Applications

Human
Christopher

of Proton MRS to Study

Brain Metabolism
C. Hanstock

and Peter S. Allen

1. Introduction
Magnetic resonance spectroscopy (MRS) provides information
that is rarely obtainable by other noninvasive means, or even by
invasive methods using radioactive labels. For example, it provides the means to monitor in time and in space changes in various metabolic pools and allows one to think in terms of the
biochemistry of these pools. In this sense, MRS is quite unique,
and, although it cannot be said to be highly specific in diagnosing
individual diseases, it nevertheless enables changes in many critical and characteristic parameters to be observed noninvasively
for a broad range of metabolic abnormalities. By its nature MRS
lends itself more toward the evaluation of diffuse brain diseases
rather than that of focal lesions. For example, typical applications
of MRS have been (1) to assess the regional distribution of neuronal dysfunction or death, (2) to evaluate distributions in the
oxidative state of the brain, or (3) to detect regions of membrane
abnormality. This list is growing as new MRS technology emerges.
The adoption of MRS as a routine diagnostic and patient management tool in clinical medicine has, however, been quite slow when
compared to the rapid acceptance of magnetic resonance imaging
(MRI) several years ago. To understand this one must acknowledge that not only is MRS more demanding technically than MRI,
but the clinical significance of spectroscopic findings has not always been recognized prior to the in vivo application of the nuclear
magnetic resonance (NMR) spectroscopic techniques. The significance of N-acetylaspartate
(NAA) in brain is a case in point
(Blakely, 1988). Although its exact role in neurons is still not fully
From
Neuromethods,
vol. 33 Cell Neurobiology
Techmques
Ed A A Boulton,
G B Baker, and A N Bateson 0 Humana
Press Inc

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understood,
it was the MRS demonstration
of correlations
between
NAA variations
and changes in the clinical measures that gave
rise to its acceptance as a putative
neuronal marker.
Several atomic nuclei afford a measurable
NMR signal in vivo,
namely,
H, 13C, 23Na, and 31P, their observability
depending
not
only on the intrinsic
NMR observability
of each nucleus (i.e., on
its magnetic
moment and natural abundance)
but also on the concentrations
of the metabolites
within which these nuclei reside
Notwithstanding
the valuable data acquired from the other nuclei,
it is generally
accepted that the principal
successes of MRS in
studying
brain have arisen from H spectroscopy
This is primarily because of the ubiquity
of the proton and because of its large
magnetic
moment.
For example,
evaluations
of major dysfunctions of neuronal
and glial metabolism
in progressive
degenerative diseases,
in dementia,
in encephalopathies,
and in the
detection
and lateralization
of the epileptogenic
focus in temporal lobe epilepsy (Hugg et al., 1992; Gadian et al., 1993; Hugg et
al., 1993) fall into this category.
Moreover,
evaluations
of the
regional
distribution
of glycolytic
activity through
the measurement of lactate has provided
(Hanstock
et al., 1988) information
on the degree of aerobic and anaerobic metabolism
during hypoxia,
anoxia, and ischemia (Behar et al., 1983; Ikeda et al., 1990). This
review will therefore focus on developments
in H spectroscopy.
An illustration
of a typical H spectrum from brain is illustrated
in Fig. 1, the separation
of resonances from different metabolites
arising primarily
as a result of the chemical shielding
interaction
(Gunther,
1995). It is apparent
from this spectrum
that several
major resonance peaks can be clearly resolved, i.e., from N-acetyl
groups (NA) at 2 02 ppm, and from the methyl groups of creatme
(Cr) at 3.05 ppm and choline (Cho) at 3.2 ppm. These resonances
are from methyl protons not coupled to any other protons and, as
a result of the zero coupling,
appear as singlets. The overwhelming majority
of MRS work on brain has made use of these three
resonances and this is described more fully in Subheading
2.
Beyond the successful exploitation
of the methyl singlets of NA,
Cr, and Cho, H MRS development
is now probing
the measurement of metabolites
with coupled H spins. The resonance peaks
from these metabolites
exhibit multiplet
structures that often overlap and thus complicate
spectral interpretation.
Notable
among
metabolites
with coupled
spins are glutamate
(Glu), glutamine
(Gin), aspartate
(Asp), and y-aminobutyric
acid (GABA).
These

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349
N-Acetylaspartate

N-Trimethyl
(Chqphosphoryl-Cho.
glycerophos~horyl-ChoI

PR
CHZ
AH,
I+

Cr / PCr

H
HN
NHR
NC/
I

CH,COOH

'c'
HOOC"

CH,-I;I-CH,

3.2

2.8

2.4

2.0

Chemical Shij? (ppm)

Fig. 1. Proton MR spectrum from normal human brain (PRESS, TE 80
ms) showing the three prominent singlet peaks arising from the Ntrimethyl compounds, from Cr/PCr and from NAA (including other NAcetyl compounds). The resonances from coupled spin multiplets
between 2.0 and 2.7 ppm are not observable at this value of TE.

metabolites
are of particular
interest because of their roles in neurotransmission
and for Glu and Asp in their excitotoxicity.
Consequently,
the ability
of H MRS to detect changes
in their
concentrations
may provide insights into the etiology
of neurodegenerative
disease that could not have otherwise been obtained
(VanderKnaap
et al., 1992). It must be borne in mind, however,
that the limitations
in spatial resolution
and sensitivity
inherent
in MRS will prevent the discrimination
between the amino acids
residing in different cellular subpools, e.g., intracellular
vs extracellular
synaptic
compartments.
However,
the observation
of
changes in GABA concentrations
resulting from antiepileptic
treat-

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ments (Preece et al., 1991; Confort-Gouny et al., 1993) have begun

to be reported. The observation of Gln, on the other hand, could
shed light on its activity as a Glu precursor, as well as its role in
the metabolism of ammonia in diseases where abnormalities of
ammonia are present (Preece et al., 1991; Ross, 1991; Kreis et al.,
1992b; Confort-Gouny
et al., 1993). Other coupled spin metabolites that are being studied include myo-inositol, a sugar involved
in several mechanisms including a secondary messenger system
(Berridge et al., 1989); glucose, the basic substrate of brain
metabolism, and the observation of which has been reported under
both normal (Gruetter et al., 1992) and hyperglycemic conditions
(Kreis et al., 1992a); and lactate, whose concentration has been
shown to increase as a result of anaerobic metabolism. The development of methods for observing coupled spins is covered in Subheading 3.
2. localized

H Spectra of Methyl

Singlets in Human

Brain

2.1. Methods
Localized single voxel H spectroscopy studies of the human
brain have increasingly relied on the use of two localization pulse
sequences, namely, the STEAM (stimulated echo-acquisition
mode) (Frahm et al., 1989a) and the PRESS (point resolved spectroscopy) (Gordon et al., 1984; Bottomley, 1987) schemes. Both of
these techniques allow for localization to a volume whose dimensions, and orientation in space are defined by the three orthogonal slice-selective pulses present in each of the sequences. The
location of that volume is at the intersection of these slices. In
addition to the localization pulses, additional pulses are usually
included in both of the sequences to bring about water suppression. This can be done either by frequency selective saturation
(Haase et al., 1985; Frahm et al., 1989a) or by inversion nulling
(Patt et al., 1972). Water suppression is necessary because of the
large difference in concentration between the water (50 M) and
the metabolites (up to -10 mM) to be measured.
As well as the methods for acquiring single voxel spectra, there
are several methods that allow a spatial mapping of a metabolite
over a predefined brain slice. These fall into the category of spectroscopic or chemical shift imaging methods (Brown et al., 1982;
Maudsley et al., 1983; Pykett et al., 1983; Dixon, 1984). Through
postprocessing, such techniques can provide either a complete H

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spectrum from each adjacent voxel within a slice or, alternately,

an image representing the concentration map of a single metabolite resonance throughout the slice. Generally, the much longer
acquisition period required for these methods, as well as the signal processing, extracts a significant time penalty.
For the majority of studies reported in the literature, the spectroscopic pulse sequences are applied using a circumscribing
radiofrequency
(RF) head coil, often a birdcage coil (Hays et al.,
1985; Tropp, 1989; Vu110 et al., 1992), which facilitates the acquisition of preparatory NMR images used for volume selection or registration. A much smaller number of studies have used a surface
coil for both transmission and signal reception (Ackerman et al.,
1980), primarily to take advantage of the higher receiver sensitivity of the surface coil for selected volumes that lie close to the
surface of the head. In addition, the lower RF power requirements
of surface coil transmission are advantageous in studies performed
at higher magnetic field strengths (3-4 T), where typical RF power
levels of a circumscribing coil would have exceeded safety guidelines (Athey, 1992).
One consequence of the time required to execute all the RF and
gradient pulses necessary for localization ofH spectroscopy in vivo
is that a significant part of the available signal can be lost through
various relaxation mechanisms. The reported resonance peak ratios
of the various metabolites are therefore weighted by the respective
transverse relaxation rates (T,s> (Hanstock et al., 1988; Frahm et al.,
1989b) of the metabolites in question. The variation in T2sbetween
the metabolite resonances thus makes the concentration ratio measurement strongly dependent on the spin-echo time or TE that was
used in the experiment. Because there is also a magnet field dependence of Tz, care must be exercised when comparing data from one
field strength/laboratory
to another. The TE chosen for the pulse
sequence also affects any additional signal loss resulting from
molecular diffusion in any field inhomogeneity, a loss which is governed by the nature of the pulse sequence used.
Methods for absolute concentration quantification have been
reported (Kreis et al., 1993a), and have made use of both internal
(e.g., Cr, water) and external (water) concentration references. Such
methods make use of corrections for differences in the T2 relaxation rates of metabolites and for partial volume effects caused
by regions of CSF falling within the selected volume.
For metabolites that are freely mobile, the fundamental factor
affecting the sensitivity or signal-to-noise (S/N) of the methyl sin-

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glet peaks is the total metabolite concentration present in the

selected volume. Bound metabolites cannot be observed by a typical in vivo MRS spectrometer. The MRS visible concentration
determines both the minimum volume size accessible in a given
time and the minimum amount of signal averaging that will be
required to give an adequate S/N in a time tolerable for the subject. A second factor affecting S/N is the magnet field homogeneity within the selected volume. Manual and automatic shim
routines allow for optimization of field homogeneity, however,
one cannot loose site of the fact that placement of the voxel adjacent to certain structures, e.g., near to bone or air interfaces, can
substantially limit ones ability to shim. This becomes more serious at higher magnetic field strengths where the dlstortlons m
the field homogeneity at tissue interfaces due to susceptibility
effects become more significant. The increase in signal strength
and hence the improvement in S/N obtained by using a higher
magnetic field is partially offset by the reported shortening of
metabolite T2s, particularly where longer TE experiments are
described. Typical acquisition times for single volume spectra are
in the 2-10 min range, whereas for a spectroscopic image the
acquisition time may be over 1 h.
2.2. Distribution
of the Resonances
in Human
Brain

NA,

Cr, and Cho

The distribution of the three most easily observable metabolite

resonances in the proton spectrum of the human brain, shown in
Fig. 2, has been studied on both the macroscopic and the cellular
levels. For example, several studies have demonstrated the differences in metabolite levels between gray and white matter, whereas
others have reported the metabolite complement in different cell
types grown in culture, as a guide for in vivo observations.
The NA resonance has been proposed and has gained considerable acceptance as an index of the neuronal pool size, since a
growing body of evidence suggests that the amino acid NAA is
confined to neurons (Birken et al., 1989; Urenjak et al., 1992;
Urenjak et al., 1993). The only reported exception has been that
detected in oligodendrocyte type-2A progenitor cells (O-2A) cultured in vitro (Urenjak et al., 1992; Brand et al., 1993; Urenjak et
al., 1993). Because this latter cell type would not be expected to
contribute significantly to MR spectra acquired from healthy adult

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MRS
NAA + N-Ace@?

Cho

.i
I

3.5

2.5

Chemical Shift (ppm)

Fig 2 Proton MR spectrum from human brain acquired at 3 T using
the PRESS pulse sequence with an intermediate
TE = 30 ms, from the
temporal lobe of a normal volunteer. Because of the intermediate
nature
of TE, the overlapping
multiplet resonances of the coupled spin metabohtes can be clearly seen in the 2-O-2.7 ppm region of the spectrum.

brain, it may be an issue in studies of developing brain and in

injured brain. Specifically, injured brain has been shown to have
increased activity of platelet derived growth factor and fibroblast
growth factor, both of which induce O-2A adult cells to exhibit
characteristics of O-2A perinatal cells (Wolswijk et al., 19921, which
in turn may modulate the NA peak measured by MRS.
In normal brain the distribution of NAA is reported to be 525% higher in gray matter than in white matter (Kreis et al., 1993a;
Michaelis et al., 1993; Hetherington et al., 1994a; Kreis, 1994), possibly indicating an increased concentration in the nerve cell bodies compared
to the axons. It has also been suggested that this

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apparent concentration gradient may be due to the higher axonal

activity of the enzymes NAA-aminohydrolase
and L-aspartate-Ntransferase (Burri et al., 1991). The action of these enzymes would
result in a faster turnover of NAA, an important element in its
proposed role as an acetyl reservoir in lipid synthesis (Matalon et
al., 1989; Burri et al., 1991; Kunnecke et al., 1993; Petroff et al.,
1994). The NA resonance peak at 2.02 ppm has signal contributions from N-acetylaspartylglutamate
(NAAG) as well as NAA.
Immunohistochemical
studies have shown that while NAA and
NAAG both stain positively to carbodiimide in the brain, they
appear to be exclusive in location, with NAAG in mterneurons and
NAA in pyramidal neurons (Moffett et al., 1993). Because both NAA
and NAAG are exclusive to neuronal elements, then their sum, as
measured by in vivo MRS, continues to remain a potential marker
for the overall neuronal pool within the voxel of interest.
Because of its ubiquity and suggested uniform distribution in
normal brain (Frahm et al., 1989a), many MRS studies have used
total Cr (Cr + PCr) as an internal concentration reference when
exploiting metabolite peak ratios as a means to quantify apparent
concentration changes. In contrast, several quantitative MRS measurements of direct concentration in vivo (Kreis et al., 1993a;
Michaelis et al., 1993; Hetherington et al., 1994a; Kreis, 1994) and
in tissue extract (Petroff, 1989) indicate that there is a variation in
the total Cr concentration levels, where gray matter has a 25-30%
higher concentration than white matter. Moreover, it has been
reported that creatine concentration increases in the proportion
1:2:4 between neurons:astrocytes:oligodendrocytes
grown in culture (Urenjak et al., 1992; Urenjak et al., 1993) the latter pair of
these being located predominantly
in gray matter. While these
three cell types occupy a significant proportion of the cytoplasmic space in normal brain, studies of injured brain have revealed
that elevated levels of macrophages may be observed Petroff et
al., 1992; Lopez-Villegas et al., 1995). This is relevant because macrophages in culture have been shown to possess elevated PCr concentrations when activated, and would contribute to the total Cr
pool measured by H MRS (Seguin et al., 1990; Seguin et al., 1991).
The MRS peak designated as Cho is the sum of contributions
from several choline derivatives including free choline, phosphorylcholine, and glycerophosphorylcholine,
as well as those of
noncholine origin from betaine and carnitine. Several quantitative MRS studies comparing the Cho concentration in gray and

Applrcations

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MRS

white matter have found there to be no significant difference

between them (Kreis et al., 1993a; Michaelis et al., 1993; Hetherington
et al., 1994a; Hetherington et al., 1996). Owing to the involvement
of cholines with membrane lipids, particularly myelin in brain
tissue, it has been suggested that choline containing compounds
would be expected to rise in conditions of membrane disruption,
as may be experienced following brain injury (Brenner et al., 1993;
Szigety et al., 1993).
2.3. Observation
of Spectral Changes of NA, Cr, and Cho
from H MRS in Human Subjects
2.3.1,

Brain

Development

and Aging

Several studies have been performed that explored the changes

in the II MR spectrum resulting from early development and from
aging of the brain (Kreis et al., 199313;Chang et al., 1996; Ashwal
et al., 1997). The most marked changes occur in the first 6 mo after
birth, when a rapid increase in the NA/Cr ratio is observed,
accompanied by a decrease in the Cho/Cr ratio at a similar rate.
The factor of 2 increase in the NA intensity from birth to adult
brain was interpreted as neuronal development
(Kreis et al.,
1993b), whereas the elevated Cho was thought to reflect accelerated myelination in the first few months of life. For a group of
adults in the age range 19-78, a quantitative study estimating
metabolite concentrations in frontal white matter found that while
the NA was relatively stable, there were increases with aging in
the Cr and Cho resonances in gray matter, whereas in white matter, no significant changes in metabolite concentrations were
observed (Chang et al., 1996).
2.3.2.

Neurodegenerative

Diseases

Neurodegenerative
diseases such as Alzheimers disease (AD),
amyotrophic lateral sclerosis (ALS), and multiple sclerosis (MS),
which involve the regional loss of neuronal tissue, are potentially
fertile areas for study by H NMR. The modulation of the NA peak
has been the focus of attention owing to its postulated role as a
neuronal marker. Observations of a decline in the NA peak, typically reported as changes in the NA/Cr or NA/Cho ratios, have
been ascribed to a depletion in neurons. It is important to bear in
mind, however, that in order to observe a significant decrease in
these ratios, a rather substantial decrease in neurons per unit vol-

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ume is required.
The reason for this stems from the large cytoplasmic
volume
occupied by neurons (-80%) compared
to glia,
coupled to the fact that the neuronal metabolite
pool has all three
metabolites
present, whereas the glia contribute
only to the Cr
and Cho peaks.
MRS studies of AD have examined
tissue extracts from brain
regions that encompassed
a range of senile plaques. In one study,
significant
decreases in the NA intensity
for AD brain samples
were observed compared
to controls, with the largest decreases
correlating
with the largest number
of senile plaques (Klunk
et
al., 1992). In a second study, whereas decreases in the NA intensity (20-30%) were observed u-t samples from cortical gray matter
regions, no changes were observed in the cortical white matter
samples (Kwo-On-Yuen
et al., 1994). In contrast, an in vivo study
using spectroscopic
imaging
methodology
reported
significant
decreases in the NA/Cr
and NA/Cho
ratios for selected volumes
in white matter, but no differences in the Cho/Cr
ratio between
the AD brain and controls
In the posterior
centrum
semiovale,
however,
NA/Cho
and Cho/Cr
ratios were both increased
and
no change in the NA/Cr
ratio was observed (Meyerhoff
et al.,
1994a). The authors concluded
that their data suggest diffuse
axonal loss accompanied
with membrane
alterations
in both gray
and white matter.
The application
of MRS techniques
to the study of the rapid
neurodegeneration
resulting
from ALS has also received attention recently. Initial
reports focused on the neuronal
loss m the
primary motor cortex and showed significant
decreases in the NA/
Cr ratio (Pioro et al., 1994; Jones et al., 1995; Gredal et al., 1997)
Similar conclusions
have been made from studies of the bramstem,
with a strong correlation
between upper motor neuron and bulbar function loss based on neurological
testing and the degree of
NA/Cr
depletion
(Cwik et al., 1997). A strong correlation
between
the extent of motor cortex depletion
and brainstem
depletion
resulting
from ALS, in addition
to a progressive
reduction
of the
NA/Cr
ratio has been reported in abstract form (Hanstock
et al.,
1997) when longitudinal
measurements
were made at 2-3-mo
intervals
over a 1-yr period.
In vivo MRS studies from brain regions that were hyperintense
on MRI and associated with MS lesions revealed that the NA/Cr
ratio could be decreased by up to 30%, with the largest reductions
observed
for those patients
who were most severely affected

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(Arnold et al., 1990a; Matthews et al., 1991; Miller et al., 1991;

VanHecke et al., 1991; Arnold et al., 1992; Bruhn et al., 1992; Arnold
et al., 1994; Davie et al., 1994; Husted et al., 1994; Pan et al., 1996a).
Changes in the NA/Cho ratio showed a similar pattern, with a
reduction of similar magnitude to that reported for the NA/Cr
ratio. These observations did not depend on whether the lesions
were acute or chronic. Longitudinal studies for chronic lesions
found that while MR images showed little change in the extent of
the lesions studied, MRS measurements of the NA/Cr ratio
showed further decreases 12-18 months after the initial examination (Arnold et al., 1994). In contrast, studies of acute MS lesions
reflect a significant decrease in the NA/Cr ratio at the onset of
lesion development, which decreases further over a l-4 mo period,
followed by a recovery toward control values in the subsequent
4-8 month period (Davie et al., 1994). Reductions in the NA/Cr
ratio have also been observed in normal appearing white matter
in patients with either acute or chronic MS lesions, with the magnitude of the reductions being intermediate between control and
lesion values (Davie et al., 1994; Husted et al., 1994). False assignments of MS lesions based on NA/Cr ratio measurements has been
overcome by the use of MR image segmentation to estimate the
gray:white mix in selected MRS voxels (Hetherington et al , 1996;
Pan et al., 1996a).
2.3.3.

lschaemia

The modification of metabolite concentrations caused by the

interruption of blood flow to the brain has been evaluated by in
vivo MRS in the case of stroke and cardiac arrest. All studies of
chronic lesions resultmg from stroke have demonstrated that there
is a significant decrease in the NA peak relative to the peaks of
other metabolites, thereby suggesting neuronal loss (Graham et
al., 1992; Petroff et al., 1992; Sappey-Marinier et al., 1992; Graham
et al., 1993; Gideon et al., 1994; Hetherington et al., 1994b). In the
acute stages of lesion development, however, NA levels were
shown to remain in the normal range, and remain so for the first
l-2 wk following the event (Graham et al., 1993). Using spectroscopic imaging and taking advantage of a magnetic field strength
of 4.1 T, the spatial distribution of the NA resonance intensity was
determined
following
a stroke (Hetherington
et al., 1994b).
Extracting subspectra from a region within the 6-wk-old infarct
showed a total absence of NA, whereas a volume adjacent to the

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infarct showed only a decrease in the NA compared to that in an

equivalently located volume in the contralateral hemisphere.
In a study of coma, resulting from a variety of insults in newborns, infants, and children, occipital NA/Cr was lower in the
infants and children, whereas Cho/Cr was elevated in all groups
when compared to age-matched controls (Ashwal et al., 1997). The
extent of NA/Cr and Cho/Cr ratio changes were further increased
in those patients who had elevated lactate. Patient outcome and
recovery was shown to correlate strongly with the extent of these
abnormal metabolite concentration ratios. A similar study for adult
subjects in a coma, and currently under review, has shown that
serial NA/Cr ratio measurements declined following ischemia
with the rate and extent of reduction being predictive of outcome
(Penn et al., 1997). Moreover, postmortem studies on the nonsurvivor
group showed that the largest decreases in the NA/Cr ratios correlated with the largest loss of nerve cell bodies and axons following
histological examination and cell volume estimation.
2.3.4. Cancer
MRS studies of cancer in brain has taken place at both in vivo
and in vitro levels of investigations. Whereas in vitro studies of
cultured human tumor cell lines and excised tumor tissue has
facilitated the identification of tumor-borne metabolites, in vivo
applications have enabled comparisons of metabolite pools to be
measured between regions located within the tumor, adjacent to
the tumor, and in contralateral brain. By using the MRS data from
the contralateral region as an intraindividual
control, the metabolite pool changes occurring adjacent to the tumor, which result
from the effects of, for example, compression and peritumor
edema, have been evaluated. Moreover, the metabolic milieu of
the tumor tissue, and the effects on metabolism as a response to
forms of therapy have also been investigated.
The presence of all three singlet metabolite peaks, which are
routinely observed in normal brain (NA, Cr, Cho), has been demonstrated in extract studies of excised tumor tissue in vitro (Peeling et al., 1992; Kotitschke et al., 1994; Florian et al., 1995; Carpmelh
et al., 1996). However, the relative proportions of these peaks
reflect the differentiation
of tumor types. The presence of a NA
peak was considered to stem from residual brain that had been
infiltrated by the tumor and was therefore contaminating
the
excised tissue sample (Peeling et al., 1992; Carpinelli et al., 1996).

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Glioblastoma multiforme exhibited an elevated Cr/Cho ratio when

compared to either differentiated
or anaplastic astrocytomas
(Carpinelli et al., 1996). Conversely, in tissue derived from astrocytoma, gliobastoma on malignant melanoma, the choline level
was reported to vary not only between different tumor types, but
also between tumors of the same type and level of malignancy,
and also between samples from within the same tumor (Kotitschke
et al., 1994).
In vivo studies of tumor metabolism as a means to assess tumor
grade and type have been the focus of several reports and reviews
(Arnold et al., 1990b; Kugel et al., 1992; Barker et al., 1993; Ott et
al., 1993; Usenius et al., 1994a,b; Negendank et al., 1996; Preul et
al., 1996). All report significant changes in the metabolite ratios,
where, as a general rule, the NA/Cr ratio decreased and the Cho/
Cr ratio increased. These data were interpreted in early studies as
a decrease in NA intensity (loss of neurons in the sampled volume), and an increase in Cho (increase in lipid metabolism or
mobilization) (Arnold et al., 1990b; Kugel et al., 1992; Barker et
al., 1993; Ott et al., 1993). However, as a result of quantitative concentration measurements for astrocytomas in vivo (grades I-IV),
it was realized that the Cho concentration remained relatively
constant and that the Cr concentration decreased from grade I-II
tumors through to grade IV (Usenius et al., 1994a,b). Confirmation of these in vivo data was provided by the examination of
tissue extracts derived from excised tumor.
In a large 15 site study, an attempt was made to evaluate and
classify several tumor types based on their Cho/Cr, NA/Cr and
Cho/NA ratios (Negendank et al., 1996). Whereas all tumor types
had ratios distinguishable from normal brain, classification was
imprecise due to the large data scatter, astrocytomas being a particular problem. Using a method described as metabolic profiling, Preul et al. report the accurate classification of 90/91 tumors,
compared to 71/91 correct diagnoses obtained using the primary
preoperative clinical tests of CT, conventional MRI, and conventional angiography (Preul et al., 1996). This spectroscopic metabolite profiling requires the concentrations of seven metabolites to
be estimated, six from the tumor (Cho, Cr, NAA, alanine, lactate,
and lipid) and Cr from normal contralateral brain (used as a reference). The ratios of the six tumor to contralateral Cr are then
expressed as a ratio profile which was shown to be characteristic
for each of the tumor types examined grade II (low grade) astro-

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cytoma, grade III (anaplastic) astrocytoma, grade IV astrocytoma

(glioblastoma multiforme), meningioma, and metastases from lung
and breast cancer).
Metabolite ratios in regions adjacent to tumors are reported to
be dependent on the presence or absence of peritumor edema
(Kamada et al., 1994a,b). If peritumor edema is present, then a
significant decrease in the NA/Cr ratio was observed, moreover,
this decrease returns to normal values as the edema dissipates.
The large decrease in the T, relaxation rate for the metabolites
that was observed to accompany edema is suggested as responsible for some of the changes in metabolite ratios.
Several studies have examined the effects of radiation therapy
on the metabolite composition of tumor tissue, and also on that of
the normal brain (Szigety et al., 1993; Sijens et al., 1995, Usenius et
al., 1995). Decreases in the tumor Cho were observed following
radiation therapy, which were suggested to result from a decrease
in cell density (increased interstitial space) (Sijens et al., 1995).
The decrease in NA intensity observed m normal brain, which
had received a substantial dose of radiation (Szigety et al., 19931,
was recently confirmed by more careful quantitative measurements (Usenius et al., 1995).
2.3.5. HIV
Changes in the metabolite ratios observed in spectra obtained
from patients with HIV, where dementia has been diagnosed,
showed significant reductions in the NA/Cr ratio, and increases in
the Cho/Cr ratio for both gray and white matter regions (Menon et
al., 1990; Chong et al., 1993; Laubenberger et al., 1996; Tracey et al.,
1996). For HIV-positive asymptomatic patients, one study showed
that the NA/Cr was only slightly decreased, while the Cho/Cr ratio
was unchanged (Laubenberger et al., 1996). Conversely, another
study using patients in the early stages of HIV infection showed a
significant elevation of the Cho/Cr ratio, but with no change in the
NA/Cr ratio, suggesting that the elevation of Cho may be a marker
prior to the onset of dementia (Tracey et al., 1996). A study showing changes in the NA/Cr ratio for children with AIDS, and grouped
according to the clinical parameters of encephalopathy (AE) or
nonencephalopathy CANE), showed that in the basal ganglia both
the AE and the ANE groups had reduced NA/Cr ratios compared
to controls (Pavlakis et al., 1995>, whereas in white matter only the
AE group had an NA/Cr ratio lower than the controls.

Applications

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367

3. The Development
of Techniques to Measure
Metabolites with Coupled Spins
3.1. introduction
The ubiquity of the proton gives rise at one and the same time
to enormous analytical potential (because each and every metabolite has a proton spectrum), as well as to serious problems of analytical discrimination (because the small chemical shift range of
the proton often leads to unmanageable overlap of metabolite
spectra, particularly at 1.5 T). The richness of the proton spectrum from brain is illustrated by the 2-3 ppm section of a 300MHz spectrum from a cat brain extract shown in Fig. 3, where the
narrowness of the lines in aqueous solution, coupled to the
enhanced chemical shift dispersion at - 7 T, give rise to a partial
resolution of all of the metabolite multiplets. However, during in
vivo application of MR spectroscopy, differences in magnetic susceptibility on a microscopic spatial scale within the tissue milieu
give rise to resonance linewidths (- 0.1 ppm) which tend to obscure
the finer chemical shift separations and particularly the multiplet
splittings. Although brute force enhancement of the chemical shift
dispersion by increasing the magnetic field strength is a viable
option in analytical applications of NMR spectroscopy in vitro, it
is not an option in vivo because of the concomitant increase in RF
heating and because of technological difficulties in manufacturing large-bore whole-body magnets capable of generating very
high magnetic fields
When trying to extract concentration
information
for the
severely overlapping resonances of brain metabolites that are often
less concentrated than the NA, Cr, and Cho covered in Subheading 2., four broad options are available in vivo. The first option is
to carry out a detailed numerical modeling of the whole in vivo proton spectrum (Provencher, 1993; Provencher et al., 1995; Stanley
et al., 1995), using as fitting parameters the relative metabolite
concentrations and as the basis functions, predetermined
individual metabolite spectra. The second option 1s to move to as high
afield strength as possible (Mason et al., 1994; Gruetter et al., 1996;
Pan et al., 1996b), in the hope that the concomitant increases in
signal-to-noise ratio (SNR) and chemical shift dispersion will
clarify the spectrum sufficiently for metabolite quantification. In
the many cases of metabolites with coupled proton spins, a third

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Hanstock and Allen

NAA

NAA

3.2

GABA

GABA

2.8

2.4

2.0

1.6

Chemical Shift

(ppm)

Fig. 3. A limited region (2-3 ppm) of the 300 MHz proton spectrum
from an acid extract of cat brain.

option is to acquire more information by way of a spectvuEly twodimensional (2-D) spectrum (Berkowitz et al., 1988; Hurd et al., 1991;
Brereton et al., 1994; Dreher et al., 1995; Ryner et al., 1995; Ziegler
et al., 1995; Kreis et al., 1996) that separates into the second NMR
dimension the unique coupling information of all the metabolites
present. The fourth option, also viable only for the metabolites
with coupled spins, is to reduce the information content of the spectral acquisition by editing the one-dimensional
(1-D) spectrum
(Rothman et al., 1984; Dumoulin, 1985; Hetherington et al., 1985;
Williams et al., 1986; Hanstock et al., 1987; Hanstock et al., 1988;
McKinnon et al., 1988; Sotak et al., 1988; Brereton et al., 1990;
Knuettel et al., 1990; Trimble et al., 1990; Thomas et al., 1991;
Rothman et al., 1992; deGraaf et al., 1993; Rothman et al., 1993), so

Applications

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as to observe only a single multiplet from a single metabolite while

suppressing the signal from all but that predefined metabolite.
The technical problem of spectral discrimination is exacerbated by the issue of spatial encoding, which is basic to all in
vivo MRS studies. The spectral discrimination problem has in
general been addressed in the two limiting cases of either single
voxel localization or multiple voxel spatial maps (spectroscopic
imaging or chemical shift imaging, CSI). In the former limit, either
a complete 1-D spectrum or an edited spectrum is acquired from
the single voxel of interest, In the latter limit, multiple 1-D spectra are obtained, each localized to an individual voxel, (Brown et
al., 1982; Adalsteinsson et al., 1993; Meyerhoff, 1994b; Hwang et
al., 1996) and often rendered into an image for a single peak. For
example, the phosphocreatine (PCr) peak in the 31Pspectrum or
the Cho, Cr, and NA methyl singlets in the proton spectrum readily
provide metabolite images because they are strong and have a
sufficiently long T,. However, for the weaker, broad, and overlapping multiplets of coupled spin systems, e.g., the amino acid
neurotransmitters in the proton spectrum of brain, it is questionable if at 1.5 T this methodology can give rise to quantitative maps
of concentration that are free from overlap artifacts. The edited
multiplet is, nevertheless, still a viable option at 1.5 T for producing either a single voxel measurement or a two-spatial-dimension
concentration map of a single coupled-spin metabolite resonance.
3.2. Metabolic

Specificity

The MRS signal will be most metabolite specific if its identification is made to depend not on the usual single chemical shift
value of one of the resonances of the metabolite of interest, but
instead on a combination of all the chemical shift differences,
together with the indirect scalar couplings, associated with as
many coupled spin multiplets of the target metabolite as possible.
3.2.1.

Scalar

Coupling

The indirect scalar interaction (Abragam, 1961; Gunther, 1995)

is that which couples together the spins of the protons of neighboring molecular groups in the target metabolite molecule, e.g.,
the proton spins of the CH, the CH,, or the CH, groups etc., each
of which has a different chemical shift value. The strength (and
sign) of the scalar coupling interaction,f, which in turn determines
the multiplet splitting, is governed by the electronic structure of

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all the bonds between the two atoms whose nuclei are coupled and
through which the coupling information must be transmitted. As
such, J depends on bond hybridization, bond lengths and angles,
and, particularly, on the electronegativity
of substituents other
than protons bonded to the carbon atoms involved. A thorough
discussion of the determinants of J values can be found in any one
of a number of textbooks (Gunther, 1995). In NMR terms, the
magnitude of J determines the frequency splitting of the multiplets in a 1-D spectrum and it determines the frequencies of evolution of the various coherences that evolve between the pulses in a
multiple pulse experiment. The number of identical spins in each
of the coupled groups will determine the multiplicity of the multiplet in the 1-D spectrum, as well as the number of coherence
terms involved in the evolution of the density operator between
pulses (Ernst et al., 1987).
The complexity introduced into the nuclear spin dynamics by
the scalar coupling (and therefore into the response to a pulse
sequence) is heavily dependent on the relative strengths of the
scalar coupling and the chemical shielding. When the scalar coupling J is weak compared with the chemical shift difference 6
between the protons of the two (or more) coupled groups, e.g.,
for the CH to CH, coupling in lactate (called an AX, system) where
J = 7.8 Hz and 6 = 178 Hz, giving 6/J = - 23 (>> 1) at 1 5 T, the
edited multiplet behavior is independent of the magnetic field
strength B, and the mathematical analysis is straightforward using
product operator methods (Adalsteinsson et al., 1993). Under conditions of strong coupling, e.g., for the inequivalent methylene
proton coupling in citrate (called an AB system) where J = 15.4 Hz
and 6 = 8.3 Hz, giving 6/J = - 0.5 (I 1) at 1.5 T, the edited multiplet behavior becomes quite field dependent and difficult to evaluate analytically (McClung et al., 1986; Kay et al., 1988; Wilman et
al., 1995a).
Many of the multiplet chemical shifts and scalar couplings of
brain metabolites are listed by Behar and Ogino (Behar et al., 1991)
and although some of these fall under the category of weak coupling even at 1.5 T, e.g., the AX, systems of lactate and alanine,
and the A,M, coupling in the A,M,X, system of y-aminobutyric
acid (GABA), several are clearly strongly coupled even at the high
field of 4 T, namely, the ABX systems of the aspartate groups of
aspartate (Asp) and NAA and the M,N, coupling of the AM,N,Q
spin system of myo-inositol (Ins>. Between these extremes fall sev-

Applications

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MRS

365

era1 metabolites whose coupling cannot be described as weak, being

strong at 1.5T, but relaxing more towards the weak limit at 4 T.
Into this category fall the important metabolites of Glu and Gln,
collectively designated Glx, as well as the A,B, system of the two
methylene groups in taurine (Tau) and the N2Q coupling in the
AM,N,Q spin system of Ins. However, because of the high current interest in Glx, it is worth discussing this case in more detail.
The challenge with Glu and Gln arises not only because the similarity of their molecular structures produces multiplet chemical
shifts that are very similar for both molecules, but also because
the steric effects in the two Glx molecular structures produce
inequivalencies within both pairs of methylene protons in those
molecules and give rise to strong negative J couplings between
the protons of each of the methylene groups. These strong couplings cause the multiplet structure (and hence the overall spectral lineshape) to be quite sensitive to variations in pulse sequence
(both timings and pulse shapes) and magnetic field strength.
Under such circumstances a quantitative interpretation of the spectral intensity at any single frequency is not at all straightforward
and requires a detailed understanding of its origin.
3.2.2.

Numerical

Modeling

The identification of a metabolite, and the measurement of its

concentration, from a numerical modeling of the complete 1-D
proton spectrum relies on being able determine its individual contribution to the spectrum, usually at the single chemical shift value
of one of its multiplets. It has been practiced with some degree of
success by several workers (Behar et al., 1991; Provencher, 1993;
Provencher et al., 1995; Stanley et al., 1995). However, when spectral overlap occurs the spectral intensity at a single chemical shift
value is no longer a unique measure of a single metabolite and
one may have to assume that any changes observed in the intensity are because of only one of the contributing metabolites changing with pathology. Greater metabolite specificity may be obtained
by seeking consistency between the changes of more than one
multiplet of the metabolite in question at each of their characteristic chemical shift values. This makes the numerical fitting routines very dependent on a detailed understanding of the pulse
sequence dependence and the magnetic field dependence of all
the multiplet lineshapes. Even for a single metabolite, one cannot
assume that the shape and relative intensities of the multiplets

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remain the same at all echo times, even if all those multiplets were
to have the same relaxation times. This is illustrated quite strikingly in NAA (Wilman et al., 19961, by a comparison of the NA
singlet (2.02 ppm) and the strongly coupled aspartate ABX multiplet (2.6 ppm). Because of the strong coupling, the echo time
dependence of the aspartate multiplet is itself field dependent and,
moreover, at any field strength it is markedly different from that
of the uncoupled singlet, which depends only on transverse
relaxation. A lack of appreciation of this point could suggest that
the two NAA resonances were reflecting different concentrations
of NAA. The case of Glx is significantly more involved than NAA
in this regard.
3.2.3.

Spectroscopy

at High

Field

In Vivo

The luxury of a high field magnet (4 T, for example) clearly

mitigates several of the more severe difficulties associated with
strongly coupled spin systems at 1.5 T. Glx is a case in point. At
4.1 T, the team at the University of Alabama (Mason et al., 1994;
Pan et al., 1996b) have approximated the Glx response by means
of a weak couplmg approach in which the spin system is regarded
as a AM,X, system. In this approximation, the Alabama group
neglected the inequivalencies of the methylene protons on the C3
and C4 carbons CM2and X,, respectively) and assumed that 6,,/
J - 38 and 6,x/J MX - 6.6 correspond to the weak coupling limit.
Ir?Fomparison, a full calculation at two different field strengths,
namely, 1.5 T and 4 T (Allen et al., 1997), using all the J couplings
listed in that reference, illustrates some of the consequences of
making the weak coupling approximation even at 4 T. Nevertheless, the Alabama group have been able to provide quantitative
estimates of Glu m a number of human subjects.
The high field strength of 4 T has also enabled Gruetter et al.
(1996) to separate from the water peak, and subsequently observe
the 5.23 ppm peak of glucose. A comparison of the normally sought
3.44 ppm glucose peak, however, showed that even at 4 T the 3.44
ppm peak is still partially overlapping a 3.49 resonance assigned
to myo-Ins.
3.2.4.

2-D

Spectroscopy

In Vivo

The exploitation of 2-D spectroscopic methods to unravel complicated 1-D spectra is standard practice in chemical applications
of high-spectral resolution NMR (Ernst et al., 1987). For example,

Applicatrons

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MRS

367

J-resolved spectroscopy can provide a spectral map that separates

the multiplet structure from the chemical shift structure along
orthogonal axes of presentation, thereby eliminating the cause of
much overlap. COSY (Gunther, 1995>, on the other hand, gives a
2-D spectral map in which the peaks along the diagonal reflect
the 1-D spectrum and the off- diagonal peaks represent all the
connectivities. The exploitation of the same techniques in vivo
would be an ideal proposition. However, some of the constraints
of working in vivo have proved to be substantial handicaps. For
example, the extensive data acquisition period (previously 30 min
or more, but now as short as 15 min) which accommodates the
incrementation of the so-called t, interval, renders the technique
quite susceptible to subject motion, a serious issue when those
subjects are from neurodegenerative
brain patient populations.
Moreover, the longer values of the f, increment that are needed to
provide the appropriate sweep width at in vivo field strengths,
push out the total t, periods to values that can be comparable to or
greater than T, for metabolites in vivo. Signal loss due to transverse relaxation is therefore also a significant problem in vivo.
Nevertheless in certain casesJ-resolved spectroscopy has had some
success at 1.5 T, as recently demonstrated both in human brain
(Ryner et al., 1995) and human muscle (Kreis et al., 1996).
3.2.5.

Spectral

Editing

In Vivo

The question of whether or not to edit for a particular metabolite

resonance is one whose answer is dependent both on the metabolite
and on the magnetic field strength available. Overlap is the key
issue and even in the midst of some very crowded spectral regions,
e.g., 2.0 to 3.0 ppm and 3.3 to 4.3 ppm, the decision of whether or
not to edit is a subjective one. Once the decision to edit has been
made, the criteria by which a viable editing sequence must be judged
are as follows. First, the sequence must provide excellent background discrimination against overlapping resonances. Second, It
must be sufficiently fast to have a low vulnerability to motion artifacts, and third, the sequence length must be sufficiently short to
ensure that all editing and localization procedures can be accomplished well within T, in order to preserve signal strength.
A simple form of editing and one which has been used very
successfully in the weak coupling limit by the Yale group
(Rothman et al., 1984; Hetherington et al., 1985; Hanstock et al.,
1988; Rothman et al., 1992; Rothman et al., 19931, is that of difeer-

368

Hanstock and Allen

ence spectroscopy. Their principal applications have been to the lactate AX, system and the A,M,X, spin system of GABA. Although
this method takes advantage of the existence of scalar coupling
between groups within the target metabolite molecule, its metabolite specificity stems primarily from the uniqueness of the two
chemical shift values associated with the two coupled multiplets.
The Yale group has combined difference spectroscopy with surface coil (Bendall et al., 1985) and with ISIS (Ordidge et al., 1986)
localization techniques, as well as with several water-suppression
strategies (Rothman et al., 1993). The method is highly metabolite
specific and the intrinsic signal loss from the method is small when
the multiplicity is low. Its strength is in its simplicity, and it has
been applied most notably to groups of epilepsy patients to monitor the efficacy of GABA-enhancing drugs (Petroff et al., 1995;
Petroff et al., 1996). It is, however, quite vulnerable to background
subtraction artifacts arising from patient motion between subtracted scans, hardware instabilities, and minor differences in spin
dynamics owing to differences in the two pulse sequences. Certain arbitrary adjustments in one spectral intensity have been used
(Rothman et al., 1993) to optimize background cancellation. As a
result the efficacy of the singlet background elimination is much
more modest than that of the multiple quantum filters treated
below. Other groups have also used this technique to monitor
GABA (Preece et al., 1995; Keltner et al., 19961, though the latter
reference incorporates difference spectroscopy into a PRESS
sequence. The PRESS variant of difference spectroscopy has also
been proposed for lactate editing (Bunse et al., 1995). A variant of
the difference method (reported for a Glx phantom-only experiment [Lee et al., 19951) uses differential transverse relaxation at 4
T to provide the difference between two spectra. It is based on the
estimation of a particularly short T, for Glx (-50 ms), in contrast
to the T,s (-few hundred milliseconds) of other metabolites that
are present in the 2.00-3.00 ppm range of the proton spectrum.
An alternative to the dfirence spectroscopy approach is multiple
qtlantum coherencefiltering (Ernst et al., 1987; Gunther, 1995; Lee et
al., 1995). By using magnetic field gradients to filter out all but a
single order of multiple quantum coherence (MQC), the goal is to
produce a single shot editing method, which in addition to being
highly metabolite specific, is also far less vulnerable to patient
motion than editing methodologies that subtract successive scans.
The term single shot does not exclude signal averaging. It is

Applrcations

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369

simply meant to convey the notion that all information is obtained

from a single sequence. MQC filters also provide far greater background discrimination
against uncoupled spin magnetization,
such as the intense singlets of water, NA at 2.02 ppm, Cr at 3.02
ppm, and Cho at 3.2 ppm, which can easily be made as much as
three orders of magnitude in phantoms (McKinnon et al., 1988;
Wilman et al., 1993). In vivo, however (Keltner et al., 19971, singlet suppression has fallen far short of this.
Although MQC filters surpass difference editing in several
respects, they clearly suffer from weaknesses of their own. For
example, one weakness is the potential signal loss associated with
an MQC filter. This signal loss can arise for two main reasons.
The first is because of the limited inherent yield of the filter. The
more coherences there are to share the spin information,
the
smaller the magnetization that can be derived from any one. The
second loss mechanism is transverse relaxation, the seriousness
of which stems from the short T,s of metabolites in vivo relative
to the length of the filter sequence. Another weakness, and one
shared with difference editing, is the difficulty in suppressing
unwanted coupled-spin background when specific multiplets of
the target metabolite spectrum cannot be excited selectively without exciting background multiplets at the same time. This is a problem that is worse at low fields (1.5 T), and for backgrounds arising
from larger groups of coupled spins. Finally, because of the reliance on the careful manipulation of coherences by the RF pulses,
MQC editing in vivo is probably more demanding of RF pulse
integrity than is difference editing. The issue of B, inhomogeneity
caused by surface coil transmission (Shen et al., 1991) has been
thoroughly dealt with by Garwood and coworkers (Garwood et
al., 1991; deGraaf et al., 1995a,b) through the development of adiabatic pulses. The issue of self-refocussing in a spatially uniform
B, in order to maintain the relative phases of all coherences was
described by Geen and Freeman (Geen et al., 1991).
Procedures to mitigate the weaknesses mentioned above can
be illustrated by reference to two problems that have dominated
the MQC filter literature over the last decade. The first is the measurement of lactate in the presence of a lipid background and the
second is the measurement of the amino acid neurotransmitters.
Lactate measurement benefits from a simple weakly coupled spin
system, a long T, (Blamire et al., 1994; vanderToorn et al., 19951,
and a chemical shift difference between the coupled methine and

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methyl groups (2.78 ppm or -178 Hz at 1.5 T), which facilitates

selective excitation of the methine spins without perturbation of
the lipid spins. Because of lactates simple coupled-spin system,
it has been possible to derive a procedure to deal with the inherent yield problem (Trimble et al., 1990), which combines different
orders of coherence and recoups the full magnetization, thereby
giving an inherent yield of unity. To improve background lipid
suppression and maintain inherent yield, this procedure has been
incorporated into a series of sequences elegantly exploiting even
more spectral selectivity by the Johns Hopkins group (He et al.,
199513; 1996) and applied at 4.7 T to the measurement of lactate
and Iproplatin in tumors in rats and mice (He et al., 1995a). However, with the amino acid neurotransmitters,
the coupled-spin
systems are more complex, having a greater number of coupled
spins as well as strong coupling in several cases. Nevertheless,
strategies for mitigating signal loss due to both inherent yield and
to transverse relaxation have been proposed for the weakly
coupled GABA (Wilman et al., 1995b) and the strongly coupled
Glx (Thompson et al., 1997) and demonstrated in vivo on the normal human brain (Keltner et al., 1997; Thompson et al., 19971,
though the level of the success falls short of that achieved in the
simple lactate case.
The performance of MQC filters on phantoms is in little doubt,
largely because of the narrow linewidths and long T,s. The translation of this performance to an in vivo capability has not yet been
so well demonstrated. The crux of the matter seems to be the
incorporation of spatial encoding into the filter sequence without
undermining the filter specificity and sensitivity.
When the spin system is amenable to its use, e.g., lactate and
GABA, difference spectroscopy is simple and easy to use. Bearing
in mind its vulnerability to small patient movements, it provides
a broad measure of metabolite changes due to pathology or drug
therapy. When the spin system does not provide well-separated
multiplets, which are also weakly coupled, MQC filtration looks
much more promising. However, it is ironic that as one pushes
the MQC filter to the most demanding of tasks, e.g., Glx with its
coupled spin background, one finds oneself taking refuge in techniques such as spectral modeling, which one originally developed
the filter to avoid. Nevertheless, it should be realized that after
MQC filtration, the residual spectrum contains many fewer components than the unfiltered spectrum and, moreover, it is the larg-

Applications

of Proton

est of the background

most efficiently.

MRS

peaks (i.e., the singlets) that are suppressed

Acknowledgments
The authors are grateful to the Medical Research Council of
Canada for ongoing support of their spectral editing program.

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