Effects on detection of radical formation in skin due to solar irradiation measured by EPR spectroscopy.
In various research projects, oxidative stress in irradiated skin was investigated by measuring the production of free radical using EPR spectroscopy. However, comparison of the obtained measuring results proved to be difficult as different preparation parameters were used for those measurements. In the present study the influence of the preparation parameters on the detected radical production was methodically investigated. For this purpose, porcine skin was exposed in situ to UV and VIS-NIR radiation, respectively, while being measured in an X band EPR spectrometer. Prior to the measurements, the skin had been treated with the spin trap N-tert-Butyl-α-phenylnitrone (PBN) and the spin marker 3-(Carboxyl)-2,2,5,5-tetramethyl-1-pyrrolidinyloxy (PCA). The two methods were investigated for quantitative comparability, for advantages and disadvantages and for errors potentially affecting the evaluation of the results. A significant influence of the preparation parameters (concentration and amount of substance) on the detected radical formations could be found. This influence had a nonlinear effect on the detected radical production. 120μl of incubated amount for 1M PBN and for PCA at a concentration of 0.6 and 1.5mM were determined to be the optimum parameters. The incubated skin samples were 1cm in diameter and 300μm thick. Between 22 and 37°C the incubation temperature showed no significant influence on the detected radical production. For the first time it could be demonstrated for PCA-incubated skin that the radiation-induced radical production depends exclusively on the irradiation dose, provided the preparation parameters and the spectral region are kept constant. In addition, the radical production in the UVB-UVA and VIS-NIR spectral regions was measured in PCA- and PBN-treated excised porcine skin. It was found that PBN and PCA provide comparable results for the relative quantity and kinetics of radical production.
Center of Applied Cutaneous Physiology (CCP), Department of Dermatology, Charité-Universitätsmedizin Berlin, Charitéplatz 1 in 10117 Berlin, Germany. Electronic address: stephanie.albrecht@charite.de.
Stephanie
S
Albrecht
Center of Applied Cutaneous Physiology (CCP), Department of Dermatology, Charité-Universitätsmedizin Berlin, Charitéplatz 1 in 10117 Berlin, Germany.
Sebastian
S
Ahlberg
Beuth University of Applied Sciences Berlin, Luxemburger Straße 10 in 13353 Berlin, Germany. Electronic address: beckers@beuth-hochschule.de.
Ingeborg
I
Beckers
UV-Technik, Vogelsbergstr. 27 in 63456 Hanau, Germany. Electronic address: kockott@t-online.de.
Dieter
D
Kockott
Center of Applied Cutaneous Physiology (CCP), Department of Dermatology, Charité-Universitätsmedizin Berlin, Charitéplatz 1 in 10117 Berlin, Germany. Electronic address: juergen.lademann@charite.de.
Jürgen
J
Lademann
Center of Applied Cutaneous Physiology (CCP), Department of Dermatology, Charité-Universitätsmedizin Berlin, Charitéplatz 1 in 10117 Berlin, Germany.
Victoria
V
Paul
Center of Applied Cutaneous Physiology (CCP), Department of Dermatology, Charité-Universitätsmedizin Berlin, Charitéplatz 1 in 10117 Berlin, Germany.
Leonhard
L
Zastrow
Center of Applied Cutaneous Physiology (CCP), Department of Dermatology, Charité-Universitätsmedizin Berlin, Charitéplatz 1 in 10117 Berlin, Germany. Electronic address: martina.meinke@charite.de.
Martina C
MC
Meinke
D000818:Animals; D003497:Cyclic N-Oxides / Q000737:chemistry; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods*; D005609:Free Radicals / Q000737:chemistry / Q000302:isolation & purification*; D018384:Oxidative Stress / Q000528:radiation effects*; D012867:Skin / Q000737:chemistry* / Q000378:metabolism / Q000528:radiation effects; D013113:Spin Labels; D013552:Swine
D003497:Cyclic N-Oxides; D005609:Free Radicals; D013113:Spin Labels; C029217:phenyl-N-tert-butylnitrone
Kinetics of radical production; Oxidative stress; Spin marker and spin traps; UV and VIS-NIR radiation