Contributors i

Biomedical Magnetic Resonance:

Proceedings of the International Workshop
Biomedical Magnetic Resonance:
Proceedings of the International Workshop

Edited by
NR Jagannathan
Professor and Head
Department of NMR
All India Institute of Medical Sciences
New Delhi, India

Foreword
Richard R Ernst
Nobel Laureate

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Biomedical Magnetic Resonance: Proceedings of the International Workshop

© 2005, NR Jagannathan

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Dedicated to
My Parents
and
Family
 Contributors

Ackerstaff E Bhujwalla ZM
Department of Radiology Department of Radiology
Johns Hopkins University School of Medicine Johns Hopkins University School of Medicine
Baltimore, USA Baltimore, USA

Artemov D Binesh Nader

Department of Radiology Department of Radiological Sciences
Johns Hopkins University School of Medicine David Geffen School of Medicine at UCLA
Baltimore, USA Los Angeles, USA

Baert R Bolan PJ
Institute for Biodiagnostics Centre for Magnetic Resonance Research
National Research Council of Canada University of Minnesota Medical School
Winnipeg, Canada Minneapolis, USA
Barker PB Carmichael D
Department of Radiology Department of Obstetrics and Gynaecology
Johns Hopkins University School of Medicine
University College London
Baltimore, USA
London, UK
Becker ED
Chung HK
National Institutes of Health
G. E. Healthcare Technologies
Bethesda, USA
Waukesha, USA
Bendahan D
Centre de Résonance Magnétique Chung YL
Biologique et Médicale Department of Basic Medical Sciences
Faculté de Médecine de Marseille St. George’s Hospital Medical School
Marseille, France London, UK

Bezabeh T Cozzone PJ
Institute for Biodiagnostics Centre de Résonance Magnétique
National Research Council of Canada Biologique et Médicale
Winnipeg, Canada Faculté de Médecine de Marseille
Marseille, France
Bhandari M
Department of Urology Crawford TO
Sanjay Gandhi Post-Graduate Department of Neurology
Institute of Medical Sciences Johns Hopkins University School of Medicine
Lucknow, India Baltimore, USA
viii Biomedical Magnetic Resonance: Proceedings of the International Workshop

Damon BM Gambini A
Vanderbilt University The Kennedy Kreiger Institute
Institute of Imaging Science Johns Hopkins University School of Medicine
Nashville, Tennessee Baltimore, USA

De Bruhl N Garwood M
Department of Radiological Sciences Centre for Magnetic Resonance Research
David Geffen School of Medicine at UCLA University of Minnesota Medical School
Los Angeles, USA Minneapolis, USA

DelaBarre L Gillies RJ
Centre for Magnetic Resonance Research Arizona Cancer Centre
University of Minnesota Medical School Tucson, USA
Minneapolis, USA
Glunde K
Devasahayam N Department of Radiology
Centre for Cancer Research Johns Hopkins University School of Medicine
National Cancer Institute, NIH Baltimore, USA
Bethesda, USA
Gore JC
Ellermann J Vanderbilt University
Department of Radiology Institute of Imaging Science
University of Minnesota Medical School Nashville, Tennessee
Minneapolis, USA
Govil G
Emory TH Tata Institute of Fundamental Research
Department of Radiology Mumbai, India
University of Minnesota Medical School
Minneapolis, USA Gowda NGA
Centre of Biomedical Magnetic Resonance
Everson LI Sanjay Gandhi Post-Graduate
Department of Radiology Institute of Medical Sciences
University of Minnesota Medical School Lucknow, India
Minneapolis, USA
Griffiths JR
Fatemi SA Department of Basic Medical Sciences
The Kennedy Kreiger Institute St. George’s Hospital Medical School
Johns Hopkins University School of Medicine London, UK
Baltimore, USA
Grodd W
Gadian DG Radiology Clinic
Radiology and Physics Unit Abteilung Fur Neuroradiologie
Institute of Child Health, University College University of Tuebingen
London, UK Tuebingen, Germany
 Contributors ix

Gulbahce E Khetrapal CL
Department of Pathology and Centre of Biomedical Magnetic Resonance
Laboratory Medicine Sanjay Gandhi Post-Graduate
University of Minnesota Medical School Institute of Medical Sciences
Minneapolis, USA Lucknow, India

Gullapalli RP Kochupillai N
Department of Radiology All India Institute of Medical Sciences (Rtd.)
University of Maryland Medical System New Delhi, India
Baltimore, USA
Komoroski RA
Gupta A Department of Radiology
Centre of Biomedical Magnetic Resonance University of Arkansas for Medical Sciences
Sanjay Gandhi Post-Graduate Little Rock, USA
Institute of Medical Sciences
Lucknow, India Kossoff EH
Department of Neurology
Han S Johns Hopkins University School of Medicine
Department of Hepatology Baltimore, USA
David Geffen School of Medicine at UCLA
Los Angeles, USA Krishna MC
Centre for Cancer Research
Hennig J
National Cancer Institute
Department of Diagnostic Radiology
National Institutes of Health
University Hospital Freiburg
Bethesda, USA
Freiburg, Germany
Kumar A
Horska A
Department of Psychiatry
Department of Radiology
David Geffen School of Medicine at UCLA
Johns Hopkins University School of Medicine
Los Angeles, USA
Baltimore,USA

Huda A Lean CL
Department of Physics Institute for Magnetic Resonance Research,
California State University St Leonards, NSW
Fresno, USA Australia

Jagannathan NR Lindquist DM
Department of NMR Department of Radiology
All India Institute of Medical Sciences University of Arkansas for Medical Sciences
New Delhi, India Little Rock, USA
x Biomedical Magnetic Resonance: Proceedings of the International Workshop

Mathur R Nandini V
Division of Radiopharmaceuticals Department of Anaesthesiology
and Radiation Biology St Johns Medical College
Institute of Nuclear Medicine Bangalore, India
and Allied Sciences, Delhi, India
Nelson MT
Matsumoto K Department of Radiology
Centre for Cancer Research University of Minnesota Medical School
National Cancer Institute, NIH Minneapolis, USA
Bethesda, USA
Ordidge RJ
McIntosh A Department of Medical Physics and
Centre for Magnetic Resonance Research Bioengineering
University of Minnesota Medical School University College London
Minneapolis, USA London, UK

Meisamy S Pathak AP
Centre for Magnetic Resonance Research Department of Radiology
University of Minnesota Medical School Johns Hopkins University School of Medicine
Minneapolis, USA Baltimore, USA

Mishra AK Peebles D
Division of Radiopharmaceuticals Department of Obstetrics and Gynaecology
and Radiation Biology University College London
Institute of Nuclear Medicine London, UK
and Allied Sciences, Delhi, India
Raghunand N
Mitchell JB Arizona Cancer Centre
Centre for Cancer Research Tucson, USA
National Cancer Institute, NIH
Bethesda, USA Raghunathan P
Fluorosis Research Foundation
New Delhi, India
Mountford CE
Department of Magnetic Resonance in Medicine
University of Sydney, Sydney and Raman V
Institute for Magnetic Resonance Research, Department of Radiology
St Leonard’s, NSW, Australia Johns Hopkins University School of Medicine
Baltimore, USA
Murugesan R
Centre for Cancer Research Röll S
National Cancer Institute, NIH Siemens Medical Solutions
Bethesda, USA Erlangen, Germany
 Contributors xi

Russel P Snyder CJ
Institute for Magnetic Resonance Research, Centre for Magnetic Resonance Research
St Leonards, NSW University of Minnesota Medical School
Australia Minneapolis, USA

Saini RS Solaiyappan M
Medical Solutions Division Department of Radiology
Siemens Ltd Johns Hopkins University School of Medicine
New Delhi, India Baltimore, USA

Schenker KV Styczynski A
MRI Division Centre for Magnetic Resonance Research
Bruker Bio-Spin, AG University of Minnesota Medical School
Faellanden, Switzerland Minneapolis, USA

Sharma R Subramanian S
Department of Radiodiagnosis Centre for Cancer Research
All India Institute of Medical Sciences
National Cancer Institute, NIH
New Delhi, India
Bethesda, USA
Sharma RK
Thomas MA
Division of Radiopharmaceuticals
Department of Radiological Sciences
and Radiation Biology
David Geffen School of Medicine at UCLA
Institute of Nuclear Medicine
Los Angeles, USA
and Allied Sciences
Delhi, India
Tuttle TM
Sharma U Department of Surgery
Department of NMR University of Minnesota Medical School
All India Institute of Medical Sciences Minneapolis, USA
New Delhi, India
Van Zijl PCM
Smith ICP Department of Radiology
Institute for Biodiagnostics Johns Hopkins University School of Medicine
National Research Council of Canada Baltimore, USA
Winnipeg, Canada

Smith M Vaughan JT
Department of Radiology Centre for Magnetic Resonance Research
Johns Hopkins University School of Medicine University of Minnesota Medical School
Baltimore, USA Minneapolis, USA
xii Biomedical Magnetic Resonance: Proceedings of the International Workshop

Wang X Zhou J
Department of Obstetrics and Gynaecology Department of Radiology
University College London Johns Hopkins University School of Medicine
London, UK Baltimore, USA
Yee D
Department of Medicine Zhou J
University of Minnesota Medical School Department of Radiology
Minneapolis, USA University of Maryland Medical System
Baltimore, USA
Yue K
Department of Medicine
University of Hawaii
Honolulu, USA
 Foreword

What an exciting International Workshop that has been organized by Professor N.R.
Jagannathan! More and more, magnetic resonance imaging (MRI) is becoming the most
versatile and most informative diagnostic tool of medical research and clinical practice. It
was a long pathway from the original conception of magnetic resonance in nuclear
physics in the forties of last century to today’s perfection of MRI in our most advanced
medical institutions, such as the All India Institute of Medical Sciences. Both,
sensitivity and inherent information contents were originally quite meager; and none of
the pioneers could conceive the incredible development that took place during the past 60
years.
Magnetic resonance, or NMR (Nuclear Magnetic Resonance) as it is called by more
knowledgeable scientists, owes its power to the omnipresence of nuclear spins in virtually
all living and dead materials. These nuclear spins serve as natural ‘spies’ for reporting on
their immediate environment. They are interacting very weakly only with the surrounding
material. Thus, they remain ‘unseen’ but are capable nevertheless of gathering most valuable
information. Much technological advancement and sophistication was necessary for reliably
recording the feeble spies’ messages. In addition, advanced digital data processing and
artificial intelligence were required for their interpretation.
Today, NMR is indispensable in chemical and biomolecular research for visualizing
molecular structures, interactions, and processes. Today, MRI is a mature tool in the hands
of clinicians and medical researchers. The teething problems are largely overcome, although
further development and improvements are still needed and are indeed quite feasible.
The involvement of many of the most capable scientists in the instrumental, chemical,
biological, and medical fields gives us hope for further progress.
We scientists, responsible for the development of these marvelous tools, are quite
proud of ‘our’ immortal achievements. Perhaps, we do not realize that we have just been
extremely lucky in our endeavors. Quite accidentally, we found a golden egg, while other
compatriots working extremely hard gain very little compensation, recognition, or joy.
The Indian cities provide plentiful of speaking examples of such tragic injustice.
Many of us might just shrug their shoulders, being used to what they might call
‘eternal fate’. Indeed, we have developed handy philosophies that allow us to endure the
conflicts, to calm our conscience, and to continue our daily profitable business.
xiv Biomedical Magnetic Resonance: Proceedings of the International Workshop

I am convinced that our serendipitous success in magnetic resonance loads responsibility

upon our shoulders. We cannot change the situation in our crazy world immediately, but
we can conceive models of a better, sustainable, and just future. We can put the germs
of foresight, of justice, and of determination to remedy the worst iniquities into the brains
of our students who will be the leaders of tomorrow. Let us hope that they will do a
better job than we have done so far (outside of magnetic resonance)!

Richard R Ernst
Nobel Laureate
Laboratorium für Physikalische Chemie
ETH Hönggerberg HCI
8093 Zürich, Switzerland
 Preface

This volume consists of contributions from the invited speakers of the “International Workshop
on Biomedical MR” organized by the Department of NMR, All India Institute of Medical
Sciences, New Delhi from January 12-15, 2005 as a satellite event of the International
Conference on Magnetic Resonance in Biological Systems (ICMRBS). The workshop covered
exploding areas of the applications of magnetic resonance techniques in biomedicine.
Clinical imaging has shown faster advancement during the last two decades and especially
the field of MR continues to progress at an impressive speed that requires periodic up-
date. The organizers of the ICMRBS felt that it would be ideal to organize a workshop
on biomedical applications of MR in this part of the world where the use of MRI is still
at its infancy.
The workshop addressed various aspects of MRI and MRS starting from the fundamental
physics to the latest advances. Several eminent scientists including Prof. Richard R Ernst,
Nobel Laureate, delivered lectures in the workshop. Gathering of experts from various
countries like Australia, Canada, France, Germany, India, Switzerland, UK and USA enabled
the participants of the workshop to interact and exchange freely their scientific ideas. In
addition, we had participants from Bangladesh, Canada, Israel, Nepal, Russia, Singapore
and USA.
This volume contains twenty-nine chapters and is organized in such a fashion so as to
maintain continuity of thought process with several illustrations. The authors have tried
to make complex methodologies understandable and interesting to knowledgeable readers
who may not be specialists. In particular, it is hoped that this volume will prove valuable
to graduate students and young scientists who plan to enter the fascinating area of
biomedical magnetic resonance.
The organizers would like to thank the Director and Dean of the All India Institute of
Medical Sciences. The Department of Science and Technology and the Department of
Biotechnology of the Government of India, the Indian National Science Academy,
International Union of Pure and Applied Biophysics (IUPAB), International Society for
Magnetic Resonance in Medicine (ISMRM), National Magnetic Resonance Society of India
(NMRS), Indian Biophysical Society (IBS), Siemens Ltd., Bruker Spectro-Spin, and German
Remedies, Kodak India Ltd., Varian Inc., USA and Amersham Health are thanked for
financial support to the workshop. I acknowledge the help and assistance rendered by my
colleagues and staff, in particular Mr Virendra Kumar, Mr KA Danishad, and Dr Uma
Sharma in the preparation of this volume.
Above all, I thank all the speakers who contributed enthusiastically their lecture notes
in the form of an article. This volume has emerged only out of their enthusiasm and
cooperation. The efficiency of the publishers Jaypee Brothers Medical Publishers (P) Ltd.,
and their cooperation is greatly acknowledged. Last, but not least, I would like to thank
my parents and my family for their patience, understanding, and encouragement.

NR Jagannathan
 Sponsors

International Workshop on Biomedical MR

All India Institute of Medical Sciences, New Delhi, India
1. All India Institute of Medical Sciences, New Delhi, India.
2. Amersham Health, India.
3. Bruker BioSpin, India and Switzerland.
4. Department of Biotechnology, Government of India.
5. Department of Science and Technology, Government of India.
6. German Remedies, India.
7. Indian Biophysical Society (IBS).
8. Indian National Science Academy, New Delhi, India.
9. International Society for Magnetic Resonance in Medicine (ISMRM)
10. International Union for Pure and Applied Biophysics (IUPAB)
11. Kodak India Ltd.
12. National Magnetic Resonance Society of India (NMRS).
13. Siemens Ltd., India.
14. Varian Inc., USA
 Contents

1. Historical Perspectives of MRI ............................................................................................. 1

Edwin D Becker
2. Introduction to Biological NMR Spectroscopy ............................................................. 12
Girjesh Govil
3. Fundamentals of Magnetic Resonance Image Production ......................................... 29
P Raghunathan
4. Contrast Mechanisms in MRI ............................................................................................. 42
John C Gore, Bruce M Damon
5. RF Pulse Sequences Including Rapid Imaging Sequences ....................................... 52
Rao P Gullapalli, Jiachen Zhuo
6. Overview of an MRI System .............................................................................................. 71
RS Saini
7. In vivo MR Spectroscopy .................................................................................................... 77
Stefan Röll
8. Multi-dimensional NMR Spectroscopy and Editing in vivo ................................... 86
M Albert Thomas, Nader Binesh, Kenneth Yue, Hyun-kyung Chung,
Steven Han, N DeBruhi, A Aude, Anand Kumar
9. Ultrafast and Parallel Imaging ......................................................................................... 110
J Hennig
10. fMRI: Neuroscientific and Clinical Applications ....................................................... 129
Wolfgang Grodd
11. Appropriate Functional Magnetic Resonance Imaging for the
Study of Evolving Yogis ................................................................................................... 138
N Kochupillai, V Nandini
12. Perinatal MR Studies in Animal Models ..................................................................... 141
RJ Ordidge, DW Carmichael, X Wang, DM Peebles
13. Magnetic Resonance in the Developing Brain ........................................................... 155
David G Gadian
14. MRS in Brain Pathlogies ................................................................................................... 161
Peter B Barker, Mari Smith, A Gambini, SA Fatemi,
TO Crawford, EH Kossoff, A Horská
15. Application of Proton MRS in the Study of the
Pathology of Human Cancers Other than Brain ........................................................ 171
Cynthia L Lean, Peter Russell, Carolyn E Mountford
xx Biomedical Magnetic Resonance: Proceedings of the International Workshop

16. 31P MR Spectroscopy of Muscle Energy Metabolism in Humans:

Physiological and Clinical Applications ...................................................................... 192
D Bendahan, PJ Cozzone
17. MRI and MRS of Nuclei other than 1H ....................................................................... 217
Diana M Lindquist, Richard A Komoroski
18. MRI/MRS to Predict and Monitor Response to Anticancer Therapies ................ 233
Robert J Gillies, Natarajan Raghunand
19. In vivo Cellular and Molecular Imaging of Cancer .................................................. 247
Zaver M Bhujwalla, Ellen Ackerstaff, Dmitri Artemov,
Kristine Glunde, Arvind P Pathak, Venu Raman, Meiyappan Solaiyappan
20. Body MR Imaging ............................................................................................................... 257
Raju Sharma, Shiva Gamanagati
21. Applications of MR Spectroscopy in Neuromuscular Diseases ............................. 313
Uma Sharma, NR Jagannathan
22. EPR Imaging for Biomedical Applications ................................................................... 330
R Murugesan, N Devasahayam, K Matsumoto, S Subramanian,
JB Mitchell, Murali C Krishna
23. Studying Mobile Proteins and Peptides in vivo using MRI and MRS ............. 353
Jinyuan Zhou, Peter CM van Zijl
24. Magnetic Resonance of Biofluids and Tissues ............................................................ 362
Ian CP Smith, Tedros Bezabeh, Racquel Baert
25. MR Metabolomics: Studies of Gene Function and Novel Anticancer Drugs ... 378
JR Griffiths, YL Chung
26. Metallopharmaceuticals as Exogenous Contrast Agents ........................................... 393
RK Sharma, R Mathur, AK Mishra
27. High-resolution MR Spectroscopy in Clinical Medicine ......................................... 403
GA Nagana Gowda, Ashish Gupta, M Bhandari, CL Khetrapal
28. Clinical Assessment of Breast Cancer using
1H MRS at High Magnetic Field .................................................................................... 412

M Garwood, A McIntosh, PJ Bolan, S Meisamy, CJ Snyder,

A Stycznski, L DelaBarre, J Ellermann, E Gulbahce, TM Tutte,
LI Everson, TH Emory, MT Nelson, D Yee, JT Vaughan
29. MR Applications in Experimental and Animal Model Systems ........................... 423
Kurt V Schenker

Index ......................................................................................................................................... 447


Historical Perspectives of MRI
INTRODUCTION
Nuclear magnetic resonance imaging
(usually abbreviated MRI, or sometimes in
clinical applications, just MR) has become a
very widely used and extremely valuable
medical diagnostic method and a valuable
tool for a broad range of biomedical
research studies.* As a medical technology,
MRI is a multi-billion dollar industry, with
thousands of clinical scanners available in
every major hospital in the world.
For radiologists and other clinicians, MRI
burst onto the scene in the early 1980s, when
commercial clinical instruments became
available. MRI appeared as a new method
closely related superficially to X-ray compu-
ted tomography (CT), which had been
developed a few years earlier as a diagnostic
tool for examining internal human anatomy.
In fact, early clinical MR images were
evaluated as adjuncts to CT. For chemists,
however, MRI was a brilliant, but logical,
extension of the nuclear magnetic resonance
(NMR) instruments and methods that had
been unraveling molecular structures for 30
years. And for physicists, MRI was one more
application of the phenomenon of NMR that
was discovered in 1938.
*MRI is also widely employed in such areas as plant physiology, food technology, and materials research.
This presentation does not delve into these applications.
Historical Perspectives of MRI 1
1
Edwin D Becker
In this article, I will try to describe the
evolution of biomedical MRI in the context
of the long-range, overall development of
NMR itself. I will discuss a few important
NMR concepts and methods and show how
they eventually came to play key roles in
today’s MRI applications. I have drawn
heavily on information contained in the 200
historical articles in the Encyclopedia of
NMR.1
ORIGINS OF NMR
In the mid-1920s, the advent of quantum
mechanics provided a coherent explanation
of many chemical and spectroscopic obser-
vations in terms of quantized energy levels
and the concept of a spin of the electron
and of many atomic nuclei. In rough terms,
spin can be pictured as a rotation of the
electron or nucleus about an axis, much as
the earth rotates about its polar axis. Because
nuclei and electrons are charged particles,
such a motion would be expected to gene-
rate a magnetic moment, and these particles
could interact with an externally applied
magnetic field. In 1933, Otto Stern (Nobel
Prize, 1943) and his co-workers found that
a beam of hydrogen molecules in a highly
2 Biomedical Magnetic Resonance: Proceedings of the International Workshop
evacuated container could be deflected by
an inhomogeneous magnetic field. 2 The
beam split into two components, depending
on whether the nuclear magnetic moments
of the hydrogen nuclei were oriented
parallel or antiparallel to the magnetic field.
Stern’s molecular beam technique was able
to provide estimates of the magnitude of
nuclear magnetic moments and spawned
increasingly more elegant experiments,
particularly in the laboratory of II Rabi at
Columbia University. In seeking ways to
improve the precision of the measurement,
Rabi was led to the idea that in a magnetic
field a nucleus with spin orientation parallel
to the field would exist in a different energy
level from one with antiparallel orientation,
and that absorption of a quantum of energy
at precisely the correct frequency could
cause nuclei to “flip” from one orientation
to the other. The energy v needed to satisfy
the Bohr relation
hν = E2 – E1 = (γ/2π)B0 ...(1)
turns out to be in the radiofrequency (RF)
range, generally 1 to 900 megahertz (MHz),
depending on the magnetic field strength. In
equation 1, h is Planck’s constant, Ei are the
magnetic energy levels, γ is the nuclear
magnetic moment divided by the nuclear
spin quantum number, and B0 is the strength
of the applied magnetic field. In December
1937, Rabi and co-workers subjected a beam
of LiCl molecules to an RF of 3.518 MHz as
the molecules passed through a static
magnetic field. They varied the strength of
the magnetic field and found that at one
particular value the beam was deflected and
its intensity on the detector abruptly
dropped.3 This was the first observation of
nuclear magnetic resonance (NMR), the term
“resonance” describing the very precise
relation in equation 1 that had to be fulfilled
among the magnetic field, the magnetic
moment of the lithium nucleus, and the RF.
Rabi received the Nobel Prize in 1944 for
the discovery of NMR.
The NMR in molecular beams provided
physicists with an elegant method to
measure nuclear magnetic moments with
much higher precision than could be
obtained previously. However, the practical
use of NMR really stems from its first
observation in bulk materials—solids, liquids
and even gases at normal pressures. The
physicist CJ Gorter had made two
unsuccessful attempts to observe NMR in
bulk materials (1937 and 1943),4 and it was
not clear that the weak NMR signals could
be detected. The signal from a molecular
beam arises from all nuclei in the beam; but
in bulk materials, the theory of absorption
and induced emission makes it clear that
the signal would come from only the
difference in the populations in the two
energy levels—about 1 in 100,000. However,
just after the World War II two groups
succeeded in detecting NMR. On December
15, 1945, Purcell, Torrey and Pound (MIT/
Harvard) found the resonance of hydrogen
nuclei in a large sample of paraffin wax in
a waveguide cavity.5 About a month later,
Bloch, Hansen and Packard (Stanford) used
a different experimental approach to find
the resonance in a small sample of liquid
water. 6 These experiments, carried out
independently and concurrently, were
published in January 1946 and earned Bloch
and Purcell the Nobel Prize in 1952. Purcell’s
address on this occasion presaged the
myriad applications that would be found
for NMR:7
“I remember in the winter of our first
experiments, just seven years ago, looking
on snow with new eyes. There the snow
lay around my doorstep—great heaps of
protons quietly precessing in the earth’s
magnetic field. To see the world for a
moment as something rich and strange is

the private reward of many a discovery.
But I’m afraid it has little bearing on the
sober question we, as physicists, must ask
ourselves: What can we learn from all this
about the structure of matter?”
APPLICATIONS IN PHYSICS AND
CHEMISTRY
Once NMR could be studied in ordinary
substances, myriad applications soon
emerged. As the details of NMR were
elucidated through theoretical and experi-
mental work in the late 1940s and early
1950s, it became apparent that the method
could be used for much more than measur-
ing nuclear magnetic moments. Shortly after
the initial publication, Bloch showed that
relaxation of the nuclear spins occurs after a
burst of RF power (an RF pulse). Relaxation
processes are characterized by a time T1,
measuring the transfer of energy from the
spins to their surroundings (the lattice), and
a time T2, measuring the interchange of
energy among the spins themselves. Soon
thereafter, Bloembergen, Purcell and Pound
showed that these relaxation processes are
intimately related to random molecular
motion (Brownian motion), a result that
would ultimately be a very important factor
in the feasibility of biomedical MRI, as we
see later.
About 1950, several investigations made
it clear that the nuclear resonance frequency
depends on magnetic shielding from the
electrons around the nucleus, hence on the
specific chemical environment. This chemical
shift is the cornerstone of the use of NMR to
elucidate molecular structure. Other pheno-
mena, including spin-spin coupling and the
nuclear Overhauser effect, were soon dis-
covered that further enhanced the use of
NMR in chemistry. Soon there were
Historical Perspectives of MRI 3
hundreds of NMR spectrometers dispersed
in almost every chemistry laboratory
throughout the world, and an industry
developed to make dramatic improvements
in technology, particularly higher and more
homogeneous magnetic fields and vastly
improved sensitivity. This piece of infra-
structure would later play a key role in the
rapid commercialization of MRI.
Methods for obtaining NMR spectra were
vastly improved with the advent of pulse
Fourier transform (FT) techniques that were
realized just as the computer revolution
permitted their incorporation into NMR
instruments. The pulse FT approach resulted
in a dramatic gain in effective sensitivity.
Later, the development of two-dimensional
FT methods provided a conceptually new
way of correlating information for chemical
applications and also played a key role in
the realization of MRI. Richard Ernst
received the Nobel Prize in 1991 for his
development of FT and 2D FT methods.
BIOLOGICAL APPLICATIONS OF NMR
Since water constitutes a large percentage
of all biological tissues, animals and humans
have been a natural target for NMR studies.
Bloch, probably, carried out the first biologi-
cal NMR experiment when he put his finger
into an NMR probe and found, not
surprisingly, a strong signal from the water
protons. However, the first real NMR
investigations of biological samples, begun
in 1950, aimed at studying the water content
and the relaxation properties of water in
bits of tissue excised from plants, animals
and humans. Over the next 20 years, scores
of studies of 1H, 2H, 17O and 23Na NMR in
cells and tissues agreed on one common
and striking feature—in nearly all cases
studied, the relaxation times of water nuclei
and the diffusion constants of water
4 Biomedical Magnetic Resonance: Proceedings of the International Workshop
molecules are much lower than the values
observed for free water, probably, the result
of restricted and anisotropic motion of
water molecules in the vicinity of biological
macromolecules.
In 1971, Raymond Damadian made an
important discovery that would have long-
term consequences.8 With the background
of observations of cellular water, Damadian
anticipated possible differences in NMR
relaxation times for water in normal cells
and cancer cells. He measured T1 and T2 in
pieces of tissue excised from normal rats
and from fast-growing tumors that had been
implanted into other rats, and found that
each of the eight tumor samples had longer
relaxation times than any of the normal
samples. However, a number of further
studies in several laboratories showed that
with slow-growing mouse tumors and with
excised human tissues the distinction
between normal and malignant tissue was
not so clear-cut. Although tumors tended
to have longer relaxation times (partly, at
least, because of higher water content), there
was such a wide variability that relaxation
measurements could not be used as a
definitive method to distinguish among
tissues that are normal, malignant, or
pathological but not malignant. Never-
theless, the attention of a number of NMR
investigators had been drawn to the
possibility of using this method in medical
diagnosis.
Meanwhile, in a number of laboratories,
attention was being given to NMR studies
within a living animal—initially worms and
a rat tail, which fit into standard NMR tubes,
but later to small animals that could be
accommodated in a large bore magnet. Little
useful information could be obtained from a
spectrum of the entire animal, but localization
by means of surface coils permitted spectra
to acquired, particularly with 31P NMR, that
gave insights into physiological function.
Surface coils, however, were limited to
studies of tissue near the surface, not deep
within the body. The need for better
localization methods for NMR research and
the potential for using NMR information
obtained in the living human body for
medical diagnosis provided the impetus for
development of new techniques.
INVENTION OF NMR IMAGING
The breakthrough came in 1971, with Paul
Lauterbur’s conception of the use of linear
magnetic field gradients to localize NMR
signal information along one dimension and
to construct a two- or three-dimensional
image from a collection of such one-dimen-
sional data sets.9 Lauterbur recognized that
the basic resonance equation (1) would be
modified when a magnetic field gradient
Gx is applied along direction x:
hν = (γ/2π)B0(1+Gxx) ...(2)
The resonance frequency thus provides a
measure of the location of the signal along
the x direction for a non-uniform macro-
scopic object such as an animal or a human.
Lauterbur first demonstrated the application
of this idea a year later, using a commercial
NMR spectrometer (Varian Model A-60) and
a sample consisting of two capillaries filled
with water inside a 5 mm diameter sample
tube filled with D2O. He used combinations
of the normal x and y shim coils to generate
magnetic field gradients in various direc-
tions in the xy plane, then applied a “back
projection” method to generate a 2D image
from these several data sets. These results
were published in a short paper in Nature
early in 1973.10 His paper emphasized the
basic physics and the key role of the inter-
action of the static and RF fields. To reflect
the importance of these two factors, he
coined the name zeugmatography from the

Greek word zeugma, “that which is used for
joining.” He showed that the experimental
image could be made dependent on relaxa-
tion times, and he correctly forecast the
future wide applicability of the technique:
“The variations in water contents and
proton relaxation times among biological
tissues should permit the generation, with
field gradients, large compared to internal
magnetic inhomogeneities, of useful zeugma-
tographic images from the rather sharp
water resonances of organisms, selectively
picturing the various soft structures and
tissues. A possible application of consider-
able interest at this time would be to the in
vivo study of malignant tumours, which have
been shown to give proton nuclear magnetic
resonance signals with much longer water
spin-lattice relaxation times than those in
corresponding normal tissues.”
“The basic zeugmatographic principle
may be employed in many different ways,
using a scanning technique, as described
above, or transient methods. Variations on
the experiment, to be described later, permit
the generation of two- or three-dimensional
images displaying chemical compositions,
diffusion coefficients and other properties
of objects measurable by spectroscopic
techniques. Although applications employing
nuclear magnetic resonance in liquid or
liquid-like systems are simple and attractive
because of the ease with which field gradi-
ents large enough to shift narrow resonances
by many line widths may be generated,
NMR zeugmatography of solids, electron
spin resonance zeugmatography, and analo-
gous experiments in other regions of the
spectrum should also be possible. Zeugmato-
graphic techniques should find many useful
applications in studies of the internal
structures, states, and compositions of micro-
scopic objects.”
A very different approach to MRI was
initiated concurrently and independently by
Historical Perspectives of MRI 5
Peter Mansfield.11 Mansfield and his co-
workers were exploring high resolution
NMR in solids. In 1972, Mansfield devised
a method using pulsed magnetic field
gradients to create a diffraction pattern that
could provide information on atomic sepa-
rations in crystals. The first demonstrations
of the feasibility of the concept were given
in a paper in the Journal of Physics in 197312
and at a Colloque AMPERE in Krakow,
Poland in September 1973. There Mansfield
learned of Lauterbur’s initial imaging paper
and recognized that his method could also
be used for imaging of general macroscopic
objects, not just crystalline solids. Mans-
field’s recollection of his initial reaction to
Lauterbur’s paper is interesting:11
“I was struck by the stark contrast
between our two approaches to imaging. In
my pulsed approach, I had formally pro-
posed the optical analogy of plane-wave
scattering through a mathematical frame-
work where reciprocal lattice space or k-
space was introduced and in which the k
vector was proportional to the product of
time and gradient vector. The concept of k-
space in MRI, of course, plays an important
role these days in categorizing different
imaging sequences in terms of the k-space
trajectories.”
Mansfield’s laboratory soon showed how
a magnetic field gradient and frequency-
selective pulses could be used to select
specific slices in a three-dimensional object
to permit 2D imaging of each slice; and later,
he developed the technique of echo-planar
imaging, which provides images much more
rapidly than the conventional methods.
Lauterbur and Mansfield shared the Nobel
Prize in 2003 for their independent develop-
ment of MRI.
OTHER EARLY APPROACHES TO
NMR IMAGING
Lauterbur’s initial paper did not attract wide
6 Biomedical Magnetic Resonance: Proceedings of the International Workshop
attention in the NMR community, but his
presentations at two NMR conferences had
dramatic effects.
2D FT Method
At the Experimental NMR Conference
(ENC) in Raleigh, North Carolina, in April,
1974, Richard Ernst heard Lauterbur’s talk
on zeugmatographic imaging and imme-
diately recognized that the two-dimensional
NMR method he was beginning to develop
could be used with switched magnetic field
gradients to obtain images. In Mansfield’s
terminology, Ernst’s method scanned k-
space in a different and more efficient
manner than Lauterbur’s initial back projec-
tion method. Shortly thereafter, Kumar,
Welti and Ernst13 obtained an image, again
of two tubes of water, with repetitive
experiments using an x gradient that is
stepped through successive time periods
after the exciting RF pulse is applied (so-
called phase encoding), while the y gradient
is applied during data acquisition (frequency
encoding). As this 2D imaging method began
to be used in more sophisticated practical
applications, it became clear that distortions
could be introduced with the variable time
periods for the phase encoding step. This
artifact was overcome by Edelstein, Hutchi-
son, Johnson and Red path14 by using a
constant time but variable strength field
gradient for phase encoding. With this so-
called spin warp refinement, the basic
Lauterbur concept, implemented in Ernst’s
2D manner, has become the standard
method for MRI diagnostic imaging.
Sensitive Point Method
Other approaches to NMR imaging, deve-
loped in the mid-1970s, were initially useful;
but ultimately, proved to be inefficient for
most practical imaging applications. One
arose from a talk by Lauterbur in January
1974, at the meeting of the International
Society of Magnetic Resonance (ISMAR) in
Bombay (now Mumbai). Two members
of the audience from the University of
Nottingham—Waldo Hinshaw and William
Moore—were inspired to begin a new app-
roach as soon as they returned home. They
developed a simple method that was easy
to implement with equipment then avai-
lable, the sensitive point method. They
applied oscillating magnetic field gradients
in the x, y and z directions concurrently
across the sample, so that only the sample
in a very small volume remains in resonance,
hence contributes to the imaging signal. By
altering the electrical parameters of the coils
used to create the gradients, the sensitive
point could be moved through the sample
and eventually produce a 3D image. 15
Within a few months, this simple method
had been developed to produce images of
small objects such as fruits and vegetables.
The problem is that a signal is generated
from only one point at a time, a very
inefficient procedure that requires a lengthy
period for data acquisition for a large
sample. Improvements were made to
generate a sensitive line, rather than a point,
and this method was used in early human
imaging, as described later, but this techni-
que was never developed extensively in
commercial application.
FONAR
A somewhat similar method that relied on
obtaining signal from only one small portion
of sample at a time was developed by
Raymond Damadian. Following his obser-
vation that relaxation times in excised tissue
from some tumors were longer than those
in normal tissue, Damadian began to think
about obtaining NMR information from

within a human body. In a patent application
in 1972 that covered his work on excised
tissue, he also speculated on a large in vivo
NMR system in which the RF could some-
how be focused into a narrow beam:16
“In Figure 2 (not shown here) an electro-
magnet shown in cross-section is designed
to have sufficiently large dimensions to hold
a mammal or human being to be examined.
A transmitter probe is provided with a beam
focusing mechanism for focusing the radiated
magnetic energy from the radiofrequency
generator into a beam having a narrow cross-
section. This probe is slidably mounted on a
helical track and positioned so that the
radiated beam is orthogonal to the direction
of the field provided by the electromagnet.
The transmitter probe is moved on the track
by means not shown so that the probe may
scan the entire body to be examined. Also
mounted on the track is a receiver probe of
the same design as the transmitter probe
which detects a beam having the same cross-
sectional width as the beam radiated by the
probe...”
Focusing RF of several meter wavelength
into a narrow beam flies in the face of well-
known optical principles, Damadian turned
his attention to the design and construction
of a large, low field magnet where the static
field B0 could be shaped with suitable shim
coils to obtain a small homogeneous volume,
with the homogeneity rapidly falling off
outside this volume. Damadian called the
method “field focusing NMR” and coined
an acronym FONAR. FONAR is similar to
the sensitive point method in obtaining data
inefficiently, from only one small volume at
a time. Moreover, FONAR has the addi-
tional disadvantage that the location of the
homogeneous volume is fixed by the design
of the magnet, so the subject must be physi-
cally moved in three orthogonal directions
Historical Perspectives of MRI 7
to scan a macroscopic volume, and the
magnet must be large enough to accommo-
date this movement.
In spite of these disadvantages, Dama-
dian and co-workers were able to construct
a magnet for animal studies and obtain an
image of the thoracic cavity of a mouse in
1976, with an estimated resolution of 3 mm.17
They also showed the image of a mouse in
which a tumor had been implanted in the
upper thorax. By 1977, they had constructed
a 0.05 T magnet of 53 inch diameter, with
an NMR frequency of 2 MHz, and were
able to demonstrate an image of a human
chest.18 With the very low field and point-
by-point method, the signal/noise was poor
and it took several hours to complete a scan.
The resolution was poor; but major features,
such as the heart and lungs, could be
identified. Damadian pursued this method
with the establishment of the FONAR
Corporation, which produced a few instru-
ments based on the FONAR method.
However, by 1981, the FONAR Corporation
joined other companies in adopting the
Lauterbur field gradient principle as
embodied in the Ernst 2D method with spin
warp modification.
DEVELOPMENT OF HUMAN MRI
The first live human NMR image was
published by Mansfield and Maudsley in
1977—a line scan of the internal anatomy of
a finger, an appendage small enough to be
inserted into a standard NMR magnet.19
Contrast between bone, bone marrow,
nerve, artery and other tissues was demon-
strated. Also, in 1977, Andrew et al20 used
the multiple sensitive point method and a
larger gap electromagnet to image a hand,
while Hinshaw, Bottomley and Holland
examined Bottomley’s wrist and discussed
potential medical uses.21
Serious application to most anatomical
studies required a magnet capable of holding
8 Biomedical Magnetic Resonance: Proceedings of the International Workshop
a human being. The necessary scale-up from
traditional NMR sample tube sizes of 5 to
20 mm diameter to nearly a one meter bore
for a whole human body was a daunting
task, which could be accomplished only with
a corresponding reduction in the magnitude
of the magnetic field. Most early efforts
were directed toward electromagnets with
an air core, designed for a field of about
0.1 T (4 MHz for 1 H NMR), except for
Damadian’s 0.05 T superconducting magnet.
In 1978, Mansfield et al observed the first
image of the human abdomen,22 and Clow
and Young reported the first NMR image
of a section through a human head. 23
Features such as the eyeballs and ventricles
in the brain were clearly visible. By 1980,
Moore and his colleagues at Nottingham
were able to demonstrate one advantage of
NMR imaging over X-ray computed tomo-
graphy (CT) in showing the first sagittal
and coronal sections of a human head.24 In
another paper, the same year they showed
that NMR images could reveal a wide range
of pathology in the head and commented
on the “dramatic ... new advance in neuro-
radiological capability with its potential
impact on clinical management.”25 Their
clinical results provided examples that
included tumors, aneurysms, circulatory
malformations and chronic sinus infection.
As these successful applications became
more widely known, interest accelerated
among a number of companies in developing
MRI instruments commercially for a poten-
tially very large and lucrative market in
medical diagnostic imaging. There is a
fascinating story—too lengthy to be presen-
ted here—of the concerted efforts in several
companies in the USA and Europe to rapidly
convert laboratory prototypes to clinically
useful instruments. Superconducting mag-
nets with a 1 meter bore were developed
initially at 0.5 T; but by the early 1980s, a
field of 1.5 T (about 60 MHz for proton
NMR) became the industry standard. By
October, 1987, sufficient clinical data had
been obtained for the National Institutes of
Health in the United States to convene a
Consensus Conference, in which a “jury” of
radiologists and other clinical experts could
assess the state of the art and make
recommendations on optimum uses of MRI.
The following excerpts from the Conference
Statement highlight the prevailing views:26
“Even in the short period of its use, it
has proved to be unusually rewarding in
the detection, localization, and assessment
of extent and character of disease in the
central nervous, musculoskeletal, and
cardiovascular systems. In the brain, for
example, it has a proven capacity to define
some tumors and the plaques of multiple
sclerosis provided by no other technique. It
is a competing imaging method in the
evaluation of many other organs. Additional
prospective studies comparing MRI with
other diagnostic methods are essential in
those areas where the method has shown
promise but where its precise role has not
yet been defined. This consensus develop-
ment conference does not purport to include
all of the applications of MRI to the pediatric
patient, a subject that will require separate
consideration... The full potential of MRI
has not been reached, and continuing
refinement of equipment, contrast agents,
and software may be anticipated...”
The final sentence quoted was prophetic.
MRI has become widely available and is
regarded as an indispensable diagnostic
modality. New generations of magnets have
been developed at both higher and lower
magnetic fields for specific purposes. Faster
scanning methods have become available,
and the quality of images has steadily

improved. Moreover, imaging methods have
been used to study function, as well as
anatomy, and imaging principles have been
used for localized in vivo NMR spectroscopy,
as described in a number of other articles
in this book.
SOME ANTECEDENTS OF MRI METHODS
I would like to close this account by recalling
some of the early methods and technological
developments in NMR which occurred long
before the invention of NMR imaging in the
early 1970s but which were crucial to the
development of MRI as we now know it.
Chemical Applications
One important aspect that is sometimes
forgotten is the long history of chemical
applications of NMR. Once the chemical shift
and spin coupling had been discovered
about 1950, chemical applications dominated
commercial development of NMR, just as
imaging would do three decades later.
During this 30-year period, NMR became
an invaluable tool for chemical research only
because of:
• improvements in the homogeneity of B0
• introduction of a field/frequency lock to
stabilize B0
• continued increase in the magnitude of
B0 in order to accentuate chemical shift
differences and to improve sensitivity
• improvements in probe design and
electronic circuitry that increased sensiti-
vity by orders of magnitude
• introduction of pulse Fourier transform
methods to speed up data collection and
further enhance sensitivity
• introduction of multiple pulse experi-
ments, including two-dimensional NMR,
that greatly expanded the scope of NMR
Historical Perspectives of MRI 9
Lauterbur’s initial demonstration of
imaging was carried out on a Varian A-60
spectrometer that was widely used for
chemical analysis. Indeed, his experiment
was simple to implement: He had only to
twist the x or y electric shim control from
its optimal position for maximum field homo-
geneity to an extreme position where it
introduced the usually unwanted magnetic
field gradient that is at the heart of imaging.
Almost all early imaging experiments in
academic labs used magnets developed
commercially primarily for chemical
applications. The rapid scale-up of magnets
to human size and the development of
software designed for MRI were remarkable
achievements, but they were possible only
because of the advanced technology already
available for chemical NMR.
The First NMR “Image”
There is no question that Lauterbur first
conceived the idea of employing field
gradients in separate directions and using
the resulting NMR data to construct two-
or three-dimensional images of macroscopic
objects. However, he was not the first to
apply a magnetic field gradient to discri-
minate objects along a single axis. In fact,
what is probably the first one-dimensional
NMR image was obtained by Herman Carr
in Purcell’s lab at Harvard and reported in
Carr’s PhD thesis in 1952.27 There is an
interesting story behind this experiment:
Long before Fourier transform NMR
methods had revolutionized the field,
physicists were aware of the Fourier
transform relation between a slow passage
continuous wave (cw) NMR spectrum and
the free induction decay (FID) following a
90o RF pulse. Carr and others made “mental”
Fourier transforms of FIDs. For two or three
10 Biomedical Magnetic Resonance: Proceedings of the International Workshop
lines of equal intensity, it was not too
difficult to make the mental transform and
picture the cw spectrum. However, for
systems with unequal line separations and
unequal intensities, the process was more
difficult. A “hot” topic at the time was the
recently discovered 1H chemical shift in
ethanol (CH3CH2OH), which displayed a
readily interpretable cw spectrum—three
lines of intensities 3:2:1. The corresponding
FID, however, was a complicated beat
pattern. To convince visitors of the equi-
valence of the two responses, Carr made
what in current NMR imaging would be
called a “phantom”, three pieces of rubber
of volumes 3:2:1, which were placed in a
magnetic field gradient that simulated the
chemical shifts in ethanol. The FID obtained
from this phantom agreed with that from
liquid ethanol itself, thus verifying Carr’s
interpretation.
Echoes
Whatever pulse sequence is used to obtain
an NMR image, a spin-echo is invariably
involved in some way. The discovery of
the spin echo by Erwin Hahn in 1949 ranks
as a milestone in NMR,28 since it introduced
a principle of reversibility that might at first
appear to be physically impossible. The spin-
echo has proved to be widely applicable in
all sorts of NMR experiments. The physics
of the spin-echo will be covered elsewhere;
here I comment only on its accidental dis-
covery. Hahn was investigating the use of
short RF pulses when he observed what he
calls “a weird signal” that appeared when
no pulse was being applied.29 He initially
considered it an artifact but persisted in
exploring the phenomenon and soon recogni-
zed that he had observed a refocusing of
magnetization that had apparently decayed
to zero. Carr and Purcell investigated the
spin echo in detail and developed a more
effective pulse sequence,30 later improved
by Meiboom and Gill (the CPMG sequ-
ence),31 that formed the basis for making
reliable measurements of T 2 . Carr and
Purcell also introduced the 180°-τ-90° pulse
sequence to measure T1. These sequences
have had enormous impact in MRI, as T1-
and T2-weighted images play a critical role
in diagnosis.
The pulse sequences for diffusion-
weighted images can also be traced back to
the early 1950s when the CPMG sequence
was used to measure diffusion in liquids
under the influence of a steady magnetic
field gradient. A much more effective
method was introduced in 1963 when
Stejskal and Tanner32 showed that a pulsed
field gradient offered independent control
of the magnitude and length of the gradient
without interfering with the measurement
of the spin echo. In fact, the pulsed field
gradient had been introduced originally by
Hahn and co-workers33 in 1955 to avoid
spurious echoes, an application that survives
in imaging methods as crushers and spoiler
gradients.
Other applications of echoes abound in
imaging. The stimulated echo from a 90o-τ-
90o-τ-90o pulse sequence was also discovered
by Hahn and now appears in imaging
methods. The gradient-recalled echo (GRE) was
first reported by Herman Carr in his PhD
thesis in 1952.27 Rather than reversing the
direction of the gradient to create the echo,
he rotated the sample in the gradient (the
first example of sample spinning). However,
there was little application of the GRE until
it was introduced in the form of a pulsed
gradient as a key element of imaging
methods.
CONCLUSION
This short chronology of some early deve-
lopments in NMR only scratches the surface

in describing the conceptual advances, the
innovative techniques that have been intro-
duced, and the extremely wide range of
applications for NMR that have come about
in the 60 years since the discovery of NMR
in bulk materials. Modern MRI and its close
relative, localized NMR spectroscopy, are
feasible only because this whole array of
technology has now come together to create
the powerful methods we now employ.
There is no sign of any abatement in further
rapid advances in this field.
REFERENCES
1. Becker ED (Ed) Historical Perspectives, Vol. 1
of Grant DM, Harris RK (Eds) Encyclopedia of
Nuclear Magnetic Resonance Chichester,
England: John Wiley and Sons, 1996.
2. Estermann I, Stern O. Z Phys 1933; 85:17; Frisch
R, Stern O Z Phys 1933; 85:4.
3. Rabi II, Zacharias JR, Millman S, Kusch P. Phys
Rev 1938; 53:318.
4. Gorter CJ. Physica (The Hague) 1936; 3: 995;
Gorter CJ, Broer LJF. Physica (The Hague) 1942;
9: 591.
5. Purcell EM, Torrey HC, Pound RV Phys Rev
1946; 69: 37.
6. Bloch F, Hansen WW, Packard M Phys Rev
1946; 70: 474.
7. Purcell EM, Les Prix Nobel 1952.
8. Damadian R. Science 1971; 171: 1151.
9. Lauterbur PC. “One path out of many—how
MRI actually began”. Ref. 1, 445-49.
10. Lauterbur PC. Nature (London) 1973; 242: 190.
11. Mansfield P. “A personal view of my involve-
ment in the development of NMR and the
conception and development of MRI”. Ref. 1,
478-81.
12. Mansfield P, Grannell PK. J Phys C 1973; 6:
L422.
13. Kumar A, Welti D, Ernst RR. J Magn Reson
1975; 18: 69.
Historical Perspectives of MRI 11
14. Edelstein WA, Hutchison JMS, Johnson G,
Redpath T. Phys Med Biol 1980; 25: 751.
15. Hinshaw WS. Phys Lett 1974; 48A: 87.
16. Damadian RV. US Patent 3 789 832 (Filed March
17, 1972, Issued February 5, 1974).
17. Damadian R, Minkoff L, Goldsmith M, Stanford
M, Koutcher J. Science 1976; 194: 1430.
18. Damadian R, Goldsmith M, Minkoff L. Physiol
Chem Phys 1977; 9: 97.
19. Mansfield P, Maudsley AA. Br J Radiol 1977;
50: 188.
20. Andrew ER, Bottomley PA, Hinshaw WS,
Holland GN, Moore WS, Simaroj C. Phys Med
Biol 1977; 22: 971.
21. Hinshaw WS, Bottomley PA, Holland GN.
Nature (London) 1977; 270: 722.
22. Mansfield P, Pykett IL, Morris PG, Coupland
RE Br J Radiol 1978; 51: 921.
23. Clow H, Young IR. New Scientist 1978; 588.
24. Holland GN, Hawkes RC, Moore WS J Comput
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25. Hawkes RC, Holland GN, Moore WS,
Worthington BS. J Comput Assist Tomogr 1980;
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26. Magnetic Resonance Imaging: National Insti-
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Statement, Office of Medical Applications of
Research, NIH, Bethesda, MD, 1987.
27. Carr HY PhD Thesis, Harvard University,
Cambridge, MA, 1952. See also “Early Years of
Free Precession Revisited,” Reference 1, 253-
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28. Hahn EL. Phys Rev 1950; 77: 297.
29. Hahn EL. “Pulsed NMR—A personal history,”
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30. Carr HY, Purcell EM. Phys Rev 1954; 94: 630.
31. Meiboom S, Gill D. Rev Sci Instrum 1958; 29:
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12 Biomedical Magnetic Resonance: Proceedings of the International Workshop
Introduction to
Biological NMR Spectroscopy
Soon after its discovery, NMR became an
indispensable tool in chemical and physical
sciences. For several reasons, its applications
to life sciences had to wait another three
decades. New techniques in excitation and
detection, better probe designs, break-
through in electronics, advancement in
magnet technology and development in
computer software and hardware, led to
new frontiers in NMR, and this in turn
opened its applications in biological sciences.
Today, NMR is an indispensable tool in
structural determination of large biological
molecules, understanding biochemistry of
cellular systems, imaging and spectroscopy
of whole organs (MRI and MRS), clinical
medicine, pharmacology and several other
areas of life sciences.
The foundations of modern biology were
laid during the period 1940-1970. For
example, it was established that nucleic acids
(DNA and RNA) are carrier of genetic
information. The information coded in the
form of sequence of DNA bases is translated
into specific proteins, which are responsible
for functions of cells. Cells, in turn, are
responsible for the chemistry and functions
of whole organs. During the last 40 years,
2
Girjesh Govil
there has been major progress in decoding
of gene sequences of various organisms,
sequencing and structure determination of
macromolecules such as proteins, carbo-
hydrates and nucleic acids, cellular metabo-
lism and understanding of how organs
function. In this brief presentation, I shall
show how NMR has made impact on
modern areas of life sciences, focusing on
topics not covered by other speakers in this
workshop.
CLASSICAL VIEW OF NMR
SPECTROSCOPY
We note that NMR is observed with nuclei
having spin (I). In the presence of magnetic
field (B0), the angular momentum of such
nuclei causes a precessional motion of their
magnetic moment (μ) around the z-axis, with
an angular velocity ω0 = -γB0 (radians/sec).
Under thermal equilibrium, more nuclei are
aligned parallel to B0 and there is a net
magnetization (Mz) in the z-direction. If we
apply an oscillating radiofrequency (RF) field
(B1) in the x-direction, with a frequency equal
to ω0, then the net magnetization will tend
to flip from the z-direction to the xy plane
depending on the strength and duration of
 Introduction to Biological NMR Spectroscopy 13
the RF pulse. The flip angle is given by θ =
γB1t (radians), where B1 is the strength of
the RF field and t is the time duration of
the applied field. Modern NMR techniques
use RF pulses, which are characterized by
the flip angles that they produce. Thus, a
π/2 (90°) pulse flips the net magnetization
from the z-axis to xy plane and produces a
transverse magnetization (Mxy). A π (180°)
pulse inverts the net magnetization to the
negative z-direction. Pulses with small flip
angles rotate only a portion of magnetization
vector (M) in the transverse plane. Most
experiments are conducted using more than
one pulse applied at specified periods of
time. The motion of M may appear complex
as we have the Larmor precession and the
spin flip superimposed. It is conceptually
simpler to describe the motion of the NMR
nucleus in a rotating frame of reference.
One can imagine the viewer standing at the
origin of the reference frame and rotating
with an angular frequency equal to the
Larmor precession. Such a description
greatly helps in understanding the behavior
of net magnetization.
Observation of NMR Spectrum: There are
three basic elements of an NMR spectro-
meter. A magnet with a very homogeneous
magnetic field is used to lift the degeneracy
between nuclear energy levels. Higher the
field, larger is the gap between the ground
and the excited states, and higher is the
population difference between the two
states. Thus, higher magnetic fields help in
better sensitivity. We will see later that it
also helps in a better dispersion of NMR
lines. Thus, in solution studies on bio-
molecules, one tries to use the highest
possible magnetic field. Spectrometers using
B0 as high as 23.5 T are available, which
correspond to a 1H resonance frequency of
1000 MHz. One generally uses 1H resonance
frequency rather the magnitude of the field
to indicate the magnetic field of the
spectrometer. The sample volume used is
around 5 mL, which is placed in a sample
tube of 5 mm diameter. The pole gap is
relatively narrow. When working with
animals and humans, lower magnetic fields
are used for safety consideration (usually
less than 4 T), but the pole gap is much
larger to accommodate the animal. In
addition to the main magnetic field, electro-
nic coils are used to achieve high level of
field homogeneity. Field gradients are used
to change the field to suit the requirement
of the experiment.
The second component of NMR equip-
ment is an arrangement for generating RF
powers at desired frequencies. Modern
NMR experiments are usually performed,
using strong RF fields in the range 0.01 to
0.4 T or several hundred kilowatts. These
RF pulses are applied for short durations,
with a very precise and specific time frame.
A pulse programmer helps to attain such an
objective. RF pulses may cause heating and
proper arrangements for maintaining the
temperature are made.
The signal is detected by a receiver coil
and then processed by a computer. Several
types of editing procedures are built in the
computer software, all of which are part of
the spectrometer console. For optimum
signal-to-noise (S/N) ratio, there has to be
a very precise coupling between the
excitation and detection hardware.
A Simple Pulse Sequence for NMR Excita-
tion and Detection: The simplest pulse
sequence used in NMR is the free induction
decay (FID) following an excitation pulse as
illustrated in Figure 2.1. A 90° pulse is used
14 Biomedical Magnetic Resonance: Proceedings of the International Workshop

Fig. 2.1: A simple pulse sequence used in NMR. The FID signal is detected as a function of time. Instead of a
90° pulse, one can use a softer pulse (flip angle less than 90°), which allows system to return to equilibrium
at a faster rate

to excite the sample, which tips the orienta-
tion of the net magnetization from the z-
axis to the xy plane. This pulse establishes
“phase coherence” as immediately after the
pulse, all nuclei rotate together (coherently)
around the z-axis, in the transverse plane.
A signal is induced in the detector coil,
which is called the FID. This signal decays
with a particular time constant (T2) and
signal is detected as a function of time. Since
the transmitter pulse is several order of
magnitude stronger than the detected signal,
the collection of the signal is delayed by a
few milliseconds for the receiver coil to
recover and respond to the FID. The
excitation and detection can be repeated
several times to get a good time-averaging
and improve S/N. The signal S(t) received
in the single-pulse FID experiment (as well
as in other pulse experiments) is in the time
domain. The Fourier transform (FT) of S(t)
gives the spectrum as a function of frequency
S(ν).
NMR NUCLEI IN LIFE SCIENCES
All living systems have five major elements,
C, H, N, O and P. Of these, the naturally
abundant isotopes of C (12C) and O (16O)
do not have magnetic moments. The more
abundant isotope of N (14N) has spin 1. For
O, 17O has magnetic moment, but its spin is
5/2. Nuclei with spin > ½ have quadrupole
moments. Such nuclei give relatively broad
signals. Fortunately, we have four spin ½
nuclei, 1H, 13C, 15N and 31P, which serve as
reporter groups and provide us with a
 Introduction to Biological NMR Spectroscopy 15
Table 2.1: Important NMR nuclei in life sciences
Nucleus Natural Resonance frequency
abundance at 10 T (MHz)
1H 99.985 425.53
13C 1.11 106.97
15N 0.37 43.10
31P 100.00 172.25
wealth of information about biological
systems. Their NMR properties are listed in
Table 2.1.
It is this rich arsenal of information
carriers, which makes NMR such a powerful
method. For example, P is present in
relatively smaller number of biological
molecules. However, some of these, such as
adenosine triphosphate (ATP) are involved
in providing energy for essential processes
of life. 31P is the nucleus of choice for
studying bioenergetics in cells and organs.
In muscle and brain, another energy-rich
compound is phosphocreatine (PCr). When
sufficient oxygen and fuels are available to
facilitate ATP synthesis, abundant amount
of PCr is produced. During energy crisis
(hypoxia, ischemia or cell damage), PCr
serves to maintain ATP levels so that cellular
work can continue. However, if ATP is not
synthesized and PCr stores are depleted,
some of the cellular functions can no longer
occur.
Both H and C are elements present in all
biological molecules. 1 H and 13C NMR
provide information on all classes of
biological systems. These spectra are
relatively more complex, but are rich in
information. Even 1H resonance in a simple
molecule like water (which constitutes almost
75% of living systems) provides us with a
wealth of information on biological organs
as will be discussed extensively in this
workshop. The natural abundance of both
13C and 15N are low. Both 13C and 15N are
Relative Chemical shift
sensitivity range (ppm)
100.0 15
0.02 250
0.0004 1700
6.6 430
stable isotopes and are available at reason-
able costs. Gene cloning methods can be
used to prepare reasonable quantity of
compounds enriched in these isotopes for
NMR studies. 13C or 15N labeled substrates
can be used in studies of metabolism.
In addition to above, use has been made
of certain other nuclei for specialized
applications. 2H (deuterium) has been used
in studies on biological membranes. 19F has
been used for studying drug metabolism.
Nuclear polarized 3He and 129Xe have been
used in imaging of lungs. Use of 7Li and
23
Na has also been reported.
NMR PARAMETERS
It may appear from the above discussion
that each atomic nucleus having a magnetic
moment shall give a single line spectrum.
This is true if we look at the 1H NMR in
water. However, what makes NMR useful
is the fact that nuclei are not isolated and
electronic clouds in their environment
modify their behavior. Let us discuss some
of these properties.
Chemical Shift (δ): Figure 2.2 shows 1 H
NMR spectrum of a small DNA duplex
containing 12 base pairs. It is obvious that
the spectrum has several hundred lines. The
31 P spectrum like wise has several lines.
These arise because different 1H or 31P are
in different chemical environments. The
electrons modify the magnetic field seen by
the nucleus, and the field (Bn) at the site of
16 Biomedical Magnetic Resonance: Proceedings of the International Workshop

Fig. 2.2: 1H and 31P NMR spectrum of a self- complementary DNA duplex dissolved in water and having the
sequence GGTAGIACTACC. Positions of different 1H resonances are marked with reference to the shift of
sodium 2,2- tetradeutero-3-trimethyl-silylpropionate used as standard. Standard chemical nomenclature has
been used for labeling different atoms in the oligomer.

the nucleus is slightly different from the
one (B0) applied.
Bn = B0 (1 – σ)
σ is called the shielding or the screening
constant. The differential screening of nuclei
due to electrons and other local fields in
the vicinity of the nuclei under study is
called chemical shift. One has to apply higher
magnetic field (at fixed frequency) to get
the resonance.
Several short- and long-range factors
influence σ and a theoretical calculation is
difficult. However, it is possible to break-
down contributions from various factors and
estimate changes in values of σ, rather than
their absolute values. For example, one may
compare the chemical shift values of the
same protons in an ordered state of a
macromolecule and the values in a random
coil. The difference can then be correlated
with structural changes, and the associated
changes in the magnetic fields arising from
conformational differences. Instead of
absolute value of σ, one uses the difference
from a standard compound to express
chemical shifts (δ = σref – σ, or (ν–νref)/ν).
The values of δ are of the order of 10–6 and
are, therefore, expressed in parts per million
(ppm). In this form, δ is a dimensionless
quantity. However, chemical shifts are also
expressed as differences in frequencies in
Hz (Δν = B0Δσ). In this form, the shift is
proportional to the applied field. For
example, on a 100 MHz spectrometer, 1 ppm
corresponds to 100 Hz; while on a 1000 MHz
instrument, it is equal to 1000 Hz.
Let us look at the spectrum in Figure
2.2, more carefully. One finds that different
 Introduction to Biological NMR Spectroscopy 17
1H have different shifts and these are
characteristic of the chemical groups. The
thymine CH3 protons come at around 1.7
ppm and are at the highest field. The signals
in the 12-15 ppm range are due to hydrogen
bonded imino protons. What is more, even
protons with similar chemical groupings
such as the two thymine methyls have
different chemical shifts.
Peak Intensities: The next thing we notice
is that the CH3 proton lines have much
larger amplitude than the cytosine H5, H6
protons. The intensities in NMR spectra are
directly proportion to the number of protons
giving rise to the signal and provide relative
number of 1 H in the chemical groups.
However, amplitudes may be different if
resonance lines have different line widths.
NMR spectrometers have a built-in subro-
utine to provide integrated areas of various
lines and thus to provide relative intensities.
Scalar Coupling (Jij): A careful look at the
cytosine H5, H6 proton signals reveals that
each of them are doublets separated by
about 7 Hz. This splitting arises because the
H5 and H6 protons interact with each other
through an interaction called scalar coupling.
Such an interaction (Jij) between nuclei i and
j, occurs through the electronic cloud joining
the two atoms. It depends on the number
and the nature of chemical bonds joining
atoms i and j, hybridization of the bonding
orbitals, and other stereochemical factors.
J values between directly bonded nuclei are
large. For example, J value for directly
bonded 13C and 1H is around 125 Hz for
saturated carbon atoms (sp3 hybridized C),
150 Hz for ethylenic C, and 220 Hz for
acetylenic C. Three bond coupling J (A-B-
C-D) usually follow a cos2ϕ dependence on
the dihedral angle (ϕ) around the B-C bond.
J values are relatively small when the
number of intervening bonds is more than
three. In the case of cytosine, the interaction
occurs via the H5-C5-C6-H6 fragment and
is about 7 Hz. On the other hand, coupling
between T(CH3) and the T(H6) protons is a
four bond interaction and its magnitude is
small.
Dipolar Couplings (Dij): Nuclei also interact
directly due to their proximity in space, as
each nuclear magnet exerts a magnetic field
on other spins in its close proximity. This is
called direct magnetic dipole-dipole inter-
action or dipole coupling and results in the
splitting of the resonance of each interacting
1
H into two, with a separation:
ΔB = 3μ (3cos2 θij –1) (μ0/4π)/rij3
Here, μ0 is the permeability of the medium,
μ is the nuclear magnetic moment, rij is the
distance between nuclei i and j and θij is the
angle between the internuclear vector (rij)
and the applied field, B0 . Thus, dipolar
interaction contains geometrical information
about the molecule. In solutions, because of
the rapid motion of the molecules, the angle
θ varies with time. The time average of the
term (3cos2 θij–1) is zero and, therefore,
dipole-dipole interaction is not observed
directly. However, if a molecule is dissolved
in an anisotropic liquid crystalline solvent,
then the value of this term may not average
to zero, and the geometrical information
can be retrieved. This interaction is also a
dominant contributor to relaxation rate,
which is manifested in the form of nuclear
Overhauser effect (NOE).
Relaxation Rates and Line-widths: Imme-
diately after the application of B0 field, the
population of the two nuclear spin energy
levels is equal, the net magnetization Mz
= 0, and the system is in a non-equilibrium
18 Biomedical Magnetic Resonance: Proceedings of the International Workshop
state. The build up of equilibrium magneti-
zation obeys a first order rate equation:
dMz/dt = (M0–Mz)/T1
T1 is called the spin-lattice or the longi-
tudinal relaxation time. Immediately after
excitation by a 90° pulse, the net magneti-
zation in the z-direction is zero, and Mz
returns to its equilibrium value with the
same time-constant.
The built up or decay of the z-magneti-
zation requires exchange of energy between
nuclear spins and the surrounding (lattice).
In liquids, the dominant process for such an
energy exchange for spin ½ nuclei is the
magnetic dipole-dipole interaction. Rota-
tional and translational motions of a
molecule in a liquid cause a fluctuating
magnetic field. The components in the trans-
verse plane with component at the Larmor
frequency can stimulate transitions between
the two states of the nuclear spin system.
The magnetic energy received by the
environment is transformed into thermal
energy. Both intra- and inter-molecular
interactions can contribute to relaxation. For
13C, the relaxation is dominated by intra-
molecular 1H-13C dipole-dipole interaction.
For 1H, the relaxation mechanism involves
1H-1H dipole-dipole interactions, both inter-
molecular (particularly with solvents) and
intramolecular.
The magnetization in the xy plane (Mxy)
decays with a different time-constant T2,
which is called spin-spin or transverse
relaxation time. An important mechanism
for T2 relaxation is energy transfer within
the nuclear spin system. Transition in the
spin state of a nucleus changes the magnetic
field at the nearby nuclei and contributes to
shortening of the life-time of the spin state.
The relaxation times depend on two factors:
the strength of the interaction responsible
for energy transfer and the time-scale of
the motion causing the fluctuations in the
local magnetic fields. For non-viscous
liquids, such as pure water, T1 and T2 are
almost equal. However, in general, the
transverse magnetization decays faster than
the longitudinal magnetization.
The line-width of the resonance signal
at half-height is related to T2 and is, in fact,
equal to 2/T2 . The decay of transverse
magnetization can also be caused by in-
homogeneities in the magnetic field (ΔB0).
One therefore defines another time
relaxation time T2*
1/T2* = 1/T2 + γΔB0/2
The line-width is given by 2/T2*.
We will learn later that relaxation
parameters T1, T2 and T2*, even in a simple
molecule such as water, are influenced by
factors such as its interaction with other
molecules in the tissue, flow and diffusion,
paramagnetic molecules and other factors.
These properties form the basis of medical
imaging by NMR.
Quadrupolar Interactions: Nuclei with spin
quantum number > ½ possess a charge
distribution, which is not spherically
symmetric. These nuclei, therefore, have a
quadrupole moment Q. This can interact
with the electric field gradient (q) at the
nucleus and contribute to relaxation. In case
of deuterium ( 2 H), the value of Q is
relatively small (Q = 2.77 × 10–3) and, there-
fore, NMR signals are relatively sharp. In
fact, the quadrupolar coupling constants
(e2 qQ) can be used for obtaining useful
information in certain biological problems.
On the other hand, 14N and 17O resonances
are fairly broad.
 Introduction to Biological NMR Spectroscopy 19
Nuclear Overhauser Effect (NOE): The NOE
is defined as the fractional change in the
intensity of the signal of the nucleus i, when
the transition due to another spin j is
perturbed. NOE arise from dipolar-dipolar
interaction between the two spins (i and j)
and also depend on molecular motion. When
intramolecular dipole-dipole interactions
dominate the relaxation, NOEs are inversely
proportional to the sixth power of the inter-
nuclear distances. In a molecule, if one
distance is known (e.g. H5-H6 distance in
cytosine), then the other distances can be
estimated. This is one of the key information
in determining macromolecular structures.
Chemical Exchange: Another factor, which
influences NMR signals, is exchange between
different chemical entities. For slow
exchange (on the NMR time-scale), we see
signals from the two species involved in
chemical exchange. When the exchange rate
is fast, one gets a spectrum, which is the
time-average of spectra for the two
exchanging species. This information can be
used to obtain thermodynamic and kinetic
information for the exchange reaction.
DIFFICULTIES IN STUDYING
BIOLOGICAL SYSTEMS BY NMR
Why it took 30 years for NMR to move
from chemical to biological systems. There
are several problems which one encounters
when dealing with biological systems.
Sensitivity: By its very nature, NMR is a
relatively insensitive technique. In earlier
days, one required solutions of 100 mM
concentrations to obtain a decent NMR
spectrum. With the advent of FT technology,
advances in probe designs, magnet and
computer technology, the limit has come
down to mM concentrations. The techno-
logies are improving every year leading to
new advancements in biological research.
With the availability of cryoprobes and other
technical advances in NMR, the concen-
tration limit may go down to nM ranges.
Resolution: We have seen, that even a small
oligomer gives a very large number of NMR
signals. The 1H signals lie in a narrow range
of 15 ppm. Thus, it is difficult to resolve all
resonances, particularly for nuclei, which are
in very similar chemical environment. In the
above example, one notices the crowding
of signal in the sugar 1H regions. Therefore,
it is preferable to use higher fields for
biomolecular studies and for in vivo
spectroscopy.
Assignments: To make meaningful conclu-
sions, one needs to know which signal is
associated with which nucleus. In the
example above, there are two thymines and
indeed one gets two 1H signals in the CH3
region. But how do we find as to which
signal belongs to which thymine? Assign-
ment of signals is one of the first steps in
applications on biological systems.
Water Signal: In most chemical applications,
NMR studies are conducted using
deuterated solvents such as D2O or CDCl3.
However, for biological applications, studies
have to be performed in H2O, which is the
milieu of living systems. D2O is toxic. Even
in studies on macromolecules, D2O is avoi-
ded, to prevent deuterium exchange with
the labile NH and OH protons, which
provide valuable information on hydrogen
bond networks. With two protons, the
molar concentration of water protons in
aqueous solutions is almost 110 M, against
which signals from mM concentrations of
the molecules of interest have to be picked.
Fortunately, FT NMR has given us power
to suppress undesirable resonances. Two
such molecules from our view-point are
water and fat.
20 Biomedical Magnetic Resonance: Proceedings of the International Workshop
Broad Lines: The line-widths in most organic
compounds are around 0.3 Hz. The lines in
biological macromolecules are broader
because of shorter T 2 due to their high
molecule weight, which leads to lower
mobility. The line-width increases with the
molecular weight and, therefore, studies on
large proteins are not only complicated by
problems of resolution and assignments but
also by the higher widths of NMR signals.
Some solutions to such problems have been
suggested recently. The line-widths in cells
and organs are even more.
2D NMR SPECTROSCOPY
Several problems discussed in the previous
section have been resolved through the use
of two-dimensional (2D) and multi-
dimensional NMR spectroscopy. Special
techniques of pulse NMR allow spectra to
be recorded with two independent
frequency dimensions F1 and F2. The signals
in such spectra are characterized by two
frequencies (f1, f2). Depending on the pulse
sequences used, the two frequencies may
correspond to the same nuclei (homonuclear
2D) or different nuclei (heteronuclear 2D).
The diagonal peaks in a homonuclear 2D
NMR (f1 = f2) correspond to the 1D NMR.
The off-diagonal peaks (commonly called
cross peaks) have important information
about the relationships between different
spins. Different interactions between the
spins can be picked up depending on the
pulse sequence.
A general scheme of 2D NMR is shown
in Figure 2.3. A second time domain is
created by performing 1D experiments with
various incremental times during the
evolution period t1, during which the nuclear
spins interact with each other through
various type of interactions. The various 2D
NMR experiments differ essentially in the
nature of mixing during the period t1. Some
critical experiments in biomolecular studies
are J-Correlated Spectroscopy (COSY) (and
several advanced versions of COSY such as
HMQC, TOCSY), Nuclear Overhauser
Spectroscopy (NOESY) and ROESY. The
basic pulse sequences for such experiments
are shown in Figures 2.3A to D. Spin-echo
and inversion recovery experiments, which
are extensively used in MRI are also multi-
pulse experiments. Recently, such experi-
ments have also found applications in MRS.
New pulse sequences are being developed
every year and it is difficult to keep count
of the ever-increasing acronyms in the
literature of pulse NMR.
The following advantages of 2D
spectroscopy are obvious:
1. Since we can spread the peaks in two
dimensions, cross peaks have a better
dispersion of signals. In heteronuclear
experiments, the larger chemical shift of
the second nucleus helps in resolving
signals from protons with very similar
chemical shifts.
2. Homonuclear correlation parameters
between nuclear spins, such as J and
NOE can be obtained, which in turn, can
be used for structural determination.
3. Correlations between various NMR
nuclei (1 H, 13C, 15N and 31 P) can be
established. This helps in assignments.
4. Information on chemical exchange can be
obtained using ROESY experiments.
5. Pulse sequences have been developed
which suppress signals from water or
other undesirable species.
NMR AT DIFFERENT LEVELS OF
BIOLOGICAL SYSTEMS
Information transfer in biological systems,
flows from 1D information present in DNA
 Introduction to Biological NMR Spectroscopy 21

Figs 2.3A to D: Use of pulses to generate 2D spectrum. (A) General scheme of 2-dimensional NMR; it usually
consists of (i) a period during which the spin system of interest is prepared; for example, by generation of
transverse magnetization through a 90° pulse, (ii) an evolution period t1 during which it develops under
factors such as Larmor precession or J/D couplings, (iii) the detection time t2. (B) By a systematic variation of
evolution time a second time axis (t1) is created. (C) Pulse sequence for a simple COSY spectrum consists of
two 90° pulses separated by the evolution time t1. (D) Pulse sequence for a NOESY spectrum
22 Biomedical Magnetic Resonance: Proceedings of the International Workshop
sequence, through m-RNA, t-RNA, and
ribosomal RNA to amino acid sequences in
proteins. The 3D structures of proteins are
responsible for activities of cells. Though
all cells are derived from the same genomic
material (paternal gene from spermatozoa
and maternal gene from the ovum), the
initial fertilized egg cell retains its properties
only during the first few rounds of division.
They later differentiate into cells for
different organs, which have different
properties and functions. This is achieved
by differential expression of the genes and
thereby, changes in the cellular levels of
different proteins. Further, cells from the
same organ may behave differently, if
subjected to stresses such as disease, changes
in diet, chemical and environmental factors,
etc. For example, if one ingests a toxin or a
drug, the protein levels may not change,
but it may inhibit a critical enzyme and
reduce its activity.
Thus, a biologist is interested in knowing
sequence, the 3D structure and function of
biological molecules, organization and
functions of macromolecular assemblies,
cellular functions as well as chemistry of
whole organs. NMR has found applications
at all these diverse levels of biological
processes.
MACROMOLECULAR STRUCTURES
AND ORGANIZATIONS
One of the most rewarding areas of NMR
is study of the three-dimensional structures
of biological macromolecules (proteins,
nucleic acids and sugars), their dynamic
behavior and their structure-function rela-
tionship. The structures of a large number
of such molecules have been reported, but
our knowledge is far from complete. As
yet, we do not know the total number of
proteins or even protein folds involved in
biological functions of living systems. The
human genome project has witnessed a
major breakthrough, raising the hope that
biological processes will be understood at
molecular level. At this stage, we do not
know the exact gene sequences which are
expressed. However, several proteins with
similar functions have similar 3D structures
(e.g. kinases, dehydrogenases) and, there-
fore, knowledge of the structure of a parti-
cular class of protein from one organism,
may help us to understand a particular
reaction in the cell or organ of another.
NMR and X-ray crystallography are the
major techniques used to unravel 3D
structures of biological molecules. Several
NMR parameters provide structural infor-
mation. These include distance information
from NOE, orientation constraints from
dipolar couplings (using liquid crystalline
solvents), backbone and side chain dihedral
angles from scalar couplings, secondary
structural elements from chemical shift
changes, location of hydrogen bonds from
chemical shifts or exchange studies, etc. The
structural parameters are normally fed into
structural algorithms, which provide struc-
ture(s) consistent with the structural para-
meters. Unlike crystallography, NMR does
not provide the structure directly. At this
stage, only medium size macromolecules and
molecular assemblies have been studied
using NMR. One advantage of NMR
methods is that the structures are obtained
in water solutions and are not dictated by
crystal forces. It has been realized that
biological macromolecules are not rigid and
have internal motions, which in fact, plays
a dominant role in their functions. NMR
has proved advantageous in exploring
motions of biomolecules.
 Introduction to Biological NMR Spectroscopy 23
The next level at which NMR has helped
is in understanding macromolecular
organization. In recent years, studies have
been made on complex macromolecular
assemblies, such as protein-nucleic acid
complexes, biological membranes, glyco-
proteins and glycolipids. The use of recently
developed high resolution solid-state NMR
has made a major impact on such studies.
STUDIES OF BODY FLUIDS,
TISSUES AND CELLS
Cell function is the result of an intricate
network of functioning proteins, small
molecules and ions and external factors,
which may activate or control enzyme
functions. Though enzyme functions have
been studied in vitro, one understands in
vivo biological functions only by working at
cellular level. NMR has proved invaluable
to understand cell metabolism and factors,
which control and modify such actions. NMR
of cells, tissues, and body fluids has,
therefore, become an important area of
research in biological and medical sciences.
Metabonomics is a recent term coined for
this field, which is aimed to understand at
a molecular level, the behavior of cellular
systems and aberrations in their function as
a result of diet, stresses and changes during
cell development and differentiation. The
non-invasive nature of NMR measurements,
offer several advantages over conventional
techniques. While most analytical techniques
are capable of detecting compounds for
which the technique has been designed,
NMR is not biased towards a particular
compound. It does not require precise
selection of analytical conditions to obtain
quantitative information from endogenous
metabolites. It can simultaneously detect
molecules which are expected, unexpected
or which are difficult to assay by standard
chemical methods. The only problem is
related to the low sensitivity of the method,
limited at this stage to mM ranges.
Cell metabolism is a highly intricate net-
work of coupled reactions. These include
biosynthetic (which usually require energy)
and oxidative pathways (from which living
systems obtain energy). Energy stored in
the form of fatty acids, or sugars is released
during their oxidation and stored in the
form of ATP or PCr. There are three major
stages in energy-producing metabolism,
glycolyis, Krebs cycle and oxidative phos-
phorylation. During glycolysis, glucose is
metabolized to pyruvate through a series
of reactions. One molecule of glucose is
oxidized to two molecules of pyruvate, two
molecules of NAD are reduced to NADH
and there is a net production of two
molecules of ATP. Certain organisms
survive entirely on glycolysis as energy
source. In these cases, pyruvate may be
fermented to alcohol or some other molecule
and simultaneously, NADH is oxidized back
to NAD. During anaerobic conditions in
brain or in spermatozoa, the final product
is lactic acid. In presence of oxygen, pyru-
vate is converted to acetate, which is then
further metabolized in the Krebs cycle. The
two carbons are oxidized to CO 2 and
coenzymes FAD and NAD are reduced. In
the final stage of oxidative phosphorylation,
which occurs in mitochondrial membrane,
the reduced coenzymes are oxidized and
energy released is stored in the form of
ATP. The energy pathways are controlled
at several stages. For example, if there is
excess of ATP, then glucose metabolism
stops and it is stored in the form of gluco-
gen. This simple picture may be modified
in different organs or organisms or under
stresses such as disease.
24 Biomedical Magnetic Resonance: Proceedings of the International Workshop
Study of cell metabolism under aerobic
conditions (presence of oxygen) require
special modifications in the probe and in
sample handling. However, glycolysis,
inactive cells, cell free extracts, body fluids
and tissues can be analysed using a normal
probe in a high resolution NMR spectro-
meter. I shall spend some time on cellular
NMR since this subject is extremely impor-
tant in following the area of magnetic
resonance spectroscopy (MRS) of whole
organs, and is not covered by other
speakers.
STUDIES ON SPERMATOZOA
As an illustrative example, we discuss
studies on spermatozoa, the paternal cells
in reproductive biology of mammals.
Importance of such studies in fertility
regulation, artificial insemination, and in
understanding cellular and molecular
biology of spermatozoa is obvious. From
the view point of NMR, spermatozoa are
extremely friendly, as they can survive for
a long time under anaerobic conditions and
do not settle because of their motility.
General features of sperm morphology,
and its diversity in different animals has
been the subject of extensive investigations
using microscopic techniques. Its overall
structure can be divided into three parts;
(a) head, which contains the paternal
chromatin and has a cap-like cover called
acrosome, which participates in egg penetra-
tion and fusion, (b) mid-piece containing
mitochondria and which contributes to most
of the NMR signals, and (c) tail which is
responsible for the movement in the seminal
fluid (motility). Motility and fertilizing
ability are the major markers of sperma-
tozoan activity, which can be beneficially or
detrimentally influenced by a variety of
physical and chemical factors. Epididymis,
is the male reproductive organ. It acts as a
highly efficient storage system and enables
spermatozoa to mature and preserves/
enhances their motility and fertilizing
capacity. There are three main parts of
epididymis: caput is the birthplace of
spermatozoa, corpus where they are in semi-
matured stage and cauda where they are
fully matured. During the passage through
epididymis, spermatozoa undergo major
morphological and biochemical changes. The
next stages in the cycle of spermatozoa are
capacitation, a process where spermatozoa
are activated, and acrosomal reaction where
the cap region delivers hydrolytic enzymes
so that it can fuse (fertilize) with the egg.
These two processes take place in the female
reproductive tract. Changes in the bio-
chemistry of spermatozoa in all the five
steps have been followed using NMR.
Identification of Small Molecular Weight
Compounds: 1H and 13C NMR allows direct
detection of the molecules present in the
cells (Figs 2.4A and B). It may be noted that
macromolecules do not contribute to such
spectra as their lines are relatively broad
and concentrations are low. Typically, 105
cells suspended in Dulbecco medium have
been used for obtaining 2D homonuclear
DQF-COSY and 1H-13C HSQC spectra. The
wealth of information available in such
spectra is obvious. The chemical composition
and relative concentrations of molecules in
cells changes dramatically, not only from
species to species but also within samples
from the same group of animals. The signals
and levels of amino acids such as Ala, Arg,
Asp, Gly, Glu, Ile, Leu, Lys, Met, Pro, Thr,
Val, and molecules such as lactate (Lac), m-
inositol, glycerophosphorylcholine (GPC),
hypotaurine and uridinediphosphoglucose
(UDP) could be identified and their
concentrations measured. The relative
 Introduction to Biological NMR Spectroscopy 25

Figs 2.4A and B: 2D 1H-13C (J-correlated) HMQC spectra, from sperm cells derived from two different regions
of goat’s reproductive tract. As the cells mature, there are marked changes in their chemical composition,
which is reflected in these spectra

concentrations of these molecules change as metabolic intermediates can be followed in

a result of maturation. Two unexpected
molecules detected by NMR are β-Ala and
hypo-taurine, which have been reported for
the first time in goat spermatozoa (Fig. 2.4).
Metabolism in Cells: Cells derive their
energy from ATP, which in turn is synthe-
sized by biochemical pathways mentioned,
earlier. The overall reactions in glycolyis
under anaerobic conditions is represented
by:
Glc + 2ADP + 2Pi
= 2Lac + 2ATP + 2H+ + 2H2O
Metabolism can be followed by a
decrease in the 13 C (Glc) signal or an
increase of 13C (Lac) signal. The levels of
real time. For example, when cells are fed
with glucose (Glc) labeled at position 1, the
label appears at the corresponding carbon
atoms in the various metabolites. In glyco-
lysis, a decrease in the glucose signal, and
build up of signals from lactate (arising from
C-3 methyl) can be monitored with time
(Fig. 2.5).
31
P NMR is particularly useful to detect
ADP, PCr and ATP levels and intra- and
intercellular pH (Fig. 2.6). Using these levels,
effect of various external factors on
glycolysis can be determined. In the case of
spermatozoa, the energy released in glyco-
lysis and trapped in the form of ATP is
needed for its biochemical requirements,
26 Biomedical Magnetic Resonance: Proceedings of the International Workshop

maturation, capacitation, motility and

Fig. 2.5: 13C NMR spectrum, from cells undergoing
glycolysis. 13C labeled Glc has been used. The label
goes to other intermediate and final product of the
reaction (lactate)
fertilization. Cells, which are dormant, (for
example, when stored under frozen condi-
tions or starved) have a low level of ATP
and higher level of ADP and P i. When
starved cells are fed glucose, the ATP level
builds up. Inactive cells do not consume Glc
and there is no increase in ATP/ADP ratio.
Such cells also do not show motility or
fertilizing capability. Inorganic Pi exists in
the cells in the form of H2PO4– or HPO4–
depending on the pH, and the 31Pi signal is
at a position which is the time average of
the chemical shifts of the various forms of
the tri-basic acid. The measurement of pH
is a quick check on the metabolic activity of
the cell (Fig. 2.6).
Biochemical Changes during Cell Matu-
ration: The changes in the biochemical
constituents of spermatozoa have been
analyzed. When the cells are born in caput
region, one sees mainly ADP signal with
very little ATP. ATP levels increase as the
cells mature. There are also changes in the

Fig. 2.6: 31P NMR spectrum of active cells provides vital information on bioenergetics, including levels of
ATP, PCr and intracellular pH. A similar methodology is followed in MRS
 Introduction to Biological NMR Spectroscopy 27
levels of several compounds during cell
maturation as spermatozoa move from caput
to cauda region. The total inositol (m-
inositol + s-inositol) decreases with the cell
maturation, while the relative concentration
of GPC and amino acids Arg, Glu and Gln
increases. The signal for the unusual amino
acid hypotaurine, which is an important
compound for sperm survival capacitation
and fertilization, is very weak in caput
region. The amount of lactate, creatine and
phosphocreatine is much less in cauda cells
compared to caput while that of Glu, Gln,
GPC and hypotaurine is higher (Fig. 2.4).
Effect of Argon on Activity of Spermato-
zoa: We can learn about effect of certain
compounds on metabolic pathways, which
is of potential value in drug discovery and
in study of inhibition of cell growth. For
example, L-Arg is an important molecule in
sperm metabolism and is known to increase
spermatogenesis. Administration of Arg to
oligospermic and asthenospermic patients
leads to both an improvement in sperm count
and in motility. NMR studies show that Arg
increases Glc and Fru consumption by cells
with a concomitant increase of Lac pro-
duction and decrease of pH. It reduces
damage due to the presence of ionizing
radiation and reverses impairment caused
by glycolytic inhibitors (potential contracep-
tives) and the extent of lipid per-oxidation
induced by UV radiation, freezing and
oxidizing agent. L-Arg has a high degree of
specificity in its catalytic activity as
structurally related amino acids such as L-
ornithine, L-Lysine and nitroarginine do not
act as activators. NMR studies have helped
in establishing that Arg stimulates the nitric
oxide synthesis in spermatozoa, which is a
key factor in the enhancement of metabolic
activity. It has been concluded that the
mechanism of action of Arg is primarily
through increased biosynthesis of nitric
oxide in spermatozoa.
APPLICATIONS IN DRUG DEVELOPMENT
During the last decade NMR has been
extensively used for the detection of
metabolic disorder, biochemical basis of
drug and xenobiotic metabolism, as a
pathological tool, toxicological processes,
using biological fluids such as sweat, urine,
aqueous humour, amniotic fluid, seminal
plasma, cerebrospinal fluid, synovial fluid,
stool and blood plasma. Drugs may interact
at a series of organizational level, ranging
from changes in genetic expression,
biochemical regulation, or by inducing
synthesis of drug metabolizing enzymes. In
such cases, genomic and proteomic
approaches may be useful. In many cases,
toxic effects of a drug may be indirect, but
drug-induced perturbations result in changes
in relative concentrations of key molecules.
In such cases, analysis of body fluids reflects
abnormal composition. Pharmaceutical
applications include study of drug metabo-
lism and the metabolite distribution in body
fluids, and characterization of the composi-
tion and purity of drugs. Solid-state NMR
studies provide information on the
polymorphism of drug powder and confor-
mation of drugs, etc.
FRONTIERS IN NMR SPECTROSCOPY
IN LIFE SCIENCES
During the last 30 years, NMR has made
major impact in life sciences. The
potentialities of this technique have been
summarized in Table 2.2. It may be noted
that in the last 14 years, four Noble Prizes
have been awarded to scientists whose
work has brought new revolutions to this
area of science.
ACKNOWLEDGMENTS
I am grateful to Dr Sudha Srivastava for her
assistance in preparation of this manuscript. The
help of National Facility for High Field NMR located
28 Biomedical Magnetic Resonance: Proceedings of the International Workshop

Table 2.2: Frontiers of NMR in life sciences

Year Research areas opened Major researchers
1946 NMR observed in bulk material Bloch, Purcell et al.
1952 Nobel Prize (Physics) Bloch and Purcell
1950-60 NMR developed as a tool in chemistry Several workers
1965 First FT NMR experiment Ernst and Anderson
1970-1980 T1, T2 in tissues Damadian
NMR zuegmatography Lauterbur
Fourier imaging Ernst
Echo planar imaging Mansfield, Edelstein
2D NMR Jeener, Ernst
1980-90 NMR angiography Dumoudin
MRI as a clinical tool Several workers
Macromolecular structure, dynamics Wuthrich, Bax
3D NMR Several workers
1991 Nobel Prize (Chemistry) Ernst
1990- BOLD: f MRI Ogawa, Ugurbil
MRS in cells and organs Shulman
2002 Noble Prize (Chemistry) Wuthrich
2003 Nobel Prize (Medicine) Lauterbur, Mansfield

in TIFR and supported by the DST, DBT and CSIR,
is gratefully acknowledged.
FURTHER READING
1. Early development of NMR spectroscopy in
chemical sciences is elegantly covered in:
JA Pople, HG Schneider, HJ Bernstein. High
Resolution Nuclear Magnetic Resonance, McGraw
Hill Publications 1959.
For more theoretical treatment see:
A Abragam. Principles of Nuclear Magnetism.
Oxford: Clarendon Press 1961.
2. The basic principles of NMR spectroscopy have
been described in several text books. For a
somewhat simplified version see:
H Gunter NMR Spectroscopy: Basic principles,
concepts, and applications in chemistry, 2nd edn.
Chichester John Wiley and Sons 1995.
3. An excellent book for theory and experimental
pulse FT NMR spectroscopy is:
G Bodenhausen, H Kogler, RR Ernst. Principles
of Nuclear Magnetic Resonance in One and Two
Dimensions, New York; Oxford University Press
1985.
4. For protein and macromolecular NMR one may
consult:
J Cavanagh, WJ Fairbrother, AG Palmer, NJ
Skelton. Protein NMR Spectroscopy: Principles
and Practice. San Diego Academic Press Inc.,
1996.
K Wuthrich. NMR of Proteins and Nucleic Acids.
New York; John Wiley and Sons, 1986.
G Govil and RV Hosur. Conformation of Biological
Molecules: New Results from NMR, Berlin;
Springer-Verlag 1982.
5. For cells, body fluids and metabonomics: No
good books, but some recommended articles
are:
Sudha Srivastava, Girjesh Govil. Applications of
NMR to the study of cells and body fluids. Current
Organic Chemistry 2001; 5: 1039-57.
Cath O’Driscoll. Metabonomics on the map.
Chemistry in Britain 2002; 25-28.
JP Shockcor, Elaine Holmes. Metabonomic
Applications in Toxicity Screening and Disease
Diagnosis. Current Topics in Medicinal
Chemistry 2002; 2: 35-51.
JK Nicholson, J Connelly, JC Lyndn, E Holmes.
Metabonomics: a platform for studying drug toxicity
and gene function, Nature Reviews 2002; 1: 153-
61.
RG Shulman, F Hyder, DL Rothman. Biophysical
basis of brain activity: Implication for neuroimaging.
Q Rev Biophysics 2002; 35: 287-25.
 Fundamentals of Magnetic Resonance Image Production 29
Fundamentals of Magnetic
Resonance Image Production
INTRODUCTION
Having been introduced to the basics of
magnetic resonance spectroscopy in the
earlier chapter, we now ponder the question,
‘how is the magnetic resonance signal
rendered into a clinically useful ‘MRI’
image?’ Let us start with a brief recapitula-
tion of the basic physics.
The frequency equivalent (ω0) of the
energy that the precessing macroscopic
nuclear magnetization absorbs at ‘reso-
nance’, familiarly known as the Larmor
frequency, is determined by the exact value
of the external magnetic field (B0) that it
experiences, namely,
ω0 = γB0 ...(1)
In Eq. (1), γ is the ratio of field-frequency
match for a nuclear spin species, and is called
the gyromagnetic ratio. For the 1H nucleus
(proton), it has the value 42.3 MHz T -1. As
a result of the resonant RF energy absorp-
tion, the net macroscopic magnetization is
flipped away from its fully relaxed equili-
brium direction along the axis of B0, labeled
the z-axis, to create a rotating magnetization
in the xy plane. This produces an electrical
signal that decays due to relaxation pre-
3
P Raghunathan
cesses (characterized by time constants T1
and T2) that eventually restore the magneti-
zation to its equilibrium value along the
z-axis. This electrical signal is, in fact, our
magnetic resonance signal, called the free
induction decay (FID).
Being a waveform, the FID corres-
ponding to each resonating 1 H nuclear
species (e.g. water, fat, etc.) is a composite
of three important properties, viz., the signal
strength or amplitude, the signal frequency
(ω), and the signal phase (φ). While the
signal amplitude is a measure of the number
of resonating nuclei (we shall call this the
proton density), ω and φ may be exploited
together to ‘code’ a resonance signal at each
time-domain point. The answer to the
question raised in the opening paragraph
should be sought, therefore, in terms of how
ω and φ become spatially encoded when the
MR signal is produced from in vivo tissues.
LINEAR MAGNETIC FIELD GRADIENTS
The traditional ‘high resolution’ magnetic
resonance signal, observed by placing a (bio)
chemical specimen in a highly homogeneous
B0 magnetic field, may be said to have zero
spatial dimensionality. However, since the
30 Biomedical Magnetic Resonance: Proceedings of the International Workshop
Larmor frequency (Eq. 1) is proportional to
the magnetic field strength, it should be
possible to label or encode precisely the
spatial origin of a resonant spin by using a
magnetic field that varies linearly with position.
The italicized entity is what we call a
magnetic field gradient. It was this brilliant
piece of lateral thinking in the early seven-
ties, due to Lauterbur,1 that introduced
spatial dimensionality into magnetic
resonance signals by the deliberate imposi-
tion of a small linear magnetic gradient on
to the B0 field. Following the first coarse
gradient-modified NMR experiments for
imaging two spatially separated water
samples, a demonstration of the first human
tissue images using NMR was published.2
Several novel magnetic field gradient
implementation strategies were soon
reported. Whereas Lauterbur’s experiment1
used narrow-band ‘continuous wave’ RF in
a d.c. magnetic field gradient to acquire
linear ‘projections’ of the signal along
different directions which were later
‘reconstructed’ into an image, other early
strategies employed rapid a.c. magnetic field
gradients to define ‘sensitive lines’ or
‘sensitive points’ that were rastered spatially
to acquire image data directly.3,4 Useful as
they were in early research prototypes in
so far as they required rather moderate data
acquisition rates and digital electronics, they
were unacceptable for practical medical
imaging. On the other hand, current imaging
methods fall overwhelmingly in the
category of pulsed field-gradient ‘Fourier
imaging’ and exploit properties of Fourier
transforms as shown by Ernst et al5 and
others.6 Our present discussion is, therefore,
confined to the concepts of pulsed gradient
Fourier imaging techniques. By the mid
1980’s, even as intense activities were taking
place in laboratory level technology design,7
system modeling, 8 etc., full-fledged
commercial MRI scanners started appearing
on the world market, and diagnostic MR
imaging protocols became a standard part
of radiological practice.
Coding to Create ‘Spatial Frequencies’
and ‘Spatial Phases’
In the presence of linear magnetic field
gradients, the Larmor frequency equation
(Eq. 1) becomes modified to
ωi = γ(B0 + G.ri), r = x, y, or z ...(2)
where ωi is now a ‘spatial frequency’, i.e.,
the frequency of the resonant 1H nucleus at
position r i , and G is the ‘gradient’ – a
constantly sloping vector representing the
total gradient amplitude and direction, and
usually expressed in mT m-1 or G cm-1. For
example, along the z-direction, the ‘z-gradi-
ent’ becomes defined as Gz = dB0/dz. A
typical magnetic field gradient is a small
superposition on the main B 0 field to
produce a uniformly graded change of upto
± 25 mT m-1 about the magnetic iso-center
(where the field corresponds to B0). It is
applied for short time periods and is there-
fore called a gradient pulse. It is, therefore,
easy to see how a 1-D image profile is made
from such ‘space-encoding’ frequencies. The
MR signal is simply acquired in the presense
of a field gradient, and the intensity plotted
versus frequency, with the frequency axis
‘scaling’ for, or being proportionately
equivalent to, the spatial position of the
resonant 1H nuclei.
Images in two or three dimensions are
made by an extension of the above coding
concept, using additional magnetic field
gradients along the y and z axes. For
imaging in two dimensions to produce, say,
an (xy) map, we recognize the important
fact that a precessing spin experiencing a
 Fundamentals of Magnetic Resonance Image Production 31
gradient pulse of a duration Δt along the
second axis (the y-axis) will accumulate a
phase along that axis:
φy = γGyyΔt ...(3)
Phase changes are cyclic, and anything that
undergoes cyclic changes has a frequency.
Therefore, by bringing about changes in the
spin phase using phase encoding gradients
(e.g., G y in Eq. 3), we are essentially
achieving another dimension in frequency-
encoding.
Physical Gradients of the MRI Scanner
Three sets of independent gradient coils are
used in a scanner with their axes oriented
along the x-, y- and z- directions. Direct
current, pulsed for several milliseconds
through any of these coils, produces the
gradient pulse Gx , Gy or Gz. In the case of
a superconducting magnet, which is used in
most mid-field and all high-field (>1.5 T)
MRI scanners, the z-direction is conven-
tionally defined along its long axis. The
three gradient coils may be activated to
produce a slice selection gradient (Gsl), a
frequency encoding gradient (Gfr), and a
phase encoding gradient Gph. Each of these
may be chosen to dertermine a certain tomo-
graphic plane of the human anatomy and
will be assigned, through the operating
software, to one of these three ‘logical’
operations. For example, in Figure 3.1, we
show the orientation of the logical frequency
encoding and phase encoding gradients for
the axial, sagittal and coronal tomographic
planes when the logical ‘slice selection’
gradient is taken perpendicular to the plane.
Because of the availability of the three
gradient coils, controlled alterations in the
effective magnetic field can be generated
along any orthogonal axis. Furthermore,
oblique imaging axes can also be defined
by pulsing more than one gradient coil; a
45° oblique axis would use equal amplitudes
of two orthogonal gradients.
To accomplish MR imaging, the three
gradient coils must be activated suitably for
(i) slice selection, (ii) phase encoding, and
(iii) frequency encoding or ‘readout’. We
shall discuss these modular operations in
this order, which is the usual sequential
order of their appearance in standard MRI
pulse sequences.
Slice Selection
The first step in MR image production is
the selection of an anatomical slice. To do
this, it becomes necessary to confined the
RF excitation selectively to the ‘region of
interest’ (ROI) of the tissue under diagnostic
examination. This may be achieved by
applying a bandwidth-limited RF pulse in
conjunction with the magnetic field gradient
Gsl; for example, Gsl could be Gz, applied to
resonate a perpendicular plane in the ROI.
The frequency selective RF pulse is essen-
tially a pulse tuned to have a small band-
width Δω, which is typically 1-2 kHz around
Fig. 3.1: (Top) The orthogonal x,y and z gradient field
directions inside the MRI magnet.
(Bottom) The logical frequency encoding and phase
encoding gradients for the axial, sagittal and coronal
planes, with the logical slice selection direction taken
along z.
(A to P: Anterior to Posterior, S to I: Superior to Inferior,
L to R: Left to Right)
32 Biomedical Magnetic Resonance: Proceedings of the International Workshop
the central Larmor frequency. When such a
pulse impinges on the tissue in presence of
Gsl, 1H nuclear magnetization in a thin 2-D
slice of the tissue becomes resonant, or will
become ‘excited’. The short time-period for
which the RF pulse stays ‘on’ (given by t),
and its magnetic field strength (given by
B1), together determine the angle by which
the tissue magnetization is flipped on to the
resonant plane, namely,
α = γB1t ...(4)
We may, thus speak of a 30°, 90° or 180°
flip angle. Using these principles, different
2-D slices of the tissue may be excited by
changing the central frequency of the
selective RF pulse.
Each of the three mutually perpendicular
tomographic slice orientations, familiar to
the radiologist as the axial (or transverse),
sagittal, or coronal orientations (Fig. 3.1), is
selectable by a particular magnetic gradient
defined as the ‘logical’ slice selection
gradient. The gradient orientation is always
perpendicular to the required slice plane,
so that every 1H nuclear spin contained in
the slice experiences the same gradient field.
Orthogonal slices are those in which only
the x, y or z gradient is used as Gsl. Oblique
slices are vectorial combinations having more
than one physical gradient during the RF
pulse transmission.
This overall ability to generate an image
in a plane with arbitrary orientation is a
major advantage of the MR imaging moda-
lity. In any case, for diagnostic examinations,
the slice selection direction is always
perpendicular to the viewing surface. The
strength of Gsl (in mT m-1) determines both
the thickness and the position of a slice,
regardless of the orientation. The slice
thickness, d, becomes defined by the
bandwidth of frequencies, Δω, excited by
the RF pulse:
Δω = γΔ[Gsl.d] ...(5)
In the clinical setup, where multi-slice
imaging is often a requirement for disease
diagnosis, it is found practical to hold Δω
fixed and then define d by changing the
strength of Gsl. To produce a thinner slice,
we would require a large Gsl. Once Gsl is
thus, set according to the required d value,
the central Larmor frequency required to
bring the desired slice into resonance is
calculated using Eq. 1. Multi-slice imaging
thus uses the same Gsl for each slice but a
unique RF excitation pulse. Each RF pulse
has the same bandwidth but a different
central frequency capable of selective
excitation.
Phase Encoding
Having excited the spins in a selected
imaging plane as described above, we must
do a similar selective excitation of spins to
define their xy positional coordinates. It has
been found that rather than varying fre-
quency along both the imaging co-ordinates,
a phase (φ) variation in one of the directions
– the y direction – of the 2-D image plane
leads to a more distinctive coding.5 In effect,
after the spins have been excited within a
slice, the phase encoding gradient (Gph) is
pulsed for a short period Δt to alter the
phase slightly along each y column of the 2-
D data grid (or matrix). When the phase
gradient is switched off, each spin reverts
to precessing at its original frequency. The
net effect is that each position within a given
y direction has a slightly different phase
angle (φ), according to Eq. 3. In other
words, each spin phase is slightly ahead of
the preceding one.
At this point, all spins will emit signals
with the same frequency but the signals
between rows are out of phase. G ph is
 Fundamentals of Magnetic Resonance Image Production 33
progressively stepped up and played out
for each x position in the matrix, e.g., 256
times for a 256 × 256 picture data array.
Gph, applied perpendicular to both Gsl and
G fr, in the only gradient that changes
amplitude in a series of programmed steps
during the data acquisition loop of a stan-
dard 2-D imaging sequence.
Frequency Encoding
Following the slice selection and phase
encoding modules we apply the Gfr gradient
for producing a space-encoded dispersion
of the MR signal which is collected as an
‘echo’. (The description of an echo, and the
need for producing it, are discussed in the
next section). As the echo signal is detected
by the RF receiver coil – the ‘probe’ of the
MRI scanner – it is simultaneously digitized
by an analog-to-digital converter (ADC) for
later Fourier transformation. We note that
Gfr is switched on perpendicular to the Gsl
direction, and must be applied for each value
of Gph.
In Figure 3.2 we illustrate the processes
of ‘selecting’ an anatomical slice, and then
Fig. 3.2: A slice of thickness Δωsl, selected in the
transverse orientation (top), and the xy imaging plane
(bottom). For clarity, the spatial frequencies (wavy
lines) and phases (arrows) are shown only in alternate
elements of the pixel array
encoding that slice to produce a (ω, φ)
‘spatial frequency – spatial phase’ data
matrix. This matrix constitutes an array of
pixels. Image contrast contributed by each
pixel will be later seen to be related to the
characteristics of tissue in the corresponding
voxel (volume element). Transverse magneti-
zation within each pixel contributes a specific
signal waveform, i.e., specific frequency,
phase and amplitude, to the composite RF
signal. Each composite RF signal, or echo, is
the sum of RF components from all the
voxels in the image plane.
Dephasing FID, and Its ‘Rephasing’ to
Create an ‘Echo’
This section is an interlude to examine what
an ‘echo’ signal is, and why we need to
create it. Admittedly, the MR signal used
for imaging is present immediately after the
RF pulse and is, in fact, the FID. This signal,
however, decays or ‘dephases’ with a time
constant T2 or T2* depending on the chemical,
physical and magnetic environment of the
tissue 1H nuclei. In order to give ourselves
sufficient time for spatially encoding the MRI
signal, the FID must be delayed from the
RF pulse and recovered by rephasing it a
little later. The is done by creating an echo.
Two types of rephased echoes are in use in
MR imaging: the spin-echo (SE), and the
‘gradient-recalled’ echo or, simply, the
gradient-echo (GE).
Spin-Echo
Magnetization in the xy plane starts to
dephase immediately following the initial
90° RF excitation. Dephasing is the disper-
sion of the individual spin moments that
comprise the macroscopic magnetization
vector in the xy plane, and it occurs both
due to the intrinsic spin-spin interactions
34 Biomedical Magnetic Resonance: Proceedings of the International Workshop
amongst the precessing spins and due to
local magnetic field in homogeneities.
The way we compensate for dephasing
is called rephasing. This may be achieved
by the reversal of the phase of the spin
moments by a refocusing 180° RF pulse. As
a result, the dispersing spin phases now flip
over in the transverse (xy) plane and pro-
gressively rephase to form a spin echo (SE)
signal. The time between the initial 90° RF
pulse and the maximally rephased echo is
labeled TE (‘time-to-echo’). In the SE signal,
any inhomogeneities present in the static B0
field and any magnetic susceptibility
contributions to the tissue ROI from the
surrounding structures are corrected (i.e.,
get refocused), and the signal intensity
decays exponentially in time due to the
intrinsic spin-spin interactions only, as
exp (–TE/T2).
We note, however, that a sequence of
the 90° and 180° RF pulses alone do not
constitute the imaging sequence. For doing
this, both phase encoding and frequency
encoding gradients must be applied as well,
inevitably increasing the minimum achiev-
able TE. Additionally, the slice selection
gradient must also be applied to define the
image plane. These features of a basic SE
imaging sequence are illustrated in Figure
3.3, where the important parameters TE and
TR (which is the ‘time-to-repeat’ the imaging
sequence in a repetitive scan) are also shown.
To acquire multi-slice images, the 90° and
180° RF pulses are repeated with different
RF center frequencies to excite similar slices
centered at different positions along the
direction of the slices selection gradient (see
Eq. 5). A series of different slices can be
excited and detected in an ‘interleaved’
manner. That is, more than one slice is
excited (and the echo digitized) in one TR
period. One Gph is played out per slice per
TR. When longitudinal recovery of the spin
moments towards equilibrium is occurring
in one excited slice, a spin-echo can be
aquired from another slice.
Conventional SE imaging is associated
with long image acquisition times since the
pulse sequence repetition time, TR, must be
sufficiently long to allow recovery of the
magnetization to thermal equilibrium prior
to the next 90° RF pulse. Reducing TR
should, in principle, lead to a saving in time
in a repetitive image scan. Such a time –
saving is possible through use of the
gradient echo, as we shall see below.
Gradient-Echo
To understand the gradient-echo (GE), we
recall that an initial, small flip-angle (<90°)
RF pulse (α-pulse) may be used to flip the
precessing tissue magnetization into the (xy)
plane, producing the FID. Instead of using
an inverting RF pulse to rephase the
decaying spin moments, we now employ a
gradient reversal strategy by using a
‘dephase-rephase’ readout gradient pair.
That is, a negative gradient (-G x) is first
used, which is immediately followed by a
positive gradient of the same amplitude
Fig. 3.3: The basic spin-echo imaging sequence
 Fundamentals of Magnetic Resonance Image Production 35
(+Gx). This gradient reversal rephases the
spin moments and forms an echo the
gradient-echo (GE) – for signal detection.
The GE is particularly advantageous for
short TR, small RF pulse angle (α < 90°)
imaging protocols because of the shorter
time necessary for gradient reversals,
compared with 180° RF pulse flips of the
magnetization. However, unlike in the SE
sequence, magnetic gradient reversal does
not rephase the so-called ‘T2* - dephasing’
due to magnetic field and magnetic
susceptibility inhomogeneities. If the echo
delay TE from the initial α-RF pulse is
varied, an exponential decrease in signal
intensity, exp (-TE/T2*) with a time constant
T2* will be observed, with T2* < T2.
The gradient-echo imaging sequence is
shown in Figure 3.4. Its application shows
how the signal is moved away from the RF
pulse and how the data is allowed to range
over both negative and positive values of
spatial frequencies in a signal data acquisi-
tion. At the same time that this dephasing
frequency encoding gradient is applied, the
Fig. 3.4: The basic gradient-echo imaging sequence
phase encoding gradient Gph is also switched
on so that spin information in the ortho-
gonal direction gets space-encoded.
Use of the Parameters TR and
TE in Producing Image Contrast
In the previous sub-section two important
timing parameters, TE and TR, have been
introduced. The parameters can be varied
to achieve contrast sensitivity in MR images.
The MR image intensity represents the tissue
characteristics of a specific slice through the
patient’s body. Primarily, the intensity
depends on two factors, one of which
represents an exponential signal decay due
to the T2 relaxation process, and the other
an exponential signal re-growth due to the
T1 relaxation process. Taking the case of
the most frequently used SE imaging
sequence, the quantitative dependence of
the MR echo intensity, S, on T1 and T2 may
be written as 9,10
  TR    TE  ...(6)
S = AM0 1 – exp  –   exp  – 
  T1    T2 
Where A is a factor related to the
amplification of the MR receiver, and M0
represents the equilibrium magnetization or
the proton number density (also called
simply the proton density).
The above equation tells us that the signal
intensity could be ‘tuned’ by specific
combinations of TR and TE. One can
therefore, vary the relative contributions or
‘weighting’ of T1 and T2 due to the tissue
components by an appropriate selection of
TR and TE. For example, reduction of TR
introduces T1-weighting, while for longer
TR values, the signal intensity depends less
on T1-weighting and more on T2-weighting.
Also, as TE is lengthened, the image
becomes more T2-weighted.
36 Biomedical Magnetic Resonance: Proceedings of the International Workshop

The gradient-echo intensity may be Acquisition of Two-Dimensional

written in terms of α and T2* as Raw Data for Imaging

  TR   During each data acquisition cycle of

1 – exp  –  duration TR, typical pulse sequences such
A
M

  T1    TE 
S  sin 
0

exp  –
 TR   T2 *  ...(7) as those depicted in Figure 3.3 show that a
1 – cos  exp  – 
 T1  number of individual events occur concur-
rently such as the application of RF pulses
It may be seen from Eq. 7 that in GE- to the patient’s body, tuning magnetic
based imaging sequences in general, the gradient pulses on and off, and digitizing
contribution of α and TR determines the and storing the RF signals emitted by the
amount of T1-weighting, while TE deter- tissue 1H nuclei. Most of these events are
mines the amount of T2*-weighting. Also associated with, and determine, the spatial
use of low flip angles leads to more rapid characteristics of the image. Each imaging
scans, if somewhat weaker signals compared cycle produces one composite time-domain
with SE sequences. sinusoidal signal, which has a component
Typical 1H nuclear T1 and T2 values for coming from each individual voxel.
various human tissues at B0 values of 1.0T Although the individual sinusoids are phase
and frequency encoded, it is not possible to
and 1.5 T are set forth in Table 3.1. Whereas
unscramble them from one composite signal
the T2’s are relatively independent of B0,
waveform. It is necessary to acquire a series
T1’s are found to be generally shorter at
of composite signals (or ‘views’) by repeat-
the lower B0 value. By choosing TE and TR ing the data acquisition cycle. Let us,
appropriately, these T1 and T2 values can therefore, briefly chronicle the series of
be exploited for selective imaging of a tissue events leading to the filling up of the entire
type and, therefore, for delineating a 2D data matrix.
pathology (e.g., tumor). It is this unique After the slice has been selected by the
flexibility provided by MRI in image simultaneous application of the initial RF
acquisition, and the ability to introduce pulse and the Gsl gradient, the raw data
dynamic levels of soft tissue contrast for acquisition cycle starts. During each cycle,
probing tissue proton density as well as lasting for TR, 1H nuclear spins in individual
tissue distribution, that renders it such an voxels become phase and frequency encoded
important modality in diagnostic radiology. as per the following sequence of
programmed steps. Phase encoding is
Table 3.1: Typical 1H nuclear T1 and T2 values of produced by Gph that is pulsed on briefly
living human tissue at 1.0 T and 1.5 T for a time Δt and then turned off. The
Tissue T1 (ms) T2 (ms) frequency encoding G fr gradient is then
1.0 T 1.5 T 1.0 T 1.5 T switched on, and stays on with a constant
amplitude during the buildup, peaking and
Grey matter 810 920 100 100
decay of the signal echo. The ADC window
White matter 680 780 90 90
is also left open during this period so that
Fat 240 260 85 80
Muscle 730 860 45 50
the echo is ‘read out’, i.e., sampled and
Cerebrospinal fluid 2500 3000 1400 1420 digitized. During each cycle, the sequence
Blood 525 600 260 260 of gradient activity and data acquisition is
so adjusted that the echo peak occurs at
 Fundamentals of Magnetic Resonance Image Production 37
time TE, and at the instant of its formation
the spin voxels is each row are in phase.
Because the digital signal comprises the sum
of all the precession induced time domain
signals of the slice-selected 1H nuclei, it is
Fourier transformed to yield a spatial
frequency distribution and thus a one-
dimensional proton spatial distribution.
With each repetition of the acquisition
cycle, the amplitude of Gph is incremented
stepwise, to impart a unique phase change
along the phase encoding direction, whereas
Gfr has the same amplitude every time it is
applied during readout. As the rate of phase
change for each spin moment along the
phase encoding axis has a unique frequency
distribution, a second Fourier transfor-
mation is used to yield projections along
this axis.
The data matrix is filled out in this
fashion, and the rows become distinctly
coded by their linear variation in the phase
angle (Eq. 3) while the columns become
coded by their linear variation in preces-
sional frequency (Eq. 2). The entire 2D data
matrix, thus becomes parsed into an array
Fig. 3.5: Fourier transform relationship between the ‘raw data’ spatial frequency matrix and the final image
matrix. The latter is a 256 × 256 matrix; the pixel intensities show T1 – and T2 – weighting according to the TR,
TE pulse sequence parameters used
of distinct pixels, with each pixel in turn
being associated with the proton density
and relaxation time characteristics of the
tissue in that voxel.
Image Reconstruction and the
‘Display’ Matrix
The image data matrix (or display matrix)
is obtained via the 2D Fourier transfor-
mation of the raw data matrix, and this is
performed by the array processor of the
MRI scanner. The array of spatial frequencies
thus becomes converted into final image
amplitudes, and this is depicted in Figure
3.5. The image matrix is a frequency map of
the 1H nuclear signal intensity from a volume
element weighted by the T1 and T2 values
of the tissues contained within the volume.
The immense diagnostic utility of suitably
weighted MR images is extensively
documented in the literature9-11, and just a
couple of prototypical illustrations are given
involving pathologies of the brain (Figs 3.6A
and B) and spine (Figs 3.7A and B).
As implemented in most clinical MRI
scanners, image reconstruction creates two
38 Biomedical Magnetic Resonance: Proceedings of the International Workshop

Figs 3.6A and B: Mid-brain sagittal MRI: (A) T1– weighted (TR = 600 msec; TE = 25 msec), demonstrating only
cortical atrophy; (B) T2– weighted (TR = 2500 msec; TE = 90 msec), showing multiple ‘bright’ foci both in the
white matter and in the corpus callosum, due to Multiple Sclerosis

Figs 3.7A and B: Sagittal spine MRI: (A) T2– weighted and (B) T1 – weighted images. The extramedullary
soft tissue mass shows up better (black arrow) in the T1– weighted image
 Fundamentals of Magnetic Resonance Image Production 39
important image types called magnitude and
phase images. They arise basically due to
the fact that the transverse magnetization
component that is responsible for producing
the MR signal is a mathematically complex
quantity. The phase image becomes
significant whenever extraneous factors
contribute to some phase errors across the
imaging plane. The most important of these
factors is patient motion, especially if it
occurs in the presence of an applied gradient.
The usual method for separately recove-
ring magnitude and phase images is to
measure the in phase or real (R) and
quadrature or imaginary (I) components of
the complex magnetization by comparing the
echo signal to a ‘clock’ reference RF
waveform and demodulating. Each
component, R and I, may then be utilized in
the usual 2D reconstruction procedure to
create either the absolute magnitude or
‘modulus’ (M) image according to M =
(R 2 + I 2 ) 1/2 , or the ‘phase’ (P) image
according to P = arctan (1/R). The modulus
reconstruction sacrifices phase information
and, in the process, eliminates phase errors.
It represents the actual magnetization levels
within each voxel and is therefore used
regularly for displaying SE and GE diagnos-
tic images. The phase image occasionally
comes in handy, e.g., for depicting blood
flow, or for high-lighting magnetic suscepti-
bility or other phase-dependent information.
‘k-space’: Raw Data
Matrix by Another Name
The concept of k-space is important for a
proper understanding of the subtleties
involved in Fourier imaging.12,13 The raw
data that are acquired in MR imaging are
digital samples of the Fourier transform of
the ‘object’, which in our case is defined as
the arrangement of the transverse magneti-
zation (due to the tissue 1H nuclei) in space.
Each digital data sample is one Fourier
coefficient, or one spatial frequency
component. The variable we use to denote
spatial frequency is ‘k’. Therefore, the 2-
dimensional space in which the FT operation
is defined is called ‘k-space’, with its
components being labelled kx and ky. If x
and y represent the two space variables
defining an object, kx and ky represent the
two spatial frequency variables. If the units
of x and y are, say, cm, then the units of kx
and ky would be cycles cm–1 or, simply,
cm–1. Spatial frequency is the rate of change
of frequency as a function of distance. A
more appropriate term than ‘spatial
frequency’ would be ‘spatial phase’ since kx
and ky actually express the phase ‘advance’
or ‘retardation’ the spin moments experience
per unit tissue length in the x- and y-
directions.
The symbol ‘k’ is a carry-over from the
earlier ‘wave vector’ concept used in optics
and crystallography, and there exists a
transparent analogy with the familiar
reciprocal space concept in crystal physics. Just
as in the diffraction limit in optics, it is the
wavelength λ = 2π/k that decides the spatial
resolution achievable in MRI, and not the
wavelength of the RF at the Larmor fre-
quency. The axes kx and Ky constitute the
k-space grid of ‘raw data’ which is the
Fourier inverse, or the diffraction pattern, of
the MR image. We can examine this
diffraction pattern with appropriate ‘low
frequency’ and ‘high frequency’ filters and
convince ourselves of the following
properties which are of crucial importance
in clinical MR imaging: (i) low spatial
frequencies become encoded in the central
area of k-space (around k=0), and these
determine gross image features and image
contrast; (ii) the high spatial frequencies,
40 Biomedical Magnetic Resonance: Proceedings of the International Workshop
which are present at the peripheries of k-
space, contain edge information and control
spatial resolution.
All MRI imaging sequences strive to fill
k-space with optimum trade-offs in scan
time, signal-to-noise ratio, and resolution
with minimal artefacts. Just exactly how
magnetic gradient trajectories fill k-space is
what differentiates the wide variety of pulse
sequences in use today. As a particularly
important example of fast k-space filling,
we shall briefly discuss echo planar imaging
(EPI) in the next section.
Echo-Planar Imaging (EPI)
The EPI is a ‘snapshot’ strategy to map the
multiple phase-encoded lines of k-space after
only one RF excitation. In other words, it is
a technique wherein the complete data set
required to reconstruct an image is acquired
from the MR signal produced by a single
RF excitation of the selected slice, provided
that the magnetic gradients creating the
sampling trajectory could be switched
rapidly enough and with sufficient magni-
tude. The original concept, due to Mans-
field,14 employed a continuous scan path
zigzagging rectilinearly through the entire
k-space plane. This requires an ingenious
manipulation of the encoding gradients
whereby Gx is inverted after each line-scan
while Gy is applied very briefly in ‘blips’
after completion of each line. Inversion of a
gradient reverses the scan direction in k-
space. Gx is preferably strong and rapidly
switched to obtain very fast echoes (1 per
millisecond), while Gy is blipped to produce
pulses coinciding with the zero crossings of
the G x gradient. Programmed to act in
synchrony, these gradients generate the fast
zigzagging route through the k-plane. The
MR signal is continuously sampled during
this k-space traverse, and the method is
therefore, ultrafast and efficient.
The EPI may be implemented in the GE
or SE version. If a 180° RF pulse is inserted
between the initial excitation pulse and the
start of signal sampling, the sequence
becomes the SE EPI.
The EPI is somewhat limited by the fact
that signal-to-noise ratio is relatively poor.
Each line must be digitized within a very
short time, since all lines have to be scanned
before the T2 decay of the spin moments
following excitation. Short acquisition time
for each line requires a large RF receiver
bandwidth and, consequently, noise is
higher. Spatial resolution is also usually
limited.
The above limitations notwithstanding,
today’s rapid and spectacular developments
in the applications of MRI to real-time
functioning of the brain and other organs
have become possible mainly due to ultra-
quick and ingenious strategies of mani-
pulating k-space.
CONCLUSION
We have discussed the basic principles of
space-encoding the frequencies and phases
of time domain in vivo MR signals to
produce clinically useful MR images. Linear
magnetic field gradient pulses, when
imposed on the main magnetic field in a
controlled manner, achieve (i) selective
activation of tomographic tissue slices, and
(ii) the encoding of spin-echo or gradient
echo MR signals from an activated slice into
spatially resolved frequency and phase
dimensions. The 2D raw data acquired after
scanning a slice plane are shown to comprise
spatial frequencies which are then Fourier-
transformed into a 2D MR image. The ‘k-
space’ concept is introduced for developing
a comprehensive understanding of the 2D
raw data array. Fast MRI pulse sequences
are distinguishable, in principle, by how
rapidly magnetic gradient trajectories fill
 Fundamentals of Magnetic Resonance Image Production 41
k-space and, as a particularly important
example, echo-planar imaging (EPI) is
discussed briefly.
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Contrast Mechanisms in MRI
INTRODUCTION
Magnetic resonance imaging is a very
versatile technique and may be used to
produce images that depend on several
different tissue biophysical properties. Some
of these are affected by pathological and
physiological changes, which gives rise to
the high contrast and exquisite sensitivity
of many clinical images. In this short review
we provide an overview of the nature of
the underlying information that may be
obtained from the most common types of
images, and explain the physical origins of
the contrast portrayed using images produ-
ced by different approaches. It should be
emphasized that there are many qualitatively
different types of MR image, and the signals
acquired and mapped spatially may be
manipulated to portray or accentuate diffe-
rent tissue biophysical properties. The speci-
fic pulse sequence used for image acquisition
(i.e. the pattern of radiofrequency and
gradient waveforms used) determines the
precise manner in which the image depends
on different underlying characteristics,
which may be regarded as analogous to the
stains used in conventional microscopy. We
here describe only those mechanisms
4
John C Gore, Bruce M Damon
commonly available to modulate images
based on so-called proton (1H) NMR. While
NMR signals from other nuclei (notably
sodium) may in principle be used to create
images, the high natural abundance and
favorable nuclear properties of protons
(hydrogen nuclei) make them by far and
away the most suitable for producing high
quality images.
The large majority of imaging studies
rely on acquiring images of mobile hydro-
gen nuclei that are contained mainly in water
molecules. Water comprises approximately
80 percent of most soft tissues, which
therefore contain roughly 90 molar water
protons. Thus, most images portray some
property of the water within tissues, which
indirectly then reveals information on tissue
structure or function. Many physiological
or pathological processes of interest modu-
late the behavior of the water in an “MRI-
visible” manner, albeit often in non-specific
fashion. A second important type of contrast
is also available from mobile non-water
protons contained mainly within lipids with
different chemical shift to water protons,
and this forms the basis of separate fat and
water imaging, which we will not consider
further here.

CONTRAST AND RELAXATION
The primary properties of tissues that
modify the signals measureable using NMR
are the density of nuclei, the relaxation times
of the nuclear magnetization (which, as
detailed below, describe the times for
recovery of the nuclear magnetization back
to equilibrium after disturbance by applied
radiofrequency pulses), the magnetic homo-
geneity of the environment in which the
nuclei reside, and the rate of transport of
molecules (whether due to flow or via ran-
dom Brownian motion) within the medium.
Each of these may in turn be affected by
subtle changes in the properties of the
tissue, such as the confinement of water
within compartments, restrictions to water
molecular diffusion, or physico-chemical
effects such as tissue pH and the presence
of certain trace elements. Although in
principle the effects of many of these
influences are understood, it is often not
straightforward to interpret changes in NMR
signals in terms of any single or simple
underlying causes. Tissue is an extremely
heterogeneous medium, and the NMR signal
in an image voxel represents the summed
behavior of a large number of molecules
within a variety of cellular environments
and undergoing a wide variety of interac-
tions, so there often is no direct or specific
interpretation of the variations within
tissues depicted in MR images.
It may be recalled that each hydrogen
nucleus (proton) may be considered to
possess spin which in turn gives rise to a
magnetic dipole moment. When a large
number of such magnetic nuclei are placed
in an external and static magnetic field the
majority tend to align themselves in the
direction of the field, giving rise to a
measureable macroscopic magnetization. MRI
Contrast Mechanisms in MRI 43
involves disturbing this magnetization using
oscillating magnetic fields that alternate at
a specific resonant radiofrequency (RF), the
Larmor frequency, that is proportional to
the strength of the applied static magnetic
field (= 42.6 MHz per Tesla applied field).
The disturbed magnetization induces small
electrical signals in coils of wire tuned to
this same frequency as it recovers back to
equilibrium. At any instant during this
recovery there may exist components of the
magnetization along the applied field
direction (by convention the z direction) and
orthogonal to this direction (the xy plane).
The latter are responsible for the signals
measured during any acquisition. The
relaxation times T1 and T2 and their corres-
ponding rates R1 (=1/T1) and R2 (=1/T2)
denote the characteristic time constants of
the recovery back towards equilibrium of
the z and xy components respectively. In
equilibrium the z component is M0, which
largely depends on the water content of
the tissue. All NMR signals used for record-
ing images are proportional to M0 and the
simplest type of image is a map of this
equilibrium magnetization. An example is
shown in Figure 4.1, in which it can be seen
that grey matter in the brain is brighter
mainly because it contains more water. In
practice the range over which M0 or water
density within soft tissues vary is small, so
images that depend largely on M0 often do
not show high contrast or dynamic range.
The most commonly used types of image
have a contrast that portrays the hetero-
geneous distribution of NMR relaxation
rates. By modifying the timings of RF pulses
used to acquire NMR signals, images can be
produced in which the signals depend in
different ways on T 1 and/or T2 . Many
different pathologies affect these relaxation
times, often to a large degree, and thus
44 Biomedical Magnetic Resonance: Proceedings of the International Workshop
Fig. 4.1: Sagittal MR image that is T1 weighted and
in which contrast is mainly dependent on relaxation
times. The corpus callosum, for example, is bright,
whereas the cerebrospinal fluid is dark mainly
because of their different relaxation times compared
to the surrounding brain
such images are sensitive to a variety of
clinical disorders and may exhibit very high
contrast. For example, Figure 4.2 shows a
brain tumor in an image that depends
strongly on T2. Here the relaxation time of
the cancerous tissue is more than 100 percent
increased compared to the normal brain.
To understand the nature of the infor-
mation portrayed by such images, it is
worthwhile discussing what factors may
affect the relaxation times of water within
tissues. For proton NMR, after being distur-
bed by an RF pulse, the nuclear magneti-
zation does not spontaneously recover very
fast, but relies almost entirely on interactions
between the water molecules and the
surrounding medium to re-equilibrate. R1 is
called the spin-lattice relaxation rate. The
“lattice” denotes the molecular environment
surrounding a hydrogen nucleus, and
Fig. 4.2: T2-weighted MR scan of the brain showing
a large brain tumor and associated edema
includes the remainder of the host (water)
molecule as well as other solute and solvent
molecules. Spin-lattice relaxation occurs
because of magnetic interactions between
the nuclear spin dipoles and the local
randomly fluctuating magnetic fields that
exist on an atomic scale inside any medium.
These originate mainly from neighboring
magnetic nuclei such as other hydrogen
protons (e.g. within a water molecule, each
hydrogen affects the neighbor) and are
modulated by the motion of other surroun-
ding dipoles in the lattice which have
components fluctuating with the same
frequency as the resonance frequency. The
recovery is very efficient when there is a
local fluctuating field that can provide a
magnetic perturbation at the Larmor fre-
quency. For example, each proton in a water
molecule has a neighboring proton that is
also a magnetic dipole which generates a
magnetic field at the neighboring proton of
about 5 gauss (0.5 milli Tesla). This field is

constantly changing in amplitude and
direction as the water molecule rotates
rapidly and moves about in the liquid. It
also changes as a result of intermolecular
collision, translation, or chemical dissociation
and exchange. The magnetic field experi-
enced by any nucleus will fluctuate with a
frequency spectrum that is dependent on
the molecular tumbling due to the random
thermal motion of the host and surrounding
molecules. The mean strength of the local
field is determined by the strength of the
magnetic dipoles in the medium and how
close they approach to the hydrogen nuclei.
The component of the frequency spectrum
which is equal to the resonant frequency,
(or, for reasons beyond our discussion, twice
as high) is effective in stimulating an energy
exchange to induce recovery back towards
equilibrium, i.e., T1 relaxation. In liquids the
characteristic frequencies of thermal motion
are of the order of 1011 Hz or higher, much
greater than NMR frequencies of 107–108
Hz. Consequently, the component of the
frequency spectrum from molecular motion
that can induce T1 relaxation is small and
the process is slow. As the molecular motion
becomes slower, either due to lower
temperature, or increased molecular size,
the intensity of the fluctuations of the
magnetic field at the resonance frequency
increases, reaches a maximum, and then
decreases again as the energy of the motion
becomes increasingly concentrated in
frequencies lower than the NMR range.
Thus, R1 passes through a maximum value
when the molecular tumbling rate matches
the Larmor frequency as the molecular
motion becomes slower. It may be noted
from this that relaxation times depend on
the frequency of the NMR measurement,
and thus are usually shorter at low field
strengths. Thus the contrast at higher fields
Contrast Mechanisms in MRI 45
is different from that at lower fields. The
effect of the molecular motion is usually
expressed by a correlation time, τ c ,
characteristic of the time of rotation of a
molecule or of the time of its translation
into a neighboring position. Relaxation rates
in simple liquids are affected, for example,
by viscosity, temperature, and the presence
of dissolved ions and molecules, which alter
the correlation times of molecular motion
or the amplitudes of the dipolar inter-
actions.
Whereas T1 is sensitive to radiofrequency
components of the local field, T 2 is also
sensitive to low frequency components.
When an ensemble of nuclei is excited with
RF a transverse component of magnetization,
orthogonal to the applied field direction,
may develop, and it is this component that
rotates with the Larmor frequency and
induces the MRI signal in a receiver coil. T2
reflects the time it takes for the ensemble
to become disorganized and for the trans-
verse component to decay. Since any growth
of magnetization back toward equilibrium
must correspond to a loss of transverse
magnetization, all contributions to T1 relaxa-
tion affect T2 at least as much. In addition,
components of the local dipolar fields that
oscillate slowly, at low frequency, may be
directed along the main field ( Bo ) direction
and thus, can modulate the precessional
frequency of a neighboring nucleus. Such
frequency perturbations within an ensemble
of nuclei result in rapid dephasing of the
transverse magnetization and accelerated
spin-spin relaxation. Since the low frequency
content of the local dipolar field increases
monotonically as molecular motion pro-
gressively slows, although T1 passes through
a minimum value, T2 continues to decrease
and then levels off, so that T1 and T2 then
take on quite different values.
46 Biomedical Magnetic Resonance: Proceedings of the International Workshop
In the picture developed above, relaxa-
tion results from the action of fluctuating
local magnetic fields experienced by protons,
which stimulate the return to equilibrium
of an excited population of spins. In pure
water the dominant source of such effects
is the dipole-dipole interaction between
neighboring protons, mainly between
hydrogen nuclei in the same water molecule.
The tumbling of each water molecule then
causes the weak magnetic field produced
by each proton to fluctuate randomly, and
at the site of a neighboring proton these
random alterations in the net field produce
relaxation. The time scale characteristic of
the dipolar interaction reflects molecular
motion and clearly is expected to influence
the efficacy of relaxation. Qualitatively,
when there is a concentration of kinetic
motion in the appropriate frequency range,
relaxation will be efficient. We can envisage
other types of motion which will be too
rapid or too slow to be effective. The key
important descriptor is the correlation time,
τc which measures the time over which the
local fluctuating field appears continuous
and deterministic. It represents the time it
takes on average for the field to change
significantly.
In simple liquids such as water the
molecular motion is rapid and on average
is isotropic. The motions are so fast that
relaxation is not very efficient—the dipolar
fields fluctuate too rapidly to be very effi-
cient, and the motion averages out any net
effects of the local fields (so-called motional
averaging). In pure water, T 1 is several
seconds long, whereas in tissues water
relaxation times are often substantially
under 1 second. In pure water most of the
relaxation is accounted for by the effects of
intra-molecular dipolar effects from protons
within the same H2O molecule. In tissue,
water relaxation times are shorter than in
pure water because of the presence of large
macromolecules and chemical species that
promote relaxation. Proteins and other
macromolecular structures produce, via
protons on their surfaces, dipolar fields that
fluctuate relatively slowly and which do not
average out on the NMR timescale, and
these are efficient sites for relaxation. Water
molecules or single protons may exchange
between these sites and the rest of tissue
water, thereby spreading the effect. Proteins
are often considered to contain one or more
layers of hydration, which is often loosely
termed the “bound water” fraction, and this
may play an important role in mediating
interactions between the bulk aqueous
medium and the macromolecular surface.
Some specific chemical side groups, notably
hydroxyl and amide moieties, are known
to be efficient conduits for relaxation, so
when these occur in high abundance the
relaxation will be fast, but in general the
large array of environments and chemical
species within tissue usually make it
impossible to identify specific interactions
that dominate. Figure 4.3 shows MR images
that are sensitive to relaxation times of two
eggs – one boiled and one fresh. In the
boiled egg the proteins are cross-linked and
heat denatured, and this induces gross
changes in the water relaxation times
compared to the fresh egg, even though the
elemental composition has not altered.
Solutions of macromolecules and biologi-
cal tissues are chemically heterogeneous and
thus, water in such media may experience a
wide variety of different environments and
chemical species with which to interact. Even
in simple protein solutions, there may be
different ranges and distributions of
correlation time, coupling strengths and
molecular dynamics, that affect the local
dipolar fields experienced by water protons.

In general, larger proteins produce more
efficient relaxation per unit mass than
smaller molecules. An even greater variety
of different scales and types of constituents
occur within cells and whole tissues. Tissues
contain diverse, freely tumbling solute ions
and molecules such as small proteins and
lipids as well as relatively immobilized or
even rigid macromolecular assemblies such
as membranes and mitochondria. Tissues are
also spatially inhomogeneous, containing
many different types of cell or structure,
and there may exist multiple compartments
that are not connected or in which water
transport is restricted. Nonetheless,
although tissues are markedly heterogeneous
at the cellular level, NMR relaxation will
still reflect the average character of local
dipolar fields experienced by water protons.
Multi-compartment Systems and the
Effects of Exchange
If at any time only a small proportion of
water protons are in close juxtaposition to
efficient relaxation sites, then the average
Fig. 4.3: T2 -weighted MR images of two eggs. The right egg is fresh and has a long T2, whereas the left egg
has been boiled. The heat denatures and cross-links the macromolecules in the egg which drastically reduces
the relaxation times of the egg white
Contrast Mechanisms in MRI 47
water proton relaxation will depend on how
effectively, and at what rate, these effects
are spread through the rest of the water
population. Such exchange processes have
profound effects on the observable NMR
relaxation phenomena. In a time of 50 msec,
water molecules diffuse distances of the
order of 20 μm, so that they sample many
different environments on the cellular level
within the timescale of relaxation.
In many (though by no means all)
situations, very rapid exchange occurs
between bulk water and bound and inter-
facial water in biological systems. Consider
the situation in which there are two such
populations in rapid exchange; one is a bulk
solvent phase with relaxation time Ta and
the other is a small hydration layer fraction
whose relaxation time Tb is much shorter
because it is in an efficiently relaxed environ-
ment associated with the “dry” solute
material. If one gram of the dry weight
biological material directly hydrates and
decreases the relaxation times of C grams
of water, and this then exchanges rapidly
48 Biomedical Magnetic Resonance: Proceedings of the International Workshop
with the solvent phase, then the average
relaxation rate is
 1 1  1 1
    C(g/ h)  –  ...(1)
 T av Ta  Tb Ta 
in which g is the number of grams of dry
weight biological material in the sample and
h is the total weight of water in the sample.
Small variations in the ratio of g/h (i.e. in
the ratio of dry matter to water) may then
markedly affect the relaxation rate since
(1/Tb – 1/Ta) is large. This is believed to be
the origin for many increases in T1 or T2 in
various pathologies, such as edematous
changes following insults to tissue, or in
rapidly dividing cells that have higher
water fractions. Changes in tissue water
content in general will affect relaxation. In
addition, changes in macromolecular
composition other than proteins will also
affect relaxation—for example, changes in
glycogen within muscle or liver may make
significant contributions to the variation of
relaxation within these tissues.
It should be mentioned in passing that
the effect of exchange on the observable
transverse relaxation is different when the
NMR frequencies in the various environ-
ments are unequal. This is important when
considering what additional processes
shorten T2 compared to T1, because several
surface groups e.g. amides, on proteins may
exchange protons with water and have
different NMR chemical shifts. This fre-
quency difference causes a further increase
in the transverse relaxation rate that
depends on the presence of such exchanging
species. The contributions to exchange
generally are pH dependent: for example,
it is conjectured that the increases seen in
relaxation time in exercising muscle (Fig.
4.4) are partially due to changes of pH and
osmolarity within muscle cells that increase
the contributions of chemical exchange
processes to relaxation.
Magnetization Transfer
Relaxation in tissues is affected by inter-
actions that occur between water protons
and protons at or near the surface of macro-
molecules. Longitudinal proton magneti-
zation can be exchanged between water and
neighboring nuclei (whether interfacial
water that is hydrogen bonded to the
surface or protons within other chemical
groups that are part of the macromolecule)
by direct through-space dipolar couplings
as well as by so-called chemical exchange of
protons. Chemical dissociation of protons
occur at rates that are pH dependent,
providing possible interchanges between
water and surface sites (hydroxyls, amides,
and so forth) as intact water molecules move
constantly in and out of the hydration layers
of macromolecular surfaces and exchange
protons. Imaging methods have been
devised that are specifically sensitive to the
rate of exchange of magnetization between
the different pools, and special methods
have been devised by which, from a series
of images, the sizes of different pools may
be measured. For example, the application
of an RF pulse off-resonance from water
may nonetheless affect the magnetization
of other protons or protons within tissues
that have very broad resonances (e.g.
because they are largely immobilized and
“solid-like”) and this saturation of the off-
resonance protons may be transferred to
the on-resonance water protons via magne-
tization transfer. The resultant image is
altered to a degree that depends on the
exchange rate and the ratio of the sizes of
the different proton populations. Quanti-
tative images on derived MT quantitites may
be useful for detecting changes in various
disorders, such as demyelination of white
matter.

Fig. 4.4: T2 sensitive echo planar images through a human leg. The image on the right was obtained after
2 minutes dorsiflexion exercise after which the relaxation time of the anterior tibialis muscle increases
Paramagnetic Relaxation
The administration of exogenous agents such
as gadolinium chelates reduces relaxation
times where they localize and have found
widespread use in clinical imaging. Such
paramagnetic agents affect the amplitude
and time scale of variation of the local
magnetic fields experienced by water
molecules. Paramagnetic agents are materials
that on the atomic scale generate extremely
strong local magnetic fields. Each agent
possesses a large magnetic dipole moment
compared to, say, the proton. Transition and
rare earth metal ions are examples of these.
The origin of their strong local fields lies in
the fact that they contain unpaired electrons
viz. particles with spin and electric charge
(and therefore a magnetic moment) that have
not been “matched” (paired off) in a chemical
bond with spins of opposite character, so
that there is a net residual magnetic dipole
moment. The electron spin magnetic dipole
is 658 times greater than the proton
essentially because it has a smaller radius
but the same charge, so any water molecules
that approach close to a paramagnetic ion
containing unpaired electrons will experience
Contrast Mechanisms in MRI 49
an intense interaction that can promote
relaxation.
Contrast materials are often introduced
intravenously. If they leak out from the
blood into the extracellular water space
within tissues then the tissue relaxation rate
is reduced. This happens in several normal
tissues and in the brain when the blood
brain barrier is disrupted. Otherwise the
effect depends on the degree to which water
can exchange with the water in the blood
stream.
Susceptibility Contrast and
BOLD Effects
Another important factor that may modulate
MRI contrast is the magnetic homogeneity
of the local environment in which water
molecules reside. In particular, if there are
variations in the magnetic susceptibility
within the sample then the transverse
magnetization, and therefore the measured
signal, decays more rapidly. Variations in
the susceptibility arise, for example, at
interfaces between air and tissue. Within
tissues there may also be small-scale
50 Biomedical Magnetic Resonance: Proceedings of the International Workshop
variations in susceptibility due to the
presence of metals within tissues, such as
iron-containing proteins such as hemosiderin
and hemoglobin. MRI may be usefully
employed for the non-invasive detection of
iron deposition in tissues in normal and
pathological states.
A specific example of susceptibility
variations arises from the vasculature within
tissues, because of the presence of
hemoglobin in the circulation. This particular
variation leads to the so-called BOLD
(Blood Oxygen Level Dependent) effect
which has been found very useful for studies
of brain activation and tissue oxygenation.
In the brain, the physical origins of BOLD
signals are reasonably well understood,
though their precise connections to the
underlying metabolic and electrophysio-
logical activity need to be clarified further.
It is well established that increasing neural
activity in a region of cortex stimulates an
increase in the local blood flow in order to
meet the increased demand for oxygen and
other substrates. The increase that is
produced actually exceeds that which is
needed so that, at the capillary level, there
is a net increase in the balance of oxygena-
ted arterial blood to de-oxygenated venous
blood. Essentially, the change in tissue
perfusion exceeds the additional metabolic
demand so the concentration of deoxy-
hemoglobin within tissues decreases. This
decrease has a direct effect on the signals
used to produce MR images. While blood
containing oxyhemoglobin is not very
different in terms of its magnetic suscepti-
bility to the rest of tissues or water, it
transpires that de-oxyhemoglobin is signi-
ficantly paramagnetic (other paramagnetic
materials include the agents used for MRI
contrast materials such as gadolinium), and
thus deoxygenated blood differs substan-
tially in its magnetic properties from
surrounding tissues. When oxygen is not
bound to hemoglobin, the difference
between the magnetic field applied by the
MRI machine and that experienced close to
a molecule of the blood protein is much
greater than when the oxygen is bound. On
a microscopic scale, replacing deoxygenated
blood by oxygenated blood makes the local
magnetic environment more uniform. The
longevity of the signals used to produce
MR images is directly dependent of the
uniformity of the magnetic field experienced
by water molecules – the less uniform the
field, the greater the mixture of different
signal frequencies that arise from the sample,
and therefore the quicker the overall signal
decays. The result of having lower levels of
deoxyhemoglobin present in blood in a
region of brain tissue is therefore, that the
MRI signal from that region decays less
rapidly, and so is stronger when it is
recorded in a typical MR image acquisition.
This small signal increase is the BOLD signal
recorded in fMRI. It typically is around
1 percent or less, though this depends on
the applied field strength (one of the reasons
why higher field MRI systems are being
developed). As can be predicted from the
above explanation, the magnitude of the
signal depends on the changes in blood flow
and volume within tissue, as well as the
change in local oxygen tension, so there is
no simple relation between the signal change
and any single physiological parameter.
Thus, fMRI does not report absolute changes
such as the units of flow obtained with
positron emission tomographic (PET)
imaging. Furthermore, as neurons become
more active, there is a time delay before
the necessary vasodilation can occur to
increase flow, and for the wash-out of
deoxyhemoglobin from the region. Thus, the
so-called hemodynamic response detected
by BOLD has a delay and a duration of

several seconds following a stimulating
event.
Effects of Spin Motion
Water molecules are in constant motion and
there are various ways in which their
movements may affect the signals used to
produce MR images. For example, Brownian
motion of molecules is random and gives
rise to the self-diffusion of water within an
aqueous compartment. In the presence of
applied magnetic field gradients Brownian
motion causes the net signal from water to
attenuate to a degree that depends on the
rate of diffusion. Experimental measure-
ments typically involve a specific time scale
over which the effects of diffusion affect
the signal: in imaging this is typically several
milliseconds. MR measurements of diffusion
rely on the fact that the net distance that a
molecule moves away from a starting posi-
tion increase with time – in free diffusion,
the mean squared distance moved is
proportional to time and the self-diffusion
coefficient D. If, during the time of the
measurement, water that is initially freely
diffusing encounters an obstructing boun-
dary (such as a membrane that is not
permeable) then its ability to move away
from the original position is reduced and
thus the apparent diffusion coefficient or ADC
is reduced. The manner in which ADC falls
below the intrinsic value of D is a measure
of the sizes and permeabilities of the cellular
compartments in which water resides.
Theory and experiments have shown that
in whole tissues the ADC is affected by the
sizes of the intra- and extra-cellular water
spaces, and it alters when fluid shifts
between them, as in ischemia, seizure and
other conditions. Images can be acquired
which depict areas of higher ADC as darker
than areas of lower ADC, and such images
Contrast Mechanisms in MRI 51
are very sensitive to physiological and
pathological perturbations such as brain
ischemia. In addition, changes in the cellu-
larity of tissue, such as those that occur with
malignancy, affect the contrast in diffusion
weighted images and measurements of the
ADC may be useful for monitoring the res-
ponse of tumors to treatment. Furthermore,
when the tissue has an anisotropic internal
structure e.g. white matter tracts or muscle
fibers, the ADC is greater in some directions
than others. By measuring the different
components of the diffusion tensor at each
location, information may be obtained about
the directions of the constituent structures,
and these may then be traced within the
tissue. For example, this approach has been
used to map white matter tracts within the
brain and fiber bundles within muscles.
CONCLUSION
The MR images may be produced that reflect
several different biophysical properties of
tissues. Relaxation rates largely reflect the
concentration of macromolecules and may
be modified by specific physico-chemical
effects. Diffusion images largely depict the
number and density of boundaries that
restrict water diffusion within tissue. BOLD
effects are dominated by changes in tissue
blood volume and oxygenation. There are
other, less common contrast mechanisms that
are under investigation or have been used
for specific purposes, such as the use of T1ρ
imaging, the study of protons with ultra-
short T2, and the effects of coupled spins in
lipids on image brightness. Much work
remains to be done to clarify the precise
contributions of different tissue components
and the manner in which specific pathologies
affect the NMR signal in complex media but
the contrast in practical clinical images will
often depend on the general processes
described above.
52 Biomedical Magnetic Resonance: Proceedings of the International Workshop
RF Pulse Sequences Including
Rapid Imaging Sequences
The contrast seen in MR images between
different tissue types is mainly because
different tissues have different relaxation
properties.1 Taking these intrinsic properties
into consideration and a clever manipulation
of the MR pulse sequence parameters
produces the desired contrast in the images.
For example pulse sequences that have
shorter repeat time (TR) tend to exhibit
contrast that brings out the T1 differences
between the tissues and manipulation of the
echo time (TE) tends to highlight the T2
differences between the tissues. Scan time
for a given pulse sequence depends on the
type of contrast and the desired resolution
and is given by TR × No. of phase encoding
steps × No. of averages. For example, a T2
weighted sequence with a TR of 2000 ms
and a 256 × 256 resolution will take 8 mins
and 32 s to complete for one average. Clearly
T2-weighted sequence take fairly long to
complete compared to T 1 -weighted
sequences since they use shorter TR’s. Such
long scan times can be problematic for
several reasons. First, the quality of the scan
relies on the patient being still during the
scan process and, the chance of the patient
not moving increases as the scan time
5
Rao P Gullapalli, Jiachen Zhuo
increases. Second, depending on the
condition of the patient he or she may not
be able to tolerate such long scan times and
third, there is a danger of having to repeat
the scan if in fact the original scan ends up
with motion artefacts or to terminate the
scanning in which case diagnosis will be
made using substandard images. Several
innovations over the last decade or so have
taken place to circumvent this problem. The
innovations have come in the form of
gradient technology improvement, novel
pulse sequence design and like any other
technology MR has also benefited from the
advances in computer engineering
technology.
Spatial encoding in MR involves the
application of a linear gradient over a period
of time. In the phase direction this gradient
is stepped from –ky to +ky to provide the
same delta phase shift to the spins from
one excitation to the next. This means that
if the k-space consists of a total of 256 views,
the views go from –128 to +127 since there
is also a zero in between. This also means
that the views that are closer to the center
of k-space see the least amount of phase
dispersion whereas the views that are at
 RF Pulse Sequences Including Rapid Imaging Sequences 53

the extreme ends of the k-space are subjected The high frequency components, which are
to a more severe phase dispersion. Since at the periphery of k-space on the other
the starting phases of the views, closer to hand provide resolution to the images. One
the center of k-space receive the least amount should remember that k-space is band
of phase dispersion, the echo signal is the limited in terms of frequencies in each of
highest for these views. This also suggests the directions. Of course, in order to obtain
that these views will have preserved in them a high resolution image one could sample
the low frequency content. Signals that have longer and longer. It should be remembered
low frequency information also contribute that the gain in resolution comes at a cost
towards the signal to noise of the image. in terms of scan time and a loss of signal to
Conversely echoes that are encoded with noise. Hence, an optimum balance between
the largest phase encode gradient, such as sampling time and signal to noise should be
the 128th view will have faced a severe de- maintained when it comes to resolution.
phasing of the echo signal. The large amount All images as seen in MR are a result of
of de-phasing provided by the phase encode a two-dimensional Fourier transform of the
gradient essentially removes all the low raw data that is obtained from the RF
frequency components within the signal and receiver. The realization of the source of
the read gradient captures only the high contrast and resolution and the improve-
frequency components of the excited slice. ment in gradient technology has allowed
Essentially low frequency components get many an investigator to develop novel pulse
encoded in the center of k-space and higher sequences that provide snapshot images.
and higher frequency components are
encoded as one travels to the periphery of
the k-space. This concept is crucial and forms
the basis for the design of novel pulse
sequences.2,3
Thus as the number of k samples increase,
the spatial resolution of the image is
increased. In order to construct a complete
image from k x and k y all possible
combinations of k must be sampled along
each axis for the desired resolution. In order
to gain an understanding of pulse sequence
design it is necessary for one to understand
the source of contrast and resolution within
an image. The configuration of a typical
k-space is seen in Figure 5.1 along with the
location of the different frequency
components and their contribution to the
Fig. 5.1: Configuration of k-space showing the
image. The center of k-space, which mainly
distribution of different frequency components and
contains low frequency components the main sources that contribute to contrast and
provides the contrast in the resulting image. resolution
54 Biomedical Magnetic Resonance: Proceedings of the International Workshop

You may recall that a standard spin-echo
sequence takes 8 minutes and 32 seconds
to complete the acquisition of a T2-weighted
image at a TR of 2 sec and 256 × 256
resolution. This sequence is shown in Figures
5.2A and B along with its associated scheme
of filling k-space. Only one line in k-space
is filled for every TR. Recall that higher
spatial frequencies are encoded at the
periphery since maximum de-phasing occurs
through the use of a large phase encode
gradient. Lower spatial frequencies are
encoded during the time that the phase
encode gradient is small or absent.
The limitations of such a sequence is that
only one line in k-space gets encoded per
TR and hence the duration of the scan is
TR × desired resolution. If the desired
contrast is T2-weighting with an echo time
of 100 ms then one has to wait 50 ms to
apply the refocusing pulse followed by
another 50 ms to capture the echo. During
both the wait periods either the signal is
decaying or is being build-up to form the
echo. Rather than waiting for this decay
and build-up during the imaging sequence
it is possible with the new high-speed and
self-shielded gradient technology to
generate multiple echoes during that time.
That is, echoes may be generated every few
milliseconds, say every 10 ms, for as long
as the signal might last. While one could
use this sequence to obtain images at TE’s
in multiples of 10 ms, one could also use
these echoes cleverly to cover k-space with
the goal of minimizing scan time. For

A B
Figs 5.2A and B: (A) Pulse diagram for a spin-echo sequence, and (B) its corresponding k-space filling
scheme. The slice-selection process leaves one at the center of k-space. The x- and y-gradients then moves
one across or up and down the k-space depending on the polarity of the individual gradients. In this example,
although the x- and y- gradients are applied simultaneously we have shown for simplicity, the vector moving
along the x-direction first and then moving up to the maximum point indicating the application of maximum
phase-encode gradient. The 180o slice-selective refocusing pulse moves this vector through the origin to the
other side of k-space. At this point a positive read-gradient is applied and simultaneously the analog to digital
converter is turned on which reads out the first line in k-space. This process is continued until each of the lines
is read-out by appropriate scaling of the phase-gradient during each TR
 RF Pulse Sequences Including Rapid Imaging Sequences 55

example, one could construct a multi-echo
sequence using 16 refocusing rf-pulses with
an echo spacing of 10 ms. Such a sequence
could potentially generate 16 different k-
spaces that provided 16 different images
with different T2-weightings. Alternatively,
one could construct a single k-space from
the different echoes by forcing the desired
echo (or T2-weighting) to occupy the center
of k-space and those echoes farthest from
this desired weighting to occupy the
periphery of k-space. The T2-weighting of
the echo that is at the center of k-space
would dictate the contrast in the image
whereas the rest of the echoes would
provide resolution to the image. Which of
the 16 echoes occupies the center of k-space
is determined by the T2-weighting desired
in the image. An example of a four-echo
scheme is shown in Figures 5.3A and B
where the fourth echo is used to encode
the center of k-space, the first echo encodes
the periphery of k-space and the other two
echo make up the intermediate space. The
number of refocusing pulses used is typically
called the echo train length (ETL) or the
TURBO-factor. The ETL or the TURBO-
factor gives an indication of the speed of
such a sequence when compared to a tradi-
tional spin-echo sequence.4,5 For example a
16 ETL sequence would be 16 times faster
or have a TURBO-factor of 16 compared to
a conventional spin-echo sequence. Such class
of sequences is called Fast Spin-Echo (FSE)
or TURBO spin-echo sequences. The example
shown in Figures 5.3A and B has a TURBO
factor of 4. Please note that while in this
example the fourth echo was chosen to be
the center of k-space any of the other echoes
could have been used to encode the center
just by changing the phase encode gradients
to obtain the desired contrast.
The inherent advantage of FSE is its
speed compared to a conventional spin-echo
sequence. This advantage has to be taken in
context with other issues that arise when
using FSE. Since the number of RF-pulses
required to produce an FSE image is much

A B

Figs 5.3A and B: (A) Pulse diagram of a four-echo-fast spin-echo sequence and (B) the k-space associated
with this sequence. It should be noted in this sequence that after every echo is read the phase encode
gradient is rewound prior to the application of the next refocusing pulse. Please follow the k-space traversal
diagram at the center of k-space but after the first refocusing pulse. For the second half of k-space the
polarities of the phase gradient change
56 Biomedical Magnetic Resonance: Proceedings of the International Workshop
greater than a conventional spin echo
sequence, heat deposition (measured as
specific absorption rate (SAR)) to the patient
becomes a factor and increases linearly with
the number of refocusing pulses used per
unit TR. This becomes even more of a
limiting factor at field strengths greater than
1.5 Tesla since SAR increases as square of
the field strength. At higher field strengths
either longer inter-echo spacing needs to be
used or refocusing RF pulses with less power
need to be used. Another issue is the
contrast of the images themselves. When
comparing an FSE image with a standard
spin-echo sequence, it is obvious that the
FSE images have a smooth look to them.
This is because there is some amount of T2
smoothing that occurs since several echoes
were responsible for the creation of the FSE
image. Hence when relating an FSE image
to a standard spin-echo image the echo time
is referred to as the effective echo time or
Teff.5 One would also notice that the FSE
images have a somewhat reduced contrast
(such as between gray and white matter)
since the images are subject to magnetization
transfer effects due to the application of a
series of 180o pulses.6 Fat is bright on T2-
weighted images using FSE. This is due to
the fact that the multiple train of closely
spaced refocusing pulses essentially
eliminates the J-coupling modulation
enabling the fat to be bright.7
In clinical practice, FSE is routinely used
for T2-weighted imaging. A traditional fluid
attenuation inversion recovery (FLAIR)
sequence would take unreasonably long
period of time to complete the scan due to
its requirement of long inversion time and
echo time even at low resolution. 8 FSE
makes such imaging possible as it cuts down
the scan time to within 4 minutes depending
on the resolution required. It is also used
to image the abdomen as it allows one to
minimize on respiratory artifacts by
obtaining images within a breath-hold.
Currently, single shot FSE sequences are
used routinely for cholangiopancreatography
(MRCP) where they have proven to be an
excellent alternative for ERCP and also in
diffusion-weighted imaging where the goal
is to create diffusion anisotropy maps or
perform fiber tractography.9-11
The above sequence type shows how
refocused spin echoes can be used to make
up the k-space required for image formation
with a given contrast. One could also do
the same using refocused gradient echoes
where the echo is reversed several times
using the read-out gradient. Such an
acquisition is commonly called the echo-
planar imaging (EPI) technique. 12 Each
reversal of the gradient creates another
echo, which can be phase encoded to a
different line in k-space. Figures 5.4A and B
show a typical single-shot EPI sequence
where all the k-space data is acquired after
a single RF-excitation. Notice that every
alternate line in k-space is read in the
opposite direction. This is due to the nature
of the acquisition where every other echo is
obtained with reversed polarity of the read
gradient. In such an acquisition, one has to
reverse these lines in k-space prior to 2D-
fourier transform. Any off-resonance signal
(such as fat) will encounter a position shift
due to the long duration of data sampling
(50-100 ms). Fat that is 225 Hz away from
water frequency will be shifted by about 12
pixels for a read-out time of 50 ms and will
appear as an artefact. Hence it is necessary
to minimize these off-resonance artefacts by
good shimming over the region of interest
and also by suppressing these off-resonance
 RF Pulse Sequences Including Rapid Imaging Sequences 57

A B
Figs 5.4A and B: (A) Pulse diagram for a single-shot spin-echo EPI sequence and (B) its traversal in k-space.
Following a single excitation, the data is read-out by reversing the read gradient while at the same time
‘blipping’ the phase gradient. Every alternate line then needs to be reversed, and phase corrected prior to
Fourier transform

signals using chemical saturation one could use magnetization preparation

techniques.13 Care also needs to be taken to
minimize phase errors that might arise due
to minor mismatches in the behavior of the
gradient while it traverses through
maximum positive polarity to a maximum
negative polarity. Any eddy currents that
might be induced in the process could
introduce phase errors that would need to
be accounted for prior to the image
formation. Ghosts from such a mismatch in
phase will show up as an additional image
with reduced intensity that is shifted by
half the field of view since half the lines in
k-space are different from the other half.
Such a ghost is commonly referred to as the
N/2 ghost. Minimizing the phase errors
reduces the energy in the ghost image and
puts it back into the main image. As in
conventional SE imaging, the T2 contrast is
controlled by varying the echo time of the
EPI sequence. Since this technique is a single-
shot technique the TR is essentially infinite.
To obtain images of other types of contrast,
prior to every shot.
Single-shot imaging is useful when
temporal resolution is important since the
scan time for a slice is little more than the
read-out time (50-100 ms). The cost for this
increased temporal resolution comes with a
sacrifice in spatial resolution. Hence the use
of this technique is predominantly to
determine function. It is used in conjunctions
with techniques such as diffusion-weighted
imaging, perfusion-weighted imaging, and
functional MRI for brain mapping.14-18 Other
variants of EPI are available where a balance
is sought between the temporal and the
spatial resolution in images. Multi-shot EPI
sequences that acquire k-space in more than
one excitation but in far fewer steps than a
regular gradient echo have been designed.
Such EPI sequences face similar challenges
as single-shot sequences in terms of phase
errors due to mismatched echoes. In
addition they are more sensitive to flow and
motion artifacts. The main advantage of
58 Biomedical Magnetic Resonance: Proceedings of the International Workshop
multi-shot EPI is that the requirements for
the gradients are not as stringent as for single-
shot EPI.19 In such situations multi-shot EPI
provides a better alternative to standard
gradient-echo sequences when temporal
resolution is required. One should remember
that the potential for artifacts increases with
the number of potential discontinuities in k-
space. To accurately correct for these artifacts
it may be necessary to obtain an additional
navigator echo that can be used to minimize
these artifacts.20
All the above sequences described above
including the EPI sequence obtain data in a
rectilinear fashion and the corresponding
k-space essentially follows a raster scanning
system. Several variations from this raster
scanning have been successfully implemented
by various investigators. For example it is
possible to sample k-space radially by
turning on the x- and y-gradients simul-
taneously with varying proportions. The
reconstruction of such a data would then
use projection reconstruction techniques as
done in CT.21 Perhaps the most useful and
promising acquisitions among the variants
of EPI is the spiral scan where the coverage
of k-space is accomplished in a spiral fashion
starting from the center of the k-space.22,23
Figure 5.5 shows a typical k-space trajectory
used in spiral imaging. Both the x- and
y-gradients spiral out starting at the center
of k-space. This technique makes efficient
use of the available gradients as the
gradients are smoothly varying and is
inherently less sensitive to motion.24 Since
the acquisition is not Cartesian in nature,
the raw data obtained needs to be
regridded to a Cartesian co-ordinate system
prior to Fourier transform. Other variants
on spiral imaging such as the Rossette has
also been proposed for efficient sampling
of k-space.25 Multi-shot and 3D versions of
these sequences have also been explored and
show excellent promise for the future.26
A relatively new sequence that incorpo-
rates both spin echoes and gradient echoes
is called the GRASE. Figures 5.6A and B
shows a typical GRASE acquisition and its
associated k-space traversal. GRASE can be
thought of as a combination of FSE and
EPI.27,28 For each spin echo formed one could
generate several gradient echoes. Such an
acquisition has several advantages. Firstly,
the received signal is kept alive for a longer
duration due to refocusing; secondly,
susceptibility artifacts are much reduced
compared to EPI; thirdly, the acquisition
will have generally a better signal to noise
compared to EPI; and finally, the acquisition
is less prone to run into SAR limitations
compared to a similar FSE acquisition since
the 180o pulses are spaced further apart.
While the use of GRASE has been shown
for several applications, its use might
increase as higher and higher field strength
magnets become part of the main stream.29
In general, GRASE might be thought of as
a class of sequences that encompasses both
FSE and EPI. In FSE for each refocusing
pulse there is a gradient-echo, whereas in
spin-echo EPI for each refocusing pulse there
are multiple gradient echoes. So one could
think of FSE as a GRASE factor of 1 whereas
in the example shown in Figures 5.6A and
B the GRASE factor is 3 where one obtains
three echoes per refocusing pulse. Similarly,
the GRASE factor in a single-shot EPI
sequences would be the number of gradient
echoes obtained to cover k-space.
STEADY-STATE IMAGING PHYSICS
Gradient-echo sequences can also be used
with similar scanning parameters as the spin
echo sequences but the image quality is
 RF Pulse Sequences Including Rapid Imaging Sequences 59

rather poor due to susceptibility effects at
longer echo times and artifacts due to flow.
However, the biggest advantage of gradient
echo type scans is the ability to perform
fast imaging or steady-state imaging. Steady
state imaging involves the use of excitation
pulses usually less than 90o at a very rapid
rate usually at the rate where TR<<T2 of
the object being imaged. As the TR is
shortened such that it gets comparable to
the T2 of the tissue, the MR signal persists
from one excitation to the next. So as the
excitation process continues from TR to TR,
the signal strength and contrast of the
resultant image are governed by the history
of the spins over several TR’s.

Fig. 5.5: k-space filling scheme for a spiral EPI scan. In this case both the x- and y-gradients spiral out from the
center of k-space. The sequence is less sensitive to flow artifacts and makes efficient use of the gradients.
60 Biomedical Magnetic Resonance: Proceedings of the International Workshop
When using conventional sequences, the
TR is generally much longer than the T2 of
the tissues being imaged. In other words,
the transverse relaxation process is complete
prior to application of the next RF pulse to
encode the next phase-encode view. Hence
the chance of any spins having a history of
the previous excitation is negligible.
However, in steady state imaging where
TR approaches or becomes less than the T2
of the imaging region the history of the
spins persists for several TR’s since the
magnetization has not had a chance to
B the encoded lines are shown in k-space for clarity.
completely decay. During these first few
TR’s or RF excitations, the magnetization
will be found in a different direction each
time resulting in different signals during
each TR interval (for a mathematical
treatment of steady-state spin physics,
please see the appendix). This creates
stimulated echoes whose amplitude is half
that of a regular spin-echo.30,31 Depending
on the spacing of the pulses and the
relaxation properties of the tissues under
consideration this stimulated echo could
interfere with the desired echo and cause
Figs 5.6A and B: (A) Pulse diagram for a 12 echo
GRASE sequence, and (B) its trajectory in k-space.
The true spin-echo forms the center of k-space
whereas the two gradient echoes on either side of
the spin echo encode the periphery of k-space. Only
 RF Pulse Sequences Including Rapid Imaging Sequences 61

interference. Figures 5.7A and B show the
effect of closely spaced RF pulses when they
are unequally spaced (a) and when they are
equally spaced (b). Typically n RF pulses
applied to a spin system can generate up to
(3n-1 - 1) echoes following the last RF pulse.
The stimulated echoes produced are typically
T 2 -weighted and their origin is from
previous RF pulses. It is necessary then to
de-phase this unwanted signal prior to the
application of the next RF pulse.
As seen in Figure 5.8, after several
excitations the RF and the magnetization
come to a steady state wherein the
magnetization due to each RF pulse is exactly
compensated by changes due to precession
and relaxation during the short TR interval.
However artifacts begin to develop as the
TR is reduced and manifest as bright bands
(commonly called FLASH bands) parallel to
the frequency encode direction. The
brightest of these bands occur around the
physical center of the phase encode gradient
since the spins in this region receive the
least amount of dephasing during the
application of the phase encode gradient.
As a result these spins preserve the history
of previous excitations and introduce some
T2-weighting into the images. These bands
also occur in places where the phase
encoding pulse is able to produce a complete
2 π rotation of magnetization. The strength
of these bands is a function of T1, T2, TR,
and flip angle. To appreciate the contrast
mechanisms in steady state imaging, it is
essential to understand that the image

B
Figs 5.7A and B: (A) Three RF pulses spaced unequally between them will produced five echoes one of
which is the stimulated echo, (B) A train of equally spaced RF pulses produce several echoes
62 Biomedical Magnetic Resonance: Proceedings of the International Workshop
Fig. 5.8: The transverse magnetization approaches
a steady state after a few excitations. The approach
to steady state is a complex function of the repeat
time (TR), flip angle, and the relaxation properties of
the tissue being imaged
contrast and the signal strength are
determined by the history of not only the
RF pulses but also the gradients applied
along the way. With appropriate manipu-
lation of RF pulse phase and flip angle along
with appropriate use of gradients for
spoiling, one could achieve images with a
desired contrast using steady-state imaging
techniques.32-34
The banding artifacts as described above
can be eliminated in a couple of different
ways. The different steady state techniques
that have been developed over the years
have either manipulated gradients only or
have manipulated both RF and gradients to
arrive at a desired contrast. One way would
be to simply rewind the phase encode
gradient following the read out gradient
pulse and prior to the next excitation. This
would ensure that the phase of the spins
after each TR remains the same irrespective
of the location within the imaging plane.
This is what is typically done in GRASS,
FISP, and FAST imaging sequence types.
These acronyms are used by different
manufacturers and stand for Gradient
Recalled Acquisition in the Steady-State
(General Electric), Fast Imaging with Steady-
state Precession (Siemens), Fast Acquisition
in the Steady-State (Picker International,
currently Philips Medical System)
respectively. The resulting contrast in the
image turns out to be a complicated function
of T 1 and T 2 . Alternatively, one could
employ a large de-phasing pulse following
the readout gradient to crush any residual
magnetization prior to the next RF excitation
pulse. In this case, since the transverse
magnetization is completely destroyed prior
to the application of the next RF pulse, only
spins that have had the chance to recover
would be pulled down to the transverse
plane after each excitation. Typically spins
with short T1 would have recovered the
most and hence the resulting image would
be heavily T1-weighted.31,34,35
A third method to ensure that artifacts
do not occur is to change the phase of the
RF pulse at each excitation. A semi-random
change in the phase from one RF excitation
to the next will ensure that the steady state
condition will be spoiled leading to mainly
T1 contrast. The semi-random phase shift of
the excitation pulse could be complemented
with a rewinding phase encode gradient
after the read-out gradient is played out.
This will ensure that the resulting image is
T1-weighted without the banding artifacts.
Such a technique is commonly called RF-
spoiled or spoiled gradient echo. Depending
on the manufacturer such a sequence goes
by the name of Spoiled GRASS (SPGR, GE),
Fast Low Angle Shot (FLASH, Siemens) and
Radio-Frequency FAST (RF-FAST, Picker
International).36,37
It should be noted that contrast derived
from both the above-described types of
sequences are influenced by TR, TE, and
the flip angle along with tissue specific
 RF Pulse Sequences Including Rapid Imaging Sequences
relaxation properties. Contrast manipulation
is done through the appropriate choice of
these sequence parameters. TR determines
the optimum flip angle to be used in order
to obtain the maximum signal to noise ratio
as given by the Ernst angle. Long TE’s
provide images with T2*-weighting whereas
shorter TE’s produce images with high
signal-to-noise ratio (SNR) with minimal
motion artifacts. The choice of an
appropriate flip angle will provide images
ranging from proton density weighted (low
flip angles) to highly T1-weighted images
(higher flip angles). As a general rule for
T1-weighting in the images one could use a
short TR (50 ms), short TE (4 ms) and a
high flip angle (50 degrees). Of course one
could use even a shorter TR and a higher
flip angle for even higher T1-weighting but
the reader should be cautioned that the SNR
will be greatly diminished. To obtain proton
density weighting one could use a long TR,
short TE and a small flip angle. For T2*-
weighting a long TR, long TE and a small
flip angle is preferred. The small flip angle
and the long TR ensures minimization of
any T1 effects in the image.32-34
In the case of GRASS, FISP, and FAST,
the free induction decay of the signal coming
from the RF is sampled, while the spin-echo
and the stimulated echo components
emanating from the string of RF pulses are
kept away from the sampling window. Since
the RF pulses are fired at constant intervals
the spin-echo and the stimulated echo
overlap. One can then ignore the FID part
of the signal and move the sampling
window so as to capture the spin-echo
signal, which has more T 2 -weighting
associated with it. The sequence diagram
for such a pulse sequence resembles that of
the sequence for GRASS except that the slice
63
and read gradients are reversed. While the
first a pulse will pull down some magneti-
zation onto the transverse plane, the next a
pulse will form the spin-echo and the
subsequent a pulses will form a combination
of spin and stimulated echoes. The
stimulated echo will be coincident with the
spin-echo since the RF pulses are spaced
equidistantly in time. The effective TE of
such a sequences is (TR+TE) since the echo’s
origin is truly from a previous RF excitation.
Subsequent echoes will have contributions
from even earlier RF excitations. One
should expect an image which is more
heavily T 2 -weighted compared to that
obtained from GRASS or FAST imaging
sequence types albeit with much lower signal
to noise ratio. Such rapid imaging sequences
that produce T 2 -weighted images are
commonly called Contrast Enhanced-FAST
(CE-FAST), PSIF (reverse of FISP) and
Steady State Free Precession (SSFP, GE).31,32
For more details on the different acronyms
used for different flavors of gradient echo
sequences please see reference 38.
TrueFISP
Improvement in gradient coil technology has
produced coils with higher and higher peak
gradients while at the same time reducing
the rise times. These gradients are also
capable of performing at high duty cycles
with excellent eddy current compensation.
Because of this, techniques such as FISP
which were first described in 1986 have
become practical in the clinical setting as
this technique demands high duty cycle.34
The TrueFISP (Siemens) sequence, also
known as Fast Imaging Employing Steady-
state Acquisition (FIESTA, GE) and Balanced
Fast Field Echo (Balanced FFE, Philips), uses
fully balanced gradients in all directions
64 Biomedical Magnetic Resonance: Proceedings of the International Workshop
(slice, read, phase encoding directions) to
refocus all the transverse magnetization as
shown in Figure 5.9.39 This allows for the
steady state of both the longitudinal and
transverse magnetization to be maintained.
Therefore TrueFISP can be used with very
large flip angle without spin saturation
problem. Due to the intrinsic high SNR, it is
then possible to use the highest available
receiver bandwidth to achieve even shorter
TRs.
Since the signal in TrueFISP is a mixture
of echoes refocused from all the previous
RF excitations with different amplitudes, the
signal behavior is a complicated function of
the flip angle, TR, T1, T2. Nevertheless, the
image contrast of TrueFISP is generally
believed as T2-T1-weighted.40 So TrueFISP
has found its most applications in cine heart
imaging or coronary arteries imaging due
to its high contrast between blood and
myocardium41. Further, the ultra-fast speed
also makes it very suitable for breath hold
imaging in order for higher resolution,
shorter imaging time and hence less motion
artifacts.
Due to the fact that TrueFISP signal
depends on coherences set up from several
RF pulses prior, it is very sensitive to off-
resonance effects. Hence the use of long
TR’s could lead to image quality problems
Fig. 5.9: Pulse diagram for a typical TrueFISP
sequence. Gradients in all three directions are fully
balanced
since phase errors accumulate with longer
TR’s. Steady state magnetization is critical
for TrueFISP which also minimizes the off-
resonance effect. However, it takes several
TR’s (equivalent to a few T1’s of the tissue
being imaged) to reach this steady state.
Techniques are available through the use of
steady-state preparation pulses to bring the
magnetization to a steady state in a shorter
time.42
DESS (Dual Echo in the Steady State)
Another sequence called the DESS is a
variation of the fast gradient echo sequences
such as FLASH or GRASS which uses long
readout gradient to focus a second gradient
echo as shown in Figure 5.10 to introduce
T2-weighting to the image.43 The first echo
has mixed T1/T2-weighting with high SNR
and the second echo is strongly T2-weighted
but with low SNR due to T2 decay. Signals
from both echoes are then averaged to
achieve a reasonable T2-weighting and high
SNR. One main problem with DESS is its
sensitivity to motion due to the prolonged
TR. 3D-DESS has been routinely used in
cartilage imaging for a high contrast noise
ration between the thin sheet of cartilage (low
T2) and joint fluid (high T2).
By adjusting the echo times for the two
echoes, one could also vary the relative
Fig. 5.10: Pulse sequence diagram for DESS
sequence
 RF Pulse Sequences Including Rapid Imaging Sequences
phase of fat and water, thereby making the
sequence sensitive to chemical shift differen-
ces. This sequence has been used to identify
adenomas of the adrenal glands by
simultaneous acquire in-phase/opposed-
phase images.44
Magnetization Prepared Sequences
Other forms of steady state imaging that
make clever use of steady state sequences
for a desired contrast are also available.
One could prepare the longitudinal magneti-
zation such that the magnetization has a T1
or a T2 history prior to rapidly imaging the
magnetization. 45-47 Such sequences are
generally termed magnetization prepared
rapid acquisition techniques. One such
sequence uses an inversion pulse followed
by a delay prior to capturing all the views
required to make a single image. The delay
after the inversion pulse is to let the
magnetization recover to a point where the
contrast between any two tissues is the
greatest and follow this up with a rapid
gradient-echo imaging scheme. Such a
sequence commonly called magnetization
prepared rapid acquisition gradient-echo
(MP-RAGE), provides a highly T1-weighted
scan with the T1 contrast being far superior
to the one obtained with a regular T 1 -
weighted sequence. Similarly one could also
obtain a very highly T2-weighted image by
preparing the longitudinal magnetization
such that it has a T2 history. Such a sequence
is called driven equilibrium fourier trans-
form or DEFT acquisition.48 The preparation
part of this sequence consists of a normal
spin echo(90x-180y-90-x) pulses. The phase
of the second 90o pulse would be adjusted
such that the transverse magnetization,
which is T 2 -weighted is now in the
longitudinal direction. This longitudinal
65
magnetization is then imaged with a rapid
gradient echo sequence that captures the T2
contrast. For more detailed theoretical
descriptions of the above techniques the
reader will find references 47, 49 and 50
helpful.
BURST Imaging
Burst imaging is another novel technique
derived from the behavior of steady state
pulse sequences. In this technique a series
of RF pulses are applied in the presence
of a small x-gradient. 51,52 This is followed
by a 180 o pulse that refocuses all the
echoes that are then read-out using the same
x-gradient to sample all the echoes as shown
in Figure 5.11. A y-gradient is also applied
simultaneously to move the echoes to
various parts of k-space.53,54 The inherent
signal-to-noise of this scan is low compared
to an EPI sequence since the full echo is
never sampled and the echo-intensity is
distributed through k-space.51 Theoretically,
the SNR of such a scan is reduced by a
factor of √N where N is the number k-
space lines sampled in the burst acquisition.
While the utility of burst imaging is limited
due to its poor SNR it can be useful in special
applications of fMRI where the acoustic
noise of the scanner needs to be low. Burst
acquisitions tend to have low acoustic noise
compared to EPI acquisitions since there is
no rapid switching of gradients.
Fig. 5.11: Pulse sequence diagram for BURST
sequence
66 Biomedical Magnetic Resonance: Proceedings of the International Workshop

Parallel Imaging can greatly benefit from this technique. But

non-Cartesian k-space sampling, such as
While significant progress has been made
spiral or radial sampling, remains a
in the area of pulse sequence design more
challenge. Although a generalized SENSE
innovations are expected especially with
(GSENSE) could be used, the reconstruction
the advent of new techniques such as
is very computationally expensive. Gene-
simultaneous acquisition of spatial
rally, the SNR of images acquired by parallel
harmonics (SMASH) and sensitivity
imaging is decreased because SNR is propor-
encoding (SENSE).55,56 Both these techniques
tional to the square root of acquisition time.
exploit phased-array coil technology in
The decrease will be square root of the
different ways with a common goal of
reduction factor in parallel imaging. Further,
reducing scan time. SMASH takes into
unlike conventional scan, SNR in parallel
consideration the inherent geometry of the
imaging varies significantly over the whole
elements of the phased array coil to
FOV due to coil geometry.59 Nevertheless
substitute for the normal phase encode
the techniques are very promising and
gradients. Essentially the RF coils are used
steady-state imaging techniques can greatly
to spatially encode different positions with
benefit from parallel imaging.
the application of each phase encode
The above discussions on various pulse
gradient. Whereas in SENSE the distance
sequences is designed to provide an
between samples in the phase encode
overview of the breadth of techniques
direction is reduced while maintaining the
available in MR. The techniques discussed
maximum k-values. This produces aliased
above by no means form an exhaustive list
images for each of the coils since the FOV
of techniques available. However, it is hoped
is reduced. A sensitivity matrix for each of
that the reader will find the article useful in
the coil is then derived based on the signal
understanding the basic concepts of data
contribution from a number of positions in
acquisition in MR and will lead him or her
the full FOV image. This sensitivity matrix
to choose the appropriate pulse sequence
is then applied to the actual images. Both
for the desired diagnosis. While it appears
techniques are sensitive to coil geometry
that most of the developments in pulse
and extensive calibration is required for
sequence design have come from people
these techniques to work in a robust manner.
with technical background, these
New generations of SMASH such as Auto-
developments would not be possible
SMASH and GRAPPA (GeneRalized Auto-
without the driving force from clinicians to
calibrating Partially Parallel Acquisition) have
provide better diagnosis using the versatility
greatly reduced the effort for coil sensitivity
of MR. In the future we will see many more
calibration and could work with essentially
techniques that will be derived purely on a
arbitrary coil configurations.57,58 An obvious
clinical need that will assist both in the
advantage of parallel imaging is the
diagnosis and follow-up of disease.
acceleration in imaging that if a reduction
factor 2 is used, then only half the phase
encoding steps are needed and imaging time APPENDIX
is reduced by half. Typically, all sequences A set of simultaneous equations for the
including steady-state imaging sequences vector components of the magnetization can
 RF Pulse Sequences Including Rapid Imaging Sequences 67

be derived for a system that has reached Mz- = Mo(1-E1)[1-E2cosϕ-E2cosα(cosϕ-E2)]/D

steady state after applying a series of a
pulses at constant intervals TR for any
tissue with a given T1 and T 2 relaxation
times (Figs 5.12A to C).
Mx-, My-, Mz- = vector components of total
magnetization prior to the application of RF
pulse.
Mx+, My+, Mz+ = vector components of total
nuclear magnetization just after the RF
pulse.
Mo = Total available magnetization
E1 = exp(-TR/T1); E1(t) = exp(-t/T1)
E2 = exp(-TR/T2); E2(t) = exp(-t/T2)
D = (1-E1cosα) (1-E2cosϕ) - (E1-cosα) (E2-
cosϕ) E2
Mx- = Mo(1-E1)sinα sinϕ/D
My- = Mo(1-E1)[(1-E2 cosϕ)sinα]/D
Mx+ = Mx-
My+ = Mo(1-E1)(1-E2cosϕ)sinα/D
M z+ = M o (1-E 1 )[E 2 (E 2 -cosϕ)+(1-E 2 cosϕ)
cosα]/D
The above equations provide information
on the magnetization just after each RF
pulse. However, of interest would be the
evolution of the magnetization throughout
the TR interval. Of particular interest in
imaging is the dependence of this
magnetization (which does not vary
constantly) on resonance frequency offset
determined by local field inhomogeneities
and due to the applied gradients. Matsui et al
developed the following equations for
instantaneous magnetization as a function of
time and frequency offset.50

A B C
Figs 5.12A to C: Free precession of the steady state signal. (A) At time t=0 the magnetization is rotated by a
RF pulse with an angle α. (B) This magnetization precesses about the main field determined by the local
inhomogeneities at an angle ϕ between time t=0 and t=TR and is a function of the relaxation properties of the
signal. (C) The magnetization returns to its steady state
68 Biomedical Magnetic Resonance: Proceedings of the International Workshop
Q = Mo(1-E1)/D = R/{P-Qcos[(δω)(TR)]}
P = 1-E1(TR)cosα - E2(2TR){E1(TR)-cosα}
Q = E2(TR){1-E1(TR)}{1+cosα}
R = Mo[1-E1(TR)]
Mx and My as a function of time and
frequency offset can be shown to be
Mx (t,δω) = Qsinα[E2(t)sin[(δω)t] + E2(TR+t)
sin[δω(TR-t)]}
My (t,δω) = Qsinα[E2(t)cos[(δω)t] - E2(TR+t)
cos[δω(TR-t)]}
The magnitude of the transverse
magnetization given by the above equations
is proportional to the sum of the squares of
the x- and y- magnetization.
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Overview of an MRI System
INTRODUCTION
The MRI system consists of the following
components, which operate in relation to each
other to produce images of various organs of
the patient for diagnosis. (i) Magnet system,
(ii) Gradient system, (iii) RF system,
(iv) Patient table, (v) Control system, (vi) Main
computer, (vii) RF and magnet shielding,
(viii) Phantoms, and (ix) Supporting systems
(Fig. 6.1).
The MR scanner is located inside the
scanning room, which RF shielded. The
magnet produces the main magnetic field,
B0. The gradient coils are used to generate
gradients in B 0 in the three-dimensional
imaging area. The RF coil produces B 1
magnetic field to excite the proton spins
present in the human tissues. The gradient
and RF coil are placed inside the magnet.
The RF coil also acts like a receiver and
picks signal generated from the patient
body. Patient is positioned inside the magnet
on a computer controlled patient table. The
RF shielding filters the signals from radio
and television transmitter, reaching the
scanner. Also, it prevents the RF signal
produced in the scanner, to spread outside.
To avoid magnetic field to spread beyond
the permissible area, magnetic shielding is
Overview of an MRI System 71
6
RS Saini
also used sometimes around the scanning
room. Phantoms are used for quality
assurance as a substitute to patients during
standardization.
The operation and control of the above
components is synchronized by control
system in association with the main
computer, which gets instructions from the
operator through software of the system
developed. The following sections describe
the various components in some detail.
MAGNET SYSTEM
There are mainly three types of magnets
presently in use in imaging scanner. These
are as follows.
Permanent Magnet
This type of magnet is always magnetized.
The advantage of these magnets is non-
requirement of any energy source for its
magnetization. These magnets are highly
sensitive to thermal instability and require
proper arrangements for temperature
control. Only limited field strength can be
achieved with permanent magnets and these
magnets are very heavy. A 0.2 T magnet
weighs approximately 11 ton.
72 Biomedical Magnetic Resonance: Proceedings of the International Workshop

MR SYSTEM OVERVIEW

Fig. 6.1: Figure gives a general overview of the major components of the
MR system and their interconnections

Resistive Magnets
These are electromagnets and require an
electric current to pass continuously through
a loop of wire. They remain magnetized as
long as electric current is flowing through
them hence require continuous electric
supply. As the wire is resistive it causes
heat generation, which needs to be
dissipated by making a cooling arrangement.
Generating very high magnetic fields with
these types of magnets is not practical, as it
requires high electric energy and elaborate
cooling arrangements.
Superconductive Magnets
Superconductive magnets are the most
commonly used magnets in MR systems.
Superconductivity is achieved by immersing
the coil made up of Niobium-Titanium wire
in liquid helium. The liquid helium keeps
the coil temperature at about 4.2 K. At this
temperature the resistance of the wire is
effectively zero and no heat loss occurs when
electric current flows through it. Hence an
electric current, if made to flow through
this wire once, will keep on flowing perma-
nently without the need of continuous
electric supply. This current in the coil will
produce a constant magnetic field.
If due to some reasons the temperature
of the wire rises above liquid helium
temperature by few degrees, it will result
in loss of the superconductivity. The loss of
the superconductivity is known as “quench”.
In this case there will be rapid heat loss
due to resistance to current flow, resulting
in sudden boil off of liquid helium. This
will render the magnet discharged and most
of the liquid helium escaping to the
atmosphere.
Overview of an MRI System 73
High magnetic fields can be produced
using superconductive magnets. Most
commonly used magnets are 1.5 T. These
magnets produce highly homogeneous field.
These magnets are very costly and use
cryogen (liquid helium, etc.) which is very
expensive.
Magnet Shielding
Since the magnetic field can be dangerous
to patients with metallic implants, peace
makers, etc. and machinery, all efforts are
made to restrict the magnetic field within
scanning room. There are generally two
ways to achieve this. Firstly, using heavy
(tons of) ferromagnetic pieces around the
magnet. These iron pieces will cause
magnetic field to get concentrated in them
and does not allow them to spread. This
type of shielding is called passive shielding.
Secondly, new magnets use another
superconducting coil inside, which produces
a field of opposite polarity to the main B0
in the area outside the imaging area. This
opposite polarity field effectively reduces
the strength of the magnetic field outside.
This type of shielding is called active
shielding.
SHIM
The field produced by all type of magnets
contains some inhomogeneity. To make the
field homogenous a process called shimming
is used. This is achieved in two ways. First,
by placing small iron or magnet pieces in
the magnetic field arranged in particular
manner. This is called passive shim.
Secondly, another coil is kept within the
magnet and small currents are made to flow
74 Biomedical Magnetic Resonance: Proceedings of the International Workshop
through them making the field homo-
geneous. This is called active shim. Shim
system and software of the MR system
control the current through the coil.
REFRIGERATOR SYSTEM
In superconductive magnets the coil is
immersed in liquid helium. The liquid helium
container is well insulated from all sides to
avoid heat dissipation to the container. But
some heat flow always takes place and
liquid helium keeps on boiling. These helium
vapors escape to atmosphere, bringing the
level of liquid helium down slowly. Hence
the magnet requires liquid helium refilling
after a certain period (generally few
months). To minimize flow of heat from
outside atmosphere to liquid helium
container the magnet also contains shields
which are being cooled down by
refrigerator systems. This refrigerator
system captures the heat going into the
magnet and does not allow it to reach
helium container. With latest refrigerator
system, the liquid helium boil off is reduced
and refilling of the magnet is required after
few years.
MONITORING SYSTEM
Various parameters like temperature,
pressure and liquid helium level in the
magnet are continuously monitored and
appropriate action is automatically taken if
any parameter is varied beyond permissible
limit. In case the parameter is not being
brought under limits an alarm is generated
for information. This system also has quench
buttons to discharge the magnet
immediately in case of an accident.
RF SYSTEM
This system has transmitter and receiver
section. A transmitter is used to produce
RF to excite the protons from the subject.
The transmitter is made up of RF generator,
which produces a small RF signal generally
a sine-wave of a particular frequency. This
signal is then shaped in desired form using
modulator and waveform generator,
generally a sinc pulse. This pulse is then
amplified using a RF amplifier. The
amplified pulse is radiated by an antenna
(namely RF coil) on to the patient body.
Absorbing this RF pulse excites the protons
inside the patient body. After excitation of
the protons the RF pulse transmission is
stopped. Now the excited protons slowly
come back to their normal condition. While
coming back to their normal status the
proton release the absorbed RF energy back.
This RF energy is captured by the receiver
coil kept around the patient body. RF coils
can be used for transmission as well as
receiving the RF energy. Generally,
transmission of RF energy is done by body
coil; while for receiving the signal from
patient body different receive-only coils are
used. The body coil is permanently fixed in
the scanner. Other receiver coils can be
connected as and when required, depending
upon the area to be examined. Various types
of coils are available. Coil, which surrounds
the imaging area, is known as volume coil,
while those kept adjacent to imaged area
are called surface coil. When these coils
contain multiple numbers of receiving
elements in the form of an array they are
known as array coil.
The receiver section of the scanner then
processes the received signal, which is very
small. The receiver section generally

contains amplifier, de-modulator and
digitizer. Few components required for
signal cleanup are also present in this section.
RF Shielding
The transmitted signal is of a very high
strength; it can interfere with the local radio
and television transmission. Similarly, local
radio and television transmission can
interfere with the very low strength signal
received from patient body. To avoid this
interference the MR scanning room is
enclosed inside a RF shielding.
Filter
All electrical connections going and coming
out of scanning room have to pass through
a filter. This is to avoid interference from
outside RF signals.
GRADIENT SYSTEM
Gradient system is used to exactly specify
the area to be examined. Slice selection and
spatial information is possible due to the
gradient system. By this system additional
linear electromagnetic fields are produced
to systematically vary the magnetic field in
the imaging area. This is achieved by
passing currents through gradient coils. As
imaging takes place in three dimensions,
there are three sets of coils for x, y and z
directions. All these three sets of coils are
mounted on one cylindrical surface and fixed
inside the magnet.
The current strength, duration and timing
depend upon the type of study to be
performed. System operator and the
software decide about these parameters.
These parameters once selected are passed
on to gradient pulse generator. The pulse
Overview of an MRI System 75
produced is then amplified in gradient
power amplifier and fed to the gradient
coil.
Sudden and frequent change of magnetic
fields due to gradients causes eddy currents
to be produced in various metallic parts in
the magnet. To take care of the effects of
these eddy currents, special circuitry is
incorporated in the gradient system. Now
day’s “active shielded” gradients are
available, which minimize the production
of eddy currents.
The rocking sound of pounding noise
heard during MR examination is due to the
vibration of the gradient coils. Since these
coils are kept in magnetic field, high current
flow through them during scanning causes
them to vibrate.
Gradient strength (in mT/m), duty cycle,
rise time and slew rate are parameters which
determine the performance of the gradient
system.
Patient Table
Patient table is also a very important part
of MR scanner. This table is generally
computer controlled and is used to take
patient into imaging area. The movement
of the table can be controlled through the
operating console. Connections for the
various coils are incorporated in the table
itself. Few coils can be kept permanently
connected on the table.
Imager
The raw data (signal) received from the
subject is being sent to the imager. Here
this data is mathematically analyzed by
performing Fourier transform. By this
process the image data is created, which is
transferred to the main computer for display
76 Biomedical Magnetic Resonance: Proceedings of the International Workshop
and further processing. This procedure in
new systems is performed by software while
in earlier systems dedicated hardware was
used for this purpose.
Control System
During the scanning process the desired
data to various systems (e.g., RF, gradient,
etc.) was supplied from the control system.
This section controls the sequence in which
different section of the scanner operate.
Control system gets these instructions from
the main computer. This system also
monitors the performance of the different
components of the scanner and raises alarm
in case of some abnormality is observed.
Small signal parts of RF and gradient system
are generally contained in control system.
MAIN COMPUTER
The main frame computer contains the
software along with the system data,
necessary to run the MR scanner. The system
operator gives input to the scanner through
the main computer while scanned images
are displayed on the computer monitor. This
computer also has large storage capacity for
scanned images. Additionally various
archiving system like CD writer, MOD, etc.
are connected to it. For filming the images,
a camera is connected to this computer.
The monitor, for display of images, and
keyboard/mouse for data entry constitute
the operating console. From this console the
operator runs the scanner and does various
processing on the acquired patient images.
PHANTOMS
These are being used to test the performance
of the scanner. Phantoms are used as a
substitute to the patient, for quality
assurance programs. They contain material,
which can produce MR signal. Water is the
most commonly used material in the MR
phantom.
SUPPORTIVE SYSTEM
There are a number of supportive systems,
which help the above-described main
components of the MR scanner to perform
their activities. Few of these systems are
helpful in performing special MR studies.
Details of some of these systems are given
below.
i. Physiological measurement unit: This
system is used to display physiological
parameters of patient (e.g. ECG, pulse,
etc.) during scanning. One of these
parameters can be used to control the
scanning sequence to acquire special
images like in cardiac imaging.
ii. Injector: Injector is used to inject
contrast agent to the patient for special
contrast enhanced studies.
iii. Intercom: This system enables operator
to give instructions to the patient while
scanning (e.g. holding the breath, etc.).
Also, CCD camera installed with the
system monitors the patient movement.
iv. Chiller: Chiller circulates cold water
through various sections of the scanner
to dissipate the heat generated while
scanning and during other operation.
v. Additional console: One or more
additional consoles can be connected
to the system. These facilitate
independent post-processing of the
scanned images. Acquisition can not be
done on these consoles.
vi. Camera: Camera is connected to the
main computer through network or by
other means. This is used to make films
from the scanned images.

In vivo MR Spectroscopy
INTRODUCTION
This presentation details the methods and
applications of applying MR spectroscopy
(MRS) to living organisms, especially to
humans, for diagnosing pathological
processes or even predicting outcome of
therapeutic measures.
PRINCIPLES, REQUIREMENTS AND
RESTRICTIONS
The task of in vivo MRS is the detection of
metabolites of low concentrations within a
clinically feasible acquisition time. Given
typical metabolite concentrations in mM
range, four orders of magnitude lower than
the typical in vivo concentration of water, it
is obvious that in vivo MRS methods will
not reach the temporal or spatial resolution
achieved by MR imaging. Nevertheless, its
chemical profile allows for a specific tissue
characterization.
As a consequence of the detection limit,
in vivo MRS has a strong focus on detecting
the chemical shifts of protons of small
molecules such as choline containing
compounds, creatine, N-acetyl aspartate,
myo-inositol, glutamate, glutamine, lactate,
and a few others. The detection of 31P nuclei,
providing insight into the energy
metabolism of the living cell, is even more
In vivo MR Spectroscopy 77
7
Stefan Röll
demanding. While the concentrations of
many of the 31P-metabolites are similar to
those of molecules detectable by their
protons, the sensitivity of detection scales
with the third power of the gyromagnetic
ratio of the nucleus, i.e. 31P nuclei provide
less than 7 percent of the signal as the same
amount of 1H nuclei. The sensitivity for
detecting carbon is only 1.6 percent relative
to proton, and as only 13C is MR-visible
occurring with a natural abundance of
1.1 percent, the in vivo detection of natural
abundant 13C is at the borderline of clinical
feasibility. Apart from technically enhancing
SNR, e.g. by NOE and decoupling, clinical
MRS of enriched compounds or labelled
medication appears to be a possibility. The
same holds true for the detection of 19F or
7
Li containing compounds, the natural
concentrations of which are far below the
detection limit. However, while some of the
outlined methods are generally applicable,
this presentation focuses on 1H MRS.
The above mentioned small molecules are
detectible by 1H MRS in the liquid phase,
with T2 values of some hundred milliseconds
and linewidths of a couple of Hertz. In a
bound state, such molecules might only be
detected indirectly when exchanging
magnetization with protons in the liquid
state, via “magnetization transfer”
78 Biomedical Magnetic Resonance: Proceedings of the International Workshop
experiments. Larger macromolecules
exhibiting low T2 values become visible in
spectra as baseline modulations.
An expensive way of lowering the
detection limit of MRS is increasing field
strength. For clinical studies, field strengths
ranging from 0.2 up to 7 T have been used
today. Within this range, noise increases
approximately linearly with B0, while signal
amplitudes increase quadratically. However,
the expected linear net-increase in SNR can
be only observed if the same field
homogeneity is reached. As susceptibility
based distortions increase with increasing
B0, many areas of the human body show a
less homogeneous B0 at high B0 compared
to low B0 despite shimming. This is the major
source of signal loss at high fields. As T1
increases and T2 decreases with increasing
B0, there is also the tendency of relaxation
counter balancing field-dependent SNR gain;
however, the variation of in vivo relaxation
properties is minor for many metabolites of
interest for 1H MRS.l,2 Apart from SNR, the
other user-relevant quantity positively
affected by increased field-strength is
spectral resolution. The chemical shift
dispersion Δf of a spectral line with respect
to a reference line scales linearly with B0.
To allow for comparison of spectra obtained
from different B0 scanners, the ordinate of
spectra is usually scaled by the relative
dispersion with respect to B0, in parts per
million: Δf 106 / f0. So while the spectral
positions of two identical singlet lines
remain identical at B0 and 2B0, they appear
half as wide or twice as good separated at
2B0.
LOCALIZATION TECHNIQUES
For studying, e.g. calf muscle or tumors close
to the body surface, it may be sufficient to
use a small surface coil to excite and detect
signals from the superficial region of
interest. However, for reaching region of
interests within the body in brain, prostate
or breast MRS, additional localization by
slice selection or phase encoding is required.
With single “voxel” spectroscopy (SVS), a
single spectrum from a defined location is
acquired; chemical shift imaging (CSI) or
spectroscopic imaging (SI) refers to the
simultaneous acquisition of multiple voxels.
The location of the spectra is conveniently
planned on anatomic MR reference images,
acquired beforehand. While since the
introduction of MRS many localization
techniques have been invented and tested,
only the few most robust ones have
prevailed in clinical practice. Those are
described below.
Single Voxel Spectroscopy
The single voxel spectroscopy sequence
STEAM (stimulated echo acquisition mode)
detects the “stimulated echo” generated by
three consecutively applied RF-pulses of 90o
flip-angle each (Fig. 7.1).3,4 Each of the RF-
pulses is slice-selective. Since their
underlying gradient pulses are applied along
mutually orthogonal directions, these pulses
define a cuboidal subvolume, i.e. the voxel:
The first pulse selects a slice within the
sample, the second orthogonal pulse selects
a pencil-shaped cuboid from within the slice,
and the third pulse cuts the voxel out of the
pencil shape. However, the three pulses will
not only generate the desired stimulated
echo, but seven other “coherence pathways”
will be populated: FIDs generated by each
of the three pulses, and spin-echoes
generated by any two of the pulses need to
be eliminated since magnetization of those
signals has not experienced complete

Fig. 7.1: SVS STEAM sequence; spoiling gradient
pulses are shaded black, slice selection pulses are
in grey; TE: echo time, signal decay determined by
T2; TM: mixing time, signal decay determined by T1;
signal acquisition starts at formation of the stimulated
echo
localization. Two techniques are successfully
used: “Spoiling” gradients (shaded grey in
Figs 7.1 and 7.2) dephase the unwanted
signals during each acquisition. An often
critical issue is the complete spoiling of the
FID of the third pulse immediately before
acquisition. If more than one acquisition is
performed, than phase cycling can be used
as a second method for eliminating the
unwanted coherences. The phases of the
RF-pulses and data acquisition are for each
accumulation modified in such a way that
the unwanted echoes add de-constructively,
while the stimulated echo adds coherently.
At least eight phase cycles are required to
selected the desired echo of a three-pulse
sequence.
No gradients are applied during
acquisition of the stimulated echo, so that a
1-D Fourier-transform transforms the time-
domain data into the frequency-domain
spectrum. It is crucial to use precisely the
echo peak as the first sample of the time-
In vivo MR Spectroscopy 79
domain data. The amplitude of this sample
only depends on T 2 , while any signal
dephasing due to field inhomogeneity (T2*)
is rephased. Hence, the frequency domain
signal area which is correlated with
metabolite concentration becomes indepen-
dent of field homogeneity. Secondary
reasons for using the echo maximum as the
first time-domain sample are the well-
defined signal behavior of weakly coupled
signals such as lactate and that a first order
phase correction during data post-processing
is not needed.
These remarks on localization and signal
acquisition apply also to the spin-echo SVS
sequence, also called PRESS for (point
resolved spectroscopy). Whereas during the
STEAM sequence half of the originally
excited magnetization is dephased, the
90o – 180o – 180o spin-echo sequence (Fig.
7.2) utilizes the entire excited magnetization.
As today selection profiles of 90o and 180o
pulses are of virtually equal high quality,
and RF-heating is usually not an issue in
MRS due to long TR, the two-fold sensitivity
advantage makes the spin-echo sequence,
Fig. 7.2: SVS spin-echo sequence; spoiling gradient
pulses are shaded black, slice selection pulses are
in grey; TE: echo time, signal decay determined by
T2; signal acquisition starts at formation of the second
spin-echo
80 Biomedical Magnetic Resonance: Proceedings of the International Workshop
the sequence of choice compared to STEAM.
A minor additional advantage of the spin-
echo sequence is a lower sensitivity against
diffusion, due to shorter intervals between
spoiling gradient pulses. An advantage of
the STEAM sequence is shorter minimum
echo times during which magnetization
decays by T2; during the second and the
third RF-pulse of the STEAM sequence, i.e.
the “mixing period”, the magnetization of
the stimulated echo is stored as longitudinal
magnetization decaying only by T 1 .
Furthermore, some coupled resonances
show higher signals at intermediate echo-
times when detected by STEAM.5
When used for detection proton signals,
these SVS techniques are used in
combination with a water suppression
sequence module. In the brain the water
signal is 105 times more intense than typical
metabolite signals. Given that the dynamic
range of the receiver is sufficient, a smooth
water signal could be removed from the
spectrum in a post-processing step.
However, tiny modulations of the water
signal can appear as severe distortions with
respect to the metabolite signal scale. Hence,
the water signal is preferably suppressed
experimentally, usually by frequency-
selective excitation in combination with
dephasing. Among often used sequence
modules applied before localization are
CHESS and WET, and among those applied
during the sequence are MEGA and DRY-
STEAM.1
Chemical Shift Imaging
CSI sequences mostly rely on phase-encoding
as it is known from imaging sequences, for
resolving the metabolite concentrations from
multiple voxels. In a conventional CSI
experiment, the phase encoding gradients
are incremented with each acquisition, while
the readout period is again reserved solely
for resolving the spectroscopic dimension.
An extreme example is the 3D-CSI sequence
shown in Figure 7.3, achieving spatial
resolution along all spatial dimensions by
phase-encoding. The spatially resolved
spectra are obtained from the k-space data
by 4-D Fourier transform. Unlike the
position of a slice selection profile, the
position of a CSI-grid realized by phase-
encoding can be modified retrospectively
by fractions of the voxel size. In a 2D-CSI
sequence, one spatial dimension is selected
by a slice-selective RF-pulse, while only the
other two dimensions are phase-encoded.
To minimize the above mentioned field
dependent T2* signal loss, it is necessary to
minimize the delay TD between the center
of the pulse (when the echo maximum is
formed) and the start of the acquisition.
Such 2D- or 3D-CSI sequences of only a
short acquisition delay are especially useful
Fig. 7.3: CSI FID sequence; the slice selection pulses
is shaded grey; the delay TD, during which signal
decays by T 2 *, is minimized; the experiment is
repeated to realized each of the Nx Ny Nz phase-
encoding steps, assuming full k-space acquisition

to avoid heavy T2-weighting in non proton
applications where often T2-values are low.
Another strategy for minimizing T2* loss
in CSI is generating an echo. Adding phase
encoding to the SVS sequences during a
period when magnetization is transverse,
one arrives at hybrid 2D- or 3D-CSI
sequences. Those are successfully used
especially in brain 1H MRS. The volume
selected by the slice selective pulses covers
a region of interest of the inner brain which
is further resolved by phase encoding, while
the selection of intense subcutaneous or bone
lipid signals can be avoided. Another
advantage of hybrid CSI sequences is that
the FOV defined by phase-encoding can be
made small without the risk of signal
folding, as long as the selection profile is
sufficient to effectively suppress any outside
signals.
An important limitation of the usage of
slice selection pulses in SVS or hybrid-CSI
sequences is the chemical shift selection
artefact. As the spectroscopic signals precess
with different Larmor frequencies, thus
superimposing another frequency offset to
the one caused by the slice selection
gradient, they are excited at different
positions. Quantitatively, the position shift
of the slice center Δz for 2 signals of a
frequency difference Δf is given by
Δz = 2π Δf/γ Gslice
= 2π Δf tpulse slice_thickness/BWTP.
The frequency difference Δf depends
linearly on the field strength B0. To minimize
chemical shift displacement, one seeks to
maximize the slice selection gradient Gslice,
by designing pulses of a high band-width-
time product BWTP, and by realizing the
pulses with minimal pulse duration tpulse
using maximum transmitter voltage. The
latter can be achieved by using local transmit
In vivo MR Spectroscopy 81
coils. As a consequence of the effect, the
VOIs superimposed over the anatomical
images as shown in the graphical user
interface for positioning are only precisely
correct for one frequency offset or one
spectroscopic signal. It should be left to the
user of a spectroscopy sequence to decide
to which signal the precise positioning of
VOIs refers to; e.g. instead of using the
system resonance frequency which is usually
the one of the water signal, the user may
choose an spectroscopic intermediate signal
as e.g. the choline signal, to reach the
smallest average deviation for all signals of
interest. As a consequence of the chemical
selection artefact in hybrid CSI, the voxels
at the borderline of the VOI are not
completely excited, and may not be
quantitatively evaluated without further
measures (note however that the effect only
occurs with slice selection but not with
phase encoding, so that the CSI-grid is not
shifted!). One way of handling the effect is
numerical correction. Another possibility is
exciting a larger VOI than planned, and
avoiding the excitation of outer volumes by
saturation pulses which can be designed for
higher bandwidth than selection pulses. A
third possibility is the usage of spectral-
spatial selection pulses.
The minimal measurement time of a
conventional CSI experiment discussed so
far is given by the number of phase encoding
steps along each direction, times TR, i.e.
TA = Nx Ny Nz TR.
By reduced phase encoding covering
only an spherically or elliptically shaped
inner portion of k-space, the CSI
measurement time is significantly reduced.
This strategy is advantageous when e.g. a
spherically symmetric Hamming k-space
filter is applied for reducing voxel-bleeding
82 Biomedical Magnetic Resonance: Proceedings of the International Workshop
effects. Especially when low intensity
metabolite signals are surrounded by
disturbing signals of several orders of
magnitude higher intensity such as lipid
signals, it is desirable to control voxel-bleed-
ing, to avoid signal contamination of each
spectrum of the CSI dataset (Figs 7.4A to
D). The price to pay for reduced voxel-
bleeding is the concomitant reduction of
spatial resolution. The “effective voxel-size”
of filtered CSI data is approximately 1.5
times larger along each CSI-dimension than
the “nominal voxel-size” obtained by
dividing the FOV by the number of phase-
encoding steps, the precise value depending
on the shape of the filter function. However,
in the filtered case spatial resolution is
determined by the filter function and not
by the decision whether full or an elliptical
portion of k-space is phase-encoded. In this
case, the most efficient encoding technique
realizes the filter function during data
acquisition, to avoid attenuating outer
k-space during the filtering step and thus
wasting measurement time. One example of
Figs 7.4A to D: The effect of voxel bleeding: In the
case of k-space filtering by a spherical Hamming filter,
only citrate and choline signals from within the
phantom container are visible (A), the point-spread-
function shows only minor sidelobes (C), in the
unfiltered case; lipid signals from another outside
container appear (B), the point-spread-function
shows significant contributions originating from
outside areas
such a technique is acquisition weighting;
e.g. during an experiment of 4 averages,
only the innermost portion of k-space four
times while the outer portions are
acquired once, the number of averages
of intermediate k-space portions are
determined by the filter function. Then, for
same SNR of 3D-CSI experiment of 8 phase
encoding steps along each direction, NA =
4, TR = 1.5 s, requiring by conventional
encoding more than 50 minutes, is obtained
within less than 10 minutes.
The question whether to use a SVS or a
CSI technique depend among many factors
on the size and the spatial distribution of
the pathology. However, it can be generally
said that CSI techniques are more efficient
than SVS techniques since multiple voxels
are covered simultaneously, without a major
loss in sequence sensitivity; only inner voxel
dispersion causes a loss of CSI signal of a
few percent. However, whether a CSI
technique can be applied will depend
critically on whether a sufficient field
homogeneity can be reached over the larger
volume of interest.
Matrix Spectroscopy
Using multiple element receive “matrix”
coils, the sensitivity of clinical MRS can be
substantially increased. The development of
multi-channel hardware is on-going for 1H
detection, and it may be transferred for
detecting other nuclei. The problem of com-
bining the data from the single receive
elements is more involved for complex MRS
data than for magnitude imaging data, but
was described already by Roemer et al. 7
One arrives from the complex signals Si of
the coil elements i at a combined signal Scomb
by
Scomb = λ(r) Σi Wi(r) Si(r)

The purpose of the complex weighting
factors Wi is two-fold. They contain the
appropriate phase correction terms for
ensuring coherent complex signal addition
and avoiding signal cancellations.
Furthermore, they ensure the maximum SNR
of the combined data by an additional
weighting of the signals Si with their signal
intensities. This self-weighting property of
the combination algorithm leads to the
additional advantage that contributions of
low SNR data are virtually negligible, thus
facilitating the selection of coil elements.
After computing the sum term, a
normalization of signal amplitudes over all
locations is to be carried out in the case of
CSI data. Various choices of λ(r) have been
suggested. Data-inherent procedures well-
known from imaging such as sum-of-squares
normalization can be applied also to
spectroscopy. Even better homogeneity is
achieved when in a pre-scan imaging
experiment, the sensitivity profiles of the
selected coil elements are compared to the
profile of the large and homogeneous body
coil. Using this pre-scan data the CSI data
A B
Figs 7.5A and B: Upon coherent, appropriately weighted combination of each channel’s data, the multiple
array brain coil increases signal to noise especially of cortical voxels (A) as compared to a single-array head
coil (B)
In vivo MR Spectroscopy 83
can be normalized to the reception profile
of the body coil.
Figures 7.5A and B show an example
where the sensitivity of an MR exam of
cortical and white matter voxels was tripled
by using a 23 channel coil, as compared to
the usage of a single element receive coils.
The potential of clinical matrix 1H spectro-
scopy appears high when considering its
possible realization at very high field
strengths such as 3T or 7T.
Fast-CSI
Especially in the case of 3D CSI, the
conventional CSI experiment described
above may takes too long. The goal of fast
CSI techniques is to reduce the minimum
measurement time. With respect to sequence
sensitivity, i.e. the SNR reached per unit
measurement time, the conventional CSI
experiment serves as a benchmark. As
usually spectroscopic experiments are
limited by the need of reaching sufficient
SNR, fast techniques are hardly of practical
importance when sensitivity is sacrificed.6
84 Biomedical Magnetic Resonance: Proceedings of the International Workshop
On the other side, since e.g. matrix
spectroscopy enhances the sensitivity, fast
CSI matrix sequences not compromising with
sensitivity are useful, e.g. to buy spatial
resolution without prolonging acquisition
times.
Among the fast sequences8,9 which almost
reach the sensitivity of the conventional
sequence are those which use an echo-planar
readout module, designed to simultaneously
encode spectral and spatial dimensions
during signal acquisition (Fig. 7.6). Thus,
minimal measurement time of such an EPI-
CSI experiment is reduced compared to a
conventional CSI experiment by the number
Fig. 7.6: 2D CSI sequence with an EPI readout
module. If data is acquired during both, negative and
positive symmetric readout gradient lobes, the dwell-
time determining the spectral bandwidth is equal to
the duration of a single gradient lobe. Note that due
to the first half negative gradient lobe, signals
acquired in the center of the subsequent lobes have
experienced no gradient encoding as the effect of
positive and negative lobes cancel. For non-central
samples, one can substitute the effect of the readout
gradients by a phase encoding gradient applied
before readout; however, the samples acquired
during negative readout lobes reach the identical
k-space positions as during positive lobes in reverse
order. Hence, to process EPI-CSI data, an additional
time reversal of one half of the data is required
of phase encoding steps along one direction,
i.e. a typical factor of 16.
POST-PROCESSING
The task of MRS post-processing is to
evaluate at least relative metabolite
concentrations and to display results. It
appears hardly possible to device a general
purpose algorithm for absolute metabolite
quantitation, due to a number of parameters
usually not determined during a clinical
MRS exam (such as T1 and T2 values of
metabolite signal in pathological tissue), and
due to the fact that a ground truth for
verifying in vivo results is hardly available.
Hence, for each metabolite signal an
intensity value in arbitrary units is usually
obtained by a prior-knowledge based fitting
procedure, which can be compared to other
intensities of the same spectrum.
Quantitation of short TE 1H clinical spectra,
showing a large number of partially poorly
resolved signals and possibly artefactual
Fig. 7.7: Typical metabolite signals visible by short
TE brain MRS; signal properties are detailed, e.g.
in.10 Due to the multitude of partially overlapping
signals in the presence of a slowly varying baseline,
quantification of signal areas is challenging

lipid signals, is one of the most ambitious
problems of MRS post-processing (Fig. 7.7).
REFERENCES
1. R de Graaf. In vivo NMR spectroscopy Wiley,
Chichister, 1998.
2. T Ethofer, I Mader, U Seeger, A Ludolph, W
Grodd, U Klose. Comparison of metabolite T1
relaxation times in different brain regions at
1.5 and 3 Tesla. Proc. ISMRM, 2003;434.
3. EL Hahn. Spin echoes. Phys Rev. 1950;80(4):
580-94.
4. J Frahm, KD Merboldt, W Hänike. Localized
proton spectroscopy using stimulated echoes.
J Magn Reson 1987;72:502-508.
5. J Hennig. Coupling effects in volume selective
1H spectroscopy of major brain metabolites.
Magn Reson Med 1991;21:82-96.
6. R Pohmann, MV Kienlin, A Haase. Theoretical
evaluation and comparison of fast chemical
shift imaging methods. J Magn Reson
1997;129:145-60.
In vivo MR Spectroscopy 85
7. P Roemer, W Edelstein, C Hayes, S Souza, O
Mueller. The NMR phased array. Magn Reson
Med 1990;16:192-225.
8. J Scheidler, H Hricak, D Vigneron, K Yu, D
Sokolov, L Huang, C Zaloudek, S Nelson, P
Carroll, J Kurhanewicz. Prostate cancer:
Localization with three-dimensional proton MR
spectroscopic imaging—clinicopathologic study.
Radiology 1999;213:473-80.
9. T Scheenen, D Klomp, S Röll, J Fütterer, J
Barentsz, A Heerschap. Fast acquisition-
weighted three-dimensional proton MR
spectroscopic imaging of the human prostate.
Magn Reson Med 2004;52:80-88.
10. V Govindaraju, K Young, A Maudsley. Proton
NMR chemical shifts and coupling constants
for brain metabolites NMR Biomed 2000;
13:129-53.
11. S Neubauer. High-energy phosphate meta-
bolism in normal, hypertrophied and failing
human myocardium Heart Failure Rev 1999;
4:269-80.
86 Biomedical Magnetic Resonance: Proceedings of the International Workshop
Multi-dimensional NMR
Spectroscopy and Editing in vivo
M Albert Thomas, Nader Binesh, Kenneth Yue, Hyun-kyung Chung,
Steven Han, N DeBruhi, A Aude, Anand Kumar
INTRODUCTION
The NMR spectral resonances due to methyl,
methylene and methine protons of
N-acetyl aspartate (NAA), glutamate/
glutamine (Glx), creatine (Cr), choline (Ch),
myoinositol (mI), GABA, aspartate (Asp),
N-acetyl aspartyl glutamate (NAAG) and
other metabolites have been clearly
identified in human brain using three-
dimensional (3D) localized, water-
suppressed one-dimensional (1D) 1H MR
spectra recorded in human tissues. 1-10
Spectral overlap of macromolecules, GABA
and glutathione with the methyl resonances
of NAA, Cr, NAAG and methylene
resonances of glutamate/glutamine has also
been reported.7,11-12 Attempts have been
made to quantify glutamine and glutamate,13
glucose,14 and other metabolites with only
minimal success due to the difficulty in
extracting this information from a region
with many overlapping resonances. At low
magnetic fields such as 1.5 T MRI, it is diffi-
cult to resolve multitude of peaks existing
over a small spectral range of approximately
300 Hz. Using spectral-editing techniques,
it is possible to select a particular J-coupled
metabolite. 11,15-18 However, one major
8
drawback of editing techniques is that only
one metabolite is selected at a time assuming
that the multiplets of the J-coupled meta-
bolites are well separated. Using the LC-
model postprocessing algorithm with a
basis-set of 10-20 different metabolite
spectra in vitro, quantification of several
metabolites in human brain has been
reported recently.19-21
In contrary to the spectral editing
techniques which optimize one metabolite
at a time, 2D MRS can unambiguously
resolve many overlapping peaks non-
selectively as demonstrated by Ernst and
co-workers two decades ago. 22 Better
dispersion of several metabolite peaks and
improved spectral assignment make the
proposed technique more attractive. It now
seems natural to explore the clinical
potentials of 2D MRS techniques of human
tissues in vivo. 2D MR spectroscopy
facilitates converting a crowded, over-
lapping 1D MR spectrum to a better
resolved 2D spectrum through the addition
of a spectral dimension. Instead of a
standard 1D spectrum plotting intensity
versus a single-axis (i.e., chemical shift), 2D
NMR spectroscopy techniques produce a 2D
 Multi-dimensional NMR Spectroscopy and Editing in vivo
spectrum plotting intensity versus two axes,
the dimensions of which depend on the
specific 2D NMR technique.22
There have been several attempts during
the last fifteen years in implementation and
evaluation of 2D NMR spectroscopy on the
MRI scanners and NMR spectrometers.
Sotak et al made use of 2D-zero-quantum
(ZQ) spectra on a 2 T scanner to visualize
the lactic acid resonances in the presence of
interfering lipid resonances in phantom
models and mouse tumors.23 Crozier et al
used a multiple quantum edited 2D sequence
to visualize the excitatory neurotransmitters
glutamine and glutamate in phantom and
rat brain, although this work was carried
out on a 4.7 T spectrometer.24 Other high-
resolution spectrometer work has been
focused toward spatially localized 2D
studies and in vivo studies.25-32 Desmoulin
and Seelig showed a localized SECSY study
of a rat.25 van Zijl et al recently utilized 2D
{ 1 H- 13C}- spectroscopy to visualize 13C-
labeled glucose, glutamine, glutamate, and
lactate in the cat brain in vivo.26 Other recent
studies have attempted 2D DQ spectroscopy
in human tissues.33-34 Different versions of
the localized COSY sequence have also been
implemented previously.35-45 McKinnon and
Bosiger proposed a conventional COSY
sequence with hard RF pulses (90°-t1-90°)
followed by three volume selective 180° RF
pulses. 35 Haase and co-workers
implemented a COSY combined with an
outer volume suppressing sequence, namely
LOCUS. 36 Many previous attempts to
develop localized 2D COSY spectra yielded
only the phantom results35-38 or, rat brain
spectra using a surface coil without a built-
in localization sequence.46-48 A new gradient
enhanced COSY in combination with VOSY
used the STEAM sequence for volume
87
localization. 49 A human brain 2D COSY
spectrum using a 2 T MRI scanner was
recorded in a gross occipital volume of 5 ×
6 × 8 cm3 which required in a total sampling
duration of 1 hour and 42 minutes. Homo-
nuclear decoupled in vivo 1H MR spectra
using constant time chemical shift encoding
were presented by Leibfritz and co-
workers.50 Non-localized versions of COSY
spectra have also been recorded in rat brain
and rabbit kidney by other researchers using
the high field NMR spectrometers.51-52
A major goal of this presentation is to
give an overview of recent progress with
the implementation of several localized 2D
MRS sequences on the whole body 1.5 T
and 3 T MRI scanners.
SPATIALLY RESOLVED MULTI-DIMEN-
SIONAL 1H MR SPECTROSCOPY AND
EDITING
Single-and Multi-voxel-based One-
dimensional 1H MR Spectroscopy
High concentration of water in biological
tissue makes MR imaging quite sensitive.
However, this leads to a major problem in
MR spectroscopy since the metabolites are
tens of thousands of times lower in
concentration than water. Thus, in order to
obtain an acceptable spectral quality from
the metabolites, MRS techniques must
incorporate ways of minimizing or
eliminating the water signals. Other basic
challenges in clinical 1D MR spectroscopy
are lipid suppression and optimal spatial
localization of the volume of interest (VOI)
including single versus multiple volume
elements (voxel).
First, water suppression can be extended
from changes in hardware for data
acquisition (digitizers), pulse sequences, to
88 Biomedical Magnetic Resonance: Proceedings of the International Workshop
postprocessing software. The primary
contribution stems from an addition of three
frequency selective (water as the target
frequency) radiofrequency (RF) pulses with
arbitrary flip-angles followed by a dephasing
gradient to the pulse sequence. These pulses
are commonly called the CHESS (chemical
shift selective saturation) pulses.4,53 Instead
of CHESS water saturation, an inversion
recovery scheme can also be employed using
a 180° frequency selective pulse on water
such that excitation of the metabolites can
be prolonged until the crossing of the water
signal at the null point with known T1
values.54 Second, quite often the lipid signals
from either within the volume of interest
or outside of the volume of interest hold a
similar challenge like water as described
above. In order to suppress the lipids, the
same two approaches utilized in water
suppression in terms of pulse sequence
modification can also be applied here.55 The
most common approach is the use of
frequency selective (targeted at lipids)
pulses in conjunction with spatial selective
around the edges of the brain called the
outer volume saturation (OVS) slabs. Of
course, post-processing software can also
contribute to elimination of lipid signals for
a more accurate spectrum.
There is a fundamental difference in
which localization is done in imaging and
spectroscopy. Imaging relies on acquisition
of the entire proton signal (primarily water
and fat) and the differences in frequencies
of the signal are attributed to the spatial
shifts introduced via the application of the
frequency encoding gradient during
sampling to reproduce the selected slice
within a plane. A frequency encoding
gradient cannot be used in spectroscopy
during sampling because the differences in
frequencies of metabolites sought after are
due to the small chemical shifts and a
frequency encoding gradient would corrupt
this basic information. However, the
demand for volume definition remains
whether single voxel or multiple voxel
spectroscopy is done since the metabolite
concentration information is sought from a
particular voxel and not the whole brain.
The most common localization procedure is
the use of slice selection and phase encoding
gradients. These serve to demarcate the
dimensions of the voxel and are operated
for a very short duration. The spins then
return to the influence of the external
magnetic field.
The two most popular pulse sequences
for defining a small (ranging from 1 cm3 to
8 cm 3 ) volume within the anatomy are
termed as PRESS and STEAM.2,4,56 Both the
techniques use frequency selective RF pulses
along with magnetic field gradients to isolate
a volume; however, the timing and the
sequence of the pulses has consequences on
the sensitivity and the strength of the
signal.57 Stimulated Echo Acquisition Mode
or STEAM also uses the frequency selective
90 o RF pulses in conjunction with the
magnetic field gradients (90oss). The first
90 oss along with the negative lobe for
selection of a slice is identical to the PRESS
sequence. However, after a duration TE/2,
the spins from a column of the chosen slice
are returned to the longitudinal direction
by the application of another 90oss in an
orthogonal plane. These spins with both the
RF pulses now remain in the longitudinal
direction for a period called the mixing time,
TM. The last 90 o ss is applied in the
remaining orthogonal direction after TM.
After an equivalent amount of time, i.e.,
TE/2, there is the formation of a stimulated
 Multi-dimensional NMR Spectroscopy and Editing in vivo
echo from the desired volume. This echo is
different from the “race-track” echo analogy
of the spin echo. This stimulated echo
corresponds to the analogy of “pancake”
echo.57
Point Resolved Spectroscopy or PRESS
(90oss-180oss-180oss) involves the selection
of a slice from a given volume with the use
of a frequency selective 90o RF pulse in
conjunction with a magnetic field gradient
(90oss). However, the use of the gradient
dephases the spins from the chosen slice
which are now in the transverse plane.2,57
Hence, a negative lobe is added immediately
after the RF pulse to rephrase the phase-
scrambled magnetization during this
excitation. The FID produced from this slice
is ignored since the objective is to elicit
signals from a small voxel within the slice.
After a duration of TE 1/2, a frequency
selective 180o RF pulse with a magnetic field
gradient (180oss) in a plane orthogonal to
that of 90oss is used. This creates an echo
Fig. 8.1: 1D MRS protocol including water and fat suppression and signal acquisition
89
from the column chosen by the two RF
pulses at a time TE1/2 after the 180o pulse.
This echo is echoed again by another 180oss
pulse in the remaining orthogonal plane and
the second echo is actually from the desired
volume which is acquired and processed.57
Hence, PRESS is nothing but a double-spine
echo based MRS sequence. Figure 8.1
summarizes different protocols including
both single- and multi-voxel localized MRS
including global CHESS water suppression
and OVS. The single-voxel based STEAM
and PRESS are denoted in Figure 8.1 as
STEAMSV and PRESSSV.
Spectroscopic imaging (SI), also called
chemical shift imaging (CSI), enables
distilling both chemical shift as well as spatial
information from the acquired signal.58-59
Because of the latter challenge, it has several
similarities in mode of data acquisition with
MR imaging like the use of phase encoding
gradients. However, the data processing in
this case requires both spatial and time
90 Biomedical Magnetic Resonance: Proceedings of the International Workshop
Fourier transforms to decode the
information. The PRESSCSI and STEAMCSI
sequences in Figure 8.1 can be used where
the basic PRESS and STEAM sequences
remain the same; however, the field
gradients are modified for spatial encoding.
The first slice selection gradient along with
the RF pulse is the same but spatial
information along the two dimensions of
the chosen slice are encoded via phase
encoding gradients. Phase encoding
gradients do not change the frequency of
the spins in the received signal since they
are not on during signal acquisition. Instead,
they serve to momentarily change the phase
of the spins. The spins return to the same
frequency under the influence of the external
magnetic field after the gradients are
switched off but retain the phase memory
difference relative to the spatial dimension.
These phase encoding gradients are repeated
several times between the remainder of the
RF pulses each time repeating the entire
sequence but with the phase encoding
gradient changing slightly. This successive
change is done in a stepwise fashion with
the gradient amplitude changing thus;
several samples of a frequency are
produced. The data can then be Fourier
transformed to allocate signal intensities of
different frequencies to appropriate voxels.
Chemical Shift Correlated
MR Spectroscopy
For MR spectroscopy of a particular nucleus,
namely hydrogen (1H), one counts on the
differences in internal spin interactions which
are due to differing inter and intramolecular
environments. These differences in the
molecular environment are of several types
and are hence, responsible for different
features of a spectrum or an image. The
chemical shift expresses the local electronic
environment and is predominantly an intra-
molecular interaction, Molecular motion also
influences this shift significantly. Thus, a
primary manifestation and measure of the
chemical bond between the nuclear spins,
i.e., the nuclear-nuclear coupling via the
bonding electrons, is the exclusively intra-
molecular J-coupling, which is independent
of the applied magnetic field. Exploitation
of this interaction is the crucial link to the
molecular chemistry of the compound under
study. A map correlating the peaks that
belong together in the same spin system is
quite important and a 2D spectrum provides
such a map. Two-dimensional NMR
spectroscopy enables converting a crowded,
overlapping 1D MR spectrum to a better-
resolved 2D spectrum through the addition
of a spectral dimension.22 The problems due
to the overlap in the 1D spectrum
introduced by the multiplicity of various
peaks due to J-coupling could be removed
by separating the interactions due to
chemical shift and indirect spin-spin coupling
(J) along the two dimensions in 2D MR
spectroscopy. Instead of a standard 1D
spectrum plotting intensity versus a
singleaxis (i.e., chemical shift), 2D NMR
spectroscopy techniques produce a 2D
spectrum plotting intensity versus two axes,
the dimensions of which depend on the
specific 2D NMR technique.
Before applying these multi-dimensional
MR techniques in human tissues, each
sequence needs to be converted first into
localizing a volume of interest (VOI) using
three orthogonal slice-localizing RF pulses.
A second task is to minimize the number of
RF pulses essential for creation of Hahn’s
spin-echo or coherence transfer echoes.
Shown in Figure 8.2 are different modules
 Multi-dimensional NMR Spectroscopy and Editing in vivo
of 2D MRS protocol. Similar to 1D MRS,
global water suppression by CHESS and fat
suppression using OVS can be performed
prior to volume localized 2D NMR. In
addition to repeated spectral acquisition for
signal averaging, the sequence has to be
repeated multiple times for encoding the
second spectral dimension.
L-COSY
Figure 8.3 depicts a localized two-
dimensional correlated spectroscopy (L-
COSY) sequence, a double-echo based
(coherence-transfer-echo and spin-echo).60,61
The VOI was localized in one shot by a
combination of three slice-selective RF pulses
(90o-180o-90o), a single volume localizing MRS
sequence called CABINET.60 In contrast to
the commonly used PRESS sequence (90o-
180o-180o) with 100 percent efficiency, the
Fig. 8.2: 2D MRS protocol including volume localization, 2D spectral sampling, etc.
91
CABINET sequence retains only 50 percent
net signal from the localized volume due to
a selection of only N-type echo enabled by
the B o gradient crusher pulses. 60 A 50
percent efficiency of the CABINET sequence,
same as STEAM, was confirmed with the
simulated 1D spectra of selected metabolites
using GAMMA library.63 While using the
inhomogeneous RF pulses delivered by a
surface coil or other types, one has to
consider the influence of flip-angle errors.
In the CABINET sequence with the flip
angle distribution of (ϕo, 2ϕo, ϕo), the total
signal amplitude from the VOI will be scaled
by a factor of sin4ϕ. The maximum
attenuation coefficient for the PRESS and
STEAM sequences will be sin5ϕ and sin3ϕ,
respectively. The last slice-selective 90o RF
pulse acted also as a coherence transfer pulse
for the 2D spectrum. An incremental period
92 Biomedical Magnetic Resonance: Proceedings of the International Workshop
Fig. 8.3: 2D L-COSY sequence
(t1) for the second dimension was inserted
immediately after the formation of the Hahn
spin-echo. In addition to the slice-selective
180o RF pulse, there were refocusing B o
gradient crusher pulses before and after the
last 90o RF pulses. Each time point of the
pulse sequence is marked as shown in Figure
8.1 to evaluate the time course of
magnetization that will take into account
the rotation after each RF pulse and
evolution during the Bo gradient pulses and
the incremental period.
A theoretical analysis to calculate the
amplitudes and phases of L-COSY diagonal
and cross peaks in a weakly coupled two
spin (I=1/2, S=1/2) system is discussed here.
A product operator formalism was used to
evaluate the time course of the magneti-
zation. 22,60 The amplitude and phase
characteristics at different time points of an
L-COSY spectrum are derived here. Prior
to the first 90o RF pulse, the spin-state is
explained by
σo ∝ Iz ...(1)
where I is the spin under consideration and
S is its J-coupled partner. After the rotation
by the first 90o RF pulse, the spin-state is
explained by
σ1 ∝ Iy ...(2)
After an evolution during the interval of
2Δ, the spins evolve under the influence of
J coupling, but the chemical shift will be
refocused resulting in an echo characterized
by
σ2 ∝ Iy cos(2πJΔ) – 2IxSzsin (2πJΔ) ...(3)
The spin-state (σ 2) after the evolution
due to both chemical shift (ω1(I)) and J-
coupling with its coupled partner during
the t 1 evolution can be calculated as
discussed earlier.60
A second pair of Bo gradient crusher
pulses was transmitted around the last slice-
selective 90o RF pulse. The spin state after
an evolution during the last set of B o
gradient crushers,
σ5 ∝ -0.5 cos(2πJΔ) [Iycos (ω1(I)t1) cos(πJt1) +
Ixsin (ω1(I)t1) cos (πJt1)+ 2IzSxcos (ω1(I)t1) sin
(πJt1)-2IzSy sin (ω1(I)t1) sin(πJt1)] + 0.5 sin
(2πJΔ) [Iycos (ω1(I)t1) sin(πJt1) + Ix sin (ω1(I)t1)
sin (πJt1) - 2IzSxcos (ω1(I)t1) cos (πJt1)+2IzSy
sin (ω1(I)t1) cos(πJt1)] ...(4)
Eqn. (4) was calculated under the
assumption that the gradient crushers after
the last sliceselective 90o RF pulse. It is also
evident from Eqn. [4] that the coherence
transfer from spin I to S is characterized by
2IzSx and 2IzSy. The 2D signal acquired after
the last crusher gradients is given by,
s(t1, t2) = Tr {(Ix) σ5} exp(-iω2 (I)t2) exp(-t1/
T2) exp (-t2/T2) [1-exp (-TR/T1)] ...(5)
A similar equation can also be calculated
for S spin resulting in a coherence transfer
to I spin. A double Fourier transformation
along both t1 and t2 axes will result in a
two-dimensional MR spectrum as a function
of two frequency variables (F1, F2) described
by,
S(F1,F2) =   s(t1,t2) dt1dt2 ...(6)
Fourier transformation of the standard
acquisition dimension converts each FID to
 Multi-dimensional NMR Spectroscopy and Editing in vivo
a frequency spectrum. The 2D data set
S(t1,t2) becomes S(t1,F2). A second Fourier
transform applied to the data along the
incremented echo time dimension t1 converts
the oscillating phases of each of the coupled
metabolite peaks to their respective
frequency components. The 2D data set
S(t1,F2) then becomes S(F1, F2). The final 2D
spectral matrix has the information of both
chemical shift and J-coupling along the F2
dimension and only J-coupling along the F1
dimension. Ernst and co-workers reported
that the diagonal peaks were dispersive
where as the cross peaks were absorptive
when two hard 90o RF pulses were used to
acquire the basic 2-pulse COSY spectrum.62
One drawback of including the gradient
pulses is due to the mixed line shapes in the
2D NMR spectra.60 Eqn. [4] clearly indicates
that both the diagonal and cross peaks of
an L-COSY spectrum have mixed phases
along the F 1 axis. In contrast to the
amplitude modulation in conventional COSY,
the phase modulation in L-COSY is caused
by the evolution during the Bo gradient
pulse before the last 90o RF pulse.60 Pure
phase L-COSY spectrum can be recorded
using a quadrature detection method
along the F1 axis described by Doddrell and
co-workers41 that will require two separate
P- and N- type spectral acquisition and
recombination of the two data sets.
Relaxation during the gradient pulses before
the last 90o RF pulse will cause further losses
in signal intensities.
The L-COSY sequence has been
implemented on the GE (General Electric
Healthcare Technologies, Waukesha, WI)
and Siemens (Siemens Medical Systems,
Erlangen, Germany) 1.5 T and 3 T MRI
scanners. Shown in Figure 8.4 (Plate 1) is a
2D L-COSY spectrum recorded in a grey
93
matter brain phantom using a Siemens 3 T
MRI scanner. The brain phantom had several
metabolites at physiological concentrations
as shown below: 8.9 mM NAA, 0.7 mM
GABA, 2.1 mM Asp, 0.9 mM Ch, 7 mM Cr,
1 mM Glc, 12.5 mM Glu, 2.5 mM Gln, 2 mM
GSH, 4.4 mM mI, 0.4 mM Lac, 3.6 mM PCr,
0.6 mM PCh, 1.8 mM Tau, 0.3 mM Thr,
1 mM PE and 1 mM DSS in a phosphate
buffer solution to maintain pH of 7.2. 2D L-
COSY spectra were recorded using the
following parameters: 27 ml voxel, TE = 20-
30 ms, TR = 2s, total number of scans = 768
(96 Δt1 increments and 8NEX/Δt1). The total
duration for each 2D scan with water
suppression was approximately 24 minutes.
Before zero-filling to (2048 × 256), the 2D
raw data was apodized using skewed
squared sine-bell filter along t2 and squared
sine-bell filter along t1. The resultant 2D
spectrum was displayed in the magnitude
mode.
L-SECSY
Three-dimensional (3D) localized spin-echo
correlated spectroscopic (L-SECSY) was
derived from the basic SECSY originally
reported by Nagayama et al.64 Modification
of L-COSY by inserting equal incremental
periods before and after the last 90o slice-
selective RF pulse in Figure 8.3 results in L-
SECSY. The diagonal peaks of L-SECSY lie
on a line at F1 = 0 and the cross peaks are
symmetrically disposed on both sides of the
diagonal. Similar to L-COSY, the singlet
resonances appear on the diagonal. The cross
peaks can be identified on a line cutting
through the diagonal with an angle of 45o.
Shown in Figure 8.5 (Plate 1) is a 2D L-
SECSY spectrum of a gray matter brain
phantom, same as the one used for L-COSY.
A Siemens 3 T MRI scanner and the
94 Biomedical Magnetic Resonance: Proceedings of the International Workshop
following parameters were used: 27 ml
voxel, TE = 20 ms, TR = 2 s, 1024 complex
points along t2, total number of scans = 1024
(64 Δt 1 increments and 16NEX/Δt1). The
total duration for each 2D scan with water
suppression was approximately 34 minutes.
The 2D raw data was apodized with squared
sine-bell filters along both the dimension.
The resultant 2D spectrum was displayed
in the magnitude mode.
A major advantage of L-SECSY over L-
COSY is that a smaller F1 size is needed
since there is a partial refocusing of chemical
shift along F 1. Hence, the number of t1
increments can be smaller than that used
for L-COSY. However, the T2 losses are
more severe in L-SECSY due to the extended
t1 evolution.
Multiple-quantum (MQ) Filtered
COSY and SECSY
Single-quantum (SQ) resonance of water
protons and SQ coherences of metabolite
protons are used in conventional MRI and
MRS, respectively. Multiple-quantum (MQ)
coherence based MRS has been used to select
a particular J-coupled metabolite in human
tissues in vivo. In particular, lactate-editing
double-quantum (DQ) and zero-quantum
(ZQ) MRS techniques were proposed by
Sotak et al and Doddrell and co-workers.23,24
Ryner et al implemented the first volume
localized DQ- and ZQ-filtered COSY and
SECSY sequences on a 1.5 T MRI scanner.65
In contrast to the dispersive phase characte-
ristics of the basic COSY diagonal peaks,
the double-quantum filtered COSY offers
superior resolution of the multiplets adjacent
to the diagonal peaks.22 Slightly modified
single-volume localizing DQ-filtered COSY
and SECSY sequences were implemented
recently using the following sequence:
(90°SS-TE/2-t1-90°SS-TM-90°SS-TE/2-k*t1-t2)
...(6)
The TE-crushers and TM crushers were
distributed using the following values: 4, 3
and 10 as suggested by Shaw et al.66 DQ-
filtered COSY was implemented using k=0.
A DQ-filtered COSY of a gray matter brain
phantom is shown in Figure 8.6 (Plate 1). A
Siemens 3 T Trio MRI scanner with the body
RF coil transmit and a circularly polarized
extremity receive coil. No water suppression
was enabled and the following parameters
were used: 27 ml voxel, TR/TE = 2 s/20
ms, TM = 6 ms, 8 NEX/t 1 and 96 t 1 .
Compared to L-COSY, the singlets due to
the N-methyl protons of NAA and Cr, N-
methylene protons of Cr and tri-methyl
protons Ch were clearly eliminated as
expected and the multiplets adjacent to the
diagonal were better resolved.
DQ-filtered SECSY was implemented
using k = 1 to split the evolution period,
first half before and the second half after
the last 90os pulse. A DQ-filtered SECSY of
a gray matter brain phantom using a Siemens
3T Trio MRI scanner with the body RF coil
transmit and a circularly polarized extremity
receive coil is shown in Figure 8.7 (Plate 1).
Again, no water suppression and the
following parameters were used: 27 ml
voxel, TR/TE = 2 s/20 ms, TM = 6 ms, 8
NEX/t1 and 96t1. Compared to L-SECSY,
the singlets due to the N-methyl protons of
NAA and Cr, N-methylene protons of Cr
and tri-methyl protons Ch were clearly
eliminated and the multiplets adjacent to
the diagonal were better resolved.
ZQ-filtered COSY and SECSY can also
be easily derived from the DQ-filtered
COSY/SECSY sequences by modifying the
amplitudes of TE- and TM-crushers. One
drawback of ZQ-filtered COSY/SECSY is due
 Multi-dimensional NMR Spectroscopy and Editing in vivo
to an inability in differentiating ZQ
coherences from the longitudinal
magnetization during TM resulting in full
return of water and singlet peaks in the
filtered 2D spectra. Even though MQ filtered
COSY/SECSY enable a semi-selective
spectral editing, a major concern is due to
the signal losses. The first two 90° RF pulses
excite all the MQ coherences (ZQ, SQ, DQ,
etc.) and there is a severe loss of signal due
to the selection of either ZQ or DQ
coherences in the 2D ZQ-filtered and the
2D DQ-filtered COSY/SECSY sequences.
Compared to the localized 2D J-resolved
sequence, there is a minimum of 50 percent
signal loss due to the third 90° RF pulse.
When the gradient pulses are used to
enhance the desired MQ pathway, there is
an additional loss of signal due to the loss
of certain pathways. Consequently, this
leads to severe losses of signals that is
evident in the DQ-filtered COSY/SECSY
shown in Figure 8.6 and Figure 8.7 compared
to L-COSY and L-SECSY.
Exchange 1H MR Spectroscopy
(EXSY) in vivo
In addition to three RF pulses necessary for
a single volume localizing STEAM or PRESS
sequence, either an off-resonance/
continuous wave (CW) saturation or
selective inversion RF pulse is essential in
the 1D 1H MR exchange spectroscopy.67-72
A localized two-dimensional 1 H MR
chemical exchange spectroscopic (L-EXSY)
sequence has been implemented using three
slice-selective 90° RF pulses (90oSS-TE/2-t1-
90 o SS -TM-90 o SS -TE/2-t2). 73 The second
spectroscopic encoding during t1 to monitor
the chemical exchange was an integral part
of the single-volume localization, thereby
eliminating a need for any additional RF
95
pulses. Even though single and higher order
multiple quantum coherences can be
dephased using the TM-crusher, the zero-
quantum coherences are indistinguishable
from the longitudinal magnetization leading
to J coupled 2D cross peaks similar to that
of COSY. With a fixed TM, the magnetization
exchange can be detected after the last 90°
pulse during the detection period (t2). The
experiment is repeated with equidistant
incremental period (t1) and the resulting 2D
raw data matrix is given by s(t1, TM, t2). A
double Fourier transformation yields a
frequency encoded 2D data matrix, S(F1,
TM, F2). Considering two chemical species
A and B, the magnetization precessing with
a frequency of δA during the evolution time
(t1) is transferred to δB during the detection
time (t 2 ). Exchange of magnetization is
manifested by cross peaks in the 2D
spectrum at the following frequencies (F1 =
δA, F2 = δB) and (F1 = δB, F2 = δA). Non-
exchanging magnetization components
appear at the following frequencies on the
diagonal (F1 = F2).
Two-dimensional L-EXSY sequence has
been implemented on both GE and Siemens
3 T and 1.5 T MRI/MRS scanners recently.73
Figure 8.8 (Plate 2) is a composite 2D L-
EXSY spectrum using TM = 7.5 ms recorded
from a corn oil phantom containing
approximately 82 percent poly- and mono-
unsaturated, and 14 percent saturated
triglycerides and a muscle phantom
containing 10 mM Cr, 10 mM PCr, 3 mM
Ch, 3 mM phosphocholine (PCh) and 10 mM
carnosine (Car) with pH=7.2. The following
parameters were used to acquire the 2D L-
EXSY spectra: TR = 2 s, TE min = 22 ms, Δt1
= 1.6 ms, Δt2 = 0.4 ms, 1024 complex points
along t2 and 64 along t1, and 8 number of
excitations (NEX) per Δt1. Following were
96 Biomedical Magnetic Resonance: Proceedings of the International Workshop
the dominant diagonal peaks (F2 = F1): (i) the
oil phantom: the olefinic protons at 5.4 ppm,
the poly-methylene protons (CH2)n at 1.4
ppm and methyl protons at 1.0 ppm. (ii) the
muscle phantom: (i) the residual water at
4.8 ppm, the methyl and methylene protons
of Cr/PCr at 3.0 ppm and 3.9 ppm, tri-
methly protons of Ch/PCh) at 3.2 ppm and
imidazole protons of Car at 7.0 ppm and 8
ppm, respectively. Two pairs of cross peaks
were identified at the following locations:
(F2 = 5.4 ppm, F1 = 2.9 ppm), (F2 = 2.9 ppm,
F1 = 5.4 ppm), (F2 = 5.4 ppm, F1 = 2.2 ppm),
and (F2 = 2.2 ppm, F1 = 5.4 ppm). The first
two cross peaks are due to the indirect spin-
spin (J) coupling between the methylene
protons connecting olefinic groups and
olefinic protons in polyunsaturated fatty acyl
chains. 74 The last two cross peaks were
possibly due to J-coupling between CH2 next
to an olefinic group and the olefinic
protons.75 In agreement with the assignments
reported by Szczepaniak et al,76 the diagonal
peaks of glycerol backbone protons were
also identified at (F2 = F1 = 4.3 ppm), (F2 =
F1 = 5.3 ppm), and the cross peaks (F2 = 5.3
ppm, F1 = 4.3 ppm), (F2 = 4.5 ppm, F1 = 4.0
ppm), (F2 = 4.3 ppm, F1 = 5.3 ppm) and (F2
= 4.0 ppm, F1 = 4.5 ppm). The diagonal and
cross peaks of the methylene and methyl
protons of saturated fatty acids overlapped
with unsaturated fatty acids in the aliphatic
region (0.9 - 4 ppm). In the 2D spectra of
the muscle phantom, there were also weak
cross peaks between the imidazole and
aliphatic protons of carnosine.
A 2D L-EXSY spectrum recorded in the
soleus muscle of a healthy volunteer is
shown in Figure 8.9 (Plate 2) using TM of
300 ms. Two different exchange cross peaks
were recorded in human calf muscle: first
peak, between the mobile tissue water and
total creatine pools, and a second peak,
possibly between the olefinic and
magnetically equivalent polymethylene
protons of unsaturated lipids. These two
peaks are marked as 1 and 2 in Figure 8.9.
In the J-coupled metabolites and lipids,
coherent magnetization transfer leads to
COSY-type cross peaks. At TM = 300 ms,
these cross peaks were absent. As explained
two decades ago, dipolar interactions
between neighboring protons of different
molecular species modulated by random
tumbling leads to cross relaxation and
magnetization transfer between the protons
of different species.22 A non-equilibrium
state between the magnetically coupled
water and metabolites is created
immediately after the second 90oss pulse in
the 2D L-EXSY sequence. A 2D L-EXSY
spectrum records transfer of magnetization
within protons of one molecule or between
two different molecular species effected by
the intra- and inter-molecular magnetization
exchange mechanisms. It was also reported
earlier that magnetization may be exchanged
among molecular species by coherent and
incoherent transfer processes.22 The build
up rates of 2D L-EXSY peaks have been
further investigated in human muscle after
recording several 2D spectra with TMs
varying between 100 ms and 2 s.
CLINICAL APPLICATIONS OF 2D L-COSY
In the following section, our recent findings
on three clinical applications of 2D L-COSY
in i) a liver disorder and a neurological
disorder, namely late-life major depression,
and breast cancer in vivo will be discussed.77-
79 Two-dimensional L-COSY spectra of these
three patient cohorts were recorded using
a GE 1.5 T MRI scanner (GE Healthcare
Technologies, Waukesha, WI) equipped with
echo-speed-gradients operating at a slew
 Multi-dimensional NMR Spectroscopy and Editing in vivo
rate of 120 T/m/s. A body RF coil was
employed for RF transmission, and a single
3” surface coil in the first two groups and
a phased-array breast coil for reception in
the third group involving breast cancer
patients.
Hepatic Encephalopathy (HE)
Clinical features of HE range from subtle
changes in sleep patterns, anxiety, and
personality changes to profound cognitive
impairment, confusion, disorientation, and
finally coma. They are graded from 1-4,
depending on the clinical severity.80 Over
the past few decades, the focus has shifted
to a sub-group of patients with cirrhosis
who show no overt clinical signs and
symptoms of encephalopathy, but who
perform poorly on psychometric tests when
compared to other patients with cirrhosis
and healthy controls.81-86 These patients are
categorized as having “subclinical” HE
(SHE). 87-90 This group of patients is
identified conventionally on the basis of a
battery of neurocognitive tests and/or
electroencephalographic abnormalities.
Significant findings in this group of patients
with SHE are impairment in tests of
psychomotor speed, concentration, visual
attention, tracking, visuo-spatial and fine
motor skills while verbal memory skills are
relatively well preserved.86-91 However, it
is not always possible to detect SHE by
psychometric tests alone.82 Thus, a true
single gold standard for the detection of
SHE is still not defined.
Ross et al first reported a triad of
signature changes observed in HE with 1H
MRS of brain white matter: a significant
reduction in the myoinositol to creatine ratio
(mI/Cr), a decline in the choline to creatine
ratio (Ch/Cr), and an increase in the
97
glutamine/ glutamate to creatine ratio (Glx/
Cr) in the parietal lobe.92-93 These changes
have also been reproduced in the studies of
minimal HE patients with volumes localized
in other regions of the brain. 94-96 One-
dimensional MRS, used in these studies,
suffers from the spectral overlap of various
metabolites resonating within a limited
spectral range, which may lead to inaccurate
quantification. In addition, some metabolites
at physiological low concentration are also
not visible. Hence, there is a need to address
these limitations with 1D MRS. 2D L-COSY
was investigated to record the changes in
cerebral metabolite levels in this group of
patients compared to healthy controls. A
total of 18 patients (10 females and 8 males)
with a mean age of 53.3 ± 10.6 years
undergoing evaluation for liver transplan-
tation and 21 healthy controls (13 females
and 8 males) with a mean age of 52.7 ± 12.6
years have been investigated. The diagnoses
of the patients were alcoholic cirrhosis (n =
3), primary biliary cirrhosis (n = 2), chronic
hepatitis B cirrhosis (n = 1), autoimmune
hepatitis (n = 2), cryptogenic cirrhosis (n =
1), and chronic hepatitis C cirrhosis (n = 9)
including a history of concomitant alcoholic
liver disease in 2 patients.
A 3 × 3 × 3 cm3 voxel was placed on the
anterior cingulate region, since the
neuropsychological tests have indicated an
alteration predominantly in the frontal tasks
that require attention and mental flexibility.
A CHESS sequence was used for water
suppression. The following parameters were
used: minimal TE = 30 ms, TR = 2 s, total
number of scans = 768 (96 Δt1 increments
and 8 NEX/Δt1), spectral width, F1 = 625
Hz and F2 = 2500 Hz. The total duration for
each 2D scan was approximately 25 minutes.
98 Biomedical Magnetic Resonance: Proceedings of the International Workshop
Table 8.1. Discriminant Analysis on MRS peak ratios
Peaks Subjects Predicted
Healthy
mICh Healthy (21) 19
Tau
Ch Patients (18) 3 15
NAA
Figures 8.10A and B (Plate 2) show the
2D L-COSY spectra of (A) a 44-year-old
patient and (B) a 45-year-old healthy human
subject, recorded in the anterior cingulate
using a voxel size of 27 ml. The 2D cross
peaks due to NAA, Cr, Glx, mI, Asp, PE,
PCh, Tau, ThrLac and GABA are marked.
The mean ratios (± SD) of the metabolites
with respect to Cr_d in SHE patients and
healthy controls are summarized in Figure
8.11. Statistically significant differences (p <
0.05) were seen in the mean values of ratios
calculated from the cross peaks: Glx/Cr_d,
mICh/Cr_d, mI/Cr_d. In addition, the
mean ratio of the diagonal peak of choline
(Ch_d/Cr_d) showed statistically significant
Fig. 8.11: Metabolite ratios of HE patients and age-
matched healthy controls calculated from the 2D peak
volumes
Predicted Correct Total
Patients Prediction Percentage
2 90.5 percent
86.9 percent
83.3 percent
reduction in patients compared to healthy
controls. The ratios of the cross peaks of
NAA, Cr, Asp, PE, GABA, PCh, and ThrLac
with respect to Cr_d did not show
significant differences, but Tau/Cr_d ratio
showed an increase (p=0.053) in the patients
when compared to the healthy group. A
stepwise discriminant analysis was
performed on the MRS ratios. The first ratio
was found to be mICh/Cr_d, which on its
own could correctly classify the patients and
healthy controls at 69 percent. Addition of
more ratios improved the predictability with
four ratios in the order of mICh/Cr_d, Tau/
Cr_d, Ch/Cr_d and NAA/Cr_d. The total
correct classification from these four
variables was 87 percent (Table 8.1).
Addition of a fifth ratio did not improve
the classification any further.
Late-life Major Depression
Late-life major depression is one of the most
common mental disorders in the elderly. It
is associated with considerable medical and
psychosocial morbidity.97 Both magnetic
resonance imaging (MRI) and magnetic
resonance spectroscopy (MRS) have been
utilized to study the anatomical and
biological substrates of depression in the
elderly.98-100 Late-life mood disorders are
associated with a decrease in brain volume
in critical brain regions such as the
 Multi-dimensional NMR Spectroscopy and Editing in vivo
prefrontal lobe, the hippocampus, and the
head of the caudate nucleus, when compared
with nondepressed control subjects.101 MRS
studies demonstrate an increase in the ratios
of Ch and mI with respect to Cr in the
white matter regions with NAA levels
comparable to those seen in agematched
controls.100 However, “first generation” MRS
studies used single voxel, onedimensional
(1D) 1H MR spectroscopy. While NAA, Cr,
Ch, and mI can be measured using 1D MRS,
overlap of the resonances of these
metabolites hinders their precise
measurements. Further, this limitation results
in poor reliability of detecting other
metabolites that are at lower concentrations.
On the other hand, two-dimensional (2D)
spectroscopy can overcome these problems
by the improved resolution enabled by the
second spectral dimension, 60-61 thus
providing a more reliable method for
measuring metabolites. 101 Our recent
findings on comparing relative metabolite
levels in patients diagnosed with major
depressive disorder (MDD) to healthy
elderly subjects are presented in this section.
Our study samples comprised 16 healthy
female controls (mean age of 72 ± 7 years)
and 12 female patients diagnosed with major
depression (mean age of 72 ± 8 years). All
depressed patients met DSM-IV criteria for
major depressive disorder. Figure 8.12 (Plate
3) shows a 2D L-COSY spectrum recorded
in the frontal white matter region of a 61yo
patient diagnosed with major depression.
The 2D metabolite ratios of 12 patients and
16 controls are summarized in Figure 8.13.
Even though increase of metabolite ratios
such as mI/Cr_d, Glx/Cr_d, GABA/Cr_d
and PE/Cr_d, and decrease of NAA_d/
Cr_d were observed in patients compared
to elderly controls, none of these changes
99
were statistically significant. The volumes
of white matter, gray matter, and CSF in
the MRS voxels used for 2D L-COSY were
calculated in 16 healthy women and 12
women patients using the 3D SPGR MRI
data, and the mean values were 63.7 percent,
30.7 percent and 5.6 in healthy women, and
60 percent, 33.1 percent and 6.9 percent in
elderly patients. Our preliminary study
demonstrates the feasibility of applying the
2D L-COSY method in elderly subjects with
and without a clinical brain disorder.
Human Breast Cancer
Magnetic resonance spectroscopy is a
potentially useful and minimally invasive
technique for breast tissue classification. A
number of prior investigators have applied
MRS to characterize breast tissue, with
promising results. MRS of human breast
tissues shows the resonances due to water,
choline, nucleotides, saturated and
unsaturated fatty acids.103-110 Mackinnon
et al used one-dimensional (1D) MRS to
distinguish 102/106 fine-needle biopsy
samples of benign or normal tissue from
Fig. 8.13: The metabolite ratios of elderly depressed
female patients and female elderly controls
100 Biomedical Magnetic Resonance: Proceedings of the International Workshop
samples of breast carcinomas.103 Kvistad
et al, using in vivo 1D MRS, identified
choline-containing compounds in 9/11
patients with breast carcinomas, 2/11
patients with benign breast lesions and 5/
7 healthy breastfeeding volunteers.104 Cecil
et al diagnosed 19/23 confirmed cancers and
13/15 benign processes correctly by 1D in
vivo MRS.105 Jagannathan et al monitored
the responses to chemotherapy, indicated
by both choline levels and the ratios of
water-to-fat, in malignant breast tumors
using in vivo MR spectroscopy.106,107 Yeung
et al reported a sensitivity of 92 percent
and specificity of 83 percent using the choline
peaks in MRS of breast carcinoma and
benign lesions in vivo.108 Mountford et al
evaluated the spectra of fine-needle aspirates
of breast tissue using a statistical
classification strategy,111 and Jong-Kanglee
et al analyzed breast tissue spectra in vivo
using artificial neural networks.109 A recent
report by Leifbritz and co-workers has used
1
H MRS and self-organizing maps in
visualizing biochemical changes in breast
cancer.110
A recent study using two-dimensional
localized correlated spectroscopy showed
elevated water-to-fat (lipid) ratios in breast
cancer and the presence of choline peaks in
some breast cancer patients. 112 Previous
investigators have also identified significant
differences in these quantities between
malignant breast tumors and benign tumors
or healthy breast tissue103-111 using one-
dimensional spectroscopy. The additional
cross-peaks detectable in COSY spectra may
provide valuable supplementary
information, and may thereby yield more
accurate discriminations among different
types of breast tissue than the water-to-fat
or choline-to-fat ratio alone. A classification
scheme based on the information available
in 2D COSY spectra could potentially assist
the physician in diagnosing suspicious
radiographic findings. The aim of the
present study was to evaluate the efficacy
of a statistical pattern classification method
namely, Classification and Regression Trees
(CART), in discriminating malignant breast
tumors and healthy breast tissues using the
2D L-COSY spectra.
A total of 34 women participated in the
present study. These human subjects
included 13 patients with biopsy-proven
invasive breast carcinomas (13 MRS voxels
in the lesions) and 21 healthy controls (30
MRS voxels predominantly in fatty tissue
regions). The cancer patients were referred
to the study from the Iris Cantor Center
for Breast Imaging. The average age of the
cancer patients was 51 years (range: 26-72
years), and that of healthy controls was 44
years (range: 23-68 years). The following
parameters were used to acquire each 2D
L-COSY spectrum: repetition time (TR) of 2
seconds, minimal echo time (TE) of 30 ms,
spectral width of 2500 Hz along the first
and 625 Hz along the second dimensions,
8-12 excitations per Δt1, and 40 increments
of Δt1. Total duration for recording a 2D L-
COSY was approximately 10-16 minutes after
3-5 minutes of optimization of transmitter
and receiver, and the static field (B o )
homogeneity. A two-step phase-cycling was
imposed on each RF pulse with receiver
cycled through addition/subtraction. No
water suppression was used while recording
the 2D L-COSY spectra. The MRS voxel size
was varied from 1 × 1 × 1 cm3 to 2 × 2 × 2
cm3. At least one spectrum was acquired
from the suspicious lesion for each patient.
For healthy controls, more than one voxel
were located in the fatty tissue.
 Multi-dimensional NMR Spectroscopy and Editing in vivo
Fig. 8.14A: Axial MRI of a 42-year-old healthy
breast with a voxel of 2D L-COSY
Shown in Figures 8.14A and B (Plate 3)
are the MRS voxel placement and the
corresponding 2D L-COSY spectra recorded
in a 42-year-old healthy woman. The voxel
size was 1.5 × 1.5 × 1.5 cm3. The 2D COSY
spectrum showed the following diagonal
peaks (F2 = F1) for the protons of water
(WAT), and the methyl, methylene and
olefinic protons of saturated and unsaturated
fatty acids, FMETD, FAT, and UFD. There
were symmetric 2D cross peaks of UFR,
UFL and TFGR between the methylene and
triglyceryl backbone protons of saturated
and unsaturated fatty acids. There were also
other unresolved cross peaks between the
methyl and methylene protons of fatty acids.
On the other hand, the 2D COSY spectrum
recorded in the affected side of a 62 year
old patient diagnosed with invasive breast
carcinoma showed drastically increased
water and reduced fat as shown in Figure
8.15 (Plate 3) in conformity with the earlier
1D MRS reports.104,106 Unlike the 1D MRS
findings, the 2D L-COSY spectrum showed
significant reduction of both diagonal and
101
cross peaks of unsaturated and saturated
lipids.
There was also a diagonal peak due to
the trimethyl protons of choline (CHO) in
breast carcinoma that was not detected in
any of the healthy breasts investigated so
far. The 2D cross peaks between the two
methylene protons of CHO were not detec-
table at (F2 = 4.0 ppm, F1 = 3.5 ppm) and (F2
= 3.5 ppm, F1 = 4.0 ppm) in breast tissues
contrary to the peaks detected in brain using
larger voxel size (27 ml). The rectangular areas
marked in the 2D spectra were used for
volume integration. Eighteen metabolite ratios
were calculated using the 2D L-COSY spectra
of invasive carcinoma, and healthy fatty
tissues. There was a significant increase
(p<0.05) of the following ratios in the
carcinomas compared to healthy fatty
tissues: WAT/FAT, WAT/FMETD, WAT/
UFD, CHO/FAT, CHO/UFD, WAT/UFR,
WAT/UFL, WAT/TFGR, CHO/UFR and
CHO/UFL. The decline of two ratios, CHO/
FMETD and FAT/TFGR, was also signi-
ficant. Group of three diagonal peak ratios,
namely WAT/FAT, WAT/CHO and CHO/
FAT predicted 11 out of 13 carcinomas and
28 out of 30 fatty regions correctly. The
analysis showed same prediction when three
cross peak ratios, namely WAT/UFR, WAT/
UFL and WAT/TGFR were used as a sepa-
rate group. However, analysis of combined
six ratios predicted 29 out of 30 fatty tissues
correctly with no change in the prediction
of carcinomas. CART analysis of CHO/
FMETD alone predicted all the carcinomas
and fatty tissues with 100 percent accuracy.
MULTI-VOXEL BASED 2D 1H MR
SPECTROSCOPY
One of the major concerns in the single-
voxel localized 2D MRS is due to the long
102 Biomedical Magnetic Resonance: Proceedings of the International Workshop
data acquisition (20-30 minutes) necessary
for the spectral averaging combination with
the second spectral encoding steps. When
CSI or SI is added to 2D MRS, there will be
further increase of time due to additional
stepping of the phase-encoding
gradients.58,59 The metabolite images can be
reconstructed from the 2D spatial and 2D
spectral data projecting the cross peak
volumes into the corresponding spatial
images, a methodology termed as cross peak
imaging (CPI). 113,114 A combination of
16 x 16 spatial, 64 spectroscopic encoding
steps and repetition time (TR) of 1 s will
result in a total duration of 4.6 hours.114
Recent implementation of circular sampling
combined with 2D J-resolved spectroscopy
was demonstrated by Renshaw and
coworkers using a 4 T whole body MRI
scanner resulting in a total duration of 2
hours.115
Two different multi-dimensional MRS
sequences have been proposed recently with
a drastic reduction of total duration. First,
Spielman and coworker introduced the first
application of time-varying gradients during
the read-out time to acquire multi-voxel
based 2D J-resolved spectral data in phantom
solutions containing lactate and ethanol
using a 1.5 T MRI scanner.116 Spiral-based
k-space trajectories were used in conjunction
with a 2D J-resolved sequence. By preceding
the spiral read-out with a 2D preparation
scheme, 128 spectral encoding with TR of
2 s resulted in a total duration of 17
minutes. Second, an asymmetric variant of
echo-planar spectroscopic imaging (EPSI)
consisting of a train of trapezoidal read-out
gradients, each followed by short refocusing
pulse was implemented by Meyer et al.117
A constant-time based COSY was used and
the total acquisition was 34 minutes.
Application of this technique was demonst-
rated in rat brain using a 4.7 T MRI scanner
and the cross peak images of mI and Tau
were recorded only. The 2D spectra derived
from EPSI are complicated by artifacts
contributed by strong coupling of various
metabolite protons unavoidable at both 1.5 T
and 3 T MRI scanners. The artifacts in these
multi-voxel based 2D MRS sequences need
to be simulated and further work is
necessary in the future to demonstrate the
clinical potential of these multidimensional
MR spectroscopic imaging methods.
CONCLUSION
Broadband excitation and detection of the
J-coupled metabolites in human tissues
noninvasively have been demonstrated using
different 2D MRS techniques. 2D cross peaks
of more than ten J-coupled metabolites have
been observed using L-COSY recorded in
the frontal and occipital regions of human
brain.60,61,77-78 Other researchers have also
reported the implementation of different
versions of 2D COSY.118-121 Simultaneous
acquisition of COSY from multiple volumes
of interest was proposed by Delmas and
co-workers118 and two-voxel localized COSY
spectra of rat brain were recorded in a total
duration of 42 minutes using a Bruker 4.7 T
MRI scanner. A different pulse-sequence
(ISIS-COSY) was also proposed by Welch
et al,121 where the volume was localized by
outer-volume suppressed ISI (OSIRIS). Two
major drawbacks of ISIS-COSY compared
to L-COSY were: (i) eight shots are
necessary to achieve the VOI and (ii) five
RF pulses are necessary to record the single
voxel localized COSY leading to more
dependence on the B1-field inhomogeneity.
In contrast to a recently implemented ISIS-
COSY sequence, 121 the 2D techniques
discussed in this chapter are one-shot based.
 Multi-dimensional NMR Spectroscopy and Editing in vivo
It has been shown recently that one-
dimensional MR spectra processed using the
LCModel algorithm and due to severe
overlap of several spectral resonances in 1D
MRS in vivo, LC Model uses the individual
phantom spectra to come up with the
absolute concentrations of high and low-
concentrated metabolites. 19,21 In the
frequency domain, 2D L-COSY shows the
unambiguous presence of 2D cross peaks
originating from several J-coupled
metabolites. However, further work is
necessary in order to ascertain the
superiority of 2D L-COSY over the
quantitation of 1D MR spectra using LC-
Model. Additionally, different 2D MRS
techniques discussed in this chapter need
further improvements in terms of voxel
sizes, coil design, etc. A major limitation
with the poor signal-to-noise ratio (SNR)
achievable in 2D MRS and a long signal
acquisition is necessary even with a voxel
size of 27 ml. Also, due to the differential
T 2 * -weighting during the incremental
duration of 2D L-COSY, absolute
quantitation of several metabolites is further
complicated by lack of T2 and T1 values in
the literature for different proton groups
(methyl, methylene, methine, etc.) in any
given metabolite. Regarding the application
of these multidimensional MRS techniques
in different pathologies, targeting a specific
metabolite or selected metabolites in
different pathologies may be more
advantageous than nonselective excitation/
detection of several metabolites. Another
major concern is that the 2D spectra are
complicated by artifacts contributed by
strong coupling of various metabolite
protons unavoidable at both 1.5 T and 3 T
MRI scanners. As discussed earlier, the
artifacts in these multi-voxel based 2D MRS
103
sequences need to be further evaluated.
Further work is necessary to demonstrate
the clinical potential of both single and
multi-voxel based multidimensional MR
spectroscopic imaging methods.
ACKNOWLEDGMENTS
This work was supported by the grants from the
National Institute of Health (MH065695) and the US
Army Breast and prostate cancer research programs.
Scientific support of the following people is gratefully
acknowledged: Dr. Lawrence Ryner and Dr. T.N
Venkatraman in the implementation of several 2D
MRS sequences, Dr. Shida Banakar and Tai Dou and
Ashwin Thomas during the preparation of this
chapter, and Mr. Art Ambrosio during the
preparation of brain phantoms.
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ABBREVIATIONS
1. MRI- Magnetic Resonance Imaging
2. MRS- Magnetic Resonance Spectroscopy
3. VOI- Volume of Interest
4. CHESS- Chemical Shift Selective Suppression
5. RF - Radio Frequency
6. OVS- Outer Volume Suppression
7. STEAM- Stimulated Echo Acquisition Mode
8. PRESS- Point Resolved Spectroscopic Sequence
9. STEAMSV- single-voxel based STEAM
10. PRESSSV- single-voxel based PRESS
11. CSI- Chemical Shift Imaging
 Multi-dimensional NMR Spectroscopy and Editing in vivo 109

12. SI- Spectroscopic Imaging 22. CT-COSY- Constant time-based COSY

13. STEAMCSI- Multi-voxel based STEAM 23. SS- Slice-Selective
14. PRESSCSI- Multi-voxel based PRESS 24. GAMMA- General Approach to Magnetic
15. 1D- One-dimensional Resonance Mathematical Analysis
16. 2D- Two-dimensional 25. U-FLARE- Ultra-fast Low Angle Rapid
17. 3D- Three-dimensional Acquisition with Relaxation Enhancement
18. L-COSY- Localized Correlated Spectroscopy 26. CABINET- Coherence transfer based spin-echo
19. VOSY- Volume Localized Spectroscopy spectroscopy
20. SECSY- Spin-echo Correlated Spectroscopy 27. ISIS- Image-selected in vivo spectroscopy
21. CT-PRESS- Constant time-based PRESS 28. OSIRIS- outer volume suppressed ISIS

Ultrafast and Parallel Imaging
INTRODUCTION
Terms like “fast“ and “ultrafast“ derive
meaning only from the context within which
they are being used. The extinction of the
dinosaurs within a few thousand years is
ultrafast in geological terms, building up a
research laboratory in one year or writing
a paper in one week would be regarded as
fast in their respective context. In
photograpy an imaging speed of 2 ms
(corresponds to 1/500 shutter speed) is
considered to be fast and 250 μs (1/4000
shutter speed) to be ultrafast. In the
assessment of the performance of a technical
apparatus like an imaging system there are
actually two scales against which the
fastness of the performance is been
measured. On one hand there is the inherent
time scale for the actual application for
which the system is being used. In this
context one would call a measure slow, if
the measurement speed actually interferes
and limits the measurement. A fast technique
would be one which is comfortably fast
compared to the inherent timescale, and
ultrafast would be all implementations
where the imaging speed can be ignored.
For MR imaging this implies that fast
imaging includes quite a significant range
of acquisition times:
9
J. Hennig
• For diagnostic imaging, where high and
very high-resolution (typically better than
one millimeter) and a significant volume
coverage (20 to 30 cm) is required and
where a specific contrast behavior is
necessitated by actual indication, imaging
times below one minute are already fast.
• For the imaging of the abdomen, where
breathing motion is a problem, fast
imaging necessitates breathhold
acquisition for mulitslice imaging
covering for example the whole liver.
• In cardiac imaging where the motion of
the beating heart needs to be overcome
in addition to the overall breathing
motion, the borderline between fast and
ultrafast lies in a range of one heartbeat
for the acquisition of several slices.
• Finally in functional brain imaging where
one wants to observe cortical signal
changes anywhere in the brain, one needs
fast imaging in the order of one second
to cover the whole brain in observations
based on hemodynamic responses. In
order to be able to follow neuronal
activity directly one would very much
like to be much faster.
Complementary to this application
defined performance scale, one can define
fast and ultrafast imaging on the basis of

technical limits of the system. In this context
a fast imaging technique is one which can
be realized with reasonable technical effort
and ultrafast imaging would be anything
which pushes the limits of technology. There
is, of course, a strong correlation between
the application related and the technical scale
systems: technical performance will define
the range of problems, which can be
addressed. Vice versa, once imaging speed
will allow to adress a new range of
applications, one will immediately try to
push the envelope further to a point where
performance is not a limiting factor anymore.
In the following, a discussion will be given
about fundamental limitations of MR
imaging followed by examples of fast and
ultrafast imaging in various fields of
applications.
FUNDAMENTALS OF FAST AND
ULTRAFAST IMAGING
The fundamental challenge for using
magnetic resonance for imaging purposes is
related to the fact that the wavelengths of
the radiofrequency field useful the
observation is far longer than the desired
spatial resolution. The Larmor frequency at
1.5 Tesla is 64 MHz corresponding to
wavelengths of a bit less than 5 m in air.
The strong dielectric moment of water
compresses the wavelengths by roughly a
factor of 9. But the ~60 cm in tissue is still
by far outside the range which would allow
imaging based on wave optical principles.
The Rayleigh criterion for imaging by wave
optical principles states that the limit of
spatial resolution is roughly equivalent to
the wavelength of the radiation used. This
applies not only to photographic imaging
using light, but also to conventional X-ray
imaging, Computer Tomography, PET
Ultrafast and Parallel Imaging 111
(Positron Emission Tomography), SPECT
(Single Photon Emission Computer
Tomography) and even to ultrasound, all
of which are based on applications of optical
principles.
In MR spatial information has to be
derived in a fundamentally different way
by encoding the signal in an indirect way.
In practically all MR imaging techniques in
use today this is being achieved by
translating the spatial domain into a
frequency domain while the application of
linear gradient (Fig. 9.1, Plate 4).
This basic principle has been introduced
by Paul Lauterbur1 in 1972 who together
with Sir Peter Mansfield received the 2003
Nobel Prize for this outstanding achieve-
ment. Although tremendous progress has
been made in the last 30 years in developing
all kind of new MR sequences with very
different performance characteristics, all of
these are based on this common principle
of spatial encoding using gradients. Without
going into details of any specific pulse
sequence some very fundamental limitations
on gradient based imaging can be derived
from signal theory:
Data acquisition in MR imaging is
performed using the principles of Fourier
NMR. This means MR signal is acquired
sequentially in time. The frequency spectrum
encoding the spatial information is derived
by Fourier transformation from the time
signal. In order to acquire data for an image
of matrix size N × M one therefore needs
to sample N × M data points in time. The
sampling speed defined by the dwell time
DW between successive (complex) data
points is related to the bandwidth BW of
the spectrum via the sampling theorem:
DW=1/BW ...(1)
The total ‘pure’ data collection time is
then given by:
112 Biomedical Magnetic Resonance: Proceedings of the International Workshop
Tacq = N × M × DW
= (N × M)/BW ...(2)
According to the Larmor condition, BW is
translated into the field of view FOV by
FOV = BW/(γG) ...(3)
Faster sampling therefore is invariably
related to a higher sampling bandwidth and
will also require stronger gradients in order
to achieve some desired field of view. In
the regime around several 10th and 100th
of megahertz used for MR imaging physio-
logical noise produced by the body as well
as instrumental noise is evenly distributed
over the spectral range BW defining the
image. The signal-to-noise (SNR) will thus
be proportional to the square root of the
inverse bandwidth and therefore according
to (2) proportional to the square root of the
acquisition time:
SNR ~ √(1/BW) ~ √Tacq ...(4)
This means that the larger the spectral
width, the more noise will contribute to the
signal. To give an example, lets consider a
MR image of the head acquired by 1.5 Tesla
with 5 mm slice thickness, 1 mm in-plane
resolution and acquired with a bandwidth
of 200 Hz pro pixel (50 KHz spectral width
for 256 × 256 matrix) with a SNR of about
200. According to (2) the pure sampling time
Tacq for acquiring the data for an image
will be 256 × 256/BW = 1.28 s. Tacq is the
pure time necessary for data acquisition and
ignores all additional times required for
preparing the signal for the data readout.
Reducing the acquisition time by a factor of
100 to 12.8 ms (which is still modest
compared to the shutter speed of even the
cheapest photocamera) will lead to an
inherent reduction of the SNR by a factor
of 10 and thus to an image of very poor
quality. This illustrates that the ultimate
limitation of MR imaging even with a most
ingenious pulse sequence and ignoring all
technical limitations set by the instrument
will be defined by the signal-to-noise. No
matter how clever a pulse programmer may
be, SNR will set the ultimate limits for
ultrafast imaging.
Technical Limits
Of course, in practice the imaging speed is
not dependent on the data acquisition time
alone, but will also be affected by the time
required for signal generation by RF-pulses,
by contrast considerations and also by the
performance of the gradient system. This is
a consequence of the fact that a static
magnetic field gradient can never be shaped
such that image information for a 2D image
can be uniquely encoded into a static
magnetic field profile. As shown in (Fig.
9.1, plate 4) unambiguous encoding by a
gradient is easily achieved in one dimension.
As long as the change in the magnetic field
along the axes of the gradient is
monotonous, every point in space is
uniquely assigned to the corresponding
Larmor frequency. For a constant gradient,
the transformation scale will be linear, but
even for other field shapes, an unambiguous
assignment between resonance frequency
and spin location can be made as long as
the field change is monotonous. Of course
the shape of the magnetic field needs to be
known in order to make the correct
transformation. Measurement of the field
profile is the basis of the multiple techniques
for distortion corrections published in the
literature.
Thus an infinite range of magnetic field
profiles will lead to unambiguous spatial
assignment of the spin positions This is
fundamentally different for two (or more)

dimensions. As illustrated by Figure 9.2,
(Plate 5) it is a fundamental property of any
continuous field profile that a unique
attribution of the field strength, and
therefore the frequency, is only reached at
the extreme points (maximum and minimum)
of the field from Peano’s theorem. It follows
therefore that for any positions in between
there will be an infinite number of points
with equal values. Therefore, it is not
possible to encode a two dimensional image
in a single measurement using a constant
gradient even with arbitrarily shaped
magnetic field gradients. The commonly
used procedure for resolving this ambiguity
problem is to acquire data sequentially
under time variant gradients. Intuitively the
approach used by Paul Lauterbur in his
original paper illustrates this principle very
nicely. In the back projection approach he
used (called zeugmatography at that time),
measurements are performed sequentially
such that projections of the objects in
different directions of field gradients were
measured, the image could then be
reconstructed from these projections by
Fig. 9.3: Correspondence of the image domain in the xy-plane to
k-space by two-dimensional-Fourier transformation (2DFT)
Ultrafast and Parallel Imaging 113
appropriate algorithms like the Radon
transformation. Over the years this concept
has been generalized by the principles of
Fourier imaging introduced by Ernst 2 in
which a two dimensional sampling domain
called k-space is defined by Fourier
transformation of the image (Fig. 9.3).
The spatial coordinates x and y are
related to the k-space coordinates according
to Eq. [3] by the following relations:
kx = γtGx x = ω/γGx ...(4a)
ky = γtGy x = ω/γGy ...(4b)
The task of acquiring an image is then
transformed into a problem of finding
strategies for efficient sampling for the data
in the k-space domain.
The task of designing a strategy to design
a fast or ultrafast imaging sequence can thus
be translated into the task to cover k-space
as efficiently and as rapid as possible. In
view of the hundreds of available pulse
sequences it may be surprising, that there
are only two ‘vehicles’ available to perform
this task (Fig. 9.4). First of all transverse
magnetization will move around k-space with
114 Biomedical Magnetic Resonance: Proceedings of the International Workshop

Fig. 9.4: Vehicles for moving around k-space: application of gradients will lead to a trajectory, the direction of
which will be defined by the gradients, the gradient amptitude will define the velocity of moving around
k-space (left). A refocusing pulse will bring spins to a position at the other side of k-space symmetrically
around the origin. The origin is indicated by a cross and is normally at the center of the k-space acquisition
window.

a velocity proportional to the amplitude of In the conventional approach of Fourier

the gradients and in a direction defined by
the direction of the gradients. Furthermore,
a refocusing pulse can be used to jump across
k-space and to reach a position on the
opposite side. In practical implementations
one has to consider that a refocusing pulse
with a flip angle not exactly equal to 180°
will leave some magnetization at the original
position. It will also generate coherent
z-magnetization which will be awakened by
any further RF pulse and generate further
k-space trajectories from the original
position.3 This can very rapidly lead to
signals consisting of superpositions of
spins traveling along different k-space
trajectories, which will lead to severe image
artifacts.
imaging data acquisition is performed line
by line after preparing a spin system to an
appropriate starting position by a sequence
of pulses and gradients preceding the actual
data acquisition. This is the approach used
in the conventional spin-echo imaging as
well as in various gradient-echo imaging
approaches. After introduction of actively
shielded gradients and with the develop-
ment of more and more powerful gradient
power supplies, the limitation of data
acquisition under a constant gradient could
be alleviated and more efficient ways like
spiral imaging or EPI could be realized in
which larger parts or even all of the k-space
data are acquired after a single excitation
of the spin system.
 Ultrafast and Parallel Imaging 115

FROM FUNDAMENTALS TO PRACTICAL induced current I is proportional to the

LIMITS: GRADIENT PERFORMANCE AND speed of change ΔB in the local magnetic
RADIOFREQUENCY PULSES field:
In the earlier part it has been shown, that I ~ ΔB/Δt = ΔG Δx/Δt ...(5)
SNR sets quite stringent limits to the
where ΔG/Δt is the gradient slew rate and
sampling speed even without any considera-
Δx the distance to the gradient isocenter
tion of the actual physics or even technical
illustrates, that nerve stimulation depends
implementations. In the ‘real’ world the
imaging speed will be further reduced by on the gradient slew rate as well as on the
the fact, that the total acquisition time Ttot size of the gradient system. It should be
not only consists of the duration spent for noted, that for a typical gradient system
data acquisition, but will also include some with a linear range of 40 cm (corresponding
additional times necessary for preparation to Δx= ± 20 cm), the largest field change
of the spin system, for maintaining stable will occur outside of the linear range by
steady state conditions and for multi excita- about 30-50 percent depending on the
tion sequences for additional waiting times gradient coil design (Fig. 9.5). The maximum
necessitated by spin recovery rather than field will be about 30-50 percent higher than
the technical limitations of gradient and RF the gradient specifications. For a gradient
performance. amplitude of 25 mT over 40 cm, the field
In discussing technical limits, gradient change ΔBlin at the end of the linear volume
performance now-a-days has already will be 5 mT, but the maximum field change
reached a status, where limits are ΔBmax can be as high as 7.5 mT. A slew rate
determined by safety considerations related of 100 T/m/s this corresponds to a mean
to nerve stimulation rather than by technical
performance. Likewise, the limits with
respect to radiofrequency pulses are
determined by safety considerations due to
patient heating. This especially applies at
high fields, which as we have seen are
essential for fast imaging due to the
improved SNR. In the following, a brief
description of the pertinent safety issues
will be given. The discussion will focus on
physiological effects, and does not
necessarily reflect the legal status, which is
different in different countries and which is
not always derived from a rational
discussion of exposure issues alone. Fig. 9.5: Typical variation of the magnetic field B
With respect to physiological limits of produced by a gradient along x. The gradient will
produce a linear change of the field in the range
gradient performance, the relevant
between -ΔXlin and ΔXlin (typically ± 20 cm). This will
physiological mechanism is the involuntary lead to an overshoot at positions ± ΔXmax, where the
nerve stimulation caused by electrical field change ΔBmax can be 30-50% higher than the
currents created by the gradients. The value ΔBlin at the edge of the linear range
116 Biomedical Magnetic Resonance: Proceedings of the International Workshop
rate of field change of ΔB/Δt = 20T/s at
Δxlin= 20 cm, but ΔB/Δt = 30 T/s at Δxmax.
State-of-the-art gradient power supplies
today easily deliver sufficient power to
exceed the physiological nerve stimulation
limits. The technical challenge in gradient
design today thus is not so much to further
increase gradient power, but rather to design
gradient coils such, that ΔBmax does not
exceed ΔB lin overly much. For specific
purposes like diffusion imaging of the head,
shorter gradient coils designed specifically
for that purpose can increase the tolerable
performance of a body coil by a factor of 2
or more.
How these limits translate into perfor-
mance of ultrafast imaging sequences, which
use gradients to cover k-space will be
described later.
With respect to radiofrequency (RF)
power, the physiological limits are defined
by the amount of energy, which is absorbed
by the patient and which will lead to
heating. The specific absorption rate (SAR)
depends on the energy submitted by the
patient, but also on the frequency of RF
and the size of the patient. The absorbance
of the human body has a maximum at
around 120-150 MHz, which corresponds to
3 T field strength. Therefore the increase in
SAR between 1.5 T and 3 T is rather steep
and scales with roughly the square of the
field strength. The further increase at even
higher frequencies is somewhat less
dramatic, but in any case, SAR will continue
to go up with the field.
Different exposure limits are valid for
whole body transmission compared to
exposure of only parts of the body (head).
Since thermo regulation is assumed to be the
relevant mechanism to deal with RF heating,
the time constant, over which RF exposure
is measured, is rather long. The current IEC-
standards are based on average exposure
over 6 min, with separate provision for short
term peak exposure, where three times the
average power can be applied over 10 s.
These time constants are rather awkward
in comparison with the typical acquisition
times used in MRI. Taking the limits literally,
the average 6 min-‘dose’ (1 W/kg body
weight~ 75 W for a 75 kg patient) could be
applied over 2 min (~ 225 W average
power), provided, that no RF is applied over
the rest of the 6 min period. Likewise and
somewhat worrisome, there is no lower limit
for the exposure time. Consequently, one
could apply the permitted 225 W short term
exposure limit over 10 s over any arbitrarily
short period like 2.25 kW over 1 s, 22.5 kW
over 0.1s or a staggering 225 kW over 0.01s.
Although no hard data exist for such
ultrashort exposure, one would be hesitant
to apply such excessively high RF power to
a patient. So fortunately it is at current
technically infeasible to reach such high
power levels.
The actual RF power (RFP) necessary for
a RF pulse will depend on a scaling constant
c depending on the size and design of the
transmitter coil and the Q-factor of the
(loaded) coil. Variable parameters are the
duration TD and a shape factor ‘s’ related
to the shape of the RF pulses used and–last
but not least–the flip angle α of the pulse:
RFP = c s α2/TD ...(6)
A conservative value for a 180°-pulse of
1 ms duration at 3 T applied by the whole
body transmitter coil is about 5 kW or 5kW
× 1ms = 5 Ws per pulse. In other words,
450 such pulses could be applied over 10 s.
It is therefore clear, that SAR is not a serious
consideration, if a single image is to be

acquired within the 10 s averaging time, for
a single image with 128 pulses the pulse
duration could be shortened to 0.28 ms.
For such an implementation, the
limitations would be given by the maximum
voltage tolerated by the RF coil rather than
by SAR considerations. Acquiring a single
image within 10 s is, however, not a very
sensible way to do fast imaging and what
be called sudden imaging rather than fast
imaging. In most applications fast imaging
implies short repetition times. Figure 9.6A
(Plate 6) shows the number of pulses np,
which can be applied over 10 s as a function
of the flip angle for a pulse of 0.5 and 1 ms
duration. The complimentary display in
Figure 9.6B (Plate 6) shows the minimum
pulse spacing, if pulses are applied conti-
nuously. It is quite clear, that for sequences
requiring continuous application of RF pulses
(like gradient-echo sequences), the maximum
allowed flip angle will get rather small at
very short TR. Alternatively, for sequences
requiring high flip angles (like spin-echo
sequences), additional waiting times have
to be built in order to limit the total number
of pulses to the SAR limits. It should be
emphasized, that these diagrams are for
demonstration only and relate to a
conservative ‘worst-case’ scenario in body
imaging at 3 T. For smaller transmitter coils
and partial body exposure (like in a
transmit/receive head coil) and at lower
fields SAR is much less of a problem.
From Theory to Practice: Gradient Based
Fast and Ultrafast Imaging Sequences
Echo-planar imaging has been introduced
by P. Mansfield in 19774 as the very first
ultrafast imaging sequence. Even today EPI
still is the ‘workhorse’ for most ultrafast
imaging applications especially in the brain.
Ultrafast and Parallel Imaging 117
Its pulse program and k-space trajectory are
shown in (Fig. 9.7). Originally sinusoidal
gradient waveforms have been used for the
readout gradient GR and a constant gradient
GP was used in order to minimize the
power requirements on the gradient
systems. With current state-of-the-art
technology, a rectilinear sampling with
blipped phase encoding gradient is,
however, more commonly used. As shown
by the k-space trajectory, EPI requires two
distinct modes for gradient operation: while
data are sampled in each line, a constant
and high gradient GR is required to optimize
sampling speed, whereas very fast switching
is necessary at the end of each k-line.
Technically high and constant gradient
amplitude requires high current in the
gradient coil, whereas fast switching
necessitates high voltage. In terms of nerve
stimulation only the switching periods are
relevant, since a constant gradient does not
create any stimulation problems.
In order to optimize data acquisition for
EPI one needs to balance two opposing
requirements: From equations (2) and (3) it
is clear, that the sampling speed is increased,
when gradients with high amplitude are
being used during data sampling. High
gradients will require faster switching at
the end of each k-line, when the readout
gradient has to be reversed. Fig. 9.8 gives
some examples for the performance of EPI
that given gradient slew rates. It is clear,
that eventually the time necessary for
switching will dominate and the overall
acquisition time may even increase with
higher gradients. Using non-rectilinear
sampling, data acquisition can be performed
even during the gradient reversal. In the
ultimate limit, the minimum achievable
acquisition time will then be given by the
switching times alone. Even then and with
extremely fast switching (slew rate 200 T/
118 Biomedical Magnetic Resonance: Proceedings of the International Workshop

Fig. 9.7: Pulse program (top left) and k-space trajectory (bottom left) for an idealized EPI-experiment with
rectilinear sampling of k-space using constant gradient GR for sampling each k-line along kx and a blipped
gradient GP to advance by one line during reversal of GR. The figures on the right show a non-rectilinear k-
space trajectory with sinusoidal GR and a constant gradient GP

m/s) the acquisition time will be longer than
~20 ms. Typical acquisition times used today
are in the order of 50-60 ms for a 64 × 64
image. Even with a slew rate of 1000 T/s/
m and 1000 mT/m gradient amplitude the
acquisition time would still be in the order
of 10-15 ms. This illustrates, that the ‘cost’
of increasing imaging speed with EPI in
terms of gradient power and switching
behavior grows dramatically. Accelerating
data acquisition by brute force thus cannot
be expected to significantly reduce the EPI
acquisition times much below the current
20-50 ms.
As an alternate and in several ways more
efficient way to cover k-space, spiral imaging
has found new interest over the last years.
As shown in Fig. 9.9, the k-space trajectory
is much smoother compared to EPI. In
practical terms this means, that the demand
on the gradient system is evenly distributed
along the data acquisition. The alternation
between fast switching and sampling and a
constant gradient in EPI is thus avoided. In
the literature several approaches have been
described, how to optimize the spiral
trajectory within the boundary conditions
defined by the gradient slew rate and

Fig. 9.8: Minimum acquisition time Tacq for EPI with
rectilinear sampling for 64 k-lines, 3 mm spatial
resolution and gradient slew rate 100T/m/s (top) and
200 T/m/s (bottom), (a) represents rectilinear
sampling, (b) gives the tie used for gradient switching,
(c) represents the tie required for data acquisition.
When under-the-rap sampling is being used, the
minimum total time will be given by (b) for values of
GR, where the switching time is longer than the
acquisition time
amplitude.5,6 In general and as a consequence
of the even distribution of the gradient load
along the k-space trajectory, spiral imaging
will be somewhat faster than EPI for most
gradient systems.
Spiral imaging has been first introduced
back in 1985,7 but it has been dormant for
a long time for several reasons. Initially the
Ultrafast and Parallel Imaging 119
Fig. 9.9: Sequence diagram and
k-space trajectory for spiral imaging
most important problem related to the fact,
that image quality very crucially depends
on the exact k-space trajectory used (which
is true for most nonectilinear sampling
techniques). If the gradients do not follow
exactly the prescribed trajectory, extremely
severe image degradation will occur. With
the typical eddy current behavior of early
gradient systems it was impossible to realize
a robust and reproducible spiral trajectory.
In addition and from a practical point of
view, spiral images can not be directly
generated from the acquired data by the
120 Biomedical Magnetic Resonance: Proceedings of the International Workshop
very fast Fast Fourier reconstruction
algorithm. Many approaches to optimize the
quality and speed of image reconstruction
for spirals and other nonrectilinear
k-space sampling strategies have been
suggested.8-14 Only in recent times computers
have become sufficiently fast to perform
image reconstruction within reasonable
reconstruction times. For applications in
fMRI, where several hundreds or thousands
of images are acquired within a few seconds
or minutes, image reconstruction times are
still a practical limit on the standard
computer platforms of current MR systems.
It can be expected, that this problem will
vanish in the future, when even faster
computers will be introduced. A more subtle
problem relates to the artifact behavior. The
most common source of imperfections in
single shot sequences are the signal decay
due to T2* effects and off-resonance effects.
Both of these mechanisms will develop along
the k-space trajectory leading to a strong
dependence of the resulting artifacts on the
actual trajectory used.
Figure 9.10 shows simulations of the
resulting artifacts on a typical range of
parameters. Typical observations for EPI
are:
• a signal attenuation with 1/T2* due to
the fact, that the center of k-space is
sampled at a finite echo time (25 ms in
the example shown)
• at shorter T2* edge artifacts will appear.
Note, that the images look pretty good,
as long as the overall acquisition time is
in the order of the T2* of the tissue.
• off-resonance effects will lead to a shift
in the phase encoding direction. This will
create typical distortion artifacts at areas
of local field inhomogeneities.
Spiral imaging overall signal intensity
remains constant due to the acquisition of
the k-space center at the beginning of data
acquisition. Spiral images do not show
distortions, since both T2* and off-resonance
effects will lead to blurring rather than to
a displacement. This can be good news or
bad news: if the acquisition time is very
short, no distortions will occur and only
minor blurring will be visible. At longer
Tacq, however, image quality will
deteriorate.
The distortion caused by EPI at typical
acquisition times can be quite pronounced.
For EPI-based fMRI of the brain the
displacement can easily reach 1 cm or more.
Recently stable algorithms based on
measurements of the local field distribution
have been suggested, which can be applied
to correct for these effects. 15-19 The
misalignment in uncorrected EPI as well as
the quality of correction performed
according to19 is demonstrated in Figure
9.11, Plate 6. It should be emphasized, that
the distortion correction is performed fully
automatic during the scan without any user
interaction. For spirals, distortion correction
is more tricky and can only be performed
on the raw k-space data, whereas the
displacement matrix necessary for EPI-
correction can be easily applied to the final
images.
It should be noted, that other k-space
trajectories have been suggested for
different purposes. Radial trajectories offer
some quite interesting possibilities with
respect to their tolerance to radial under-
sampling and are quite attractive for
imaging at ultrashort echo times.20-24
The main application for EPI is in BOLD-
based fMRI, where its ‘natural’ sensitivity
to T2* combined with the possibility to cover
the whole brain in less than 1 s are ideally
suited to observe the hemodynamic res-
ponse to cortical activation. Spiral imaging
 Ultrafast and Parallel Imaging 121

Fig. 9.10: Artifact behavior of EPI vs. spiral imaging based on a total acquisition time of 50 ms respectively.
The first row shows the excellent representation of a 4x4 box in the ideal case of no signal decay and perfect
on-resonance of the signal. With shorter T2* EPI images will eventually develop edge artifacts. Additionally a
pixel shift will occur for off-resonance spins. Both signal decay as well as off-resonance effects will lead to
blurring in spiral imaging

has also been used for fMRI.25-27 In order to
generate T2* -sensitivity, this requires either
a delayed start of data acquisition or the
use of ‘outside-in’ sampling, where the
center is sampled at the end of data
acquisition .
Outside of fMRI, EPI (and to some extend
spirals and other sequences) are mainly used
as imaging modules in contrast modified
sequences. Probably most important is the
combination with diffusion preparation for
mapping of the apparent diffusion
coefficient. For early detection in stroke this
is probably the clinically most useful
application of such techniques. In addition
to that, the measurement of diffusion
anisotropy for neuronal fiber tracking has
found considerable interest in the neuro-
science community,30 although its robust
clinical application has still to be established.
Fast Imaging with Multipulse Sequences:
Gradient Echoes
Gradient-echo sequences apply a periodic
sequence of multiple pulses at a pulse
spacing, which typically is much sorter than
the T1 and T2 of the tissue. Depending on
the refocusing behavior on transverse
magnetization created by preceding pulses,
gradient echoes are know to occur in
different varieties: Spoiled gradient echoes
(spoiled FLASH, GRASS, FFE), where only
the available z-magnetization is observed,
un-spoiled gradient echoes, where part of
122 Biomedical Magnetic Resonance: Proceedings of the International Workshop
the transverse magnetization is retrieved and
fully balanced gradient echoes (balanced
SSFP; trueFISP, FIESTA, balanced FFE),
where all magnetization is fully refocused.
An overview of the intrinsic signal behavior
is given in.31
In spoiled FLASH the steady state trans-
verse magnetization Mtr depends on the
flip angle α, T1, T2 and the repetition time
TR:
Mtr=M o sinα (1-exp(-TR/T1)) exp(-TE/
T2)/(1-cosα exp(-TR/T1)) ...(7)
For tissues with specific T1 and T2, the
maximum signal is achieved at the Ernst-
angle αE:
cosαE = exp (-TR/T1) ...(8)
Mtr then yields:
Mtr(αE) = Mo exp(-TE/TR) sinαE/(1+cos
αE)
Fig. 9.12 shows, that for TR<<T1 the
Ernst angle will be very small and the steady
state signal will be only a few percent of
the available magnetization. Therefore
ultrafast imaging with T1-weighted FLASH
carries a heavy penalty in signal-to-noise.
Nevertheless it is used especially for high-
Fig. 9.12: Steady state signal intensity lral (left vertical
scale) and Ernst angle α (vertical scale right) for data
acquisition with spoiled FLASH as a function of
TR/T1
field animal imaging because of the good
image contrast and the quite robust image
behavior.
For short TR and TR<<T2 the signal in
fully balanced gradient echo sequences can
be shown to be
Mtr = Mo sinα/((T1/T2+1) –
cosα (T1/T2-1)) ...(8)
The optimum flip angle is then given by
cosαSSFP = (T1/T2-1)/(T1/T2+1); ...(9)
The signal intensity for αSSFP is then given
by
Mtr ~ ½ M0 √(T2/T1) ...(10)
Remarkably, the signal intensity does not
depend on TR, therefore a constant signal
amplitude can be maintained even at
arbitrary short TR. Since the acquisition time
Tacq will always be shorter than TR, faster
imaging will require higher bandwidth and
thus yield lower SNR according to Eq.[4].
A serious consideration for balanced
SSFP is the fact, that it takes quite some
time (roughly in the order of T1) until the
signal steady state is reached. Before that,
signal fluctuation will occur, which may
seriously affect image quality. An initial
pulse with flip angle α/2 and preceeding
the periodic sequence by TR/2 will stabilize
the signal for on-resonance spins, but not
for off-resonance magnetization. Various
approaches have been published, how to
improve the initial signal behavior. 32
Nevertheless, balanced SSFP is not really
suitable for single shot imaging. The main
are of applications are based on 3D-versions
and/or repetititive measurements during
continuous acquisition.
Practical limitations for balanced SSFP
will be determined by SAR. Optimum flip
angles are quite appreciable (αSSFP = 90° for
T2/T1=1). According to the earlier discussion
(see Fig. 9.6B) the practical limit of TR will
 Ultrafast and Parallel Imaging 123

be in the order of 1-3 ms at very low flip

angles. Since one k-line is acquired per TR,
the minimum total imaging time for trueFISP
will be in the order of 150-400 ms depending
on the matrix size of the image. T1-weighted
snapshot FLASH can be somewhat faster,
since the low flip angles can be realized at
shorter pulse duration. Additionally
snapshot FLASH does not necessarily
require a rewinder for the phase encoding
gradient.

Fast Imaging with Multipulse

Sequences: RARE, TSE, FSE
The possibility to move from one k-line to
the next by means of a 180°-refocusing pulse Fig. 9.13: High resolution hyperecho TSE (0.5 x 0.5
x 1.5 mm) acquired at 3T (Siemens Magnetom Trio)
has already been mentioned in the original
EPI-paper by Mansfield4 and called RF-EPI.
Practical implementations were unsuccessful,
however, due to the generation of multiple
refocusing pathways, whenever the
refocusing flip angle is not exactly equal to
180°. The resultant spurious echoes generate
quite severe image artifacts. This problem
was solved by the introduction of the phase
encoding rewinder gradient, which brings
magnetization back to the same point in k-
space before each refocusing pulse.33 This
and the introduction of actively shielded
Fig. 9.14: Single shot TSE (HASTE) acquired at 3T
gradient systems, which take care of (Siemens Magnetom Trio)
unwanted signal dephasing due to eddy
current, has enabled the use of RARE (TSE, For application in ultrafast imaging, single
FSE.) for T2-weighted imaging. Normally shot RARE can be applied with echo spacing
high-resolution imaging can be performed in the order of around 4 ms (see Fig. 9.14).
with acquisition times of 1-5 min at In order to minimize T2-dependant signal
extremely high image quality (see Fig. 9.13). loss, such single shot applications are
This and the inherent T2-contrast, which is normally performed with partial Fourier
sensitive to many pathological tissue encoding, such that the center of k-space is
conditions including tumors and inflam- acquired early in the echo-train. Such
matory disease has made RARE(TSE,FSE.) single shot HASTE-acquisitions show very
to the diagnostically most useful MR imaging decent image quality with very good SNR
sequence today. and no artifacts. From Fig. 9.6A it is
124 Biomedical Magnetic Resonance: Proceedings of the International Workshop
clear, that quite appreciable waiting times
have to be accepted between successive
applications of HASTE especially when 180°-
refocusing pulses are being used. Luckily, it
has been shown early on, that the refocusing
flip angle can be quite considerably reduced
with only a minor penalty in SNR. For
constant refocusing flip angles SNR will still
be 50 percent at α = 60° or 1/9th of the
original RF power (see Eq.6).
The very benign and robust signal
behavior and the excellent tissue contrast
make RARE (TSE, FSE…) very attractive
especially in the rapidly developing field of
high field imaging at 3 T or even higher. At
the same time, SAR dramatically increases
with field strength. Consequently, the
development of strategies to optimize RARE
(TSE, FSE…) without any signal loss has
found widespread interest. The hyperecho-
technique has been introduced first as a very
generic way to recover fill magnetization
after arbitrary pulse sequences.34 Applied
to RARE (TSE, FSE…) it can be used to
retrieve the full SNR at the particular echo
used for encoding the k-space center. In
recent years further improvements leading
to asymmetric Hyperechoes (called TRAPS
= TRAnsition into Pseudo Steady state)
have been developed, which allow to
dramatically reduce SNR by a factor of 3-5
for conventional multishot applications and
up to a factor of 10 for single shot HASTE/
RARE.35 By calculating flip angles such, that
the envelope of the echo train assumes a
Gaussian shape, this will not only improve
SAR, but also lead to a better point-spread
function and thus to improved image
quality.36 It has been shown, that an overly
fast variation of the refocusing flip angles
will lead to a loss in image resolution,37 but
this can be easily avoided by use of a careful
and proper implementation.
The necessity to come back to the same
point in k-space before each refocusing pulse
plus the finite duration of such pulses leads
to a quite significant ‘overhead’ in terms of
the time efficiency of the experiment.
Therefore it is clear, that RARE (TSE,
FSE…) is not well-suited for ultrafast,
repetitive measurements. On the other hand,
the extremely benign signal behavior leads
to very high image quality even at
moderately fast implementations with
acquisition times in the order of 250-500
ms. This very robust signal behavior allows
the application everywhere in the body and
not just for head imaging.
BEYOND THE LIMITS: PARALLEL
IMAGING
From the earlier discussions it seems like
the development of fast MRI has reached a
status of completion, where even dramatic
further increase in gradients and RF
performance will lead to only minor
improvements in imaging speed. Therefore
image acquisition in times below 10-20 ms
per 2D-slice appear to be infeasible at least
for imaging in humans. These limitations
have been shown earlier to be of a rather
fundamental nature, which can not be
overcome by further hardware development
or by inventing just another sequence.
Whenever fundamental limits are
reached, it is wise to question the underlying
concepts. The basic cornerstone of MR
imaging is spatial encoding by gradients and
sampling the signal sequentially while going
around k-space. It has been known for quite
sometime, that the spatial selectivity of coils
can be used to discriminate signals from
different parts of an object even during
simultaneous data sampling. The real
breakthrough to parallel imaging started,

however, with the introduction of the
SMASH-technique by Sodickson.38 The basic
concept is rather simple at least in
retrospective. If coils with approximate
sinusoidal sensitivity are put together, than
different linear combinations of the signals
sampled separately can be generated, which
correspond to different lines in k-space. The
reduction in the number of lines, which have
to be consecutively sampled, is reduced in
proportion to the number of separate coils.
In the basic concept the field of view is
determined and fixed by the hardware of
the coil arangement. Following the original
developments, various improvements like
GRAPPA,39 autoSMASH40 and others have
been found leading to a more general way
to simultaneously acquire multiple k-lines
without the inherent limitations of SMASH.
On a parallel track and immediately
following the original presentation of
SMASH, the SENSE-approach has been
introduced by K.Pruessmann.41 SENSE also
is based on data acquisition using separate
coils with different spatial sensitivity. Data
recombination is, however, performed in the
image domain rather than on the k-space
data. Various improvements have been
found for SENSE also.842-45 Maybe the most
important current development in parallel
imaging is related to the fact, that parallel
data acquisition strategies can not only be
applied in the spatial domain. The
algorithms can also be used to improve
sequential data acquisition in a technique
called k-t-BLAST.46 It can indeed be shown,
that parallel techniques are expecially well
suited to be applied along data, which do
not show excessive changes along one
particular domain in the dataset. In k-t-
BLAST this scarcely sampled domain is the
time domain for the sequential observation
of images of moving objects, where typical
Ultrafast and Parallel Imaging 125
changes affect only a fraction of the total
data. An even more extreme example is
found in mapping of the magnetic field by
sequential acquisition of EPI-images with
different echo times, where parallel
reconstruction factors by a factor of 4-8 can
be easily realized without any loss in
information content.19
In imaging applications the improvement
in imaging speed the reduction factor
depends on the number of independent
coils, which is always smaller than the
number of coils actually used. Strong
overlap between the sensitivity profiles of
the coils will lead to a redundancy in the
data and thus to errors in the coefficients
used to separate the acquired signals. This
will lead to increased noise but also to
artifacts caused by signal misattribution. On
the other hand, strong separation between
sensitivity profiles will improve signal
separation, but at the cost of reduced SNR.
At the higher frequencies at higher fields,
the separation of the coil sensitivies can be
shown to improve and higher reduction
factors become feasible. In general parallel
imaging will suffer from a penalty in SNR,
which at best is proportional to the square
root of the reduction factor. In a very
elegant paper, the theoretical limits of SNR
to be gained by parallel imaging have
already been established.48 These limits are,
however, based on data acquisition with
equal weights for all data points. Except for
steady state sequences like FLASH and
trueFISP, many fast imaging techniques used
today are based on the acquisition of a
signal, which decays with T2* (EPI, SPirals)
or T2 (TSE). Parallel imaging will reduce
the length of the echotrain. As shown in
Fig. 9.15 this will lead to discarding low
intensity signals at the end of the echotrain.
For an echo train length corresponding to 2
126 Biomedical Magnetic Resonance: Proceedings of the International Workshop
T2, reducing the acquisition by one half will
already lead to a slight increase in SNR.
Depending on the decay constant, SNR can
thus even improve with parallel imaging
approaches. This is illustrated in Fig. 9.16,
where parallel acquisition for hyperecho TSE
with considerably reduced number of data
leads to improvement in SNR by more than
50 percent.47 In practical terms the reduced
number of data will not only improve SNR,
but lead to a further reduction in SAR, which
Fig. 9.15: Effect of reducing the length of the echotrain
by parallel imaging. SNR is proportional to the square
root of the are under the curve. At sufficiently fast
signal decay, SNR can thus increase, when the less
important data at the end of the echotrain are
discarded (shaded area)
Fig. 9.16: TSE image with full acquisition (left)
compared to an image acquired with GRAPPA at a
reduction factor of 2 (right). Image resolution is
identical. For deep gray matter SNR is improved by >
50% with parallel imaging
is of particular interest especially in high
field MR as discussed above.
Similar arguments also apply to other
single shot techniques like EPI and spiral
imaging, where the reduced acquisition
window leads not only to an improvement
in SNR, but also to much better image
quality as shown in Fig. 9.10.
The number of coils to be used in parallel
imaging will increase with decreasing coil
size compared to the observed field of view.
In the extreme case, N × M independent
and very small coils can be used to acquire
a N × M image, where the signal in each
pixel is uniquely defined by the signal in
one coil. Such a massive parallel imaging
approach has already been demonstrated in
principle.49,50 The penetration depth of such
coil arrays will be in the order of the size
of a single coil, so medical applications may
be limited to the surface of the body. In
terms of the ultimate technical limits, the
imaging speed of such massive parallel
imaging arrays are determined by the
sampling speed. Theoretically a ‘shutter
speed’ in microsecond range could be
achieved by reconstructing one image per
sampling point. The SNR of such
implementations will, however, be a severe
challenge.
CONCLUSION
The limits of imaging speed are in the order
of a few 10th of milliseconds, when
conventional principles of gradient encoded
Fourier imaging are being used. Parallel
imaging approaches allow further reduction
of the image acquisition time. In the ultimate
limit using as many RF coils as there are
data points in the image, imaging times in
the microsecond regime could be realized
but with coil arrays being sensitive only at

their immediate surface. The ultimate and
realistic limits of fast imaging will be
determined by SNR. For medical imaging
further ‘lateral thinking’ will be required to
escape the boundary conditions of current
imaging techniques. Hyperpolarization with
the possibility to increase SNR by several
orders of magnitude may offer totally new
opportunities to redefine imaging speed in
future MR applications.51,52
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29. Preston AR, Thomason ME, Ochsner KN,
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30. see Proc. XIIth Ann.Meeting ISMRM, Kyoto,
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31. Scheffler K. A pictorial description of Steady-
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the signal behavior in the transition to driven
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33. Hennig J, Nauerth, A. et Friedburg, H: RARE
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34. Hennig J, Scheffler K. Hyperechoes, Magnet
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35. Hennig J, Speck O, Scheffler K. Optimization of
the signal behavior in the transition to driven
equilibrium in steady state free precession
sequences, Magn Reson Med 2002;48:801-09.
36. Hennig J, Weigel M, Scheffler K. Multiecho
sequences with variable refocusing Flip Angles:
Optimization of Signal Behavior using smooth
Transitions between Pseudo Steady States
(TRAPS), Magn Reson Med 2003;49:527-35.
37. Busse RF. Reduced RF power without blurring:
correcting for modulation of refocusing flip
angle in FSE sequences. Magn Reson Med
2004;51:1031-37.
38. Sodickson DK, Manning WJ. Simultaneous
acquisition of spatial harmonics (SMASH): Fast
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39. Griswold MA, Jakob PM, Heidemann RM,
Nittka M, Jellus V, Wang J, Kiefer B, Haase A.
Generalized autocalibrating partially parallel
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Sodickson DK. AUTO-SMASH: A self-
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Simultaneous Acquisition of Spatial Harmonics.
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43. Weiger M, Pruessmann KP, Boesiger P. 2D
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44. Pruessmann KP, Weiger M, Bornert P, Boesiger
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45. Weiger M, Pruessmann KP, Osterbauer R,
Bornert P, Boesiger P, Jezzard P. Sensitivity-
encoded single-shot spiral imaging for reduced
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46. Tsao J, Boesiger P, Pruessmann KP. k-t BLAST
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Magn Reson Med 2003;50:1031-42.
47. J. Hennig, M. Weigel, T. Thiel Optimizing SAR-
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Electrodynamics and ultimate SNR in parallel
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10158-163.

fMRI: Neuroscientific and
Clinical Applications
INTRODUCTION
Activation of cortical areas is accompanied
by a depolarization of neurone membrane
potentials. Maintaining and re-establishing
this potential requires an increased supply
of energy and oxygen, which causes increa-
sed blood flow and oxygen use in these
activated cerebral regions. Neuronal activa-
tion leads to more increase in local blood
flow than expected from oxygen consump-
tion, and thus to an increase of oxygenated
hemoglobin in the capillaries of activated
brain tissue. These changes of the local blood
supply and the magnetic properties of blood
can be visualized via magnetic resonance
imaging (MRI). Over a decade ago Ogawa1
and Turner et al2 were the first to show
that changes in oxygenation of blood lead
to MRI signal changes caused by alterations
in the magnetic susceptibility of blood
during deoxygenation. This functional mag-
netic resonance imaging (fMRI) usually called
BOLD (blood oxygenation level dependent)
imaging was first exploited by Kwong et al
in 19913 for activation of cortical regions
during visual stimulation. Since then fMRI
has rapidly grown so that today numerous
studies are performed not only to assess
10
Wolfgang Grodd
normal human brain functions with sensory,
motor, and emotional tasks but also during
cognitive processing and furthermore to
elucidate changes in localization, organiza-
tion and connectivity in a variety of inborn
and acquired brain diseases. 4-8 In this
presentation some fMRI studies on the orga-
nization and reorganization of the sensori-
motor system and on language production
in normal subjects and in children with early
acquired unilateral brain lesions will be
discussed, to demonstrate its neuroscientific
and clinical value. However, before starting
with these results some practical issues
concerning the realization of fMRI exami-
nations will be addressed.
PRACTICAL CONSIDERATIONS
In order to successfully perform a functional
MRI examination, initially it should be asked
whether the chosen experimental design and
tasks can easily be performed within the
magnetic field of the MR apparatus. Second-
ly, the study design (block, parametric and/
or event-related design etc.) and protocol
should be determined in detail including a
definition of activation and rest periods and
an appropriate imaging regimen (including
130 Biomedical Magnetic Resonance: Proceedings of the International Workshop
imaging sequences, matrix size, number of
slices, number of acquisitions, number of
activation and rest periods, etc.). In addition,
special attention is required to ensure that
any additional experimental equipment does
not lead to imaging artefacts. One should
keep in mind that electronically applied
stimulation signals may be distorted by the
MR tomograph (MRT). For visual
stimulation, the subject can view pictures or
moves during the measurements, which may
be projected onto a screen to the subject via
a mirror or prismatic glass (Fig. 10.1, Plate
7). Photic stimulation can also be provided
by using special stereo-goggles where each
eye piece displays an image via LCD or
fibre optics. Commercial MRI systems are
equipped with speakers or headphones
which can be used for auditory stimulation.
With respect to acoustic stimuli, i.e. the
acquisition of vocal utterances by the patient
or volunteer, the measurement noises
generated by the tomograph, especially with
EPI measurements, may mask the sound.
Sensory stimuli such as tactile examination
of the skin may present problems in that
most of the electromagnetic components of
a stimuli apparatus cannot be used in the
magnetic and electromagnetic field of the
tomograph. In addition to these passive
sensory stimuli, patients or volunteers can
also be instructed to perform certain tasks
in order to cause stimulation. The simplest
methods are to execute certain defined
movements, i.e. hand or finger movements;
however, cognitive tasks are suitable as well.
As a rule, the volunteer should repeatedly
perform a task during acquisition to
accurately determine the difference in signal
between rest and activation phases, thus
reducing the effect of random signal
fluctuation. For simple motor tasks, the
instructions may be given verbally over the
intercom of the MRI. Alternatively, it is
frequently more practical to use visually
projected texts or symbols for instructing
the volunteer or patient. Finally, the subject
or patient must carefully and comfortable
be positioned within the scanner. Figure
10.1, Plate 7 illustrates such an experimental
setup with a visual presentation for
performing a cognitive task. First, a picture
is generated on a personal computer (PC)
using a stimuli presentation program. Then
a LCD video projector is used to project the
picture on a screen at the foot end of the
patient table. The picture is projected upside
down, so that the patient in the tomograph
can view it right side up via a reversing
mirror. Finally, the projector is enclosed in
a wire cage to reduce interference during
image generation.
To control the timing of the applied
sequence, it is recommended to use a
separate PC which receives and registers
the trigger signals from the tomograph
indicating the start of each measurement.
This registration allows synchronising the
measurement sequence with the stimulus
presentation. The diagram in Figure 10.1,
Plate 7 shows that the interaction of MRT
with the subject is based on recording the
physiological responses during the measure-
ments. Therefore, the subject should always
be able to communicate with the examiner,
e.g. to signal the recognition of a word in
a memory test. The easiest method is the
use of a key switch with fibre optic cable
connection. In such case, it is important to
remember that the motor task has to be
performed also during rest phases, so that
the results are not masked by the primary
motor activation. The key signals should be
recorded and saved by a PC via an electro-
optic transducer. Using the MRI trigger
signals and the sequence of images pre-
 fMRI: Neuroscientific and Clinical Applications
sented, an electronic protocol can be gene-
rated. In addition, cardiac and respiratory
responses may be collected to indicate
whether a certain picture or stimulus has
been recognised during an examination.
APPLICATIONS
fMRI of Sensorimotor Somatotopy
In 1873 Jackson9 was the first to postulate
that distinct cortical activation centers serve
for different types of movements after
having observed isolated movements of
different body parts during epileptic convul-
sions. He hypothesized that the size of the
cortical area which represents movements,
is not proportional to the size and strength
of the muscle group involved but to the
degree of differentiation and specialization
for the type of movement. Since then
numerous experimental studies were
performed and a functional differentiation
of pre- and post-central gyri was achieved
by Sherrington and coworkers.10 In 1936-37
Foerster as well as Penfield and Boldrey
reported a somatotopic order of movements
in the human precentral gyrus comparable
to the somatotopic representation of somato-
sensory stimuli in the postcentral gyrus.11,12
Later more precise electrical stimulation in
monkeys13,14 suggested that the concept of
a somatotopic organization of the primary
motor cortex (M1) is probably only adequate
in a gross approximation. The representation
areas of finger and hand movements in M1
show strong overlap indicating that
movements of different representational
sites share atleast some neural substrates.
Similar functional imaging studies
showed a high overlap of the represen-
tational sites in M1.15,16 Kleinschmidt et al17
showed that subtraction of maps of
individual finger movements from those of
131
the other fingers yielded a somatotopic
representation of the individual fingers and
concluded that somatotopy within the hand
area of M1 is represented as “quantitative
predominance of certain movement or digit
representation embedded in an overall joint
hand area”. Using PET Grafton et al 18
reported distinct activation maxima in M1
of shoulder, elbow, wrist, and finger
movements if an individual analysis was
performed, but this distinction was lost after
grouped analysis. Therefore we performed
an fMRI study to map foot, elbow, fist,
thumb, index finger and lip movements in
30 healthy subjects. For each movement type
confidence intervals of representational sites
in M1 were evaluated.19 In order to improve
the precision of anatomical location and to
optimize the mapping of cortical activation
sites, we compared the assessment of
locations in the conventional 3D-system and
an own 2D-projection method. In addition
to the use of activation maxima the location
was determined by the center of gravity of
the activation cluster within the precentral
gyrus (Fig. 10.2, Plate 8). Both methods
showed a high overlap of their confidence
intervals but only the 2D-projection method
revealed statistically significant distinct
locations for the elbow and index finger
movements and for index finger to thumb
movements. Increased degree of complexity
of finger movements resulted in a spreading
of the location in the direction of the arm
(Fig. 10.3, Plate 8).
fMRI of Speech Production
An important neuroscientific field is the
study of language processing and compre-
hension in the cognitive and speech motor
domain. Commonly cortical areas like of the
left inferior frontal lobe (Broca´s area), the
132 Biomedical Magnetic Resonance: Proceedings of the International Workshop
left superior temporal lobe (Wernicke´s
area), and the primary motor cortex bila-
terally are primarily attributed to language
functions. But by disentangling syntax,
semantic and prosody aspects in respect to
speech motor control yielded in a more
refined view. Penfield and Rasmussen
already reported on arrest of vocalization
and speech after left as well as right hemis-
pheric electrical stimulation.20 To evaluate
lateralization of speech production at the
level of the primary motor cortex an fMRI
examination was performed during a speech
task (continuous silent recitation of the
names of the months of the year). As control
conditions, non-speech tongue movements
and silent singing of a well-known melody
with the syllable ‘la’ as its carrier were
considered.21 Tongue movements produced
symmetrical activation at the lower primary
motor cortex. During automatic speech a
strong functional lateralization to the left
hemisphere emerged within the same area.
In contrast, singing yielded a predominant
right-sided activation of the motor region
(Fig. 10.4). Functional lateralization of speech
production therefore seems to include the
pre-central gyrus as well as Broca´s area.
Cortical Reorganization in Unilateral
Brain Lesions
Reorganization of motor and language
functions after early brain injuries is not
only determined by the maturational stage
of the central nervous system at the time of
the insult (“timing”), but also by the struc-
tural properties, location, and extent of the
lesion. To address the impact of different
lesion extents on the type of reorganization
induced we examined a cohort of patients
with lesions of uniform structure and
location (unilateral periventricular defects)
and similar timing (early 3rd trimester of
pregnancy) by fMRI.22 Twelve young adult
patients with congenital hemiparesis (Fig.
10.5) due to unilateral lesions in the
periventricular white matter and without
any history of epileptic seizures were studied
as well as 10 age-matched controls. The goal
was to examine the localization of the motor
system and of language processing especially
in respect to its motor part, the speech pro-
duction.23 The severity of structural damage
to hand motor projections of the cortico-
spinal tract was assessed on semi-coronal
MRI reconstructions along anatomical
landmarks of cortico-spinal tract (Fig. 10.6).
The functional integrity of these crossed
cortico-spinal projections in the affected
hemisphere, as well as the presence of any
abnormal ipsilateral projections to the paretic
hand, was examined by transcranial mag-
netic stimulation (TMS). Cortical activation
during simple voluntary hand movements
was studied by functional magnetic
resonance imaging (fMRI).
Motor Function
The severity of damage to cortico-spinal
projections of the pyramidal tract, as
assessed on semi-coronal reconstructions,
correlated with motor impairment of the
paretic hands, and also with the type of
cortico-spinal tract reorganization. Patients
with small lesions (SL; n = 4) and only mild
hand motor impairment possessed intact
crossed cortico-spinal projections to the
paretic hand (TMS), whereas in those with
large lesions (LL; n = 6) and more severe
hand motor impairment, no motor response
could be elicited by TMS of the affected
hemisphere. Evidence for compensatory
recruitment of the unaffected hemisphere
was found in both subgroups: In the SL
 fMRI: Neuroscientific and Clinical Applications 133

Fig. 10.4: Top: Axial T1-weighted images with individual activation clusters at the level of the basal ganglia in
one representative subject during left aprosodic (automatic) speech, middle vertical tongue movement and
right during syllable singing. Note the left hemispheric activation during speech (regions 3,4 and 5), and the
right hemispheric activation during singing (area 4 and 5). Bottom: Corresponding group results of the
distribution of activated voxels in left and right area 4 and 5 from 10 subjects

group, fMRI demonstrated ipsilateral
activation of premotor areas, without any
abnormal projections to the paretic hand
originating from these sites (TMS). In the
LL group, such abnormal ipsilateral
projections to the paretic hand were indeed
found (TMS), and fMRI confirmed cortical
activation of an abnormal ipsilateral hand
motor representation in the primary
sensorimotor region of the unaffected
hemisphere (Fig. 10.7). Two patients with
intermediate-sized lesions presented
combined features of both groups (SL, LL).
Language Function
In the second part of our study seven young
adult patients were studied. 24 All had
attended main-stream schools, without any
obvious cognitive or language deficits. The
cortical organization of language was
assessed by fMRI using two different
activation tasks: To monitor language
perception, a narrative story was presented.
For the assessment of language production,
134 Biomedical Magnetic Resonance: Proceedings of the International Workshop

Fig. 10.5: Axial T2-weighted images of 12 patients included in this study with right-sided
hemiparesis due to left-sided periventricular leukomalacia

Fig. 10.6: Reconstruction of semi-coronal plane along the course of hand motor projections, adopted from [2].
(A) Axial and b) parasagittal image with the hand knob identified by its omega shape (axial) and its hook sign
(inset in (B). Both sign were used for the upper landmark and the anterior third of the posterior limb of the
internal capsule (dashed line in (C)) as the lower landmark. (D) The semi-coronal plane of this patient shows
enlargement of the right lateral ventricle (black arrow) and hypointensity of the adjacent white matter, which
corresponds well with a moderate hand motor impairment (score 2)
 fMRI: Neuroscientific and Clinical Applications 135

Fig. 10.7: Top: Individual structural and functional MRI results (paretic hand movement) for two representative
patients from each group. Left side: Semi-coronal reconstructions (from T1-weighted 3D-data) following
anatomical landmarks of pyramidal tract somatotopy, illustrating the severity of structural damage to hand
motor projections. A white “figure-of-eight coil”-symbol indicates hemispheres from which MEP’s in the paretic
hand could be elicited. Right side: Individual fMRI activation patterns (SPM99, p <0.05 corrected) during
paretic hand movement. The functional data are displayed on individual surface reconstructions calculated
from normalized 3D-data sets. Bottom: Schematic illustration of cortico-spinal tract organization in the three
subgroups of patients (P = paretic hand), and TMS results from one representative patient from each group.
Vertical dashed line = time of the TMS stimulus

the subjects were instructed to silently next word starting with the last letter of
generate word chains, the first word starting the previous word, and so on. Hemispheric
with a letter given by the examiner, the asymmetries of fMRI activation for each task
136 Biomedical Magnetic Resonance: Proceedings of the International Workshop
were assessed by determining the number
of activated voxels (SPM99, p<0.05,
corrected for multiple comparisons) in each
cerebral hemisphere. From these voxel
counts, asymmetry ratios were calculated
as (R-L)/(R+L), yielding a range from –1 (=
exclusive left-hemisphere activation) to +1
(= exclusive right hemisphere activation).
The relationship between the lesion and the
facial portion of the pyramidal tract, we
used the same approach as depicted above
to correlate pyramidal tract lesions to motor
impairment. Neurons projecting to the lower
face are located in the lower half of the
central region; in the internal capsule, their
axons pass through the genu (Fig. 10.8, Plate
9). Therefore, the course of facial pyramidal
tract projections can be depicted on a coronal
reconstruction that includes these landmarks
(see Fig. 10.8D, Plate 9). The severity of
damage was quantified by measuring the
distance from the most lateral part of the
lesion to the interhemispheric fissure; again,
the correction for different brain sizes was
achieved by dividing this value by the
distance measured from the lateral wall of
the lateral ventricle in the intact hemisphere
(x/y in Fig. 10.8D, Plate 9). Figure 10.8A
displays the asymmetry ratios for our
patients and the controls. In the controls,
strong leftward asymmetries were observed
for language production (mean –0.930, SD
0.096), whereas the asymmetries for
language perception were weaker and more
variable (mean –0.264, SD 0.447). The
patients did not differ from the controls in
respect to the asymmetry of language
perception (mean –0.053, SD 0.259, p = 0.366,
Mann-Whitney); during language pro-
duction, however, all patients showed a
significant shift of activation to the undama-
ged right hemisphere compared with the
controls (mean +0.291, SD 0.572, p = 0.001).
Representative examples for the activation
patterns in one patient and one control are
given in Figure 10.8B, Plate 9. The degree
of rightward asymmetry during language
production correlated with the total lesion
volume (r = 0.750, p = 0.026, Spearman corre-
lation rank) and, more strongly, with the
lateral extent of the lesion in the recons-
tructed semi-coronal plane (r = 0.893, p =
0.003, Figure 10.8C, Plate 9).
CONCLUSION
The presented fMRI results on functional
anatomy of the motor system and of cortical
networks engaged in language production
as well as the detected cortical reorganiza-
tion after acquired brain lesions should
convince the reader that the fMRI method
can reliably be used to address neuroscienti-
fic questions and safely be applied in a
clinical setting. With the latter prospect we
might gain a deeper insight in neuronal
mechanism of functional reorganization and
compenzation after localized brain damage,
which will be of relevance for the design of
new therapies and rehabilitation regimens.
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138 Biomedical Magnetic Resonance: Proceedings of the International Workshop
Appropriate Functional Magnetic
Resonance Imaging for the
Study of Evolving Yogis
INTRODUCTION
Yoga is a system of physical of psycho-
spiritual practices in vogue in South Asian
sub-continent for more than three thousand
years. Currently, it is gaining global popu-
larity in view of its perceived benefits in
promoting the psycho-physical health and
well-being. There have been progressive
efforts to examine the efficacy and
significance of yoga practice by objective
scientific methods with variable but limited
success. However, truly comprehensive and
scientific studies on the phenomenon of yoga
have been slow to emerge due to the
predominantly subjective nature of yogic
experience as well as the scarcity of scientific
insights relevant to its biology. Non-avai-
lability of techno-scientific tools and concepts
appropriate for the study of yoga has been
another stumbling block in the scientific
study of yoga. However, a quantum leap;
out of that predicament is now being fuelled
by recent conceptual advancements in
neurobiology during the past “decade of
the brain”. Also, major advances in in vivo
brain imaging, made possible by techno-
11
N Kochupillai, V Nandini
logical advances in functional MRI (fMRI)
and PET scanning, have considerably
advanced the scope for in-depth scientific
studies on yoga. However, several specific
techno-scientific issues relevant to such
studies still remain to be defined and
resolved to frame meaningful questions and
answer them appropriately, about the
science of yoga. The important among such
issues include ‘neurobiological definition’ of
the psycho-physical correlates of yogic
progression in evolving yogis.
fMRI AND NEURO-BIOLOGIC DEFINITION
OF YOGIC PROGRESS
The most significant advancement in the
study of the functioning of the human brain
has been the development of the concept of
“Blood Oxygenation Level Dependent”
(BOLD) and its successful exploitation to
image functionally activated brain regions
through MRI.1,2 Ongoing developments in
the techno-science of fMRI seems to open
up3 hitherto unimagined prospects to study
human brain in situ to understand the
neurobiologic correlates of purely subjective
 Appropriate Functional Magnetic Resonance Imaging for the Study of Evolving Yogis
experiences such as sense-perception,
cognitive and affective behavioral states, as
well as differing states of consciousness
including sleep. These developments hold
the promise of meaningfully studying the
scientific dimensions of yogic practices and
their neurobiologic impact. Since changes in
neuronal activity evoked by behavioral
cognitive and affective states result in area
specific changes in oxygen consumption of
the brain, BOLD effects would occur in brain
as a result of yogic practice, both long-term
and short-term. On the basis of published
studies on yogis such changes may be global
or regional. By virtue of the nature of the
psycho-physical techniques use in different
types of yogic practices, several anatomically
and functionally identifiable regions of the
brain can undergo activation or suppression
during yog-sadhana. Asana, pranayama, and
dhyana can all, presumably, induce global
or focal activity shifts, in anatomically and
functionally indefinable regions of the brain.
However, such activities may involve any
part of the living human brain; large, small
or even microscopic-cortical, sub-cortical or
even the phylogenitically older hind-brain
regions! fMRI studies on practicing yogis,
in their differing states of yogic progression,
would therefore demand functional-imaging
competence of far greater sensitivity,
specificity and sophistication than what is
presently available. There are, however,
reasons to believe that progress in fMRI
would eventually succeed in all these and
would also succeed even in documenting,
and analyzing shifting connectivity as well
as dynamic temporal interactions between
brain regions, in evolving yogis. As and
when such possibilities emerge, studies on
practicing and evolving yogis would yield
new insights into the science of yoga, as a
psycho-spiritual practice that promote the
139
health and well-being of human mind and
body.
Subjective World of Yogic Experience
and its Neuro-biological Objectivisation
An effective approach to achieve objecti-
visation of the predominantly subjective
experience along the path of progress in
yoga is by nudging neurobiologists to adopt
clearly definable systems of yoga practice
and to try and understand their neuro-
biological correlates (basic). From such
understanding, studies can be designed to
answer specific questions. A striking
example of such an approach can be seen in
the case of ‘koodiyattom’ an art form which
is more than a thousand year old, and which
has been declared to be ‘Heritage of
Humanity’ by the UNESCO. In this art form
a yogically trained and attuned artist,
through a long and intense ‘dhyana’ like
‘involvement’ with an emotively gripping
theme from scriptures. He develop creative
abilities of artistic expression of that emotive
theme in what amounts to a supra human
achievement of willfully expressing through
subtle, incredibly varied and artistically
transformed ‘involuntary emotional expres-
sions’ through what is truly voluntary
movements of facial muscles! What one
experience in a ‘koodiyattam’ performance
by a yogically gifted artist like Ammannoor
Madhava Chakyar, is a sharing with Mr
Chakyar of an intensely felt personal and
yet ‘artistically transformed and voluntarily
expressed’ emotion through the universal
and visually expressed language of human
facial expression! Mr Chakyar’s language of
facial expressions of emotions has the
authenticity of involuntariness! Yet he can
summon these at will—as it where he is
recollecting the overwhelming emotion in
140 Biomedical Magnetic Resonance: Proceedings of the International Workshop
yogic tranquility, to borrow an expression
from Shakespeare! Obviously, Mr Chakyar,
through life long yoga and sadhana,
successfully erased the voluntary—
involuntary dichotomy in facial expression
and added a 3rd aesthetic dimension to the
expression language of face by transforming
it to artistic experience! Here we have an
excellent paradigm to study the yogically
engineered anatomy and physiology of
human facial expression, in all its psycho-
physical and even spiritual dimensions. But
before we can venture into it by studies
involving ‘koodiyattam’ artists at different
stages of their professional training, we need
to bridge gaps in the techno-scientific
approach to it, particularly in the realm of
imaging living and functioning human brain!
Brainstem: The Psychosomatic
Generator-cum-Switch Board of
Yogic Transformation
Those who go through yogic progression
with neurobiological insights can discern that
psycho-physical practices like pranayama
and dhyana are both energizing and
transforming in a psycho-somatic sense—
and that the major anatomical substrate for
such transformation is brain stem. Therefore,
a area that need to be studied to unravel
the biology of yoga would be the brain stem.
On the basis of known anatomy of the
various functionally and anatomically
discreate centers of the brain stem, one can
say that pixel size requirement to study the
functional anatomy of brain stem with fine
discrimination are not met, in a technical
sense by the currently available imaging
technology. The range of dimensional
specificity/requirement in this regard may
vary from few microns to several milli-
meters. On the basis of known and emerging
functional anatomy of brainstem techno-
scientific competence for such sensitive and
discriminant study of the living brain stem
would help unravel a variety of interesting
variations from the ‘normal’ in early
evolving yogis. Examples of such yogis
include the Tum-mo yoga trainees with the
neuro-biologically altered bodily thermo-
genesis and practitioners of pranayama
dhyana based Patanjali yoga and related to
psycho-physical training and evolution it is
not contextual or relevant here to elaborate
details in this regard. Suffice it to state that
the major impact of these practices on our
mind-body system has been scientifically
documented to some extent. Therefore,
functionally and anatomically definable sub-
cortical systems involved in these yogic
phenomena can be studied appropriately if
technical requirements of the type referred
to above are achievable to facilitate mini or
micro-functional imaging of brainstem of
evolving yogis. It is earnestly hoped that
imaging science would soon come up with
appropriate science and technology to permit
scientific studies on one of the most
enigmatic facets of the mind body gestalt
of man!
REFERENCES
1. Logothetis NK, Guggenberger H, Peled S, Paul
J. Functional imaging of the monkey brain.
Nature Neurosci 1999; 2: 555-62.
2. Kwong KK, Belliveau JW et al. Dynamic
magnetic resonance imaging of human brain
activity during primary sensory stimulation.
Proc Natl Acad Sci USA 1992; 89: 5675-79.
3. Grodd WG. Recent advances in functional MRI
In NR Jagannathan (Ed): Recent Advances in
MR Imaging and Spectroscopy. New Delhi:
Jaypee Brothers Medical Publishers (P) Ltd,
2005.

Perinatal MR Studies
in Animal Models
RJ Ordidge, DW Carmichael, X Wang, DM Peebles
INTRODUCTION
Perinatal hypoxia-ischemia (HI) remains an
important cause of death and permanent
disability, affecting 3 to 5 per 1000 live births
in the United Kingdom with subsequent
moderate or severe neonatal encephalopathy
(NE) in between 0.5 and 1 per 1000 live
births.1 NE is a major problem worldwide
as upto 60 percent of affected infants die
and atleast 25 percent of survivors suffer
long-term neurodevelopmental effects.2 It
has been shown that intrapartum hypoxia is
responsible for 10 percent of cases of cerebral
palsy.3,4
The understanding the processes of HI
damage and the development of potential
therapies for perinatal HI has largely been
left to University based researchers since
the relative number of individuals affected
is small in comparison with the major causes
of disability and morbidity, and the pharma-
ceutical industry make these the priority
areas for research and development. The
causes and effects on individual babies are
also highly variable and it is ethically
unacceptable to perform large scale trials
on these neonates. However, the effect of
injury can persist over the lifetime of the
individual involved and the cost of care can
Perinatal MR Studies in Animal Models 141
12
be considerable. Therefore, it is highly desir-
able that all potential therapies should be
appropriately tested. Because of these
reasons, the use of animal models has found
widespread application in developing
potential therapies for perinatal HI. Such
models provide the opportunity to create
reproducible injuries in groups of animals
that either receive or are denied therapy so
that the outcome can be determined with
the appropriate statistical significance. The
surgical procedures required to produce
reproducible HI injury of the animal brain
are time-consuming and post-injury the
animals require intensive care. Therefore, it
is particularly desirable to use the minimum
number of animals to achieve a statistically
significant result. Through the noninvasive
nature of in vivo MRI and MRS, it is possible
to follow the time-course of the progression
of injury in these animals thereby avoiding
the use of invasive procedures, such as
histopathology, that requires sacrifice of
animals at various stages through the injury
process and greatly increases the number
of animals required.
In this chapter, as an example of the use
of animal models, the role of a chicken
embryo model is described to monitor the
142 Biomedical Magnetic Resonance: Proceedings of the International Workshop
pre-birth injury process associated with
hypoxia in brain in vivo without the
confounding effects of maternal physiology.
Intrauterine growth restriction is associated
with newborn encephalopathy5 and may
increase susceptibility to perinatal hypoxia-
ischemia. Infection has also been shown to
be a major etiology in fetal brain injury5,6.
Therefore, the synergistic effects of hypoxia
and either growth retardation or pre-
treatment with a bacterial endotoxin are
evaluated using this model.
HYPOXIC INJURY IN CHICKEN
EMBRYO BRAIN
Effective hemodynamic and metabolic
compensation mechanisms are thought to
make the fetal brain less vulnerable to HI
than at later stages of development.7,8 These
mechanisms are important because their
failure, for example during severe HI during
labor, can result in brain injury with the
long-term consequence of cerebral palsy.3
The fetal hemodynamic response to
hypoxemia, defined as an insufficient
delivery of oxygen to the brain, has been
well characterized in humans, and more
recently, the chick embryo.9,10 However, less
is known about fetal cerebral metabolic
responses.
Therefore, proton magnetic resonance
spectroscopy (1H MRS) was used to measure
changes in cerebral metabolite concentration
during acute hypoxia in the chick embryo
at Day 19 of incubation. MR visible meta-
bolites of interest are the N-acetyl aspartate
(NAA) resonance which is only present in
viable neurons11 and, therefore, provides
an estimate of the concentration of such
cells, and the Lactate (Lac) resonance which
is a product of anaerobic metabolism and
also an alternative metabolic substrate for
the developing brain.12
Studies in the adult rat and neonatal
piglet show a close correlation between
reductions in apparent diffusion coefficient
(ADC) and Adenosine tri-phosphate (ATP)
(which is essential for cellular energy
metabolism), measured by 31P spectroscopy
or biochemically. 13,14 Hence, diffusion-
weighted MR imaging (DWI) was used to
measure the ADC of brain water before,
during and after the hypoxic episode to
provide a putative marker of cellular energy
failure.
T2*-weighted MRI is commonly used to
monitor cerebral deoxyhemoglobin levels in
activated human and animal brain regions
using the blood oxygenation level depen-
dent (BOLD) contrast effect.15 Therefore,
T 2 *-weighted MR imaging was used to
measure changes occurring during the
hypoxic episode.
Materials and Methods
Fertilized white leghorn chicken eggs were
incubated at 38.5º C and 50 to 60 percent
humidity. Studies were performed on
incubation day 19 (hatching occurs on day
21).
NMR Experimental Procedure
The chick embryo were anesthetised with
either 5.0 mg ketamine, dropped onto the
chorio-allantoic membrane through a hole
in the shell, or immoblized in a similar
fashion using 0.5 mg tubocurarine one hour
prior to the start of the insult. A non-
magnetic thermistor wire (Luxtron) was
positioned for continuous monitoring of
temperature in vivo. The egg was placed
into a plastic bag, adapted with an inlet
and an outlet tubing system for gas supply
and exhaust, through which room air was
circulated (5 litres per minute). Inside the

bag, the egg was placed onto a 7 × 5.5 cm
(elliptical) surface coil and temperature was
regulated to maintain the eggs at approxi-
mately 35 to 36°C (either by circulation of
heated air or heated water through piping).
The arrange-ment was then placed within
the magnet bore of a 7 Tesla Bruker Biospec
imaging spectrometer (Bruker Medical
GmbH, Ettlingen, Germany).
MRI and MRS
Multi-slice gradient - echo MR scout images
were obtained in three orthogonal planes
to determine the position of the chicks head
within the egg. Baseline MR measurements
were taken prior to the insult, then data
acquired throughout the duration of the
insult and post-insult. The egg and bag were
then transferred to a 2.5 cm diameter
circular surface coil with the head placed
immediately over the coil center. Global
shimming was performed and further
gradient-echo scout images acquired to
establish geometrical locations for the
subsequent acquisitions. Volume-localized
1
H MRS spectra were acquired using a point-
resolved spectroscopy sequence (PRESS).16,17
Protocol A
This was used to collect high quality 1H
MRS. PRESS was implemented with an echo
time (TE) of 270 ms, repetition time (TR)
5 s, and the spectra were obtained from a
6 × 6 × 6 mm3 volume containing mostly
tissue from the cerebral hemispheres. A
signal average of 256 echoes was used for
the baseline measurement (total acquisition
time 21.3 minutes), and 128 echoes was used
in subsequent acquisitions (total acquisition
time 10.7 minutes).
Protocol B
This was used to investigate the acute
Perinatal MR Studies in Animal Models 143
cerebral metabolic response and ADC/T2*
changes that accompany hypoxia. 1H-MRS
were obtained using PRESS with an echo
time (TE) of 135 ms, repetition time (TR) of
2 s, and the spectra were obtained from a
4 × 4 × 4 mm3 volume.
ADC measurements were obtained using
spin-echo EPI (four images, one with no
diffusion weighting (b~0) and three with
diffusion weighting (b = 875) applied along
the three Cartesian axes; TE = 110 ms, TR
= 2.5 s, 128 × 128 matrix; 3.5 cm field of
view). ADC maps were calculated by
performing a log/linear fit for each image
pixel and combined to obtain the trace ADC
value.
Absolute T2* values were measured using
the fast low angle single shot (FLASH)
method with echo times of 6.3, 23.4 and
33.4ms (128 × 128 matrix, TR = 100 ms, field
of view = 3.5 cm; total scan time for one
image = 12 s; one average for 6.3 ms echo
time, three averages for 23.4 and 33.4 ms
echo times).
The spectra were analyzed using an
automated linear combination modeling
method, whereby in vivo spectrum are fitted
with a weighted sum of spectra from in
vitro solutions of the compounds (the basis
set). The basis set was acquired using the
same pulse sequence, echo time, and under
the same conditions as in the in vivo spec-
trum, to ensure that effects of J-modulation,
eddy currents, and partial volume are
modelled correctly.18,19 The spectral data of
choline (Cho), NAA, Lac, alanine, taurine,
beta-hydroxy butyrate, asparatate, gluta-
mate, glutamine, inositol, taurine and GABA
(gamma amino butyrate) were analyzed.
Hypoxia and Recovery
After collection of baseline 1H spectra and
ADC images the oxygen concentration of
the gas supply to the egg was lowered to a
144 Biomedical Magnetic Resonance: Proceedings of the International Workshop
value of either 4 or 8 percent of fractional
inspired oxygen content (FiO2), as measured
by an oxygen analyzer (Servomex 570A,
Sussex, UK), by mixing with nitrogen.
Hypoxia was maintained for a period of
either 40 or 60 minutes, followed by a return
to normoxia for two hours. During hypoxia
and recovery 1 H spectra, T 2* -weighted
images and ADC images (each taking 10
minutes to collect) were measured as
required by the study. A group of ‘control’
embryos were subjected to the same
protocol but remained in normoxia for the
duration of the experiment.
Validation Studies
In a separate group of embryos that were
not placed in the magnet, but otherwise
maintained in identical conditions, blood was
sampled from the chorioallantoic artery for
measurement of pH, PO2, PCO2 and lactate
during either normoxia or hypoxia (FiO2 =
8 percent for 40 minutes). The results clearly
demonstrated that the correct conditions
were present during the insult where cellular
energy metabolism would be expected to
be severely compromised.
RESULTS OF MR STUDIES
Transient Mild Hypoxia Studied by 1H MRS
Nine unhatched chicken embryos (incuba-
tion day 19) were anesthetized with
ketamine (as above) and maintained at
constant temperature (35 - 36°C). Baseline
1 H MRS was performed according to
protocol A and seven embryos were studied
during 40 minutes of hypoxia (FiO2 = 8%)
followed by 2 hours of recovery (normoxia).
Two embryos were studied as controls.
Seven embryos survived the examination
period; two died during hypoxia.
1H Spectroscopy
Spectra were well resolved and had
excellent signal-to-noise ratio (see Fig. 12.1).
In the baseline data prominent peaks could
be observed corresponding to choline (Cho),
creatine and phosphocreatine (Cr) and NAA
together with numerous other resonances.
Particularly noteworthy are the relatively
high levels of Lac observed under normal
conditions (see Discussion).
In all embryos, hypoxia instigated a
significant rise in cerebral Lac/NAA which
returned to baseline during recovery, as
demonstrated by the hypoxia spectrum in
Figure 12.1.
Conclusion
1H MRS shows great sensitivity in detecting
the consequences of cerebral hypoxia in the
chicken embryo. Subsequent histopathology
studies revealed that there was no
detectable neuronal damage caused by the
mild hypoxic insult used in this study.
MR Changes in Cerebral Metabolism
During Transient Acute Mild/Moderate
Hypoxia in the Chick Embryo
The fetal brain is known to be less vulne-
rable to hypoxia-ischemia than at later stages
Fig. 12.1: Cerebral 1H spectra (TE = 270 ms) from
the brain of a 19 day chick embryo in vivo at baseline
and following 30 minutes of hypoxia. Cho = choline;
Cr = creatine and phosphocreatine; NAA = N-acetyl-
aspartate; Lac = lactate

of development. Acute hypoxia (FiO2 = 8%
for 40 minutes, as discussed earlier) caused
an increase in intracerebral lactate in chick
embryos at day 19 of incubation.20 There-
fore, we wished to establish whether
compensatory mechanisms can be over-
whelmed by a global hypoxic insult with a
failure of cellular energy homeostasis and
confirmed cell death (via histopathology).
On incubation day-19, ten embryos recei-
ved 0.5 mg of tubocurarine followed by 60
minutes of hypoxia (FiO2 = 4%). MRI/MRS
measurements of 1H MRS, ADC (measured
using EPI) and T2* were performed using
protocol B, which can be repeated entirely
every 15 minutes. Histology was performed
on a second group that received the same
hypoxic insult.
Figure 12.2 shows that hypoxia was
associated with a significant rise in the Lac/
Cr ratio from 0.3 ± 0.3 to 4.3 ± 1.1 (mean ±
SD) by the end of 1 hour hypoxia. The
NAA/Cr ratio was not significantly changed
throughout the procedure. In most animals
lactate was completely cleared by 120-
Fig. 12.2: Metabolite response versus time of cerebral lactate/creatine and NAA/creatine ratios for one
hour of hypoxia (shown by bar) with FiO2 = 4% (n = 10)
Perinatal MR Studies in Animal Models 145
minute post-hypoxia. Figure 12.3 (graph at
bottom) shows that a reduction in the
average trace ADC values of 10 ± 5 % (mean
± SD) was consistently measured during
hypoxia, revealing changes to the cellular
energy statue of the embryos. Upon return
to normoxia the ADC values returned to
baseline within 30 minutes. Representative
ADC trace maps are shown for three time
ponts and clearly show a reduction in the
chick embryo brain during hypoxia. Figure
12.3 (graph at bottom) shows that the change
in T2* value followed a similar time course
to the changes in ADC, with a maximal
reduction of 31 ± 19 percent, however, the
response appeared to be faster than the ADC
response. On return to normoxia, a slightly
increased T2* compared to baseline persists
upto 30 minutes post-insult which may
indicate hyperperfusion following hypoxia.
Also in Figure 12.4, representative T 2 *-
weighted images (FLASH, TE = 23.4 ms)
are shown acquired before, during and after
hypoxia and clearly show a reduction in
signal amplitude during hypoxia.
146 Biomedical Magnetic Resonance: Proceedings of the International Workshop
Fig. 12.3: Temporal response of cerebral trace ADC
(lower graph) within the embryonic brain, before
during and after hypoxia (one hour; FiO2 = 4 percent;
n = 10). The upper three images show actual trace
ADC maps with a visible decline during the hypoxic
period
Histology did not show any significant
neuronal damage in chicks that had been
subject to hypoxia with FiO2 = 4 percent
(although a small amount of apoptosis
occurs naturally in perinatal brain).
Conclusion
Compared with a milder insult, mild/mode-
rate acute hypoxia caused a decrease in
ADC, compatible with a depletion in high-
energy metabolites and T2* values suggest
hyperperfusion post-hypoxia. However, the
results from MRI/MRS suggest while acute
hypoxia (with FiO2 = 4 percent) caused
metabolic disturbances and cellular energy
deficits it constitutes a relatively mild insult.
Histology data confirmed that the brain cells
could repair and recover from this injury,
as no significant neuronal damage was
observed.
The advantage of acquiring ADC trace
maps is that they provide excellent spatial
resolution that could better define regional
Fig. 12.4: Temporal response of change in cerebral
T2* (lower graph) within the embryonic brain, before
during and after hypoxia (one hour; FiO2 = 4%; n =
10). The upper three images show T2*-weighted
images (TE = 23.4 ms) with a visible reduction in
signal amplitude during the hypoxic period
changes compared with 1H MRS. In neonatal
piglet brain, a correlation was demonstrated
between reduced cerebral ADC and loss of
high energy phosphorous metabolites. 21
Therefore, reduced ADC is likely to offer a
similar surrogate index of cellular energy
failure in this model.
1
H MRS OF TRANSIENT CEREBRAL
HYPOXIA IN A CHICKEN EMBRYO MODEL
OF FETAL GROWTH RETARDATION
Intrauterine growth restriction may increase
cerebral damage susceptibility to perinatal
hypoxia-ischemia. On the basis of the
previous study, where 1H MRS was shown
to be a sensitive marker for hypoxic injury,
the chicken embryo model was used to study
the effects of chronic hypoxia and protein
insufficiency during incubation upon 1 H
metabolite levels. In vivo MRS was also used
to compare the cerebral metabolic response
to acute hypoxia in growth-restricted (GR)
with that observed in normally-developed
chicks.

Forty-five eggs were incubated for 19
days. In 20 embryos, growth-restriction was
induced by removing 10 percent of the total
albumen on the first day of incubation and
reducing ambient oxygen concentration to
14 percent from day 10 until the time of
study. Twenty-five embryos were incubated
normally.
At day 19, baseline measurements 1H
spectra were obtained with normal hypoxic
conditions, and then hypoxia was induced
in 11 of the normally grown and eight of
the GR embryos by reducing the ambient
FiO2 level to 8 percent for 40 minutes.
Protocol A was followed to acquire high
quality 1H spectra. Following restoration of
normoxia, observations continued for a
further two hours.
Figure 12.5 shows, on the right-side
graph, normal and GR baseline peak area
ratios of NAA, inositol (In), and, on the
left-side graph, a peak at 1.15 ppm (tentati-
vely assigned to β-hydroxybutyrate (β-HB)),
with respect to total creatine (Cr).
Fig. 12.5: Right hand graph: Cerebral NAA/creatine and inositol/creatine ratios for normal (n = 8) and growth
restricted (n = 11) chick embryos. Left hand graph: Cerebral β-hydroxybutyrate/creatine ratios for normal and
growth-restricted chick embryos (levels of min, IQR, median and max are shown by horizontal lines in
ascending order)
Perinatal MR Studies in Animal Models 147
Compared to the normally-developed
embryos GR Naa/Cr and In/Cr were both
significantly reduced (t-test: p<0.005)
whereas β-HB/Cr was significantly incre-
ased (Mann-Whitney Rank Sum test: p
< 0.0001). Cerebral Lac/Cr was similar for
GR and normally-developed embryos.
Figure 12.6 shows Lac/Cr before, during
and after the hypoxic insult. In both groups
Lac/Cr increased during hypoxia, and
returned towards baseline values upon
restoration of normoxia. In the GR group,
the level of Lac/Cr elevation was
significantly lower (*p <0.01 Mann Whitney
Rank Sum test; normal (n = 8) vs growth
restricted (n = 11)) than in the normally-
developed group.
Conclusion
Decreased cerebral NAA/Cr and In/Cr in
the GR embryos may reflect reduced
neuronal and glial cell density respectively.22
Further studies are required to confirm the
identity of the peak at 1.15 ppm which we
148 Biomedical Magnetic Resonance: Proceedings of the International Workshop

Fig. 12.6: Cerebral lactate/creatine ratios before, during and after hypoxia (40 minutes, FiO2 = 8%) for normal
(n = 8) and growth restricted (n = 11) chick embryos. The period of hypoxia is shown by a bar. Significant
differences are indicated by a star(*) next to the data; p < 0.01 Mann-Whitney Rank Sum test

assigned to β-HB. However, β-HB may act
as an alternative energy substrate and
elevation would be consistent with enhanced
ketosis consequent to impaired nutrition.
The acute hypoxic insult caused an elevation
of cerebral lactate (presumably due to
increased anaerobic glycolysis) which was
attenuated in the GR group. The reason for
the attenuation is as yet difficult to elucidate.
However, such a response may suggest that
GR embryos find it less easy to recruit
anaerobic glycolysis in order to replenish
ATP in response to a hypoxic insult.
TRANSIENT CEREBRAL HYPOXIA IN A
CHICKEN EMBRYO MODEL WITH
BACTERIA INFECTION
Having established the synergetic effect of
growth retardation and hypoxia in the
previous study, this section aims to investi-
gate whether prior exposure to endotoxin
worsens the degree of cerebral injury
observed in chick embryos in vivo after an
acute global hypoxic insult and whether this
effect is mediated by changes in cerebral
metabolism.
Chick embryos (19 days incubation)
received either 0.4 ml of saline (control
group) or lipopolysaccharide (LPS, 3mg)
from Salmonella typhimurium. Four hours
later they were subject to either normoxia
(for embryos that would subsequently be
sacrificed for histopathology) or hypoxia
(FiO2 = 4%) for one hour (for embryos to
be studied by MR). 1H MRS and trace ADC
and T2* were repeatedly-measured before,
during and for 2 hours post-insult according
to Protocol B. Data was then analyzed and
combined to obtain the trace ADC.
MR data shows that compared to base-
line values brain Lac/Cr ratios increased
dramatically by the end of hypoxia and then
returned to baseline by 2 hours (Fig. 12.7).
In contrast, ADC fell during hypoxia, indica-
ting a change in cell membrane homeostasis,
and then recovered (Fig. 12.8). However,
these changes were not significantly
 Perinatal MR Studies in Animal Models 149

Fig. 12.7: Cerebral lactate/creatine ratios plotted against time for chick embryos that had received hypoxia +
LPS (n = 9) and hypoxia alone (n = 10). The hypoxic period is shown by a bar (one hour; FiO2 = 4%)

different between chick embryos exposed In a related study, histopathology

to hypoxia alone (n = 10) and to both LPS
and hypoxia (n = 9). Figure 12.9 shows that
the magnitude of the change in T2* (decline)
for the group of embryos that experienced
hypoxia alone was significantly greater than
that for the embryos that experienced both
hypoxia and LPS (p <0.05).
performed 12, 24 and 48 hours post-hypoxia
indicated that only the combination of
hypoxia and LPS (but not each alone)
resulted in significant cell death although
this discrepancy with MR data may be as a
result of apoptosis occurring that had been
triggered by the insult.

Fig. 12.8: Cerebral trace ADC values plotted against time for chick embryos that had received hypoxia + LPS
(n = 9) and hypoxia alone (n = 10). The hypoxic period is shown by a bar (one hour; FiO2 = 4%)
150 Biomedical Magnetic Resonance: Proceedings of the International Workshop

Fig. 12.9: Change in cerebral T2* values plotted against time for chick embryos that had received hypoxia
+ LPS (n = 9) and hypoxia alone (n = 10). The hypoxic period is shown by a bar (one hour; FiO2 = 4%)

Conclusion the slightly higher ADC values at all time

Combined exposure to LPS and hypoxia
results in significant neuronal cell death,
which could not be induced by either insult
alone. Pre-exposure to LPS does not worsen
the degree of cerebral lactate accumulation
or cellular energy deficit observed during
and shortly after acute hypoxia. The
difference between the histo-logical outcome
of the two groups is not explained by the
changes in cerebral metabolism during acute
hypoxia, but rather appears to be due to
LPS enhancing effects on the cellular
response in post-hypoxic brain. However,
an interesting finding was the different T2*
response to hypoxia in the two groups, with
a lower T2* change occurring in the group
that experienced both hypoxia and LPS
group. This could be related to LPS induced
fever prior to hypoxia which might elevate
blood volume thereby reducing the size of
response to the additional challenge. Indeed,
points observed in the hypoxia and LPS
group in Figure 12.6 might indicate increa-
sed cerebral temperature since cerebral ADC
values are known to be temperature
dependent.
Discussion
Chicken Embryo Model
The chick embryo in vivo model is easy to
implement and produces MRS, MRI and
ADC data of good quality. The model is
adaptable to allow infection, malnourish-
ment and transient- and chronic-hypoxia
investigations and is also suitable for
putative therapeutic strategies.
High cerebral lactate concentrations
observed in the chick-embryo, compared to
the mammalian neonate, may be due to the
relative degree of hypoxemia at this stage
of incubation, which provides the stimulus
for insertion of the beak into the air cell at

around 21 days. However, high cerebral
lactate levels have also been observed in
fetal rat brain.23
The brain of the chick-embryo is
sufficiently large at day 19 of incubation to
allow collection of MRS information from a
6 × 6 × 6 mm3 voxel placed almost entirely
within brain tissue. Baseline 1 H spectra
obtained during normoxia exhibited peaks
similar in amplitude and chemical shift to
those obtained from the brain of the human
neonate 24 and fetal sheep.25 The presence
of a large peak corresponding to NAA, a
molecule found almost exclusively in
neurones, confirmed that the signal obtained
from the chick-embryo was of cerebral
origin. Other prominent peaks can be
assigned to choline-containing compounds
(Cho), Cr and Lac.
Infant Studies
Studies of asphyxiated infants using
phosphorus magnetic resonance spectro-
scopy (31P MRS) have shown the develop-
ment of a second phase of cerebral energy
failure during a 48 hour period following a
presumed intrapartum primary transient
phase of HI.26,27 The severity of this delayed
or “secondary” energy failure has been
shown to be related to the chances of fatality
or persistent neurodevelopmental impair-
ment at 1 and 4 years.26,28,29
Neonatal Piglet Model
We have previously employed a piglet
model of secondary energy failure following
transient cerebral HI in the newborn piglet.30
This model employs continuous observations
of cerebral energy metabolism using 31P
MRS, and was used to follow the develop-
ment of secon-dary cerebral energy failure
Perinatal MR Studies in Animal Models 151
which showed reductions in [phospho-
creatine (PCr)]/ [inorganic phosphate (Pi)]
and [nucleotide triphosphates (NTP); mainly
ATP]/ [exchangeable mobile phosphate pool
(EPP)]. These changes became more
profound until 48 to 72 hours when the study
was terminated. This provides a well-
characterized post-natal model for testing
interventions designed to minimise cerebral
injury in newborn babies following presu-
med perinatal asphyxia. One advantage of
the piglet model is that the animal is
sufficiently large to make it feasible for the
surgical procedure necessary to induce HI
to be performed. Such studies have proved
invaluable in the assessment of hypothermia
as a potentially viable therapy for reducing
the effects of neonatal HI during the period
of secondary energy failure, and provided
information on the optimum cooling
strategy.
Findings of Chicken Embryo Studies
During exposure of the embryonic chick to
acute hypoxia at both levels of FiO2 (8 and
4%), a rapid increase in the cerebral Lac/Cr
ratio was seen, which peaked soon after
the return to normoxia and then fell back
to baseline values within two hours post-
insult (Fig. 12.2). It is likely that the
observed increase represents intracellular
Lac produced within the brain by anaerobic
respiration. If elevation of cerebral Lac
during hypoxia represents a failure of
oxidative phosphorylation and consequential
reliance on anaerobic glycolysis to maintain
ATP levels, changes would be expected in
the 1H MRS Cr peak. But this peak also
includes signal from phosphocreatine (PCr)
and so, naively, one might expect that
dephosphorylation of PCr during hypoxia
will lead to no net change in the Cr
152 Biomedical Magnetic Resonance: Proceedings of the International Workshop
resonance. However, due to the relatively
long echo time used in our study (270 ms)
and the shorter relaxation time of PCr
compared to Cr (T2 = 143 ± 41 ms for PCr
and T2 = 297 ± 41 ms for Cr 31 ) significant
dephosphorylation should have been
observable but was not. Taken together with
the results of histopathology, our data shows
that at both levels of FiO2 (8 and 4%) the
hypoxic insult on its own was severe enough
to affect energy metabolism but mild
enough not to cause lasting cellular damage.
In addition to being a product of
anaerobic glycolysis, lactate is known to
provide an alternative metabolic substrate
for the developing brain.12 We conclude that
the increase in Lac could be a component of
a co-ordinated metabolic response to match
cellular energy demand with production
rather than representing a failure of cellular
energy metabolism.
In neonatal brain, reduction in ADC is
observed during and after cerebral hypoxia-
ischemia in both animal and human
studies.21,32-34 The exact mechanism is unclear
but is thought to be related to a net shift in
water from the extracellular to the
intracellular space during hypoxia-ischemia,
as a result of cell membrane depolarization
due to energy failure and a reduction in
intracellular water mobility. Reductions in
ADC were measured in embryos that
received hypoxia with FiO2 = 4 percent and
all the embryos that received both hypoxia
and bacterial infection, where significant cell
death was confirmed by histopathology. The
Lac and ADC changes in these two groups
were indistinguishable. The lack of progno-
stic discrimination may be due to the time
difference of 2 days between MR studies
and histopathological analysis of the hatched
chick. During the intervening period we
speculate that those embryos exposed to LPS
and hypoxia suffer secondary cerebral
energy failure due to their weakened state
resulting in cellular death, but those that
have just received the insult make a full
recovery with no detectable permanent
damage.
T2 * values dropped by upto 31 ± 19
percent during the hypoxic insult (Fig. 12.4),
which reflects increased blood flow to
the brain coupled with the expected
increase in deoxyhaemoglobin. The T 2 *
changes were more rapid than ADC
changes, and slightly elevated values post-
insult may indicate hyperperfusion with
return to normal oxygenation levels. Whilst
T 2* -weighted imaging may be useful to
follow the time course of hemodynamic
changes, it is not a prognostic indicator of
the insult outcome. However, in the embryos
that suffered both LPS and hypoxia, the
reduced decline in T2* values may indicate
that brain meta-bolism was already impaired
by LPS induced fever.
The data of transient cerebral hypoxial
in chicken embryo model shows the
expected finding that in the growth retarded
embryos, neuronal density is reduced (as
estimated by NAA). Such changes would
also be expected to be visible in embryos
that have suffered neuronal damage by
severe hypoxia, although the changes are
not immediate and the hypoxic insult
delivered in our studies is relatively mild.
However, paradoxically the GR embryonic
brain shows a reduced increase in Lac during
hypoxia compared with normally-developed
embryos. The observation of an increased
peak, tentatively assigned to β-HB, in the
GR embryos is significant. β-HB may act as
an alternative energy substrate and
elevation would be consistent with enhanced
ketosis due to impaired nutrition (protein
insufficiency and reduced ambient oxygen

concentration during incubation) and the
associated low levels of blood glucose. The
low levels of glucose may then make it less
easy for GR embryos to use anaerobic
glycolysis to replenish ATP in response to
hypoxia.
CONCLUSION
We have demonstrated the value of in vivo
MR studies in perinatal animal models to
better understand the factors and processes
associated with hypoxia at birth. The hypoxic
insult alone is tolerated by the embryos,
but when applied in combination with other
detrimental factors, the brain damage that
occurs may be greatly exacerbated.
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born encephalopathy: the Western Australian
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Gluckman PD. Increased vulnerability to
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8. Newman JP, Peebles DM, Hanson MA. Adeno-
sine produces changes in cerebral haemo-
dynamics and metabolism as assessed by near
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9. Sutterlein MW, Seelbach-Gobel B, Oehler MK
et al. Doppler ultrasonographic evidence of
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low oxygen saturation according to pulse
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20.
10. Mulder AL, van Golde JC, Prinzen FW, Blanco
CE. Cardiac output distribution in response to
hypoxia in the chick embryo in the second half
of the incubation time. J Physiol 1998; 508: 281-
87.
11. Salibi N and Brown MA. Clinical MR spectro-
scopy first principles. 1998; New York: Wiley-
Liss.
12. Jones CT, Rolph TP. Metabolism during fetal
life: a functional assessment of metabolic
development. Physiol Rev 1985; 65:357-430.
13. Thornton JS, Ordidge RJ, Penrice J, Cady EB,
Amess PN, Punwan S, Clemence M, Wyatt JS.
Temporal and anatomical variations of brain
water apparent diffusion coefficient in perinatal
cerebral hypoxic-ischaemic injury: relationships
to cerebral energy metabolism. Magn Reson
Med 1998; 39:920-27.
14. Back, M Hoehn-Berlage, K Kohno, KA
Hossman. Diffusion nuclear magnetic resonance
imaging in experimental stroke: correlation
with cerebral metabolites. Stroke 1994; 25:494-
500.
15. Moonen CTW, Bandettini PA (Eds), Functional
MRI; 2000; Berlin: Springer-Verlag.
16. Ordidge RJ, Bendall MR, Gordon RE, Connelly
A. Volume selection for in vivo biological
spectroscopy. In: Magnetic Resonance in
Biology and Medicine, Govil, Khetrapal and
Saran (eds), McGraw-Hill, New Delhi, India,
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17. Bottomley, PA. Spatial localization in NMR
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18. Provencher SW. Estimation of metabolite
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NMR spectra. Magn Reson Med 1993; 30: 672-
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19. Cady EB, Amess P, Penrice J, Wylezinska M,
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Priest A, Miller SL, Blanco CE, Mulder TL, Ordi-
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 Magnetic Resonance in the Developing Brain
Magnetic Resonance in
the Developing Brain
INTRODUCTION
New magnetic resonance techniques provide
increasingly powerful ways of identifying
the structural correlates of normal brain
function and of functional abnormalities in
disease. There are two general magnetic
resonance approaches to examining brain
structure-function relationships. The first
uses functional imaging methods to identify
regions of the brain that are activated on
performance of specific tasks. The second
general approach follows the more tradi-
tional way of relating structure to function;
it involves the identification of focal abnor-
malities in brain structure or physiology,
which can then be related to cognitive
function as measured by neuropsychological
testing. This article reports on the ways in
which we have been using these two appro-
aches in our programme of research on focal
brain damage and its functional consequen-
ces in groups of children with brain disease.
The groups include children with intractable
epilepsy, children with specific types of
cognitive impairment, and children with
sickle cell disease who have a very high
risk of stroke. The examples that are given
below, taken from our own work, illustrate
155
13
David G Gadian
the importance of detecting brain abnormali-
ties that might previously have gone
undetected, and of obtaining quantitative
measures that can be related to particular
aspects of brain function. The acquisition
and analysis methods that we have used
include, in addition to conventional neuro-
radiological assessment, volumetric mea-
surements, T 2 relaxometry, voxel-based
morphometry, 1H MRS, diffusion imaging
and perfusion imaging, as well as functional
MRI.
CHILDHOOD EPILEPSY
Epilepsy affects about 1 percent of children.
The majority become seizure-free on
medication, but in upto 25 percent of these
children the seizures remain drug-resistant.
In selected cases, seizure relief can be
effected by surgical resection of the epilepto-
genic focus, and the results can prove
remarkably beneficial.
Presurgical identification of the seizure
focus has been greatly facilitated in recent
years by improvements in MR techniques
for the detection of the underlying focal
pathology. Many research groups have made
contributions to this area of research. Our
156 Biomedical Magnetic Resonance: Proceedings of the International Workshop
own contributions have included the intro-
duction of 1H MRS1,2 and of T2 relaxometry3
for the detection of temporal lobe patho-
logy, both in adults (in collaboration with
colleagues at the Institute of Neurology,
London) and in children. Together with
volumetric measurements of the hippocampi
(see, for example Van Paesschen et al4),
these techniques add greatly to the
sensitivity and specificity of conventional
visual inspection of MRI scans, even when
these scans are optimized for visualization
of the hippocampi. For example, T2 relaxo-
metry (a procedure whereby the T2 value is
computed from a series of MR images
acquired at different echo times) has proved
to be an objective and highly sensitive means
of detecting abnormalities of the hippo-
campus.3,5 1H MRS provides complementary
information; in particular, it commonly
detects the presence of additional, relatively
widespread temporal lobe pathology than
can be extremely difficult to detect on MRI.
The dominant signals seen in 1H spectra
of the brain are normally from N-acety-
laspartate (NAA), creatine plus phospho-
creatine (Cr), and choline-containing
compounds (Cho). While much remains to
be learned about the functions of NAA, it
is believed to be present in the brain
primarily within neuronal cells, and so its
signal provides a means of reporting on
neuronal loss or damage. Consistent with
this, a reduction in the NAA signal (or in
the ratio of the NAA signal to the Cr and
Cho signals) is commonly seen in disorders
where neuronal loss or damage can be
expected. In our studies of temporal lobe
epilepsy, we have acquired spectra from a
2 × 2 × 2 cm region within each temporal
lobe, positioned so that there is little
contribution from the hippocampus.1,2 These
spectra often show abnormalities, indicating
that there is relatively widespread pathology
that is additional to any more focal damage
to the hippocampi. This diffuse pathology
is not necessarily evident on MRI.6 The pre-
sence of such MRS abnormalities in areas
that appear normal on MRI is intriguing,
and some of the implications are discussed
below, in relation to our understanding of
the cognitive impairments that may be
present in these children.
Functional MRI studies can also be
invaluable in the study of epilepsy. In some
cases, fMRI can play a role in helping to
identify the seizure focus through the
detection of abnormal cortical activation that
may take place at sites of seizure activity.7
In addition, the use of fMRI to identify brain
regions that are activated with motor or
language or memory tasks can aid in
neurosurgical planning of candidates for
epilepsy surgery8,9, and can provide impor-
tant neuroscientific information about any
reorganization of brain function that may
occur in response to brain injury.9
CHILDREN WITH MEMORY IMPAIRMENTS
The methods that we have adopted for the
investigation of temporal lobe abnormalities
in epilepsy are useful for studying memory
impairments also, given the importance of
temporal lobe structures in memory function.
An important conclusion from our
extensive studies of patients with temporal
lobe epilepsy is that many of them, including
some with well-lateralized seizures, have
bilateral pathology. This has implications for
our understanding of the cognitive impair-
ments observed in such patients. An
illustration of this is provided by a study in
which we found unexpected deficits in
verbal learning and recall amongst a series
 Magnetic Resonance in the Developing Brain
of adult patients who had undergone right
temporal lobe surgery for relief from child-
hood-onset epilepsy.10 Given our obser-
vations of bilateral pathology in pre-surgical
cases of temporal lobe epilepsy, we hypo-
thesized that the unexpected deficits in the
patients with right-sided resections may
reflect previously unsuspected pathology of
the left hemisphere. Subsequent MRS and
T2-mapping studies showed that this was
indeed the case.10
We have seen particularly striking effects
of bilateral temporal lobe pathology in a
number of children with a severe memory
disorder that we now refer to as develop-
mental amnesia. Our initial report11 descri-
bed our findings in three young patients,
all of whom showed a substantial reduction
in hippocampal volume bilaterally. These
patients had a profound impairment of
episodic memory with relatively preserved
semantic memory, a pattern of function that
provoked extensive interest and debate
within the cognitive neuroscience commu-
nity. Interestingly, 1H MRS showed much
more modest abnormalities than those seen
in many of our cases of temporal lobe
epilepsy; this is consistent with the notion
that the pathology is quite focal, and in turn
is consistent with the selectivity of the
resulting functional deficits.
We went on to show that this pattern of
memory impairments may occur as a
previously unrecognized consequence of
perinatal hypoxic-ischemic injury.12 Some of
the evidence for this came from another
method of analysis of MRI scans, namely
voxel-based morphometry.13 This method
provides an automated means of comparing
brain images from different groups of
subjects. Several steps are involved: these
include normalization of the images to a
standard template; segmentation of the
157
resulting images into grey matter, white
matter and cerebrospinal fluid; spatial
smoothing; and finally statistical analysis of
group data on a pixel-by-pixel basis. In the
children with developmental amnesia, voxel-
based morphometry identified bilateral
reductions in grey matter, not only in the
hippocampus, but also in the regions of the
putamen and the ventral part of the thala-
mus13 (abnormalities of the putamen and
thalamus were not visible on conventional
neuroradiological assessment of the scans).
This pattern of neuropathology of putamen,
thalamus, and hippocampus is consistent
with some of the known effects of acute
perinatal hypoxic-ischemic injury, and
strengthened the argument that the
pathology and associated memory impair-
ments in these children can be attributed to
the hypoxic-ischemic episodes that these
children had suffered early in life. Presum-
ably, the degree of hypoxia-ischemia had
been sufficient to produce selective damage
to these particularly vulnerable regions of
the brain, but was not sufficient to result in
the more severe neurological and cognitive
deficits that can follow hypoxic-ischemic
injury.
AN INHERITED SPEECH AND
LANGUAGE DISORDER
Voxel-based morphometry has also made a
critical contribution to our investigations of
structure-function relationships in members
of a family with an inherited speech and
language disorder. The disorder is caused
by a mutation in the FOXP2 gene14, and
manifests itself as a pronounced verbal and
orofacial dyspraxia. In the affected members
of the family, voxel-based morphometry
revealed bilateral brain abnormalities in a
number of motor- and language-related
regions of the brain, including the caudate
158 Biomedical Magnetic Resonance: Proceedings of the International Workshop
nucleus (which also showed bilateral reduc-
tions in volume on volumetric measure-
ments), putamen, cerebellum, and inferior
frontal gyrus.15,16,17 Further studies were
carried out using functional MRI to examine
regions of the brain that were activated with
covert verb generation and with overt verb
generation and word repetition. In the
affected family members, many of the
regions that were morphologically-abnor-
mal were also found to be functionally-
abnormal on functional MRI, with a pattern
of activation that was more posterior and
more extensively bilateral.18 Together, these
structural and functional MRI studies have
provided some fascinating insights into the
links between the genetic abnormality and
the resulting speech and language disorder.
CHILDREN BORN PREMATURELY
Advances in perinatal care have led to the
survival of increasing numbers of children
born very prematurely. However, these
children have an increased risk of cognitive
and learning difficulties. We set out to
establish whether volumetric measurements
and in particular voxel-based morphometry
might enable us to identify the neural
correlates of such difficulties. Following
initial studies in which we showed that a
sub-group of these children had mild
episodic memory impairments impaired with
modest reductions in hippocampal volume19,
we went on to use voxel-based morpho-
metry to identify a neural correlate of
calculation difficulties in a group of children
born preterm; the technique revealed an
area in the region of the left intraparietal
sulcus where children with calculation
difficulties had less grey matter than child-
ren who did not have these difficulties.20
We have also shown an association between
visuospatial deficits and grey matter density
in right extrastriate cortex.21 More recently,
we have gone on to show that preterm
children are at risk of declining IQ over-
time even if they have not suffered obvious
neurological damage and that the decline is
associated with specific neural regions.22 On
the basis of all of these studies, it is evident
that voxel-based morphometry provides a
powerful approach to identifying the subtle
differences in brain structure that underlie
the various types of cognitive impairment
that may be associated with very premature
birth.
CHILDREN WITH SICKLE CELL DISEASE
While stroke is primarily a disease of
adulthood, it can also occur in children.
Certain groups of children are at particular
risk; for example in children with sickle cell
disease, stroke is 250 times more common
than in other children, with an incidence
similar to that for a general population of
elderly adults. Diffusion and perfusion
imaging techniques now have a major role
to play in the investigation of patients with
acute stroke, and we set out to evaluate
whether these techniques, in particular
perfusion imaging, might also play a role in
investigating the hemodynamic effects of
chronic cerebrovascular disease, as seen in
children with sickle cell disease.
An important imaging feature of acute
stroke is that the area of perfusion abnorma-
lity is commonly greater than the area of
diffusion change. Given the accepted
interpretation of the diffusion changes that
are seen in acute stroke, such areas of
diffusion-perfusion mismatch presumably
reflect a fall in regional cerebral blood flow
that is not sufficient to cause energy failure,
cytotoxic edema, and inevitable infarction.
 Magnetic Resonance in the Developing Brain
These mismatch areas may well be at risk
of subsequent damage, however, and have
attracted considerable interest as a target
for therapeutic intervention in patients with
acute stroke. In our studies of children with
sickle cell disease, we have concentrated on
chronic abnormalities rather than acute
changes. Nevertheless, in line with the acute
studies, we also frequently detect regions
that display perfusion abnormalities but no
other neuroimaging abnormality.23 On the
basis of these and additional findings, we
believe that perfusion imaging could make
important new contributions to our under-
standing of the pathophysiology of sickle
cell disease, and that it could play an
important role in the development and
evaluation of strategies for the prevention
of stroke in this at-risk population of
children.
DISCUSSION
As stated at the outset, there are two
general ways in which MRI can be used for
the investigation of brain function. For
studies of children with brain disease,
functional MRI obviously provides an
excellent approach to identifying the sites
underlying preserved functions, for example
as an aid to surgical decision-making or in
the investigation of cerebral reorganization.
However, for the investigation of impaired
function, the more traditional approach of
relating structural damage to cognitive
impairment can prove more informative,
particularly with the development of impro-
ved techniques that allow us to identify focal
pathophysiology with increasing sensitivity
and specificity. This is especially important
in the case of neurodevelopmental disorders,
as many such disorders involve relatively
159
subtle morphological abnormalities that may
not be visible on conventional neuroradio-
logical assessment of MRI scans. The
investigations described in this article
illustrate the complementary roles that the
different magnetic resonance approaches
play in the study of brain structure/function
relationships. In addition to helping us to
understand the structural basis of a range
of neurological and cognitive problems, one
of our long-term hopes is that early MR
findings in high-risk groups of children will
be predictive of functional outcome, and will
as a result provide the opportunity for more
timely intervention.
ACKNOWLEDGMENTS
This article draws on the results of a multidisciplinary
programme of research at the Institute of Child
Health and Great Ormond Street Hospital for
Children, and I would like to acknowledge all of the
many colleagues involved in this programme. I also
thank the Wellcome Trust for its support.
REFERENCES
1. Connelly A, Jackson GD, Duncan JS, King MD,
Gadian DG. Magnetic resonance spectroscopy
in temporal lobe epilepsy. Neurology 1994; 44:
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3. Jackson GD, Connelly A, Duncan JS, Grunewald
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MRS in Brain Pathologies
Peter B Barker, Mari Smith, A Gambini, SA Fatemi,
INTRODUCTION
Proton magnetic resonance spectroscopy
(MRS) of the brain is now well established in
both research and clinical applications.1 This
presentation will focus on some recent tech-
nical developments in magnetic resonance
spectroscopic imaging (MRSI) of the human
brain, involving the use of 3 Tesla magnets
and phased-array receiver coils, and also
present some recent studies in acute
disseminated encephalomyelitis (ADEM).
METHODS
Technical Development
Metabolites detectable by in vivo spectro-
scopy are present in the brain are present
in millimolar concentrations (typically 1-10
mM), about a factor of 104 lower than the
water signal concentration used for magnetic
resonance imaging (MRI). For this reason,
spatial resolution in MRS or MRSI is much
lower than in MRI, and scan times are often
lengthy because of the time averaging
required. Therefore, technical developments
that can increase sensitivity and/or decrease
scan time are particularly important for MRS
and MRSI. Some approaches to increase
sensitivity are the use of high magnetic field
MRS in Brain Pathologies 161
14
TO Crawford, EH Kossoff, A Horská
strengths, 2,3 and the use of improved
radiofrequency coil technology. In particular,
it has been shown that the use of phased-
array coil technology can improve signal-
to-noise for brain imaging; while at the same
time, maintaining the brain coverage of a
conventional volume head coil resonator.4
With the increasing commercial availability
of phased-array coils, and their routine use
for brain MRI, it is logical to explore their
utility for brain MRS and MRSI.
In addition to improved sensitivity,
phased-array coils offer opportunities for
MRSI scan time reduction through parallel
imaging approaches such as “sensitivity-
encoding” (SENSE). However, these advant-
ages are also accompanied by more complex
demands on data processing.
Single Voxel MRS
For single voxel MRS, each channel of the
phased-array coil detects the (same) signal
from the localized voxel, weighted by the
sensitivity of the coil at that region in space.
The amplitude of the signal will also depend
on the gain of each receiver amplifiers for
channel; and in general, the phase of the
signal will also be different in each channel.
To combine the signal from all channels in
162 Biomedical Magnetic Resonance: Proceedings of the International Workshop

an optimal way, the phase difference

between the channels must be corrected,
and then a weighted average performed.
Optimal SNR is obtained when the channels
with the highest SNR have the greatest
weight,4 i.e.
N
 exp(– in ) × an ×Sn (t) Fig. 14.1: Single-voxel spectra of a phantom
n=1
s(t) = N ...(1)
containing 65 mM NAA in a 6-element phased-array
 an head coil recorded at 3 Tesla. Note that the signal
n=1 amplitude and phase of each channel is different

where s(t) is the combined time-domain

signal, sn(t) is the time-domain signal from
the nth of N channels, and a n is the
magnitude of the coil sensitivity at the
location of the localized volume. φn is the
phase error of the nth channel. There are
several ways that a n and φ n can be
determined—for instance, they can be
measured from complex sensitivity maps
calculated from magnetic resonance images
(MRI) recorded using the individual phased-
array elements. Alternatively, the phase and
amplitude information is also contained in
the MRS data itself, sn(t), and, provided
that the SNR is sufficient, can be readily
obtained from the first point of the free
Fig. 14.2: Phased-array single-voxel spectroscopy of
induction decay {a = |sn(t=0)|, and φn =
a phantom containing common neurochemicals at
arg[s n (t=0)]}. Equation (1) can then be physiological concentrations at 3 Tesla (PRESS, TR
written 2000 msec, TE 35 msec). Top row—single channel
N data, SNR = 24:1, bottom row—all 6 channels
 Sn  (t =0) × Sn (t) combined, SNR = 49:1
n=1
s(t) = N ...(2)
 |Sn (t =0)| can be seen that the amplitude and phase of
n=1
the NAA resonance is different in each
A high SNR estimate of an and φn can be channel. Figure 14.2 shows PRESS spectra
obtained if a non-water-suppressed signal is from a phantom that contains physiological
also available from the localized volume, concentrations of neurometabolites; the
which is often the case. Otherwise, the water- spectrum from the single channel (with the
suppressed signal may be used lowest sensitivity) has an SNR of 24:1,
Figure 14.1 shows an example of single- whereas the combined dataset from all 6
voxel PRESS spectra recorded at 3.0 Tesla channels (equation 2) has an SNR of 49:1, i.e.
from a phantom containing N-acetyl aspar- about a 100 percent improvement. Theoreti-
tate (NAA) using a 6-channel head coil. It cally, if the SNR was equal in all N channels,

combining the channels should lead to a
SNR improvement of N so long as the
channels are properly combined (i.e. phase
errors are accounted for) and the noise is
uncorrelated. In practice, noise correlation
between different channels of the phase-
array may be quite significant, so SNR gains
may be somewhat lower than expected; also,
depending on the voxel location and the
phased-array geometry, one channel may
have much greater SNR than the others,
and the combined SNR will be very close to
that of the best channel. However, SNR is
still higher with the phased-array than with
volume head coils because of their individual
smaller sizes; in particular, for superficial
voxel locations near the surfaces of the brain,
SNR gains can be large compared to
standard quadrature birdcage volume coils.
MR SPECTROSCOPIC IMAGING WITH
PHASED-ARRAY COILS
For MRSI with phased-array coils, it is desir-
able to generate a single, combined MRSI
dataset with uniform sensitivity and optimal
SNR at each point in space. A similar
approach as in single-voxel spectroscopy can
be applied for combining multichannel data
on a pixel-by-pixel basis. Sensitivity and
phase information is contained within the
MRSI data, as in the case of SV spectroscopy.
However, in practice, it may be more conve-
nient to measure an(x,y) and φn from sensiti-
vity maps generated by MRI, as opposed to
directly using the MRSI data.
The data processing algorithm is sche-
matically illustrated in Figure 14.3; multi-
channel data are processed in the spatial
domains to produce the individual (time-
domain) MRSI data from each channels. The
combined time-domain MRSI signal [for
each point (x, y) in the MRSI slice] is then
given by
MRS in Brain Pathologies 163
...(3)
where bn(t, x, y) is the MRSI data from the
nth (out of a total of N) receiver coil after
spatial filtering and Fourier transformation
in the spatial domains, an(x, y) is the complex
sensitivity of the nth receiver channel at point
(x, y), and φn = arg [an(x, y)]. an(x, y) is
calculated from the complex division of the
gradient echo images recorded alternately
with reception using the phased-array and
body RF coils, then degraded to the
resolution of the MRSI dataset using a
Gaussian function. After s(t, x, y) has been
calculated, further processing (time domain
filtering, Fourier transformation, etc.) can
be applied as in conventional, single-channel
MRSI to generate the final metabolic image.5
Figure 14.4 shows an example of one slice
from a multi-slice MRSI study recorded using
a 6-channel phased-array coil on a 3.0 Tesla
system. The scan parameters were repetition
time (TR) 1600 msec, echo time (TE) 140
msec, water suppression with 3 frequency
selective pulses of flip angle 83°, 89° and
160° respectively,6 8 octagonal outer volume
saturation (OVS) lipid suppression pulses,7
3 slices, 15 mm thick, 2.5 mm gap, 24 × 18
cm field-of-view (FOV), 32 × 24 phase-
encoding steps, with a circular k-space
sampling, 1 signal average, nominal voxel
size 0.8 cm3, and a scan time 15 minutes.
The sweep width was 2000 Hz and 512
points were collected. Signal-to-noise ratios
in spectra from deep and superficial
locations in the brain were measured, and
compared to scans recorded using the same
conditions using a 1.5 T system and a
conventional volume head coil. It was found
that SNR improved anywhere from 55 to
164 Biomedical Magnetic Resonance: Proceedings of the International Workshop

Fig. 14.3: Schematic diagram of the data processing scheme for phased-array MRSI. MRSI data from each
channel of the phased-array are processed in the spatial domains before combination with phase- and
amplitude-correction from B1 field maps created from MRI scans. Combined, time-domain MRSI data is then
further processed by filtering, Fourier transformation and magnitude calculation before constructing metabolic
images by integration of peak areas

142 percent depending on location, with the
greater part of the improvement due to the
higher sensitivity of the phased-array coil,
rather than the higher field strength.8
SENSE-MRSI
As is well known from MRI, the non-
uniform sensitivity profiles of phased-array
coils can be used to decode spatial infor-
mation from the MR signal.9 This then allows
the number of phase-encoding steps to be
reduced, resulting in shorter scan times
without sacrificing spatial resolution. A
number of approaches for parallel imaging
have been proposed. One of the attractive
features of parallel imaging is that it is a
general way of decreasing scan time, and
can be used with any type of MRSI pulse
sequence.
Parallel encoding has been previously
described for proton MRSI of the human
brain.10 Since MRSI scan times are usually
long (e.g. 15 to 30 minutes), acceleration
factors of 2 or more offer substantial scan
time savings. Since MRSI typically involves
phase-encoding in 2 or even 3 dimensions,
 MRS in Brain Pathologies 165

Fig. 14.4: Phased-array MRSI of the human brain recorded at 3 Tesla. T1-MRI localizer, and metabolic images
of Cho, Cr and NAA are shown. Selected spectra from deep and superficial voxel locations are shown, and
signal-to-noise ratio improvements compared to conventional MRSI performed at 1.5 Tesla on the same
subject are given. Frontal lobe hypointensity in the Cr and NAA images is due to field inhomogeneity effects
from the sinuses

the use of SENSE-MRSI in multiple dimen-
sions offers the possibility of very large
reductions in scan time (e.g. factors of 4 or
even 8). However, minimum scan times will
almost certainly be determined by signal-to-
noise requirements (SNR decreases with the
square root of the scan time) so very high
acceleration factors may not be viable unless
working under high SNR conditions. Our
implementation of SENSE-MRSI involves an
acceleration factor (M) of 2, used in one
direction of a 2D-MRSI scan. Multichannel
data is combined according to
S(t, x, y) = A(x, y)–1 b(t, x, y) ...(4)
where b(t, x, y) is a vector of dimension N
containing the folded MRSI data, A(x, y) is
the coil sensitivity profile matrix of dimen-
sion N × M, and s(t, x, y) (the combined,
output MRSI) is a vector of dimension M.
Equation 4 can be solved using standard
matrix inversion techniques. Note that s, A
and b are all of complex data type, and that
the SENSE-reconstruction in this case is
applied on time domain data. While it may
also be performed on the frequency domain
data, the advantage of applying SENSE to
the time domain data is that some of the
time domain filtering used on the MRSI data
may be spatially dependent (e.g. we apply
a susceptibility correction as a time-domain
phase ramp prior to performing a high-pass
water-suppression filter).
Figure 14.6 shows an example of a
SENSE-MRSI scan recorded with similar
parameters to that of Figure 14.5; the number
of phase-encoding steps in the left-right
dimension was reduced to 16, and the field-
of-view (FOV) in this direction was reduced
to 12 cm, lowering scan time to 10 minutes,
166 Biomedical Magnetic Resonance: Proceedings of the International Workshop

Fig. 14.5: SENSE-MRSI of the human brain recorded at 3 Tesla. T1-MRI localizer, and metabolic images of
Cho, Cr and NAA are shown. Selected spectra from deep and superficial voxel locations are shown, and
signal-to-noise ratio improvements compared to conventional MRSI performed at 1.5 Tesla on the same
subject are given. Some lipid contamination is apparent in the spectra; this is due to the very intense lipid
signals detected because of their proximity to the phased-array coils. The bright rim in the NAA image is also
due to pericranial lipid contamination

but also resulting in fold-over in the input

MRSI data (b). After SENSE reconstruction,
fold-over is largely eliminated (apart from
some residual lipid signals) and uniform
sensitivity MRSI data obtained (Fig. 14.6).
SNR is better than conventional MRSI at
1.5 T, although not as good as in Figure
14.5, mainly because of the shorter scan
time. Lipid contamination is a potential Fig. 14.6: Metabolite ratios (NAA/Cho, NAA/Cr and
problem with SENSE-MRSI, since pericranial Cho/Cr) in ADEM and Control Subjects: (A) Basal
Ganglia Lesions, (B) White Matter Lesions, (C)
lipid is located close to the phased-array
Normal-Appearing White Matter (NAWM). NAA/Cho
coils (and thus detected with high and NAA/Cr are both significantly reduced in both
sensitivity), and the reduced FOV folds the white matter and basal ganglia lesions compared to
lipid into the brain. Therefore, accurate controls, while Cho/Cr ratios were unchanged. NAA/
unfolding is required to eliminate artifactual Cr was also significantly reduced in normal-
appearing white matter of ADEM cases compared to
residual lipid signals, and SENSE-MRSI will
that in control subjects, suggesting more widespread
undoubtedly benefit from improved lipid metabolic brain involvement in ADEM than seen on
suppression techniques.11 conventional MRI

In summary, SV-MRS and MRSI are
possible with phased-array receiver coils and
offer the advantages of improved SNR and
decreased MRSI scan times through the use
of parallel encoding. These advantages come
at the relatively low cost of somewhat more
complicated data handling/processing
requirements.
PATIENT STUDIES
This section presents some recent results of
the use of proton MRSI in the diagnosis of
acute disseminated encephalomyelitis
(ADEM). ADEM is a rare, inflammatory
demyelinating disease that principally
involves the brain and spinal cord.12 It affects
children and young adults, commonly after
an infectious disease or immunization. The
pathogenesis is not fully understood, but
the hypothesis is that of a transient
autoimmune reaction against myelin or
other autoantigens.12 In the past ADEM was
observed commonly after typical childhood
infectious diseases such as measles, rubella
and varicella, with 25 percent of mortality
rates in ADEM after measles and 35-40
percent of permanent neurological sequelae.
Now-a-days, because of a better infectious
disease control in developed countries,
ADEM is seen most frequently after non-
specific upper respiratory tract infections.12
Typically, ADEM is a monophasic disease
characterized by focal neurological signs and
symptoms, with 50 percent of the cases
having a complete recovery, usually within
a few weeks or months. However, the other
50 percent show permanent deficits.
Approximately 10 percent of ADEM cases
have recurrent illness (multiphasic ADEM)12
that clinically and otherwise is difficult to
distinguish multiple sclerosis.
In the acute phase of ADEM, lesions
MRS in Brain Pathologies 167
pathologically are characterized by areas of
perivenous demyelination, with infiltration
of inflammatory cells, edema and sometimes
hemorrhage.12 Histopathology studies have
nearly always been confined to the most
severe cases of ADEM, either postmortem
or brain biopsy studies. Relatively little is
known about the pathology of lesions in
patients with monophasic disease and good
clinical outcome.
Prognosis in the acute phase of ADEM is
arguably as important as diagnosis. It has
been reported that poor clinical outcome
correlates with severity of abruptness of
onset,12 but there are no specific prognostic
factors to predict the degree of recovery
and the possibility of recurrence.
Because of its noninvasive nature and
its ability to measure the levels of several
metabolites believed to be related to
neuronal integrity (NAA) and myelination
(choline— Cho), MR spectroscopy is a
promising technique for the diagnosis of
ADEM, and to improve understanding of
the underlying pathophysiology. 13,14 This
study was undertaken to characterize brain
metabolism in the acute phase of ADEM,
and to investigate if brain MRS has any
prognostic value in predicting which patients
will have monophasic disease and good
clinical outcome, versus those with either
multiphasic ADEM and/or permanent
neurological deficits.
Materials and Methods
Eleven patients with ADEM (M:4, F:7, mean
age: 8.8 yr) and 11 age-matched controls
(M:5, F:6, mean age: 9.8 yr) were evaluated
with routine brain MRI and MRSI within 7
days of symptom onset. MRI and neurologi-
cal exam were repeated at least 6 months
later in all cases. MRSI was performed at 1.5
Tesla using a multislice, spin-echo technique
168 Biomedical Magnetic Resonance: Proceedings of the International Workshop

with CHESS water suppression and outer- regions (p = 0.02 in basal ganglia, p< 0.01 in
volume lipid suppression 15,16 (TR/TE = white matter). Cho/Cr was unchanged in
2300/280 ms, 4 axial slices, thickness 15 mm, lesions compared either to contralateral
nominal voxel size 0.8 cm3 ). Regions of brain regions or control subjects. In normal-
interest were chosen in both ADEM lesions, appearing white matter (NAWM) in ADEM
and normal-appearing white matter in patients, NAA/Cr and Cho/Cr were signi-
standardized supratentorial brain regions. ficantly reduced (p < 0.01 and p = 0.013)
The same regions were also measured in compared to controls. Patients with multi-
control subjects. Peak areas were measured phasic disease and/or poor clinical outcome
by integration and metabolite ratios were showed a significant reduction of NAA/
Cho (p = 0.02), and also a trend for an
calculated.
increase of Cho/Cr in NAWM versus pati-
The ADEM patients were classified into
ents with monophasic disease.
2 groups depending on clinical outcome; the
“good” outcome group had monophasic
Discussion
disease progression and no permanent
neurological deficit on follow-up exami- At presentation, brain lesions in ADEM pri-
nation, while the “poor” outcome group had marily showed reduced NAA/Cr and NAA/
either multiphasic disease progression (with
at least one relapse), and/or significant
residual neurological deficit on follow-up
clinical examination. Statistical analysis
between groups was accomplished using
student’s t-test with correction for multiple
comparisons. The level of significance was
set at p < 0.05.

Results
Metabolite ratios for lesions and normal-
appearing white matter in ADEM patients,
and control data from the same regions are
presented in Figure 14.6. Figure 14.7 shows
an example of MRI and MRSI data in one
ADEM case; this patient had monophasic
disease and good clinical outcome. In total,
6 patients had a monophasic disease with
good recovery, while 5 had either multi-
phasic disease and/or permanent deficits.
N-acetyl-aspartate (NAA)/creatine (Cr) and Fig. 14.7: FLAIR MRI, metabolic images of Cho and
NAA, and spectra from lesion and contralateral white
NAA/choline (Cho) ratios in lesions within
matter in an ADEM case. There is a large T 2
the basal ganglia and white matter of hyperintense region in the right occipital white matter
patients were reduced both versus controls which is characterized by a prominent decrease in
(p < 0.01) and versus contralateral brain NAA, but without much change in Cr or Cho

Cho ratios, while Cho/Cr was within normal
limits. This pattern seems to be consistent
with lesions that primarily consist of vaso-
genic edema with neuronal/axonal dysfunc-
tion, rather than overt inflammatory
demyelination (which is often accompanied
by an increase in Cho in other patho-
logies).17-19 Since inflammatory demyelina-
tion is a common pathological finding in
ADEM lesions, the absence of an elevation
of Cho in ADEM is perhaps surprising. In
MS, elevation of the Cho peak is believed
to be either due to the accumulation of
myelin breakdown products18 or due to
inflammation.20 It would appear that the
degree of myelin breakdown or inflamma-
tion in ADEM (with good outcome) may be
insufficient to cause significant accumulation
of Cho, and irreversible white matter
damage. MRI T2 hyperintensity in ADEM
lesions would, therefore, appear to reflect a
greater component of vasogenic edema as
opposed to irreversible demyelination. This
is also consistent with the lack of peri- or
intra-lesional contrast enhancement, sugges-
ting a rather mild inflammatory process
without any breakdown of the blood-brain
barrier.
Brain involvement may be more exten-
sive than suggested by conventional MRI,
since NAWM also showed abnormal
metabolism (low NAA/Cr). These changes,
apparently due to lower NAA rather than
increased Cr, were found in both white and
gray matter regions, and would appear to
indicate the presence of a more diffuse
pathological involvement in ADEM than
suggested by conventional MRI. NAA
reductions in regions of normal MRI
appearance have also been reported in MS.21
In white matter, NAA is believed to be
primarily located within axons. 22 The
reduction of NAA in ADEM (both in lesions
MRS in Brain Pathologies 169
and in normal appearing white matter)
would appear to be due to temporal axonal
dysfunction rather than axonal loss, at least
in those cases that showed improvement on
follow-up scans and favorable neurological
outcome.
Finally, ADEM patients with recurrent
disease or poor neurological outcome
showed lower ratios of NAA/Cho (and a
trend for increased Cho/Cr) compared to
those with good outcome, suggesting a
possible prognostic role for proton MRSI in
ADEM. We interpret these findings as
indicating a more severe degree of
pathology (i.e. greater degree of neuroaxonal
loss/dysfunction, more intense inflamma-
tory demyelination) occurring in those
patients who subsequently develop recurrent
disease and/or permanent neurological
deficits. While these preliminary findings
will require confirmation in larger studies
with long-term clinical follow-up, they do
nevertheless suggest a role for MRSI in
offering prognostic information in ADEM.
It should also be pointed out, although
beyond the scope of the current article, that
proton MRSI may be helpful in distin-
guishing ADEM from other pathologies that
may sometimes be considered in the
differential diagnosis; for instance, multiple
sclerosis, brain tumors, or mitochondrial
diseases.13,23
ACKNOWLEDGMENTS
This work was supported in part by P41RR15241,
R21CA/RR91798, R21-EB00991 and Philips Medical
Systems.
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11. Smith M, Barker PB. Simultaneous water and
lipid suppression in multi-slice brain MR spec-
troscopic imaging. In: ISMRM Kyoto, Japan,
2004.
12. Garg RK. Acute disseminated encephalomye-
litis. Postgrad Med J 2003;79:11-17.
13. Bizzi A, Ulug AM, Crawford T et al. Quanti-
tative proton MR spectroscopic imaging in acute
disseminated encephalomyelitis. AJNR 2001;
22:1125-30.
14. Harada M, Hisaoka S, Mori K, Yoneda K, Noda
S, Nishitani H. Differences in water diffusion
and lactate production in two different types
of postinfectious encephalopathy. J Magn Reson
Imaging 2000;11:559-63.
15. Duyn JH, Moonen CT. Fast proton spectroscopic
imaging of human brain using multiple spin-
echoes. Magn Reson Med 1993;30:409-14.
16. Golay X, Gillen J, van Zijl PCM, Barker PB.
Scan time reduction in proton magnetic reso-
nance spectroscopic imaging of the human
brain. Magn Reson Med 2002; 47:384-87.
17. Kruse B, Barker PB, van Zijl PCM, Duyn JH,
Moonen CTW, Moser HW. Multislice proton
MR spectroscopic imaging in X-linked adreno-
leukodystrophy. Ann Neurology 1994; 36:595-
608.
18. Davie CA, Hawkins CP, Barker GJ et al.
Detection of myelin breakdown products by
proton magnetic resonance spectroscopy
[letter]. Lancet 1993;341:630-31.
19. Helms G. Volume correction for edema in
single-volume proton MR spectroscopy of
contrast-enhancing multiple sclerosis lesions.
Magn Reson Med 2001;46:256-63.
20. Brenner RE, Munro PM, Williams SC et al. The
proton NMR spectrum in acute EAE: The
significance of the change in the Cho:Cr ratio.
Magn Reson Med 1993;29.
21. Arnold DL. Magnetic resonance spectroscopy:
Imaging axonal damage in MS. J Neuroimmunol
1999;98:2-6.
22. Barker PB. N-acetyl aspartate—a neuronal
marker? Annals of Neurology 2001;49:423-24.
23. Lin DDM, Crawford TO, Barker PB. Proton
magnetic resonance spectroscopy in the diag-
nostic evaluation of suspected mitochondrial
disease. AJNR 2003;24:33-41.
 Application of Proton MRS in the Study of the Pathology of Human Cancers
Application of Proton MRS in the
Study of the Pathology of Human
Cancers Other than Brain
Cynthia L Lean, Peter Russell, Carolyn E Mountford
INTRODUCTION
Proton MRS of human tissue and fine needle
aspirate biopsies can determine both the
pathological status and, for malignant tissues,
key prognostic variables with accuracies
approaching 95 percent.1 The MR method is
fast, accurate and reliable and offers an
adjunct to the routine histopathological
diagnosis of human cancers. The technology
was extended to in vivo H MRS in the 1990’s
and is approaching clinical use for some
organs in the body including the breast and
prostate.
Limitations of Histopathology
Histopathology has been the medical
diagnostic gold standard for much of the 20th
Century, providing diagnostic and prognostic
information for human diseases including
cancer. Current histopathology comprises
many decades of experience by medical
specialists who rely on pattern recognition
in human tissues and cells using light
microscopy.
171
15
The principal limitation to histopathology
is the restricted range of morphological
changes that tissues can express, each in a
continuum, yet from which pathologists are
expected to identify patterns specific for
individual diseases. These patterns overlap
and are susceptible to subjective assessment.
Moreover, sampling errors are inherent at
several levels in routine diagnostic services
at least partly due to the cost of examining
multiple sections of tissue. The skill, expe-
rience and thoroughness of surgeons and
pathologists play a major role in ensuring
detailed diagnostic and prognostic infor-
mation.
PROTON (1H) MRS ON BIOPSIES EX VIVO
Proton magnetic resonance spectroscopy (1H
MRS) on human biopsies is a powerful tool
for the detecting, diagnosing and monitoring
human cancers with a precision and prog-
nostic capability not previously available.
MRS reports on the chemical composition
of cells, the changes that occur in the ageing
process,2 disease processes3 and the host
immune response.4 It was established in the
172 Biomedical Magnetic Resonance: Proceedings of the International Workshop
late 1970s using an AKR mouse model5 that
MRS could identify changes during tumor
development, which were not morphologi-
cally manifest. This initial finding has now
been substantiated over a period of 25
years.6,7
The MRS can detect small populations of
abnormal cells with high accuracy, and
identify and subcategorize preinvasive states.
MRS often detects changes to cellular
chemistry in human tissues prior to these
changes being apparent under the light
microscope and, hence, discernable by current
methods. The MRS methodology is also of
clinical importance in that it can identify the
extent of abnormality in preinvasive states,
particularly in patients with a predisposition
to cancer. Accurate diagnosis and characte-
rization of preinvasive states is at present a
key missing link in the diagnostic chain and
would allow optimal management and
minimal therapy for many patients in whom
cancer has not fully developed.
Biopsy Collection and Handling
The methods used to collect, store and pre-
pare biopsies prior to MRS examination are
critical for maintaining the high diagnostic
accuracy of the MRS method. While these
methods can differ and have been docu-
mented for each individual organ, there are
common features that need to be considered.
Polypropylene vials used to store biopsies
prior to MRS must be tested for leeching of
chemicals into the supporting buffer under
storage conditions. The presence of these
chemicals may cause cell death and conta-
minate the MR spectrum. For collection of
fine needle aspiration biopsies (FNAB)
syringe barrels also need to be tested for
chemical leakage.
Biopsies must be immediately placed in
vials containing phosphate buffered saline
in D 2O (PBS/D2O) and frozen in liquid
nitrogen. The accuracy of the MRS method
decreases with the length of time the
specimen remains at room temperature.
According to the type of tissue from which
the biopsy was taken there is a finite time
the biopsy can be stored frozen at –70oC
before MRS examination for diagnostic
accuracies to be maintained. For all organs
examined to date, MRS analysis within six
weeks of biopsy collection is sufficient.
When preparing frozen samples for MRS
analysis it must be considered that tissues
may leak variable amounts of potentially
diagnostic metabolites into the storage buffer
upon thawing (Fig. 15.1).8 In many labora-
tories, preparation of tissue samples for
Fig. 15.1: 1H MR (8.5 T, 37oC) spectra showing leakage
of metabolites into collection during freezing storage
and thawing of prostate tissue. Spectrum A shows
biopsy tissue transferred to 200 µL fresh buffer
immediately after thawing. Spectrum B shows the
collection/storage buffer immediately after thawing and
removal of the biopsy tissue. Reprinted from NMR in
Biomedicine, 2003;16:96-101. Bourne R, Dzendrow-
skyj T, Mountford C. Leakage of metabolites from
tissue biopsies can result in large errors in quanti-
tation by MRS. With permission from John Wiley and
Sons limited.
 Application of Proton MRS in the Study of the Pathology of Human Cancers 173

MRS analysis includes washing thawed tissue
in fresh buffer to reduce the amount of
residual water. This has been shown to result
in the loss of large and unpredictable
amounts of possibly diagnostic metabolites
prior to MRS and to reduce the reproduci-
bility of the MR analysis. The problem is
overcome by minimizing the volume of
storage buffer, omitting tissue washing and
performing the MRS measurements on the
tissue immersed in the original storage
buffer.8
Statistical Classification Strategy (SCS)
Magnetic resonance spectroscopy is known
to report on pools of chemicals in cells and
tissues as they alter (many at the same time)
with ageing and the development of disease
processes,2,7,9-25 Many of these biochemical
processes occur in parallel but do not neces-
sarily alter in the same direction. Thus,
managing complex databases which include
large volumes of biomedical data including
spectroscopy, pathology and patient statistics
requires a special methodology. The lack of
specialist methods to overcome this problem
was recognized by Dr Ian Smith (National
Research Council, Canada, NRC) in the late
1970s.
A three-stage statistical classification
strategy (SCS) was developed at the Institute
for Biodiagnostics (IBD), NRC by Dr Ian
Smith and Dr Ray Somorjai specifically to
address these issues (Fig. 15.2). The method
has allowed automated analysis of both ex
vivo and in vivo spectroscopic data generat-
ing highly accurate and reliable mathematical

Fig. 15.2: Statistical Classification Strategy (SCS):A schematic road-map of how the SCS method is developed
for individual data bases. Abbreviations are GA_ORS: genetic algorithm based optimal region selection; LDA:
linear discriminant analysis; LOO: leave one out (method of cross validation); coeff: coefficients. Reprinted
from Chemical Reviews, 2004;104: 3677-704. Mountford C, Doran S, Lean C, Russell P. Proton MRS Can
Determine the Pathology of Human Cancers With A High Level of Accuracy. American Chemical Society
174 Biomedical Magnetic Resonance: Proceedings of the International Workshop
classifiers for a variety of clinical applica-
tions. For a review see Lean et al 2002.1
Described below is the current state of
1H MRS on biopsies of the prostate, breast,
liver, thyroid, oesophagus and in vivo for
the prostate, breast and thyroid.
PROTON MRS ON BIOPSIES
Prostate
Prostate carcinoma is the most common
cancer in men in Australia26,27 and the United
States.28 Histological examination is the defi-
nitive standard for diagnosis and classifi-
cation of prostate neoplasms.29-31
The IBD/NRC undertook the first MRS
study (8.5 T) of prostate biopsies and
analysed the data using the SCS method
(Table 15.1). The accuracy for distinguishing
benign prostatic hyperplasia (BPH) from
cancer was 96.6 percent.32 It was notable in
this study that no prostate intraepithelial
neoplasia (PIN) or proportional volumes of
disease states in tissue specimens were
reported. As the accuracy of the SCS method
is dependent upon correct and detailed
histological data being provided to the
computer-based strategy the possibility that
variant pathologies may have been included
in developing this classifier needed to be
considered. In addition the prostate is a
complex organ with four different functional
zones and mixtures of glandular and stromal
tissues present in varying quantities in a
given tissue biopsy.
A second study by Swindle et al 33
involved MRS spectral analysis of biopsy
specimens that were subsequently serially
sectioned. An error rate of 8 percent in the
routine hospital diagnosis was identified
due to incomplete sampling of the tissue.
Distinct proton MRS spectra (8.5 T) from
glandular BPH, stromal BPH, PIN, and
invasive adenocarcinoma were recorded in
this study (Fig. 15.3, Table 15.2). Spectra
from tissues with different proportions of
adenocarcinoma present (5 % and 50%)
were also found to be spectrally unique (Fig.
15.3). Resonances from lipid, amino acids,
citrate, choline and creatine were assigned
in the spectra of specimens containing
adenocarcinoma and PIN, albeit at different
levels (Table 15.2). The spectra of BPH
specimens from glandular and stromal areas
of the prostate were different with citrate
(the marker for apparently healthy tissue)
not identified in the spectrum from stromal
BPH. It can be seen that there is a gradation
in the spectra from benign stromal and
glandular BPH through PIN to frankly
malignant tissue with increasing volumes of
tumor was reported.
The Swindle study33 further divided the
specimens into BPH from patients with and
without cancer elsewhere in the prostate
and compared both groups with
adenocarcinoma. Histologically confirmed
carcinomas were determined by MRS with
a sensitivity of 100 percent and a specificity
of 94 percent but the accuracy decreased
rapidly when routine hospital histology
reports were used for correlation. Depleted
citrate and elevated choline levels alone
were not accurate markers of malignancy,
since citrate levels remained high when a
small amount of malignant disease was
present. Citrate resonances were present in
the spectra of tissues containing 95 percent
BPH and 5 percent adenocarcinoma. A
recently developed mathematical regression
method34 allows small volumes of carcinoma
to be identified with accuracies approaching
100 percent for the prostate cohort.
Although histological examination remains
the standard in the assessment of human
prostate disease, the use of MRS as an adjunct
to aid in the characterization of prostate
lesions on the basis of their biochemical
 Application of Proton MRS in the Study of the Pathology of Human Cancers 175

Table 15.1: Summary of classifiers and spectral regions using SCS

Biopsy type Classification Spectral Regions Accur Ref

(n) (ppm)

Thyroid (n = 107) Normal v Spectral identity not retained 99.0% 44

Malignant
Brain (n = 206) Control v 23/55 subregions 94.4% 87

Malignant
Astrocytoma High v 0.81-0.85, 1.71-1.75, 2.16-2.20, 2.46-2.50, 95.7% 88

(n = 91) Low grade 2.54-2.58, 3.02-3.06, 3.51-3.55 #

Breast FNA Benign v 1: 0.87-0.92; 1.20-1.25; 1.63-1.67; 1.79-1.82; 96.1% 36

(n = 140) Malignant 1.95-1.98;2.85-2.87;2.95-2.97;3.19-3.33

2: 1.19-1.23; 1.32-1.38; 1.79-1.82; 1.96-2.00;
2.13-2.15; 2.70-2.76; 2.92-2.94;2.97-2.99
Lymph node 1: 0.43-0.51; 0.64-0.77; 1.10-1.20; 1.56-1.59 95.0% 36

involvement 2: 0.44-0.51; 0.67-0.71; 1.13-1.19;1.56-1.60;

1.86-1.96
Prostate (n = 87) Benign v 3.46-3.52; 3.40-3.46; 2.50-2.56; 2.14-2.20; 96.6% 32

Malignant 1.84-1.90; 1.12-1.18

Residual cancer 0.77-0.82; 0.94-1.00; 1.32-1.37; 1.59-1.71;
after radiotherapy
2.24-2.27; 3.19-3.28. 91.4% 89

Liver (n=122) Normal v HCC 1.34-1.37; 2.28-2.33; 2.83-2.85 100% 39

Cirrhotic v HCC 3.00-3.03; 3.56-3.60; 3.66-3.68 98.4% 39

39
Normal v 1.52-1.57; 2.03-2.08; 3.63-3.67 92.1%
Cirrhotic
Ovary (n=56) Normal v Cancer 1.47, 1.68, 2.80, 2.97, 3.17, 3.34 98% 90

Oesophagus Normal v Cancer 3.49-3.57, 3.23-3.28, 0.92-1.12 100% 6

(n = 105) Normal v Barretts 3.50-3.54,1.93-2.06, 1.37-1.43 100% 6

Barretts v Cancer 3.05-3.13, 2.51-2.57, 1.45-1.49, 1.21-1.29 98.6% 6

Melanoma Metastatic v Not yet available 91

Benign
In vivo at 1.5T

Brain Grey v White Not yet available 98% Unpub-

matter lished
Brain Pain v no pain 0.72-1.44, 1.64-1.83, 2.39-2.84, 3.00-3.12, 96% 92

3.17-3.42
Soft tissue Normal mesen- 93% 93

sarcoma chymal v soft

tissue sarcoma
176 Biomedical Magnetic Resonance: Proceedings of the International Workshop

Table 15.2: Resonances in one-dimensional 1H MR spectra

Molecules Abbr Species Resonance assignment

(ppm)

Amines
Choline, phosphocholine, Chol -N(CH3)3 3.2
glycerophosphocholine
Spermine, spermidine, PA -NCH2 3.1
polyamines
Creatine, phosphocreatine Cr -NCH3 3.0
Lipids
Triaclglycerol (fatty acyl chain) Lip CH3-CH2-CH2- 0.90
Lip CH3-CH2-CH2- 1.33
Lip -OOC-CH2-CH2 1.6-1.7
Lip CH3-CH2-CH2- 2.02
Lip CH=CH-CH2- 2.08
Lip -OOC-CH2-CH2 2.3
Lip CH=CH- 5.32
Amino acids
Alanine Ala CH3-CH- 1.49
Glutamate, glutamine Glu, Gln -CH-CH2-CH2-COO-/NH3+ 2.2
Glutamate, glutamine Glu, Gln -CH-CH2-CH2-COO-/NH3+ 2.6
Isoleucine Ile -CH3 0.97
Leucine Leu -CH3 0.97
+
Lysine Lys H3N -CH2-CH2-CH2 1.7
Lysine Lys H3N+-CH2-CH2-CH2 3.03
Threonine Thr CH3-CHOH- 1.33
Threonine Thr CH3-CHOH- 4.25
Valine Val -CH3 1.03
Other
Lactate Lac CH3-CH- 1.33
Citrate Cit -OOC-CHAHB-C(OH) 2.4-2.7
Taurine Tau H3N+-CH2-CH2-SO3- 3.25
Taurine Tau H3N+-CH2-CH2-SO3- 3.43

composition may provide additional diagno-
stic and prognostic information.
Breast
Breast cancer is the most common cancer to
affect women in Western countries with 80-
100 newly diagnosed patients per 100 000
inhabitants per year.35 The combination of
physical examination, mammography, and
fine needle aspirate cytology or needle core
biopsy (triple assessment) is currently the
preferred method for the preoperative
 Application of Proton MRS in the Study of the Pathology of Human Cancers
Fig. 15.3:1H MR (8.5 T, 37oC) spectra of prostate biopsy
specimens, 256 acquisitions, sweep width 3,597 Hz,
and a pulse repetition time of 2.14 seconds. The water
peak was suppressed by selective gated irradiation.
(A) adenocarcinoma (50% of the tissue made up of
malignant tissue), (B) adenocarcinoma (5% of the
tissue made up of malignant tissue) (C) Prostatic
intraepithelial neoplasia (PIN) (D) stromal BPH (95%
stromal, 5% glandular) and (E) glandular BPH (85%
glandular, 15% stromal) are compared. Reprinted
from Radiology 2003; 228:144-151. Swindle P,
McCredie S, Russell P, Himmelreich U, Khadra M,
Lean C, L., Mountford CE. Pathologic characterization
of human prostate tissue with proton magnetic
resonance spectroscopy. With permission from
Radiological Society of North America
diagnosis of clinically or radiographically
detected breast lesions. Although triple
assessment has a high probability of
detecting all malignant lesions, its
177
suboptimal specificity results in diagnostic
uncertainty, requiring open biopsy to
exclude malignancy in many women.
In a study of 218 FNAB specimens from
191 consecutive patients undergoing diag-
nostic biopsy or definitive treatment (i.e.
lumpectomy, quadrantectomy, or mastec-
tomy) for histologically proven invasive
breast cancer, 1H MRS identified invasive
carcinoma (n = 106) based on the intensity of
the 3.25 ppm resonance (choline-containing
metabolites) standardised to the resonance
intensity at 3.05 ppm, which contained
contributions from creatine, phosphocreatine,
and lysine (P< 0.0001, Mann-Whitney test).24
MRS of FNAB specimens correlated with the
final histological diagnosis for 96 percent of
benign lesions. The 4 percent false positive
rate compares with the rate of other moda-
lities. They were, however, from four young
women with fibroadenoma. The MRS of
FNAB specimens (Fig. 15.4) correlated with
a malignant histological diagnosis in 95
percent of cases.
One of the most revealing studies into the
diagnostic power of 1H MRS analyzed by
the SCS method is the recent study of FNAB
from breast tumors. Using the SCS method
the distinction between benign and malig-
nant biopsy specimens gave a sensitivity of
95 percent and specificity of 96 percent with
an overall accuracy of 96.1 percent (Table
15.1). However, from the same spectra
obtained from tissue in the primary tumor
and using the SCS method, lymph node
involvement could also be predicted with
an overall accuracy of 95 percent (Table
15.1). Similarly, vascular invasion in the
definitive surgical specimen was predicted
with an overall accuracy of 94 percent.36
Thus, the application of MRS to the exami-
nation of FNAB from the breast has the
178 Biomedical Magnetic Resonance: Proceedings of the International Workshop
Fig. 15.4: 1 H MR (8.5 T, 37 o C) Spectra (256
acquisitions) of a FNAB taken from an invasive
carcinoma of the breast and normal tissue. By visual
inspection the distinction between normal breast and
invasive carcinoma is based on an increase in N-
trimethyl resonance at 3.25 ppm normalised to that
of creatine at 3.05 ppm (peaks indicated by arrows)
(Reprinted from Radiology, 1997; 204, 661-666.
Mackinnon WB, Barry B, Malycha P, Gillett D, Russell
P, Lean CL, Doran S, Barraclough B, Bilous M and
Mountford CE: Fine needle biopsy of benign breast
lesions distinguished from invasive cancer by proton
magnetic resonance spectroscopy. With permission
from the Radiological Society of North America.)
potential to revolutionise breast cancer
management by providing both diagnosis
and prognostic information prior to surgery.
It is important to note that the entire tissue
specimen needs to be examined histologically
at section intervals as small as 100 μm for
accurate correlation with MRS data. A good
example of this is ductal carcinoma in situ
(DCIS) of the breast where differences in
pathology may exist in between two adjacent
tissue sections 100 μm apart (Fig. 15.5,
Plate 9).
Visual inspection of MR spectra from
DCIS found that specimens that had
breached the basement membrane (< 1 mm)
in one or more foci (i.e. microinvasion) and
those with extensive comedonecrosis had
spectra similar to frankly malignant lesions.
Other cases of DCIS, without microinvasion,
contained far less chemical activity and were
more readily correlated with benign
specimens.24
Liver
A common internal malignancy worldwide
is primary hepatocellular carcinoma (HCC).37
In 1998 it was the most frequent tumor in
males38 and the most common in Asia and
Africa. The annual incidence in Western
countries is 4 per 100,000.38 HCC almost
always occurs as a sequel to cirrhosis and is
increasingly prevalent because of the
upsurge in cases of chronic hepatitis B and
hepatitis C.
Biopsies obtained from apparently
normal liver, cirrhotic liver and HCC, were
examined by 1H MRS at 8.5 T (Fig. 15.6).
The correlative histopathology from 54
consecutive patients undergoing partial or
total hepatectomy was obtained from Royal
Prince Alfred Hospital39 in Sydney where
the Australian National Liver Transplant
Unit is located.
Distinct MR spectra were recorded from
HCC, cirrhotic, and apparently healthy liver.
Resonance assignments were made from two-
dimensional (2D) 1H, 1H COSY (Fig. 15.7)
and 1H, 13C HSQC and are summarized in
Table 15.2.39
SCS-based classification of 1H MR data
from the liver biopsies distinguished normal
liver from HCC with 100 percent accuracy
(Table 15.1). The SCS method also distin-
guished cirrhotic liver from HCC with an
accuracy of 98.4 percent (Table 15.1). The
distinction between normal and cirrhotic liver
was, however, less accurate at 92 percent
(Table 15.1). The histological distinction of
HCC from tumor look-alikes can, however,
 Application of Proton MRS in the Study of the Pathology of Human Cancers
Fig. 15.6: 1H MR spectra (8.5 T, 37oC) of liver biopsy
specimens: A) Normal Liver; B) Cirrhotic Liver; and
C) Hepatocellular Carcinoma (HCC). Spectra were
acquired with water suppression using selective gated
irradiation, sweep width of 3600 Hz, 8K data points,
256 accumulations and a repetition time of 2.3
seconds. (Reprinted from Pathology 2002;34:417-
422. Soper R, Himmelreich U, Painter D, Somorjai R,
Lean C, Dolenko B, Mountford C, Russell P. Pathology
Of Hepatocellular Carcinoma And Its Precursors
Using Proton Magnetic Resonance And Statistical
Classification Strategies. With permission from Taylor
and Francis: http://www.tandf.co.uk/journals/titles/
00313025.html)
be difficult. SCS analysis of MRS offers a
new and accurate supplementary means for
the objective diagnosis of HCC. Extended
into the in vivo situation this technology
would provide clinically significant pre-
operative information.
Thyroid
Thyroid nodules are common and are
estimated as being clinically evident in up to
10 percent of the population. While the vast
179
majority (90-95%) of solitary thyroid
nodules are benign, 40-42 the exclusion of
follicular thyroid malignancy remains a
significant diagnostic problem. Preoperative
FNAB cytology, although accurate in
identifying papillary, medullary and
anaplastic carcinomas, is unable to reliably
distinguish benign from malignant follicular
neoplasms. Thus, biopsy material obtained
following thyroidectomy is required for
diagnosis resulting in many women having
their entire thyroid removed to exclude
malignancy. Histologically, follicular
adenomas and carcinomas are often
indistinguishable and the identification of
capsular or vascular invasion at the edge of
the lesion is required to confirm malignancy.
In a study of 53 consecutive patients with
thyroid nodules, 1D 1H MRS distinguished
normal thyroid tissue from proven carcinoma
of all types with a sensitivity and specificity
of 100 percent (p<0.0001, Student t-test).7 The
samples were obtained intraoperatively i.e.
with no blood supply to the organ. The basis
of the discrimination was altered cellular
chemistry reflected in the resonance intensity
ratio at a chemical shift of 1.7 ppm (arising
from lysine) and lipid at 0.9 ppm (Fig. 15.8).
The lipid spectral profile is much weaker in
adenoma than carcinoma showing the same
trend previously observed for uterine
cervix.18
Histologically benign follicular lesions, as
currently defined, were found to span both
the normal and malignant spectral patterns
(Fig. 15.8). The biological significance of the
span of MR spectral ratios recorded for
follicular lesions is unclear, although it would
support the concept that there is a progression
from benign to malignant, preceding any
histological evidence of malignancy.
Molecular genetic studies have recently
supported this concept.43
180 Biomedical Magnetic Resonance: Proceedings of the International Workshop

Fig. 15.7: Typical 2D 1H, 1H COSY spectra from biopsy

samples of A) Normal liver; B) Chirrotic liver; and C) HCC;
48 accumulations, 200 experiments. Spectra were
processed using a Sine bell and a Lorenzian-Gaussian
(LB = –30, GB = 0.20) window function , in the t1 and t2
domains, respectively. (Reprinted from Pathology
2002;34:417-422. Soper R, Himmelreich U, Painter D,
Somorjai R, Lean C, Dolenko B, Mountford C, Russell P.
Pathology of Hepatocellular Carcinoma and its
Precursors using Proton Magnetic Resonance and
Statistical Classification Strategies. With permission from
Taylor and Francis: http://www.tandf.co.uk/journals/titles/
00313025.html)

A second study22 involved MRS of FNAB
of thyroid tissues with the aim of addressing
the inability of the fine needle cytology to
accurately discriminate between follicular
adenomas and carcinomas. MRS on FNAB
offers the prospect of a technique that more
accurately reflects the actual biology of
follicular thyroid tumors, allowing more
accurate diagnosis of thyroid cancers and
reducing the need for unnecessary surgery
performed solely for diagnostic purposes.
It is possible to obtain an accurate 1H MR
spectrum from as few as 106 thyroid cells
from a FNAB.22
In a study of FNAB and tissue specimens
from 70 patients undergoing thyroidectomy
for solitary or dominant thyroid nodules, a
close correlation between fine needle MR
spectra and tissue spectra for a range of
benign and malignant neoplasms has been
demonstrated (Fig. 15.9).22 The sensitivity
or probability of correctly identifying
thyroid cancer on the basis of MRS assessed
on FNAB was 95 percent. However, again
these FNAB’s were obtained intraoperati-
vely.
The application of SCS allowed further
refinement of the discriminatory potential
 Application of Proton MRS in the Study of the Pathology of Human Cancers 181

of MR spectral analysis of thyroid tissues.

Fig. 15.8: Ratio of resonances at 1.7 ppm and 0.9
ppm for normal thyroid tissue, follicular neoplasms and
malignant thyroid biopsies. Histologically proven
follicular cancers all have a ratio below 1.1, whereas
“non-malignant” follicular neoplasms fall into groups,
comparable with either normal or carcinomatous
tissue. A separation of ratios above and below 1.1 is
observed with no overlap between normal and
malignant tissue. (Reprinted from World Journal of
Surgery, 1994; 18, 512-517, Delbridge, L., Lean, C.L.,
Russell, P., May, G.L., Roman, S., Dowd, S., Reeve,
T.S. and Mountford, C.E. Proton magnetic resonance
and human thyroid neoplasia. II: Potential avoidance
of surgery for benign follicular neoplasms. With
permission from Springer-Verlag.)
When tissue data were subjected to SCS a
sensitivity and specificity of 100 percent for
the diagnosis of malignancy was achieved
for the training set and a sensitivity and
specificity of 98 percent and 100 percent,
respectively for a test set of samples of
known malignancy44 (Table 15.1).
In order for the MRS methods to be of
clinical use and prevent surgery the biopsies
needed to be taken in the surgeons rooms
i.e. with blood supply to the organ. Due to
the high level of vascularization of the
thyroid some of the FNAB specimens
contained blood contamination and the
accuracy dropped to below acceptable levels.
Thus for the MRS methods to be clinically
applicable the method needed to be
developed for diagnosis in vivo.
Based on the knowledge of which reso-
nances were diagnostic from ex vivo MRS of
follicular adenomas, MR images at particular
chemical shifts were collected and compared

Fig. 15.9: Two directional plots to compare the resonance intensity ratio (1.7/0.9 ppm) obtained from FNB and
resected tissue specimens from the same solitary nodule. A O, Normal thyroid; the difference in the MRS ratio
between FNB and tissue bipopsy is 0.75± 0.79 (mean ±SD. , Malignant thyroid; the difference is 0.19 ± 0.12
(mean ± SD). B: , Follicular lesions; the MRS ratio is 1.1 or more; the difference is 0.31 ± 0.27 (mean ± SD).
, Follicular lesions; the MRS ratio is less than 1.1; the difference is 0.19 ± 0.24 (mean ± SD). (Reprinted from
Journal of Clinical Endocrinology and Metabolism 1995; 80:1306-1311, Lean CL, Delbridge L, Russell P, et
al. Diagnosis of follicular thyroid lesions by proton magnetic resonance on fine needle biopsy. Copyright
1995, The Endocrine Society.)
182 Biomedical Magnetic Resonance: Proceedings of the International Workshop
for biopsies (obtained intraoperatively) of
benign follicular adenoma; histopatho-
logically benign follicular adenoma with no
capsular invasion but with an ex vivo MR
spectrum consistent with malignancy; folli-
cular carcinoma confirmed by secondary
tumors. The intensity of the methyl image
was variable. No signal was observed for
benign tissue while intense signals were
observed for carcinoma. Scattered areas of
signal were observed for the benign
adenoma that was, by MRS criteria, malig-
nant indicating that the pathology was
heterogenous and chemically consistent with
malignant (Fig. 15.10, Plate 10).45 See later
in this review for the role of MR in vivo.
Esophagus
Adenocarcinoma of the lower esophagus in
the Western world is rising and accounts
for more than 40 percent of esophageal carci-
nomas in males.46 The condition is thought
to result from gastric reflux and Barrett’s
oesophagus is thought to be a precursor47
which increases the risk of developing
malignancy 40 to 50 fold.
Histopathology can accurately distinguish
normal from invasive carcinoma in the
esophagus. The accurate prediction of the
behavior of dysplastic Barrett’s epithelium
is, however, not possible.48,49 MRS has been
considered as a possible objective technique
to identify Barrett’s patients at risk of prog-
ressing to adenocarcinoma. In a study investi-
gating 72 consecutive patients, 29 non-cancer-
bearing and 43 cancer-bearing, 1H MRS of
esophageal biopsies combined with the
SCS strategy provided a robust diagnosis
with a high degree of accuracy for discrimi-
nating normal epithelium from esophageal
adenocarcinoma and Barrett’s esophagus
(Table 15.1).6
The spectra of Barrett’s epithelium from
a non-cancer-bearing patient and from a
cancer-bearing patient were unique by
visual inspection. Histologically, these tissues
are indistinguishable yet the MR spectra
group the specimens from cancer-bearing
patients with adenocarcinomas. There is
substantial evidence supporting the existence
of an adenoma-carcinoma sequence in the
esophagus.50-52 These MRS data support the
MR method being able to identify a field
change consistent with the presence of an
adenoma carcinoma sequence.
PROTON MRS IN VIVO
Clinical applications of MRS in vivo have
improved significantly over the last decade.
Single voxel and magnetic resonance
spectroscopic imaging (MRSI) studies of
brain and prostate have shown that the
method can identify metabolite levels which
may be correlated with human disease
states. It may be expected that when
expensive and detailed pathological
examination of biopsy material is correlated
with in vivo spectra this methodology will
also generate accuracies approaching 100
percent.
Prostate
The use of serum prostate specific antigen
(PSA) testing and transrectal ultrasound
(TRUS) guided biopsy has allowed a
significant increase in screening for prostate
cancer with increased numbers of patients
identified as having disease at an earlier
stage.53 The decision on how to manage
prostate disease once identified can cause a
dilemma for both the doctor and patient as
many men will die with, but not of the
disease. Whilst there is a high level of
accuracy achievable using MRS to analyze a
 Application of Proton MRS in the Study of the Pathology of Human Cancers 183

biopsy specimen, can this information be method (9.4 T) and conventional 1D

obtained in vivo? spectrum (8.5 T) both obtained on an intact
The UCSF group led by Kurhanewicz, biopsy specimen (Figure 15.11A and B
Nelson and colleagues, has spearheaded respectively). This is compared with the
the in vivo MR prostate program in the spectrum obtained from a healthy prostate
USA.54-56 In Europe, Heerschap and collea- in a 3 T whole body scanner (Fig. 15.11C)
gues have worked in parallel to put prostate (Bourne R., Stanwell P., Mountford C.,
MR spectroscopy in the clinic in vivo.57 The unpublished data) but using only a surface
implementation of prostate MRS will phased array coil. Whilst the 3 T in vivo
conceivably be the most difficult of all the data show broader resonances, the chemical
signature of the healthy tissue is apparent.
in vivo applications due to the complexity of
It is expected that with the introduction of
the zonal anatomy of the prostate and the
endorectal coils for use at 3 T the resolution
often multifocal nature of prostate cancer.
and signal to noise will be sufficient for
As with the brain, single voxel spectroscopy
was initially developed. An endorectal coil
was used which allowed relatively high
resolu-tion anatomical images in a reason-
able amount of time.54-61
Using the UCSF method, lipid was
suppressed, both inside the voxel of interest
and outside the voxel of interest. High levels
of citrate indicated the presence of healthy
tissue. Malignant tissue was characterized
by low levels of citrate together with
increased levels of compounds involved in
phosphotydylcholine and phosphotidyle-
thanolamine synthesis and hydrolysis
(choline, phosphocholine, glycerolphos-
phocoline, ethanolamine and phospho-
ethanolamine) contributing to the in vivo
measurement of choline.
The goal now is to marry the knowledge
acquired using high field 8.5 T MRS on tissue
specimens with meticulous histological
examination33 and the accurate registration
method of MRSI. It remains to be seen, how-
ever, whether this information can be Figs15.11A to C: 1H MR spectra of healthy prostate
obtained at 1.5T or whether the higher field (A) Ex vivo MAS at 9.4 T (B) Ex vivo at 8.5 T (C) In vivo
3T magnet is needed to provide the required 3 T whole body scanner using a surface coil.
resolution. (Reprinted from Chemical Reviews 2004; 104:3677-
3704. Mountford C, Doran S, Lean C, Russell P. Proton
In Figure 15.11 the spectra from healthy
MRS Can Determine the Pathology of Human
prostate tissue are shown. The comparison Cancers With A High Level of Accuracy. American
is made between the magic angle spinning Chemical Society)
184 Biomedical Magnetic Resonance: Proceedings of the International Workshop
highly accurate pathological diagnosis of
prostate disease.
A major breakthrough came with the
development of MRSI (see Vigneron and
Nelson for review).62 The MRSI method
produces a three-dimensional map of conti-
guous volumes of about 0.24–0.34 cc voxels
that map the entire prostate. The goal now
is to marry the knowledge acquired using
high field 8.5 T MRS on tissue specimens
with meticulous histological examination33
and the accurate registration method of
MRSI. This combined with analysis of the
data with the SCS method should allow
diagnosis of relatively small lesions.
Breast
If the spatial location(s) and pathology of
breast lesions could be determined prior to
surgery it would allow preoperative
decisions regarding patient management.
Improvements in dynamic contrast enhance-
ment features of breast tumors and the
appreciation of morphological charac-
teristics of these lesions has improved the
accuracy of the MRI method.63-72
The first report of in vivo 1H MRS at
1.5 T of breast tumors was by Sijens and
colleagues. 73 They used point-resolved
spectroscopy (PRESS) and their work
suggested that cancers had higher water-
to-fat ratios than normal tissue. 73 This
observation was later refuted in a study by
Roebuck74 using a stimulated-echo acquisi-
tion mode (STEAM) sequence. The study
by Roebuck showed the presence of a broad
composite resonance at 3.2 ppm, containing
choline in 7 of 10 malignant tumors and
also in 1 of 7 benign lesions. One particular
false positive was a large tubular adenoma,
a relatively rare tumor, but at large size
considered to be a borderline malignant
lesion.75
In other studies undertaken at 1.5 T,
Yeung76 showed the presence of the compo-
site choline resonance at 3.2 ppm in 22 of 24
malignant lesions and 1 of 6 benign lesions
(sensitivity and specificity of 91.7 and 83.3%,
respectively). Kvistad and colleagues 77
reported the same composite resonance at
3.2 ppm in 9 of 11 malignant breast cancer
lesions and 2 of 11 benign lesions (sensitivity
and specificity of 81.8 and 75.9%, respec-
tively). Of note was the report this compo-
site resonance containing choline was
observed in 5 of 7 lactating women but in
none of 11 apparently normal volunteers.
Similar accuracies were reported by Cecil78
(sensitivity and specificity of 82.6 and 86.7
percent, respectively); and Jagannathan and
colleagues (sensitivity and specificity of 78.2
and 85.7 percent, respectively).79 Of these
studies, Kvistad and colleagues77 were the
only group where 10 normal volunteers
were examined. None were false positives,
for summary see Table 15.3.
Our more recent study (Figs 15.12A and
B), which included 40 apparently healthy
volunteers, 3 lactating women and 14 women
with carcinoma of the breast, questioned
the reliability of using the composite choline
resonance at 3.2 ppm as a marker for cancer
as addressed. Two of the lactating volun-
teers and 3 of the 40 apparently healthy
volunteers recorded the 3.2 ppm composite
resonance. Eleven of the 14 cancer patients
also recorded the presence of the resonance
at 3.2 ppm. This resulted in a sensitivity of
79 percent and a specificity of 88 percent.
For summary see Table 15.3.
It was demonstrated by Aboagye and
colleagues that total choline-containing
phospholipid metabolite levels increase with
progression from normal to immortalized
to oncogene-transformed to tumor derived
cells.80 It has also been shown that the MR
spectra from fibroblasts81 contains low levels
of choline and creatine. Immortalized and
aging cells 2 contain increasing levels of
choline and to a much lesser extent creatine.
 Application of Proton MRS in the Study of the Pathology of Human Cancers
Thus for the MRS method to provide
the accuracies recorded at the higher fields
strength of 8.5 T on biopsy material more
resonances than the composite at 3.2 ppm82
need to be resolved for accurate diagnosis
in vivo 1. This was achieved by careful
referencing of the spectra and post acquisi-
tional precessing of the data in the following
manner; the 2048 data points were zero filled
to 8192 data points and a Gaussian function
of 1.5 Hz was applied with a 155 echo offset
(Fig. 15.13).83 Using this methodology proton
MRS in vivo distinguished the volunteer
cohort from breast cancer patients with 100
percent specificity. Spectra from lactating
women and control volunteers ceased to be
false positives when the spectra were post-
processed and the resonance at 3.28 ppm,
yet to be definitively assigned but consistent
with taurine, myo-inositol or possibly GPC,
the chemical shift of which can change with
pH. 84 In vivo 2D spectroscopy should
provide a definitive assignment.85
This did not, however, remove the 20
percent false negative rates due to the
intense lipid signal masking diagnostic mole-
185
Figs 15.12A and B: (A) L to R Sagittal
short tau inversion recovery (STIR)
fast-spin echo MR image (42/155/
4000); Sagittal T1 -weighted spin
echo MR image (12/520); Sagittal
gadolinium-enhanced MR image
(2.1/9.1) shows the tumor and voxel
used for SVS in patient 16. The
pathology at surgery was mixed
tubular/lobular carci-noma. (B)
Corresponding 1 H in vivo proton
spectrum (135/2000, 256 signal
averages). (Reprinted from Euro-
pean Radiology 2004 online edition
September. Stanwell P, Gluch L,
Clark D, et al. Specificity of choline
metabolites for in vivo diagnosis of
breast cancer using 1H MRS at 1.5 T.
With permission from Springer-
Verlag.)
cules. This remains an ongoing problem to
be solved.
Thyroid
As described earlier MRS of thyroid tissue
biopsies obtained intraoperatively provided
an accurate guide to the pathology. Further-
more CSI identified tumor heterogeneity not
apparent using conventional light micro-
scopy.
A purpose-designed multi-ring surface
coil was built for undertaking in vivo MRS
of the thyroid.86 A coil was wound for both
1.5 T and 3 T scanners. The MR spectrum
collected using this coil at 3 T from a solitary
thyroid nodule was comparable to that
obtained at 8.5 T ex vivo from a thyroid
biopsy of the same pathology (Figs 15.14A
to C). The clinical future of MRS of the
thyroid at 3 T therefore, looks promising
for diagno-sing disease accurately.
CONCLUSION
The MRS is poised to supplement the already
significant contribution of magnetic reso-
186 Biomedical Magnetic Resonance: Proceedings of the International Workshop

Table 15.3: Summary table of breast in vivo data

Study Roebuck74 Kvistad77 Yeung76 Cecil 78 Jagannathan79 Stanwell83

Magnet GE Signa Picker EPI Philips GE Signa Siemens GE Signa

1.5 T II 1.5 T Gyroscan 1.5 T Magnetom 1.5 T
1.5 T 1.5 T
Coil Home-built Home-built Unspecified Unspecified Proprietary Home-
single breast single breast proprietary phased array bilateral built
receive-only receive- receive- multicoil receive- single
multicoil only coil only double- only coil breast
breast coil transmit-
receive
multicoil
Pulse STEAM PRESS PRESS STEAM STEAM PRESS
sequence
No of cases Carcinoma 10 11 24 23 46 21
Benign 7 11 6 15 14 0
Volunteers 0 11 0 0 43
Lactation 0 7 0 0 1 3
Sensitivity* 70.0% 81.8% 91.7% 82.6% 78.2% 80.0%

Specificity* 85.7% 75.9% 83.3% 86.7% 85.7% 86.0%

*Sensitivity and specificity determined as the ability of MRS to predict malignancy based on the presence
or absence of choline (3.2 ppm).
Fig. 15.13: Typical proton single voxel spectrum (135/
2000, 256 signal averages) from a patient with
histologically confirmed invasive ductal carcinoma is
compared with a typical spectrum from one of the three
lactating volunteers and one of the false-positive non-
lactating volunteers (3/40) (2.0-3.7 ppm shown).The
spectra are processed by zerofilling the 2048 data points
to 8192 data points, applying a Gaussian apodization
function of 1.5 Hz with a 15% echo offset before fast
Fourier transformation. In the spectrum from the cancer
bearing patient, resonances can be assigned to creatine-
containing compounds (3.04 ppm), phosphocholine (3.22
ppm) and glycerophosphocholine/taurine/myo-inositol
(3.28 ppm). In the spectra from the lactating volunteer
and the false-positive non-lactating volunteer the
dominant resonance is centred at 3.28 ppm consistent
with glycerophosphocholine/taurine/myo-inositol. The
spectra were referenced to the methylene resonance of
lipid at 1.33 ppm and water at 4.74 ppm. (Reprinted from
European Radiology 2004 online edition September.
Stanwell P, Gluch L, Clark D, et al. Specificity of choline
metabolites for in vivo diagnosis of breast cancer using
1H MRS at 1.5 T. With permission from Springer-Verlag.)
 Application of Proton MRS in the Study of the Pathology of Human Cancers
Figs 15.14A to C: 1 H MR on benign follicular
adenomas of the thyroid. (A) In vivo T1 weighted axial
image; (B) Corresponding in vivo 1H 3 T MR spectrum.
(Undertaken at the National Research Council of
Canada, Winnipeg. Data was obtained using a
specially designed multi-ring surface coil); (C) Ex vivo
1H 8.5 T MR spectra from a biopsy specimen of the
same pathology. (Reprinted from Chemical Reviews
2004; 104:3677-3704. Mountford C, Doran S, Lean
C, Russell P. Proton MRS Can Determine the
Pathology of Human Cancers With A High Level of
Accuracy. American Chemical Society.)
nance imaging (MRI) for the clinical manage-
ment of malignant disease. As well as
accurately reporting pathological processes
in tissue, spectroscopy also identifies
important subsets of disease states that are
not morphologically manifest. The MRS
method in vivo is currently being developed
and should be capable of accuracies appro-
aching those employing MRS on excised
biopsy specimens.
ACKNOWLEDGMENTS
The authors wish to acknowledge the long-term
contributions from the other clinical and scientific
directors of the IMRR, Prof Martin Tattersall, Dr
Uwe Himmelreich, Dr Peter Malycha, and Mr Peter
Stanwell. We gratefully acknowledge the contri-
butions made by our colleagues at the NRC,
Winnipeg, Dr ICP Smith, Dr Ray Somorjai, Dr Brion
Dolenko, Dr Slavek Tomanek, Dr Scott King and Dr
S Nikulin. We thank Dr Roger Bourne and Dr Philip
Katelaris for their contribution to the prostate pro-
187
gram and Dr David Clarke for his contri-bution to
the breast program.
A special thank you to Sinead Doran for
assistance in preparing and collating this manuscript.
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59. Chelsky M, Schnall M, Seidmon E, Pollack H.
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60. Schick F, Bongers H, Kurz S, Jung WI, Pfeffer
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65. Kvistad KA, Rydland J, Vainio J.et al Breast
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192 Biomedical Magnetic Resonance: Proceedings of the International Workshop
P MR Spectroscopy of
31
Muscle Energy Metabolism in
Humans: Physiological and
Clinical Applications
INTRODUCTION
Thirty years ago, Hoult et al reported for
the first time that phosphorus metabolites
could be observed in vivo using 31P magnetic
resonance spectroscopy (MRS) opening
promising opportunities of understanding
muscle energetics in vivo under strictly non-
invasive conditions.1 From that time, MRS
technology has rapidly evolved with the
development of RF surface coils in 1980,2 the
availability of high-field wide-bore super-
conducting magnets and methodological
developments (dedicated pulse sequences,
spatial localization of NMR signal, etc.)
making MRS a tool of choice for investi-
gating muscle energetics noninvasively in
animals and humans. So far, a large number
of publications has been devoted to the
investigation of muscle energetics in
a variety of conditions ranging from
diseases 3-5 to training regimens 6-8 as
compared to normal conditions. Such studies
have provided interesting information
16
D Bendahan, PJ Cozzone
regarding not only pathologies and
metabolic changes associated with training
but also about what normally occurs
throughout muscle contraction in terms of
balance between energy production and
consumption. In this presentation, we intend
to provide key information to the non-
specialist in order to understand what 31P
MRS can tell us about muscle energetics.
We will concentrate on results obtained in
humans and the issue of muscle fatigue is
not in the scope of this paper.
MR TECHNIQUES
MR spectra as well as MR images are gene-
rated by placing samples in a powerful
magnetic field and then exciting them with
a radiofrequency energy. Susceptible nuclei
(in the present case 1H in water molecules or
31
P in high-energy phosphate compounds)
are boosted to a higher energy state, pro-
ducing a detectable signal when the
excitation stops. This signal declines over
 31
P MR Spectroscopy of Muscle Energy Metabolism in Humans
time via two concurrent processes described
as relaxation. T1 relaxation (longitudinal or
spin-spin relaxation) involves the loss of
energy to surrounding nuclei with similar
resonance frequencies and roughly
determines signal intensity for a given
repetition time. T2 relaxation (transverse or
spin-lattice relaxation) results from
interaction between the excited nuclei and
any perturbing magnetic field with no
transfer of energy. Signal width is inversely
proportional to T2. The non-discriminatory
nature of T2 relaxation mechanisms greatly
increases the probability of such interactions
thus making T2 much shorter than T1 in
heterogeneous solutions. Signals obtained
in 31P MR spectra and 1H MR images are
highly dependent on both T1 and T2 values
together with nuclear density, i.e. the
number of free rotational nuclei of the tissue
investigated. For a given experimental time,
compounds with shorter T1 will end up with
higher intensities in MR spectra, while signal
width in spectra is inversely proportional
to T2 values. Regarding MR images, T1, T2
and nuclear density will determine the
contrast of cellular and subcellular
structures.
Informational Content of a
31
P MR Spectrum
Measurement of phosphorylated com-
pounds’ concentrations in living cells is not
easy. Traditional methods such as percuta-
neous needle biopsy and freeze clamping
exhibit limitations, especially related to
alteration of anatomic integrity and partial
degradation of phosphorylated metabolites
during extraction and analysis. In addition,
repeated measurements cannot be performed
on the same muscle, making impossible the
achievement of high-time resolution kinetics.
193
Compared to analytical methods, 31P MRS
offers the opportunity of measuring
noninvasively and continuously with high
time-resolution, the concentration of
phosphorylated compounds involved in
muscle energetics. In addition, comparison
of direct biochemical measurements with 31P
MRS findings suggests that the two methods
give comparable results.9
A typical 31P MRS spectrum exhibits six
to seven peaks corresponding to phospho-
creatine (PCr), inorganic phosphate (Pi), the
three phosphate groups of ATP (in position
α, β and γ) and phosphomonoesters (PME)
(Figs 16.1A and B). The Pα signal of ATP
displays occasionally an upfield shoulder
corresponding to NAD+/NADH. In between
the PCr and Pi signals, the phosphodiester
signal is sometimes observed. This signal is
usually assigned to glycerophosphory-
lcholine and glycerophosphoryethanolamine
which can be detected as a small peak in
normal muscle spectra (mainly from lower
limb) and as a larger peak in patients with
muscle dystrophy10 indicating membrane
breakdown. Given the low sensitivity of the
technique, the free metabolically active ADP
concentration, which is only a tiny fraction
of its total intracellular concentration, cannot
be measured. It can, however, be calculated
using the creatine kinase equilibrium where
the total creatine content is taken as either
42.5 mM or considering that phospho-
creatine represents 85 percent of the total
creatine content.11 Similarly, AMP concen-
tration can be calculated using the adenylate
kinase equilibrium. 11 In the absence of
biochemical data, ATP is often assumed to
be normal and is used as the equivalent of
an internal standard in order to calculate
the concentrations of other metabolites.
Apart from the dynamic measurements
of high-energy phosphate compounds, 31P
194 Biomedical Magnetic Resonance: Proceedings of the International Workshop

Figs 16.1A and B: Typical series of 31P MR spectra recorded in humans forearm flexor (A) and thigh muscles
(B). In A, MR spectra have been recorded at 4.7 T (Biospec 47/30 Bruker) during a standardized rest-exercise-
recovery protocol with a time resolution of 15 s. In B, MR spectra have been recorded at 1.5 T(Siemens Vision
Plus) after a standardized bicycle exercise protocol with a time resolution of 2 s. On the higher panel of each
figure, is represented a single spectrum with the corresponding assignments. Ref : Reference compound
(phenyl phosphonic acid), PME (phosphomonoesters), Pi (inorganic phosphate), PDE (phosphodiesters),
PCr (phosphocreatine), phosphate groups of ATP in position α, β and γ

MRS offers the only noninvasive way of
assessing intracellular pH. Indeed, under
conditions of physiological pH and consi-
dering one of the pKa, i.e. 6.75, two forms of
Pi coexist (H2PO4– and HPO42–). These two
forms are exchanging so fast that only a single
Pi signal is detected. However, the chemical
shift of this single signal is a weighted
average of both monobasic and dibasic
forms. Due to this sensitivity of the Pi
chemical shift to pH, it is possible, with
appropriate calibration curves, to translate
any Pi shift in terms of intracellular acidosis
or alkalosis.
The quantitative measurements of high-
energy phosphate compounds and pH allow
 31
P MR Spectroscopy of Muscle Energy Metabolism in Humans
to compute a number of derived metabolic
parameters such as the oxidative phos-
phorylation potential and the free energy of
ATP hydrolysis in vivo. All those parameters
reflect the regulation and control of energy
metabolism, ion transport and muscular
contraction.
Also, magnesium concentration has been
calculated from changes in ATP chemical shift
on the basis that the chemical shift of the
resonance corresponding to the beta group is
sensitive to the intracellular magnesium
concentration.12-14
Fat, fibrous tissue blood and extracellular
fluid contribute no significant signal and
mitochondrial metabolites are too tightly
bound to interfere.
Technical Considerations
The requirement for magnetic field homo-
geneity generally dictates that the muscle
examined be positioned at magnet center and
remain in a fixed position during data
collection. In that respect, dedicated ergo-
meters have been designed in each laboratory
in order to investigate exercising muscles
within superconducting magnets. So far,
adductor pollicis,15 forearm and wrist flexor
muscles,5,16,17 calf18,19 and thigh muscles20-23
have been investigated using 31P MRS. MRS
recordings have been sometimes coupled to
other noninvasive techniques such as
electromyography 24,25 and gas exchange
measurements.21 Given the low magnetic
resonance sensitivity of 31P (6% of proton)
and the low tissue concentrations of some
of the relevant metabolites, MR signals are
time-averaged over a period ranging from
a few seconds to several minutes depending
on the required signal-to-noise ratio and
the desired time resolution. In addition, MR
signal is detected with a surface coil over a
195
relatively large muscle volume proportional
to the surface coil radius making this signal
a weighted average of the muscle fibers
existing within the sampling volume. This
has to be taken as a comparative item with
histological and biochemical analyses which
are often carried out on very small samples
of tissues which may not give a represen-
tative picture of the biochemical state of the
muscle. However, care has to be taken not
to turn this advantage into a drawback
while sampling exercising and non-exercising
muscles at the same time. This can be
achieved when MRS is combined to MRI
(Figs 16.1A and B) for (i) proper localization
of the coil, and (ii) proper determination of
the sensitive volume of the coil.
Yet only a small number of centers
around the world utilize MRS routinely for
investigations of metabolic changes
surrounding muscle contraction. The labor
intensive nature of this type of study and
the necessity to combine efforts of scientists
from different areas (biochemistry, physio-
logy, physics, medicine) may be important
in this regard in addition to the cost of the
equipment. Studies involving hundreds of
subjects have been rarely reported but in
that respect MRS does not differ than other
techniques. In terms of time, a typical rest-
exercise recovery protocol almost requires
an hour and several additional hours are
necessary for data processing. Initially
manual, data processing has become
automatic quite rapidly and recent analytical
procedures performed in the time domain
and incorporating prior knowledge have
significantly improved the automatic
analysis.26
ATP HOMEOSTASIS AND ATP SOURCES
One of the main contributions of 31P MRS
to muscle physiology is its ability to measure
196 Biomedical Magnetic Resonance: Proceedings of the International Workshop
sequentially the variations of concentrations
in PME, Pi, PCr, ATP as well as intracellular
pH during muscle contraction and recovery.
Biochemical reactions taking place in the
myocyte are then directly reflected by specific
perturbations on the spectrum. Muscle energy
production results in PCr and pH changes
which in turn would modify ADP and Pi
concentrations both of which ultimately
stimulate ATP production. Typical series of
spectra recorded in exercising flexor muscles
(4.7 T) and in thigh muscles (1.5 T) are dis-
played in Figure 16.1. During contraction,
the free energy stored in ATP (the ubiquitous
substrate for energy production) is converted
into mechanical energy via the interaction of
two muscle proteins, actin and myosin.
Muscle work is linked to hydrolysis of ATP.
However, in normal subjects, the intra-
cellular concentration of ATP remains
unchanged during moderate exercise
indicating an adequate balance between
energy demand and supply.27 A net ATP
decrease has been only reported when
exercise intensity was by far larger28 and
interestingly such a net ATP consumption
has rarely been reported in patients with
metabolic disorders.29-31
As illustrated in Figure 16.2, the balance
between ATP demand and ATP supply is
ensured by the three main sources of ATP
within the muscle and each of them can be
accurately quantified from the PCr and pH
time-dependent changes in exercising
muscle and throughout the recovery period.
PCr Changes in Exercising Muscle
PCr is largely recognized as an intracellular
buffer (some authors have also reported PCr
as an energy shuttle between production and
consumption sites) allowing to compensate
for any shortfall in either non-oxidative or
oxidative ATP production. Across the rest
to work transition, ATP in working muscle
Fig. 16.2: Schematic drawing of the
main pathways involved in energy
production. The asterisks refer to the
compounds either detected or
calculated from MRS data
 31
P MR Spectroscopy of Muscle Energy Metabolism in Humans
is maintained by phosphocreatine break-
down in a reaction catalyzed by creatine
kinase. After a few seconds (if not imme-
diately) both glycolysis and oxidative
phosphorylation are activated. 32,33 As
illustrated in Figures 16.3A and B, PCr
changes in exercising muscle (constant
intensity) with respect to time (t) follows a
first order exponential kinetics from the
resting PCr concentration (PCr rest) to a
steady-state end-of exercise value (PCrend)
with a kinetic constant k:
d(PCr)/dt = PCrend + ΔPCr exp(-kt) ...(1)
with ΔPCr referring to the difference
between the resting PCr concentration and
PCrend.
In a recent paper, a double exponential
function has been used in order to take into
account a biphasic PCr kinetics including a
secondary slow component.34
197
d(PCr)/dt = PCrend + ΔPCr1 exp(-kt) -
ΔPCr2 {exp[-k(t-TD)]–1} ...(2)
with ΔPCr 1 and ΔPCr2 representing the
asymptomatic values to which ΔPCr is
projecting at the end of each phase.
From the PCr time-dependent changes,
the involvement of PCr breakdown in
energy production can be easily calculated
as a decreasing contribution. In addition,
considering PCr as the major energy source
at the onset of exercise,35 the initial rate of
PCr consumption can be calculated as k*
ΔPCr (a derivative of equation # 1 at time
zero) and can be used to infer the energy
cost of contraction when power output (W)
is taken into account.
Energy cost = (k* ΔPCr)/W ...(3)
The comparative analyses of PCr changes
and oxygen consumption have provided
interesting information related to the regula-
Figs 16.3A and B: Time-dependent
changes in PCr in exercising muscle.
In A, time zero refers to the exercise
onset and changes can be fitted with
a monoexponential function. In B, the
exercise duration is longer and a
second exponential phase can be
detected. A biexponential function
has been used to fit the data
198 Biomedical Magnetic Resonance: Proceedings of the International Workshop
tion of PCr breakdown with respect to
oxygen availability. A direct proportionality
between the kinetics of pulmonary oxygen
consumption and PCr hydrolysis has been
demonstrated. Changes in PCr at the start
of exercise has been analyzed and found
comparable to the onset of oxygen
consumption kinetics.21,36,37 In addition, a
correlate of the slow component in oxygen
consumption, indicating an increased oxygen
cost for a constant work-rate, has been
reported for the time-dependent PCr
changes further illustrating the similarity
between oxygen and PCr changes.34,38 The
steady-state level to which PCr falls, i.e.
PCrend is also positively correlated with the
steady-state level of oxygen consumption
during exercise suggesting that PCr
hydrolysis and more likely inorganic
phosphate accumulation could be a potent
regulator of oxidative phosphorylation as
suggested previously. 39,40 However, this
tight coupling between PCr hydrolysis and
regulation of aerobic ATP production could
be modified by altering oxygen avail-
ability.34,41-43 Interestingly, it has been repor-
ted that any change in oxygen availability
was immediately translated at the PCr level
in exercising muscle either as a faster
decrease (switch from hyper to hypoxic
conditions) or a faster increase (switch from
hypoxia to normoxia and switch from
hypoxia to hyperoxia). 41 Two opposite
hypotheses could be formulated regarding
the effects of oxygen availability upon PCr
changes in exercising muscle. If PCr kinetics
follow oxygen consumption as previously
suggested, PCr changes should be slowed
in hypoxia due to the fact that oxygen has
to be present as the terminal electron
acceptor within the electron transport chain.
On the other hand, if PCr hydrolysis is a
process largely dependent upon the
metabolite signals arising from the contrac-
tile sites; then, for a given ATP demand,
PCr kinetics may remain unchanged
regardless of oxygen availability. Recent
experiments aiming at analyzing the effects
of using varied fractions of inspired oxygen
(FiO 2 ) on PCr kinetics have provided
interesting information. During an incre-
mental exercise, the reduction of oxygen
availability accelerates the occurrence of
fatigue, whereas the extent of PCr reduction
was not modified. 44 However, for an
identical work output, PCr hydrolysis was
larger when oxygen supply was lower
suggesting that this greater PCr hydrolysis
might have contributed to the earlier time
to exhaustion. Similar experiments conduc-
ted throughout a steady-state exercise have
provided additional information regarding
the time-dependent changes in PCr and
oxygen. PCr and oxygen kinetics remain
comparable regardless of oxygen availability
at least during the primary phase. An
additional phase (the slow component) can
be observed for both PCr and oxygen unless
oxygen supply is made hyperoxic. All
together, these data indicate that PCr
changes at the onset of exercise are not
modulated by the oxygen driving pressure.
On the contrary, after this initial phase,
reducing oxygen supply can accelerate PCr
kinetics and precipitate the occurrence of
fatigue; while increasing the oxygen, driv-
ing pressure (hyperoxia) eliminates the
additional PCr consumption (slow compo-
nent or phase II) suggesting that PCr kinetics
after the initial phase are regulated by
microvascular and intracellular PO2.41
pH Changes in Exercising Muscle,
Buffering Components and Glycolytic
ATP Production
On initiation of muscle contraction or at
times when the workload or rate is
 31
P MR Spectroscopy of Muscle Energy Metabolism in Humans
increased, glycogen breakdown is rapidly
activated to provide substrate for glycolysis.
Pyruvate that is not used as a substrate by
the mitochondria accumulates and the
combined production of lactate and ATP
hydrolysis lowers pH.45,46 The extent of the
change in pH is a balance among lactic acid
production proton efflux, ATP hydrolysis
and the pH raising effect of PCr breakdown.
Interestingly, all these components could be
quantitatively analysed using 31P MRS.
With a high enough time-resolution such
that an initial alkalosis (dpH/dt) can be
measured at the onset of exercise, a composite
buffering capacity (βc) including bicarbonate
and non-bicarbonate compounds is
calculated according to equation 4 taking
into account the rate of proton consumption
due to PCr breakdown. (γdPCr/dt)
represents the proton stoichiometric coeffi-
cient of the coupled Lohman reaction, i.e.
PCr breakdown coupled to ATP hydrolysis:47
βc = [γd(PCr)/dt]/[d(pH)/dt] ...(4)
The contribution (βx) of inorganic phos-
phate (Pi) and sugar-phosphate (PME) to βC
can be determined using the standard
formula of a buffer dissociation with K being
the dissociation constant:
βx = [2.303 (H+) K (X)]/[K +(H+)]2 ...(5)
where (X) is the concentration of either Pi
or PME and K the corresponding
dissociation constant. The buffering capacity
of tissue alone mainly including imidazole
groups of histidine can then be calculated
as the difference between βx and βc. In an
“open” muscle system, the buffer capacity
due to bicarbonate is between 30 slykes at
resting pH and 3 slykes at pH close to 6.48
Exercising muscle is usually assumed as a
“closed” system48-50 and buffer capacity due
to bicarbonate is set to zero.
199
These values are used for the deter-
mination of the glycolytic rate of ATP produc-
tion which can be calculated differently
according to the type of exercise. Under
ischemic conditions, given that ATP supply
is mainly supported by PCr breakdown and
glycolytic ATP production, the glycogenolytic
flux can be calculated using a two-step
method, using measurements of PCr content
only.51 In the first step, the total rate of ATP
turnover is calculated from the derivative of
equation 1 at time zero. Then glycogenolytic
flux can be calculated as the simple difference
between the total rate of ATP turnover and
the rate actually calculated at any given time-
point of exercise as indicated in Figure 16.4,
Plate 10.
When ATP demand is supported by both
non-oxidative and aerobic sources (mixed
exercise), the calculation of the glycolytic rate
takes into account each component of the
H+ balance, i.e., overall H+ production asso-
ciated with non-oxidative ATP production,
H+ efflux, and H+ uptake associated with
PCr breakdown (Eqn. 6).30,52
As previously described,48 the rate of
+
H efflux (EfH+) cannot be directly estimated
during exercise but has to be analyzed from
the coupled analysis of pH changes (usually
an additional acidosis, VpH) and PCr resyn-
thesis during the initial part of the recovery
period following exercise. Indeed, as
illustrated in Figures 16.5A and B, pH
recovers back towards its resting value
despite a continuous proton load (H+PCr)
from PCr resynthesis:
EfH+ = βVpH + H+PCr ...(6)
[CRMBM5]
Taking into account both phenomena, a
first-order rate constant linking the rate of
H+ efflux to the extent of pH changes can be
calculated and used to infer the rate of
200 Biomedical Magnetic Resonance: Proceedings of the International Workshop

proton efflux during exercise from any given

pH value.
Combining equations 4 and 6, the total
proton generation in exercising muscle can
then be calculated as follows:
T = EfH+ + γdPCr/dt + βd(pH)/dt ...(7)
Considering that the glycolytic gene-
ration of 1 mole of ATP is accompanied by
the production of 2/3 moles of lactic acid
(i.e. protons), 45 when considering ATP
supply coupled to ATP hydrolysis, glycolytic
ATP turnover (GATP) can be calculated as:
GATP = 3/2 T ...(8)
In terms of glucosyl equivalents,
glycolytic ATP production (G D) is also
related to lactate synthesis with a
stoichiometry of 3ATP/glucosyl unit and 1.5
ATP/lactate:
GD = GATP/3 ...(9)
It is generally accepted that glycolytic
ATP production is controlled through
feedback inhibition of phosphofructokinase
(PFK) by metabolites issued from ATP
hydrolysis such as ADP, Pi, AMP, through Figs 16.5A and B: Typical PCr (A) and pH (B) time-
activation of glycogen phosphorylase by dependent changes after a muscle exercise. The
horizontal line refers to the end of exercise. One can
accumulation of Pi.53-55 In that context, it is
observe the initial alkalosis at the onset of exercise
unclear why glycolytic flux is low at rest or indicating an imbalance between the proton load due
in anoxic muscle even when extensive to PCr resynthesis and proton efflux
transformation to phosphorylase A is
achieved by adrenaline infusion56 or when papers have examined the activation of
Pi concentration is maintained high after glycolysis in vivo11, 58-61 and its relationship
ischemic stimulation. 57An alternative to oxygenation.59
mechanism would be a feed forward or open During ischemic exercise, Conley et al
loop involving calcium (in other words, reported a constant rate of glycolytic ATP
muscle contraction) thereby controlling both production in the face of increasing concen-
energy supply and demand.11 Given that trations (well above their respective Km) of
31P MRS allows continuous monitoring of potential controllers such as Pi and ADP
both the putative feedback signals involved and a gradual decrease of PCr. 58,59 In
in glycolytic ATP production and the rate addition, proton accumulation was directly
of non-oxidative ATP production, several proportional to the number of stimulation
 31
P MR Spectroscopy of Muscle Energy Metabolism in Humans
regardless of the stimulation frequency.58,59
Through a similar ischemic exercise, a fast
increase of the glycolytic ATP rate followed
by a plateau was reported by Kemp et al,
while both Pi and ADP was increasing
constantly.11 Both results illustrate that the
main control of glycolytic ATP production
is not achieved either by the feedback
inhibition at the PFK level or as a result of
glycogen phosphorylase activation by
increasing Pi level, but rather through an
open loop likely involving calcium and
ensuring parallel control of both glycolytic
energy production and ATP use. In Conley’s
experiments the constant rate of glycolytic
ATP production coupled to the decreasing
level of PCr would suggest that energy cost
of muscle contraction changes over time as
suggested in other studies. 62 Another
important point related to glycolytic ATP
production is related to its relationship with
pH changes and its chronological activation.
Given that glycolytic ATP production is
coupled to lactate accumulation, one could
conclude that pH and glycolytic ATP
production both vary in the same way.
However, one should keep in mind that in
muscle cells, changes in proton concentration
are modulated by a series of events including
buffering processes, proton efflux, ATP
hydrolysis and PCr changes.11,63 Compara-
tive experiments of pH changes throughout
various exercise modalities have clearly
shown a clear dissociation between pH
changes and glycolytic ATP produc-
tion.58,59,64 On the one hand, glycolytic ATP
production can be measured with no
corresponding pH changes.59 On the other
hand, from a detailed investigation of H+
balance, Roussel et al clearly illustrated
through a comparative analysis of moderate
and high intensity exercise that non-
201
oxidative ATP production preceded a change
in pH for both protocols and that changes
in pH were not recorded as long as sources
and sinks for H+ approximately balanced.64
In addition, regardless of the protocol, a
significant acidosis systematically occurred
while the rise in H+ generation exceeded
the rise in H+ efflux at a nearly constant H+
uptake associated with phosphocreatine
breakdown. These results illustrate that
intracellular acidosis in exercising muscle
does not occur exclusively as a result of
nonoxidative ATP production but, rather,
reflects changes in overall H+ balance.64
Another interesting issue related to
anaerobic glycolysis activation is linked to
oxygen availability. Non-oxidative glycolytic
ATP production has long been characterized
as an anaerobic process although quite a
few studies have clearly documented that
anaerobic glycolysis occurred under fully
aerobic conditions. 53,65,66 The effects of
oxygenation on glycolytic rate of ATP
production has been analysed using the
dynamics of PCr and Pi signals in the 31P
MR spectra during stimulation and
recovery.59 From the comparative analyses
of aerobic vs ischemic experiments, it has
been shown that total glycolytic ATP
production was similar regardless of the
oxygen availability, whereas PCr changes
and Pi and ADP accumulation in ischemic
muscle. 59 Interestingly, the relationship
between glycolytic ATP production and Pi
over the exercise period differ between
ischemic and aerobic conditions, whereas
glycolytic flux was proportional to stimu-
lation number independent of the presence
of oxygen.59 From these results, it is clear
that glycolytic flux is varying with stimula-
tion number and not with either Pi or ADP
indicating that glycolytic control by
202 Biomedical Magnetic Resonance: Proceedings of the International Workshop
metabolite feedback is minor in the
regulatory process of glycolysis; and further,
illustrating a major role for calcium in the
control of glycolysis via a cascade of events
controlling both ATP demand and ATP
supply. This conclusion has been modulated
by the same group on the basis of repeated
ischemic exercises separated by an ischemic
period at rest with an initial exercise period
of varying duration.60,61 They showed that
increasing the exercise duration in the first
exercise bout led to larger PCr, Pi and ADP
changes and foremost resulted in an earlier
activation of glycolysis during the second
ischemic exercise.60,61 These results illustrate
the key role of metabolite concentrations
for the activation of glycolysis in contracting
muscle.
PME Changes in Exercising Muscle,
Glycogenolytic and Glycolytic ATP
Productions
The PME signal in 31P MR spectra comes
from hexose phosphates, i.e. glycolytic inter-
mediates such as glucose-6-phosphate (G-6-
P) and fructose-6-phosphate (F-6-P).
Alternatively, the IMP and AMP could
contribute to the PME signal, but only when
ATP homeostasis is compromised and a net
ATP degradation is measured. An accumu-
lation of PME occurs normally during
exercise likely as an imbalance between
glycogenolytic and glycolytic fluxes.11,67
Indeed, glycogenolytic flux often exceeds
glycolysis flux58 and glycogenolysis supplies
glycolysis with most of its substrate G-6-P.
Glycogenolyis flux could then be quantified
from the accumulation of PME [d(PME)/dt]
and taking into account the glycolytic flux
(GATP, equation 8):11,59
Gf = Glycogenolytic flux
= GD + d(PME)/dt ...(10)
The analysis of PME accumulation during
exercise has delineated whether glycolysis is
activated or not as a mass-action response to
glycogenolysis. Actually, PME accumulation
occurs before the significant activation of
glycolysis11,61 indicating that the glycogenoly-
sis production of PME is not sufficient to
trigger glycolysis flux and that the two
pathways differ in their sensitivities to the
signals that control flux, i.e. metabolites such
as Pi and ADP and calcium. Instead, the
glycolytic pathway is controlled at one or
more sites downstream of glycogenolysis.60
Pathological changes in PME encompass
lower and higher accumulation of PME
occurring in proximal and distal glycogenoses
respectively.27,68 In the former situation, PME
does not accumulate as a result of impaired
glycogenolysis whereas in the latter
situation, a reduced PFK activity (or another
enzyme downstream of the glycolytic path-
way) enlarges the already existing imbalance
between glycogenolysis and glycolysis and
accounts for the larger accumulation of PME.
Together with these abnormal changes,
exercise-induced acidosis is either limited68
or does not exist27 again as a result of the
reduced activities of either glycogenolysis
and/or glycolysis.27,68
PCr and pH Changes in Muscle after
Exercise, Aerobic ATP Production
Following exercise, PCr and pH both recover
back to their respective value with a lag time
for pH. As previously underlined, this lag
time accounts for the proton load from PCr
resynthesis and this can be used in order to
calculate proton efflux63 which is modulated
by blood flow and activities of various
transporters. The PCr recovery kinetics
provide information regarding oxidative
 31
P MR Spectroscopy of Muscle Energy Metabolism in Humans
ATP production. Indeed, it has been clearly
demonstrated that PCr resynthesis in
recovery is purely aerobic,69,70 one of the
illustrations being the absence of PCr resyn-
thesis during the post-stimulation ischemic
period; whereas all the potential controllers
of glycolysis, i.e. Pi and ADP, were by far
larger than their respective Km. 70 In
addition, the rate of PCr recovery is slowed
down in mitochondrial disorders71-73 and
cardiac failure74,75 as a result of impaired
mitochondrial respiration, improved in
athletes7,76-78 consistent with an increased
mitochondria content and/or activities of
enzymes involved aerobic ATP production.
Also, it has been shown that PCr recovery
kinetics can be accelerated with increased
oxygen supply 42,43 (at least in exercise-
trained subjects) and following antiasthenic
treatment.73 The independence of recovery
data with respect to stimulation frequency
and exercise intensity 69,79 is also of
importance for the utilization of recovery
data as indices of aerobic capacity.
Comparative analyses of in vivo MR data
and in vitro measurements of mitochondrial
enzymes’ activities have shown good
agreements.80-82 Overall, PCr recovery data
are considered as robust measures of
mitochondrial function as long as the depen-
dence with end-of-exercise parameters such
as intracellular acidosis is properly taken
into account.16,77,83 The PCr recovery kinetics
is usually considered as a monoexponential
function with PCr increasing from the end-
of exercise value (PCrrest – ΔPCr) to the
resting value with a kinetic constant k.
d(PCr)/dt = PCrrest – ΔPCr exp(-kt) ...(11)
Similarly, to the exercise situation, the
initial rate of PCr resynthesis illustrating
the rate of aerobic ATP production (OxATP)
can be calculated from the time-derivative
of equation 11 at time zero.69
203
OxATP = k ΔPCr ...(12)
In some studies, a double exponential
function has been used in order to fit the
PCr time-dependent changes in recovery
from exercise, but the superiority of this fit
has never been clearly demonstrated.84,85
Considering that ADP is the main controller
of aerobic ATP production in muscle cells,
the apparent maximum aerobic capacity
(Vm), i.e. the theoretical maximum capacity
(never reached for physiological concentra-
tions) 86 for a muscle to produce ATP
aerobically, can be calculated from ADP
concentration and the initial rate of PCr
resynthesis. Interestingly, a plot of the Vi
dependence upon ADP can be built over
the entire recovery period using successive
rates of PCr resynthesis (Vi) and corres-
ponding ADP concentrations. As illustrated
in Figure 16.6, the relationship between Vi
and ADP can be fitted to the Hill equation:
Vi = Vm ([ADP]/Km)n/
1 + ([ADP]/Km)n ...(13)
with Km being defined as the ADP concen-
tration at half maximum rate.
Most of the time, the Hill coefficient n has
been taken as 1 and the equation 13 is
equivalent to the Michaëlis-Menten equation
(equation 14):
Vi = Vm (ADP) / [(ADP) + Km] ...(14)
from which the apparent maximum aerobic
capacity can be calculated as follows:
From equation 14, the apparent maxi-
mum aerobic capacity can be defined as:
Vm = Vi [1 + Km/(ADP)] ...(15)
Alternatively, considering that PCrrest
could be the theoretical maximum amount
of PCr consumed, the apparent maximum
aerobic capacity can be defined as follows
from equation 12:
204 Biomedical Magnetic Resonance: Proceedings of the International Workshop
Fig. 16.6: Rate of PCr time-dependent changes after
a muscle exercise with respect to ADP fitted with the
Hill equation (equation #13) taking n as 2.2 (A) or 1
(B). When the hill coefficient n is 1, the Hill equation
corresponds to the Michaëlis-Menten equation
OxATP = k PCrrest ...(16)
More recently, it has been proposed that
n could be more than 1 (i.e. 2.2)87 indicating
an amplification of mitochondrial sensitivity
to cytosolic ADP, but additional studies are
needed in order to assess the value of n in
vivo and the corresponding control mecha-
nism of aerobic ATP production.
Aerobic ATP production (OxATP) during
exercise has been calculated using different
methods. Once the coefficients of the Hill
equation are obtained through a fit of Vi vs
ADP using equation 12, Vi can be calculated
at any time-point of exercise, as far as ADP
is known and considering that this equation
holds during exercise. 59 Aerobic ATP
production can also be indirectly calculated
from the energy cost of contraction (equation
3), the rate of PCr breakdown (dPCr/dt;
equation 1) and the glycogenolytic contri-
bution (Gf; equation 10) to energy produc-
tion.11
At any time:
EC = d(PCr)/dt + Gf + OxATP ...(17)
Considering that EC remains unchanged
during the entire exercise period, any change
in either d(PCr)dt or G f must be
compensated by a corresponding change in
OxATP:
At any time:
OxATP = EC – [d(PCr)/dt + Gf] ...(18)
This hypothesis related to the constancy
of EC has been independently checked by a
comparative analysis of OxATP calculated at
end of exercise from equation 18 and the
same variable calculated at the onset of
recovery from equation 12.69
The role of oxygen in modulating the PCr
recovery data has been investigated on the
basis of comparative analyses of hyperoxic,
hypoxic and normoxic conditions. 42,43 In
trained subjects, hyperoxia increased the rate
of PCr recovery, whereas hypoxia slowed
these kinetics 42 although magnitude of
changes in exercising muscle for PCr and pH
were not affected. This result illustrates that
the rate of PCr recovery would be limited by
oxygen availability at least in trained subjects.
Aerobic energy production depends on both
the oxygen gradient between the capillary
and the cell and by the rate of mitochondrial
respiration itself. While the results obtained
in trained subjects suggested a limitation of
the PCr recovery rate by oxygen and not
by mitochondrial metabolic limits, similar
experiments conducted in sedentary subjects
has shed light to the understanding of this
modulation. 43 On the contrary to what
occurred in exercise-trained subjects, the rate
of PCr recovery in sedentary subjects
remained unchanged under hyperoxia as
compared to normoxia, whereas hypoxia
similarly decreased the rate constant.43 The
comparative analysis of sedentary and
trained subjects indicated that oxygen limits
mitochondrial metabolism in trained
subjects; whereas in sedentary subjects, the
 31
P MR Spectroscopy of Muscle Energy Metabolism in Humans 205

rate of PCr recovery is limited by mitochon-

drial capacity and not oxygen availability.
In other words, hyperoxia enhances the
oxygen driving gradient from blood to
muscle thereby resulting in an elevated
intracellular oxygen partial pressure. This
raised intracellular PO2 improves aerobic
ATP production in trained subjects who did
not display metabolic limitation but not in
sedentary subjects who most likely have
reduced aerobic capacity. The oxygen
driving gradient is reduced under hypoxia,
even if a compensatory increased in muscle
blood flow may exist88 and compromised
aerobic metabolism in both sedentary and
trained subjects.43

REPRODUCIBILITY OF
MEASUREMENTS
For meaningful comparisons data must be
reproducible from study to study and it is
important to assess repeatability in groups
of subjects and patients. Foremost, one have
to keep in mind that metabolic changes in a
control population is highly heteroge-
neous.11,15,16,89 The usual standardization
procedures such as measurements of maxi-
mum voluntary contraction does not reduce
this between-subject variability.11,16,89 In that
respect, it is clear that a set of factor which
could influence metabolic changes in exer-
cising muscle has to be taken into account
and ad-hoc standardization procedures that
would reduce the inter-subject variability
have to be used for proper comparisons
among various groups of subjects.
Between-subject Variability
In a limited number of subjects, it has been
shown that neither fasting nor carbohydrate
loading significantly modified the extent of
Figs 16.7A and B: Typical PCr (A) and pH (B) time-
dependent changes recorded throughout a
standardized rest-exercise (shaded area)—recovery
protocol in a group of 18 subjects. Measurements
(black and white symbols) have been recorded seven
days apart. Results are presented as means ± SD
and one can observe the very good reproducibility of
measurements
metabolic changes in exercising muscle.89
Similarly, severe metabolic acidosis induced
by ammonium chloride loading had no effect
on changes recorded during exercise.89 Aging
is another potential accounting factor that
should be taken into account. The studies
based on measurements of enzymatic acti-
vities have suggested a decrease oxidative
capacity with age.90,91 Results from MRS
investigations differed regarding that physi-
cal activity has been taken into account or
not.92 Aging has been related to both a 50
206 Biomedical Magnetic Resonance: Proceedings of the International Workshop
percent reduction in the PCr recovery rate
and a reduced mitochondrial content with
no changes regarding the metabolic changes
in exercise.93 When physical activity has been
taken into account, no deficit of aerobic
capacity has been reported, whereas the PCr
depletion was not modified and the pH was
more alkalotic in older subjects94 in agree-
ment with previous studies conducted in
sedentary95 and moderately active subjects.96
These results would imply a primary role of
disuse in the decline of oxidative capacity
rather than an inherent age-related defect. In
addition, the recent observation of increased
maximum aerobic capacity in older subjects
as a result of training would be in keeping
with the hypothesis that the decline in
oxidative capacity would mainly result from
a reduction in habitual physical activity
rather than of aging per se.97 However, this
conclusion must be moderated on the basis
of two points. First, the oxidative capacity
per mitochondrial volume measured in older
subjects after a training period was still
below the value reported in young adult
muscle93 and adaptive mechanisms were
distinct from what has been reported so far
in young especially regarding resistance
training.97
Another factor that should be taken into
account regarding the between-subject
variability is related to high body fat stores.
Indeed, changes in fiber types proportions
that could affect muscle energetics have been
reported in obese subjects98 and high-energy
phosphate metabolism has been investigated
using 31P MRS in order to test this hypo-
thesis. Studies conducted in moderately-
obese women (BMI range: 27 to 30 kg/m2)99
and in prepubertal girls with a familial
predisposition to obesity100 have clearly
shown that muscle energetics was not
altered. In addition, a weight-reduction
program had also no effect on mitochondrial
function thereby suggesting that a low
substrate oxidative capacity of skeletal
muscle is not involved in the pathogenesis
of obesity on the contrary to what has been
suggested earlier.101
Within-subject Variability
Beside the between-subject variability, the
within-subject variability is also important
to analyse. Several studies have clearly
demonstrated a very low within-subject
variability as illustrated in Figures 16.7A
and B and indicating a very high repro-
ducibility.73,89,102,103 For instance, variation
coefficients calculated from repeated
experiments were 5-10 percent for metabolic
indices at rest such as ADP and PCr/Pi and
3-15 percent for variables measured during
exercise and in the recovery period.102
Standardization Procedures
This within-subject variability may also be
used in order to understand muscle ener-
getics and to initiate ad-hoc standardization
procedures that could be used as reliable
comparative methods between groups of
subjects. It has been shown in normally
active adults that an index of PCr consump-
tion [(PCr + Pi)/PCr] (measured with 31P
MRS) was highly correlated with power
output normalized to the volume of muscle
(measured with MRI) in the plantar flexor
compartment indicating that combined MRS
and MRI measurements could offer a way
of reliably compare subjects with different
body size.104 Similarly, expressing metabolic
changes such as PCr breakdown and intra-
cellular acidosis in exercise muscle with
respect to power output has been reported
to offer an interesting standardization proce-
dure independent of exercise protocols
and anthropometric measurements.105 In
 31
P MR Spectroscopy of Muscle Energy Metabolism in Humans
agreement with such an approach, it has
been shown, in exercising muscle, that the
extent of PCr breakdown was linearly linked
to the intracellular acidosis regardless of
exercise protocols. 16,105 In other words,
changing the exercise intensity results in
changing both PCr consumption and
intracellular acidosis, but these parameters
still remain linked through a linear relation-
ship.16,105 Interestingly, averaged end-of-
exercise (ADP) did not change among
protocols as illustrated in Figure 16.8,
Plate 10 suggesting that various combi-
nations of PCr and pH are multiple solu-
tions for muscle fibers to reach a given level
or range of ADP, which in turn will act as
a regulator of energy production as pre-
viously described.69,106
PATHOLOGICAL CHANGES
ASSESSED WITH 31P MRS
Given its ability to follow high-energy phos-
phate compounds and pH changes during
transitions from rest to exercise and exercise
to rest, 31P MRS has a clear potential for
delineating the metabolic abnormalities of a
particular myopathy thereby providing
unique diagnostic indices. Skeletal muscle
makes up about 40 percent of body mass. Its
metabolism largely influences whole-body
homeostasis and disorders in other organ
systems should have an impact on muscle
energetics. 31P MRS have not only provided
key information for primary metabolic
disorders affecting muscle energy produc-
tion (Table 16.1) but also for secondary
problems of muscle metabolism associated
with common conditions such as heart
failure, renal failure and thyroid disease.
As illustrated by the results in Table 16.1,
the specificity of MRS data does not reside in
any single measurement but in the pattern of
207
abnormalities that distinguish particular
types of primary or secondary muscle
diseases. For instance, the PCr recovery rate
measured after exercise is slow in a large
variety of disorders indicating that in all of
them oxidative ATP production is impaired
(given that PCr kinetics throughout the
recovery period is exclusively oxidative).
However, with 31P MRS data one could go
further that and determining the causative
factors of this impaired aerobic energy
production. The lack of acidosis in McArdle
disease27 indicates that impaired glycogeno-
lysis and then lack of substrate is the
causative factor of impaired oxidative ATP
production. On the contrary, in mitochondrial
myopathy, the combined analysis of PCr and
pH time-dependent changes point towards a
mitochondrial deficiency as accounting for
the impaired muscle energetics.29,72,107 A final
example of the specificity of MR results is
related to the impaired aerobic energy
production in idiopathic inflammatory myo-
pathy.108 In that particular case, the reduced
rate of PCr recovery is related to an abnormal
rate of pH recovery illustrating that impaired
blood flow might be responsible for the
abnormal aerobic production as a result of
the muscle inflammatory process.108
The intracellular concentrations of many
ions such as sodium, potassium, calcium and
protons are linked through the actions of
the several sarcolemmal transporters and
the maintenance of the intra- extra-cellular
ionic gradients requires energy so MRS can
provide important clues about ionic
homeostasis.
Deficits in ATP production caused direc-
tly by gene defects in skeletal muscle or
indirectly by hormonal changes, virus
infection, inflammatory process, renal
failure, ischemic disease, respiratory disease
etc. could alter the normal MRS pattern
208 Biomedical Magnetic Resonance: Proceedings of the International Workshop

Table 16.1: Pathological changes measured using 31P MRS

Pathological Rest ATP PCr Intracellular Rate of PCr Ref

situation depletion depletion acidosis recovery

Mitochondrial Reduced PCr/Pi No Larger Limited Slow together 29, 72, 107

myopathy with faster rate

of pH recovery
McArdle disease Increased PCr/ATP No Larger Limited Slow 27, 117

PFK deficiency Increased PCr/ATP Yes Larger Limited Slow 118, 119

Dystrophy Increased pH and no Larger Normal Normal 120, 121

(Duchenne and PDE

Becker)
Myotonic Decreased No Larger Limited Slow 122, 123

dystrophy phosphorylation
potential
sIBM Increased pH Normal Normal Normal Normal 124, 125

Increased Pi/ATP
Malignant Increased PDE No Larger Excessive Normal 126, 127

hyperthermia
IIM (Poly and Low PCr/ATP No Larger Normal Slow together 108, 128

dermatomyositis) and Pi/ATP with rate of pH

recovery
Renal failure Increased Pi/ATP No Larger Normal Slow 30, 129

Chronic respiratory No Larger Excessive Slow 130-132

failure
Congestive heart No Larger Excessive Slow 133, 134

failure
Peripheral vascular No Larger Excessive Slow together 11, 135, 136

disease with rate of pH

recovery
Thyroid disease No Larger Smaller Normal together 137

(Hypo) with slow rate of

pH recovery
Thyroid disease Excessive Normal 138

(Hyper)
Essential Smaller Normal together 139

hypertension with faster rate

of pH recovery

thereby providing one or more diagnostic
indices. However, one should be cautious
with the interpretation of the results. For
instance, two brothers with genetic defects
of MELAS showed similar reduction in
maximum aerobic capacity, while one was
with 85 percent mutated DNA and the other
with only 4 percent mutated DNA.109 This
result could be interpreted as a discrepancy
between in vivo and in vitro measurements
or as indicative of the absence of a clear
threshold for the A3243G mutation in
 31
P MR Spectroscopy of Muscle Energy Metabolism in Humans
skeletal muscle. These results have impor-
tant implications for the understanding
of the phenotypic expression of mtDNA
disease.
An additional step into the understanding
of 31P MR metabolic indices resides in the
quantitative analysis initially developed by
Kemp et al.106 With this type of analysis,
one can further distinguish between reduced
muscle mass and/or reduced muscle
efficiency and reduced oxidative capacity as
accounting factors of abnormal exercise-
induced metabolic changes. Indeed, for a
given muscle mass and a given mechanical
power output, the initial rate of ATP synthesis
(equation 3) is by definition inversely
proportional to metabolic efficiency.110 As
illustrated earlier, both aerobic and non-
oxidative contributions to energy production
can be calculated from the analysis of PCr,
Pi, PME and pH dynamics. These changes
depend on mechanical work, muscle mass
and metabolic efficiency (the latter two
variables are combined as effective muscle
mass).30 Generally speaking, greater PCr
and pH changes should be recorded if the
ratio of work to effective muscle mass is
increased or if aerobic ATP synthesis is
decreased. Such a comparison has been
performed in a study of dialyzed uremic
patients. 30 Raw data recorded in this
group of subject illustrated a larger PCr
consumption, while exercise duration and
the corresponding power output was
reduced. pH time-dependent changes were
normal and the analysis of the recovery
period disclosed a 50 percent reduction of
the maximum aerobic capacity. From the
initial increase in the PCr consumption rate,
one can infer a reduced effective muscle
mass. Given that muscle mass (measured
separately) was similar in both groups of
subjects, reduced metabolic efficiency can
209
be suspected as accounting for the greater
exercise-induced metabolic changes. In
addition, considering that both oxidative
and glycolytic contributions to energy
production were increased in absolute terms
but normal when energy cost was taken
into account, it can be proposed that reduced
muscle efficiency rather than impaired
aerobic ATP production likely accounts for
the greater PCr changes in exercise.30
FUTURE INVESTIGATIONS
Employing non-MR based techniques simul-
taneously with MRS can aid in data inter-
pretation and will certainly broaden the
scope of muscle investigation. Near infrared
spectroscopy (NIRS) provides data on state
of tissue oxygenation but reliability has still
to be proven mainly on the basis of compa-
rative and/or combined analyses using MRS
and NIRS.111 Electromyography is another
technique of interest which can be used to
study correlation between metabolic and
electrical changes and provide interesting
features regarding muscle fatigue.112 Com-
bined with MRS, MR imaging techniques can
also help understanding muscle energetics.
For instance, MRS data showing altered
metabolites concentrations need to be
interpreted with caution. Indeed, metaboli-
tes’ concentrations may be altered, but the
presence of fatty or fibrous infiltration can
introduce false-positive results in a sense
that data might not represent concentrations
in the actual muscle fibers. Functional MRI
can also be of interest for understanding
muscle activation during exercise. Based on
T2 changes due to uptake or redistribution
of fluid within the exercising muscle,
functional MRI is considered as a semi-
quantitative method of assessing muscle
recruitment during exercise.113 For instance,
210 Biomedical Magnetic Resonance: Proceedings of the International Workshop
in McArdle’s disease, abnormal glycogeno-
lysis is coupled to abnormal exercise-induced
T 2 changes. 114,115 . Similarly, subnormal
exercise-induced T2 changes increase can be
recorded in other metabolic myopathies.116
However, muscle functional MRI is still
underutilized although its use is certainly
well suited in the context of muscle ener-
getics and for documenting muscle activation
pattern in pathological conditions and in
subjects with very highly trained to exercise.
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Bank WJ. Effects of thyroid hormones on skeletal
muscle bioenergetics. In vivo phosphorus-31
magnetic resonance spectroscopy study of
humans and rats. J Clin Invest 1988;81:1695-701.
138. Erkintalo M, Bendahan D, Mattei JP, Fabreguet-
tes C, Vague P, Cozzone PJ. Reduced metabolic
efficiency of skeletal muscle energetics in
hyperthyroid patients evidenced quantitatively
by in vivo phosphorus-31 magnetic resonance
spectroscopy. Metabolism 1998;47:769-76.
139. Dudley CR, Taylor DJ, Ng LL, Kemp GJ, Ratcliffe
PJ, Radda GK et al. Evidence for abnormal Na+/
H+ antiport activity detected by phosphorus
nuclear magnetic resonance spectroscopy in
exercising skeletal muscle of patients with essen-
tial hypertension. Clin Sci (Lond) 1990;79:491-
7.

MRI and MRS of
Nuclei other than 1H
INTRODUCTION
The basic principles of magnetic resonance
imaging (MRI) and magnetic resonance
spectroscopy (MRS) have been treated
elsewhere and will not be discussed in depth
here (see, for example, reference 1). A
number of isotopes are well suited for MRI
or MRS studies in vivo. Table 17.12 gives the
properties of some endogenous and
exogenous nuclei potentially available for
in vivo studies. The isotopes in Table 17.1
fall approximately into two categories. One
category contains nuclei of moderate or high
natural abundance and sensitivity (e.g. 19F),
which are observable in vivo without isotopic
enrichment. The second contains nuclei
typically only observable with isotopic
enrichment (e.g. 13 C) or special signal
enhancement (e.g. 3He). Phosphorus-31 MRS
is treated in another article in this procee-
dings and thus will not be discussed here.
Of the remaining nuclei, we restrict our-
selves to those that have significant in vivo
applications, focusing primarily on 7Li and
19
F, with which we are most familiar.
MAGNETIC RESONANCE IMAGING
The general physical principles of MRI apply
to all nuclei. However, adjustments to the
MRI and MRS of Nuclei other than 1H 217
17
Diana M Lindquist, Richard A Komoroski
MR experiment must be made to account
for the differing resonance frequencies for
nuclei other than protons. The most direct
adjustment must be made to the gradient
that is used for spatial encoding. In an MR
imaging experiment, the resolution Δx in a
single dimension is given by
Δx = 1/γGt ...(1)
where γ is the magnetogyric ratio, which is
different for each nucleus; G is the strength
of the applied gradient; and t is the sampling
time. Since γ’s for the heteronuclei are
smaller than for protons, either the gradient
strength G, or the sampling time t, must be
increased to achieve the same spatial
resolution for these nuclei as is common for
protons. However, G is limited by the
hardware and t is limited by the relaxation
time of the nucleus.
In practice, these limitations generally are
not important because the greatest limitation
on resolution is the available signal, which
is determined by the nuclear sensitivity and
concentration. For example, protons are
present in water in tissue in the range 80-
100 M, whereas even under the best of
research conditions, the concentrations of
218 Biomedical Magnetic Resonance: Proceedings of the International Workshop

Table 17.1: NMR properties of selected nuclei2

Nucleus Spin Natural Magnetogyric Relative Frequency
abundance ratio sensitivity at 1.5 T
(%) (MHz/T)
1H 1/2 99.985 42.58 100 63.8
2H 1 0.015 6.54 0.000145 9.8
3He 1/2 0.00013 32.43 0.0000575 48.7
7Li 3/2 92.58 16.55 27.2 24.8
10B 3 19.58 4.58 0.389 6.9
13C 1/2 1.108 10.71 0.0176 16.1
15N 1/2 0.37 4.31 0.000385 6.5
17O 5/2 0.037 –5.77 0.00108 8.7
19F 1/2 100 40.05 83.28 60.1
23Na 3/2 100 11.26 9.25 16.9
27Al 5/2 100 11.09 20.6 16.6
31P 1/2 100 17.23 6.63 25.8
87Rb 3/2 27.85 13.93 4.88 20.9
129Xe 1/2 26.44 11.78 0.56 17.6
133Cs 7/2 100 5.58 4.74 8.4
195Pt 1/2 33.8 9.15 0.336 13.7

exogenous nuclei, such as 7Li or 19F, will be
on the order of 10 mM or less. Under these
conditions, even 19F will have a poor signal-
to-noise ratio (SNR), despite its relatively
good sensitivity (83% that of protons).
Localized Spectroscopy
Localized spectroscopy refers to the acqui-
sition of spectral data from a restricted
region of interest within a subject. There
are several methods by which this can be
accomplished. The simplest method is to use
a surface coil, in which case the region
sampled is determined by the excitation
profile of the coil. In general, this is an area
approximately equal to a hemisphere with
the same radius as the coil. Although this
can be useful if the region of interest is
near the surface and easily accessible, it
suffers from the inhomogeneous excitation
produced by the coil.
A different method of localization, which
can be used with either volume or surface
coils, uses gradients and radiofrequency
pulses to choose the volume element, or
voxel, that is excited. For simple single voxel
and multivoxel spectroscopy, the read-out
gradient is off during the data acquisition
period to preserve the spectral information.
In these sequences, localization can be
one-, two-, or three-dimensional, depending
on the available SNR and the study
requirements. An example of a three-
dimensional localization scheme is the point-
resolved spectroscopy sequence (PRESS),
which is shown in Figure 17.1. It consists of
three RF pulses applied simultaneously with
orthogonal gradients. Each RF/gradient
combination produces the selective excitation
of a “slice” of spins. The excitation of three
such slices orthogonal to each other results
in the selection of the voxel at the inter-
section of the slices. The resolution

considerations described above for MRI also
apply to MRS.
Equipment Considerations
In general, a clinical MR imager does not
include the hardware or software necessary
for heteronuclear studies. This hardware can
include an additional transmitter and recei-
ver, although many modern MR scanners
now use broadband receivers that are
capable of sampling the heteronuclear signal.
Since each nucleus resonates at a different
frequency, nucleus-specific radiofrequency
coils must be built. The imaging and
spectroscopy software must be modified to
include the effects of γ, as discussed above.
SPECIFIC NUCLEI
Fluorine-19
Of the non-hydrogen isotopes available for
in vivo study with MR, one of the most
used is 19F. Fluorine-19 is a spin-1/2 nucleus,
is 100 percent naturally abundant, has a
relative sensitivity that is 83 percent that of
protons and has short spin relaxation times.3
These properties, combined with the fact
that there is no naturally occurring fluorine
Fig. 17.1: A version of the PRESS sequence used
for in vivo localized spectroscopy of rat brain. Shaped
gradients were used to improve the slice profile68
MRI and MRS of Nuclei other than 1H 219
in tissue, make it nearly ideal for in vivo
studies of drugs or other exogenous
compounds. Over 150 fluorinated drugs are
in use clinically,4 so the potential usefulness
of 19F MRS for in vivo monitoring of these
drugs is substantial.
Although no comprehensive review
describing the biomedical uses of 19F MRS
has recently appeared, there have been a
number of focused reviews of studies on
psychoactive medications, such as fluoxetine,
and the anticancer drug 5-fluorouracil
(5-FU). Several mini-reviews5-8 of 19F MRS
are included in more general reviews of
MRS in psychiatry. The most recent general
review is by Wolf et al4 which describes
primarily the usefulness of 19F MRS in
studying 5-FU.
Psychiatric Medications
Many psychoactive drugs contain fluorine.
These include the anti-depressant fluoxetine
and the drugs trifluoperazine and fluphe-
nazine, which are used in the treatment of
schizophrenia. The concentrations of
these drugs in brain are on the order of
10-200 μM, which is significantly higher than
serum levels. Because serum levels of these
medications tend to provide little infor-
mation about the clinical response to or side
effects from these drugs, in vivo measure-
ments in the target tissue can be beneficial.
Fluorine-19 magnetic resonance spectroscopy
has been used to study these fluorine-
containing drugs in vivo in both human
subjects and animal models,9-26 and has been
reviewed.5-9
Initial work focused on detecting and
quantifying compounds containing a trifluo-
romethyl group, such as trifluoperazine,
fluphenazine, or fluoxetine.10-16 The struc-
tures of these and similar compounds are
shown in Figure 17.2. Figure 17.3 shows an
in vivo spectrum from the head of a patient
220 Biomedical Magnetic Resonance: Proceedings of the International Workshop

on fluoxetine. More recently, work has
focused on the pharmacokinetics and
distribution of these and other
medications.17-19 One difficulty with these
types of studies is that in general the in vivo
spectrum consists of a single peak due to
the overlapping reso-nances of the fluorine-
containing drug and its metabolites. Despite
this limitation, several studies have
identified brain metabolites in vivo20-21 and
correlated brain concentrations with clinical
response.22,23
Trifluoperazine has been the subject of
several in vivo studies.13,15,20 In unlocalized
studies we observed signals from 6 clinically
responding patients on TFP doses of
10-120 mg/day.15 A good correlation was
found between brain concentration and

Fig. 17.2: Structures of some psychoactive drugs containing fluorine.

 MRI and MRS of Nuclei other than 1H 221

Fig. 17.3: Fluorine-19 spectrum from the head of a patient on fluoxetine. The peak labeled ‘standard’ arises
from a vial of 12 mM 2,2,2-(trifluoroethyl)-p-toluene sulfonate in CDCl3. The chemical shift scale is in ppm from
CCl3F.

daily dose. Interestingly, despite repeated
attempts, we could not observe a signal
from a nonres ponder who was on the very
high dose of 120 mg/day. This result
suggests that in vivo 19F NMR may have a
role in assessing nonresponse to antipsy-
chotic medication.
More recent work in our lab to identify
trifluoperazine metabolites in rat brain
suggests that the primary in vivo signal
arises from the parent drug and 5-7 meta-
bolites. 20,27 Figure 17.4 shows the 19 F
spectrum of the extract of brain tissue from
a rat dosed with trifluoperazine. Other
metaboli-tes of trifluoperazine may be
visible in the in vivo spectrum as well.20
Fluoxetine metabolizes to the therapeuti-
cally active compound norfluoxetine in the

Fig. 17.4: In vitro 19F spectrum of the extract of brain from a rat dosed with trifluoperazine.20 See reference 20
for compound names. Unidentified peaks are marked with asterisks. The chemical shift scale is in ppm from
CCl3F.
222 Biomedical Magnetic Resonance: Proceedings of the International Workshop
brain. The 19F chemical shift of norfluoxetine
is very close to fluoxetine and the two
compounds are not resolved in vivo (Fig.
17.3).10,15 In vitro 19F NMR studies of extracts
of both human and rat brain confirmed that
the in vivo signal arises roughly equally from
fluoxetine and norfluoxetine.10 A study of
22 patients on fluoxetine showed that brain
concentrations continued to rise long after
clinical effects were evident and leveled off
after a 6-8 month period. 16 The drug
accumulated to about 20 times the level in
the plasma in these patients. 16,24 No
correlation was seen with clinical response,
although more recent studies have found
such correlations.22
Other psychiatric medications that have
been studied with in vivo 19F MRS include
the trifluorinated drugs dexfenfluramine,25
paroxetine,22 fluvoxamine,17,19,23 fluphena-
zine,13 as well as the monofluorinated anti-
psychotics melperone26 and haloperidol.18
5-fluorouracil
5-Fluorouracil is a primary drug used in the
treatment of many cancers. The role of 19F
MRS in studying the metabolism of 5-FU
was reviewed by Wolf et al4 Since that
review there have been several studies of
the 5-FU prodrug capecitabine.28-30 Improve-
ments in localization techniques have
allowed the in vivo detection of 5-FU and
α-fluoro-β-alanine in tumor and liver tissue
with a time resolution of 4 minutes at
1.5 T.31 The in vivo spin-lattice relaxation
times (T1) of 5-FU and fluoronucleotide have
been measured recently.32
Anesthetics
Because many inhaled anesthetics contain
fluorine atoms, 19F MRS has been used in
several pharmacokinetic studies of anesthe-
tics in animals.33-36 Fluorine-19 MRI has also
been used, but with less success.37,38 Lock-
wood et al acquired 19 F MRS data from
volunteers to determine the wash-out time
of isoflurane.39,40 No further human experi-
ments have been done, presumably because
of the difficulties involved.39
OTHER APPLICATIONS
Fluorine-19 MRI and MRS have been used
for a variety of other applications, including
measuring lung volume,41-43 oxygen partial
pressure,44,45 tumor oximetry,46 imaging the
gastrointestinal tract, 47 catheter visuali-
zation,48 and metabolism of anti-inflam-
matory drugs.49
Lithium-7
Naturally occurring Li consists of the 7Li
and 6Li isotopes at 92.58 percent and 7.42
percent abundances, respectively (Table
17.1).2 Like the other alkali metals, both Li
isotopes are quadrupolar. The problems of
achieving adequate SNR and resolved
resonances can be exacerbated by quad-
rupolar effects that occur with nuclei that
have spins greater than ½. For quadrupolar
nuclei, the interaction of the nuclear
quadrupole and the electric field gradient
arising from the local electronic environment
usually provides a strong relaxation mecha-
nism for the spin states, often resulting in
short-lived signals with broad resonance
lines. This is particularly true in biological
systems, where ionic motion is restricted.
However, both Li isotopes are weakly
quadrupolar relative to 23Na or 39K. Both
isotopes yield narrow spectral lines and long
spin-lattice relaxation times (T1). Lithium-7
(I=3/2) is the isotope of choice for most
studies because of its higher natural isotopic
abundance and magnetic moment, and more
favorable relaxation properties than 6 Li

(I=1).2 Relatively little biological MRS work
has been done with 6Li, because of its low
sensitivity and very long T1s.50,51
The chemical shift range of the Li cation
is small because of its simple electronic
structure and aqueous chemistry. Spectra
usually display a single resonance from all
environments, which complicates inter-
pretation of 7 Li NMR signals from
heterogeneous biological systems.
Lithium, usually administered orally as
Li2CO3, remains the treatment of choice for
acute mania and for prophylaxis in bipolar
(manic-depressive) disorder. Despite its
widespread clinical use and extensive
research into its mechanism of action, it is
not known how Li exerts its clinical effects.
We currently know little about the environ-
ment of Li in vivo.
An in vivo 7Li spectrum of brain typically
consists of two resonances, the second from
a vial of aqueous Li standard with position
shifted from the in vivo resonance by a shift
reagent. Quantitation can be performed
relative to a separate Li phantom, with the
small, shifted standard for calibration. High
quality spectra can be obtained in about
20 minutes at 1.5 T. Usable unlocalized
spectra can be obtained in as little as
3 minutes. Most 7Li MRS work in vivo has
centered on humans. Early work in humans
involved surface-coil-localized in vivo 7Li
studies of Li pharmacokinetics, and corre-
lation of brain and serum concentrations.
Further work continued to improve quanti-
tation, spatial localization, and measurement
of spin relaxation times. We direct the
reader to earlier reviews for detailed
treatments of both methodology and early
results.5-8,50-56
Since the magnitude of the pharmacologic
effect of a drug depends on the concen-
tration at receptor sites in the target tissue,
MRI and MRS of Nuclei other than 1H 223
the brain Li concentration is of great
interest.56 The relationship between serum
and brain Li concentration at steady state is
an issue that has received considerable
attention. It has been explored in a relatively
large group of bipolar patients.57 The mean
brain-to-serum ratio of Li for that group
was 0.80 ± 0.19. Over a large serum-
concentration range, the correlation with
brain concentration was weak but statis-
tically significant, whereas in the therapeutic
range the correlation was not significant.
Another report found a weak correlation
between plasma and brain Li concentrations
for longer-term therapy, but no correlation
for shorter-term therapy.58 The above results
suggested that serum Li, the parameter
usually monitored during treatment, is less
than ideal as a measure of Li therapy. A
recent steady-state study59 on a 3 T MR
scanner confirmed the absence of a strong
correlation between steady-state brain and
serum Li levels.
Moore et al60 measured steady-state
brain and serum Li levels as a function of
age in 27 children and adults. Consistent
with most previous work, they found a
weak but significant correlation of brain Li
level with that of serum (r = 0.60, p < 0.001).
They also found a correlation of brain-to-
serum concentration ratio and age (r = 0.44,
p < 0.02). Based on this result, they
suggested that children may require higher
maintenance serum levels of Li to insure
therapeutic brain concentrations.
In vivo 7Li MRS is powerful tool for the
study of Li pharmacokinetics.61,62 The half-
life for Li was 28 hours in brain, and
16 hours in serum. It has also been
demonstrated that brain Li followed serum
Li, in attenuated form, over a daily cycle.
Figure 17.5 shows the time course of Li
levels after termination of therapy for a
224 Biomedical Magnetic Resonance: Proceedings of the International Workshop
patient.63 Whereas Li was not detected in
muscle 5-6 days after termination, brain Li
was still detectable. The reader is referred
to previous reviews for detailed discussions
of early pharmacokinetic results.5-8,50-56
The Li distribution in human brain may
shed light on this cation’s mechanism of
action in bipolar disorder, either thera-
peutically or regarding neurotoxicity. We
previously reviewed progress made in the
development of 7Li MRI and spectroscopic
imaging (SI), both in humans and in
animals.51 Since then Girard et al64 reported
a 7Li SI scan of human head at 4.3 cm
resolution in the axial plane (with no slice
selection) taken in 55 min. The distribution
of Li in the head appeared relatively
uniform. However, spatial resolution in all
three dimensions will need to be signi-
ficantly improved for the technique to be
clinically or mechanistically useful. Sensiti-
vity improvements from higher magnetic
fields and better RF coils may brighten this
picture.
Many Li studies are best carried out in
a convenient animal model like the rat,
Fig. 17.5: Li levels in brain, muscle, and serum
after temination of Li therapy in a patient.63
where dosing is flexible and experimental
conditions can be closely controlled. Earlier
work on the development of 7Li MRS and
MRI in animal models has been reviewed in
several places.50,51,55
Recently our laboratory has built on
earlier work to resolve several issues best
addressed in an animal model. Our previous
work in rats emphasized low-resolution 7Li
MRI to map the relative distribution of Li
in brain and muscle tissue.65-67 Such studies
required substantially higher Li concen-
trations than used clinically in humans. These
high concentrations were generally in the
toxic range and their clinical relevance was
limited. The core therapeutic range of Li in
serum for humans is now considered to be
0.6-1.0 mM.
We very recently reported the appli-
cation of 7Li MR point-resolved spectroscopy
(PRESS) to measure Li concentrations and
spin-relaxation times in rat brain. 68 We
improved signal-to-noise ratio by using a
more sensitive RF coil and by sampling a
larger voxel than that in our previous
imaging studies, allowing measurements
over the entire range of therapeutic Li
concentration. The method involves use of
an external standard phantom and takes into
account T1, T2, and 7Li MR visibility.68 For
spin-3/2 nuclei such as 23Na, studies of many
tissues have shown that ion concentrations
determined by MR are less than those
determined by other analytical methods.
Lithium-7 is generally considered less likely
to display reduced MR visibility. Previous
7Li-MR estimates of brain Li concentrations
in humans assumed 100 percent visibility.
However, in an in vivo MRI study we
determined that 7Li does display some-
what reduced visibility in rat brain in vivo
(but not in muscle), on the order of

10-25 percent.67 The detailed origin of the
reduced 7Li visibility is not known.
Using that approach,68 we investigated
the relationship of brain and serum
concentrations in the rat at two treatment
times.69 Measurements were made at about
7 days to insure steady state and at about
16 days because that roughly corresponds
to the delay in onset of Li’s therapeutic
efficacy after initiation of treatment. No
significant change was observed for either
brain or serum concentration, or their ratio,
at 16.1 days vs. 6.6 days. However, the
brain Li concentration at 16.1 days did
correlate significantly with the corres-
ponding concentration at 6.6 days (r = 0.61,
p = 0.001, N = 26).
Brain Li strongly correlated with serum
Li at both 6.6 days (r = 0.95, p < 0.000001,
N = 34) and 16.1 days (r = 0.95, p < 0.000001,
N = 25). Figure 17.6 shows a plot of brain
Li concentration vs. serum Li concentration
at 6.6 days. Both brain and serum
concentrations of Li at both treatment
durations correlated with dose. There are a
number of factors responsible for the
substantially better correlations in animal
Fig. 17.6: Plot of brain Li concentration vs. serum Li
concentration at 6.6 days in the rat. The solid line
represents the regression equation, [Li] in brain =
0.01078 + 0.97315 × [Li] in serum. The dashed lines
represent the 95 percent confidence interval.69
MRI and MRS of Nuclei other than 1H 225
studies than for humans, including reliable
drug delivery over a relatively wide dose
range, as well as controlled diet, age, and
weight.
The average brain/serum Li ratio was
0.98 ± 0.24 at 6.6 days and 0.93 ± 0.16 at
16.1 days. Most previous studies found
average ratios of 0.5-0.8, although a recent
study60 found 0.92 for adult patients, a value
very similar to that found by us for rats.
Beyond the fact that our values are for rats
and all previous values are for humans, the
major difference is that in rats the reduced
7
Li MR visibility was taken into account in
estimation of the brain Li concentrations.
Spin relaxation times are of interest both
for accurate quantitation and as probes of
the cellular environment. In vivo spin
relaxation of 7Li has been reviewed.51 The
7Li T in vivo ranges from about 3 to 8 s,
1
depending on conditions. Our recent loca-
lized, spin-relaxation studies of 7Li in rat
brain under controlled conditions found a
narrower range for T1 (2.4 – 5.1 s, mean
3.3 ± 0.9 s) at 4.7 T.68 Very few previous
results of any sort are available for T2.51
Previous T2 results in rats were unlocalized
and hard to interpret. Since biexponential
behavior from the presence of both intra-
and extracellular Li might be expected for
T2, measurements ideally should be loca-
lized to a single tissue type to eliminate
apparent biexponential behavior from multi-
ple tissues with differing T2s. In practice, it
is difficult to detect unambiguously the pre-
sence of biexponential decay. In our recent
study in rats,68 we found T2 values in the
range of 53 to 113 ms (mean 82 ± 20 ms).
Very recently Ramaprasad70 obtained 7Li
spectroscopic images of 3.1 × 3.1 × 10 mm3
resolution on rat brain and muscle at 7 T.
In vivo 7 Li MRS is likely to remain
primarily a research tool, or be used to
226 Biomedical Magnetic Resonance: Proceedings of the International Workshop
measure Li levels in conjunction with MRS
of other isotopes such as 1H or 31P.71
Sodium-23
Sodium-23 MRI is potentially a powerful
technique for visualizing endogenous,
naturally abundant sodium in vivo. Appli-
cations of the technique have been reviewed,
although not recently.72 Of critical impor-
tance is the distribution of Na between the
intra- and extracellular environments. One
approach to this question has been via
multiple quantum techniques.73 The reader
is directed to recent applications of 23Na
MRI to myocardial infarction, 74 brain
tumors,75 cerebral ischemia, 76 and other
organs.77
Hyperpolarized Gases—3He and 129
Xe
An MR experiment has inherently low
sensitivity compared to other spectroscopic
techniques, such as optical spectroscopy.
This is due to the small population difference
between the nuclear energy levels, even at
the highest magnetic field strength. This
population difference in a static magnetic
field is given by
N hB 0
 ...(2)
N -
2k BT
in which N+ is the number of spins parallel
to the field, N– is the number of spins anti-
parallel to the field, h is Planck’s constant,
B 0 is the magnetic field strength, k B is
Boltzmann’s constant, and T is the abso-
lute temperature.
The spin polarizations for 3He and 129Xe
at 1.5 T and room temperature are 7.8 ×
10-6 and 2.8 × 10-6 respectively. Recently,
optical methods of increasing the spin
polarization of 3 He and 129 Xe, termed
“optical pumping,” have proven very
successful in increasing the polarization to
upto 70 percent (an increase by a factor of
about 100,000), which allows these
hyperpolarized gases to be used for MR
imaging. This prepolarization of the nuclei
not only increases the signal that can be
obtained from the MR experiment, but also
imposes some restrictions on the techniques
used to measure that signal. For example,
since the polarization is not the result of
the magnetic field, the available signal
disappears with each RF pulse. Thus, small
flip angle experiments are used to maintain
as much of the signal as possible for as long
as possible. Fortuitously, the relaxation
times of both 3He and 129Xe are long in
vivo, which means that it is possible to
acquire 3 He or 129Xe images using fast
imaging techniques.
Two methods are used to achieve
hyperpolarization: Spin exchange optical
pumping and metastability exchange optical
pumping. In both techniques, the noble gas
is placed in a chamber with the exchanging
atoms. The spin exchange method uses alkali
metal vapors whereas the metastability
method uses metastable atoms. The chamber
containing the noble gas and the exchanging
medium is placed in a magnetic field and
illuminated with laser light of the appro-
priate frequency for optical absorption by
the metal vapor or the metastable atoms.
This causes spin polarization of the vapor
or the metastable atoms. This energy is
transferred through collisions to the noble
gases, which causes them to become spin
polarized. For a more complete description
of the process, refer to the review by
Kauczor et al78
Hyperpolarized 3He and 129Xe have been
used primarily for visualizing spaces, such

as the lungs, that are poorly imaged with
protons. The first human studies using
hyperpolarized helium were done by
Kauczor et al.79 Xenon was first used by
Albert et al.80 In general, helium is preferred
over xenon because of its larger magnetic
moment, smaller relaxivity, and greater
polarization.81 In addition, helium has no
side effects, other than reduced oxygenation
due to reduced oxygen levels in the inhala-
tion mixture, whereas xenon has known
anesthetic properties, which may not be
desirable.81
Recently, hyperpolarized helium has been
compared to Xe-133 scintigraphy to measure
lung defects.82 The authors found that the
two methods gave similar results. Hyper-
polarized helium has been used for MR
microscopy of the rat lung, providing
images with resolutions on the order of
5 × 10–3 mm3.83 The relaxation time of the
hyperpolarized helium can be used to
measure the alveolar pO 2 . 84 Other
applications of hyperpolarized helium in
humans have been reviewed recently85 and
include spin density imaging, dynamic
ventilation imaging, and diffusion weighted
imaging.
Unlike helium, xenon is very soluble in
lipids and water and has a large chemical
shift change when dissolved or adsorbed.86
These properties have been exploited in
studies of lung function,86 perfusion,87 blood
oxygenation, 88 and tumors. 89 Hyper-
polarized xenon has also been used to image
tissues, particularly brain.90
Carbon-13 and Nitrogen-15
In vivo 13C MRS can provide a great deal of
information about metabolic pathways in the
brain and other tissue. However, the low
natural abundance and low sensitivity of
13
C make its MRS technically challenging
MRI and MRS of Nuclei other than 1H 227
in vivo. Isotopic enrichment is a necessity,
both to improve SNR and to follow distri-
bution of label along metabolic pathways.
Despite the difficulty, however, 13C has been
used in several studies of brain,91 muscle,92
and liver 93 metabolism. The challenges
involved in, and the applications of, 13C in
brain have been reviewed by Gruetter et
al.91 Carbon-13 has been used in the clinical
setting to examine various disorders,
including Alzheimer’s disease and epilepsy.94
However, the complexity of the exam limits
its availability to research sites.
Similar considerations apply to 15N,
which has been used in vivo to follow ammo-
nia transport and glutamine metabolism.95,96
OTHER NUCLEI
A number of other nuclei have been used
for in vivo imaging or spectroscopy. Several
involve very specific applications in limited
situations, whereas others hold promise for
more general applicability. Other applica-
tions include 27Al for measuring gastro-
intestinal transit times, 47 10B for locating
boron neutron capture agents,97 17O for
imaging cerebral metabolic rate of oxygen,98
87
Rb for measuring ion transport in tissue,99
133Cs for probing the intracellular environ-
ment,100,101 195Pt for monitoring the fate of
the anticancer drug carboplatin in tissue,102
and 2H for following water diffusion and
flow, and lipid metabolism.103,104
FUTURE PROSPECTS
Limitations on the clinical applications of
MRS of heteronuclei are slowly being
alleviated. Multinuclear capability is now
more commonly available on clinical
scanners. This trend is driven largely by
the desire for 31P MRS capability, and will
improve prospects for other nuclei. High-
magnetic-field clinical scanners, now in
228 Biomedical Magnetic Resonance: Proceedings of the International Workshop
operation at up to 8 T, are becoming
increasingly widespread. As with all MR
techniques, higher strength magnets will
lead to increased signal, which will aid in
heteronuclear studies. For heteronuclei this
translates into either higher resolution
spectroscopic exams or lower detection
limits. More sensitive detection coils,
isotopic enrichment, and novel post-
processing methods can further reduce the
minimum detectable concentration in vivo,
permitting more rapid measurements or
better resolution in localized studies.
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 MRI/MRS to Predict and Monitor Response to Anticancer Therapies
MRI/MRS to Predict and Monitor
Response to Anticancer Therapies
INTRODUCTION
Over the last decade, the changes in
diagnostic imaging have been nothing short
of revolutionary. What used to be a
discipline focused on anatomy has been
transformed to one that is concerned with
increasingly sophisticated measures of tissue
function and molecular expression. This has
occurred largely through advances in both
magnetic resonance imaging (MRI) and
positron emission tomography (PET). While
there has been much discussion regarding
the application of imaging to clinical trials,
a clear vision for its use remains to be clearly
delineated. Therapeutic clinical trials can be
impacted by imaging in two distinct areas:
diagnosis and segmenting patients for
inclusion, and monitoring therapeutic
response.
Diagnosis and staging is the classical
venue for application of imaging. Current
imaging of cancers with MRI, ultrasound,
CT or PET is an important part of the
oncologist’s armamentarium in making
informed diagnoses, staging and therapeutic
decisions. However, the imaging information
used in therapeutic decision making in
233
18
Robert J Gillies, Natarajan Raghunand
clinical practice is primarily limited to
anatomical extent and size of lesions.
Notable exceptions are scintigraphic bone
scans and FdG PET staging, which generate
contrast due to tissue function. Hence, the
current practice of oncology does not take
advantage of more cutting-edge imaging of
tumor function, especially in the field of
MRI. An extension of this is the use of
imaging data as inclusion criteria in clinical
trials. While inclusion criteria generally
include imaging confirmation of disease
extent, more cutting edge approaches might
be useful to identify patients whose tumors
contain the biochemical targets. This will be
discussed in the section entitled “MRI/MRS
to predict therapeutic response”.
Monitoring therapy is becoming an area
wherein functional MR imaging is having a
significant impact, especially in the use of
dynamic contrast-enhanced MRI for anti-
angiogenic agents. However, the applica-
tions of MR approaches to monitoring
therapy responses are not limited to DCE.
A large number of approaches have been
proposed, including 1H MRS, 31P MRS,
diffusion MRI and DCE-MRI with more
specific contrast reagents. Out of this
234 Biomedical Magnetic Resonance: Proceedings of the International Workshop
multitude of possibilities, the right choice
of experiment and acquisition parameters
requires educated forethought taking into
account the drug’s mechanism of action and
the question to be asked. It is our contention
that the right choices can be better made
with appropriate preclinical studies in
appropriate animal models. This will be
discussed in the section below, entitled
“MRI/MRS to monitor therapeutic
response”.
MRI/MRS TO MONITOR
THERAPEUTIC RESPONSE
The rationale driving this aspect of MR
research is that MR imaging or spectroscopy
can provide early, identifiable and
quantifiable endpoints in response to drug
actions.16 These endpoints are often, but not
always, mechanistically connected to known
drug targets. Because this has historically
been a major focus of research activity, no
attempt will be made to comprehensively
review the field. Instead, readers are
directed to recent reviews on the sub-
ject.17,21,25,49,56 In this section, we will
describe some of our own work in using
diffusion MRI, dynamic contrast MRI, and
MRS to monitor response to therapies in
humans and animal models.
Diffusion MRI
In the simplest experiments, diffusion MRI
involves the use of balanced gradient pulses,
which serve to refocus spins that are
physically stationary. This is shown in
Figure 18.1, wherein two gradient pulses of
strength, g, and duration, δ, are separated
by an evolution time, Δ. Since the gradient
is applied along a direction, the effect of
the first pulse is to impart a spatially-
dependent phase upon the recipient spins.
If spins do not move during the evolution
time, they are completely rephased upon
application of the second pulse. However,
if they do move, they will fail to refocus
and signal is lost. The rate at which signal
is lost is dependent on g, δ and Δ, such
that65
S = So e–bD
2 2 2
where, b = δ γ g (Δ – δ/3)
In practice, gradients of increasing strength
are used, which increases the strength of
the diffusion weighting, or b-factor. Signal
decreases exponentially with increasing b-
factor (Fig. 18.1).
In pure water, the signal will decay with
a single exponential and fitting this will
yield ca. 3.2 × 10-5 cm2 sec-1, which is the
true diffusion coefficient of water, D at 37oC.
In tissues, however, water is restricted by
numerous membranous boundaries, which
are more numerous and closer together in
the intracellular space. Hence, the measured
values do not reflect true diffusion, but the
amount of diffusional restriction. Hence, the
measured value is termed the apparent
diffusion coefficient, ADC. Since diffusional
restriction is more pronounced in the
intracellular space, the ADC will decrease
with increasing cellularity. 64 Hence,
diffusion MRI is sensitive to changes in cell
volume fraction. Such changes occur with
multiple modes of cell death, including
apoptosis.8
Diffusion MRI of animal tumors was first
shown to be affected by chemotherapy in
the mid-1990s by Ross and Chenevert at
Michigan 62 and by Evelhoch’s group at
Wayne State.76 Since that time, there have
been a number of studies to investigate this
phenomenon in both preclinical animal
models and in humans.21,37 In the following
 MRI/MRS to Predict and Monitor Response to Anticancer Therapies 235

Fig. 18.1: Standard diffusion weighting protocol. Diffusion weighting is imparted by a matched pair of pulsed
field gradients that will refocus the phase of stationary spins. The magnitude of diffusion weighting is quantified
in the ‘b-factor’ which embodies terms for the gradient strength, g, duration, δ, and evolution time, Δ. Signal
decays exponentially with increasing b-value. The faster the decay with b, the higher the apparent diffusion
coefficient, ADC. The in vitro curve is shown for a pure water solution. The in vivo curve is shown for a prostate
cancer xenograft. See reference 34.

sections, we describe our published and
unpublished results investigating the effect
of chemotherapy on the apparent diffusion
in clinical and preclinical tumors. Each
paragraph is identified by the lead
investigator on the project, and their contact
information is given at the end of this article.
Preclinical Studies
Jean-Philippe Galons
We began examining this approach in 1998.22
These early studies investigated xenografts
of MCF-7 human breast cancer tumors that
were treated with taxol or with vehicle
alone. Both drug-sensitive (MCF-7/s) and
drug-resistant (MCF-7/D40) tumors were
examined. These studies showed clear and
unequivocal increases in the ADC within
two days of treatment in those tumors
destined to respond to drug (MCF-7/s
treated with taxol) and no change in ADC
for those tumors that did not respond (MCF-
7/D40 treated with taxol or vehicle, MCF-
7/s treated with vehicle). Representative
results are shown in Figure 18.2. This figure
shows histograms of the ADC values for
taxol- and vehicle-treated MCF-7/s tumors
two days after initiation of therapy. As
shown, there are significant and substantial
increase in the ADC by this time point. The
use of drug-resistant tumors was significant
because it showed that the response was
dependent on tumor response and did not
result from systemic effects of drug on the
host.
236 Biomedical Magnetic Resonance: Proceedings of the International Workshop
Fig. 18.2: ADC histograms in response to taxol.
Diffusion data can be represented as histograms
wherein the percent of pixels are plotted at different
values of ADC. As shown in this graph, there is a
movement to higher b-values upon treatment with
taxol in drug-sensitive tumors. See reference 22.
Dominique Jennings
In a further study, we examined the effect
of different doses of another taxane
(docetaxel, or TaxotereTM) on the diffusion
and response of LnCaP prostate cancer
xenografts. 34 In these studies, tumor
response to drug was quantified by both
the tumor growth delay (TGD) and the
levels of secreted prostate-specific antigen
(PSA) by these tumors. The purpose of this
study was to ask if a quantitative relation-
ship existed between the magnitude of the
change in ADC and the magnitude of the
response. Interestingly, there were linear
relationships between tumor volume and
PSA both before and after therapy, yet the
slope of the relationship was much shallower
after therapy commenced. This may have
resulted from selective killing of PSA-
secreting cells and outgrowth of cells with
low PSA levels. Hence, only TGD could be
used to assess response. These analyses
showed that there was no clear relationship
between the magnitude of the change in
Fig. 18.3: Changes in ADC and TGD in response to
Taxotere TM. Changes in the mean ADC were
measured 2 and 4 days following treatment of LnCaP
prostate cancer xenografts in response to different
doses of TaxotereTM. Additionally, tumor response was
measured as the tumor growth delay. As shown, ADC
increased at all doses, and increased further between
days 2 and 4. In contrast, TGD increased mono-
tonically with dose. See reference 34.
ADC and the magnitude of the response.
While TGD showed a monotonic dose-
dependence, the increase in the ADC
appeared to be a step function, wherein it
responded almost maximally for all non-
zero doses (Fig. 18.3). Our interpretation of
this observation is that the maximal change
in ADC may not be related to the TGD
response. We further hypothesize that the
duration of elevated ADC values may be
related to response, and this remains to be
tested.
David Morse
More recently, we have been investigating
the tumor biology of the ADC response and
its relationship to other MR-visible para-
meters. This was examined in breast cancer
xenografts (as above) treated with
TaxotereTM. It should be noted that MCF-
7/D40 tumors are not resistant to
Taxotere TM , as they are for taxol. This
 MRI/MRS to Predict and Monitor Response to Anticancer Therapies
system was used to compare the effects of
therapy on ADC and choline containing
compounds by in vivo and in vitro 31P and
1 using diffusion MRI to predict or monitor
H magnetic resonance spectra (MRS). When
the magnitudes of the ADC and MRS results
were compared via ROC analysis, there was
a strong, but inexact, correlation. We
interpret these results to indicate that there
is a disconnect between cellular responses
reported by MRS of choline were distinct
from the responses that are reported by the
ADC changes. Hence, in response to
therapy, these two indicators react
differently, which is not the case in
untreated tumors, where the ADC is lower
and choline is higher in volumes with high
cellularity.29
Bénédicte Jordan
In a more recent study,35 we have been
examining the response of HT-29 colon
cancer xenografts to a novel HIF-1α inhibi-
tor, PX-478,73 using both diffusion MRI and
dynamic contrast enhanced (DCE) MRI
(discussed later). These studies were
designed to determine which MR visible
parameters were most sensitive to treatment
with this drug prior to a clinical trial.
Quantification of sensitivity was achieved
by determining the dynamic range of the
response, defined as the magnitude of
change divided by the natural variance of
the parameter. Variance was determined by
repeated measurements of vehicle-treated
controls. In this study, significant changes
in the ADC were observed by 24 hours,
which was the earliest documented time to
reach significance for this parameter.
Although the DCE-MRI changes occurred
earlier (< 2 hr) than the changes in ADC,
the dynamic range of ADC changes was
greater.
237
Human Studies
Very few clinical trials have been performed
therapeutic response. Most of these have
involved brain neoplasms either for
diagnosis/prognosis, 10,13,19,23,29,41,69 or for
therapeutic monitoring.14,46 Clinical studies
outside of the brain are fewer, and have
been limited to diagnosis/prognosis in
breast,60 liver,11,33,55 and bone18,43 neoplasms.
As described below, we have been applying
diffusion MRI to measure chemotherapy
response in breast cancer patients with
metastases to liver or bone. Such patients
are often treated with third-line therapies,
with attendant increases in morbidity.
Hence, early detection of non-response
could prevent treatments with toxic
therapies.
Rebecca Theilmann
In this study, breast cancer patients with
liver lesions were examined prior to, and at
4, 11 and 39 days following commencement
of therapy. Diffusion was measured with
three b-values (150, 300 and 450 × 10-3 sec
mm -3) using echo planar DWI during a
single breath hold.70 A representative image
is shown in Figure 18.4. Analyses of the
changes in mean diffusion coefficients using
regions of interest (ROI) showed that, on
average, responders had significant increases
in their ADC values by day 11, while non-
responders did not. Data were also analyzed
to see if the magnitude of the diffusion
change related to the magnitude of response
and showed that there was a significant
correlation on day 4, for smaller (< 8 mm3)
lesions, but not for larger lesions. Pearson
correlations coefficients on day 11 just
238 Biomedical Magnetic Resonance: Proceedings of the International Workshop

missed significance (p = 0.07). We hypo-
thesized that analyses would be improved
with better image registration, as motion
between scans prevented pixel-by-pixel fits.
Hence, we have developed an automated
segmentation scheme based on region
growing followed by fuzzy Sobel edge
detection and a modified Kass snake.42 Such
analyses compared favorably to radiologist-
identified ROIs, and reduced the time
required by orders of magnitude. An
extension of these analyses will allow
segmentation and pixel-by-pixel fits, which
should yield more detailed interpretations
of the ADC changes.
Dominique Jennings
Because of the aforementioned motion
problems, and because breast cancer often
metastasizes to bone, we have recently
turned our attention to analyses of diffusion
in bone lesions. The prognosis for such an
approach is tempered by the fact that bone
lesions can be lytic or blastic, and
histological analyses reveals that the tumor
cells are relatively disperse. Hence, the
magnitude of ADC changes would be
expected to be variable and somewhat less
that those observed in the liver. To improve
matters, though, diffusion images from bone
can be acquired at higher resolution
compared to the liver (Figs 18.4A and B).
Since they are self-registered, images can
be analyzed on a pixel-by-pixel basis to
generate ADC maps. Although these studies
are only preliminary with only 8 patients,
data have shown that responding patients
have small but statistically significant
increases in their ADC values by day 11,
and non-responding patients do not.
Mechanistic Studies
Because of the importance and utility of
diffusion MRI in cancer and in monitoring
ischemia, a number of researchers have
examined the mechanisms behind the ADC
changes.32,44,64 We have been investigating
this both by correlations to mechanisms of

Figs 18.4 A and B: Diffusion weighted images of breast cancer metastases. Diffusion weighted
images were obtained from breast cancer patients with metastases to either liver or bone. In
the liver, due to motion, images were collected during a single breath hold using echoplanar
imaging (see reference 70). In the bone, images were collected with a fast spin echo sequence,
to yield higher resolution (unpublished observations)
 MRI/MRS to Predict and Monitor Response to Anticancer Therapies
cell death and by the use of a novel in vitro
bioreactor system.
David Morse
In the first of these studies, we demon-
strated that, under our experimental
conditions, docetaxel kills breast cancer
xenografts via both lytic necrosis and mitotic
catastrophe, and not apoptosis.48,50,51,52 The
drug resistant line, MCF-7/D40, is not
resistant to docetaxel, and was killed by
the same mechanisms as the other two cell
lines used in this study (MCF-7/s and MDA-
mb-231). Furthermore, the spatially variant
changes in ADC within these tumors could
be registered to spatially-variant modes of
cell death, showing that, in this system, the
increases in ADC were not due to apoptosis,
but were related to other modes of cell
death, namely lytic necrosis and mitotic
catastrophe. These were shown to involve
the destruction of plasma membrane
integrity, which would be consistent with
and increase in ADC. This is significant
because previous reports had ascribed the
diffusion change to apoptosis,30 and it was
necessary to determine if the diffusion
changes were specific for this mode of cell
death or not.
Jean-Philippe Galons and
Theodore Trouard
Cells grown in an NMR-compatible hollow-
fiber bioreactor (HFBR) can reach tissue
densities in the extra-fiber space.3,26 Because
of the orientational geometry in these
reactors, magnetic susceptibility gradients
exist that can be exacerbated through the
addition of susceptibility reagents, such as
those containing gadolinium. These gradi-
ents are anisotropic and addition of
Gd-DTPA to the perfusate results in a
239
susceptibility-induced shift in the extra-
cellular water, which uncovers a resonance
identifiable as intracellular. Hence, the
apparent diffusion coefficient of intracellular
water can be measured directly, and
monitored in response to ischemia, osmotic
shock or chemotherapy. Although scalar
ADC values are completely dependent on
the acquisition conditions (specifically, Δ),
measured values on cultured C6 glioma cells
are consistent with predicted values (ca. 1 ×
10–5 cm2 sec-1).
Dynamic Contrast-enhanced MRI
Dynamic contrast-enhanced (DCE) MRI
involves monitoring the time-dependent
signal intensity increases following the bolus
injection of a contrast reagent. If the arterial
input function (AIF) is known, these
intensity changes can be modeled and used
to extract valuable parameters, such as
extraction fraction or transfer constants.71
If the AIF is not known and the injections
are reproducible, data can be analyzed using
an area-under-the-curve approach.15 Too
much has been written on the application of
DCE-MRI to monitor therapeutic response
to be comprehensively reviewed here.
Instead the readers are referred to the work
of Padhani, 57,58,59 Knopp 31,39,40 and
Brasch9,27,61 for application in the clinic, and
to Bhujwalla, 4,5,7 Neeman, 1,9,53,54 and
Nalcioglu66,67,68 for preclinical applications.
Bénédicte Jordan
Our approach to this problem has been to
incorporate DCE-MRI acquisitions into the
armamentarium of techniques used to
investigate MR visible parameters in animal
models prior to clinical trials of new drugs.
This paradigm has been applied to a novel
class of redox drugs.
240 Biomedical Magnetic Resonance: Proceedings of the International Workshop
PX-478 is a Melphalan analog. All
available evidence indicates that it inhibits
the activity of the hypoxia-inducible factor,
HIF-1α, through transcriptional repression.74
HIF-1α is a transcription factor that is
upregulated by a number of stress responses,
including hypoxia. This transcription factor
induces the expression of a number of stress
response genes, including vascular endo-
thelial growth factor, and a number of
glycolytic enzymes.63 The maximum tole-
rated dose is 120 mg/kg, and it has shown
dramatic activity against a number of
tumors, including HT-29 colon cancers.
Other tumors, such as A549, are resistant.
PX-12 is a redox inhibitor of thioredoxin,
TRx-1. Thioredoxin is a secreted redox
protein that is upregulated in a number of
cancers.2,28 In clinical trials, inhibition by PX-
12 causes stasis in tumors that overexpress
TRx.38
These studies used a “standard” protocol
for imaging of tumor-bearing mice treated
with experimental drugs. In this setting,
animals are imaged prior to and at times
following single treatment at 80 percent of
the maximum tolerated dose (MTD) of the
drug under study, or the vehicle control.
Times vary from 1 hr up to 48 hr post-
therapy or post-vehicle control. This
generates both the time courses of responses
and the natural variance of normal
(untreated) values. Imaging modalities
included DCE-MRI with low MW contrast
media (Omniscan), and DCE with high MW
contrast media (albumin-DTPA-Gd), in
addition to diffusion MRI (vide supra) and
single-voxel 1H MRS.
Small MW contrast reagents (SMWCR)
provided equivocal results, with measurable
changes occurring within 4 hr of therapy.
This is in contrast to other studies which
have shown more significant differences in
SMWCR-measured K trans following anti-
angiogenic therapies.12 Initial experiments
with 1H MRS showed significant changes in
the choline resonance, but only at 24 hours.
These results were confirmed with in vitro
spectroscopy of tumor extracts.
DCE MRI with large MW contrast
showed dramatic changes in vessel
permeability determined from slope of the
enhancement curve within 2 hr of either
PX-1236 or PX-47835 treatment. These changes
relaxed back to pre-therapy baseline by 48
hours. Changes in vascular permeability
were not accompanied by changes in vascular
volume, determined from intercept of the
enhancement curve. These results were
contrasted to the effects of Avastin (anti-
VEGF antibody), which decreased both
permeability and the apparent vascular
volume. Treatment of PX-478 resistant A549
tumors showed that the measured changes
were dependent upon the tumor respon-
siveness. Interestingly, the time course of
changes in permeability induced by PX-12
followed the time course of changes in
serum TRx-1, and not that of VEGF.
Clinical trials of PX-12 have begun with
interesting results. As mentioned above, this
drug is most efficacious in tumors that
already overexpress TRx-1. Data from
patients shows that PX-12 decreased secreted
VEGF and that the magnitude of response
was directly correlated to pretherapy serum
VEGF levels (A. Baker, unpublished
observations).
Implications
This preclinical study is a paradigm for the
first phase of a “standard” protocol for
endpoint determination. On the basis of
these studies, the phase I/II clinical trial of
PX-478 will include both diffusion and DCE-
MRI for therapy response monitoring. The
 MRI/MRS to Predict and Monitor Response to Anticancer Therapies 241

correlation between either DCE-MRI and/
or diffusion MRI and ultimate clinical res-
ponse to this drug has not been established.
At a more profound level, however, it
can be postulated that patients with high-
VEGF tumors will exhibit a greater response
than those with more indolent, non-VEGF
expressing tumors and that permeability to
a macromolecular contrast agent is a bio-
marker for VEGF. Hence, high permeability
from DCE-MRI could be used as an inclusion
criterion in clinical trials with this drug. Such
segmentation of patients would lead to
higher efficiencies in clinical trial design,
with substantial savings in terms of time
and money. This is discussed further in the
following section.
MR IMAGING TO PREDICT
THERAPEUTIC RESPONSE
The entire field of pharmacogenomics is
predicated on identifying patients whose
cancerous lesions contain an intended drug
target prior to treatment. 45,47,72,75 It is
expected that, if a drug is efficacious, those
patients with the intended target will have
a higher response rate, compared to those
who do not.
Of all of the MR-visible parameters, the
one currently with the highest connectivity
to a molecular target is DCE-MRI and
VEGF. This is illustrated in Figure 18.5,
which shows the HIF-1α axis. As shown,
there are a number of activities that can
lead to stabilization of this transcription
factor besides hypoxia. Elevation of HIF-1α
should induce expression of VEGF in most
systems, and elevation of VEGF should
increase permeability. However, going
backwards, it must be realized that high
permeability can be affected by other factors
besides VEGF, such as nitric oxide, NO.20
Thus, permeability would be predicted to
be a sensitive, yet non-specific, biomarker
for VEGF. Consequently, permeable tumors
would be expected to have a higher
proportion of VEGF-expressing phenotypes,
compared to the general population.

Fig. 18.5: The HIF-1α axis. HIF-1α is stabilized by hypoxia, as well as (counter-clockwise from 12 o’ clock):
alterations in microtubule distribution (ìtubes), cyclo-oxygenase-2 (Cox-2), activities of insulin-like growth
factor (IGF) or human EGF receptor-2 (HER-2), hypoxia, phosphatidylinositol 3-phosphate kinase (PI3K), heat
shock protein-90 (HSP-90), histone deacetylase (HDAC) and thioredoxin (TRX). These can be inhibited by
drugs indicated in italics. One of the proteins induced by HIF is vascular endothelial growth factor, VEGF,
which increases vessel permeability
242 Biomedical Magnetic Resonance: Proceedings of the International Workshop
This opens the question: Under what
circumstances can DCE-MRI be used as a
biomarker for VEGF expression? For large
MW contrast reagents (e.g. Gd-BSA) in
animal studies, the answer appears to be
clear. In two important studies, both
Neeman’s and Bhujwalla’s groups have
shown that VEGF expression (and
reduction) were matched by increases (and
decreases) in vascular permeability to Gd-
DTPA-albumin.1,6 In human patients using
low MW contrast media, however, the data
appear to be more equivocal. For example,
George et al measured VEGF levels
compared to DCE-MRI data in colon cancer
patients and reported a weak correlation.24
However, if the data in this study were
analyzed differently, e.g. if pretreatment
DCE-MRI were used to segment colorectal
patients prior to therapy, the proportion
expressing high VEGF would have increased
to 45 percent (4/9) compared to 18 percent
(5/28) in the general population. Hence, use
Fig. 18.6: Power in clinical trials. This graph represents
the numbers of patients required to achieve 90
percent power, given the assumptions outlined in
the text. If the percent of the patient population
harboring a target is 10 percent, 159 patients will be
required. If 60 percent of the patients harbor the target,
only 22 will be required (Courtesy: James Ranger-
Moore)
of pretherapy DCE-MRI to identify a
potential responding population would have
excluded 19 non-responding patients, while
missing only one.
James Ranger-Moore: The effect of using DCE-
MRI in therapy trial inclusion criteria on
savings in time and money would be
substantial. This is illustrated in Figure 18.6,
which shows a power calculation of the
numbers of patients required to reach 90
percent confidence. This analysis was made
assuming α = 0.05 with two-sided Fisher’s
Exact test, that ‘Target-positive’ tumors have
a response rate of 70 percent, that ‘Target-
negative’ tumors have a response rate of 5
percent and that there is a 5 percent
spontaneous regression rate. As shown, if
the proportion of target-positive (e.g. VEGF-
expressing) tumors can be increased from
10 to 60 percent, the number of patients
required to obtain 90 percent power can be
reduced from 159 to 22.
ACKNOWLEDGMENTS
• Bénédicte Jordan—All MR experiments with PX-
478 in animals.
<bjordan@email.arizona.edu>.
<benedicte.jordan@rema.ucl.ac.be> (current
address)
• David Morse—In vivo and in vitro MRS and
diffusion MRI in response of breast cancer
xenografts to taxotere. Mecha-nisms of cell death.
<dmorse@email.arizona.edu>
• Jean-Philippe Galons—diffusion in taxol with drug
resistance. Mechanisms of diffusion.
<jgalons@U.Arizona.EDU>
• Theodore Trouard—Diffusion in ische-mia.
Mechanisms of diffusion.
<trouard@email.arizona.edu>
• Dominique Jennings—Pre-clinical and clinical
diffusion response to therapy.
<djenning@email.arizona.edu>
• Garth Powis—Developer of PX-478, Molecular
pharmacologist and research director of Arizona
Cancer Center <gpowis@azcc.arizona.edu>
 MRI/MRS to Predict and Monitor Response to Anticancer Therapies
• Amanda Baker—Molecular experiments with PX-
478 <ABaker@azcc.arizona.edu>
• NIH U54 CA90831, CA77575
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In vivo Cellular and
Molecular Imaging of Cancer
Zaver M Bhujwalla, Ellen Ackerstaff, Dmitri Artemov, Kristine Glunde,
Arvind P Pathak, Venu Raman and Meiyappan Solaiyappan
INTRODUCTION
The twenty-first century has witnessed an
explosion of molecular biology techniques,
amazing advances in imaging, and the
design of unique imaging probes. Despite
the tremendous strides made in these areas
of science, the cure for cancer remains
beyond our grasp. Cancer is a complex
disease and the apparent impenetrability of
the disease is largely due to the multiple,
often redundant pathways, which appear
to evolve through the genetic instability of
cancer cells. The ability to identify and
image key common pathways specific to
cancer cells, and the ability to image the
effectiveness and outcome of strategies
designed against these targets is critically
important in the treatment of this disease.
The MR methods provide a vast array
of capabilities to characterize function. These
imaging techniques are also easily
translatable into the clinic, and are therefore,
compatible with ‘bench to bedside’
applications. The MR component of the Johns
Hopkins University (JHU). In vivo Cellular
and Molecular Imaging Center (ICMIC)
Program spans molecular, cellular, preclinical
human tumor model, and clinical imaging.
19
A major focus of this program is to develop
transgenic tumor models to understand
cancer. The application of MR to transgenic
cells or animals whose genetic composition
has been altered by addition of foreign
DNA, or transgene, has provided unprece-
dented opportunities for understanding and
characterizing specific targets and pathways
in cancer. To understand and exploit
molecular pathways in cancer for therapeutic
strategies, it is essential not only to detect
and image the expression of these pathways,
but also to determine the impact of this
expression on function at the cellular level,
as well as in vivo. The MR studies of
transgenic cells and mouse models can,
therefore, be used to determine the
consequences of overexpression, under-
expression or complete inactivation of the
gene under investigation. These studies
enhance the understanding of the function
of these genes and how they influence the
malignant phenotype. Information derived
from such transgenic models can also lead
to the development of therapies for cancer
treatment. During the course of this lecture
specific examples will be provided to
demonstrate how the applications of MR to
transgenic cells and tumor models has
increased our understanding of cancer.
248 Biomedical Magnetic Resonance: Proceedings of the International Workshop

SOME EXAMPLES OF THE APPLICATION membranes. PtC together with other

OF MR SPECTROSCOPY TO
TRANSGENIC MODELS
The elevation of phosphocholine (PC) and
total choline-containing compounds is one
of the most widely-established characte-
ristics of cancer cells, which has been
demonstrated in breast,1 prostate and brain
tumors2,3 as well as leukemia.4 Phospho-
choline is both a precursor and a breakdown
product of phosphatidylcholine (PtC), the
most abundant phospholipid in biological
phospholipids such as phosphatidylethano-
lamine and neutral lipids forms the
characteristic lipid bilayer structure of
cellular membranes and regulate membrane
integrity.5,6 The biosynthesis and hydrolysis
of PtC (see Fig. 19.1) are essential processes
for mitogenic signal transduction events in
cells. 7-9 Products of membrane choline
phospholipid metabolism (MCPM) such as
PC, diacylglycerol, and arachidonic acid
metabolites may function as second
messengers essential for the mitogenic

Fig. 19.1: Schematic of the biosynthesis (solid lines) and hydrolysis (dashed lines) of phosphatidylcholine
(PtC), including all enzymes involved in choline phospholipids metabolism. Phosphorylation of choline to
phosphocholine (PC) by choline kinase is the first step in the biosynthesis of PtC. PC is then converted to PtC
via intermediates involving the rate-limiting enzyme CTP:phosphocholine cytidyltransferase and
phosphocholine transferase. Hydrolysis of PtC is affected by three major PtC-specific enzymes, phospholipase
C, phospholipase D and phospholipases A1 and A2. MR-detectable metabolites are glycerophosphocholine
(GPC), phosphocholine (PC), and free choline. Abbreviations: ADP, adenosine diphosphate; ATP, adenosine
triphosphate; CDP, cytosine diphosphate; CMP, cytosine monophosphate; CTP, cytosine triphosphate; PPi,
pyrophosphate. Adapted from 24
 In vivo Cellular and Molecular Imaging of Cancer
activity of growth factors particularly in the
activation of the ras-raf-1-MAPK cascade and
protein kinase C pathway.7-9 The regulation
of MCPM can occur through growth factor
stimulation,8,10 cytokines,11 oncogenes,12,13
chemical carcinogens14 or requirements for
eicosanoid production. These eicosanoids
impact on cell motility, invasion, vascular
characteristics and metastatic dissemina-
tion.15-17 The altered MCPM observed in
cancer cells can, therefore, impact upon
several of the phenotypic characteristics of
solid tumors, and may provide targets for
cancer treatment.
The use of transgenic cells and tumor
models has provided unique insights into
the aberrant choline phospholipid meta-
bolism typically observed in cancer cells.
Transgenic interventions that increase the
malignant characteristics of cancer cells are
found to increase phosphocholine or total
choline levels, whereas the reverse is
observed in transgenic interventions that
reduce the malignancy of cells. Mutations
of the ras-oncogene family are frequently
detected in cancers. Ronen et al have studied
NIH-3T3 cells transfected with mutant ras-
oncogene with MRS and detected signi-
ficantly higher PC levels in the ras-transfected
cells compared to the parental cell line.13
In our program we have performed the
following two studies using transgenic
models to further understand choline
phospholipid metabolism.
i. Human cancers that contain a p53
mutation are more aggressive, and
more often fatal. We characterized the
choline phospholipids of HCT116
human colon cancer cells with (p53+/+)
and without p53 (p53-/-). We observed
that loss of p53 function resulted in
increased PC and total choline, which
were consistent with increased
249
malignancy.18 Examples of these data
will be presented in the lecture.
ii. We also studied transgenic breast
cancer cell lines stably transfected with
a metastasis suppressor gene (nm23).
Transfection with nm23 led to
increased glycerophosphocholine (GPC)
and decreased PC compared to vector-
transfected control cells, consistent
with a reduction of the metastatic
phenotype of the nm23 transfected
cells.19 To understand the impact of
nm23 genes on tumor physiology and
metabolism, we performed a 31P NMR
spectroscopic study of tumors formed
in the mammary fat pad of severe
combined immunodeficient (SCID) mice
by MDA-MB-435 human breast
carcinoma cells transfected with cDNA
encoding wild type nm23-H1 protein.
Tumors formed by MDA-MB-435 cells
transfected with the empty vector were
used as controls. As shown in Figure
19.2, the in vivo 31P NMR spectra of
transgene tumors formed by wild type
forms of nm23-H1 (MDA-MB-435-1β)
exhibited a significantly higher amount
of PDE relative to PME levels when
compared with control tumors (MDA-
MB-435-Vβ). Nm23-expressing MDA-
MB-435 cells had significantly lower PC
levels and higher GPC levels compared
to empty vector-transfected or wild
type MDA-MB-435 cells, as determined
by high-resolution MR spectroscopy of
tumor extracts. The effect of nm23
transfection on the attenuation of
metastasis was confirmed by histologi-
cal analysis of lung sections obtained
from tumor bearing animals (Figs 19.3A
to D, Plate 11). These changes in
phospholipid metabolism detected
following nm23 transfection demons-
trate that choline phospholipid meta-
bolism mediated processes are involved
in invasion and metastasis.
250 Biomedical Magnetic Resonance: Proceedings of the International Workshop

Figs 19.2A and B: Representative fully relaxed 31P NMR spectra obtained from (A) an MDA-MB-435-Vβ tumor
(empty vector) and (B) an MDA-MB-435-1β tumor. Peak assignments are (1) 3-APP (2) phosphomonoester
(PME), (3) Inorganic phosphate (Pi), (4) phosphodiester (PDE), (5) phosphocreatine (PCr), (6) γ-nucleoside
triphosphate (NTP), (7) α-NTP (set to -10 ppm), (8) β-NTP. Adapted from 19

Overall, these as well as other data will
be presented to demonstrate that diverse
molecular alterations such as nm23 expres-
sion, p53 expression, and malignant
transformation arrive at a common endpoint
in choline phospholipid metabolism. These
data are evidence of the important role of
choline phospholipids and their metabolites
in cancer, and provide a strong rationale
for targeting enzymes in choline phospho-
lipid metabolism of cancer cells.
Antisense RNA’s20 and ribozymes21 have
been used widely to inhibit gene expression.
The design of these probes is arbitrary to
some extent because of the uncertainty of
how these molecules cause an inhibitory
effect within a cellular environment. In
addition, the expression of antisense RNA’s
or ribozymes does not guarantee an
inhibitory effect. It is possible to circumvent
these problems with the use of small
interfering RNA (siRNA) of 21-23 nucleo-
tides (nt) which mediate effective degrada-
tion of the endogenous complementary
homologous RNA. 22 The availability of
siRNA technology to target specific gene
expression has proved very successful,23 and
provides new opportunities to target genes
 In vivo Cellular and Molecular Imaging of Cancer
in the choline phospholipid as well as other
critical pathways identified by MR methods.
During the course of the lecture examples
will be given of siRNA targeting of the
enzyme choline kinase, which is one of the
enzymes over-expressed in breast cancer
cells,24 and its effect on MR spectra.25,26
These data will demonstrate how MR of
transgenic models can be used to identify a
key target, to image the effectiveness of the
delivery of therapy, and to use imaging to
determine the outcome of treatment.
SOME EXAMPLES OF MR IMAGING
APPLICATIONS TO TRANSGENIC
MODELS
A tumor is a complex organization of cells
with different genotypic as well as
phenotypic characteristics. One of the most
verified and widely accepted concepts about
cancer is that the malignant cell arises from
genetic alterations. However, it is possible,
that several of the subsequent lethal
phenotypic traits of cancer, such as invasion
and metastasis, may arise from the unique
physiological environment of solid tumors
which is frequently characterized by areas
of poor blood flow, hypoxia, high lactate
levels and low extracellular pH (pHe). MR
imaging and spectroscopic imaging of
transgenic tumor models provide unique
opportunities to characterize the dynamics
between vascularization and metabolism and
understand their role in cancer invasion,
metastasis and drug resistance. We have
studied, with combined MRI and MRSI and
optical imaging, a transgenic prostate cancer
model which expresses green fluorescent
protein under hypoxia.
In normal and cancer cells, a critical
response to hypoxia is the induction of the
hypoxia-inducible transcription factor (HIF-
251
1).27 HIF-1 consists of two subunits, HIF-1α
and HIF-1β. While HIF-1β is a common
subunit for several transcription factors,
HIF-1α is unique to HIF-1 and its expression
is highly regulated by cellular oxygen
concentration.28 Under nonhypoxic condi-
tions, HIF-1α is subject to ubiquitination and
proteasomal degradation. Under hypoxic
conditions, ubiquitination of HIF-1α is
dramatically reduced and the activity of its
transcriptional activation domain incre-
ases.27,29 HIF-1 can act as a transcriptional
activator for VEGF, inducible nitric oxide
synthase, heme oxygenase 1, glucose
transporter (GLUT1), and the glycolytic
enzymes aldolase A, enolase 1, lactate
dehydrogenase A and phosphoglycerate
kinase 1. Each of these target genes contains
hypoxia response elements (HREs) which
include one or more HIF-1 binding sites
with the core sequence 5’-RCGTG-3’.30 Thus,
HIF-1 mediates the adaptive response of
cells to hypoxia.
The presence of a HIF-1 binding site is
necessary but not sufficient to constitute a
functional HRE. By placing green fluorescent
protein (GFP) expression under the control
of an HRE, it is possible to indirectly detect
HIF-1 activity in tissues. Recently Shibata et
al31 described a vector where the HRE of
human VEGF-A was isolated and ligated to
the luciferase reporter gene. Luciferase
activity in cells transfected with this vector
increased over 500-fold in response to
hypoxia, reaching levels comparable to that
obtained by a full length CMV promoter
(which results in constitutive overexpression
of luciferase) linked to the luciferase reporter
gene. This vector, with the luciferase
reporter replaced with an enhanced green
fluorescent protein (EGFP) gene (schematic
shown in Figure 19.4, Plate 11), was kindly
provided by Dr J Martin Brown from
Stanford University.
252 Biomedical Magnetic Resonance: Proceedings of the International Workshop
PC-3 prostate cancer cells stably
transfected with HRE-EGFP, under (a)
normoxia and (b) 14 h after treatment with
10 micromolar cobalt chloride, which mimics
hypoxia are shown in (Fig. 19.5A, Plate 11).
A dramatic increase in fluorescence can be
observed in (Fig. 19.5B, Plate 11). Data will
be presented to demonstrate fluorescence
under hypoxic conditions. We have
generated solid tumors derived from PC-3
cells stably transfected with the hypoxia
response element (HRE) of human vascular
endothelial growth factor (VEGF-A) ligated
to the EGFP gene to detect regions of
hypoxia using optical imaging.
We are currently using a multi-para-
metric approach of combined vascular,
spectroscopic and optical imaging to obtain
vascular, metabolic, and hypoxia maps, from
co-localized regions within a tumor. Data
from these studies performed in our
program will be presented to demonstrate
that regions of low vascular volume are
typically hypoxic and also exhibit high
permeability, most likely due to increased
expression of VEGF. Data of combined
vascular and metabolic MR and optical
imaging of EGFP will also be presented to
demonstrate how such an approach can be
used to understand the dynamics of the
relationship between vascularization and
metabolism in tumors.
Another example of the use of transgenic
cancer cells to understand tumor vasculari-
zation is the use of VEGF overexpressing
cancer cell lines grown either in vivo, or
characterized in culture. VEGF-A is a potent
angiogenic and permeability factor and its
expression has been linked to metastasis in
clinical studies.32-34
VEGF-A is a multifunctional cytokine
that is expressed as several isoforms
consisting of polypeptides of different sizes
(121, 145, 165, 183, 189 and 206 amino acid
residues) which are formed from the same
gene by alternative splicing. These isoforms
have distinct but overlapping functions in
angiogenesis. Neeman and colleagues 35 have
used C6-pTET-VEGF 165 tumors in which
over-expression of VEGF165 was modulated
in vivo by addition or withdrawal of
tetracycline in the drinking water to study
the effect of sustained expression of VEGF
on lymphatic drain. VEGF over expressing
tumors showed high blood volume fraction
and high vessel permeability as detected by
contrast enhanced MRI using biotin-BSA-
GdDTPA. The rate of change in contrast
enhancement was highest at early time
points within the tumor, and was detected
progressively at later time points further
from the tumor, in accordance with radial
outward convection of the contrast agent.
We have created stable clones of human
breast and prostate cancer cells over-
expressing VEGF 165. Using solid tumors
derived from these transgenic cells, we have
investigated the effect of VEGF overexpres-
sion on vascularization as well as the
relationship between vascular and metabolic
parameters in a human prostate cancer
model, using combined MRI and MRSI.
VEGF 165 overexpression significantly
increased vascular volume and permeability.
In (Figure 19.6, Plate 12) vascular volume is
displayed in red and permeability in green.
The areas of yellow represent regions with
high vascular volume as well as high
permeability. In previous studies we have
routinely observed that regions of high
vascular volume were not very permeable,
and that regions of high permeability were
usually associated with dead or dying
regions of the tumor.36 The implication of
 In vivo Cellular and Molecular Imaging of Cancer 253

these observations is that macromolecular

therapeutic agents will not leak out easily
in well vascularized viable regions, but will
leak out in dead or doomed to die regions
of the tumor. In the VEGF165 overexpressing
tumors, however, regions of high vascular
volume were also highly permeable.
Data from the JHU ICMIC Program will
be presented to demonstrate that VEGF
overexpressing tumors exhibited patterns of
co-localized vascular volume, permeability,
total choline and lipid which were distinct
from control tumors. These data suggest
that VEGF overexpression can influence the
choline and lipid metabolism of tumors. (A)
We are currently also characterizing the
invasiveness of the VEGF overexpressing
transgenic cells under conditions of hypoxia
and normoxia in our MR compatible cancer
cell invasion assay. We have developed and
characterized an invasion assay system to
dynamically track the invasion of cancer cells
and simultaneously characterize oxygen
tensions, and physiological and metabolic
parameters.37 We can carefully regulate
oxygen tensions to below 1 percent to
evaluate the impact of the hypoxic tumor
environment on cancer cell invasion.
Perfluorotripropylamine (FTPA) doped
alginate beads are placed within the cells in (B)
the MR tube to directly measure oxygen Figs 19.7A and B: Expanded 1H MR image of sample
tensions in the sample. Typical invasion and showing the Matrigel layer for (A) DU-145 and (B)
metabolic data obtained with this assay from MatLyLu cells at comparable time points. MR images
a highly invasive (MatLyLu) and a less were obtained with TR 1s, TE 30 ms, FOV 40 mm,
slice thickness 2 mm, and an in-plane resolution of
invasive (DU-145) prostate cancer cell line
0.078 mm. Representative 1H MR spectra are from
are shown in Figures 19.7A and B.37 We 0.31 mm thick localized slices from within the sample.
routinely obtain localized proton spectra Adapted from 37
with a slice thickness of 0.31 mm.
Data from the JHU ICMIC Progam will metastasis detected in patients with cancers
be presented to demonstrate that under secreting high levels of VEGF.32,33
hypoxia, VEGF overexpression increased It is also possible to use MRI to detect
invasion. The increased invasion detected gene expression as well as receptor
may explain, in part, increased rates of expression in transgenic models. In one of
254 Biomedical Magnetic Resonance: Proceedings of the International Workshop
the first examples of the use of MRI to detect
gene expression, Weissleder and colleagues
used 9L glioma cells genetically engineered
to express the modified transferrin receptor,
ETR, with a knocked-down negative
feedback regulation domain. Tumors
derived from these cells exhibited significant
shortening of T2* following administration
of transferrin-monocrystalline iron oxide
nanoparticles.38
Detecting the expression of a specific
reporter is another example of the appli-
cation of MR imaging to trangenic cancer
cells and tumor models. Data will be
presented to demonstrate an MR based
approach developed by Artemov et al39 to
detect HER-2/neu receptor expression in a
transgenic HER-2/neu overexpressing tumor
model. A two-step labeling procedure
permits separation of the targeted contrast
agent complex into two components with
relatively low molecular weight (160 kDa
for monoclonal antibody (mAb), and 70 kDa
for avidin), which improves delivery.
Receptors are prelabeled with biotinylated
anti-HER-2/neu mAb, and probed with a
GdDTPA-avidin conjugate 12 h later. A
maximum labeling efficiency of 50 gado-
liniums per receptor was estimated, from
an average of 12.5 Gd3+ per avidin and 4
biotins per mAb. Positive contrast in HER-
2/neu overexpressing tumor xenografts was
detected in T1-weighted MR images using
this approach.39 Such an approach can be
extended to image gene expression with MRI
which, combined with the functional imaging
capabilities of MR methods, can provide
insight into the functional effects of a gene
of interest.
CONCLUSION
In summary, recent advances in the develop-
ment of targeted contrast agents have
significantly increased the versatility of MR
in studying transgenic models of cancer.
Although MR techniques provide a wealth
of structural and functional information,
sensitivity remains an issue. Developing
strategies to improve the sensitivity of
detection of molecular targets in MR
molecular imaging of cancer is, therefore,
critically important. One of the most exciting
aspects of MR is the ability to perform multi-
parametric imaging. Multi-parametric
imaging in combination with the ability to
visualize gene and receptor expression of
transgenic models of cancer cells and tumor
models will provide major advances in our
understanding of cancer.
ACKNOWLEDGMENTS
Work from the authors program was supported
through funding by NIH grants 2R01 CA73850, 1R01
CA90471, 2R01 CA82337 and P50 CA 103175.
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INTRODUCTION
Body MR imaging applications are growing
at a rapid pace. Body MR imaging continues
to be one of the largest areas of growth in
terms of the percentage of examinations
performed. Recently, outstanding advances
in body MR methods have resulted in an
increase in the use of MR for patients with
a variety of suspected diseases. For example,
new contrast agents are now available for
use in hepatic imaging, and advanced clinical
trials for intravascular contrast agents are
taking place. Faster pulse sequences that
result in improved image quality with higher
resolution are available; these have helped
to control problems of motion in the chest
and abdomen. Entirely new applications that
barely existed few years ago, such as MR
cholangiopancreatography (MRCP), adrenal,
and breast MR imaging, are widely reques-
ted by referring physicians.
The complexity of these newer appli-
cations remains a challenge for both radio-
logists and technologists. This article
presents a summary of the most widely used
applications in body MR imaging, but is by
no means a complete text on body MR
imaging.
MR IMAGING OF LIVER
The advantages of MR imaging in the
investigation of liver disease are well
Body MR Imaging 257
20
Body MR Imaging
Raju Sharma, Shiva Gamanagati
documented. The lack of ionizing radiation
of MR imaging and the safety of gadolinium
chelates are important considerations.
Moreover, recent developments in MR
imaging, including fast scanning techniques
and new MR imaging contrast agents, enable
improved detection and characterization of
many liver lesions so that a definitive dia-
gnosis can be made noninvasively. Lesion
characterization and determination of the
extent of disease often have important
prognostic and therapeutic implications.
Liver Imaging Protocol
The routine protocol for screening MR
imaging of the liver consists of axial T1-
weighted (T 1 W) and T2 -weighted (T2 W)
sequences. Depending on the clinical
question, acquisitions in the coronal or
sagittal plane should be added; for example,
if there is a question of a sub-diaphragmatic
process, these additional planes may be
more helpful than the axial plane.1-4
T1-weighted sequences: Breath-hold spoiled
GRE (SGE) is widely used as a T 1 W
sequence for evaluating the liver. This
sequence allows imaging of the entire liver
during a single breath hold. Other advan-
tages include good signal-to-noise ratio,
strong T1 weighting, and minimal magnetic
susceptibility effects. Three-dimensional (3D)
258 Biomedical Magnetic Resonance: Proceedings of the International Workshop
GRE imaging is being investigated as a T1-
weighted sequence to provide accurate 3D
segmental and vascular anatomy for surgical
planning, including assessment prior to liver
resection and transplantation.
T2-Weighted sequences: Breathing-averaged T2-
weighted conventional or fast spin-echo,
with optional fat suppression, is the most
commonly used T2-weighted sequence. If
the respiratory pattern is irregular, turn off
respiratory triggering and add fat satura-
tion. Phase correction is helpful. Flow
compensation is also helpful but reduces the
number of slices that can be acquired in a
given respiratory interval. Fast spin-echo
(FSE) techniques are compatible with breath-
hold acquisitions by using long echo train
lengths of approximately 20 and reduced
matrix array size. By using a high band-
width acquisition (e. g., 32 kHz), acquisition
times are further reduced. Using this
method, approximately eight axial sections
can be acquired throughout the liver in 20
to 30 seconds. Fat suppression improves the
quality of both conventional and FSE T2-
weighted sequences. There are two reasons
for this. First, moving fat during respiration
causes phase artifacts that are propagated
through the entire image. By using fat
suppression, the signal of the moving
anterior abdominal wall is suppressed, and
thus it reduces respiratory artifacts. Second,
suppression of the high signal fat (especially
with FSE or turbo spin-echo) allows for a
greater dynamic range of signal intensities
to be displayed in the images. This increases
conspicuity of focal lesions in the liver.
If FSE T2 is of poor quality, use axial
single-shot FSE (SSFSE) or Half-Fourier
acquisition single-shot turbo spin-echo
(HASTE) with breath holding. HASTE has
been shown to be effective for characterizing
cysts and hemangiomas with long T2 times.
HASTE is not effective for tumor detection
due to poor contrast resolution for lesions
with moderate T2 times. It should not be
used as the primary sequence for liver tumor
detection.
In-phase, out-of phase sequence: This is useful to
look for fatty liver, focal fat or focal sparing.
Dynamic gadolinium-enhanced sequence: This
can be performed as either in-phase FLASH
or as out-of-phase FLASH with the addition
of fat saturation to eliminate artifact at fat-
organ boundaries. Repeat the scan multiple
times with breath holding as follows:
• Pre-injection.
• Arterial phase (inject gadolinium, flush
immediately with 30 cc saline and start
scan halfway through the flush during
breath holding).
• Portal venous phase.
• Delayed (If cirrhotic liver, then a delay
of 2 to 3 minutes is adequate; if there is
a lesion that might be hemangioma or
cholangio CA then repeat every 2 to 3
minutes out to 10 minutes).
Use of contrast media in MRI of the liver:
The liver may be assessed using para-
magnetic contrast agents such as gadolinium
agents, which increase the intensity of
normal hepatocytes or superparamagnetic
agents such as superparamagnetic oxides,
which decrease the intensity and appears
dark.
To be effective contrast agents should
exhibit selective or differential accumulation
within the different tissue compartments
being assessed.
Extracellular gadolinium chelates: Gadolinium
chelates are non-specific extracellular
contrast agents that rapidly equilibrate
between the intravascular and extracellular
spaces after injection. Gadolinium is

delivered in the liver through the hepatic
arteries and then by the portal vein, and
distributes fairly, rapidly in normal and
abnormal tissues. This fast distribution
requires rapid imaging in order to maximize
differential enhancement between normal
liver parenchyma and lesions. On high field
scanners, using 2D or 3D SPGR sequences,
the whole liver could be assessed in one
breath hold. Increased SNR is achieved
using phased array coil, whilst CNR is
improved using fat suppression.5,6
The portal venous phase images are
obtained approximately 60 seconds after the
injection of contrast material. During this
time, the hepatic parenchyma returns a high
signal, because the portal vein supplies 75
to 80 percent of the blood to the liver. This
phase will decrease conspicuity of
hypervascular lesions as both lesion and
normal tissues are filled with contrast. As
most liver tumors are hypovascular relative
to the liver parenchyma, they will be
visualized as hypointense lesions relative to
the enhancing liver on the venous phase.
Delayed or equilibrium phases are
obtained 3 to 5 minutes after the injection
of contrast media. Most liver lesions become
less conspicuous on these images, but some
tumors with interstitial space such as
cholangiocarcinoma will accumulate more
contrast material than the liver and return
a high signal.
Liver-specific Contrast Agents
These are divided into hepatocyte-selective
and Kupffer’s cell-selective contrast agents.
The former encompass the chelated com-
pounds Mn-DPDP, Gd-BOPTA and Gd-EOB,
whereas the latter consist of the group of
superparamagnetic iron oxide particles.7-9
Body MR Imaging 259
The hepatocyte-specific contrast agents
are ionic metal chelates with a relatively
pronounced hydrophilicity, a weak protein
binding and a residual capacity of lipophi-
licity. The gadolinium formulations, Gd-
BOPTA and Gd-EOB-DTPA, enter the
hepatocytes by way of the adenosine tripho-
sphate (ATP) dependent, membrane bound,
multispecific organic anion transporters
located in the sinusoidal and canalicular side
of the hepatocytes. Both are cleared rapidly
from the plasma with a varying degree of
hepatobiliary elimination. Mn-DPDP is a
manganese derivative and demonstrates a
more complicated pharmacokinetic path-
way. Its hepatocellular uptake involves a
complex process of dissociation, transmetal-
lization, selective manganese uptake and
elimination. On the basis of the paramag-
netic properties of gadolinium and
manganese ions, the above mentioned
hepatobiliary contrast agents enhance the
T1 signal.
The superparamagnetic iron oxide (SPIO)
consists of nanoparticles typically composed
of an iron core of 3 to 5 nm in size. The
core is covered with a dextran that increases
the particle size between 50 nm and
150 nm. After IV administration the particles
are cleared from the plasma by the reticulo-
endothelial system of the liver (80%) and
spleen (12%). The particles are eliminated
in the lymph nodes and bone marrow. The
plasma half life of the nanoparticles is 8 to
10 minutes. Nevertheless the superparamag-
netic effects last for several days, providing
an imaging period of several hours after
administration. The super-paramagnetic
properties of the iron core are responsible
for a significant disturbance of the local
magnetic field around the particles resulting
in a pronounced shortening of both T2 and
260 Biomedical Magnetic Resonance: Proceedings of the International Workshop
T 1 relaxivity. The principle of contrast
enhancement is based on the presence of
Kupffer’s cells within the normal hepatic
parenchyma, but these cells tend to be
absent in neoplastic lesions of the liver. Thus
SPIO particles, lead to a decrease in signal
intensity in tissue containing Kupffer’s cells,
whereas a relative signal enhancement can
be observed in tissues devoid of Kupffer’s
cells such as metastases.
Although reticuloendothelial system and
hepatocyte selective contrast agents provide
additional functional imaging, they are still
non-specific because even malignant hepa-
tocytes may still possess functioning anion
transporters and hepatocellular adenomas
may contain Kupffer’s cells.
Diffusion Imaging
Diffusion imaging of the brain is routinely
used for early detection and characterization
of stroke. The clinical utility of diffusion
imaging in the abdomen is not yet clear,
although potential applications in the
characterization of tumors and infiltrative
diseases exist. Differences in the mean
apparent diffusion coefficients have been
demonstrated for hepatocellular carcinoma,
metastasis, and hemangiomas; these were
all higher than that of normal liver. Single-
shot echo-planar imaging has been evaluated
with and without a small diffusion-sensi-
tizing gradient, and it has been compared
with breath-hold T2-weighted fast spin-echo
sequences. This study demonstrated better
lesion-to-liver signal intensity with echo-
planar imaging than with fast spin-echo
imaging; the use of the diffusion-sensitizing
gradient caused suppression of high signal
intensity from vessels and resulted in better
conspicuity of small lesions. This sequence
depicted more metastatic deposits, compared
with T 2 -weighted fast spin-echo sequ-
ences.10-12
Focal Lesions
Benign Liver Lesions
Incidental benign liver lesions are very
common and the diagnosis is possible in
the majority of cases using MR imaging,
without the need for surgery or biopsy13.
Hepatic Cysts
Simple cysts are derived from biliary
endothelium. They contain thin serous fluid,
not bile, and are lined by a single layer of
epithelium. They occur in 5 to 14 percent of
the population.13 Simple cysts are usually
solitary to few in number, but innumerable
cysts may occur in autosomal-dominant or
recessive polycystic disease. Like other cysts,
the criteria for diagnosis of hepatic cyst
include sharp margins with no definable
walls. On MR, cysts show internal signals
on T1 and T2 sequences that follow cerebro-
spinal fluid (CSF) (Figs 20.1A and B). After
contrast administration, cysts do not show
enhancement. The differential diagnosis of
a hepatic cyst includes cystic metastases such
as leiomyosarcoma and hemangioma. Note
that so-called ‘‘cystic’’ metastases are usually
pseudocystic and tend to be internally
inhomogeneous.
Hemangioma
Hemangioma is the most common benign
hepatic neoplasm, occurring in 5 to 20
percent of the population. Most lesions grow
slowly and are uncommonly diagnosed in
children. Hemangiomas consist of large
endothelial-lined vascular spaces separated
by fibrous septa. They can range in size
from a few millimeters to more than 20 cm.
 Body MR Imaging 261

A B
Figs. 20.1A and B: Polycystic liver disease
A. Axial T1W image showing multiple lowsignal intensity focal lesions. Note: largest lesion in left lobe is
isointense
B. Axial T2W image showing that focal lesions are hyperintense. Note: largest lesion in left lobe is showing low
signal intensity area inside suggestive of bleed inside the cyst

Although usually solitary, multiple heman- dynamic breath-hold acquisition over 5 to

giomas are commonly encountered. On MR,
hemangiomas have a characteristic appea-
rance of a 1 to 3 cm lesion that is extremely
bright (‘‘light bulb’’) on T2 images.
Lesions larger than about 3 cm tend to
be less homogeneously bright, due to
internal fibrous septa or scarring. The tumor
margin is well defined, and may be
somewhat lobulated. No peritumoral edema
is present. Long TE images (TE 120–180
milliseconds) will demonstrate that heman-
giomas have a signal similar to CSF, whereas
most malignant tumors will not be as bright
as CSF. There are two differential diagnostic
considerations for hemangioma: a cyst and
a hypervascular hepatic metastasis.
Differentiation of hemangioma from a cyst
is unimportant because both are benign.
Metastatic leiomyosarcoma in the liver may
frequently (75%) have an appearance that is
similar to hemangioma. Because of this
overlap, characteristics at contrast enhanced
MR have been used to further differentiate
hemangiomas from metastases. Suspected
hemangiomas should be evaluated using a
15 minutes, in a manner similar to that per-
formed for dynamic CT evaluation.
Following rapid contrast injection, a GRE
T1-weighted sequence is acquired in 20 to
30 seconds (FMPSPGR, 2D FLASH). This is
repeated once per minute, until the lesion
has filled in completely or nearly completely,
or until a diagnosis can be rendered. Using
this protocol, hemangiomas demonstrate
hyperintense peripheral nodular enhance-
ment, with either complete or partial filling
in 5 to 15 minutes. The specificity of these
criteria approaches 100 percent. Three
enhancement patterns were reported on
serial gadolinium-enhanced spoiled GRE
images in a multicenter study describing the
MR imaging findings of small (<1.5 cm),
medium (1.5 to 5.0 cm), and large (>5.0 cm)
hemangiomas (1) immediate homoge-neous
enhancement after contrast (Type 1), (2)
peripheral nodular enhancement with
centripetal progression to homogeneity
(Type 2) (Figs 20.2 A to D), and (Figs 20.3A
to D) peripheral nodular enhancement with
centripetal progression and a persistent
262 Biomedical Magnetic Resonance: Proceedings of the International Workshop

A B

C D
Figs 20.2A and D: Hemangioma of liver
A. Axial T1W image showing low signal intensity lesion in right lobe
B. On axial T2W image, lesion is uniformly hyperintense
C. Dynamic contrast study, arterial phase image showing, peripheral nodular enhancement of the lesion
D. Delayed phase image showing, centripetal filling in of the lesion

central hypointense region (Type 3). The
Type 1 enhancement pattern was observed
only in small tumors. Type 2 and Type 3
enhancement patterns were observed in
tumors of all sizes.13-15
Focal Nodular Hyperplasia (FNH)
FNH may occur in 2 to 5 percent of the
population. It is rarely symptomatic,
although lesions may grow to more than 10
cm in diameter. Lesions are solitary in about
70 percent of patients. Multiple lesions of
FNH have been described in patients with
certain brain neoplasms and vascular
malformations of various organs.16 FNH is
a nonencapsulated firm nodule of normal
hepatocytes with a distinct central scar and
thin radiating fibrous septa containing
 Body MR Imaging 263

A B

C D
Figs 20.3A to D: Hepatocellular carcinoma of liver
A. Axial T1W (FLASH) image showing, isointense focal lesion within the caudate lobe in background of
cirrhotic liver
B. On T2W image, focal lesion is isointense to mildly hyperintense to liver parenchyma
C. Arterial phase image of dynamic study showing, early uniform enhancement of the lesion. Note:
inhomogeneous enhancement of liver parenchyma
D. Venous phase image showing, contrast wash out and peripheral well defined capsule

Kupffer’s cells and primitive bile ductules.
Intratumoral calcification, fat, hemorrhage,
and necrosis are extremely rare. FNH
should be differentiated from adenoma,
because the two lesions occur in a similar
patient population. An additional important
differential consideration is fibrolamellar
HCC. On MR images, FNH is typically
isointense to liver on T1 images, and slightly
hyperintense on T2 images. On T1 images,
an internal low signal area that corresponds
to a central scar is present in more than 50
percent of cases. The scar is usually
hyperintense to liver on T2 images, but may
264 Biomedical Magnetic Resonance: Proceedings of the International Workshop
be hypointense. A central scar, best seen on
T 1 images, is highly suggestive of the
diagnosis. Other than the central scar, the
lesions are homogeneous on both T1 and T2
images. FNH lesions are highly vascular.
Unlike the central scars in hemangioma, the
central scar in FNH is vascular and is
surrounded by collagenous fibers. Following
contrast administration, because of their
prominent vascularity, FNH lesions tend to
enhance early, and may be isointense to liver
as early as 1 to 2 minutes after contrast
injection, except for the central scar.
FNH can be confused with fibrolamellar
hepatocellular carcinoma (HCC). Fibro-
lamellar HCC is a rare form of HCC seen in
young patients without underlying liver
disease. Fibrolamellar HCC, which has a
better prognosis than conventional HCC, is
typically a large mass with a central scar
that is low signal intensity on T2W image
(T2WI). After gadolinium administration,
there is intense heterogeneous enhancement
on the arterial phase and the central scar
may enhance on the delayed images. If there
is any question that a lesion may be
fibrolamellar HCC, biopsy is mandatory.17,18
Hepatocellular Adenoma
Adenoma is a rare hepatic neoplasm that
occurs almost exclusively in the liver that
has abnormal metabolism due to exogenous
steroids or, less commonly, congenital
abnormalities of carbohydrate metabolism.
Over 90 percent of cases occur in women of
reproductive age, and lesions may regress
with withdrawal of oral contraceptives or
other steroids. Adenomas are composed of
cords of hepatocytes that occasionally form
bile. Adenomas lack bile ductules, portal
tracts, and hepatic veins, which result in
necrosis and hemorrhage. Adenomas are
sharply defined masses with smooth
borders. Unlike FNH, adenomas are usually
heterogeneous due to the presence of
hemorrhage, necrosis, or fat. Hepatic
adenomas may infrequently under-go
malignant transformation.19 In addition, as
they enlarge, they may be the source of
spontaneous intra-abdominal hemorrhage.
For these reasons, surgeons will typically
resect solitary liver adenomas. The MR
appearance of adenomas is somewhat
variable, but the typical tumor demonstrates
slight increased signal on T 1 images
(probably due to fat content) and moderate
increased signal on T2 images. As opposed
to FNH, they are more frequently hetero-
geneous in their internal signal. This is, in
part, due to the tendency of this tumor to
undergo spontaneous internal hemorrhage
with areas of calcification and fibrosis.
Biliary Cystadenoma
This tumor presents typically as a sympto-
matic large hepatic mass characterized by
large cystic spaces surrounded and separa-
ted by thickened, nodular, enhancing walls.
Although imaging features might overlap
with a pyogenic or parasitic abscess, this is
usually not a clinical dilemma. MR imaging
studies clearly demonstrate cystadenoma as
an ‘‘aggressive’’ and complex neoplasm.20
Biliary Hamartoma
Biliary hamartomas are benign biliary
malformations. They are estimated to be
present in about 3 percent of patients. They
can be solitary or multiple, but their size is
almost invariably less than 1 cm in diameter.
On MR imaging, biliary hamartomas are
always small in size (usually 1 cm) and well
defined. They show low signal on T 1 -
weighted images and high signal on

T2-weighted images, reflecting their high
fluid content. Although this appearance
resembles that of simple cysts, biliary
hamartomas show a thin rim of enhance-
ment. There is no central enhancement,
however, on immediate and delayed post-
gadolinium images. This thin enhancing rim
represents compressed hepatic parenchyma
surrounding the lesion. It may be difficult
to distinguish biliary hamartomas from
metastatic lesions because of the presence
of this rim enhancement. Rim enhancement
in biliary hamartomas does not spread
centrally, whereas enhancement in meta-
stases most often spreads centrally.21
MRI Characteristics of Common
Malignant Lesions
Metastases
The most common cause of malignant liver
lesions is metastatic disease; they out
number primary hepatic neoplasms. The
most common primary sites that metastasize
to the liver are colon, stomach, pancreas,
breast, and lung. The MRI characteristics
vary with each tumor, but are generally
classified as hypervascular or hypo-
vascular.22,23
Hypervascular metastases are usually
from renal cell carcinoma, pancreatic islet
cell tumors, breast carcinoma, melanoma,
thyroid carcinoma, sarcomas, chorio-
carcinoma, and carcinoid tumors. These
lesions draw their blood supply from the
hepatic artery, which is reflected in the
enhancement pattern seen on dynamic gado-
linium imaging. Hypervascular metastases
generally have a high signal intensity on
T 2 -weighted images. Dynamic serial
gadolinium-enhanced sequences, especially
those from the hepatic arterial-dominant
phase, are particularly useful in the detection
Body MR Imaging 265
and characterization of these metastases.
Frequently, centripetal contrast enhancement
is observed, as well as irregular or peri-
pheral washout. These lesions may show
marked peripheral rim enhancement during
the arterial phase with delayed peripheral
or central washout in the portal venous and
equilibrium phases.
Hypovascular hepatic metastases usually
arise from colon carcinoma, pancreatic
carcinoma, transitional cell carcinoma, and
lung carcinoma. These lesions derive little
blood supply from the hepatic artery or
portal vein and are best imaged in the portal
venous phase of gadolinium-enhanced
imaging. These lesions will have low signal
compared to liver parenchyma. Hypovas-
cular metastases usually have a low signal
intensity relative to liver on T1- weighted
images and are nearly isointense on T2-
weighted images.
Near Isovascular Metastases
Near isovascularity refers to lesion
enhancement that is comparable to the liver
on early-and late-phase gadolinium-
enhanced images. This pattern of metastases
is observed when lesions are poorly
depicted on gadolinium-enhanced images
but may be well demonstrated on noncon-
trast T1 , non-contrast T2 , or both. Liver
metastases from primary tumors of the
colon, thyroid gland, and endometrium may
demonstrate this type of enhancement
pattern, and they are observed most often
in the setting of chemotherapy treated
lesions.24
Metastases: Hyperintense on
T1- weighted Images
Despite the fact that high signal intensity
on T1-weighted images is not specific for
266 Biomedical Magnetic Resonance: Proceedings of the International Workshop
malignancy, this appearance can be seen in
a number of metastases and is presumably
related to the internal content of a para-
magnetic substance such as melanin and
extracellular methemoglobin, or high protein
content. This appearance has been observed
in metastases from melanoma, colon cancer,
ovarian cancer, multiple myeloma, and
pancreatic mucinous cystic tumor.25,26
Fatty Liver with Metastases
MR imaging is very useful in the evaluation
of metastases in the setting of fatty liver.
In-phase T1-weighted images tend to show
good conspicuity of low-signal liver lesions
against a background of liver that may be
slightly higher in signal than normal because
of the presence of fat. On out-of-phase
images, the liver parenchyma drops in signal
and the metastases do not, which provides
a different signal relationship with higher-
signal lesions against a background of low-
signal liver.27
Important Differential Diagnosis
Metastases Versus Hemangiomas
The differentiation of metastases from
hemangiomas has been extensively discus-
sed in the literature. On MR images,
metastases and hemangiomas generally are
distinguished on the basis of lesion morpho-
logy, enhancement, and T2 signal. Small
hypervascular metastases (<1.5 cm) and small
hemangiomas (type I enhancement) show
intense and homogeneous enhancement on
hepatic arterial-dominant– phase images.
However, hemangiomas tend to retain
contrast or fade to isointensity with liver
parenchyma, whereas hypervascular meta-
stases tend to fade to a signal lower than
that of liver. In addition, small heman-
giomas should be almost invariably of high
signal on T 2 -weighted images, whereas
small metastases often are near isointense
on T2-weighted images.
Hepatocellular Carcinoma
Hepatocellular carcinoma (HCC) is the most
common primary liver neoplasm. HCC
usually occurs in the settings of chronic liver
disease and cirrhosis, which include hepatitis
B and C, aflatoxins, alcoholism, and hemo-
chromatosis. Alpha fetoprotein is elevated
in 80 percent of cases. The liver responds to
chronic insults and inflammation by forming
regenerative nodules. Some of these regene-
rative nodules become dysplastic and evolve
into HCC. This sequence of dedifferation
of regenerative nodules to dysplastic
nodules to HCC has important imaging
implications. HCCs present as a solitary liver
mass in 50 percent of cases, multifocal liver
masses in 40 percent of cases, and diffuse
infiltration in 10 percent of cases. They have
a propensity for vascular invasion, which is
seen in 30 to 50 percent of cases. The most
important factors affecting long-term
survival are the presence of vascular
invasion, the number of tumors, and size
greater than 5 cm. MRI has become the
modality of choice to characterize the
number and size of lesions and to assess
vascular invasion. The T 1 and T2 signal
intensity of HCC is highly variable. On MR,
the T 1 appearance of HCC ranges from
hypointense to slightly hyperintense,
depending on fat content, copper deposition
within the tumor, and the degree of
differentiation. On T2 images, most HCC
demonstrate increased signal compared to
the surrounding liver, although the tumors
tend to be inhomogeneous. The pattern and
degree of enhancement with gadolinium is
directly related to tumor differentiation,
histologic subtype, and degree of vascularity.

Well-differentiated HCC has minimal
arterial enhancement that washes out on
portal venous images. As stated earlier, well-
differentiated HCC contains Kupffer cells
and will take up ferumoxides. Moderate and
poorly differentiated HCCs show marked
hypervascularity and therefore enhance on
the arterial phase. In a cirrhotic liver, any
abnormal enhancement should be
considered suspicious for HCC. HCCs are
highly vascular lesions and may infrequently
present with spontaneous hemorrhage. Most
commonly, lesions are well circumscribed
with the appearance of a well-defined
‘‘capsule’’27-29 (Figs 20.3A to D).
Fibrolamellar HCC is a distinctive type of HCC
that occurs in younger patients (mean age
of 20 years). Although it is a malignant
lesion, the prognosis is better than that for
typical HCC, with 25 percent of patients
having resectable lesions. Alpha-fetoprotein
levels are usually not elevated. Fibrolamellar
HCC is typically a well circumscribed lesion
that is hypointense on T1 images and hyper-
intense on T2 images. A central scar may be
present. Central calcifications are present in
one third of lesions. The differential
diagnosis includes adenoma or FNH.30
Cholangiocarcinoma
Cholangiocarcinoma is a malignant tumor
arising from bile duct epithelium. Risk
factors include Clonorchis sinensis infection,
prior exposure to Thorotrast, primary
sclerosing cholangitis, and Carolis disease.
The prognosis is poor and patients typically
die within months of diagnosis. Although
not technically considered an intraheptic
neoplasm, the most common type of cholan-
giocarcinoma is hilar cholangiocarcinoma or
Klatskin tumor. These tumors may be small
and obstruct the biliary tree. As the tumor
Body MR Imaging 267
grows, the hilum of the liver may be
involved, resulting in biliary obstruction.
The most prominent feature with central
cholangiocarcinoma on MR is usually
intrahepatic biliary duct dilatation. Morpho-
logic changes may occur late in the disease
process, with atrophy of the left lobe of the
liver compared with the right lobe. Dynamic
gadolinium-enhanced imaging demonstrates
a characteristic pattern of slow, gradual
enhancement best seen on the equilibrium
phase. Intrahepatic cholangiocarcinoma is a
rare primary malignant liver neoplasm.
These lesions usually present as a large
solitary mass without biliary obstruction.
Dynamic gadolinium imaging demonstrates
peripheral enhancement on the arterial
phase, and incomplete central enhancement
on the delayed images. Ten percent of
patients with sclerosing cholangitis may have
associated cholangiocarcinoma.31
Diffuse Liver Diseases
Fatty Liver
Fatty liver has many causes, including
obesity, diabetes mellitus, malnutrition, or
exposure to ethanol or other chemical toxins.
In fatty liver, fat accumulates in hepatocytes,
either focally or diffusely. On CT or ultra-
sound, focal fatty change can be difficult to
differentiate from a neoplasm. With MRI,
the confidence of diagnosing fatty liver is
greatly improved by using chemical shift
techniques. The most useful technique
involves comparison of in-phase (water +
fat) and opposed-phase (water – fat) images.
Fatty liver, on the other hand, will have
lower signal intensity on opposed-phase
images because of cancellation of water and
fat signals within voxels. Fat-suppressed
images can also be helpful in cases of
moderate to severe fatty infiltration. Fatty
268 Biomedical Magnetic Resonance: Proceedings of the International Workshop
liver will have lower signal intensity with
fat suppression, although the drop is not as
much as on opposed phase images. In the
setting of diffuse fatty infiltration, neoplasms
are more difficult to detect by CT or
ultrasound. On the contrary, a diffuse fatty
liver may help MRI detect a tumor. On non–
fat-saturated T1WI, the fatty liver may have
higher intensity than a normal liver, which
increases the contrast relative to the low
signal intensity of tumors. On fat-saturated
T2WI, the fatty liver has lower intensity than
a normal liver. Again, the contrast between
the liver and high intensity tumors
increases32-34.
Iron Overload
Iron accumulation in liver is seen in hemo-
chromatosis, transfusion overload, hemolytic
anemias, and cirrhosis. Intracellular iron is
usually superparamagnetic. It reduces T2
and T2* relaxation times, and results in low
signal intensity on both T 2WI and T 2 *-
weighted images (T2*WI). This makes MRI
a more sensitive and specific method for
diagnosing iron overload; gradient recalled
echo (GRE) T2*WI have greatest sensitivity.
Normally, liver has higher signal intensity
than skeletal muscle on both T1WI and and
T2WI.
Hemochromatosis is an autosomal recessive
disorder with a homozygous prevalence of
0.25 to 0.50 percent. In hemochromatosis,
iron overload results from long-term
increased iron absorption through the
intestine. Reticuloendothelial (RE) function
may be deficient. Iron accumulates in liver,
pancreas, and heart, as well as the paren-
chyma of other organs. In liver, the iron is
incorporated preferentially into the hepa-
tocytes rather than Kupffer cells of the RE
system.35,36
Hemochromatosis is associated with
increased incidence of hepatocellular
carcinoma, diabetes mellitus, and myocardial
dysfunction. Magnetic resonance imaging
detects low signal intensity on GRE T2*WI
as evidence of excessive hepatic iron in
preclinical hemochromatosis without
cirrhosis, when treatment is crucial before
irreversible damage occurs. As the disease
progresses, low T2* signal intensity can be
seen in the pancreas and heart, but normal
signal of the spleen is preserved. Magnetic
resonance imaging can be used longitudi-
nally to monitor disease progress and to
evaluate response to treatment. Liver iron
concentration can be measured by calcu-
lating T2 relaxation time or intensity ratios.
In transfusional iron overload, iron deposits in
RE cells of the liver, spleen, and bone
marrow. In contradistinction to hemo-
chromatosis, iron overload in RE is of little
clinical significance. Magnetic resonance
imaging shows low signal intensity of liver,
spleen, and bone marrow on GRE T2*WI,
sparing the heart and pancreas. This finding,
as well as a clinical history of multiple
transfusion, helps to differentiate transfusion
overload from primary hemochromatosis.
Paroxysmal nocturnal hemoglobinuria, due to
renal filtration and tubular absorption of
free hemoglobin, they may have renal
cortical low signal, which is independent of
transfusion. Reduced signal intensity of the
renal cortex is a characteristic finding in
patients with intravascular hemolysis.
Cirrhosis
Cirrhosis is a pathologically-defined entity
characterized by irreversible chronic injury
of the hepatic parenchyma, extensive
fibrosis, and regenerative nodules. The
leading cause of cirrhosis has been alcohol
abuse, followed by chronic viral hepatitis,
hemochromatosis, autoimmune chronic

hepatitis, primary biliary sclerosis, drug
toxicity, etc. However, the incidence of
hepatitis C has increased dramatically in
recent years.
Magnetic resonance imaging provides
comprehensive evaluation of a cirrhotic liver,
including diagnosis, assessment of disease
progression, as well as detection of dys-
plastic nodules and hepatocellular carci-
noma. The findings in cirrhosis are liver
surface nodularity, regenerative nodules,
change of hepatic morphology, fatty
changes, and iron deposition, as well as
ascites, splenomegaly, varices, and colla-
terals. These signs are usually seen in
advanced cirrhosis. The natural course of
cirrhosis progresses from compensated stage
(Child’s class A) to decompensated stage
(Child’s class B or C), then to end-stage
cirrhosis. In cases of compensated cirrhosis,
hypertrophy of the lateral segment and
caudate lobe are predominant findings on
longitudinal MRI exam. Atrophy of the
medial segment and the right lobe are the
predominant findings in cases of progressive
cirrhosis. These MRI findings help predict a
patient’s clinical outcome. Although the
cause of the segmental morphologic changes
is unclear, it is thought to be related to
altered blood flow in cirrhotic liver. Many
findings of early cirrhosis are caused
primarily by preferential atrophy of the
medial segment of the left lobe. As this
segment atrophies, enlargement of the hilar
portal space occurs. This has been described
as an early MRI sign of cirrhosis. Medial
segment atrophy also causes an expanded
gallbladder fossa, another MR sign of
cirrhosis. This finding was reported to have
98 percent specificity, although the
sensitivity was 68 percent. Other imaging
signs, such as ratio of the transverse
diameter of the caudate to right lobe (C/
RL >0.65), have been used to determine
cirrhosis.
Body MR Imaging 269
Regenerative nodules are regenerated
hepatocytes surrounded by fibrotic septa.
Regenerative nodules are hypointense in
relation to hyperintense inflammatory septa
on T 2WI and gadolinium-enhanced MR
images. They have variable intensity on
T1WI and can be iso- or hypo-intense on
T2WI, but are not hyperintense on T2WI,
with the exception of those in chronic hepatic
vein thrombosis. In contradistinction to
HCC, regenerative nodules are not hyper-
intense on hepatic arterial-phase gadolinium-
enhanced images. Dysplastic nodules are
premalignant and are found in 15 to 25
percent of cirrhotic livers. These nodules
are commonly hyperintense on T1WI and
hypointense or isointense on T2WI. Like
regenerative nodules, they are not hyper-
intense on T2WI. Occasionally, dysplastic
nodules may be hyperintense on early post-
gadolinium.37-41
Budd-Chiari Syndrome
Budd-Chiari syndrome results from obstruc-
tion of the hepatic venous outflow, causing
portal hypertension, ascites, and hepatic
failure. Most often there is atrophy of the
peripheral liver, and hypertrophy of the
caudate lobe and central liver. It is best
diagnosed by multiphasic gadolinium enhan-
ced MR images, which show a thrombosed
or absent hepatic vein and altered hepatic
enhancement. In acute onset of the Budd-
Chiari syndrome, the periphery of the liver
has moderately low signal on T1WI and
moderately high signal on T2WI relative to
the central liver. Severe congestion causes
decreased peripheral enhancement during
the arterial and portal venous phase. In the
subacute setting, the T 1 and T 2 signal
characteristics are similar to those with acute
disease. During early and late phases of
contrast enhancement, there is hetero-
geneously increased enhancement at the
270 Biomedical Magnetic Resonance: Proceedings of the International Workshop
periphery. In chronic disease, signal diffe-
rences between the central and peripheral
part of the liver on T1WI and T2WI and
post-gadolinium contrast images are
minimal. Varices may be present in chronic
Budd-Chiari syndrome. Curvilinear intra-
hepatic collaterals and capsular collaterals
are characteristic. In patients with chronic
Budd-Chiari syndrome, hyperplastic nodules
may develop. These nodules are hyper-
intense on T1WI, variable on T2WI, and en-
hance early on post-gadolinium images.42,43
MR IMAGING OF DISEASES OF BILIARY-
PANCREATIC SYSTEM
With recent technologic advances, such as
breath-hold, dynamic contrast-enhanced
imaging, and MR cholangiopancreatography
(MRCP), MR is increasingly used as a
primary imaging modality for the pancreas
and biliary system.
MR Protocol
The standard MR protocol includes coronal
half-Fourier acquisition single-shot turbo-
spin-echo (HASTE), axial in-phase and
opposed-phase gradient-recalled echo (GRE),
axial T 2 turbo-spin-echo (TSE) with fat
suppression, or turbo-inversion-recovery
through the liver and pancreas. Two-dimen-
sional (2D) spoiled GRE with fat suppression
and three-dimensional (3D) spoiled GRE
with fat suppression through the pancreas
are also obtained. After administration of
gadolinium contrast material at 2 mL/sec,
we obtain breath-hold dynamic 3D spoiled
GRE sequence in arterial, interstitial, and
equilibrium phases through the pancreas.
Dynamic post-gadolinium axial T1-weighted
SGE with fat suppression are obtained at 15
to 20 seconds (arterial–capillary phase), 45
seconds (early interstitial phase), and 90
seconds (late interstitial phase or equilibrium
phase) after the start of contrast injection.
If islet cell tumor is suspected, the first
phase should be obtained in the arterial
phase, which occurs approximately 10 sec,
before the interstitial phase. The dynamic
study can be performed either by care bolus
or test bolus technique. A 2D spoiled GRE
sequence with fat suppression can be used
instead of a 3D spoiled GRE sequence for
the dynamic study. With the contrast-
enhanced dynamic GRE sequence, one
should try to include both liver, and
pancreas, so that liver lesions can also be
studied at the same time. An MRCP
sequence may be added as necessary before
administration of contrast material. MR
cholangiopancreatography is performed
using half-Fourier rapid acquisition with
relaxation enhancement (RARE). A thick-
slab technique is used to provide a global
view of the biliary tree and pancreatic duct.
Thin slice technique is useful for evaluation
of detailed anatomy.
The Indications of Pancreatic MRI are as
follows: 45-48
• Detection and staging of pancreatic
neoplasms to avoid ‘‘understaging’’ on
the basis of CT
• Detection of small non-organ-deforming
pancreatic ductal adenocarcinomas
• Detection of islet cell tumors
• Evaluation of acute and chronic pancrea-
titis
• Characterization of suspected paren-
chymal abnormalities found on CT or
US (e g, focal fatty infiltration)
• Detection of choledocholithiasis, pan-
creatic duct calculi, and cholangio-
carcinoma
• Possible improved ability to distinguish
chronic pancreatitis from neoplasm

• Equivocal findings on US and CT, or if
iodinated contrast media are contraindi-
cated.
Normal MR Imaging Appearance
of the Pancreas
The pancreas is unique in that it has the
highest T1 signal intensity of intra-abdo-
minal organs on the fat-suppressed sequence
higher than or at least equal to liver signal
intensity. This is attributed to the presence
of a large amount of aqueous protein within
the glandular elements of the pancreas,
abundance of endoplasmic reticulum of the
protein-producing pancreatic acinar cells,
and high content of paramagnetic ions such
as manganese within the pancreas. The
signal intensity of the pancreas is also higher
than that of liver on the arterialcapillary
phase of the gadolinium-enhanced fat-
suppressed T1-weighted SGE sequence. In
addition, the pancreatic head is easily
differentiated from the duodenum on this
sequence because of its higher signal
intensity. 49,50 On delayed images, the
pancreas has similar signal intensity
compared with the liver.
The T2-weighted sequences, with fluid
being bright, allow for optimal visualization
of the anatomy and pathology of the
common bile duct, cystic duct, and pan-
creatic and hepatic ducts in the physiologic
state, without the pressure distension of the
ducts that is associated with direct contrast
injection, as is required in ERCP. Unlike
ERCP, the T2-weighted MRCP sequences can
assess areas proximal to an obstruction.
Biliary and pancreatic ductal stones and
strictures 51,52, pancreatic cystic lesions,
complex peripancreatic fluid collections, and
islet cell tumors are well-visualized on these
T 2 -weighted sequences. The T 2 signal
Body MR Imaging 271
intensity of the normal pancreas is quite
variable, ranging from isointense to liver to
as high as abdominal fat.47 It is difficult, at
times, to differentiate normal parenchyma
from adenocarcinomas on T 2 -weighted
images, and generally tumors are best
appreciated on the fat-suppressed T 1 -
weighted dynamic enhanced SGE sequen-
ces53. The addition of fat suppression to the
T2-weighted sequences is useful for depic-
ting islet cell tumors and liver metastases.54
Other uses of T2-weighted sequences include
detection of low signal intensity fibrosis and
iron deposition associated with hemochro-
matosis, which result in a very low signal
intensity gland.55
The two best sequences for detecting
focal pancreatic mass lesions and increasing
the confidence with which pancreatic
pathology are excluded are the precontrast
fat-suppressed T 1-weighted SGE and the
immediate (arterial–capillary phase) post-
gadolinium fat-suppressed T1-weighted SGE
sequences.56,57 The T1-weighted GRE in-and
out-of-phase sequences are most useful in
the detection and characterization of
lymphadenopathy, stranding of the peripan-
creatic tissue planes in acute pancreatitis,
hepatic fatty infiltration, fluid collections
containing subacute blood or concentrated
proteins, and adrenal adenomas.
Clinical Applications
Acute Pancreatitis
The diagnosis of acute pancreatitis is
typically based on clinical and laboratory
findings; however, CT is often used as the
imaging modality of choice in confirming
the clinical suspicion and for detecting
potential complications. MR may be more
sensitive than is CT in the detection of acute
pancreatitis, particularly in mild cases.47
272 Biomedical Magnetic Resonance: Proceedings of the International Workshop
Currently, the role of MRI in the assessment
of severe acute pancreatitis is unclear. In
patients with severe pancreatitis are
generally too ill to cooperate for an MRI
examination47. In cooperative patients, MRI
may be helpful in elucidating the underlying
causes of pancreatitis, including chole-
docholithiasis, pancreas divisum, and
pancreatic carcinoma. In cases of mild to
moderate acute pancreatitis, the T1-weighted
sequence with fat suppression, which is
helpful in the evaluation of masses and
chronic pancreatitis, is relatively insensitive
to acute edema.57 On fat-suppressed T 1-
weighted sequences in the setting of mild
acute pancreatitis, the T1 signal intensity of
the pancreas often remains high. The
diagnosis of acute pancreatitis is made on
the presence of morphological changes,
including focal or diffuse enlargement and
peripancreatic inflammatory changes. Low
signal intensity peri-pancreatitic inflam-
matory stranding and fluid in the retro-
peritoneal fat is well shown on the
T1-weighted GRE sequence53, whereas peri-
pancreatic edema and fluid collections are
best demonstrated on the fat-suppressed T2-
weighted HASTE or SS-FSE sequence. With
acute pancreatitis on MRCP, the main pan-
creatic duct retains a smooth contour, but
may be compressed due to the surrounding
edematous parenchyma.51 In cases of severe
acute pancreatitis, there is loss of the normal
homogeneous appearance of the pancreas
on precontrast fat-suppressed T1-weighted
SGE sequence. Delayed enhancement of the
pancreas is often seen due to edema of the
gland. The use of IV contrast is important
to evaluate for the presence of pancreatic
necrosis, which is associated with increased
morbidity and mortality. Pancreatic necrosis
may be better delineated on MRI than on
CT, because of the greater sensitivity of MRI
to gadolinium than of CT to iodinated
contrast. In addition, MRI has an advantage
over CT because gadolinium may be
administered to patients with impaired renal
function. Pancreatic pseudocysts are
encapsulated collections of pancreatic fluid
that can occur in acute or chronic
pancreatitis. They are best depicted on a
combination of T 2 -weighted HASTE or
SS-FSE and contrast-enhanced T1-weighted
SGE sequences. These lesions are usually
homogeneous, may be unilocular or multi-
locular, and often communicate with the
pancreatic duct. MRCP is far more sensitive
than is ERCP in the detection of pseudo-
cysts, because less than 50 percent of
pseudocysts fill with contrast injected at
ERCP. ERCP better demonstrates the site
of communication with the pancreatic duct
than does MRCP. Unlike ERCP, the internal
consistency of pseudocysts—including the
presence of hemorrhage, proteinaceous fluid,
or necrotic debris can be characterized with
MRI to help guide drainage procedures
more effectively than with CT. Disruption
of the pancreatic duct is best shown on the
thick-slab T2-weighted RARE or thin-slice
T2-weighted HASTE or SS-FSE sequence.
Chronic Pancreatitis
Chronic pancreatitis is a chronic inflam-
matory process of the pancreas with irre-
versible exocrine and endocrine dysfunction.
It is characterized by permanent replace-
ment of normal pancreatic parenchyma with
atrophy, fibrosis, and calcification, as well
as ductal dilatation, strictures, and cal-
culi.47,58 MRI is less sensitive than is CT for
the detection of calcification associated with
chronic pancreatitis. MRI, however, is more
sensitive for the early detection of chronic
pancreatitis prior to the development of

calcifications by examining the pre-contrast
fat-suppressed T1-weighted sequence for a
low signal intensity gland.58 The usual high
signal intensity of the pancreas on fat-
suppressed T 1 -weighted sequences is
reduced due to replacement of normal
pancreatic parenchyma by chronic
inflammation and fibrosis, resulting in the
loss of aqueous protein within the glandular
elements of the pancreas. In addition,
dynamic post-gadolinium images demon-
strate abnormalities associated with chronic
pancreatitis. Unlike the normal pancreas that
exhibits rapid uniform enhancement, the
pancreas in chronic pancreatitis shows
decreased heterogeneous enhancement on
the immediate (arterial–capillary phase) post-
gadolinium images and delayed enhance-
ment of the gland compared with normal
pancreas. MR imaging is able to delineate
well the biliary and pancreatic abnormalities
associated with chronic pancreatitis,
including biliary and pancreatic ductal
dilatation, strictures, irregularities,
sacculation and ectasia of the secondary
radicals, pseudocysts, intraductal calculi, and
morphologic changes such as parenchymal
atrophy or focal pancreatic enlargement.
These findings—except for parenchymal
atrophy and focal pancreatic enlargement—
are best depicted with thin-slice T2-weighted
HASTE or SS-FSE and thick-slab RARE.59,60
Pancreatic intraductal calculi are best seen
on T 2 -weighted sequences, depicted as
filling defects within the high signal intensity
main pancreatic duct The normal main
pancreatic duct is approximately 2 mm in
diameter, and the side branches draining
the pancreatic lobules are usually not
depicted, even with secretin administra-
tion.45 Dilated side branches are the most
prominent and specific feature of chronic
pancreatitis and are well demonstrated using
T2-weighted MRCP sequences.
Body MR Imaging 273
Pancreatic Adenocarcinoma
Adenocarcinomas of the pancreatic head
usually present earlier than do adeno-
carcinomas of the pancreatic body or tail,
because of their proximity to the common
bile duct.53 Those involving the body and
tail usually present late, with a high
percentage of metastases at the time of
diagnosis. The primary role of MRI is in the
detection of small lesions that are potentially
curable and to avoid ‘‘understaging’’ on the
basis of the CT examination. In addition,
MRI is also helpful to detect or exclude
pancreatic cancer in patients with a promi-
nent pancreatic head or uncinate process on
CT or ultrasound examinations. Using
current fast scanning techniques, dynamic
gadolinium-enhanced MRI has been shown
to be superior to contrast enhanced helical
CT in detection of smaller pancreatic masses,
accurate assessment of local tumor extension,
and assessment of resectability of pancreatic
tumors.48,61-63 Pancreatic adenocarcinoma is
difficult to discern from normal pancreas
on T 2 -weighted or T 1 -weighted GRE
sequences without fat suppression, because
most are minimally hypointense relative to
normal pancreatic parenchyma. 53 The
detection and characterization of pancreatic
tumors, especially when small and non-organ
deforming, is best performed by a combi-
nation of precontrast and immediate post-
gadolinium fat-suppressed T1-weighted SGE
images.45,47,48,60,61 Pancreatic adenocarcinoma
is typically a desmoplastic hypovascular
tumor, appearing as a focal low signal
intensity mass on the precontrast fat-
suppressed T1-weighted SGE images and
during the immediate postgadolinium
arterial – capillary phase images. Because of
the presence of abundant amount of fibrous
stroma and interstitial fluid within the tumor
274 Biomedical Magnetic Resonance: Proceedings of the International Workshop
and relative sparse vascularity, there is late
enhancement of the tumor as gadolinium
diffuses across capillaries. 50,53 In adeno-
carcinomas involving the pancreatic head,
reduction in T1 signal intensity may be seen
distal to the tumor. This is attributed to
obstruction of the main pancreatic duct, with
subsequent chronic inflammation, leading to
progressive fibrosis and parenchymal
atrophy. In such cases, the distinction
between adenocarcinoma and chronic
inflammation of the pancreas can be difficult
on unenhanced fat-suppressed T1-weighted
images. The tumor may be better demons-
trated in the arterial– capillary phase of
enhancement as a rim-enhancing low signal
intensity mass in a background of slightly
greater enhancing chronically inflamed
pancreatic parenchyma. 47,48 In addition,
morphological changes such as effacement
of the fine lobular contours of the pancreas
with loss of the well-defined internal archi-
tecture favor the diagnosis of carcinoma. In
general, the distinction between pancreatic
cancer and chronic pancreatitis on the basis
of MRI is not difficult. Both entities show a
low T1 signal intensity pancreatic mass, with
the same degree and delay in enhancement
and associated dilatation of the pancreati-
cobiliary ducts. This is attributed to the
abundant fibrosis in both conditions. Both
pancreatitis and pancreatic carcinoma can
cause peripancreatic changes, including
infiltration of the fat planes that surround
the superior mesenteric vessels with features
of vessel encasement.60 In cases of masses
involving the pancreatic head, the diagnosis
of chronic pancreatitis is favored if there is
preservation of the morphology of the pan-
creas without loss of the usual marbled
appearance of the gland. In addition, an
irregularly dilated main pancreatic duct with
intra-ductal calcifications is more common
in chronic pancreatitis, as opposed to smooth
dilatation of the pancreatic duct in an
obstructing adenocarcinoma.64 On the other
hand, the presence of lymphadenopathy and
liver or peritoneal metastases favor the
diagnosis of pancreatic cancer. CT, like MRI,
has difficulties in the differentiation of focal
chronic pancreatitis from tumor. MRI criteria
of unresectability are similar to the esta-
blished CT criteria. At most institutions,
these include involvement of the superior
mesenteric artery, celiac trunk or the
superior mesenteric vein–portal vein
confluence; and peritoneal or hepatic
metastases.53
Islet Cell Tumors
The three most common islet cell tumors
are insulinomas, gastrinomas, and nonfunc-
tioning islet cell tumors. The use of fat-
suppressed T1-weighted, T2-weighted, and
dynamic gadolinium enhanced images is
necessary, because some lesions are seen on
one sequence and not the others. These
tumors have long T1 and T2 relaxation times,
exhibiting low signal intensity on T 1 -
weighted images and high signal intensity
on T2-weighted images. They can be seen
as low signal intensity masses on a
background of high signal intensity pancreas
on fat-suppressed T 1 -weighted images.
They are well depicted on dynamic contrast
enhanced imaging during the arterial phase
because of their hypervascularity53,65,66, and
on the fat-suppressed T2-weighted sequen-
ces. The enhancement pattern of islet cell
tumors can be homogeneous, ring-like, or
diffusely heterogeneous on the immediate
postgadolinium T1-weighted SGE images.
Functional tumors, predominately gastri-
nomas and insulinomas, tend to be small at
presentation, usually less than 2 cm in size.
Nonfunctioning tumors are usually large at

presentation, with increased incidence of
calcification, cystic degeneration, and central
areas of necrosis or hemorrhage.67 Small islet
cell tumors may be distinguished from
ductal adenocarcinomas on the basis of
higher T2 signal intensity and more uniform
hyperintense enhancement on the immediate
post-gadolinium fat-suppressed T1-weighted
SGE images, hypervascular hepatic meta-
stases, lack of vascular encasement, absence
of pancreatic duct obstruction, presence of
tumor thrombus, and preservation of the
usual high T1 signal intensity of the adjacent
pancreas.67
Cystic Neoplasms
Cystic neoplasms of the pancreas can be
divided into two subgroups: serous cysta-
denoma and mucinous cystic neoplasms.
Serous cystadenoma is a benign neoplasm,
often referred to as microcystic cysta-
denoma. It typically consists of clusters of
small cysts <2 cm in diameter, although a
macrocystic variant has been reported.68 On
T2-weighted images, serous cystadenoma is
a lobulated mass with very high signal
intensity.69 The walls of each septa may be
imperceptible. Stellate calcification may be
visible as a signal void, although MR is less
sensitive to calcium than CT. After contrast
administration, diffuse faint contrast en-
hancement can be seen because of wall
enhancement within small cysts. There is
considerable overlap in the imaging
appearance of serous and mucinous cystic
neoplasm; therefore, unless the lesion has a
completely typical appearance for serous
adenoma, it should be surgically-removed.
Mucinous cystic neoplasm is often
referred to as macrocystic cystadenoma or
cystadenocarcinoma. Such lesions should be
considered either premalignant or frankly
Body MR Imaging 275
malignant. These lesions commonly occur
in the body or tail of the pancreas in women
between the ages of 40 to 70 years.
Mucinous cystic tumors typically consist of
clusters of cysts, with each cyst >2 cm in
diameter. On T2-weighted images, muci-
nous cystic neoplasms have high signal
intensity and internal septations.69 There is
no contrast enhancement within the cystic
component of the mass. On T 1-weighted
images, the cyst may be hyperintense due
to mucin content. The walls of the cystic
mass may be variable in thickness and there
may be nodular enhancing areas. Thick walls
and the presence of mural nodules are
suggestive of malignancy, although absence
of these findings does not exclude malig-
nancy. Since it is often very difficult to
differentiate between mucinous adenoma
and adenocarcinoma by imaging, mucinous
tumors usually require resection.
Intraductal papillary mucinous tumor
(IPMT) is a subtype of the mucinous cystic
neoplasms. The tumor arises from the
epithelium of the main pancreatic duct or
its branches, and grows into the pancreatic
duct in a papillomatous pattern. The tumor
produces copious amounts of mucin; thus
the tumor is often referred to as mucin
hypersecreting tumor of the pancreas. The
hallmark of the main-duct type IPMT is
dilatation of the main pancreatic duct, which
may be indistinguishable from chronic
pancreatitis. On MRCP, pancreatic duct
dilatation is well depicted, and branch-duct
type IPMT appears as a cluster of small cysts
with or without septation.70 Communication
between the cysts and main pancreatic duct
can often be visualized, which may help in
differentiation from a pseudocyst. Intra-
ductal papillary mucinous tumor has variable
malignant potential. Findings that favor
malignant IPMT include the presence of
276 Biomedical Magnetic Resonance: Proceedings of the International Workshop
filling defects or papillary projections within
the dilated pancreatic duct or cysts and
dilatation of the main pancreatic duct,
>15 mm for main duct type and any dilation
for branch-duct type.71
Solid and Papillary Epithelial Neoplasms
Solid and papillary epithelial neoplasm of
the pancreas is a rare tumor that occurs
mainly in women under 40 years of age.
The mass is usually large (>10cm) and
commonly exhibits cystic and hemorrhagic
degeneration from necrosis with a thick rim
of soft tissue. On T1-weighted images, the
hemorrhagic component appears hyper-
intense72, and the soft-tissue rim enhances
after administration of gadolinium contrast.
MR Cholangiopancreatography
MR cholangiopancreatography (MRCP) is
still a rapidly evolving technique, but has
been already accepted as clinically useful
and is widely used to evaluate biliary or
pancreatic diseases in a noninvasive way.
This technique uses MR imaging to visualize
stationary or slow-moving fluid, such as
bile, displaying them as high signal intensity.
Heavily T2-weighted sequences are generally
used for MRCP with single shot echo-train
spin-echo technique achieving the most
widespread use.
MRCP Versus ERCP
Direct cholangiography methods (ERCP and
PTC) offer certain advantages that cannot
be reproduced with MR methods. Both
ERCP and PTC yield images with higher
spatial resolution, and both techniques have
therapeutic as well as diagnostic appli-
cations. However, even with the higher
spatial resolution of these invasive methods,
visualization of bile duct morphology with
MRCP equals or exceeds that of ERCP73,74
without the associated morbidity or morta-
lity. The risks of ERCP include pancreatitis,
sepsis, hemorrhage, duodenal and bile duct
perforation, and death. 75 Biliary-enteric
anastomoses and obstructions can also make
ERCP difficult, if not impossible. After a
failed or incomplete ERCP, MRCP should
be considered the diagnostic test of choice.
In this group of patients, MRCP has been
shown to have a sensitivity of 97 percent, a
specificity of 100 percent, and an accuracy
of 98 percent for the diagnosis of pan-
creaticobiliary disease. ERCP has advantages
over MRCP, which include direct therapeutic
interventional procedures that may be
performed concurrent with diagnostic
imaging. Unlike ERCP, MRCP produces
images of the ducts in their natural state,
because it does not involve distention of
the ducts by injected contrast medium.
MRCP Pulse Sequences
Bile and pancreatic fluid are rich in protons,
resulting in a T2 relaxation time that is much
longer than that of surrounding tissues. On
heavily T2- weighted sequences, fluid within
the biliary tree, gallbladder, and pancreatic
duct is bright, while background tissues are
suppressed. Signal from background fat,
which is usually high on fast T2- weighted
sequences, can be decreased with the use of
fat-suppression techniques. Magnetic reso-
nance cholangiopancreatography is usually
performed using one of the hybrid rapid
acquisition with relaxation enhancement
(RARE) sequences or their derivatives. The
introduction of single-shot RARE techniques
and half-Fourier acquisition single-shot
turbo-spin-echo (HASTE) imaging has
reduced imaging times to within a single
breath-hold. These breath-hold techniques

are best applied in two forms: a single, thick
coronal (or axial) slab and a multislice, thin
coronal sequence.76
Pediatric applications
Magnetic resonance applications in the
pediatric population are expanding. Mag-
netic resonance cholangiopancreatography
rarely requires anesthesia; ERCP and PTC
cannot be performed on children without
anesthesia. However, conscious sedation is
required when MRCP is performed on young
or uncooperative patients. Respi-ratory-
triggered MRCP enables evaluation of major
and minor bile ducts, even in uncooperative,
young patients. Images can be produced in
which the common bile duct and hepatic
ducts are clearly seen in infants as young as
3 days old.77
Biliary Atresia
Biliary atresia and neonatal hepatitis are the
most common causes of prolonged conju-
gated hyperbilirubinemia in the neonate.78
Because the treatment for biliary atresia and
neonatal hepatitis is very different (surgical
in the former and medical in the latter),
definitive diagnosis is necessary. Ultrasound,
generally recommended as the initial
imaging test, excludes the presence of a
choledochal cyst, which may also be a cause
of neonatal jaundice. Jaw et al79 found that
the common hepatic ducts and common bile
duct could be readily visualized in control
subjects and infants with neonatal hepatitis,
whereas infants with surgically confirmed
biliary atresia failed to demonstrate a visible
common bile or hepatic duct on MR cholan-
giogram. The failure to visualize the
extrahepatic biliary ducts on MR cholangio-
graphy and presence of high signal intensity
periportal thickening has also been shown
Body MR Imaging 277
by Giubaud et al80 to be strongly associated
with the presence of biliary atresia children
with biliary atresia confirmed by biopsy also
demonstrated significant high signal inten-
sity periportal thickening on MR imaging,
which correlated with periportal fibrosis
found at histologic examination. Magnetic
resonance cholangiopancreatography may
also reveal a triangular-shaped area of high-
signal intensity within the porta hepatis
caused by cystic dilation of the bile duct
proximally.81
Cystic Diseases of the Bile Duct
Congenital cystic lesions of the bile duct
can be classified according to Todani’s
classification system.82 type I, choledochal
cyst; type II, diverticulum of extrahepatic
ducts; type III choledochocele; type IV,
multiple segmental cysts (Fig 20.4); and type
V, Caroli’s disease. MRCP can be effective
and comparable with ERCP for the evalu-
ation of these lesions. Also, the combination
of MRCP and gadolinium-enhanced T 1-
weighted images is useful to diagnose
associated findings, such as gallstone disease
and cancer.
Fig. 20.4: Coronal thick slab (HASTE) image
showing, fusiform dilatation of the common bile duct
suggestive of type I choledochal cyst
278 Biomedical Magnetic Resonance: Proceedings of the International Workshop
Congenital Variants of the Biliary System
MRCP can demonstrate accurately various
variants, such as a low cystic duct insertion,
a medial cystic duct insertion, a parallel
course of the cystic and hepatic ducts, and
an aberrant right hepatic duct.83
Stone Disease
Biliary calculi are seen as relative areas of
decreased signal surrounded by bright bile.
Many studies have investigated the use
of MRCP for the detection of choledo-
cholithiasis, and excellent results have been
reported, with sensitivities of 85 to 100
percent, specificities of 90 to 99 percent, and
accuracies of 89 to 97 percent. 84,85 The
accuracy of MRCP in diagnosing hepatoli-
thiasis is 96 percent.86 Intrahepatic bile duct
calculi may be seen as well-defined filling
defects or as castlike areas of low signal
that conform to the wall of the duct.86 On
thin-slice source images, stones appear as
signal void lesions and can be detected as
small as 2 mm in dilated and nondilated
ducts.
There are several pitfalls and mimickers
of stones with MRCP. Intraductal air bub-
bles (pneumobilia) may mimic the appea-
rance of stones. An important differentiating
feature from stones is that air bubble filling
defects lie on the nondependent portion of
the bile duct against the wall on axial images.
Blood clots may appear indistinguishable
from bile duct stones. Other pitfalls that
may mimic bile duct stones include (1)
tortuosity of the bile duct running in and
out of the imaging plane; (2) merging of the
cystic duct into the CBD when observed en
face on coronal images, which may result in
a round hypointense focus; (3) metallic clips;
and (4) extraductal compression from the
right hepatic or the gastroduodenal artery,
which may result in a signal void focus (5)
bile flow within the CD may give false
impression of calculus. Correct diagnosis
usually can be achieved by careful attention
to the exact location of these foci and
interpretation of thick-slab MRCP or MIP
reconstructed images in conjunction with the
thin-slice source images.87,88
Primary Sclerosing Cholangitis
Primary sclerosing cholangitis is charac-
terized by chronic fibrosing inflammation
of the biliary system of unknown etiology.
The diagnosis of primary sclerosing cholan-
gitis is made by cholangiographic findings
supported by histologic results. The imaging
appearance of primary sclerosing cholangitis
is characterized by multiple, irregular
strictures and saccular dilatations of the
intrahepatic and extrahepatic bile ducts
producing a beaded appearance. The
conventional imaging modality for the
diagnosis of primary sclerosing cholangitis
is ERCP. MRCP has been shown to be useful
for the diagnosis and follow up of primary
sclerosing cholangitis.89,90 Diagnostic challen-
ges include that subtle changes of mild
primary sclerosing cholangitis may be
difficult to detect by current MR imaging
techniques, and cirrhosis may cause
distortion of the intrahepatic bile ducts and
mimic primary sclerosing cholangitis.
Strictures
With MRCP, ducts are seen in their passive
state because contrast material does not
need to be injected forcefully to opacify
them. Thus, images reflect the diameter of
strictures more closely than they do with
invasive cholangiography. Magnetic reso-
nance cholangiopancreatography has been
reported to have a sensitivity of 100 percent
and a specificity of 98 percent for the
diagnosis of strictures.91 However, reduced

spatial reso-lution and the inability to
evaluate lack of distensibility during MRCP
limits visualization of subtle strictures.
Cholecystokinin may also have a role for
evaluating the distensibility of the biliary
tree at MRCP. Extraductal metallic surgical
clips, intravascular metallic coils, or gas in
the stomach or duodenum may cause signal
loss in the adjacent extrahepatic bile ducts.
This may lead to a false positive diagnosis
of ductal narrowing. Also, MIP recons-
tructed images can overestimate the length
of strictures. This pitfall can be avoided by
evaluating source images and the amount
of bile duct dilatation proximal to an
apparent stricture.
Obstruction at the Ampulla of Vater
Both benign and malignant disease can cause
an obstruction at the ampulla of Vater with
dilatation of the common bile duct and
pancreatic duct. Causes include ampullary
carcinoma, inflammatory stenosis, sphincter
of Oddi dysfunction, and impacted stones.
MRCP is helpful in determining both the
degree of obstruction and the luminal
margins of the tumor. Magnetic resonance
cholangiopancreatography also aided in
determining the degree of decompression
after biliary stent placement.
Cholangiocarcinoma
Classic MRCP findings of cholangio-
carcinoma include an abrupt site of biliary
obstruction with marked dilatation proxi-
mally. In some cases, it may be difficult to
distinguish duct wall thickening caused by
pyogenic cholangitis from that caused by
cholangiocarcinoma. Cholangiocarcinoma
usually arises from the extrahepatic bile
ducts, but may also arise from the intra-
hepatic ducts. 92,93 When intrahepatic,
cholangiocarcinoma occurs as a focal or
Body MR Imaging 279
infiltrating lesion. MRCP is superior to CT
for defining the proximal extent of biliary
tract tumors. Hepatocellular carcinoma rarely
manifests by intrahepatic bile duct obstruc-
tion, but this tumor should be considered
in the differential diagnosis of cholangio-
carcinoma if there is an intrabiliary tumor
with an associated hepatic mass. 94 MR
provides an “all-in-one” approach for
evaluating malignant obstructions. The
origin, size, and site (hilar, intrahepatic, and
subhilar) of the tumor, definition of tumor
margins, and stage of disease can be
depicted without need for other imaging
modalities such as CT or ERCP. Lympha-
denopathy, infiltration by hepatic metasta-
ses, mesenteric vascular involvement, distant
metastases, and occlusion of segmental and
subsegmental bile duct branches can also be
seen. In some cases, MRCP is sufficient for
planning appropriate therapeutic interven-
tions. Grading of hilar cholangio-carcinoma
by MRCP may also prevent patients from
undergoing an unnecessary ERCP when
endoscopic drainage is not the optimal
treatment segmentally occluded ducts.95
Post-biliary-enteric Anastomosis
Surgical alterations of the gastrointestinal
tract after biliary-enteric anastomoses can
make ERCP difficult or impossible. MRCP
should be considered a first-line diagnostic
method in these patients because it is capable
of providing information about the biliary
tree, which cannot be obtained by invasive
procedures. Anastomotic strictures are
usually caused by postoperative scarring
and, less often, by ischemia. MRCP can
evaluate anastomotic sites and create a
complete “roadmap” of the bile ducts.96
Post-laparoscopic Cholecystectomy
MRCP can evaluate the biliary tract proximal
and distal to a duct lacerated at cholecystec-
280 Biomedical Magnetic Resonance: Proceedings of the International Workshop
tomy. ERCP may result in incomplete
visualization of the biliary tree, showing
only a cutoff sign of the distal bile duct, so
that PTC is required to visualize the proximal
biliary system. Information about anatomy
proximal to the transection is important for
determining the strategy for reconstructive
surgery. This information can be provided
by MRCP, reducing the need for PTC.
Post-liver Transplant
MRCP has a lower spatial resolution than
conventional cholangiography, limiting
depiction of strictures. However, in the case
of liver transplantation patients, this does
not represent a significant limitation because
the lesions have a benign origin.96 These
strictures can be classified either as anasto-
motic or non-anastomotic. Non-anastomotic
strictures usually involve the hepatic duct
bifurcation, peripheral ducts, or both, and
occur in approximately 8 percent of patients.
Non-anastomotic strictures are important
because they may indicate rejection. MRCP
enables early visualization of intrahepatic
duct dilatation, which can occur as a result
of stricture formation.
Evaluation of the Pancreas
MRCP has been slower to develop as a
diagnostic tool for the pancreas because of
the smaller caliber of the pancreatic duct.
Advances in breath-hold imaging, surface
coils, and the use of pharmacologic agents
has allowed MR pancreatography to become
a useful means for evaluating pancreatic
ductal anatomy and function. The use of
breath-hold imaging with surface coils
reduces blurring artifacts and improves
visibility. Visualization of the pancreatic duct
is further improved following the adminis-
tration of secretin.
Pancreas Divisum
Pancreas divisum has important implications
for ERCP, as cannulation of the major papilla
opacifies only the dorsal duct, resulting in
incomplete visualization of pancreatic ductal
structures. Lack of opacification of ductal
structures in the body or tail of the pancreas
at ERCP also may erroneously suggest
occlusion of the main pancreatic duct, lead-
ing to an incorrect diagnosis of pancreatic
carcinoma. In contrast, MRCP is excellent
for diagnosing pancreas divisum because this
technique can show the ventral and dorsal
ducts simultaneously without need for
cannulation of the major and minor
papillae.97,98
MR Imaging of Carcinoma
of the Gallbladder
Ultrasound, CT, and MR are the primary
means of imaging gall-bladder carcinoma.99-
102 This neoplasm has three major patterns
of presentation patho-logically and on cross-
sectional imaging: (1) focal or diffuse mural
thickening; (2) an intraluminal polypoid
mass, usually larger than 2 cm, originating
in the gallbladder wall; and (3) most
commonly (45–65%) a subhepatic mass
replacing or obscuring the gallbladder, often
invading adjacent liver. On CT and MR,
polypoid cancers enhance homogeneously
after administration of contrast medium, and
the adjacent gall-bladder wall may be
thickened. Polypoid gallbladder carcinomas
do not usually show necrosis or calcification
on CT. Carcinoma as a gallbladder fossa
mass is the most common form of
gallbladder cancer and presents as solid
masses with variable echogenicity and may
be homogeneous or inhomogeneous. The
mass may be difficult to separate from the
liver sonographically, especially when there

is direct hepatic invasion. The absence of a
gallbladder and the presence of gallstones
can be helpful clues to the diagnosis.103
Infiltrating carci-nomas that replace the
gallbladder often show irregular contrast
enhancement with scattered regions of
internal necrosis on CT and MR.103
Magnetic resonance cholangiopancreato-
graphy is becoming the primary method for
evaluating patients with a variety of biliary
and pancreatic diseases. The absence of
ionizing radiation and safety of this techni-
que make MRCP an excellent diagnostic tool.
MR Imaging of the Kidneys
and Adrenal Glands
The practice of urology has changed greatly
in recent years. Selection of the appropriate
surgical technique for the individual case
depends highly on renal anatomy, tumor
size, tumor location, anatomy of obstruction,
and the relationship of the diseased organ
to surrounding viscera. The role of pre-
operative imaging has evolved from pro-
viding diagnostic confirmation to providing
a roadmap for selection and application of
technique. Gadolinium chelates have been
shown to be exceedingly safe, may be
administered to patients without concern
for contrast induced nephrotoxicity, and are
well tolerated in those patients with a
history of iodinated contrast allergy. Finally,
the ability of MR imaging to detect both
gross and microscopic fat provides for
accurate characterization of adrenal and
renal masses.
Protocol
Comprehensive examination of the kidneys
entails evaluating the renal vasculature,
parenchyma, and collecting system. Pre-
Body MR Imaging 281
contrast imaging includes an axial breath-
hold T1-weighted gradient-echo (FLASH)
sequence performed in and out of phase.
Used in conjunction with a frequency-
selective fat suppressed T 1 -weighted
sequence, this also allows differentiation of
fat from hemorrhage, both of which may
occur in a renal or adrenal mass. A coronal
breath-hold T2-weighted half-Fourier single-
shot turbo spin-echo sequence is performed
to help characterize cystic lesions of the
kidney, to assess for hydronephrosis, and
to determine if an adrenal mass is present.
The coronal plane is advantageous in
evaluating exophytic lesions that occur at
the poles of the kidneys. These may not be
optimally demonstrated in the axial plane.
To evaluate the renal vasculature, a high-
resolution breath-hold fat-suppressed three-
dimensional T1-weighted FLASH sequence
is performed in a coronal-oblique plane
before and after the intravenous adminis-
tration of gadolinium. Using the proper scan
delay and acquiring the low spatial fre-
quency (high contrast) lines of k-space
during peak arterial enhancement are critical
to minimize venous contamination and to
prevent scanning before sufficient contrast
reaches the aorta. Optimization of the
arterial phase may be determined by using
fluoroscopic triggering , an automated bolus
detection technique104, a timing run, or with
a ‘‘best guess’’ method. Evaluation of the
renal parenchyma is performed with a
second breath-hold three-dimensional fat-
suppressed T1-weighted spoiled GRE sequ-
ence in the axial plane.105 This is performed
before and after the administration of
intravenous gadolinium. The postcontrast
acquisition is obtained after the MR
venogram, approximately 3 to 5 minutes
282 Biomedical Magnetic Resonance: Proceedings of the International Workshop
after the gadolinium bolus. MR urography
may be performed with T2- weighted turbo
spin-echo sequences using a thick slab
projection technique or with multiple
contiguous thin sections. Alternatively, a
delayed three-dimensional T 1 -weighted
GRE sequence after the administration of
gadolinium contrast material can be
performed106.
Renal Masses
The most important aspect in analyzing a
renal mass is to demonstrate the presence
or absence of enhancement. An enhancing
mass implies a vascular mass, consistent with
a neoplasm. Once a lesion has been shown
to demonstrate enhancement, it is necessary
to characterize it as a surgical lesion (renal
cell carcinoma, oncocytoma, or transitional
cell carcinoma) or a nonsurgical lesion
(metastases, lymphoma, or angiomyo-
lipoma).
Renal Cell Carcinoma
Renal cell carcinoma is the most common
renal neoplasm accounting for 80 to 85
percent of all malignant renal tumors and
for 2 percent of all cancers.107 Renal cell
carcinomas have variable signal intensity on
T1- and T2-weighted sequences.With regard
to the background renal parenchyma, they
are often slightly hypointense on T 1 -
weighted images and isointense to slightly
hyper-intense on the T2-weighted images.
Renal cell carcinomas, however, may
demonstrate any signal intensity depending
on their content of hemorrhagic material.
The diagnosis of renal cell carcinoma rests
on demonstrating enhancement within a
renal mass. The prognosis of renal cell
carcinoma is related to the tumor stage. MR
imaging has been shown to be accurate for
the staging of renal cell carcinoma and more
accurate than CT for the evaluation of tumor
extension into the renal vein and inferior
vena cava.108 It is important to demonstrate
the most cephalad extent of thrombus in
the inferior vena cava, because the surgical
approach is altered if thrombus approaches
the right atrium. For this the multiplanar
capability of MR imaging is ideally suited.
In addition, it is often possible to differen-
tiate enhancing tumor thrombus from bland
thrombus. MR imaging does not offer any
advantage when compared with CT for
evaluating retroperitoneal adenopathy.
Angiomyolipoma
Angiomyolipoma (renal hamartoma), a
benign tumor, is composed of varying
amounts of fat, smooth muscle, and blood
vessels. They are uncommon lesions with a
prevalence of 0.3 to 3 percent. Patients are
usually asymptomatic and angiomyolipomas
are usually incidentally discovered when the
patient is imaged for another reason. When
large, angiomyolipomas may exert mass
effect on the adjacent organs and cause
symptoms. In addition, patients with large
Angiomyolipomas may present with acute
flank pain caused by spontaneous hemor-
rhage. Angiomyolipoma is the only renal
tumor that may be characterized on the basis
of its tissue composition and signal charac-
teristics. The relative amounts of fat, smooth
muscle, and vessels within the tumor
establish its MR imaging appearance. The
diagnosis of angiomyolipoma rests on
demonstrating the presence of macroscopic
fat within the lesion. The hemorrhagic cysts
may also show similar signal characteristics.
It is imperative to compare the T1-weighted
images obtained with frequency-selective
fat-suppression with those obtained without
fat-suppression, to establish the presence or
absence of macroscopic fat. Some angio-
myolipomas contain only a tiny amount of
macroscopic fat and a concerted effort

should be made to identify even small
amounts of fat. In rare instances these lesions
may not contain any fat. In such cases, the
diagnosis of angiomyolipoma cannot be
made and the lesion is indistinguishable
from a renal cell carcinoma. Caution should
be used in diagnosing a renal mass as an
angiomyolipoma if it loses signal on out-of-
phase imaging, because clear cell carcinoma
of the kidney may show identical find-
ings.109 Clear cell carcinoma does not contain
bulk fat, however, and does not lose signal
on frequency selective fat-suppressed T1-
weighted images.
Lymphoma
Lymphoma may involve the kidneys by
hematogenous spread, in which a single mass
or multiple bilateral masses are present, or
by direct extension of retroperitoneal
lymphoma. Generally, most patients with
renal lymphoma have systemic involvement
and the diagnosis should not be difficult,
given the appropriate clinical history. The
MR imaging appearance of lymphoma is
nonspecific; however, the most common
appearance is that of multiple homogeneous
solid masses that may be well defined, but
tend to have infiltrative margins with the
kidney. When lymphoma diffusely infiltrates
a kidney, the kidney enlarges, but maintains
its reniform shape.110
Simple and Complex Renal Cysts
The appearance of complex cystic renal
masses is diverse and the proper manage-
ment of these lesions is frequently not
clear cut. Bosniak111-114 has proposed a classi-
fication system designed to help categorize
cystic lesions into surgical and nonsurgical
cases. Although the classification scheme is
based on CT criteria, the same approach
Body MR Imaging 283
provides a useful framework for MR
imaging.115 When evaluating a complex cystic
renal mass on an MR examination, it is
necessary to analyze the various components
of the lesion. This includes the number of
septae, the thickness of the wall or septae,
the interface of the lesion with the kidney,
the contents of the lesion, and most
importantly, the presence or absence of
enhancing soft tissue components. MR
imaging is ideally suited for characterizing
hemorrhagic cysts, particularly in those
patients who cannot receive iodinated
contrast secondary to a history of renal
failure or allergy. This is especially true in
patients with acquired cystic disease of
dialysis or with autosomal-dominant poly-
cystic kidney disease, in which hemorrhagic
cysts are very common. Using a subtraction
algorithm, it is possible to demonstrate that
these lesions do not enhance, and thereby
characterize them as benign hemorrhagic
cysts A limitation of MR imaging in
characterizing cystic renal masses is the
inability to depict calcification within the
wall or septum of a lesion. With CT, it is
sometimes difficult to determine enhance-
ment of a heavily calcified lesion.
MR Imaging of the Adrenal Glands
With MR imaging, it is possible to charac-
terize some adrenal lesions by means of
their signal characteristics on different pulse
sequences or by their enhancement charac-
teristics. These include adenoma, myelo-
lipoma, hematomas, and cysts. In addition,
the multiplanar capability of MR imaging
allows improved depiction of the relation-
ship of the adrenal gland to the kidney. It
is sometimes difficult to differentiate an
exophytic lesion arising from the upper pole
of the kidney from an adrenal lesion, a
relationship not optimally demonstrated
284 Biomedical Magnetic Resonance: Proceedings of the International Workshop
with conventional axial images. The capa-
bility of obtaining images in innumerable
planes is an advantage of MR imaging.
Adrenal Adenoma
The incidence of adrenal adenomas in the
general population is estimated at 2 to 8
percent.116 The adrenal gland is also the
most common site of metastases perunit
weight of any organ. Within the oncologic
population, it is common to find an adrenal
mass, and a frequent clinical problem is to
determine the etiology of such a lesion. MR
imaging accurately can distinguish an
adenoma from a metastasis in most cases.
Various MR techniques have been proposed
to characterize adrenal masses as adenoma
or a metastasis. The easiest, fastest, and
most reliable way to diagnosis an adrenal
adenoma, however, rests on demonstrating
intracellular lipid within the mass (lipid-rich
adenoma). By using chemical shift techniques
(breath-hold T1- weighted GRE images in
phase and out of phase) it is possible to
characterize lipid-rich adenomas. These
adenomas contain intracellular lipid and
water protons within the same imaging
voxel. On out-of-phase images, the signal
from these protons cancel each other out
and result in signal loss when compared
with the in-phase images. Frequently, signal
loss on opposed-phase imaging is obvious.
Adrenal Cortical Carcinoma
Adrenal cortical carcinoma is a rare neo-
plasm that most commonly occurs in the
fourth to fifth decades.They are typically
large (>5 cm) at presentation, may contain
varying degrees of hemorrhage and necrosis,
and often contain calcium.116 The most
common hormone produced is cortisol,
which manifests as Cushing’s syndrome. The
signal intensity of adrenal cortical carci-
nomas is variable and they generally are
heterogeneous on T 1 - and T 2 -weighted
sequences (Figs 20.5A to C). This is
secondary to necrosis and hemorrhagic
components that are common within these
lesions. After the administration of
gadolinium, the viable portion of the tumor
enhances. Adrenal cortical carcinomas may
grow to be very large and may directly
invade adjacent organs including the kidney,
liver, spleen, pancreas, and diaphragm. At
times, it may be difficult to determine the
exact organ of origin, especially when a
normal adrenal gland cannot be identified.
Imaging in the coronal or sagittal plane is
very helpful in showing the relationship of
the tumor to its surrounding structures and
demonstrating the suprarenal location of an
adrenal mass. Adrenal cortical carcinoma has
a predi-lection to invade the adrenal veins,
grow into the renal vein and inferior vena
cava, and extend cephalad toward the heart.
Gadolinium-enhanced MR imaging can
clearly demonstrate the venous extension
of an adrenal cortical carcinoma.
Pheochromocytoma
Pheochromocytomas are neoplasms of the
adrenal medulla that produce catechola-
mines. Pheochromocytomas are extra-
adrenal, bilateral, or malignant 10 percent
of the time. Although patients may be
symptomatic, the symptoms are nonspecific,
and include palpitations, headache, sweat-
ing, and hypertension. The MR imaging
appearance of pheochromocytoma has
classically been described as markedly
hyperintense on T 2 -weighted sequen-
ces117,118 (Fig. 20.6). Subsequently, it has been
demonstrated that pheochromocytomas may
have variable signal on T 2 -weighted
sequences, especially when they are greater
than 5 cm.118 MR imaging is more useful in
 Body MR Imaging 285

A B

C
Figs 20.5A to C: Adrenal carcinoma
A. Axial T1W (FLASH) image showing, large mass lesion in right adrenal area with hyperintense areas inside
suggestive of hemorrhagic areas
B. Axial T2W image showing, heterogeneous appearance of the mass lesion
C. Coronal T2W image showing that mass is arising separate from the right kidney

identifying an adrenal mass in a patient who adrenal pheochromocytomas (paraganglio-

is clinically thought to have a pheochro- mas) in the retroperitoneum along the
mocytoma than in characterizing an adrenal paraspinal muscles. Confirmation with
mass as a pheochromocytoma. Furthermore, nuclear medicine studies is useful in
MR imaging is useful in identifying extra- equivocal cases.
286 Biomedical Magnetic Resonance: Proceedings of the International Workshop

A B
Figs 20.6A and B: Adrenal pheochromocytoma
A. Axial T1W (FLASH) image showing, isointense mass lesion in right adrenal area
B. Axial T2W image showing that mass is markedly hyperintense

Myelolipoma discovered on cross-sectional imaging.

Patients with these lesions are usually
Adrenal myelolipoma is a rare nonfunc-
asymptomatic unless they are large enough
tioning benign neoplasm that contains a
to produce mass effect on adjacent organs.
variable amount of hematopoietic tissue and
Adrenal cysts have been subdivided into
fat. In general, they are asymptomatic and
four main categories: (1) endothelial
are incidental findings at ultrasound, CT,
(angiomatous or lymphangiectatic); (2)
or MR imaging, but may cause pain if they
epithelial; (3) pseudocysts; and (4) parasitic.
hemorrhage or are large enough to exert
Pseudocysts may be post-traumatic or
mass effect on the adjacent organs. The
postinfectious. At MR imaging, simple
diagnosis of myelolipoma rests on the
adrenal cysts are usually hypointense on T1-
demonstration of macroscopic fat within an
weighted and hyperintense on T2-weighted
adrenal mass. With MR imaging, the fatty
images. Some pseudocysts may contain
portion of the lesion is hyperintense on T1-
hemorrhage, however, and their signal
weighted images. This is nonspecific and
intensity on different pulse sequences can
can be seen in any lesion that contains
vary. The wall of an adrenal cyst should be
hemorrhage. Just as in diagnosing a renal
thin, without nodular or enhancing compo-
angiomyolipoma, it is necessary to perform
nents. In addition, calcification may occur
a frequency-selective fat-suppressed T 1-
within the wall, which is depicted better
weighted sequence and compare it with the
with CT than with MR imaging. If only
non-fat-suppressed T1-weighted sequence.
unenhanced MR images are obtained it may
Adrenal Cysts, Pseudocysts, be difficult, if not impossible, to differentiate
and Hematomas an adrenal cyst or pseudocyst from a classic
pheochromocytoma, both of which are
Cysts and pseudocysts of the adrenal gland hyperintense on T2-weighted sequences. In
are rare and are usually incidentally this situation, a gadolinium-enhanced study

can reliably distinguish these two entities
because a pheochromocytoma enhances and
an adrenal cyst or pseudocyst does not.
MR Imaging in the Evaluation
of Bladder Cancer
Bladder cancer is a common tumor of the
urinary tract, accounting for 6 to 8 percent
of male malignancies and 2 to 3 percent of
female malignancies.119 The most common
histologic cell type is transitional cell carci-
noma, accounting for 90 percent of all
primary tumors of the urinary bladder.
Cross-sectional imaging aids in determining
tumor stage, including detection of local
extension of the tumor, lymph node involve-
ment, and distant metastasis.120 Presently,
magnetic resonance (MR) imaging is the
modality of choice in imaging of urinary
bladder neoplasms, and staging accuracy of
MR imaging in bladder cancer ranges from
73 to 96 percent. These values are 10 to 33
percent higher than those obtained with
computed tomography (CT).
Technique
Techniques of Bladder MR Imaging: Lack of
bladder distension may limit the detection
of small tumors secondary to detrusor
muscle thickening. Overdistension of the
bladder may result in patient motion and
can decrease sensitivity for plaque-like
lesions. Optimal bladder distension is
achieved by instructing the patient to void
approximately 2 hours before imaging.121
Currently, a 1.5 T MR scanner with a
phased-array pelvic coil is commonly used
for imaging of the urinary bladder. The
typical urinary bladder protocol includes T1-
weighted spin-echo images obtained in the
axial plane and T2-weighted fast spin-echo
images also obtained in the axial plane.
Subsequently, fast multiplanar spoiled
Body MR Imaging 287
gradient-echo images with fat suppression
are obtained in the axial plane before, at 20
seconds (arterial phase), and at 70 to 115
seconds (venous phase) after gadopentate
dimeglumine injection (0.1 mmol/kg). Sagit-
tal or coronal T2-weighted images may be
obtained, if the anteroposterior or infero-
superior extent of disease needs to be
evaluated. Sagittal and coronal gadolinium-
enhanced images are also recommended for
the evaluation of the bladder tumors, when
the tumor is located in the base or the dome
of the bladder.
Bladder Cancer
Pathologically, 90 percent of the bladder
tumors are epithelial, and 90 percent of the
epithelial tumors are transitional cell
carcinomas. At the time of presentation, 30
percent of patients have multifocal disease,
and there may be widespread areas of
metaplasia and carcinoma in situ.119
Staging and Management of Bladder Cancer:
Local extension of the tumor (T), lymph node
involvement (N), and distant metastasis (M)
are the indicators used in staging. Treatment
options and prognosis depend on the clinical
stage of the bladder tumor. It is critically
important to differentiate between super-
ficial and invasive disease.119 Superficial
tumors are treated with local endoscopic
resection, with or without adjuvant
intravesical instillations of chemotherapeutic
agents; while invasive tumors are treated
by curative cystectomy and palliative
chemo- and/or radiation-therapy.122
CT has been a valuable tool for the
evaluation of bladder tumors, but MR
imaging has been shown to be superior in
the detection of superficial and multiple
tumors, and in staging accuracy in detecting
extravesical tumor extension and surround-
ing organ invasion, especially with dynamic
contrast administration.123-129
288 Biomedical Magnetic Resonance: Proceedings of the International Workshop
Morphologic features: Tumors can arise
anywhere in the bladder, but they are most
commonly located in the lateral wall. When
a tumor is located around the ureteral
orifices, it may produce partial or complete
blockage of one or both ureters, resulting
in hydroureter and hydronephrosis. 130
Tumors manifest a variety of patterns of
growth, including papillary, sessile, infiltra-
ting, nodular, mixed, and fat intraepithelial
growth. Because the bladder does not have
a distinct basement membrane, it is difficult
to detect the invasion of the lamina propria
by imaging. 131 However, the detrussor
muscle can be well evaluated with MR
imaging; therefore, the depth of muscle
invasion can be assessed. The fat tissue
around the bladder can be seen clearly on
MR imaging, and the interface between the
bladder wall and the surrounding fat can
be evaluated for extravesical tumor spread.
MR imaging features: Urinary carcinomas have
intermediate signal intensity, equal to that
of muscle on the T1-weighted images. T1-
weighted images are used to assess
perivesical fat invasion and lymph node
involvement. T2 -weighted images aid in
determining the depth of tumor infiltration
into the bladder wall. On T 2 -weighted
images, urine has high signal intensity, and
the bladder wall appears hypointense.
Bladder tumors have the same, or slightly
higher, signal intensity as the bladder wall
on T2 -weighted images. An intact, low-
signal-intensity muscle layer at the base of
the tumor is classified as stage Ta or T1; a
disrupted low-signal-intensity muscle layer
without infiltration of perivesical fat is
classified as stage T2b.132
Urinary bladder carcinomas and their
metastases develop neovascularization133;
therefore, these tumors enhance earlier than
the normal bladder wall.
In the arterial phase of contrast enhance-
ment, bladder tumors enhance more than
the muscle of the bladder.
Based on MR imaging features, it is not
possible to differentiate between different
histologic cell types of bladder tumors
MR Imaging of Prostate
Staging of Prostate Cancer
To date, the American Urologic Association
staging system, based on that of Whitmore
and Jewett, is the one most widely used in
North America.134,135
Technique of Prostate MR Imaging
MR imaging and MR spectroscopic imaging
allow a ‘‘one-stop shop’’ for detailed anato-
mic and metabolic evaluation of the prostate
gland. Neither TRUS nor CT can offer this
simultaneous coverage and tissue detail. MR
spectroscopic imaging is a method of
demonstrating normal and altered tissue
metabolism, and is therefore fundamentally
different from other imaging modalities that
only assess abnormalities of structure. MR
imaging, and especially MR spectroscopic
imaging, are relatively new technologies in
continued evolution. T1-weighted MR imag-
ing T1-weighted images of the pelvis aid in
the detection of postbiopsy hemorrhage,
lymphadenopathy, and bone metastases.
T2-weighted images in the axial and coronal
planes are the cornerstone of MR imaging
in prostate-cancer evaluation. These images
clearly depict the distinction between the
peripheral zone and the central gland, allow
the detection of tumor as low T 2 signal
intensity foci in the peripheral zone, and
facilitate staging by demonstration of
extracapsular extension and seminal-vesicle
invasion.136-139

Prostatic Anatomy as Seen on MR Imaging
The zonal anatomy of the prostate cannot
be distinguished on T 1-weighted images
because of uniformly intermediate signal
intensity. The prostatic zones are well
demonstrated on T2-weighted images. The
anterior fibromuscular stroma is of low T1
and T2 signal intensity. The peripheral zone
is of high T2 signal intensity, similar to or
greater than the signal of adjacent peri-
prostatic fat. The anatomic or true capsule
surrounding the peripheral zone appears as
a thin rim of low signal intensity on T2-
weighted images. The central and transition
zones are of lower T2 signal intensity than
the peripheral zone, possibly because of
more compact smooth muscle and sparser
glandular elements.140
Identification of Prostate Cancer by
Magnetic Resonance Imaging
It is generally accepted that the direct MRI
manifestation of prostatic cancer is the
presence of low signal intensity (SI) on T2
weighting. This is typically seen as an island
of low signal enclosed by high signal from
surrounding benign peripheral zone tissue.
False positive MRI studies were caused by
dense fibromuscular stroma. The causes of
false-negative studies were thought to be
multiple but chiefly reflected similarity in
water content with surrounding tissue,
particularly adjacent BPH. MRI is highly
reliable in identifying palpable tumors, with
a sensitivity of 96 percent.136,138,139
Staging of Prostate Cancer by
Magnetic Resonance Imaging
The critical medical problem presented once
the diagnosis of prostate cancer has been
made is the ascertainment of resectability.
Body MR Imaging 289
Disease confined to the gland (A-B,T1-2) is
resectable for cure. Disease outside the gland
(C,T3), whether penetrating the capsule or
invading the seminal vesicles, is unlikely to
be cured by surgery or other means. The
criterion for extracapsular disease was the
presence of a low-signal region on T2 scan
transgressing the capsule or periprostatic
venous structures adjacent to the peripheral
zone. Invasion of the seminal vesicle was
likewise considered to be present if low T2
SI was seen.
A further issue relevant to consideration
of curative surgery for patients with
clinically localized prostatic cancer concerns
the presence of tumor in the vicinity of the
neurovascular bundle (NVB). NVB is
located between the prostatic capsule and
Denonvillier’s fascia, at the 5- and 7-o’clock
positions just outside the posterolateral
margins of the prostate and at the
anterolateral borders of the rectum, carries
the pelvic nerves innervating the corpora
cavernosa. Interruption of the neurovascular
bundles is responsible for the certainty of
permanent impotence associated with
traditional radical prostatectomy. Preser-
vation of one bundle will usually preserve
potency. Imaging is performed with T 1
weighting to assess the integrity of the
periprostatic fat plane and to outline the
NVB and with T2 weighting to identify
prostatic cancer directly.141-143
Newer Techniques of Magnetic
Resonance Imaging
The phased-array coil is one of the
enhancements recently introduced in an
effort to improve the resolution of pelvic
MRI. A combination of fast spin-echo pulse
sequences and phase-array coils provides
an opportunity to combine a high signal-to-
290 Biomedical Magnetic Resonance: Proceedings of the International Workshop
noise ratio with more signal averages
obtained in a shortened imaging period.
The use of the endorectal surface coil
has been the subject of much interest for
improving the resolution of MRI prostatic
imaging. An improvement in staging
accuracy of 16 percent over body coil
technology is described in early experience.
Criteria for capsular stage C disease were
infiltration of periprostatic fat, obscuration
of periprostatic veins, involvement of the
neurovascular bundle, disruption of the
capsule, and a bulge in the capsular contour
defined as bulging in the glandular surface,
making an angle with adjacent peripheral
zone.144-146
MR Imaging of Female Pelvis
MR imaging is becoming an increasingly
important tool in the diagnosis of benign
and malignant disease of the female pelvis.
MR imaging is now routinely used in the
diagnostic work-up of infertility, including
mullerian anomalies, and chronic pelvic pain.
Both CT and MR imaging can be used to
determine the origin of and to characterize
a pelvic mass. In obstetrics, MR imaging is
used to assess maternal complications of
pregnancy and to identify or confirm fetal
anomalies.
MR Imaging Technique
It is also advisable to have the patient void
before the examination to limit deformation
of adjacent organs by an enlarged bladder.
Although imaging using the body coil is
certainly adequate for most diagnoses, the
highest resolution images are obtained using
a phased array. Saturation bands placed
along the anterior and posterior body wall
fat are useful to diminish near field artifact.
Smaller field-of-view sagittal, axial, and
coronal T 2 -weighted images are then
obtained because they contain the most
information about the pelvic organs. A pulse
sequence such as fast or turbo spin-echo,
yields the highest resolution images. After
all important T2 imaging is obtained, the
need for further imaging is variable. In the
case of benign disease of the uterus, no
further imaging is necessary. When charac-
terizing a pelvic mass or staging a pelvic
malignancy, dynamic, axial, or sagittal
images of the pelvis should be obtained at
20 seconds, 40 seconds, and 3 to 5 minutes
following the administration of intravenous
contrast medium. Fat saturation, although
not essential, is useful for highlighting areas
of enhancement.
Normal Anatomy
The uterus has three distinct zones: (1) the
high T2 signal endometrium, (2) the low T2
signal junctional zone, and (3) the inter-
mediate T2 signal myometrium. The endo-
metrium, composed of high signal glands,
may measure upto 1.5 cm in thickness in
women of menstrual age and 5 mm in
postmenopausal women. The junctional zone
has been shown to consist of compacted
myometrial cells with a high nuclear to
cytoplasmic ratio.147 The junctional zone
should measure no more than 11 mm in
thickness.148 In women of menstrual age,
the ovaries should be identified in virtually
every case. They are ovoid structures
containing high signal follicles. In postmeno-
pausal women the ovaries are atrophied and
identified in only 40 percent of cases.
Benign Conditions
Mullerian anomalies: Although often initially
diagnosed using HSG, MR imaging should
be performed when surgical therapy is

considered to characterize more accurately
the anomaly. It is particularly important to
differentiate a septate from a bicornuate
uterus, each of which requires different
therapy149 (Figs 20.7A and B).
Minimal failure of fusion leads to the
arcuate uterus with a U-shaped internal
contour of the fundal portion of the endo-
metrial canal. This is an entirely benign
variant and has no effect on fertility. Failure
of septal resorption is the cause of the
septate uterus. In contradistinction to the
bicornuate uterus, the external contour is
flat or minimally (<1 cm) concave. The
septum may be of variable length and
contain fenestrations. This anomaly is often
treated with hysteroscopic metroplasty.
Failure of development of the urogenital
sinus leads to an absence of the caudal two-
thirds of the vagina and an obstructed cervix
and uterus. The differential diagnosis is
imperforate hymen. On axial T2-weighted
MR imaging of the pelvic floor, the vagina
is absent. A small low T2 signal scar may be
present. Hematometrocolpos manifests as a
heterogeneous, but predominantly high T2
Figs 20.7A and B: Bicornuate uterus
A. Axial TRUE-FISP image showing, two uterine horns separated by myometrium
B. Coronal TRUE-FISP image showing enlarged uterine size with two horns
Body MR Imaging 291
signal mass filling the uterine canal and
markedly thinning the myometrium. Treat-
ment in the young woman often involves
the formation of a neovagina.
Fibroids: Fibroids, or leiomyomata, are
present in an estimated 40 percent of women
over 40 years of age. Symptoms associated
with fibroids include bleeding, pain, and
urinary incontinence. Their presence can also
contribute to infertility. MR imaging is of
value in the symptomatic patient when
uterine salvage therapy is considered. Using
axial and sagittal T2-weighted imaging, most
fibroids, including pedunculated ones, can
be identified with confidence. They should
be described in terms of size, location, and
signal intensity. Any fibroid that impresses
on the endometrial canal is considered to
have a submucosal component and can be a
source of bleeding. Based on T 2 signal,
fibroids can be classified further into groups
depending on their degree of cellularity.150
The most common group is the ordinary
fibroids, of relatively homogeneous low T2
signal and composed of collagenous material
292 Biomedical Magnetic Resonance: Proceedings of the International Workshop
(Fig. 20.8). Cellular fibroids contain less
collagen and are of intermediate T2 signal
intensity. Degenerating fibroids are of very
bright T2 signal intensity When large or
pedunculated they can be difficult to
differentiate from an ovarian neoplasm.
Degenerating fibroids do not respond well
to uterine artery embolization because of
central necrosis.151
Adenomyosis: Adenomyosis is a common
cause of pelvic pain in women of menstrual
age. It is defined as extension of endometrial
glandular tissue more than one third of the
depth of the myometrium with adjacent
muscular hypertrophy. Adenomyosis cannot
be diagnosed using CT. MR imaging remains
the diagnostic test of choice.152,153 On T2-
weighted MR imaging, adenomyosis appears
as poorly defined low T2 signal intensity
confluent with and widening the junctional
zone (>11 mm).154 High T2 signal intensity
glands are often seen within the diseased
area. Treatment options have traditionally
been limited to hysterectomy.
Endometriosis: Laparoscopy remains the test
of choice for diagnosing and staging
Fig. 20.8: Uterine fibroid. Axial T2W image showing,
hypointense mass lesion arising within the
myometrium and causing indentation over the uterine
cavity
endometriosis. Transvaginal US and MR
imaging are useful only when the diagnosis
of an endometrioma is suspected. Endo-
metriomas may be unilocular or multilocular
and are predominantly high signal on T1-
and low or mixed signal on T2-weighted
images.155 On T2-weighted images, inter-
mediate signal shading, caused by T 2
shortening of blood products, is often seen
within the mass.156
Polycystic ovarian disease: The triad of hirsu-
tism, obesity, and amenorrhea comprises
Stein-Leventhal syndrome. Although the
biochemical underpinnings of this syndrome
are variable, most patients have elevated
levels of luteinizing hormone. The classic
US and MR imaging appearance of a
polycystic ovary is increased central stroma
with peripheral location of follicles.157 The
ovary is enlarged (>4 cm maximal diameter)
in one third of cases.
Dermoid: Mature teratoma or dermoid is an
ovarian mass composed of tissue arising
from the endoderm, mesoderm, and ecto-
derm. On MR imaging, fat can be identified
using fat-suppression pulse sequences.158 The
chemical shift artifact consisting of an
adjacent black and white line at the edge of
the teratoma in the frequency encoding
direction is also diagnostic of macroscopic
fat. On T2-weighted images, the speckling
artifact is diagnostic. It is composed of
myriad chemical shift artifacts occurring
within the Rokitansky nodule.
Gynecologic neoplasms: Most gynecologic
oncologists stage pelvic neoplasms according
to the FIGO system (International Federation
of Gynecology and Obstetrics). Performing
a single MR exam of the pelvis can replace
all of the ancillary examinations with the
exception of the chest radiograph. MR
imaging should consist of sagittal and axial

T2 -weighted images, optimally obtained
with a multicoil array and fast-pulse sequen-
ces and dynamic images immediately post-
intravenous administration of gadolinium
pentetic acid (Gd-DTPA). These contrast-
enhanced images better characterize cervical
and ovarian masses and can separate them
from adjacent structures, such as the bladder
and rectum.
Endometrial cancer: MR imaging is recommen-
ded when locally advanced disease is
expected based on physical examination
findings and in the patient with a difficult
physical examination because of obesity,
prior radiation, or surgery. Sagittal and axial
T2-weighted images and contrast-enhanced
images should be performed and scrutinized
for extension of hyperintense tumor into or
through the myometrium. Preservation of a
subendometrial band of enhancement
virtually excludes myometrial extension.147
Invasion of greater than 50 percent of the
myometrial wall thickness is strongly
indicative of involved lymph nodes and a
poorer overall prognosis.159 Metastases can
extend by the fallopian tubes to the ovaries
causing development of an adnexal mass.
Cervical cancer: Cervical cancer is usually a
disease of women of menstrual age that
plateaus at age 40. Any cervical mass that is
greater than 1.5 cm or presumed to extend
beyond the cervix should be examined using
MR imaging. CT scanning lacks the contrast
resolution On T2 -weighted MR imaging,
cervical cancer is usually hyperintense to
the dark cervical stroma. The preservation
of the black ring of the cervical stroma, no
matter how thin, virtually excludes para-
metrial extension. These patients are candi-
dates for surgical cure (stage IIA). Those in
whom the black line is broken and a mass
extends beyond the expected confines of
the cervix are usually treated primarily with
Body MR Imaging 293
brachytherapy (stage IIB). In cases of
advanced disease, sagittal T2-weighted or
Gd-DTPA enhanced T1-weighted images
show invasion of the rectum, bladder, and
vaginal fornices.148,160
Ovarian cancer: Ovarian cancer is a disease
of perimenopausal women with its peak
incidence between the ages of 45 and 55
years. Most tumors are of epithelial origin
and are serous or mucinous cystadeno-
carcinomas. A recent radiologic diagnostic
oncologic trial showed that US, CT, and
MR imaging are equal in the detection and
overall staging of ovarian cancer.161 MR
imaging has a slightly higher detection rate
for peritoneal metastases. 162 In routine
practice, CT is usually preferred because it
is well accepted by the patient and provides
a rapid overall view of the abdomen and
pelvis that is well understood by referring
physicians. MR imaging, however, is
particularly useful in the detection of
recurrent disease because its tremendous
contrast sensitivity yields excellent identi-
fication of even very small cystic implants.
Musculoskeletal MRI
Over the past 10 to 15 years, magnetic
resonance imaging (MRI) has had a major
impact on musculoskeletal imaging. Excellent
soft-tissue contrast, spatial resolution, and
multiplanar imaging are among the major
advantages of MRI. Although noncontrast-
enhanced MRI is sufficient for the evaluation
of many musculoskeletal conditions, the
addition of either intravenous (IV) or intra-
articular gadolinium-based contrast agents
is, at times, necessary to improve the
evaluation of certain disease processes.163
Bone Tumors
The superior contrast discrimination pro-
vided by magnetic resonance (MR) imaging
294 Biomedical Magnetic Resonance: Proceedings of the International Workshop
has proved extremely valuable in the assess-
ment of bone and soft-tissue tumors.164-167
In the evaluation of tumors, a combi-
nation of both T 1 and conventional T 2
weighting, fat-suppressed T2-weighted fast
spin-echo, or gradient-echo techniques is
essential. With short T1 inversion recovery
(STIR) imaging sequences, T1 and T2 contrast
are additive. 168,169 T 1 -weighted images
provide excellent contrast for identification
of marrow, cortical, and soft tissue involve-
ment. In particular, T1 -weighted images
allow differentiation between fat and tumor,
as well as definition of muscle planes and
separate anatomic compartments.
Gadolinium has been used to enhance
contrast on T1-weighted images to better
characterize osseous and soft-tissue tumor
involvement.170-172 On gadolinium enhanced
images, regions of low signal intensity are
thought to represent areas of nonviable
tumor or necrosis. Gadolinium-enhanced
images may also be useful for differentiating
peritumoral edema from underlying tumor
and recurrent tumor from scar or fibrosis.173
Only the areas of solid tissue show
significant enhancement. This information
may be quite useful in directing biopsy to
the solid enhancing portions of the lesion
that harbor the diagnostic tissue, as opposed
to the cystic, necrotic, or hemorrhagic
nondiagnostic components.
Dynamic-enhanced MRI: The response of
high grade osteosarcoma and Ewing’s
sarcoma to chemotherapy with dynamic
enhanced MRI has been studied by several
investigators who proposed evaluation
based on rather complicated schemes
relating to factor analysis of dynamic MRI
sequences174 and regions of interest.175-177
Shapeero and Vanel178 have proposed a less
cumbersome method for using dynamic
contrast enhancement to evaluate the degree
of tumor necrosis and tumor response to
chemotherapy. For osteosarcoma, the
absence of early enhancing nodules or the
presence of only one small early enhancing
nodule suggests greater than 90 percent
tumor necrosis, representing a good
response to the chemotherapy Masses with
early and persistent enhancement suggest a
poor response to chemotherapy in both
osteosarcoma and Ewing’s sarcoma.
T 2 -weighted images are valuable in
distinguishing muscle from tumor and can
increase diagnostic specificity in evaluating
marrow infiltration, seen as areas of low
signal intensity on T1-weighted images.
Although each MR examination must be
tailored to the patient and pathology in
question, certain general guidelines can be
followed. The longitudinal extent of tumors
can be seen on T 1 -weighted coronal or
sagittal images. T1- and T2-weighted images
(including fat-suppressed T2-weighted fast
spin-echo or STIR sequences) in the axial
plane provide the most important images in
delineating the relationship of a tumor to
adjacent neurovascular structures and
compartments essential information in
preoperative limb salvage planning. Prior
to imaging the particular region of interest,
a large field-of-view localizer using an
increased diameter surface coil or body coil
may be necessary to exclude skin lesions or
to accurately determine the proximal and
distal extension of a large mass. For lesions
of the appendicular skeleton, it is crucial
that at least one long axis sequence be
performed through the entire involved bone
to evaluate for the presence of skip meta-
stases. Skip metastases represent a second
site of disease in the same bone as the
primary tumor separated by an area of
normal marrow. The presence of skip lesions
is very important to determine because they
may significantly worsen patient prognosis

and must be removed if limb salvage sur-
gery is to be successful.
General MR Appearances
Direct multiplanar imaging facilitates
improved preoperative and pretherapy
evaluation of the exact extent of a lesion.
It is often not possible to distinguish benign
from malignant lesions of bone and soft
tissue on the basis of MR signal charac-
teristics alone. Both benign and malignant
lesions may demonstrate areas of low signal
intensity on T 1 -weighted images and
increased signal intensity on T2-weighted
images (Figs 20.9A to D). Malignant lesions
tend to be more extensive, involving
marrow, cortical bone, and soft tissues; but
these criteria do not always distinguish
benign from malignant lesions. No
correlations have been shown matching T1
and T2 relaxation times with corresponding
histopathology. 179 If the neurovascular
bundle is involved, the lesion is likely to be
malignant. A fluid-fluid level is a nonspecific
finding in bone and soft-tissue lesions.
Tumors with fluid-fluid levels include
fibrous dysplasia, simple bone cysts,
malignant fibrous histiocytoma (MFH),
osteosarcoma, aneurysmal bone cysts,
hemangioma, and synovial sarcoma.180
Staging: Proper staging of osseous neo-
plasms is essential for planning effective and
appropriate therapy. There are two widely
used staging systems for primary tumors of
osseous origin: the system developed by
Enneking and the Musculoskeletal Tumor
Society in 1985, and that developed by the
American Joint Committee on Cancer. These
systems are very similar and emphasize the
same basic prognostic factors.181
Tumor grade: The first component of the
Enneking staging system is tumor grade. In
the Enneking system, grade takes into
Body MR Imaging 295
account not only histologic appearance but
also radiologic assessment and clinical
behavior. Grade 0 lesions have a benign
biologic behavior. They may have an
indolent or aggressive course. It is important
to remember that, for these lesions, the
radiographic appearance may be more useful
for determining biologic activity than the
histologic grade.182 Grade 2 lesions are high-
grade malignant neoplasms. Grade 1
tumors, classified as low-grade malignant
neoplasms, have a biologic behavior in
between these two extremes.
Site: The second component of the staging
system is site. T0 lesions remain within their
capsule or pseudocapsule and involve only
one compartment. Extension of the primary
tumor or its reactive zone beyond the
pseudocapsule without extracompartmental
spread characterizes T1 lesions. These lesions
are limited by anatomic barriers, such as
articular cartilage, cortical bone, joint
capsule, or tendons and ligaments. 182,183
Extension of the reactive zone beyond the
compartment of origin also obligates a T2
designation because this area may often
contain satellite nodules or tumors projec-
tions.184 Neurovascular involvement also
defines a lesion as T2.
Soft tissue mass: Although CT and MRI can
both provide information regarding the
presence of soft tissue extension, most
studies clearly indicate the superiority of
MRI in defining the precise extent of
extraosseous disease.176,185 The presence of
a soft tissue mass indicates involvement of
a second compartment, which increases the
tumor stage and has significant implications
not only regarding staging but also therapy.
Involvement of major neurovascular struc-
tures is critical information to the orthopedic
296 Biomedical Magnetic Resonance: Proceedings of the International Workshop

A B

C D
Figs 20.9A to D: Pelvic bone chondrosarcoma
A. AP Plain radiograph of pelvis showing large mass lesion involving the right sided pelvic bones associated
with large soft tissue component having chondroid type calcification
B. Coronal STIR image showing true extent of the lesion
C. Axial T1W image showing that mass is hypointense having intrapelvic component as well
D. Axial T2W image showing, heterogeneous appearance of the lesion

oncologist in surgical planning. A fat plane
of varying diameter surrounds virtually all
major neurovascular bundles, aiding in
radiologic determination of tumor involve-
ment.
Cortical involvement: Early cortical involve-
ment or localization of a lesion in the cortex
is important to recognize. Subtle cortical
disruption suggests early extracompart-
mental extension and a more aggressive
lesion.
Metastases: The final component of the
Enneking staging system is the presence or
absence of distant metastases (M0 or M1).
No differentiation is made between regional
metastases, such as to regional lymph nodes,
and distant metastases, because they both
carry the same prognosis. 182,183 Skip
metastases, as in the case of osteogenic
sarcoma (Figs 20.10A and B), also require a
designation of M1 even without associated
distant metastases The crucial role that the
 Body MR Imaging 297

A B
Figs 20.10A and B: Osteosarcoma of femur
A. AP and lateral radiograph of left femur showing, sclerotic lesion arising from distal metaphysis associated
with osseous matrix within the soft tissue component
B. Coronal STIR image showing true extent of the lesion, associated soft tissue component and the cortical
discontinuity

radiologist plays in the staging process, in
determination of both the extent of tumor
involvement and the presence of metastases,
is obvious.
Musculoskeletal infection: The initial imaging
of osteomyelitis typically consists of conven-
tional radiographs; however, this method
is insensitive and may take up to 10 days to
reveal an abnormality. Radionuclide studies
can be helpful, but are often nonspecific,
and cannot differentiate neoplasm, infection,
and trauma reliably. Non-contrast enhanced
MRI is a cost-effective, sensitive, and specific
study for detecting osteomyelitis. The
reported rates of sensitivity and specificity
for MR detection of osteomyelitis range from
86 to 98 percent and 77 to 100 percent,
respectively. 186 Standard sequences may
include a combination of T1-weighted, T2-
weighted, and/or short T 1 inversion
recovery (STIR) or T2-weighted sequences
with chemical fat suppression. T1-weighted
images are generally best for evaluating
marrow infection. T1-weighted sequences
will show infection as intermediate or low
signal within the bone marrow. Infection is
high in signal on T2 -weighted and STIR
images.186 Chronic and acute osteomyelitis
298 Biomedical Magnetic Resonance: Proceedings of the International Workshop
can be distinguished, as well. Acute disease
causes poor definition of soft tissue planes,
lack of cortical thickening, and indistinct
transition between normal and abnormal
bone marrow. Chronic osteomyelitis will
show cortical thickening and a more readily
defined distinction between the normal and
abnormal tissues.186
In both osteomyelitis and soft tissue
infections, MRI can depict the extent of
infection accurately. Determining the
presence of necrotic tissue or abscess is also
important in treatment planning. Necrotic
tissue is treated surgically, whereas viable
tissue is treated with antibiotics. The
addition of IV contrast will readily demon-
strate areas of necrosis or abscess.
Distinguishing viable from necrotic tissue
will also aid in biopsy or fine-needle
aspiration for culture. If surgery is contem-
plated, the excellent spatial resolution of
MRI will aid in surgical planning. MRI is
also very sensitive for detection of septic
arthritis. Very small joint effusions are
detected readily. The addition of contrast
may reveal enhancement and hypertrophy
of the synovium. These findings may also
be seen in transient synovitis, but the
presence of adjacent signal abnormality
within the bone is more specific for septic
arthritis.186
Marrow Disorders
Conventional radiographic techniques,
insensitive to many marrow infiltrations and
tumors, are limited in providing accurate
bone marrow characterization. Frequently,
there is significant trabecular or cancellous
destruction before disease progression is
detected on standard radiography.
MRI has the major benefit of imaging
bone marrow directly. Multiplanar MR
imaging provides the excellent spatial and
contrast resolution necessary to differentiate
the signal intensities of fatty (yellow)
marrow elements from hematopoietic (red)
marrow elements. MR imaging may thus
become the diagnostic gold standard for
diseases that involve or target the bone
marrow.
The normal distribution and MR
appearance of bone marrow changes with
age. 187-190 An understanding of these
variations is important in examining MR
patterns in appendicular skeletal locations
and determining whether they are potential
disease processes or normal variations of
marrow.
Protocol
The protocol for an MR survey examination
for marrow evaluation uses T1 -weighted
coronal images of the pelvis and proximal
femurs, which are adult sites of red marrow
concentration. These images are acquired
with large (40 cm) fields of view to include
assessment of lumbosacral spine marrow.
Transcoronal STIR images are obtained to
null fat signal and identify abnormal T1 or
T2 prolongation. Fat-suppressed T2 weighted
FSE may be used when thin slice or multi-
planar imaging is required in a limited
period of time.
Axial T1-weighted, fat-suppressed T 2-
weighted FSE, or STIR images may be
obtained at specific sites of suspected
pathologic processes and are important in
determining cross sectional marrow involve-
ment. T1-weighted images are particularly
valuable in evaluating blastic processes,
which are low in signal on STIR and T2*-
weighted images. Gadolinium enhanced
axial images may improve the visibility of
lesions, especially in cases with soft tissue
or cord involvement. Sagittal T1-weighted
and STIR images are routinely acquired to
evaluate suspected spinal malignancies.

Leukemia
In both children and adults, leukemic
marrow involvement is homogeneous,
diffuse, and symmetric. Focal infiltration is
more commonly seen in myelogenous
leukemia. On T1-weighted images, leukemic
hypercellularity is seen as low signal
intensity replacement of higher signal
intensity marrow fat. Due to the greater
proportion of hematopoietic marrow in
children, there is an overlap in the
appearance of normal low signal intensity
cellular hematopoietic marrow and low to
intermediate intensity hypercellular leuke-
mic marrow. Conventional T 2-weighted
images may show increases in signal
intensity in acute leukemia. Unlike the
situation with metastatic disease, however,
T2-weighted images may not be sensitive to
leukemic hypercellularity. Short T1 inversion
recovery imaging techniques offer superior
contrast for demonstrating increased signal
intensity in leukemic marrow, exceeding that
displayed by normal hematopoietic cells.
Nulling of fat signal intensity facilitates the
detection of both focal and diffuse leukemic
infiltrates.
In primary myelofibrosis, T1-weighted
images show patchy marrow involvement
with low signal intensity on T1- and T2-
weighted images. T2*-weighted images have
been used to evaluate areas of susceptibility
where fibrous tissue and hemosiderin have
been identified. With STIR techniques, the
imaging characteristics of areas of involve-
ment are identical to those of normal
hematopoietic marrow (ie, intermediate to
mild increased signal intensity). T 2 * -
weighted images, however, do not display
typical red marrow imaging characteristics
(ie, isointensity with surrounding fat mar-
row).191,192
Body MR Imaging 299
Lymphomas represent neoplastic proli-
feration of the lymphoid cells that normally
reside in primary lymphoid tissue such as
lymph nodes. The two major types of
lymphomas are Hodgkin’s disease and non-
Hodgkin’s lymphoma.
MR imaging, has the advantage of being
able to sample a large volume of marrow,
thus making it possible to detect marrow
involvement. Since identification of marrow
tumor in patients with lymphoma affects
both staging and treatment, this is
potentially one of the more important
clinical applications for MR imaging of the
body. Staging is of particular importance in
Hodgkin’s disease, because bone marrow
involvement portends a potentially fatal
outcome and is therefore treated with more
aggressive multi-agent chemotherapy. MR
imaging can also identify bone marrow
involvement not appreciated at the time of
diagnosis.193-195
Myeloma
Imaging is important in the diagnosis of
multiple myeloma. Involvement of the
marrow is characteristically multifocal—
a finding clearly demonstrated on MR
scans.196 The patchy asymmetric process of
focal deposits is distinguishable from
metastases by signal intensity characteristics
and morphology. Multiple myeloma may
also present as a complete marrow replace-
ment, simulating leukemia. MR imaging of
the spine in early myeloma may show
involvement in both symptomatic and
asymptomatic patients. Areas of involve-
ment demonstrate low signal intensity on
T 1 -weighted images and bright signal
intensity on T2-weighted, STIR, and Gd-
DTPA enhanced images.197 Short T1 inver-
sion recovery images demonstrate greater
300 Biomedical Magnetic Resonance: Proceedings of the International Workshop
lesion contrast than that seen on corres-
ponding conventional T2-weighted images.
Metastatic Disease of the Marrow
Marrow invasion occurs by hematogenous
dissemination, and the high frequency of
metastases to pelvic bones and vertebrae is
attributed to the abundant vascular supply
afforded by the vertebral venous plexus,
which serves as a major venous pathway.198
Metastatic disease has been successfully
detected on MR T 1 -weighted and STIR
images, even when bone scans are negative
or equivocal. MR imaging provides an
excellent means of assessing soft tissue
involvement, including adenopathy, and
Gd-DTPA enhancement is useful in the
assessment of pathologic fractures and
epidural spread. Metastatic lesions may
show isointensity with adjacent vertebral
body marrow on non-fat-suppressed
contrast enhanced images.199 The morpho-
logy of the posterior vertebral body cortex
should be evaluated for convexity, a sign
associated with metastatic tumor extension.
A chronic, benign compression fracture
frequently demonstrates fat marrow signal
intensity (i e, bright on T1-weighted images,
dark on STIR images). In more acute
compression fractures, subchondral reaction
often parallels the endplate without the more
central or heterogeneous involvement seen
in metastatic disease. The “bull’s-eye sign”
(a focus of high signal intensity in the center
of a lower signal intensity osseous marrow
abnormality, seen on T1-weighted images)
as a specific indicator of normal hemato-
poietic marrow and not metastatic dis-
ease. 200 However, both the halo sign (a
peripheral rim of increased signal intensity
around an osseous lesion seen on T 2 -
weighted images) and diffuse signal
hyperintensity on T2-weighted images have
been found to correlate with metastatic
disease. The halo sign, most commonly seen
in osteoblastic metastases from prostate
cancer, is thought to occur secondary to
trabecular destruction with a resultant fluid-
filled gap.201
Whole-body MR Imaging
Whole-body imaging can be afforded by
radiographic skeletal survey, whole-body
CT, scintigraphy and, more recently, by PET.
Each technique has advantages and
disadvantages, which include cost, sensiti-
vity, radiation dose and acquisition time. A
recent modification is the, whole-body
turbo-STIR or TRUE-FISP MRI technique, a
powerful screening tool.
Technique
Whole body imaging can be performed
either by using TRUE-FISP or by turbo-STIR
technique. For the MR examination, the
patient is positioned on the rolling table
platform. The body phased-array coil is
placed at the region of the largest diameter
of the patient with manual movement of
the rolling table platform. Then the surface
coil is positioned in the isocenter of the
magnet by using the automatic movement
of the original patient table. During the
examination, there is no further movement
of the patient table.202
Coronal images are acquired at seven
consecutive stations by sequential table-top
movement. Axial images are acquired in a
similar way by sequential table-top move-
ment through up to seven stations. Sagittal
images are also acquired for the spine.
Sagittal T1-weighted images are acquired to
facilitate detection of sclerotic metastases,
which are often poorly visualized on STIR
weighted scans.

APPLICATIONS
Oncologic Applications
Whole body screening MRI is essentially an
oncologic tool, with primary roles in
assessing spread of disease to the skeleton
as an alternative to bone scans 203-206, in
assessing total tumor burden 207,208 , in
assessing patients with suspected multiple
myeloma, and in evaluating patients
presenting with skeletal metastases without
a known primary tumor.209 Employing fat
suppression, proton - or fluid-rich tumor
deposits become dramatically hyperintense
and conspicuous against a hypointense
background of suppressed signal within fat-
containing structures.
In the assessment of skeletal metastases,
MRI allows direct tumor visualization, in
contrast to observing the secondary response
of induced osteoblastic activity in scinti-
graphy. In this role, whole body turbo-STIR
has been shown to be more accurate than
bone scans, In its role in the assessment of
total tumor burden, screening whole body
turbo-STIR MRI is best suited to the
assessment of tumors that spread to the
skeleton, liver and brain, and soft tissue
sarcomas. Each site is well visualized by
this technique.
Non-oncologic Applications
Recognizing the role of MRI in the assess-
ment of localized muscle ailments, we
recently described the use of whole body
screening turbo-STIR MRI in the assessment
of patients with polymyositis.210 In this role,
whole body MRI allows assessment of
symmetry of muscle involvement, shows the
extent of inflammation, and may identify
an involved muscle group to be targeted
for diagnostic biopsy. In pediatrics, small
children may be screened by whole body
Body MR Imaging 301
MRI in a single field of view. Such limited
coverage allows high-resolution scanning
without a significant impact on acquisition
time. Whole body MRI in children referred
with suspected child abuse, as a single non-
ionizing study may allow the assessment of
the integrity of the entire skeleton, brain
and intra-abdominal viscera.
Limitations and Controversies
Whether or not whole body turbo-STIR MRI
is a reliable tool for simultaneously
evaluating the viscera, hence fulfilling a
wider role in patient assessment including
screening medical examinations, is more
controversial. In such a role, whole body
MRI is as likely to detect insignificant
findings as it is to detect critical pathology.
Such an application would place immense
pressure on the reader to ultimately
determine the validity of encountered
pathologies, and in many cases would
generate psychological trauma to the patient
quite unnecessarily.
Fetal MRI
The Role of MRI in Fetal Diagnosis
There is a growing body of literature on
the use of MRI and has documented its
usefulness in confirming or expanding upon
US findings. In contradistinction to US, MRI
visualization of the fetus is not significantly
limited by maternal obesity, fetal position,
or oligohydrominos, and visualization of the
brain is not restricted by the ossified skull.
It provides superior soft tissue contrast
resolution and the ability to distinguish
individual structures such as lung, liver,
kidney, bowel, and gray and white matter.
Multiplanar imaging is easier with MRI than
it is with US, where it is often a challenge
to obtain images in three planes. MRI
302 Biomedical Magnetic Resonance: Proceedings of the International Workshop
provides a large field-of-view, facilitating
examination of fetuses with large or complex
anomalies, and visualization of the lesion
within the context of the entire body of the
fetus.
MRI has significant disadvantages
compared with US. It tends to be more
expensive, can be difficult for those suf-
fering from claustrophobia, is not as readily
available, and provides less spatial
resolution. Furthermore, MRI is exquisitely
sensitive to fetal motion. Fast imaging
techniques have overcome this to some
extent, and the average MRI sequence,
which historically required 15 minutes, now
takes approximately 20 seconds to acquire.
Technical Considerations
Safety
While the long-term safety of MRI has not
been fully established, it is believed, in
general, that the benefit it provides in fetal
management far outweighs any theoretical
risks. No adverse biological effects have
been reported at the energies currently
delivered with MRI.211 Whether or not to
obtain written consent reflects institutional
bias: at Children’s Hospital Boston, consent
is no longer obtained for performance of
routine fetal MRI, while at Massachusetts
General Hospital, it is. Most imaging occurs
at or after 18 weeks’ gestation, when fetal
size and motion first allow reasonable
visualization with current MRI techniques,
avoiding exposure during the peak of
organogenesis. Gadolinium is not adminis-
tered.
Technique
A 1.5 T magnet is used. Imaging at or after
18 weeks’ gestation is preferred. Beyond
this period, the fetus is large enough to
image many anomalies, and motion is
usually not as problematic. The mainstay of
fetal MR imaging has been fast single-shot
T2-weighted imaging. Body coils and larger
phased-array coils have been used most
commonly. Smaller surface coils are
sometimes useful for directed imaging of
specific parts of fetal anatomy if the area of
interest is not too far from the surface.
Typically, 10 to 15 slices are acquired in 20
to 25 seconds. As fetal motion is unpredic-
table, shorter sequences are optimal. An
initial fast localizing sequence is performed,
and sequences are then acquired in ortho-
gonal planes relative to each previous
sequence.
For Imaging fetus, single-shot fast spin-
echo (SSFSE) technique, Half-Fourier turbo-
spin-echo technique or True-FISP Techniques
are employed.211 The T1-weighted technique
has had particular utility in the brain, looking
for hemorrhage, and in the abdomen, where
the T1-weighted conspicuity of the liver has
been reported to be helpful in assessing liver
position in fetuses with congenital diaphrag-
matic hernia (CDH).212
Prenatal MRI of the Central Nervous
System, Head and Neck
The most common use of fetal MRI has been
to evaluate central nervous system (CNS)
abnormalities, where it has been found to
influence or change management and
counseling in upto 50 percent of cases. MRI
has been particularly useful in looking at
the posterior fossa, corpus callosum, and
gray-white matter.
The mainstay of fetal CNS imaging has
been fast single-shot T2 -weighted image
techniques. Normal anatomic structures can
be seen routinely in second - and third-
trimester fetuses. The single most common
indication for fetal MRI has been evaluation
of ventriculomegaly, where MRI is used to

evaluate for associated abnormalities and
underlying etiologies that are not as readily
depicted with US. Agenesis of the corpus
callosum, in particular, which is commonly
associated with ventriculomegaly, can be a
difficult second-trimester diagnosis with US
but is often readily depicted with MRI. MRI
has been found to be helpful in evaluating
posterior fossa cystic lesions and in
distinguishing between the Dandy-Walker
malformation and variant, posterior fossa
arachnoid cyst, and mega cisterna magna,
holoprosencephaly and encephaloceles (Fig.
20.11).211,213-215
The fetal scalp, neck, and airway can be
difficult to evaluate fully with US because
of artifact related to adjacent bony
structures as well as fetal position. MRI can
be effective in evaluating abnormalities
related to these structures and can provide
information complementary to US. The fetal
airway is fluid-filled and, therefore, discre-
tely bright on T2-weighted images.
Fig. 20.11: Fetal encephalocele. HASTE (T2W) image
showing herniation of brain material to the outside,
in occipital region of the fetus
Body MR Imaging 303
Ultrasound and MRI can be similarly
complementary in evaluation of sacrococ-
cygeal teratoma. Ultrasound, with its fine
spatial resolution, often shows the details
of the association of the lesion with the
caudal aspect of the fetal spine and can also
show its cystic and solid nature.
Whether MRI adds value in the routine
management of neural tube defects (NTDs)
has been argued. MRI may be more accurate
than US in the setting of NTDs in the
presence of maternal obesity. MRI can detect
subtle NTDs when US shows only the
characteristic associated Chiari II malfor-
mation and fails to show the spinal lesion.
When in utero surgery is not a consideration,
it is yet established whether MRI changes
diagnosis or management in NTD.
Prenatal MRI of the Fetal Body
After the CNS, the fetal thorax has received
the most attention by those performing fetal
MRI. Its role has been adjunctive to US,
helping to define the anatomy of large
masses, examine lesions with an atypical US
appearance, and aid in the prognosis of
CDH. Its emerging impact on fetal manage-
ment and counseling for chest masses is
approaching its use for CNS abnorma-
lities.212,216
Congenital chest masses have charac-
teristic, though overlapping, MRI appea-
rances. The appearance of CCAM varies
depending on whether the lesions are
microcystic or macrocystic. The lesions can
have multiple large cysts with discrete walls
or can be solid lesions with scattered small
cysts. Congenital cystic adenomatoid
malformations tend to be higher in T 2-
weighted signal intensity than normal lungs.
Bronchopulmonary sequestration can have
an appearance similar to CCAM. Indeed,
combined lesions are common. Pulmonary
304 Biomedical Magnetic Resonance: Proceedings of the International Workshop
sequestration, found predominately in the
left lower lobe, often appears as a bright
echogenic mass on US and as a well-
demarcated, wedge-shaped region of high
signal intensity on T2-weighted MRI.
MRI may be particularly useful in the
assessment of pregnancies complicated by
oligohydramnios, which can limit the
diagnostic sensitivity of US. Poutamo et al,
performed MRI on 24 fetuses with oligo-
hydramnios or genitourinary anomalies
diagnosed by US and found that provided
the correct diagnosis in 12 of 24 (50%).
However, US, provided the correct dia-
gnosis in 3 of 24 (12.5%).217
Fetal MRI is useful in the assessment of
the fetal gastrointestinal tract and the MRI
appearances of a number of abnormalities
have been described, including omphalocele,
gastroschisis, obstruction, intestinal and
esophageal atresias, and hiatal hernia. MRI
has become a valuable adjunct to US in the
antenatal evaluation of conjoined twins in
whom postnatal separation is being
anticipated.211,218
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Applications of MR Spectroscopy in
Neuromuscular Diseases
INTRODUCTION
Neuromuscular diseases constitute a
significant proportion of neurological
disorders and are an important cause of
morbidity and mortality. Clinical evaluation,
investigation, diagnosis and treatment of
diseases of muscle therefore constitute an
important area of medicine, and in particular
clinical neurology. Precise diagnosis of
several neuromuscular diseases such as
mitochondrial myopathy is often difficult
using routine histological and immuno-
histochemical techniques. Abnormality in
skeletal muscle metabolism is common in
neuromuscular diseases and study of
metabolism may provide additional
biochemical markers for diagnosis.
Catheterization and needle biopsy
techniques coupled with various other
analytical methods were used to study the
muscle metabolism and relative importance
of substrates stored in the muscle cells.1,2
Nuclear magnetic resonance (NMR)
spectroscopy has emerged as a powerful tool
to gain an insight into tissue metabolism.
The technique exploits magnetic properties
of nuclei such as hydrogen (1H), phosphorus
( 31 P) and carbon ( 13 C) to generate the
21
Uma Sharma, NR Jagannathan
spectrum of metabolites present in cells and
spectrum can be obtained both in vivo and
as well as in in vitro condition. In vivo 31P
NMR spectroscopy provides noninvasive
measure of muscle metabolism and is
applicable in monitoring metabolic changes
throughout a period of rest, exercise and
recovery.3-8 The inherent sensitivity and the
presence of 1 H in most metabolites of
interest make 1H NMR spectroscopy as a
dynamic method for the comprehensive
biochemical characterization of tissues.
However, in vivo 1 H NMR is less
informative, since, due to abundance of
water and fat in muscle tissues, only few
metabolites such as choline (Cho), creatine
(Cr) and carnosine (Car) are observed in
the spectrum. On the contrary, under in vitro
conditions various one-dimensional (1D) and
two-dimensional (2D) NMR techniques can
be applied to characterize the comprehensive
metabolic profile of tissues and absolute
quantification of the concentration of various
metabolites other than Cho and Cr are
possible thus providing an in-depth
understanding of the tissue metabolism.9-12
13
C, a comparatively an insensitive nuclei,
also has been useful in monitoring glycogen
314 Biomedical Magnetic Resonance: Proceedings of the International Workshop
metabolism since it has higher abundance
of 13C and is also present in higher concen-
trations in muscle cells.13
In this presentation, we review the work
carried out in the study of the muscle meta-
bolism using in vivo and in vitro NMR
spectroscopy techniques, in addition to the
work carried in our laboratory. A brief
description of the various biochemical
pathways (skeletal muscle metabolism) of
energy generation in skeletal muscle during
contraction and resting state are initially
presented.
SKELETAL MUSCLE METABOLISM
Muscle generates energy to carry out the
work using similar metabolic pathways as
in other cells however, energy generation
occurs at a larger scale. Hydrolysis of
adenosine–triphosphate (ATP) through the
following reaction which is catalyzed by
myosin ATPase generates the energy for
muscle contraction.
ATP + H2O ADP + Pi
Muscle has very small amount of ATP
which can provide energy only for a short
time but there is a reserve supply of readily
available energy in the form of the
compound phosphocreatine (PCr). PCr acts
like an energy shuttle between the sites of
ATP production and utilization through the
following reaction which is catalyzed by the
enzyme, creatine kinase
PCr + ADP+ H+ ATP + Cr
In this reaction the phosphoryl group of
PCr is transferred to ADP and ATP is
regenerated.
In resting muscle most of the energy is
ATP and is obtained through mitochondrial
respiration or oxidative phosphorylation.
During exercise or anaerobic (ischemic)
contraction, glycogenolysis and PCr supply
ATP for contraction. When energy meta-
bolism is switched from aerobic to anaerobic,
lactic acid (Lac) is produced and accumu-
lated in the cytoplasm, thus leading to a
large decrease in the intracellular pH.
Skeletal muscle has the ability to utilize a
number of substrates such as carbohydrate
glucose (Glc), fatty acids, ketone bodies and
amino acids etc as energy source. Glc is
assimilated from the blood and is stored as
glycogen in the muscle tissue. At rest muscle
derives energy preferentially through
oxidation of free fatty acids and ketone
bodies.
Glucose Metabolism
Glc is being oxidized to release energy
through three main stages in muscle, the
first is glycolysis followed by Krebs cycle
and the oxidative phosphorylation. Glc in
muscle tissue is obtained from the following
sources: (i) blood, (ii) breakdown of muscle
glycogen stores, and (iii) through gluco-
neogenesis pathway in which Glc is
produced from non-carbohydrate precursors
such as Lac, propionic acid, glutamine (Gln)
and alanine (Ala). The glycolysis pathway
is composed of ten chemical reactions, each
catalyzed by a different enzyme, details of
these reactions are presented in many text
books. 14,15 Glycolysis does not involve
oxygen and very little energy is produced,
in fact only two ATP molecules per molecule
of Glc are produced through this pathway.
The end products of glycolysis are pyruvate
(Pyr) and nicotinamide-adenine-dinucleotide
(NADH). Yet, to produce more ATP mole-
cules, NADH is reoxidized to NAD+ and
Pyr is oxidized to CO2 and H2O, in the
presence of oxygen through Krebs cycle
 Applications of MR Spectroscopy in Neuromuscular Diseases
followed by oxidative phosphorylation in
mitochondria. Oxygen is not necessarily
always plentiful for generation of ATP
through Krebs cycle and oxidative phos-
phorylation in muscle tissues. In these
situations energy is generated through high
rate of glycolysis.
Since, NAD+ acts catalytically and is
present in small amounts and there is need
to convert NADH to NAD+ for glycolysis
to proceed further. Lactate dehydrogenase
enzyme catalyzes the reduction of Pyr to
Lac through the following reaction and the
NADH is reoxidized to NAD+.
Pyruvate + NADH + H+ Lactate
+ NAD+
The production of Lac from Glc is known
as anaerobic glycolysis. Thus reoxidation of
NADH allows continued ATP production
from glycolysis for rapid muscle contraction.
Muscle tissue has higher levels of lactate
dehydrogenase and hence NADH can be
rapidly reoxidized in this way and this in
turn permits glycolysis to proceed at a very
fast rate. Muscle cells act in an economic
manner, the Lac formed in this manner is
not wasted but leaks out into blood which
transports it to liver and it is again
converted into Glc in liver by Cori cycle.14,15
Thus, glycolysis provides ATP in the absence
of oxygen and allows skeletal muscle to
perform at high levels.
Krebs Cycle
The Pyr produced through glycolysis enters
in the mitochondria where an irreversible
oxidative decarboxylation of Pyr occurs. In
this reaction CO2 is released (decarboxy-
laton) and a pair of electrons is transferred
to NAD+ (oxidation), and an acetyl group
is transferred to Co.A and acetyl Co.A is
315
formed. The acetyl group of acetyl Co.A is
fed into the Krebs cycle (citric acid cycle)
and three molecules of CO 2 are pro-
duced.14,15 Reactions of Krebs cycle take
place in the mitochondria, small organelles
located in the cytoplasm of the cells, are
therefore called main energy generating
units where most of the ATP is produced
through Krebs cycle.14,15 The enzyme system
that carries out citric acid cycle functions in
a cyclic manner. To begin a turn of the cycle,
acetyl Co. A donates its acetyl group to the
oxaloacetate to form citrate. Citrate is then
transformed to isocitrate which through
dehydrogenation reaction yields α-keto
glutarate (KG) (α-KG). The latter then
undergoes loss of CO2 and ultimately yields
succinate (Succ). Succ is then enzymatically
converted in three steps into oxaloacetate
with which the cycle began. In this process
three molecules of NAD+ and one molecule
of flavine-adenine-dinucleotide (FAD) are
reduced to NADH and FADH 2 . The
oxidation of NADH and FADH 2 in an
electron transport system in inner
mitochondrial membrane is the next step
for ATP generation. This drives conver-sion
of ADP and Pi to ATP. This complete process
is called oxidative phosphorylation.
Fatty Acid Metabolism
The triacylglycerols play an extremely
important role in providing energy in
organs like liver, heart and resting skeletal
muscle. Long chain fatty acid component of
triacylglycerols has approximately 95 percent
of available energy while only 5 percent
energy resides in glycerol portion. Fatty acid
is being transported into mitochondria
through the formation of fatty acyl-carnitine
where they are oxidized to generate energy.
The reaction is catalyzed by the enzyme
316 Biomedical Magnetic Resonance: Proceedings of the International Workshop
carnitine acyl transferase, present on the
outer surface of the inner membrane of
mitochondria. Fatty acid oxidation occurs
through two main stages. In the first stage
fatty acids undergo oxidative removal of
successive 2-carbon units through a set of
enzymatically catalyzed reactions in the form
of acetyl Co. A. This process starting from
the carboxyl end of the fatty acid chain is
repeated several times according to the fatty
acid chain length. In the second stage, acetyl
Co. A derived from fatty acid oxidation are
oxidized to CO2 and H2O through citric acid
cycle in a similar manner as acetyl Co. A
derived from oxidation of Pyr. Long chain
even and odd number fatty acids both are
oxidized in a similar manner. However in
odd chain amino acid the products of the
last pass through of oxidation are propionyl
Co. A and acetyl Co. A. Propionyl Co. A is
converted to succinyl Co. A through a set
of reactions which is then oxidized through
citric acid cycle.
Amino Acids Metabolism
Amino acids are the building blocks for the
biosynthesis of proteins. However, during
fasting or in diabetes when carbohydrates
either unavailable or not properly utilized
or if in excess of the body’s need for protein
synthesis, they undergo oxidative degrada-
tion. In these circumstances the α-amino
groups of amino acids are enzymatically
transferred from the amino acid to the α-
carbon atom of α-KG, leaving behind the
corresponding α-keto acid analog of the
incoming amino acid and in turn amination
of the α-KG occurs and is converted into
glutamate (Glu). The reaction is catalyzed
by enzymes called transaminases or
aminotransferases as given below.
L-α-amino acid + α-KG α-keto
acid + L-Glu
The α-keto acid thus formed enters TCA
cycle to undergo oxidation and yields CO2
and H2O. The α-KG is the common acceptor
of amino groups from most of the amino
acids. During fasting and in diabetes muscle
degrades 3-5 fold more branched chain
amino acids than in normal condition. Ala
yields Pyr directly on transamination with
α-KG which enters citric acid cycle through
the formation of acetyl Co. A. Threonine
(Thr), glycine (Gly), serine (Ser) and cysteine
(Cys) are metabolized through the formation
of Pyr followed by acetyl Co. A. Part of the
carbon backbone of the amino acids
phenylalanine (Phe), tyrosine (Tyr), Lys,
tryptophan (Trp) and leucine (Leu) yield
acetoacetyl Co.A, which is then converted
into acetyl Co. A. Arginine (Arg), histidine
(His), Glu, Gln and proline enter the citric
acid cycle via α-KG. The carbon skeletons
of methionine, isoleucine (Ile) and valine
(Val) are utilized by pathways which yield
succinyl Co. A, an intermediate of Krebs
cycle.
During metabolism of amino acids
ammonia is produced in muscle. Ala plays
a special role in transporting ammonia to
the liver in a nontoxic form through glucose-
alanine cycle. In this cycle ammonia is
converted into the amino group of Glu by
the action of glutamate dehydrogenase. The
Glu so formed now transfers its α-amino
group to Pyr, a readily available product of
muscle glycolysis, by the action of alanine
transaminase. The Ala so formed escapes
into blood and is carried to the liver. Here
again through transamination reaction Ala
transfers its amino group to α-KG to yield
Glu which then undergoes deamination to
again yield α-KG and ammonia. The
ammonia so released is converted by the
liver into urea and excreted.
 Applications of MR Spectroscopy in Neuromuscular Diseases
IN VIVO MRS
31P MRS
In vivo phosphorus magnetic resonance
spectroscopy (31P MRS) has been the method
of choice for repetitive, noninvasive dynamic
studies of muscle energy metabolism in
normal and diseased state. Muscle
metabolism has been extensively
31
investigated using P NMR spectroscopy
and several review articles4,16,17 have been
published. Skeletal muscle has the capacity
of changing the rate of metabolism at will
just by exercising at different levels of work.
Information is obtained by measuring the
relevant phosphorylated compounds in
different conditions, i.e. at rest, during
controlled work and during the subsequent
phase of recovery. One of the major
advantages of 31P NMR is the measurement
of few phosphorus containing metabolites
that offers information on the functionality
and regulation of the central metabolic
pathways of phosphate which are vital to
the energy status of the cell. Investigation
protocols of skeletal muscle therefore in
general include acquisition of spectrum at
rest, during exercise and during recovery
after contraction.
Figure 21.1 shows the 31P MR spectra
recorded from the calf muscle of a normal
volunteer in resting state which is simple
with only few resonances and is easy to
comprehend. This is due to the fact that in
in vivo MRS only metabolites that are free
or rapidly exchanging are observed in the
spectra. The five major peaks observed in
the spectra corresponds to α-, β- and γ
phosphates of ATP, phosphocreatine (PCr)
and inorganic phosphate (Pi). The area under
each peak is directly proportional to the
concentration of the metabolite. The spectral
distance between Pi and PCr peaks varies
317
Fig. 21.1: 31P in vivo MR spectrum of calf muscle of a
normal volunteer using a surface coil with a repetition
time of 3 s (Reproduced with permission from
reference 38)
with alteration in pH in muscle tissue and
therefore provides information about the
intracellular pH. The peaks due to phospho-
monoesters (PME) and phosphodiesters
(PDE) are also often observed in 31P MR
spectra.
Several review articles have been
published documenting the efficacy of 31P
MR spectroscopy in the study of muscle
metabolism in several neuromuscular
disease.16,17 In vivo 31P MRS has been used
in the diagnosis and monitoring of muscle
metabolism in mitochondrial disease.18,19
Several reports exist in the literature in
which large number of patients with mito-
chondrial diseases have been studied.7,20-22
About 35 to 85 percent of patients with
mitochondrial encephalopathies have been
reported to have abnormally low PCr/Pi at
rest. Under aerobic condition, patients with
mitochondrial disease show a more rapid
fall in muscle energy state per unit work
than do normal controls consistent with the
318 Biomedical Magnetic Resonance: Proceedings of the International Workshop

limited oxidative metabolism. The most
sensitive variable for mitochondrial
abnormality during exercise was the change
in PCr level.21
Some mitochondrial disorders have lactic
acid accumulation and low serum pH. While
in some a relative resistance to intracellular
acidosis has been observed in exercising
muscle of patients with mitochondrial
diseases.7,20,21 The mechanism of this pheno-
menon may be related to adaptation of
intracellular buffering systems (including
proton extrusion mechanism) in response to
chronic overproduction of Lac. 31 P MR
studies of the recovery rates of PCr provide
valuable quantitative indices of mitochon-
drial dysfunction. The initial rate of recovery
can provide a measure of maximal oxidative
rate in the tissues and thus a sensitive and
reliable index of mitochondrial dysfunction
in in vivo.23 Approximately 90 percent of
patients with mitochondrial myopathies
showed abnormal recovery kinetics.
A high muscle pH at rest has been
observed in patients with muscle dystro-
phies. 24-27 In patients with limb girdle
muscular dystrophy (LGMD), the degree of
fat replacement in calf muscle correlated
inversely with cytosolic pH and directly
with PCr/ATP.26 In patients with Becker
muscular dystrophy (BMD) an early drop
in the level of PCr or PCr/Pi ratio and
reduced acidosis was reported. 25,28 A
reduced glycolytic or glycogenolytic activity
has been assumed as the cause of reduced
acidosis but the cause of defect in glucose
metabolism common to dystrophies and
myotonic dystrophy, remains unclear.25,29,30
1H MRS
Volume localized proton MRS have been
used by several investigators to study the
processes like glycolysis and lipid meta-
bolism in muscle.31-33 Recently Boesch et al
reviewed the use of 1H MRS in the muscle
tissue.34 Figure 21.2 shows the in vivo proton
MRS from the calf muscle of a normal
volunteer recorded in our laboratory. In this
spectrum various resonances due to residual
water (at 4.7 ppm), terminal methyl of lipids
(0.8-1.0 ppm), acyl chain methylene [-(CH2)
n-: 1.1 – 1.6 ppm], α- and β- methylene of
the lipid chain (2-2.8 ppm) and the oleifinic
protons of the polyunsaturated fatty acids
(-HC=CH-: 5.5 ppm) are observed. In
addition, the resonances due to N-methyl
protons of Cr and PCr resonate at 3.03 ppm,
while the N-CH3 of carnitine (Car) and Cho
containing compounds resonate at 3.22 ppm.
Peaks at 7.1 and 8.1 ppm are due to the C-
4 and C-2 ring proton of histidyl moiety in
carnosine.
Fig. 21.2: 1H in vivo MR spectrum using STEAM pulse
sequence from calf muscle of a normal volunteer
observed at an echo time (TE) of 135 ms with
repetition delay of 3s. The voxel size was 20×20×20
mm3 (Reproduced with permission from reference
38)
 Applications of MR Spectroscopy in Neuromuscular Diseases
Schick et al have shown that the spectra
from muscle tissue exhibit two compartments
of lipid signals with a difference of 0.2 ppm
in their resonance frequencies.33 These two
compartments arise from intramyocellular
(IMCL) and extramyocellular (EMCL)
compartments of lipids. In vivo 1H MRS was
used in the study of polio patients by our
group.35 The spectra were obtained at a long
echo time of 270 ms to characterize clearly
the IMCL and EMCL components of the
lipid. The study revealed that mildly
paralyzed patients are comparable with
control subjects in relation to the presence
of IMCL while moderate and severely
paralyzed patients were comparable in
relation to the absence of IMCL. In addition,
there is reduction or complete absence of
Cr, Car and Cho metabolites in severely
paralyzed patients.
IN VITRO NMR SPECTROSCOPY OF
MUSCLE TISSUE
In vitro MR spectroscopy plays an important
role in understanding the biochemical
processes and pathogenesis of the muscle
weakness observed in different neuro-
muscular diseases. We, in our laboratory,
are involved in the study of muscular
dystrophies like LGMD, DMD, BMD and
metabolic myopathies such as mitochondrial
myopathy (MM). Identification of meta-
bolically pertinent substances from the
perchloric acid (PCA) extracts of human
skeletal muscle tissue of patients in the
above neuromuscular diseases and normal
controls were carried out using 1- and 2-D
proton and heteronuclear NMR techni-
ques.36-38 In addition, the concentration of
metabolites in these groups of patients were
calculated and compared with the controls
and among different neuromuscular diseases
319
and in the following section some details
are presented from our study.
Human Muscle Biopsy
Muscle specimens from patients under
regional anesthesia were obtained from the
vastus lateralis muscle of the quadriceps in
thigh. A cylindrical piece of muscle meas-
uring 2-3 cm in length was taken and was
divided into four parts. For NMR studies
one piece was quickly frozen and stored in
liquid nitrogen until the perchloric acid
extraction procedure was carried out. Other
pieces were used for diagnosis of disease
using histological, enzyme histochemical and
immunohistochemical studies.
Muscular dystrophy was diagnosed
based on histopathological characteristics
such as variation in fiber size, ring and
whorled fibers, more fiber splitting, degene-
ration and necrosis along with the presence
of lobulated fibers in some cases. Enzyme
histochemistry showed that the good fiber
differentiation was present in 46 percent
cases. Muscle dystrophies wherein immuno-
histochemistry were normal for dystrophin
1,2 and 3 as well as normal for sarcoglycans
(α, β, γ and δ), merosin and emerin were
then diagnosed as LGMD in conjunction
with clinical features (i.e. muscle involved).
Perchloric Acid Extraction
The PCA extracts from muscle specimens of
patients with LGMD (n = 11), DMD (n = 9),
BMD (n = 4), MM (n = 5), WNHL (n = 11)
and normal subjects (controls) who came
for orthopedic surgery (knee and hip
replacement) and who did not show any
muscle disease were prepared as described
by Payen et al.39 The wet weight of muscle
tissue taken was in the range of 250 - 400
mg. The frozen biopsy specimen was
320 Biomedical Magnetic Resonance: Proceedings of the International Workshop
pulverized in a liquid nitrogen chilled
mortar and pestled to a fine powder. The
tissue was homogenized with 0.6 N of PCA
(10 ml/gm wet weight). After centrifugation
at 10,000 rpm for 20 min at 4°C, the pH of
the supernatant was adjusted between 7-8
with potassium hydroxide using pH strip.
The precipitate of KClO4 was removed by
centrifugation at 10,000 rpm for 20 min at
4°C and the resulting supernatant was
lyophilized. The lyophilized powder was
then dissolved in D2O solvent to carry out
NMR experiments.
NMR Spectroscopy Study
NMR experiments were carried out at DRX-
400 MHz (Bruker, Switzerland) spectrometer
equipped with 5 mm multinuclear broad
band inverse probe at 298 K. Sodium tri-
methyl- silyl –[2,2,3,3-H4] propionate (TSP)
was added as an internal standard for
chemical shift and quantification of concen-
tration. Two-dimensional double quantum
filtered (2D-DQF) COSY and Total Corre-
lation Spectroscopy (2D-TOCSY) experiment
were carried out using the standard para-
meters. In addition, 1H-13C heteronuclear
multiple quantum coherence (HMQC)
experiments were also recorded using
standard parameters for confirmation of
assignments.
Quantification of Metabolites
The concentration of metabolites was
determined by comparing metabolite fit
integral with the fit integral of TSP as
described earlier.11 The significance of the
various concentration values was analyzed
using student ‘t’ test. Probability values of
(5 %) were recorded as significant (p<0.05).
Metabolite Assignments
Various proton resonances in the spectra of
muscle tissue extracts are assigned to specific
metabolites using 2D DQF COSY, TOCSY,
spin multiplicities, relative peak intensities.
In addition, the chemical shifts of meta-
bolites were compared with that reported
in literature in muscle tissue extracts40 and
also with other tissue extracts9-12 wherever
applicable. The assignments of compounds
like Car, Cr and Cho containing compounds
were made by comparing their chemical
shifts with those of standard compounds.
Figures 21.3 A to D show the representative
1D 1H NMR spectrum of the perchloric acid
extract of the muscle tissue of a DMD patient
while Figure 21.4 shows the TOCSY
spectrum. In all, about 37 compounds could
be identified in the muscle tissue spectra of
DMD36, LGMD patients37 and MM patients.
However, in some DMD patients 56 meta-
bolites could be identified and similarly in
few of the LGMD samples 44 metabolites
could be identified unambiguously. Few
resonances still remain to be assigned.36,37
A total of fourteen amino acids were
identified unambiguously in the spectra of
patients of most neuromuscular diseases.
Resonances of many organic acids such as
Lac, Ace, Succ, α-KG and sugars such as
Glc were also observed in the spectra of all
the muscle diseases studied. The resonances
due to glycerophosphoryl choline (GPC),
Cho, phosphoethanol amine (PE), phos-
phoryl choline (PC) were also present in all
the diseases and controls. The N (CH3) and
CH2 protons of Cr and PCr resonate at 3.02
ppm and 3.93 ppm, respectively. The
N(CH3) of Car appear at 3.23 ppm along
with N(CH3) of GPC and PC (Fig. 21.3B).
The CH protons of CHOH group of Car at
4.56 ppm showed connectivity with its CH2
protons at 2.45 and 3.45 ppm, respectively.
Resonances of H4, H5 and H6 protons of
indoxyl sulphate were also observed in
 Applications of MR Spectroscopy in Neuromuscular Diseases
Fig. 21.3A: 400 MHz 1D proton NMR spectrum
showing aliphatic region (expanded from 0.5 to 3.0
ppm) of the perchloric acid extract of skeletal muscle
tissue of a DMD patient in D2O at 298 K (Reproduced
with permission from reference 38)
Fig. 21.3C: 400 MHz 1D proton NMR spectrum
showing aliphatic region (expanded from 5.1 to 6.6
ppm) of the perchloric acid extract of skeletal muscle
tissue of DMD patient in D2O at 298 K. The pH of the
sample was in the range of 6 to 7 (Reproduced with
permission from reference 38)
LGMD, DMD and MM patients. In the
downfield region, resonances due to base
protons of nucleotides were clearly seen.
The singlet from the H2 and H8 protons of
ATP were observed at 8.24 and 8.50 ppm,
respectively (Fig. 21.3D). The H1´ of
deoxyribose sugar of ATP resonate at 6.15
ppm (Fig. 21.3C). Proton resonances of
321
FIG. 21.3B: 400 MHz proton NMR spectrum showing
downfield region (expanded from 2.9 - 4.7 ppm ) of
perchloric acid extract of skeletal muscle tissue from
DMD patient in D 2O at 298 K (Reproduced with
permission from reference 38)
Fig. 21.3D: 400 MHz 1D proton NMR spectrum
showing aliphatic region (expanded from 6.4 to 9.1
ppm) of the perchloric acid extract of skeletal muscle
tissue of DMD patient in D2O at 298 K (Reproduced
with permission from reference 38)
guanosine-di-phosphate (GDP), guanosine–
mono-phosphate (GMP), guanosine-tri-
phosphate (GTP) and inosine-mono-
phosphate (IMP) were assigned according
to the chemical shift positions reported in
the literature12 and by spiking experiments.
The resonances due to nicotinamide-adenine-
dinucleotide (NAD) and nicotinamide-
322 Biomedical Magnetic Resonance: Proceedings of the International Workshop

Fig. 21.4: Phase-sensitive two-dimensional total correlation spectrum (2D-TOCSY) (expanded region) of
perchloric acid extract of skeletal muscle tissue from DMD patient at 298 K in D2O (Reproduced with permission
from reference 38)

adenine-dinucleotide phosphate (NADP) significantly reduced in DMD and LGMD

protons were assigned by their unique
chemical shift position.
Metabolite Concentration in Muscle
Diseases and Controls
The concentration of various metabolites in
muscle tissue of patients with LGMD, DMD,
BMD, MM, WNHL and controls were
determined and compared (see Table 21.1).
The concentration of glycolytic product Lac
together with glycolytic substrate Glc were
patients compared to controls. Similarly, a
significant decrease was noticed in the
concentration of amino acids (Glu+Gln) and
Ala in DMD and LGMD patients compared
to controls. In addition, the concentrations
of total Cr (PCr+Cr) content, Cho and GPC/
PC/Car were also found to be significantly
reduced in DMD patients in comparison to
controls while in LGMD patients the
reduction was observed only in Cho concen-
tration. However, there is no significant
 Applications of MR Spectroscopy in Neuromuscular Diseases 323

Table 21.1: The concentration (Mean ± SD, mM/Kg wet weight) of metabolites estimated in the muscle
tissue extracts of normal subjects and patients with neuromuscular diseases

Subjects Metabolites Concentration (mM/Kg wet weight)

Glc Lac Ala Glu+Gln Cho GPC/Car Cr/PCr Prop Ace

Controls 2.3±1.4 24.6±13.6 4.2±2.4 12.6±6.7 0.9±0.3 2.5±2.4 26.4±10.8 2.4±2.1 3.5±2.8
LGMD 0.7±0.2 12.8±4.9 1.3±0.7 5.3±2.8 0.4±0.3 1.04±0.7 24.2±9.6 1.3±0.7 3.8±2.3
DMD 0.6±0.3 4.7±2.7 1.0±0.6 1.4±0.7 0.3±0.2 0.5±0.3 3.4±2.6 1.6±1.1 0.9±0.6
BMD 2.5±0.7 21.1±0.7 4.1±0.6 16.8±3.2 1.0±0.7 4.1±3.3 19.5±6.4 2.9±1.9 1.5±1.1
MM 3.8±1.5 41.7±14.7 6.1±1.5 22±5.4 1.3±0.6 4.8±0.5 27.5±9.8 1.8±0.8 1.5±1.0
WNHL 3.9±0.2 54.2±19.6 6.2±1.8 19.4±7.8 1.2±0.8 5.1±2.8 25.6±14.3 2.3±1.0 1.9±0.8

P-values
Control vs. LGMD 0.02 0.04 0.009 0.02 0.01 0.1 0.4 0.1 0.3
Control vs. DMD 0.008 0.002 0.005 0.004 0.0006 0.03 0.005 0.2 0.01
Control vs. MM 0.03 0.03 0.05 0.03 0.05 0.03 0.1 0.1 0.1
Control vs. WNHL 0.04 0.003 0.05 0.04 0.05 0.03 0.1 0.1 0.1

difference in the concentration of these Muscle Metabolism in

metabolites between controls and BMD
patients.41 A significant reduction in the
concentration of Lac, Glu+Gln and GPC in
DMD patients were noticed compared to
LGMD patients. Patients with DMD also
showed significantly lowered concentration
of Glc, Lac, Ala, Glu+Gln, and Cr+PCr
metabolites in comparison to BMD patients.
A comparison of the metabolite concen-
tration of mitochondrial myopathy, WNHL
and controls were also carried out. The
concentrations of Lac, Glc, Glu+Gln, Ala
were significantly higher in MM patients in
comparison to the control group (Table 21.1).
In addition, GPC was higher in MM patients
than in controls. WNHL patients also
showed significantly higher concentration
of Glc, Lac, Ala and Glu+Gln compared to
controls. However, there was no significant
difference in the concentration of these
metabolites between the WNHL and the
MM group of patients.42
Muscular Dystrophies
Earlier studies on LGMD, DMD and BMD
patients reported the metabolic profile of
phosphorus containing metabolites using 31P
MR spectroscopy. 24-30 In recent years
investigations of various muscle dystrophies
using in vitro proton NMR have been
reported. 36,37 A study using 1 H NMR
spectroscopy and pattern recognition
approach on mdx mouse was reported which
showed that dystrophic tissue had distinct
metabolic profiles for cardiac and other
muscle tissues.43 Advantage of using in vitro
proton NMR is that a more comprehensive
metabolic profile of muscle tissues in various
muscle disorders can be obtained. A brief
summary of the several interesting obser-
vations for various neuromuscular diseases
studied in our laboratory are presented
below.36-38
High-speed muscle contractions require
rapid generation of high-energy phosphate
324 Biomedical Magnetic Resonance: Proceedings of the International Workshop
ATP, which is achieved through glycolysis
pathway using Glc as energy substrate
obtained preferentially through breakdown
of muscle glycogen stores. After depletion
of muscle glycogen store, Glc is either
obtained through blood or fatty acids are
used for energy generation. In terms of the
number of ATPs produced per oxygen
molecule, glycogen or Glc are used more
efficiently from than that of fatty acids.
Significant reduction in the concentration of
the energy substrate Glc was noticed in
DMD and LGMD patients.36,37 Therefore in
LGMD and DMD patients, the decrease in
the concentration of Glc may probably
appear to be one of the reasons for the
energy deficit. In a detailed biochemical
study, Nishio et al have also shown
reduction in the concentration of Glc in
DMD patients with low creatine kinase
activity.44 This reduction was suggested due
to caloric shortage in the degenerated
muscle which cannot supply enough gluco-
neogenic substrate Ala. Our observation of
lowered concentration of gluconeogenic
amino acids, Ala and Gln in DMD patients
agree with the biochemical results of Nishio
et al.44 Recently, deficiency of myophos-
phorylase enzyme was noticed in one LGMD
case by Nishio et al 45 suggesting glyco-
genolytic defect. In muscular dystrophy, the
muscle tissue is infiltrated by fat; there is
also fiber atrophy or fibrosis in the diseased
muscle. As a consequence of increased
quantities of fatty tissue, decreased concen-
tration of several metabolites in per gram
wet weight of muscle tissue in comparison
to controls can be expected.
Lowered concentration of Lac observed
in LGMD and DMD patients from in vitro
proton NMR may be attributed to lowered
levels of Glc or reduced glycogenolysis or
a defect in glycolytic processes. 46
Biochemical studies documented significant
reduction in the specific activities of various
muscle enzymes, lactate dehydrogenase,
aldolase and pyruvate kinase in muscle
biopsies from DMD patients supporting
reduced anaerobic glycolytic activity.47,48
The H-type of the LD isoenzyme which is
more aerobic was found to predominate in
DMD patients.49 The abnormal patterns of
LD isoenzymes in dystrophic muscle was
suggested as reflection of a change in the
metabolism of the diseased muscle to be
more aerobic.50,51 31P MR studies revealed
increased intracellular pH in the muscle of
DMD/BMD patients.24-28 Our results using
in vitro NMR suggests that glycolysis or
glycogenolysis processes are depressed in
LGMD and DMD patients resulting in
energy deficit that may be the underlying
reason for muscular weakness in these
patients.36,37 It may be noticed that reliable
quantitation of Lac concentration could be
affected by immediate shift from aerobic to
anaerobic metabolism in tissues as soon as
it is removed from the body. Presence of
α-KG, an intermediate of Krebs cycle only
in DMD patients also suggests a predo-
minant oxidative metabolism for energy
generation in diseased muscle.36
Oxidation of fatty acids is also an
important source of energy in both the
skeletal and cardiac muscle at rest.52 Car is
necessary for the transport of long-chain
fatty acids into mitochondria where β-
oxidation of these molecules occur. The
concentration of Car was found to be
significantly less in DMD patients from
in vitro NMR. The decrease in the
concentration of Car may affect the transport
of fatty acids. Increased concentration of
free fatty acids and ketone bodies was
reported in DMD patients indicating that
 Applications of MR Spectroscopy in Neuromuscular Diseases
these may be used as energy source sparing
muscle protein. 44 In our study we also
observed Ace, propionate, 3- hydroxy
butyric acid (3-HB) and Succ compounds in
the muscle tissue extracts indicating the use
of fatty acids for energy generation,
however, the concentration of acetate was
significantly less which may be the
consequence of decreased Car concentration
in DMD patients.
Gln was also significantly reduced in
LGMD and DMD patients. Gln is a major
gluconeogenic precursor,53,54 serves as a fuel
for tissues with a high cell turn over rate,
and play a pivotal role in the regulation of
protein synthesis.55 In healthy adult human
subjects, increase in the plasma Gln
concentration was shown to stimulate
protein synthesis56 and with experimental
decrease protein wasting was reported.57 It
may be hypothesized that the decreased
concentration of Gln, besides resulting in
decreased Glc concentration, may also be
the underlying reason for protein wasting
and protein breakdown in the muscle of
LGMD and DMD patients.
Another interesting observation of our
study is the decreased concentrations of the
peak at 3.23 ppm due to GPC/phosphoryl
choline (PC)/Car and the peak at 3.20 ppm
due to choline in DMD patients. PC is the
precursor of membrane phospholipid
lysolecithin and is formed in the cell from
choline by the activity of enzyme choline
kinase. While GPC is the catalytic product
of lysolecithin which again gets converted
into PC by phospholipase activity. Burt
et al have suggested that water soluble
metabolite GPC regulates phospholipid
composition by inhibiting the enzyme
lysolecithinase, of which it is a catalytic
product.58 It is known that the properties
of membranes are fundamentally dependent
325
on the composition of phospholipid as well
as the relative proportions of different
phospholipids and the ratio of phospholipids
to cholesterol. Any alteration in the compo-
sition of phospholipids might profoundly
affect the cellular behavior as these are
important structural components of mem-
branes and also play an important role in
regulating the activities of membrane bound
enzymes. 59 Thus, several biochemical
processes appear to play an important role
in the decrease of glucose concentration
observed in DMD patients.
Skeletal Muscle Metabolism in
Mitochondrial Myopathy
The morphological abnormalities are
observed in mitochondrial myopathies but
changes in mitochondrial structure are not
disease specific and the presence of such
changes in muscle biopsy does not
necessarily indicate impaired mitochondrial
metabolism. Diagnosis of muscle myopathies
is often challenging and usually requires
muscle biopsy for morphologic and
biochemical characterization of mitochon-
drial abnormalities. In our study, eleven
patients were suspected to have mito-
chondrial myopathy according to clinical
symptoms, but the histological, histochemical
and electron microscopy techniques per-
formed on muscle biopsies were inconclusive
and no proper diagnosis of myopathy could
be made, these were categorized as WNHL
(histopathology within normal histological
limits) patients.42 Estimation of the meta-
bolite levels in patients with proven mito-
chondrial myopathy was carried out and
compared with WNHL patients and controls
in order to understand the biochemical basis
of mitochondrial myopathy and to evaluate
the utility of in vitro NMR in distinguishing
mitochondrial myopathies from control
group.42
326 Biomedical Magnetic Resonance: Proceedings of the International Workshop
Elevated concentration of Lac in both
mitochondrial myopathy and WNHL
patients were observed compared to
controls, however there is no significant
difference in the concentration of this
metabolite in MM and WNHL group.
Higher levels of Lac in these patients may
be attributed to higher rates of glycolysis
to derive energy. In mitochondrial diseases,
since there is defect in the respiratory chain
of enzymes impairing the citric acid cycle.
So energy is derived via anaerobic pathway,
resulting in increased Lac levels and
subsequently intracellular acidosis which
was observed in biopsies of mitochondrial
myopathy patients. 31P in vivo MR studies
also documented low PCr/Pi ratio at rest in
mitochondrial myopathy patients indicating
impaired oxidative metabolism in these
patients.5,60
The concentration of Glc and gluco-
neogenic amino acids Gln and Ala in MM
patients were observed higher compared to
controls while there was no significant
difference in the concentration of these
metabolites between MM and WNHL
patients. It appears that in MM patients due
to deranged citric acid cycle, there is
limitation in the amount of energy liberated
per molecule of Glc oxidized. Hence, to
provide a given amount of energy, much
more Glc must undergo glycolysis anaero-
bically compared to aerobic conditions. This
could be the reason for higher levels of Glc
noticed in these patients. Since, MM patients
have higher rates of glycolysis, there is also
need to generate more amount of Glc. This
is achieved by gluconeogenesis, i.e.
generation of Glc from other compounds
such as Glu, Ala and Lac etc, which could
possibly be the reason for higher levels of
these metabolites.
These results indicate similar metabolism
in WNHL and MM patients suggesting that
mitocondrial functions may be impaired in
WNHL patients as well. Clinical symptoms
of WNHL patients were also similar to MM
patients confirming the clinician’s suspicion
of mitochondrial myopathy in WNHL
patients.
CONCLUSION
In vivo 31P MR spectroscopy provides
information about energy metabolism of
muscle. In some disorders like mitochondrial
myopathies, measurement of PCr or PCr/Pi
ratio at rest, during exercise and during
recovery could play an important role in
the understanding mitochondrial function
that would be useful in the diagnosis.
In vitro 1D and 2D proton NMR analysis
perchloric acid extract of skeletal muscle
tissue of controls and patients with DMD,
BMD, LGMD, MM and WNHL revealed
differences in the concentration of several
metabolites among the various neuro-
muscular diseases and control group. DMD
showed lowest concentration of Lac,
Glu+Gln and GPC compared to BMD and
LGMD. Concentration levels of several
metabolites are also significantly different
among MM patients and muscular dystro-
phies which can supplement diagnosis.
Significantly higher concentration of Glc,
Lac, Ala and Glu+Gln was observed in
patients with MM compared to DMD, LGMD
and BMD. Levels of Glc and Lac were found
to vary among disease groups and controls
suggesting alteration in the carbohydrate
metabolism. Presence of high concentration
of Car, Ace and propionate suggest
utilization of free fatty acids as an energy
source in skeletal muscle tissue. Alteration
in the concentration of membrane precursors
GPC, Cho reflects changes in membrane
metabolism in normal and diseased state.
In vitro 1H MRS has been found useful in
 Applications of MR Spectroscopy in Neuromuscular Diseases
the diagnosis of mitochondrial myopathy in
eleven patients in whom no histopathological
abnormalities were observed. The concen-
trations of several metabolites were found
to be significantly different among
dystrophies and mitochondrial myopathies
which can aid in unambiguous diagnosis of
these diseases on the basis of underlying
biochemistry. These preliminary results
imply that the quantitation of metabolites
of skeletal muscle tissue using in vitro 1H
NMR spectroscopy has the potential to
provide important additional information to
the histological methods in the diagnosis of
different neuromuscular diseases.
ACKNOWLEDGMENTS
Authors thank Prof Chitra Sarkar, Dr MC Sharma
and Dr Surinder Atri for many useful discussions.
US acknowledge the award of Senior Research
Associateship in the Scientist’s pool scheme of Council
of Scientific and Industrial Research, Government of
India.
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330 Biomedical Magnetic Resonance: Proceedings of the International Workshop

22
EPR Imaging for
Biomedical Applications
R Murugesan, N Devasahayam, K Matsumoto,
S Subramanian, JB Mitchell, Murali C Krishna

INTRODUCTION We have developed hardware and software

Electron paramagnetic resonance (EPR) is a
sensitive technique for the study of free
radicals.1,2 EPR Imaging (EPRI), an adjunct
to EPR spectroscopy, is rapidly emerging as
an important tool for spatially resolved EPR
studies. 3,4 EPRI is a functional magnetic
resonance imaging modality, because EPR
spectral parameters can reflect useful
physiological information. For example, EPRI
has been applied, to study tissue metabolic
redox state, to monitor pO2 in normal as
well as tumor tissues, and to measure tissue
pH and viscosity.5-8 But, unlike MRI which
has found a very valuable place in clinical
practice; biomedical EPRI is still in its
relative infancy. The progress in the field
of EPRI is much slower than in MRI, because
EPRI is technically much more demanding
than MRI.9 Nevertheless, with the recently
available non-toxic free radical probes, EPRI
has now demonstrated its capability to
investigate biomedical problems. We have
focused on developing EPRI instrumen-
tation, optimized for spatial and spectral-
spatial imaging of small animals, using
exogenously administrated spin probes.10-20
for different methodologies of free radical
imaging, especially motivated towards
developing EPRI as a viable technology for
in vivo oxygen measurements. In this article,
we briefly outline the principles and
biomedical applications of EPRI, by drawing
examples from our work, mainly for the
sake of easy illustration, although several
groups are involved in this area of research.
Pioneering work by different researchers is
referred to in our publications cited in this
review.
Development of EPR instrumentation for
biological applications has continued to be
a challenging task. Although a commercial
L-band imaging system has now become
available, specialized instrumentation is still
required for many applications. 21 Some
important technical problems, which make
EPRI more difficult to achieve in practice
than MRI are as follows.
1. Paramagnetic centers, produced in vivo
via endogenous processes, are present
in subnanomolar concentrations,
compared to the large concentrations
(~110 M) of water protons utilized in
 EPR Imaging for Biomedical Applications
NMR imaging. In addition, the free
radicals formed, in vivo, are transient and
hence inadequate for imaging.
2. Due to the large value of the electron
magnetogyric ratio, in a magnetic field
of 1 T (a common field strength for
clinical MRI), the EPR frequency is
28 GHz, while the NMR frequency is
only 42.6 MHz. The microwave frequency
of EPR leads to strong absorption by
biological samples. Hence biomedical,
free radical imaging by EPR is to be
carried out using much lower magnetic
field strengths, to obtain sufficient tissue
penetration. In our lab the EPRI
technologies are being developed at a
resonant frequency of 300 MHz (magnetic
field around 10 mT), which can penetrate
a few centimeters into tissues, allowing
at least small animals, such as mouse can
be investigated.
3. Another major difference between EPRI
and MRI is that the electron relaxation
times of free radicals are very short,
typically between 0.1 μs and 1 μs, six
orders-of-magnitude shorter than those
encountered in clinical MRI. As a result,
most EPR detection is implemented using
‘‘continuous wave’’ (CW) detection
methods. The line widths associated with
EPR signals are three orders of
magnitude larger than NMR signals, and
hence EPR imaging requires more
powerful gradients. In addition, the EPR
spectra of most widely used spin probes
such as nitroxides contain multiple lines
due to hyperfine splitting, leading to
reduced sensitivity as well as hyperfine
artifacts in imaging. In spite of these
difficulties, instrumental and software
techniques have been developed to make
EPRI a viable technology for biomedical
investigations.
331
EPR IMAGING MODALITIES
EPRI by Projection Reconstruction
EPR imaging requires measurements of the
spatial distribution of free radicals within a
sample. This is achieved by applying
magnetic field gradients at several angles,
and recording the EPR spectrum for each
gradient angle. The amplitude of the signal
is proportional to the number of spins in a
plane perpendicular to the gradient. For
example, to produce an XY plane image of
an object, the magnetic field gradient is
rotated in the XY plane and several EPR
spectra (projections) are recorded, as the
gradient angle is incremented up to 180º in
small steps, say 10° each. This procedure,
known as frequency encoding, causes the
resonance frequency to be proportional to
the position of the spin.
ν = γe (Bo + x Gx) = νo + γε x Gx
x = (ν - νo) /(γε Gx) ...(1)
A set of projections thus collected can
then be put together using the method of
back projection, which distributes the
measured spin densities evenly along the
line normal to the axis it was acquired on.
There is however, a blurred artifact across
the whole image. This can be corrected using
a technique called filtered back projection
[FBP], which convolves each of the profiles
with a filter.
CW EPRI Technique
In CW EPRI, spatial encoding of the spins is
accomplished by volume excitation of the
sample in the presence of static magnetic
field gradients. The sample is continuously
irradiated with low intensity electromag-
netic radiation and the resonant response
of the unpaired electrons is measured by
slowly sweeping the applied magnetic field,
332 Biomedical Magnetic Resonance: Proceedings of the International Workshop
in the presence of the gradient. EPR spectra,
collected in a polar grid under the presence
of static gradients, are then used to generate
the EPR images by employing the projection
reconstruction (PR) method. The schematics
shown in Figure 22.1 gives the general layout
of a CW EPRI scanner that operates at 300
MHz.14 There are three major modules, the
magnet-gradient coil assembly, the radio
frequency signal detection bridge, and the
data acquisition system. A brief description
of these modules in our CWEPRI instrument
is as follows.
Description of RF CW EPRI Scanner
Module 1
Magnet At 300 MHz, for a free electron
system, the magnetic field B0 at resonance
is 10.6 mT. This field is provided by a set
of air-core, water-cooled coils in a
Helmholtz configuration. The coils enclose
a cylindrical volume of 30 cm diameter and
34 cm length. The magnet current is
provided by a DC power supply capable of
providing 30 A current at 12 V. Long term
current stability of this power supply is
critical for artifact free imaging. The active
volume in the magnetic field is a 3-cm
diameter spherical volume with field
homogeneity of ± 200 ppm, reasonable for
small animal imaging.
Gradient The imaging gradient system
consists of a set of three mutually orthogonal
gradient coils Gx, Gy, and Gz. A Maxwell
pair of coils produces the Gz gradient while
two pairs of saddle coils orthogonal to each
other provide the Gx and Gy gradients. The
gradient linearity is better than 1 percent
over 3 cm DSV. These coils are powered by
a set of three digitally controlled bipolar
power amplifiers, capable of generating a
maximum gradient of approximately 50 mT/
m in the three axes.
Field Modulation Super imposition of a low
frequency sinusoidal modulation on the
Zeeman field for phase sensitive detection,
required for the CW mode of spectral
acquisition is provided by a set of additional
Helmholtz coils, incorporated within the
gradient coil system. The center of the
modulation field, origin of the gradients,
and the center of the Zeeman field are
coincident and require no electrical offset
corrections. The modulation coils are
powered using a custom-built amplifier that
is capable of delivering a modulation of up
to 5.0 G and limited to a frequency of 12.5
kHz.
Field Sweep Magnetic field sweep is achieved
using an IEEE-488 bus by setting the field
to a given value and incrementing the
current as a function of time. However, the
number of spectral points in a sweep is
limited because, for each field increment, a
‘handshake’ period of 10 ms is required for
the computer to communicate with the
power supply through the IEEE bus. During
this time, the computer is also tied up.
Therefore, either a slow scan with more
points for increased spectral resolution, or
a fast scan with fewer number of points,
for increased temporal resolution may be
opted. However, to achieve rapid field
sweep with minimal spectral distortion, the
magnet power supply is modified to include
a sweep controller which enables all the
sweep instructions to be addressed by the
PC, only once, after which the field sweep
can be set to proceed independently of the
computer. With this setup, spectral sweeps
of up to 50 G can be performed in 4 s
without any detectable spectral distortions.
For example, a 5–8 G sweep that is required
 EPR Imaging for Biomedical Applications
for small animal imaging using narrow single
line spin probes could be accomplished in 4
s when 256 points are used for digitizing
the EPR spectrum (projection). Thus, a two-
dimensional image data collection with 16
projections (~256 points each, 4 s per scan)
can be completed in about 1 min and a three
dimensional image with 81 projections (~256
points each, 4 s per scan) can be collected in
less than 6 min.
Resonator An imaging coil must resonate, or
efficiently store energy, at the Larmor
frequency. Imaging coils are composed of
an inductor, or inductive elements, and a
set of capacitive elements. The resonant
frequency of an RF coil is determined by
the inductance (L) and capacitance (C) of
the inductor capacitor circuit. We have
developed suitable resonators known as
parallel coil resonators [PCR] for RF in vivo
EPR imaging for both the CW and time
domain modalities.13 The resonators are 25
mm in diameter, and either 25 or 50 mm in
length. Schematics of the PCR is presented
in Figure 22.2, Plate 12.
Module 2
RF Detection Bridge The performance of the
EPR imager critically depends on the
functioning of the RF bridge, which acts as
a precision reflectometer. The resonator is
loaded with the sample and tuned to the
resonance frequency of 300 MHz and the
coupling is adjusted to ‘‘critical’’ value. The
‘‘balanced’’ bridge condition is perturbed
by the EPR resonance absorption. A
sinusoidal modulation of the resonance
frequency and subsequent phase sensitive
detection produces the EPR signal.
CW EPRI method is sensitive to
physiological motion of the animal and hence
an efficient automatic frequency control
333
(AFC) feature is highly desirable. In addition
to animal motion, thermal drifts of the
resonator frequency and RF source
instabilities also affect the bridge balance.
The AFC circuit in our CW EPRI scanner,
(Fig. 22.1) uses a low level FM modulation
of the RF source, which is converted to AM
modulation, and after phase-sensitive
detection, produces an error signal to adjust
the RF source. During in vivo EPR
measurements, the animal motion can affect
also the resonator coupling. Hence, automatic
coupling control (ACC) is needed to further
improve the quality of projections. In an ideal
situation, signals from both AFC and coupling
changes need to be separated from the true
EPR signal, and used to provide the feedback
loop error signals for appropriate dynamic
corrections. Similarly, implementation of
automatic phase control (APC) can enhance
the SNR, because the phase error can have a
marked effect on the noise level. Hence APC
can particularly improve the consistency of
the projections of large size objects in lengthy
3D data acquisition schemes.
Module 3
Data Acquisition The data acquisition system
(DAS) is built around a commercially
available lock-in amplifier and a PC to
control the spectrometer/imager. The PC,
via the IEEE bus addresses all operations
of the spectrometer such as: setting up the
number of data points, modulation
frequency and amplitude, signal
amplification level, detector reference phase,
center field, sweep width, sweep time, the
number of gradient steps, the gradient
amplitude and orientation, etc. Once these
parameters are specified, the software
calculates the gradient amplitudes to be set
on the x, y, and z axes. Based on these
334 Biomedical Magnetic Resonance: Proceedings of the International Workshop

Fig. 22.1: Schematic diagram of 300 MHz EPR imaging system showing the individual submodules consisting
of magnet, gradient coil system, analog sweep unit, modulation unit, RF bridge, RF synthesizer, SR850lock-
in amplifier, acquisition computer, and the communication links.

inputs, the program automatically and
sequentially sets the required gradient
currents on the three gradient amplifiers,
acquires projection data, and stores the
projections in the computer memory. It also
collects a zero-gradient spectrum to
deconvolve the projections, if required, to
reduce line broadening.
Image Reconstruction
The image reconstruction sequence involves
the following: individual projections are
base-line corrected, either by averaging a
predetermined (user chosen) number of
initial points in the spectrum, or by
averaging the entire first derivative
spectrum. The resulting profiles are
subjected to a smoothing filter and the
spectrum is integrated. The integrated
profiles are then subsampled to 64, 128, or
256 points. In 2D image processing, the
filtered profiles are subjected to either a
Ram-Lak or Shepp Logan filter and the
images reconstructed via a single stage PR
method. For 3D imaging, the smoothed
profiles are subjected to a second derivative
filter and are further subjected to a
Savitzky– Golay smoothing filter. The
resulting profiles are then used to
reconstruct 3D image using a two-stage back-
projection algorithm. If necessary, it is also
possible to deconvolve the projections with
the zero-gradient spectrum prior to the
application of the smoothing filters, for
resolution enhancement. To display 3D
images from the projection data, the surface-
rendering program VOXELVIEW® in a
Silicon Graphics Indigo2 workstation was
used. The VOXELVIEW software also allows
slicing of the 3D image through axial,
coronal, and sagital planes and performing
distance, area, and volume measurements
on the image. Slices of 3D images of normal
and tumor bearing legs of a 3CH mouse is
presented in Figure 22.3, Plate 12.
 EPR Imaging for Biomedical Applications 335

Limitations of CW EPR Technique gradients were Fourier transformed to get

the projections and images were constructed
CW EPRI techniques utilizes the inherent
using the standard FBP method.
advantages of phase-sensitive detection and
Projection reconstruction using the free
do not impose any restriction on the line
induction decay (PR-FID) has demonstrated
width of the species under examination. But,
the capability of fast radio frequency
the image resolution in this method depends
electron paramagnetic resonance imaging of
on the line width and the gradient strength.
small objects. Nevertheless, the implemen-
The time required for CW EPRI depends
tation of pulsed EPR methods at radio-
on the rate of the magnetic field scan. The
frequencies (RF) for in vivo applications is a
spectral acquisition times (~500 ms –10 s)
formidable task, because of the following
are comparable to those of physiological
challenges:
motions such as heartbeat, peristalsis,
1. Narrow pulses are required to have
respiration, etc. which can result in both
broad spectral coverage. The narrow
increased noise and image artifacts. Artifacts
pulses should encompass several cycles
associated with sweep rates, magnetic field
of the incident frequency of the RF to
modulation, and power saturation can also
have reasonable selectivity in excitation.
distort the spectra. The pulse techniques can
The pulses should be amplified signi-
offer better temporal solution. Nevertheless,
ficantly, with minimal rise times
the recent studies using the oscillating
associated with pulse amplification, to
magnetic field gradients have open the
provide the necessary nutation of the
possibility of very fast CW EPRI.22,23
spin systems in the resonator.
Pulsed RF EPRI 2. The resonator should be able to
accommodate the object under study in
The potential of time-domain EPR techniques
terms of physical dimension as well as
to obtain better temporal resolution as well
spectral bandwidth. Additionally, the
as the Fellgett multi-channel sensitivity
dead time should be minimal. The
advantage for in vivo studies, using RF FT
performance of a pulsed EPR spectro-
EPRI was demonstrated for the first time in
meter mainly depends upon the spectro-
our lab.10,11 In these studies, free induction
meter dead time, excitation bandwidth
decays (FIDs) following the pulse excitations
and sensitivity. The dead time of the
were collected in the presence of static
spectrometer is a critical parameter,
magnetic field gradients in a polar
because it severely affects the choice of
coordinate frame, Fourier transformed, and
spin probes that could be studied by
then used for image reconstruction by back
pulsed EPR. Although there are several
projection techniques. The short T2* of the
factors that contribute to the dead time,
EPR spin probes ruled out the possibility of
for low frequency operation the major
gradient switching. Hence imaging schemes
source is the ring down time constant of
such as the spin-warp, spin-echo or
the resonator, which is given by
gradient-echo techniques could not be used
for EPRI, even for narrow line, paramagnetic τr = QL/ω ...(2)
contrast agents. Hence the FIDs, collected Here QL is the loaded quality factor
in the presence of static magnetic field of the resonator. The dead time, which
336 Biomedical Magnetic Resonance: Proceedings of the International Workshop
is inversely related to the carrier
frequency ω, poses serious problem for
the successful detection of the time-
domain EPR responses at RF. Useful data
can be collected only after the input pulse
power decays down to the detector noise
level. Therefore for weak signals, data
can be collected only after about 300 to
400 ns (dead-time) following the trailing
edge of the pulse. Spin-spin relaxation
times of most spin probes, such as
nitroxides, are in the nanosecond time
range; therefore, for these spin probes,
the signal in the time domain may not
persist beyond the resonator dead time.
3. The first stage amplifier should possess
rapid recovery times, low noise, and high
gain; all these are necessary to detect
rapidly decaying signals of the electron
spins. Moreover, to exploit the faster
relaxation behavior of electron spins for
signal enhancement, rapid digitization
Fig. 22.4: Block diagram of the time-domain RF FT EPR spectrometer
and averaging strategies should be
implemented.
We have addressed these challenges and
fabricated a broadband time-domain EPR
spectrometer suitable for in vivo appli-
cations.11 Figure 22.4 shows a block diagram
of this time-domain RF EPR spectrometer,
operating typically at 300 MHz. Though in
principle similar to NMR imaging, the
implementation of time-domain EPRI for in
vivo applications requires developmental
efforts in all aspects of the spectrometer,
namely, the transmit arm, the receive arm,
the resonator, and the data acquisition
system. In the following a brief description
of the important modules of the RF FT EPR
imager is presented.
Diplexer
A single coil probe was used for the
experiments. Therefore a transmit/receive
 EPR Imaging for Biomedical Applications
diplexer is preferred over the circulator, to
avoid excessive insertion losses. The diplexer
used in our study is of a standard (¼)λ
configuration with a series pin-diode switch
assembly in the receiver path. These diodes
are connected in a back-to-back configuration
to minimize the transients in the RF paths.
Such an arrangement provides high speed
switching (~about 5 ns) necessary for time-
domain RF FT EPR experiments. Receiver
isolation during transmit mode is 25 dB with
a transmit insertion loss of 2 dB. The
insertion loss during receive mode is as low
as 0.5 dB. This provided the necessary
sensitivity in terms of excitation as well as
desired bandwidth for imaging experi-
ments.
Resonator
Several factors need to be considered in the
development of resonators for time-domain
EPR imaging methodology for in vivo
applications. While in MRI, the frequency
bandwidth is about 20 kHz to obtain high-
resolution images of large-size objects, in
EPRI the required bandwidth is at least 10
MHz, even for a resonator of 25-mm bore
size. Desirable features of a resonator for
such time-domain EPR applications are:
(1) ability to accept narrow and intense
pulses and convert to B1 with high efficiency;
(2) short resonator ring-down time;
(3) optimal Q-profiles with adequate spectral
bandwidth for imaging studies; (4) adequate
B1 field homogeneity; and (5) potential to
be scaled up for larger objects. We have
designed parallel coil resonators for the RF
FT EPR experiments to obtain: optimal,
homogeneous B1 field within the resonator
for a given transmit power. Loops of
conducting material spaced at equal
intervals, along the cylindrical axis are
337
connected in parallel to obtain large volume
coils. These parallel coil resonators offer an
attractive alternative to bird-cage, solenoi-
dal, and loop-gap resonators. The design is
of particular advantage for studying large-
size objects, because the usable volume can
be readily increased and the construction is
inexpensive and relatively simple to
fabricate. The resonator bandwidth readily
encompasses a spectral bandwidth of 15
MHz. These dimensions are adequate to
accommodate an experimental animal, such
as a mouse, for imaging studies. The loaded
Q of the coil is maintained at 25. Q reduction
in these resonators can be achieved by
overcoupling. In addition, a resistance in
parallel (~3.3 kV) is also used to minimize
the dead time and also help reach the
desired spectral bandwidth. Pulses down
to 30 ns give a power spectrum almost close
to the theoretical expectation. We have also,
in addition, investigated surface coil
resonators for RF EPRI by connecting loops
of the required diameter to a ½ length semi
rigid coaxial line to allow for seperation
between the coil, and the tuning and
matching capacitors.
Signal Detection
A mixer was used for phase-sensitive
detection. The induction signals from the
receiver arm are mixed with a local
oscillator ~350 MHz. The intermediate, 50
MHz signal (IF) is isolated using a low pass
filter and utilized for digitization and
recovery of the FID. The first stage amplifier
in the receive arm is designed to avoid
overload and also to facilitate fast recovery
to detect and amplify the weak FID signals.
After further amplification and filtering
followed by, rapid digitization and coherent
averaging provide adequate SNR.
338 Biomedical Magnetic Resonance: Proceedings of the International Workshop

Data Acquisition System digitized waveforms from the sampler and

adds them into a 32-bit buffer. The summing
EPR spectroscopy and imaging suffer from
and digitizing operations overlap so that 4
the loss of sensitivity resulting from
k wave forms can be summed at a rate of
unfavorable Boltzmann factor incurred by
238000 per second. However, in view of
the low frequency operation. The advantage
the cycle time (~50 kHz) to take care of the
of sensitivity enhancement by Time-domain
relaxation, the full capability of the digitizer/
EPR may be utilised to offset the loss in
summer is seldom used. The summed signal
sensitivity in the RF regime of EPR
was downloaded to a host computer for
spectroscopy and imaging. One of the
further data processing.
advantages of FT EPRI is its potential for
The inadequate onboard memory and the
improved temporal resolution over the
data download time to the host computer
continuous wave technique. The scan time
of the custom-built averager led to longer
is eventually determined by the spin probe
image collection times. Hence, we were
clearance rate and the required temporal
interested in developing a low-cost data
resolution. For meaningful interpretation of
acquisition system, satisfying the require-
the EPR images, the imaging protocol must
ments of time-domain EPR, as a reasonable
be faster than the clearance of the exo-
alternative. In this context, a 500 MS/s
genously administered spin probe. Reduced
sampling/summing board ~EG and G 9826-
image acquisition times can further enable
500, was found to have the necessary speed
examination of temporal changes in pO2 as
for both data acquisition and summation.
well as spin probe distribution. Therefore,
The digitizer has an eight-bit resolution with
high-speed data acquisition and averaging
an on-board memory sufficient to sum 16384
system is very essential to fully exploit the
FIDs. The minimum retrigger rate of the
faster electron spin relaxation times and
board is as high as 1.66 MHz allowing rapid
achieve the optimal sensitivity and temporal
sampling and summing of the transient data.
resolution, needed for in vivo studies.
We have designed a dual channel data
During the earlier stages of the
acquisition system, with two EG and G 9826
development of our pulsed RF EPR imager,
digitizer boards and integrated it in our
high-speed data acquisition/averaging
pulsed RF EPR spectrometer.16
systems, suitable for EPR applications, were
not commercially available. Hence, high-
Image Reconstruction
speed data acquisition systems were deve-
loped in house as well as custom-built.12,24 The FIDs are first filtered by a digital
The digitizer/summer of the customer-built Butterworth bandpass filter of appropriate
averager consists of four interleaved flash window (approximately 610 MHz) and also
analog-to-digital converters.12 Each unit has by resolution/sensitivity enhancing filters,
a digitizing speed of 500 MS/s and a as needed, finally, are zero filled up to 64
minimum retrigger period of 4.2 μs. The k points and Fourier transformed to obtain
sampling unit can provide 1 GS/s in single- the projections. Image reconstruction from
or double-channel modes with an 8-bit these projections is performed using PR
vertical resolution. The summer captures the method.
 EPR Imaging for Biomedical Applications
Artifact-free Imaging
In pulsed RF-EPRI, a 90° rectangular pulse
is used for the excitation of the spins. Such
a hard pulse leads to artifacts in images
because of its nonuniform excitation profile
across the sample. The Fourier transform of
the rectangular pulse has the characteristic
sinc-shaped [sin (x)/x] excitation profile. This
may not be optimal for imaging applications,
because it introduces ‘sinc-like’ side lobes,
symmetric about the central frequency of
excitation, which get progressively weaker
with resonance offset. For a pulse width tp,
the central lobe, which zero-crosses at
frequencies ±1/2tp, is centered on the carrier
frequency, and uniform excitation is possible
only for half this range. With such a profile,
the excitation becomes nonuniform and the
resulting sensitivity loss, especially in the
outer regions of the images can be signifi-
cant.
Shaped pulses of short-duration may help
to achieve more uniform excitation profiles
over the extended frequency ranges often
needed for EPR imaging applications. Of
the various types of excitation schemes for
uniform excitation, the sinc pulse is easy to
implement. The sinc pulse represents the
inverse Fourier transform of a rectangular
profile and, therefore, should produce a
uniform excitation profile. The advantage
of sinc pulse over a rectangular pulse is that
the former essentially exchanges power in
the center of the excitation band with power
at the edges. In addition, the Q-profile of
the large bandwidth, low Q, resonator can
also distort the EPR images. In smaller
objects or low resolution imaging applica-
tions, where the gradient values are low,
such distortions are not too evident. But, as
the bandwidth requirement increases, this
effect can be quite significant. It can distort
339
the image and also can lead to errors in
quantitative parameter estimation. To
address this problem and to obtain artifact
free imaging, we have evaluated three
different waveforms and their experimental
excitation profiles. We have shown that using
a tailored sinc pulse, excitation as well as
the resonator Q-profile artifacts in pulsed
RF-EPRI can be readily corrected.19
Limitations of Pulsed RF EPRI
Pulsed RF EPR imaging based on the PR-
FID technique is a pure frequency encoding
technique. Being a frequency encoding
method, the PR-FID requires accurate
phasing of the sampled magnetization. In
this context, the spectrometer dead time
plays a crucial role in pulsed RF EPRI.
Besides limiting the sensitivity, the dead
time also leads to considerable distortion in
spectral features, when the FID is not
properly phase-corrected. The magnetization
components accumulate phase errors in
proportion to their observed frequencies.
The missing time interval may be too large
for proper data reconstruction at high offset
frequencies when the dead times are
comparable to the phase memory times.
Hence imaging of large size objects by the
pure frequency encoding PR-FID technique
is prone to artifacts arising from
spectrometer dead time. In addition, the
T2* of the FIDs depends on the spectral
range according to the equation
1/T2* = 1/T2 + γe ΔB/2 ...(3)
where ΔB is the variation of magnetic
field over the region of interest, T2 is the
spin-spin relaxation time, and γe is the
electron gyromagneticratio. Such a T 2 *
dependance can lead to a differential loss
in the integrated intensity among the
various projections, causing artifacts in the
340 Biomedical Magnetic Resonance: Proceedings of the International Workshop
image, reconstructed using FBP. This
particular artifact will be significant for
oblong objects under large gradients. In
addition, Attempt to increase the resolution
by increasing the gradients will lead to
further reduction of T2* according to Eq.
(3).
Constant Time Pulsed RF EPRI
A novel pure phase encoding technique has
been proposed to enhance the resolution of
solid-state NMR imaging. This technique
known as single-point imaging (SPI), also
referenced as constant time imaging, has
been later used for in vivo 11B NMR imaging.
SPI has been widely applied to solid-state
NMR imaging and shown to provide artifact
free images. But, the requirement of one
excitation per pixel of the SPI image leads
to very long image collection times, limiting
the usefulness of the methodology. Although
the high resolution capability of the SPI
modality has been recognized nearly 15
years ago in 1D EPRI, no further efforts
have been made, perhaps due to the
limitation posed by the long data acquisition
time. But, by implementing fast data
acquisition hardware and software, we have
shown that well resolved, distortion-free,
2D EPR images of phantoms approximating
the physical dimensions of the internal
organs of mice could be obtained in
reasonable time. In what follows we
describe briefly the principles of this
modality.
Principles of Constant Time Imaging
A schematic description of 1D SPI method
is shown in Figure 22.1. In this sequence, a
short, intense and nonselective radio-
frequency (RF) pulse generates the
transverse magnetization. The spatial
information is encoded in reciprocal space,
S(k), where k = (½ π) γGt, by amplitude
cycling of the applied phase gradient G. At
each gradient value, a single complex data
point is acquired in quadrature at a fixed
time t = tp following the RF pulse. The SPI
signal intensity, S(tp) at time t = tp, is related
to the local electron spin density, ρ(z) along
the gradient direction, Z by
S(tp) = ∫-∝ ρ(z) exp [iγG.Zt]dz ...(4)
In the k-space formalism the above equation
may be written as
S(k) = ∫-∝ ρ(z) exp [i2πk.Z]dz ...(5)
Thus the signal intensity, S(k) and the
spin density, ρ(z) have a Fourier relationship
and therefore direct Fourier image
reconstruction algorithm can be applied. In
SPI, the k-space is traversed by stepping
the gradient amplitude while keeping the
encoding time tp constant. A 2D image is
collected by similarly traversing in another
Cartesian direction. Thus in a 2D SPI
experiment using free induction decay (FID),
both spatial dimensions are phase encoded.
Because there is no frequency-encoding
gradient, the SPI images are free from
distortions due to the magnetic field
inhomogeneity, susceptibility variations and
chemical shift. The point spread function
(PSF) and hence the resolution is unaffected
by T 2 * because the magnetization is
measured as a function of applied gradient
and not as a function of time, in contrast to
PR-FID method. The pixel resolution, ΔZ is
given by,
ΔZ = FOVz / N = 1.2π / iγGz max tp ...(6)
where N is the number of samples of
k-space, measured in Z direction, and γe is
the gyromagnetic ratio of the electron. The
resolution, even for short T2* species, is
limited mainly by the maximum gradient
 EPR Imaging for Biomedical Applications
strength. Even for spin probes with broad
EPR signals, image resolution independent
of line width can be achieved.15 Although
the pure phase encoding method produces
high-resolution images with minimal
artifacts, the SPI sequence is time inefficient
because it requires N2 excitations for a 2D
image of N2 pixels. Thus the 2D pure phase
encoding requires N times more phase
encode steps than a 2D frequency-phase
image. Long data collection time of SPI may
limit its use for in vivo imaging. But, efficient
data acquisition system (DAS) may solve
this problem. Towards this direction, we
have focussed further improvement the
speed of our DAS. At present, we can collect
2D SPI imagres (64 × 64) in 5 seconds.
Fig. 22.5: The schematics for data collection for single
point (constant time) EPR imaging. Time-domain
responses (FIDs) are collected in presence of
stationary gradients after applying a 90o pulse. The
phase-encoding gradients are incremented in equal
intervals between ± Gmax. and applied in nested loops
in a Cartesian raster. Although only a single time
point is needed for image reconstruction, the whole
FID (approx. 1500 points) is collected at 500 Ms/s
digitization rate. Each data point in the FID undergoes
a pseudo echo phase modulation (similar to the
gradient recalled echo imaging sequence in MRI). A
one-dimensional gradient ramping and the
corresponding pseudo echo are shown on the right
hand side. Fourier transformation of each echo leads
to a one-dimensional image profile.
341
SPIN PROBES FOR EPRI
Spectroscopy and imaging can have different
requirements; the former generally needs a
characteristic set of lines whereas the latter
requires a signal as intense as possible to
aid detection. Therefore a single spectral
line is preferable for imaging applications.
But for in vivo applications, it is insufficient
to simply select a probe which has a single
spectral line. The probe must have a chemical
significance to its application. A few
desirable application-dependent properties
of a useful spin probe for biological
applications are:
i. Suitable solubility in the chosen
medium
ii. Narrow spectral lines.
iii. A simple EPR spectrum (in some types
of imaging or oximetry a single line is
desirable).
iv. Sharp lines with no unresolved
splittings, to provide maximum sensiti-
vity to detection,
v. Chemical, thermal and metabolic
stability.
vi. Non-toxic for in vivo applications.
vii. EPR parameters sensitive to physio-
logical conditions
The capability of EPR imaging has been
hampered by the lack of spin probes fulfilling
the above criteria. In fact, the multiple lines
of nitroxyls cause hyperfine-based limita-
tions in image resolution and hyperfine-
based artifacts in the reconstructed image.
Solid state probes with a single-line ESR
spectrum have been tested. 25 The most
commonly used solid probes are lithium
phthalocyanine (LiPc), fusinite a type of coal
and carbohydrate chars. The line broadening
of a single crystal of LiPc covers the range
from 0.002 mT to 0.1 mT for 0 to 100 percent
oxygen respectively showing a linear
342 Biomedical Magnetic Resonance: Proceedings of the International Workshop
dependence on pO2. But these solid-state
spin probes can not be used for noninvasive
measurements.
Need for Newer Spin Traps
Unfortunately, the nitroxyl half-life is short
and there is a need to develop new spin
probes possessing longer half-lives and high
tissue selectivity. Different approaches have
been proposed to increase the nitroxyl half-
life. The half-life of a nitroxyl, measured in
the mouse tail, is prolonged to about 10 h
when it is bound to bovine serum albumin.26
The in vivo mouse Tempo-dextran half-life
is about 30 min.27 Novel single-line free
radicals based on triarylmethyl backbone
have been recently developed for EPR
imaging.28 TAM is a charged radical, highly
water soluble with a half-life in tissue of
about 60 min and a linewidth around 32
mG in hypoxia.
APPLICATIONS
Oximetry
Abnormal values of pO2 are linked to many
pathophysiological conditions (e.g., ischemic
diseases, reperfusion injury, and oxygen
toxicity). Approximately one-third of human
tumors, evaluated for oxygen status has
shown significant oxygen deficiency, and the
oxygen deficiency increases the tumor’s
resistance towards cancer treatment
modalities, including radiation and
chemotherapy. Hence, a noninvasive
technique that could accurately and
repetitively measure tissue oxygenation
would find broad application in clinical and
basic research. EPR methods (both
spectroscopic and imaging) show better
sensitivity to oxygen concentration than the
NMR methods. In EPRI, the spatial as well
as the spectral information of the
exogenously administered spin probes is to
be derived to obtain pO2 maps.
Spectral-Spatial (Spectroscopic) Imaging
Spectral-spatial imaging has been
implemented in CW EPRI by several
researchers. In this methodology one of the
image dimensions gives the spectral
information. In spectral-spatial imaging, the
magnitude of the gradient, G, for a given
projection angle α can be computed using,
G = (ΔB/ΔX) tanα ...(7)
Here, ΔB is the spectral window and the
spatial dimension is given by ΔX. Based on
this formula, gradient values are calculated
for various values of α and a set of
projections are collected. Using FBP, the
projections are converted to 1D spatial and
1D spectral image.
For a pseudo viewing angle α = 90° we
need an infinite gradient. Since an infinite
gradient is not practical, we may have to
miss some profiles close to α = 90°, but this
will not adversely affect the reconstruction.
Missing angle back-projection algorithms are
available in the literature. The missing angle
range will depend on the gradient strength
that can be applied, limited by the
capabilities of the hardware. The scan time
for each field sweep should be kept
proportional to the scan range, such that
the resultant sweep rate (G s-1) is constant.
Such a spectral spatial image can give
linewidth information and hence the oxygen
concentration can be calculated.
We have investigated the application of
continuous wave (CW) electron para-
magnetic resonance (EPR) constant-time
spectral-spatial imaging (CTSSI) for in vivo
oxymetry.29 Both, 2D and 3D spectral-spatial
imaging (SSI) studies of phantom and live
 EPR Imaging for Biomedical Applications
mouse were carried out using projection
reconstruction (PR) and constant-time (CT)
modalities using a CW EPR spectrometer/
imager operating at 300 MHz frequency.
Figure 22.6 illustrates this application with
phantom imaging. The phantom consists of
three tubes of the Oxo63 solutions (5.0 mM)
saturated with air (21% O2), 10 percent O2
and Ar (0% O2) gas at least for 1 hr at room
temperature respectively. The FOV of
spectral and spatial window were selected
as 1.0 Gauss and 3.0 cm, respectively. A set
of 16 polar projections were obtained within
71.56° pseudo-viewing angle with constant
angle increment in PR-SSI modality. The FOV
of spectral and spatial window were selected
as 1.0 Gauss and 3.0 cm, respectively. A set
of 16 polar projections were obtained within
71.56° pseudo-viewing angle with constant
angle increment in PR-SSI modality. Sweep
width was changed from 1.00 to 3.16 Gauss
depending on the projecting angle. Different
Filtered Back-Projection (FBP) programs
were employed to reconstruct the 2D
spectral-spatial images.
Although the principle of the CTSSI
experiment is based on the time-domain
EPR, EPR data in the frequency domain (CW
EPR) can easily be treated in the same
manner. The inverse FT of the acquired data
with field or frequency sweep is the only
extra step necessary to obtain the time
domain data. In the CTSSI modality, spectral
data were obtained under 16 linear steps of
field gradient increment. All spectral data
were obtained at a constant sweep width
of 16.0 G with 1024 data points. The
projections were Fourier transformed to
yield a 16 × 1024 pixel k-space matrix. 2D
spectral-spatial images were reconstructed
in accordance with the principle of CTSSI
described above. Before final FT of spectral
343
axis, the FOV of spatial window was
rescaled to 3.0 cm for all time points and
interpolated to 128 points. The central 1.0
G range of spectral axis was extracted and
interpolated to 128 points after the FT along
the spectral axis. Finally, a spectral-spatial
image represented by a 128 × 128 pixel-
matrix is obtained.
Similarly, 3D spectral-spatial images
having 1.0 Gauss × 3.0 cm × 3.0 cm FOV
were reconstructed upon 64 × 64 × 64 pixel
matrix using 16 × 16 spectral data which
obtained in the PR and CTSSI modality for
the same phantom. From the 3D spectral-
spatial images reconstructed by FBP, the 2D
spatial distributions of the signal intensity
(double integrated value) and the line width
mapping were calculated and are shown in
Figures 22.6A and B, Plate 13 respectively.
Figure 22.6C shows histogram of the line
width obtained from the area indicated on
the line width map (Fig. 22.6B, Plate 13). A
suitable 2D spatial distribution (τ = 48) was
chosen from the SPI data set (Fig. 22.6D,
Plate 13). After the 2D spatial distribution
was masked by a threshold value, spectral
shape was obtained by FFT of pseudo-time
domain only for the masked area. Then,
the pixel-wise line width was calculated (Fig.
22.6E, Plate 13). Figure 22.6F, Plate 13 shows
the histogram of the line width obtained
from indicated area (dotted square) of
Figure 22.6E, Plate 13. Both FBP and CTSSI
show similar good result although CTSSI
gives higher line width value than FBP in
this phantom study. The relatively low pixel
resolution of the spectral axis works as a
smoothing filter for FBP image. FBP will
show a better range of line width when a
larger pixel matrix is employed to represent
a better pixel resolution. FBP shows
inhomogeneity of the line width distribution
344 Biomedical Magnetic Resonance: Proceedings of the International Workshop
between outer side and inner side of the
image. On the other hand, fundamental
spectral smoothness of CTSSI is not affected
by pixel resolution. However, 3D CTSSI
gives wider line width than 2D CTSSI. This
is because each pixel has 4 neighboring
pixels leading to mutual broadening by
overlap. In addition, CTSSI shows an appa-
rent higher line width values at outer edges
of the object. Nevertheless, CTSSI shows
higher convergence for the line width values
compared with FBP and shows a greater
promise of providing reliable line width
information with relatively noisier data.
Figure 22.7, Plate 6 shows the application
of these techniques for in vivo tumor
oximetry. 2D spatial distributions and 2D
line width mappings of normal and tumor
(1 cm size of SCC tumor) bearing mouse
legs of an anesthetized are presented in this
figure. Figure 22.7A shows a cartoon of the
positioning of mouse legs. Figures 22.7B and
22.7C show the 2D spatial distributions
(double integrated values) and the line
width mapping respectively of Oxo63
injected in the tumor bearing mice obtained
by FBP. Figure 22.7D shows histogram of
the line width obtained from indicated area
(dotted square) of Figure 22.7C. Figure
22.7E and 7F show the 2D spatial
distributions (SPI, τ = 24) and the line width
mapping respectively of Oxo63 injected in
the tumor bearing mice obtained by CTSSI
modality. Figure 22.7G shows histogram of
the line width obtained from indicated area
(dotted square) of Figure 22.7F. The 2D
spatial distributions from FBP image are of
lower intensity compared with SPI obtained
in CTSSI modality which shows a better
contrast in the image. The line width
mapping from FBP image shows very little
difference between the normal and the
tumor legs. The histogram from FBP image
shows that the values are varied widely.
As described above, spatial resolution of
PR method is affected by line width. The
clear 2D distribution of Oxo63 in mouse legs
was obtained by SPI slice. The line width
mapping shows distinct difference between
the normal and tumor legs. The in vivo
CTSSI shows wider line width than phantom
study, mainly due to the amplification effect
of T2* mentioned earlier. The wide spatial
distribution of Oxo63 in the mouse legs
generates broad blurred distribution at short
τ region of SPI data set. This low frequency
contribution on the pseudo-FID may cause
an overall line width broadening. However,
the in vivo CTSSI study shows clear
differences in the line width between the
normal left hind leg and tumor-bearing right
hind leg. The line width mapping (Fig. 22.7F)
shows the distinctly hypoxic nature of the
tumor leg. The histogram also clearly shows
the difference between normal and tumor
legs (Fig. 22.7G).
Oximetry by FT PR method
In pulsed radio frequency (RF) EPR the spin
probe is excited in the time domain with a
short RF pulse and the resulting FID is of
the form, exp [-t/T2*] cos [(ω−ωo)t. Here,
T2* is a decay constant with contribution
from both natural T2 and magnetic field
inhomogeneties, and (ω−ωo) is the offset
frequency. Fourier transformation of the FID
gives the absorption spectrum from which
the line width can be measured after suitable
phase correction. The FID is governed by a
first order decay constant, T 2*, which is
related to the line width by,
Line width = (π T2*)-1 ...(8)
T2* is shortened by spin-spin interaction
between the spin probes. T2* can be further
 EPR Imaging for Biomedical Applications 345

Figs 22.7A to G: 2D spatial distributions and 2D line width mappings of normal and tumor bearing mouse legs
of a C3H mouse with 1 cm size of SCC tumor on one of the legs. (A) A cartoon of the positioning of mouse legs.
(B) 2D spatial distributions of the double integration values and (C) the line width mappings calculated from
3D PR spectral-spatial image. (D) The histogram of the line width obtained from indicated area in C. (E) 2D
spatial distributions from SPI data and (F) the line width mappings calculated from 3D CT spectral-spatial
image. (G) The histogram of the line width obtained from indicated area in F. The differences in the line width
ranges between D and G arise from the differences in T2 and T2*
346 Biomedical Magnetic Resonance: Proceedings of the International Workshop
shortened by molecular oxygen interacting
with the spin probe. Thus the observed T2*
can be written as the sum of the above
factors,
1/ T2*(observed) = 1 / T2 (intrinsic) + 1 / T2
(conc.) + 1 / T2 (pO2) ...(9)
where, 1/T2 (intrinsic) results from natural
line width, as well as the magnetic field
(B o ) inhomogeneity. Therefore, for a
particular spin probe at a fixed concentra-
tion, the effect of pO2 on T2* can be deter-
mined.
Fourier transformation of the FID
provides the absorption spectrum whose
peak intensity is dependent on (a) the
concentration of the spin probe and (b) the
line width. Since the FID is an exponentially
decaying function, prior to FT, deletion of
the initial points of the already acquired
FID (equivalent to acquisition delay) can
cause significant decrease in the intensity of
the absorption spectrum. The signals that
belong to slowly decaying components
survive longer than the ones belonging to
fast decaying components. The intensity of
the absorption spectrum when plotted as a
function of delay (time) should exhibit an
exponential decrease, with the slope related
to 1/T2* and can be expressed as:
I(t) = I(0) exp (-t/T2*) ...(10)
Since oxygen shortens the T2*, all other
conditions being identical, the slope of the
intensity as a function of acquisition delay
would be larger, higher the oxygen content.
Such data treatment, when applied to
imaging experiments provides information
related to T2* on a pixel-by-pixel basis. Thus
a single data set obtained from constructing
a spatial EPR image can also provide oxygen
maps by computing a series of images
varying in the length of ‘acquisition delay’.
The slope of intensity vs. delay time
corresponding to a given pixel in consecutive
images, then reflects the oxygen concen-
tration. A schematic representation of T2*
weighted spectroscopy is given in Figure
22.8. Calculated FIDs of two signals at
50.5 and 52.5 MHz with the lower frequency
signal corresponding to a larger line width
are presented at the top. The initial time
points were progressively deleted (0, 100,
200 and 400 ns) to obtain the corresponding
absorption mode spectra, given in the
bottom. The distinctly different slopes of
intensity attenuation as a function of delay
are also indicated. The relatively slower
attenuation of narrow line signal would
indicate that hypoxic regions could be better
distinguished by the delayed processing
scheme of ‘T2*-weighted’ EPR imaging.
Figure 22.9, Plate 14 illustrates oximetry
by RF FT PR method using a three-tube
phantom. Figure 22.9(A) shows the sche-
matics of the phantom, and Figure 22.9(B)
shows the 2D spin density images of the
phantom obtained without any T2* discri-
mination. The three tubes in the image show
almost identical spin density map. Figure
22.9(C) gives the plot of pO2 based on (1/
T2*). The differences in oxygen concentration
are clearly visible due to differential line
broadening brought out by different pO2.
It is also possible to derive pO2 from the
relaxation rates (1/T2*).
Oximetry by Time Domain
Single Point Imaging
Single point EPRI can also be used to
determine oxygen concentration in vivo. To
do so, the pO2 dependent line width must
be determined from the SPI images. In
traditional PR-EPRI this is straightforward,
as outlined in the previous section.
Unfortunately, in SPI-EPR the field of view
 EPR Imaging for Biomedical Applications 347

Fig. 22.8: A schematic representation of T2* weighted spectroscopy. Calculated FIDs (top) of two signals at
50.5 and 52.5 MHz with the lower frequency signal corresponding to a larger line width. The initial time points
were progressively deleted (0, 100, 200 and 400 ns) to obtain the corresponding absorption mode spectra,
given in the bottom. The distinctly different slopes of intensity attenuation as a function of delay are also
indicated. The relatively slower attenuation of narrow line signal would indicate that hypoxic regions could be
better distinguished by the delayed processing scheme of ‘T2* weighted’ EPR imaging.

of single-point image depends on tp as given
by Eq. (4). Therefore, a direct pixel-by-pixel
comparison cannot be done, because the
corresponding pixel represents different
spatial location. To correct for the change
in field of view, images generated for
different tp are resized. The resized images
can be compared pixel by pixel, and slopes
of the decay of pixel intensities can be
computed to arrive at pO2, after calibration
with samples of known pO2.
Pharmacokinetics
Figure 22.10, Plate 14 shows a sequence of
2-D CW EPR images of Oxo-63 distribution
in anesthetized mouse after intravenous
administration.9 The mouse was positioned
(A) in the resonator in such a manner that
the thoracic, abdominal and pelvic regions
are in the FOV. Projections corresponding
to 2-D images were collected at 2.8, 9.0,
13.0 and 16.9 minutes (B,C,D,E respectively)
after spin probe administration using a
gradient of 1.0 G/cm. The image at the first
time point was interpreted as the spin probe
localization predominantly in the two
kidneys with some intensity in other organs
in the abdominal region such as liver, spleen,
etc. Subsequent images suggest a
progressive redistribution of the spin probe
from the circulation to the bladder as
evidenced by the increasing image intensity
from the bladder. However, contours
corresponding to the two kidneys could be
seen even in the presence of intense signal
from the bladder. The physical dimension
348 Biomedical Magnetic Resonance: Proceedings of the International Workshop
of the organs, obtained from the EPR images
were in good agreement with the actual
dimensions. Figure 22.10 F shows a graph
of the decrease in the average spin count
from the kidneys, and the increase in the
spin count in the bladder as a function of
time. In this experiment the spin distribution
in thoracic region could not be clearly
visualized, perhaps due to the low temporal
resolution of the CW EPRI modality.
Figure 22.11, Plate 15 shows a sequence
of 2D EPR images by SPI modality of a
C3H mouse as a function of time after
infusion of 100 μL of 100 mM Oxo63,
administered via tail-vein cannulation. In the
first image, the spin probe is found to be
predominantly localized in the thoracic and
kidney regions. Subsequent images suggest
a progressive redistribution of the spin probe
from the circulation to the bladder.
Improvement in the speed of data collection
has enabled clear visulaisation of the spin
probe in the thoracic region.
Redox Mapping
We have used 300 MHz, CW EPR imaging,
in vivo, to probe tissue heterogeneity and
redox status in normal and RIF tumor
tissues in mice.30 Figure 22.12, Platge 15
illustrates this application using a phantom
imaging of four identical tubes containing
nitroxides at identical concentration and
volume. Different rates of nitroxide reduc-
tion were established in each tube by
adjusting the flux of O2- generated by the
aerobic reaction of 5 mM hypoxanthine (HX)
at different levels of xanthine oxidase (XO).
Tubes A, B, C and D contained 0.0, 0.4, 0.8
and 1.6 U/ml of XO for nitroxide reduction
at different rates. EPR spectral projections
were collected immediately after addition
of the enzyme. A total of 12 projections
were collected for each 2D image which took
108 seconds and a total of 8 images were
collected separated temporally at 2-3 minute
intervals. Sequential images obtained form
the same phantom as a function of time show
that, while the intensity in tube A is
invariant, there is a progressive loss in
intensity in tubes B, C and D, at rates
proportional to the enzyme content in the
individual tubes. Reduction rates were
computed for individual voxels in the image
based on the time dependent intensities and
assuming a first order kinetics. Figure 22.12C
shows a ‘redox-image’ where the intensity
of each pixel is proportional to the first
order rate constant. In this image, the
intensity corresponding to the tube A is
within the image noise threshold, whereas
the image intensities corresponding to tubes
B, C and D increase progressively reflecting
the enhanced rate of reduction. The
histograms of the reduction rates and their
frequency of occurrence were computed
from each of the four tubes individually
(Fig. 22.12D). The median reduction rate,
derived from the histogram profiles of four
tubes correspond well with the XO
concentration. This study illustrates that a
time-series of 2D images measured from an
object with spatially differing rates of spin-
loss can provide a redox map or a
pharmacokinetic image, which would be a
reliable measure of the “metabolic” activity.
Redox Status in Tumors
EPR imaging experiments were also used to
study the differences in nitroxide reduction
in normal muscle and implanted tumors.
Tumors were grown by implanting RIF-1
cells in the hind leg of C3H mouse. The
spin probe, 3-carbamoyl proxyl (3-CP) was
administered by a tail vein cannulation.
 EPR Imaging for Biomedical Applications
Sequence of data sets were collected to
compute pharmacokinetic image or the
redox map can be computed. Figure 22.13A,
Plate 16 shows representative spatial images
obtained from such experiments on several
mice with tumors at different sizes (5 days,
7 days, 10 days, 12 days, 14 days after tumor
implantation). Images for each mouse at
different times after spin probe administ-
ration are shown with the corresponding
times indicated in the panel. The left hand
side in each panel represents the image of
spin probe distribution in normal leg and
the right hand side that from the tumor
bearing leg. The spatial images obtained
form such experiments show that the spin
probe accumulates in normal and tumor
muscle readily and the reduction of the
nitroxide proceeds rapidly as a function of
time. The accumulation of the spin probe in
the tumor was found to be significantly
altered as a function of tumor size.
The nitroxide reduction rates in the
tumor bearing leg and normal leg were
derived from the time dependent variation
of nitroxide levels from each voxel. The
image of the tumor leg showed a gradual
increase in nitroxide distribution area and
heterogeneity in the distribution of reduction
rates. Interestingly, the position of the lower
reduction rate was observed in the center
of tumor on days 12 (size 118.44 ± 8.53 mm2
s.e.m) and 14 (size 132.43 ± 11.65 mm2 s.e.m)
consistent with regions of necrosis in the
tumor core. This suggests that the image of
reduction rate may be related to the
physiological reducing capacity, oxygen
concentration level (hypoxia) and necrosis.
The differences in the reduction rates of
nitroxide in the region containing the normal
and tumor legs were further examined by
making redox measurements calculated as a
349
function of days after tumor implantation
and the results shown in Figure 22.13B, Plate
16. As can be seen the reduction rates
increased in both the normal leg and the
tumor bearing leg as a function of time after
tumor implantation.
FUTURE DIRECTIONS
Multimodality Approach
In EPRI, the spatial as well as the spectral
information of the exogenously administered
spin probes is obtained. Hence, EPR images
present the in vivo visualization of the spin
probes and often do not provide anatomical
information. In contrast, MRI is a well-
established modality that gives superior
anatomical information. Overhauser MRI
(OMRI) is a double resonance technique that
couples the advantages of MRI with the
sensitivity of EPR, by making use of the
Over hauser effect. By saturating the EPR
transition of a paramagnetic agent, the NMR
signal intensities of the coupled water
protons are enhanced by a significant factor
by means of the Overhauser effect. The
Overhauser enhancement (OE) depends on
the line width of the paramagnetic agent,
which in turn depends on oxygen concen-
tration, making it possible for coregistration
of both the anatomical structures and the
corresponding oxygen status. Using the
narrow-line trityl-based contrast agent, we
have demonstrated the feasibility of
obtaining morphological images of high
resolution, coregistered with oxygen
concentration in-formation.31 Based on these
images, the pO 2 status of any specific
location in the tumor can be interrogated
accurately and repeatedly, thus providing a
useful approach to evaluate the importance
of oxygen status in tumor physiology.
350 Biomedical Magnetic Resonance: Proceedings of the International Workshop
A crucial aspect of EPRI concerning its
applicability to human studies is the
powerdeposition caused by the EPR B1 field,
which may cause undesired heating.
Developing new spin probes with narrower
line widths, compared with those currently
used, will help in decreasing the RF power
deposition. We have explored the application
of correlation spectroscopy employing
stochastic excitation and the Hadamard
transform to time-domain FT-EPR spectros-
copy in the RF band. Our results show
decrease in the total acquisition time, and
an improved FID SNR, compared to the
conventional coherent averaging approach.
These techniques have the potential to
reduce the RF pulse power to the levels
used in CW EPR while retaining the
advantage of time-domain EPR methods.
These methods have the potential to
facilitate the progression to in vivo FT-EPR
imaging of larger volumes.
Recent development of narrow line spin
probes for functional imaging and the
potential of the pulsed technique to provide
better sensitivity and temporal resolution
for such narrow line spin probes, offer the
attraction for the development of low
frequency pulsed EPR instrumentation. The
possibility of co-registering anatomic images
(MRI) with functional images (EPRI)32 is
likely to add an extra impetus to research
in this field.
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1. EPR Imaging and In Vivo EPR. G.R. Eaton, S.S.
Eaton and K. Ohno, Eds., CRC Press, Inc., Boca
Raton, 1990.
2. Kuppusamy P. EPR Spectroscopy in biology
and medicine, Antioxidants Redox Signaling,
2004;6:583-85.
3. MC Krishna, N Devasahayam, JA Cook, S
Subramanian, P Kuppusamy, JB Mitchell.
Electron paramagnetic resonance for small
animal imaging applications Ilar J 2001;42:209-
18.
4. Krishna MC, Subramanian S, Kuppusamy P,
Mitchell JB. Magnetic resonance imaging for in
vivo assessment of tissue oxygen concentration,
Seminars in Radiation oncology, 2001;11:58-69.
5. Kuppusamy P, Afeworki M, Shankar RA,
Coffin D, Krishna MC, Hahn SM, Mitchell JB,
Zweier JL. In vivo electron paramagnetic
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oxygenation in a murine model, Cancer
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6. Krishna MC, Afeworki M, Devasahayam N,
Cook J, Subramanian S, Mitchell JB. In vivo free
radical detection and imaging by EPR: Non-
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7. Van Dam GM, Afeworki M, Subramanian S,
Krishna MC, Hoekstra HJ, Cook JA, Russo A,
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Barth, ME, Teicher BA, Grdona D. Pharmaco-
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9. Yamada KI, Murugesan R, Devasahayam N,
Cook JA, Mitchell JB, Subramanian S, Krishna
MC. Evaluation and comparison of pulsed and
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detection and imaging of free radicals, J Magn
Reson, 2002;154(2):287-97.
10. Murugesan R, Cook JA, Devasahayam N,
Afeworki M, Subramanian S, Tschudin R,
Larsen JA, Mitchell JB, Russo A, Krishna MC. In
vivo imaging of a stable paramagnetic probe
by pulsed-radiofrequency electron paramag-
netic resonance spectroscopy. Magn Reson Med
1997;38:409-14.
11. Murugesan R, Afeworki M, Cook JA,
Devasahayam N, Tschudin R, Mitchell JB,
Subramanian S, Krishna MC. A broadband
pulsed radio frequency electron paramagnetic
resonance spectrometer for biological
applications. Rev Sci Instru 1998;69:1869-876.
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12. Subramanian S, Murugesan R, Devasahayam
N, Cook JA, Afeworki M, Pohida T, Tschudin
RG, Mitchell JB, Krishna MC. High-speed data
acquisition system and receiver configurations
for time-domain radiofrequency electron
paramagnetic resonance spectroscopy and
imaging. J Magn Reson, 1999;137:379-88.
13. Devasahayam N, Subramanian S, Murugesan
R, Cook JA, Afeworki M, Tschudin RG, Mitchell
JB, Krishna MC. Parallel coil resonators for
time-domain radiofrequency electron
paramagnetic resonance imaging of biological
objects. J Magn Reson 2000;142:168-76.
14. Koscielniak J, Devasahayam N, Moni MS,
Kuppusamy P, Yamada K, Mitchell JB, Krishna
MC, Subramanian S. 300 MHz continuous wave
electron paramagnetic resonance spectrometer
for small animal in vivo imaging. Rev Sci Instru
2000;71:4273-281.
15. Subramanian S, Devasahayam N, Murugesan
R, Yamada K, Cook J, Taube A, Mitchell JB,
Lohman JAB, Krishna MC. Single-point
(constant-time) imaging in radiofrequency
Fourier transform electron paramagnetic
resonance. Magn Reson Med 2002;48:370-79.
16. Devasahayam N, Murugesan R, Yamada K,
Reijnders K, Mitchell JB, Subramanian S,
Krishna MC, Cook JA. Evaluation of a high-
speed signal-averager for sensitivity
enhancement in radio frequency Fourier
transform electron paramagnetic resonance
imaging. Rev Sci Instru 2002;73:3920-925.
17. Pursley RH, Kakareka J, Salem G,
Devasahayam N, Subramanian S, Tschudin RG,
Krishna MC, Pohida TJ. Stochastic excitation
and Hadamard correlation spectroscopy with
bandwidth extension in RF FT-EPR. J Magn
Reson 2003;162:35-45.
18. Taube AG, Subramanian S, Murugesan R,
Devasahayam N, Mitchell JB, Krishna MC,
Cook JA. An application system for automation
of constant-time radio frequency electron
paramagnetic resonance imaging. Comp
Method Pro in Biomed. 2003;72:127-38.
19. Devasahayam N, Murugesan R, Matsumoto K,
Mitchell JB, Cook JA, Subramanian S, Krishna
MC. Tailored sinc pulses for uniform excitation
and artifact-free radio frequency time-domain
EPR imaging. J Magn Reson 2004;168:110-17.
20. Johnson CA, McGarry D, Cook JA,
Devasahayam N, Mitchell JB, Subramanian S,
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imaging. Ann Oper Res 2003;119:101-18.
21. ELEXSYS E540: The L-band spectrometer for
EPR imaging, BRUKER Instruments, Inc.
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22. Ohno K, Watanabe M. Electron paramagnetic
resonance imaging using magnetic-field-gra-
dient spinning, J Magn Reson 2000;143:274-79.
23. Deng YM, He GL, Petryakov S, Kuppusamy P,
Zweier JL. Fast EPR imaging at 300 MHz using
spinning magnetic field gradients. J Magn
Reson 2004;168:220-27.
24. Pohida TJ, Fredrickson HA, Tschudin RG,
Fessler JF, Krishna MC, Bourg J, Harrington F,
Subramanian S. High-speed digitizer/averager
data acquisition system for Fourier-Transform
electron paramagnetic resonance spectroscopy.
Rev Sci Instru 1994;65:2500-504.
25. Afeworki M, Miller NR, Devasahayam N, Cook
J, Mitchell JB, Subramanian S, Krishna MC.
Preparation and EPR studies of lithium
phthalocyanine radical as a oxymetric probe,
Free radical bio med 1998;25:72-78.
26. Kuppusamy P, Wang P, Zweier J, Krishna M,
Mitchell J, Ma L, Trimble C, Hsia CJ. Electron
paramagnetic resonance imaging of rat heart
with nitroxide and polynitroxide-albumin,
Biochem, 1996;35:7051.
27. Yordanov AT, Yamada K, Krishna MC, Mitchell
JB, Woller E, Cloninger M, Brechbiel MW. Spin-
labeled dendrimers in EPR imaging with low
molecular weight nitroxides, Angew Chem Int
Ed. 2001;40:2690-692.
28. Ardenkjaer-Larsen JH, Laursen I, Leunbach I,
Ehnholm G, Wistrand LG, Petersson JS, Golman
K. EPR and DNP properties of certain novel
single electron contrast agents intended for
oximetric imaging, J Magn Reson 1998;133:1-
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29. Matsumoto K, Chandrika B, Lohman JAB,
Mitchell JB, Krishna MC, Subramanian S.
Application of continuous-wave EPR spectral-
spatial image reconstruction techniques for in
vivo oxymetry: Comparison of projection
reconstruction and constant-time modalities,
Magn Reson Med, 2003;50:865-74.
30. Yamada KI, Kuppusamy P, English S, Yoo J,
Irie A, Subramanian S, Mitchell JB, Krishna
MC. Feasibility and assessment of non-invasive
in vivo redox status using electron paramagnetic
resonance imaging, Acta Radiol 2002;43:433-40.
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31. Krishna MC, English S, Yamada K, Yoo J,
Murugesan R, Devasahayam N, Cook
JA, Golman K, Ardenkjaer-Larsen JH,
Subramanian S, Mitchell JB. Overhauser
enhanced magnetic resonance imaging for
tumor oximetry: Coregistration of tumor ana-
tomy and tissue oxygen concentration, Proc
Nat Acad Sci USA 2002;99:2216-221.
32. Hyodo F, Yamada K, Yasukawa K, Ichikawa
K, Matsumoto S, Utsumi H. Co-registration
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clinical MRI, Free radical bial med 2002;33:7
Suppl. 1.
 Studying Mobile Proteins and Peptides In vivo using MRI and MRS
Studying Mobile Proteins
and Peptides in vivo
using MRI and MRS
INTRODUCTION
Proteins constitute 18 percent of the total
mass of a typical mammalian cell, but only
a fraction of these are sufficiently mobile to
be MRS-detectable. Behar et al 1,2 and
Kauppinen et al3,4 first detected and identi-
fied several macromolecular peaks in brain
proton MR spectra at low frequency
(0 - 4 ppm). These signals were attributed
to the aliphatic protons from mobile
proteins, polypeptides, and lipids.1-6 The
backbone water-exchangeable amide protons
of proteins, resonating in the 6-10 ppm
range,7 are more difficult to detect because
water suppression schemes may suppress
signals of these protons due to exchange
with saturated water protons. Recently,
using water-flip-back approaches and
reduced saturation pulse sequences, Mori
et al 8 were able to observe a composite
resonance around 8.3 ± 0.5 ppm in the
proton spectra of cancer cells and cat brain
in vivo, which was assigned to these amide
protons. 8-10 The signal intensity of this
resonance was sensitive to pH changes, in
353
23
Jinyuan Zhou, Peter CM van Zijl
accordance with knowledge gained from
in vitro protein studies using high-resolution
MRS. The 8.3 ppm resonance in the human
brain was reported at 4.0 T by Chen and
Hu.11
These MR-detectable proteins and pep-
tides are present at a very low concentration,
which has prohibited their use in clinical
diagnosis and prognosis. In order to increase
detection sensitivity, it would be useful if they
could be detected via the water resonance,
especially for imaging purposes. The first
requirement for such detection is a magnetic
interaction with the water protons, which
applies because of the exchange between
amide protons and water. Recently, using
small molecules in solution (e.g., urea),
Balaban et al 12-14 demonstrated such a
chemical exchange saturation transfer (CEST)
enhancement scheme in which saturation was
transferred from exchangeable solute protons
to water. In addition, several paramagnetic
CEST (PARACEST) agents have been
reported to enlarge the frequency shift of the
exchanging site and reduce the direct
saturation of bulk water.15,16 Because the
354 Biomedical Magnetic Resonance: Proceedings of the International Workshop
water pool is much larger than the saturated
solute proton pool, each exchanging solute
proton is replaced by a nonsaturated water
proton. A few seconds of irradiation then
leads to enhancement if the exchange rate
and saturation efficiency are sufficiently high.
Especially large enhancements in sensitivity
(up to 500,000,17) were demonstrated for
amide protons on cationic polymers, such as
poly-lysine and dendrimers, which have a
favorable exchange rate range (10-300 s-1)
under physiological conditions. These results
suggested that it should be possible to detect
the amide protons of tissue proteins and
peptides in the water signal, via the amide
proton resonance at 8.3 ppm. We recently
demonstrated this capability, which was
called “amide proton transfer” or APT imag-
ing10,18 which should depend on endogenous
protein concentration, proton exchange rates,
pH, temperature, and several other factors.
DIRECT DETECTION OF THE
INTERACTION BETWEEN AMIDE
PROTONS AND WATER: WATER
EXCHANGE (WEX) SPECTROSCOPY
There are two approaches to study the
interaction between water protons and amide
protons. One is to magnetically label the
water protons and study magnetization
transfer (MT) to the proteins, the other is the
inverse pathway. The first approach has been
demonstrated in vitro using so-called water
exchange (WEX) spectroscopy,19,20 in which
water magnetization is selectively labeled
and its transfer properties to the water-
exchangeable protons are monitored as a
function of time after labeling (mixing time,
tm). Recently, WEX spectra were acquired
in situ in the rat brain9,10 to verify that the
composite resonance at 8.3 ppm in vivo is
indeed due to proteins in tissue. The acquired
in situ WEX spectra (Figs 23.1A and B) at
4.7 T show an initial increase (10-50 ms) in
the resonance at 8.3 ppm, confirming its
exchange-related character. At longer tm,
signals at low frequency appear, which we
attribute to the intramolecular nuclear
Overhauser effect (NOE). It is important to
stress that, when using the WEX approach,
only signals from more mobile macromole-
cules (proteins, peptides, some lipids) will
be visible, while those of small brain
metabolites are not.9 It is interesting to see
that the in vivo proton spectrum of endo-
genous proteins and peptides at high
frequency is very simple and resembles
protein spectra obtained in vitro in the sixties
and seventies.7
The WEX data can also be used to assess
the exchange properties of the amide
protons. Figure 23.2 shows the normalized
signal integral values of the protons at about
8.3 ppm as a function of the mixing time,
tm. These spectra were used to study amide
proton exchange rates, which were
determined to be 28.6 ± 7.4 s -1 for
normocapnia and 10.1 ± 2.6 s-1 (n = 5)
postmortem (10). Because no significant
change in amide intensity at tm = 300 ms
was seen in the first two hours postmortem,
it was concluded that this rate change is
due to the pH change upon death. To
calibrate the exchange rate changes in terms
of intracellular pH (pH i ) changes,
phosphorus spectroscopy was also perfor-
med. The measured pHi values were 7.11 ±
0.13 at normocapnia and 6.66 ± 0.10 post-
mortem (n = 7). Using the fact that amide
proton exchange is predominantly base-
catalyzed21,22 for pH > 5, it was deduced
that:
k = kb [OH]= kb × 10pH – pK W
= 5.57 × 10pH–6.4 ...(1)
in which pKw = 15.4 at 37oC.23
Using this
result, an average pK value of 17.2 for the
 Studying Mobile Proteins and Peptides In vivo using MRI and MRS 355

Figs 23.1A and B: Normocapnic (A) and postmortem (B) WEX spectra for rat brain as a function of mixing time,
tm Volume 16´12´4 mm3, SW 3000 Hz, 1024 points, 256 scans, TR 4 s, and TE 8 ms. (Reproduced, with
permission, from Zhou J, Payen J, Wilson DA, Traystman RJ, van Zijl PCM. Nature Med 2003;9:1085-1090)

amide groups contributing to the composite
resonance was calculated, in good agreement
with literature values for amide protons.
INDIRECT DETECTION OF AMIDE
PROTONS THROUGH THE WATER
SIGNAL (APT MRI AND MRS)
In Situ Rat Brain APT Effects in
the Water Signal
Figures 23.3A and B, Plate 17 show in situ
MT spectra of the rat brain during different
physiological conditions. In these so-called
z-spectra, the water signal intensity
reduction is acquired as a function of offset
of the saturation pulse with respect to the
water resonance frequency. It is important
to understand that the signal intensities in
these z-spectra reflect the effect of
conventional MT based on solid-like tissue
structure, direct water saturation, and APT.
In order to selectively assess the APT effect,
one can define an MT-ratio asymmetry
parameter:10,13 MTRasym = Ssat/S0 (negative
offset) - Ssat/S0 (positive offset). If the solid-
like MT effects were symmetric with respect
to the water resonance, the additional APT
would give rise to a positive MT difference.
Interestingly, the rat brain asymmetry curves
(Fig. 23.3C) show a varying MT difference
that is initially slightly positive and then
becomes negative. This result supports a
report24 that the solid-like MT effect is asym-
metric with respect to the water resonance,
with the MT center frequency in the aliphatic
range. An additional series of experiments in
which the irradiation range was extended
(Fig. 23.3D, n = 3) shows that the MTRasym
curve becomes constant and negative at about
-3 percent for higher offsets. When subtract-
ing the normocapnic MTRasym plot from the
postmortem curve, the maximum signal
356 Biomedical Magnetic Resonance: Proceedings of the International Workshop

Fig. 23.2: Fitting of proton exchange rates using the average amide proton integrals (n = 5) as a function of
mixing time. Integrals were normalized to the normocapnic value at tm = 300 ms. (Reproduced, with permission,
from Zhou J, Payen J, Wilson DA, Traystman RJ, van Zijl PCM. Nature Med 2003;9:1085-1090.)

change is found at an offset of 3.5 ppm
from water (Fig. 23.3E). Using 4.75 ppm for
the water resonance, this corresponds
exactly to the spectral frequency of the
composite amide proton resonance at 8.3
ppm (Fig. 23.1), indicating that this
difference originates predominantly from
the amide protons of the mobile proteins
and peptides. Therefore, we have in zero
order:
MTRasym (3.5 ppm) =
MTR’asym (3.5 ppm) + APTR
in which MTRasym (3.5 ppm) is the inherent
asymmetry of the solid-phase MT effect
associated with immobile macromolecules
and membranes, and APTR is the proton
transfer ratio for the amide protons
associated with mobile cellular proteins and
peptides. APTR can be assessed under the
assumption that MTRasym (3.5 ppm) remains
unaltered in the same experiment or by
comparison with normal tissue.
Using a two-site exchange model (a small
pool for amide protons and a large pool for
bulk water protons) and assuming instanta-
neous amide proton saturation, the APTR can
be derived to be:25
k [amide proton] –R t
APTR = (1 – e 1W sat )
[water proton] R1W
(2) (3)
in which k is the exchange rate for all amide
protons participating in the effect, R1W the
average spin-lattice relaxation rate of water,
and tsat is the saturation transfer time. The
water proton concentration [water proton]
= 2 × 55 M × water content. In the
 Studying Mobile Proteins and Peptides In vivo using MRI and MRS
postmortem study,10 which focused on the
first two hours after cardiac arrest,
negligible changes in the combined effects
for water content and R1W were found. The
amide proton content also remained
constant in this early postmortem period
(Fig. 23.2). Thus, using ΔAPTR (-1.90%) and
the measured exchanged rates at
normocapnia and postmortem, the total
amide proton concentration from all proteins
and peptides could be calculated to be about
71.9 mM, leading to APTR values at the
3.5ppm offset of 2.94 percent at normocapnia
and 1.04 percent postmortem.
pH Imaging of the Ischemic Rat Brain
The APT approach for pH imaging has been
applied to brain ischemia following middle
cerebral artery occlusion in rats.10,25 The
MTRasym spectra for the contralateral and
ipsilateral regions of interest (Fig. 23.4C,
Plate 18) were found to compare very well
with those for normocapnic and postmortem
brain (Fig. 23.3C, Plate 17), respectively,
indicating that ischemic brain has a similar
pH decrease to postmortem brain, typically
about 0.5 units.26 The pH-weighted images
(i.e. the MTRasym (3.5 ppm) images) were
acquired using frequency-labeling offsets of
± 3.5 ppm (40 scans), in which B 0
inhomogeneity was corrected using field
maps. When combining Eqs. [1] and [3], and
using the experimental data that the amide
proton content does not change appreciably
in the first hours post-occlusion, it can be
seen that APTR is a direct function of pH,
with a dependency on field strength
(because of R1W). For the rat, using the post-
mortem calibration data above, it was
deduced that APTR = 5.73 × 10pH–9.4, which
was used to generated absolute pH images
(Fig. 23.5, Plate 18). The area of infarction
357
visible on the right side of both the pH-
weighted and pH images is located in the
caudate nucleus, a region commonly affected
by infarction following MCA occlusion, as
confirmed by the diffusion-weighted image
and by histology, which acquired 8 hours
later. No infarct is visible on the T2-weighted
image
One question that arises is whether it is
useful to make pH-weighted images if
diffusion images already show the initial
ischemia. Present hospital policy in advanced
academic centers for acute stroke treatment
is to acquire MRI images and assess regions
of reduced diffusion constant (expected to
go on to infarction) and zones of reduced
flow (penumbra). If the reduced flow zone is
much larger than the diffusion-affected area
(so-called perfusion/diffusion mismatch),
treatment with clot lysing agents such as tPA
may commence. However, such treatment has
a large risk of causing hemorrhage, while it
is uncertain that the penumbra will progress
to infarction. The thought is that a better point
of reference for treatment will be when
oxidative metabolism becomes impaired. At
this point, pH changes will also occur and if
this can be imaged in acute stroke, the
treatment risk may be reduced by only
applying treatment if a large part of the
perfusion/diffusion mismatch shows reduced
pH (pH/perfusion mismatch criterium).
APT Imaging of Rat Brain Tumors
APT contrast also reflects the tissue content
of labile amide protons of mobile proteins
and peptides and should thus be able to
provide an assessment of endogenous
protein/peptide content. This possibility was
recently demonstrated18 in a 9L rat brain
tumor model. The MTRasym curves in brain
areas with tumor and tumor-related edema
358 Biomedical Magnetic Resonance: Proceedings of the International Workshop
Figs 23.6A to C: MT spectra, MTRasym spectra, and
ΔPTR spectra for the 9L brain tumor model (tumor
age 10 or 11 days; n = 5). Tumor (open circle),
peritumoral edema (diamond), and contralateral
region (solid circle). The MTRasym spectrum in tumor
is different from those for postmortem normal brain
and ischemic brain, and the magnitude of ΔPTR is
positive at the offset range of 2-5 ppm. (Reproduced,
with permission, from Zhou J, Lal B, Wilson Da,
Laterra J, van Ziji PCM. Magn Reson Med
2003;50:1120-1126. Wiley-Liss, Inc).
(Figs 23.6A to C) show an increase in signal
intensity over the complete z - spectrum.
Interestingly, when looking at the MTRasym
spectra, the tumor regions show an increase
in proton transfer ration over a range of
offsets between 2 - 5 ppm, while the
peritumoral regions do not (Fig. 23.6B). In
addition, the maximum change in the tumor
MTRasym spectra was found to be at an offset
of 2 - 3.5 ppm. It is important to note that
the intracellular pH of tumors remains
neutral to slightly alkaline, as measured by
31P MRS using the resonance of inorganic
phosphate.27,28 The increased MTRasym or
APTR in tumor can therefore in principle be
attributed increased protein/peptide content
or increased intracellular pH in tumor with
respect to normal tissue. In addition, tissue
water content and relaxation rates are two
possible contributing factors. Increased
protein/peptide content is the most likely
explanation due to the fact that only a small
pHi increase (< 0.1 unit) is often detected in
tumor, which is insufficient to explain the
large APT change. The flexible side chains
of proteins have a composite amide
resonance around 6.8 ppm in the proton
spectrum (Figs 23.1A and B), which also
leads to the increased MTRasym in tumor at
the 2 ppm offset from water in these tumors.
For imaging, we concentrated on the APT
effect of the backbone amide protons at the
offset of 3.5 ppm. Figure 23.7 compares the
acquired APT image, namely the MTRasym
(3.5 ppm) image, with several common MR
image types and histology. The APT image
was acquired using frequency-labeling
offsets of ± 3.5 ppm (16 scans), and B 0
inhomogeneity was corrected using field
maps. It is important to note that the APT
images show a clear boundary of the tumor,
which has good qualitative agreement with
histology.
 Studying Mobile Proteins and Peptides In vivo using MRI and MRS 359

Fig. 23.7: Comparison of several images for a 9L glioma (10 or 11 days) in a rat. The glioma (arrow) is visible
in all the MR images, as confirmed by histology, but it is much clearer in the APT-weighted image. Arrow head:
peritumoral tissue and open arrow: CSF. (Reproduced, with permission from Zhou J, Lal B, Wilson DA, Laterra
J, van Ziji PCM. Magn Reson Med 2003;50:1120-1126. Wiley-Liss, Inc)

CONCLUSION magnetic fields, where the contribution of

APT imaging is an entirely novel MRI
technology, never before realized. This tech-
nology will allow several new applications
of non-invasive tissue assessment and is a
technology that will work better at higher
direct water saturation at resonance
frequencies of the amide protons is reduced.
One of these applications is noninvasive
cancer imaging, since the concentration of
proteins/peptides is altered in some tumor
360 Biomedical Magnetic Resonance: Proceedings of the International Workshop
types. Another is pH imaging. A method to
image pH changes via endogenous contrast
would allow immediate noninvasive clinical
use of this important physiological indicator,
for instance in the case of acute stroke. APT
imaging, which detects changes in protein/
peptide amide proton content and pH,
explores a completely new area of contrast
and is eminently clinically translatable.
Future work will have to realize this potential
in the clinical setting.
ACKNOWLEDGMENTS
The authors thank Drs Phillip Zhe Sun, Jean-Francois
Payen, David A. Wilson, Richard J. Traystman,
Bachchu Lal, and John Laterra for cooperation in
this project. This study was supported in part by
grants from NIH/NIBIB (EB02634 and EB02666) and
the Whitaker Foundation.
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362 Biomedical Magnetic Resonance: Proceedings of the International Workshop
Magnetic Resonance of
Biofluids and Tissues
Ian CP Smith, Tedros Bezabeh, Racquel Baert
INTRODUCTION
Applications of high-resolution NMR to
biomedical problems have been hindered
by the common view that the spectra would
be difficult to obtain, the resonances would
be broad, and overlaps of many broad
resonances would make the spectra difficult
to interpret. This has proved to be quite
incorrect, and high-resolution NMR has been
very successfully applied to biological fluids,
tissue ex vivo, and living organisms.
One reason for this success was the rapid
increase in availability of NMR instruments
operating at high magnetic field. Detection
sensitivity increases with the magnitude of
the applied magnetic field, from linearly to
the 1.75 power, depending on the nature of
the specimen. Concomitantly, the spectral
separation in Hertz between chemically
different species increases linearly with the
field. A wide variety of multinuclear and
multidimensional techniques have led to
increased sensitivity and spectral simpli-
fication. Finally, the increasing Megahertz
available per unit cost has made the
accessibility of high-field NMR much greater.
Some of the earliest applications to
complex biomedical systems were made in
the 1970s, principally with 31P NMR. A long
24
period followed with emphasis placed on
microorganisms and cell extracts. The
observation of well-resolved 31P spectra
from humans in vivo stimulated a new
interest in NMR of tissue. NMR of biofluids
evolved slowly but steadily during this time.
Due to its popularity, this application has
recently been given a name of its own,
metabolomics. However, reader beware:
Metabolomics science is not new, only the
name has changed. Reviews of applications
to both clinical fluids and tissue have
appeared recently.1-7
BIOLOGICAL FLUIDS
A large number of biological fluids is accessi-
ble for MRS study ex vivo. Early applications
of 1H-MRS analysis demonstrating its utility
involved analysis of urine and serum.10-12
A large number of other biofluids, including
cerebrospinal fluid (CSF),13 amniotic fluid,14,15
synovial fluid, 16,17 seminal plasma, 18,19
bile,20,21 and tissue extracts22,23 have since
been examined.8,24,25 1H-MRS analysis of bio
fluids offers several advantages. It requires
little or no preparation of the sample. High-
quality spectra, suitable for quantitative
measurements, may be obtained in under
15 min. Potentially all 1 H-containing
 Magnetic Resonance of Biofluids and Tissues 363

metabolites (including acids, bases, and

neutral compounds) may be measured by
applying a suitable spectroscopic technique.
In a single measurement, all of the more
usually represented metabolites may be
studied simultaneously.
Biological fluids contain compounds of
interest at relatively low concentrations in
water. The 1H resonance due to water is
roughly 106 times more intense than the
resonance of the compounds of interest, thus
creating a serious problem of dynamic range.
Two approaches to resolve this difficulty
have been applied. One is the increasing
dynamic range of the analog-to-digital
convertors (ADCs), reaching values as high
as 32 bits (232 = 4.3 × 109, a significant range
of discrimination). Furthermore, it has been
found that saturation of the H2O resonance
by simple irradiation, or by a variety of
pulse sequences, serves to suppress this
resonance to tolerable amplitudes. One or
both of these methods may be applied.
A second problem is the wide variety of Figs 24.1A and B: 1H-NMR spectrum (360 MHz) of
human blood plasma; the strong resonance due to
compounds present in biological specimens.
H2O has been reduced by irradiation at its frequency
The high magnetic fields presently available, before commencing the spectral acquisition: (A) with
up to 19 T (800 MHz for 1 H), serve to no spectral manipulation; (B) after multiplication of
diminish this problem to a tractable one. the free induction decay by a sine-bell function to
A further problem is the simultaneous minimize broad resonances.
presence of species of high and low mole-
cular weight. High-molecular-weight species BIOLOGICAL TISSUE
rotate slowly, and thus have very broad
resonances which may obscure those of the For many years it was believed that NMR
low-molecular-weight species. Fortunately, of tissue was a hopeless pursuit. The seminal
broad resonances are usually indicative of article by Mountford et al,26 demonstrating
1
short transverse relaxation times (T2), and H spectra of good resolution from the
these may be used in data manipulation to mammary tissue of rat, stimulated a
remove the confounding effect of the resurgence of interest. High-resolution 1H
resonances due to high-molecular-weight spectra (360 MHz) of human colon tissue
species such as proteins, nucleic acids, and were reported. The transverse relaxation
membrane lipids. This is demonstrated in times (T2) of the lipid CH2 resonance at
Figures 24.1A and B, where manipulation 1.3 ppm appeared to be indicative of the
of the free induction decay leads to total metastatic potential of the hyperplastic
suppression of the broad resonances due to tissue. The race was on! Since this initial
proteins in plasma. observation, a wide variety of studies has
364 Biomedical Magnetic Resonance: Proceedings of the International Workshop
been reported on human tissue ex vivo,
suggesting that the high information content
of the spectra should lead to effective clinical
use of NMR in vivo.
Tissue differs drastically from biological
fluids in that it is macroscopically solid. This
leads to dificulty in optimizing the homo-
geneity of the applied magnetic field for
optimal spectral resolution. A technique has
been reported that minimizes this problem.27
Figure 24.2 shows the positioning of a piece
of human tissue within a capillary tube facili-
tating the optimization of field homogeneity
and allowing an estimation of the volume
of the specimen. This method has been used
in most quantitative studies to date.
Ex vivo tissue is not dead tissue; it has,
however, begun a progression toward death.
In studies of human tissue ex vivo, great
care must be taken to ensure that the spectra
of the many specimens are taken at similar
Fig. 24.2: Diagram of the positioning of a biopsy
specimen within a standard NMR tube of outside
diameter 5 mm. (Reproduced by permission from
Kuesel et al.)
times after removal from the host. One way
to ensure this is to freeze the samples at
liquid N2 temperature within minutes of
excision. These frozen specimens are stable
for weeks. Likewise, a rigid protocol for
warming the specimens and obtaining their
spectra must be followed.
NMR spectra contain a wealth of infor-
mation—some vital, some useful, some
useless. It is often dificult to decide by visual
inspection which regions of the spectra will
be most useful in classifying the specimens.
Ratios of peaks can be useful, but much time
must be spent before the most useful ratios
are found. In our experience computerized
methods to select the most discriminatory
region, and to perform the classification,
are robust and accurate. The most exhaustive
application of these methods is described in
Somorjai et al.28 Recently, a description of
the regional selection process has appea-
red. 29 A summary of applications and
methods is given in Lean et al.4
For spectral classification, multivariate
analysis has proved to be very effective. A
variety of methods, such as linear discri-
minant analysis, quadratic discriminant
analysis, neural networks, and genetic
algorithms may be used. Very often the
simplest method, linear discriminant ana-
lysis, is sufficient. In the most dificult cases,
a number of methods may be used and
finally entered into a meta classifier.28 The
combi-nation of methods is known as the
statistical classification strategy.4
It is crucial that the NMR spectra be
adequately prepared for the classification
process. They must be normalized for area,
adjusted for chemical shift, and the most
discriminatory regions determined. Data
must be divided into a training set, in which
the method is calibrated, and a test set, in
which the accuracy of the method is tested.
Regrettably, this has been done very rarely
in the literature to date, yielding promising
 Magnetic Resonance of Biofluids and Tissues
results but with a method that is not robust.
To enhance robustness, the leave-one-out
method should be implemented. The
procedure is challenged by leaving out one
data set, classifying it by exposure to the
existing classifier, leaving another out and
classifying it, until all data sets have been
thus treated. Finally, a sufficient number of
data sets must be used if the classifier is to
be accurate, specific, and robust. The
number required depends strongly on the
types of spectra compared.
Using the procedures described above,
experimental results from which no obvious
discriminators can be seen, and in which
there is a wide variation in spectra from a
given class, can be accurately classified. The
reader is referred to the articles.2-6 The
remainder of this article presents an
overview of the medical applications of high-
resolution MRS ex vivo. Specifically, progress
has recently been made towards the
diagnosis of inborn errors of metabolism,
the measurement of renal injury, diagnosis
of neurological disorders and cancer.2
INBORN ERRORS OF METABOLISM
High-field 1H spectroscopy of biofluids
offers great potential for furthering the
under standing of disease processes in
humans. Historically, one of the most
successful clinical applications of MRS has
been the detection of a large number of
metabolic disorders. In these disorders, the
reduction or absence of activity of an
enzyme or cofactor can have dramatic
consequences for metabolism and its control.
Many inherited metabolic disorders result
in the accumulation of large amounts of
organic intermediates produced proximal to
the defective enzyme step, which eventually
spill into the blood and urine. 1H-MRS has
been used to study the urinary excretion of
such compounds. Initial studies demons-
365
trated the ability to diagnose a number of
metabolic disorders including histidinemia30,
citrullinemia,31 argininosuccinic acid lyase
deficiency, 31,32 ornithine carbamoyl trans-
ferase deficiency,31 cystinuria,33 oxalic aci-
duria,33 alkaptonuria,30 multiple acyl CoA
dehydrogenase deficiency (glutaric aciduria
type II),34 methylmalonic aciduria,35 propio-
nic aciduria, 35 porphyria, 33 5-oxopro-
linuria,33,36,37 homocysteinemia,38 trimethyl-
aminuria,39 and Fanconi syndrome.33
More recently, both 1H-and 13 C-MRS
have been used to identify and quantify the
presence of elevated levels of galactonate
in the urine of 27 patients with uridyltrans-
ferase-deficient galactosemia. 40 750 MHz
1
H-MRS has also been used recently to
examine the elevated levels of 2-hydro-
xyglutaric acid in the urine of a patient with
the rare condition of 2-hydroxyglutaric
aciduria.41 Maschke et al42 reported the use
of 1H-MRS (300 MHz) for the analysis of
trimethylamine (TMA) in the urine of a
patient with trimethylaminuria and of other
members of his family. In this study, 300
MHz 1H-MRS was shown to have sufficient
sensitivity, resolution, and linearity to allow
the diagnosis of trimethylaminuria in a
classical one-dimensional spectrum of the
urine of the patient (Figs 24.3A and B).
Moreover, this technique is easier than the
established classical biochemical methods
that primarily use gas chromatography. The
increased TMA signal, and the decreased
trimethy-lamine-N-oxide (TMAO), as com-
pared to a normal healthy subject, are clearly
seen at 2.90 ppm and 3.27 ppm, respectively.
Although urine appears to be the
specimen of choice for the diagnosis of
inborn errors of metabolism, the use of CSF
has also been reported.43,44 Derangements
of organic acid concentrations in CSF may
occur independent of the systemic meta-
366 Biomedical Magnetic Resonance: Proceedings of the International Workshop

bolism, and thus, analysis of metabolites in

CSF may be important in the diagnosis and
follow-up of these patients. Figures 24.4A
to C show examples of the diagnostic power
of 1H-MRS, for relevant parts of the spectra
from CSF samples of patients with isolated
3-methylcrotonyl-CoA-carboxylase
deficiency, Canavan disease, and histi-
dinemia.
A significant amount of data in the
literature indicates that 1H-MRS of urine,

Figs 24.3A and B: 1H-MRS spectra (300 MHz) of

urine samples: (A) a patient with trimethylaminuria;
(B) a healthy subject.42

A C

Figs 24.4A and B: 1H-NMR spectra (600 MHz) of CSF from patients with: (A) isolated 3-methylcrotonyl-CoA-
carboxylase deficiency; (B) Canavan disease; and (C) histidinemia44
 Magnetic Resonance of Biofluids and Tissues
and possibly of CSF, is capable of diagnosing
a vast number of inborn errors of meta-
bolism. MRS not only provides information
on endogenous biochemical processes, but
is also capable of rapid quantitation of
metabolites, using a small volume that
requires little pretreatment. Furthermore, it
is not necessary to preselect the metabolites
of interest, as the technique allows simul-
taneous measurements of a range of compo-
nents. There exists also, the possibility for
the detection of some novel markers of these
diseases, or insights into the underlying
defects of these disorders.
RENAL INJURY
Early studies of 1H-MRS of urine demons-
trated that in situations where renal damage
is present, the urinary profile of the low-
molecular-weight metabolites is altered.45,46
Furthermore, in studies of the effects of
region-specific nephrotoxins, the application
of 1H-MRS urinalysis indicates that abnormal
patterns of metabolites are associated with
different sites of nephrotoxic actions. Renal
proximal tubular toxins cause glycosuria,
lactic aciduria, and amino aciduria; renal
papillary toxins produce abnormal excretion
patterns; and renal papillary necrosis
produces an initial increase in TMAO and
dimethylamine (DMA) excretion followed
by a subsequent increase in N,N-dimethyl-
glycine, succinate, and acetate, and a decre-
ase in TMAO and 2-oxoglutarate.47,48 This
information is useful when assessing renal
disease progression or nephrotoxicity asso-
ciated with therapeutic agents. Subsequently,
1
H-MRS urinalysis has been used as a non-
invasive diagnostic technique for tubular and
papillary distortions in glomerulonephri-
tis,49,50 and for the assessment of the degree
of chronic renal failure.51
The abnormal urinary excretion of
specific enzymes and proteins as possible
367
markers of renal allograft rejection has been
studied extensively, 52,53 and found to be
relatively nonspecific. Foxall et al54 used 1H-
MRS to investigate the pattern of metabolic
changes associated with renal allograft
dysfunction. As shown in Figures 24.5A to
E, the spectra of normal human urine
showed signals for creatinine, glycine,
citrate, alanine, lactate, and N-methylated
meta-bolites in the chemical shift range of
3.1 – 3.3 ppm. The spectra of patients’ urine
collected following renal transplantation
were considerably different. Compared to
normal urine, the spectral pattern of urine
from patients with an immediate functioning
graft, urinary tract infection, renal tubular
ischemia or with a nonfunctioning graft are
widely different. Further studies associated
an increased excretion of TMAO with
biopsy confirmed acute graft rejection.55 It
has also been shown that 1H-MRS of urine
can be used to predict rejection of a kidney
much earlier than the usual chemical analysis
made in kidney biopsies.76 Thus, unlike the
excretion of enzymes or proteins, 1H-MRS
provides both diagnostic and prognostic
information.
Since urine has been studied most
extensively, a large number of the reso-
nances in the spectrum of normal human
urine have been assigned. A study was
performed to standardize the analytical con-
ditions, to quantify the major metabolites
present in urine of normal subjects, and to
evaluate changes due to physiological condi-
tions such as feeding. 56 In this study,
1H-MRS, operating at 300 MHz, studied
serial urine samples from 50 normal subjects.
In all specimens, creatinine, lactate, alanine,
citrate, DMA, TMAO, glycine, and hippurate
were found to be present. All metabolites
were quantified on the basis of peak heights
and were expressed as millimoles per mole
368 Biomedical Magnetic Resonance: Proceedings of the International Workshop
Figs 24.5A to E: 1H-NMR spectra (500 MHz) of (a)
normal human urine, and urine collected from four
patients 3 days following renal transplantation
showing: (b) immediate functioning graft; (c) urinary
tract infection; (d) renal tubular ischemia; and (e) non-
functioning graft. Abbreviations are as follows: Ac D
acetate, Ala D alanine, Ch D choline, Cit D citrate,
DMA, GLC D glucose, Gly D glycine, Lac D lactate,
myo-Inos D myo-inositol, Pep D peptides, P D
acetaminophen (paracetamol) metabolites, Suc D
succinate, TMAO, V D cyclosporin A drug metabolite54
of creatinine. The study of metabolic profiles
in serial samples allowed evaluation of intra-
individual variability and physiological
changes due to feeding. The results suggest
that every subject is characterized by a
typical profile that does not change with
time under physiological conditions and is
independent of feeding. Thus, a 1H-MRS
urinary profile may be considered as an
individual’s metabolic fingerprint. Changes
in the 1H-MRS urinary profile of an indivi-
dual may be indicative of metabolic changes
or disturbances in renal function.
NEUROLOGICAL DISORDERS
The use of high-resolution 1H-MRS to evalu-
ate brain metabolism through the metabolic
profile of CSF constitutes a potentially
powerful strategy to aid the differential
diagnosis of neurological diseases. Although
the biochemical composition of CSF has been
well characterized by standard biochemical
techniques, a number of studies have been
done to evaluate the effectiveness of
1
H-NMR spectroscopy for examination of
CSF as an aid to the biochemical diagnosis
of central nervous system diseases. Early
studies reported the assignments of a
number of resonances. 13,43 Since then, a
number of pattern-recognition approaches
and discriminant analyses have been used
to separate samples into different classes,
and these used to differentiate between
normal controls and subjects with various
neurological disorders.57-59
Koschorek et al 58 reported that the
spectra of CSF of normal controls and sub-
jects with tumors or multiple sclerosis (MS)
can be perfectly separated, whereas those
from subjects with disk herniations can be
separated approximately 90 percent of the
time using principal component analysis
(PCA).
 Magnetic Resonance of Biofluids and Tissues
500 MHz 1H-MRS spectra of postmortem
CSF specimens from control subjects and
patients with Alzheimer’s disease were
examined in a similar study.60 PCA achieved
partial separation of the groups; more formal
statistical analysis suggested that citrate by
itself was the best discriminator.
Spectra showing the metabolic brain
profile induced by degenerative dementia
are reported to be considerably different
from those of control patients.61 An increase
in many metabolites has been observed, and
for some (leucine–isoleucine) the concen-
tration is increased three-fold. Furthermore,
some abnormal and unassigned resonances
have been observed. These qualitative MRS
abnormalities were not found to be corre-
lated with any medical treatments.
In the CSF of patients with Huntington’s
chorea, 400 MHz 1H-MRS analysis demons-
trated a significant increase (approximately
60%) in the pyruvate concentration as
compared to controls. However, in serum
samples, no variation in this metabolite was
detected.61
In studies of CSF from patients with MS,
the findings among studies differ. Lynch et
al62 reported that there were no significant
differences between the levels of most
metabolites, with the exception of acetate
and formate which were increased and
decreased respectively in patients with MS
as compared to controls. Moreover, in 93
percent of patients with actively progressing
MS, an unknown singlet peak at 2.82 ppm
was found; it was presumed to be an N-
methyl compound. Nicoli et al61 similarly
reported that MS induced only slight
modifications in the 1H-MRS spectra, but
these modifications differ from those
reported earlier: An increase in lactate and
fructose, and a decrease in creatinine and
phenylalanine concentrations. Further, the
unknown N-methyl compound described
369
previously was not found in the CSF of any
of the patients with MS. Interestingly, Aasly
et al63 observed significantly lower levels
of lactate and glutamine in MS patients as
compared to controls. When the MS group
was divided into two sub-groups (chronic
progressive and relapsing–remitting forms
of MS), no significant differences were
found in any of the parameters measured
in the CSF, although there was a tendency
toward lower levels of lactate and glutamine
in the relapsing–remitting groups. What
accounts for the different results in these
studies is unclear; it may simply reflect the
lack of a standardized method for specimen
prepara-tion and recording 1H-MRS spectra
from CSF. A standardized method for high-
resolution 1H-MRS of CSF needs to be
adopted.44
CANCER
Increased research in the field of cancer
diagnosis and treatment over the past
several years included a search for an
inexpensive, accurate, and noninvasive
screening test for early malignancy. It had
been hoped that a simple screening test of
plasma, using 1 H-MRS, might reveal a
malignant condition. However, these investi-
gations have been unsuccessful,64,65 and
attention has shifted toward the assessment
of various tissue specimens, and extracts of
tissue specimens, to determine whether
1H-MRS can differentiate between malignant
and nonmalignant diseases.
Colon Cancer
Colon cancer was the first disease to be
approached in detail by 1H-NMR.66 A large
number of spectra were obtained from colon
tumors, and from tissue at the surgical
margins of the removed colonic region to
serve as normals. A very wide variety of
370 Biomedical Magnetic Resonance: Proceedings of the International Workshop
spectra was obtained, and the transverse
relaxation time (T2) of the lipid resonance
at 1.3 ppm in the 1H spectrum was used as
an indicator of metastatic potential, whereas
the intensities of resonances due to choline-
containing compounds correlated with the
degree of invasion of the cancer (Dukes A,
B, C, or D). Subsequent studies on cultured
human cell lines of different degrees of
invasive capability showed that two-
dimensional COSY 1H spectra could be
discriminatory67 (Fig. 24.6).
More recent studies of colon tissue
revealed another critical element—the purity
of the colon tissue. If care is not taken to
exclude both muscle and subepithelial fat
Fig. 24.6: 1 H-NMR (360 MHz) COSY spectrum
(magnitude mode, symmetrized) of excised tissue in
400 µL phosphate-buffered saline in D 2O, for a
specimen of human ovarian serous carcinoma, poorly
differentiated. Multiple cross-peaks are attributable
to cell–surface fucosylation, namely Thr/Fuc I (1.33–
4.27 ppm), which also contains a contribution from
the amino acid threonine, Fuc II (1.25 – 4.28 ppm)
Fuc IIb (1.19 – 4.20 ppm), and Fuc III (1.41 – 4.30
ppm), and correlated with tumor grade and loss of
cellular differentiation72
from the specimen, resonances from these
underlying tissues may confound the
analysis. A definitive study was performed
using well-defined specimens, and rigorous
analytical methods, showing that 1H-NMR
can yield very high levels of sensitivity and
specificity for the detection of cancer.22 More
recently, this approach has been applied to
the evaluation of other colonic disorders,
such as Crohn’s disease and inflammatory
bowel disease.68 The chemical discriminatory
power of 1H-NMR is expected to provide
valuable insight into the progression of colon
disease from dysplasia to malignancy, and
to interpret the progression in terms of the
oncogenic genes expressed during the
progression.
Thyroid Cancer
A problem in thyroid cancer is the detection
of follicular malignancy. Regular histopatho-
logical procedures cannot yield a conclusion.
Only thyroidectomy, and analysis of the
entire gland, can yield a definitive diagnosis
of invasive disease. However, the patient
has already lost the thyroid gland. 1H-NMR
studies on thyroid biopsies by both surgical
and needle approaches demonstrated a high
level of sensitivity to thyroid cancer, and
provided a serious test of the multivariate
methods for spectral analysis.28 This appro-
ach is now used in Australia to determine
the therapy applied to neoplasms of the
thyroid (C.E. Mountford, personal communi-
cation).
Brain Tumors
A variety of human brain neoplasms have
been studied by this technique. Figure 24.7
shows the 1H-MR spectra of tissue from
three different brain tumor types, and a
patient with epilepsy. Relatively simple data
analysis allowed accurate classification of
 Magnetic Resonance of Biofluids and Tissues
these cancers, and gave cause for hope for
a method to determine their degree of
aggressivity.29,69 The resonance at 1.3 ppm
due to lipid was seen to be indicative of the
degree of necrosis (dead tissue) in the
tumor. 70 Many in vivo studies of brain
Fig. 24.7: 1H-NMR spectra (360 MHz) of biopsy
specimens of human brain tumors, astrocytoma and
the more aggressive glioblastoma. The large peak at
4.8 ppm is due to residual H2O
371
tumors have now been reported,71,77 and it
is expected that 1H-MRS will be a valuable
technique for the planning of treatment for
brain tumors. For a good overview, see
Danielsen and Ross.77
Ovarian Tumors
1
H-NMR spectra of ovary biopsy specimens
have shown good sensitivity to the presence
of malignant disease by both resonance
ratio72 and multivariate methods.73 In the
latter study, sensitivity of the tumor to drug
therapy was also detected by the method.
It is hoped that in vivo use of this method
will help detect ovarian cancer at an early
stage, and define its potential resistance to
therapy.
Prostate Cancer
Screening of prostate tissue is currently done
by transurethral or transcolonic biopsy. In
both methods, the sampling is not represen-
tative of the entire gland. 1 H-NMR of
prostate tissue has demonstrated remark-
able discriminatory power.74 Benign disease
can be distinguished from cancer with sensi-
tivity of 100 percent, and specificity of 95.5
percent. Furthermore, the method appears
to be capable of distinguishing the stromal
and glandular forms of benign prostatic
hypertrophy (Figs 24.8A and B). Transrectal
1H-MR spectra in vivo show similar detail,
which promise to yield an accurate whole-
gland diagnosis.75,78 A subsequent biopsy
study on patients before and after radiation
therapy indicated that 1H MRS can be useful
in determining the success of radiation in
clearing the prostate of cancer.80 It is hoped
that this method can also be applied to in
vivo studies of the entire organ.
Biliary Tree
Recently high-resolution MRS of bile has
been used to distinguish benign from
372 Biomedical Magnetic Resonance: Proceedings of the International Workshop
Figs 24.8A and B: 1H-NMR spectra of biopsy
specimens from human prostate gland suffering from
benign prostatic hyperplasia. The compositions of the
tissue specimens were determined histologically to
be (A) 95% stromal, 5% glandular, and (B) 90%
glandular, 10% stromal74
malignant disease of the biliary tract. 8
Figure 24.9 shows a 1H spectrum of normal
bile. For a cohort of thirty patients with
either benign or malignant disease, the
spectra were classified by means of the
statistical classification strategy described
earlier4,28,29 to yield an accuracy of 100
percent. Clearly a larger number of subjects
must be studied in order to develop a truly
robust classifier, one that is able to classify
accurately specimens presented blindly to
the method, but given the simplicity of the
measurements and analysis it appears that
this could be an extremely valuable
diagnostic of biliary cancer.
Head and Neck
Poor treatment outcome remains high in
patients with squamous cell carcinoma of
the head and neck regions. Treatment
methods vary, but none has been guided
by reliable prognostic indicators to date. A
recent 1H MRS study of biopsies from fifty-
eight patents treated concurrently, some
with failure and some with success, showed
significant prognostic value. 9 Using the
ratios of spectral intensities of choline to
creatine and CH2 to CH3, a sensitivity of
83 percent and a specificity of 82 percent
were obtained. Figures 24.10A and B show
the 360 MHz 1H spectra of head and neck
tumors from (A) control and (B) failed cases.
As with the biliary tree example above, a
larger cohort must be studied to develop a
representative data base, and a robust
statistical analysis must be made.
Breast
Breast cancer is the second most prevalent
killer of women. The most common method
Fig. 24.9: Representative Proton MR spectrum (360
MHZ, 25°C) of normal bile. Abbreviations: AAc,
Acetoacetate; Ac, Acetate, Ala, Alanine, CA, Cholic
acid; Cho, choline-containing compounds; Chol,
Cholesterol; DCA, deoxycholic acid; Gln, glutamine,
Gly, Glycine; HOD, deuteriated water; Ile, Isoleucine;
Lac, Lactic acid; Lip, Lipid; TSP, 3-(trimethylsilyl)
propionate sodium salt. Assignments do not imply
that these are the only substances contributing to a
particular peak8
 Magnetic Resonance of Biofluids and Tissues
Figs 24.10A and B: 360 MHz 1H MR spectra of head
and neck tumors (A) control (B) failed case.
Abbreviations: Chos, choline-containing compounds;
Cr, creatines; Gln, glutamine, Glu, glutamic acid; HOD,
deuteriated water; Lac, lactic acid; Leu, leucine; Lip,
lipid; Tau, taurine; Val, valine. Assignments do not imply
that these are the only substances contributing to a
particular peak9
of screening is X-ray mammography, which
has a detection accuracy of approximately
70 percent. If lesions are detected, two
approaches may then be followed; fine
needle biopsy or open biopsy, the former
being the less invasive. Diagnosis then
follows via conventional histology.
1
H MRS of fine needle biopsies yields
spectra which are significantly different for
normal and cancerous tissue. The ratios of
the areas of resonance due to choline and
creatine fall into two groups, with some
overlap. Application of advanced statistical
methods, including use of the genetic
algorithm to choose the most discriminating
regions of the spectra, and linear discri-
minant analysis, led to a specificity and
sensitivity of 94 and 98 percent, with a high
degree of robustness.79 An extra benefit
from this sophisticated analysis is the ability
to determine with high accuracy, from only
a one pulse 1H MR spectrum of a biopsy of
373
the primary tumor, the involvement of
distinct lymph nodes (accuracy of 95%) and
the degree of vascularization of the primary
tumour (accuracy 92%). There is now consi-
derable activity involving the use of in vivo
MRS to accomplish the same ends.
CONCLUSION
The range of applications of 1H-NMR of
biological fluids is wide, and is growing
steadily. The study of the composition of
physiological fluids, and the changes thereof
in disease, are amenable to study by MRS.
Although a greater availability of instru-
ments, standardized methodology, a larger
database of spectral changes correlated with
pathological conditions, and more NMR-
trained individuals in the clinical environ-
ment are necessary for routine use, the
discriminating power of MRS can and will
be exploited to attack difficult problems in
the clinic.
The ex vivo 1H-NMR of tissue technique
has also enjoyed a remarkable degree of
success in distinguishing cancer tissue from
normal or otherwise abnormal tissue. The
methods thus developed are leading to
extension of the studies to human subjects
in vivo with a high degree of accuracy.
These studies are more time-consuming, but
it is expected that the savings in lives and
treatment cost will readily justify the effort.
With more automated methods for MRS or
commercial MRI instruments, we should see
clinical MRS as a standard technique very
soon.
ABBREVIATIONS AND ACRONYMS
ADC Analog-to-digital Convertor
COSY Correlated Spectroscopy
CSF Cerebrospinal Fluid
DMA Dimethylamine
MRS Magnetic Resonance Spectroscopy
374 Biomedical Magnetic Resonance: Proceedings of the International Workshop
MS Multiple Sclerosis
NMR Nuclear Magnetic Resonance
PCA Principal Component Analysis
RF Radiofrequency
S/N Signal-to-noise
TMA Trimethylamine
TMAO Trimethylamine-N-oxide
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Ex vivo 1H-NMR Spectroscopy. J Neurochem
1995;64:1655-61.
70. AC Kuesel, KM Brière, WC Halliday, GR
Sutherland, SM Donnelly, ICP Smith. Mobile
Lipid Accumulation in Necrotic Tissue of High
Grade Astrocytomas. Anticancer Res 1996;16:
1485-90.
71. MC Preul, Z Caramanos, DL Collins, J-G
Villemure, White, G Unsgard. Cerebrospinal
Fluid Lactate and R LeBlanc, A Olivier, R
Pokrupa, DL Arnold. Accurate Noninvasive
Diagnosis of Human Brain Tumors by using
Proton Magnetic Resonance Spectroscopy. Nat
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72. WB Mackinnon, P Russell, GL May, CE Mount-
ford. Characterization of Human Ovarian
Epithelial Tumors (Ex vivo) by Proton Magnetic
Resonance Spectroscopy. Int J Gynecol Cancer
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73. JC Wallace, GP Raaphorst, RL Somorjai, CE
Ng, M Fung Kee Fung, M Senterman, ICP
Smith. Classification of 1 H-MR Spectra of
Biopsies from Untreated and Recurrent Ovarian
Cancer Using Linear Discriminant Analysis.
Magn Reson Med 1997;38:569-76.
74. P Hahn, ICP Smith, L Leboldus, C Littman, RL
Somorjai, T Bezabeh. The Classification of
Benign and Malignant Human Prostate Tissue
by Multivariate Analysis of 1H-MR Spectra.
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75. JK Kurhanewicz, DB Vigneron, SJ Nelson, H
Hricak, JM Macdonald, P Konety, P Narayan.
Citrate as an In vivo Marker to Discriminate
Prostate Cancer From Benign Prostatic
Hyperplasia and Normal Prostate Peripheral
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77. ED Danielsen, B Ross. Magnetic Resonance
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78. FV Coakley, J Kurhanewicz, Y Lu, KD Jones,
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Gluch, CL Lean, P Russell, BH Barraclough, DJ
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80. C Ménard, ICP Smith, RL Somorjai, L Leboldus,
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378 Biomedical Magnetic Resonance: Proceedings of the International Workshop

25
MR Metabolomics: Studies
of Gene Function and
Novel Anticancer Drugs
JR Griffiths, YL Chung

INTRODUCTION being widely utilized, both for research and

The publication of the human genome in
the last decade of the 20th century opened
a new era in biology, and it has had a
particularly marked effect on medical
research. The genetic specification of a
human being – something that had once been
assumed to be of almost infinite complexity
– turned out to consist of 20 - 25,000 protein-
coding genes. Once the genome was
perceived as a finite, manageable problem,
the way was open for a paradigm shift in
our attitudes to many areas of research,
particularly medical research. The
transcriptome, the totality of mRNA
transcripts in a cell, organism or disease
state could also be considered in totality,
and within a few years chip-based
transcriptome assays have become a routine
tool in medical research. Next, attention
focused on the proteome, and although
current analytical methods are not capable
of “solving” the entire proteome
(particularly not if post-translational
modifications of the polypeptides are to be
included) proteomic methods are already
for medical testing.
The youngest and least developed of
these four “-omics” sciences is metabolomics,
which aspires to an understanding of the
totality of the small molecule metabolites
present in living cells, organisms or disease
states. In the same way as the genome acts
through the proteome, so the proteins have
their effects through the metabolome, and
in order to complete our knowledge of
ourselves we will eventually have to
understand human metabolomics. Again, this
may be a more manageable problem (at least
in humans) than had generally been
assumed—it has been estimated that the
human metabolome consists of as few as
2,000 compounds.1
Many analytical methods can be used for
metabolomics, notably mass spectroscopy.
Currently, for reasons that will be
discussed, high-resolution NMR spectros-
copy is one of the most effective ways to
study the metabolome, particularly as the
data produced can be correlated with those
from MRS studies performed in vivo. Before
 MR Metabolomics: Studies of Gene Function and Novel Anticancer Drugs
considering the results that have been
produced with this approach, we should
review the problems and opportunities
presented by metabolomics. Most of the
published, systematic work is concerned
with the metabolomes of plants2 or micro-
organisms such as yeast,3,4 and there has
been little systematic work on animal
metabolomes, still less in humans. However,
the power of this approach in human disease
and particularly in drug development is
obvious, and studies on simplified systems
are already showing useful results.
Like the transcriptome and proteome
(but unlike the genome), the metabolome is
state-specific. Whereas an organism’s
genome can be considered as an absolute,
the transcriptome in one of its cells will
vary continuously according to its
physiological state, as different genes are
expressed and the mRNA molecules are
spliced. There will be still more variation in
the proteome because of post-translational
modifications to the peptides translated from
the spliced mRNA molecules. The
metabolome is yet more labile; metabolite
concentrations can change within seconds
in response to nerve signals (e.g. the Ca2+
induced activation of glycogenolysis when
a muscle receives a signal to contract) but
in an unperturbed cell or tissue they usually
settle to a metabolic steady state. It is this
state-specificity that gives the “-omics”
methods their strengths in medicine: for
instance, one can consider the totality of
changes in gene expression or protein
content caused by a disease such as cancer
or even by a drug used to treat it.
Measuring the state of the metabolome will
enrich our understanding of the data from
the other –omics methods.
379
THE ROLE OF NUCLEAR MAGNETIC
RESONANCE METHODS IN
METABOLOMICS
Metabolomics presents several methodologi-
cal challenges beyond those of the other -
omic sciences. Ideally, to specify the instan-
taneous state of the metabolome, we should
like to know about the rates at which
metabolites flow through all the metabolic
pathways (“metabolic fluxes”). There is no
way to measure pathway fluxes on a large
scale at present, so it is necessary to measure
the steady-state concentrations of the
metabolites. This need for precise measure-
ment of large numbers of metabolite
concentrations, ideally with an accuracy of
better than a few percent, is technically
quite challenging. Many metabolites are
present at very low concentrations and some
are extremely labile. There is no current
method that will simultaneously measure
the concentrations of thousands of
metabolites in a manner analogous to
transcriptomics or even proteomics (although
mass spectrometry methods may eventually
do this), but by using NMR we can measure
sub-sets of the metabolome, termed
“metabolic profiles”.5 As studies of the entire
metabolomes of microorganisms such as
yeast are completed, it will be possible to
correlate metabolic profiles of human cells
or tissues with them. Even without such a
framework, useful information can be
obtained by interpreting the profiles in the
light of conventional biochemical knowledge.
NMR-Derived Metabolic Profiles
High-resolution NMR of cell or tissue ext-
racts is currently a very attractive technique
for obtaining metabolic profiles without the
need for elaborate sample preparation. A
380 Biomedical Magnetic Resonance: Proceedings of the International Workshop

Fig. 25.1: 1H NMR spectrum of tumor cell extract. Peaks assigned as: sodium 3-trimethyl-silyl-2,2,3,
3-tetradeuteropropionate (1-included as a chemical shift reference and for quantification), isoleucine (2), valine
(3), lactate (4), alanine (5), acetate (6), glutamate (7), succinate (8), glutamine (9), creatine and phosphocreatine
(10), phosphocholine (11), scyllo-inositol (12), myo-inositol (13), glycine (14), creatine (15), phosphocreatine
(16), glucose (17), tyrosine (18), ATP and ADP (19), NADH (20), formate (21). For detailed peak assignments
see References 37,38

simple, one-dimensional NMR spectrum can
quantify a substantial number (typically
30-50) of metabolites, many of which show
variations in disease (Fig. 25.1). The main
constraint on NMR visibility is concentra-
tion—only metabolites present at high
concentrations (typically in the millimolar
range, but with a lower limit of around
10 μM) in the cell will be detectable in a
reasonable time by this rather insensitive
method. A metabolite must also have at least
one resonance that can be resolved from its
neighbors. In difficult cases, one can use
methods such as the many types of 2-D NMR
to resolve peaks. Shifting the pH of the
sample is also often able to resolve a peak.
 MR Metabolomics: Studies of Gene Function and Novel Anticancer Drugs
Brindle6 has pointed out two important
reasons why these simple NMR-derived
profiles can give useful insights into the
metabolome. First, the metabolic pathways
within a cell interconnect so richly that each
metabolite is only a few removes from all
other metabolites. Thus, any metabolic
perturbation, e.g. a gene knockout, or the
action of a drug on a single target protein,
is likely to have an indirect effect on at
least one of the NMR-detectable metabolites.
Second, the fact that NMR measures nume-
rous metabolites simultaneously, in the same
sample, means that each was subject to an
identical preparation and assay procedure,
and thus the ratios of their concentrations,
one to another, can be measured with great
precision. The pattern of metabolite signals
in the profile is often very characteristic of,
for instance, a disease state, and this allows
computer-based pattern recognition
methods to give diagnostic information. In
contrast, individual metabolite concentra-
tions, obtained by conventional analytical
methods, are usually subject to several
percent error. Even if several replicates of
each sample are analyzed the ratios between
the mean concentrations of metabolites are
known much less precisely than those in an
NMR metabolic profile. For this reason, even
though the number of metabolites used can
be greatly increased by incorporating mean
concentrations obtained from replicate
individual assays, the pattern recognition
process (e.g. in the FANCY method, see
below) is often less successful.
SAMPLE PREPARATION FOR
METABOLIC PROFILES
Extraction
Metabolic profiling studies of genes
involved in cancer are typically performed
381
on extracts made by grinding a sample with
perchloric acid (for water-soluble
metabolites) or organic solvent (for lipids).
The extracted samples are then freeze-dried
and reconstituted in deuterated solvents
prior to analysis. Extractions can be made
of cultured cells or experimental tumors
grown from human cancer cells in rodents,
or even from frozen biopsies taken from
patients.7,8 In all cases, it is important that
the sample should be frozen or the acid
added as quickly as possible, since
metabolism continues post-mortem and
concentrations of some metabolites (e.g.
glucose and lactate, if the glycolytic pathway
is active) rapidly depart from the steady-
state values that originally existed in vivo.
A cell culture plate can be extracted by
pouring off the culture medium and rapidly
washing the cells with saline (to prevent
chemicals from the culture medium from
contaminating the profile), adding an aliquot
of perchloric acid and scraping the cells into
the acid. Experimental tumors in animals
can be rapidly frozen by the freeze-clamp
method in which they are crushed between
the metal jaws of a hand-held clamp that
have been pre-cooled in liquid nitrogen.
Provided appropriate precautions are taken,
and the samples are then kept deep-frozen
until the extraction is performed, these
methods give “metabolic snapshots” that
reflect the steady-state metabolism of the
tissue concerned at the moment when the
sample was taken.
Magic Angle Spinning NMR
By using Magic Angle Spinning (MAS) NMR,
one can obtain well-resolved spectra from
solid tissue samples without having to
extract them (Fig. 25.2). This method avoids
artefacts induced by the extraction process,
but gives less well-resolved peaks than
382 Biomedical Magnetic Resonance: Proceedings of the International Workshop
Fig. 25.2: HRMAS 1H spectrum of an HT29 tumour.
Peaks assigned as: lipids (1), lactate (2), alanine (3),
glutamate (4), creatine + phosphocreatine (5), choline-
containing compounds (6), taurine (7), creatine +
phosphocreatine (8). For detailed peak assignments
see References 37,38
would be obtained from a solution (since
not all the broadening processes are
eliminated). It also risks “scrambling” or
degradation of some metabolites while MAS
rotor is being packed with tissue and the
spectrum is being taken, because the
enzymes in the tissue will still be active,
even if all procedures are performed at 4°C.
One solution to this problem is MAS of deep-
frozen specimens. Some years ago, we
demonstrated the practicability of 13C MAS-
NMR at –24°C of biological tissues that had
been kept deep-frozen since excision,9 but
this method does not seem to have been
widely employed.
The MAS method is less satisfactory for
cultured cell models. If, for instance, one
were to perform MAS on a pellet of cells
(even if deep-frozen), obtained by spinning
down a suspension culture, the cells in the
pellet would rapidly consume all available
oxygen and become hypoxic during the
centrifugation procedure, and the energy-
pathway changes that ensued would
dominate the metabolic profile.
MRS Profiling in vivo
MR spectroscopy performed in vivo is
usually termed MRS. It has the unique
ability to monitor metabolites noninvasively
in situ, either in an experimental animal or
a human subject. Any MR spectrum can thus
be regarded as a metabolic profile that
indicates the relative concentrations of the
metabolites in the living organism, without
the need for extraction or biopsy. However,
NMR peaks from living tissue are not so
well resolved as they are in a spectrum from
an extract or even in an MAS specimen.
Another consideration is that the metabolic
state of a cancer growing in situ is influenced
by that of the host animal, whereas that of
cells growing on a culture plate can be
standardized – although they are kept in
highly artificial conditions.
METABOLIC PROFILING STUDIES ON
GENETICALLY MODIFIED CELLS
In recent years, the creation of genetically
modified cells, and even genetically
modified experimental tumors has become
much easier, and a large range of cancer
cell lines (along with the corresponding
parent or “wild-type” unmodified cells) is
already available. The use of small
interfering RNA (RNAi), in particular, makes
it simple to knock down the expression of
selected genes, and there are methods (e.g.
transfection of a cancer cell with an RNAi-
expressing virus) that allow one to make
such a knock down persistent, and then
grow the modified cells as tumors in
experimental animals. These methods are
ripe for exploitation in the field of
metabolomic studies since they will permit
examination of the action of individual gene
products on the metabolome.
 MR Metabolomics: Studies of Gene Function and Novel Anticancer Drugs
NMR-derived metabolic profiles can be
used in both cultured cells and experimental
tumors to study the effect of knocking out,
suppressing (e.g. by RNAi) or overex-
pressing a gene. The basic method involves
comparing the metabolic profile of the
modified cell (or tumor) with that of a
matched control – normally the parent cell
in which the modification was performed.10
The same approach can be used with mutant
cells obtained by any other means – e.g. a
“natural” mutation- provided that a suitable
control cell that differs only in the gene in
question can be compared with the parent
cell.
An obvious use for this method is to
study the control of metabolic pathways. A
putative control protein can be eliminated
from a pathway and one can then observe
the metabolic consequences. Raamsdonk et
al 3 devised a method termed FANCY
(Functional Analysis by Co-responses in
Yeast) for using NMR-derived metabolomic
data to infer the function of silent genes
(those whose deletion has no obvious
phenotypic effect) in the yeast genome. This
approach involves pattern recognition of the
metabolomic effects of knockouts of genes
known to be in particular pathways (the
training set) followed by assignment of an
unknown gene to a pathway on the basis of
the metabolomic consequences of its
deletion.
UTILISING METABOLOMIC METHODS
TO STUDY ANIMAL AND HUMAN
GENE MODIFICATIONS
Methods such as FANCY are aimed at large-
scale attacks on the metabolome of an
organism, preferably one like yeast that
can be manipulated quickly in the
383
laboratory.11,12 If they are successful we
should eventually have a library of
metabolomic patterns associated with all (or
a large fraction) of the genes in that genome.
Until that is achieved, can such methods be
usefully employed on mammalian cells or
even human cells?
It is, in fact, possible to infer much from
the metabolic profiles obtained from gene
modifications induced in cultured animal or
human cells, or in cells or tissues from
knockout animal models. The method has
been used on naturally occurring mutations,
as in genetic diseases like muscular
dystrophy,13 but more recently it has been
employed to study the effect of artificial
gene modifications. Combining proteomic
and metabolomic data in such studies is
particularly powerful. We have performed
a number of collaborative studies of this
type with Xu, Mayr and colleagues, using
knockout mice lacking the protein kinase
Cδ isozyme, to study the role of that
enzyme in cardiac muscle14,15 and vascular
smooth muscle cells, 16 and thus to
investigate its role in cardiovascular diseases.
The abnormalities induced in the proteome
by the gene knockout (e.g. an overex-
pression of enzymes in oxidative pathways
and under-expression of glycolytic enzymes
and glucose transporters) were found to be
associated with analogous changes in the
concentrations of oxidative and glycolytic
metabolites.
A METABOLIC PROFILE STUDY
ON HIF-1 MODIFIED TUMORS
Our first use of metabolic profiling by NMR
was in the field of cancer, in order to
investigate the HIF-1 pathway, which
enables cells (both normal and malignant)
to sense and respond to hypoxia. HIF-1,
384 Biomedical Magnetic Resonance: Proceedings of the International Workshop
which is overexpressed in many cancers, is
a dimeric transcription factor that
upregulates genes involved in glucose
uptake, glycolytic metabolism and many
other pathways. We studied tumors (grown
as xenografts in nude mice) in which HIF-
1 activity is blocked because the β subunit
is deficient, using the 1H NMR/perchloric
acid extract method outlined above. We also
used 31P MRS in vivo to compare the
metabolic profile of HIF-1 β deficient tumors
with the profile of the wild-type tumors.
The HIF-1 β deficient tumors had only 20
percent of the normal ATP as well as a
number of other statistically-significant
abnormalities in the metabolic profile: low
betaine (36%), phosphocholine (32%), choline
(40%) and glycine (49%).10
The hypothesis we have put forward to
explain these findings is that HIF-1
deficiency impairs the cancer cell’s ability
to perform anabolic synthesis of purine
molecules such as ATP. One of the effects
of HIF-1 in the “normal” cancer cells is
increased glycolysis, and it may also enhance
the “bleeding off” of glycolytic intermediates
for anabolic synthesis of important
metabolites required in cell replication.17 One
of these metabolites, glycine (which is
synthesised from 3-phosphoglycerate via
serine), is an essential intermediate in the
synthesis of new ATP molecules. In the HIF-
1 β deficient cells, glycolysis is not
upregulated, and we hypothesised that
because of this glycine synthesis cannot be
upregulated and glycine levels fall. An
alternative pathway for glycine synthesis is
from phosphocholine and choline, via
betaine. All these metabolites are depleted,
consistent with the hypothesis that they are
being used to synthesise glycine as an
intermediate in ATP synthesis.10
METABOLIC PROFILING BY NMR IN THE
DEVELOPMENT OF ANTICANCER DRUGS
Metabolic profiling has not yet been widely
used for anticancer drug discovery, but there
are several possible applications. Many novel
chemotherapeutics are intended to inhibit a
particular gene or its protein product, so
one can investigate the effect of this
inhibition on the metabolome by using cells
(or experimental tumors) with appropriate
gene knockouts or knockdowns and
compare these results with the actions on
the metabolome of the candidate drug. Such
studies could be used to suggest new drug
targets. Metabolic profiling could also be
used to refine the results of high-throughput
screens (do all the candidate drugs induce
the expected metabolic changes?), to indicate
mechanisms of drug action (are the changes
induced by the drug in the metabolic profile
consistent with the putative mechanism?),
or to detect side-effects (do the changes in
the metabolic profile suggest that the
candidate drug also acts on unintended
pathways?).
MRS in vivo also has much to offer for
monitoring anticancer drugs during the
development process, both at the preclinical
level on cultured cells or experimental
animals and in clinical trials on patients. It
is sometimes possible to monitor the
changing concentrations (i.e. pharma-
cokinetics, PK), and metabolism of the
anticancer drug itself, and this method will
be discussed later, but MRS is mainly used
to follow the action of the drug on the
metabolism of the cancer itself (an aspect of
PD). This ability to perform noninvasive
chemical assays at the site of action of a
drug is a unique advantage of MRS.
Monitoring the action of novel therapeutic
 MR Metabolomics: Studies of Gene Function and Novel Anticancer Drugs
agents on tumors in laboratory animals was
in fact one of the first uses to be proposed
for MRS in cancer.18 Most of the published
chemotherapeutic work by NMR has been
performed on routinely-used drugs, but it
can also offer important insights into the
actions of novel anticancer agents both in
the laboratory and in clinical trials on
patients, and thus, help to accelerate the
drug development process.
With regard to clinical trials, there has
been a trend to move away from the tradi-
tional approach in which a Phase I trial was
solely concerned with establishing the
toxicity and safe dose level of an anticancer
drug. Now-a-days, every effort is made to
produce data on the efficacy of the drug
and to verify its mechanism of action, even
in a Phase I trial. Many elaborate MRI and
PET protocols and even tissue biopsies are
used to maximise the data obtained at the
Phase I/II stage, and MRS methods could
have a role here. Understanding whether a
candidate drug is acting on its intended
target is made more difficult because many
of the anticancer agents currently under
development are not intended to cause
tumor shrinkage when used as single agents
(as they usually are in Phase I or Phase II
clinical trials). This means that conventional
clinical indications of response or even
sophisticated imaging methods will be
inadequate. Noninvasive MRI methods
(particularly, in the case of antivascular and
antiangiogenic agents, dynamic contrast-
enhanced MRI) are therefore being adopted
as surrogate pharmacodynamic (PD)
biomarkers. MRS could provide an
alternative PD method for trials of such
drugs.
The following sections will provide some
examples of PD studies performed by MRS
and metabolic profiling both on conventional
385
Table 25.1: Phospholipid metabolite levels in mouse
carcinoma at 48 hours after vehicle or 5-FU treatment
Metabolite Control 5-FU treated
PE 0.56 ± 0.11 0.95 ± 0.29*
PC 0.76 ± 0.11 0.61 ± 0.12
GPE 0.25 ± 0.06 0.36 ± 0.10*
GPC 0.82 ± 0.24 1.82 ± 0.61*
Data are expressed as Mean + S.E.M
Metabolite concentrations in μmol/g tissue.
*Significantly different from control group.
cytotoxic agents and on novel drugs that
were in the process of development.
MRS Studies of a Conventional Cyto-
toxic Agent – 5-Fluorouracil
5-Fluorouracil (5-FU) is a widely-used cyto-
toxic drug in medical oncology. Its PK and
metabolism have been extensively studied
in vivo in animals and patients by 19 F
MRS19,20 and its PD has been monitored by
31
P MRS. Street et al,21 used in vivo and in
vitro 31 P MRS to examine the
pharmacodynamic effect of 5-FU on a mouse
mammary carcinoma model. An increase in
NTP/Pi and PCr/Pi ratios was observed in
vivo 48 hours after 5-FU treatment. The in
vivo PE to PC ratio was also elevated relative
to controls and this was found to be due to
an increase in PE (Table 25.1; as confirmed
by in vitro 31P MRS). In addition, increases
in GPC and GPE were also observed 48
hours after 5-FU treatment.21
MRS Studies of a Cyclin-Dependent
Kinase Inhibitor-R-
Roscovitine (CYC202)
CYC202 (R-roscovitine, Cyclacel Ltd,
Dundee, Scotland) inhibits the cyclin-
dependent kinases 1, 2 and 7 and thus,
blocks cell cycle progression. In vivo 31P MRS
386 Biomedical Magnetic Resonance: Proceedings of the International Workshop
was used to examine xenografts treated with
CYC202, to gain insights into the biochemical
changes that are associated with cell cycle
disruption.22
CYC202 treatment for 4 days caused
inhibition of tumor growth. 31P MRS of the
tumors in vivo showed a significant decrease
in the intracellular pH and also in NTP/
TotP (total phosphorus signal) ratio. A
significant increase in the Pi (inorganic
phosphate)/TotP and the Pi/NTP ratios
were also observed post-treatment, whereas
no significant changes were seen in the
vehicle group. As well as reducing cell
proliferation, CYC202 impaired tumor
bioenergetics and decreased tissue pH. If
CYC202 comes to clinical trial, these 31P MRS
effects may provide a non-invasive marker
for assessing tumor response in patients.
MRS Studies on 17AAG,
an Inhibitor of HSP90
The heat-shock protein HSP90 is of interest
as an anticancer target because it helps
maintain the shape of many oncogenic
proteins. Inhibiting single oncogenes with
“magic bullet” drugs has proved somewhat
disappointing as the cancers often become
resistant; a drug that acts indirectly on a
large number of potentially oncogenic
proteins may be able to overcome this
problem. The HSP90 inhibitor 17AAG was
developed with this strategy in mind and is
currently under trial as an anticancer agent.
A surrogate marker of tumor response was
needed for the clinical trial, and noninvasive
MRS methods were therefore investigated.
The actions of 17AAG on several cultured
human colon cancer cell lines and on the
HT29 human colon tumor xenograft in nude
mice were studied in vivo and in vitro using
31 P MRS. 23 Treatment with an inactive
analogue was used as a control. In all the
cell lines studied, there were significant
increases in phosphocholine and glycero-
phosphocholine levels following 17AAG
treatment.
In vivo, after 4 days of 17AAG treatment
the HT29 tumors showed significant growth
delay and an increased in ratios of phos-
phomonoester/phosphodiester (PME/PDE),
PME/total phosphorus (TotP) and PME/β-
nucleoside triphosphate (β-NTP), as well as
a decrease in the β-NTP/TotP ratio (Fig.
25.3). The elevated PME observed in vivo
was due to increased levels of phos-
phoethanolamine and phosphocholine as
shown in vitro by 31P MRS of tumor extracts
(Fig. 25.3). A significant inverse correlation
was found between the percentage of
change in PME/PDE ratio and the
percentage of change in tumor size following
17AAG treatment. These 31P MRS changes
suggested that it would be possible to
monitor tumors in patients during the Phase
1 clinical trials of 17AAG, and such a study
is currently under way.
Pharmacokinetics and Metabolism
MRS also has the unique ability, in some
cases, to monitor the changing concentration
of the anticancer drug itself (PK) as well as
its metabolism and detoxification. The first
MRS studies 19 on an anticancer drug,
5-fluorouracil (5-FU), used 19F MRS on rat
tumors to monitor 5-FU PK, activation to
toxic fluoronucleotides and the appearance
of detoxification products from the liver.
There have been many other animal studies
on 5-FU and its derivatives (for a review,
see Reference 20) and this widely used drug
has also been monitored by 19F MRS in
patients.24,25 A recent clinical study26 has
shown that the detoxification products of
 MR Metabolomics: Studies of Gene Function and Novel Anticancer Drugs 387

Figs 25.3A and B: Expanded in vitro 31P MR spectra of extracts from (A) a control and (B) a 17AAG-treated
HT29 tumour. C and D: In vivo 31P MR spectra of an HT29 tumour (C) before and (D) after 17AAG treatment.
Peak assigned as: phosphomonoester (PME), comprised mainly of phosphocholine (PC) and
phosphoethanolamine (PE); inorganic phosphate (Pi); phosphodiester (PDE), comprised of
glycerophosphocholine (GPC) and glycerophosphoethanolamine (GPE); phosphocreatine (PCr), α-, β- and
γ-nucleoside triphosphates (α-, β-, γ-NTP).
Note that the ppm scale on A and B is referenced to methylenediphosphonic acid, whereas the ppm scale
for the in vivo spectra in C and D is referenced to PCr. The box drawn on C indicates the region of the in vivo
spectrum corresponding to the in vitro spectra shown in A and B

5-FU are conjugated and excreted via the
bile duct after storage in the gall bladder.
In another recent study it proved possible
to monitor the toxic fluoronucleotides in
tumors in patients using a conventional 1.5 T
MRI instrument; surprisingly, these toxic
species were also found in the apparently
normal livers of two patients who failed to
develop liver metastases.27
MRS studies using other nuclei to monitor
anticancer drugs have also been performed
on tumors in vivo. 1H MRS has been used to
388 Biomedical Magnetic Resonance: Proceedings of the International Workshop
monitor iproplatin,28 2H MRS to monitor
misonidazole, 29 13 C MRS to monitor
temozolomide, 30 31 P MRS to monitor
ifosfamide 31 and 195Pt MRS to monitor
carboplatin.32 Apart from 19F, however, only
31
P has been used to monitor an anticancer
drug in patients. 33 In general, the low
sensitivity of MRS militates against its use
for routine PK studies, despite its unique
ability to monitor an unlabelled drug at its
site of action. It is rarely possible to monitor
a concentration change of more than 10-
fold with MRS, which means that the
elaborate modelling studies often performed
on PK data obtained from body fluids can
rarely be performed using data obtained
by MRS. There have been few PK studies
by MRS on drugs in development since few
novel drugs contain suitable nuclei (e.g.
fluorine or phosphorus). In principle, one
might study a fluorinated analogue of the
lead compound in a series (see Reference
34) in which the drug signal was used to
create an image) but the presence of the
fluorine atom(s) might well alter the PK.
Another possibility would be preparation
of a 13C-labelled drug sample,30 but the cost
of this, bearing in mind that because of the
low sensitivity of 13C MRS, almost all mole-
cules of the drug would have to be labelled25
is too great for routine use.
Table 25.2: Rate constants (k) of 5′DFUR, capecitabine and its intermediary metabolites
Tumour Capecitabine 5′DFCR/5′DFUR
group (breakdown) (build-up)
2T10 0.028 –0.026
+ 0.003 + 0.003
Control 0.027 –0.018
+ 0.003 + 0.004
Data are expressed as Mean + S.E.M.
*Statistically different from control.
PHARMACOKINETIC MEASUREMENTS
OF A 5-FU PRO-DRUG, CAPECITABINE
A PK study by 19 F MRS on the oral
anticancer drug capecitabine, a prodrug of
5-fluorouracil (5-FU) was performed
recently. The final activation step in the
tumor is from 5′deoxy-5-fluorouridine
(5′DFUR) to 5-FU, catalysed by thymidine
phosphorylase (TP). Previous studies have
shown that tumor response is strongly
correlated with tumor TP, so we monitored
the PK of capecitabine in 2T10 human
bladder tumors engineered to over-
expressed TP, compared with wild type and
empty vector controls.35 19F spectra were
acquired after a single dose of capecitabine
(Fig. 25.4) or 5’DFUR. In the TP-overexpres-
sing 2T10 tumors the rate constant of the
breakdown of the intermediate molecules
5’DFCR + 5’DFUR was significantly faster
than in controls (Fig. 25.4 and Table 25.2)
and the rate constant of the breakdown of
5’DFUR was also doubled compared with
controls (Table 25.2).
This study confirmed that the rate of
5’DFUR conversion is related to TP expres-
sion. 19F MRS measurement of the rate
constants for the interconversion of capecita-
bine and its metabolites could be a non-
invasive surrogate for measuring TP levels
in patients and predicting response to
5′DFCR/5′DFUR 5’DFUR
(breakdown) (breakdown)
0.012* 0.021*
+ 0.001 + 0.001
0.006 0.010
+ 0.001 + 0.003
 MR Metabolomics: Studies of Gene Function and Novel Anticancer Drugs 389

(A) Time course of the accumulation and breakdown of

5’DFCR/5’DFUR In TP over-expressing and control tumors

(B) In vivo 19F MRS

Fig. 25.4: A: In vivo 19F MR spectra of a tumour 52-72 minutes after an injection of capecitabine. B: Time
course of the accumulation and breakdown of 5′DFCR/5′DFUR. Spectral peaks assigned as: 5′-deoxy-
5-fluorocytidine (5′DFCR); 5′-deoxy-5-fluorouridine (5′DFUR); 5-fluorouracil (5-FU); α-fluoro-β-alanine (FBAL).

capecitabine. A recent 1.5 T and 3 T 19F laboratory animals or on patients – can be

MRS study36 has detected capecitabine, as
well as its metabolites and catabolites, in
livers of patients with colorectal cancer, so
this method might be applicable in clinical
trials or even for choosing therapies for
individual patients.
CONCLUSION
Several magnetic resonance methods – high-
resolution NMR of extracts, MAS-NMR of
biopsies and MRS in vivo either on
used to produce metabolic profiles of cancer
cells or experimental tumors. The recent
development of relatively simple methods
for knocking out or knocking down genes
has made it easy to produce matched pairs
of cell lines or implanted tumors, one with
and one without the gene, or alternatively,
with the gene overexpressed. Metabolic
profiling of these paired cell lines and
tumors is proving to be a highly productive
method for assessing gene function.
390 Biomedical Magnetic Resonance: Proceedings of the International Workshop
The same strategy can also be applied to
drug development, since many drugs are
designed to inhibit one or a few gene
products. Metabolic profiling methods could
be used to search for potential drug targets,
to assess whether a candidate drug induces
the expected metabolic change or to look
for unwanted metabolic side effects.
Because of its unique ability to obtain
chemical information non-invasively from a
drug’s site of action, MRS performed in vivo
could have a special role in the later stages
of drug development, particularly for PD
(and even in some cases PK) in early-phase
(I and II) clinical trials. However, it should
be remembered that although most of the
current generation of 1.5 T clinical MRS
instruments can easily be adapted to
perform MRS, they have significant
limitations when used on tumors outside
the brain (brain tumors rarely respond to
conventional chemotherapy, and are not
usually included in early-phase trials). The
main problem is the low sensitivity of MRS
at 1.5 T (equivalent to that of the 60 MHz
NMR instruments used for laboratory work
in the 1960s). Outside the brain, only large
tumors can be studied, and to maximise
sensitivity it is preferable to study a
superficial lesion, using a surface coil.
Motion is also a serious problem, and most
thoracic (e.g. lung) tumors and many
abdominal tumors are currently inaccessible.
Lastly, signal from adjacent fat often
obscures features of interest in the spectrum.
For these reasons, it seems unlikely that
current MRS spectroscopy could be used as
the primary biomarker – i.e. as surrogate
for response – in a clinical trial. Neverthe-
less, even if it could only be used on a
proportion of the patients, MRS data could
be used to enrich the information obtained
in a trial.
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1-15.

Metallopharmaceuticals as
Exogenous Contrast Agents
INTRODUCTION
Developments in molecular science are
shifting the detection horizon of medical
diagnosis and therapeutics to the earliest
physiological and biochemical manifestations
of disease. This emerging new paradigm
for healthcare contrasts with the
conventional assessment, in which the
disease comes to the notice of the physician
when the patient presents either with the
symptoms or with anatomical lesions, which
are visible with various diagnostic
modalities. These new diagnostic and
therapeutic markers stand at the core of
‘molecular imaging’. Molecular imaging is
the in vivo diagnosis of complex pathological
processes by detection of unique biological
signatures at the subcellular level. This
approach differs from the ‘traditional’
imaging approaches, in which diagnosis have
relied primarily on registering and
interpreting intrinsic differences in image
contrast between normal and abnormal
tissues, which are the anatomic expression
of disease. However, molecular imaging1
does not aim at replacing existing imaging
technology and interpretation. Rather, it is
directed towards extending and improving
26
RK Sharma, R Mathur, AK Mishra
diagnostic accuracy by visualizing the
molecular components or processes that are
the underlying causes of disease. A wide
range of medical imaging techniques like
positron emission tomography (PET), 2-4
optical imaging5-8 and magnetic resonance
imaging (MRI)9-12 can be used for molecular
imaging and the choice will be determined
by the specific molecular process that is
being targeted. Magnetic resonance imaging
(MRI) offers the unique opportunity to
extract both anatomic and physiological
information simultaneously. MR is,
therefore, emerging as a particularly
advantageous modality for molecular
imaging.
The MRI allows one to tailor the imaging
study to the anatomic part of interest and
to the disease process being studied. It is a
method of obtaining images of the body in
thin slices giving spatial distribution of the
intensity of water proton signal in the
volume of the body.13 Thus, over the years
MR has emerged as an exquisitely powerful,
robust and versatile technique methodology
in diagnostic clinical medicine and
biomedical research. Its prime advantage is
that it permits the simultaneous acquisition
of multiparametric qualitative/quantitative
394 Biomedical Magnetic Resonance: Proceedings of the International Workshop
information of living organism at various
levels of organization. Although the spatial
resolution of MR is high and it also offers
physiological sensitivity, production of
detailed images that provide an insight into
how physiological functions occur in situ is
somewhat tricky affair. Image contrast in
MRI is primarily achieved by exploiting the
selection of appropriate pulse sequences. It
can also be made sensitive to diffusion,
perfusion and macroscopic blood flow. The
potential of the MRI procedures can be
enhanced by their use in conjunction with
the metallo-pharmaceuticals in both clinical
and experimental setting. A MR contrast
agent is basically an imaging sensitive
substance that improves the resolution of
the MRI images by increasing the brightness
in various parts of the body where they
reside. The contrast media actually enhances
the image quality by enhancing the
relaxation rate of the protons in the tissue
thereby altering the signal intensity in the
image relative to the areas not affected by
the contrast agent. Since the signal intensity
depends on the amount of water in the
given place and on the magnetic relaxation
times T1 and T2, which in turn are influenced
by a range of factors.14,15 Deducing these
factors in each case is the ultimate goal of
the radiologist trying to diagnose the
problem by looking at the MR image. With
its dependence on the more biologically
variable parameters of proton density,
longitudinal relaxation time (T 1 ), and
transverse relaxation time (T 2), variable
image contrast can be achieved by using
different pulse sequences and by changing
the imaging parameters. Signal intensities
on T 1, T 2 , and proton density-weighted
images relate to specific tissue characteristics.
For example, the changing chemistry and
physical structure of hematomas over time
directly affects the signal intensity on MR
images, providing information about the age
of the hemorrhage. Moreover, with MR’s
multiplanar capability, the imaging plane can
be optimized for the anatomic area being
studied, and the relationship of lesions to
eloquent areas of the brain can be defined
more accurately. Flow-sensitive pulse
sequences and MR angiography yield data
about blood flow, as well as displaying the
vascular anatomy. Even brain function can
be investigated by having a subject perform
specific mental tasks and noting changes in
regional cerebral blood flow and
oxygenation.
A particularly powerful paradigm for
functional MR imaging of micro-vascular
hemodynamics incorporates paramagnetic
materials that create significant image
contrast. These include exogenous
(transition/lanthanide metal chelates) and
endogenous agents (like deoxygenated
hemoglobin) for mapping cerebral blood
volume/ flow and neuronal activity.16 In
this presentation we shall be focusing
primarily on the chemistry behind the
exogenous contrast agents.
EXOGENOUS CONTRAST AGENTS
Paramagnetic Contrast Agents
General Considerations
Spin-lattice relaxation time T1 and spin-spin
relaxation time T 2 may be shortened
considerably in presence of paramagnetic
species. The resulting effects can be seen
best in NMR spectroscopy. While shortening
of T1 leads to increase in signal intensity,
shortening of T2 produces broader lines with
decreased intensity.17 The net result is a
nonlinear relationship between signal
intensity and the concentration of the
 Metallopharmaceuticals as Exogenous Contrast Agents 395

contrast agent.18 At low concentrations, an

increase in contrast agent provides an
increase in signal intensity due to effect on
T1 until the optimal concentration is reached.
Further increase in concentration reduces
the signal because of effect on T2. Therefore,
in clinical practice it is possible to achieve
less than optimal contrast effect and even
produce a negative contrast effect. This
dictates the use of agents that have a
relatively greater effect on T1 than on T2, as
well as the use of pulse sequences that
Fig. 26.1: Nitroxide radical as contrast
emphasize the changes in T1. Paramagnetic
species are species, which have unpaired
electrons. It may be simple substance (i.e. metal ions, their electronic configuration and
molecular oxygen), stable radical (i.e. magnetic moment.
nitroxide radical) Figure 26.1 or metal ion There are more transition metals and
(i.e. many transition metal ions). lanthanide metals with unpaired spins, but
Radicals generally cause damage to the for the metal to be effective as a relaxation
living tissues. Therefore, they are not agent the electron spin-relaxation time must
suitable candidates for medical MRI match the Larmor frequency of the protons.
purposes. The paramagnetic effect of oxygen, This condition is met better for the Fe3+,
although demonstrable, seems too weak for Mn 2+ and Gd3+ ions (about 10 -8 - 10-10s,
practical applications.19 Paramagnetic metal others have 10-11 - 10-12s).20
ions do show suitable effect which depends The main problem with paramagnetic
on number of unpaired electrons in the ion. heavy metal ions in their native form is their
Table 26.1 shows some of the paramagnetic toxicity. Investigation has focused on the

Table 26.1: Some important paramagnetic ions

Atomic Ion 3d 4f Magnetic

no. moment (BM)
24 Cr3+ 3.8
— — — — —
25 Mn2+ 5.9 (weak field)
— — — — —
26 Fe3+ 5.9 (weak field)
— — — — —
29 Cu2+ 1.7 – 2.2
— — — — —
63 Eu3+ — — — — — — — 6.9
64 Gd3+ — — — — — — — 7.9
3+
66 Dy 5.9
— — — — — — —
396 Biomedical Magnetic Resonance: Proceedings of the International Workshop
development of stable paramagnetic ion
complexes. Both the metal ion and the ligand
usually exhibit substantial toxicity in the
unbound state. Together, however, they
may create a thermodynamically and
kinetically stable compound, which is much
less toxic. Complexation of the metal ion
with organic ligand, while considerably
decreasing toxicity, may alter paramagnetic
properties of the metal. Chromium-EDTA
complex was the first such agent tried, but
problems with synthesis and long-term
stability prevented its clinical application. 21
Gadolinium-DTPA complex, a renally
excreted chelate with a very high formation
constant (Table 26.2), 22 had sufficiently
favorable properties to be approved by Food
and Drug Administration of USA for use in
cranial disease diagnostics in mid-1988.
Another, relatively new type of para-
magnetic contrast agents is so-called
superparamagnetic iron oxide (SPIO) based
colloids. They consist of nonstoichiometric
microcrystalline magnetite cores, which are
coated with dextrans or siloxanes. Use of
these colloids as tissue-specific contrast
agents is now a well-established area of
pharmaceutical development.23-25
Table 26.2: Gd-DTPA formation constant = 1023
Compound
Gd-DTPA
Gd-EDTA
Gd-CI3
Meglucamine diatrizoate
Common X-ray contrast agent
Half life in urine = 21.0 min and in blood = 19.6 min after intravenous injection
Gadolinium (III) Complexes
Most widely used contrast agents are based
on Gd(III) cation because of its long
electronic relaxation time and 4f7 electronic
configuration is equally attractive for the
synthesis of “smart CA”. There are two
classes of chelating agents, acyclic DTPA and
macrocyclic DOTA, that are most widely
exploited for the complexation with Gd(III)
because they form stable complexes and do
not allow the Gd(III) cation to dissociate
under physiological conditions, chelates are
essential to avoid the undesirable
biodistribution and the relatively high
toxicity of the free metal ion. All the
complexes derived from these chelating
agents to use as contrast in MRI are
summarized in (Figs 26.2A and B).
The relationship between the thermo-
dynamic stability of the complex and the
acute toxicity in vivo seems to be more
complex, as demonstrated by study26 on that
matter. Authors investigated stability of the
series of complexes of several ligands with
Gd3+ versus acute toxicity on mice. They
also attempted to measure the rate of
transmetallation of Gd 3+ by Cu 2+ and
LD 50 with intravenous dose in rat
(mmol/kg body weight)
10
0.3
0.4
18
 Metallopharmaceuticals as Exogenous Contrast Agents 397

Fig. 26.2A: Gadolinium complexes with macrocyclic Fig. 26.2B: Acyclic chelating agents with different
chelating agents used as contrast agents functional groups as Gd-based MR contrast agents

selectivity of different ligands towards Gd3+
as compared to Zn3+, Cu3+, Ca3+. Iron (III)
was not considered because it is tightly
bound in vivo by the storage proteins ferritin
and hemosiderin and is essentially unavai-
lable for interaction with Gd3+ complexes.
The main competitor to Gd3+ was found to
be Zn2+, and the most important thermo-
dynamic criterion of toxicity is the selecti-
vity of the ligand for Gd 3+ over other
398 Biomedical Magnetic Resonance: Proceedings of the International Workshop
endogenous metal ions. Zinc trans
metallation was found to be the most likely
mode of both acute and sub chronic toxicity
in experiments on rats. Complexes whose
structure make in vivo trans metallation
reactions much slower than renal excretion
rates have significantly improved toxicities
than would be predicted by thermo-
dynamics. Slower clearance from the body
is likely to significantly increase the toxicity
of any Gd3+ complex shows other ligands
often used in gadolinium studies.
Gd3+ has been shown to inhibit Ca2+
binding to mammalian cardiac sarcoplasmic
reticulum. The mechanism of toxicity could
involve hemodynamic disruption. Potential
improvement also lies in the idea of
covalently coupling the ligand to protein to
generate tissue-specific contrast agents.27
Ca2+ plays a very important role in signal
transduction and its presence or absence is
seen to be affecting the relaxivity of the
DOPTA-Gd complex.28
Understandably, search for new potential
ligands for Gd3+ complexation is still hot
area of investigation. The complexes are
evaluated against Gd-DTPA, the only
approved compound for human use so far.
Criteria include thermodynamic stability,
rates of excretion, toxicity, lipophilicity,
biodistribution, percent change in MR signal
intensity. Some of the complexes are slightly
better than Gd-DTPA in particular tests,
others are a little worse. However, neither
of them got as much attention as Gd-DTPA
itself.29 This complex is far not be the best
choice as of today, but it is relatively well-
known choice, widely used in many MRI
facilities on a daily basis. The Gd-DTPA
complexes have been over the period been
improvised chemically by using different
substituents and derivatives like the DTPA-
bis (amide),30 silyl lewis X mimetic31 with
the aim to obtain better relaxation and hence
a better contrast.
Iron Oxide Nanocompounds
Nanoparticles are typically defined as the
particles in the nanometer size range
(ranging typically from 10–200 nm) with
colloidal properties so that the particles do
not settle out of the solution. The size,
charge, hydrophilicity and composition of
the nanoparticles can be manipulated to
achieve optimal uptake within a tumor and
specific targeting ligands (i.e., peptides,
monoclonal antibodies, small molecules) can
be attached to the surface for specificity.
The iron oxide nanoparticles tend to be
magnetic or superparamagnetic in nature
with the particle approximately 20 nm being
the later. For the purpose of acting as MRI
contrast agent the iron oxides can be
classified as MION (mononcrystalline iron
oxides) and CLIO (cross linked iron oxides)
They are basically the primary iron oxide
nanoparticles (the core of the iron oxide
crystal consists of and iron oxide crystal
coated with a polymer. This class of
compounds normally consists of dextran
coated iron oxide preparations. The CLIO
particles represent a stabilized dextran
coated iron oxide nanoparticle in which the
iron oxide core is caged in the cross-linked
dextran coating. These compounds consist
of nonstoichiometric microcrystalline
magnetite cores, which are coated with
dextrans (in ferumoxides) or siloxanes (in
ferumoxils). In case of SPIO (superpara-
magnetic iron oxide) the nanoparticle have
a size greater than 50 nm (coating included)
where as cases where the size is smaller
than 50 nm the nanoparticles are termed as
USPIO (Ultra small superparamagentic iron
 Metallopharmaceuticals as Exogenous Contrast Agents 399
oxides). Lumirem and Endorem are
examples of SPIO where the former contains
silica coated particles 300 nm in diameter
and the later contains magnetite particles
with 150 nm diamter. They are being used
for the gastrointestinal tract imaging and
for the liver and spleen disease detection.
Sinerem is an example of USPIO and is
composed of magnetite particle with 30 nm
diameter and is currently being used for
tumor detection. The particle size greatly
affects the physicochemical and the
pharmacokinetic properties. SPIO agents are
much more effective in MR relaxation than
their paramagnetic counterparts. Since they
are nonstoichiometric, there was and still is
much interest in studying these compounds
with all the vast array of modern physical-
chemical methods: single crystal X-ray
diffraction, powder X-ray diffraction,
Mossbauer spectroscopy, transmission
electron microscopy, dynamic light
scattering, atomic adsorption spectroscopy,
spectrophotometry, electron microscopy,
superconducting quantum interference
devices, etc.32-35
The compositions and physiochemical
properties of nonstoichiometric magnetite’s
are continuously variable between those of
Fe3O4 and Fe2O3. Conceptually, these cation-
deficient, inverse-spinel phases are formed
by partial oxidation of Fe(II) in stoichio-
metric magnetite 24. The Fe2+ content is
typically 8-15 mol percent. The lattice
parameters of these colloids also fall between
those of Fe3O4 and Fe2O3.
The particles are usually of varying sizes
from several to several hundred nanometers.
They are irregular in shape and highly light-
absorbing. They have no magnetic hysteresis
at ambient temperatures, which is charac-
teristic of superparamagnetic materials.
Mossbauer spectra are characteristic of small
(<10 nm) magnetic domains, which undergo
superparamagnetic relaxation or collective
magnetic excitation on the Mossbauer time
scale.33 There is no evidence of covalent
bonding between the iron surface and the
surrounding dextran.35
The SPIO compounds are promising
contrast agents since their properties may
be fine tuned for the specific application.
They are non-toxic and rapidly cleared from
the organism. Experiments have been
successful in receptor-specific SPIO deli-
very.36
Porphyrins have been known for decades
as indicators of various metabolic disorders
and disease states.37,38 They are used in
photodynamic therapy of tumors. 39 Low
toxicity of metalloporphyrins and their
selective retention in tumors have led
recently to their study as an MRI contrast
media. 40-45 Theoretical treatment of
relaxivity of some of the metalloporphy-
rinshas been studied by Mercier. The
proposed mechanism via which porphyrins
accumulate in tumors is through selective
uptake by benzodiazepine receptors
abundant in tumor cells but not in normal
cells.46 Mangafodipir trisodium (MnDPDP)
was shown to be useful in establishing the
diagnosis of acinar cell carcinoma, a rare
pancreatic exocrine neoplasm.47 MnDPDP,
which specifically labels liver tumors over
healthy liver tissue, helps determine if
surgery on hepatic tumors is a viable
option. 48-50 This is a fairly new area of
development, but metalloporphyrins of
Mn(III) and Fe(III) show favorable pro-
perties as MRI contrast agents for tumor
detection.
Gastrointestinal Contrast Agents
The gastrointestinal tract cannot be reliably
studied by MRI without the use of contrast
400 Biomedical Magnetic Resonance: Proceedings of the International Workshop
agents. Oral contrast agents may drama-
tically improve utility of MRI for gastro-
diagnostics. The only clinically approved
agents for that purpose are soluble iron
compounds. (ferrous gluconate, ferric
ammonium citrate) and Gd-DTPA. There is
a problem with dosage of iron salts, which
may not exceed the levels above those when
iron supplementation is used. There are no
particulate agents approved for oral use yet.
Gadolinium(III) DTPA has been used to
depict the lumen of the digestive organs in
rats.51 It is effective in the primary diagnosis
of Crohn’s disease and in differentiating
nonactive from active forms of the
disease.52,53 Gadolinium(III) DOTA has been
used to examine the movement of material
exiting the stomach and for diagnosis of
delayed gastric emptying 54,55 zeolites
containing gadolinium have been shown to
be an inexpensive, non-toxic alternative to
X-rays and barium salts for imaging of the
GI tract.56,57 Further, hectorite clay that has
undergone ion exchange with gadolinium
ions has been used as an oral contrast agent
for GI tract imaging,58 superparamagnetic
iron oxide agents have been used to image
the GI tract and assess ulcerative colitis.59,61
Contrast Agents For Angiography
MR angiography usually requires a rapid
bolus injection of a paramagnetic ECF agent
since these agents rapidly redistributes from
the vascular space into the interstitium.
Following such a bolus injection the initial
gadolinium concentration in blood can easily
be ten times higher than the steady state
concentration. The benefit of such a high
gadolinium concentration is that the
resulting T1 of blood is extremely short
typically less than 100 ms thus giving rise
to a very large blood background contrast
heavily T1 weighted images. This effect is
successfully utilized in contrast enhanced
MR angiography.62,63 Various iron oxide
nanoparticles are suitable contrast agents for
MRI angiography.64,65 Superparamagnetic
iron oxide particles have been shown to
accumulate in human atherosclerotic plaques
which indicates potential for noninvasive
assessment of active atherosclerotic
disease.66
Other Contrast Agents
Examples of contrast agents other than
paramagnetic involve rather mechanical
action than changing the characteristics of
the surrounding tissue. They are thus, similar
to agents used in X-ray methods. One recent
example successfully utilized vege-table oil
for rectal MRI applications. 67 Other
possibilities include insufflation of air to
achieve better contrast of intestinal walls.68
CONCLUSION
We have summarized the various exogenous
contrast agents available for MRI in this
chapter. Contrast agent enhanced MRI is an
invaluable tool to the diagnosis of cancer
and many other diseases, and recently in
molecular imaging of experimental animals.
With improved targeted delivery of these
agents, the ability to diagnose diseases will
become more sensitive, accurate, and
ultimately simplified. The activatable agents
that respond to biological phenomena by
altering the intensity of signal enhancement
in a conditional fashion are steps toward
unraveling the complex connectivity of
developmental biological systems. In order
to further the usefulness of contrast agents,
chemistry needs to be used to develop
agents that are responsive to biological
 Metallopharmaceuticals as Exogenous Contrast Agents 401
phenomena and directed to specific regions.
These two improvements are closely related
in that many biologically significant
molecules are located in specific parts of
the body. Further, these agents may
represent the prelude to complete, non-
invasive, medical examinations that are safe,
fast, and accurate.
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High-resolution MR Spectroscopy
in Clinical Medicine
GA Nagana Gowda, Ashish Gupta, M Bhandari, CL Khetrapal
INTRODUCTION
Applications of MR in biomedicine have
grown exponentially during the last over
two decades as magnetic resonance imaging
(MRI). Although MR spectroscopy, unlike
MRI, has the potential of providing informa-
tion on a wide range of biological processes
in molecular level, its potentials in bio-
medicine have not been fully realized. While
in vivo spectroscopy applications to human
are still constrained by the problems
associated with sensitivity, resolution and
shorter T2 relaxation times, in vitro appli-
cations to specimens such as body fluids do
not suffer from such constraints as it is
possible to study these in high and more
homogenous magnetic fields. With reference
to this, this article presents high-resolution
MR spectroscopy applications to body fluids
in the diagnosis of diseases with emphasis
on malabsorption syndrome, bacterial
infections, and in the assessment of liver
graft function.
Malabsorption syndrome is associated
with impaired absorption of nutrients from
upper small intestine. D-xylose absorption
test is one of the common diagnostic
methods for screening the patients of this
27
disease.1,2 When given orally, D-xylose is
absorbed from proximal small intestine and
is partly metabolized in the liver. 23-50
percent of the absorbed D-xylose is excreted
in urine in healthy subjects. Urinary excre-
tion of D-xylose is less than 20 percent in
patients with malabsorption syndrome. Of
the several available colorimetric methods
for estimation of this pentose sugar, most
widely used method employs use of
p-bromo aniline.3 However, this method is
laborious and it requires stringent conditions
of temperature, reagents and reaction times.
Furthermore, the error is governed by the
stability constant of the colored complex. In
addition, if the urine sample contains other
sugar(s) such as glucose, it may lead to
further error since this sugar also form
colored complex.4 In order to circumvent
these problems, the application of high
resolution MR spectroscopy for the
assessment of malabsorption syndrome is
explored and the results are presented.5
Infection from bacteria such as E-coli,
K. pneumonia and P. aeruginosa is the leading
cause of urinary tract infection (UTI).6 In
the clinical diagnosis, conventional urine
culture method is routinely employed.
However, this is time-consuming and
404 Biomedical Magnetic Resonance: Proceedings of the International Workshop
labour-intensive. It takes nearly 24 h
incubation to obtain an accurate colony
count. In addition, 12-24 h is needed for
organism identification and susceptibility
testing which further delay the treatment.7,8
For routine applications, a method which
requires minimum sample, rapid, automated,
specific and noninvasive is required. Several
bacterial screening tests have been applied
for the rapid diagnosis of UTI of which, the
most common approach is the nonculture
method which uses reagent dipsticks to
detect the presence of nitrite and leucocycte
esterase.9,10 The drawback of this method
is that it yields false negative results for
bacteria which do not produce nitrite11 and
false positive results in presence of ascorbic
acid, drug interference and over growth
with nitrite-producing bacteria. 12 The
potential of MR spectroscopy as an
alternative diagnostic tool is explored for
simultaneous cell counting as well as identi-
fication of P. aeruginosa.13
Normally liver graft function is assessed
by monitoring serum lactate, aspartate
aminotransferase (AST), alanine transferase
(ALT), bilirubin, blood urea, glucose, arterial
blood gases including bicarbonate and
international normalized ratio (INR) and
color Doppler imaging. In human liver, toxic
ammonia is converted into urea through
urea cycle. Its small proportion also gets
metabolized into glutamine.14 In acute liver
cell failure, there is impairment in urea cycle,
resulting in abnormally high levels of blood
ammonia and decreased urea levels.
Increased ammonia level triggers glutamine
synthesis.15,16 Usually blood ammonia levels
are measured biochemically but the accuracy
of ammonia measurement is constrained by
time, temperature and hemolysis of the
sample drawn, besides high cost. Monitoring
of glutamine levels in blood and urine along
with urea levels in urine by 1H MR spectros-
copy is reported here. The utility and unique
application of MR spectroscopy in liver graft
function monitoring is demon-strated.17
METHODS
Malabsorption Syndrome
Thirty-five patients with suspected malab-
sorption syndrome underwent conventional
medical tests such as total serum protein,
albumin, blood hemoglobin, upper gastro-
intestinal endoscopy and duodenal or jejunal
biopsy. Pre-test urine specimens were
collected from patients after overnight fast.
The post-xylose test urine samples (after oral
injestion of xylose) were collected for the
first five hours after administration of D-
xylose. The post-xylose specimens were used
to estimate excreted xylose using colori-
metric and MR methods, independently.
Estimation using colorimetric method is
based on the conversion of xylose to furfural
in an acidic medium and subsequently into
a colored complex whose optical density
(OD) is measured at 520 nm and the
concentrations of the xylose were calculated
using the standard protocol. 4,18 One-
dimensional 1H MR experiments on post-
xylose test urine were performed with
water suppression by presaturation. For
confirming the assignments of the individual
proton signals from D-xylose in urine,19
two-dimensional MR experiments were per-
formed. In vitro experiments were
performed on standard solutions of xylose
in the presence and absence of glucose using
colorimetry and MR under identical
conditions for comparison of the results.
Bacterial Infection
Nicotinic acid metabolism experiments were
performed on the following standard
 High-resolution MR Spectroscopy in Clinical Medicine
bacterial strains: P. aeruginosa (ATCC-25922,
NCTC-10662); E. coli NCTC-10418; ATCC-
25923, K. pneumonia NCTC-9633; ATCC-
13883, Enterobacter ATCC-13048; Acinetobacter
ATCC-19606; Proteus mirabilis ATCC-49565;
Citrobacter ATCC-8090; Enterococcus faecalis
ATCC-19433; Streptococcus gp B ATCC-13813;
and Staphylococcus aureus ATCC-12600.
Bacterial growth media were first prepared
by addition of nicotinic acid (1.1 mg/ml) in
sterile urine and bacteria of about 3 × 107
cfu. The media were divided into two parts
of 1.0 ml each. One part was used for
counting the bacterial cells by growing them
on Mac Conkey agar medium on culture
plates and counting the colonies grown after
incubation. The other part was kept for
incubation at 37oC with shaking at a speed
of 150 rpm, for 6 hours. Subsequently, the
bacterial suspensions were removed and the
supernatant parts were subjected to MR
experiments. MR experiments were also
performed on the bacterial media of
P aeruginosa varying from about 103 to 107
cfu/ml in multiple of 10. Control samples in
triplicate (without bacteria) were also made
under similar conditions and subjected to
MR experiments. Experiments on the urine
samples from 30 patients of P. aeruginosa
infection were performed. Nicotinic acid (1.0
mg/ml) was added to the urine, incubated
at 37oC and the supernatant was taken for
the MR experiments as explained above in
the case of standard bacterial strains. For
unambiguous assignment of the metabolic
product of the nicotinic acid induced by P.
aeruginosa, homonuclear and heteronuclear
two-dimensional experiments were
performed.
Assessment of Liver Graft Function
The blood and urine samples of the patients
undergoing liver graft were collected one
405
day before and at every 24 hrs after the
transplant for a period of 7 days. Blood
samples were centrifuged at 5000 rpm,
20oC and the supernatant was decanted to
obtain the serum. These body fluids samples
were subjected to one dimensional MR
experiments with water suppression by
presaturation. Two-dimensional experiments
were performed for the confirmation of the
assignments made using one-dimensional
spectra. Assessment of the liver graft was
based on biochemical parameters obtained
from conventional method.
RESULTS AND DISCUSSION
Malabsorption Syndrome
Typical spectra of urine sample before and
after xylose-test are shown in Figure 27.1.
Quantity of D-xylose estimated in same urine
specimens was higher with MR than
colorimetric method in patients without
malabsorption syndrome [median 1.58 (0.72
to 3.20) versus 1.03 (0.32 to 1.89) g, p <0.0001,
Wilcoxon’s signed rank test). Xylose
estimated by colorimetry from standard
additions showed a maximum error of 20
percent, while that estimated by MR
showed 7 percent. The quantity estimated
by MR in the presence of glucose showed
8 percent error. MR is less prone to error as
compared with colorimetry in estimating
D-xylose. Quantification of D-xylose using
colorimetry and its potential limitations are
reported in detail in literature. 3,4 It is
reported that the concentrations of the
standard xylose samples estimated using
identical procedure show widely different
results. These varied results have been
attributed to temperature, time and light
dependence of the resultant colored
complex. 3 The results presented here
406 Biomedical Magnetic Resonance: Proceedings of the International Workshop

Fig. 27.1: One dimensional 1H MR spectra of typical (a) pretest and (b) postxylose urine specimen of a patient
with suspected malabsorption syndrome. D - xylose signals of both the isomers (α and β) are clearly seen in
the postxylose urine (figure 1b) which are absent in the pretest urine (figure 1a). The assignments of signals
of α and β isomers are made in figure 1b. The structures of α- and β-D - xylose are shown with the numbering
of all the nonlabile protons.

confirm that colorimetry significantly underestimates the quantity of D-xylose

underestimates quantity of D-xylose
excreted as compared with MR in the same
urine specimens. Colorimetry is less specific
though marginally more sensitive in
diagnosis of malabsorption syndrome, as it
excreted in urine, particularly in those
without malabsorption syndrome.
In the spectra of mixture of D - xylose
and glucose all the signals of α- and β-D -
xylose, except proton 1 from both the
 High-resolution MR Spectroscopy in Clinical Medicine
isomers, overlap with the glucose signals.
Further, proton 1 of β−xylose cannot be used
for quantification for the reason of partial
saturation during water suppression and
hence, in such cases, proton 1 of the α−
xylose at 5.2 ppm is the only signal left for
the quantitative estimation of the total
xylose. Therefore, glucose was estimated
using the ratio of the isomers as 1 : 1.9
which requires area of only proton 1 of the
α isomer. There was no significant difference
between the xylose estimated in the presence
of glucose using the ratio (maximum error
8%) and that estimated using the average
area of the signals (maximum error 7%). On
the other hand, in the colorimetric method
the presence of glucose is shown to interfere
with the quantification of the xylose
concentration leading to misleading results.20
Thus colorimetric method may pose a serious
problem in patients with uncontrolled
diabetics mellitus having glycosuria.
Bacterial Infection
Figure 27.2 shows one dimensional 1 H
spectra of incubated bacterial media of
E. coli K. pneumonia, and P. aeruginosa. In
the spectra of P. aeruginosa media, Nicotinic
acid signals were totally absent and, simul-
taneously, new set of signals were seen.
Using two-dimensional experiments, DQF-
COSY and HMBC, the new set of signals in
the media of P. aeruginosa were assigned to
6-hydroxynicotinic acid (6-OHNA).
Pseudomonas group of bacteria metabolize
NA giving rise to 6-OHNA as follows:
COOH
H2O
H2
N HO
Nicotinic acid 6-Hydroxynicotinic acid
407
Therefore, addition of small quantity
(about 1 mg/ml) of nicotinic acid to the
urine specimen containing the micro-
organism P. aeruginosa yields 6-OHNA after
incubation for about 6 hours.
Analyses of the MR spectra of the
bacterial media with variable cell count of
P. aeruginosa strain showed that the intensity
of nicotinic acid signals gradually decreased,
while, the intensity of the signals of the
6-OHNA increased with increasing number
of bacterial cells. This clearly establishes that
the 6-OHNA produced depends on the
number of bacteria.
In the spectra of E. coli NCTC-10418;
ATCC-25923, K. pneumonia NCTC-9633;
ATCC-13883, Enterobacter ATCC-13048;
Acinetobacter ATCC-19606; Proteus mirabilis
ATCC-49565; Citrobacter ATCC-8090; Enter-
ococcus faecalis ATCC-19433; Streptococcus gp
B ATCC-13813; and Staphylococcus aureus
ATCC-12600 showed that they do not
metabolize nicotinic acid. This suggests that
only P. aeruginosa metabolises nicotinic acid
among the bacteria causing UTI and thus
nicotinic acid metabolism is specific to P.
aeruginosa.
29 out of 30 patients of UTI showed
metabolism of nicotinic acid to 6-OHNA as
observed by the reduction of the intensity
of nicotinic acid signals with concomitant
appearance of 6-OHNA signals. However,
in one of the samples, 6-OHNA could not
be detected under similar conditions owing
to its less concentration. The concentrations
of 6-OHNA determined from the MR
spectra for 29 patients were in good agree-
COOH
ment with that obtained from conventional
method. Thus from a single MR experiment,
N the identity of the bacteria as well as the
viable bacterial count could be obtained.
408 Biomedical Magnetic Resonance: Proceedings of the International Workshop

Fig. 27.2: Parts of 1H MR spectra highlighting the signals of substrate (nicotinic acid, marked as NA) and its
metabolite 6-OHNA. The spectra shown are of sterile urine [marked as “medium”], growth medium [sterile
urine with 1.1 mg/ml nicotinic acid, marked as “medium +NA”] and the remaining ones are in the growth media
after incubation for 6 hours in presence of 107 cfu/ml of E. coli (ATCC-25923, NTCC-10418), K. pneumonia
(ATCC-13883, NCTC-9633) and P. aeruginosa (ATCC-25922, NCTC-10662)
 High-resolution MR Spectroscopy in Clinical Medicine 409

Fig. 27.3: Part of the 1H MR spectra of serum, showing levels of glutamine ( within dotted lines) in mg/dL on
different days: (a) 1 hour before transplantation, (b) 5 hours after transplantation on day 1, (c-f) from day 2 to
day 5 post transplantation, respectively. The numbers on the extreme left denote the corresponding INR, ALT,
AST (in international units) and Lactate (LAC, mg/dL) as observed by routine biochemical methods. ND: not
detected
410 Biomedical Magnetic Resonance: Proceedings of the International Workshop
Assessment of Liver Graft Function
The serum spectra of a typical nonfunctional
liver graft is shown in Figure 27.3. The urine
urea after transplantation continuously
decreased with concomitant increase of
serum glutamine. Increased levels of
glutamine in serum was compatible with
persistent abnormal graft function in-terms
of other parameters such as ALT, AST and
blood lactate levels. Further, reduced urea
levels correlated with rising glutamine
levels. During the study period, the patient
remained normoglycemic. Arterial blood
gases pH varied between 7.34 - 7.53 and
bicarbonate values ranged between 20.1 -
27.6 mmol/L. After day 3 of transplantation,
the urine also showed high glutamine (454
mg/dl). This is the period when the patient
developed PV and HA thrombosis which
continued even after re-exploration till
day 5. Following liver transplantation, the
patient had progressive graft dysfunction,
as evidenced by persistently abnormal INR,
transaminases, blood lactate.
Serum glutamine of blood that was
almost undetectable before transplantation
increased to abnormal levels of 27 - 79 mg/
dL (normal 5 - 10 mg/dL)21 posttransplan-
tation. A simultaneous abnormal excretion
of urinary glutamine posttransplantation
(day 2 - 5) was observed (83 - 455 mg/dL).
High levels of glutamine in both blood and
urine in the patient simulate concordant non
1 9. Koenig C, Tick L, Hanna B. Analysis of the
H MR spectroscopy parameters, which
signify graft failure. In addition, compared
to pretransplant levels in urine, urea level
abruptly decreased posttransplantation and
remained at very low level. This observation
along with rising glutamine levels in serum
and urine suggest impairment in the urea
cycle. In conclusion, the biochemical para-
meters particularly urine urea is a sensitive
and direct indicator of liver function which
can be measured rapidly and noninvasively.
Increase of serum/urine glutamine was
noticed only under progressive dysfunction
of the liver graft. Therefore, urine urea
levels after liver graft may serve as a quick
marker for the graft assessment.
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3. Goodhart JM, Kingston GR. Modification of
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4. Roe JH, Rice EW. A photometric method for
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8. Pappas PG. Laboratory in the diagnosis and
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12. Jones C, McPherson D, Stevens D. Inability of
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Gowda GA, Ayyagari A, Bhandari M, Khetrapal
CL. 1H NMR Spectroscopy in the Diagnosis of
Pseudomonas aeruginosa induced Urinary Tract
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14. Kuntz E, Kuntz HD. Hepatology Principles and
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15. Waniewski RA. Physiological levels of ammonia
regulate glutamine synthesis from extracellular
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16. Suarez I, Bodega G, Fernandez B. Glutamine
synthetase in brain: effect of ammonia. Neuro-
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Nagana Gowda GA, Bhandari M, Khetrapal
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CL. A new dimension of 1H NMR spectroscopy
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try, 6th edition, Gowenlock Ah et al. (eds).
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19. Benesi AJ, Falzone CJ, Banerjee S, Farber GK.
NMR assignements for the aldopentoses.
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20. Kohout P. Small bowel permeability in dia-
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Kralove). 2001;44:101-04.
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Am J Neuroradiol 2001;22:834-37.

Clinical Assessment of
Breast Cancer using 1H MRS at
High Magnetic Field
M Garwood, A McIntosh, PJ Bolan, S Meisamy, CJ Snyder,
A Stycznski, L DelaBarre, J Ellermann, E Gulbahce, TM Tutte,
LI Everson, TH Emory, MT Nelson, D Yee, JT Vaughan
INTRODUCTION
Breast cancer is uncontrolled proliferation
of cells, most of which are believed to arise
from the terminal ductal lobular unit. The
lifetime risk to American women is 1 in 8 to
1 in 7,1. The most obvious risk factors for
breast cancer are female gender and age.
Male breast cancer accounts for only
1 percent cases of breast carcinoma.2 Other
relatively strong risk factors include family
history, BRCA1 and BRCA2 gene mutations,
mantle radiation exposure usually for
treatment of lymphoma, dense breast tissue,
and certain benign breast histologies found
at biopsy, atypical ductal or lobular
hyperplasia, lobular carcinoma in situ (LCIS),
and radial scar.
Breast cancer is a diverse disease of many
varying histologic types, including ductal
carcinoma in situ (DCIS), medullary, cribri-
form, tubular, and mucinous adenocarci-
nomas, with the majority being invasive
ductal carcinoma not otherwise specified
(IDC NOS). These histological subtypes have
28
prognostic importance, with IDC NOS
making up 70 percent of cases with less than
50 percent 10-year survival, while tubular,
mucinous, and cribriform have 10 years
survivals of greater than 80 percent. Nodal
status is the most important factor in
determining prognosis.3 Other important
prognostic factors include tumor size, grade,
hormone receptor status.4
In the Swedish Two County Study, which
began in 1977, Tabar et al showed the
significant positive impact of screening
mammography on breast cancer mortality.5
During the first eight years of the trial,
screening mammography was shown to
decrease breast cancer mortality by
32 percent. Mammography is limited,
however, by its sensitivity which varies from
33-43 percent.6,7 especially in young women
with dense breast tissue. 8 The specificity
ranges from 93-99 percent in high risk
women.6
Ultrasound is another imaging modality
used to determine the nature of breast
 Clinical Assessment of Breast Cancer using 1H MRS at High Magnetic Field
disease. It is often used when there is a
mammographic abnormality, a palpable
lesion, and during pregnancy. This modality
avoids the exposure to ionizing radiation.
The specificity and sensitivity ranges from
80 to 93 and 33 to 50 percent, respectively,
in high risk women.6,7
The imperfect specificity and sensitivity
of these modalities leads to missed cancers
and benign biopsies. Magnetic resonance
imaging (MRI) has been proposed as an
alternative diagnostic to improve screening
performance in high-risk women.9 Addi-
tionally MRI may be used to follow response
to neoadjuvant chemotherapy,10-15 to detect
local recurrences16 and occult primaries,17
and to improve preoperative staging.18,19
Importantly, MRI scanning is noninvasive
and exposure to a strong static magnetic
field in the range typically used for magnetic
resonance scanning (~1-10 Tesla) results in
no known long-term biologic effects. In
addition to providing high-resolution images
of soft tissue anatomy, MRI offers the
possibility to measure multiple physiologic
and functional properties of tissue. The use
of MRI in detecting breast cancer was first
reported in 1985.20
Commonly exploited sources of image
contrast in MRI rely on the existence of
differences in the water relaxation rates
described by the longitudinal and transverse
relaxation rate constants, T 1 and T 2 .
Differentiation based on the uniqueness of
T1 or T2 in different tissues is often sufficient
to allow good separation of normal from
malignant tissues in many parts of the body.
However, standard imaging methods (or
“pulse sequences”) designed to detect
differences in the relaxation rates usually
perform poorly in breast because the T1 and
T2 values of malignant lesions are often very
413
similar to those of normal and benign breast
tissues. Thus, reliable detection of breast
cancer by MRI requires intravenous injection
of a paramagnetic reagent (Gd-DTPA) that
enhances image contrast. 21 Due to the
greater blood volume, leakiness of the lesion
vasculature, and extracellular space, Gd-
DTPA accumulates and thus, shortens T1
more in the lesions than in normal (fibro-
glandular) tissue.
Many studies have shown that contrast-
enhanced MRI offers nearly 100 percent
sensitivity for detecting breast cancer, but
unfortunately its ability to differentiate
benign from malignant breast lesions appears
less impressive, with published values for
specificity varying in the 30 to 90 percent
range. 22 Low specificity is a significant
drawback because ~70 percent of breast
lesions found in the general population are
benign and their detection results in
unnecessary surgical procedures, costs, and
patient anxiety. Although recent reports
support the use of MRI for screening women
with a hereditary predisposition to breast
cancer (e.g., see9,23), the variable specificity
of contrast-enhanced MRI limits its utility
for screening and diagnosing breast cancer
in the general population, despite its
impressive sensitivity.7
A related technique, magnetic resonance
spectroscopy (MRS), offers a noninvasive
way to measure a metabolic profile of the
tumor, providing a “fingerprint” of cancer.
The ability to detect metabolic alterations
noninvasively in cancer cells can be exploited
to enhance our understanding of their
aberrant metabolic and biochemical pro-
perties. Furthermore, the metabolic profile
that can be measured by MRS may provide
information that can be used to improve
the accuracy of diagnosis and may provide
414 Biomedical Magnetic Resonance: Proceedings of the International Workshop
biomarkers for identifying a response to
treatment. From previous studies of cells, it
has been shown that MRS is able to detect
changes in the levels of choline compounds
and lipids during malignant transformation
and progression and in altered resistance
to chemotherapy (e.g, see24-26). By exploiting
the high sensitivity of proton (1H) MRS in
studies of fine-needle biopsy specimens, an
intense signal from total choline-containing
compounds (tCho) can be detected in
invasive cancers that distinguishes them
from benign breast lesions.27
The precise mechanism(s) as to why
neoplastic tissues exhibit elevated tCho
levels remains unanswered. The largest
molecular component contributing to the
tCho signal arising from neoplastic tissue is
typically phosphocholine, a known precursor
of membranes. Thus, the increased tCho level
in neoplastic tissues may be a reflection of
increased membrane turnover by replicating
cells. Elevated tCho may also reflect a
change in the enzyme activities and fluxes
in biosynthetic and catabolic pathways in
which choline compounds serve as both
precursors and catabolites.28,29
Building upon considerable research on
cells, tissues, and cancer models, near the
end of the last decade a few groups were
the first to explore the use of in vivo 1H
MRS to diagnose breast cancer30,31 and to
monitor response to chemotherapy,32 with
highly encouraging results. In testing the
ability of 1H MRS to predict malignancy,
the presence (or absence) of malignant cells
was based on the presence (or absence) of
a tCho signal in the measured spectrum of
the tumor. The MRS data obtained in these
early in vivo studies were acquired at a low
magnetic field by today’s standards (1.5
Tesla). With diffuse and small lesions in
particular, the detection tCho signal is
limited by the signal-to-noise ratio (SNR)
of the MR spectrum. Because SNR increases
at least linearly with magnetic field,33,34 the
ability to detect tCho in diffuse or small
lesions can be improved by using one of
the 3 Tesla MR systems that are increasingly
found in radiology departments these days.
Below we provide an overview of our
recent progress in developing and testing
1H MRS techniques at high magnetic field
(4 Tesla), with the goals of diagnosing breast
cancer and monitoring response to treat-
ments noninvasively.
TECHNIQUES TO LOCALIZE
TUMOR SPECTRA
Several different techniques have been
developed to obtain a spectrum from a
selected tissue region, which in this case is
the tumor. The most common class of
technique that is used to localize spectra in
breast is known as single-voxel spectroscopy
(SVS). With a SVS method, the pulse
sequence performs localization using a
procedure originally developed for MRI to
excite only the 1H nuclei contained within a
chosen planar slice.35 In brief, slice-selection
is achieved by applying a radiofrequency
(RF) pulse in the presence of a spatially-
dependent magnetic field (i.e., a B0 gradient)
that is used to establish a spread of nuclear
precession frequencies along a chosen spatial
axis. When a RF pulse is applied in the
presence of this B 0 gradient, signals are
generated from only those nuclei that
precess in the magnetic field within a certain
range of frequencies; thus, only a “slice” of
nuclei will respond to the RF pulse. With
SVS, a cube-shaped volume or “voxel” is
demarcated using a minimum of three RF
pulses, each of which is applied using a
 Clinical Assessment of Breast Cancer using 1H MRS at High Magnetic Field
different orientation (x, y, or z) of the B0
gradient. SVS methods are designed in such
a way that only the nuclei that experience
all RF pulses (e.g., only the nuclei within
the intersection of the three orthogonal
slices) yield coherent signal. Although all
SVS techniques require at least three RF
pulses and three gradients to demarcate the
volume of interest, the use of more than
three pulses can be advantageous for certain
applications. For studies of breast cancer,
we perform localization with a SVS method
chosen for its ability to provide high quality
1H spectra reproducibly. The technique is
known as localization by adiabatic selective
refocusing (LASER).36 LASER accomplishes
localization with six RF pulses.
An alternative technique to localize MRS
signals is known as chemical shift imaging
or spectroscopic imaging (MRSI). 37,38 A
recent pilot study successfully showed the
feasibility of using 1H MRSI to map the
distribution of tCho in breast. 39 In the
future, MRSI is likely to be the method of
choice for breast studies, provided
remaining technical obstacles currently
limiting the quality of breast MRSI can be
overcome.
OBTAINING QUANTITATIVE
INFORMATION FROM 1H MRS
Several previous studies performed with
single-voxel 1H MRS at 1.5 T have shown
the feasibility of using this technique to
distinguish malignant from benign les-
ions.30,32,40-42 These studies were based on
the hypothesis that tCho is detectable only
in malignancies. A pooled analysis of these
five studies showed that this tCho
detectability criterion can be used to identify
malignancies with 83 percent sensitivity and
85 percent specificity. 43 This qualitative
415
approach is promising, but it is only appli-
cable if the MRS measurement sensitivity is
invariant from subject-to-subject. In similar
studies, we found that the increased sensiti-
vity at 4 T allows detection of tCho in benign
lesions as well as in normal fibroglandular
tissue, and thus, a procedure to quantify
the tCho signal in the breast is required.44
Quantification of metabolite levels is
routinely performed in 1H MRS of the brain,
but it is more difficult to perform in the
breast due to the heterogeneous distribution
of the glandular and adipose tissues.
Although the MR spectroscopist can usually
plan a voxel to include mainly glandular
tissue or tumor, voxels of typical size (1-2
mL) nearly always contain some adipose
tissue as well. The amount of included
adipose tissue can vary greatly depending
on the architecture of the gland and/or
lesion. Although two groups have pre-
viously reported quantification of tCho
levels using external phantom referencing
methods,30,45 this approach is likely to be
error prone since variable adipose tissue
content in the voxels cannot be accounted
for.
The precision in measuring tCho is
limited primarily by SNR. Several factors
affecting SNR change from subject-to-subject.
Examples of factors that vary include voxel
size, partial volume of adipose tissue in the
voxel, distance between the voxel and the
receiver coil, and tissue-induced ohmic
losses. It has been observed that these
factors are responsible for inter-subject SNR
variation on the order of 100-fold in 1 H
MRS studies of breast.44 Thus, there is a
need for quantitative procedures to deter-
mine tCho levels, rather than qualitative
assessment (i.e., presence or absence of tCho
signal), when making a differential dia-
gnosis. In other words, the difference in
416 Biomedical Magnetic Resonance: Proceedings of the International Workshop
tCho signal intensity between malignant and
benign lesions would need to be a factor of
>100 for the qualitative approach to be
reliable for diagnosis, but the difference in
tCho signal is not typically this large.
Therefore, a procedure to calibrate the tCho
signal is required to yield a tCho concen-
tration, [tCho].
The two basic elements of a quantitative
MRS methodology are the referencing
strategy and the spectral fitting technique.
Our preferred referencing strategy uses
water as an internal reference signal.44 This
approach models the breast as a two-
compartment tissue (i.e., lipid and aqueous
fractions), with the assumption that tCho
molecules reside only in the aqueous
fraction. Accordingly, the water referencing
approach compensates for the partial volume
of adipose tissue in the voxel and naturally
leads to a molal concentration (mmol/kg)
for the water-soluble metabolite, tCho. The
fitting technique is based on the TDFD
approach,46 which allows flexible lineshape
definition using a time domain model and
has excellent frequency-selection properties
since the residuals are evaluated and
minimized in the frequency domain. The
ability to select a narrow frequency range
is crucial for fitting weak signals in the
presence of very large ones, as is the case
in breast spectra containing large lipid
signals. Previous studies using this method
have shown that breast malignancies
typically present with [tCho] >1 mmol/kg.44
The ability to estimate the error of the
[tCho] measurement is another advantage
of the quantitative approach. A common
method to estimate the error in quantitative
MRS is based on the Cramer-Rao minimum
variance bound,47 although other approaches
may be better suited to breast MRS, such as
the boot-strap method.48 In the author’s
experience, sometimes the tCho signal is not
detectable or has low SNR due to small
lesion size, deep location, large adipose
tissue content, and/or low cellularity. In
such cases, the MRS measurement may be
indeterminate, in which case the error
assessment must make this known to the
reader. When using MRS for breast cancer
diagnosis, for example, it is important to
know when the [tCho] measurement is
indeterminate since in this case it should
not be a factor in the decision-making
process. The error estimation should provide
an indication of whether a weak or absent
tCho signal is truly reflecting low [tCho] in
the lesion, in which case the lesion is
probably benign, or is caused by a technical
limitation, such as inadequate SNR because
the lesion is too small and/or has a low
aqueous fraction, for example. To achieve
this, a minimum detectable level (MDL) of
[tCho] can be calculated.44 In any measure-
ment in which the MDL is greater than the
threshold [tCho] value that is indicative of
malignancy (>1 mmol/kg water), it is
obvious that the MRS measurement is
indeterminate and as such the information
should not be used in making the diagnosis.
Besides the procedures described above,
research has revealed additional technical
requirements for performing high quality
breast MRS, beyond those commonly
encountered in brain MRS. Firstly, it was
discovered that metabolite peaks such as
tCho are sometimes obscured by the
abundance of mobile lipids which produce
spurious sidebands that interfere with
metabolite signals. To solve this problem,
echo-time averaging was found to be
necessary. 49 Secondly, large shot-to-shot
frequency shifts associated with respiratory
 Clinical Assessment of Breast Cancer using 1H MRS at High Magnetic Field
motion of tissues in the chest and abdomen
occur at high magnetic fields. If not
corrected, these frequency shifts significantly
reduce spectral resolution and increase the
error in assessing the tCho signal.50 Finally,
to obtain high quality spectra of breast, the
RF detector coil must be specifically
designed and optimized for the breast.
With these tools in hand, high quality
1
H spectra of breast lesions can be acquired
fairly routinely. Representative examples are
shown here for two types of benign lesions,
cyst and fibroadenoma, and an IDC. In the
cyst (Fig. 28.1, Plate 19), the tCho level was
too low to be quantified. The simulated peak
(dashed line) provides an estimate of the
tCho signal that would have been necessary
to obtain a reasonably accurate measure-
ment (i.e., the MDL), with an error of ±0.4
mmol/kg in this case. In the fibroadenoma
and IDC (Figs 28.2, Plate 19 and 28.3, Plate
20), tCho was measurable, and the fit of the
tCho resonance and the residual are shown
above and below each full spectrum. In each
case, the tissue location giving rise to the
spectrum shown is indicated by the box
drawn on the image. The anatomical images
depict a slice from the high-resolution 3D
image dataset acquired approximately 5
minutes after Gd-DTPA injection. The Gd-
DTPA enhancement kinetics are para-
meterized using a color overlaid on the
anatomical images. These color-coded maps
are based on the three-time point (3TP)
algorithm described by Degani et al.51 This
method uses images acquired at three time
points–precontrast, immediately post-
contrast, and delayed postcontrast–to
characterize the “wash-in” and “wash-out”
phases of the Gd-DTPA uptake curve. The
brightness of each image pixel is determined
by the amount of enhancement during the
wash-in phase. The color of each pixel is
417
determined by the wash-out phase: pixels
showing decreased signal are colored red,
those with little or no change are colored
green, and those with increased signal are
colored blue.
BREAST CANCER TREATMENT
Treatment of breast cancer has changed
dramatically in the last 40 years, due to
advances in surgical technique, chemo-
therapy, radiation therapy, and radiographic
detection. Throughout the last century,
breast cancer treatment was mainly surgical,
and was based on local control of the
disease. With this local control in mind, most
surgeons performed radical mastectomies,
as popularized by Halsted.52 However, it
became apparent that smaller surgeries could
also achieve excellent local control, and
several large studies showed no survival
benefit to radical surgical excision when
compared to procedures that left the breast
intact.53,54 Local control remains important
as recurrences were substantially reduced
with whole breast irradiation after lumpec-
tomy when compared to women who
received no radiation treatment.55
Once it was understood that initial
surgical technique did not affect overall
survival, additional systemic strategies were
necessary to reduce the risk of recurrence
in distant organs. Both chemotherapy and
hormone therapy had proven to be effective
in women with advanced breast cancer, so
these treatments were applied to women
with respectable disease. Both types of
systemic therapy were effective at reducing
recurrence and death from breast cancer56,57
highlighting the need to provide appropriate
systemic treatment, even in apparently early
stage breast cancer.
Systemic therapy, either hormonal or
chemotherapy, can be given prior to surgery
418 Biomedical Magnetic Resonance: Proceedings of the International Workshop
in women with large tumors.58,59 This type
of “neoadjuvant” therapy can reduce tumor
size prior to surgery. While neoadjuvant
chemotherapy provides no survival
advantage over post-surgical adjuvant
chemotherapy,58 it does allow clinicians to
assess the response of tumor to a particular
drug and increases the number of women
who can receive breast conserving therapy.
Archer et al showed that tumor apoptosis
can be detected almost immediately after
the administration of chemotherapy.60 Thus,
response to specific chemotherapeutic agents
could be identified in patients treated with
neoadjuvant chemotherapy. Armed with the
knowledge that malignant and benign
lesions have differing biochemical profiles,
and that these changes can be detected quite
early, we hypothesized that quantitative 1H
MRS can detect early specific changes in
breast lesions that directly correlate with
apoptosis and subsequent tumor response
to neoadjuvant treatment.
Predicting Response to Neoadjuvant
Chemotherapy using 1H MRS
Primary systemic therapy (PST), also known
as neoadjuvant chemotherapy, given prior
to surgery offers several advantages over
standard post-operative chemotherapy. The
ability to immediately detect response to a
specific choice of therapeutic regimen would
be ideal, since it would allow for optimal
individualization of the regimen with
improved likelihood of achieving a
pathologic complete response. With the
above MRS tools in hand, we performed a
study designed to determine whether early
changes in [tCho] could provide a biomarker
of clinical response as soon as 24 hours after
the first dose of doxorubicin-based PST for
locally advanced breast cancer.61 Of the first
13 patients who successfully completed the
protocol without technical problems, the
change in [tCho] between baseline and 24
hours after the first dose of PST showed
significant positive correlation with the
change in lesion size measured at the end
of four cycles of PST (R = 0.79, p = 0.001).
The change in [tCho] within 24 hours was
significantly different between responders
and nonresponders (p = 0.007), based on
RECIST criteria.62 These results suggest that
the change in [tCho] within 24 hours after
the first dose of PST can serve as an early
indicator for predicting clinical response to
doxorubicin-based chemotherapy in locally-
advanced breast cancer. A representative
set of MR data from an objective responder
is shown here (Fig. 28.4a, Plate 20 and 28.4B
and C, Plate 21 and 28.4D, Plate 22).
FUTURE PROSPECTS
With the increasing availability of high field
(>3 Tesla) systems and improved
methodology, spectral quality can be
significantly better than that of previous
years. It is clear that high field MR scanners
will have an increasing role in the clinical
assessment of cancer in the near future.
From our experience, we feel that now is
the appropriate time to distribute the latest
technology and to begin multicenter trials
to evaluate the use of MRS as an adjunct to
MRI in diagnosing breast cancer and for
assessing response to primary systemic
therapy in locally advanced breast cancer.
ACKNOWLEDGMENTS
This research was supported by the DOD Breast
Cancer Research Program DAMD 17-01-1-0331, NIH
grants RR08079, CA92004, CA94002, RR00400, PHS
Cancer Center Support Grant P30 CA77398, the
Tickle Family Land Grant Endowment in Breast
Cancer Research, and the Lillian Quist-Joyce Henline
Chair in Biomedical Research.
 Clinical Assessment of Breast Cancer using 1H MRS at High Magnetic Field
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 MR Applications in Experimental and Animal Model Systems 423

29
MR Applications in Experimental
and Animal Model Systems
Kurt V Schenker

INTRODUCTION MRI takes a rather unique position when

Imaging techniques such as magnetic
resonance imaging (MRI), nuclear
tomographic imaging or X-ray computed
tomography have become indispensable in
clinical use. Applications of these techniques
in biomedical and pharmaceutical research
have become equally vital and its significance
in many research fields has grown
considerably over that last few years.1 This
is in particular true for MR methods. The
main driving forces are the advances in
instrumentation and methodology, and the
increasing availability of (transgenic) animal
models.
comparing the different imaging modalities.
Table 29.1 collates some relevant properties
of various imaging techniques. MRI covers
the widest range of applications and has a
number of distinct advantages over other
techniques: Excellent image resolution and
contrast behavior, gives access to many
physiological, functional and metabolic
parameters, mostly noninvasive, multi-
parametric character, scalable from mouse
to man and from basic research to clinical
use. There are also disadvantages, mainly
the inherent low sensitivity. The choice of
imaging modality depends primarily on the

Table 29.1: Comparing key characteristics of animal imaging techniques

Technique Spatial Penetration Time Target

resolution depth resolution applicationc)
MR 10-100 μm no limit msec-min A, P, M
CT 50 μm no limit sec-min A, P
Ultrasound 50 μm 5-50 mm min A, P
Nucleara) 1-2 mm no limit min P, M
Opticalb) 1-3 mm 5-50 mm sec-min P, M
a). PET (positron emission tomography), SPECT (single photon emission computed tomography).
b). FMT (fluorescence-mediated molecular tomography), FRI (fluorescence reflectance imaging), GFP (green
fluorescence protein), NIR (near infrared imaging).
c). A: Anatomical/structural imaging, P: Physiological/functional imaging, M: Molecular imaging.
424 Biomedical Magnetic Resonance: Proceedings of the International Workshop
specific question, and different imaging
techniques are in general complementary
rather than competitive.
In this article a number of animal
research MRI and MRS applications are
presented. It seems not an easy task to make
this a representative overview, however.
Looking at the current literature and from
personal experience one still can derive a
few generally valid trends:
• The number of MRI/MRS publications
based on small animal work (mouse, rat,
etc.) is about three times larger compared
to work based on larger animals (rabbit,
cat, dog, monkey, etc.). The main reasons
are that more small animal models are
available, in particular using transgenic
animals, the better cost efficiency and
lower cost for instrumentation. This ratio
shifts to about nine to one in favor of
the small animal applications when
looking at academic and industrial users
and include unpublished work.
• MR spectroscopy applications account for
not more than a few percent of all animal
MR applications. The clear dominance of
MR imaging may surprise since in vivo
MR spectroscopy has been praised as an
outstanding tool to gain new insights into
physiology for more than twenty years.
Nevertheless, MRS seems to catch up
lately as higher magnetic fields become
available and affordable, and newer
instrumentation and methodology make
the application of MRS protocols easier
and more promising.
• 1H MRI/MRS applications outnumber
heteronuclear applications even more.
The low sensitivity in heteronuclear
experiments is clearly the limiting factor.
Again, technological advances in instru-
mentation are helping here a lot and
researchers become increasingly
interested in heteronuclear applications,
primarily with the nuclei 3He, 13C, 19F,
23
Na, 31P, and 129Xe.
• MRI and MRS plays its most important
role in the wide field of neurosciences.
This has been the case since the early
days of MRI. With advancing technology
and methodology other foci have arisen,
in particular applications to the cardio-
vascular system. MRI and MRS have
proven to be a valuable tool in almost
any other area of life sciences, too, e.g.
muscle physiology, cancer research,
phenotyping and screening of animals,
toxicology, nutrition science, etc.
• In the pharmaceutical industry, i.e. in the
drug discovery and development process,
MR methods have become established
tools from target identification to clinical
trials, fulfilling the “bench-to-bedside”
model. MR techniques can contribute
greatly to a reduction in costs and deve-
lopment time for new drugs.2,3
The examples presented below shall
highlight a number of facets of MR techni-
ques, its animal models and how they can
contribute to a better understanding of life.
The reader shall bear in mind that this collec-
tion of applications is somewhat arbitrary,
many aspects will be alluded, many more
will remain untouched.
DIFFUSION, fMRI AND METABOLIC
PROFILE IN TRANSGENIC MICE WITH
CEREBRAL AMYLOIDOSIS: AN
ALZHEIMER’S STUDY4,5,6
Alzheimer’s disease pathology correlates
strongly with the formation of cerebral
amyloidosis. However, it is not clear how
amyloid formation compromises brain
function and leads to dementia. MR imaging
 MR Applications in Experimental and Animal Model Systems
and spectroscopy experiments with
transgenic mice may address different
aspects of the pathophysiology in
Alzheimer’s disease in a noninvasive way.
The transgenic mouse lines APP23 and
PS2APP express high levels of amyloid
precursor protein leading to amyloid
plaques, mimicking the pathophysiologic
processes in Alzheimer’s disease.
The transgenic mouse line APP23 has
been used for a brain MR diffusion study,4
looking for correlations between amyloi-
dosis and changes in the apparent diffusion
coefficient (ADC) in different brain areas.
Quantitative functional MR imaging was
applied to characterize brain function using
the same transgenic mouse line to evaluate
the age-dependent impairment of
somatosensory response.5 Transgenic mice
of the line PS2APP were observed through-
out their life-span in search of morphologic
and metabolic changes.6
The diffusion and functional MR
experiments were performed on a 7 Tesla,
16 cm bore MRI system (BRUKER BIOSPEC
70/16), the 1H MR spectroscopic experiments
were done on a 4.7 Tesla, 40 cm bore MRI
system (BRUKER BIOSPEC 47/40).
Restriction of Diffusion by Amyloidosis4
The purpose of this study was to assess the
ADC of water in brains of APP23 transgenic
mice using diffusion-weighted MR imaging
(DWI) followed by immunohistochemical
analysis of cerebral amyloidosis. DWI is
sensitive to microscopic motion of water
molecules and can detect disturbances in
ion and water homeostatsis by measuring
the ADC in tissues.
The basic pulse sequence used for DWI
is depicted in Figure 29.1, typically a normal
spin-echo sequence to which a pair of
425
diffusion encoding magnetic field gradients
G D is added. The signal contribution of
mobile water spins will be dephased as they
move along the gradient direction. If this
motion is incoherent the two diffusion
gradient lobes will lead to a net signal
attenuation. For stationary spins the effects
of the two gradient lobes will compensate,
i.e. their net signal contribution will be
unaffected by the diffusion gradients. The
signal attenuation can be quantified as
S(b)/So = exp{-b × ADC}
where S o refers to the pixel intensity
obtained without diffusion gradients and
S(b) to the intensity obtained with a pair of
diffusion gradients of strength g, duration
δ and time separation Δ. Then b is given by
b = (γ × g × δ)2 × (Δ - δ/3)
γ being the magnetogyric ratio. If the DWI
experiment is repeated for a number of
different b values (five in this study), ADC
values can be determined for each individual
image pixel, rendering an ADC map.
Fig. 29.1: Pulse sequence for spin-echo diffusion-
weighted MR imaging: A 90° excitation and 180°
refocusing RF pulse generate a spin-echo signal SE,
spatial encoding is obtained by standard slice, phase
and read gradients (GS, GP and GR). The diffusion
encoding (GD) is obtained by a pair of gradients of
equal strength g and duration δ, applied before
and after the refocusing RF pulse, separated by the
time Δ.
426 Biomedical Magnetic Resonance: Proceedings of the International Workshop
Figures 29.2A to C show images of the
mouse brain acquired with a DWI pulse
sequence. The echo time was TE = 50 ms,
the repetition time TR = 1325 ms, the
resolution was 128 x 64 pixels with a field-
of-view of 25.6 x 25.6 mm2, i.e. an in-plane
resolution of 200 μm × 400 μm, which is
typically sufficient for DWI applications and
also allows to keep the scan time short. The
total acquisition time for the five b values
measured to produce the ADC maps
represented in Figure 29.3, Plate 22 was
7 minutes. The slice thickness was 1 mm,
the inter-slice gap 0.6 mm. The diffusion
encoding gradient was the applied along
the slice direction, i.e. the experiment is
sensitive to the incoherent motion
component of water perpendicular to the
image plane. Animals were anesthetized
with isoflurane, intubated and artificially
ventilated. This helps to avoid motion
artefacts to which DWI sequences are rather
sensitive. For further data processing
regions-of-interest (ROI) were defined: ROI
1 for the lateral and medial caudate
putamen, ROI 2 for the neocortex,
additionally subdivided into medial cortex
(2a), dorso-lateral cortex (2b), and ventro-
lateral cortex (2c).
The color-coded ADC maps in Figure
29.3, Plate 22 show each a single slice of a
Figs 29.2A to C: Sections through the mouse brain with regions-of-interest (ROI) selected for quantitative
data analysis indicated (see text). Courtesy M. Rudin, Novartis Inst. for Biomedical Research, copied from.4
6-month-old wild-type (control) mouse (A),
a 6-month-old transgenic mouse APP23 (B),
a 25-month-old wild-type mouse (C) and a
25-month-old transgenic mouse APP23 (D).
The color red encodes high diffusivity, blue
encodes a reduced diffusivity. The images
reveal a substantial ADC decline in the
medial and dorso-lateral cortex of the aged
APP23 mice compared to the age-matched
control mouse or young APP23 and control
animals. In the dorso-lateral cortex of the
6-month-old APP23 mouse few areas with
reduced ADC are visible.
One day after the DWI experiments mice
were sacrificed and brains were prepared
for immunohistochemistry. APP23 mice
revealed robust compact amyloid-β plaques
in the neocortex but only few and diffuse
amyloid in the caudate putamen. No
amyloid deposition was detected in
6-month-old APP23 mice and wild-type
control animals (Fig. 29.4, Plate 23).
Observations suggest that the ADC
reduction in the neocortex of the aged APP23
mice is related to the amyloid deposition in
the brain of these animals. However, ADC
values were not reduced in all cortical areas
containing amyloid. For example, the ventro-
lateral part of the neocortex did not show
a reduction in ADC although this region
exhibited a significant amyloid load.
 MR Applications in Experimental and Animal Model Systems
Interestingly, this brain region develops
more diffuse amyloid compared to the
medial and dorso-lateral parts of the
neocortex. Thus, one may argue that not
only the amount of amyloid deposition but
also its nature (compact versus diffuse)
determines diffusivity within cerebral tissue.
AGE-DEPENDENT IMPAIRMENT OF
SOMATOSENSORY RESPONSE IN
APP23 MICE5
Functional MR imaging (fMRI) methods are
suited to measure neuronal function non-
invasively. fMRI probes secondary changes
associated with neuronal activation such as
regional changes in cerebral blood volume
(CBV), or cerebral blood flow (CBF), or a
change in the ratio of oxygenated versus
de-oxygenated hemoglobin (blood
oxygenation level dependent contrast,
BOLD contrast). The goal of these
experiments was to assess the functional
response in distinct areas in the
somatosensory cortex of transgenic APP23
mice at different ages by using the CBV-
fMRI method upon electrical stimulation of
the animal’s paw.
Intravascular paramagnetic contrast
agents that maintain a steady-state blood
concentration permit to measure relative
CBV values with a good temporal
resolution. Neuronal activity leads to an
increase in CBV, i.e. a larger amount of
paramagnetic contrast agent in the
respective brain area. The relevant effect of
the paramagnetic contrast agent is to reduces
the T2 relaxation time. Hence a T2 sensitive
pulse sequence will lead to a decrease of
the signal intensity. If Spre, S0 and S(t) denote
the signal intensity before and after
administration of the contrast agent and
during the fMRI experiment, respectively,
427
we can express the activation induced
percentage change in CBV as
ΔCBV%(t) = ln {S(t)/S0}/ln{S0/Spre} × 100
Animals were anesthetized with isoflu-
rane and actively ventilated. As a contrast
agent 50 mg/kg ENDOREM™ (ferumoxides,
11.2 mg Fe/ml; Laboratoire GUERBET,
Roissy, France) was injected intravenously.
Twenty minutes after injection the plasma
concentration of ENDOREM has reached a
steady state, the plasma half-life was
measured to be 3.5 hours. A single slice
including the somatosensory cortices was
recorded in the fMRI studies using a fast
spin-echo sequence (see Figs 29.5A and B).
The sequence parameters were: TR = 1135
ms, inter-echo time TE = 6.7 ms, effective
echo time TEeff = 80 ms, echo collection factor
was 32. The field-of-view was 25.6 × 25.6
mm2 with a resolution was 128 × 128 pixels,
the slice thickness was 1 mm. With four
averages the acquisition time per image
amounted to 21 s. Moreover, high-resolution
anatomical MR images were acquired onto
which the colored fMRI activation maps
were superimposed (see Figs 29.6A to C,
Plate 23).
For somatosensory stimulation pairs of
small needle electrodes were implanted
subcutaneously in each hind paw. Electrical
stimulation was performed by using constant
current pulses with 0.5 ms pulse length at a
frequency of 1.5 Hz. The stimulation
paradigm consisted of a resting period of
105 s followed by a stimulation period of
105 s. This basic module was repeated three
times with progressive current strength of
0.5 mA, 1.0 mA and 2.0 mA.
Figure 29.5 shows statistically derived
ROIs overlaid on the spin-echo images
applying a correlation threshold of r>0.3 and
a minimum cluster size of 10 pixels. The
428 Biomedical Magnetic Resonance: Proceedings of the International Workshop
Figs 29.5A and B: Statistically derived ROIs overlaid
on spin- echo images 0.96 mm posterior to the
Bregma for a 25-month-old control (A) and an age-
matched APP23 mouse (B). Courtesy M. Rudin,
Novartis Inst. for Biomedical Research, copied from.5
ROIs are consistent with the known
topographic location of the murine hind limb
region within the primary somatosensory
cortex S1. In these ROIs transient signal atte-
nuations upon stimulation of 4 to 6 percent
have been observed, depending on the
amplitude of electrical current applied.
For quantitative analysis of fMRI response
three ROIs have been defined (see Fig. 29.6
B, Plate 23): (1) the active ROI comprising
the S1HL region contralateral to the
stimulated hind paw as obtained from the
statistical map (see Fig. 29.6A, Plate 23),
(2) the equivalent topographical location on
the ipsilateral side, and (3) a ROI comprising
ipsilateral hippocampal structures used as a
signal reference. For each stimulation period
the CBV increased within the first images
after onset, peaked at ~60 s, and declined
at the end of the stimulation period.
Maximal ΔCBV percent amplitudes for the
individual periods were 11.0 ± 1.0%, 13.6 ±
2.0%, and 17.7 ± 2.4% for electrical current
strength of 0.5 mA, 1.0 mA, and 2.0 mA,
respectively, i.e. CBV changes in the
activated areas increased with increasing
current amplitude. No activation was
observed in the S1HL region ipsilateral to
the stimulated paw as well as in the
reference ROI.
Figure 29.7 shows the temporal profile
of Δ CBV percent changes in the S1HL
contralateral region for 6-, 13-, and 25-
month-old control and APP23 animals.
Analysis of maximal amplitudes for 6-month-
old wild-type control mice revealed ΔCBV
percent values for the individual periods of
8.4 ± 2.0%, 11.5 ± 2.0%, and 17.6 ± 2.0% of
the baseline values. The corresponding
values for 13 months and 25-month-old
control animals were 6.7 ± 1.0%, 10.7 ± 1.0%,
and 15.2 ± 2.0% and 7.6 ± 2.0%, 12.6 ± 2.0%,
and 14.6 ± 2.0% for electrical current strength
of 0.5 mA, 1.0 mA, and 2.0 mA, respectively.
There was no significant dependence of the
fMRI response on the age of wild-type
animals.
 MR Applications in Experimental and Animal Model Systems 429

In APP23 mice of age 6 and 13 months

ΔCBV percent values were identical within
error limits to those of age-matched control
animals for stimulation current amplitudes
of 0.5 mA and 1.0 mA. At 2.0 mA stimulation
the CBV response in transgenic mice was
slightly weaker as compared with wild-type
animals, yet these decreases were not
statistically significant (see. Figs 29.7A and
B). In 25-month-old APP23 mice the CBV
did not increase with increasing stimulation
current, with maximal ΔCBV percent values
of 7.9 ± 1.0%, 8.0 ± 1.0%, and 6.7 ± 1.0% for
stimulation current amplitudes of 0.5 mA
and 1.0 mA. At 2.0 mA, respectively (see
Fig. 29.7 C).
The on-line monitoring of trans-
cutaneously measured blood carbon dioxide
(PtcCO 2 ) values over the course of the
experiment (see Fig. 29.7 D) for 25-month-
old mice revealed a slight increase in PtcCO2
of the order of 3.5 mmHg for both animal
groups. However, no significant difference
in PtcCO2 between control and APP23 mice
or between stimulation and resting periods
have been detected. This rules out the
possibility that the observed differences are
attributed to hyper- or hypo-capnia.
In conclusion, these experiments have
demonstrated that amyloidosis in APP23
mice leads to an impaired hemodynamic
response in the somatosensory cortical area
which can be assessed quantitatively by
Figs 29.7A to D: Time course of ΔCBV percent
using fMRI in combination with a
calculated from the contralateral ROI defined in Fig.
standardized sensory stimulus. This 29.5 B for 6 months (A), 13 months (B), and 25 months
functional deficit becomes more severe with (C) old APP23 and control mice. The bars at the
increasing age of the animals and with bottom of the panels represent the stimulation periods
increasing amplitude of the stimulation of 105 s duration with progressive stimulation current
current. Two factors might contribute to the amplitudes of 0.5 mA, 1.0 mA, and 2.0 mA. Data are
given as mean ± SEM. On-line monitoring of blood
compromised hemodynamic response: An carbon dioxide (PtcCO2) of 25-month-old control and
impaired neuronal excitability because of APP23 animals is shown in (D). Courtesy M. Rudin,
amyloid related neurodegenerative Novartis Inst. for Biomedical Research, copied from.5
430 Biomedical Magnetic Resonance: Proceedings of the International Workshop

processes, in particular loss of synaptic
connectivities, and/or severe cerebral
amyloid angiopathy (CAA) compromising
the vascular reserve capacity.
ALTERED METABOLIC PROFILE IN
THE FRONTAL CORTEX OF PS2APP
TRANSGENIC MICE6
Metabolic profiling in human Alzheimer’s
disease patients by MR spectroscopy (MRS)
revealed a decrease of N-acetyl-aspartate
(NAA), indicating a neuronal loss. An
increase of the inositol MRS signal has also
been reported for patients with probable
Alzheimer’s disease and a transient recovery
of NAA in patients treated with donepezil
has been described. Based on these findings
in humans 1 H MRS experiments with
PS2APP transgenic mice as an Alzheimer’s
disease model were performed. Animals
were anesthetized with isoflurane and
positioned on a cradle with their heads
immobilized in a stereotaxic holder. A
72 mm diameter birdcage coil was used for
RF excitation, and an actively decoupled
surface coil positioned on the head of the
animal for MR signal reception.
The sensitive voxel for localized 1 H
spectroscopy was selected in the frontal
cortex and was positioned on a sagittal and
horizontal scout image acquired for each
animal (see Fig. 29.8). Great care was taken
to reposition the voxel always at the same
location fully within the brain, and to avoid
the ventricles. The size of the voxel was 1.7
× 1.7 × 3.4 mm3, the sensitive volume was
10 μl.
A STEAM pulse sequence with the
VAPOR water suppression scheme was
employed to acquire the localized 1H spectra.
The sequence parameters were: TE = 8 ms,
TM = 7 ms, TR = 2000 ms, number of
averages 1024, leading to a total scan time
of 35 minutes. Spectra shown in Fig. 29.9
have been acquired in a 20-month-old
PS2APP mouse and an age-matched control
mouse. The major metabolites NAA, creatine
(Cr), choline (Cho), taurine (Tau) and inositol
(Ins) can be readily identified. The typical
line width of all spectra was 18 Hz (0.09
ppm).
In view of the limited signal-to-noise
ratio of MR spectroscopy, it is important to
assess the reproducibility of the measure-
ment method. Therefore, 9 animals approxi-
mately 22 months old were subjected to two
measurements each about one month apart.
The NAA concentration measured in
individual animals is shown in Figure 29.10.
A large variability between individual

Figs 29.8A and B: Voxel position for localized 1H MR spectroscopy in frontal cortex,
defined in sagittal and horizontal image planes. Voxel size is 1.7 x 1.7 x 3.4 mm3, voxel
volume is 10 µl. Courtesy M. von Kienlin, Hoffmann-La Roche Ltd, copied from.6
 MR Applications in Experimental and Animal Model Systems 431

Figs 29.9A and B: Localized 1H spectra from a 10 µl voxel as defined in Fig. 29.8 of 20-month-old mice: (A)
from a control wild-type mouse, (B) from a transgenic PS2APP mouse. The main metabolites are: N-acetyl-
aspartate (NAA, 2.0 ppm), creatine (Cr, 3.0 ppm), choline (Cho, 3.2 ppm), taurine (Tau, 3.4 ppm), inositol (Ins,
3.8 ppm), glutamate and glutamine (Glu and Gln, 2.0-2.5 ppm), lactate (Lac, 1.3 ppm) and lipids (Lip, 0.8-1.8
ppm). Courtesy M. von Kienlin, Hoffmann-La Roche Ltd, copied from.6

Fig. 29.10: The NAA concentration measured twice

in 9 animals at approximately a one month interval
shows the good reproducibility of localized 1H MRS
assessment in individual animals. The coefficient of
variance estimated for NAA is 6.9 percent. The four
animals with the highest NAA signal were controls
(open symbols), the five with the lowest NAA signal
were PS2APP mice (solid symbols). Courtesy M. von
Kienlin, Hoffmann-La Roche Ltd, copied from.6

animals is evident, with NAA
concentrations, which ranged from 6 to 12
mM. Within the measurement interval of
one month the value measured for NAA in
each animal remained very stable. The four
animals with the highest NAA concen-
trations were controls, the five with the
lowest NAA concentrations were PS2APP.
The main question to be addressed was
whether some significant difference could
be detected in the metabolic profile of the
animal in the end stage of the pathophysio-
logical process. The box charts in Figure
29.11 illustrate the findings for four major
cerebral metabolites in 24-month-old mice.
In the PS2APP mice, there is a statistically
significant decrease of both the NAA/Cr
(P<0.01) and Glu/Cr (P<0.002) ratios com-
pared to control animals. No significant
difference was detected for any of the other
metabolites.
For the analysis of the cortical plaque
load, thioflavin-S staining was performed
in the 24-month-old PS2APP mice directly
after the last MRS session. The same areas
of the frontal cortex as those investigated
432 Biomedical Magnetic Resonance: Proceedings of the International Workshop

Fig. 29.11: Box-plot of the main metabolites measured in 24-month-old PS2APP (hashed, n=12) and control
mice (open squares, n=11). Both NAA and Glu are significantly lower in the PS2APP mice, no significant
differences were found for Ins or Gln. Courtesy M. von Kienlin, Hoffmann-La Roche Ltd, copied from.6

by MRS were stained and documented. The
12 PS2APP brains analyzed revealed a high
inter-individual variability resulting in
significant differences in amyloid plaque
load. Also the MRS analysis showed
astonishingly high variability of metabolite
levels within the PS2APP mouse group. In
order to investigate whether these variations
are predictive for the terminal plaque
pathology, the metabolic profiles were
correlated with the histological data. The
markers inositol, glutamine, taurine and
choline did not show any trend or
correlation with plaque load. The neuronal
marker NAA and glutamate showed a
negative correlation with plaque-covered
area, the former reaching significance
(P<0.05).
The main observation of this study is
the reduction of NAA and glutamate in
PS2APP mice. This finding is consistent with
the general view of a loss of functional
neurons in Alzheimer’s disease. Figures
29.12A and B shows the NAA/Cr and Glu/
Cr ratios throughout the life-span of
PS2APP and control mice from age 4 to 24
months. In control animals the NAA/Cr
ratio is stable around a mean value of 0.96
(see Fig. 29.12 A). In the PS2APP group,
however, there is a trend towards reduced
NAA starting at 16 months. This trend
reaches significance at 20 months, and
continues further at 24 months. Linear
regression of the evolution starting at
12 months yields a reduction of NAA/Cr
of –17% per year in the PS2APP mice,
compared to –4.3% in controls. The
evolution of glutamate shows a similar
pattern: In the control group the Glu/Cr
remains more or less stable with an average
value of 1.42, while there is a more
pronounced decline in the PS2APP mice at
older age. Computation of the slope of linear
decline starting from age 12 months yields
 MR Applications in Experimental and Animal Model Systems
Figs 29.12A and B: Temporal evolution of (A) NAA/
Cr and (B) Glu/Cr over time for control mice (squares)
and PS2APP mice (circles). While these metabolites
remain stable during the first 12 months of the animals
life-span, both drop significantly in PS2APP mice in
the subsequent 12 months. Courtesy M. von Kienlin,
Hoffmann-La Roche Ltd, copied from.6
a loss of –9.9 percent per year in the controls,
while it is –35 percent per year in the
PS2APP mice.
QUANTITATIVE BODY COMPOSITION
ANALYSIS BY T2 MR RELAXOMETRY7
The purpose of the experiments described
here was to quantitatively measure the body
composition, i.e. the fat and lean mass in
mice and rats, in an obesity study. The
433
experiments are based on T 2 relaxation
measurements of the fat and water protons.
No imaging or spatial resolution is involved.
The measurements are astonishingly simple
yet have proven to be extremely efficient,
accurate and reproducible.
Five animal groups were scanned in order
to evaluate the effects of different feeding
schemes and two appetite suppressants. The
experiments were performed on a 4.7 Tesla,
40 cm bore MRI system (BRUKER BIOSPEC
47/40) with awake animals, 700 mice and
400 rats.
A multi-echo CPMG pulse sequence
represents a most efficient means to
determine T2 relaxation times. Figure 29.13
depicts the sequence employed to acquire
the data presented here: A 90° excitation
pulse followed by 256 refocusing pulses
(180°) of 200 μs and 400 μs duration,
respectively, generating 256 spin-echoes. The
inter-echo delay was chosen TE = 2.5 ms
allowing to follow the T2 decay over a time
window of 640 ms (= 256 × 2.5 ms). Two
averages with a repetition time of TR = 10 s
have been acquired. The pulses were of
rectangular shape and no slice, read nor
phase encoding gradients were switched
making the sequence completely silent. Due
to the short inter-echo interval the sequence
is highly insensitive to motion of the
animals.
The whole animal preparation and MR
data acquisition took less than two minutes
per animal. All MR relaxometry experiments
could be carried out with non-anesthetized
mice and rats thus avoiding any undesired
effects potentially interfering with the goal
of the study. The animal throughput was
between 30 and 40 animal per hour.
The whole procedure for a series of
measurements involved the following steps:
434 Biomedical Magnetic Resonance: Proceedings of the International Workshop
Fig. 29.13: CPMG pulse sequence used for T2 MR
relaxatiometry, i.e. the determination of T2 decay of
fat and water protons. A 200 µs excitation pulse (exc)
is followed by 256 refocusing pulses (ref n) of 400 µs
duration, rendering 256 spin-echoes (SE n).
• Automatic shimming of the magnetic field
with FASTMAP using a tap water filled
phantom once per day.
• Measurement with a reference phantom
before and after a series of experiments
for the purpose of calibration and
verification of instrument performance.
• Manual tuning and matching of the RF
coil for each animal or sample.
• Automatic RF pulse power adjustment
for every animal or sample.
• Acquire data with the CPMG multi-echo
sequence.
The quantification of the body
composition was achieved by analysis of T2
distributions. Only the maximal amplitude
points of each spin-echo of the CPMG signal
train were extracted. Plotting these
amplitude points versus TE time renders a
signal decay envelope as depicted in Figure
29.14 A. An inverse Laplace transformation
was applied to the 200 time domain data
points in the echo time range 10–400 ms.
The resulting relaxograms from in vivo and
phantom studies always showed two fully
separated peaks at approximately 40 ms and
230 ms (see Fig. 29.14 B) which have been
assigned to tissue water and fat, respectively.
Integration of the two peaks yielded a
quantitative measure of the signal contri-
butions of the two constituents.
Figs 29.14A and B: (A) Envelope of a typical signal
train obtained from a rat submitted to whole-body 1H
MR relaxometry using the CPMG multi-echo
sequence shown in Fig. 29.13. (B) T2 relaxogram
resulting from the signal train depicted in (A) after
inverse Laplace transformation. The two peaks at T2
relaxation times of approximately 40 ms and 230 ms
represent tissue water and body fat, respectively. The
area under the peaks is proportional to the total
number of protons in each constituent. Courtesy B.
Kuennecke, Hoffmann-La Roche Ltd, copied from.7
The absolute quantification of the
measurements is based on phantom
experiments. Lean tissue was simulated with
a 1% agar agar gel supplemented with 20
mM CuSO4, resulting in an average T2 of
the water protons of 45 ms. A plain 0.75
percent agar agar gel rendering a water
proton T2 of 230 ms was used to model the
fat. Furthermore, the actual coil sensitivity
varying with different coil loading was
accounted for by multiplication with the
transmitter voltage required for the 90°
excitation pulse. In the context of this study
 MR Applications in Experimental and Animal Model Systems 435

lean tissue (or fat-free mass) designates the

sum of water and dry matter and it was
assumed that lean mass and water content
are strongly correlated and hence body
weight (BW) may be expressed in terms of
fat and water contents alone
BW = fat + β × water
where β represents the lean mass-to-water
ratio. Rewriting this equation in terms of
MR signal intensities S fat and S water and
proton densities 1Hρfat and 1Hρwater of fat and
water leads to
BW=αinst× (1Hρfat × Sfat + β × 1Hρwater × Swater)
with αinst representing the general sensitivity
of the MR instrument. This concept allows
a self-calibration of the fat and water signals
to be performed by means of multiple
regression analysis of body weights and fat
and water signal intensities obtained from
cross-sectional analysis of larger cohorts.
Figure 29.15 A shows a correlation of
body weights (x-axis) with the fat and lean Figs 29.15A and B: (A) Cross-sectional body
masses and their sum (y-axis) as determined composition analysis data from 450 rats, correlation
by MR relaxometry. This calculated body between actual body weight and the relaxometrically
weight (sum of fat and lean mass) is highly determined fat and lean mass and their sum. (B)
correlated (r>0.986) with the actual body Correlation between relaxometrically determined fat
and lean mass. Courtesy B. Kuennecke, Hoffmann-
weight. The correlation between lean mass
La Roche Ltd, copied from.7
and body weight or fat mass and body
weight is significantly lower. Notably, there
is almost a complete lack of correlation
between fat and lean mass in individual single awake mouse and rat, respectively.
animals as can be seen from Figure 29.15 B. The coefficients of variance for water and
The reproducibility of the relaxometric fat were 2.5 percent and 2.0 percent for the
measurements was tested with agar agar mouse and 1.7 percent and 1.2 percent for
phantoms. A coefficient of variance better the rat. The linearity of relaxometric
than 0.25 percent was achieved for ten determination of body composition was
consecutive experiments. Long-term tested with phantoms with variable contents
reproducibility tested over half a year of agar agar gels. Linearity was assessed
rendered a coefficient of variance better than up to 525 g of MR signal producing mass
5 percent. The reproducibility under in vivo which corresponds to an animal weight of
conditions was determined from repeated 650 g. Correlation of water and fat mass
measurements taken over a day from a with the respective signal integrals in the
436 Biomedical Magnetic Resonance: Proceedings of the International Workshop

relaxograms showed excellent linearity for

both components (r>0.998).
To study the effect of centrally-acting
appetite suppressants sibutramine
(MERIDIAN™, Knoll, Germany) and
dexfenfluramine Sprague-Dawley rats of
3 weeks of age were fed ad libitum with a
high caloric diet. After an initial growth
phase of 10 weeks the 50% of the animals
with the largest body weight were
randomized into five groups (n = 9).
Thereafter, group I was continued to be
fed with high fat diet ad libitum and treated
with placebo (tap water). Group III and IV
had also unlimited access to high fat diet
but in addition received daily doses of
3 mg/kg p.o. of appetite suppressants
(sibutramine for group III, dexfenfluramine
for group IV). Group V was also treated
with dexfenfluramine akin to group IV but
with a daily dose of 10 mg/kg. The animals
Figs 29.16A to C: Longitudinal study of body
of group II were pair-feed with those of
composition in obese rats. Five groups of animals
group III and received a reduced amount have been examined (for the definition of the groups
of food corresponding to the food intake of see text). The three panels show the changes in body
individual peers in group III. The changes weight (A), lean mass (B) and total body fat (C)
in body weight, lean mass and body fat as observed after three weeks of treatment. Courtesy B.
quantified by MR relaxometry after three Kuennecke, Hoffmann-La Roche Ltd, copied from.7
weeks of treatment is documented in Figure
29.16. experiments animals were anesthetized with
Auxiliary experiments for the determi- 2 - 2.5 percent isoflurane in NO2/O2.
nation of body fat composition were The 13C spectra (see Fig. 29.17) show
performed using 13C MR spectroscopy (at individual signals from carboxylic (1), olefinic
50 MHz). Individual animals were (2,3), methylene (5) and methyl (6) moieties
positioned on a 40 mm diameter surface of fatty acid chains and the three carbon
coil such that the abdominal fat pads lay on moieties in glycerol (4). The olefinic carbons
the coil. 13C spectra were recorded with a can further be subdivided: signal 2 at
pulse-acquire sequence, number of averages 130 ppm represents the resonances of mono-
were 128, repetition time was 5 s. Spectra unsaturated fatty acids as well as the “outer“
were 1 H decoupled with a WALTZ-16 olefinic carbons in poly-unsaturated acids,
sequence using a birdcage resonator of signal 3 at 128 ppm represent the “inner“
195 mm diameter. For the 13C MRS olefinic carbons in poly-unsaturated fatty
 MR Applications in Experimental and Animal Model Systems
Fig. 29.17: Proton-decoupled 13C MR spectrum
obtained in vivo from the abdominal fat depots of a
rat. The spectrum is dominated by glycerides, which
give rise to six distinct groups of resonances:
1 carboxylic, 2 olefinic, 3 poly-olefinic, 5 methylene,
6 methyl carbons of esterified fatty acids, and 4 the
three carbon moieties in glycerol, respectively.
Courtesy B. Kuennecke, Hoffmann-La Roche Ltd,
copied from.7
acids. On the assumption that the vast
majority of poly-unsaturated fatty acids in
adipose fat is doubly unsaturated and that
the 13C spectrum has been acquired from
fully relaxed spins the average carbon chain
length and the degree of unsaturation of
the fat’s constituents can be quantified from
the signal integrals.
With S1 to S6 denoting the integrals of
the respective 13C signals, k being a
calibration constant, CL the average chain
length, and FAS, FAm and FAd the fractional
contribution of saturated, mono-unsaturated
and doubly-unsaturated fatty acids we can
write
S1 =S4 =S6 = k × ( FAS + FAm + FAd)=k × 1
S2 = k × (2 × FAm + 2 × FAd)
S3 = k × 2 × FAd
S5 = k × ((CL - 2) × FAs + (CL - 4) × FAm +
(CL - 6) × FAd
437
Rearrangement of these equations yields
CL = (S5 + 2 × S1 + S2 + S3)/S1
FAS = (S1 - S2/2)/S1
FAm = (S2 - S3)/( 2 × S1)
FAd = S3/(2 × S1)
Using the integrals S1 to S6 obtained from
the 13C spectrum of adipose tissue in rats as
listed in Table 29.2, the average chain length
evaluates to CL = 16.3 ± 1.6 and the frac-
tional constituents of saturated, mono-
unsaturated and doubly-unsaturated fatty
acids to FAS = 27 ± 3 percent, FAm = 22 ±
2 percent and FAd = 51 ± 3 percent.
In conclusion this application demons-
trates that MR relaxometry using a
conventional high field MR scanner provides
a convenient means for rapid noninvasive
body composition analysis in awake mice
and rats. This method was shown to have
excellent reproducibility, linearity and
sensitivity. Body composition analysis by
MR relaxometry has proven a high-
throughput modality suited for large-scale
screening. New concepts for calibration for
absolute quantification of lean mass and total
body fat content based on in vivo 13C MR
spectroscopy of body fat and 3D MR whole
body imaging (latter not presented here)
have successfully been implemented.
TYPE 2 DIABETES AND MUSCLE
LIPID METABOLISM8
Obesity and type 2 diabetes mellitus share
insulin resistance as a common feature. A
good correlation has been found between
intramyocellular lipid (IMCL) concentration
and insulin resistance which is the main
reason for the interest in IMCL levels.
Volume-selective 1H MR spectroscopy is
currently the only methodology that enables
non-invasive monitoring of IMCL levels in
438 Biomedical Magnetic Resonance: Proceedings of the International Workshop

Table 29.2: Quantification of signal integrals obtained from 13C MR spectroscopy of adipose fat in rats.

Resonance (*) Assignment Signal integral [au]

S1 carboxylic 1.00 ± 0.05

S2 olefinic 1.46 ± 0.07
S3 poly-olefinic 1.02 ± 0.05
S4 glycerol 1.03 ± 0.05
S5 methylene 11.86 ± 0.6
S6 methyl 0.96 ± 0.05

(*) 13 C MR signal of lipid resonances 1-6 as depicted in Fig. 29.17.

vivo. The aims of this study were to 1) rendering a chemical shift for the EMCL
establish an animal model in rats for IMCL methylene protons of 1.5 ppm. If the
detection, and 2) systematically investigate magnetic field B0 is oriented perpendicular
the influence of age, gender, muscle type, to the muscle fibers this chemical shift would
and rat strain on IMCL levels. be 1.0 ppm (see Fig. 29.19)
The most relevant spectroscopic signals
of muscle tissue stem from total creatine
(tCr) at 3.05 ppm and from the methylene
and methyl resonances of fatty acyl chain
protons at 0.8-1.7 ppm (see Fig. 29.18), one
from (IMCL) and one from extramyocellular
lipids (EMCL) which are separated by a
frequency shift of 0.25 ppm. This frequency
shift has been explained by magnetic
susceptibility differences between these two
compartments, and the anisotropic spatial
arrangements of muscle fibers.
The chemical shift of the EMCL resonance
has been shown to be orientation-dependent,
which is most likely due to the arrangement
of adipocytes along the muscle fibers. IMCL,
on the other hand, is stored in the cytoplasm
of muscle cells within spherical droplets,
resulting in no spatial dependence of their
chemical shift on the magnetic field
direction. This orientation dependence is Fig. 29.18: 1H MR spectrum in rat muscle soleus
essential for the separation of EMCL and (muscle fiber oriented parallel to the magnetic field
IMCL by MR spectroscopy. The best B0) from a voxel of (A) 18 mm3 and (B) 8 mm3. In (A)
signals due to IMCL and EMCL are present, while in
separation between the two signals is
(B) the EMCL signals are no longer detectable.
achieved when the investigated muscle lies Courtesy C. Neumann-Haefelin, Aventis Pharma Ltd,
roughly parallel to the magnetic field B0, copied from.8
 MR Applications in Experimental and Animal Model Systems 439

Experiments were performed in Wistar PRESS sequence. Voxels were selected in

and Sprague-Dawley rats, and in male obese
Zucker Diabetic Fatty rats. For measure-
ments the animals were anesthetized with
isoflurane, body temperature was main-
tained at 37°C with a heating pad, and the
animals were placed in a device for
reproducible and accurate aligning of the
muscle fibers in the magnet. Spectra were
acquired on a 7 Tesla, 31 cm bore magnet
(BRUKER BIOSPEC 70/30). A resonator was
used for RF transmission, for signal
detection an actively decoupled surface coil
was placed under the hind leg of the rat. A
gradient-echo sequence was used to acquire
scout images for an accurate and repro-
ducible positioning of the animals. Localized
1
H spectroscopy was performed using a
the muscle soleus and the muscle tibialis of
the rat hind leg, voxel size was 2 x 2 x
2 mm 3 (see Fig. 29.20). A CHESS water
suppression scheme was applied prior to
voxel excitation, spectra were acquired with
TE = 17 ms and TR = 1000 ms. The total
measurement time per spectrum was
17 minutes.
The results of this study show that IMCL
and EMCL resonances can be separated in
the rat hind leg in vivo using the orientation
dependent shift of the EMCL signal after
proper alignment of the muscle. By reducing
the voxel size the contribution of the EMCL
can be minimized. The study demonstrates
that the main physiological factor influencing
IMCL levels is age. Substantial differences

Fig. 29.19: A schematic representation of muscle fibers with the isotropic intramyocellular lipid (IMCL) droplets
and the anisotropic extramyocellular lipid (EMCL) within adipocytes. The methylene protons of EMCL resonate
at 1.5 ppm when the B0 field is aligned in parallel to the muscle fibers or at 1.0 ppm when the orientation is
perpendicular where as the IMCL methylene protons resonate at 1.3 ppm. Measurements in this study were
all done with the magnetic field oriented parallel to the muscle fibers.
440 Biomedical Magnetic Resonance: Proceedings of the International Workshop

Figs 29.20A and B: Typical voxel localization in muscle soleus (1) and muscle tibialis anterior (2) shown in a
sagittal (A) and transverse (B) slice of the rat hind leg in a gradient echo image. The box in the sagittal image
(A) indicates the slice position shown in image (B). The rat is lying on its side on the surface coil with the inner
aspect of the hinge facing upward. Courtesy C. Neumann-Haefelin, Aventis Pharma Ltd, copied from.8

were found between muscle types: IMCL
levels in muscle soleus were significantly
influenced by gender and rat strain, while
no such influence was detected in muscle
tibialis [Figs 29.21 and 29.22 (Plate 24)]. In
female rats, the age related decline of IMCL
levels overlaps with effects related to sexual
maturity. In conclusion, this study shows
that before investigating diseases such as
insulin resistance and diabetes as well as
the effects of drugs on these diseases, normal
IMCL levels for a chosen rat strain and
gender at a given age must be determined.
STUDIES ON THE MOUSE
AND RAT HEART9,10
MRI is inherently a rather slow imaging
technique. For the acquisition of a 2D image
matrix of say 256 x 256 points a typical scan
time in the minute range is required. Of
course this is very long compared to the
timeframe of a beating heart or respiration
motion and thus blurred images of these
body parts would result. An obvious
solution to the problem is to make the
acquisition faster. This has been successfully
implemented on newer MRI hardware.
Scan times from 20 seconds down to
20 milliseconds are nowadays available,
sufficiently short for a breath-hold scans or
even to freeze out cardiac motion.
Nevertheless, fast imaging techniques are
often compromising the image quality and/
or resolution.
An image data acquisition (again we look
at a 256 x 256 matrix) is usually composed
of 256 individual phase-encoding steps,
during each 256 points are collected, which
last only a few milliseconds. The individual
phase-encoding steps are separated by the
TR time, which is in the order of a second
or so. If we now can synchronize the phase-
encoding steps with the periodic cardiac
and/or respiration motion we may get
unblurred images. A combination of fast
imaging techniques and synchronization may
further improve the situation.
 MR Applications in Experimental and Animal Model Systems 441

Figs 29.21A and B: Typical 1H spectra observed in muscle soleus in male (A) and female (B) Wistar rats at age
4, 6, 10, and 17 weeks. A clear age dependence of the IMCL level is detected. Courtesy C. Neumann-
Haefelin, Aventis Pharma Ltd, copied from.8

Clinical MRI techniques for cardiac
applications have now been implemented
most successfully and MRI is seriously
challenging the gold standards in cardiology
like X-ray DSA or ultrasound techniques. In
rodent applications things are complicated
by the fact that rat hearts beat at a rate of
around 300 per minute (bpm), mouse hearts
even around 600 bpm and it may be higher
in diseased animals. Figure 29.23, Plate 24
proves that one still can get cardiac images
with an astonishingly high resolution from
juvenile mice weighing between 20 g and 2 g
only.9 These images are frames from a
movie, i.e. the data acquisition was
synchronized in such a way that the cardiac
cycle can be resolved into individual images
with a time resolution of approximately 4
milliseconds and renders spatial an in-plane
resolution of 100 μm. This is obviously a
sound technical basis to be used in cardiac
rodent models.
These models allow e.g. quantification
of ventricular volumes and mass, assessment
of global and regional wall motion,
measurement of myocardial perfusion and
regional blood volume, assessment of flow
and tissue velocities, and detailed visuali-
zation of cardiac morphology. Figure 29.24
gives an example of a quantitative evaluation
of the myocardial motion, comparing
infarcted and healthy control mice.10
UNDERSTANDING THE
MONKEY BRAIN11-17
In the early 1990s brain functional MRI
(fMRI) has become a popular and significant
tool for studying the operational organi-
zation of the (human) brain.11,12 The basic
concept behind brain fMRI is that neuronal
activity will lead to an increase in glucose
use and blood supply at the site of
activation. These changes are much greater
442 Biomedical Magnetic Resonance: Proceedings of the International Workshop

Fig. 29.24: Infarcted (bottom) and control (top) mice were scanned with magnitude and phase contrast
imaging techniques rendering vector maps. The infarcted myocardium can clearly be recognized. The vectors
depicted in the diagrams represent the myocardial motion. These images were acquired with a FLASH
gradient echo sequence with velocity compensation in all three gradient directions. In order to accomplish
additional motion encoding, the spin phase was prepared using bipolar gradient pulses which resulted in a
linear dependence between the voxel velocity and the spin phase. This method provides accurate quantification
of the velocity magnitude and direction of the murine myocardium at a spatial resolution of around 200 µm
and a temporal resolution of 10 ms. The data were acquired at 7 Tesla on a BRUKER BIOSPEC 70/20 system.
Courtesy J. Ruff, Axel Haase, University of Wuerzburg.
 MR Applications in Experimental and Animal Model Systems
than those in oxygen consumption, thus
resulting in an increase of the oxygen level
in those areas of activation. MRI is capable
to map hemodynamic variations, such as
alterations in cerebral blood volume (CBV),
cerebral blood flow (CBF), and blood
oxygenation level dependant (BOLD)
response with a relatively high temporal and
spatial resolution. The BOLD contrast relies
on a change in concentration of deoxy
hemoglobin (dHb) which is paramagnetic
and acts as an endogenous contrast agent.
The local increased in oxygen supply results
in an equilibrium shift form dHb to oxy-
hemoglobin, i.e. a lower dHb concentration
rendering an increase in MRI signal.
Mainly BOLD fMRI has generated much
interest as a tool for mapping brain
activation and as a means of studying
dynamics of neuronal networks. 13 For a
number of reasons the visual cortex became
one of the main application areas of brain
fMRI research. In Figure 29.25, Plate 25 the
concept of such an experiment is outlined.
The stimuli used here to generate neuronal
activity in the visual cortex of monkeys were
polar-transformed checkerboard patterns,
rotating at different speeds, changes of
rotation direction at different rates and
covering smaller or larger field of vision.
To study the stimulus-induced activation in
the visual cortex a multi-slice T2* sensitive
gradient-recalled echo-planar imaging (GE-
EPI) sequence to image the entire brain was
used. The EPI data representing the BOLD
contrast signal changes are superimposed
on a high resolution 3D anatomical image
rendering the functional map.
So far brain fMRI can be rather straight-
forward. However, BOLD responses can
only measure hemodynamic changes or
intravascular magnetic susceptibility
443
changes, leaving many questions regarding
its relation to the actual neuronal activation
unanswered. In order to get a better
understanding of the underlying mecha-
nisms experiments with great spatial and
temporal resolution and simultaneous
measurements of intracordical field poten-
tials proved to be extremely valuable.
Furthermore, high magnetic field strength
and a very careful implementation of the
experiments is pivotal to the outcome of
the experiments.14,15 (See Figs 29.26, Plate
25, 29.27, Plate 26 and 29.28, Plate 27)
Diffusion tensor imaging (DTI) is another
technique that has attracted much attention
and there is no doubt that it can greatly
contribute to the understanding of the
functional architecture of the brain.16 DTI
has the ability to visualize anatomical
connections between different parts of the
brain, noninvasively and on an individual
(rather than a statistical) basis. Diffusion
weighted imaging is based on the concept
that the signal of spins, i.e. water molecules,
will dephase as they randomly move along
the diffusion gradient direction, resulting
in a signal attenuation. This signal
attenuation quantitatively depends on the
diffusion coefficient and the gradient
strength (also see section 2.1 and Fig. 29.1).
In an ideal isotropic environment without
any restriction for the diffusion motion the
signal strength can be described by the
simple equation given in section 2.1. In
surroundings like brain white matter,
diffusion is highly anisotropic reflecting the
organization of white matter in bundles of
parallel fibers. As a consequence the signal
attenuation will be more pronounced when
the diffusion gradient is aligned with the
fiber orientation as compared to a
perpendicular orientation where diffusion
444 Biomedical Magnetic Resonance: Proceedings of the International Workshop
motion is hindered by the tissue structure.
The diffusion tensor D is a description of
the diffusion properties for each individual
voxel. Equivalent to the isotropic case the
signal attenuation in an anisotropic medium
may be described by the equation
S(bij)/S0 = exp{-∑ bij × Dij}
D is a 3 x 3 matrix with diagonal elements
(Dxx, Dyy, Dzz) that describe the mobility in
perpendicular directions and off-diagonal
elements describing the coupling between
different directions (Dxy, Dxz, Dyz). So the
task is to determine D for every voxel. This
is achieved by repeating DWI acquisitions
for a set of diffusion gradient strengths and
orientations. At least six different diffusion
gradient directions must be chosen and at
least one experiment with very low diffusion
weighting is necessary as a reference. In
fact, the choice of an optimal set of diffusion
gradient directions is still a matter of
contention. The representation of DTI data
is not straightforward. An image ought to
convey information about diffusivity and
the degree anisotropy as well as its spatial
orientation (See Figure 29.29, Plate 27).
Figure 29.30B, Plate 28 shows one attempt
to achieve this. It is important to note that
diffusion imaging is truly quantitative
method that gives direct insight into the
voxel-averaged microscopic physical
properties of tissue.
A very different way of “fiber tracking”
can be done with MnCl 2 . 17 The axonal
transport of the radioactive isotope 54Mn2+
was first studied using histological methods.
Although these studies were carried out
with the goal of understanding the regional
specificity of Mn 2+ distribution, they
indicated the usefulness of Mn2+ as an
anterograde neuronal tract tracer. The
divalent manganese (Mn2+) has an ionic
radius similar to that of the calcium ion
(Ca2+), which can enter cells through calcium
pathways such as voltage gated calcium
channels and is confined to the intracellular
compartment. In MRI experiments injections
of MnCl 2 allow the determination of
projections and connectivities in the brain.
Mn2+ is a paramagnetic contrast agent. It is
actively transported along fibers and axons,
e.g. the optic nerve and tract with a speed
of a few mm per hour. The concentration of
Mn2+ required for these investigations was
found not to have toxic effects. Figure 29.31,
Plate 28 gives an example of MRI studies
with MnCl2 injections. Such paramagnetic
tracer studies may also be used to validate
and further develop noninvasive fiber
tracking techniques, such as diffusion tensor
MRI. In contrast to histological validation
methods, Mn 2+ enhanced tracts can be
readily acquired under identical conditions,
and in exact spatial co-registration with DTI
where as histological methods are prone to
tissue distortions or destruction during the
procedures. The Mn2+ loaded tracts show a
signal enhancement in T1 weighted images.
This signal enhancement can be observed
over a period of approximately two to three
days.
ACKNOWLEDGMENTS
The author thanks all the research groups that have
generously provided images and data for this article
and workshop lecture. These are in particular (in the
order of appearance in the article):
• Novartis Institute for Biomedical Research, Basel,
Switzerland; Markus Rudin, Nicolau Beckmann,
Martin Rausch, Thomas Mueggler and co-
authors.
• MR Imaging and Spectroscopy Group, F.
Hoffmann-La Roche Pharmaceuticals Ltd, Basel,
Switzerland; Markus von Kienlin, Basil
Kuennecke, and co-authors.
• LO-MRI lab, Aventis Pharma Ltd, Frankfurt,
Germany; Hans-Paul Juretschke, Claudia
Neumann-Haefelin, and co-authors.
 MR Applications in Experimental and Animal Model Systems
• Biophysics Group, University Wuerzburg,
Germany; Axel Haase, Joerg Streif, and co-
authors.
• Max Plank Institute for Biological Cybernetics,
Tuebingen, Germany; Nikos Logothetis and co-
authors.
• Bruker BioSpin MRI Ltd, Ettlingen, Germany;
Martin IIg and his applications team.
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630-42.

A 2D CSI sequence with an EPI
1H MRS in studies of fine-needle
biopsy specimens 414
RF pulses 88
1.5 T and 3 T MRI scanners 87,
102, 103
1D 1H MR exchange spectroscopy
95
1-D Fourier-transform 79
1D MR spectra 103
1D MRS 91
1D MRS protocol including water
and fat suppression and signal
acquisition 89
1D spectra 91
1H MR (8.5 T, 37º C) spectra
showing leakage of meta-
bolites into collection during
freezing storage and thaw-
ing of prostate tissue 172
1H MR (8.5 T, 37ºC) spectra (256
acquisitions) of a FNAB
taken from an invasive
carcinoma of the breast 178
1 techniques 90
H MR (8.5 T, 37ºC) spectra of
prostate biopsy 177
1H MR spectra (8.5 T, 37ºC) of
liver biopsy 179
1H MR spectra highlighting the
signals of substrate and its
metabolite 6-OHNA 408
1 extract of skeletal muscle
H MR spectra of serum, showing
levels of glutamine 409
1H MRS 156, 233
1H NMR spectroscopy 313
1H spectra, 144
1H spectroscopy 144
1HNMR spectrum of a small
DNA duplex 15
2 D sequence 87
2 T MRI scanner 87
2 T scanner 87
23 channel coil 83
2D COSY spectra 87, 100, 101
readout module 84
2D DQ spectroscopy 87
2D DQ-filtered COSY/SECSY
sequences 95
2D J-resolved spectroscopy 102
2D L-COSY 103
2D L-COSY method 99
2D L-COSY sequence 92
2D L-COSY spectra 99, 100
2D L-EXSY sequence 96
2D L-EXSY spectrum 96
2D metabolite ratios 99
2D MRS protocol 91
2D MRS protocol including
volume localization, 2D
spectral sampling, etc. 91
2D MRS sequences 87, 103
2D MRS techniques 86, 102
2D NMR spectra 93
2D NMR spectroscopy 20
2D NMR spectroscopy techniques
86
2D NMR spectroscopy
2D NMR technique 87, 90
2D spectrum 90, 91
2D-CSI sequence 80
2D-MRSI scan 165
2D-zero-quantum (ZQ) spectra 87
2-pulse COSY spectrum 93
31P and 1H magnetic resonance
spectra (MRS) 237
31P magnetic resonance
spectroscopy (MRS) 192, 233
ATP homeostasis and ATP
sources 195
between-subject variability
205
future investigations 209
informational content of a
31P MR spectrum 193
MR techniques 192
pathological changes
assessed with 31P MRS 207
Index
PCr and pH changes in
muscle after exercise, aerobic
ATP production 202
PCr changes in exercising
muscle 196
pH Changes in exercising
muscle, buffering components
and glycolytic ATP production
198
PME changes in exercising
muscle, glycogenolytic and
glycolytic ATP productions
202
reproducibility of measure-
ments 205
standardization procedures
206
technical considerations 195
within-subject variability 206
31 P MR spectra recorded in
humans torearm flexor 194
31 P spectroscopy 142
3D CSI 82, 83
3D-CSI sequence 80, 81
4.7 T spectrometer 87
400 MHz 1D proton NMR
spectrum showing aliphatic
region 321
expanded from 0.5 to 3.0 ppm,
of the perchloric acid
tissue 321
expanded from 5.1 to 6.6 ppm,
of the perchloric acid
extract of skeletal muscle
tissue 321
expanded from 6.4 to 9.1 ppm,
of the perchloric acid
extract of skeletal muscle
tissue 321
400 MHz 1D proton NMR spec-
trum showing downfield
region 321
448 Biomedical Magnetic Resonance: Proceedings of the International Workshop
expanded from 2.9 - 4.7 ppm,
of perchloric acid extract
of skeletal muscle tissue
321
4-D Fourier transform 80
Acquired signal 89
Acquiring ADC trace maps 146
Acquisition of two-dimensional
raw data for imaging 36
Acute disseminated
encephalomyelitis 161
Acyclic chelating agents with
different functional groups
as Gd-based MR contrast
agents 397
ADC values 145
ADC changes 238
ADC histograms in response to
taxol 236
ADC images 144
ADC maps 143, 238
ADC trace maps 145
ADC values 235, 238
Advantage of L-SECSY over L-
COSY 94
Advantage, 2D spectroscopy 20
Ambient FiO2 level 147
Antecedents of MRI methods 9
chemical applications 9
echoes 10
the first NMR “image” 9
Apparent diffusion coefficient
(ADC) 142
Applications in drug
development, NMR 27
Applications in physics and
chemistry NMR 3
Applications of echoes 10
APT imaging 354, 359
APT imaging of rat brain
tumors 357
Assessment of liver graft
function 405, 410
Automated linear combination
modeling method 143
Axial MRI of a 42 year old
healthy breast with a voxel
of 2D L-COSY 101
Axial T1-weighted images with
individual activation clusters
at the level of the basal
ganglia 133
B Chemical shift selection artefact 81
Bacterial infection 407
Bacterial infection MR spectro-
scopy 404
Balanced fast field echo
(balanced FFE, Philips) 63
Balanced gradients 63
Baseline 1H MRS 144
Basic gradient-echo imaging
sequence 35
Biological applications of NMR 3
Blood oxygenation level
dependent (BOLD) contrast
effect 142
Body MR imaging 257
Bohr relation 2
Breast cancer treatment 417
Burst imaging 65
b-values 237
C of animal imaging
Cabinet sequence 91
Catheterization 313
Cerebral 1H spectra
Cerebral lactate/creatine ratios
before, during and after
hypoxia for normal growth
restricted chick embryos 148
Cerebral lactate/creatine ratios
plotted against time for
chick embryos that had
received hypoxia + LPS (n =
9) and hypoxia alone 149
Cerebral trace ADC values plot-
ted against time for chick
embryos that had received
hypoxia 149
Change in cerebral T 2* values
plotted against time for chick
embryos that had received
hypoxia + LPS (n = 9) and
hypoxia alone (n = 10) 150
Changes in ADC and TGD in
response to taxotere 236
Chemical exchange saturation
transfer (CEST) 353
Chemical shift 90
Chemical shift displacement 81
Chemical shift imaging 89, 415
CHESS (chemical shift selective
saturation) pulses 88
Chicken embryo model 150
Choline peaks in MRS of breast
carcinoma 100
Classical view of NMR
spectroscopy 12
Classification and regression
trees (CART) 100
Coding to create ‘spatial
frequencies’ and ‘spatial
phases’ 30
Coherence transfer echoes 90
Coherence transfer pulse 91
Coherence-transfer-echo 91
Coherent complex signal
addition 83
Combined exposure to LPS and
hypoxia 150
Comparing key characteristics
techniques 423
Concentration of metabolites
estimated in the muscle tissue
extracts, neuromuscular dise-
ases 323
Continuous wave (CW)
saturation 95
Contrast 52
Contrast agents for angio-
graphy 400
Contrast and relaxation 43
Contrastenhanced MRI for
detecting breast cancer 413
Control system 76
COSY sequence 87
COSY spectra 87, 100
Critical experiments in
biomolecular studies 20
HMQC 20
J-correlated spectroscopy
(COSY) 20
NOESY 20
ROESY 20
TOCSY 20
Cross peak imaging (CPI) 102

Crushers and spoiler gradients 10
CSI FID sequence 80
CSI sequences 80
CSI signal 82
CSI techniques 82, 83
CSI-grid 80, 81
Cuboidal subvolume 78
D oximetry by FT PR method
Data-inherent procedures 83
DCE-MRI 233
DCE-MRI to monitor
therapeutic response 239
Dephasing FID, and its ‘rephasing’
to creat an ‘echo’ 33
DESS (dual echo in the steady
state) 64
Development of human MRI 7
Difficulties in studying biological
systems by NMR
assignments 19
broad lines 20
resolution 19
sensitivity 19
water signal 19
Diffraction pattern, of the MR
image 39
Diffusion MRI 233
Diffusion weighted images of
breast cancer metastases 238
Diffusion-weighted MR imaging
(DWI) 142
Discriminant analysis on MRS
peak 98
DQ-filtered COSY 94
DQ-filtered COSY/SECSY 94
DQ-filtered SECSY 94
Duty cycle 63
Dynamic contrast enhanced
(DCE) MRI 237, 239
E data generating 173
Echo planar DWI 237
Echo planar imaging (EPI) 40
Echo times 80, 100, 143, 163
Echoes 79, 143
Echo-planar imaging (EPI)
technique 56
Echo-planar spectroscopic
imaging (EPSI) 102
Echo-times 80
Effect of voxel bleeding 82
Effects of spin motion 51
Electron paramagnetic resonance
(EPR) 330
EPR imaging modalities 331
applications 342
oximetry 342
344
oximetry by time domain
single point imaging
346
pharmacokinetics 347
redox mapping 348
redox status in tumors 348
spectral-spatial (spectro-
scopic) imaging 342
artifact-free imaging 339
constant time pulsed RF EPRI
340
CW EPRI technique 331
data acquisition system 338
description of RF CW EPRI
scanner 332
diplexer 336
EPRI by projection recons-
truction 331
image reconstruction 334
image reconstruction 338
limitations of CW EPR
technique 335
limitations of pulsed RF EPRI
339
need for newer spin traps 342
principles of constant time
imaging 340
pulsed RF EPRI 335
resonator 337
signal detection 337
spin probes for EPRI 341
Ex vivo and in vivo spectroscopic
Exogenous contrast agents 394
F sequences, RARE, TSE, FSE 123
Fast imaging employing
steadystate acquisition
(FIESTA, GE) 63
Fast low angle shot 62
Fast low angle single shot
Index 449
(FLASH) method 143
Fast sequences 84
Fetal brain injury 142
Fetal MRI 301
Field-of-view (FOV) 163
Filter 75
Findings of chicken embryo
studies 151
Fine needle aspiration biopsies 172
Flip-angle errors 91
fMRI 156
fMRI and neuro-biologic definition
of yogic progress 138
brainstem, the psychosomatic
generator-cum-switch board
of yogic transformation 140
subjective world of yogic expe-
rience and its neuro-biological
objectivasation 139
Fourier imaging 39
Fractional inspired oxygen
content (FiO2) 144
Fractions of inspired oxygen
(FiO2) on PCr kinetics 198
Free induction decay (FID) 29
Free precession of the steady
state signal 67
Frontiers in NMR spectroscopy
in life sciences 27
Functional magnetic resonance
imaging (fMRI) 129
applications 131
cortical reorganization in
unilateral brain lesions 132
fMRI of sensorimotor
somatotopy 131
fMRI of speech production 131
language function 133
motor function 132
practical considerations 129
Fundamentals of fast and
ultrafast imaging 111
fast imaging with multipulse
sequences, gradient echoes 121
fast imaging with multipulse
gradient based fast and ultra-
fast imaging sequences 117
gradient performance and
radiofrequency pulses 115
technical limits 112
450 Biomedical Magnetic Resonance: Proceedings of the International Workshop
G prostate cancer 371
Gadolinium (iii) complexes 396
Gadolinium complexes with
macrocyclic chelating agents
used as contrast agents 397
GAMMA library 91
Gastrointestinal contrast agents
399
Gd-DTPA formation constant 396
General scheme, 2D NMR 20
Glycolysis flux 202
Gradient coils 71
Gradient crusher pulses 92
Gradient pulse 30
Gradient Strength 217
Gradient system 75
Gradient-echo (GE) 33, 34
Gradient-echo scout images 143
Gradient-modified NMR 30
Gradient-recalled echo 33
Gradient-recalled echo (GRE) 10
Graph, cerebral NAA/creatine
and inositol/creatine ratios
for normal (n = 8) and
growth restricted (n = 11)
chick embryos 147
Graph, cerebral β-hydroxy-
butyrate/creatine ratios for
normal and growth-restric-
ted chick embryos 147
Gyromagnetic ratio 29
H I
Hahn’s spin-echo 90, 92
Heavy T2-weighting in non
proton applications 81
HIF-1α inhibitor 237
High-resolution NMR 362
biliary tree 371
biological fluids 362
biological tissue 363
brain tumors 370
breast 372
cancer 369
colon cancer 369
head and neck 372
inborn errors of metabolism
365
neurological disorders 368
ovarian tumors 371
renal injury 367
thyroid cancer 370
High-resolution NMR to
biomedical problems 362
Homonuclear correlation
parameters 20
Hybrid CSI sequences 81
Hypoxia 144
Hypoxia-inducible factor HIF-1α
240
Hypoxic injury in chicken
embryo brain 142
1H MRS of transient cerebral
hypoxia in a chicken embryo
model of fetal growth retar-
dation 146
hypoxia and recovery 143
materials and methods 142
MR changes in cerebral meta-
bolism during transient acute
mild/moderate hypoxia, chick
embryo 144
MRI and MRS 143
NMR experimental
procedure 142
protocol A 143
protocol B 143
results of MR studies 144
transient mild hypoxia
studied by 1H MRS 144
validation studies 144
Image reconstruction and the
‘display’ matrix 37
Imager 75
Imaging techniques, animal
models 423
age-dependent impairment of
somatosensory response
in APP23 mice 427
altered metabolic profile in the
frontal cortex of PS2APP
transgenic mice 430
diffusion, fMRI and metabolic
profile in transgenic mice
with cerebral amyloidosis
424
quantitative body composition
analysis by T2 MR
relaxometry 433
restriction of diffusion by
amyloidosis 425
studies on the mouse and rat
heart 440
type 2 diabetes and muscle
lipid metabolism 437
understanding the monkey
brain 441
Important NMR nuclei in life
sciences 15
In vitro NMR spectroscopy of
muscle tissue 319
human muscle biopsy 319
perchloric acid extraction 319
In vivo 1H MRS 171
In vivo MR prostate program 183
In vivo MR spectroscopy 100
In vivo MRS 77
In vivo MRS, studies of muscle
energy metabolism 317
1H MRS 318
31P MRS 317
Indirect detection of amide pro-
tons through the water sig-
nal (APT MRI and MRS) 355
Indirect spin-spin coupling 90
Infant studies 151
Intramolecular J-coupling 90
Invention of NMR imaging 4
Iron oxide nanocompounds 398
ISIS-COSY sequence 102
Isotopes, suited for MRI or MRS
studies in vivo 217
J
J-coupling 90, 93
J-modulation, eddy currents 143
K
K-space 39, 40, 53, 80-82
K-space associated with sequence
55
K-space filling scheme 54
K-space filling scheme for a
spiral EPI scan 59
L
Lac/Cr ratio 145
Lactate-editing double-quantum
(DQ) and zero-quantum
(ZQ) MRS techniques 94

Larmor frequency 29, 30, 81
Larmor frequency equation 30
LC model algorithm 103
L-COSY sequence 93
L-COSY spectrum 92, 93, 100
Linear magnetic field gradients 29,
30
Localization techniques 78
chemical shift imaging 80
fast-CSI 83
matrix spectroscopy 82
post-processing 84
single voxel spectroscopy 78
Localized spectroscopy 218
Localized spin-echo correlated
spectroscopic (L-SECSY) 93
Localized two-dimensional 1H
MR chemical exchange
spectroscopic (L-EXSY)
sequence 95
Localized two-dimensional
correlated spectroscopy 91
M Marrow disorders 298
Macromolecular structures and
organizations 22
Magnetic field gradient 30
Magnetic resonance imaging 217
Magnetic resonance imaging
(MRI) 233
Magnetic resonance spectro-
scopic imaging (MRSI) 182
Magnetic resonance spectroscopic
imaging (MRSI), the human
brain 161
methods 161
single voxel MRS 161
technical development 161
MR spectroscopic imaging
with phased-array coils 163
patient studies 167
discussion 168
materials and methods
167
results 168
sense-MRSI 164
Magnetic resonance
spectroscopy 29
Magnetic resonance spectroscopy
(MRS), measure a metabolic
profile of the tumor 413
Magnetic resonance techniques
155
an inherited speech and
language disorder 157
childhood epilepsy 155
children born prematurely 158
children with memory
impairments 156
children with sickle cell disease
158
Magnetization prepared
sequences 65
Magnetization transfer 48
Magnetogyric ratio 217
Magnets, imaging scanner 71
magnet shielding 73
permanent magnet 71
resistive magnets 73
SHIM 73
superconductive magnets 73
Main frame computer, MR
scanner 76
Malabsorption syndrome MR
spectroscopy 404
leukemia 299
metastatic disease of the
marrow 300
myeloma 299
Maximum tolerated dose 240
Metabolic profile study on HIF-
1 modified tumors 383
Metabolic profiling by NMR in
the development of
anticancer drugs 384
Metabolic profiling studies on
genetically modified cells 382
Metabolite ratios 99, 166
Metabolite response versus time
of cerebral lactate/creatine
and NAA/creatine ratios
hypoxia 145
Metabolite signal 84
Metabolite signals visible by
short TE brain MRS 84
Metabolomic methods to study
animal and human gene
modifications 383
Mid-brain sagittal MRI 38
Monitoring system 74
MQ filtered COSY/SECSY 95
MQ pathway 95
Index 451
MR imaging applications to
transgenic models 251
MR imaging in the evaluatioan
of bladder cancer 287
MR imaging of carcinoma of
the gallbladder 280
MR imaging of diseases of biliary-
pancreatic system 270
clinical applications 271
acute pancreatitis 271
chronic pancreatitis 272
cystic neoplasms 275
islet cell tumors 274
MR cholangiopancreato-
graphy 276
MRCP pulse sequences
276
indications of pancreatic MRI
270
MR protocol 270
normal MR imaging appear-
ance of the pancreas 271
MR imaging of female pelvis 290
adenomyosis 292
cervical cancer 293
dermoid 292
endometrial cancer 293
endometriosis 292
gynecologic neoplasms 292
MR imaging technique 290
ovarian cancer 293
polycystic ovarian disease 292
MR imaging of liver 257
diffuse liver diseases 267
Budd-chiari syndrome 269
cirrhosis 267
fatty liver 267
iron overload 267
diffusion imaging 260
focal lesions 260
benign liver lesions 260
biliary cystadenoma 264
biliary hamartoma 264
focal nodular hyperplasia
(FNH) 262
hemangioma 260
hepatic cysts 260
hepatocellular adenoma
264
important differential
diagnosis 266
452 Biomedical Magnetic Resonance: Proceedings of the International Workshop
cholangiocarcinoma
267
hepatocellular carci-
noma 266
metastases versus
hemangiomas 266
liver imaging protocol 257
liver-specific contrast agents
259
MRI characteristics of com-
mon malignant lesions 265
fatty liver with metastases
266
metastases 265
metastases, hyperintense
on T1-weighted images
265
near isovascular meta-
stases 265
MR imaging of prostate 288
identification of prostate cancer
by magnetic resonance imag-
ing 269
newer techniques of magnetic
resonance imaging 289
prostatic anatomy as seen on
MR imaging 289
staging of prostate cancer by
magnetic resonance imaging
289
technique of prostate MR
imaging 288
MR imaging of the adrenal glands
283
adrenal adenoma 284
adrenal cortical carcinoma 284
adrenal cysts, pseudocysts and
hematomas 286
myelolipoma 286
pheochromocytoma 284
MR imaging of the kidneys and
adrenal glands 281
angiomyolipoma 282
lymphoma 283
protocol 281
renal cell carcinoma 282
renal masses 282
simple and complex renal cysts
283
MR imaging to predict
therapeutic response 241
MR in biomedicine 403
MR scanner 71, 75
MR spectroscopy 77, 86
MR spectroscopy assessment of
malabsorption 403
MR spectroscopy to transgenic
models 248
choline phospholipid
metabolism 249
MR tomograph (MRT) 130
MR visible parameters 239
MR-detectable proteins 353
MRI system 71
MRI/MRS to monitor therapeutic
response 234
diffusion MRI 234
mechanistic studies 238
preclinical studies 235
dynamic contrast-enhanced
MRI 239
implications 240
MRS post-processing 84
MRS signals 415
MRS studies of a coventional
cytotoxic agent - 5-fluoro-
uracil 385
MRS studies of a cyclin-
dependent kinase inhibitor-
R-Roscovitine (CYC202) 385
MRS studies on 17AAG, and
inhibitor of HSP90 386
MRS voxel size 100
MRS voxels 100
Multi-compartment systems and
the effects of exchange 47
Multi-dimensional 1H MR
spectroscopy and editing 87
chemical shift correlated MR
spectroscopy 90
clinical applications of 2D L-
COSY 96
exchange 1H MR spectro-
scopy (EXSY) in vivo 95
hepatic encephalopathy (HE)
97
human breast cancer 99
late-life major depression 98
L-COSY 91
L-SECSY 93
multiple-quantum (MQ) fil-
tered COSY and SECSY 94
single-and multi-voxel-based
one-dimensional 1H MR
spectroscopy 87
Multiple array brain coil 83
Multiple-quantum (MQ)
coherence 94
Multi-slice gradient - echo MR
scout images 143
Multi-voxel based 2D 1H MR
spectroscopy 101
Musculoskeletal MRI 293
bone tumors 293
general MR appearances 295
musculoskeletal infection 297
Mutation in the FOXP2 gene 157
N
NAA/Cr ratio 145
N-acetyl aspartate (NAA)
resonance 142
Needle biopsy techniques
coupled 313
Neonatal encephalopathy 141
Neonatal piglet model 151
Neuromuscular diseases 313
Newborn encephalopathy 142
Nitroxide radical as contrast
395
NMR nuclei in life sciences 14
NMR parameters 15
chemical exchange 19
chemical shift 15
dipolar couplings 17
nuclear overhauser effect
(NOE) 19
peak intensities 17
quadrupolar interactions 18
relaxation rates and line-
widths 17
scalar coupling 17
NMR properties of selected
nuclei 218
NMR spectral resonances 86
NMR spectrometers 87
NMR spectroscopy, noninvasive
measure of muscle metabolism
313
NMR spectroscopy study 320
metabolite assignments 320
metabolite concentration in
muscle diseases and controls
322

muscle metabolism in
muscular dystrophies 323
quantification of metabolites
320
skeletal muscle metabolism in
mitochondrial myopathy 325
NMR spectroscopy techniques
314
NMR-compatible hollow-fiber
bioreactor (HFBR) 239
NMR-Derived metabolic profiles
379
Novel pulse sequences 53
Nuclear magnetic resonance
(NMR) 1, 2, 313
Nuclear-nuclear coupling 90
O primary sclerosing cholangitis
Observation of NMR spectrum 13
Obtaining quantitative informa-
tion from 1H MRS 415
One dimensional 1H MR spectra
of suspected malabsorption
syndrome 406
One-dimensional (1D) and two-
dimensional (2D) NMR
techniques 313
Origins of NMR 1
Other early approaches to NMR
imaging 5
2D FT method 6
fonar 6
sensitive point method 6
Outer volume saturation (OVS)
163
Outer volume saturation (OVS)
slabs 88
Overview of the major
components, MR system and
their interconnections 72
P frequency encoding 33
Parallel imaging 124
Paramagnetic CEST
(PARACEST) agents 353
Paramagnetic contrast agents 394
Paramagnetic ions 395
Paramagnetic relaxation 49
Partial volume 143
Patient table 75
PCr kinetics 198
Pediatric applications, MRI 277
biliary atresia 277
cholangiocarcinoma 279
congenital variants of the
biliary system 278
cystic diseases of the bile
duct 277
evaluation of the pancreas 280
obstruction at the ampulla of
vater 279
pancreas divisum 280
post-biliary-enteric anasto-
mosis 279
post-laparoscopic cholecystec-
tomy 279
post-liver transplant 280
278
stone disease 278
strictures 278
Perinatal hypoxia-ischemia 141,
142
pH imaging of the ischemic rat
brain 357
Phantoms 76
Pharmacokinetic measurements
of a 5-FU pro-drug,
capecitabine 388
Phase 31
Phase cycles 79
Phase encoding gradients 31
Phased-array coil technology 66
Phase-sensitive two-dimensional
total correlation spectrum
322
expanded region of perchloric
acid extract of skeletal
muscle tissue 322
Physical gradients of the MRI
scanner 31
phase encoding 32
slice selection 31
Point resolved spectroscopy
PRESS , 79, 89, 143, 184, 218
Point-resolved spectroscopy
sequence (PRESS) 88, 91, 143,
218
Power in clinical trials 242
Index 453
Predicting response to
neoadjuvant chemotherapy
using 1H MRS 418
Prenatal MRI of the central ner-
vous system, head and neck
302
prenatal MRI of the fetal body
303
PRESS spectra from a phantom
that contains physiological
concentrations of neuro-
metabolites 162
Primary motor cortex (M1) 131
Prostate MR spectroscopy 183
Proton (1H) MRS on biopsies
ex vivo 171
biopsy collection and
handling 172
proton MRS on biopsies 174
breast 176
esophagus 182
liver 178
prostate 174
thyroid 179
proton MRS in vivo 182
breast 184
prostate 182
thyroid 185
statistical classification strategy
(SCS) 173
Proton density 29
Proton efflux 202
Proton magnetic resonance
spectroscopy (1H MRS) 142,
161, 171
Proton MRS in vivo 185
Proton MRSI in the diagnosis of
acute disseminated encephalo-
myelitis (ADEM) 167
Pulse diagram for a 12 echo
GRASE sequence 60
Pulse diagram for a single-shot
spin-echo EPI sequence 57
Pulse diagram for a spin-echo
sequence 54
Pulse diagram of a four-echo-
fast spin-echo sequence 55
Pulsed field-gradient 30
Pulsed gradient Fourier imaging
techniques 30
Pulse-sequence (ISIS-COSY) 102
454 Biomedical Magnetic Resonance: Proceedings of the International Workshop
PX-478 using both diffusion MRI
237
Q Signal-to-noise ratio 63, 162
Quantitation of short TE 1H clinical
spectra 84
R 88
Radio-frequency FAST 62
Ratio of resonances at 1.7 ppm
and 0.9 ppm for normal
thyroid tissue 181
Reciprocal space concept in
crystal physics 39
Recovery time 89
Redox inhibitor of thioredoxin 240
Refrigerator system 74
Relaxation properties 52
Repetition time 100, 143, 163
Rephased echoes 33
Resonances in one dimensional
1H MR spectra 176
RF coil 71
RF pulses 78, 80, 87, 91, 92, 95,
218
RF shielding 75
RF system 74
RF-FAST, Picker international 62
ROC analysis 237
S Small MW contrast reagents
Sagittal MR image that is T1
weighted, contrast
dependent on relaxation 44
Sagittal short tau inversion
recovery (STIR) fastspin
echo MR image 185
Sagittal spine MRI 38
Sample preparation for metabolic
profiles 381
extraction 381
magic angle spinning NMR
381
MRS profiling in vivo 382
Sampling time 217
SECSY 87
Select a particular J-coupled
metabolite 86
SENSE-MRSI scan 165
Sensitivity encoding (SENSE) 66
Signal frequency 29
Signal phase 29
Signal strength or amplitude 29
Simple pulse sequence for NMR
excitation and detection 13
Simulated echo acquisition mode
Simultaneous acquisition of spa-
tial harmonics (SMASH) 66
Single volume localizing MRS
sequence 91
Single-array head coil 83
Single-quantum (SQ) resonance
of water protons 94
Single-voxel PRESS spectra
recorded at 3.0 tesla from a
phantom containing N-acetyl
aspartate (NAA) using a 6-
channel head 162
Single-voxel spectroscopy 414
Skeletal muscle metabolism 314
amino acids metabolism 316
fatty acid metabolism 315
glucose metabolism 314
krebs cycle 315
Slice center 81
Slice selection gradient 81
Slice selection profile 80
Slice selection pulses 81
Slice-selective RF-pulse 80
(SMWCR) 240
Specific absorption rate (SAR) 56
Specific Nuclei 219
5-fluorouracil 222
carbon-13, 277
fluorine-19 219
future prospects 227
hyperpolarized gases—3He
and 129Xe 226
lithium-7 222
nitrogen-15 227
other nuclei 227
10
B 227
133Cs 227
17O 227
195Pt 227
27Al 227
2H 227
87
Rb 227
sodium-23 226
Spectra from healthy prostate
tissue 183
Spectra of fine-needle aspirates
of breast tissue 100
Spectral editing techniques 86
Spectroscopic imaging (MRSI)
415
Spectroscopic imaging (SI) 89
Spherically symmetric Hamming
k-space filter 81
Spin echo 33, 78, 89, 91, 143
Spin phase 31
Spin-echo sequence 79
Spin-echo SVS sequence 79
Spoiled gradient echo 62
Spoiled GRASS 62
Spoiling gradients 79
SQ coherences of metabolite
protons 94
Standard diffusion weighting
protocol 235
Steady-state imaging physics 58
STEAM (stimulated echo
acquisition mode) 78
STEAM sequence 80, 87
Stern’s molecular beam technique
2
Stimulated echo 10, 78
Stimulated-echo acquisition
mode (STEAM) 184
Studies of body fluids, tissues
and cells 23
Studies on spermatozoa 24
biochemical changes during
cell maturation 26
effect of Argon on activity of
spermatozoa 27
identification of small mole-
cular weight compounds 24
metabolism in cells 25
Summary of classifiers and spec-
tral regions using SCS 175
Summary table of breast in vivo
data 186
Sum-of-squares normalization 83
Supportive system, MR scanner 76
additional console 76
camera 76
chiller 76
injector 76
intercom 76

physiological measurement
unit 76
Susceptibility contrast and bold
effects 49
SVS sequences 81
SVS spin-echo sequence 79
SVS STEAM sequence 79
SVS techniques 80, 82
T Three-pulse sequence 79
T1 and T2 values of metabolite
signal in pathological tissue 84
T1 values 88
T2 relaxometry 156
T2*-weighted images 144
T2*-weighted MRI 142
T2-weighted MR scan of the brain
44
Technical problems, EPRI 330
Techniques to localize tumor
spectra 414
TE-crushers 94
Temporal response of cerebral
trace ADC within the embryo-
nic brain, before during and
after hypoxia 146
show actual trace ADC maps
with a visible decline during
the hypoxic period 146
show T2*-weighted images (TE
= 23.4 ms) with a visible
reduction in signal amplitude
during the hypoxic period 146
transient cerebral hypoxia in
a chicken embryo model
with bacteria infection 148
The role of nuclear magnetic
resonance methods in
metabolomics 379
Three RF pulses spaced unequally,
produced five echoes one
stimulated echo 61
Time-dependent changes in PCr
in exercising muscle 197
TM crushers 94
Trace ADC value 143
Train of equally spaced RF
pulses produce several
echoes 61
Trajectory in k-space 60
Transcranial magnetic
stimulation (TMS) 132
Transverse magnetization
approaches 62
Traversal in k-space 57
TrueFISP 63
TrueFISP (Siemens) sequence 63
Tumor growth delay 236
Two directional plots to compare
the resonance intensity ratio
181
Two-dimensional L-EXSY
sequence 95
Two-dimensional NMR spectro-
scopy 90
Typical 2D 1H COSY spectra
from biopsy 180
Index 455
Typical PCr and pH time-
dependent changes affer a
muscle exercise. 200
Typical proton single voxel
spectrum 186
U
Use, parameters TR and TE
producing image contrast 35
Use, pulses to generate 2D
spectrum 21
V
Volume of interest (VOI) 90
Volume-localized 1H MRS spectra
143
VOSY 87
Voxel size 101
Voxel-based morphometry 157
Voxel-bleeding effects 81-82
W
Water exchange (WEX)
spectroscopy 354
Whole-body MR imaging 300
non-oncologic applications 301
oncologic applications 301
X
X-ray computed tomography
(CT) 1
Z
ZQ coherences 95
ZQ-filtered COSY and SECSY 94