The nitroblue tetrazolium reduction

The first quantitative NBT test was developed by Baehner and Natham in 1968 to detect neutrophil dysfunction in patients with chronic granulomatous disease (CGD). The neutrophil membranes of CGD patients lack the specific cytochrome 245 which is a key component in generating superoxide by the cell, following bacterial stimulation of the cell. As a result the patients neutrophils are incapable of responding with a respiratory burst, failing to generate the free radicals necessary for the antimicrobial function of this cell. 

Principle The respiratory burst of phagocytic cells can be assessed by incubating a suspension of the cells in an isotonic solution of the yellow oxidised nitroblue tetrazolium (NBT) dye. During this process, the soluble dye interacts with the cytoplasmic components associating with the oxidant species generated. Although the NBT test is not a specific marker for superoxide production, NBT reduction by activat- 

In normal healthy individuals, the spontaneous reduction of NBT dye by neutrophils is very low (less than 10% of the cells are positive). A low basal NBT response is an important negative control during the performance of any NBT test, as it monitors the non-stimulatory nature of the test reagents and procedure. Stimulated NBT tests are carried out as a positive control. By deliberately stimulating cells with specific stimulating agents, such as phorbol myristate acetate (PMA) one can activate cells from normal individuals to reduce NBT in a dose-responsive manner. 

Sterile endotoxin free water for irrigation (Travenol). 

Ten times and single strength HBSS (Hanks balanced salt solution). 

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In anaesthetised new-born pigs injury of moderate severity (1.9 to 2.1 atm) induced by the lateral fluid percussion brain injury technique staurosporine (10" M/1) blocked the nitroblue tetrazolium reduction after phorbol 12,13-dibutyrate and blunted the nitroblue tetrazolium reduction observed after lateral fluid percussion brain injury (1 1 to 15 2 versus 1 1 to 5 1 pmol/mm after lateral fluid percussion brain injury in the absence versus presence of staurosporine) (Armstead 1999). 

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] ... 

Systems used to detect Oj include cytochrome c reduction (M4,M11,S4), nitroblue tetrazolium reduction (Bll,S3), adrenalin (M24,S19), hydroxyamine (M3), and a hydroxyamine derivative (E2,K14,K15). NADH/lactate dehydrogenase (B15), 6-hydroxydopamine (H10), sulfite (F8.G13), pyrogallol (M3), tetranitro-methane (M19), and dianisidine (G13.M24) are frequently employed. NAD(P)H oxidation has also been employed to generate Oj. In this system, NAD(P)H is the 0 detector (P2). SOD activity has also been estimated using hematoxylin autooxidation and chemiluminescence of a luciferin analog (K8,N6). In the case of illuminated flavin, nitroblue tetrazolium was used for the Oi detection (S3). 

Nishikimi et al., (1972) developed an assay for superoxide dismutase, using phenazine methosulfate which is structurally related to flavin. In this assay phenazine methosulfate is reduced by NADH, and on reoxidation, O2 was generated. The O2 was detected with nitroblue tetrazolium, and maximum inhibition of blue formazan formation by superoxide dismutase was 95% indicating that 5% of the reduction was due to direct interaction between the nitroblue tetrazolium and the reduced phenazine methosulfate. Addition of around 30 ng copper/zinc superoxide dismutase resulted in 50% inhibition of formazan formation. The assay was also used to locate the enzyme on polyacrylamide gels. 

Recently, the superoxide dismutase activity of low molecular mass copper chelates in the indirect coupled assay systems has been dispute It was demonstrated that copper in CuSO and Cu(II)(gly)2 prevents the ferricytochrome c and nitroblue tetrazolium reduction. This is not virtually new, as it is a well known phenomenon that Cu(II)-salts lead to a reoxidation of ferrocytochrome c and that they are potent inhibitors of xanthine oxidase which is often used as Oj" -generator in indirect SOD assay systems Although the indirect assays may be sometimes inadequate for the measurement of the SOD-activity, there are no doubts that low molecular mass copper chelates have their superoxide dismutase during pulse radiolysis. 

Naked monosodium urate crystals stimulate oxygen radical release by human polymorphonuclear leukocytes. Very recently, two papers dealing with superoxide anion generation by human neutrophils exposed to monosodium urate have been published. From the comparison of the data derived from nitroblue tetrazolium and cytochrome c reduction, it has been suggested that radical production in response to urate crystals is compartmentalized and occurs predominantly in the intracellular space. Microcrystalline sodium urate-induced oxygen consumption and nitroblue tetrazolium reduction were present for at least 15 min after stimulation of the cells. To enhance extracellular radicals neutrophils were converted to secretory non phagocitic cells by use of cytochalasin... 

The Nitroblue tetrazolium test (NET), as applied to the infective problems (30) presents an intriguing development. This phenomenon of reduction of colorless Nitroblue tetrazolium to blue/ black forazan by the neutrophil of normal man in vitro (7) was applied to the phagocyte in vivo during bacterial infection in infants by Park in 1968 (46). Later confirmation was obtained with adults (27,41) in bacterial infection and in fungal infection (5,46). 

Phagocytosis, bactericidal activity and nitroblue tetrazolium reduction by the rabbit neutrophil in protein malnutrition, Indian J. Med. Res. 62 42. 

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... 

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 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. 

Phagocytosis and the reduction of nitroblue tetrazolium to formazan was impaired by chromium(III) but not chromium(VI). These effects represent a decrease in the functional and metabolic activity of the macrophage (Johansson et al. 1986a, 1986b). Mice exposed to chromium trioxide mist at concentrations of 1.81 and 3.63 mg chromium(VI)/m3 intermittently for i2 months developed perforations in the nasal septum, hyperplastic and metaplastic changes in the larynx, trachea, and bronchus, and emphysema (Adachi 1987 Adachi et al. 1986). 

Figure 6-17. Reactions involved in coupling oxidation of lactate with the reduction of p-nitroblue tetrazolium chloride.

In this test, p-hydroxybutyrate in the presence of NAD is converted by P-hydroxybutyrate dehydrogenase to acetoacetate, producing NADH. Diaphorase catalyzes the reduction of nitroblue tetrazolium (NBT) by NADH to produce a purple compound and its absorbance is read in a special meter that provides a digital readout. 

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