Thiamine Oxidative Transformations Catalyzed by Copper Ions and Ascorbic Acid

I. I. Stepuro,^1,2 T. P. Piletskaya,¹ V. I. Stepuro³, and S. A. Maskevich³

¹Institute of Biochemistry, Academy of Sciences of Belarus, Grodno, 230017 Belarus; fax: (0152) 33-2141.

²To whom correspondence should be addressed.

³Y. Kupala State University of Grodno, ul. Ozheshko 22, Grodno, 230023 Belarus; fax: (0152) 44-8461.

Submitted May 26, 1997; revision submitted September 6, 1997.

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In aqueous solutions containing Cu(II) ions and ascorbic acid, thiamine was observed to be oxidized to the fluorescent products thiochrome and oxodihydrothiochrome in neutral and acid media. At high initial concentrations of thiamine, thiochrome was practically the only product of thiamine oxidation. Catalase inhibited the oxidation rate by approximately by 30-fold, whereas superoxide dismutase reduced the rate by only 2.5-fold. Aliphatic alcohols, glucose, and high concentrations of ascorbic acid effectively inhibited the production of thiochrome. The yield of thiochrome was also decreased in the presence of aliphatic amino acids (especially by histidine) and by human serum albumin (HSA). With complete binding of copper ions by HSA, no formation of fluorescent products was observed. In neutral and acidic media under the action of hydroxyl radicals, thiamine formed a tricyclic semiquinone form which was then oxidized to thiochrome by superoxide anion or H₂O₂. Ascorbic acid played the main role in the reduction of Cu(II), whereas the contribution of superoxide anions was less significant. Cu(I) interacted with H₂O₂ to form hydroxyl radicals. The addition of H₂O₂ both to thiamine and to the mixture of thiamine and Cu(II) ions did not lead to significant production of thiochrome in neutral and acidic media.

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KEY WORDS: thiamine, thiochrome, ascorbic acid, copper ions, fluorescence, hydroxyl radicals, superoxide anion, hydrogen peroxide, amino acids, catalase, superoxide dismutase.

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