Biological Magnetic Resonance Volume 18 In Vivo EPR (ESR) AContinuationOrderPlanisavailableforthisseries.Acontinuationorderwillbringdeliveryofeach newvolumeimmediatelyuponpublication.Volumesarebilledonlyuponactualshipment.Forfurther information pleasecontactthe publisher. Biological Magnetic Resonance Volume 18 In Vivo EPR (ESR) Theory and Application Edited by Lawrence J. Berliner University ofDenver Denver, Colorado KLUWER ACADEMIC/PLENUM PUBLISHERS NEW YORK, BOSTON, DORDRECHT, LONDON, MOSCOW LibraryofCongressCataloguing-in-Publication Data In vivo EPR(ESR): theory & applications/edited by LawrenceJ. Berliner. p. cm.- (Biological magnetic resonance; v. 18) Includes bibliographical references and index. ISBN0-306-47790-4 I. Electron paramagnetic resonancespectroscopy. 2. Electron paramagnetic resonance spectroscopy. I. Berliner, LawrenceJ. II.Series. QP519.9.E433152oo3 572'.36-dc22 2003054690 ISBN: 0-306-47790-4 © 2003 KluwerAcademic/Plenum Publishers, New York 233SpringStreet,New York, New York 10013 http://www.wkap/nl 10 9 8 7 6 5 4 3 2 Ac.I.P. record for this book isavailable from the LibraryofCongress All rights reserved No partofthis book may be reproduced, stored inaretrieval system,ortransmitted in any form orbyany means,electronic, mechanical, photocopying, microlilming, recording,or otherwise, without written permission from the Publisher, with theexceptionofany material suppliedspecilically forthe purposeofbeingentered andexecutedonacomputersystem, for exclusive use by the purchaserofthework. Permissionsfor bookspublished in Europe: [email protected] Permissions for books published in the United StatesofAmerica: [email protected] To my mother Foreword The field of in-vivo EPR (ESR) has grown tremendously over the past two decades to the extentthat the need for acomplete volumeon all ofthe techniques and advances was sorely needed. This volume represents the combined effort of the world's true experts in their respective specializations. Like a major film, the compilationandproductionofthisbookwastrulyalongundertaking-almosttwo to three years. This was further complicated by a move from Columbus, Ohio to Denver, Coloradothatalsocontributedtosomedelays inproduction. Ifitwerenot for the help of my friend and colleague, Harold Swartz, the quality and completenessofthis text would nothave beendone so well. Iamtruly grateful to himfor his unselfishgenerosityoftimeand advice. Iamespeciallyindebtedtoall ofmyfriends whoauthoredchaptersinthisbookfortheirhardworkandpatience. Hal Swartz, alongwith LarryPiette,BarryCommoner,andahandfulofothers, really started exploringthis field in the sixties and seventies. They inspiredmany othersto have the courage to tackle seemingly impossibleproblems. This book is dedicated to theseearlypioneersas well as the contributorswhoseexcellentwork isfound herein. Lawrence1. Berliner Denver,Colorado vii Preface History ofinVivo EPR HaroldM. SwartzandLawrence1.Berliner EPRCenterfortheStudyofViableSystems, DeparmentofRadiology, DartmouthMedical School, Hanover, NH03755andDeparmentofChemistryandBiochemistry, Universityof Denver, Denver, CO 80208 Thedevelopmentofin vivo EPRis partofa continuumofdevelopments towards more applications and importance in the biomedical sciences. The initial uses of EPR were in the realm of physics but soon also began to involve chemistry and then biochemistry, especially in regard to metalloproteins. But within ten years of the work of Zavoisky (1946) establishing the feasibility ofEPR spectroscopy, the technique was applied not only to biological problems, but also directly to biological systems. In the following we summarize some ofthe key devel~pments, especially as they relate to the development of in vivo EPR. We.consider in vivo EPR itselfthrough the 1980s. By 1990 it became clear that in vivo EPR was a useful and continually developing technique, as indicated by the balance of the book, which provides summaries of the developments 0f the last ten years andsomeprojectionsofthefuture courseofinvivoEPR. THE BEGINNING Perhaps the proximate origins ofin vivo EPR studies are in the initial studies ofisolated cells and tissues. These studies provided insights into the ix PREFACE opportunities and problems of doing EPR in biological systems and provided some ofthe motivation to carry such studies forward into the fully functional situation: in vivo. These aspects have been reviewed recently in moredetail(Swartz, 1998). There were several attempts by the 1950's to study cells and tissues by EPR. The studies proceeded almost independently in the Soviet Union and in western countries. Some rationale came from the proposition by Michaelis in the 1930'sthat many enzymatic steps proceeded by a series of one-electron oxidation-reduction steps,which wouldresult in theoccurrence of free radicals (Michaelis, 1932). Another strong motivation came from speculations that free radicals might be involved in the development of cancer, which led to an early emphasis on EPR studies ofmalignant cells and tissues Swartz(1972). The early studies were complicated by low sensitivity ofavailable EPR spectrometers, combined with the problem of nonresonant absorption of microwaves, which led to further loss of sensitivity and .sometimes significant heating of the sample. These problems were solved to some extent by three different approaches, but almost inevitably these led to differentresultsand, hence,controversy. 1) Removal ofthe waterfrom cells and tissues by drying - usually by freezing first (i.e. lyophilization). This not only eliminated the problem of non-resonantabsorption ofmicrowave, butalso provided ameans to greatly increase the amount of biologically relevant material. Unfortunately, the drying process also gave rise to many changes on the systems that became recognized and understood only after considerable time and effort had been expended on studies ofdried systems Heckley (1972). The changes were in partbiological, involvingalterations in the systems that occurred during the process of drying, but the most important effects were artifacts due to physicochemical factors. The latterwere especially related to the generation of observable free radicals by interactions of dried cell constituents with molecular oxygen. The generation of free radicals by mechanical forces associated with freezing DIbert (1962) and processing ofthe samples also were importantsourcesofartifacts in somecases. 2) Minimizing dielectric losses by freezing the sample (the dielectric constant of ice is much less than that of liquid water). This also had the advantage of stopping most biochemical processes. However, as noted above, the mechanical forces associated with freezing can generate free radicals. This also gave rise to much confusion in early studies because of the ease with which frozen organic free radicals undergo microwave power saturation. The freezing process did not always stop all reactions; some reactions could proceed, especially ifthe sample was not kept at a very low temperature. Exposure of frozen samples to visible and UV light was anotherpotential sourceofartifactual free radicals. PREFACE xi 3) Study wet tissues in special sample holders and/or resonant cavities designed to minimize nonresonant microwave losses. The development of the aqueous flat cell was a key factor, which made direct observations of wet samples possible. This sample holderkeptthe dimensions ofthe sample to an acceptable limitand then the samplewas oriented so itwas in a region ofminimum electric field and maximum magnetic field. Lateran analogous sample holder, the "tissue cell," was developed which had a removable quartz plate where thin slices ofsolid tissues could be accommodated. With these techniques, however, problems of alteration of biological functions during the time of observation became a potential limitation. In addition, adequate perfusion was difficult to achieve in the limited volume in the sample holders, hence metabolism often depleted nutrients and/or altered the local environment. Sensitivity often was problematic due to the limited amount of sample. Some approaches to minimize these problems included the use oflarger volume multimode cavities and time averaging techniques to increase signal to noise. Because the aqueous nature of these samples resulted in fairly substantial losses of microwave power, power saturation was not a problem. Subsequently there have been considerable improvements in resonators, sample holders, and EPR spectrometers. It is now possible to successfully study significantly larger aqueous biological samples at 9 GHz, when proper attention is paid to maintaining an appropriate environmentfor thecellsortissues. The first studies of lyophilized cells and tissues appear to have been done by Commoner and colleagues (Commoner et aI, 1954). In that paper they describe experiments done in 1952 at9GHzon 250 mgoffreeze-dried bacteria (Pseudomonas jluorscens) in which they detected a small but reproduciblesingletatg== 2.0which theyattributed to free radicals involved in metabolism. Similar studies then were carried out "successfully" on mammalian tissues consisting of freeze-dried samples of7 different rabbit tissues (Hollocher and Commoner, 1961). In retrospect it is not clear whether these were really free radicals that were present in the tissues before lyophilization or ifthey were generated bythe lyophilization process itself. They also noted the presence ofstable free radicals due to melanin in pigmented cells and these undoubtedly were present prior to lyup~li1ization. Investigators in the Soviet Union began their extensive experimentation on cells and tissues about the same time, relying almost exclusively on lyophilized materials. A considerable body ofintriguing data and some interesting speculations resulted, for example, the suggestion that EPR might be useful in understanding cancer and a numberofother pathological states. Eventually, however, it also became clear that the approach was fraught with difficulties and is was probably not possible to draw useful conclusions despite this extensive body of work. The EPR signals in lyophilized biological materials were shown to bedue to the exposure ofthe xii PREFACE freeze-dried sample to traces ofoxygen and water, particularly due to the ascorbyl radical and itsproducts(Ruugeetai, 1976). Commoner recognized some ofthe disadvantages associated with using dried materials and turned to using aqueous preparations in order to study biological samples undermore physiologicallyrelevantconditions. The first EPR signals attributed unambiguously to free radicals in live cells were associated with photosynthesis, which required the presence ofchlorophyll (Commoneretai, 1956). Subsequentlythey obtained EPR signals from wet, functioning submitochondrial particles. They were able to relate these to enzyme function ofthe systems in these particles, which helped to initiate the continuingand a very fruitful useful line ofinvestigations using EPRto study redox enzymes. Then, with subsequent instrumentation development, they reported EPR spectra in survivingmammaliantissues that were clearly free radicals associated with function (Commonerand Temberg, 1961). The signals were 15-20 Gauss wide singletsat g=2.004 - 2.005. Similarsignals were reported in living E. coli (Isenberg and Baird, 1962). In general, the more metabolically active tissues such as liver, kidney, and heart had the most intense signals while other tissues had much smaller EPR signals (Mallard and Kent, 1966). The EPR signals in the tissues were stable for at least several hours ifthe tissues were kept at 00 C and tended to disappear with increased temperature, showing an apparent heat of activation of 15 kcaVmole for the decay process. They noted that the nature of their apparatus resulted in the tissues being severally hypoxic but reported no effects on the EPR signal from equilibrating the tissues with oxygen. This and other observations (such as the relatively intense EPR signals seen in submitochondrial particles) lead to the conclusion that much of the EPR signal of cells resides in the mitochondria and that its relative intensities may reflect the number of mitochondria in the cells. Commoner also reported some oftheearliestattempts to relate the EPR signals oftissues to pathophysiology, including a study of human tissue by liver biopsy (Commoner, 1961). They found thatthe liver in obstructivejaundice had an increased EPR signal while in non-obstructive jaundice the intensity was about the same as normal, and suggested that this observation might lead to the first diagnostic application ofEPR in medicine. They also reported an early study of the EPR signal in experimental tumors and found that, contraryto the sometimespredicted increase in free radicals intumors, there appeared to be a decrease. It has been suggested that this may reflect that tumorcellstendto have less mitochondria. The low sensitivity and potentially compromising physiological conditions associated with the use of wet samples at 9 GHz stimulated a search for other methods to deal with these problems. A number of investigators, including Commoner, carried out experiments on quickly frozen samples. To the surprise of some, the much more complex EPR