Spin trapping limitations

There are several limitations on the use of the spin trapping technique when quantitative results are required. These are ... 

It is unfortunate that typical concentrations of free-radical species present in biological systems are only at the limit of e.s.r. detection sensitivity and, of course, there are major technical difficulties in studying whole animals in this manner. Therefore, the most successful e.s.r. experiments have adopted the approach of spin trapping in which very reactive and thus transient radical species are converted to long-lived adducts via reaction with a trap such as a nitrone, e.g. Equation 1.1 ... 

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-... 

This article concerns a simple expedient whereby short-lived reactive free radicals may be transformed into more persistent paramagnetic species, thus enabling esr techniques to be applied to systems in which the concentration of the reactive radical remains below normal detection limits. The principle is a simple one. It depends upon the addition to the reaction system of a small quantity of a diamagnetic substance (the spin-trap ) having a particularly high affinity for reactive radicals the product of this trapping reaction must be a particularly persistant free radical (the spin adduct ) whose concentration will build to readily detectable levels (>ca. 10—7—10-6 M). The general reaction is represented by equation (1). 

MNP has undoubtedly been the most widely used aliphatic nitroso spin trap, but there is one severe limitation on its use. It decomposes under the influence... 

Schmid and Ingold (1978) extended their investigation by allowing pairs of spin traps to compete for n-hexyl radicals (from bis-heptanoyl peroxide). It was emphasized that the error limits in these experiments were large (ca. 30%), since the onset of the decay of certain spin adducts was evident at very low adduct concentrations. 

Very recently, rate constants for scavenging of hydroxyl radicals by DMPO, and by the nitrone [18c], have been determined (Marriott et al., 1980) (see Table 5). As might be expected, the figures are close to the diffusion-controlled limit. The report of this work includes a concise and informative discussion of some of the difficulties with, and limitations of, the spin trapping method, especially where these relate to reactions involving hydroxyl radicals. 

Spin trapping is an often-used technique in the study of possible radical production in biological systems (for reviews see Kalyanaraman, 1982 Mason, 1984 Mottley and Mason, 1989), particularly by the detection and monitoring of spin adducts of the hydroxyl and hydroperoxyl ( OOH) radicals in view of their relation to possible damage mechanisms. This is a large area of research which it is not possible to cover in a limited review, and the treatment will therefore be restricted to a discussion of the electron transfer properties of biochemical systems (for a review on the application of the Marcus theory to reactions between xenobiotics and redox proteins, see Eberson, 1985) and... 

The quite negative reduction potentials of spin traps (Table 2) make them less amenable to participation in the radical anion mechanism, as first established in the cathodic reduction of benzenediazonium salts at a controlled potential in the presence of PBN (Bard et al., 1974). In fact, the lower cathodic limit of the spin trapping method is set not by the nitrone but by the spin adduct formed. 

Thus there is little doubt that the hydroxyl radical, if generated by an unambiguous method such as pulse radiolysis, can be trapped by PBN or DMPO, even if the former has several deficiencies, among them low trapping efficiency and short half-life of HO-PBN. The problem in hydroxyl radical trapping thus rests with the possible competition from the nucleophilic addition-oxidation mechanism, as exemplified in reaction (69) for DMPO and Ox-Red as a general one-electron redox system, or the inverted spin trapping mechanism (70). The treatment to follow will mostly be limited to DMPO. 

We have shown that, with the exception of DMPO, all spin traps studied exerted a limited negative inotropic and chronotropic effect. This effect was only present at the highest concentrations studied [116]. Bolli et al. [104-108] have previously reported that intracoronary infusions of PBN at concentrations greater than 20 mmol/1 blood caused profound toxicity, as manifest by the rapid, complete and persistent loss of cardiac contractile function, in the absence of ischemia and reperfusion, in open-chest dogs. These authors subsequently used PBN at much lower concentrations ( 1.6 mmol/1) in all of their experiments, and questioned the validity of results obtained in previous studies where the... 

Although the detection limit for ESR spectroscopy per se is extremely low, the use of electrochemical cells filled with solvents that have high dielectric constants results in considerable losses in the cavity of the ESR spectrometer. This in turn increases the limit of detection. In the case of electrode reactions that have only very small stationary concentrations of radicalic intermediates, detection may be impossible. The use of spin traps may help. These compounds are rather simple organic molecules that react easily with radicals forming adducts (see Fig. 5.118). The molecular structure of the intermediate may be deduced from the known structure of the spin trap and the observed ECESR spectrum. Unfortunately, this technique doesn t necessarily trap the major reaction intermediate rather, it only traps those which react easily with the spin trap. Consequently, misinterpretations are possible. 

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