2.1.

Volunteers

The measurements of the carotenoid antioxidants in the skin were carried out over the course of $12\text{months}$ on ten healthy volunteers (2 male, 8 female) aged between 23 and $50\text{years}$ at the Center of Experimental and Applied Cutaneous Physiology (CCP) of the Department of Dermatology and Allergology, Charité—Universitätsmedizin Berlin. The measurements were performed daily at the same time, except for on weekends and holidays, and at the same position on the palm of the left hand. Before each measurement, volunteers were interviewed about their diets and the influence of possible stress factors, such as illness, smoking, alcohol consumption, fatigue, etc., using questionnaires. All volunteers had skin type 2.²³

The volunteers were asked not to use dermatological or cosmetic products containing antioxidants during the course of the experiment.

Approval of the experiments had been obtained from the Ethics Committee of the Charité—Universitätsmedizin Berlin. The study was conducted according to the ethical rules stated in the Declaration of Helsinki Principles.

2.2.

Raman Spectroscopic Measurements

Resonance Raman spectroscopy was used for the fast in vivo determination of the concentration of carotenoid antioxidant substances, such as beta-carotene and lycopene, in human skin.

The radiation of an ${\mathrm{Ar}}^{+}$ laser, which operated at $488\mathrm{nm}$ , was filtered and focused onto the skin. The weak Raman scattered signal from the skin was filtered, collected by a lens system of an optical imaging system, and transferred into a fiber bundle to a spectrograph. The skin spectrum was recorded by a charge-coupled device (CCD) camera and visualized on a PC. The small Raman peak, which corresponded to the concentration of carotenoids in the skin, could easily be detected on a large fluorescence background. The software subtracted the fluorescence background and determined the intensity of the Raman peak, which corresponded directly to the concentration of carotenoids present in the skin. The obtained concentrations were measured in arbitrary units (a.u.) for all volunteers and were thus comparable between the subjects. The measured Raman peaks were usually more than ten-fold higher than the quantitative limit of the measurements, which was determined as a noise signal (averaged around $0.003\mathrm{a.u.}$ ). One measurement, needed for determination of the concentration of carotenoids in the skin, lasted $5\mathrm{s}$ . This measurement was performed three times on the palm of the left hand to determine the scattering of the measured values of the cutaneous concentration of carotenoids. The average value was subsequently determined and analyzed. Thus the total time of the measurements was $15\mathrm{s}$ for one volunteer for each day.

The superficial penetration depth $(\sim 200\mu \mathrm{m})$ of the blue laser light at $488\mathrm{nm}$ into the skin confined the Raman measurements to the epidermis.

The experimental arrangement for the measurements of carotenoid antioxidant substances have been described in detail by Darvin ¹⁷

2.3.

Statistical Analysis

The average value and the standard deviation of the carotenoid concentrations measured daily were determined for ten volunteers. The confidence interval including approximately 68% of the measured values was evaluated as an average value ± standard deviation.

The Wilcoxon test (SPSS 13.0) was utilized for the statistical analysis of the seasonal differences regarding the content of carotenoids in the skin of the ten volunteers.

3.1.

Seasonal Influence on the Monthly Concentration of the Carotenoids in the Skin

The average monthly values of the ten volunteers, which were obtained from the daily values measured over the period of $12\text{months}$ , are shown in Figs. 1a and 1b . The results show that in the summer and autumn months, the average values of cutaneous carotenoids were higher than during the spring and winter months.

Fig. 1

Average monthly values of the carotenoid antioxidant substances in the skin of ten volunteers during a $12\text{-}\text{month}$ period. (a) Volunteers 1 to 5 and (b): volunteers 6 to 10.

This “seasonal increase” of the cutaneous carotenoids, which characterizes an increase of the carotenoid concentration in the skin during the summer and autumn months compared with the winter and spring months, is summarized in Table 1 for each of the ten volunteers. The average “seasonal increase” of the carotenoid content in the skin for all volunteers was determined to be $(1.26\pm 0.21)$ -fold. This increase was found to be statistically significant $(\mathrm{p}=0.001)$ .

Table 1

The seasonal increase, confidence interval, and the quantity of strong variations in the content of carotenoids in the skin of ten volunteers (2 male, 8 female) are shown. Areas A and B correspond to the winter-spring and summer-autumn months, respectively.

The seasonal variation of the carotenoid antioxidant level in the skin can be explained by a higher consumption of fruit and vegetables, which are naturally rich in carotenoids, in the daily ration of the volunteers during the summer and autumn months, as well as by a decreased occurrence of diseases.

Different volunteers had different individual levels of carotenoid concentrations in their skin. The average values of the individual carotenoid levels in the skin are presented in Fig. 2 as an average value ± standard deviation.

Fig. 2

Average annual values of the carotenoid concentrations in the skin of all volunteers.

The analysis of the questionnaires showed that, for example, volunteer 1 usually ate a very large amount of fruit and vegetables each day. Volunteers 2 and 3 ate small amounts of fruit and vegetables, while volunteer 3 was also a strong smoker. Their low cutaneous carotenoid levels can be explained by the consumption of only small amounts of carotenoid-rich food. The almost three-fold difference between the carotenoid levels of volunteers 1 and 3 strongly correlated with their lifestyle, in particular with their nutrition. There were almost no observed changes in the cutaneous concentration of the carotenoids during the one year for volunteer 3. The relatively constant level and very small “seasonal increase” $(1.12\pm 0.04)$ of the carotenoid concentration for this volunteer suggests that the “seasonal increase” was mainly caused by nutrition, which included increased uptake of carotenoid-rich foodstuffs. The influence of external factors such as increased sun radiation, smoking, etc. gave rise to the temporary decrease in the level of carotenoids in the skin, which subsequently leveled.²²

Volunteers 4 to 10 had moderate levels of carotenoids in the skin. Their diets remained relatively constant regarding the amount of fruit and vegetables in the daily ration, as did the amounts of influencing stress factors.

The volunteers, whose values are presented in Figs. 1 and 2, neither altered their diet nor their stress status significantly during the $12\text{-}\text{month}$ investigation period with the exception of volunteer 5. Their average level of carotenoids in the skin remained almost constant, excluding the presence of the “seasonal increase.” Thus the recovery and degradation processes of the cutaneous carotenoids balanced each other under physiological conditions.

Volunteer 5 had a low initial concentration of carotenoids in the skin. Taking these low values into consideration, this volunteer started to increase his daily consumption of fruit and vegetables consistently for three months after the beginning of the measurements. The volunteer significantly minimized smoking and tried to exclude the influence of possible stress factors. The results presented in Fig. 1a show the average monthly concentration of carotenoids in the skin for this volunteer. As can be seen, the level of carotenoids increased approximately two-fold compared with the initial values as a result of the changes in lifestyle.

From the average monthly values presented in Fig. 1 and the average annual level presented in Fig. 2, it becomes clear that the individual carotenoid antioxidant level of the volunteers reflect their lifestyle. This became much more obvious when the daily measured values of the volunteers were analyzed over the course of $12\text{months}$ .

3.2.

Relation between the Daily Carotenoid Concentration in the Skin and the Influence of Stress Factors

As a typical example, Figs. 3a and 3b show the average daily values of the carotenoid concentration in the skin measured during a $12\text{-}\text{month}$ period for volunteer 6.

Fig. 3

Average daily values of the carotenoid concentrations in the skin during the course of one year (volunteer 6). (a) The average value ± standard deviation (confidence interval) is shown. (b) Areas lying outside the confidence interval are shown.

Two different areas marked “A” and “B” in Fig. 3a corresponded to the winter-spring months (area “A”) and to the summer-autumn months (area “B”), where the minimum and maximum of measured values were obtained (“seasonal increase”). Each cross-hatched region in Fig. 3a corresponds to the confidence interval, which is equal to the average value ± standard deviation interval. The presented confidence interval contains approximately 68% of the measured values.

Questionnaires were used to find possible explanations for the appearance of untypically strong increased or decreased carotenoid values lying outside the confidence interval [see Fig. 3a]. These periods, when the daily values were significantly higher or lower than the average concentrations, were marked by numbered areas from “I” to “IV” in Fig. 3b. During the evaluation of the questionnaires, it was found that these distinctive values correlated to strong stress factors, such as illness, fatigue, smoking, and alcohol consumption, to which the volunteers were exposed (when the values were less than average), or to the uptake of a high amount of food rich in carotenoids, as well as a reduced influence of stress factors (when the values were higher than average).

Table 1 shows the quantities of the strong increases/decreases in the level of cutaneous carotenoids for each of the ten volunteers, which were measured daily during the $12\text{-}\text{month}$ period. These specific events were directly correlated with the lifestyle changes of the volunteers, which had been noted in the questionnaire.

The periods of strong changes in the carotenoid concentration in the skin, marked by the numbered areas from “I” to “IV” in Fig. 3b, are presented with a higher magnification in Fig. 4 .

Fig. 4

Magnification of the numbered areas I to IV from Fig. 3b with strong variations in the carotenoid concentrations in the skin (volunteer 6).

Additionally, in Table 2 , extracts from the corresponding questionnaires are shown. The synopsis of the data reveals a distinct correlation between the state of health, a diet rich in fruit and vegetables, and the level of carotenoid antioxidants in the skin of volunteers. The current values of the concentration of carotenoids in the skin were compared with the previous values and then labeled with appropriate symbols (↔, ↑ or ↓), which are shown in the last column of Table 2. If there were no changes in the carotenoid concentration within the typical spread in values, this is marked with the symbol (↔). An increase is shown with the symbol (↑), while a decrease is shown with (↓). The paired symbols in the last column of Table 2 correspond to the group of events and indicate the slight increase [see for example I (1 to 4), I (8 to 10), and I (30 to 32)] or slight decrease [I (21 to 27), II (3), II (16 to 20), III (24 to 30), and III (4)] of the concentration of carotenoids in the skin. In all cases of illness and sickly feeling [see I (21 to 29), II (4 to 6), III (4 to 14), IV (10, 11) in Fig. 4 and in Table 2], the level of carotenoids in the skin strongly decreased compared with the initial level.

Table 2

Extracts from the completed questionnaire of volunteer 6, corresponding to the numbered areas I to IV shown in Figs. 3b and 4 (↔ is no changes, ↑ is an increase, and ↓ means decrease of the carotenoid concentration in the skin), are shown.

Additionally, other stress factors such as large amounts of alcohol and smoking reduced the level of carotenoids in the skin [see IV (3) in Fig. 4 and in Table 2].

The same effect was observed for stress in the workplace, such as long phases of physical and mental exertion without regular breaks [see II (2, 3) in Fig. 4 and in Table 2].

It was found that the level of carotenoid antioxidants in the skin of volunteers who had some periods of reduced stress conditions increased, even if their nutrition remained unchanged.

Stress is commonly accompanied by the production of high amounts of free radicals, which are immediately neutralized by the antioxidative system of the human body. This interaction gives rise to the neutralization of free radicals and, as a result, to the degradation of carotenoid antioxidants of the body.

The uptake of high amounts of food rich in carotenoids, such as fruit and vegetables, increased the level of carotenoids in the skin [see I (19, 20), II (13, 15), and III (22, 23) in Fig. 4 and Table 2]. The kinetics of these findings are supported by results obtained previously, where the influence of carotenoid-rich nutrition on the cutaneous level of carotenoids was shown.^{24, 25} In Fig. 5 , as an additional example, a long-term kinetic of the carotenoid concentration in the skin is shown for volunteer 7. Again, the values lying outside the confidence interval (marked areas in Fig. 5) corresponded to significant events, such as stress or antioxidant-rich supplementation, which were also interpreted in detail but not shown in this work. Similar tendencies were observed for all volunteers.

Fig. 5

Average daily values of the carotenoid concentrations in the skin of volunteer 7 over the course of $12\text{months}$ . Confidence interval and areas with strong variations in the carotenoid concentrations in the skin are shown.

Analysis of the questionnaire showed correlations between the level of carotenoids in the skin and the influence of different stress factors, which were detected inside the confidence interval (see, for example, results presented for volunteer 6 in Figs. 3 and 4 and Table 2).

Significant decreases in the carotenoid antioxidant levels of the skin, which lay outside of the confidence interval, were found more often in the winter-spring months than in the summer-autumn months $(\mathrm{p}<0.05)$ for all volunteers. Additionally, significantly stronger increases were observed during the summer-autumn months $(\mathrm{p}<0.05)$ [see, for example, Figs. 3a and 5]. Figure 6 shows the difference between the quantitative amounts of increases and decreases for both areas A and B, which was averaged for ten volunteers. As can be seen, the obtained difference was positive for area B and negative for area A. These results demonstrate that the incidences of illnesses, which gave rise to the fall of carotenoids in the skin, occurred more often during the winter-spring months compared with summer-autumn months.

Fig. 6

Difference between the quantitative amounts of increases and decreases in the level of cutaneous carotenoids measured during winter-spring months (area A) and during summer-autumn months (area B), averaged for ten volunteers.

3.3.

Kinetics of Carotenoids in the Human Skin

The increase and decrease of concentrations of carotenoids that were caused by different events showed different kinetics. The carotenoid decrease was detected immediately on the day after the volunteers were exposed to stress, while the increase needed up to three days to peak (see Fig. 4 and Table 2). The increase in the cutaneous carotenoid concentration due to an antioxidant-rich supplementation or the decrease as a result of stress occurred much more quickly than the leveling of the carotenoid concentration back to the initial level subsequent to increasing or decreasing events.

A decrease in carotenoids was caused by the influence of free radicals and ROS, which were produced in the skin as a result of the direct influence of stress factors, and could give rise to oxidative stress.^{10, 14, 26} In this case, carotenoids in the skin decreased rapidly. This effect was also observed by in vivo analysis of the degradation kinetics of beta-carotene and lycopene during UV and IR irradiations of the skin.^{22, 27}

The increase in the level of carotenoids occurs relatively fast $\left(1\text{to}3\text{days}\right)$ in comparison to the slow process of stratum corneum renewal $\left(2\text{to}3\text{weeks}\right)$ . Increases in the carotenoid levels seem to be caused by numerous processes, which act simultaneously and independently of each other. The first one is the diffusion of the carotenoids from the blood and the hypodermis and the dermis to the epidermis of the skin. This process dominated in our opinion. The second one is the transport of carotenoids from the blood and the hypodermis and the dermis into the sweat glands, and then with the sweat onto the skin surface. This hypothesis is supported by the results that the highest concentration of carotenoids in the skin was detected on body sites with a high density of sweat glands, i.e., on the forehead and the palm,¹⁷ and by our measurements of concentration of carotenoid substances in human sweat (unpublished data).

This assumption is also supported by the measurements of lipophilic antioxidant vitamin E in the sebum and its delivery onto the skin surface by the exuded sebum.²⁸ A similar process can be assumed for lipophilic carotenoids, which are bound to the lipid structures that are present in sweat.