Martina C. Meinke

Determination of optical properties of human blood in the spectral range 250 to 1100 nm using Monte Carlo simulations with hematocrit-dependent effective scattering phase functions.

The absorption coefficient mu(a), scattering coefficient mu(s), and anisotropy factor g of diluted and undiluted human blood (hematocrit 0.84 and 42.1%) are determined under flow conditions in the wavelength range 250 to 1100 nm, covering the absorption bands of hemoglobin. These values are obtained by high precision integrating sphere measurements in combination with an optimized inverse Monte Carlo simulation (IMCS). With a new algorithm, appropriate effective phase functions could be evaluated for both blood concentrations using the IMCS. The best results are obtained using the Reynolds-McCormick phase function with the variation factor alpha = 1.2 for hematocrit 0.84%, and alpha = 1.7 for hematocrit 42.1%. The obtained data are compared with the parameters given by the Mie theory. The use of IMCS in combination with selected appropriate effective phase functions make it possible to take into account the nonspherical shape of erythrocytes, the phenomenon of coupled absorption and scattering, and multiple scattering and interference phenomena. It is therefore possible for the first time to obtain reasonable results for the optical behavior of human blood, even at high hematocrit and in high hemoglobin absorption areas. Moreover, the limitations of the Mie theory describing the optical properties of blood can be shown.

Model function to calculate the refractive index of native hemoglobin in the wavelength range of 250-1100 nm dependent on concentration

The real part of the complex refractive index of oxygenated native hemoglobin solutions dependent on concentration was determined in the wavelength range 250 to 1100 nm by Fresnel reflectance measurements. The hemoglobin solution was produced by physical hemolysis of human erythrocytes followed by ultracentrifugation and filtration. A model function is presented for calculating the refractive index of hemoglobin solutions depending on concentration in the wavelength range 250 to 1100 nm.

Optical properties of platelets and blood plasma and their influence on the optical behavior of whole blood in the visible to near infrared wavelength range

The optical parameters absorption coefficient, scattering coefficient, and the anisotropy factor of platelets (PLTs) suspended in plasma and cell-free blood plasma are determined by measuring the diffuse reflectance, total and diffuse transmission, and subsequently by inverse Monte Carlo simulation. Furthermore, the optical behavior of PLTs and red blood cells suspended in plasma are compared with those suspended in saline solution. Cell-free plasma shows a higher scattering coefficient and anisotropy factor than expected for Rayleigh scattering by plasma proteins. The scattering coefficient of PLTs increases linearly with the PLT concentration. The existence of physiological concentrations of leukocytes has no measurable influence on the absorption and scattering properties of whole blood. In summary, red blood cells predominate over the other blood components by two to three orders of magnitude with regard to absorption and effective scattering. However, substituting saline solution for plasma leads to a significant increase in the effective scattering coefficient and therefore should be taken into consideration.

Radical Production by Infrared A Irradiation in Human Tissue

The influence of the ultraviolet (UV) irradiation of the sun on the formation of free radicals in human skin is well investigated. Up to now, only small amounts of data are available stating that infrared (IR) irradiation can produce free radicals in the skin. In the present study, the formation of free radicals in human skin, subsequent to IRA irradiation (600–1,500 nm), has been demonstrated by means of two different methods. Firstly, the radical formation was detected indirectly by the degradation of the cutaneous carotenoid antioxidants β-carotene and lycopene, which was investigated in vivo by resonance Raman spectroscopic measurements. Secondly, the direct observation of produced radicals subsequent to IRA irradiation of the skin was performed in vitro by electron paramagnetic resonance spectroscopy. Taking into account the results of the present study and previous UV light studies, it can be expected that also solar irradiation in the visible spectral range will produce free radicals in the human skin. Therefore, the current sun protection strategies should be reconsidered. Furthermore, it was shown in the present study that the side effect in the form of radical formation could be significantly reduced by increasing the protection system of the human organism in form of the antioxidant network.

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

Penetration and storage of particles in human skin: Perspectives and safety aspects

The application of particles in dermatology and cosmetology represents an emerging field and is closely connected with the question of risk assessment as the potential for, and consequences of, penetration of such particles into the living tissue has not been determined conclusively. In the medical sector, extensive research activities are in progress to develop particles, which can be used as efficient carriers for drug delivery through the skin barrier. In contrast, in cosmetic products, particles are mostly required to remain on the skin surface to fulfill their beneficial effect. Whereas the intercellular penetration of particles seems to be unlikely, the hair follicle has been shown to be a relevant penetration pathway for particles as well as an important long-term reservoir. It has been demonstrated that the penetration depth of the particles can be influenced by their size resulting in the possibility of a differentiated targeting of specific follicular structures. In the present review, the follicular penetration mechanisms and storage properties of particles are discussed.

Determination of the complex refractive index of highly concentrated hemoglobin solutions using transmittance and reflectance measurements

The complex refractive index of highly concentrated hemoglobin solutions as they appear in red blood cells are determined in the wavelength range of 250 to 1100 nm using transmittance and Fresnel reflectance measurements. The determined real parts of the refractive indices are on average 0.02 units higher than the values found in the literature. The wavelength dependence of the measured data in the UV region differs from the calculated data using the Kramers-Kronig relation.

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.