Spin traps DMPO

TEMPO, and any of its (not too bulky) derivatives, is comparable in molecular mass with the spin trap DMPO, so the tumbling in water at ambient temperature should again average out all anisotropy. The spectrum is even simpler (namely, three identical lines see the high-temperature traces in Figure 10.4) than that of the hydrox-yl-DMPO adduct because only the 14N nuclear spin contributes to the spectrum ... 

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]. 

However, it has been shown that the incubation of SOD with hydrogen peroxide in the presence of spin-trap DMPO resulted in the appearance of the ESR spectrum of DMPO OH... 

The data with the spin trap DMPO were very similar to those of Tauber et al. 127, ns) measuring the formation of ethylene from methional or KMB. Unfortunately, as with the use of oxidizable organic molecules, the specificity for OH of the spin adduct identified in the experiments with ESR is not clear. A critical, cautionary review of this approach has recently appeared... 

Figure 16.10. Chemical structure of spin-trap DMPO (diamagnetic), reacting with hydroxyl radical ( OH), showing the formation of spin-adduct DMPO-hydroxyl radical ( OH) and typical DMPO-OH adduct EPR signal.

Spin traps also exhibit site-specific radical reactions [56]. The water-soluble spin trap DMPO can only trap the OH produced extracellularly, whereas the lipophilic spin trap PBN can trap intracellular OH (Fig. 1). Spin adducts can also undergo enzymatic and chemical reduction to hydroxylamines[57]. The localization of spin adducts and hydroxylamines is dependent upon their partition coefficients. 

Figure 2. Detection of 02- in BAEC upon treatment with CsA. Left representative experiment of superoxide detection in supernatants of BAEC measured by electron spin resonance (ESR) using the superoxide-sensitive spin trap DMPO. The superoxide generator DMNQ was used as a positive control. Right flow cytometiy detection of 02 with die probe DHE. BAEC were preincubated for 1 hour with 5 pM DHE and then incubated for 2 hours with the indicated doses of CsA. Data are represented as mean intracellular fluorescence of ethidium (oxidized fluorescent form of DHE) (n = 7). p < 0.05 vs CsA vehicle (0 pM CsA).

In an effort to determine whether other radical species are generated on the globin, EPR spin-trapping experiments were carried out using the spin trap DMPO (5,5-dimethyl-1-pyrroline-iV-oxide). In these experiments, unlike those carried out with myoglobin, no significant signals were observed (101). At the time the interpretation... 

The spin trap, DMPO, is most often used to determine the presence of reactive oxygen species and, in particular, it is erroneously used to establish ... 

Spin-traps DMPO (5,5-dimethyl-l- pyrroline-N-oxide), MNP (2-methyl-2-nitroso-propane, dimer), dG (2 -deoxyguanosine monophosphate), 8-HOdG standard (8-hydroxy-2 -deoxyguanosine) and deferoxamine mesylate were purchased from Sigma phosphate buffer (pH 7.4), hydrogen peroxide (H2O2) (30%), were purchased from Merck. All other chemicals used were of analytical quality. 

By determination of the parameters of the spin adduct spectrum it is often possible to identify the nature of the primary trapped radical, or at least to determine the type of radical trapped. Table 1 gives examples of the range of hyperfine coupling constants obtained for a variety of trapped species for the two most commonly used spin traps DMPO (dimethyl-pyridine N-oxide) and PBN (a-phenyl-N-t-butyl nitrone). Care has to be taken, however, because the coupling constants vary with solvent polarity (for example, in water the DMPO adduct of the t-butoxy radical has < (N) = 1.48 and fl(H) = 1.60 mT while in toluene < (N) = 1.3 and fl(H) = 0.75 mT). 

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 ... 

In related work, the reactions of hydrogen peroxide with iron(II) complexes, including Feu(edta), were examined.3 Some experiments were carried out with added 5.5"-dimethyl-1-pyrroline-N-oxide (DMPO) as a trapping reagent fa so-called spin trap) for HO. These experiments were done to learn whether HO was truly as free as it is when generated photochemically. The hydroxyl radical adduct was indeed detected. but for some (not all) iron complexes evidence was obtained for an additional oxidizing intermediate, presumably an oxo-iron complex. 

Scheme 43 Spin trapping of superoxide with DMPO and DEPMPO...

Recendy, PEN, a-4-pyridyl-oxide-N-t-butyl nitrone (POEN) or 5-5,dimethyl-1, pyrroline-N-oxide (DMPO) were evaluated in models of experimental shock (endo-toxic, traumatic and mesenteric artery occlusion in rats). All three nitrones, when given prior to the insult intraperitoneally, were protective. When the nitrone s spin trapping ability was inactivated by exposure to solar light and air, they were no longer efficacious (Novelli, 1992). 

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. 

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]. 

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-... 

Radical cations of the most popular spin traps PBN and DMPO have been generated by the methods of ionizing radiolysis and laser flash-photolysis in solid matrices (435-437). As a polar solvent with high solvating ability for... 

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],... 

There is some evidence, from EPR spectroscopy and analysis of spin-trapped adducts, to suggest that OH may indeed be formed by activated neutrophils. However, caution must be exercised in interpreting such data because 02"-generated adducts may decay to form adducts that resemble those generated directly from -OH. For example, 5,5-dimethyl-l-pyrroline-l-oxide (DMPO) can react with C>2 to form DMPO-OOH, and with OH to form DMPO-OH formation of the latter adduct in phagocytosing neutrophils is taken as evidence for OH formation. However, two facts must be considered ... 

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. 

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