Download An In Vivo Proton Magnetic Resonance Spectroscopy Study of

VOL22.NO. 4, 1996
An In Vivo Proton Magnetic
Resonance Spectroscopy
Study of Schizophrenia
Patients
by Jeff A. Stanley, Peter C.
Williamson, Dick J. Drost,
R. Jane Rylett, Tom J. Carr,
Ashok Malta, and R. Terry
Thompson
Abstract
The level of the ] H metabolites in
the left dorsolateral prefrontal
region of schizophrenia patients at
different stages of illness were measured in vivo using a short echo
time spectroscopy technique. During both the early onset and chronic
stages, normal A/-acetylaspartate levels were observed, which suggests
that these patients had no significant neuronal cell damage and/or
loss. The in vivo measurements of
glutamate in thefirst-episode,drugnaive patients failed to provide convincing evidence for the involvement of the glutamatergic system in
the dorsolateral prefrontal region.
Significant differences in the glutamine levels were observed in the
acutely medicated and chronic patients; however, the interpretation of
these differences requires further
study.
Schizophrenia Bulletin, 22(4):
597-609,1996.
Evidence from several studies has
recently implicated the excitatory
glutamatergic system in the pathophysiology of schizophrenia
(Deutsch et al. 1989; Carlsson and
Carlsson 1990; Wachtel and Turski
1990; Uras and Cotman 1993). For
example, phencyclidine (PCP) and
ketamine, which are specific noncompetitive antagonists of the N-methylD-aspartate (NMDA) glutamate subtype receptors, induce psychosis
resembling schizophrenia in normal
controls and exacerbate psychosis in
schizophrenia patients (Javitt and
Zukin 1991; Krystal et al. 1994). In
several postmortem brain studies,
evidence of glutamatergic dysfunction in schizophrenia has been
reported, including abnormalities in
the binding density of glutamate sub-
type receptors in various brain
regions (Utas and Cotman 1993);
decreased release of glutamate in
synaptosomes prepared from cortex
tissue of schizophrenia patients
(Sherman et al. 1991); and a reduction
of messenger ribonucleic acid (RNA)
that encodes non-NMDA glutamate
receptors in hippocampal tissue
observed in patients compared with
controls (Harrison et al. 1991).
Obtaining information in vivo on
the biochemistry of patients with
schizophrenia can potentially contribute further understanding of the
chemical pathology that gives rise to
the glutamatergic dysfunctions
observed in postmortem studies.
Magnetic resonance spectroscopy
(MRS) is a noninvasive and nondestructive technique that can provide
such information (Bottomley 1989;
Kauppinen et al. 1993). MRS can
assess the viability of neuronal cells
by quantifying the peptide A/-acetylaspartate (NAA) (Arnold et al. 1990;
De Stefano et al. 1995), reliably quantify the in vivo relative concentration
of the excitatory neurotransmitter
glutamate (de Graaf and Bovee 1990;
Provencher 1993; Stanley et al.
1995a); and quantify additional cerebral metabolites including glutamine,
phosphocreatine plus creatine (PCr +
Cr), and choline-containing compound (Chot). Moreover, the localized region(s) where the in vivo
metabolic information is obtained
may be as small as several cm3 (Ernst
et al. 1989) and can be positioned in a
specific area of the brain such as the
dorsolateral prefrontal region. Consequently, phosphorus (31P) and proton
Reprintrequestsshould be sent to Dr.
PC Williamson, Dept. of Psychiatry,
University Hospital, 399 Windermere Rd.,
Box 5339, London, Ontario, N6A 5A5,
Canada.
SCHIZOPHRENIA BULLETIN
598
('H) MRS have been applied in several studies to investigate the biochemistry in vivo of patients with
schizophrenia (O'Callaghan et al.
1991; Pettegrew et al. 1991; Calabrese
et al. 1992; Sharma et al. 1992; Buckley et al. 1994; Nasrallah et al. 1994;
Stanley et al. 1994,1995b; Renshaw et
al. 1995).
In this in vivo study, 'H metabolite
levels from the dorsolateral prefrontal region of first-episode, drugnaive, and acute and chronic medicated schizophrenia patients were
compared to metabolite levels from
controls of comparable age, gender,
education, and parental education
levels. It was hypothesized that differences in levels of glutamate and /or
glutamine would be evident if the
glutamatergic system was involved
in the pathophysiology of schizophrenia. Differences in levels of NA A
in patients would suggest neuronal
cell damage or loss in the prefrontal
region. Examination of levels of
metabolites before and after medication might indicate some of the metabolic effects of these agents.
Methods
Subjects. Twenty-nine patients
with schizophrenia ranging in age
from 16 to 49 years participated in
this study. There were 13 firstepisode, drug-naive schizophrenia
patients, of whom 11 were classified
as paranoid and 2 as undifferentiated. The length of illness (the time
between the onset of positive symptoms and the MRS examination) for
the drug-naive patients ranged from
1 month to 6 years. None had been
exposed to any antipsychotic medication before the MRS examination.
However, within the 24 hours before
assessment, seven patients had
received 1 to 3 mg of lorazepam. The
remaining patients included 12 acute
medicated schizophrenia patients (of
whom 8 had been previously examined'as first-episode, drug-naive
patients) and 12 chronic medicated
schizophrenia patients. The acute
medicated patients with a mean
length of illness of 2 ± 2 years
included 10 classified as paranoid
and 2 as undifferentiated. The
chronic medicated patients group,
with a mean length of illness of 17 ±
6 years, had six diagnosed as paranoid, two as undifferentiated, and
four as residuals. All medicated
patients were receiving antipsychotic
medication. Eighteen of these
patients were also on anticholinergic
medication for side effects. The mean
length of time on medication for the
acute medicated patients was 14 ± 10
weeks.
The diagnoses of all the patients
were established with the Structured
Clinical Interview for DSM-1H-R
(SCID; Spitzer and Williams 1985)
administered by a psychiatrist. The
diagnoses of the drug-naive patients
were reconfirmed with the treating
psychiatrist 6 months after the MRS
experiments were done. Both the
Scale for the Assessment of Negative
Symptoms (SANS; Andreasen 1984a)
and the Scale for the Assessment of
Positive Symptoms (SAPS; Andreasen 1984b) were also administered
by a psychiatrist without any knowledge of the 'H MRS results. Education level was rated on a 4-point scale
(1 = s grade 10; 2 = grade 11-13; 3 =
1-3 years college or university; 4 =
more than 3 years college or university). Parental education ratings were
evaluated for the most-educated parent; however, three were evaluated
for the most-educated adoptive parent. Clinical information is summarized in table 1. The handedness of
each subject was defined by the hand
used to write and throw a ball. All
were right-handed.
Twenty-four nonschizophrenia
controls ranging in age from 16 to 53
were recruited by advertisement.
Each control subject was evaluated
with the SCID by a psychiatrist. Controls were of gender, age, education
level, parental education level, and
handedness (all were right-handed)
comparable to the patients. Subject
characteristics of the control group
are shown in table 1.
Patients and controls were free of
any history of head injury, drug or
alcohol abuse, or serious medical illness based on the information provided during the SCID interview. No
gross abnormalities were detected on
routine clinical magnetic resonance
(MR) images that were also collected
as part of the study on each subject.
None of the drug-naive patients, all
of the medicated patients, and 13 of
the 24 controls had undergone at
least one previous magnetic resonance imaging scan.
J
H MRS. The in vivo 1H MRS experiments were conducted using a
circularly polarized head coil on a
whole body MR unit (Helicon SP system, Siemens AG, Erlangen, Germany) with a static magnetic field of
1.5 Tesla. The STEAM sequence
(stimulated echo acquisition mode;
Frahm et al. 1989,1990) with an echo
time of 20 ms provided the single
voxel localization technique. The
mixing time interval was 30 ms.
Three Gaussian-shaped radio frequency pulses (CHESS pulses; Haase
and Frahm 1985), separated by
spoiler gradients, were placed at the
beginning of the STEAM sequence to
suppress the large contribution of the
water MR signal. The 'H MR signal
was obtained from 2 x 2 x 2 cm3 volume of interest (VOI) located in the
left dorsolateral prefrontal region of
the subjects. The VOI was positioned
599
VOL22.NO. 4, 1996
Table 1. Subject characteristics
Age
Group
Gender
(yrs)
Control group
Schizophrenia
patient groups
First-episode,
drug-naive
Acute medicated
Chronic medicated
24M/0F
32±11 2
11 M/2F 26±7
10M/2F 26 ±7
11 M/1 F 41 ±5
Parental
Education education
level1
level1
Length of
Illness
(yrs)
Length of
time on
medication
(yrs)
SANS
score
_
_
37 ±13
28 ± 14
26 ±12
3.0 ± 0.92
2.5 ± 0.92
_
2.3 ± 0.9
2.4 ± 0.7
2.6 ± 0.8
2.3 ± 0.8
2.2 ± 0.8
2.0 ± 0.9
2.0 ±1.9
—
2.5 ± 2.4
0.8 ±1.9
16 ±8.0
17 ±7.0
SAPS
score
_
30+ 11
9± 16
8 ± 11
Note.—Values are mean 11 standard deviation; M = males; F = females; SANS •= Scale for the Assessment of Negative Symptoms (Andreasen 1984a);
SAPS - Scale for the Assessment of Positive Symptoms (Andreasen 1984b).
'Education level: 1 « * grade 10. 2 • grade 11-13, 3 • 1-3 years college or university, 4 = more than 3 years college or university.
2
ln a two-tailed f-test, these values are not significantty different when compared with combined patient groups.
with a set of sagittal and coronal 'H
MR images as shown in figure 1. The
dashed lines in figure 1 represent the
anterior (a), posterior (b), lateral (c),
and medial (d) surfaces of the three
dimensional VOI (white box). The
magnetic field homogeneity was
maximized with a global head shim
followed by a localized shim on the
VOI. The interpulse repetition time
was 1,500 ms, and 450 acquisitions
were averaged. For each water suppressed 'H spectrum acquired, a
water unsuppressed ] H spectrum
was also collected (55 acquisitions)
by setting the amplitudes of the
CHESS pulses to zero voltage. Further details of the spectroscopy protocol are discussed by Stanley et al.
(1995a).
Spectral Processing. The data files
were coded such that the operator
had no knowledge of the subject's
status. A time domain deconvolution
technique, QUALITY (de Graaf et al.
1990), was first applied to the MR
signal to restore the spectral lineshapes to pure Lorentzian. The time
domain signal was then multiplied
by a Lorentzian-to-Gaussian transformation to enhance the apparent spectral resolution (Ferrige and Lindon
1978). The MR signal was then zero
filled, Fourier transformed, and
phased with Oth order and 1st order
(< one dwell period). No spline
function was applied to the baseline.
The data were processed on a personal computer using the NMR-286
software (Soft Pulse Software, Box
504, Guelph, Ontario, N1H 6K9,
Canada).
There is a direct relationship
between the area under a spectral
peak and the concentration of the
metabolite associated with that peak.
Therefore, Gaussian functions were
fitted to each spectral peak between
1.88 and 3.45 ppm (parts per million)
and the areas under the functions
were used to calculate the 'H
metabolite levels. To resolve the issue
of quantifying complex 'H spectra
with multiple peaks that overlap
each other (de Graaf and Bovee 1990;
Provencher 1993; Stanley et al.
1995a), a priori knowledge was incorporated into a frequency domain
nonlinear least-squares-fitting algo-
rithm (Marquardt 1963). The a priori
knowledge included information on
chemical shifts, relative amplitudes,
and linewidths of the peaks for each
metabolite. The quantified 1 H
metabolites included NAA, glutamate, glutamine, gamma-aminobutyric acid (GABA), aspartate, Nacetylaspartylglutamate (NAAG),
PCr + Cr, Chot, glucose, taurine,
scy//o-inositol, and two macromolecule resonances at 2.12 and 2.9 ppm
(Kauppinen et al. 1992; Behar and
Ogino 1993; Behar et al. 1994; Stanley
et al. 1995a). To calculate the relative
metabolite levels, the integral value
of the phased water peak from the
unsuppressed water 'H spectrum,
which represents the total MR visible
water content in the VOI, was used
as an internal standard to normalize
the metabolite peak areas (Christiansen et al. 1993). The quantified
areas of the spectral peaks were not
corrected for any attenuation due to
spin-spin (TJ or spin-lattice (T,)
relaxation; therefore the quoted relative metabolite levels have arbitrary
units. Complete details on the implementation of the a priori knowledge
SCHIZOPHRENIA BULLETIN
600
Figure 1. The location of the volume of Interest (VOI) in the left
dorsolateral prefrontal region
ables from the multiple regression
analysis. For the eight patients who
were examined twice (first as drugnaive then as acute medicated
patients) a two-tailed, paired f-test
was also used to compare the SANS
and SAPS scores and metabolite levels
between pre- and on-medication measures. Pearson product moment correlations of age, length of illness, length
of time on medication, equivalent
dose of chlorpromazine, and SAPS
and SANS scores along with levels of
NAA, glutamate, glutamine, PCr +
Cr, and Chot were evaluated for the
controls and the combined patients.
Results
C)
Sagittal and corona) 1H magnetic resonance Images with the 2 x 2 x 2 cm3 VOI (the white box) positioned In the left dorsolateraJ prefrontal region. The dotted lines in (a) and (b) represent position of the
corona) Images In (c) and (d) while the dotted lines In (c) and (d) represent the position of the sagittal
Images in (a) and (b).
into the fitting routine and on testing
the efficacy of quantifying in vivo 'H
MR spectra has been reported by
Stanley et al. (1995a)
Statistical Analysis. A two-tailed ttest was performed to determine any
significant differences in age, education level, and parental education
level when comparing the combined
patients with the control group. The
quantified ] H MR parameters were
modeled in a stepwise multiple
regression analysis with subject
group (i.e., patient and control), age,
gender, education level, and parental
education level as independent parameters. This method enabled us to
compare differences in the NAA, glutamate, glutamine, PCr + Cr, and
Cho, levels between patient and control groups while adjusting for the
covariables of age, gender, education
level, and parental education level.
Probability values of < 0.05 were considered statistically significant and a
threshold value of p = 0.2 was used to
enter or remove independent vari-
A typical processed in vivo TH spectrum acquired from the left dorsolateral prefrontal region of a control is
shown in figure 2. The spectrum contains no dominating broad spectral
resonances that underlie the baseline
noise over the 1.88 to 3.45 ppm spectral region. The signal-to-noise ratio
(i.e., the signal of the NAA peak at
2.02 ppm over the root mean square
of the noise) is approximately 80 (figure 2), which is adequate to reliably
quantify the spectrum (Stanley et al.
1995a). The quantification of this
spectrum is displayed in figure 2b as
a sum of all spectral peaks of the
metabolites superimposed on the
acquired spectrum. The difference
between the two is shown as the
residual plot in figure 2b.
Instructions to perform specific
physical or psychological tasks were
not given to the subjects during the
collection of the data. Therefore, in
general, the observed metabolite levels reflect steady-state values at rest.
The absolute difference in metabolite
levels of NAA, glutamate, glutamine,
PCr + Cr, and Cho, between the three
patient groups and controls is shown
in figure 3. In the controls, the coeffi-
601
VOL. 22, NO. 4, 1996
Figure 2.
An In vivo 1H magnetic resonance (MR) spectrum collected with the STEAM sequence
suppressed
water peak
a)
/^'<'W»^oAA/V»/*-vV'>***V«^^
y\
12.0
10.0
4.0
8.0
3.6
6.0
3.2
-2.0
2.8
2.4
2.0
1.6
-4.0
1.2
-6.0
0.8
Chemical Shift (ppm)
Figure (a) contains a typical processed in vrvo 1H MR spectrum from the left dorsolateral prefrontal region acquired with the STEAM (stimulated echo acquisition mode) sequence (TE = 20 ms) and (b) shows the same spectrum with the frequency region expanded. The result of modeling the spectrum with a priori
The spectral peaks are N-acetyiaspartate (NAA), glutamate (G)u), glutamlne (Qln), gamma-amino-butyric acid (GABA), NAACHJ (methyl resonance from the
NAA molecule), aspartate (Asp), phosphocreatjne plus creatine (PCr + Cr), chollne-containing compounds (Cho,), glucose (Gte), myixnosrto) (myo-lns),
scyflo-inositol (scyflo-lns), and taurine (Tau). ppm = parts per million.
cients of variation for NAA, PCr +
Cr, and Cho, were approximately 10
percent, and the coefficients of variation for glutamate and glutamine
were 22 and 33 percent, respectively.
Quantifying the metabolite levels of
GABA, aspartate, NAAG, taurine,
scy//o-inositol, and glucose was considered less reliable because their low
in vivo concentration levels resulted
in coefficients of variation i 30 percent (Stanley et al. 1995a), and therefore these ^H metabolites were not
tested in the statistical analysis.
There were no significant differences when comparing the ^H
metabolite levels of the first-episode,
drug-naive and acute medicated
patients with the control group. The
glutamate levels tended to be greater
in the acute medicated patients compared with the controls; however,
this difference did not reach significance (p = 0.10 where age and gender
were the covariates). In the paired ttest for the eight drug-naive patients
who were also examined as med-
SCHIZOPHRENIA BULLETIN
602
Figure 3.
Absolute metabolite level difference between schizophrenia patients and controls
I—
5 r
Controls
^SSI Drug-naive
Acute medicated
2
Chronic medicated
II \
B
0)
-1
0)
-2
o
c
CD
(5
-3
Q
-4
"o
(/>
< -5
L
NAA
Glu
Gin'
PCr+Cr
Cho.
NAA = AAacetytaspartate; Glu = glkitamate; Gin « glutamlne; PCr + Cr = phosphocreatine plus creatine; Cho, = choline-containing compounds; error bars
are ± 1 standard deviation.
'Values are relative to that of the controls (i.e., mean metabolite level of patient - mean metabolite level of controls).
'Significantly different when comparing the chronic medicated patients and the controls (p » 0.013).
icated patients, glutamine levels (p =
0.020) and SAPS scores (p = 0.006)
were both significantly reduced in
the on-medication measures compared with the premedication measures. When comparing the metabolite levels of the chronic medicated
patients and the control group, the
only significant difference was the
increase in glutamine in the chronic
medicated patients (p = 0.013 where
age, gender, education level, and
parental education level were the
covariates). Age, education levels,
and parental education levels of the
patients with schizophrenia were not
significantly different from the controls. These results are summarized
in table 1 and figures 3 and 4.
Combining the schizophrenia
patients together, the length of illness, length of time on medication,
equivalent dose of chlorpromazine,
and SANS and SAPS scores were not
significantly correlated (after applying a Bonferroni correction for multiple comparisons) with the levels of
NAA, glutamate, glutamine, PCr +
Cr, or ChOj, except for a significant
positive correlation between the glu-
VOL. 22, NO. 4, 1996
603
Figure 4. Palrwise comparison of the glutamine levels between
the premedication and on-medication measurements of the eight
schizophrenia patients
9 r
§
I
-2.
iE
ca
2
2
O
33
CD
>
jg 2
<D
1
Pre-medication
measurement
On-medication
measurement1
A solid line connects the premedteatlon and on-medlcatlon measurements of the same patient.
'On-medlcation measurements are significantly different than the premedication measurements
(p = 0.020).
famine level and the length of illness
(r = 0.53, p = 0.0007, figure 5). The
glutamine level and age correlation
was not significant in the controls.
Discussion
Assessing Neuronal Damage and
Loss. NAA levels in the left dorsolateral prefrontal region did not differ
between any of the patient groups
and the controls. In terms of the total
concentration of free amino acids in
mammals, NAA is second only to
glutamate (Tallan 1957). This is
reflected on the dominant spectral
feature of NAA (figure 2) that gives
rise to a reliable measure (Stanley et
al. 1995a). While the function of NAA
in the central nervous system (CNS)
has not been elucidated (Birken and
Oldendorf 1989), it has been established that NAA is found exclusively
in mature neurons and neuronal
processes (Matalon et al. 1988; Birken
and Oldendorf 1989; Urenjak et al.
1993). Decreased NAA levels have
been observed by MRS in numerous
cerebral pathologies involving neuronal cell damage and loss (Arnold et
al. 1990; Menon et al. 1990; Graham
et al. 1992; Klunk et al. 1992; Cendes
et al. 1994). Recently, recovery of
NAA levels has been reported in
patients with acute CNS damage (De
Stefano et al. 1995). The absence of a
difference in NAA levels in the left
dorsolateral prefrontal region between
schizophrenia patients and controls
suggests there is no cell damage or
loss in this part of the brain.
A previous localized in vivo 'H
MRS study of the left frontal region
(Buckley et al. 1994) also observed no
significant differences in the percent
of NAA signal between schizophrenia
patients (who included first- episode,
drug-naive and medicated patients)
and controls. However, when only
male patients were tested, a 23 percent
decrease in the NAA was observed
compared with the male controls. The
subjects used in this study were
mostly males, so a gender effect could
not be completely assessed. These
findings are in contrast to other MRS
findings in the temporal lobes, which
show marked reductions in NAA in
schizophrenia patients compared with
controls (Buckley et al. 1994; Nasrallah et al. 1994; Renshaw et al. 1995).
MRI volumetric studies have shown
that deficits in gray matter have been
less striking in the frontal lobes than
in the temporal lobes (Breier et al.
1992; Zipursky et al. 1992; Buchanan
et al. 1993).
Involement of the Glutamatergic
System in Schizophrenia. Based on
in vitro studies, the glutamate concentration in the frontal cortex is
approximately 9.0 mmol/kg wet
weight (w. wt.) and the concentration
of glutamine is approximately twofold to threefold smaller (Perry et al.
1971; Erecinska and Silver 1990).
Using the identical acquisition and
processing protocol as this study, our
group has reported glutamate and
glutamine concentration levels in the
left dorsolateral prefrontal region of
approximately 9.4 and 4.8 mmol/kg
w. wt., respectively (Stanley et al.
1995a). This would suggest that the
bulk of the in vivo concentration of
SCHIZOPHRENIA BULLETIN
604
Figure 5.
12
Glutamine level versus length of illness
r
10 c/T
'c
•
•
o
o
o
•
13
I
_
o
8 -
o
CO
•
*
*
1
CD
_ l
CD
I*
o
0
o
iS
*
O
o
' o
1
0
^
1
1
I
1
I
.
0
.
.
1
.
.
.
.
1
.
.
.
.
1
10
15
20
Length of Illness (years)
.
.
.
.
1
25
.
.
.
.
1
30
Scatter plot of the glutamine levels of the drug-naive (*), acute medicated (O), and chronic medicated (0) patients. The solid Hne represents the regression
Bne ( r » 0.53, p = 0.0007).
glutamate and glutamine is observable with this short echo MRS technique. This observation is consistent
with the 'H MRS study by Kauppinen and Williams (1991), in which
79 percent of the total glutamate concentration was estimated as MR-visible. Approximately 80 percent of the
total glutamate concentration is
found in glutamatergic neurons
(large compartment) and approximately 2 to 20 percent in glial cells
(small compartment) (Erecinska and
Silver 1990). The larger glutamate
compartment has been described as a
slow turnover pool of glutamate (i.e.,
the metabolic pool) derived from glucose precursors, and the smaller glutamate compartment as a raster
turnover pool of glutamate (i.e., the
neurotransmitter pool) that serves as
a substrate to glutamine (Erecinska
and Silver 1990). Glutamine plays an
important role in the recycling of the
neurotransmitter glutamate in the
smaller compartment (Nicholls and
Attwell 1990). For instance, following
the release of glutamate by calciumdependent exocytosis, excess glutamate is transported into glia and is
subsequently converted to glutamine
by glutamine synthetase. Following
release from glial cells, glutamine
may then enter the presynaptic neuron and serve as a precursor for
glutamate by mitochondrial glutaminase. Overall, the localization of
glutamine is found predominantly in
glial cells (Erecinska and Silver 1990;
605
VOL. 22, NO. 4, 1996
Urenjak et al. 1993).
In this study, the in vivo glutamate
and glutamine levels observed in the
left dorsolateral prefrontal region did
not differ between the first-episode,
drug-naive schizophrenia patients
and controls. This finding suggests
that there are no abnormalities in the
metabolic and (with less confidence)
the neurotransmirter pool of glutamate at the early-onset stage of schizophrenia before treatment. Additionally, this does not provide support for
the involvement of the prefrontal
glutamatergic system in schizophrenia. However, this study would have
found significant differences for our
sample size only if those differences
had been greater than 20 percent
because of the precision in quantifying the in vivo level of glutamate.
In the chronic medicated patients,
glutamine levels were, however,
increased compared with controls.
Since glutamine is predominantly in
glial cells, increased glial cell volume
in the chronic medicated patients is
one possible interpretation of our
data. However, the failure to find any
differences on NAA levels would suggest that there is no change in the proportion of neurons to glial cells. Glutamine is directly associated with the
recycling of the neurotransmirter glutamate (Nicholls and Attwell 1990).
Recently, Pellerin and Magistretti
(1994) have shown that the glucose
uptake is directly dependent on the
glutamate uptake into glia. It is
unclear which metabolic process or
processes are responsible for the
increase observed in the steady-state
level of glutamine. This alteration
could be in keeping with the
decreased glucose uptake observed in
the prefrontal lobe of schizophrenia
patients from positron emission
tomography studies (Buchsbaum
1990) if glutamine was not being converted to glutamate in the neurons.
Nevertheless, further investigation is
required to determine the physiological significance of the observed increase in glutamine in these patients.
Levels of glutamine in the combined patient group were correlated
positively with the length of illness,
while there was no age glutamine
correlation from the controls. There
also were no significant correlations
between the equivalent dose of chlorpromazine or the length of time on
medication. Considering only the 12
chronic medicated patients (i.e., the
patient subgroup with the greatest
range in length of illness), a stronger
relationship between glutamine and
length of illness was observed (r =
0.61, p = 0.035). This would suggest
that the abnormal levels of glutamine
have a stronger dependency on the
progression of the illness than on the
medication treatment.
The decreased glutamate and
increased glutamine levels that were
reported in the first preliminary in
vivo 'H MRS study on first-episode,
drug-naive patients by Stanley et al.
(1992) were not reproduced in this
study. The sampling of a larger subject population and advancements
made in the spectral quantification
procedure may account for these discrepancies. In this study, a priori
knowledge on the spectral peak
arrangement for each metabolite was
incorporated into the quantification
fitting algorithm, increasing the precision and accuracy of our results (de
Graaf and Bovee 1990; Provencher
1993; Stanley et al. 1995a).
Effects of Treatment The 1H
metabolites observed in the firstepisode, drug-naive patients and the
acute medicated patients were not
significantly different from the controls. Glutamate levels tended to be
higher in the acute medicated
patients, but this finding was not sta-
tistically significant. However, of the
eight first-episode, drug-naive
patients who also participated as
acute medicated patients, the glutamine levels were significantly
decreased in the on-medication measurements compared with the premedication measurements. Additionally, during this acute on-medication
period (i.e., with a mean length of
time on medication of 14 ± 8 weeks),
the SAPS scores also were significantly decreased. This finding would
suggest that during the initial period
of neuroleptic treatment, the antipsychotic medication, whose function is to block dopamine receptors,
has altered directly or indirectly the
steady-state level of glutamine in the
prefrontal region. It may also suggest
that the antipsychotic medication has
influenced the glutamatergic system
since glutamine is directly associated
with the recycling of the neurotransmitter glutamate.
There is little support in the literature for this interpretation. Antipsychotic drugs have been reported to
affect the dopaminergic activity in the
nigrostriatal and limbic structures but
not the glutamatergic activity in the
prefrontal region after acute and
chronic administration in animals
(Yamamoto and Cooperman 1994).
However, Pehek and colleagues
(1991) and Daly and Moghaddam
(1993) found increased extracellular
concentrations of glutamate in the
prefrontal cortex following acute
administration of antipsychotic
drugs. Further in vivo MRS studies
on larger patient populations are
required to confirm the involvement
of the glutamatergic system in the
prefrontal region during acute administration of antipsychotic drugs.
Limitations. The sagittal and coronal images in figure 1 suggest that
within the dimension of the VOI, the
SCHIZOPHRENIA BULLETIN
606
observed ] H MR signal is dominated
by white matter compared with gray
matter. The identical spectroscopy
protocol was used in the repeated
measures study, and the volume of
gray matter for the 8 cm3 VOI was
estimated at approximately 30 percent (Stanley et al. 1995a). If metabolite differences are only present in the
gray matter of schizophrenia patients, then the observed intensity of
the difference is reduced because of
sampling relatively less gray matter
volume. However, acquiring ] H spectra with a smaller VOI to reduce the
partial white matter volume decreases the signal-to-noise ratio,
which reduces the reliability of the
quantification. To account for the ] H
signal from macromolecules (i.e., signal from mobile proteins and polypeptides), two macromolecule resonances were incorporated in the fitting routine (Stanley et al. 1995a).
These two resonances are dominant
spectral peaks that are observed in
spectra of macromolecules in vivo
(Behar et al. 1994). In the controls, the
two macromolecule resonances combined accounted for 3 ± 2 percent of
the total signal compared with 28 ± 2
percent for NAA. We do acknowledge that there are inaccuracies associated with this approach that may
result in overestimating the levels of
certain metabolites (Stanley et al.
1995a). The in vivo T, (spin-lattice
relaxation time) of glutamine is
approximately 2,100 msec (Hanicke
et al. 1993), which is greater than the
interpulse repetition time used and
implies that the observed glutamine
signal is partially saturated. A
decrease in T, of glutamine could
account for the observed increase;
however, the T, would have to decrease significantly by about 34 percent to observe the 30 percent
increase in glutamine level. Differences in the T2 values of the
metabolites between patients and
controls could not be detected because the relative linewidths (i.e., T2
values) of the metabolites were held
constant in the quantification technique (Stanley et al. 1995a).
References
Andreasen, N. Scale for the Assessment of Negative Symptoms (SANS).
Iowa City, IA: The University of
Iowa, 1984a.
Andreasen, N. Scale for the Assessment of Positive Symptoms (SAPS).
Iowa City, IA: The University of
Iowa, 1984b.
Arnold, D.L.; Matthews, P.M.; Francis, G.; and Antel, J. Proton magnetic
resonance spectroscopy of human
brain in vivo in the evaluation of
multiple sclerosis: Assessment of the
load of disease. Magnetic Resonance in
Medicine, 14:154-159,1990.
Behar, K.L., and Ogino, T. Characterization of macromolecule resonances
in the 'H NMR spectrum of rat brain.
Magnetic Resonance in Medicine,
30:38-44,1993.
Behar, K.L.; Rothman, D.L.; Spencer,
D.D.; and Petroff, O.A.C. Analysis of
macromolecule resonances in H
NMR spectra of human brain. Magnetic Resonance in Medicine, 32:294-
302,1994.
Birken, D.L., and Oldendorf, W.H.
N-acetyl-L-aspartic acid: A literature
review of a compound prominent in
^-NMR spectroscopic studies of
brain. Neuroscience and Biobehavioral
Reviews, 13:23-31,1989.
Bottomley, P.A. Human in vivo NMR
spectroscopy in diagnostic medicine:
Clinical tool or research probe? Radiology, 170:1-15,1989.
Breier, A.; Buchanan, R.W.; Elkashef,
A.; Munson, R.C.; Kirkpatrick, B.;
and Gellad, F. Brain morphology and
schizophrenia: A magnetic resonance
imaging study of limbic, prefrontal
cortex, and caudate structures.
Archives of General Psychiatry,
49:921-926,1992.
Buchanan, R.W.; Breier, A.; Kirkpatrick, B.; Elkashef, A.; Munson,
R.C.; Gellad, F.; and Carpenter, W.T.,
Jr. Structural abnormalities in deficit
and nondeficit schizophrenia. American Journal of Psychiatry, 150:59-66,
1993.
Buckley, P.F.; Moore, C; Long, H.;
Larkin, C; Thompson, P.; Mulvany,
F.; Stack, J.P.; Ennis, J.T.; and
Waddington, J.L. H-l magnetic resonance spectroscopy of the left temporal and frontal lobes in schizophrenia: Clinical, neurodevelopmental,
and cognitive correlates. Biological
Psychiatry, 36:792-800,1994.
Buchsbaum, M.S. The frontal lobes,
basal ganglia, and temporal lobes as
sites for schizophrenia. Schizophrenia
Bulletin, 16(3):379-389,1990.
Calabrese, G.; Deicken, R.F.; Fein, G.;
Merrin, E.L.; Schoenfeld, F.; and
Weiner, M.W. 31-Phosphorus magnetic resonance spectroscopy of the
temporal lobes in schizophrenia. Biological Psychiatry, 32:26-32,1992.
Carlsson, M., and Carlsson, A. Schizophrenia: A subcortical neurorransmitter imbalance syndrome? Schizophrenia Bulletin, 16(3):425-432,1990.
Cendes, F.; Andermann, F.; Preul,
M.C.; and Arnold, D.L. Lateralization
of temporal lobe epilepsy based on
regional metabolic abnormalities in
proton magnetic resonance spectroscopic images. Annals of Neurology,
35:211-216,1994.
Christiansen, P.; Henriksen, O.; Stubgaard, M.; Gideon, P.; and Larsson,
H.B.W. In vivo quantification of brain
metabolites by 'H-MRS using water
as an internal standard. Magnetic Resonance Imaging, 11:107-118,1993.
607
VOL. 22, NO. 4, 1996
Daly, D.A., and Moghaddam, B.
Actions of dozapine and haloperidol
on extracellular levels of excitatory
amino acids in the prefrontal cortex
and striatum of conscious rats. Neuroscience Letters, 152:61-64,1993.
de Graaf, A. A., and Bovee, W.M.M.J.
Improved quantification of in vivo ^H
NMR spectra by optimization of signal acquisition and processing and
by incorporation of prior knowledge
into the spectral fitting. Magnetic Resonance in Medicine, 15:305-319,1990.
de Graaf, A.A.; van Dijk, J.E.; and
Bovee, W.M.M.J. QUALITY: Quantification improvement by converting
lineshapes to the Lorentzian type.
Magnetic Resonance in Medicine,
13:343-357,1990.
De Stefano, N.; Matthews, P.M.; and
Arnold, D.L. Reversible decreases in
N-acetylaspartate after acute brain
injury. Magnetic Resonance in Medicine, 34:721-727,1995.
Deutsch, S.I.; Mastropaolo, J.;
Schwartz, B.L.; Rosse, R.B.; and Morihisa, J.M. A glutamatergic hypothesis
of schizophrenia: Rationale for pharmacotherapy with glycine. Clinical
Neuropharmacology, 12:1-13,1989.
Erecinska, M., and Silver, I.A. Metabolism and role of glutamate in mammalian brain. Progress in Neurobiology,
35:245-2%, 1990.
Ernst, T.; Hennig, J.; Ott, D.; and
Friedburg, H. The importance of the
voxel size in clinical 'H spectroscopy
of the human brain. NMR in Biomedicine, 2:216-224,1989.
Ferrige, A.G., and Lindon, J.C. Resolution enhancement in FT NMR
through the use of a double exponential function, journal of Magnetic Resonance, 31:337-340,1978.
Frahm, J.; Bruhn, H.; Gyngell, M.L.;
Merboldt, K.D.; Hanicke, W.; and
Sauter, R. Localized high-resolution
proton NMR spectroscopy using
stimulated echoes: Initial applications to human brain in vivo. Magnetic
Resonance in Medicine, 9:79-93,1989.
Frahm, J.; Michaelis, T; Merboldt,
K.D.; Bruhn, H.; Gyngell, M.L.; and
Hanicke, W. Improvements in localized proton NMR spectroscopy of
human brain and water suppression,
short echo times, and 1 ml resolution.
Journal of Magnetic Resonance, 90:464-
473,1990.
Graham, G.D.; Blamire, A.M.;
Howseman, A.M.; Rothman, D.L.;
Fayad, P.B.; Brass, L.M.; Petroff, O.A.;
Shulman, R.G.; and Prichard, J.W.
Proton magnetic resonance spectroscopy of cerebral lactate and other
metabolites in stroke patients. Stroke,
23:333-340,1992.
Haase, A., and Frahm, J. Multiple
chemical-shift-selective NMR imaging using stimulated echoes. Journal of
Magnetic Resonance, 64:94-102,1985.
Hanicke, W.; Michaelis, T; Merboldt,
K.D.; and Frahm, J. On the use of a
fully automated data analysis
method for in vivo MRS: Metabolite
concentrations and relaxation times
from proton spectra of human brain.
In: Proceedings of the 12th Annual
Meeting of the Society of Magnetic Reso-
nance in Medicine. Berkeley Springs,
CA: Society of Magnetic Resonance
in Medicine, 1993. p. 977.
Harrison, P.J.; McLaughlin, D.; and
Kerwin, R.W. Decreased hippocampal expression of a glutamate receptor gene in schizophrenia. Lancet,
337:450-152,1991.
Javitt, D.C., and Zukin, S.R. Recent
advances in the phencyclidine model
of schizophrenia. American Journal of
Psychiatry, 148:1301-1308,1991.
Kauppinen, R.A.; Kokko, H.; and
Williams, S.R. Detection of mobile
proteins by proton nuclear magnetic
resonance spectroscopy in the guinea
pig brain ex vivo and their partial
purification. Journal of Neurochemistry,
58:967-974,1992.
Kauppinen, R.A., and Williams, S.R.
Nondestructive detection of glutamate by ] H nuclear magnetic resonance spectroscopy in cortical brain
slices from the guinea pig: Evidence
for changes in detectability during
severe anoxic insults. Journal of Neurochemistry, 57:1136-1144,1991.
Kauppinen, R.A.; Williams, S.R.;
Busza, A.L.; and van Bruggen, N.
Applications of magnetic resonance
spectroscopy and diffusion-weighted
imaging to the study of brain biochemistry and pathology. Trends in
Neuroscience, 16:88-95,1993.
Klunk, W.E.; Panchalingam, K.;
Mossy, J.; McClure, R.J.; and Pettegrew, J.W. W-acetyl-L-aspartate and
other amino acid metabolites in
Alzheimer's disease brain: A preliminary proton nuclear magnetic resonance study. Neurology, 42:1578-1585,
1992.
Krystal, J.H.; Karper, L.P.; Seibyl, J.P.;
Freeman, G.K.; Delaney, R.; Bremner,
J.D.; Heninger, G.R.; Bowers, M.B.;
and Charney, D.S. Subanesthetic
effects of the noncompetitive NMDA
antagonist, ketamine, in humans.
Archives of General Psychiatry,
51:199-214,1994.
Marquardt, D.W. An algorithm for
least-squares estimation of non-linear
parameters. Society of Industrial and
Applied Mathematics Journal, 11:431441,1963.
Matalon, R.; Michals, K.; Sebesta, D.;
Deanching, M.; Gashkoff, P.; and
Casanova, J. Aspartoacylase deficiency and N-acetylaspartic aciduria
in patients with Canavan disease.
American Journal of Medical Genetics,
29:463-471,1988.
Menon, D.K.; Baudouin, C.J.; Tomlinson, D.; and Hoyle, C. Proton MR
spectroscopy and imaging of the
SCHIZOPHRENIA BULLETIN
608
brain in AIDS: Evidence of neuronal
loss in regions that appear normal
with imaging. Journal of Computer
Assisted Tomography, 14:882-885,1990.
Nasrallah, H.A.; Skinner, T.E.;
Schmalbrock, P.; and Robitaille, P.M.
Proton magnetic resonance spectroscopy (H-l MRS) of the hippocampal formation in schizophrenia: A
pilot study. British Journal of Psychiatry, 165:481-485,1994.
Nicholls, D., and Attwell, D. The
release and uptake of excitatory
amino acids. Trends in Pharmacological
Sciences, 11:462-468,1990.
O'Callaghan, E.; Redmond, O.; Ennis,
R.; Stack, ].; Kinsella, A.; Ennis, J.T.;
and Waddington, J.L. Initial investigation of the left temporoparietal
region in schizophrenia by 31P magnetic resonance spectroscopy. Biological Psychiatry, 29:1149-1152,1991.
Pehek, E.A.; Yamamoto, B.K.; and
Meltzer, H.Y. The effects of clozapine
on dopamine, 5-HT, and glutamate
release in the rat medial prefrontal
cortex. Schizophrenia Research, 4:323,
1991.
Pellerin, L., and Magistretti, P.J. Glutamate uptake into astrocytes stimulates aerobic glycolysis—A mechanism coupling neuronal activity to
glucose utilization. Proceedings of tlie
National Academy of Sciences of the
United States of America, 91:1062510629,1994.
Perry, T.L.; Hansen, S.; Berry, K.;
Mok, C; and Lesk, D. Free amino
acids and related compounds in
biopsies of human brain. Journal of
Ncumdiemistry, 18:521-528, 1971.
Pettegrew, J.W.; Keshavan, M.S.; Panchalingam, K.; Strychor, S.; Kaplan,
D.B.; Tretta, M.G.; and Allen, M.
Alterations in brain high-energy
phosphate and membrane phospholipid metabolism in first-episode,
drug-naive schizophrenics: A pilot
study of the dorsolateral prefrontal
cortex by in vivo phosphorus 31
nuclear magnetic resonance spectroscopy. Archives of General Psychiatry, 48:563-568,1991.
Provencher, S.W. Estimation of
metabolite concentrations from localized in vivo proton NMR spectra.
Magnetic Resonance in Medicine,
30:672-679,1993.
Renshaw, PR; Yurgelun-Todd, D.A.;
Tohen, M.; Gruber, S.; and Cohen,
B.M. Temporal lobe proton magnetic
resonance spectroscopy of patients
with first-episode psychosis. American Journal of Psychiatry, 152:444—446,
1995.
Sharma, R.; Venkatasubramanian,
P.N.; Barany, M.; and Davis, J.M. Proton magnetic resonance spectroscopy
of the brain in schizophrenic and
affective patients. Schizophrenia
Research, 8:43-49,1992.
Sherman, A.D.; Davidson, A.T.;
Baruah, S.; Hegwood, T.S.; and
Waziri, R. Evidence of glutamatergic
deficiency in schizophrenia. Neuroscience Letters, 121:77-80,1991.
Spitzer, R., and Williams, J. Structured
Clinical Interview for DSM-III-R. New
York, NY: New York Psychiatric Institute, 1985.
Stanley, J.A.; Drost, D.J.; Williamson,
PC.; and Thompson, R.T. The use of a
priori knowledge to quantify short
echo in vivo 'H MR spectra. Magnetic
Resonance in Medicine, 34:17-24,
1995a.
Stanley, J.A.; Williamson, PC; Drost,
D.J.; Canr, T.J.; Rylett, R.J.; Malla, A.;
and Thompson, R.T. An in vivo study
of the prefrontal cortex of schizophrenic patients at different stages of
illness via phosphorus magnetic resonance spectroscopy. Archives of General Psychiatry, 52:399-406,1995b.
Stanley, J.A.; Williamson, PC; Drost,
D.J.; Carr, T.J.; Rylett, R.J.; and
Merskey, H. "In Vivo Proton MRS in
Never Treated Schizophrenics." Presented at the Annual Meeting of the
American Psychiatric Association,
Washington, DC, May 1992.
Stanley, J.A.; Williamson, PC; Drost,
D.J.; Carr, T.J.; Rylett, R.J.; and
Thompson, R.T. Membrane phospholipid metabolism and schizophrenia:
An in vivo P-31-MR spectroscopy
study. Schizophrenia Research, 13:209215,1994.
Tallan, H.H. Studies on the distribution of N-acetyl-L-aspartic acid in
brain. Journal of Biological Chemistry,
224:41^5,1957.
Uras, J., and Cotman, C.W. Excitatory amino acid receptors in schizophrenia. Schizophrenia Bulletin,
19(l):105-117,1993.
Urenjak, J.; Williams, S.R.; Godown,
D.G.; and Noble, M. Proton nuclear
magnetic resonance spectroscopy
unambiguously identifies different
neural cell types. Journal ofNeurosciencc, 13:981-989,1993.
Wachtel, H., and Turski, L. Glutamate: A new target in schizophrenia?
Trends in Pharmacological Sciences,
11:219-220,1990.
Yamamoto, B.K., and Cooperman,
M. A. Differential effects of chronic
antipsychotic drug treatment on
extracellular glutamate and
dopamine concentrations. Journal of
Ncuroscience, 14:4159-4166,1994.
Zipursky, R.B.; Lim, K.O.; Sullivan,
E.V.; Brown, B.W.; and Pfefferbaum,
A. Widespread cerebral gray matter
volume deficits in schizophrenia.
Archives of General Psydiiatry, 49:195205,1992.
Acknowledgments
This study was supported by grant
MH-50768 from the National Institute
of Mental Health and grant MT-12078
609
VOL. 22, NO. 4, 1996
from the Ontario Mental Health Foundation, Medical Research Council of
Canada. The authors thank Mr. John
Potwarka for computer software
development assistance.
The Authors
Jeff A. Stanley, Ph.D., is a Senior
Announcement
Research Fellow, Laboratory of Neurophysics, University of Pittsburgh
Medical Center, Pittsburgh, PA. Peter
C. Williamson, M.D., is Associate
Professor of Psychiatry; Dick J. Drost,
Ph.D., is Associate Professor of Medical Biophysics; R. Jane Rylett, Ph.D.,
is Professor of Physiology; Tom J.
Carr, M.D., is Clinical Assistant Pro-
fessor of Diagnostic Radiology and
Nuclear Medicine; Ashok Malla,
M.D., is Associate Professor of Psychiatry; and R. Terry Thompson,
Ph.D., is Associate Professor of Medical Biophysics, Nuclear Medicine
and Magnetic Resonance, St. Joseph's
Health Centre, University of Western
Ontario, London, Ontario, Canada.
The Pacific Clinics and the Alliance
for the Mentally 111 will sponsor a
conference entitled Integrating Approaches to Intervention With Persons Who Have Schizophrenia to be
held in Pasadena, California, January
30 and 31 and February 1,1997. This
meeting focuses on exercises, demonstrations, and role plays to teach
usable skills from the neurobiological, recovery, and family psychoeducational approaches to schizophrenia.
A model for integrating the interventions into a unified view of person,
illness, and family is presented.
For more information about the
conference, please contact:
Sarah Shatford
Pacific Clinics
909 South Fair Oaks
Pasadena, CA 91105
Telephone: 818-577-6697