Biomedical applications of EPR spectroscopy

Simon K. Jackson and Philip E. James

DOI: 10.1039/b709469h

1. Introduction

EPR spectroscopy can provide useful and even unique information pertinent to the study of oxidative stress and consequent disease settings. The parameters that can be measured include (a) oxygen-centred radicals (by spin trapping); (b) carbon-centred radicals (by spin trapping and sometimes by direct observation); (c) sulphur-centred radicals (by spin trapping and sometimes by direct observation); (d) nitric oxide (by spin trapping); (e) molecular oxygen (using oxygen sensitive paramagnetic materials); (f) redox state (using metabolism of nitroxides); (g) thiol groups (using special nitroxides); (h) pH (using specific nitroxides); (i) perfusion (using wash out of paramagnetic tracers) and (j) redox active metal ions (chromium, manganese). For an excellent review the reader is referred to ref. 1. In this chapter we highlight recent biomedical applications of EPR spectroscopy. This field is vast and growing; we concentrate here on the use of EPR spectroscopy in studying reactive oxygen and nitrogen species, damage to biomacromolecules and the major disease settings associated with such damage. We also review the application of particular techniques that have evolved primarily for the biomedical field.

2. Reactive oxygen species

Perhaps one of the more influential developments during recent times has been the synthesis and characterization of better DEPMPO-type spin traps for the detection of hydroxyl and superoxide radicals. 5-(2,2-dimethyl-1,3-propoxy cyclophosphoryl) -5-methyl-1-pyrroline N-oxide (CYPMPO) is a cyclic nitrone that is colourless, crystalline and freely soluble in water. In practical terms it is useful to know that this is stable as the solid or in aqueous solution and does not develop an EPR signal for at least 1 month under ambient conditions. It has readily assignable EPR spectra for both hydroxyl and superoxide adducts with no conversion from the latter to the former (as tested in vitro in UV-illuminated H2O2 solution and hypoxanthine/xanthine oxidase model systems).

Mito-DEMPO, a new DEPMPO analogue bearing a triphenylphosphonium group, was synthesized via a novel NH2-reactive DEPMPO. The half life of the superoxide adduct generated in intact mitochondria was >40 min. This exhibits an eight-line EPR spectrum with partial asymmetry. The mito-DEPMPO adduct formed from glutathionyl centered radicals (DEPMPO-SG) is 3-times more persistent than that of the parent DEPMPO adduct. Thus the EPR parameters of mito-DEPMPO adducts are distinctly different and highly characteristic in the case of superoxide, hydroxyl, glutathionyl and carbon-based radicals, and in many cases mito-DEPMPO nitrone and its analogues are more effective than most nitrone spin traps. It has long been recognized that redox status is critical to health and is offset in disease, and EPR techniques have contributed significantly to this understanding. There is a plethora of examples where the antioxidant capacity of compounds, cells, and tissues has been tested. Tempol (4-hydroxy-2,2,6,6-tetramethyl-1-piperidinyloxy) has long been known to protect experimental animals from the injury associated with oxidative and inflammatory conditions. In the latter case, a parallel decrease in tissue protein nitration levels has been observed. Protein nitration represents a shift in nitric oxide actions from physiological to pathophysiological, and potentially damaging pathways involving oxidants derived from this species such as nitrogen dioxide and peroxynitrite. In infectious diseases, protein tyrosine nitration of tissues and cells has been taken as evidence for the involvement of nitric oxide-derived oxidants in microbicidal mechanisms. To examine whether tempol inhibits the microbicidal action of macrophages, its effects on Leishmania amazonensis infection in vitro (RAW 264.7 murine macrophages) and in vivo (C57Bl/6 mice) was tested. The results indicated that tempol exacerbated L. amazonensis infection by a dual mechanism involving down-regulation of iNOS expression and scavenging of nitric oxide-derived oxidants. Thus, the development of therapeutic strategies based on nitroxides should take into account the potential risk of altering host resistance to parasite infection. This work also highlights the synchrony between oxidative and nitrosative stress, and how EPR techniques can (with appropriate understanding) yield important insights; further examples are summarized in relevant sections below. Antioxidant capacity was also tested in studies using flow-injection EPR to investigate hydroxyl radical scavenging activity of Gd(III) containing MRI contrast media and more recently of C60 and newly synthesized fulleropyrrolidine derivatives encapsulated into liposomes.

3. Reactive nitrogen species

One of the major advantages of EPR is the specific detection of radical species. Nowhere is this better observed than in the case of nitric oxide (NO) rather than its oxidative metabolites. Thus there is considerable overlap with the cardiovascular field (below). In most cases, the spin trap will react with NO only, although great care must be taken when undertaking such experiments to ensure that the NO metabolites themselves do not generate NO in the system that can then be spin trapped. In particular nitrite is a concern, where it is critical that pH and oxygenation are maintained. An excellent example of where this has been utilized to maximum benefit is in the understanding that NO production from nitrite occurs primarily in tissues, and not in blood, and primarily by the reductase activity of tissue xanthine oxidase and aldehyde oxidase. In addition, this study also highlights the use of 15N isotope trapping to specifically identify the source of the nitrogen in NO (the isotope gives rise to a characteristic and readily identifiable spin trapped adduct).

The options in terms of NO spin traps include nitronyl nitroxides and their derivatives (cPTIO, tPTIO) that can be used to report on NO in general or, as in the former case, from intracellular locations. These compounds have been used ubiquitously in cardiovascular research to...