Spin trapping using DMPO

It is conceivable that DMPO/ OH is formed from oxidation of DMPO (to a radical cation) by the ferryl species, followed by hydrolysis, and/or from trapping of OH derived from H2O2 as follows  

Another caveat in spin trapping with DMPO is that the DMPO/ OH could be formed from the reduction of DMPO-OOH. However, if this were to occur, the DMPO/ OH will be inhibited by SOD and not by catalase. 

An alternate pathway for production of OH has recently been described that is independent of iron via the reaction between 0 2 and NO forming the peroxynitrite ( OONO)  

It is not clear whether production of NO during ischemia and reperfusion leads to deleterious or beneficial effects. There is conflicting evidence on the role of NO during reperfusion [122-124], 

The best way to prove the existence of the hydroxyl radical is to perform kinetic competition experiments with hydroxyl-radical scavengers [125]. Using the kinetic criterion, we can also exclude the intermediacy of the ferryl oxidant. However, such experiments in isolated heart models are, in practice, very difficult. 

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Thus, superoxide itself is obviously too inert to be a direct initiator of lipid peroxidation. However, it may be converted into some reactive species in superoxide-dependent oxidative processes. It has been suggested that superoxide can initiate lipid peroxidation by reducing ferric into ferrous iron, which is able to catalyze the formation of free hydroxyl radicals via the Fenton reaction. The possibility of hydroxyl-initiated lipid peroxidation was considered in earlier studies. For example, Lai and Piette [8] identified hydroxyl radicals in NADPH-dependent microsomal lipid peroxidation by EPR spectroscopy using the spin-trapping agents DMPO and phenyl-tcrt-butylnitrone. They proposed that hydroxyl radicals are generated by the Fenton reaction between ferrous ions and hydrogen peroxide formed by the dismutation of superoxide. Later on, the formation of hydroxyl radicals was shown in the oxidation of NADPH catalyzed by microsomal NADPH-cytochrome P-450 reductase [9,10]. 

Reported rate constants for the reaction of 02 with GSH have varied from 102 to > 105 M 1 s. A re-examination of this reaction by spin trapping with DMPO established that earlier studies had been confounded by the direct reduction of the DMPO/ OOH adduct to DMPO/ OH by GSH. Taking account of this reaction, the revised rate constant was reported to be 200 M-1 g-i.25i.2S2 other workers have examined, for example, the effects of GSH and N-acetyl-L-cysteine on lipid peroxidation 253 and the role of GS in the toxicity of the diabetogenic agent alloxan.254 Direct EPR has been used to detect binuclear Cu(II) complexes of homocysteine. The interactions of such complexes with blood-vessel linings may account for the link between elevated homocysteine and atherosclerosis.255... 

Kochany and Bolton (1992) studied the primary rate constants of the reactions of hydroxyl radicals, benzene, and some of its halo derivatives based on spin trapping using detection by electron paramagnetic resonance (EPR) spectroscopy. The competitive kinetic scheme and the relative initial slopes or signal amplitudes were used to deduce the kinetic model. Based on a previously published rate constant (4.3 x 109 M 1 s ) in the pH range of 6.5 to 10.0 for the reaction of hydroxyl radicals with the spin trap compound 5,5 -d i methy I pyrro I i ne N-oxide (DMPO), rate constants for the reaction of hydroxyl radicals with benzene and its halo derivatives were determined. 

Detection of primary radical anions in microsomal incubations generally is possible only under anaerobic conditions. In the presence of air a redox cycle is set up, as referred to above. Thus, in initially aerobic systems, the primary radical can be detected only after a lag time that corresponds to the time required to deplete oxygen in the medium. During this lag time the superoxide radical is generated, which can be spin trapped using the nitrone 5,5 -dimethylpyrroline-l-oxide (DMPO) [187]. 

We also attempted to detect another key member of ROS, hydroxyl radical based on the formation of spin-trapped aduct (DMPO-OH) using ESR (Fig. 3). By circulating the spin trapping agent in the system, we observed the UV-dependent development of DMPO-OH signal with time. While we could detect the presence of hydroxy radicals in the circulating water, no signal could be detected in the water samples isolated from the photochemical reaction apparatus. 

Recently, evidence for the formation of hydroxyl radicals on BDD has been reported [5] by means of spin-trapping using 5,5-dimethyl- 1-pyrroline-N-oxide (DMPO). 

ESR studies on the initial free radicals were carried out by using MNP(2-methyl-2-nitrosopropane) or DMPO (5,5-dimethylpyrroline N-oxide) as the spin-trapping agent. The reactions are shown as ... 

Spin trapping experiments have been performed recently in a fuel cell inserted in the ESR resonator ("in situ" cell), using DMPO and a-(4-pyridyl-l-oxide)-N-ferf-butylnitrone (POBN) as the spin traps [78,82,83], These experiments allowed the separate examination of spin adducts at the anode and cathode sides. 

A spin trap is a diamagnetic compound that reacts with a radical by addition of the radical functionality typically to a double bond in the trap, thus forming a new radical that is more stable (better, less unstable) than the original radical. By far the most common class of spin traps are nitrone compounds that, upon addition of the primary radical, produce a stable aminoxyl radical (Figure 10.1). The compound DMPO is the paradigmatic spin trap it is readily available, widely used, and its EPR spectra are relatively easy to interpret. Some of its radical adducts have unpractically short lifetimes. 

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]. 

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-... 

Special spin-trapping techniques are also available for the detection of short-lived radicals in both homogeneous and heterogeneous systems. For instance, a-phenyl A-ferf-butyl nitrone (PBN), ferf-nitrosobutanc (f-NB), -(4-pyridyl A-oxidc) A-ferf-butyl nitrone (4-POBN), or 5,5-dimethyl-l-pyrroline A-oxidc (DMPO) can be made to react with catalytic intermediates to form stable paramagnetic adducts detectable by ESR [135], Radicals evolving into the gas phase can also be trapped directly by condensation or by using matrix isolation techniques [139],... 

The last few years have seen numerous applications of spin trapping to biological systems, and in these the trapping of hydroxyl radicals has assumed some importance. This work has been confined almost exclusively to nitrone scavengers 4 the fact that the hydroxyl adduct [6] of DMPO is much more persistent than that [7] of the commonly used nitrone, benzylidene-t-butylamine-N-oxide ( phenyl t-butyl nitrone ,3 or PBN) [3], may be due to a fragmentation reaction, with subsequent oxidation of the cr-hydroxybenzyl radical, as shown. 

Work by Harbour, Chow and Bolton (1974) on the spin adducts of superoxide (or HOO )13 with nitrones paved the way for a number of investigations of superoxide and hydroperoxyl radical chemistry. Harbour and Bolton (1975) used DMPO to trap superoxide formed by spinach chloroplasts in the presence of 02. The signal strength was greatly enhanced when methylviologen was present, consistent with the hypothesis that this bis-pyridinium dication accepts an electron from the primary acceptor of photoprotein I, and then transfers it to molecular oxygen. 

In this type of spin traps, 5,5-dimethyl-l-pyrroline-Af-oxide (DMPO) deserves particular mention. DMPO is widely employed as a spin trap in the detection of transient radicals or ion-radicals in chemical and biological systems (see, e.g., Siraki et al. 2007). Characteristic ESR spectra arising from the formation of spin adducts are used for identification of specific spin species. In common opinion, such identification is unambiguous. However, in reactions with superoxide ion (Villamena et al. 2004, 2007b), carbon dioxide anion-radical (Villamena et al. 2006), or carbonate anion-radical (Villamena et al. 2007a), this spin trap gives rise to two adducts. Let us consider the case of carbonate anion-radical. The first trapped product arises from direct addition of carbonate anion-radical, second adduct arises from partial decarboxylation of the first one. Scheme 4.25 illustrates such reactions based on the example of carbonate anion-radical. 

The sequence of events following the reaction of metMb with hydrogen peroxide has also been investigated through the use of spin trapping agents. Initial studies of this type with 5,5-dimethylpyrroline N-oxide (DMPO) led to the identification of Y103 as the primary site of DMPO adduct formation, a modification that was blocked by specific iodination of this residue 187). The identity of the radical trapped in this reaction... 

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