Tetrazolium ions, reduction

In aqueous solutions of tetrazolium salts, the formation of a precipitate was observed due to the nature of the formazan. In aqueous-alcoholic solutions, however, no precipitation was observed due to the solubility of the formazan in organic solvents. In such solutions (containing primary or secondary alcohols), the radiolytic reduction is found to be more effective, because the a-hydroxyalkyl radicals that form in the reaction of OH radicals and H atoms with the alcohol, also reduce the tetrazolium ion (Kriminskaya et al. 1987). 

Formazans are stable in alkaline solution. Alkaline hydrolysis of functionalities on formazans such as nitriles, esters, and amides leads to the acids (Section 7.3.1.1). The case of 3-nitroformazans (198) is unique. Reaction with hydroxide ion gives 3-hydroxy formazan (199) which can be readily oxidized to the tetrazolium betaine. In the presence of hydrosulfide, a reduction of the nitro group takes place giving 3-aminoformazan (200) with traces of the 3-mercaptoformazan (201), which by contrast is the main product when ammonium polysulfide is used (Scheme 30).45,346... 

Hydroxyl radical may hydroxylate tyrosine to 3,4-dihydroxyphenylalanine (DOPA). DOPAs are the main residues corresponding to protein-bound reducing moieties able to reduce cytochrome c, metal ions, nitro tetrazolium, blue and other substrates (S32). Reduction of metal ions and metalloproteins by protein-bound DOPA may propagate radical reactions by redox cycling of iron and copper ions which may participate in the Fenton reaction (G9). Abstraction of electron (by OH or peroxyl or alkoxyl radicals) leads to the formation of the tyrosyl radical, which is relatively stable due to the resonance effect (interconversion among several equivalent resonant structures). Reaction between two protein-bound tyrosyl radicals may lead to formation of a bityrosine residue which can cross-link proteins. The tyrosyl radical may also react with superoxide, forming tyrosine peroxide (W13) (see sect. 2.6). 

A variety of in vitro assays have been developed that minimize or avoid live animal experimentation. Several capitalize on the sodium channel s affinity for these toxins. Neuronal cell lines lyse in the presence of veratridine, a sodium channel activator, and ouabain, which prevents removal of the excessive sodium ions allowed in by veratridine. In the presence of both these drugs, a sodium channel blocker such as saxitoxin rescues the cells. Cellular viability can then be measured by adding tetrazolium salts that are metabolized by living cells to a colored product. Alternatively, isolated cellular membranes, typically from brain tissue, are used to bind radiolabeled saxitoxin. After incubating receptor and radioligand in the presence of a test sample, any radiolabeled saxitoxin bound to the cell membranes are deposited onto filters by vacuum pressure. Radioactivity from the labeled saxitoxin is then measured with a signal reduction indicating the presence of saxitoxin. 

Amin (2001) reported a colorimetric method for vitamin E in pure form and multivitamin capsules based on the reduction of tetrazolium blue to formazan derivative by vitamin E in alkaline medium. The reaction mixture was incubated at 90 2 C for 10 min and the absorbance of the reaction product was monitored at 526 nm with relative standard deviation of 0.7%-1.5%, a limit of detection 0.012 mg/E and sample throughput of approximately 6/h. The reduction of tetrazolium blue to formazan derivative requires 3 h at room temperature and the color developed was stable for 3 h. EDTA was added to sample solution for masking any metal ions during analysis. 

Acetazolamide and chlorthalidone have also been shown to photosensitize the reduction of nitro blue tetrazolium in phosphate-buffered saline solution. The reaction is more efficient under deoxygenated conditions and in the presence of superoxide dismutase. These results indicate that direct electron transfer occurs from either the excited state of 7 or 8 to the substrate, especially oxygen. No doubt, superoxide ion could be involved as an intermediate when oxygen is present. On the basis of these results, a photochemical reaction mechanism of acetazolamide and nitro blue tetrazolium is postulated as shown in Figure 63.9. 

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