Uptake of topically applied 5-aminolevulinic acid and production of protoporphyrin IX in normal mouse skin: dependence on skin temperature

The temperature dependence of the uptake phase of 5-aminolevulinic acid (ALA) and the following production phase of protoporphyrin IX (PpIX) in normal mouse skin was investigated. A cream containing 20% ALA was topically applied on the skin for 10 min. The amount of ALA-induced PpIX was evaluated by measuring the fluorescence of PpIX from the treated skin. No measurable amount of PpIX was found in the skin immediately after 10 min application of ALA. The penetration of ALA into the skin was almost temperature independent while the following production of PpIX was found to be a strongly temperature-dependent process. Practically no PpIX was formed in the skin as long as skin temperature was kept low (12 degrees C).     

Development of a HPLC method for determination of four UV filters in sunscreen and its application to skin penetration studies

This study describes the development, validation and application of a high‐performance liquid chromatography (HPLC) method for the simultaneous determination of the in vitro skin penetration profile of four UV filters on porcine skin. Experiments were carried out on a gel‐cream formulation containing the following UV filters: diethylamino hydroxybenzoyl hexyl benzoate (DHHB), bis‐ ethylhexyloxyphenol methoxyphenyl triazine (BEMT), methylene bis‐ benzotriazolyl tetramethylbutylphenol (MBBT) and ethylhexyl triazone (EHT). The HPLC method demonstrated suitable selectivity, linearity (10.0–50.0 μg/mL), precision, accuracy and recovery from porcine skin and sunscreen formulation. The in vitro skin penetration profile was evaluated using Franz vertical diffusion cells for 24 h after application on porcine ear skin. None of the UV filters penetrated the porcine skin. Most of them stayed on the skin surface (>90%) and only BEMT, EHT and DHHB reached the dermis plus epidermis layer. These results are in agreement with previous results in the literature. Therefore, the analytical method was useful to evaluate the in vitro skin penetration of the UV filters and may help the development of safer and effective sunscreen products.     

有色粉体材料对太阳辐射吸收性能的UV-Vis-NIR光谱法表征

进一步研究了粉体材料对太阳辐射吸收能力的定量表征方法.结果表明,当Vcolored/V(TiO₂)(有色粉体与TiO₂的体积比)<5%时,lg(1/R)(R是试样对太阳辐射的反射率)和Vcolored/V(TiO₂)呈直线关系.从直线的斜率和截距可得出粉体材料的吸光系数和最大穿透厚度.该方法具有普遍性,适用于各种有色粉体的测定.     

Fluorescence staining of oral cancer using a topical application of 5-aminolevulinic acid: fluorescence microscopic studies

INTRODUCTION: Topical application of 5-aminolevulinic acid (5-ALA) by means of a rinsing solution has been shown to be a promising new procedure in the diagnosis of oral malignancies. However, for assessing the reliability of this method regarding fluorescence-guided tumor resections and photodynamic therapy, further information on the distribution and penetration depth of 5-ALA-induced protoporphyrin IX (PPIX) in the tissue is needed. METHODS: 24 patients suffering from oral cancer were included in this investigation. Biopsies were taken immediately after fluorescence examination and either used as native sections for immediate fluorescence microscopic examination (n = 3) or shock frozen in liquid nitrogen and prepared as frozen sections (n = 46). Fluorescence imaging and digital image processing were utilized in order to determine the presence of PPIX in regions of various histologies as well as the penetration depth of PPIX into solid tumor. RESULTS: PPIX fluorescence in the tissue was limited to the epithelium. Both normal and dysplastic epithelium showed PPIX fluorescence. In the stroma, no PPIX fluorescence was found. In some cases (n = 3/4) invasive carcinomas did not show PPIX fluorescence, while the adjacent or overlying normal epithelium was strongly fluorescent. The penetration depth of PPIX after topical application of 5-ALA was found to be limited to less than 1 mm. CONCLUSION: PPIX fluorescence induced by topical application of 5-ALA can be very useful in the determination of superficial tumor margins. However, due to the limited penetration depth there is a risk of not accurately recognizing the infiltration depth of solid tumors. The aim of further investigations will be to assess the tissue distribution and depth of penetration of PPIX following systemic application of 5-ALA.     

New vegetation indices for remote measurement of chlorophylls based on leaf directional reflectance spectra

Directional reflectance (R) spectra from 380 to 780 nm for nadir illuminated leaves of four different plants (croton, Codiaeum variegatum; spotted eleagnus, Eleagnus pungens Maculata; Japanese pittosporum, Pittosporum tobira and Benjamin fig, Ficus benjamina Starlight) were acquired at a viewing angle of 30 degrees from the nadir direction. Chlorophyll-a and -b content of leaves covered a range of 1-60 and 0.5-21 microg/cm(2), respectively. In contrast with previous results from hemispherical reflectance measurements, directional reflectance data does not correlate well with chlorophyll concentration. This is mainly due to the external reflectance (R(E)) at the leaf epidermis, caused by the mismatch of the refractive index at the air-epidermis and epidermis-inner layer boundary. The external reflectance can be identified with the blue flat reflectance between 380 and 480 nm. The inner reflectance (R(I)), obtained by subtracting the external reflectance from the measured spectra, was found to be linearly related to the logarithm of the chlorophyll content. Good fitting of the log (Chl) versus R(I)(lambda) curves were obtained for R(I) in the green band (around 550 nm) and close to the inflection point in the red edge (around 700 nm). The coefficient of determination, r(2), of curve fitting improved (up to 0.97) when the normalised inner reflectance NR(I)(lambda)=R(I)(lambda)/R(I)(lambda(0)), with lambda(0)>or=750 nm, was used instead of the absolute reflectance. The best indices for Chl, Chl-a and Chl-b determination were R(I)(542)/R(I)(750), R(I)(706)/R(I)(750) and R(I)(556)/R(I)(750), respectively. However, since the content of Chl-a relative to Chl-b was almost constant for the plants investigated, the two last indices must be further validated on leaves with a high variability in the Chl-a:Chl-b ratio. The error in the determination of chlorophyll content was found to be of the order of 10%. This value was lower than those obtained by applying the vegetation indices previously suggested. Therefore, the normalised inner reflectance in the green and in the red edge represents a more suitable index for the chlorophyll determination than those up to now used.     

Synergistic Effect of Ultrasound and Thiazone–PEG 400 on Human Skin Optical Clearing In Vivo

In this paper, we propose a new physical method in combination with mixed solution of thiazone and polyethylene glycol 400 (thiazone PEG 400 solution) penetration into tissue to assess the skin optical clearing. Four treatments were performed: (1) control group (C); (2) polyethylene glycol 400 (PEG400); (3) 0.25% thiazone (0.25%T); (4) 0.25% thiazone and 5-min ultrasound (0.25%T/SP). The diffuse reflectance spectra and imaging depth of human skin in vivo at different times were measured by spectroscopy and optical coherence tomography (OCT). The optical clearing efficacy of skin was qualitatively and quantitatively analyzed. The results showed that the diffuse reflectance at 540 nm of samples at 10 min after being treated by 0.25%T/SP decreased by approximately 15.51%, whereas, 0.46%, 4.73% and 5.75% were received in C, PEG400 and 0.25%T, respectively. And at 60 min, the decrease in diffuse reflectance of samples in 0.25%T/SP is about 2.22-fold, 1.20-fold compared with that of the samples in PEG 400 and 0.25%T, at 540 nm, respectively. Simultaneously, 0.25%T/SP results in 41.33% increase in OCT 1/e light penetration depth after 60 min. There was a significant difference in the optical clearing effect on skin between ultrasound-mixed solution of thiazone in combination with PEG 400 and the mixed solution (P < 0.05).     

Kinetics of Photobleaching of Protoporphyrin IX in the Skin of Nude Mice Exposed to Different Fluence Rates of Red Light

Abstract— The purpose of the present study was to determine the kinetics and the fluence rate dependency of the photo-bleaching of protoporphyrin IX (PpIX) in normal skin of Balb/c nude mice after systemic and topical application of 5-aminolevulinic acid (ALA). ALA was administered systemically (200 mg/kg body weight, i.p.) and topically (20% w/w ALA cream) to the mice. Fluences of up to 40 J/cm² were delivered by a dye laser (636 nm) at fluence rates of 37.5, 75, 150, 300 and 500 mW/cm². The photo-bleaching rate was constant within this range of fluence rates. This result suggests that there is no oxygen effect for PpIX photobleaching in this region for the skin of Balb/c nude mice. During light exposure the fluorescence decay followed neither first- nor second-order kinetics. The decay rate was slightly faster after systemic application than after topical application of ALA, but did not depend on the time (1–8 h) between application and analysis.     

Systemic component of protoporphyrin IX production in nude mouse skin upon topical application of aminolevulinic acid depends on the application conditions

Topical application of 5-aminolevulinic acid (ALA) for protoporphyrin IX (PpIX)-based photodynamic therapy of skin cancer is generally considered not to induce systemic side effects because PpIX is supposed to be formed locally. However, earlier studies with topically applied ALA have revealed that in mice PpIX is not only produced in the application area but also in other organs including skin outside the application area, whereas esterified ALA does not. From these results, it was concluded that it is not redistribution of circulating PpIX that causes the fluorescence distant from the ALA application site, but rather, local PpIX production induced by circulating ALA. In the present study we investigate the effects of the ALA concentration in the cream, the application time, the presence of a penetration enhancer, the presence of the stratum corneum and esterification of ALA on the PpIX production in nude mouse skin outside the area where ALA is applied. For this purpose, ALA and ALA hexyl ester (ALAHE) were applied to one flank, and the PpIX fluorescence was measured in the contralateral flank. During a 24 h application of ALA, PpIX was produced in the contralateral flank. No PpIX could be detected in the contralateral flank after ALA application times ranging from 1 to 60 min. Tape-stripping the skin prior to short-term ALA application, but not the addition of a penetration enhancer, resulted in PpIX production in the contralateral flank. When ALAHE was applied, no PpIX fluorescence was measured in the contralateral flank under any application condition. The results suggest that the systemic component of PpIX production outside the ALA application area plays a minor or no role in relevant clinical situations, when the duration of ALA (ester) application is relatively short and a penetration enhancer is possibly added.     

Pharmacokinetics of 5-aminolevulinic acid-induced protoporphyrin IX in skin and blood

The fluorescence and photosensitivity of endogenously synthesized protoporphyrin IX (PPIX) is increasing used for the diagnosis and treatment of malignant and certain non-malignant diseases. A selective accumulation of PPIX can be induced by application of 5-aminolevulinic acid (5-ALA), which is a precursor of PPIX in the cellular biosynthetic pathway of heme.

The purpose of this study was to monitor the in vivo accumulation of PPIX in different locations of the skin after oral ingestion and to determine the pharmacokinetics of 5-ALA and PPIX in human blood plasma for various routes of application. At the same time we wanted to achieve an optimal treatment scheme but also study possible side-effects of 5-ALA administration.

After oral application of 5-ALA in a concentration of 40 mg kg⁻¹ body weight, the fluorescence intensities of PPIX in the skin showed maxima between 6.5 and 9.8 h depending on the location and decreased to values lower than 5% related to the maximum after a mean time of about 40 h. The measured absolute intensities of PPIX fluorescence varied strongly between different patients and different locations on one patient. In the plasma of blood samples, PPIX could be detected via its fluorescence for all studied routes of application with the exception of the ointment, where PPIX levels were below the detection limit of 1 μg l⁻¹. The highest mean concentration of 742 μg l⁻¹ PPIX in the plasma was measured 6.7 h after oral application. For inhalation of 5-ALA, a mean maximum concentration of 12 μg l⁻¹ could be detected 4.1 h after application, for intravesical instillation, the mean maximum concentration was found to be 1 μg l⁻¹ 2.9 h after application. The kinetics of 5-ALA in the plasma peaked much earlier with a maximum concentration of 32 mg l⁻¹ about 30 min. after oral administration. The 5-ALA levels did not exceed normal reference values after topical application.

The results of our experiments suggest that for a systemic application of 5-ALA side-effects in sensitive patients cannot be excluded.     

Topical use of liposomal copper palmitate formulation blocks porphyrin-induced photosensitivity in rats

Photodynamic therapy (PDT) is a new treatment modality that uses porphyrin derivatives and visible light, especially for the treatment of cancer. However, PDT with certain photosensitisers can cause prolonged skin photosensitization. This is particularly true for Photofrin II (Photofrin)-mediated PDT where patients are required to avoid direct exposure to sunlight for a period of 4-6 weeks. This is the only long-term adverse reaction to the drug. Recent studies have shown that topical copper treatment avoids this type of inflammatory reaction. In this study, we have tested the efficiency of the liposomal formulation of copper palmitate on porphyrin-photosensitized rats. Initially, adult male Sprague-Dawley rats were rendered photosensitive either by administration of Photofrin or aminolevulinic acid (ALA), a precursor of protoporphyrin IX (PpIX). Prior to this, their dorsal skin was shaved and treated topically with a cream consisting of either empty or copper palmitate-encapsulated liposomal formulation. After being kept in a dimmed light environment, the rats were exposed to visible light, and inflammatory responses were inspected. Histological studies revealed that no inflammatory cells were present at the skin sites treated with liposomal cream containing copper palmitate in the Photofrin-sensitized group while no reduction in the number of inflammatory cells was observed at the skin samples treated with the empty liposomes. In conclusion, the data demonstrate the significant protective effect of topically-applied liposome-encapsulated copper palmitate against both Photofrin and ALA-induced PpIX photosensitivity.     

5-aminolevulinic acid, but not 5-aminolevulinic acid esters, is transported into adenocarcinoma cells by system BETA transporters

5-aminolevulinic acid (5-ALA) and its ester derivatives are used in photodynamic therapy as precursors for the formation of photosensitizers. This study relates to the mechanisms by which 5-ALA is transported into cells. The transport of 5-ALA has been studied in a human adenocarcinoma cell line (WiDr) by means of [14C]-labeled 5-ALA. The rate of uptake was saturable following Michaelis-Menten kinetics (K(m) = 8-10 mM and Vmax = 18-20 nmol.(mg protein x h)-1), and Arrhenius plot of the temperature-dependent uptake of 5-ALA was characterized by a single discontinuity at 32 degrees C. The activation energy was 112 kJ.mol-1 in the temperature range 15 degrees-32 degrees C and 26 kJ.mol-1 above 32 degrees C. Transport of 5-ALA was Na+ and partly Cl(-)-dependent. Stoichiometric analysis revealed a Na+:5-ALA coupling ratio of 3:1. With the exception of valine, methionine and threonine, zwitterionic and basic amino acids inhibited the transport of 5-ALA. 5-ALA methyl ester was not an inhibitor of 5-ALA uptake. The transport was most efficiently inhibited, i.e. by 65-75%, by the beta-amino acids, beta-alanine and taurine and by gamma-aminobutyric acid (GABA). Accordingly, 5-ALA, but not 5-ALA methyl ester, was found to inhibit cellular uptake of [3H]-GABA and [14C]-beta-alanine. Protoporphyrin IX (PpIX) accumulation in the presence of 5-ALA (0.3 mM) was attenuated 85% in the presence of 10 mM beta-alanine, while PpIX formation in cells treated with 5-ALA methyl ester (0.3 mM) or 5-ALA hexyl ester (4 microM) was not significantly influenced by beta-alanine. Thus, 5-ALA, but not 5-ALA esters, is transported by beta-amino acid and GABA carriers in this cell line.     

Hyperpigmentation induced by topical 5-aminolaevulinic acid plus visible light

Photodynamic therapy (PDT) with 5-aminolaevulinic acid (ALA) is an alternative tool for the treatment of superficial non-melanoma skin cancers. Recently ALA-PDT has been employed with encouraging results also for warts, condylomata and psoriasis. In this study the effects of topical ALA plus irradiation with visible light on intact human skin have been evaluated. Five skin areas (A, B, C, D, and E) on the inner upper part of the arms of five healthy volunteers (skin types III and IV) were treated with (A) ALA 20% in base cream without irradiation, (B) only the vehicle (base cream) without ALA, (C, D and E) ALA cream at the concentrations of 5, 10 and 20%, respectively; all treatments were applied with an occlusive dressing. Four hours after ALA or vehicle application areas B, C, D and E were irradiated with a fixed dose of 40 J/cm(2). ALA penetration through the intact skin was evaluated by in vivo fluorescence determination. The effects on healthy skin were evaluated by clinical and chromometric examinations, light microscopy, immunohistochemistry and electron microscopy. RESULTS: (1) in vivo fluorescence demonstrated that ALA is able to penetrate through the intact skin, when applied with occlusive dressing and induces a classical fluorescence peak due to Protoporphyrin IX (PpIX) formation, which is the active photosensitiser. (2) Skin areas receiving ALA plus irradiation showed erythema and swelling just after the irradiative session and hyperpigmentation 48-72 h later. (3) Colourimetric data confirmed significant skin colour changes: values a* (representing the erythematous changes) increased only on the skin areas where ALA+irradiation were applied and during the 48 h after irradiation, thereafter a* began to decrease; values L* (pigmentation) increased during the 2 weeks following treatment. (4) Histopathological, immunohistochemical (S100, HMB-45) and electron microscopic findings showed an absolute increment of the number of melanocytes, which appeared clearly activated. In conclusion the application of ALA cream followed by irradiation is able to induce a pigmentation response in healthy human skin, at least in skin types III and IV. This melanocytic activation could have a potential for the treatment of skin disorders characterised by hypopigmentation.     

Sensitive high-performance liquid chromatographic method for the determination of chlorhexidine in human serum and urine

A high-performance liquid chromatographic method for the analysis of chlorhexidine in human serum and urine was developed. Chlorhexidine and the internal standard, chlorpheniramine, were extracted into chloroform, containing 5% 2-propanol, and back-extracted into dilute sulfuric acid. Chromatographic separation was achieved on a C18 column equilibrated with methanol-water (70:30, v/v), containing 0.005 M sodium heptane-sulfonate. The sensitivity of the assay was 20 ng/ml of biological matrix, using 0.5-ml samples. The application of this method to monitor chlorhexidine levels in burn patients treated topically with a chlorhexidine-containing burn cream was demonstrated.     

Photodynamically induced effects in colon carcinoma cells (WiDr) by endogenous photosensitizers generated by incubation with 5-aminolaevulinic acid.

Human adenocarcinoma cells of the line WiDr have been treated with 2 mM 5-aminolaevulinic acid (5-ALA) in the presence of 10% foetal calf serum. The treatment induces a linear accumulation of protoporphyrin IX (PpIX) for at least 7.5 h. After 7.5 h of incubation about 45% of the PpIX accumulated is cell-bound, while the rest is found in the medium (25%) or lost from the cells during washing with phosphate-buffered saline (30%). Exposure to white light at an intensity of 30 W/m2 for 18 min results in 95% reduction of clonogenicity in cells treated with 2 mM 5-ALA for 3.5 h. The enzymatic activities of enzymes located in cytosol (glyceraldehyde 3-phosphate dehydrogenase and lactate dehydrogenase) and lysosomes (acid phosphatase and beta-glucuronidase) are not influenced by a 5-ALA and light treatment inactivating about 35% of the cells. The MTT assay, which reflects mitochondrial dehydrogenase activity, but not succinate dehydrogenase, is partly inhibited by the same treatment. Treatment with 5-ALA in the absence of light increases O2 consumption by a factor of two, while the O2 consumption is inhibited when 5-ALA treatment is combined with exposure to light. In addition, 5-ALA and light exposure enhance accumulation of rhodamine 123 by 40% and reduce the intracellular ATP level by 25%. Confocal laser scanning microscopical analysis indicates granular perinuclear localization of the PpIX formed by 5-ALA treatment. In conclusion, photodynamic treatment using 5-ALA as a prodrug induces damage to mitochondrial function without inhibiting lysosomal and cytosolic marker enzymes.     

In vivo pharmacokinetics of protoporphyrin IX accumulation following intracutaneous injection of 5-aminolevulinic acid

Photodynamic therapy with 5-aminolevulinic acid (ALA) derived protoporphyrin IX (PpIX) as photosensitizer is a promising treatment for basal cell carcinomas. Until now ALA has been administered topically as an oil-in-water cream in most investigations. The disadvantage of this administration route is insuffici?nt penetration in deeper, nodular tumours. Therefore we investigated intracutaneous injection of ALA as an alternative administration route. ALA was administered in 6-fold in the normal skin of three 6-week-old female Dutch pigs by intracutaneous injection of an aqueous solution of ALA (pH 5.0) in volumes of 0.1-0.5 ml and concentrations of 0.5-2% and by topical administration of a 20% ALA cream. During 8 h fluorescence of ALA derived PpIX was measured under 405 nm excitation. For the injection the measured fluorescence was shown to be dose dependent. All injected doses of 3 mg ALA or more lead to a faster initial increase rate of PpIX synthesis and significantly greater fluorescence than that measured after topical administration of ALA. Irradiation (60 Jcm(-2) for 10 min) of the spots was performed at 3.5 h after ALA administration. After 48 and 96 h visual damage scores were evaluated and biopsies were taken for histopathological examination. After injection of 2 mg ALA or more the PDT damage after illumination was shown to be significantly greater than after topical application of 20% ALA. An injected dose of 10 mg ALA (0.5 ml of a 2% solution) resulted in significantly more tissue damage after illumination than all other injected doses.     

Topical bioadhesive patch systems enhance selectivity of protoporphyrin IX accumulation

In clinical 5-aminolevulinic acid (ALA)-based photodynamic therapy (PDT) of skin tumors it is desirable to develop vehicles that minimize the penetration of ALA through normal stratum corneum and maximize it through the compromised stratum corneum of the tumors to improve tumor selectivity. We have designed a bioadhesive patch, which may be able to achieve this aim. It induces levels of protoporphyrin IX (PpIX) in skin overlying tumors similar to those induced by the proprietary cream (Porphin) but at the same time induces less PpIX to form in normal skin and at distant sites. The mechanisms of action of the patch, as compared with that of the cream, were studied by means of Cuprophan barriers that mimic compromised tumor stratum corneum and in a mouse model with transplanted tumors.     

Topical application of 5-aminolevulinic acid and its methylester,hexylester and octylester derivatives: considerations for dosimetry in mouse skin model

Ester derivatives of 5-aminolevulinic acid (ALA-esters) have been proposed as alternative drugs for ALA in photodynamic therapy. After topical application of creams containing ALA, ALA methylester (ALA-Me), ALA hexylester (ALA-Hex) and ALA octylester (ALA-Oct) on mouse skin, typical fluorescence excitation and emission spectra of protoporphyrin IX (PpIX) were recorded, exhibiting a similar spectral shape for all the drugs in the range of concentrations (0.5-20%) studied. The accumulation kinetics of PpIX followed nearly a similar profile for all the drug formulations. The fluorescence of PpIX peaked at around 6-12 h of continuous cream application. Nevertheless, some differences in pharmacokinetics were noticed. For ALA cream, the highest PpIX fluorescence was achieved using 20% of ALA in an ointment. Conversely, 10% of ALA-Me and ALA-Hex, but not of ALA-Oct, in the cream was more efficient (P < 0.05) than was 20%. The cream becomes rather fluid when 20% of any of these ALA-esters is used in ointment, whereas 10% and lower concentrations of ALA-esters do not significantly increase fluidity of the cream. The dependence of PpIX accumulation on the concentration of ALA and ALA-ester in the applied cream followed (P < 0.002) kinetics as described by a mathematical model based on the Michaelis-Menten equation for enzymatic processes. Under the present conditions, the PpIX amount in the skin increased by around 50% by the application of ALA-Me, ALA-Hex or ALA-Oct for 4-12 h as compared with ALA for the same period. Observations of the mice under exposure to blue light showed that after 8-24 h of continuous application of ALA, the whole mouse was fluorescent, whereas in the case of ALA-Me, ALA-Hex and ALA-Oct the fluorescence of PpIX was located only at the area of initial cream application. The amount of the active compound in the applied cream necessary to induce 90% of the maximal amount of PpIX was determined for normal mouse skin. Optimal PpIX fluorescence can be attained using around 5% ALA, 10% ALA-Me and 5% ALA-Hex creams during short application times (2-4 h). Topical application of ALA-Oct may not gain optimal PpIX accumulation for short applications (<5 h). For long application times (8-12 h), it seems that around 1% ALA, 4% ALA-Me, 6% ALA-Hex and 16% ALA-Oct can give optimal PpIX fluorescence. But for long application times and high concentrations, systemic effect of ALA applied topically on relatively large areas should be considered.     

The influence of UV exposure on 5-aminolevulinic acid-induced protoporphyrin IX production in skin.

The skin of nude mice was exposed to erythemogenic doses of UV radiation, which resulted in erythema with edema. An ointment containing 5-aminolevulinic acid (ALA) was topically applied on mouse and human skin. Differences in the kinetics of protoporphyrin accumulation were investigated in normal and UV-exposed skin. At 24 and 48 h after UV exposure, skin produced significantly less protoporphyrin IX (PpIX) than skin unexposed to UV. Human skin on body sites frequently exposed to solar radiation (the lower arm) also produced less PpIX than skin exposed more rarely to the sun (the upper arm). It is concluded that UV radiation introduces persisting changes in the skin, relevant to its capability of producing PpIX from ALA. The observed differences in ALA-induced PpIX fluorescence may be the result of altered penetration of ALA through the stratum corneum or altered metabolizing ability of normal and UV-exposed skin (or both).     

Label free non-invasive imaging of topically applied actives in reconstructed human epidermis by confocal Raman spectroscopy

Raman spectroscopy has become a versatile tool for the in vivo characterization of skin. Here we describe use of Raman spectroscopy for high resolution optical cross sectioning to resolve skin constituents and administered drugs at the cellular level. Percutaneous penetration is typically studied using permeation cells with biopsies of animals or human skin. Although this technique provides valuable clinical data, little insight is gained in the microstructure of drug penetration (intercellular or transcellular) or in the mode of action of applied vehicles or penetration enhancers. Therefore, a Raman microspectroscopic method was combined with a confocal scanning setup to image the microstructure of commercially available skin models (SkinEthic^®) and the spatial distribution of penetrated actives. The models’ microstructure was scanned without any special treatment or environment such as cutting, staining, freezing, or application of vacuum. The non-invasive Raman images reveal the layered structure of stratum corneum. This in particular for lipids while water tends to be more evenly distributed. When penetration of the hydrophilic active glycerol and the lipophilic octyl methoxycinnamate, OMC, was studied, a strong correlation between the local distribution of skin constituents and the hydrophilic/lipophilic character of the active was observed.     

A novel method for the determination of synthetic colors in ice cream samples

A simple method has been developed for the extraction, separation, and determination of synthetic colors in ice cream samples. The process involves the breakdown of emulsion by neutral detergents (Triton X-100 and Tween 20) followed by extraction with petroleum ether for removal of fat. The aqueous colored solution obtained is treated with 5% acetic acid, and the uptake of color is carried out by a wool-dyeing technique. The color is eluted from the wool with 5% ammonia solution, the solution is evaporated to dryness, and the residue is dissolved in 60% ethanol for paper chromatography using trisodium citrate-ammonia-water (2 + 5 + 95, w/v/v) as the mobile phase. The colored spots from the paper chromatogram are cut and eluted with 60% ethanol, and the absorbance is measured at the respective lambda maximum corresponding to the Rf value of the appropriate standard. The recoveries of 6 colors, including sunset yellow FCF (SSYFCF), tartrazine, carmoisine, ponceau 4R, brilliant blue FCF (BBFCF), and fast green FCF from spiked samples with either detergent were found to be >90%. However, recoveries of erythrosine were 21 and 65% with Triton X-100 and Tween 20, respectively. Indigo carmine could not be recovered at all because of its fugitive property in 5% ammonia solution, which is used to strip the color from the wool. The sensitivity of the method with the use of Tween 20 is 1 ppm (1 microg/g) for the colors in spiked ice cream samples. With this method, we analyzed samples of 20 branded colored ice cream. The results showed the presence of tartrazine (8.4-43.3 ppm), SSYFCF (23.5-117.6 ppm), carmoisine (traces-53.2 ppm), erythrosine (3.5 ppm), and BBFCF (4.1 ppm) in the ice cream samples. Apart from 2 samples of tuttifruity, all of the ice cream samples showed the presence of permitted synthetic colors below the permissible level of 100 ppm established by the Prevention of Food Adulteration Act of India.