Assay superoxide

Figure 8.21 Oxygen electrode assay. Superoxide dismutase (SOD) assay in which enzyme generates H2O2 that is further reduced at the Pt-cathode to the expense of oxygen. Current generated through reduction is calibrated with respect to oxygen produced by SOD.

Biological activities of LTB4 are measured by che-motactic assay, granule enzyme release assay, superoxide generation, hamster cheek pouch vascular permeability assay, rat mesenteric venule leukocyte adherence and emigration assay, lymphocytes proliferation assay, and leukocyte adhesion assay. 

Chemical Antioxidant Systems. The antioxidant activity of tea extracts and tea polyphenols have been determined using in vitro model systems which are based on hydroxyl-, peroxyl-, superoxide-, hydrogen peroxide-, and oxygen-induced oxidation reactions (109—113). The effectiveness of purified tea polyphenols and cmde tea extracts as antioxidants against the autoxidation of fats has been studied using the standard Rancimat system, an assay based on air oxidation of fats or oils. A direct correlation between the antioxidant index of a tea extract and the concentration of epigallocatechin gallate in the extract was found (107). 

Figure 7 Differentiation of HL-60 cells by the incubation with dimethylsulfoxide [DMSO] (a), and the assay of superoxide release in the DMSO-differentiated HL-60 cells by cytochrome C method (b).

Since it might be possible that the perturbation of membrane directly stimulated the NADPH-oxidase located on the cell membrane, which is the enzyme for the production of superoxide [24], the possibility was examined by the assay using detergent (Triton X-100) instead of polymers. At 0.001% of Triton X-100, no stimulation of superoxide release from DMSO-differentiated HL-60 cells was observed. At 0.01% of Triton X-100, a... 

Colepicolo, P., et al. (1990). A sensitive and specific assay for superoxide anion released by neutrophils or macrophages based on bioluminescence of polynoidin. Anal. Biochem. 184 369-374. 

Lucas, M., and Solano, F. (1992). Coelenterazine is a superoxide anion-sensitive chemiluminescent probe its usefulness in the assay of respiratory burst in neutrophils. Anal. Biochem. 206 273-277. 

Nakano, M. (1990). Assay for superoxide dismutase based on chemiluminescence of luciferin analog. Method. Enzymol. 186 227-232. 

Oxidant production is measured with the fluorogenic substrate para-hydroxyphenylacetic acid (PHPA) in the presence of superoxide dismutase and peroxidase (9). Under these conditions, superoxide is converted to H2O2 by the superoxide dismutase, and two molecules of PHPA are converted to a fluorescent diadduct by H2O2 and peroxidase. Similar assays have been devised using homovanillic acid (16) or scopoletin (17) instead of PHPA. 

In the presence of cytochrome C, changes in transmittance at 550 nm reflect oxidant production as superoxide reduces the cytochrome C. This is usually done as an absorbance assay in a spectrophotometer, but it can be performed as a transmittance assay on the SLM fluorometer (20). 

Figure 8. Simultaneous measurement of intracellular Ca and oxidant production in neutrophils. Cells were labeled with Quin-2 and suspended at 2 x lo cells/mL buffer. At time zero, 1 nJf FLPEP was added (upper trace in each panel). In addition, the receptor blocker tBOC was added (3 x 10" M) after 30 s to stop further binding of the stimulus (lower trace in each panel). The excitation wavelength was 3A0 nm. Top panel Quin-2 fluorescence determined on channel B (of Figure 1) using a Corion A90-nm interference filter. The crossover from the superoxide assay has been subtracted. Middle panel Oxidant production (superoxide equivalents) determined by the para-hydroxyphenylacetate assay. Fluorescence was observed at AOO nm (on channel A of Figure 1).

Misra, H.P. and Fridovich, I. (1972). The role of superoxide anion in the autoxidation of epinephrine and a simple assay for superoxide dismutase. Journal of Biological Chemistry 247 3170-3175. 

C. Beauchamp and I. Fridovich, Superoxide dismutase. Improved assays and an assay applicable to acrylamide gels. Anal. Biochem. 44, 276-287 (1971). 

Popov IN, Lewin G and von Baehr R. 1987. Photochemiluminescent detection of antiradical activity. I. Assay of superoxide dismutase. Biomed Biochim Acta 46(11) 775—779. 

Lucigenin-Amplified CL as a Sensitive and Specific Assay of Superoxide Detection... 

It is extremely important that the interaction of quinones with XO (Reaction (3)) is reversible that can lead to receiving erroneous results at the measurement of superoxide production by SOD-inhibitable cytochrome c reduction [28,29] (see also Chapter 27). Lusthof et al. [30] demonstrated that 2,5-bis(l-aziridinyl)-l,4-benzoquinones are directly reduced by XO. Interestingly at quinone concentrations greater than 25pmol I 1, quinones entirely suppressed one-electron reduction of dioxygen, and cytochrome c was completely reduced by the semiquinones formed. It is well known that cytochrome c and lucigenin are effective superoxide scavengers and due to that, these compounds are widely used in the quantitative assays of superoxide detection. Nonetheless, under certain experimental conditions they can be directly reduced by XO [31]. 

Neither Suzuki et al. [206] nor Scott et al. [207] found any effect of LA on superoxide production by xanthine oxidase. Scott et al. also concluded that DHLA is incapable of reacting with superoxide. The last conclusion seems highly improbable. The ability of superoxide to react with thiols with the rate constants equal to 105 to 106lmol 1s 1 has been shown in chemical studies [208]. Dikalov et al. [209] estimated the rate constant for the reaction of DHLA with superoxide as (4.8 + 2)x 105 lmol-1 s-1 using the competition experiments with spin trap DMPO, which is very close to the previous value of (7.3+ 0.24) x 105 1 mol 1 s 1 reported for this reaction [210]. Negative results obtained by Scott et al. [207] are probably explained by the use of unreliable NBT assay for superoxide detection [211]. 

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] ... 

However, to be a quantitative assay of superoxide detection, Reaction (1) had to be an exothermic reaction, i.e., the difference between the one-electron reduction potentials of reagents AE° = / °[02 /02] / °[A /A] must be <0. In this case the rate constants of Reaction (1) will be sufficiently high (10s—109 1 mol 1 s ). Among traditionally applied assays, three compounds satisfy this condition cytochrome c, lucigenin, and tetranitromethane (Table 32.1). 

The efficiency of superoxide assays strongly depend on the nature of superoxide producers. Significant difficulties arise in the detection of superoxide in cells and tissue. Cytochrome c is unable to penetrate cell membranes and therefore, can be used only for the measurement of extracellular superoxide. Furthermore, SOD-inhibitable cytochrome c reduction is difficult to apply in nonphagocytic cells and tissue due to the complications of measuring low rates of superoxide release, direct reduction of cytochrome c by cellular enzymes, the reoxidation of reduced cytochrome by hydrogen peroxide, etc. [8], Moreover, in nonphagocytic cells superoxide is formed exclusively inside the cells and is not released outside as in phagocytes. These circumstances severely limit the number of analytical methods, which can be used for superoxide detection in vasculature. 

It has earlier been suggested to make cytochrome c a more specific reagent for superoxide detection by its acetylation or succinoylation [9-11], It was proposed that acetylation and succinoylation must cause a greater decrease in the reaction of cytochrome c with NADPH cytochrome P-450 reductase than with superoxide due to a decrease in the electrostatic charge of native cytochrome c [12]. However, the rate constant for the most selective succinoylated cytochrome c became about 10% of native cytochrome [13], making this assay even less sensitive. 

Lucigenin (bis-yV-rnethylacridinium)-amplified CL, which is produced by Reactions (10), (11), or Reactions (12), (11), is probably the most specific assay of superoxide detection. 

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