Joachim W. Fluhr, MD, Charité– Universitätsmedizin Berlin, Department of Dermatology, Center for Experimental and Applied Cutaneous Physiology, Charitéplatz 1, D-10117 Berlin, Germany, Tel.: +49 30 450 518 208, Fax: +49 30 450 518 918, e-mail: firstname.lastname@example.org
Abstract: The influence of stress factors on human skin induces the production of free radicals. Free radicals react immediately with antioxidants contained in the skin, giving rise to their depletion and with the surrounding molecules, resulting in their damage, disorganization and even destruction. High amounts of free radicals are produced in the upper skin layers, i.e. mainly in the epidermis, subsequent to sun irradiation. Irradiation of the skin in the infra-red (IR) range of the spectra, applied at physiological doses, can produce free radicals. The magnitude of destruction of antioxidants, such as carotenoids, can serve as a marker of the extent of the stress factor, characterized by the quantity of produced free radicals. In this study, measurements on the degradation of cutaneous carotenoids following IR skin irradiation of 12 healthy volunteers (skin type II), with two IR sources (standard infrared radiator = SIR and water filter infrared = wIRA) were taken using resonance Raman spectroscopy. Topical application of the antioxidant beta-carotene (2 mg/cm2) provided protection for the human skin when exposed to IR radiation. The magnitude of the degradation of dermal carotenoids after IR irradiation was significantly higher for SIR than for wIRA irradiation, for both non-treated and cream-treated skin areas. The amount of destroyed carotenoids after IR irradiation was higher in the case of pretreatment with beta-carotene than for the untreated skin, indicating that the superficial part of antioxidants is most important for protecting against external stressors. The direct comparison of beta-carotene content was significantly higher for the cream-treated compared to untreated areas for all pairs: baseline, wIRA, after wIRA, baseline SIR and after SIR. Additionally, topically applied carotenoids as a single antioxidant component are less stable than the carotenoids in the skin incorporated by nutrition and accumulated in a mixture with different antioxidant substances. Resonance Raman spectroscopy can be used for the non-invasive measurements of carotenoids, which can be rated as marker substances of redox processes.
Free radicals are produced in the human body by metabolic processes, environmental impact, during inflammation and by negative influences of light irradiation (1–4). The human organism has developed protection mechanisms to neutralize free radicals (5–7). However, if the number of radicals formed in the tissue is significantly increased, the defense mechanism of the body is not able to neutralize all these reactive molecules. In this case, chain reactions start which again form high amounts of free radicals. This is the case, if the skin is irradiated with a high dose of UV light or is in contact with environmental hazards (8–10). The action of free radicals can result in oxidative cell damage (11–13), premature skin ageing (14–17) and even the formation of skin cancer (18,19). Stratum corneum is rich in different antioxidants that are responsible for the neutralization of free radicals (20–22). Moreover, high concentration of cutaneous lycopene in humans was correlated with significantly lower values of skin roughness, measured as a combination of the depth and density of furrows and wrinkles (23).
The defense mechanism of the human body against the action of the excess amount of free radicals is based on the interaction of antioxidant substances, such as carotenoids (beta-carotene, lycopene, lutein, zeaxanthin and their isomers), vitamins (A, C, D and E), enzymes (superoxide dismutase, catalase and glutathione peroxidase) and other compounds (melanin, flavonoids, lipoic acid, selenium, coenzyme Q10, etc.). These antioxidants act as a ‘protection chain’, i.e. all antioxidants possess a synergic action, thus protecting each other against direct destruction during neutralization of free radicals and other reactive species (24–26). The side effect of these interactions with high amounts of radicals is the destruction of some compounds of the antioxidative network and, as a result, the depletion of the defense antioxidative system of the organism.
The measured kinetics showed that carotenoids can serve as marker substances for the total antioxidative network of the human skin (27). Stress factors, like irradiation and diseases, which are usually associated with the production of free radicals in the organism, can cause a decrease in the antioxidative network of the skin, measured by a decrease in the cutaneous carotenoid concentration. In contrast, the absence of influencing stress factors, as well as an antioxidant-rich supplementation, increases the antioxidative network of the human skin (28,29).
The additional topical application of cosmetic formulations that contained different antioxidants in their composition can be protective and potentially correct the free radical-induced damage of the skin. Moreover, some antioxidants may be beneficial to the skin, because of other actions, such as the effects of pigmentation suppression and bruising, stimulation of collagen production, refinement of keratinization and anti-inflammatory effects (30), improving skin hydration (31) and protection against the action of free radicals (32). For both systemically and topically applied antioxidants, protective functions on the skin and tissue are known. Antioxidants may protect not only the skin but also the topically applied formulation (as compound of the product). Tocopherol, e.g., is a frequently used antioxidant in topically applied products as free radical scavenger (33). Several studies have assessed the ability of systemically administered beta-carotene to protect the immune system and DNA from UV-induced free radical damage (15,34,35), and to increase the minimal erythema dose (36,37). The protective effect of human skin by the topical application of cosmetic formulations containing different antioxidants has been previously published in the literature (38–43). Recently, it has been shown that different carotenoids have distinguishable protective capacities on UVA-induced oxidative damage and expression of oxidative stress-responsive enzymes in a fibroblast in vitro model (44).
It is known that irradiation of the skin with UV light induces free radical production in the skin (3,45,46), because the energy of the UV photons is high enough for radical production. Recent studies have shown that infra-red (IR) radiation can also produce free radicals in the skin (47,48) despite the fact that the energy of IR photons is not high enough for direct radical induction. Thus, biological systems or specific tissues accumulate the IR energy up to a level that is sufficient to produce free radicals. The group of Krutmann could demonstrate that mitochondria produce free radicals owing to IR radiation (49,50). The energy of IR photons is much lower than that of UV photons and, therefore, IR radiation cannot directly destroy the antioxidants present in the skin and in cosmetic formulations. Moreover, cutaneous antioxidants, such as vitamins and carotenoids, do not absorb light in the IR spectral range. In this regard, in this study, an IR radiation was chosen as an exogenous stress factor that initiates the production of free radicals in the skin. Two IR sources with different spectral characteristics of excitation have been used in the study both used in clinical settings (51). SIR uses a broader band width in comparison with water filter infrared (wIRA). The aim using two sources of IR was to assess two clinically relevant sources with different band widths of IR. The concentration of the carotenoids in the skin was determined in vivo by resonance Raman spectroscopy (52).
In this study, it was investigated whether the topical application of formulation containing beta-carotene can protect the skin efficiently by the neutralization of IR-induced free radicals on the skin surface.
Materials and methods
The in vivo resonance Raman spectroscopic measurements of the kinetics of the carotenoid antioxidants, beta-carotene and lycopene in human skin subsequent to IR irradiation were taken on the flexor forearms of 12 healthy volunteers (three men and nine women). All volunteers had skin type II (53) and were aged 25–35 (mean age 29). Raman measurements were taken before and after IR irradiation.
Two areas of 4 × 4 cm2 on each flexor forearm were marked with permanent marker and then used for further investigations. One area of each arm was pretreated homogeneously with 2 mg/cm2 of an o/w formulation (cream) containing 0.2% beta-carotene before IR irradiation. The time between cream application and the first measurement was 30 min. This was carried out to increase the concentration of the antioxidant beta-carotene in the skin to approximately twofold and thus to compare the stability of the natural mixture of cutaneous antioxidants (including carotenoids) with the stability of the topically applied single compound. The other two skin areas remained untreated.
Subsequently, the pretreated and untreated skin areas were irradiated with two IR irradiation sources, the standard IR radiator (SIR) and the water-filtered IR radiator (WIRA), emitting different IR spectra. Thus, the influence of the stress factor IR irradiation on the protection efficiency of the carotenoids for human skin was investigated.
To examine the radical formation process in the formulation containing beta-carotene during IR irradiation, the formulation was placed in a quartz cuvette with a thickness of 1.0 mm to exclude the insiccation and oxidation of the investigated sample. Then, IR irradiation emitted by the SIR was performed on 12 samples. The other 12 samples were irradiated with WIRA. Additionally, control measurements without IR irradiation were taken.
During the IR irradiation, the temperature of the skin surface and of the cuvette was controlled continuously. The measurements of the skin temperature were taken with a non-contact thermometer (Rytek Schlender Messtechnik, Rüthnick, Germany).
Approval for this study had been obtained from the Ethics Committee of the Charité– Universitätsmedizin Berlin, and the volunteers had signed a written informed consent form, prior to commencement of the study.
A standard infrared radiator (SIR) Philips Infrared RI 1521 (Philips, Eindhoven, Netherlands), emitting light with a broad IR spectrum at wavelengths between 600 and 3000 nm, and a WIRA Hydrosun Medizintechnik GmbH, emitting IR light at wavelengths between 600 and 1500 nm, were used as sources of IR radiation. Both IR radiators were utilized at a power density of 190 mW/cm2 for 30 min on the human skin surface, which is recommended for medical treatment at a distance in the region of 25 cm from the skin surface.
In the in vitro experiments, a lower power density was necessary to reach the same temperature as in the in vivo measurements, because of the cooling effect produced by the blood flow. Therefore, during the in vitro measurements of the cream the power density of the IR radiation varied between 140 and 170 mW/cm2 during a 30-min application time, depending on the type of radiator.
The power densities of IR radiation on the skin and cuvette surfaces were checked every 10 min during the IR irradiation and were corrected, when necessary, by adjusting the distance between the skin surface and IR radiator. Non-contact temperature measurements were taken continuously.
Resonance Raman spectroscopic determination of the cutaneous carotenoids
Resonance Raman spectroscopy was used for in vivo determination of cutaneous carotenoid concentration. This system allows quick non-invasive measurements to be performed on human skin. The blue light at 488 nm of an argon laser excites all cutaneous carotenoids with approximately the same Raman scattering efficiency. Thus, the measured Raman peak at 527.2 nm (wavelength corresponded to the spectral Raman line at 1525 cm, which originates from the carbon–carbon double bond stretch vibration of the conjugated backbone of carotenoid molecules) supplies quantitative information about the concentration of carotenoids in the measured skin volume. The measuring system has been described previously by our group (52,54).
Taking into consideration the low penetration depth of the blue light into the skin, which is about 200 μm, and the inhomogeneous distribution of carotenoids in the skin with its maximum near the skin surface (22,55) [measured by Raman microscopy (56–58)], it can be concluded that the Raman measurements of the carotenoids are taken mainly in the epidermis.
Application of beta-carotene-containing cream on the skin surface increases the measured Raman signal from the skin and gives information about its degradation.
The results are presented as average values with their standard deviations. The statistical calculations were performed with pasw for Windows (SPSS Inc., Chicago, IL, USA). The design was selected as a descriptive, exploratory study without formulation of a-priori hypothesis. The data did not show a normal distribution. Thus, non-parametric tests (Friedman’s two-way analysis of variance) were performed followed by a pairwise comparison using Wilcoxon signed rank test. Values of P < 0.05 were considered statistically significant.
Application of beta-carotene-containing formulation
Topical application of a formulation containing 0.2% beta-carotene increased carotenoid concentrations to 1.75 ± 0.29 times on average for all volunteers. Obtained concentration of beta-carotene is close to the physiological level and scarcely higher. Therefore, the manifestation of a probable side effect of beta-carotene, such as pro-oxidative action, can be excluded. Moreover, as shown previously, prolonged topical application of a low concentration of beta-carotene is more effective for the protection of the skin (59,60).
In vitro analysis of the Influence of the IR irradiation on the beta-carotene-containing formulation
The concentration of beta-carotene in the samples was measured before and after IR irradiation by means of resonance Raman spectroscopy. At the same time, control measurements without IR irradiation were taken.
The measurements showed no statistically significant differences between the initial and final (before and after an IR irradiation) levels of concentration of beta-carotene in the irradiated samples for both the applied IR radiators (data not shown). Furthermore, no differences were found between the concentration of beta-carotene in the irradiated samples and the control samples.
In vivo analysis of the influence of the IR irradiation on the carotenoid concentration in human skin
The changes in the carotenoid concentration of the 12 volunteers before and after IR irradiation were obtained with two irradiation sources (SIR and WIRA). Different volunteers had different initial levels of carotenoids in their skin, which reflected their lifestyle. In all cases, a degradation of the carotenoids could be detected after IR irradiation with the two irradiation sources, with the degradation after irradiation being higher when SIR was used when compared to WIRA. Obtained degradations were statistically significant for both IR sources. The average destruction of carotenoids obtained after the application of SIR was 10% higher than in the case of WIRA application, which is in agreement with previously published results (61,62). Comparison of the typical degradation dynamics of carotenoids of the untreated skin areas and of the skin areas pretreated with the formulation containing beta-carotene for both IR radiators showed that a decrease in carotenoids in the skin occurs constantly during IR irradiation (data not shown).
Figure 1 demonstrates the average absolute values for all 12 volunteers of the concentration of carotenoids in their skin. The non-treated skin areas are presented in Fig. 1a and cream-treated skin areas in Fig. 1b. After the application of the beta-carotene-containing cream, the carotenoid concentration of the skin increased 1.75 ± 0.29 times on average for all volunteers. In all cases the direct comparison of beta-carotene content was higher for the cream-treated compared with untreated areas (P = 0.002 for all pairs: baseline, wIRA, after wIRA, baseline SIR and after SIR). The beta-carotene level was significantly lower in both untreated (P = 0.003) and cream-treated (P = 0.002) volunteers on SIR irradiated skin compared with wIRA.
In Fig. 2, the degradation of the carotenoids after IR irradiation is compared for three pairs of volunteers, who showed approximately the same initial concentration of carotenoids; one volunteer without cream treatment and the other after cream treatment. The degradation of the carotenoids in the skin is approximately doubled in the case of the cream-treated skin area in comparison with the untreated skin area owing to an increase by topically applied beta-carotene-containing formulation. The topically applied antioxidant is more likely to be altered by external stressor like IR irradiation in our model but is also more protective. Again, the destruction of the carotenoid content is higher when the SIR rather than the WIRA was used.
In the case of the beta-carotene-containing cream being irradiated in a cuvette with IR light, no degradation of the carotenoids could be detected. This is not surprising, because the beta-carotene did not absorb the IR radiation, and a thermal destruction at the temperature reached in the experiment could be excluded, thus no formation of radicals is to be expected.
When the human skin was subjected to IR irradiation under in vivo conditions, a degradation of carotenoids was detected. It was demonstrated by electron spin resonance (ESR) measurements that this degradation is caused by the action of free radicals that are formed in the human skin subsequent to IR irradiation (47,48). Comparing the results obtained in the in vitro and in vivo measurements, it can be concluded that biological processes in the living skin transfer the IR photons partially into free radicals. Schroeder et al. (63) demonstrated that the mitochondria participate in this energy transfer process. Additionally, it is to be expected that enzymatic processes are involved in this energy transfer mechanism. For example, formation of heat shock–induced reactive oxygen species is reported as being promoted via enzymes and mitochondrial electron transport systems under physiological temperatures (64). The absolute amount of beta-carotene, which is destroyed after having irradiated the skin with a definite IR radiation dose, is almost constant for all volunteers, independent of their initial carotenoid concentration. This means that the destruction in the case of individuals with high carotenoid levels is less than in the case of volunteers showing low carotenoid concentrations. Taking into account the positive effect of heat treatment by IR irradiation during medical therapy, for instance in wound healing, it is evident that patients with high antioxidant levels in their skin are better protected against the possible negative side effects caused by the production of free radicals than patients with low antioxidant levels. High antioxidant levels in the human organism and, consequently, in the skin, can only be obtained by a healthy nutrition, rich in fruit and vegetables. Notwithstanding the fact that the carotenoid concentration in the skin after the cream treatment had almost doubled, compared with the natural beta-carotene level, the destruction of the carotenoids after IR irradiation, in percentage, was the same for the non-treated and cream-treated skin areas. Consequently, the absolute amount of beta-carotene destroyed in the cream-treated skin areas is higher than in the non-treated skin areas. This is exemplarily shown in Fig. 2, comparing pairs of volunteers from the untreated and cream-treated groups, each having approximately the same carotenoid concentration in the skin before IR irradiation. Considering that the destruction rate for the identical IR irradiation dose used in both experiments is almost constant for the non-treated skin, it can be concluded that the high carotenoid concentration in case of cream treatment is mainly related to the topically applied beta-carotene.
This is not surprising, as the different antioxidant substances instigate a change in the protection properties of the human skin by protecting each other from the destructive action of the free radicals. The beta-carotene applied topically by the cream represents a single component, but not a mixture of antioxidants. Consequently, it is less protected (65). Therefore, it can be assumed that during the IR irradiation of the cream-treated skin areas, the topically applied beta-carotene is destroyed primarily, which results in a protection of the carotenoids, and the other antioxidant substances, accumulated in the human skin. Indeed, the total carotenoid concentration in the skin after IR irradiation in the case of the cream treatment is higher than the natural initial carotenoid concentration in the skin before the cream was applied. Consequently, topically applied antioxidants protect the natural antioxidant network of the skin.
The rate of destruction of the carotenoids was always higher in the case of irradiation of the skin with the SIR than with WIRA. This is plausible because of the missing water absorption; the WIRA penetrates deeper into the tissue (66). Consequently, the energy density per tissue volume is less in this case, i.e. the density of free radicals production is reduced.
Summarizing the results achieved by the present study, it has to be stated that in spite of the missing absorption of the carotenoids in the IR spectral range and the heat stability of the carotenoid molecules under experimental conditions, a production of free radicals could be detected under in vivo conditions indirectly by the degradation of the carotenoids. Topical application of antioxidants increased in general the measured beta-carotene level independent of baseline or post-wIRA/SIR irradiation. The degradation was higher for the SIR than for the WIRA. The relative degradation rate for a definite IR radiation dose was almost identical for all volunteers, independent of their initial carotenoid level. This means that individuals living on a healthy diet rich in fruit and vegetables are better protected than those leading a stressed lifestyle and living on antioxidant-poor nutrition.
Topical application of antioxidants, in the present study beta-carotene, protects the natural antioxidant network of the skin. The single topically applied carotenoid component was destroyed at a higher degree than the systemically applied carotenoids in the skin. This is attributed to the protection properties of the complex mixture of antioxidants in the human skin, which bring about a higher protection efficiency. Consequently, topically applied antioxidants should consist of a broad mixture of different substances similar to the composition and concentration of the natural antioxidative network of the skin (7,67).
Moreover, it was demonstrated that the Raman spectroscopic measurements are well suited to investigate the kinetics of the carotenoids in the human skin online and non-invasively.
Topical application of antioxidant substances including beta-carotene has a positive effect on the cutaneous antioxidants during the influence of a stress factor, such as IR irradiation. It was shown in the in vivo measurements taken on the skin pretreated with a formulation containing beta-carotene that free radicals, which are produced on the skin surface subsequent to IR irradiation, can be effectively neutralized by topically applied antioxidants.
The neutralization of free radicals might be further increased, if mixtures of different antioxidants at optimal concentrations would be applied. This process can be expected, when taking into consideration the synergistic action of antioxidants in neutralizing the free radicals.
Resonance Raman spectroscopy can be used for this purpose as a suitable method for the non-invasive measurements of carotenoids, which can be rated as marker substances of redox processes. The questions related to the optimization of the mixture of antioxidants used in cosmetic formulations should be investigated, additionally, in the future, and the utilization of modern non-invasive techniques, such as resonance Raman spectroscopy, is expected to be indispensable in this undertaking.
We thank the Foundation ‘Skin Physiology’ of the Donor Association for German Science and Humanities for financial support.