Superoxide detection

Release of superoxide during ORR catalysis indicates that the ferric-superoxo intermediate (Fig. 18.20) has a substantial residence time at 0.2 V (the potential of the maximum production of superoxide), suggesting that the potential of the ferric-superoxo/ferric-peroxo couple, (Fig. 18.20), is more reducing than 0.2 V. The fraction of superoxide detected at potentials >0.2 V probably reflects the fact that 02, which is a strong outer-sphere reductant [Huie and Neta, 1999], was oxidized by the mostly ferric catalytic film before it could escape the film. There are two plausible explanations for the decrease in the fraction of superoxide byproduct released at... 

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]. 

Controversial results of oxygen radical detection in mitochondria have been described in the literature. Owing to experimental difficulties many authors were obliged to work with sub-mitochondrial particles instead of the whole mitochondria. However, it is quite possible that oxygen radical production by submitochondrial particles may be artificially enhanced due to exposure to oxygen. On the other hand, some analytical methods of superoxide detection such as cytochrome c reduction cannot be used due to the direct reduction of cytochrome by mitochondrial components. 

In earlier studies [5,6] superoxide detection in mitochondria was equated to hydrogen peroxide formation. However, while it is quite possible that superoxide is a stoichiometric precursor of mitochondrial hydrogen peroxide, it is understandable that the level of hydrogen peroxide may be decreased due to the reactions with various mitochondrial oxidants. Moreover, superoxide level can be underestimated due to the reaction with mitochondrial MnSOD. Several authors [7,8] assumed that mitochondrial superoxide production may be estimated through cyanide-resistant respiration, which supposedly characterizes univalent dioxygen reduction. This method was applied for the measurement of superoxide production under in vitro normoxic and hyperoxic conditions, in spite of the finding [7] that cyanide-resistant respiration reflects also the oxidation of various substrates (lipids, amino acids, and nucleotides). Earlier,... 

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). 

SOD-inhibitable one-electron reduction of ferric cytochrome c is probably the most frequently used method of superoxide detection. 

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. 

Spin trapping has been widely used for superoxide detection in various in vitro systems [16] this method was applied for the study of microsomal reduction of nitro compounds [17], microsomal lipid peroxidation [18], xanthine-xanthine oxidase system [19], etc. As DMPO-OOH adduct quickly decomposes yielding DMPO-OH, the latter is frequently used for the measurement of superoxide formation. (Discrimination between spin trapping of superoxide and hydroxyl radicals by DMPO can be performed by the application of hydroxyl radical scavengers, see below.) For example, Mansbach et al. [20] showed that the incubation of cultured enterocytes with menadione or nitrazepam in the presence of DMPO resulted in the formation of DMPO OH signal, which supposedly originated from the reduction of DMPO OOH adduct by glutathione peroxidase. 

Another approach to this problem is a search for the other more effective spin traps. Frejaville et al. [23] demonstrated that the half-life of spin-adduct of superoxide with 5-(diethoxyphosphoryl)-5-mcthyl-l -pyrrolinc-/V-oxide (DEMPO) is about tenfold longer than that of DMPO OOH. Despite a much more efficiency of this spin trap, its hydrophilic properties limit its use for superoxide detection in lipid membranes. Stolze et al. [24] studied the efficiency of some lipophilic derivatives of DEMPO in the reaction with superoxide. These authors demonstrated a higher stability of superoxide spin-adducts with 5-(di- -propoxypho-sphoryl)-5-methyl-1 -pyrrolinc-A -oxidc (DPPMPO) and 5-(di- -butoxyphosphoryl)-5-methyl-... 

Another type of spin traps, which have been recommended for the detection of superoxide, are the derivatives of hydroxylamine. In 1982, Rosen et al. [25] showed that superoxide is able to oxidize the hydroxylamine derivative 2-ethyl-1-hydroxy-2,5,5-trimethyl-3-oxazoli-dine (OXANOH) to corresponding free radical 2-ethyl-1-hydroxy-2,5,5-trimethyl-3-oxazolidinoxyl (OXANO). Although this radical is very stable and easily identified by its ESR spectrum, it is also easily reduced by ascorbic acid and other reductants. Furthermore, OXANOH and other hydroxylamines are oxidized by dioxygen in the presence of transition metal ions to form superoxide, and therefore, superoxide detection must be carried out in the presence of chelators. 

Later on, other hydroxylamine derivatives such as 1-hydroxy-2,2,6,6-tetramethyl-4-oxo-piperidine (TEMPONEH) and l-hydroxy-3-carboxy-pyrrolidine (CP-3) have been used for superoxide detection [26]. It was found that these spin traps react with both superoxide and peroxynitrite and that they might be applied for quantification of these reactive species [27]. The CP-3 radical is less predisposed to reduction by ascorbic acid and therefore is probably more suitable for superoxide detection in biological systems. 

There is also a big uncertainty in published rate constants for Reaction (6). In Refs. [25 27] these rate constants are found to be of 103 1041 mol 1 s 1. However, these values are apparently overestimated because Bielski et al. [28] earlier showed that the rate constant for the reaction of superoxide with hydroxylamine does not exceed 301 mol 1 s 1. Thus, the use of hydroxylamines as spin traps for superoxide detection has several disadvantages. 

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. 

Another well-known CL amplifier, which is also frequently used for superoxide detection in biological systems, is luminol (5-amino-2,3-dihydro- 1,4-phthalazinedione). It has been proposed that luminol semiquinone reacts with superoxide to form the peroxide intermediate, whose decomposition is accompanied by chemiluminescence [62]. 

Although the use of luminol-amplified CL for superoxide detection in cell-free and cellular systems yielded many important data, which were confirmed by the other analytical methods, these data are more questionable than in the case of lucigenin-amplified CL. At the first stage (Reaction (14)) luminol must be oxidized to luminol semiquinone, and therefore, the whole... 

At present, other CL amplifiers are recommended for the detection of superoxide in cells and tissue such as coelenterazine (2-(4-hydroxybenzyl)-6-(4-hydroxyphenyl)-8-benzyl-3,7-dihydroimidazo[l,2-a]pyrazin-3-one]) and its analogs CLA (2-methyl-6-phenyl-3,7-dihydroi-midazo[l,2-a]pyrazin-3-one]) and MCLA [2-methyl-6-(4-methoxyphenyl)-3,7-dihydroimi-dazo[l,2-a]pyrazin-3-one]). It has been suggested that the origin of CL produced by these compounds is the oxidation of the acetamidopyrazine moiety [69,70]. Unfortunately, to our knowledge, there are still no reliable thermodynamic and kinetic data to validate the application of the above CL amplifiers for superoxide detection. Reichl et al. [71] proposed to use the photoprotein pholasin for the detection of superoxide and myeloperoxide activity in stimulated neutrophils. 

One of the oldest methods of superoxide detection is the oxidation of epinephrine [72]. This method has the typical disadvantages of oxidizable compounds due to the possibility of the nonsuperoxide-mediated oxidation of epinephrine. Still, SOD-inhibitable epinephrine oxidation might be used as a superoxide assay [72]. 

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... 

Gardner and Fridovich [85] proposed that the inactivation of aconitase might be used as an assay of superoxide formation in cells. The mechanism of the interaction of superoxide with aconitases has been considered in Chapter 21. As follows from data presented in that chapter, peroxynitrite is also able to inactivate aconitases rapidly therefore, this method cannot be a specific assay of superoxide detection. 

OXYGEN, OXIDES 0X0 ANIONS PHOTOOXYGENATION SUPEROXIDE DETECTION/MEASUREMENT SUPEROXIDE DISMUTASES SUPERACID... 

Spin trapping has been widely used for superoxide detection in various in vitro systems... 

---