivo protocols. In the pilot in vitro protocol, human peripheral blood mononuclear cells were exposed to guaraná, as well as to resveratrol, quercetin and ascorbic acid as positive controls. The effect of guaraná on cytokine levels was also evaluated in culture medium supplemented with glucose and insulin. A randomised, placebo-controlled in vivo assay was also performed to evaluate the potential influence of guaraná on the blood cytokine levels of 14 healthy volunteers supplemented for 14 days. The effect of guaraná was similar to that of resveratrol, a known anti-inflammatory molecule, decreasing IL-1β, IL-10 and Ig-γ levels and increasing IL-10 levels compared to those of the control group. The in vitro insulin supplementation potentiated the effect of guaraná on some cytokines. A decreasing effect on the blood inflammatory cytokine levels, along with an increase in IL-10 levels, was also observed in volunteers supplemented with guaraná. In conclusion, guaraná positively modulates cytokines associated with inflammatory metabolism.     

Water retention of treated stratum corneum measured by a coupling method: thermal desorption-mass spectrometry

A thermal desorption autosampler coupled to a mass spectrometer has been used to measure the in vitro water retention of human stratum corneum as a function of treatment applied. Samples were treated with ingredients possessing good hygroscopic properties which are used in the formulation of moisturizing creams. After being treated, stratum corneum samples were dried and rehydrated and then analysed. The paper describes the method used to quantify their hygroscopic capacity in terms of the percentage of water retained. Ten humectants were examined, of which urea, glycerine, ammonium lactate and a new salt of an α-hydroxy acid were found to have the highest activities. This method can also be used to quantify the water retention capacity of various types of pathological skin such as ichthyosis and psoriasis.     

Influence of cleansing on stratum corneum tryptic enzyme in human skin

Desquamation in human skin is a well-balanced process of de novo production of corneocytes and their shedding from the skin surface. The proteolysis of corneodesmosomes is an important step in the final desquamation process. In the degradation of these adhesion molecules, the stratum corneum tryptic enzyme (SCTE) plays a key role. In initial studies with extracts of porcine epidermis, SCTE was shown to be inactivated by low concentrations of sodium lauryl ether sulphate (SLES). These in vitro findings were supported by in situ results obtained by measuring the release of fluorescent dyes coupled to trypsin-specific substrates incubated on human skin cross-sections. Moreover, in further studies, it could be demonstrated that the SCTE activity in the human horny layer decreases after in vivo application of cleansing products containing SLES. After repeated washing of human volunteers with tap water, a standard market cleansing product (SLES/betaine system) or a new improved cleansing product (SLES/betaine/disodium cocoyl glutamate system), the specific SCTE activity was determined in extracts from the uppermost layers of the stratum corneum. It could be shown that after application of the new formula the remaining SCTE activity was significantly higher than after use of the standard market formula. This ex vivo approach has proven to be very helpful for measuring surfactant effects on human skin enzymes. Using this assay, we developed an improved shower gel formula, which leads to a significantly higher skin enzyme activity after application, compared to a standard market formula.     

ENZYME AND ACID HYDROLYSIS OF MALTED MILLET (Pennisetum typhoidees) AND SORGHUM (Sorghum bicolor)

Malted millet and sorghum were hydrolysed by the enzymes produced during malting. Malted and unmalted millet and sorghum were acid hydrolysed. The enzyme hydrolysed carbohydrate was mostly glucose, with only traces of disaccharides, in the case of malted millet. The yield of enzyme hydrolysed carbohydrate was however low in both millet and sorghum. Acid hydrolysis of malted millet or sorghum showed that glucose of malted grains was destroyed during acid hydrolysis more than pentoses in the malted grains.     

The enzymic breakdown of lipids to volatile and non-volatile carbonyl fragments in disrupted tomato fruits

Physical disruption of tomato fruits results in the degradation of endogenous lipids by hydrolytic and oxidative enzymes. Acyl hydrolase, phospholipase D, lipoxygenase and hydroperoxide cleavage enzymes are active in this tissue. A sequential enzymic pathway is proposed by which tissue lipids are hydrolysed to free (mainly polyunsaturated C₁₈) fatty acids which are subsequently oxidised to their hydroperoxides by lipoxygenase action. The C₁₈ hydroperoxides are cleaved (by an enzyme which is specific for 13-hydroperoxide isomers) to volatile C₆ aldehydes (hexanal or cis-3 hexenal) and non-volatile C₁₂ w-oxoacids. The non-volatile fragment from linoleic acid was identified as 12-oxo-dodec-cis 9-enoic acid.     

Effect of 2-hydroxyacids on guinea-pig footpad stratum corneum: mechanical properties and binding studies

The effect of aqueous solutions of 2-hydroxyacids of chain length C₃ to C₁₀ on the extensibility of undamaged and solvent-damaged guinea-pig footpad stratum corneum has been studied. The increase in extensibility of solvent-damaged corneum, caused by treatment with hydroxyacid, reached a maximum with 2-hydroxycaprylic acid (C₈); on undamaged corneum, 2-hydroxycaprylic acid was the only hydroxyacid studied to give a significant effect. The increase in corneum extensibility produced by 2-hydroxyacids decreases when the pH is raised from 3 to 4. This loss of effect correlates with the ionization of the hydroxyacid (p K ˜ 3.85).
The binding of radiolabelled 2-hydroxycaproic (C₆) and 2-hydroxycaprylic acids to stratum corneum has been studied. 2-Hydroxycaprylic acid binds much more strongly than 2-hydroxycaproic acid, the difference in the binding being consistent with the hydrophobic binding energy of two methylene groups. Raising the pH above 3.5 results in a large decrease in the binding of 2-hydroxycaprylic acid in line with the corresponding reduction in extensibility.
Treatment with 2-hydroxyacids results in a small increase in the water-binding capacity of solvent-damaged stratum corneum, but in a decrease in the water-binding capacity of undamaged stratum corneum. These data are discussed in terms of a possible mechanism for the plasticizing of stratum corneum.
Effet des 2-hydroxyacides sur la couche cornée du coussin des pattes de cobaye     

The ultraviolet microscopic transmission characteristics of human stratum corneum

This study has evaluated the ultraviolet light transmission characteristics of human stratum corneum at the single cell level using a low light level video microscope to measure the mean percentage transmission of light at different wavelengths and the variation in transmission across the stratum corneum. Stratum corneum was isolated by an enzymic technique and examined on a low light level UV video microscope. Quantitative evaluation of the transmitted monochromatic light for the underside of the layer was measured directly using a Kontron UNIPS image processor or indirectly with a Quantimet 920 image processor after video recording. Transmission distribution histograms were obtained from samples of stratum corneum taken from human breast, scalp, abdomen and leg. Mean transmission values were also derived and compared with diffuse transmission values obtained using the same tissue mounted on an integrating sphere. The UV microscopic transmission characteristics of enzyme separated stratum corneum clearly demonstrated that this structure was not an ideal diffuser. Uniform light intensity on the surface of the stratum corneum led to areas of transmitted intensity in close proximity that differed by factors ofthree to six fold, e.g. between regions of high (>70%) and low (<20%) brightness. However, the average transmission was found to be compatible with published data obtained by diffuse transmission spectrophotometry, taking into account the enhanced transmission arising from stratum corneum immersion in phosphate buffered saline. This was confirmed by the elevated values obtained by diffuse transmission spectrophotometry in this study for samples of stratum corneum prepared for UV microscopy being higher than these found in published data. It is obvious from these findings that viable cells in the epidermis are not exposed to a uniform incident light intensity even when this is true for the surface of skin. Studies of skin response to ultraviolet light at the single cell level must take account of the possibility of preferential exposure of specific sites in any subsequent explanation of cell sensitivity. This is in addition to the already well established cell cycle dependent ultraviolet sensitivity.     

Spectrophotometric Titration of Starch Polysaccharides with Iodine

Two methods of Spectrophotometric titration (SPT) are described. Method A describes the determination of the extinction (E) at 4 wavelengths (WL) 714, 625, 555, and 500 nm and of 16 concentrations of iodine in the range of [I] = 2.5 and 166.7 · 10⁻⁵ N I₂ or pI = 4.60 and 2.28 (pI = -log [I]) as well as the calculation of the extinction-relationship R of two WL for 6 possible pair combinations. The dependence of E respectively of log [I] in form of E- or R-curves of amyloses, amylopectins, Nägeli-amylodextrin and starches of several botanical sources are discussed under the aspect of the reaction of helical segments of molecules with iodine. Method B of the SPT defines E of the 4 wavelengths at pI 3.6 and 3.0 and calculation of the extinction relation ships R(M) and R(H) of middle (M) and high (H) concentrations of iodine. The position of the parameters R(M) and D=R(M) — R(H) of the 6 wavelength combinations in each X-Y-diagram depends on the botanical origin of potato, tapioca, wheat and regular maize starch.     

Enzymatic hydrolysis of steryl ferulates and steryl glycosides

Steryl ferulates (SF) and steryl glycosides (SG) are phytosterol conjugates found characteristically in cereals. Currently, little is known about their properties with respect to enzymatic hydrolysis. SF and SG were extracted and purified from rye and wheat bran. Their percentages of hydrolysis with different enzymes were studied using normal phase HPLC with UV detection for steryl ferulates and evaporative light scattering detection for steryl glycosides. Steryl ferulates were hydrolysed by mammalian digestive steryl esterases. It was further demonstrated that a mixture of steryl ferulates from rye and wheat was hydrolysed much more effectively than a steryl ferulate mixture from rice (commonly known as γ-oryzanol), suggesting greater bioavailability in non-rice steryl ferulates. Steryl glycosides were hydrolysed by a commercial microbial β-glucosidase preparation (cellobiase), but were not effectively hydrolysed by two other highly purified β-glucosidases. These results demonstrate for the first time the potential use of enzymes as a replacement for acid hydrolysis in analytical procedures for SG and also provide insights about the potential bioavailability of these sterol derivatives in human digestive systems. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Turnover and hydrolysis of vicine and convicine in avian tissues and digesta

The objectives of this study were to establish factors influencing the absorption, excretion and hydrolysis of vicine and convicine in chicks. Blood vicine, following the oral administration of a single dosage of vicine into the crop of young chicks, reached maximum concentrations within 3 h. It was nearly completely removed from the blood within 12 h and had a half-life of approximately 4.5 h. The accumulation and excretion patterns of vicine in the liver and kidney were similar to those of the blood except that the concentrations were much higher in these tissues, particularly the kidney. Bile also contained a very high concentration of vicine which tended to accumulate following the decline in other tissues. These results together with the appearance of vicine in the urine of colostomised birds suggest that vicine is excreted in the urine and bile. Convicine in contrast to vicine was not absorbed by the chick. In-vitro studies were carried out with tissue and digesta homogenates from the chick in order to establish the site(s) at which vicine and convicine were hydrolysed to their aglycone forms. The results demonstrated that neither vicine nor convicine were hydrolysed in the presence of liver, kidney, intestinal wall or caecal wall homogenates, digesta from the large intestine or by enzymes present in whole or ground fababeans. They were, however, slowly hydrolysed in the presence of 0.1N HCl at 37°C and very rapidly hydrolysed by digesta from the caeca. Antibiotic additions to the diets markedly reduced the in-vitro rate of hydrolysis of these compounds. The latter results suggest that vicine and convicine are hydrolysed by microorganisms in the caeca of the chick but are not hydrolysed by the micro-organisms in the gastrointestinal tract, by endogenous tissue enzymes or by enzymes present in fababeans and only minimally hydrolysed by the low pH of the stomach.     

A SIMPLIFIED ENZYMIC METHOD FOR THE DETERMINATION OF (1→3) (1→4)-β-GLUCANS IN BARLEY

A rapid simplified procedure for the enzymic determination of β-glucans is described. In this method a small sample of ground barley (0·25 g) is heated in 80% ethanol to inactivate enzymes, the β-glucan is hydrolysed by incubation for 1 h with a high concentration of purified β-glucanase and the reducing sugars produced are determined by reaction with p-hydroxybenzoic acid hydrazide. The method was calibrated using β-glucan isolated from barley and the enzymic hydrolysis was shown to be both specific and complete.