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In vivo fluorescence excitation spectroscopy and in vivo Fourier‐transform Raman spectroscopy in human skin: evidence of H 2 O 2 oxidation of epidermal albumin in patients with vitiligo
Author(s) -
Rokos Hartmut,
Moore Jeremy,
Hasse Sybille,
Gillbro Johanna M.,
Wood John M.,
Schallreuter Karin U
Publication year - 2004
Publication title -
journal of raman spectroscopy
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.748
H-Index - 110
eISSN - 1097-4555
pISSN - 0377-0486
DOI - 10.1002/jrs.1114
Subject(s) - chemistry , resonance raman spectroscopy , raman spectroscopy , in vivo , vitiligo , albumin , fourier transform infrared spectroscopy , human serum albumin , fluorescence , analytical chemistry (journal) , tryptophan , photochemistry , nuclear chemistry , biochemistry , chromatography , amino acid , biology , immunology , optics , physics , microbiology and biotechnology
Abstract Both fluorescence excitation spectroscopy and Fourier‐transform Raman spectroscopy have been utilised to study in vivo H 2 O 2 ‐mediated oxidative stress in the depigmented epidermis of untreated patients with vitiligo by following the oxidation of disulfide bridges in human epidermal albumin and the fluorescence maxima of the tryptophan residue in position 214 of this protein. In normal healthy skin, the single tryptophan in albumin fluoresces with a λ max at 295 nm which is in agreement with high concentrations (10 −3 M ) of this protein. Fluorescence analysis of the depigmented epidermis of untreated patients with vitiligo revealed three distinct patient groups: (a) one with a peak at a λ max of 280 nm (b) one with λ max in the range 300–305 nm and (c) one with three peaks with excitation maxima in the region of a λ max of 280, 290 and 300 nm. The peak at a λ max of 280 nm was in agreement with low albumin concentrations. Epidermal H 2 O 2 concentrations in the m M range, as previously documented in untreated vitiligo, were followed with in vivo FT‐Raman spectroscopy using the O—O stretch at 875 cm −1 . Here, we were able to assign the SO 2 stretch at 1040 cm −1 to cysteic acid, which was the expected peroxidation product of the disulfide bridges in albumin. Using human albumin we confirmed the oxidation of disulfide bridges as well as the concentration dependent fluorescence excitation maxima for Trp 214 in this protein. The removal of epidermal H 2 O 2 from the patients skin with a pseudocatalase (PC‐KUS) yielded the loss of the O—O stretch at 875 cm −1 together with the loss of the SO 2 stretch at 1040 cm −1 in the Raman spectrum and a shift of the λ max from 300–305 nm to 290/280 nm was observed in the fluorescence analysis. These new in vivo results add more evidence to the central role for H 2 O 2 ‐mediated oxidative stress in the pathomechanism of vitiligo. Copyright © 2004 John Wiley & Sons, Ltd.