J. Biophotonics 7, No. 9, 735–743 (2014) / DOI 10.1002/jbio.201300171
Journal of
BIOPHOTONICS FULL ARTICLE
Influence of sun exposure on the cutaneous collagen/ elastin fibers and carotenoids: negative effects can be reduced by application of sunscreen Maxim E. Darvin*; 1 , Heike Richter 1, Sebastian Ahlberg 1, Stefan F. Haag 1, Martina C. Meinke 1, Delphine Le Quintrec 2, Olivier Doucet 2 , and Ju¨rgen Lademann 1 1 2
Charite´ – Universita¨tsmedizin Berlin, Department of Dermatology, Venerology and Allergology, Center of Experimental and Applied Cutaneous Physiology (CCP), Berlin, Germany COTY LANCASTER S.A.M., R&D Center, Monaco
Received 28 October 2013, revised 4 February 2014, accepted 20 February 2014 Published online 18 March 2014
Key words: skin physiology, free radicals, antioxidants, SAAID, in vivo, sun irradiation, sun protection
Resonance Raman spectroscopy and multi-photon tomography were used in vivo to analyse the influence of sun exposure on the cutaneous carotenoids and collagen/elastin fibers. Comparing Berlin (low sun exposure) and Monegasque (high sun exposure) volunteers, it could be demonstrated that extended sun exposure significantly reduces the cutaneous carotenoids and collagen/elastin concentration (p < 0.05). The tendency towards correlation (R2 ¼ 0.41) between the dermal collagen/elastin (SAAID) and carotenoids confirms the important role of antioxidants in the protection against sun-induced negative effects. The application of sunscreen was shown to be effective, protecting cutaneous carotenoids and collagen/elastin from being damaged subsequent to sun exposure. Correlation between the SAAID values and the carotenoid concentration of the volunteers (from Berlin and Monaco) before the sunscreen application (visit 1).
1. Introduction As the biggest human organ, the skin represents the body’s barrier towards the environment and has an important physiological function. Since ancient ti-
mes, people have been endeavoring to keep their skin appearing youthful and healthy. Skin aging is determined by genetic aspects and can be accelerated by environmental factors. The most important of them is the solar UV radiation [1, 2], whose inter-
* Corresponding author: e-mail: [email protected], Phone: +49 30 450 518 208, Fax: +49 30 450 518 918
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action with the skin generates free radicals [3, 4]. If the radical concentration exceeds a critical level, these highly reactive molecules are able to destroy cells and extracellular matrix including elastin and collagen fibers. Premature skin aging (also called photoaging) [5–7], immunosuppression [8, 9] and even the development of skin diseases including skin cancer [10, 11] can be the consequences. Extensive destruction, significant alteration or diminished synthesis of the collagen and elastic fiber networks, which are the main components of dermal extracellular matrix, serve as the main hallmarks of skin aging [12–14]. For protection against the detrimental action of the free radicals, the human skin has developed a defence system that is based on the action of antioxidants. Various antioxidant substances including, inter alia, vitamins, carotenoids, and enzymes form protection chains in the skin quenching the radical activity and synergistically safeguarding each other against the destructive action of the free radicals [15, 16]. The effectiveness of the cutaneous antioxidant system can vary individually, depending on the antioxidants’ concentration and composition that is substantially influenced by lifestyle [17–19]. It was found in in vivo studies that carotenoids could serve as marker substances for the whole antioxidant status of the human epidermis [20, 21]. Cutaneous carotenoids are capable of neutralizing several attacks of free radicals and are then destroyed. It was shown in different in vivo studies that the carotenoid concentration had decreased after the irradiation of human skin with UV [22] and IR light [23] and this decrease is associated with the carotenoids’ interaction with the free radicals [24]. Thus, protection in the whole spectrum is needed to minimize the solar-induced negative effects. Modern sunscreens are capable of providing such protection efficiently. Optical noninvasive techniques are well suited to characterize different skin parameters [25]. In the study reported herein, two different optical methods were used to evaluate the influence of extended sun exposure on the concentration of cutaneous carotenoids and collagen/elastin fibers in vivo in human skin. Multi-photon tomography is well suited to analyse and quantify the collagen/elastin structure under in vivo conditions [26]. Using in vivo resonance Raman spectroscopy, the antioxidant status of the skin can be determined via the carotenoid concentration [27]. The protective capacity of one example sunscreen towards development of premature skin aging was measured. The results obtained noninvasively by the two optical methods were evaluated and compared.
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2. Materials and methods 2.1 Volunteers The study was undertaken at the Center of Experimental and Applied Cutaneous Physiology of the Charite´ – Universita¨tsmedizin Berlin. Twelve volunteers from Berlin (5 males and 7 females) and eight volunteers from Monaco (8 females) were included in the study. The volunteers were aged between 22 and 58 years. All volunteers were non-smokers of skin type II (Berlin group) and skin type III (Monegasque group) according to the Fitzpatrick classification [28]. The Berlin group was measured in June and July 2012 and the Monegasque group in July and August 2012. In addition to the Berlin volunteers, Monegasque volunteers were included in the study as the weather conditions in Berlin had been poor in the summer months of 2012. It had been raining almost every day, and the sun had been shining only occasionally.
2.2 Sunscreen samples The sunscreen used in the study was the cream LA 4032020370 provided by COTY Lancaster S.A.M, R&D Center, Monaco. It was an o/w formulation with a SPF of 30, PPD of 10 and IR protection. The cream contained UVA and UVB filters, IR reflectors and hydrophilic and lipophilic antioxidants from natural and synthetic origin. Additionally, the utilized sunscreen did not contain any anti-aging or regenerative actives (peptides). The sunscreen did not absorb and scatter light in the visible spectral range. Therefore no influence on the applied optical measurements is expected.
2.3 Study protocol The sunscreen was daily applied either to the volunteers’ right or left upper forearm areas at a concentration of approximately 2 mg/cm2 for a period of four weeks. The applications were randomized. The volunteers were instructed to desist from applying any other skin care products and sunscreens at least one week before and during the test period. The nontreated skin was used as control area (“placebo”). Volunteers were asked to stay in the direct sun with unclothed forearms minimally two hours every day. The duration of sun exposure was not controlled and therefore the applied dose was not determined. Before and after four weeks of sunscreen application the following two different skin parameters
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were noninvasively determined on the marked areas of both forearms. First, the SAAID (second harmonic generation (SHG) to autofluorescence (AF) aging index of dermis) was determined using multiphoton tomography. Second, the antioxidant status of the skin was determined by the concentration of carotenoids measured with resonance Raman spectroscopy. All measurements were performed in triplicate and the obtained mean value was used for further analyses.
2.4 Determination of the SAAID Collagen is a well-known source of tissue second harmonic generation (SHG) signal [29–31]. In the dermis, the autofluorescence (AF) is mainly determined by the elastin concentration, while the SHG is determined by the collagen concentration [32]. The collagen and elastin structure of the skin can be characterized by the SAAID [33, 34]. The SAAID is defined as (SHG-AF)/(SHG+AF). In this nomencla-
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ture, the SAAID decreases with photoaging, approaching 1 when the collagen has been completely replaced by elastic fibers. For the SAAID analysis, the average SAAID value obtained for the transition zone between the “stratum basale” and the “papillary dermis” (depth between 40 mm and 80 mm) was used. The investigations were carried out using a multiphoton tomograph (JenLab GmbH, Jena, Germany) equipped with a femtosecond titanium sapphire laser (Mai Tai XF, Spectra Physics, USA). The laser was operated at 760 nm and generated 100 fs pulses at a repetition rate of 80 MHz. For studying the structure of the dermis at different depths of the skin a sensitive photo multiplier was applied. The radiation collected from different depths was averaged over slices of 200 200 mm2. Applied power in the skin increased linearly from 10 mW in the stratum corneum to maximal value of 50 mW in the dermis (depth 70 mm) and remains maximal up to a measurement depth of 120 mm. The acquisition time was equal to 7 seconds for one slice which guaranteed a satisfactory measurement stability and picture quality. The
a)
Figure 1 Exemplary images of AF and SHG obtained by multi-photon tomography used for determining the elastin and collagen amounts to calculate the SAAID values (a) and the SAAID curves obtained before sun exposure (visit 1) and after four weeks of daily sun exposure (visit 2) for the sunscreen treated skin area (black) and untreated skin area (gray). Scale bar is 30 mm. b)
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effect of utilized parameters of irradiation on human skin was compared to daily sun exposure-induced effects [35]. During the measurements, the laser focus was moved from the skin surface into deeper parts of the skin at 10 mm increments. The maximum measuring depth into the skin was 120 mm. Exemplarily, images of the elastin (AF) and collagen (SHG) distributions in the skin used for determining the SAAID values are presented in Figure 1a. The typical SAAID curves obtained before sun exposure (visit 1) and after four weeks of daily sun exposure (visit 2) are presented in Figure 1b for the untreated and sunscreen treated skin areas of one volunteer.
2.5 Determination of the antioxidant status of human skin by the carotenoid marker substances It was demonstrated in previous in vivo studies that the carotenoids can be used as marker substances to characterize the whole antioxidant status of the human epidermis [20, 21]. In the present study, the investigations were performed in vivo on both forearms of volunteers using resonance Raman spectroscopy. This method was chosen because of the measurement quickness and high sensitivity in comparison to other available methods [36, 37]. The Raman spectrometer consisted of a base accommodating the argon laser operating at 488 nm, and the detection system. The laser radiation was led via an optical fiber to an imaging system. The imaging system produced a laser spot of 6.5 mm in diameter on
Figure 2 Typical Raman spectrum obtained from the human skin measured in vivo under an excitation at 488 nm (taken from [38]). Inlay shows the determination of the intensity of the Raman peak.
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the skin surface. The Raman measurement was performed during 5 seconds under the power density of 24 mW/cm2, which is lower than the power density of the sun. The Raman signal on the skin measured at 1525 cm1 was collected by the same imaging system and focused into another optical fiber which was connected to the detector system. The typical Raman spectrum obtained from the skin under an excitation of 488 nm is shown on Figure 2. The concentration of carotenoids was determined by the intensity of Raman peak, as shown in inlay of Figure 2. A detailed description of the utilized Raman spectrometric system was previously given by our group [27].
2.6 Statistical analysis For statistical analysis, SPSS 19.00 for Windows (SPSS Inc., Chicago, IL, USA) software was used. Data were analyzed using non-parametric tests. Firstly, the related samples, whereby Friedman’s two-way analysis of variances by ranks was performed. Differences among the means of groups were analyzed by the Wilcoxon test, considering significance at p < 0.05. Independent samples were tested using the Mann-Whitney U test.
3. Results 3.1 Analysis of the SAAID In Figure 3a, a box plot of the SAAID values of the Berlin volunteers before sunscreen treatment (visit 1) and four weeks after treatment (visit 2) is presented for the untreated and sunscreen-treated skin areas for the 12 volunteers. It can be seen that the SAAID values are quite different for the individual volunteers. The non-strongly pronounced but significant reduction (p < 0.05) was found for the untreated skin area between the two visits. No significant reduction was observed on the treated skin areas. This is not surprising as the investigations were carried out in Berlin, where the weather conditions had been poor at the time of the investigations, with cloudy skies almost every day and rare occasions of sunshine (176 sunshine hours, mean temperature 22.7 C during the study period). In Figure 3b, the box plot of the SAAID values of the Monegasque volunteers before sunscreen treatment (visit 1) and four weeks after treatment (visit 2) is presented for the untreated and sunscreen-treated skin areas. The weather conditions in
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a)
Monaco were noticeably better (358 sunshine hours, mean temperature 28.1 C during the study period). Strong differences between the sunscreen-treated and the untreated skin areas were observed. While the SAAID values measured on the untreated skin areas had distinctly declined after four weeks, the sunscreen-treated skin areas did not show declined SAAID values. These data were found to be statistically significant (p < 0.05). The differences in SAAID values obtained for both Berlin and Monegasque volunteers are depicted in Figure 3c for untreated and sunscreen-treated skin areas. Comparing the initial SAAID values (visit 1) of the Berlin and Monegasque volunteers, it was observed that the Monegasque volunteers exhibited significantly lower values than the Berlin ones (p < 0.05). This could be due to the fact that, compared to the Berlin volunteers, the Monegasque volunteers had been exposed to substantially higher UV doses in Monaco already prior to the study. The skin of the Monegasque volunteers was slightly stronger tanned.
3.2 Characterization of the antioxidant status of human skin
b)
c) Figure 3 SAAID box plot for Berlin (a) and Monegasque (b) volunteers before sunscreen treatment (visit 1) and four weeks after treatment (visit 2) for the untreated and sunscreen-treated skin areas, (c) shows the differences in the SAAID between visits 1 and 2 for both Berlin and Monegasque volunteers, *p < 0.05.
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In Figure 4a, the box plot of the carotenoid concentration values of the Berlin volunteers before sunscreen treatment (visit 1) and four weeks after treatment (visit 2) is presented for the untreated and sunscreen-treated skin areas. As can be seen from Figure 4a, the values of the 12 volunteers showed a broad spreading of the individual carotenoid concentrations. Earlier studies revealed that this variance is due to the different nutritional habits of the volunteers and the stressors they are exposed to [39–41]. It could be demonstrated that the sunscreen application influences the antioxidant protection system positively. While the antioxidant concentration in the untreated skin declined during the investigation period of four weeks significantly, the antioxidant concentration in the sunscreen-treated skin had remained unchanged. The measured decline was relatively small but statistically significant. The same investigations were carried out also with the Monegasque volunteers. These results are presented in Figure 4b. In this case, the individual volunteers showed differences in their dermal carotenoid concentrations. Nevertheless, these measurements confirmed the results obtained from the volunteers in Berlin, i.e., that the carotenoid concentration of the skin is protected by sunscreen application, while in the case of unprotected skin the carotenoid concentration is reduced.
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a)
Combining both Berlin and Monegasque groups, the statistically significant differences in the carotenoid concentration between visits 1 and 2 for the untreated skin area (p < 0.05) and between visits 2 of the untreated and sunscreens-treated skin areas (p < 0.05) are visible. The differences in the carotenoid concentration values obtained for both groups of volunteers are depicted in Figure 4c for untreated and sunscreen-treated skin areas. Comparing the initial carotenoid concentration values (visit 1) of the Berlin and Monegasque volunteers, it was observed that the Monegasque volunteers exhibited significantly lower values than the Berlin ones (p < 0.05). This could be explained again by the fact that the Monegasque volunteers had been exposed to substantially higher UV doses in Monaco already prior to the study.
3.3 Carotenoids and SAAID
b)
As antioxidants are expected to protect the collagen and elastin fibers against sun-induced free radicals, in a next step it was checked if there is a correlation between the individual SAAID values and the individual carotenoid concentrations of the Berlin and Monegasque volunteers, each, before the start of sunscreen application (visit 1). The results are presented in Figure 5a. This figure shows a correlation (R2 ¼ 0.41) between the carotenoid concentration and the SAAID in the skin. The similar correlation (R2 ¼ 0.37) was found for the SAAID values and carotenoid concentrations at the end of the study (visit 2). These results are represented in figure 5b. Both correlation curves were obtained using second order polynomial fitting.
4. Discussion
c) Figure 4 Box plot values of the carotenoid concentration of the Berlin (a) and Monegasque (b) volunteers before sunscreen application (visit 1) and four weeks after treatment (visit 2) for treated and untreated areas, (c) shows the differences in the carotenoid concentration between visits 1 and 2 for both Berlin and Monegasque volunteers, * p < 0.05.
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The determination of the SAAID is an objective method permitting the skin condition to be investigated for possible aging processes which are associated with the decrease of collagen concentration and posterior elastosis [42]. Contrary to the measurements of the skin surface structure, i.e., the depth and density of furrows and wrinkles, the SAAID measurements are not affected by hydration and mimic effects of the volunteers. For example, application of the moisturizing cream improves the skin surface structure visually, but does not influence the elastic and collagen fibers in the skin. The investigations of the volunteers in Berlin demonstrate that the applied sunscreen exhibits a positive effect even in bad weather. The results obtained from the study show a tendency towards a slightly decreased
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b) Figure 5 Correlation between the SAAID values and the carotenoid concentration of the volunteers (from Berlin and Monaco) before sunscreen application (visit 1) (a) and after the sunscreen application (visit 2) (b).
SAAID for untreated skin, whereas the sunscreentreated skin showed almost no changes in this respect. This effect was even more pronounced with the Monegasque volunteers who, due to the different climatic conditions, had been exposed to a far more intense solar radiation during the four weeks of the study. This is evidence that the elastin and collagen fibers in the volunteers’ protected skin had not degraded during the investigation period of four weeks. Thus, the application of sunscreens provides protection against sun-induced skin photoaging. The results obtained from the Monegasque volunteers reflect this situation much better than those obtained from the Berlin volunteers which is plausible as the investigations in Monaco were performed at sunny weather (mean temperature 28.1 C in Monaco versus 22.7 C in Berlin during the study period), whereas sunshine had been exceptionally rare in Berlin during the study (358 sunshine hours
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in Monaco versus 176 sunshine hours in Berlin during the study period). However, it has to be considered that the Monegasque volunteers had already been pre-tanned to some extent. Results presented in Figure 3 shows that SAAID is significantly influenced by the intensity of sun exposure. The Berlin volunteers whose initial daily sun exposure had been substantially lower than that of the Monegasque volunteers showed significantly higher SAAID values (mean 0.48 versus 0.67, p < 0.05), implying the presence of higher collagen concentrations in their skin. Analyzing the antioxidant status of the skin, it could be demonstrated that radical forming processes are responsible for damages of the collagen and elastin fibers. A reduction of the carotenoid antioxidant concentration was observed in the untreated skin of the volunteers from both Berlin and Monaco, whereas for the sunscreen-treated skin the antioxidant protection remained effective for the whole time of the study. Results presented in Figure 4 show that the carotenoid concentration is significantly influenced by the intensity of sun exposure. Berlin volunteers whose initial daily sun exposure was substantially lower than that of the Monegasque volunteers showed significantly higher carotenoid concentrations in the skin (mean 3.6 a.u. versus 2.2 a.u., p < 0.05). Nevertheless, the amount of degraded carotenoids was higher for the Berlin volunteers than for the Monegasque volunteers (Figure 4c) which again could be explained by the continuous sun exposure prior to the first measurement. A comparison of the SAAID and the carotenoid concentrations of the volunteers from Berlin and Monaco before and after the treatment disclosed a clear tendency to correlation (R2 ¼ 0.41) that, independent of age, subjects with high antioxidant concentrations in their skin exhibit less skin aging (determined in vivo in form of collagen degradation) than subjects with low antioxidant concentrations. The observed tendencies were not surprising as high antioxidant concentrations do neutralize free radicals more efficiently. Consequently, the destructive action of the free radicals on the elastin and collagen fibers in the human skin is reduced. The observance of a mere tendency instead of a clear correlation is due to the fact that the SAAID is influenced exclusively by the amount of oxidizers, i.e., the free radicals induced by solar radiation (UV and IR) in the skin, whereas the antioxidant concentration is determined not only by solar radiation, but also by individual factors like healthy nutrition (accumulation of antioxidants) and stressors (reduction of antioxidants), i.e., lifestyle conditions [17, 41, 43]. The obtained results are in agreement with a previously published correlation between the concentration of cutaneous lycopene and skin roughness measured by the depth and density of furrows and wrinkles [44].
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These results confirm the findings of Lademann et al. that a high antioxidant concentration in human skin could serve as the best protection strategy against premature skin aging [45, 46]. Still, these results show that the solar radiation plays a decisive role in premature skin aging as well known from the Refs. [8, 47, 48]. It should be additionally taken into consideration that during the measurement time (summer months) the concentration of cutaneous carotenoids was stable and reached a maximum [17] which did not influence the final results. To further clarify the fitting procedure and correlation coefficient, a larger number of volunteers should be investigated. Comparing the two different optical methods it was found that the SAAID is suited best for the evaluation of skin aging processes. This index is only determined by the collagen and elastin structures, whereas the antioxidant status is influenced by stressors like solar irradiation and nutritional habits. Additionally, it should be taken into consideration that the application of sunscreen can increase the skin hydration, which can potentially influence the optical properties of the skin. These investigations were not performed in this study.
5. Conclusion It was demonstrated that extended sun exposure gives rise to significant degradation of the collagen, elastin and the carotenoid concentrations, all of which are essential for the appearance of the skin. Sunscreen application provides a protection against sun exposure-induced degradation of cutaneous carotenoids and collagen/elastin fibers. The observed tendency towards correlation (R2 ¼ 0.41) between the SAAID and concentration of cutaneous carotenoids shows the important role of antioxidants for the protection against premature skin aging. Living conditions associated with an extended sun exposure give rise to a significant reduction of cutaneous carotenoid antioxidants (mean 3.6 a.u. for Berlin versus 2.2 a.u. for Monaco) and SAAID (mainly collagen) (mean 0.48 for Berlin versus 0.67 for Monaco) that was shown comparing the two groups of Berlin (low sun exposure) and Monegasque (high sun exposure) volunteers, respectively. Summarizing the results, it could be demonstrated that optical noninvasive methods lend themselves well for detecting and following up the progress of skin conditions, in this specific case of skin aging. Each of the two optical methods presented is focused on the analysis of different skin parameters. Which method is to be used in which study, depends on the specific task to be solved.
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Author biographies online.
Please see Supporting Information
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[36] M. E. Darvin, M. C. Meinke, W. Sterry, and J. Lademann, J Biomed Opt. 18, 061230-1–9 (2013). [37] M. E. Darvin, C. Sandhagen, W. Koecher, W. Sterry, J. Lademann, and M. C. Meinke, J Biophotonics 5, 550–558 (2012). [38] M. E. Darvin, N. N. Brandt, and J. Lademann, Opt Spectrosc 109, 205–210 (2010). [39] M. E. Darvin, W. Sterry, J. Lademann, and T. Vergou, Molecules 16, 10491–10506 (2011). [40] J. Lademann, M. C. Meinke, W. Sterry, and M. E. Darvin, Exp Dermatol. 20, 377–382 (2011). [41] S. T. Mayne, B. Cartmel, S. Scarmo, L. Jahns, I. V. Ermakov, and W. Gellermann, Arch Biochem Biophys. DOI: 10.1016/j.abb.2013.06.007 (2013). [42] K. Sellheyer, J Cutan Pathol. 30, 123–127 (2003). [43] M. E. Darvin, J. W. Fluhr, S. Schanzer, H. Richter, A. Patzelt, M. C. Meinke, L. Zastrow, K. Golz, O. Doucet, W. Sterry, and J. Lademann, J Dermatol Sci. 64, 53–58 (2011). [44] M. Darvin, A. Patzelt, S. Gehse, S. Schanzer, C. Benderoth, W. Sterry, and J. Lademann, Eur J Pharm Biopharm. 69, 943–947 (2008). [45] J. Lademann, A. Patzelt, S. Schanzer, H. Richter, M. C. Meinke, W. Sterry, L. Zastrow, O. Doucet, T. Vergou, and M. E. Darvin, Skin Pharmacol Physiol. 24, 269–273 (2011). [46] J. Lademann, S. Schanzer, M. Meinke, W. Sterry, and M. E. Darvin, Skin Pharmacol Physiol. 24, 238–244 (2011). [47] J. Krutmann, A. Morita, and J. H. Chung, J Invest Dermatol. 132, 976–984 (2012). [48] H. S. Black, Photochem Photobiol. 46, 213–221 (1987).
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Influence of sun exposure on the cutaneous collagen/ elastin fibers and carotenoids: negative effects can be reduced by application of sunscreen Maxim E. Darvin*; 1 , Heike Richter 1, Sebastian Ahlberg 1, Stefan F. Haag 1, Martina C. Meinke 1, Delphine Le Quintrec 2, Olivier Doucet 2 , and Ju¨rgen Lademann 1 1 2
Charite´ – Universita¨tsmedizin Berlin, Department of Dermatology, Venerology and Allergology, Center of Experimental and Applied Cutaneous Physiology (CCP), Berlin, Germany COTY LANCASTER S.A.M., R&D Center, Monaco
Received 28 October 2013, revised 4 February 2014, accepted 20 February 2014 Published online 18 March 2014
Key words: skin physiology, free radicals, antioxidants, SAAID, in vivo, sun irradiation, sun protection
Resonance Raman spectroscopy and multi-photon tomography were used in vivo to analyse the influence of sun exposure on the cutaneous carotenoids and collagen/elastin fibers. Comparing Berlin (low sun exposure) and Monegasque (high sun exposure) volunteers, it could be demonstrated that extended sun exposure significantly reduces the cutaneous carotenoids and collagen/elastin concentration (p < 0.05). The tendency towards correlation (R2 ¼ 0.41) between the dermal collagen/elastin (SAAID) and carotenoids confirms the important role of antioxidants in the protection against sun-induced negative effects. The application of sunscreen was shown to be effective, protecting cutaneous carotenoids and collagen/elastin from being damaged subsequent to sun exposure. Correlation between the SAAID values and the carotenoid concentration of the volunteers (from Berlin and Monaco) before the sunscreen application (visit 1).
1. Introduction As the biggest human organ, the skin represents the body’s barrier towards the environment and has an important physiological function. Since ancient ti-
mes, people have been endeavoring to keep their skin appearing youthful and healthy. Skin aging is determined by genetic aspects and can be accelerated by environmental factors. The most important of them is the solar UV radiation [1, 2], whose inter-
* Corresponding author: e-mail: [email protected], Phone: +49 30 450 518 208, Fax: +49 30 450 518 918
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action with the skin generates free radicals [3, 4]. If the radical concentration exceeds a critical level, these highly reactive molecules are able to destroy cells and extracellular matrix including elastin and collagen fibers. Premature skin aging (also called photoaging) [5–7], immunosuppression [8, 9] and even the development of skin diseases including skin cancer [10, 11] can be the consequences. Extensive destruction, significant alteration or diminished synthesis of the collagen and elastic fiber networks, which are the main components of dermal extracellular matrix, serve as the main hallmarks of skin aging [12–14]. For protection against the detrimental action of the free radicals, the human skin has developed a defence system that is based on the action of antioxidants. Various antioxidant substances including, inter alia, vitamins, carotenoids, and enzymes form protection chains in the skin quenching the radical activity and synergistically safeguarding each other against the destructive action of the free radicals [15, 16]. The effectiveness of the cutaneous antioxidant system can vary individually, depending on the antioxidants’ concentration and composition that is substantially influenced by lifestyle [17–19]. It was found in in vivo studies that carotenoids could serve as marker substances for the whole antioxidant status of the human epidermis [20, 21]. Cutaneous carotenoids are capable of neutralizing several attacks of free radicals and are then destroyed. It was shown in different in vivo studies that the carotenoid concentration had decreased after the irradiation of human skin with UV [22] and IR light [23] and this decrease is associated with the carotenoids’ interaction with the free radicals [24]. Thus, protection in the whole spectrum is needed to minimize the solar-induced negative effects. Modern sunscreens are capable of providing such protection efficiently. Optical noninvasive techniques are well suited to characterize different skin parameters [25]. In the study reported herein, two different optical methods were used to evaluate the influence of extended sun exposure on the concentration of cutaneous carotenoids and collagen/elastin fibers in vivo in human skin. Multi-photon tomography is well suited to analyse and quantify the collagen/elastin structure under in vivo conditions [26]. Using in vivo resonance Raman spectroscopy, the antioxidant status of the skin can be determined via the carotenoid concentration [27]. The protective capacity of one example sunscreen towards development of premature skin aging was measured. The results obtained noninvasively by the two optical methods were evaluated and compared.
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2. Materials and methods 2.1 Volunteers The study was undertaken at the Center of Experimental and Applied Cutaneous Physiology of the Charite´ – Universita¨tsmedizin Berlin. Twelve volunteers from Berlin (5 males and 7 females) and eight volunteers from Monaco (8 females) were included in the study. The volunteers were aged between 22 and 58 years. All volunteers were non-smokers of skin type II (Berlin group) and skin type III (Monegasque group) according to the Fitzpatrick classification [28]. The Berlin group was measured in June and July 2012 and the Monegasque group in July and August 2012. In addition to the Berlin volunteers, Monegasque volunteers were included in the study as the weather conditions in Berlin had been poor in the summer months of 2012. It had been raining almost every day, and the sun had been shining only occasionally.
2.2 Sunscreen samples The sunscreen used in the study was the cream LA 4032020370 provided by COTY Lancaster S.A.M, R&D Center, Monaco. It was an o/w formulation with a SPF of 30, PPD of 10 and IR protection. The cream contained UVA and UVB filters, IR reflectors and hydrophilic and lipophilic antioxidants from natural and synthetic origin. Additionally, the utilized sunscreen did not contain any anti-aging or regenerative actives (peptides). The sunscreen did not absorb and scatter light in the visible spectral range. Therefore no influence on the applied optical measurements is expected.
2.3 Study protocol The sunscreen was daily applied either to the volunteers’ right or left upper forearm areas at a concentration of approximately 2 mg/cm2 for a period of four weeks. The applications were randomized. The volunteers were instructed to desist from applying any other skin care products and sunscreens at least one week before and during the test period. The nontreated skin was used as control area (“placebo”). Volunteers were asked to stay in the direct sun with unclothed forearms minimally two hours every day. The duration of sun exposure was not controlled and therefore the applied dose was not determined. Before and after four weeks of sunscreen application the following two different skin parameters
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were noninvasively determined on the marked areas of both forearms. First, the SAAID (second harmonic generation (SHG) to autofluorescence (AF) aging index of dermis) was determined using multiphoton tomography. Second, the antioxidant status of the skin was determined by the concentration of carotenoids measured with resonance Raman spectroscopy. All measurements were performed in triplicate and the obtained mean value was used for further analyses.
2.4 Determination of the SAAID Collagen is a well-known source of tissue second harmonic generation (SHG) signal [29–31]. In the dermis, the autofluorescence (AF) is mainly determined by the elastin concentration, while the SHG is determined by the collagen concentration [32]. The collagen and elastin structure of the skin can be characterized by the SAAID [33, 34]. The SAAID is defined as (SHG-AF)/(SHG+AF). In this nomencla-
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ture, the SAAID decreases with photoaging, approaching 1 when the collagen has been completely replaced by elastic fibers. For the SAAID analysis, the average SAAID value obtained for the transition zone between the “stratum basale” and the “papillary dermis” (depth between 40 mm and 80 mm) was used. The investigations were carried out using a multiphoton tomograph (JenLab GmbH, Jena, Germany) equipped with a femtosecond titanium sapphire laser (Mai Tai XF, Spectra Physics, USA). The laser was operated at 760 nm and generated 100 fs pulses at a repetition rate of 80 MHz. For studying the structure of the dermis at different depths of the skin a sensitive photo multiplier was applied. The radiation collected from different depths was averaged over slices of 200 200 mm2. Applied power in the skin increased linearly from 10 mW in the stratum corneum to maximal value of 50 mW in the dermis (depth 70 mm) and remains maximal up to a measurement depth of 120 mm. The acquisition time was equal to 7 seconds for one slice which guaranteed a satisfactory measurement stability and picture quality. The
a)
Figure 1 Exemplary images of AF and SHG obtained by multi-photon tomography used for determining the elastin and collagen amounts to calculate the SAAID values (a) and the SAAID curves obtained before sun exposure (visit 1) and after four weeks of daily sun exposure (visit 2) for the sunscreen treated skin area (black) and untreated skin area (gray). Scale bar is 30 mm. b)
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effect of utilized parameters of irradiation on human skin was compared to daily sun exposure-induced effects [35]. During the measurements, the laser focus was moved from the skin surface into deeper parts of the skin at 10 mm increments. The maximum measuring depth into the skin was 120 mm. Exemplarily, images of the elastin (AF) and collagen (SHG) distributions in the skin used for determining the SAAID values are presented in Figure 1a. The typical SAAID curves obtained before sun exposure (visit 1) and after four weeks of daily sun exposure (visit 2) are presented in Figure 1b for the untreated and sunscreen treated skin areas of one volunteer.
2.5 Determination of the antioxidant status of human skin by the carotenoid marker substances It was demonstrated in previous in vivo studies that the carotenoids can be used as marker substances to characterize the whole antioxidant status of the human epidermis [20, 21]. In the present study, the investigations were performed in vivo on both forearms of volunteers using resonance Raman spectroscopy. This method was chosen because of the measurement quickness and high sensitivity in comparison to other available methods [36, 37]. The Raman spectrometer consisted of a base accommodating the argon laser operating at 488 nm, and the detection system. The laser radiation was led via an optical fiber to an imaging system. The imaging system produced a laser spot of 6.5 mm in diameter on
Figure 2 Typical Raman spectrum obtained from the human skin measured in vivo under an excitation at 488 nm (taken from [38]). Inlay shows the determination of the intensity of the Raman peak.
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the skin surface. The Raman measurement was performed during 5 seconds under the power density of 24 mW/cm2, which is lower than the power density of the sun. The Raman signal on the skin measured at 1525 cm1 was collected by the same imaging system and focused into another optical fiber which was connected to the detector system. The typical Raman spectrum obtained from the skin under an excitation of 488 nm is shown on Figure 2. The concentration of carotenoids was determined by the intensity of Raman peak, as shown in inlay of Figure 2. A detailed description of the utilized Raman spectrometric system was previously given by our group [27].
2.6 Statistical analysis For statistical analysis, SPSS 19.00 for Windows (SPSS Inc., Chicago, IL, USA) software was used. Data were analyzed using non-parametric tests. Firstly, the related samples, whereby Friedman’s two-way analysis of variances by ranks was performed. Differences among the means of groups were analyzed by the Wilcoxon test, considering significance at p < 0.05. Independent samples were tested using the Mann-Whitney U test.
3. Results 3.1 Analysis of the SAAID In Figure 3a, a box plot of the SAAID values of the Berlin volunteers before sunscreen treatment (visit 1) and four weeks after treatment (visit 2) is presented for the untreated and sunscreen-treated skin areas for the 12 volunteers. It can be seen that the SAAID values are quite different for the individual volunteers. The non-strongly pronounced but significant reduction (p < 0.05) was found for the untreated skin area between the two visits. No significant reduction was observed on the treated skin areas. This is not surprising as the investigations were carried out in Berlin, where the weather conditions had been poor at the time of the investigations, with cloudy skies almost every day and rare occasions of sunshine (176 sunshine hours, mean temperature 22.7 C during the study period). In Figure 3b, the box plot of the SAAID values of the Monegasque volunteers before sunscreen treatment (visit 1) and four weeks after treatment (visit 2) is presented for the untreated and sunscreen-treated skin areas. The weather conditions in
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a)
Monaco were noticeably better (358 sunshine hours, mean temperature 28.1 C during the study period). Strong differences between the sunscreen-treated and the untreated skin areas were observed. While the SAAID values measured on the untreated skin areas had distinctly declined after four weeks, the sunscreen-treated skin areas did not show declined SAAID values. These data were found to be statistically significant (p < 0.05). The differences in SAAID values obtained for both Berlin and Monegasque volunteers are depicted in Figure 3c for untreated and sunscreen-treated skin areas. Comparing the initial SAAID values (visit 1) of the Berlin and Monegasque volunteers, it was observed that the Monegasque volunteers exhibited significantly lower values than the Berlin ones (p < 0.05). This could be due to the fact that, compared to the Berlin volunteers, the Monegasque volunteers had been exposed to substantially higher UV doses in Monaco already prior to the study. The skin of the Monegasque volunteers was slightly stronger tanned.
3.2 Characterization of the antioxidant status of human skin
b)
c) Figure 3 SAAID box plot for Berlin (a) and Monegasque (b) volunteers before sunscreen treatment (visit 1) and four weeks after treatment (visit 2) for the untreated and sunscreen-treated skin areas, (c) shows the differences in the SAAID between visits 1 and 2 for both Berlin and Monegasque volunteers, *p < 0.05.
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In Figure 4a, the box plot of the carotenoid concentration values of the Berlin volunteers before sunscreen treatment (visit 1) and four weeks after treatment (visit 2) is presented for the untreated and sunscreen-treated skin areas. As can be seen from Figure 4a, the values of the 12 volunteers showed a broad spreading of the individual carotenoid concentrations. Earlier studies revealed that this variance is due to the different nutritional habits of the volunteers and the stressors they are exposed to [39–41]. It could be demonstrated that the sunscreen application influences the antioxidant protection system positively. While the antioxidant concentration in the untreated skin declined during the investigation period of four weeks significantly, the antioxidant concentration in the sunscreen-treated skin had remained unchanged. The measured decline was relatively small but statistically significant. The same investigations were carried out also with the Monegasque volunteers. These results are presented in Figure 4b. In this case, the individual volunteers showed differences in their dermal carotenoid concentrations. Nevertheless, these measurements confirmed the results obtained from the volunteers in Berlin, i.e., that the carotenoid concentration of the skin is protected by sunscreen application, while in the case of unprotected skin the carotenoid concentration is reduced.
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a)
Combining both Berlin and Monegasque groups, the statistically significant differences in the carotenoid concentration between visits 1 and 2 for the untreated skin area (p < 0.05) and between visits 2 of the untreated and sunscreens-treated skin areas (p < 0.05) are visible. The differences in the carotenoid concentration values obtained for both groups of volunteers are depicted in Figure 4c for untreated and sunscreen-treated skin areas. Comparing the initial carotenoid concentration values (visit 1) of the Berlin and Monegasque volunteers, it was observed that the Monegasque volunteers exhibited significantly lower values than the Berlin ones (p < 0.05). This could be explained again by the fact that the Monegasque volunteers had been exposed to substantially higher UV doses in Monaco already prior to the study.
3.3 Carotenoids and SAAID
b)
As antioxidants are expected to protect the collagen and elastin fibers against sun-induced free radicals, in a next step it was checked if there is a correlation between the individual SAAID values and the individual carotenoid concentrations of the Berlin and Monegasque volunteers, each, before the start of sunscreen application (visit 1). The results are presented in Figure 5a. This figure shows a correlation (R2 ¼ 0.41) between the carotenoid concentration and the SAAID in the skin. The similar correlation (R2 ¼ 0.37) was found for the SAAID values and carotenoid concentrations at the end of the study (visit 2). These results are represented in figure 5b. Both correlation curves were obtained using second order polynomial fitting.
4. Discussion
c) Figure 4 Box plot values of the carotenoid concentration of the Berlin (a) and Monegasque (b) volunteers before sunscreen application (visit 1) and four weeks after treatment (visit 2) for treated and untreated areas, (c) shows the differences in the carotenoid concentration between visits 1 and 2 for both Berlin and Monegasque volunteers, * p < 0.05.
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The determination of the SAAID is an objective method permitting the skin condition to be investigated for possible aging processes which are associated with the decrease of collagen concentration and posterior elastosis [42]. Contrary to the measurements of the skin surface structure, i.e., the depth and density of furrows and wrinkles, the SAAID measurements are not affected by hydration and mimic effects of the volunteers. For example, application of the moisturizing cream improves the skin surface structure visually, but does not influence the elastic and collagen fibers in the skin. The investigations of the volunteers in Berlin demonstrate that the applied sunscreen exhibits a positive effect even in bad weather. The results obtained from the study show a tendency towards a slightly decreased
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b) Figure 5 Correlation between the SAAID values and the carotenoid concentration of the volunteers (from Berlin and Monaco) before sunscreen application (visit 1) (a) and after the sunscreen application (visit 2) (b).
SAAID for untreated skin, whereas the sunscreentreated skin showed almost no changes in this respect. This effect was even more pronounced with the Monegasque volunteers who, due to the different climatic conditions, had been exposed to a far more intense solar radiation during the four weeks of the study. This is evidence that the elastin and collagen fibers in the volunteers’ protected skin had not degraded during the investigation period of four weeks. Thus, the application of sunscreens provides protection against sun-induced skin photoaging. The results obtained from the Monegasque volunteers reflect this situation much better than those obtained from the Berlin volunteers which is plausible as the investigations in Monaco were performed at sunny weather (mean temperature 28.1 C in Monaco versus 22.7 C in Berlin during the study period), whereas sunshine had been exceptionally rare in Berlin during the study (358 sunshine hours
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in Monaco versus 176 sunshine hours in Berlin during the study period). However, it has to be considered that the Monegasque volunteers had already been pre-tanned to some extent. Results presented in Figure 3 shows that SAAID is significantly influenced by the intensity of sun exposure. The Berlin volunteers whose initial daily sun exposure had been substantially lower than that of the Monegasque volunteers showed significantly higher SAAID values (mean 0.48 versus 0.67, p < 0.05), implying the presence of higher collagen concentrations in their skin. Analyzing the antioxidant status of the skin, it could be demonstrated that radical forming processes are responsible for damages of the collagen and elastin fibers. A reduction of the carotenoid antioxidant concentration was observed in the untreated skin of the volunteers from both Berlin and Monaco, whereas for the sunscreen-treated skin the antioxidant protection remained effective for the whole time of the study. Results presented in Figure 4 show that the carotenoid concentration is significantly influenced by the intensity of sun exposure. Berlin volunteers whose initial daily sun exposure was substantially lower than that of the Monegasque volunteers showed significantly higher carotenoid concentrations in the skin (mean 3.6 a.u. versus 2.2 a.u., p < 0.05). Nevertheless, the amount of degraded carotenoids was higher for the Berlin volunteers than for the Monegasque volunteers (Figure 4c) which again could be explained by the continuous sun exposure prior to the first measurement. A comparison of the SAAID and the carotenoid concentrations of the volunteers from Berlin and Monaco before and after the treatment disclosed a clear tendency to correlation (R2 ¼ 0.41) that, independent of age, subjects with high antioxidant concentrations in their skin exhibit less skin aging (determined in vivo in form of collagen degradation) than subjects with low antioxidant concentrations. The observed tendencies were not surprising as high antioxidant concentrations do neutralize free radicals more efficiently. Consequently, the destructive action of the free radicals on the elastin and collagen fibers in the human skin is reduced. The observance of a mere tendency instead of a clear correlation is due to the fact that the SAAID is influenced exclusively by the amount of oxidizers, i.e., the free radicals induced by solar radiation (UV and IR) in the skin, whereas the antioxidant concentration is determined not only by solar radiation, but also by individual factors like healthy nutrition (accumulation of antioxidants) and stressors (reduction of antioxidants), i.e., lifestyle conditions [17, 41, 43]. The obtained results are in agreement with a previously published correlation between the concentration of cutaneous lycopene and skin roughness measured by the depth and density of furrows and wrinkles [44].
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These results confirm the findings of Lademann et al. that a high antioxidant concentration in human skin could serve as the best protection strategy against premature skin aging [45, 46]. Still, these results show that the solar radiation plays a decisive role in premature skin aging as well known from the Refs. [8, 47, 48]. It should be additionally taken into consideration that during the measurement time (summer months) the concentration of cutaneous carotenoids was stable and reached a maximum [17] which did not influence the final results. To further clarify the fitting procedure and correlation coefficient, a larger number of volunteers should be investigated. Comparing the two different optical methods it was found that the SAAID is suited best for the evaluation of skin aging processes. This index is only determined by the collagen and elastin structures, whereas the antioxidant status is influenced by stressors like solar irradiation and nutritional habits. Additionally, it should be taken into consideration that the application of sunscreen can increase the skin hydration, which can potentially influence the optical properties of the skin. These investigations were not performed in this study.
5. Conclusion It was demonstrated that extended sun exposure gives rise to significant degradation of the collagen, elastin and the carotenoid concentrations, all of which are essential for the appearance of the skin. Sunscreen application provides a protection against sun exposure-induced degradation of cutaneous carotenoids and collagen/elastin fibers. The observed tendency towards correlation (R2 ¼ 0.41) between the SAAID and concentration of cutaneous carotenoids shows the important role of antioxidants for the protection against premature skin aging. Living conditions associated with an extended sun exposure give rise to a significant reduction of cutaneous carotenoid antioxidants (mean 3.6 a.u. for Berlin versus 2.2 a.u. for Monaco) and SAAID (mainly collagen) (mean 0.48 for Berlin versus 0.67 for Monaco) that was shown comparing the two groups of Berlin (low sun exposure) and Monegasque (high sun exposure) volunteers, respectively. Summarizing the results, it could be demonstrated that optical noninvasive methods lend themselves well for detecting and following up the progress of skin conditions, in this specific case of skin aging. Each of the two optical methods presented is focused on the analysis of different skin parameters. Which method is to be used in which study, depends on the specific task to be solved.
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Author biographies online.
Please see Supporting Information
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