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Tetrazolium salts in clinical biochemistry

Tetrazolium salts are widely used in cell biology and clinical biochemistry for measuring the metabolic activity of mammalian and microbial cells. Major tetrazolium
salts include
MTT (3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2H-tetrazolium bromide), INT (2-(4-Iodophenyl)-3-(4-nitrophenyl)-5-phenyl-2H-tetrazolium chloride),
TTC (2,3,5-Triphenyl-2H-tetrazolium chloride), MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium), XTT (2,3-bis-(2-
methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide), and
NBT (2,2′-bis(4-Nitrophenyl)-5,5′-diphenyl-3,3′-(3,3′-dimethoxy-4,4′-diphenylene)ditetrazolium
chloride 3,3′-(3,3′-Dimethoxy-4,4′-biphenylene)bis[2-(4-nitrophenyl)-5-phenyl-2H-tetrazolium chloride]). These dyes are somewhat heat and light unstable, and are
typically reduced by dehydrogenases, reductases, and reducing agents to formazan dyes, which display a broad spectrum of colors from dark blue, deep red, to
orange, depending on the original tetrazolium salt used for reaction. Second generation tetrazolium dyes such as XTT and MTS form water-soluble formazans and
require an intermediate electron acceptor for reduction.

MTT assay is often used to assess cell viability, cell proliferation, and drug toxicity. Although MTT reduction is primarily associated with mitochondria metabolic
activity, cytoplasmic dehydrogenases and reductases are also responsible in part for its reduction. When reduced in a cell, either enzymatically or through direct
reaction with NADH or NADPH, MTT turns to an insoluble dark blue precipitate, which can be easily extracted with DMSO for quantification.

INT is frequently used in assays for various dehydrogenases. For instance, our
lactate dehydrogenase (LDH) assay first converts L-lactate to pyruvate with
production of NADH, which then reduces INT to a cherry red formazan product that can be quantified by absorbance at 492 nm. The second reaction is facilitated
by a diaphorase, which belongs to a ubiquitous class of flavin-bound enzymes capable of catalyzing the reduction of various dyes. Either NADH or NADPH may be
used as a reductant in the diaphorase reaction.

TTC is another tetrazolium dye used to differentiate between metabolically active and inactive tissues. The white compound is enzymatically reduced to red TPF
(1,3,5-triphenylformazan) in living tissues due to the activity of various dehydrogenases while it remains as white TTC in necrotic tissues lacking active
dehydrogenases and reductases. This color contrast renders the TTC dye popular in heart research for identification of infarcted tissue caused by acute
myocardial ischemia.

NBT is used in basic and clinical immunology for sensitive detection of alkaline phosphatase (ALP) in conjunction with BCIP. In this application, NBT serves as the
oxidant and BCIP is the ALP substrate, generating a dark blue color. Since ALP is often conjugated to an antibody, the colored product reveals where the antibody
is bound. Another clinical application of NBT is for diagnosis of chronic granulomatous disease and other diseases of phagocyte dysfunction. These disorders can
involve a defect in NADPH oxidase, and therefore the phagocyte is unable to make reactive oxygen species (ROS) required for microbial killing. A high blue score
means a high level of ROS production, meaning a robust phagocyte function.
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