Nitroblue tetrazolium

Figure 7-11. Normal and pathologic patterns of lactate dehydrogenase (LDH) isozymes in human serum. LDH isozymes of serum were separated by electrophoresis and visualized using the coupled reaction scheme shown on the left. (NBT, nitroblue tetrazolium PMS, phenazine methylsulfate). At right is shown the stained electropherogram. Pattern A is serum from a patient with a myocardial infarct B is normal serum and C is serum from a patient with liver disease. Arabic numerals denote specific LDH isozymes.

Early methods of superoxide detection are well known and described in many books and reviews. They include cytochrome c reduction, nitroblue tetrazolium reduction, spin trapping, etc. (see, for example, Ref. [1]). The most efficient assays are based on the ability of superoxide to reduce some compounds by one-electron transfer mechanism because such processes (Reaction (1)) proceed with high rates [2] ... 

Nitroblue tetrazolium (NBT, 3,3 -(3,3,-dimethoxy-l,l,-biphenyl-4,4 -diyl)bis-2-(4-nitrophe-nyl)-5-phenyl-2H-tetrazolium dichloride) is reduced by superoxide to formazan as a final product, which can be measured spectrophotometrically [73]. Although the rate constant for NBT reduction by superoxide is moderately high 5.88+0.12x 104 1 mol 1 s 1 [74], the formation of formazan is not a simple one-electron transfer process, and the final product is formed as a result of disproportionation of intermediate free radicals. Similar to cytochrome c, NBT is easily reduced by the other reductants that confines its application for superoxide detection. Moreover, similar to epinephrine, NBT free radical is apparently... 

Enzymes. Enzymes too are useful labels and several have been employed, with peroxidase and alkaline phosphatase being the most popular to date. One important feature of this technique is for the enzyme to be able to convert a soluble substrate into an insoluble product in order to localize the antigen properly. Several suitable substrates are available with 3 3 -diaminobenzidine (DAB) and 3-amino-9-ethylcarbazole (AEC) being used with peroxidase and 5-bromo-4-chloro-3-indoylphosphate/nitroblue tetrazolium (BCIP/NBT) with alkaline phosphatase. 

Stopped-flow measurements with superoxide in aqueous solution at physiological pH are not possible due to its fast self-dismutation under these conditions. Therefore, the indirect assays such as McCord-Fridovich, adrenalin and nitroblue tetrazolium (NET) assays are widely used in the literature, not only for qualitative but also for quantitative detection of SOD activity of small molecular weight mimetics 52). Not going into details, we just want to stress that the indirect assays have very poor even qualitative reliability, since they can demonstrate the SOD activity of the complexes which does not react with superoxide at all. It has been reported in the literature that this is caused by the interference of hydrogen peroxide 29). We have observed that the direct reaction between complexes and indicator... 

Colorimetric methods were developed as a nonradioactive alternate method of detection. Enzymes such as HRP or AP, which are usually conjugated to the secondary antibody, can convert substrates to colored precipitates that accumulate on the blot and generate a colored signal. Such substrates include 5-bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium (BCIP/NBT) for AP and 3,3 -diaminobenzidine... 

Chronic granulomatous disease is most frequently aused by genetic deficiency of NADPH oxidase in the PMN. Patients are susceptible to infection by catalasepositive organisms such as Staphylococcus aureus, Klebsiella, Escherichia coll, Candida, end Aspergillus, k negative nitroblue tetrazolium test is useful in confirming the diagnosis. 

Add the commercially available substrate solution containing 5-Bromo-4-chloro-3-indolyl Phosphate (BCIP)/Nitroblue Tetrazolium (NBT) (SIGMA) to the blot till it is just submerged. 

The wide range of chromogenic-substrate systems available allows one to obtain excellent color contrast for double/multiple antigen detection. Chromo-genie reactions resulting in black (IGSS), yellow-brown (IPO/DAB), red-brown (IPO/AEC), blue (lAP/Fast Blue, or 5-bromo-4-chloro-3-indolyl phosphate/ nitroblue tetrazolium [BCIP-NBT]), and purple (lAP/Fast Red or New Fuchsin) can be used in different combinations (see Chapter 23). For development of these products, the following protocols are provided (see Note 16). 

Thyroid 1 uptake may be decreased. False-negative results with the nitroblue-tetrazolium test for bacterial infection. Dexamethasone, given for cerebral edema, may alter the results of a brain scan (decreased uptake of radioactive material). 

NBT, 3,3 -(3,3 -dimethoxy-l,l -biphenyl-4,4 -diyl)bis 2-(4-nitrophenyl)-5-phenyl-2H-tetrazolium dichloride, nitroblue tetrazolium NDGA, nordihydroguaiaretic acid NF- , redox-regulated transcription factor NHA, iM -hydroxy-L-arginine... 

FIGURE 3.2 Effect of retinoic acid (RA) and various lipids on cell differentiation of HL-60 cells (Tochisawa et al., 1997). HL-60 cells (5xl04) were incubated with the individual lipids for 24h, then RA was added followed by incubation for additional 72h. Nitroblue tetrazolium (NBT) reducing activity in HL-60 cells was assayed. Data shown as mean value SD. p<0.01 versus control. p<0.01 versus RA. 

A superoxide dismutase activity had been reported for the Fe-EDTA complex in contrast with the inactivity of the Cu-EDTA complex. It was shown, on the contrary, that Fe-EDTA, instead of catalysing the dismutation of OJ, interferes with the reduction of nitroblue tetrazolium and of Fe(III)-cytochrome c in the assays of the dismutase activity... 

Human ceruloplasmin inhibits lipid autoxidation induced by ascorbate or inorganic Fe It is considered an acute-phase protein with a beneficial effect in inflammation . It was suggested that ceruloplasmin acts as a scavenger of OJ radicals, as it inhibited the reduction of Fe(III)-cytochrome c and of nitroblue tetrazolium in the presence of xanthine oxidase, acetaldehyde, and dioxygen as an OJ-generating system A mechanism without reduction of Cu , similar to that... 

The evidence that O is a product of the burst has already been summarized. Another direct consequence of the formation of O is the reduction of nitroblue tetrazolium to form an insoluble deposit of blue formazan. The reduction of nitro blue tetrazolium had been observed to occur in PMNs in patients with bacterial infections. In intact cells this appears to be a consequence of the formation of O it fails to occur in PMNs from patients with chronic granulomatous disease which cannot undergo the respiratory burst it is diminished by anaerobiosis and it is inhibitable by superoxide dismutase... 

Note Superoxide reacts with nitroblue tetrazolium to produce a deep purple color measured at 560 nm. 

Note Detection is based on reduction in the purple color measured at 560 nm and produced by the reaction between superoxide and nitroblue tetrazolium. a The starred figures overstate the actual antioxidant power of the agents involved. See Table 10.4. 

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