Presence of epidermal allantoin further supports oxidative stress in vitiligo Mohammad Shalbaf 1 , Nicholas C. J. Gibbons 1,2 , John M. Wood 1 , Derek J. Maitland 3 , Hartmut Rokos 2 , Souna M Elwary 1 , Lee K. Marles 1 and Karin U. Schallreuter 1,2 1 Clinical and Experimental Dermatology, Department of Biomedical Sciences, University of Bradford, Bradford, UK; 2 Institute for Pigmentary Disorders in Association with EM Arndt University of Greifswald, Greifswald, Germany and University of Bradford, UK; 3 Department of Forensic Sciences, School of Life Sciences, University of Bradford, Bradford, UK Correspondence: Professor Karin U. Schallreuter, Clinical and Experimental Dermatology ⁄ Department of Biomedical Sciences, University of Bradford, Bradford, BD7 1DP, West Yorkshire, UK, Tel.: +44 1274 235527, Fax: +44 1274 236849, e-mail: K.Schallreuter@bradford.ac.uk Accepted for publication 11 January 2008 Abstract: Xanthine dehydrogenase ⁄ xanthine oxidase (XDH ⁄ XO) catalyses the hydroxylation of hypoxanthine to xanthine and finally to uric acid in purine degradation. These reactions generate H 2 O 2 yielding allantoin from uric acid when reactive oxygen species accumulates. The presence of XO in the human epidermis has not been shown so far. As patients with vitiligo accumulate H 2 O 2 up to mm levels in their epidermis, it was tempting to examine whether this enzyme and consequently allantoin contribute to the oxidative stress theory in this disease. To address this question, reverse transcription-polymerase chain reaction, immunoreactivity, western blot, enzyme kinetics, computer modelling and high performance liquid chromatography ⁄ mass spectrometry analysis were carried out. Our results identified the presence of XDH ⁄ XO in epidermal keratinocytes and melanocytes. The enzyme is regulated by H 2 O 2 in a concentration-dependent manner, where concentrations of 10 )6 m upregulates the activity. Moreover, we demonstrate the presence of epidermal allantoin in acute vitiligo, while this metabolite is absent in healthy controls. H 2 O 2 -mediated oxidation of Trp and Met in XO yields only subtle alterations in the enzyme active site, which is in agreement with the enzyme kinetics in the presence of 10 )3 m H 2 O 2 . Systemic XO activities are not affected. Taken together, our results provide evidence that epidermal XO contributes to H 2 O 2 - mediated oxidative stress in vitiligo via H 2 O 2 -production and allantoin formation in the epidermal compartment. Key words: allantoin – human epidermis – hydrogen peroxide – vitiligo – xanthine oxidase Please cite this paper as: Presence of epidermal allantoin further supports oxidative stress in vitiligo. Experimental Dermatology 2008; 17: 761–770. Introduction It has been well documented that the entire epidermis of patients with vitiligo is under oxidative stress because of the accumulation of mm levels of H 2 O 2 (1,2). In this con- text it has been established in vivo by fourier-transformed Raman spectroscopy that epidermal H 2 O 2 accumulation in patients with vitiligo reaches concentrations in the range of 10 )3 m (1,3) and these concentrations are sufficient to oxi- dize Met and Trp residues to Met sulfoxide and to 5-OH- Trp, respectively, in many proteins ⁄ enzymes, hormones and peptides (4–8). Several mechanisms in the epidermal compartment have been identified, which generate and degrade H 2 O 2 [for review see (9,10)]. Xanthine oxidase (XO, EC 1.1.3.22) produces H 2 O 2 in its reaction mecha- nism and therefore we were interested to test whether this enzyme could be a potential contributor to the H 2 O 2 pool in vitiligo. Xanthine oxidase belongs to the family of the mono- nuclear molybdopterin co-factor (Moco) containing proteins and is one of the two inter-convertible forms of xanthine oxidoreductase (XOR) containing xanthine dehy- drogenase (XDH, EC 1.1.1.204) and in its oxidized form it contains XO. These two enzymes have similar molecular weights and composition of redox centres (11). Under nor- mal physiological conditions, XOR is found in the cell as dehydrogenase. However, under oxidizing conditions, XDH can be immediately converted to the oxidase (12,13). This enzyme exists in different species from bacteria to man (14). In various tissues of mammals, it performs terminal oxidation of all purine bases as the rate-limiting enzyme in the purine degradation pathways (15). XO catalyses the oxidative hydroxylation of hypoxanthine to xanthine followed by xanthine to uric acid conversion accompanied by the generation of H 2 O 2 (16) (Scheme 1). Therefore this enzyme is considered as a major biological source for DOI:10.1111/j.1600-0625.2008.00697.x www.blackwellpublishing.com/EXD Original Article ª 2008 The Authors Journal compilation ª 2008 Blackwell Munksgaard, Experimental Dermatology, 17, 761–770 761