UV and storage stability of retinol contained in oil-in-water nanoemulsions.

This study was performed to examine the stability of retinol contained within oil-in-water (O/W) emulsions under UV and during storage at different temperatures. O/W emulsions were prepared using different emulsifiers and oil concentrations. The stability of the retinol contained in the O/W emulsions was investigated by measuring the percentage of residual retinol in the samples after UV exposure and storage at different temperatures (4, 25, and 40 °C). The oil concentration of the emulsion had a greater impact on UV stability than the type of emulsifier used, whereas the storage stability at different temperatures was affected by both the choice of emulsifier and the oil concentration. The storage stability of the retinol contained in the O/W emulsions may be related to the lipid oxidation properties of the emulsions rather than the latter's physical stability. Experiments with EDTA and different oil types were performed to confirm this theory.

Kinetic study on photostability of retinyl palmitate entrapped in policosanol oleogels.

Photostability of all-trans retinyl palmitate (RP) (100% bioactivity) was studied in policosanol oleogels (PCOs) (7-12% w/w policosanol in soybean oil) after UVA irradiation. RP was incorporated into PCOs at levels of 0.04%, 0.1% and 1% (w/w). PCOs efficiently protected RP from UVA-mediated degradation. Over 75% RP-activity remained in PCOs after 4 days of UVA irradiation, while 12% RP-activity remained in soybean oil. HPLC analysis showed that cis-RP was formed in liquid soybean oil after 2 days of UVA irradiation while it was absent in PCOs matrices. PCOs blocked the energy absorption from UVA and further dampened the UVA-mediated ionic photodissociation and free radical reaction due to matrix immobilization. For all samples, RP photodegradation followed a 2nd order reaction. From the reaction kinetics, it would be possible to predict the RP photodegradation rate in PCO matrices. PCOs were shown to be a promising matrix to protect RP from photodegradation.

Vitamin A and Vision.

Visual systems detect light by monitoring the effect of photoisomerization of a chromophore on the release of a neurotransmitter from sensory neurons, known as rod and cone photoreceptor cells in vertebrate retina. In all known visual systems, the chromophore is 11-cis-retinal complexed with a protein, called opsin, and photoisomerization produces all-trans-retinal. In mammals, regeneration of 11-cis-retinal following photoisomerization occurs by a thermally driven isomerization reaction. Additional reactions are required during regeneration to protect cells from the toxicity of aldehyde forms of vitamin A that are essential to the visual process. Photochemical and phototransduction reactions in rods and cones are identical; however, reactions of the rod and cone visual pigment regeneration cycles differ, and perplexingly, rod and cone regeneration cycles appear to use different mechanisms to overcome the energy barrier involved in converting all-trans- to 11-cis-retinoid. Abnormal processing of all-trans-retinal in the rod regeneration cycle leads to retinal degeneration, suggesting that excessive amounts of the retinoid itself or its derivatives are toxic. This line of reasoning led to the development of various approaches to modifying the activity of the rod visual cycle as a possible therapeutic approach to delay or prevent retinal degeneration in inherited retinal diseases and perhaps in the dry form of macular degeneration (geographic atrophy). In spite of great progress in understanding the functioning of rod and cone regeneration cycles at a molecular level, resolution of a number of remaining puzzling issues will offer insight into the amelioration of several blinding retinal diseases.

Technical brief: Pump-probe paradigm in an integrating cavity to study photodecomposition processes.

PURPOSE: Assaying photodecomposition is challenging because light must be used to initiate the photodamage and light must be used to monitor the photodecomposition. The experimental requirements are as follows: 1) During exposure of the actinic beam, continuously monitor the spectral characteristics of the sample, 2) uniformly expose the reactants to the actinic source, 3) obtain informative spectra in the presence of light scatter, and 4) achieve sufficient sensitivity for dilute reactants. Traditional spectrophotometers cannot address these issues due to sample turbidity, the inability to uniformly expose the cuvette contents to the incident beam, the inability to simultaneously perform spectral scans, and inherent low sensitivity. Here, we describe a system that meets these challenges in a practical way. METHODS: Light access to a 8.6 ml quartz integrating sphere containing 10 µM all-trans retinol in PBS was provided by three ports at right angles allowing for the following: 1) actinic light delivery from light-emitting diodes (LEDs) firing at 100 pulses/sec, 2) entry of a separate scanning beam at 100 scans/sec (10,000 µsec scan time) via an OLIS RSM 1000 ultraviolet/visual (UV/Vis) rapid-scanning spectrophotometer (RSM), and 3) light exit to the detector photomultiplier. The RSM spectral intermediate slit was partially covered to allow for a "dark" period of 2,000 µsec when no scanning light was admitted to the cuvette. During that interval, the LED was flashed, and the photomultiplier was temporarily blocked by a perforated spinning shutter disk. The absorbance per centimeter, which is increased due to the internal reflectance of the integrating sphere compared to a standard 1 cm rectangular cuvette, was calculated according to Fry et al. (2010) Applied Optics 49:575. Retinoid photodecomposition was confirmed with high-performance liquid chromatography (HPLC). RESULTS: Using the RSM to trigger the LED flash and photomultiplier shutter closure during the "dark" period allowed actinic flashes to be placed between scans. Exposure of the all-trans retinol to 366 nm flashes resulted in marked reduction in absorbance and a blue shift of the λmax. A white LED, despite its higher photon output, did not support all-trans retinol photolysis. Singular value decomposition (SVD) analysis revealed three spectral intermediates with mechanism, I -> II -> III. HPLC analysis of the reactants at the beginning and the conclusion of the light exposure confirmed the retinol photodecomposition. CONCLUSIONS: The highly reflecting cavity acts as a multipass cuvette that markedly increased the light path length and, thus, sensitivity. Triggering the LED during a dark period within the scan time allowed the actinic flashes to be interleafed between scans in a pump-probe paradigm. Furthermore, the entire sample was exposed to scan beam and actinic flashes, which is not possible in traditional spectrophotometers. Finally, the integrating cavity cuvette allowed use of turbid samples. SVD was useful for resolving spectral intermediates. Although the identity of the intermediates was not determined here, the ability to define molecular intermediates during photodecomposition reactions will allow future studies to isolate and identify the degradation products and determine the mechanism of light-induced retinoid degradation and that of retinoid-binding protein-mediated photoprotection.

Photodecomposition, photomutagenicity and photocytotoxicity of retinyl palmitate under He-Ne laser photoirradiation and its effects on photodynamic therapy of cancer cells in vitro.

OBJECTIVE: Our aim was to study photodecomposition, photomutagenicity and cytotoxicity of retinyl palmitate (RP), a principal storage form of vitamin A in humans and animals, under He-Ne laser photoirradiation. Moreover, the effect of different concentrations and timing protocol of antioxidants on photodynamic therapy (PDT) is contradictory, so the effect of RP (as antioxidant) on the PDT cytotoxicity was studied. METHODS: Photomutagenicity was tested by Ames test. Photodecomposition was studied by UV-vis spectroscopy. Cytotoxicity was measured with MTT-assay. Moreover, the effect of PDT, using hematoporphyrin derivatives (HpD) as photosensitizer under He-Ne laser irradiation (10 J/cm(2)), was studied on HeLa cells either with or without RP (1-100 µM) which incubated with the cells for short or long incubation period (1 h or 24 h) prior to PDT. RESULTS: No photodecomposition of RP alone was obseved whereas there is a little photodecomposition of RP only in presence of HpD under irradiation with He-Ne laser. Moreover, no photomutagenicity was observed in Salmonella typhimurium strains under laser irradiation in presence or absence of HpD. RP alone (1-100 µM) significantly decrease the viability of HeLa cells. Laser irradiation of HeLa cells pre-incubated with RP alone for 24 h showed further significant decrease in viability of the cells. While RP incubations for 1 h before PDT had slight effect on the cells, 24 h incubation before PDT enhanced the cytotoxicity of PDT on HeLa cells. CONCLUSIONS: RP can be used 24 h before PDT to enhance its effects. RP is not mutagenic under irradiation with He-Ne laser.

Combination of retinyl palmitate and UV-filters: phototoxic risk assessment based on photostability and in vitro and in vivo phototoxicity assays.

This study aimed to assess the phototoxic potential of combined UV-filters and retinyl palmitate (RP) in the presence or not of bemotrizinol (BMTZ), employing photostability and in vitro and in vivo phototoxicity assays. The formulations tested contained octocrylene (OCT), octyl methoxycinnamate (OMC), benzophenone-3 (BZP-3) and RP (photostable) or octocrylene (OCT), octyl methoxycinnamate (OMC), avobenzone (AVO) and RP (less photostable). Both formulations were supplemented with bemotrizinol. Photostability was evaluated by exposing, or not, formulations spread on a glass plate to UVA/UVB irradiation. The resulting products were quantified by HPLC analysis. In vitro phototoxicity of UV-filters and combinations were evaluated using 3T3 viable monolayer fibroblast cultures submitted, or not, to irradiation according to OECD TG 432. In vivo photoallergy and photoxicity were assessed by clinical studies (photopatch test). Photostability assays showed that UV-filter bemotrizinol was a better photostabilizer for RP/benzophenone-3 than for RP/avobenzone. The in vitro phototoxicity of the combination RP/avobenzone was reduced by bemotrizinol. Clinical studies did not indicate phototoxic or photoallergenic potentials in all formulations tested. It is concluded that the 3T3 NRU phototoxicity test may be considered a supplementary assay in formulation developments, since it can detect chemically unstable and potentially phototoxic combinations. However, extrapolation of in vitro positive results to human photopatch tests may be performed only to a limited extent.

Retinyl acetate-loaded nanoparticles: dermal penetration and release of the retinyl acetate.

Retinyl acetate (RA) loaded polymeric nanoparticle (NP) carriers were prepared using two different single polymers, ethyl cellulose (EC) and poly (ethylene glycol)-4-methoxycinnamoylphthaloylchitosan (PCPLC). The stability of RA to aqueous solution and UVA light was significantly improved when encapsulated with PCPLC, whilst EC encapsulation gave some improved stability in water but showed no improved photostability. Ex vivo application of free RA and the RA-loaded PCPLC NPs onto the surface of the freshly excised skin from a baby mouse indicated a significantly slower skin absorption rate for the encapsulated RA. However, 100% retention of the encapsulated RA in the skin tissue was observed after 24h. Confocal fluorescent analysis of the skin pieces applied with the RA-loaded PCPLC NPs indicated likely entry and accumulation of the PCPLC NPs and RA at the hair follicles. Release of RA from the PCPLC NP carriers was confirmed through the detection of an increasingly higher RA/PCPLC fluorescent signal ratio deeper into the dermis and away from the hair follicles.

Assessing retinol stability in a hydroquinone 4%/retinol 0.3% cream in the presence of antioxidants and sunscreen under simulated-use conditions: a pilot study.

BACKGROUND: Retinol (ie,vitamin A) is commonly used in dermatology as an adjunct to treat rhytids, acne,and dyschromia. However, vitamin A and many of its derivatives have poor photostability and are unstable in the presence of oxygen. OBJECTIVE: We aimed to assess the stability of retinol under simulated patient application conditions in a commercially available hydroquinone 4% cream containing retinol 0.3%, avobenzone (ultraviolet-A sunscreen), octinoxate (ultraviolet-B sunscreen), vitamins C and E (antioxidants), and moisturizers. METHODS: One gram of the preparation was applied as a thin film to the inside base of 4 groups of four 100-mL wide-mouthed beakers, incubated in a 37+/-2 degrees C water bath. Each experimental group consisted of 4 beakers for assays at 0.5,1,2,and 4 hours. The samples were exposed to varying combinations of full spectrum light and headspace gas (air or inert nitrogen gas [N2 ]). Retinol content was assayed via high-pressure liquid chromatography using a 1:9 water:methanol solvent system. The control group (group 5) was not exposed to full-spectrum light or headspace gas but served for comparative purposes. RESULTS: On exposure to light and room air, retinol stability was 94.4% at 0.5 hour, 94.8% at 1 hour, 92.4% at 2 hours, and 91.5% at 4 hours. The retinol contained in the preparation was stable for >or=4 hours. Samples exposed to light and N 2 gas demonstrated 96.5% and 91.3% stability at 0.5 hour and 4 hours exposure times, respectively. Samples that were not exposed to light had a stability of 99.2% (group 3, exposed to air) and 96.9% (group 4, exposed to N(2)) of the initial retinol present after 4 hours. CONCLUSION: The retinol in the hydroquinone 4%/ retinol 0.3% cream with antioxidants and sunscreens underwent <10% degradation under simulated-use conditions, including exposure to UV light, oxygen, and body temperature.

Effects of 900-MHz electromagnetic field emitted from cellular phone on brain oxidative stress and some vitamin levels of guinea pigs.

This study was designed to demonstrate the effects of 900-MHz electromagnetic field (EMF) emitted from cellular phone on brain tissue and also blood malondialdehyde (MDA), glutathione (GSH), retinol (vitamin A), vitamin D(3) and tocopherol (vitamin E) levels, and catalase (CAT) enzyme activity of guinea pigs. Fourteen male guinea pigs, weighing 500-800 g were randomly divided into one of two experimental groups: control and treatment (EMF-exposed), each containing seven animals. Animals in treatment group were exposed to 890- to 915-MHz EMF (217-Hz pulse rate, 2-W maximum peak power, SAR 0.95 w/kg) of a cellular phone for 12 h/day (11-h 45-min stand-by and 15-min spiking mode) for 30 days. Control guinea pigs were housed in a separate room without exposing EMF of a cellular phone. Blood samples were collected through a cardiac puncture and brains were removed after decapitation for the biochemical analysis at the end of the 30 days of experimental period. It was found that the MDA level increased (P<0.05), GSH level and CAT enzyme activity decreased (P<0.05), and vitamins A, E and D(3) levels did not change (P>0.05) in the brain tissues of EMF-exposed guinea pigs. In addition, MDA, vitamins A, D(3) and E levels, and CAT enzyme activity increased (P<0.05), and GSH level decreased (P<0.05) in the blood of EMF-exposed guinea pigs. It was concluded that electromagnetic field emitted from cellular phone might produce oxidative stress in brain tissue of guinea pigs. However, more studies are needed to demonstrate whether these effects are harmful or/and affect the neural functions.

Photostability and efficacy studies of topical formulations containing UV-filters combination and vitamins A, C and E.

It is already known that the photostability of a sunscreen is important for its performance on human skin. On the other hand, there are many formulations besides sunscreens containing combinations of UV-filters and daily use active substances with other claims like hydration and anti-aging effects. Vitamins A, C and E are frequently added in these kinds of products and it is not known if the UV-filters have some influence on the hydration and anti-aging effects of these vitamins on the skin as well as on their stability mainly when photounstable UV-filters like avobenzone and octyl methoxycinnamate are present in the formulation. Thus, the aim of this study was to evaluate the influence of two different UV-filters combinations, a photostable and a photounstable one, on the photostability as well as on the efficacy of a formulation containing vitamin A, C and E derivatives. The formulations that were investigated contained or not (vehicle: formulation 1) a combination of 0.6 % (w/w) vitamin A palmitate (1,700,000 UI/g), 2 % (w/w) vitamin E acetate and 2% (w/w) ascorbyl tetraisopalmitate (formulation 2) supplemented with a photounstable UV filter combination octyl methoxycinnamate (OMC), avobenzone (AVB) and 4-methylbenzilidene camphor (MBC) (formulation 3) or with a photostable UV filter combination OMC, benzophenone-3 (BP-3) and octocrylene (OC) (formulation 4). In the photostability studies, all formulations were spread onto a glass plate and exposed to UVA/UVB irradiation. The filter components and vitamins were quantified by HPLC analysis with detection at 325 and 235 nm and by spectrophotometry. To simulate the effects of these formulations daily use, all of them (formulations 1-4) were applied on the dorsum of hairless mice, which were submitted to a controlled light-dark cycle (and were not irradiated), once a day for 5 days. Transepidermal water loss (TEWL), water content of the stratum corneum and viscoelastic properties of the skin were analyzed by using different non-invasive Biophysics Techniques in order to evaluate hydration and anti-aging effects of these formulations as well as erythema to assess skin irritation. Histopathology, viable epidermal thickness as well as the number of epidermal cell layers were also evaluated. It was observed that both UV filters combinations (photounstable one containing OMC, AVB and MBC and photostable one containing OMC, BP-3 and OC) enhanced vitamin A photostability and F4 was more photostable than F3, in terms of vitamin A. In vivo efficacy studies showed that F2, F3 and F4 enhanced the viable epidermal thickness, the number of epidermal cell layers, TEWL and Uv/Ue parameter, when compared to the vehicle, which can suggest that they enhanced viable epidermis hydration and acted in cell renewal. However formulation 2 (containing only vitamins), which was the most photounstable formulation, provoked an irritation on hairless mouse skin, and consequently it cannot be considered as safe as the other formulations. It can be concluded that both UV filters combinations did not influence the hydration and anti-aging effects of the formulations containing the vitamins under study and reduced the skin irritation observed when the vitamins were present in the formulation. In addition, the photostable UV-filters combination had the highest recovery of vitamin A in the photostability studies. Finally, it could be suggested that the presence of UV-filters can be considered interesting for the reduction of skin irritation and the most suitable formulation was the one containing the combinations of vitamins A, C and E with photostable UV-filters.

Photodecomposition of vitamin A and photobiological implications for the skin.

Vitamin A (retinol), an essential human nutrient, plays an important role in cellular differentiation, regulation of epidermal cell growth and normal cell maintenance. In addition to these physiological roles, vitamin A has a rich photochemistry. Photoisomerization of vitamin A, involved in signal transduction for vision, has been extensively investigated. The biological effects of light-induced degradation of vitamin A and formation of reactive species are less understood and may be important for light-exposed tissues, such as the skin. Photochemical studies have demonstrated that excitation of retinol or its esters with UV light generates a number of reactive species including singlet oxygen and superoxide radical anion. These reactive oxygen species have been shown to damage a number of cellular targets, including lipids and DNA. Consistent with the potential for damaging DNA, retinyl palmitate has been shown to be photomutagenic in an in vitro test system. The results of mechanistic studies were consistent with mutagenesis through oxidative damage. Vitamin A in the skin resides in a complex environment that in many ways is very different from the chemical environment in solution and in in vitro test systems. Relevant clinical studies or studies in animal models are therefore needed to establish whether the pro-oxidant activity of photoexcited vitamin A is observed in vivo, and to assess the related risks.

Photoprotection of vitamins in skimmed milk by an aqueous soluble lycopene-gum Arabic microcapsule.

Riboflavin (Rf)-mediated photosensitized degradation of vitamins A and D3 in skimmed milk under illumination with a white fluorescence lamp was studied by using the HPLC technique. The photosensitized degradation of both vitamins followed first-order kinetics, and the temperature effect on the observed photodegradation rate constant allowed the determination of the activation energy Ea as being 4 and 16 kcal/mol for vitamins A and D3, respectively. The addition of lycopene microencapsulated by spray-drying with a gum arabic-sucrose (8:2) mixture (MIC) produced a reduction of ca. 45% in the photosensitized degradation rate of both vitamins. Front-face fluorescence experiments showed the same photoprotection factor in the degradation of Rf itself, indicating that the photodegradation mechanism involved Rf-mediated reactive species, such as the excited triplet state of Rf, 3Rf*, and/or singlet molecular oxygen, 1O2. The interaction of both 3Rf* and 1O2 with MIC was evaluated in aqueous solutions by using laser-induced time-resolved absorption or emission spectroscopy, and the contribution of an inner-filter effect in the presence of MIC in skimmed milk was evaluated by diffuse reflectance spectroscopy. The main operating mechanism of photoprotection is due to the deactivation of 3Rf* by the proteic component of gum arabic; thus, gum arabic based microcapsules could be used to improve the photostability of milk during its storage and/or processing under light.

Photoionization versus photoheterolysis of all-trans-retinol. The effects of solvent and laser radiation intensity.

The time-resolved formation of the retinyl carbocation from all-trans-retinol and all-trans-retinol acetate was studied by use of picosecond flash photolysis. From both precursors, the retinyl cation is produced by heterolytic C-O bond cleavage in solvents of medium polarity (acetonitrile, tetrahydrofuran, propanol with Reichardt polarity parameter ET(N) approximately 0.5) and high polarity (EtOH, MeOH, TFE, HFIP, ET(N) > 0.6) during the laser pulse (< or =5 ps) where its lifetime is >10 ns. The absorption maximum of the cation at early times (t < 100 ps) is at lambda = 590-600 nm; it shifts to shorter wavelengths (Deltalambda = 5-10 nm) within 1-10 ns. This spectral shift is suggested to be due to contact ion pair --> solvent-separated ion pair --> free-ion transformation. The quantum yield of cation formation phi(cat) is independent of excitation wavelength (213, 266 or 355 nm). Photoheterolysis proceeds via a one-quantum process. In chlorinated solvents, i.e. n-BuCl, 1,2-dichloroethane, chloroform or CCl(4), formation of the retinol radical cation (which is characterized by a peak at 610 nm and further absorption maxima at approximately 840 and approximately 940 nm) by intermolecular electron transfer to the solvent molecules was detected. The radical cation lifetime in all these solvents is 1.5-2 ns, except for CCl(4) where it is 0.25 ns. The formation of the radical cation or cation was not detected in the low polarity solvents: cyclohexane, hexane, dioxane and p-xylene. However, in solvents of medium and high polarity, at high radiation intensities the radical cation may form in addition to the cation (as a result of two-quantum ionization). DFT calculations confirm our experimental results. The rate of retinol S(1) depopulation (k = 0.3-1 x 10(9) s(-1)) is almost independent of the solvent polarity in the range from cyclohexane to methanol. In highly polar solvents (ET(N) > 0.9) the rate increases to (0.5-5) x 10(10) s(-1).

Photodegradation of retinol and anti-aging effectiveness of two commercial emulsions.

Two commercial anti-aging products, RETI C and RETI C concentrate emulsions, containing retinol and vitamin C, were studied. The concentration of vitamin A was determined over time, subjecting the creams to an accelerated stability test. Both emulsions, when stored at 25 degrees C, showed a moderate decrease over time in retinol concentration, while after storage at 40 degrees C the percentage of retinol degraded increased over time. Under UVA irradiation, the retinol degraded to a greater extent than under UVB irradiation, both in RETI C and RETI C concentrate emulsions. In order to verify the anti-aging effectiveness of the emulsions, an in vivo test on some female volunteers was carried out, evaluating the visible results of the application of the creams on the skin surface. The creams were rather unstable after storage at 40 degrees C, but they were effective in treating the signs of aging and in reducing facial wrinkles.

Photoirradiation of retinyl palmitate in ethanol with ultraviolet light--formation of photodecomposition products, reactive oxygen species, and lipid peroxides.

We have previously reported that photoirradiation of retinyl palmitate (RP), a storage and ester form of vitamin A (retinol), with UVA light resulted in the formation of photodecomposition products, generation of reactive oxygen species, and induction of lipid peroxidation. In this paper, we report our results following the photoirradiation of RP in ethanol by an UV lamp with approximately equal UVA and UVB light. The photodecomposition products were separated by reversed-phase HPLC and characterized spectroscopically by comparison with authentic standards. The identified products include: 4-keto-RP, 11-ethoxy-12-hydroxy-RP, 13-ethoxy-14-hydroxy-RP, anhydroretinol (AR), and trans- and cis-15-ethoxy-AR. Photoirradiation of RP in the presence of a lipid, methyl linoleate, resulted in induction of lipid peroxidation. Lipid peroxidation was inhibited when sodium azide was present during photoirradiation which suggests free radicals were formed. Our results demonstrate that, similar to irradiation with UVA light, RP can act as a photosensitizer leading to free radical formation and induction of lipid peroxidation following irradiation with UVB light.

UVA photoirradiation of retinyl palmitate--formation of singlet oxygen and superoxide, and their role in induction of lipid peroxidation.

We have previously reported that photoirradiation of retinyl palmitate (RP) in ethanol with UVA light results in the formation of photodecomposition products, including 5,6-epoxy-RP and anhydroretinol (AR). Photoirradiation in the presence of a lipid, methyl linoleate, induced lipid peroxidation, suggesting that reactive oxygen species (ROS) are formed. In the present study, we employ an electron spin resonance (ESR) spin trap technique to provide direct evidence as to whether or not photoirradiation of RP by UVA light produces ROS. Photoirradiation of RP by UVA in the presence of 2,2,6,6-tetramethylpiperidine (TEMP), a specific probe for singlet oxygen, resulted in the formation of TEMPO, indicating that singlet oxygen was generated. Both 5,5-dimethyl N-oxide pyrroline (DMPO) and 5-tert-butoxycarbonyl 5-methyl-1-pyrroline N-oxide (BMPO) are specific probes for superoxide. When photoirradiation of RP was conducted in the presence of the DMPO or BMPO, ESR signals for DMPO-*OOH or BMPO-*OOH were obtained. These results unambiguously confirmed the formation of superoxide radical anion. Consistent with a free radical mechanism, there was a near complete and time-dependent photodecomposition of RP and its photodecomposition products. ESR studies on the photoirradiation of 5,6-epoxy-RP and AR indicate that these compounds exhibit similar photosensitizing activities as RP under UVA light.

Photomutagenicity of retinyl palmitate by ultraviolet a irradiation in mouse lymphoma cells.

Retinyl palmitate (RP), a storage form of vitamin A, is frequently used as a cosmetic ingredient, with more than 700 RP-containing cosmetic products on the U.S. market in 2004. There are concerns for the possible genotoxicity and carcinogenicity of RP when it is exposed to sunlight. To evaluate the photomutagenicity of RP in cells when exposed to ultraviolet A (UVA) light, L5178Y/Tk+/- mouse lymphoma cells were treated with different doses of RP alone/or in the presence of UVA light. Treatment of the cells with RP alone at the dose range of 25-100 microg/ml did not increase mutant frequencies (MFs) over the negative control, whereas treatment of cells with 1-25 microg/ml RP under UVA light (82.8 mJ/cm2/min for 30 min) produced a dose-dependent mutation induction. The mean induced MF (392 x 10(-6)) for treatment with 25 microg/ml RP under UVA exposure was about threefold higher than that for UVA alone (122 x 10(-6)), a synergistic effect. To elucidate the underlying mechanism of action, we examined the mutants for loss of heterozygosity (LOH) at four microsatellite loci spanning the entire chromosome 11, on which the Tk gene is located. The mutational spectrum for the RP + UVA treatment was significantly different from the negative control, but not significantly different from UVA exposure alone. Ninety four percent of the mutants from RP + UVA treatment lost the Tk+ allele, and 91% of the deleted sequences extended more than 6 cM in chromosome length, indicating clastogenic events affecting a large segment of the chromosome. These results suggest that RP is photomutagenic in combination with UVA exposure in mouse lymphoma cells, with a clastogenic mode-of-action.

Photodecomposition of retinyl palmitate in ethanol by UVA light-formation of photodecomposition products, reactive oxygen species, and lipid peroxides.

Photodecomposition of retinyl palmitate (RP), an ester and the storage form of vitamin A (retinol), in ethanol under UVA light irradiation was studied. The resulting photodecomposition products were separated by reversed-phase HPLC and identified by spectral analysis and comparison with the chromatographic and spectral properties of synthetically prepared standards. The identified products include 5,6-epoxy-RP, 4-keto-RP, 11-ethoxy-12-hydroxy-RP, 13-ethoxy-14-hydroxy-RP, anhydroretinol (AR), palmitic acid, ethyl palmitate, and four tentatively assigned cis and trans isomeric 15-ethoxy-ARs. AR was formed as a mixture of all-trans-AR, 6Z-cis-AR, 8Z-cis-AR, and 12Z-cis-AR with all-trans-AR predominating. 5,6-Epoxy-RP, 4-keto-RP, 11-ethoxy-12-hydroxy-RP, and 13-ethoxy-14-hydroxy-RP were also formed from reaction of RP with alkylperoxy radicals generated by thermal decomposition of 2,2'-azobis(2,4-dimethylvaleronitrile). Formation of these photodecomposition products was inhibited in the presence of sodium azide (NaN3), a free radical inhibitor. These results suggest that formation of 5,6-epoxy-RP, 4-keto-RP, 11-ethoxy-12-hydroxy-RP, and 13-ethoxy-14-hydroxy-RP from photoirradiation of RP is mediated by a light-initiated free radical chain reaction. AR and the isomeric 11-ethoxy-ARs were not formed from reaction of RP with alkylperoxy radicals generated from 2,2'-azobis(2,4-dimethylvaleronitrile), and their formation was not inhibited when NaN3 was present during the photoirradiation of RP. We propose that these products were formed through an ionic photodissociation mechanism, which is similar to the reported formation of AR through ionic photodissociation of retinyl acetate. RP and all its identified photodecomposition products described above (i) were not mutagenic in Salmonella typhimurium tester strains TA98, TA100, TA102, and TA104 in the presence and absence of S9 activation enzymes, (ii) were not photomutagenic in Salmonella typhimurium TA102 upon UVA irradiation, and (iii) did not bind with calf thymus DNA in the presence of microsomal metabolizing enzymes. These results suggest that RP and its decomposition products are not genotoxic; however, photoirradiation of RP, 5,6-epoxy-RP, and AR with UVA light in the presence of methyl linoleate resulted in lipid peroxide (methyl linoleate hydroperoxides) formation. The lipid peroxide formation was inhibited by dithiothreitol (DTT) (free radical scavenger), NaN3 (singlet oxygen and free radical scavenger), and superoxide dismutase (SOD) (superoxide scavenger) but was enhanced by the presence of deuterium oxide (D2O) (enhancement of singlet oxygen lifetime). These results suggest that photoirradiation of RP, 5,6-epoxy-RP, and AR by UVA light generated reactive oxygen species resulting in lipid (methyl linoleate) peroxidation.

Dehydration reaction of hydroxenin monoacetate in carbon tetrachloride and an aliphatic alcohol under ultrasound irradiation.

A new method for the preparation of an E/Z mixture of vitamin A acetate from hydroxenin monoacetate is described. This two-step reaction was studied by changing the reaction parameters (reaction temperature, ultrasound power, and reaction time) and the alcohol used. This approach consists of the dehydration reaction of hydroxenin monoacetate under ultrasound irradiation in CCl4 and an aliphatic alcohol under an inert atmosphere. The formation of small amounts of HCl from CCl4 and an aliphatic alcohol under ultrasound irradiation is followed by the dehydration reaction of hydroxenin monoacetate. An E/Z mixture of vitamin A acetate was obtained resulting in the desired pentaenes. Some ethers derivatives were also formed as by-products, isolated and characterized. Study of the reaction mechanism is also reported here.

Quantitative analysis of vitamin A degradation by Raman spectroscopy.

Vitamin A is known to support cell growth promotion, maintenance, and differentiation of epithelial tissues. Retinol is currently used in cosmetic formulations and products to deliver these and other benefits to the skin. However, retinol is known to be unstable and, therefore, remains of great concern to the cosmetic industry. The decomposition pathways of the retinoids in general have been previously postulated and investigated mostly by high-performance liquid chromatography (HPLC) with UV/Vis spectroscopy. In our studies, we examined specific conditions at which retinol degrades and subsequently identified and quantified the products of retinol decomposition by Raman spectroscopy. We reveal which experimental settings and computational tools allow monitoring of in situ evolution of an all-trans retinol solution when submitted to UV light in the presence of oxygen.