11 Applications of Proton MRS to Study Human Brain Metabolism

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Applications of Proton MRS to Study

Human Brain Metabolism

Christopher 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 pro-

vides the means to monitor in time and in space changes in vari-

ous 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 criti-

cal 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 neu-

ronal 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 manage-

ment 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 acknowl-

edge that not only is MRS more demanding technically than MRI,

but the clinical significance of spectroscopic findings has not al-

ways been recognized prior to the in vivo application of the nuclear

magnetic resonance (NMR) spectroscopic techniques. The signifi-

cance 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 Ce ll Neurobiology Techm ques

Ed A A Boulton G B Baker and A N Bateson 0 Humana Press Inc

347





Applications of Proton MRS 349

N-Trimethyl

Chqphosphoryl-Cho.

glycerophos~horyl-ChoI

PR

CHZ

AH,

I+

CH,-I;I-CH,

Cr / PCr

N-Acetylaspartate

H

CH,COOH

HN NHR

NC/

'c'

I

HOOC

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 N-

trimethyl compounds, from Cr/PCr and from NAA (including other N-

Acetyl 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 neu-

rotransmission and for Glu and Asp in their excitotoxicity. Con-

sequently, the ability of ‘H MRS to detect changes in their

concentrations may provide insights into the etiology of neuro-

degenerative 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 extra-

cellular synaptic compartments. However, the observation of

changes in GABA concentrations resulting from antiepileptic treat-



350 Hanstock and Alien

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 metabo-

lites 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 devel-

opment of methods for observing coupled spins is covered in Sub-

heading 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 spec-

troscopy) (Gordon et al., 1984; Bottomley, 1987) schemes. Both of

these techniques allow for localization to a volume whose dimen-

sions, and orientation in space are defined by the three orthogo-

nal 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 suppres-

sion. 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 spec-

troscopic 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




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

an image representing the concentration map of a single metabo-

lite resonance throughout the slice. Generally, the much longer

acquisition period required for these methods, as well as the sig-

nal processing, extracts a significant time penalty.

For the majority of studies reported in the literature, the spec-

troscopic 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 acquisi-

tion of preparatory NMR images used for volume selection or reg-

istration. 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 sensitiv-

ity 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 guide-

lines (Athey, 1992).

One consequence of the time required to execute all the

RF

and

gradient pulses necessary for localization of’H 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 T2s between

the metabolite resonances thus makes the concentration ratio mea-

surement strongly dependent on the spin-echo time or TE that was

used in the experiment. Because there is also a magnet field depen-

dence 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 gov-

erned 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

relax-

ation 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-



352 Hanstock and Allen

glet peaks is the total metabolite concentration present in the

selected volume. Bound metabolites cannot be observed by a typi-

cal 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 sub-

ject. A second factor affecting S/N is the magnet field homogene-

ity 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 adja-

cent to certain structures, e.g., near to bone or air interfaces, can

substantially limit one’s ability to shim. This becomes more seri-

ous 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 NA, Cr, and Cho

in Human Brain

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 differ-

ences 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 consid-

erable 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) cul-

tured 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




NAA + N-Ace@?

Cho

.i L

I

3.5

I I I

3 2.5 2

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 metabo-

htes 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 5-

25 higher in gray matter than in white matter (Kreis et al., 1993a;

Michaelis et al., 1993; Hetherington et al., 1994a; Kreis, 1994), pos-

sibly indicating an increased concentration in the nerve cell bod-

ies compared to the axons. It has also been suggested that this



3.54 Hanstock and Allen

apparent concentration gradient may be due to the higher axonal

activity of the enzymes NAA-aminohydrolase and L-aspartate-N-

transferase (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 contribu-

tions 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 mea-

surements 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 cul-

ture (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 cytoplas-

mic 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 mac-

rophages in culture have been shown to possess elevated PCr con-

centrations 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, phos-

phorylcholine, and glycerophosphorylcholine, as well as those of

noncholine origin from betaine and carnitine. Several quantita-

tive MRS studies comparing the Cho concentration in gray and



Applrcations of Proton MRS 355

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 acceler-

ated 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 mat-

ter, no significant changes in metabolite concentrations were

observed (Chang et al., 1996).

2.3.2. Neurodegenerative Diseases

Neurodegenerative diseases such as Alzheimer’s 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, typi-

cally 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-




ume is required. The reason for this stems from the large cyto-

plasmic 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 inten-

sity (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 atten-

tion 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 bul-

bar 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




(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 examina-

tion (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 mag-

nitude of the reductions being intermediate between control and

lesion values (Davie et al., 1994; Husted et al., 1994). False assign-

ments 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 spectro-

scopic 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




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 new-

borns, 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 corre-

lated 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 metabo-

lite 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 dem-

onstrated in extract studies of excised tumor tissue in vitro (Peel-

ing 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).



Applicatrons of Proton MRS 359

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 astro-

cytoma, 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 vol-

ume), 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 con-

centration 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). Confirma-

tion 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 par-

ticular problem. Using a method described as metabolic profil-

ing, 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 conven-

tional angiography (Preul et al., 1996). This spectroscopic metabo-

lite 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 ref-

erence). 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-




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 respon-

sible 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 measure-

ments (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 show-

ing 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 of Proton M RS

3. The Development of Techniques to Measure

Metabolites with Coupled Spins

3.1. introduction

367

The ubiquity of the proton gives rise at one and the same time

to enormous analytical potential (because each and every metabo-

lite has a proton spectrum), as well as to serious problems of ana-

lytical 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 spec-

trum from brain is illustrated by the 2-3 ppm section of a 300-

MHz 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 sus-

ceptibility 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 manufactur-

ing 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 Subhead-

ing 2., four broad options are available in vivo. The first option is

to carry out a detailed numerical modeling of the whole in vivo pro-

ton 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 indi-

vidual 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




N

N

G B

J

G B

I

I

I I

I

3.2

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 two-

dimensional 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 spec-

tral 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 of Proton MRS

363

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 exac-

erbated 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 spec-

tra 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 over-

lapping multiplets of coupled spin systems, e.g., the amino acid

neurotransmitters in the proton spectrum of brain, it is question-

able 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 produc-

ing 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 identifi-

cation 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 neigh-

boring 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






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 cur-

rent interest in Glx, it is worth discussing this case in more detail.

The challenge with Glu and Gln arises not only because the simi-

larity 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 cou-

plings cause the multiplet structure (and hence the overall spec-

tral 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 spec-

tral 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 con-

tribution 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 spec-

tral 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 inten-

sity are because of only one of the contributing metabolites chang-

ing 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 character-

istic chemical shift values. This makes the numerical fitting rou-

tines 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




remain the same at all echo times, even if all those multiplets were

to have the same relaxation times. This is illustrated quite strik-

ingly in NAA (Wilman et al., 19961, by a comparison of the NA

singlet (2.02 ppm) and the strongly coupled aspartate ABX mul-

tiplet (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. Neverthe-

less, 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 com-

plicated 1-D spectra is standard practice in chemical applications

of high-spectral resolution NMR (Ernst et al., 1987). For example,



Applicatrons of Proton 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 trans-

verse 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 back-

ground discrimination against overlapping resonances. Second, It

must be sufficiently fast to have a low vulnerability to motion arti-

facts, and third, the sequence length must be sufficiently short to

ensure that all editing and localization procedures can be accom-

plished 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 lac-

tate 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 metabo-

lite 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 sur-

face 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 moni-

tor 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 sub-

tracted scans, hardware instabilities, and minor differences in spin

dynamics owing to differences in the two pulse sequences. Cer-

tain 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 experi-

ment [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 of Proton MRS 369

simply meant to convey the notion that all information is obtained

from a single sequence. MQC filters also provide far greater back-

ground 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, sin-

glet 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 with-

out exciting background multiplets at the same time. This is a prob-

lem that is worse at low fields (1.5 T), and for backgrounds arising

from larger groups of coupled spins. Finally, because of the reli-

ance 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 adia-

batic 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 mea-

surement 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




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 lactate’s simple coupled-spin system,

it has been possible to derive a procedure to deal with the inher-

ent 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). How-

ever, 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 nor-

mal 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 trans-

lation 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 tech-

niques 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 com-

ponents than the unfiltered spectrum and, moreover, it is the larg-



Applications of Proton MRS

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

most efficiently.

Acknowledgments

The authors are grateful to the Medical Research Council of

Canada for ongoing support of their spectral editing program.

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11 Applications of Proton MRS to Study Human Brain Metabolism