Glial dysfunction in abstinent methamphetamine

abusers
Napapon Sailasuta
1
, Osama Abulseoud
2
, Kent C Harris
1
and Brian D Ross
1,3
1
Clinical Spectroscopy Unit, Huntington Medical Research Institutes, Pasadena, California, USA;
2
Department of Psychiatry, University of Southern California, Los Angeles, California, USA;
3
Rudi Schulte
Research Institute, Santa Barbara, California, USA
Persistent neurochemical abnormalities in frontal brain structures are believed to result from
methamphetamine use. We developed a localized
13
C magnetic resonance spectroscopy (MRS)
assay on a conventional MR scanner, to quantify selectively glial metabolic flux rate in frontal brain
of normal subjects and a cohort of recovering abstinent methamphetamine abusers. Steady-state
bicarbonate concentrations were similar, between 11 and 15 mmol/L in mixed gray-white matter of
frontal brain of normal volunteers and recovering methamphetamine-abusing subjects (P>0.1).
However, glial
13
C-bicarbonate production rate from [1-
13
C]acetate, equating with glial tricarboxylic
acid (TCA) cycle rate, was significantly reduced in frontal brain of abstinent methamphetamine-
addicted women (methamphetamine 0.04 lmol/g per min (N=5) versus controls 0.11 lmol/g per min
(N=5), P=0.001). This is equivalent to 36% of the normal glial TCA cycle rate. Severe reduction in
glial TCA cycle rate that normally comprises 10% of total cerebral metabolic rate may impact
operation of the neuronal glial glutamate cycle and result in accumulation of frontal brain glutamate,
as observed in these recovering methamphetamine abusers. Although these are the first studies to
define directly an abnormality in glial metabolism in human methamphetamine abuse, sequential
studies using analogous
13
C MRS methods may determine cause and effect between glial failure
and neuronal injury.
Journal of Cerebral Blood Flow & Metabolism (2010) 30, 950960; doi:10.1038/jcbfm.2009.261; published online
30 December 2009
Keywords: acetate; glutamate; MR spectroscopy; neurochemistry; neuronalglial interaction; neurotransmitters
Introduction
The neuropathologic presentation of substance abuse
and addiction, a serious health, social, and economic
problem in the United States, involves abnormalities
of the dopaminergic and glutamatergic systems
leading to neuronalglial dysfunction (Stephans
and Yamamoto, 1994; Volkow et al, 2001, 2002). It
has been commonly observed that objective neuro-
chemical changes persist beyond the period of active
drug use, inviting the hypothesis that persistent
neurochemical abnormalities contribute to addictive
behavior and relapse (Ernst et al, 2000; Sekine et al,
2002). The timing and possible role of glial injury in
addiction and relapse is poorly understood but may
have implications for the design of pharmaceuticals
for future treatment of addiction and relapse pre-
vention. Evolving methods of noninvasive brain
analysis, in particular
13
C magnetic resonance spec-
troscopy (MRS) with
13
C glucose and
13
C acetate,
respectively (Bachelard, 1998; Bluml et al, 2001,
2002; Cerdan and Seelig, 1990; Gruetter et al, 1993;
Lin et al, 2003; Mason et al, 1999), offer new
opportunities to examine the impact of drugs of
abuse on neurons and glia separately. This pilot
study was undertaken to establish the feasibility of
applying [1-
13
C]acetate MRS to quantify glial meta-
bolic rate in frontal brain structures implicated in
drug abuse, reward, and relapse (Gass and Olive,
2008). Future studies, in which the starting substrate
would be [2-
13
C] glucose, could then be applied to
probe the impact on neuronal metabolism directly
(Gropman et al, 2009).
Glutamate (Glu) is an amino acid intimately
involved in neurotransmission and a marker of
mitochondrial oxidative status and tricarboxylic acid
(TCA) cycle function. The glutamateglutamine
cycle links glia with neurons in the mechanism of
Received 28 September 2009; revised 18 November 2009; accepted
30 November 2009; published online 30 December 2009
Correspondence: Dr N Sailasuta, Clinical Spectroscopy Unit,
Huntington Medical Research Institutes, 10 Pico Street, Pasadena,
CA 91105, USA.
E-mail: sailasuta@hmri.org
This work was supported by NIH Grant number K25DA21112 (NS)
and a Grant-in-Aid (BDR) from the Rudi Schulte Research Institute
of Santa Barbara.
Journal of Cerebral Blood Flow & Metabolism (2010) 30, 950960
& 2010 ISCBFM All rights reserved 0271-678X/10 $32.00
www.jcbfm.com
glutamate neurotransmission, the neurotransmitter
pathway overall believed to account for 80% of brain
neurotransmitter function. The functions of glial
cells in neurotransmission are often viewed as
merely supportive; however, failure of glial reuptake
of glutamate released at synapses can result in
accumulation of extracellular glutamate, leading to
neurotoxicity. Glutamate is crucial in regulating both
the development and the expression of addictive
behaviors, which requires glutamate receptor stimu-
lation in the ventral tegmental area and is associated
with enhanced glutamate release in the prefrontal
cortex (Kalivas et al, 2009; Rebec and Sun, 2005).
Using the single voxel proton MRS method, which
determines steady-state concentration of brain meta-
bolites, we found that preliminary data provide more
direct evidence of elevated frontal brain glutamate
concentration during the cerebral adaptation to
methamphetamine abuse (Abulseoud et al, 2009).
This is in addition to earlier reports of persistent
neurochemical abnormalities; elevated myoinositol,
a glial marker, and reduced N-acetylaspartate, a
neuronal marker, observed in the frontal cortex of
abstinent methamphetamine users (Ernst et al, 2000;
Sung et al, 2007). Together these results indicate that
neuronal injuries from drug abuse are likely accom-
panied by significant abnormalities in glial function
and metabolism.
The aim of this pilot study was to determine the
extent of glial metabolic abnormalities in abstinent
methamphetamine-dependent (AMD) subjects using
MRS.
13
C MRS techniques, which permit TCA cycle
rate determination directly and further allow the
dissection of glial from neuronal pathways of
glutamate metabolism based on the selective uptake
and metabolism of acetate by glia (Muir et al, 1986)
and glucose by neurons have recently been explored
in intact brains of animals and humans (Bluml et al,
2002; Cerdan, 2003; Lebon et al, 2002; Ross et al,
2003; Rothman et al, 2003; Shen and Rothman,
2002). A limitation of previous studies in relation to
the present purpose of drug abuse has been the need
to confine human
13
C MRS assays to posterior brain
regions. This brain region is distant from heat-
sensitive optical structures but purportedly of mini-
mal relevance to the neuropsychopathology of drug
abuse, which most investigators locate in frontal
and limbic pathways (London et al, 2000). These
technical limitations have recently been overcome
(Sailasuta et al, 2008) with two distinct and robust in
vivo
13
C MRS techniques adapted to the human
frontal lobe. First, the use of
13
C isotopes of which
the products can be readily observed with minimal
or absent proton decoupling. Second, the develop-
ment of low-power nuclear overhauser effects can
now be used for the same purpose. Details of these
technical advances and their efficacy in human
studies using a conventional low field 1.5 T MR
scanner have been published (Li et al, 2007;
Sailasuta et al, 2008). In this report we describe the
first such method designed to separately interrogate
glial glutamate formation and specifically, to quanti-
fy the glial oxidative rate based on the appearance of
13
C label from the unique glial precursor, acetate, into
its final product bicarbonate (HCO
3

). We have
determined the rate at which [1-
13
C]acetate appears
in cerebral bicarbonate [H
13
CO
3
] in normal frontal
brain, and shown a marked reduction in the
contribution of glial acetate oxidation to the TCA
cycle in frontal brain of AMD subjects.
Materials and methods
Subjects
A total of 21 in vivo
13
C MRS studies were performed.
Eleven subjects participated in this study approved by
institutional review board and HIPAA, of whom 6 were
normal, age- and gender-matched subjects. The six healthy
control subjects (mean age 26

1 years, one man) were

recruited from the local community. Three of the control
subjects were scanned twice on separate days to compare
13
C acetate metabolism in frontal and posterior brain. Five
female AMD subjects (mean age 317 years) recruited
from local drug rehabilitation centers and counseling
centers within the Los Angeles County were examined
only once, in the frontal brain region. All five subjects
reported methamphetamine as the drug of choice and met
Diagnostic and Statistical Manual of Mental Disorders,
fourth edition criteria for lifetime methamphetamine
dependence from the Structured Clinical Interview (First
et al, 1994). Self-report by study participants indicated the
average period of methamphetamine use was 74 days and
each had been abstinent from methamphetamine or other
illicit drugs for at least 7 days before the studies. The study
protocols were approved by the institutional review board
of the Huntington Memorial Hospital and the University
Hospital at University of Southern California. Written
informed consent was obtained from all participants.
Carbon MRS
The techniques for quantification of glial metabolic rate of
in vivo human brain
13
C MRS using [1-
13
C]acetate as
substrate have been described in detail previously (Bluml
et al, 2002). Significant modifications necessary for safe
transfer of the technique to frontal brain for this study were
as follows:
(i) To establish metabolic rates for [1-
13
C]acetate for the
frontal lobe of human brain, not hitherto examined, we
developed low rf power
13
C MRS (Sailasuta et al,
2008). To minimize RF heating in a clinical magnetic
resonance imaging system, all subjects underwent
13
C
MRS at 1.5T GE clinical scanner (GE Healthcare,
Milwaukee, WI, USA) equipped with broadband
exciter, a stand-alone proton decoupler, and a vector
signal generator (Agilent Technology E4433B, Santa
Clara, CA, USA). A half-helmet head coil was used
with the subject lying supine within the MR scanner.
Its construction was a modification of the half-head
coil used in the posterior brain
13
C MRS study from
Glial dysfunction in methamphetamine abusers
N Sailasuta et al
951
Journal of Cerebral Blood Flow & Metabolism (2010) 30, 950960
this laboratory (Bluml et al, 2001). The coil consisted
of a 4-inch proton loop and a 3.5-inch
13
C loop for
transmission and reception. Fast spin echo imaging
scan was used to confirm the consistency of the
observed volumes in all patients. For the frontal brain
study, the half-helmet coil placement was such that the
anterior portions of the frontal lobe were mainly
included in the field of view and similarly for the
posterior brain where the posterior region of the brain
was included in the field of view (Gropman et al,
2009). The coil was tuned to best match both proton
and carbon channels. Power deposition was continu-
ously monitored and remained below Federal guide-
lines for Specific Absorption Rate.
(ii) Because the method used is new, to confirm that
metabolic rates for [1-
13
C]acetate in human frontal lobe
are comparable to posterior parietal brain, values
previously reported using [1-
13
C]acetate by us, and
also recently confirmed using [2-
13
C] acetate by Lebon
et al (2002), assays of
13
C enrichment in the posterior
brain were performed in four normal subjects. To allow
for complete clearance of
13
C-enriched metabolites
from the posterior brain for three of the four normal
subjects, this examination was followed a minimum of
48 h later by examination of the frontal brain region in
each of the three normal subjects.
Magnetic resonance image was acquired at the outset of
each
13
C MRS examination, indicating regions of interest of
mixed white and gray matter of posterior parietal or frontal
and prefrontal brain structures, respectively, for
13
C MRS
acquisition. Brain volumes, approximately 100cm
3
in each
protocol, included similar proportions of white and gray
matter, were comparable and have been previously pub-
lished (Gropman et al, 2009; Sailasuta et al, 2008).
Determinations of Cerebral Bicarbonate Concentration
from Natural Abundance
13
C MRS and Maximum Rate
of HCO
3

(
max
V
HCO

3
) Production
Natural abundance
13
C MRS was acquired for 20 mins
before the start of the acetate infusion to show adjacent,
well-resolved individual resonances for bicarbonate and
for Cr +PCr (total creatine, tCr) as previously reported by
Bluml et al (2001). Because tCr has been used successfully
as an internal metabolite reference in several proton MRS
studies in abstinent methamphetamine users (Nordahl
et al, 2005, 2002; Salo et al, 2007) as well as other human
studies (see review, Xu et al, 2008), and does not become
enriched during the time scale of the present studies, we
have used this as an internal reference to quantify HCO
3

and its subsequent enrichment from [1-

13
C]acetate. To
improve signal to noise (natural abundance
13
C=only
1.1% of total) and thereby improve quantification, we
acquired additional natural abundance
13
C brain spectra
during separate imaging sessions not associated with
13
C
infusions. The timed intravenous infusion of 99%
13
C-
enriched [1-
13
C]acetate infusion was started and brain
spectra were acquired continuously thereafter for a mini-
mum of 2 h. The 6
1
2
min blocks of low-power noise nuclear
overhauser effects (0.9 W for frontal brain and 2.5 W for the
posterior brain)
13
C MRS spectra acquired were stored and
summed, as previously described (Sailasuta et al, 2008).
13
C MRS examination times of 120mins or more were well
tolerated by all subjects with no observed adverse
reactions. Maximum rate of HCO
3

production (
max
V
HCO

3
)
was calculated from the natural abundance bicarbonate
concentration at baseline and at the end of the infusion
estimated from the ratio of the areas under the two
resonances, HCO
3

(161 p.p.m.) and tCr (158 p.p.m.) using

a Marquardt fitting algorithm and assuming tCr concentra-
tion 11 mmol/g in the brain.
Infusion Protocol
The 99%
13
C-enriched [1-
13
C]acetate solution was prepared
under good manufacturing practice by the Cambridge
Isotope Laboratories (Andover, MA, USA) and shipped in
bulk to a licensed pharmacist in Minnesota, registered and
approved by the US Food and Drug Administration to ship
sterile, pyrogen-free reagents to California. The reagent was
prepared on a named-subject basis and used within 48 h
after preparation. This study was performed under the
Food and Drug Administration Investigational New Drug
Application no. 59690. To establish similar metabolic
status, subjects were fasted for 6h before the study.
Intravenous administration (through an arm vein) of 120
to 300ml of 0.4mol/L sterile, pyrogen-free solution of
[1-
13
C]acetate 99% enrichment was started (OA) at an
infusion rate of 3 mg/kg per min for 60 mins.
Fractional Enrichment of Serum [1-
13
C]Acetate
Because earlier preclinical and clinical studies have estab-
lished that
13
C acetate consumption proceeds rapidly in the
brain and the [1-
13
C]acetate resonance cannot readily be
distinguished from its primary metabolic product, 5-
13
C
glutamate on the basis of near-identical chemical shift, plasma
13
C acetate enrichment was used as a surrogate marker of the
fractional
13
C enrichment (E) of stable isotope availability for
glial oxidation. Serial blood samples were collected using
heparinized syringe before infusion and at 15min intervals
until the end of the study (at 120mins). Blood samples were
centrifuged and the resulting protein-free plasmas were stored
frozen until analysis. Plasma samples were deproteinized
using 2:1 volume ratio of 99.9% ethanol to plasma and dried
under vacuum. Samples were dissolved in 99.9% D
2
O before
proton NMR measurements using a Varian 300MHz spectro-
meter. Fractional enrichment of plasma acetate was deter-
mined under fully relaxed conditions (repetition time 18s)
from the ratio of the relative peak areas of the
13
C satellites to
the total area of the corresponding acetate H2 resonance at
1.9p.p.m. (Moreno et al, 2001).
Data Processing and Fractional Enrichment
Calculation of Cerebral Metabolites
The principal metabolic end point of this study was the
quantification of intrinsic cerebral bicarbonate (HCO
3

),
together with the progressive increase in fractional
13
C
Glial dysfunction in methamphetamine abusers
N Sailasuta et al
952
Journal of Cerebral Blood Flow & Metabolism (2010) 30, 950960
enrichment (%E) of the HCO
3

pool as the result of

complete oxidation of the sole
13
C-enriched precursor of
glial TCA cycle activity, [1-
13
C]acetate. Because
13
C creatine
and phosphocreatine do not become enriched from
13
C
acetate (Cerdan, 2003; Chapa et al, 1995) and resonate
distinctly in a portion of the brain spectrum unencumbered
by overlying resonances, we used
13
C total signal intensity
for [Cr +PCr] to quantify the initial brain bicarbonate
concentration in nonenriched baseline
13
C MR scans from
each subject. We assume there are no significant differ-
ences in T1 or T2 relaxation times between the two.
Fractional enrichment of HCO
3

is then defined as the

increase in intensity relative to that of [Cr +PCr] which is
not enriched from the infused source of [1-
13
C]acetate.
Progressively increasing intensity of
13
C HCO
3

resonance
was recorded throughout and the relative rate of enrich-
ment was expressed as percent over time, slope of initial
increase, and final %E bicarbonate. The other anticipated
cerebral metabolites of [1-
13
C]acetate, glutamine C1 and C5,
and glutamate C1 and C5, were also readily observed. An
observer-independent automatic IDL-based software devel-
oped in our laboratory (Shic and Ross, 2003) and SAGE (GE
Healthcare, Milwaukee, WI, USA)/IDL (ITT Visual Informa-
tion Solutions, Boulder, CO, USA), a commercially available
software, were used for data processing (NS, KH). Data blocks
(6
1
2
min) were first zero filled to 16K data points, applied 7Hz
Lorenzian to Gaussian lineshape transformation, Fourier-
transformed, and phase-corrected. Owing to the absence of
overlapping resonances, relative contributions of
13
C singlet of
HCO
3

(161p.p.m.), C1-acetate and C5-Glu (182p.p.m.), and

C5-Gln (178p.p.m.) were readily determined from peak
amplitudes of the observed resonances. In the case of C1-
acetate and C5-Glu, no separation of the resonances could be
achieved under near-steady-state conditions. Based on earlier
studies, the contribution of [1-
13
C]acetate cerebral flux, %E
13
C brain was considered approximately %E acetate of blood.
13
C fractional enrichments (E) of metabolic products
were calculated from:
E
met
S
post
=S
pre
1:1%
where S
post
and S
pre
are the signal intensities of the
resonances in the baseline scan and in scans after infusion
start, and 1.1% is the natural abundance of
13
C.
Determination of the Glial Acetate Oxidation Rate
(V
g ac
)
For consistency with earlier studies, results are expressed
as glial acetate oxidation rate (V
g ac
). Acetate has been
shown to be metabolized exclusively in glia, probably as a
result of the unique membrane transporter present in glia
but lacking in neurons (Muir et al, 1986; Waniewski and
Martin, 1998). During the first turn of the TCA cycle, C1
label (from C1-acetate) is transferred to form glial C5 and
C1 glutamate and transformed by glial glutamine synthase
to C5 and C1 glutamine. C1 label CO
2
(HCO
3

) is released in
the second turn of the TCA cycle (Cerdan et al, 1990).
Calculation of oxidative rate from [1-
13
C]acetate to the final
oxidative product H
13
CO
3
therefore involves no assump-
tions concerning neuronal compartmentation or flux as
proved necessary for calculations of [2-
13
C]acetate metabo-
lism (Lebon et al, 2002). Nevertheless, the two methods
appear to yield comparable estimates for average glial
metabolic rate at approximately 10% to 15% of the rate of
normal neuronal TCA cycle (Shen et al, 1999). In this study
we used the approach described previously by Bluml et al
(2002) (and Figure 1): assuming that metabolic pools that
accumulate
13
C label other than bicarbonate have constant
13
C fractional enrichment and the total influx of
13
C label
metabolites into the glial and neuronal TCA cycles is equal
to the total outflux into the bicarbonate pool, the glial
acetate oxidation rate, V
g ac
, was determined using the
following formula:
V
gac
V
total
%E
HCO3
1:1=%E
Ac
1:1
The total bicarbonate production, V
total
, is given by
V
tot
=3 V
tca
+2 V
g ac,
where V
tca
is the rate of glucose
oxidation in neuron and glia (V
nglc
+V
nglc
) (Figure 1).
Because V
g ac
< <V
tca,
V
tot
is approximately 3 V
tca
.
An additional method of calculating maximum glial TCA
cycle rate was applied, determining brain bicarbonate in
natural abundance and
13
C-enriched spectra with reference
to the adjacent Cr/PCr resonance (see above).
Figure 1 Glial versus neuronal metabolism: pathway of
13
C label
from [1-
13
C]acetate to tricarboxylic acid (TCA) cycle in human
brain. Selective metabolism of acetate and glucose occurs in glia
and neurons, respectively. Only the relevant intermediates are
shown. Glia selectively transports acetate which is then
metabolized through acetate thiokinase and reactions of the
mitochondrial tricarboxylic acid (TCA) cycle forming
13
C
glutamate and glutamine, enriched in the C1 and C5 positions,
before final oxidation and release as
13
C bicarbonate
(CO
2
=HCO
3

). Five of the relevant cerebral metabolic products

are showed in sequential
13
C brain spectra of Figure 3. The
alternative metabolic pathways of
13
C glucose metabolism,
predominantly in neurons, but not contributing to this study in
which only the single substrate, [1-
13
C]acetate is provided. Ac,
acetate; Glc, glucose; Glu, glutamate; Gln, glutamine; CO
2
,
carbon dioxide; Pyr, pyruvate; V
g glc
, glial glucose oxidation; V
g ac
,
glial acetate oxidation; a-KG, a-ketoglutarate; V
glu
, glutamate
synthesis rate; V
gln
, glutamine synthesis rate. Glucose is
metabolized in neuron and glia whereas acetate is metabolized
exclusively in glia.
Glial dysfunction in methamphetamine abusers
N Sailasuta et al
953
Journal of Cerebral Blood Flow & Metabolism (2010) 30, 950960
Statistical Analysis
Statistical analyses were performed with StatView software
(SAS Institute, Cary, NC, USA). For continuous variables, the
means and standard deviations were calculated separately
for the AMD and the normal subjects. An analysis was
performed to show that variance in the two groups was the
same using F test, followed by analysis with the two-sample
t-test. All comparisons yielding a P-value of less than 0.05
were considered statistically significant. All data are
expressed as mean

standard deviation (s.d.) in the text.

Results
Determination of Cerebral Bicarbonate Concentration
from Natural Abundance
13
C MRS
Cerebral [HCO
3

] calculated in a contiguous volume

of mixed white and gray matter of the human frontal
lobe was found to be between 11 and 15 mmol/L
(Figure 2), values consistent with literature data and
comparable to the those obtained in earlier direct and
indirect assays in simian and animal brains (Siesjo,
1964). Results obtained by direct
13
C MRS assay in
frontal brain were also indistinguishable from those
obtained in posterior brain in the same group of
control subjects (Table 1). Results for cerebral
bicarbonate concentration in the cohort of recovering
methamphetamine-abusing subjects (Table 2) fell
within this normal range indicating that metabolic
pH homeostasis is broadly normal in the recently
abstinent methamphetamine-abuse human brain.
Determination of the Maximum Rate of Bicarbonate
Production (
max
V
HCO

3
) in Human Brain
In contrast, using time course data in the two groups
of subjects, it was observed that the rate at which
Figure 2 Human
13
C MR frontal brain spectra acquired during [1-
13
C]acetate infusion: carbon-13 magnetic resonance spectroscopy
(MRS) at baseline and 120mins after the infusion (154 to 164p.p.m.) in a control and an abstinent methamphetamine-dependent
(AMD) subject.
13
C MRS spectra show HCO
3

and total Cr (tCr) chemical shift region (154 to 164p.p.m.) at baseline and at the end
of the infusion study in a normal control subject (A) and an AMD subject (B). The lower, natural abundance spectra for the normal
control (left) indicate that cerebral bicarbonate concentration is very similar to that of [Cr +PCr], i.e., 11mmol/L. The resting
cerebral bicarbonate concentration in a representative AMD subject (right) was indistinguishable from the control (HCO
3
=11mmol/
L). After 2h of [1-
13
C]acetate infusion, substantial but different increases in cerebral
13
C bicarbonate intensity were observed. Again,
by comparing the adjacent, unenriched Cr +PCr, the rate of enrichment of cerebral bicarbonate was substantially lower in the AMD
(right) than in the normal control (left). These representative data are seen to be statistically significantly different when normal and
AMA subjects are compared in Tables 1 and 2. In the control subject, the concentrations of bicarbonate determined in this way were
15mmol/g at baseline and 49mmol/g at the end of the 2h infusion study (Cr does not become enriched) whereas in the AMD subject
initial bicarbonate was 10mmol/g at baseline but reached only 25mmol/g after 2h.
Glial dysfunction in methamphetamine abusers
N Sailasuta et al
954
Journal of Cerebral Blood Flow & Metabolism (2010) 30, 950960
[1-
13
C]acetate was incorporated into the final oxida-
tion product bicarbonate was markedly different
between drug-abusing and normal subjects. Figure 2
compares
13
C MRS spectra (154 to 164 p.p.m.) from
a representative control subject (A) and a metham-
phetamine abstinent subject (B). From the start
(natural abundance) to the end of the 2h data
acquisition, there was almost fivefold increase in
13
C incorporation (fully
13
C-enriched) in the control,
whereas in the AMD subject, the full extent of
13
C
enrichment was closer to threefold. Natural abun-
dance bicarbonate concentration at baseline and at
the end of the infusion estimated was similar in the
two subjects. In the control subject, the concentra-
tions of bicarbonate determined in this way were
15 mmol/g at baseline and 49mmol/g at the end of the
2 h infusion study (Cr does not become enriched)
whereas in the AMD subject initial concentration of
bicarbonate was similar, 10 mmol/g, at baseline but
reached only 25 mmol/g after 2h. This was less than
50% of the enrichment measured in the normal
control. The baseline bicarbonate concentration is
within the range of normal values calculated from
HendersonHasselbach equation, pH=pK
a
+log
([HCO
3
]/[CO
2
]) (Agabiti et al, 1973) using brain pH
of 6.99 (Bluml et al, 1998), pK
a
=6.1 and pCO
2
of 35
to 48 mmHg in normal adults brain of 11.2 to
19.9 mmol/L. The maximum rate of bicarbonate
enrichment (
max
V
HCO

3
) in the frontal brain in a
control was 0.28 mmol/g per min compared with an
AMD subject of 0.13 mmol/g per min. This result is
highly suggestive of a persistent major abnormality
in glial oxidative mechanism in frontal brain of absti-
nent methamphetamine subjects and is consistent
with previously published work. For a precise
evaluation of the impact of AMD on glial metabolic
rate, we applied the previously used method for
calculation of V
g ac
.
Comparison of Fractional Enrichment of Bicarbonate
and Glial Acetate Oxidation Rate in the Posterior and
Frontal Brain of Normal Subjects
Five control women were scanned using the frontal
brain protocol and three of these women and one
man were scanned using the posterior brain protocol.
Summary of the results is shown in Table 1. Initial
slope of H
13
CO
3
appearance in brain was similar for
frontal and posterior brain (P=0.25), as were frac-
tional enrichment of bicarbonate at 60, 80, and
120 mins of observation. The calculated V
g ac
from
Table 1 Fractional bicarbonate enrichment and glial metabolic rate for frontal and posterior brain in controls
ID [HCO
3
]
(brain,
mmol/g)
%E
Ac
(plasma)
Initial
slope
a
%E HCO
3

at 60mins
(brain)
%E HCO
3

at 80mins
(brain)
%E HCO
3

at 120mins
(brain)
V
g ac
(mmol/g
per min)
b
V
g ac
from Bluml
et al
c
(mmol/g
per min)
Control posterior brain (N=4) 12

1.3 78 (N=1) 0.11

0.06 4.74

1.1 5.2

1.3 6.2

1.6 0.09

0.03 0.13

0.03 (N=3)
Control frontal brain (N=5) NA 78 (N=1) 0.10

0.02 5.1

0.7 5.9

0.8 7.0

1.0 0.11

0.01
d
P (frontal versus posterior brain) NA NS NS NS NS NS
NA, not available; NS, not significant.
Values are mean

standard deviation (N=number of examinations).

a
Because the time course of the appearance of HCO
3

signal amplitude was linear from the start of the infusion to 40mins, the slope of the straight line was
calculated and reported here.
b
Determined using %E at 60mins and total TCA cycle rate of 0.7mmol/g per min (Mason et al, 1995).
c
Value reported by Bluml et al (2002) determined at 60mins after start of infusion in the posterior brain.
d
V
g ac
calculated at 80 and 120mins are 0.13 and 0.16mmol/g per min.
Table 2 Fractional bicarbonate enrichment and glial metabolic rate for frontal brain in control and AMD subjects
Subjects Mean
age/
gender
[HCO
3
]
(brain,
mmol/g)
%E
Ac
(plasma)
Initial
slope
a
%E HCO
3
at 60mins
(brain)
%E HCO
3
at 80mins
(brain)
%E HCO
3
at 120mins
(brain)
V
g ac (
mmol/g
per min)
b
Control frontal brain (N=5) 26

1
5 F
NA 78 (N=1) 0.10

0.02 5.1

0.7 5.9

0.8 7.0

1.0 0.11

0.01
AMD frontal brain (N=5) 307
5 F
10
c
83 (N=1) 0.050.02 2.70.5 3.140.5 3.790.6 0.040.01
P (control versus AMD) 0.12 NA 0.002 <0.001 <0.001 <0.001 <0.001
AMD, abstinent methamphetamine-dependent; F, female; NA, not available.
Values are meanstandard deviation (N=number of examinations).
a
Because the time course of the appearance of HCO
3

signal amplitude was linear from the start of the infusion to 40mins, the slope of the straight line was
calculated and reported here.
b
Determined using %E at 60mins and total TCA cycle rate of 0.7mmol/g per min (Mason et al, 1995).
c
Natural abundance [HCO
3

] could not be determined with accuracy (low signal-to-noise ratio) from individual AMD subjects, the reported value was
determined from summed spectra from all five AMD subjects, and appeared to be indistinguishable from controls.
Glial dysfunction in methamphetamine abusers
N Sailasuta et al
955
Journal of Cerebral Blood Flow & Metabolism (2010) 30, 950960
the normal posterior brain (0.090.03 mmol/g per
min) was in excellent agreement with value pre-
viously obtained by us. Finally, the calculated rate of
frontal brain [1-
13
C]acetate oxidation (0.110.01
mmol/g per min) was indistinguishable from poster-
ior brain.
Appearance of
13
C-Enriched Metabolites of
[1-
13
C]Acetate in the Frontal Brain of Normal and AMD
Subjects
Figure 3 is a time course of the appearances of 5-
13
C
glutamate and [1-
13
C]acetate (A), 5-
13
C glutamine (B),
and HCO
3

(C) resonances in a control subject.

Similar plots were obtained for AMD (not shown).
Rapidly increased to peak amplitudes for the
enriched acetate and 5-
13
C glutamate as well as
5-
13
C glutamine as were observed at 56 mins and
slowly eliminated by approximately 120 mins. Bicar-
bonate signal amplitude reached a maximum and
remained essentially constant after 60mins. How-
ever, the pseudo-steady state for 5-
13
C glutamate
could only be indirectly verified because of the co-
resonance of the precursor [1-
13
C]acetate. Neverthe-
less, these observations confirmed that all of the
13
C
label entering the glial TCA cycle is being transferred
to bicarbonate and is not sequestered in other
metabolic pools.
Figure 4 shows stack plots of labeled metabolites
comparing results obtained in a normal control (A)
with those from a representative AMD subject (C),
over time and below, and the summed spectra of the
total brain enrichment at the end of the infusion
protocol for control (B) and AMD (D). C5 glutamate
(and acetate), C5 glutamine, C1 glutamateglutamine
and bicarbonate resonances are clearly visible. The
strikingly reduced appearance of
13
C label in bicar-
bonate is evident in Figure 2D, in the AMD subject
compared with control in Figure 2B. This result was
confirmed in the entire group of AMD. The enrich-
ment of HCO
3

estimated from the time course of the

fractional enrichment of HCO
3

was significantly
decreased in AMD (Figure 5) and the rate of acetate
oxidation in glial, V
g ac
, as well as %E HCO
3

was
quantified from the initial slope of bicarbonate
enrichment versus time, or from %E at 60, 80, and
120 mins (compare Table 2 with Table 1). Compar-
ison of bicarbonate productions between normal and
AMD subjects is shown in Figure 6. Assuming the
overall TCA cycle rate of 0.7 mmol/g per min for the
intact human brain (Mason et al, 1995), the average
rate of glial acetate oxidation in the frontal brain was
0.04 for AMD subjects compared with 0.11 mmol/g
per min in normal subjects, a reduction of 60% in
glial metabolism rate for acetate oxidation. There
were no significant differences (P=0.12) between
gender and age in the two subject groups, normal
controls and AMD subjects.
Discussion
In this pilot study we confirm the unique value of
low-power
13
C MRS applied for the first time to
methamphetamine abusers in frontal regions of the
human brain. We have discovered a hitherto un-
suspected 50% reduction in oxidative rate in the
recovering brain after methamphetamine abuse.
Although it has been known for many years that
acetate, which is a preferred glial respiratory fuel,
can be applied to defining glial TCA cycle rate of the
normal-functioning human brain in vivo, to the best
of our knowledge, this study represents the first
systematic attempt to define alterations in glial
metabolic rate as a result of neuropathology. Several
earlier studies have used
13
C MRS with
13
C glucose to
explore human brain neurochemical abnormality.
Figure 3 Time courses of glutamate C5 and acetate (A),
glutamine C5 (B), and HCO
3

(C) from a healthy subject. Signal

intensities are shown in arbitrary units. Note that glutamine C5,
glutamate C5, and bicarbonate become increasingly enriched
over time, consistent with the expected path of glial metabolism
of
13
C acetate shown in Figure 1. After significant enrichment up
to 60 to 80mins, pools of glutamate C5 and glutamine decline,
presumably as they are in turn metabolized to the final product,
13
C HCO
3

. No significant sequestration in glutamate and

glutamine occurs.
Glial dysfunction in methamphetamine abusers
N Sailasuta et al
956
Journal of Cerebral Blood Flow & Metabolism (2010) 30, 950960
[1-
13
C]glucose, a predominant neuronal respiratory
fuel, has revealed significant abnormalities of gluta-
mate enrichment and TCA cycle rate in diseases as
diverse as Alzheimers, hepatic encephalopathy,
mitochondrial disorders, and epilepsy (Bluml et al,
2001; Lin et al, 2003; Ross et al, 2003). However, in
each of these examples the neuron was the target of
investigation. Furthermore, the region of brain
examined was the posterior parietal lobe, and
suboptimal for frontal lobe study based on safety
considerations dictated by Federal guidelines for
Specific Absorption Rate. The present report appears
more relevant both as a study focused directly on the
question of deficiency in glial oxidative metabolism
and by exploring a brain region implicated in drug
abuse abnormality by almost all independent neu-
roscience techniques including executive function
and neuropsychologic metrics. Acetate is metabo-
lized exclusively in glia (Muir et al, 1986; Waniewski
and Martin, 1998); it is reasonable to assume a
single-compartment model for cerebral bicarbonate
production, that is, the rate of bicarbonate produc-
tion is equal to the rate of acetate influx into the glial
TCA cycle. Intravenous infusion of [1-
13
C]acetate
rapidly produced cerebral C5 glutamate in the first
turn of the TCA cycle where it is co-resonant and
indistinguishable from the acetate at 182p.p.m.
Glutamine C5 (at 178 p.p.m.) appeared in the second
Figure 4 Effect of methamphetamine abuse on cerebral [1-
13
C]acetate metabolism. Frontal brain
13
C metabolites observed during
[1-
13
C]acetate infusion: figure shows carbon-13 MRS (150 to 195p.p.m.) in a control and an abstinent methamphetamine-
dependent subject after enrichment with [1-
13
C]acetate. Stacked sequential carbon-13 spectra and spectra summed for the entire
infusion protocol (120mins, bottom) are shown for a control (A, B) and an abstinent methamphetamine-dependent (AMD) subject
(C, D). The spectra identified enriched cerebral metabolites glutamate C5+C1 acetate (182p.p.m.), glutamine C5 (178p.p.m.),
glutamate C1+glutamine C1 (175p.p.m.), and the final oxidation product, bicarbonate (161p.p.m.).
Glial dysfunction in methamphetamine abusers
N Sailasuta et al
957
Journal of Cerebral Blood Flow & Metabolism (2010) 30, 950960
turn of TCA cycle and glutamate C1 and glutamine
C1 (both resonates at 175 p.p.m.) in a subsequent
turn. Carbon dioxide is the final product of the TCA
cycle that appeared as bicarbonate at 161 p.p.m.
Because of our low [1-
13
C]acetate infusion dosage,
steady states were not convincingly reached in our
experimental studies, therefore to determine frac-
tional enrichment of bicarbonate, an exponential
function was fitted to the time course of the
production of bicarbonate.
The glial bicarbonate production rate was subse-
quently calculated from exponential fitting of %E at
60 mins and therefore represents the glial TCA cycle
rate. The glial TCA cycle rate determined this way
represents approximately 10% of the total TCA cycle
rate in the brain of fasted normal subjects. This rate is
similar to the previously reported value by us in the
parietal brain region using similar [1-
13
C]acetate
protocol (Bluml et al, 2002).
Information regarding abnormalities of glial cells
can typically be obtained with proton MRS. There
are few MRS studies (nine studies from 2000 to 2008)
on methamphetamine abuse that reveal abnormal-
ities in metabolite concentrations in frontal cortex
(Ernst et al, 2000), frontal white matter (Ernst
et al, 2000; Nordahl et al, 2002), and basal ganglia
(Sekine et al, 2002). Proton MRS studies show that
methamphetamine-dependent subjects had decreased
N-acetylaspartate, higher myoinositol, and elevated
glutamate in the frontal white matter. Possible
interpretations of these metabolite abnormalities in
these subjects include local loss or dysfunction of
neurons, and glial abnormalities (Abulseoud et al,
2009). From
13
C MRS, there is a significant reduction
of glial TCA cycle rate in AMD subjects compared
with non-drug users, further emphasizing the impact
of methamphetamine use on glial function.
Possible limitations to
13
C MRS: as with all
isotope techniques, there is an unknown fraction
of cerebral metabolism that will continue to consume
(unenriched) endogenous respiratory fuels, in this
case predominantly C-12 glucose. This may dilute
the resulting
13
C bicarbonate pool derived from
13
C-
enriched acetate. One correction has been applied, as
suggested by Bluml et al (2002) in expressing the
13
C
HCO
3

as a fraction of the total bicarbonate produced

in unit time. Although there is no way with the
present technique to ensure that overall cerebral
metabolic rate is normal in AMD (additional studies
using
13
C glucose will clarify the point further),
nevertheless, it is difficult to envisage a more than
50% change in intrinsic glucose metabolic rate that
would be necessary to explain the present findings.
Earlier positron emission tomography studies of
AMD argued against any significant increase in
neuronal metabolic rate in frontal brain (Kim et al,
2005, 2009; London et al, 2005). Contrary results
(small with reference to the present results) were
obtained by Wang et al (2004). There are other
limitations to our study. First, the study sample is
small; though the effect is large and statistically
robust. This is partly a consequence of the costs
([1-
13
C]acetate infusion) and technical difficulty of
the present procedures. In the future, both aspects
promise to be greatly simplified allowing large-scale
replication of the present findings in this and other
studies. Another possible limitation of the study is
the assumption that fractional enrichment of
[1-
13
C]acetate is similar between the brain and blood
(Bluml et al, 2002). Note that the enrichments of
Glu1, Gln1, as well as Gln5 were also observed but
under our non-steady-state experimental condition,
we did not generate kinetic data for metabolic rate
calculations used in more complex theoretical
models. Alternative, steady-state protocols, invol-
ving larger doses of [2-
13
C] acetate and longer
protocols may lend themselves to resolving any
remaining kinetic issues. Finally, the study of
Figure 5 Effect of methamphetamine abuse on cerebral
metabolism of
13
C acetate. Time courses of appearance of the
end product of [1-
13
C]acetate oxidation, HOC
3

are compared.
Low-power noise nuclear overhauser effects (NOE) spectra were
acquired in 6.5min blocks for up to 120mins after enrichment
by i.v. acetate (3mg/kg body weight) from frontal brain in five
abstinent methamphetamine-dependent (AMD) subjects and
five control subjects. Logarithmic fits of the averaged percent
fractional enrichment of bicarbonate (%E) were plotted as a
function of time after start of the infusion. %E in AMD (lower
trace) is shown to be lower than controls (upper trace). Standard
deviations are also shown as cross-bars.
Figure 6 Comparison of methods to express effect of abstinent
methamphetamine-dependent (AMD) in
13
C HCO
3

production
from [1-
13
C]acetate in human frontal brain. Results shown in
Figure 5 are quantified. Means and standard deviation of percent
fractional bicarbonate enrichment calculated at three different
time points (60, 80, and 120mins) and the initial slope of
bicarbonate production from control and AMD groups. P-value in
each group comparison is <0.001.
Glial dysfunction in methamphetamine abusers
N Sailasuta et al
958
Journal of Cerebral Blood Flow & Metabolism (2010) 30, 950960
short-term abstinence may limit the generalizability
of results to abuse versus craving and relapse in
study of methamphetamine. We propose that this
successful demonstration of a hitherto unrecognized
but sizeable neurometabolic adaptation is worthy of
further consideration and more sophisticated experi-
mental design that may reveal novel mechanisms
relevant to human drug abuse and many other
conditions currently ascribed to glial dysfunction.
In this preliminary study, we used a novel method
of frontal lobe
13
C MRS to explore possible defects
that may persist in the glia of drug-addicted subjects,
using as the example, recently AMD subjects.
Additional studies, using the neuronal substrate
[2-
13
C] glucose, will be necessary to determine the
cellular specificity and the frontal localization of
these neurochemical injuries. The novel capability to
dissect two major cell populations at risk in the
intact brain and in patients at varying stages of the
abuseaddictionrecovery cycle of methampheta-
mine abuse promises new insights into an intractable
clinical problem.
Acknowledgements
We thank Mentors to the K Award (NS), Alan Stacy
for advice and Thao Tran and Keiko Kanamori for the
technical and scientific insights. OA was supported
in part by research funds from Keck School of
Medicine, Division of Psychiatry, University of
Southern California.
Disclosure/conflict of interest
The authors declare no conflict of interest.
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