Analytical Sciences

Spectrophotometric Assay of Superoxide Anion Formed in Maillard Reaction Based on Highly Water-soluble Tetrazolium Salt

Hiroyuki UKEDA,*  Tomoko SHIMAMURA,* Mina TSUBOUCHI,* Yumi HARADA,* Yumi NAKAI,** and Masayoshi SAWAMURA*

*Department of Bioresources Science, Faculty of Agriculture, Kochi University, Monobe B-200, Nankoku 783-8502, Japan
**Analytical Instruments Division, JEOL Ltd., 3-1-2 Musashino, Akishima, Tokyo 196-8558, Japan

A novel highly water-soluble tetrazolium salt, WST-1 (4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate sodium salt), can be reduced to water-soluble formazan with a superoxide anion. Here, the WST-1 assay was applied to detect the superoxide anion generated during the Maillard reaction. A sample solution containing carbonyl compounds such as glyceraldehyde and glycolaldehyde (5 mM) and N^alpha-acetyl-L-lysine (10 mM) was incubated for 2 days at 37°C. The detection of a superoxide anion generated in the sample was performed by the WST-1 assay, and the result was compared with the cytochrome c assay. The reduction of WST-1 was almost perfectly (86 - 96%) inhibited by the addition of the superoxide dismutase (SOD). On the contrary, the reduction of cytochrome c was slightly (20 - 25%) inhibited by the addition of SOD. A similar result was observed for the addition of 4,5-dihydroxy-1,3-benzene disulfonic acid (Tiron). These results mean that the specificity of WST-1 to the superoxide anion was superior to that of cytochrome c. It was also possible to continuously monitor superoxide anion generation during the Maillard reaction by the coexistence of WST-1 in the reaction solution.

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