Tetrazolium compounds

Various tetrazolium compounds, introduced into enzyme research by Kuhn and Jerchel (1941) and Lakon (1942), are reduced to scarlet-red (2,3,5-triphenyl-2H-tetrazolium), purplish (neoetrazo-lium) or blue (ditetrazolium) formazans, insoluble in water and soluble in fats. Formazan generation is practically inhibited by superoxide dismutase as well as by catalase (Bromme et al. 1999). When superoxide radical generation via the xanthine-xanthine oxidase system was measured by chemiluminescence an addition of nitroblue tetrazolium immediately abolished lucigenin-enhanced chemiluminescence. Nitroblue tetrazolium did not inhibit xanthine oxidase. 

The nitroblue tetrazolium method has the main advantage of sensitivity. Oberley and Spitz (1985) used diethylenetriamine-pentaacetic acid (DETA-PAC) instead of ethylenediamine tetraacetic acid (EDTA), as they found that DETAPAC enhances the 

Dioxane proved to be the most suitable solvent for extracting the formazan formed in the nitroblue tetrazolium test (Muller et al. 1981). At 85 °C dioxane completely extracted nitroblue tetrazohum (absorption maximum at X, = 580 nm, absorption coefficient = 0.0256 ml X nmol X cm ) from the cell pellet within 15 min, and nitroblue tetrazohum diluted in dioxane was sufficiently stable. As a possible aqueous contamination under conditions of an acid pH caused a slower decrease of the optical density of the extract than did pure water, 1 N HCl as washing agent to remove remaining nitroblue tetrazolium from the cell pellet is recommended. 

Nalbandian (1982) and Nalbandian et al. (1983) modified the nitroblue tetrazohum immuno-bead test (O Donnell et al. 1979, Jabs et al. 1980) to detect cases in which faihng and absent phagocytic activity and respiratory burst intensity in ah-quots of polymorphonuclear leucocytes have been functionally restored to normal by in vitro piracetam treatment of such neutrophils. 

The combination of short-length non-denaturing isoelectric focusing and measurement of superoxide dismutase (EC 1.15.1.1) activity by water-soluble tetrazohum salt made it possible to quantify superoxide dismutase activity in a small sample (Shimazaki et al. 2002). 

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Lactate dehydrogenase (LDH) LDH released by cells with damaged membrane results in the conversion of a tetrazolium compound into a water-soluble fluorescent formazan dye Cell membrane Yes [32]... 

Among the first cell viability assays developed for HTS was the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] tetrazolium reduction assay (Mosmann 1983) that served as a milestone for this type of study. The assay offered a non-radioactive alternative to tritiated thymidine incorporation into DNA as a method of measuring cell proliferation. In many cases, the MTT assay can directly substitute for the tritiated thymidine incorporation assay (Figure 6.3). The MTT tetrazolium compound is prepared in a physiologically balanced solution, added to cells in culture, and incubated for approximately 4 hr. Viable cells convert MTT into an intensely colored formazan product that can be quantitated by recording changes in absorbance at specific wavelengths. 

Resazurin is a chemical redox indicator that functions like the tetrazolium compounds. The resazurin reduction assay approach has successfully replaced tritiated thymidine incorporation for some HTS applications (Ahmed, Gogal, and Walsh 1994 Shum et al. 2008). Resazurin can be dissolved in physiological buffers resulting in an intense blue solution that is added directly to growing... 

It has long been known that oxidation of simple formazans gives tetrazolium compounds which, even under biological conditions, are easily reduced once more to red formazans. For instance, triphenyltetrazolium chloride, prepared by oxidizing triphenyl formazan, is an approved biological indicator. 

Like the sugar formazans (9), the formazans prepared from ozone-oxidized polysaccharides readily oxidize with A-bromosuccinimide to the corresponding colorless tetrazolium compounds. 

Hexokinase procedures in which indicator reactions produce colored products are also available, enabling absorbance to be measured in the visible range. An oxidation-reduction system containing phenazine methosulfate (PMS) and a substituted tetrazolium compound, 2-(p-iodophenyl)-3-p-nitrophenyl-5-phenyltetrazolium chloride (INT), is reacted with NADPH formed in the reaction. The reduced INT is colored with maximum absorbance at 520 nm. The PMS-INT color developer must be refrigerated... 

Due to their high chemical reactivity, certain NMs can directly interact with the assay components and catalyze reactions, giving false-positive or false-negative results. For example, some NMs (e.g., single-wall carbon nanotubes, carbon black) have been shown to reduce the tetrazolium compounds in the MTT andXTT cytotoxicity assays into the colored formazan without any cells present [61, 62], Moreover, certain NPs, like gold or platinum, were found to oxidize directly NADH, the reaction substrate LDH cytotoxicity assay. 

In his excellent general survey of the chemistry of formazans and of tetrazolium salts, Nineham mentioned some 500 formazans and over 200 tetrazolium compounds, but, because of the great interest in this group, their number has since increased. 

Preparation of Tetrazolium Compounds and Their Use as Biological Indicators... 

With the objective of obtaining a biological indicator less toxic than T.T.C., and in view of a new and gradually increasing interest in tetrazolium compounds, attempts have been made to prepare them by the oxidation of sugar formazans. ... 

Dehydrogenation with iV-bromosuccinimide transforms the formazans of oxidized polysaccharides into their tetrazolium derivatives. In an alkaline medium, these colorless or cream-colored compounds readily revert to the bright-red formazans, as with the simple tetrazolium compounds. The formazans of oxidized, soluble polysaccharides (for example, dextran, or soluble starch) yield soluble tetrazolium derivatives, and those of oxidized, insoluble polysaccharides (for example, cellulose, or xylan) give insoluble compounds. 

The formulas on page 160 illustrate the transformation of the formazan of periodate-oxidized cellulose to the corresponding tetrazolium compound. 

Polysaccharide tetrazolium compounds, particularly tetrazolium papers, seem to hold some prospect for use for analytical purposes or as biological indicators. 

Tetrazolium compounds, 107-109, 115 Tetronic acid, the term (in sugar chemistry), 171... 

Citotoxicity assays using murine neuroblastoma cells (Neuro-2A) have been proposed for the assessment of ciguatoxins and brevetoxins. The simplest method is based in the ability of metabolically active cells to reduce the tetrazolium compound MTT. After incubation with serial dilutions of test samples, cells are exposed to MTT and formation of color due to MTT reduction is read using a spectrophotometer. A new cytotoxicity assay is based on a c-fos-luciferase reporter gene that uses luciferase-catalyzed light generation as an endpoint and a microplate luminometer for quantification. After... 

Eye drops containing fluprednisolone acetate have been tested [80] in a way similar to that previously described for hydrocortisone acetate preparations [63], After submitting to appropriate test conditions, the steroid extracts were chromatographed on fluorescent silica gel layers, the separated steroid ester and alcohol spots localised in UV light, eluted and determined quantitatively by UV spectrophotometry or colorimetrically with tetrazolium compounds. The stability of tablets containing oestr-4-en-17 jS-ols and the influence of the quality of the auxiliary material... 

Aqueous Non-Radioactive Cell Proliferation Assay (Promega, Madison WI, USA) Briefly, this colormetric assay measures the bioreduction of a tetrazolium compound (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt MTS) to a formazan product. The conversion of MTS is directly proportional to the number of living cells in culture. 

Kong, Q. Anticancer tetrazolium compounds and their application. Faming Zhuanli Shenqing Gongkai Shuomingshu CN 1338256, 2002 Chem. Abstr. 2002,137, 363040. 

Ehrhardt, W. C. Cheng, L. Stasney, D. Whitaker, K. A. Corrosion inhibitor mixtures with tetrazolium compounds for protection of steel and metal parts in aqueous systems. PCT Int. Appl. WO 2000066810, 2000 Chem. Abstr. 2000, 133, 353724. 

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