disfuncion glial y metanfetaminas

Glial dysfunction in abstinent methamphetamineabusers

Napapon Sailasuta1, Osama Abulseoud2, Kent C Harris1 and Brian D Ross1,3

1Clinical Spectroscopy Unit, Huntington Medical Research Institutes, Pasadena, California, USA;2Department of Psychiatry, University of Southern California, Los Angeles, California, USA; 3Rudi SchulteResearch Institute, Santa Barbara, California, USA

Persistent neurochemical abnormalities in frontal brain structures are believed to result frommethamphetamine use. We developed a localized 13C magnetic resonance spectroscopy (MRS)assay on a conventional MR scanner, to quantify selectively glial metabolic flux rate in frontal brainof normal subjects and a cohort of recovering abstinent methamphetamine abusers. Steady-statebicarbonate concentrations were similar, between 11 and 15 mmol/L in mixed gray-white matter offrontal brain of normal volunteers and recovering methamphetamine-abusing subjects (P > 0.1).However, glial 13C-bicarbonate production rate from [1-13C]acetate, equating with glial tricarboxylicacid (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 inglial TCA cycle rate that normally comprises 10% of total cerebral metabolic rate may impactoperation 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 todefine directly an abnormality in glial metabolism in human methamphetamine abuse, sequentialstudies using analogous 13C MRS methods may determine ‘cause and effect’ between glial failureand neuronal injury.Journal of Cerebral Blood Flow & Metabolism (2010) 30, 950–960; doi:10.1038/jcbfm.2009.261; published online30 December 2009

Keywords: acetate; glutamate; MR spectroscopy; neurochemistry; neuronal–glial interaction; neurotransmitters

Introduction

The neuropathologic presentation of substance abuseand addiction, a serious health, social, and economicproblem in the United States, involves abnormalitiesof the dopaminergic and glutamatergic systemsleading to neuronal–glial dysfunction (Stephansand Yamamoto, 1994; Volkow et al, 2001, 2002). Ithas been commonly observed that objective neuro-chemical changes persist beyond the period of activedrug use, inviting the hypothesis that persistentneurochemical abnormalities contribute to addictivebehavior 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 mayhave implications for the design of pharmaceuticalsfor future treatment of addiction and relapse pre-vention. Evolving methods of noninvasive brainanalysis, in particular 13C magnetic resonance spec-troscopy (MRS) with 13C glucose and 13C 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 newopportunities to examine the impact of drugs ofabuse on neurons and glia separately. This pilotstudy was undertaken to establish the feasibility ofapplying [1-13C]acetate MRS to quantify glial meta-bolic rate in frontal brain structures implicated indrug abuse, reward, and relapse (Gass and Olive,2008). Future studies, in which the starting substratewould be [2-13C] glucose, could then be applied toprobe the impact on neuronal metabolism directly(Gropman et al, 2009).

Glutamate (Glu) is an amino acid intimatelyinvolved in neurotransmission and a marker ofmitochondrial oxidative status and tricarboxylic acid(TCA) cycle function. The glutamate–glutaminecycle links glia with neurons in the mechanism of

Received 28 September 2009; revised 18 November 2009; accepted30 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: [email protected]

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.

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glutamate neurotransmission, the neurotransmitterpathway overall believed to account for 80% of brainneurotransmitter function. The functions of glialcells in neurotransmission are often viewed asmerely supportive; however, failure of glial reuptakeof glutamate released at synapses can result inaccumulation of extracellular glutamate, leading toneurotoxicity. Glutamate is crucial in regulating boththe development and the expression of addictivebehaviors, which requires glutamate receptor stimu-lation in the ventral tegmental area and is associatedwith enhanced glutamate release in the prefrontalcortex (Kalivas et al, 2009; Rebec and Sun, 2005).Using the single voxel proton MRS method, whichdetermines steady-state concentration of brain meta-bolites, we found that preliminary data provide moredirect evidence of elevated frontal brain glutamateconcentration during the cerebral adaptation tomethamphetamine abuse (Abulseoud et al, 2009).This is in addition to earlier reports of persistentneurochemical abnormalities; elevated myoinositol,a glial marker, and reduced N-acetylaspartate, aneuronal marker, observed in the frontal cortex ofabstinent methamphetamine users (Ernst et al, 2000;Sung et al, 2007). Together these results indicate thatneuronal injuries from drug abuse are likely accom-panied by significant abnormalities in glial functionand metabolism.

The aim of this pilot study was to determine theextent of glial metabolic abnormalities in abstinentmethamphetamine-dependent (AMD) subjects usingMRS. 13C MRS techniques, which permit TCA cyclerate determination directly and further allow thedissection of glial from neuronal pathways ofglutamate metabolism based on the selective uptakeand metabolism of acetate by glia (Muir et al, 1986)and glucose by neurons have recently been exploredin 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 tothe present purpose of drug abuse has been the needto confine human 13C MRS assays to posterior brainregions. This brain region is distant from heat-sensitive optical structures but purportedly of mini-mal relevance to the neuropsychopathology of drugabuse, which most investigators locate in frontaland limbic pathways (London et al, 2000). Thesetechnical limitations have recently been overcome(Sailasuta et al, 2008) with two distinct and robust invivo 13C MRS techniques adapted to the humanfrontal lobe. First, the use of 13C isotopes of whichthe products can be readily observed with minimalor absent proton decoupling. Second, the develop-ment of low-power nuclear overhauser effects cannow be used for the same purpose. Details of thesetechnical advances and their efficacy in humanstudies using a conventional low field 1.5 T MRscanner have been published (Li et al, 2007;Sailasuta et al, 2008). In this report we describe thefirst such method designed to separately interrogate

glial glutamate formation and specifically, to quanti-fy the glial oxidative rate based on the appearance of13C label from the unique glial precursor, acetate, intoits final product bicarbonate (HCO3

�). We havedetermined the rate at which [1-13C]acetate appearsin cerebral bicarbonate [H13CO3] in normal frontalbrain, and shown a marked reduction in thecontribution of glial acetate oxidation to the TCAcycle in frontal brain of AMD subjects.

Materials and methods

Subjects

A total of 21 in vivo 13C MRS studies were performed.Eleven subjects participated in this study approved byinstitutional review board and HIPAA, of whom 6 werenormal, age- and gender-matched subjects. The six healthycontrol subjects (mean age 26±1 years, one man) wererecruited from the local community. Three of the controlsubjects were scanned twice on separate days to compare13C acetate metabolism in frontal and posterior brain. Fivefemale AMD subjects (mean age 31±7 years) recruitedfrom local drug rehabilitation centers and counselingcenters within the Los Angeles County were examinedonly once, in the frontal brain region. All five subjectsreported methamphetamine as the drug of choice and metDiagnostic and Statistical Manual of Mental Disorders,fourth edition criteria for lifetime methamphetaminedependence from the Structured Clinical Interview (Firstet al, 1994). Self-report by study participants indicated theaverage period of methamphetamine use was 74 days andeach had been abstinent from methamphetamine or otherillicit drugs for at least 7 days before the studies. The studyprotocols were approved by the institutional review boardof the Huntington Memorial Hospital and the UniversityHospital at University of Southern California. Writteninformed consent was obtained from all participants.

Carbon MRS

The techniques for quantification of glial metabolic rate ofin vivo human brain 13C MRS using [1-13C]acetate assubstrate have been described in detail previously (Blumlet al, 2002). Significant modifications necessary for safetransfer of the technique to frontal brain for this study wereas follows:

(i) To establish metabolic rates for [1-13C]acetate for thefrontal lobe of human brain, not hitherto examined, wedeveloped low rf power 13C MRS (Sailasuta et al,2008). To minimize RF heating in a clinical magneticresonance imaging system, all subjects underwent 13CMRS at 1.5 T GE clinical scanner (GE Healthcare,Milwaukee, WI, USA) equipped with broadbandexciter, a stand-alone proton decoupler, and a vectorsignal generator (Agilent Technology E4433B, SantaClara, CA, USA). A half-helmet head coil was usedwith the subject lying supine within the MR scanner.Its construction was a modification of the half-headcoil used in the posterior brain 13C MRS study from

Glial dysfunction in methamphetamine abusersN Sailasuta et al

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Journal of Cerebral Blood Flow & Metabolism (2010) 30, 950–960

this laboratory (Bluml et al, 2001). The coil consistedof a 4-inch proton loop and a 3.5-inch 13C loop fortransmission and reception. Fast spin echo imagingscan was used to confirm the consistency of theobserved volumes in all patients. For the frontal brainstudy, the half-helmet coil placement was such that theanterior portions of the frontal lobe were mainlyincluded in the field of view and similarly for theposterior brain where the posterior region of the brainwas included in the field of view (Gropman et al,2009). The coil was tuned to best match both protonand 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 thatmetabolic rates for [1-13C]acetate in human frontal lobeare comparable to posterior parietal brain, valuespreviously reported using [1-13C]acetate by us, andalso recently confirmed using [2-13C] acetate by Lebonet al (2002), assays of 13C enrichment in the posteriorbrain were performed in four normal subjects. To allowfor complete clearance of 13C-enriched metabolitesfrom the posterior brain for three of the four normalsubjects, this examination was followed a minimum of48 h later by examination of the frontal brain region ineach of the three normal subjects.

Magnetic resonance image was acquired at the outset ofeach 13C MRS examination, indicating regions of interest ofmixed white and gray matter of posterior parietal or frontaland prefrontal brain structures, respectively, for 13C MRSacquisition. Brain volumes, approximately 100 cm3 in eachprotocol, included similar proportions of white and graymatter, were comparable and have been previously pub-lished (Gropman et al, 2009; Sailasuta et al, 2008).

Determinations of Cerebral Bicarbonate Concentrationfrom Natural Abundance 13C MRS and Maximum Rateof HCO3

� (maxVHCO�3 ) Production

Natural abundance 13C MRS was acquired for 20 minsbefore the start of the acetate infusion to show adjacent,well-resolved individual resonances for bicarbonate andfor Cr + PCr (total creatine, tCr) as previously reported byBluml et al (2001). Because tCr has been used successfullyas an internal metabolite reference in several proton MRSstudies in abstinent methamphetamine users (Nordahlet al, 2005, 2002; Salo et al, 2007) as well as other humanstudies (see review, Xu et al, 2008), and does not becomeenriched during the time scale of the present studies, wehave used this as an internal reference to quantify HCO3

�

and its subsequent enrichment from [1-13C]acetate. Toimprove signal to noise (natural abundance 13C = only1.1% of total) and thereby improve quantification, weacquired additional natural abundance 13C brain spectraduring separate imaging sessions not associated with 13Cinfusions. The timed intravenous infusion of 99% 13C-enriched [1-13C]acetate infusion was started and brainspectra were acquired continuously thereafter for a mini-mum of 2 h. The 61

2 min blocks of low-power noise nuclearoverhauser effects (0.9 W for frontal brain and 2.5 W for the

posterior brain) 13C MRS spectra acquired were stored andsummed, as previously described (Sailasuta et al, 2008).13C MRS examination times of 120 mins or more were welltolerated by all subjects with no observed adversereactions. Maximum rate of HCO3

� production (maxVHCO�3 )was calculated from the natural abundance bicarbonateconcentration at baseline and at the end of the infusionestimated from the ratio of the areas under the tworesonances, HCO3

� (161 p.p.m.) and tCr (158 p.p.m.) usinga Marquardt fitting algorithm and assuming tCr concentra-tion 11 mmol/g in the brain.

Infusion Protocol

The 99% 13C-enriched [1-13C]acetate solution was preparedunder good manufacturing practice by the CambridgeIsotope Laboratories (Andover, MA, USA) and shipped inbulk to a licensed pharmacist in Minnesota, registered andapproved by the US Food and Drug Administration to shipsterile, pyrogen-free reagents to California. The reagent wasprepared on a named-subject basis and used within 48 hafter preparation. This study was performed under theFood and Drug Administration Investigational New DrugApplication no. 59690. To establish similar metabolicstatus, subjects were fasted for 6 h before the study.Intravenous administration (through an arm vein) of 120to 300 ml of 0.4 mol/L sterile, pyrogen-free solution of[1-13C]acetate 99% enrichment was started (OA) at aninfusion rate of 3 mg/kg per min for 60 mins.

Fractional Enrichment of Serum [1-13C]Acetate

Because earlier preclinical and clinical studies have estab-lished that 13C acetate consumption proceeds rapidly in thebrain and the [1-13C]acetate resonance cannot readily bedistinguished from its primary metabolic product, 5-13Cglutamate on the basis of near-identical chemical shift, plasma13C acetate enrichment was used as a surrogate marker of thefractional 13C enrichment (E) of stable isotope availability forglial oxidation. Serial blood samples were collected usingheparinized syringe before infusion and at 15 min intervalsuntil the end of the study (at 120 mins). Blood samples werecentrifuged and the resulting protein-free plasmas were storedfrozen until analysis. Plasma samples were deproteinizedusing 2:1 volume ratio of 99.9% ethanol to plasma and driedunder vacuum. Samples were dissolved in 99.9% D2O beforeproton NMR measurements using a Varian 300 MHz spectro-meter. Fractional enrichment of plasma acetate was deter-mined under fully relaxed conditions (repetition time 18 s)from the ratio of the relative peak areas of the 13C satellites tothe total area of the corresponding acetate H2 resonance at1.9 p.p.m. (Moreno et al, 2001).

Data Processing and Fractional EnrichmentCalculation of Cerebral Metabolites

The principal metabolic end point of this study was thequantification of intrinsic cerebral bicarbonate (HCO3

�),together with the progressive increase in fractional 13C


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enrichment (%E) of the HCO3� pool as the result of

complete oxidation of the sole 13C-enriched precursor ofglial TCA cycle activity, [1-13C]acetate. Because 13C creatineand phosphocreatine do not become enriched from 13Cacetate (Cerdan, 2003; Chapa et al, 1995) and resonatedistinctly in a portion of the brain spectrum unencumberedby overlying resonances, we used 13C total signal intensityfor [Cr + PCr] to quantify the initial brain bicarbonateconcentration in nonenriched baseline 13C MR scans fromeach subject. We assume there are no significant differ-ences in T1 or T2 relaxation times between the two.Fractional enrichment of HCO3

� is then defined as theincrease in intensity relative to that of [Cr + PCr] which isnot enriched from the infused source of [1-13C]acetate.Progressively increasing intensity of 13C HCO3

� resonancewas recorded throughout and the relative rate of enrich-ment was expressed as percent over time, slope of initialincrease, and final %E bicarbonate. The other anticipatedcerebral metabolites of [1-13C]acetate, glutamine C1 and C5,and glutamate C1 and C5, were also readily observed. Anobserver-independent automatic IDL-based software devel-oped in our laboratory (Shic and Ross, 2003) and SAGE (GEHealthcare, Milwaukee, WI, USA)/IDL (ITT Visual Informa-tion Solutions, Boulder, CO, USA), a commercially availablesoftware, were used for data processing (NS, KH). Data blocks(61

2 min) were first zero filled to 16K data points, applied 7 HzLorenzian to Gaussian lineshape transformation, Fourier-transformed, and phase-corrected. Owing to the absence ofoverlapping resonances, relative contributions of 13C singlet ofHCO3

� (161 p.p.m.), C1-acetate and C5-Glu (182 p.p.m.), andC5-Gln (178 p.p.m.) were readily determined from peakamplitudes of the observed resonances. In the case of C1-acetate and C5-Glu, no separation of the resonances could beachieved under near-steady-state conditions. Based on earlierstudies, the contribution of [1-13C]acetate cerebral flux, %E13C brain was considered approximately %E acetate of blood.

13C fractional enrichments (E) of metabolic productswere calculated from:

Emet ¼ Spost=Spre�1:1%

where Spost and Spre are the signal intensities of theresonances in the baseline scan and in scans after infusionstart, and 1.1% is the natural abundance of 13C.

Determination of the Glial Acetate Oxidation Rate(Vg ac)

For consistency with earlier studies, results are expressedas glial acetate oxidation rate (Vg ac). Acetate has beenshown to be metabolized exclusively in glia, probably as aresult of the unique membrane transporter present in gliabut lacking in neurons (Muir et al, 1986; Waniewski andMartin, 1998). During the first turn of the TCA cycle, C1label (from C1-acetate) is transferred to form glial C5 andC1 glutamate and transformed by glial glutamine synthaseto C5 and C1 glutamine. C1 label CO2 (HCO3

�) is released inthe second turn of the TCA cycle (Cerdan et al, 1990).Calculation of oxidative rate from [1-13C]acetate to the finaloxidative product H13CO3 therefore involves no assump-tions concerning neuronal compartmentation or flux as

proved necessary for calculations of [2-13C]acetate metabo-lism (Lebon et al, 2002). Nevertheless, the two methodsappear to yield comparable estimates for average glialmetabolic rate at approximately 10% to 15% of the rate ofnormal neuronal TCA cycle (Shen et al, 1999). In this studywe used the approach described previously by Bluml et al(2002) (and Figure 1): assuming that metabolic pools thataccumulate 13C label other than bicarbonate have constant13C fractional enrichment and the total influx of 13C labelmetabolites into the glial and neuronal TCA cycles is equalto the total outflux into the bicarbonate pool, the glialacetate oxidation rate, Vg ac, was determined using thefollowing formula:

Vgac ¼ Vtotal�ð%EHCO3� 1:1Þ=ð%EAc � 1:1Þ

The total bicarbonate production, Vtotal, is given byVtot = 3�Vtca + 2�Vg ac, where Vtca is the rate of glucose

oxidation in neuron and glia (Vn glc + Vn glc) (Figure 1).Because Vg ac < < Vtca, Vtot is approximately 3�Vtca.

An additional method of calculating maximum glial TCAcycle rate was applied, determining brain bicarbonate innatural abundance and 13C-enriched spectra with referenceto the adjacent Cr/PCr resonance (see above).

Figure 1 Glial versus neuronal metabolism: pathway of 13C labelfrom [1-13C]acetate to tricarboxylic acid (TCA) cycle in humanbrain. Selective metabolism of acetate and glucose occurs in gliaand neurons, respectively. Only the relevant intermediates areshown. Glia selectively transports acetate which is thenmetabolized through acetate thiokinase and reactions of themitochondrial tricarboxylic acid (TCA) cycle forming 13Cglutamate and glutamine, enriched in the C1 and C5 positions,before final oxidation and release as 13C bicarbonate(CO2 = HCO3

�). Five of the relevant cerebral metabolic productsare showed in sequential 13C brain spectra of Figure 3. Thealternative metabolic pathways of 13C glucose metabolism,predominantly in neurons, but not contributing to this study inwhich only the single substrate, [1-13C]acetate is provided. Ac,acetate; Glc, glucose; Glu, glutamate; Gln, glutamine; CO2,carbon dioxide; Pyr, pyruvate; Vg glc, glial glucose oxidation; Vg ac,glial acetate oxidation; a-KG, a-ketoglutarate; Vglu, glutamatesynthesis rate; Vgln, glutamine synthesis rate. Glucose ismetabolized in neuron and glia whereas acetate is metabolizedexclusively in glia.


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Statistical Analysis

Statistical analyses were performed with StatView software(SAS Institute, Cary, NC, USA). For continuous variables, themeans and standard deviations were calculated separatelyfor the AMD and the normal subjects. An analysis wasperformed to show that variance in the two groups was thesame using F test, followed by analysis with the two-samplet-test. All comparisons yielding a P-value of less than 0.05were considered statistically significant. All data areexpressed as mean±standard deviation (s.d.) in the text.

Results

Determination of Cerebral Bicarbonate Concentrationfrom Natural Abundance 13C MRS

Cerebral [HCO3�] 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 andcomparable to the those obtained in earlier direct andindirect assays in simian and animal brains (Siesjo,1964). Results obtained by direct 13C MRS assay in‘frontal brain’ were also indistinguishable from thoseobtained in ‘posterior brain’ in the same group ofcontrol subjects (Table 1). Results for cerebralbicarbonate concentration in the cohort of recoveringmethamphetamine-abusing subjects (Table 2) fellwithin this ‘normal’ range indicating that metabolicpH homeostasis is broadly normal in the recentlyabstinent methamphetamine-abuse human brain.

Determination of the Maximum Rate of BicarbonateProduction (maxVHCO�3 ) in Human Brain

In contrast, using time course data in the two groupsof subjects, it was observed that the rate at which

Figure 2 Human 13C MR frontal brain spectra acquired during [1-13C]acetate infusion: carbon-13 magnetic resonance spectroscopy(MRS) at baseline and 120 mins after the infusion (154 to 164 p.p.m.) in a control and an abstinent methamphetamine-dependent(AMD) subject. 13C MRS spectra show HCO3

� and total Cr (tCr) chemical shift region (154 to 164 p.p.m.) at baseline and at the endof the infusion study in a normal control subject (A) and an AMD subject (B). The lower, natural abundance spectra for the normalcontrol (left) indicate that cerebral bicarbonate concentration is very similar to that of [Cr + PCr], i.e., 11 mmol/L. The restingcerebral bicarbonate concentration in a representative AMD subject (right) was indistinguishable from the control (HCO3 = 11 mmol/L). After 2 h of [1-13C]acetate infusion, substantial but different increases in cerebral 13C 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 andAMA subjects are compared in Tables 1 and 2. In the control subject, the concentrations of bicarbonate determined in this way were15 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 subjectinitial bicarbonate was 10 mmol/g at baseline but reached only 25mmol/g after 2 h.


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[1-13C]acetate was incorporated into the final oxida-tion product bicarbonate was markedly differentbetween drug-abusing and normal subjects. Figure 2compares 13C MRS spectra (154 to 164 p.p.m.) froma representative control subject (A) and a metham-phetamine abstinent subject (B). From the start(natural abundance) to the end of the 2 h dataacquisition, there was almost fivefold increase in13C incorporation (fully 13C-enriched) in the control,whereas in the AMD subject, the full extent of 13Cenrichment was closer to threefold. Natural abun-dance bicarbonate concentration at baseline and atthe end of the infusion estimated was similar in thetwo subjects. In the control subject, the concentra-tions of bicarbonate determined in this way were15 mmol/g at baseline and 49mmol/g at the end of the2 h infusion study (Cr does not become enriched)whereas in the AMD subject initial concentration ofbicarbonate was similar, 10 mmol/g, at baseline butreached only 25 mmol/g after 2 h. This was less than50% of the enrichment measured in the normalcontrol. The baseline bicarbonate concentration iswithin the range of normal values calculated fromHenderson–Hasselbach equation, pH = pKa + log([HCO3]/[CO2]) (Agabiti et al, 1973) using brain pHof 6.99 (Bluml et al, 1998), pKa = 6.1 and pCO2 of 35

to 48 mm Hg in normal adults brain of 11.2 to19.9 mmol/L. The maximum rate of bicarbonateenrichment (maxVHCO�3 ) in the frontal brain in acontrol was 0.28 mmol/g per min compared with anAMD subject of 0.13 mmol/g per min. This result ishighly suggestive of a persistent major abnormalityin glial oxidative mechanism in frontal brain of absti-nent methamphetamine subjects and is consistentwith previously published work. For a preciseevaluation of the impact of AMD on glial metabolicrate, we applied the previously used method forcalculation of Vg ac.

Comparison of Fractional Enrichment of Bicarbonateand Glial Acetate Oxidation Rate in the Posterior andFrontal Brain of Normal Subjects

Five control women were scanned using the frontalbrain protocol and three of these women and oneman were scanned using the posterior brain protocol.Summary of the results is shown in Table 1. Initialslope of H13CO3 appearance in brain was similar forfrontal and posterior brain (P = 0.25), as were frac-tional enrichment of bicarbonate at 60, 80, and120 mins of observation. The calculated Vg ac from

Table 1 Fractional bicarbonate enrichment and glial metabolic rate for frontal and posterior brain in controls

ID [HCO3](brain,mmol/g)

%EAc

(plasma)Initialslopea

%E HCO3�

at 60 mins(brain)

%E HCO3�

at 80 mins(brain)

%E HCO3�

at 120 mins(brain)

Vg ac (mmol/gper min)b

Vg ac from Blumlet alc (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±0.01d

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).aBecause the time course of the appearance of HCO3

� signal amplitude was linear from the start of the infusion to 40 mins, the slope of the straight line wascalculated and reported here.bDetermined using %E at 60 mins and total TCA cycle rate of 0.7 mmol/g per min (Mason et al, 1995).cValue reported by Bluml et al (2002) determined at 60 mins after start of infusion in the posterior brain.dVg ac calculated at 80 and 120 mins are 0.13 and 0.16 mmol/g per min.

Table 2 Fractional bicarbonate enrichment and glial metabolic rate for frontal brain in control and AMD subjects

Subjects Meanage/

gender

[HCO3](brain,mmol/g)

%EAc

(plasma)Initialslopea

%E HCO3

at 60 mins(brain)

%E HCO3

at 80 mins(brain)

%E HCO3

at 120 mins(brain)

Vg ac (mmol/gper min)b

Control frontal brain (N = 5) 26±15 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) 30±75 F

10c 83 (N = 1) 0.05±0.02 2.7±0.5 3.14±0.5 3.79±0.6 0.04±0.01

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 mean±standard deviation (N = number of examinations).aBecause the time course of the appearance of HCO3

� signal amplitude was linear from the start of the infusion to 40 mins, the slope of the straight line wascalculated and reported here.bDetermined using %E at 60 mins and total TCA cycle rate of 0.7 mmol/g per min (Mason et al, 1995).cNatural abundance [HCO3

�] could not be determined with accuracy (low signal-to-noise ratio) from individual AMD subjects, the reported value wasdetermined from summed spectra from all five AMD subjects, and appeared to be indistinguishable from controls.


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the normal posterior brain (0.09±0.03 mmol/g permin) was in excellent agreement with value pre-viously obtained by us. Finally, the calculated rate offrontal brain [1-13C]acetate oxidation (0.11±0.01mmol/g per min) was indistinguishable from poster-ior brain.

Appearance of 13C-Enriched Metabolites of[1-13C]Acetate in the Frontal Brain of Normal and AMDSubjects

Figure 3 is a time course of the appearances of 5-13Cglutamate and [1-13C]acetate (A), 5-13C glutamine (B),and HCO3

� (C) resonances in a control subject.Similar plots were obtained for AMD (not shown).Rapidly increased to peak amplitudes for the

enriched acetate and 5-13C glutamate as well as5-13C glutamine as were observed at 56 mins andslowly eliminated by approximately 120 mins. Bicar-bonate signal amplitude reached a maximum andremained essentially constant after 60 mins. How-ever, the pseudo-steady state for 5-13C glutamatecould only be indirectly verified because of the co-resonance of the precursor [1-13C]acetate. Neverthe-less, these observations confirmed that all of the 13Clabel entering the glial TCA cycle is being transferredto bicarbonate and is not sequestered in othermetabolic pools.

Figure 4 shows stack plots of labeled metabolitescomparing results obtained in a normal control (A)with those from a representative AMD subject (C),over time and below, and the summed spectra of thetotal brain enrichment at the end of the infusionprotocol for control (B) and AMD (D). C5 glutamate(and acetate), C5 glutamine, C1 glutamate–glutamineand bicarbonate resonances are clearly visible. Thestrikingly reduced appearance of 13C label in bicar-bonate is evident in Figure 2D, in the AMD subjectcompared with control in Figure 2B. This result wasconfirmed in the entire group of AMD. The enrich-ment of HCO3

� estimated from the time course of thefractional enrichment of HCO3

� was significantlydecreased in AMD (Figure 5) and the rate of acetateoxidation in glial, Vg ac, as well as %E HCO3

� wasquantified from the initial slope of bicarbonateenrichment versus time, or from %E at 60, 80, and120 mins (compare Table 2 with Table 1). Compar-ison of bicarbonate productions between normal andAMD subjects is shown in Figure 6. Assuming theoverall TCA cycle rate of 0.7 mmol/g per min for theintact human brain (Mason et al, 1995), the averagerate of glial acetate oxidation in the frontal brain was0.04 for AMD subjects compared with 0.11 mmol/gper min in normal subjects, a reduction of 60% inglial metabolism rate for acetate oxidation. Therewere no significant differences (P = 0.12) betweengender and age in the two subject groups, normalcontrols and AMD subjects.

Discussion

In this pilot study we confirm the unique value oflow-power 13C MRS applied for the first time tomethamphetamine abusers in frontal regions of thehuman brain. We have discovered a hitherto un-suspected 50% reduction in oxidative rate in therecovering brain after methamphetamine abuse.Although it has been known for many years thatacetate, which is a preferred glial respiratory fuel,can be applied to defining glial TCA cycle rate of thenormal-functioning human brain in vivo, to the bestof our knowledge, this study represents the firstsystematic attempt to define alterations in glialmetabolic rate as a result of neuropathology. Severalearlier studies have used 13C MRS with 13C glucose toexplore human brain neurochemical abnormality.

Figure 3 Time courses of glutamate C5 and acetate (A),glutamine C5 (B), and HCO3

� (C) from a healthy subject. Signalintensities are shown in arbitrary units. Note that glutamine C5,glutamate C5, and bicarbonate become increasingly enrichedover time, consistent with the expected path of glial metabolismof 13C acetate shown in Figure 1. After significant enrichment upto 60 to 80 mins, pools of glutamate C5 and glutamine decline,presumably as they are in turn metabolized to the final product,13C HCO3

�. No significant sequestration in glutamate andglutamine occurs.


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[1-13C]glucose, a predominant neuronal respiratoryfuel, has revealed significant abnormalities of gluta-mate enrichment and TCA cycle rate in diseases asdiverse as Alzheimer’s, hepatic encephalopathy,mitochondrial disorders, and epilepsy (Bluml et al,2001; Lin et al, 2003; Ross et al, 2003). However, ineach of these examples the neuron was the target ofinvestigation. Furthermore, the region of brainexamined was the posterior parietal lobe, andsuboptimal for frontal lobe study based on safetyconsiderations dictated by Federal guidelines forSpecific Absorption Rate. The present report appearsmore relevant both as a study focused directly on thequestion of deficiency in glial oxidative metabolism

and by exploring a brain region implicated in drugabuse abnormality by almost all independent neu-roscience techniques including executive functionand neuropsychologic metrics. Acetate is metabo-lized exclusively in glia (Muir et al, 1986; Waniewskiand Martin, 1998); it is reasonable to assume asingle-compartment model for cerebral bicarbonateproduction, that is, the rate of bicarbonate produc-tion is equal to the rate of acetate influx into the glialTCA cycle. Intravenous infusion of [1-13C]acetaterapidly produced cerebral C5 glutamate in the firstturn of the TCA cycle where it is co-resonant andindistinguishable from the acetate at 182 p.p.m.Glutamine C5 (at 178 p.p.m.) appeared in the second

Figure 4 Effect of methamphetamine abuse on cerebral [1-13C]acetate metabolism. Frontal brain 13C metabolites observed during[1-13C]acetate infusion: figure shows carbon-13 MRS (150 to 195 p.p.m.) in a control and an abstinent methamphetamine-dependent subject after enrichment with [1-13C]acetate. Stacked sequential carbon-13 spectra and spectra summed for the entireinfusion protocol (120 mins, 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 (182 p.p.m.), glutamine C5 (178 p.p.m.),glutamate C1 + glutamine C1 (175 p.p.m.), and the final oxidation product, bicarbonate (161 p.p.m.).


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turn of TCA cycle and glutamate C1 and glutamineC1 (both resonates at 175 p.p.m.) in a subsequentturn. Carbon dioxide is the final product of the TCAcycle that appeared as bicarbonate at 161 p.p.m.Because of our low [1-13C]acetate infusion dosage,steady states were not convincingly reached in ourexperimental studies, therefore to determine frac-tional enrichment of bicarbonate, an exponentialfunction was fitted to the time course of theproduction of bicarbonate.

The glial bicarbonate production rate was subse-quently calculated from exponential fitting of %E at60 mins and therefore represents the glial TCA cyclerate. The glial TCA cycle rate determined this wayrepresents approximately 10% of the total TCA cyclerate in the brain of fasted normal subjects. This rate issimilar to the previously reported value by us in the

parietal brain region using similar [1-13C]acetateprotocol (Bluml et al, 2002).

Information regarding abnormalities of glial cellscan typically be obtained with proton MRS. Thereare 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 (Ernstet al, 2000; Nordahl et al, 2002), and basal ganglia(Sekine et al, 2002). Proton MRS studies show thatmethamphetamine-dependent subjects had decreasedN-acetylaspartate, higher myoinositol, and elevatedglutamate in the frontal white matter. Possibleinterpretations of these metabolite abnormalities inthese subjects include local loss or dysfunction ofneurons, and glial abnormalities (Abulseoud et al,2009). From 13C MRS, there is a significant reductionof glial TCA cycle rate in AMD subjects comparedwith non-drug users, further emphasizing the impactof methamphetamine use on glial function.

Possible limitations to 13C MRS: as with all‘isotope’ techniques, there is an unknown fractionof cerebral metabolism that will continue to consume(unenriched) endogenous respiratory fuels, in thiscase predominantly C-12 glucose. This may ‘dilute’the resulting 13C bicarbonate pool derived from 13C-enriched acetate. One correction has been applied, assuggested by Bluml et al (2002) in expressing the 13CHCO3

� as a fraction of the total bicarbonate producedin unit time. Although there is no way with thepresent technique to ensure that overall cerebralmetabolic rate is normal in AMD (additional studiesusing 13C glucose will clarify the point further),nevertheless, it is difficult to envisage a more than50% change in intrinsic glucose metabolic rate thatwould be necessary to explain the present findings.Earlier positron emission tomography studies ofAMD argued against any significant increase inneuronal metabolic rate in frontal brain (Kim et al,2005, 2009; London et al, 2005). Contrary results(small with reference to the present results) wereobtained by Wang et al (2004). There are otherlimitations to our study. First, the study sample issmall; though the effect is large and statisticallyrobust. This is partly a consequence of the costs([1-13C]acetate infusion) and technical difficulty ofthe present procedures. In the future, both aspectspromise to be greatly simplified allowing large-scalereplication of the present findings in this and otherstudies. Another possible limitation of the study isthe assumption that fractional enrichment of[1-13C]acetate is similar between the brain and blood(Bluml et al, 2002). Note that the enrichments ofGlu1, Gln1, as well as Gln5 were also observed butunder our non-steady-state experimental condition,we did not generate kinetic data for metabolic ratecalculations used in more complex theoreticalmodels. Alternative, steady-state protocols, invol-ving larger doses of [2-13C] acetate and longerprotocols may lend themselves to resolving anyremaining kinetic issues. Finally, the study of

Figure 5 Effect of methamphetamine abuse on cerebralmetabolism of 13C acetate. Time courses of appearance of theend product of [1-13C]acetate oxidation, HOC3

� are compared.Low-power noise nuclear overhauser effects (NOE) spectra wereacquired in 6.5 min blocks for up to 120 mins after enrichmentby i.v. acetate (3 mg/kg body weight) from frontal brain in fiveabstinent methamphetamine-dependent (AMD) subjects andfive control subjects. Logarithmic fits of the averaged percentfractional enrichment of bicarbonate (%E) were plotted as afunction of time after start of the infusion. %E in AMD (lowertrace) is shown to be lower than controls (upper trace). Standarddeviations are also shown as cross-bars.

Figure 6 Comparison of methods to express effect of abstinentmethamphetamine-dependent (AMD) in 13C HCO3

� productionfrom [1-13C]acetate in human frontal brain. Results shown inFigure 5 are quantified. Means and standard deviation of percentfractional bicarbonate enrichment calculated at three differenttime points (60, 80, and 120 mins) and the initial slope ofbicarbonate production from control and AMD groups. P-value ineach group comparison is < 0.001.


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short-term abstinence may limit the generalizabilityof results to abuse versus craving and relapse instudy of methamphetamine. We propose that thissuccessful demonstration of a hitherto unrecognizedbut sizeable neurometabolic adaptation is worthy offurther consideration and more sophisticated experi-mental design that may reveal novel mechanismsrelevant to human drug abuse and many otherconditions currently ascribed to glial dysfunction.

In this preliminary study, we used a novel methodof frontal lobe 13C MRS to explore possible defectsthat may persist in the glia of drug-addicted subjects,using as the example, recently AMD subjects.Additional studies, using the neuronal substrate[2-13C] glucose, will be necessary to determine thecellular specificity and the frontal localization ofthese neurochemical injuries. The novel capability todissect two major cell populations at risk in theintact brain and in patients at varying stages of theabuse–addiction–recovery cycle of methampheta-mine abuse promises new insights into an intractableclinical problem.

Acknowledgements

We thank Mentors to the K Award (NS), Alan Stacyfor advice and Thao Tran and Keiko Kanamori for thetechnical and scientific insights. OA was supportedin part by research funds from Keck School ofMedicine, Division of Psychiatry, University ofSouthern California.

Disclosure/conflict of interest

The authors declare no conflict of interest.

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