The determination of the radical power - an in vitro test for the evaluation of cosmetic products.

OBJECTIVE: Cosmetic formulations are influenced by environmental impacts and ageing, resulting in rancidity and change of colour and structure. These changes are caused by free radicals (FRs). The sensitivity of cosmetics generating FRs is a metric for its quality and should be determined. METHODS: Electron spin resonance spectroscopy in combination with UV irradiation tested cosmetics such as creams, milks, lotions and fragrances. The probes were directly measured without expensive preparation. RESULTS: Nine formulations are tested for its radical generation and ranked corresponding to the radical power. The transformation of the FR properties of three formulations to skin is measured by the radical skin status factor (RSF) method. It shows that the higher the radical power (RP) is, the lower the radical status RSF of skin will be. CONCLUSION: The knowledge of the sensitivity of cosmetics to generate FRs is necessary for its stabilization and prevention of potential damages to skin. It is a new way in development of cosmetics which has to be considered.

The Radical Status Factor (RSF): a novel metric to characterize skin products.

A scale of skin treatments is analysed, which bases on the detection of UV-generated free radicals in pig skin. Physiological and anatomical similarities between man and pig made this animal a good model for man in many research areas. The determination of the Radical Status (free radical response) offers the possibility to see in an early stage the effect of exterior and interior influences. The detection of the skin's response after a normalized radical generation by defined UV dose combined with the application of external and internal influences enables a comprehensive and easy classification of skin products and therapies. The effect of substances, especially, applied topically on the skin, like it is usual in cosmetics and pharmacy, can be classified. The relevance of the RSF method is demonstrated with the application of numerous different treatments on the skin.

Integrated sun protection factor: a new sun protection factor based on free radicals generated by UV irradiation.

The present work uses the initial step of the whole cascade of biological effects in the skin, the creation of free radicals by means of UVA/UVB radiation, to develop a total sun protection factor. Until now, existing in vivo indices have not been fully satisfying: SPF only reflects protection from UVB light, and persistent pigment darkening is restricted to the UVA part of the sun spectrum. The quantitative measurement of free radicals generated in human skin biopsies by means of electron spin resonance X-band spectroscopy allows to determine a new total SPF. This new sun protection index covers all UVA/UVB wavelengths taking into account their effects in the epidermis as well as the dermis. Use of skin biopsies avoids exposure of human volunteers to potentially harmful radiations. The new index is always practically equal or lower than the in vivo SPF depending on the level of a product's UVA/UVB photoprotection balance. With this, we propose to name this new protection index 'integrated sun protection factor'.

UV-induced free radicals in the skin detected by ESR spectroscopy and imaging using nitroxides.

Reactive free radicals and reactive oxygen species (ROS) induced by ultraviolet irradiation in human skin are strongly involved in the occurrence of skin damages like aging and cancer. In the present work an ex vivo method for the detection of free radicals/ROS in human skin biopsies during UV irradiation is presented. This method is based on the Electron Spin Resonance (ESR) spectroscopy and imaging and uses the radical trapping properties of nitroxides. The nitroxides 2,2,6,6-Tetramethylpiperidine-1-oxyl (TEMPO), 3-Carbamoyl-2,2,5,5-tetramethylpyrrolidine-1-oxyl (PCM), and 3-Carboxy-2,2,5,5-tetramethylpyrrolidine-1-oxyl (PCA), were investigated for their applicability of trapping reactive free radicals and reactive oxygen species in skin during UV irradiation. As a result of the trapping process the nitroxides were reduced to the EPR silent hydroxylamins. The reduction rate of TEMPO was due to both the UV radiation and the enzymatic activity of the skin. The nitroxides PCM and PCA are sufficiently stable in the skin and are solely reduced by UV-generated free radicals/ROS. The nitroxide PCA was used for imaging the spatial distribution of UV-generated free radicals/ROS. As a result of the homogeneous distribution of PCA in the skin, it was possible to estimate the penetration of UVA and UVB irradiation: The UV irradiation decreased the PCA intensity corresponding to its irradiance and penetration into the skin. This reduction was shown to be caused mainly by UVA radiation (320-400 nm).

Electron spin resonance detection of UVA-induced free radicals.

Free radicals generated during UV irradiation of human skin biopsies were measured with electron spin resonance spectroscopy by using spin traps. The generation of hydroxyl and lipid radicals in skin is mainly caused by the UVA part of the solar spectrum.

In vitro and in vivo assessment of the irritation potential of different spin traps in human skin.

No clinical data are available on the acute cutaneous toxicity of spin traps which are frequently used in combination with the electron paramagnetic resonance (EPR) technique for detection of free radicals and reactive oxygen/nitrogen species. The purpose of this study was to evaluate the acute dermatotoxicity of the following spin traps in human skin: C-phenyl-N-tert.-butyl nitrone (PBN), C-(4-pyridinyl-N-oxide)-N-tert.-butylnitrone (POBN), 5, 5-dimethyl-l-pyrroline-N-oxide(DMPO), 5 diethoxyphosphoryl-5-methyl-l-pyrroline-N-oxide (DEPMPO), diethyldithiocarbamate (DDC) and N-methyl-D-glucamine dithiocarbamate (MGD). The corrosivity of the test substances was first assessed in human skin in vitro by measurement of transcutaneous electrical resistance (TER). In this assay all spin traps were non-corrosive at 500 mM concentration. Subsequently cutaneous irritation of the spin traps was determined at different concentrations (50, 250 and 500 mM) in human skin according to a routine four h human patch test in comparison to the standardized irritant sodium laurylsulfate (SLS, 20%). The response was evaluated clinically as well as by a biophysical method analyzing transepidermal water loss (TEWL). PBN and DEPMPO caused a transient and weak inflammatory reaction at 500 mM in four of 17 and in two of 17 volunteers, respectively. DMPO, POBN, DDC, MGID, and the iron complexes of DDC and MGD were clinically non-irritant at all concentrations tested and no delayed-acute inflammatory reactions were observed. However, the TEWL values were significantly increased by all spin traps except DMPO at 500 mM, indicating disturbed epidermal barrier function. We conclude that the spin traps investigated have a low potential to cause acute skin toxicity and may be used safely for in vivo EPR studies in human skin.

Electron paramagnetic resonance for everybody--MICROspec-X--a new class of electron paramagnetic resonance spectrometer.

The electron paramagnetic resonance spectroscopy is the only method for detecting free radicals. Free radicals have an increased importance in our daily life. A small transportable EPR spectrometer is presented for the popularisation of the EPR method. The technical construction and some applications are illustrated which show the usability of the spectrometer.

Cutaneous tolerance to nitroxide free radicals and nitrone spin traps in the guinea pig.

The attempts to use nitroxide free radicals and nitrone spin traps topically in skin requires analysis of their potential cutaneous adverse effects. The objective of this study was to investigate the skin irritation and sensitizing potential of nitroxides and nitrones in the guinea pig. The following unsubstituted nitroxides were investigated: 2,2,6,6-tetramethyl-1-piperidinoxyl (Tempo), 2,2, 5,5-tetramethyl-3-oxazolidinoxyl (Doxo), 2,2,5,5-tetramethyl-1-dihydro-pyrrolinoxyl (Proxo), 2,2,3,4,5,5-hexamethyl-imidazoline-1-yloxyl (Imidazo) and the nitrones: 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) and N-tert.-butyl-phenylnitrone (PBN). Cutaneous irritation was determined following the modified Draize protocol. The response was evaluated clinically as well as by a biophysical method analyzing transepidermal water loss (TEWL). The nitroxides and nitrones were classified clinically from non-irritant (Proxo, Imidazo, DMPO) to slightly irritant (Tempo, Doxo, PBN) according to the Draize protocol. In agreement with the clinical scoring, the TEWL values were significantly increased by Tempo, Doxo and PBN. TOLH, the hydroxylamine of Tempo and its major skin metabolite, did not cause skin irritation. The sensitizing effect was evaluated according to the Magnusson and Kligman test. The results showed no cutaneous hypersensitivity to all nitroxides and nitrones, indicating a weak sensitizing potential. That concludes that the nitroxides and nitrones tested in this study have a low potential of acute skin intolerance.

Cutaneous tolerance to nitroxide free radicals in human skin.

No data are available on the irritant effect of nitroxide free radicals in human skin. Nitroxides are important biomedical skin probes used in Electron Paramagnetic Resonance spectroscopy and imaging. Our purpose was to study the skin irritation potential of different nitroxide free radical structures in skin of healthy human subjects. We investigated the following nitroxides: Tempo (2,2,6,6-tetramethyl-1-piperidinoxy), Doxo (2,2,5,5-tetramethyl-3-oxazolidinoxy), Proxo (2,2,5,5-tetramethyl- -dihydro-pyrrolinoxy), and Imidazo (2,2,3,4,5,5-hexamethyl-imidazoline-1-yloxyl). Cutaneous irritation was determined in human skin following a single application and after repetitive applications in comparison to the standardized irritant sodium lauryl sulfate (SLS). The response was evaluated clinically as well as by a bioengineering method analyzing transepidermal water loss (TEWL) and skin hydration (capacitance). The nitroxides were classified clinically from nonirritant (Imidazo, Proxo), to slightly irritant (Doxo, 100 mM), or moderately irritant (Tempo 100 mM) after a single application. The TEWL values were significantly increased by Doxo and Tempo, but capacitance values were not changed significantly. In the cumulative irritation test Tempo was scored as a slight irritant (10 mM). TOLH (2,2,6,6-tetramethyl-1-hydroxypiperidin), the hydroxylamine of Tempo, which is the major skin metabolite, did not cause skin irritation after a single or repetitive applications. This may indicate that a loss of cellular reducing equivalents may be involved in the inflammation process caused by Tempo. The order of nitroxide irritation potency (Tempo > Doxo >> Imidazo = Proxo) is inverse to the order of nitroxide biostability in human skin (Imidazo = Proxo >> Doxo > Tempo). In conclusion, nitroxide free radicals are classified as nonirritant to moderately irritant in human skin. Particularly, the pyrrolidine and imidazoline type nitroxides have a low potential to cause acute or subacute skin toxicity.

Electron paramagnetic resonance studies on nitroxide radical 2,2,5,5-tetramethyl-4-piperidin-1-oxyl (TEMPO) redox reactions in human skin.

Electron paramagnetic resonance (EPR) is currently being explored for the study of living biological systems. Among biophysical and biochemical applications, the study of nitroxide radical interactions with tissue antioxidants and oxidants is of growing interest. Skin is a target organ of the EPR methodology and is frequently exposed to oxidative stress. We investigated the piperidine-type nitroxide 2,2,5,5-tetramethyl-4-piperidin-1-oxyl (TEMPO) because it is skin permeable and readily accepts electrons in biological systems. TEMPO is readily scavenged on the surface of cultured human skin. Pretreatment of skin cultures with butylhydroperoxide, which decreases intracellular ascorbate and glutathione, causes inhibition of nitroxide scavenging. Exposure of skin cultures to dehydroascorbate, which is internalized and converted to ascorbate, leads to stimulation of nitroxide scavenging. In human keratinocytes and fibroblasts, the TEMPO radical is reversibly reduced to the hydroxylamine depending on the oxygen concentration and the availability of intracellular glutathione and ascorbate. Cell exposure to the glutathione synthetase inhibitor buthionine-sulfoximine depleted intracellular glutathione and inhibited nitroxide reduction; exposure to dehydroascorbate or glutathione-monoethylester increased intracellular ascorbate or glutathione concentration and stimulated nitroxide reduction. Quantitative considerations indicate that the major reduction site of TEMPO in skin and skin cells is the cytosol ascorbate/glutathione redox cycle. We suggest that analysis of TEMPO radical scavenging by the EPR technique is a convenient method for measuring skin ascorbate and thiol-dependent antioxidant activity in vitro and in vivo.

[Theoretical principles and technical realization of high resolution nuclear magnetic resonance tomography with the example of a dedicated coil system]. / Theoretische Grundlagen und technische Realisation der hochauflösenden Kernspintomographie (KST) am Beispiel eines dedizierten Spulensystems.

The theory of high-resolution magnetic resonance imaging (MRI) and the physical properties of a dedicated coil system with its clinical application are reviewed. To evaluate the spatial resolution of the system, a phantom sample was depicted by a transverse T1-weighted sequence (time of repetition 500 ms, time of echo 25 ms, 256 x 256 matrix, 3 acquisitions, field of view 25 mm2). Relative signal intensity decrease was less using the 5-cm coil, as signal intensity field distribution depends on coil diameter. The phantom appeared as an attainable resolution of 100-microns pixel width using the 2.5-cm coil. For the 5-cm coil the pixel width was 200 microns, not accomplishing clear resolution of the phantom. Coil head choice depends on the anatomic depth of the target organ. Work-up of the skin and musculoskeletal lesions is the main indication for high-resolution MRI using surface coils.

Penetration of spin-labeled dihydrolipoate into the skin of hairless mice. Modification of epidermal and dermal polarity.

Electron paramagnetic resonance (EPR) imaging with the modulated field gradient technique is a novel method to investigate skin biophysical and biochemical properties employing specific nitroxide spin probes. Using this method, a distinct increase in polarity from epidermis towards lower dermis is observed with the spin label dit-butylnitroxide (DTBN). With proxylmaleimide a considerable increase in mobility is found, when epidermis is compared with dermal compartments. The effect of the natural antioxidant dihydrolipoate on skin membrane polarity was studied. Skin penetration of spin labeled dihydrolipoate was investigated by EPR imaging. The results indicate that dihydrolipoate also increases membrane polarity. The biophysical and biochemical changes in the epidermis and dermis as revealed by spatial imaging, provide indirect evidence for skin penetration of dihydrolipoate. This conclusion was supported by the finding that spin labeled derivatives of dihydrolipoate and lipoate were detected inside epidermis and dermis by EPR imaging. This study demonstrates the feasibility of EPR imaging to investigate pharmacodynamic and pharmacokinetic properties of spin labeled drugs in skin.

Electron paramagnetic resonance (EPR) imaging in skin: biophysical and biochemical microscopy.

Electron paramagnetic resonance (EPR) is a spectroscopic technique that allows detection of paramagnetic compounds, e.g., free radicals, in skin, and is used for measuring skin membrane fluidity and polarity. EPR imaging is concerned with spatially resolved EPR spectroscopy. We studied EPR images at X-band frequency (9 GHz) in the skin biopsies of hairless mice. Our particular imaging technique utilized a modulated field gradient to obtain cross-sectional images perpendicular to the skin surface. Employing nitroxide free radicals, this approach allows analysis of skin biophysical and biochemical features at the micrometer resolution level. By spin labeling drugs, pharmacokinetic properties of the labeled compound can be monitored in skin. We suggest that EPR imaging has a broad application potential in dermatologic research. In comparison to other spatially visualizing techniques, such as nuclear magnetic resonance (NMR) imaging and ultrasound, EPR imaging has significant advantages, such as high spatial resolution and providing specific biochemical and biophysical information that cannot be obtained by other methods in skin.

One- and two-dimensional electron paramagnetic resonance imaging in skin.

EPR imaging with modulated field gradients provides the possibility for obtaining an EPR spectrum in a selected volume. We demonstrate the feasibility of X-band (9.5 GHz) electron paramagnetic resonance (EPR) imaging in skin biopsies of hairless mice. One- (1D) and two-dimensional (2D) EPR images of the persistent free radical di-tertiary-butyl-nitroxide are measured. At a microwave frequency of 9.5 GHz (X-band), 2D images are obtained in skin biopsies with an actual point distinction resolution of 25 microns. In a biological model system, 2D images are measured at L-band frequency (2.0 GHz) with a pixel resolution of 61 microns, and a theoretical spatial resolution of 12.5 microns. In combination with the spin labeling and spin trapping technique, EPR imaging is the most direct approach to analyzing spatial distribution of physicochemical properties in skin, such as membrane fluidity and polarity, as well as detection of free radicals.