Stratum Corneum Exfoliation Effect with Hydroxy Acid according pH

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Hydroxy acid has been used to enhance anti-aging and skin moisturization by peeling effect on the skin stratum corneum, and thus it has been widely used in topical products and cosmetic products. Among them, the effect that appears most effectively in a short period of time has been reported to be effected by the pH of the cosmetic formulations. However, there are many difficulties in use due to irritation caused by pH and concerns about side effects. The purpose of this study was to evaluate the effect of applying cosmetics with (1) varying concentrations, (2) types and (3) pH of hydroxy acid on human skin. 22 healthy adults were stained with DHA (dihydroxyacetone) and DC (dansyl chloride) on the forearm, and the skin exfoliation effect was measured after application of the test products. (1) The application of GA (glycolic acid) increased the desquamation by concentration dependent. (2) the test product prepared with neutral pH showed no exfoliation effect. In contrast, SA (salicylic acid) showed a statistically significant exfoliation effect at both acidic pH and neutral pH. (3) The neutral pH SA showed excellent exfoliation effect on bot DHA and DC stained stratum corneum. These results suggest that it is possible to manufacture safe cosmetics without damaging the skin barrier, providing an opportunity to use cosmetics that are expected to exfoliate to people, whose skin is sensitive to pH.

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The outermost epidermal layer, the stratum corneum (SC), exhibits an acidic surface pH, whereas the pH at its base approaches neutrality. NHE1 is the only Na+/H+ antiporter isoform in keratinocytes and epidermis, and has been shown to regulate intracellular pH. We now demonstrate a novel function for NHE1, as we find that it also controls acidification of extracellular “microdomains” in the SC that are essential for activation of pH-sensitive enzymes and the formation of the epidermal permeability barrier. NHE1 expression in epidermis is most pronounced in granular cell layers, and although the surface pH of NHE1 knockout mice is only slightly more alkaline than normal using conventional pH measurements, a more sensitive method, fluorescence lifetime imaging, demonstrates that the acidic intercellular domains at the surface and of the lower SC disappear in NHE1 −/− animals. Fluorescence lifetime imaging studies also reveal that SC acidification does not occur through a uniform gradient, but through the progressive accumulation of acidic microdomains. These findings not only visualize the spatial distribution of the SC pH gradient, but also demonstrate a role for NHE1 in the generation of acidic extracellular domains of the lower SC, thus providing the acidification of deep SC interstices necessary for lipid processing and barrier homeostasis.
Citation: IFSCC Magazine, 11 (2008) (2) 115–119 Abstract: Hydroxy acids enjoy tremendous interest in cosmetology thanks to their skin anti-ageing and water barrier enhancing activities. One of their actions is to promote the natural stratum corneum (stratum corneum) desquamatory process. However, their use is limited due to an inherent pH-related irritancy potential which is even more exacerbated on sensitive skin. Clearly there is an opportunity for improvement. In this research we evaluated in humans the efficacy of salicylic acid, and its salts, as a corneodesmolytic agent using the dihydroxyacetone method and measuring the reduction in skin staining with treatment over time using a chromameter. Salicylic acid at 2% in a preparation of pH 3.12 significantly increased exfoliation by 10.9% compared with placebo (P < 0.05), confirming its desquamatory enhancing properties. Then the effect of vehicle cream pH on salicylic acid activity was studied. Salicylic acid at close to neutral pH (mostly in its neutralized form as salicylate, pH 6.50) exerted a corneodesmolytic activity as good as that of salicylic acid in an acidic vehicle (pH 3.12) after only two days of application. Furthermore, the performance of glycolic acid and salicylic acid salts as exfoliants were compared at pH 6.50. When these two hydroxyl acids were formulated at the same molar level in a cosmetic base (14.47mmol L-1), the salicylic acid preparation gave an 8.2% increase in stratum corneum desquamation compared with the glycolic acid preparation (P < 0.05). The corneodesmolytic effects were confirmed using a tape-stripping assay combined with a quantitative protein assay. Neutralized salicylic acid was found to enhance the removal of stratum corneum proteins significantly more than the vehicle after 25 sequential tape strippings (14%; P < 0.05). Finally, salicylic acid had no significant influence on skin water barrier properties after 22 days of treatment. In the second phase of this research we assessed the suitability of neutralized salicylic acid as an ingredient for sensitive skin. A stinging test was performed according to the Frosch & Kligman method to evaluate the influence of the formulation base-pH on stinging potential. Salicylic acid formulated at pH 6.50 induced no stinging sensation (score 0) in contrast to salicylic acid at pH 3.12 (score 19; P < 0.05). In addition, a clinical study was conducted to assess the erythema induced on volunteers’ cheeks after a single application of a neutralized salicylic acid (1%) formulation compared with placebo. Visual redness was assessed by a dermatologist and then measured with a Mexameter. No significant differences were observed. Moreover, half of the panel had sensitive skin and no correlation could be established between redness and/or abnormal sensation and sensitive skin. In conclusion, neutralized salicylic acid at a 1% concentration is a suitable exfoliant agent for subjects with sensitive skin. Keywords: exfoliation, hydroxy acids, Salicylic acid, stratum corneum Paper presented at the IFSCC Conference 2007, Amsterdam, The Netherlands.
The replacement time of the human stratum corneum of fifteen body regions was determined by measuring the days required for a strongly substantive fluorescent marker, Dansyl chloride, to disappear. Tbe horny layer was stained by a 24-h patch exposure to 5% Dansyl chloride (5-dimethylamino- 1-naphthalene-sulphonyl chloride) in petrolatum.In most body regions the renewal time for the horny layer was about 2 weeks, in agreement with the findings of other investigators.The advantage of Dansyl chloride over another compound, tetrachlorosalicylanilide, which has been used for this purpose, is that it is not an irritant.
Synopsis The evaluation of sun-product efficacy, with laboratory solar simulators or in actual sun, implicates clinical and subjective assessment of the various skin responses in terms of wavelengths constitutive of solar light. These photobiological responses vary according to skin types and particularly to basic skin melanic content, i.e. with skin colour. Now, the instrumental measurement of live skin colour has become easier to perform, fast and reliable. Based on the standard CIE-L*a*b* colour system and correlated with the human eye, this technique was used to define the skin colour domain of the caucasian population, to propose a skin colour classification, and then to objectively follow, over a three week period, the dynamics and kinetics of tanning induced by UVB, UVA and UVB +/- A multi-exposures on the three skin categories. The specific directions in the three-dimensional L*a*b* colour space of the tanning components, i.e. erythema, immediate pigmentation and constitutional melanization, as well as the resulting tanning pathways, were analysed and defined in the three-dimensional colour space, using a vectorial method. The UVB, UVA and UVB +/- A tannings were differentiated by their intensity, their hue and especially their lasting capacity: UVA tanning clearly appeared more lasting than UVB. In addition, the UVA*UVB interaction on tanning intensity was not found to be significant. With the skin colour classification and the tanning models, this comprehensive study supplies a basic tool for the colorimetric interpretation of the skin phenomena involved, provided that this interpretation is always considered in the three dimensions of the colour space. It also suggests some useful practical applications for sun product formulation and evaluation.
A simple non-radioactive technique is described. The corneal layers were stained with 5% dansyl chloride ointment during 24-48 h of occlusion. The number of newly formed corneal layers were counted on sections from punch biopsies obtained at various intervals after the staining. The rate of formation is given as layers formed per 24 hours. The technique makes it possible to detect an increase in the turnover rate when it is more than 15-32% above normal. The biological material can also be utilized for comparative studies utilizing biochemical and histological methods. The average rate of formation was 1.15+-0.09 (S.E.M.) layers per 24 hours in a group of 10 subjects.
The classical dansyl chloride test relies on visual assessment of the extinction of fluorescence in time. We introduce a microscopic and morphometric rating of this test. We show that soaps may remove the fluorescing dye from corneocytes. Therefore caution should be taken in interpreting fluorescence extinction only as an estimate of the stratum corneum renewal.
A new fluorescence comparator has been used to measure dansyl chloride-induced fluorescence from the skin surface. By taking readings twice daily, it was possible to ideality a circadian rhythm in which there was relatively depressed desquamation at night, compared with during the day. The study also demonstrated that the rate of desquamution was considerably reduced from protected sites, compared with normal unprotected sites. Other experiments demonstrated the ability of the technique to determine the enhanced rate of desquamation from uninvolved areas of skin near lesions of psoriasis, and the keratolvtic effect of preparations containing salicylic acid. It is considered that the technique adds objectivity and quantitative ability to the dansyl chloride method for characterizing desquamation.
Using a hypomitotic agent, triamcinolone acetonide, and a hypermitotic agent, retinyl propionate, we investigated the relationship between epidermal mitotic activity and stratum corneum renewal time of topically treated skin as determined by the dansyl chloride staining technique. Treatment with the base cream resulted in a reduction in renewal time compared with an untreated control site. The predicted increase in renewal time with the hypomitotic agent and reduction with the hypermitotic agent was only observed when daily treatment was commenced 2 weeks prior to and continued after dansyl chloride staining and not when treatment was started after staining. These results indicate that in order to use cell renewal methods to demonstrate changes in mitotic activity brought about by topical treatments, it is necessary to pre-treat the skin with the test material to establish full epidermal equilibrium at the changed mitotic state before labelling with dansyl chloride. Meaningful claims for effects on cell renewal of specific cosmetic ingredients should only be made after comparison with a base cream treated site, both having been allowed to equilibrate, rather than on the basis of comparison with untreated skin.
The hazards of the dansyl chloride test are not negligible. We introduce the dihydroxyacetone test as a safe substitute. Several advantages are highlighted including easy and reproducible measurements of the modifications in skin color by chromometry in the L* a* b* (luminance, hue, chroma) mode. The color variations in time as well as the fade-out of DHA-induced pigmentation by some cleansing skin care products are revealed 'in vivo' as well as 'ex vivo' on D-Squame collections of stratum corneum.
The existence of a flux of proton donors from skin (inner part of the forearm) to the electrode was observed in 12 male and female volunteers. This flux was used to collect and identify the ionic species responsible for skin acidity. It was then found that: (i) pK of these proton donors (pK = 6.13 +/- 0.07) was quasi-identical to that of trans-urocanic acid (6.10), and (ii) the amount of urocanic acid present in stratum corneum was sufficient in itself to explain the acidic level as measured with pH meter (R = 0.8484, n = 10, p = 0.00136). As a result, the contribution of other ionic species can be considered as negligible in normal human skin. The data recorded led us to identify three groups (Fast, Medium, and Slow) characterized by different skin surface pH values (low, medium, and close to neutral) and showing a pH gradient in the outer layers of the stratum corneum, or not. Data analysis suggests that these characteristics depend on urocanic acid production rate within the stratum corneum and that this production rate is self-regulated by its urocanic acid content.
There is evidence that the "acid mantle" of the stratum corneum is important for both permeability barrier formation and cutaneous antimicrobial defense. The origin of the acidic pH of the stratum corneum remains conjectural, however. Both passive (e.g., eccrine/sebaceous secretions, proteolytic) and active (e.g., proton pumps) mechanisms have been proposed. We assessed here whether the free fatty acid pool, which is derived from phospholipase-mediated hydrolysis of phospholipids during cornification, contributes to stratum corneum acidification and function. Topical applications of two chemically unrelated secretory phospholipase sPLA2 inhibitors, bromphenacylbromide and 1-hexadecyl-3-trifluoroethylglycero-sn-2-phosphomethanol, for 3 d produced an increase in the pH of murine skin surface that was paralleled not only by a permeability barrier abnormality but also altered stratum corneum integrity (number of strippings required to break the barrier) and decreased stratum corneum cohesion (protein weight removed per stripping). Not only stratum corneum pH but also all of the functional abnormalities normalized when either palmitic, stearic, or linoleic acids were coapplied with the inhibitors. Moreover, exposure of intact murine stratum corneum to a neutral pH for as little as 3 h produced comparable abnormalities in stratum corneum integrity and cohesion, and further amplified the inhibitor-induced functional alterations. Furthermore, short-term applications of an acidic pH buffer to inhibitor-treated skin also reversed the abnormalities in stratum corneum integrity and cohesion, despite the ongoing decrease in free fatty acid levels. Finally, the secretory-phospholipase-inhibitor-induced alterations in integrity/cohesion were in accordance with premature dissolution of desmosomes, demonstrated both by electron microscopy and by reduced desmoglein 1 levels in the stratum corneum (shown by immunofluorescence staining and visualized by confocal microscopy). Together, these results demonstrate: (i) the importance of phospholipid-to-free-fatty-acid processing for normal stratum corneum acidification; and (ii) the potentially important role of this pathway not only for barrier homeostasis but also for the dual functions of stratum corneum integrity and cohesion.
Transepidermal water loss (TEWL) is one of the most important biophysical parameters for evaluating the efficiency of the human skin water barrier. Different approaches exist to measure TEWL. The most commonly used methodology consists of the open chamber diffusion technique in which the water vapor pressure gradient is measured in g/h m2 according to Fick's law. A typical apparatus is the Tewameter. Recently, a portable device--the VapoMeter--became available with a humidity sensor in a closed chamber. In the present work, the closed chamber VapoMeter is compared with the open chamber Tewameter for its applicability to assess TEWL. A comparative study--including parallel in vivo measurements with both devices--was carried out on human forearm skin. It could be concluded that both instruments are reliable tools. A good correlation between recordings (r=0.503-0.966) was found with a consistent feature of measuring higher TEWL values for the Tewameter than for the VapoMeter. Probe pressure, probe temperature and relative humidity were revealed to be important parameters inducing significant differences in data outcome. From skin barrier damage experiments it became clear that the Tewameter is able to detect significantly smaller differences than the VapoMeter. In addition, the closed chamber device is currently not sensitive enough to discriminate for the effects induced by diurnal rhythm and fluctuations as a function of time. On the other hand, the small and handy VapoMeter allows more flexibility in measuring protocols and in in-use performance.
Keratolytic efficacy of topical preparations containing salicylic acid was studied in humans utilizing adhesive tape stripping and quantifying SC removal by protein analysis. In combination with tape stripping, squamometry was used to evaluate the influence of salicylic acid on skin surface scaliness and desquamation. Furthermore, skin barrier perturbation and skin irritancy was recorded and related to the dermatopharmacological effect of the preparations. In contrast to squamometry, tape stripping combined with protein analysis was sensitive in detecting keratolytic effect of salicylic acid within hours of application. Importantly, whereas the pH of the preparations only minimally influenced efficacy, local dermatotoxicity was significantly increased at acidic pH. This indicates that the quest to increase the amount of free, non-dissociated SA is, in fact, counterproductive as the more acidic preparations resulted in skin irritation and barrier disruption.
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