Electron Paramagnetic Resonance: Volume 21 / Edition 1

by Bruce C Gilbert

Overview

Electron Paramagnetic Resonance (EPR) Volume 21 highlights major developments in this area, with results being set into the context of earlier work and presented as a set of critical yet coherent overviews. The topics covered describe contrasting types of application, ranging from biological areas such as EPR studies of free-radical reactions in biology and medically-related systems, to experimental developments and applications involving EPR imaging, the use of very high fields, and time-resolved methods. Critical and up-to-the-minute reviews of advances involving the design of spin-traps, advances in spin-labelling, paramagnetic centres on solid surfaces, exchange-coupled oligomers, metalloproteins and radicals in flavoenzymes are also included. As EPR continues to find new applications in virtually all areas of modern science, including physics, chemistry, biology and materials science, this series caters not only for experts in the field, but also those wishing to gain a general overview of EPR applications in a given area. Volume 21 cover literature published during 2005 and 2006.

Product Details

ISBN-13:	9780854043736
Publisher:	RSC
Publication date:	12/16/2008
Series:	Specialist Periodical Reports - Electron Paramagnetic Resonance , #21
Pages:	350
Product dimensions:	6.15(w) x 9.20(h) x (d)

About the Author

Compiled by teams of leading authorities in the relevant subject, the series creates a unique service for the active research chemist with regular critical in-depth accounts of progress in Electron Paramagnetic Resonance (EPR).

Read an Excerpt

Electron Paramagnetic Resonance Volume 21

A Review of the Literature Published Between 2006 and 2007

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By B. C. Gilbert, M. J. Davies, D. M. Murphy

The Royal Society of Chemistry

Copyright © 2008 The Royal Society of Chemistry
All rights reserved.
ISBN: 978-1-84755-888-6

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CHAPTER 1

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 confirm that NO is directly involved (in other words, used as an inhibitor of NO activity), although in most cases EPR is not utilized to analyze the product. Other traps include DETC and MGD ferrous complexes (the latter being more water soluble) that essentially chelate NO. Cryogenic EPR is also useful for examining NO, with the "trapping agent" being an endogenous molecule such as haem or haemoglobin. Controversy has been generated in the cardiovascular field as a result of a considerable body of data that suggests that deoxyhaemoglobin reduces nitrite to NO with this eliciting hypoxic vasodilatation of blood vessels. Certainly ex vivo chemistry shows this is possible, but more recent work (alluded to above) probably negates the importance of such a mechanism in vivo. The power of using continuous-wave and pulsed electron paramagnetic resonance methods for structural analysis of ferric forms and nitric oxide-ligated ferrous forms of globins has also been tested. Interaction of NO with soluble guanylate cyclase (sGC), its primary effective receptor in vascular smooth muscle, to form ferrous nitrosyl complexes has also been studied. In vivo investigation of NO generation from nitric oxide synthase (NOS) was also undertaken in mature rat brain after injury, and oxygen-induced radical intermediates were identified in the nNOS oxygenase domain that are regulated by L-arginine, tetrahydrobiopterin, and thiols.

EPR continues to play a prominent role in understanding the radical mechanisms involved in tissue ischemia (I, the lack of oxygen to a given tissue area) and reperfusion (R, where supply is resumed). It has been recognized for some time that radicals produced during the latter phase are damaging and cause tissue injury, yet relatively little progress has been made in terms of prophylaxis. The redox status in hepatic I/R injury has been monitored in vivo in mice, and the formation and roles of plasma S-nitrosothiols in liver I/R identified. NO has also been monitored in rats following kidney transplantation and following I/R injury in kidney. Early work identified free radical formation during myocardial I/R, whereas the nitric oxide donor SNAP was shown to increase radical formation and degrade left ventricular function after myocardial I/R. Finally, evidence for nitrite derived formation of NO and subsequent signaling in post ischemic heart tissue was obtained for the first time by cryogenic EPR analysis, and confirmation that endothelium-derived NO regulates post-ischemic myocardial oxygenation by modulating mitochondrial electron transport.

4. Consequences of free radical reactions with biomolecules

Free radical damage to biomolecules is a consequence of oxidative stress and may result in tissue damage or loss of function that produce the symptoms of disease. The detection and elucidation of processes leading to such damage to biomolecues is therefore important in understanding disease pathology. Studies investigating free radical damage to some individual biomolecues is reviewed below, with the role of these processes in disease reviewed in later sections.

4.1 Damage to lipids

Lipid peroxidation remains an important index of oxidative stress and numerous studies have used this process as a measure of free radical production and tissue damage in biological systems. Lipid peroxidation has also been widely used to assess antioxidant deficiency or to evaluate new antioxidant compounds. Radical damage to lipids and membranes is often detected by non-radical lipid peroxidation products, produced as secondary events from the initiating oxidative stress. EPR spectroscopy allows the detection of radical intermediates generated during the radical chain reactions of lipid peroxidation and thereby can provide information on the possible origins of the process. Spin-trapping has been the method of choice to detect free radical intermediates during lipid peroxidation.

The antioxidant properties of many novel compounds on lipid peroxidation have been evaluated by EPR spectroscopy. Thus, the antioxidant activities of transresveratrol (trans-3,5,4'-trihydroxystilbene) and trans-piceid (trans-5,4'-dihydroxystilbene-3-O-beta-d-glucopyranoside), its more widespread glycosilate derivative, have been compared by measuring their inhibitory action on peroxidation of linoleic acid (LA) and their radical scavenging ability towards different free radicals (such as DPPH) and radical initiators. Studies using spin labelled phosphatidylcholine liposomes demonstrated that the susceptible hydroxyl groups of these compounds are located in the lipid region of the bilayer close to the double bonds of polyunsatured fatty acids, making these stilbenes particularly suitable for the prevention and control of lipid peroxidation in membranes.

Novel metalloporphyrins bearing 2,6-di-tert-butylphenol pendants as antioxidant substituents, and a long chain hydrocarbon palmitoyl group have been synthesized. The oxidation of the compounds by PbO2 lead to the formation of the corresponding 2,6-di-tert-butylphenoxyl radicals as detected by EPR. The activity of the porphyrins against lipid peroxidation was examined using in vitro lipid peroxidation induced by tert-butylhydroperoxide in respiring rat liver mitochondria, in vitro lipid peroxidation in liver homogenates of Wistar strain rats, and a model process of peroxidation of (Z)-octadec-9-enic (oleic) acid as a structural fragment of lipids.

EPR spin-trapping and spin-labelling techniques have been applied to investigate the antioxidative capacities of fullerene C60 and newly synthesized fulleropyrrolidine derivates (N-methyl (2-quinolyl) fulleropyrrolidine 60, Q-C 60 and N-methyl (2-indolyl) fulleropyrrolidine 60, I-C60) encapsulated in phospholipid multilamellar liposomes. The capacity for removal of OH and O2- and for the prevention of lipid peroxidation were found to be the most relevant biological antioxidative parameters. Spin-labelling was also used to examine a new potential antioxidant compound, spin-labelled lutein (SL-lut). The approximate location of nitroxide free radical groups of SL-lut incorporated into phosphatidylcholine liposomes was determined from their EPR spectra. Lipid peroxidation was measured by thiobarbituric acid reactive species (TBARS) compared with unlabelled lutein and SL-lut was found to be the most powerful antioxidant, significantly reducing lipid peroxidation.

Antioxidant supplements are being promoted for their potential health benefits and some of these have been investigated. An interesting study on the effect of Ginkgo biloba (Gb) supplementation in vivo on lipid peroxidation of microsomal membranes has been performed. Administration of up to 100 mg/kg/day Gb to rats did not significantly affect the activity of microsomal enzymes, the rate of generation of superoxide anion, or the iron reduction rate by rat liver microsomes. However, lipid peroxidation, assessed by the generation of lipid radicals measured by EPR spectroscopy using POBN as the spin trap was modulated. This suggests that Gb extracts at these concentrations are able to limit lipid peroxidation and scavenge lipid radicals in vivo and protect membranes from oxidative damage.

Oxidative damage to membranes and lipids continues to be assessed by EPR spectroscopy. Using EPR and sensitive spin trapping detection with 5-(diethoxyphos-phoryl)-5-methyl-1-pyrroline-N-oxide (DEPMPO), Culcasi and co-workers compared the respective roles of cigarette smoke- and gas phase cigarette smoke-derived free radicals on smoke-induced cytotoxicity and lipid peroxidation of filtered and unfiltered, machine-smoked experimental and reference cigarettes with a wide range of tar particulate matter yields. In buffer bubbled with cigarette smoke the DEPMPO/superoxide spin adduct was the major detected nitroxide. Unexpectedly a protective effect of tar particulate matter on murine 3T3 fibroblasts was observed in early (<3 h) free radical-, gas phase cigarette smoke-induced cell death, and carbon filtering decreased free radical formation, toxicity and lipid peroxidation in three cell lines (including human epithelial lung cells) challenged with gas phase cigarette smoke. These results highlight an acute, free radical-dependent, harmful mechanism specific to gas phase cigarette smoke, whose physical or chemical control may be of great interest with regard to reducing the toxicity of smoke. An interesting study challenges the putative role of iron and hydroxyl radicals in the oxidative stress-mediated cytotoxicity of the anti-cancer drugs doxorubicin and bleomycin. Using different iron chelators and measuring the formation of hydroxyl radicals by in vitro EPR, and quantifying oxidative stress and cellular damage as TBARS formation, glutathione (GSH) consumption and lactic dehydrogenase (LDH) leakage, it was found that all chelators inhibited ·OH radical formation induced by H2O2/Fe2+. However, the chelators that decreased doxorubicin and bleomycin-induced oxidative stress and cellular damage were not able to protect against H2O2/Fe2+, suggesting that the ability to chelate iron as such is not the sole determinant of a compound protecting against doxorubicin or bleomycin-induced cytotoxicity. This finding may have implications for the development of new compounds designed to protect against this toxicity.

In a study on lead toxicity, the EPR signal of ascorbyl radical in caput epididymis, cauda epididymis, testis and liver of lead acetate-treated rats revealed a significant decrease by 53%, 45%, 40% and 69% versus control tissues, respectively. In the group exposed to lead, the concentrations of lipid peroxide in homogenates of the reproductive system organs was significantly elevated versus control groups. It was assumed that the decreased EPR signal intensity was caused by decreased tissue concentrations of L-ascorbic acid. This may result from consumption of ascorbic acid by high levels of reactive oxygen species (ROS) in tissues of animals chronically exposed to lead.

The free radical reducing activity and membrane fluidity were examined by EPR spectroscopy in selenium deficient rats. Selenium deficiency caused the induction of liver microsomal cytochrome P-450 activity, and the reduction rate of nitroxyl radicals present at shallow depths in the membrane was increased. Although selenium-deficiency caused induction of liver cytochrome P-450 and chronic increases of H2O2, this did not result in oxidative liver damage. An increased level of glutathione in selenium-deficient liver was also evident, likely due to an absence of GSH-Px activity. Thus, using the EPR spin label method, this article shows that selenium deficiency causes complicated redox changes in the liver, notably, alterations in the levels of cytochrome P-450 and GSH-Px systems.

(Continues...)

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Excerpted from Electron Paramagnetic Resonance Volume 21 by B. C. Gilbert, M. J. Davies, D. M. Murphy. Copyright © 2008 The Royal Society of Chemistry. Excerpted by permission of The Royal Society of Chemistry.
All rights reserved. No part of this excerpt may be reproduced or reprinted without permission in writing from the publisher.
Excerpts are provided by Dial-A-Book Inc. solely for the personal use of visitors to this web site.

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Table of Contents

Contents

Preface Bruce Gilbert, Damien Murphy and Michael Davies, 7,
Biomedical applications of EPR spectroscopy Simon K. Jackson and Philip E. James,
EPR studies of radiation damage to DNA and related molecules David Becker and Michael D. Sevilla, 33,
Measurement of interspin distances by EPR Gareth R. Eaton and Sandra S. Eaton, 59,
Time-resolved electron paramagnetic resonance studies of transient organic radicals Dieter Beckert, 76,
EPR of paramagnetic centres on solid surfaces Damien M. Murphy and Mario Chiesa, 105,
EPR of exchange coupled oligomers Angelika B. Boeer, David Collison and Eric J. L. McInnes, 131,
Continuous wave and pulsed EPR analyses of metalloproteins Sabine Van Doorslaer, 162,
An EPR tool box for exploring the formation and properties of ordered template mesoporous materials Sharon Ruthstein and Daniella Goldfarb, 184,
High-field pulse EPR instrumentation Graham M. Smith, Paul A. S. Cruickshank, David R. Bolton and Duncan A. Robertson, 216,