On the mechanism of the multistep reduction of tetrazolium salts with special reference to the involvement of tetrazolium radicals.

Thin layer chromatography (TLC) was performed to analyze which products are formed when unstable tetrazolinyl radicals are generated during the reduction of tetrazolium salts under physiological conditions of biochemical and histochemical assays. It was found in aqueous media that irrespective the assay conditions, reduction of 2,3-di(4-nitrophenyl)-5-phenyl-2H-tetrazolium chloride (p-DNTTC) or 2,3-di(4-nitrophenyl)-5-t-butyl-2H-tetrazolium chloride (t-butyl-DNTC) always lead to production of formazan and never to generation of the corresponding 2(4-nitrophenyl)-5-phenyl- (respectively t-butyl-) tetrazoles by splitting off a nitrophenyl side chain from the tetrazolium ring as a cation. This reaction is known to occur in non-aqueous media (Neugebauer, 1973). Because p-DNTTC is analogous to tetrazolium salts that are used in biochemical and histochemical assays, it is concluded that reduction of tetrazolium salts leads to formation of formazans only under these conditions.