Maxim E. Darvin

Non-invasive in vivo determination of the carotenoids beta-carotene and lycopene concentrations in the human skin using the Raman spectroscopic method

Resonance Raman spectroscopy was used as a fast and non-invasive optical method of measuring the absolute concentrations of beta-carotene and lycopene in living human skin.Beta-carotene and lycopene have different absorption values at 488 and 514.5?nm and, consequently, the Raman lines for beta-carotene and lycopene have different scattering efficiencies at 488 and 514.5?nm excitations. These differences were used for the determination of the concentrations of beta-carotene and lycopene. Using multiline Ar+ laser excitation, clearly distinguishable carotenoid Raman spectra can be obtained which are superimposed on a large fluorescence background. The Raman signals are characterized by two prominent Stokes lines at 1160 and 1525?cm?1, which have nearly identical relative intensities. Both substances were detected simultaneously.The Raman spectra are obtained rapidly, i.e. within about 10?s, and the required laser light exposure level is well within safety standards. The disturbance of the measurements by non-homogeneous skin pigmentation was avoided by using a relatively large measuring area of 35?mm2.It was shown that beta-carotene and lycopene distribution in human skin strongly depends upon the skin region studied and drastically changed inter-individually. Skin beta-carotene and lycopene concentrations are lower in smokers than in non-smokers and higher in the vegetarian group.

Cutaneous concentration of lycopene correlates significantly with the roughness of the skin

Antioxidant substances in the skin are expected to slow down photo ageing. We therefore developed the hypothesis that high levels of antioxidant substances may be correlated to lower levels of skin roughness. By utilizing modern optical non-invasive in vivo methods, the structures of the furrows and wrinkles as well as the concentration of lycopene were analyzed quantitatively on the forehead skin of 20 volunteers aged between 40 and 50 years. In a first step, the age of the volunteers was correlated to their skin roughness. Here, no significant correlation was found. In a second step, a significant correlation was obtained between the skin roughness and the lycopene concentration (R=0.843). These findings indicate that higher levels of antioxidants in the skin effectively lead to lower levels of skin roughness, and therefore support our hypothesis.

Effect of Supplemented and Topically Applied Antioxidant Substances on Human Tissue

Systemic and topical application of antioxidant substances for the medical treatment and prophylaxis of many diseases as well as additional protection of the skin against the destructive action of free radicals and other reactive species has become very popular during the past years. Stimulated by the positive results of a fruit and vegetable diet in supporting medical treatment and in cosmetics, artificial and extracted antioxidant substances have been broadly applied. Surprisingly, not only positive but also strong negative results have been obtained by different authors. According to study reports artificial and extracted antioxidant substances support different kinds of medical therapies, if they are applied in mixtures of different compounds at low concentration levels. In the case of the application of high concentration of some single compounds, side effects were often observed. Regarding skin treatment by systemically applied antioxidant substances for cosmetic purposes, positive cosmetic effects as well as no effects, but almost no side effects, apart from a number of allergic reactions, were reported. One reason for this seems to be the lower concentration of systemically applied antioxidant substances in comparison with a medical application. Topical application of antioxidant substances is closely related to cosmetic treatment for skin protection and anti-aging. Positive results were also obtained in this case. The present review is an attempt to classify and summarize the published literature concerning the efficiency of action of systemic and topical applications of antioxidant substances, such as carotenoids and vitamins, on human organism and especially on the skin. The available literature on this topic is very extensive and the results are often contradictory. Nevertheless, there are some clear tendencies concerning systemic and topical application of antioxidant substances in medicine and cosmetics, and we summarize them in the present paper.

Bioavailability of natural carotenoids in human skin compared to blood.

Skin functions and structure are significantly influenced by nutrients. Antioxidants protect the supportive layer of the skin against any damaging irradiation effects and the action of free radicals. A lack of suitable methods means that the pharmacokinetic properties of systemically applied carotenoids transferred into the skin remain poorly understood. In this study, a natural kale extract or placebo oil were given orally to 22 healthy volunteers for 4 weeks. Carotenoid bioaccessibility was evaluated using non-invasive resonance Raman spectroscopy on the palm and forehead skin. For the analysis of the blood serum, the standard HPLC method was used. The blood and skin levels of the carotenoids increased significantly during the study but compared to the blood serum values, increases in skin were delayed and depended on the dermal area as well as on the carotenoid. Lycopene, measured as being low in the extract, increases more in the skin compared to the blood indicating that the natural mixture of the extract stabilizes the antioxidative network in the skin. After supplementation had ended, the carotenoids decreased much faster in the blood than in the skin. The delayed decrease in the skin may indicate a peripheral buffer function of the skin for carotenoids.

Formation of Free Radicals in Human Skin during Irradiation with Infrared Light

TO THE EDITOR Constant exposure to solar UV irradiation and environmental hazards produces free radicals in the skin (Darr and Fridovich, 1994; Zastrow et al., 2004). The human organism has developed a protection system against the destructive action of free radicals, consisting mostly of vitamins, carotenoids, and enzymes (Stahl and Sies, 2003; Sander et al., 2004; Darvin et al., 2006). Approximately 70% of carotenoids in human skin are b-carotene and lycopene (Hata et al., 2000), which can serve as markers for the whole antioxidative potential (Darvin et al., 2008). Recently, Darvin et al. (2007) presented a laser spectroscopic method for the noninvasive determination of carotenoids in human skin, based on resonance Raman spectroscopy (RRS). The authors suggested that IR irradiation can give rise to the production of free radicals in the skin, which they measured by the degradation of the cutaneous carotenoids b-carotene and lycopene. Schroeder et al. (2008) showed in an in vivo study that IRA (760–1,440 nm) irradiation of the skin can elicit a retrograde mitochondrial signaling response that leads to the expression of matrix metalloproteinase-1. Given that expression of matrix metalloproteinase-1 was initiated through the generation of reactive oxygen species, which originated from the mitochondrial electron transport chain, the production of free radicals in the skin subsequent to IR irradiation could be expected. This supposition can be confirmed via direct detection of free radical formation using electron paramagnetic resonance (EPR) spectroscopy (Herrling et al., 2003). Until recently—in contrast to RRS measurements—EPR analysis could be performed only in vitro. In the present study, we investigated whether IR irradiation, which is frequently used in treatment and wellness applications, could also reduce the antioxidative potential of the skin and whether this process is caused by the production of free radicals. The RRS used in this study is described elsewhere (Darvin et al., 2005). The box plot diagram in Figure 1 shows the changes in concentrations of b-carotene and lycopene measured on the flexor forearm of 12 healthy volunteers (three men and nine women, aged 25–35 years, with skin type II) before and after IR irradiation (Philips, Hamburg, Germany, Infrared RI 1521, 170 mW cm 2 for 30 minutes). The volunteers had given informed consent, and permission for the investigations had been obtained from the Ethics Committee of the Charité. The study adhered to the Declaration of Helsinki Principles. Even the initial values show a broad distribution; after irradiation of the skin by IR light, a reduction in b-carotene and lycopene concentrations was observed for all volunteers. The mean values for the absolute data for b-carotene and lycopene before and after irradiation are significantly different, Po0.005, paired t-test (SPSS, Inc., Chicago, Illinois, 16.0 for Windows). The average for the magnitude of destruction (changes relative to the initial values) in all volunteers was determined to be 30±12% for b-carotene and 37±10% for lycopene. These results are in agreement with previous investigations (Darvin et al., 2007). An increase in skin temperature was observed during irradiation, from 31.6±1.0 1C to 41.3±0.6 1C. For the direct measurement of IRinduced free radicals, EPR spectroscopy was carried out using an L-band EPR spectrometer (Magnettech, Berlin, Germany) and spin marker 3-carboxy2,2,5,5-tetramethylpyrrolidine-1-oxyl (PCA) (Sigma, Steinheim, Germany). The radical-formation process was investigated on six pig ear skin samples. The PCA marker was applied after tape stripping as described by Meinke et al. (2008). Skin samples were irradiated for 30 minutes with a power density of 115 mW cm . During this time, the skin temperature increased from room temperature 23 1C to 41.5±0.5 1C. The EPR signal of PCA was recorded every 10 minutes during IR irradiation, when the tissue samples were cooled down to room temperature, taking into consideration the strong dependence between the intensity of EPR signals and the temperature of the investigative samples (Rockenbauer et al., 2006). In Figure 2, the curves display the mean EPR signal decay in the absence of (1) and subsequent to (2) IR irradiation. Without irradiation, the signal decreased to approximately 96±3%, but with IR radiation, the IR-induced free radicals reduced the nitroxide PCA to its corresponding hydroxylamine, causing a decrease in EPR signal intensity of up to 70±6%. All measured values obtained after irradiation were found to be significantly different from those in the absence of irradiation, Po0.05, paired t-test. The reduction in PCA is directly correlated with the production of free radicals in the investigated samples (Ananthaswamy and Pierceall, 1990). This indicates that radicals were formed during IR irradiation over time. Taking into consideration the energy of UV photons, it is understandable that irradiation of the skin with UV light leads to the formation of free radicals, which can destroy the skin’s antioxidants if the dose is high enough (Stahl and Sies, 2003; Zastrow et al., 2004). However, this is not the case for IR radiation. Because of the absorption properties of the carotenoids (lo550 nm), their direct absorption Abbreviations: EPR, electron paramagnetic resonance; IR, infrared; PCA, 3-carboxy-2,2,5,5tetramethylpyrrolidine-1-oxy1; RRS, resonance Raman spectroscopy; UV, ultraviolet

In vivo skin treatment with tissue-tolerable plasma influences skin physiology and antioxidant profile in human stratum corneum.

Abstract:  The antimicrobial treatment of wounds is still a major problem. Tissue‐tolerable electrical plasma (TTP) is a new approach for topical microbial disinfection of the skin surface. The aim of the present study was to investigate the influence of TTP on a carotenoid profile in relation to skin physiology parameters (epidermal barrier function, stratum corneum (SC) hydration, surface temperature and irritation parameters). We were interested in the interaction of TTP and the antioxidative network, as well as the consequences for skin physiology parameters. These parameters are also indicative of TTP safety in vivo. For plasma application, ‘Kinpen 09’ was used (surface exposure 30–43°C) for 3 s. Beta‐carotene and water profiles were assessed by in vivo Raman microspectroscopy (skin composition analyzer 3510). Skin physiology parameters were measured with Tewameter TM 300, Corneometer CM 825, skin thermometer and Chromameter CR 300. All parameters were assessed non‐invasively on seven healthy volunteers before and after plasma application in vivo. We could show that TTP application leads to a decrease in beta‐carotene especially in the superficial SC. Skin‐surface temperature increased by 1.74°C, while the transepidermal water loss (TEWL) increase indicated an impaired barrier function. SC hydration decreased as seen in water profile especially in the superficial layers and capacitance values. A slight increase in skin redness was measurable. The induction of reactive oxygen species is probably the major contributor of TTP efficacy in skin disinfection. Skin physiology parameters were influenced without damaging the skin or skin functions, indicating the safety of TTP under in vivo conditions.

Carotenoids in human skin

Abstract:  The interaction of free radicals with antioxidants is a topic of increasing interest in the development of prevention strategies against skin ageing. Carotenoids can serve as marker substances for the complete antioxidative network of human skin. Recently, it has become possible to measure the carotenoids non‐invasively and online using resonance Raman spectroscopy. This method has been used in various studies to investigate the interaction of carotenoid antioxidants and free radicals in human skin. In this review, the results of the selected studies are summarized and compared. It could be demonstrated that the carotenoid concentration of the skin reflects the lifestyle of individuals. A high level of carotenoids can be achieved with a healthy diet rich, for instance, in fruit and vegetables. Stress factors such as illness, UV and IR radiation of the sun, and smoking and alcohol consumption reduce the concentration of the carotenoids in the skin. It could be demonstrated that premature skin ageing was less in people with a high level of antioxidants in their tissue. Consequently, the furrows and wrinkles were not so deep and dense as in the skin of individuals with a low antioxidant level. The measurements are highly suited for the development of anti‐ageing strategies and can be efficiently used in the medical diagnostics and therapy control.

Determination of the antioxidative capacity of the skin in vivo using resonance Raman and electron paramagnetic resonance spectroscopy

Abstract:  Background:  Non‐invasive measurements are of major interest for investigating the effects of stress, nutrition, diseases or pharmaceuticals on the antioxidative capacity of the human skin. However, only a few non‐invasive methods are available.

In vivo distribution of carotenoids in different anatomical locations of human skin: comparative assessment with two different Raman spectroscopy methods

Background:  The cutaneous antioxidants form an efficient protection system against the destructive potential of free radicals, produced by environmental factors, such as UV‐sun irradiation, hazardous substances and lifestyle habits. Most of the antioxidants cannot be produced by the human organism. Thus, they have to be incorporated by food and beverages.

Influence of dietary carotenoids on radical scavenging capacity of the skin and skin lipids

Nutrition rich in carotenoids is well known to prevent cell damage, premature skin aging, and skin cancer. Cutaneous carotenoids can be enriched in the skin by nutrition and topically applied antioxidants have shown an increase in radical protection after VIS/NIR irradiation. In this paper, it was investigated whether orally administered carotenoids increase the radical scavenging activity and the radical protection of the skin using in vivo electron paramagnetic resonance spectroscopy and the skin lipid profile was investigated applying HPTLC on skin lipid extracts. Furthermore, in vivo Raman resonance spectroscopy was used to measure the cutaneous carotenoid concentration. A double blind placebo controlled clinical study was performed with 24 healthy volunteers, who have shown a slow but significant and effective increase in cutaneous carotenoids in the verum group. The enhancement in carotenoids increases the radical scavenging activity of the skin and provides a significant protection against stress induced radical formation. Furthermore, the skin lipids in the verum group increased compared to the placebo group but only significantly for ceramide [NS]. These results indicate that a supplementation with dietary products containing carotenoids in physiological concentrations can protect the skin against reactive oxygen species and could avoid premature skin aging and other radical associated skin diseases.