Free Radical Threshold Value: A New Universal Body Constant.

Three evolutionary sources create 'primary' reactive oxygen species (ROS) and 'secondary' lipid oxygen species (LOS), forming the human body's 'free radical ground state'. We present evidence for the existence of a universal free radical threshold value (FRTV), defining the borderline between advantageous and adverse effects of free radicals observed above the free radical ground state. Based on standard vitamin D doses, the calculated amount of ∼3.5 × 10(12) rad/mg ROS/LOS tissue represents the tolerated number of free radicals in skin tissue - defined as FRTV. By means of quantitative ESR x-band spectroscopy, the FRTV was experimentally verified using ex vivo human skin irradiated with ultraviolet + visible (UV + VIS), UVB + UVA and VIS light. In addition, we investigated whether this threshold is also existent in internal organs by extending our experiment to fresh porcine liver. Based on the determination of ROS/LOS below and above the FRTV, ROS > LOS was characterized as beneficial and LOS > ROS as deleterious to the organism, respectively. Results of the experiments using porcine liver confirmed the appearance of the FRTV at radical generation ∼3.5 × 10(12) rad/mg. The relation ROS/LOS before and after the FRTV was consistent with the results determined for the skin. We conclude that the FRTV, theoretically calculated and experimentally confirmed, should be considered as a new 'universal body constant'.

Radical protection by differently composed creams in the UV/VIS and IR spectral ranges.

Modern sunscreens are well suited to provide sufficient protection in the UV range because the filter substances absorb or scatter UV radiation. Although up to 50% of radicals are formed in the visible and infrared spectral range during solar radiation protection strategies are not provided in this range. Previous investigations of commercially available products have shown that in addition to physical filters, antioxidants (AO) are necessary to provide protective effects in the infrared range by neutralizing already formed radicals. In this study, the efficacy of filter substances and AO to reduce radical formation in both spectral ranges was investigated after UV/VIS or IR irradiation. Optical properties and radical protection were determined for the investigated creams. It was found that organic UV filters lower radical formation in the UV/VIS range to 35% compared to untreated skin, independent of the presence of AO. Further reduction to 14% was reached by addition of 2% physical filters, whereas physical filters alone were ineffective in the UV/VIS range due to the low concentration. In contrast, this filter type reduced radical formation in the IR range significantly to 65%; similar effects were aroused after application of AO. Sunscreens which contain organic UV filters, physical filters and AO ensure protection in the complete solar spectrum.

Dose-dependent vitamin C uptake and radical scavenging activity in human skin measured with in vivo electron paramagnetic resonance spectroscopy.

Vitamin C is a potent radical scavenger and a physiological part of the antioxidant system in human skin. The aim of this study was to measure changes in the radical-scavenging activity of human skin in vivo due to supplementation with different doses of vitamin C and at different time points. Therefore, 33 volunteers were supplemented with vitamin C or placebo for 4 weeks. The skin radical-scavenging activity was measured with electron paramagnetic resonance spectroscopy. After 4 weeks, the intake of 100 mg vitamin C/day resulted in a significant increase in the radical-scavenging activity by 22%. Intake of 180 mg/day even resulted in a significant increase of 37%. No changes were found in the placebo group. A part of the study population was additionally measured after 2 weeks: in this group radical scavenging had already reached maximal activity after 2 weeks. In conclusion, orally administered vitamin C increases the radical-scavenging activity of the skin. The effect occurs fast and is enhanced with higher doses of vitamin C.

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

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. MATERIAL AND METHODS: The resonance Raman spectroscopy is well established to monitor carotenoids in the skin, but correlations with other antioxidants have not yet been described. Electron paramagnetic resonance spectroscopy used for measurements of free radicals has already been used elsewhere to investigate the reduction of applied long-living nitroxide radicals, caused by skin antioxidants and UV irradiation, but only a single or up to four volunteers were included in these studies. Therefore, in this study, the two methods were applied in parallel on 17 volunteers, and the rate constant of the nitroxide decrease was correlated with the cutaneous carotenoid concentration. RESULTS AND DISCUSSION: A correlation with R = 0.65 was found, supporting the thesis that different antioxidants protect each other and build an antioxidative network in the skin. The results also give first indications that the carotenoids serve as marker substances for the antioxidative capacity, if the nutrition is well balanced.

Radical protection by sunscreens in the infrared spectral range.

One essential reason for skin ageing is the formation of free radicals by excessive or unprotected sun exposure. Recently, free radical generation in skin has been shown to appear not only after irradiation in the UV wavelength range but also in the infrared (IR) spectral range. Sunscreens are known to protect against radicals generated by UV radiation; however, no data exist for those generated by IR radiation. This paper has investigated four different, commercially available sunscreens and one COLIPA standard with regard to radical formation in the skin after IR irradiation, using electron paramagnetic resonance spectroscopy. The use of sunscreens has led to reduced amounts of radicals compared to untreated skin. Furthermore, absorption and scattering properties and the radical protection factor of the formulations were determined to investigate their influence on the radical protection of the skin. None of these formulations contained an optical absorber in the IR range. The protection efficiency of the sunscreens was shown as being induced by the high scattering properties of the sunscreens, as well as the antioxidants contained in the formulations.

HPLC analysis of vitamin E isoforms in human epidermis: correlation with minimal erythema dose and free radical scavenging activity.

The content and composition of different vitamin E isoforms was analyzed in normal human skin. Interestingly the epidermis contained 1% alpha-tocotrienol, 3% gamma-tocotrienol, 87% alpha-tocopherol, and 9% gamma-tocopherol. Although the levels of tocotrienol in human epidermis appear to be considerably lower than reported in the hairless mouse, the presence of significant amounts of tocotrienol levels leads to speculation about the physiological function of tocotrienols in skin. Besides antioxidant activity and photoprotection, tocotrienols may have skin barrier and growth-modulating properties. A good correlation was found for epidermal alpha-tocopherol (r = 0.7909, p <.0003), gamma-tocopherol (r = 0.556, p <.025), and the total vitamin E content (r = 0.831, p <.0001) with the free radical 1,1-diphenyl-2-picrylhydrazyl (DPPH) scavenging in epidermis, as assessed by electron paramagnetic resonance (EPR) spectroscopy. In human epidermis, alpha-tocopherol is quantitatively the most important vitamin E isoform present and comprises the bulk of first line free radical defense in the lipid compartment. Epidermal tocotrienol levels were not correlated with DPPH scavenging activity. The minimal erythema dose (MED), an individual measure for sun sensitivity and a crude indicator for skin cancer susceptibility, did not correlate with the epidermal content of the vitamin E isoforms. Hence it is concluded that vitamin E alone is not a determinant of individual photosensitivity in humans.

Kinetic measurements using EPR imaging with a modulated field gradient.

EPR imaging with modulated field gradient was applied for the investigation of fast diffusion processes. Three different imaging methods are possible: spectral-temporal, spatio-temporal, and spectral-spatial imaging. The time resolution is on the order of seconds and the spatial resolution is in the micrometer region. The efficiency of this imaging technique is demonstrated for the penetration of the spin probe Tempol in the skin of hairless mice biopsies. The skin is normally protected against the penetration of water soluble substances by the horny layer, a resistive thin lipophilic layer. Overcoming this horny layer for water soluble ingredients is one of the main practical problems for the topical application of pharmaceutics which could be investigated by EPR imaging. Different images represent the penetration behavior of the water soluble Tempol in the skin after treatment with the penetration enhancer DMSO (Dimethylsulfoxide) and after removing the horny layer.