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Neuroscience Letters 570 (2014) 102–107

Contents lists available at ScienceDirect

Neuroscience Letters

jo ur nal ho me p age: www.elsev ier .com/ locate /neule t

otor cortex glutathione deficit in ALS measured in vivo with the-editing technique

. Weiduschata, X. Maoa, J. Hupfb, N. Armstrongb, G. Kanga,

.J. Langec, H. Mitsumotob, D.C. Shungua,∗

Department of Radiology, Weill Cornell Medical College, 516 East 72nd Street, New York, NY 10021, United StatesDepartment of Neurology, Columbia University, 710 West 168th Street, New York, NY 10032, United StatesDepartment of Neurology, Hospital of Special Surgery, 525 East 71st Street, New York, NY 10021, United States

i g h l i g h t s

Oxidative stress is implicated in the pathogenesis of amyotrophic lateral sclerosis.We investigated in vivo motor cortex glutathione levels in ALS.Glutathione levels were reduced by over 30% compared to healthy subjects.Glutathione might be a promising biomarker for upper motor neuron function in ALS.

r t i c l e i n f o

rticle history:eceived 17 February 2014eceived in revised form 27 March 2014ccepted 16 April 2014vailable online 24 April 2014

eywords:

a b s t r a c t

This study compared in vivo levels of the antioxidant glutathione (GSH) in the motor cortex of 11 ALSpatients with those in 11 age-matched healthy volunteers (HV). Using the standard J-edited spin-echodifference MRS technique, GSH spectra were recorded on a 3.0 T GE MR system from a single precentralgyrus voxel. GSH levels expressed as ratios to the unsuppressed voxel tissue water (W) were 31% lowerin ALS patients than in HV (p = .005), and 36% lower in ALS than in HV (p = .02) when expressed as ratios tothe total creatine peak (tCr), supporting a role for oxidative stress in ALS. Levels of the putative neuronal

agnetic resonance spectroscopymyotrophic lateral sclerosislutathionexidative stresseurodegenerationiomarker

marker N-acetylaspartate (NAA) relative to W did not differ between ALS and HV (p = .26), but were lowerby 9% in ALS than in HV (p = .013) when expressed as ratios relative to tCr. This discrepancy is attributed tosmall but opposite changes in NAA and tCr in ALS that, as a ratio, resulted in a statistically significant groupdifference, further suggesting caution in using tCr as an internal reference under pathological conditions.

© 2014 Elsevier Ireland Ltd. All rights reserved.

. Introduction

Amyotrophic lateral sclerosis (ALS) is a severe neurodegener-tive disorder characterized by progressive motor neuron loss,eading to muscle weakness and, eventually, respiratory failure.here are currently no reliable biomarkers of upper motor neuronUMN) function that can aid in early diagnosis or monitoring of dis-ase progression or treatment response. While lower motor neuron

LMN) involvement in ALS can be assessed by neurophysiologicalests, the standard evaluation for UMN dysfunction is currentlyased on clinical features, which can be difficult to interpret in the

∗ Corresponding author at: Weill Cornell Medical College; 516 East 72nd Street,ew York, NY 10021, United States. Tel.: +1 212 746 2481; fax: +1 212746 6681.

E-mail address: dcs7001@med.cornell.edu (D.C. Shungu).

ttp://dx.doi.org/10.1016/j.neulet.2014.04.020304-3940/© 2014 Elsevier Ireland Ltd. All rights reserved.

early phase of the illness and in the presence of pronounced LMNdegeneration [21].

Hypothesized pathogenic events in ALS include glutamate exci-totoxicity, mitochondrial dysfunction, protein aggregation, andoxidative stress. While it remains unclear whether these interre-lated processes are the cause or consequence of the disease, theyare all well-known triggers of apoptotic pathways and, thus, arebelieved to contribute to the degeneration of motor neurons in thedisease [7]. This study focuses on oxidative stress, which is definedas the excessive production of pro-oxidants, including reactiveoxygen and nitrogen species (ROS/RNS), relative to the cell’s antiox-idant capacity. The primary and most abundant antioxidant in the

CNS [33], nontoxic reservoir of cysteine [8], neuromodulator [22],and co-factor for many antioxidant and electrophile-scavengingenzymes [13,23] is the tripeptide thiol �-glutamylcysteinylglycineor glutathione [12]. Glutathione exists in reduced (GSH) and

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xidized (glutathione disulfide or GSSG) forms, with glutathioneeductase continuously regenerating GSH from GSSG. Loss of GSH inrain tissue leads to mitochondrial dysfunction, increased ROS/RNSroduction and widespread oxidative damage, with complete GSHepletion resulting in cell death [5].

In the present study, we sought to document the potentialnvolvement of oxidative stress in ALS in vivo by comparing levelsf GSH measured by proton magnetic resonance spectroscopy (1HRS) in the motor cortex of ALS patients with those in age-matched

ealthy controls.

. Subjects and methods

.1. Participants

This study was approved by the Institutional Review Boards ofolumbia University and Weill Cornell Medical College, and written

nformed consent was obtained from all study participants prior tonclusion.

Together, 11 subjects with possible, probable or definite ALS,iagnosed according to revised El Escorial criteria [4], and 13 age-atched healthy volunteers (HV) participated in the study. ALS

atients, who had to be free of other major neurological or psychi-tric illnesses, were recruited through the Eleanor and Lou GehrigDA/ALS Multidisciplinary Care Center of Columbia University and

he ALS Outpatient Clinic of the Hospital of Special Surgery. Func-ional status in ALS patients was assessed using the ALS Functionalating Scale (ALSFRS) and the forced vital capacity (FVC %). The tak-

ng of the commonly prescribed ALS drug riluzole was disallowedithin 3 days of the neuroimaging scans.

The normal control group consisted of healthy relatives of theLS patients and healthy subjects recruited through the research-atch.org registry. To participate, the HV subjects had to be

hysically and mentally healthy, and have no history of a significanteurological or psychiatric disorder.

General inclusion criteria for all participants consisted of thebility to lie down flat for up to 1 h, no cigarette smoking for ateast 1 month prior to study enrollment, and the ability to undergo

RI. The taking of antioxidant supplements was disallowed for 7ays before neuroimaging.

.2. Magnetic resonance neuroimaging procedures

All neuroimaging studies, which included limited structuralrain MRI examination and single-voxel 1H MRS of the motor cor-ex, were conducted on a research-dedicated 3.0 T GE MR systemith an 8-channel phased-array head coil at the Citigroup Biomed-

cal Imaging Center of Weill Cornell Medical College.

.3. Structural MRI

A three-plane, low resolution, high-speed scout imaging seriesas first obtained, followed by a series of high resolution scans, con-

isting of standard axial T1-, T2- and spin density-weighted scans,hich were used to prescribe the voxel of interest. In addition, a

1-weighted spoiled gradient-recalled echo (SPGR) volumetric scannd an axial fast fluid-attenuated inversion recovery (FLAIR) scanere performed for brain tissue segmentation, to assess structural

rain integrity and to rule out focal brain lesions.

.4. 1H MRS

In vivo spectra of GSH and other brain metabolites were obtainedy 1H MRS from a 20 mm × 25 mm × 25 mm precentral gyrus voxelhat was prescribed within the hemisphere contralateral to the

Letters 570 (2014) 102–107 103

most affected body side in the ALS patients, and within the domi-nant hemisphere in the HV subjects (Fig. 1A–C).

To derive the levels of only reduced glutathione (GSH), spec-tra were recorded using the standard J-edited spin echo differencemethod with TE/TR 68/1500 ms, as previously described [27,29].Briefly, a pair of a frequency-selective inversion pulses was insertedinto the standard point-resolved spectroscopy (PRESS) method, andapplied on alternate scans at the frequency of the GSH �-cysteinylresonance at 4.56 ppm, while avoiding excitation of oxidized glu-tathione (GSSG) �-cysteinyl at 3.28 ppm [34]. This resulted in twosubspectra (Fig. 1D, traces [a] and [b]) in which the GSH, but notGSSG, �-cysteinyl resonance at 2.98 ppm was alternatively invertedor not inverted. Subtracting these two subspectra yielded a spec-trum consisting only of the edited GSH �-cysteinyl resonance, withall overlapping resonances, including that of GSSG �-cysteinyl,eliminated (Fig. 1D, trace [c]). For each voxel, the data were acquiredin 15 min using 290 interleaved excitations (580 total), with theediting pulses on or off. The resulting raw 8-channel phased-arraycoil data were combined into a single regular 1D signal using therelative coil sensitivities derived from the unsuppressed voxel tis-sue water signal acquired with each receiver coil. The magnetic fieldhomogeneity for the acquisitions was typically ≤10 Hz, as assessedfrom the full width at half maximum of the unsuppressed water res-onance. We previously evaluated the test-retest reliability of brainGABA measurement using the J-editing technique and found it tobe high [16]. We thus postulate comparable test-retest reliabilityfor GSH detection by J-editing, since the methodology is the sameand the in vivo GSH concentrations are in the same range as thoseof GABA.

2.5. 1H MRS data processing and quantification

The area under the GSH spectral peak was obtained as illus-trated in Fig. 1D (traces [a–e]) by frequency-domain fitting of theGSH resonance in the edited spectrum to a pseudo-Voigt line-shape function using a robust and highly optimized public-domainLevenberg–Marquardt nonlinear least-squares minimization rou-tine [18]. The resulting peak areas were then expressed as ratiosrelative to the synchronously acquired and similarly fitted unsup-pressed voxel water signal (W). Levels of N-acetylaspartate (NAA),total choline (tCho) and total creatine (tCr) were likewise derivedby fitting the subspectra recorded with the editing pulses turnedoff (Fig. 1D, trace [a]).

To estimate the proportions of gray matter, white matter andCSF contained in the voxel of interest, the volumetric SPGR MRIdata were segmented using Statistical Parametric Mapping (SPM8,Wellcome Department of Imaging Neurosciences, University Col-lege London, UK). From the resulting histograms and using in-housesoftware developed in MATLAB (MathWorks, Natick, MA), a seg-mentation mask of the voxel was generated and the metabolitelevels adjusted for the proportion of CSF content of the voxel using

C = C0

1 − %CSF

where C is the corrected and C0 the uncorrected metabolite level.

2.6. Statistical analyses

After testing for data normality with Shapiro–Wilk tests, groupdifferences in metabolite levels were assessed using two-sidedindependent t-tests or Mann–Whitney tests, as appropriate. All

group comparisons were conducted both with the original data andwith values corrected for voxel CSF content.

To test for linear relationships between metabolite concen-trations and clinical indices in patients, Pearson’s correlation

104 N. Weiduschat et al. / Neuroscience Letters 570 (2014) 102–107

Fig. 1. Glutathione (GSH) detection in the primary motor cortex using proton MRS. [A] Coronal, [B] sagittal and [C] axial MR images of a human brain, with depiction of thesize, location and angulation of the voxel of interest in the primary motor cortex. [D] Demonstration of in vivo human brain GSH detection by 1H MRS: (a) and (b), single-voxels ) intere c) to o(

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ubspectra acquired in 15 min with the editing pulse on and off and 290 (580 totaldited brain GSH resonance at 2.98 ppm; spectrum (d), model fitting of spectrum (c) and (d). NAA, N-acetylaspartate; tCho, total choline; tCr, total creatine.

oefficients were determined. Analyses were performed usingPSS Version 18 (IBM PASW Statistics), except for the correla-ion analyses, which were conducted on the VassarStats websitewww.vassarstats.net, 1998–2013).

. Results

.1. Demographic and clinical characteristics

The 1H MRS data for 2 of 13 HV subjects were excluded before

RS analysis after one subject’s MRI revealed an incidental finding

nd pathological forgetfulness was noted in another.The presented results include data from 11 ALS patients (5

emales) and 11 HV subjects (7 females) (Table 1), who were well

leaved averages; spectrum (c), difference between spectra (a) and (b) showing thebtain the GSH peak area; spectrum (e), residual of the difference between spectra

matched for age (p > .05). Eight patients were categorized as proba-ble and three as possible ALS. At disease onset, 6 subjects reportedupper extremity symptoms, 3 reported lower extremity symptoms,and 2 had bulbar symptoms.

3.2. Mean neurometabolite levels and clinical correlations

Consistent with the virtually identical levels of the unsup-pressed voxel tissue water (W) between ALS ([14.7 ± 1.3] × 1011)

and HV ([14.9 ± 1.3] × 1011) (p = .72) (Table 1), correcting themetabolite levels for CSF content had no effect on the results ofgroup comparisons. Therefore, only results based on the original,uncorrected data are presented. In addition, neither gray matter

N. Weiduschat et al. / Neuroscience Letters 570 (2014) 102–107 105

Table 1Patient characteristics and precentral gyrus metabolites. Metabolite levels are given in institutional units (i.u.).

ALS group Healthy control group Mean difference in % p-Value

Mean ± SD Mean ± SDGender 5/11 female 7/11 femaleAge 61.5 ± 10.5 58.5 ± 6.6 >.05Disease duration in months 17.4 ± 13.4FVC% (n = 10) 89.9 ± 19.2ALSFRS (n = 6) 39.7 ± 4.7GSH/W (×10−3) 1.1 ± 0.3 1.6 ± 0.4 −31.3% .005*Lactate/W (×10−3) 7.8 ± 1.5 8.2 ± 1.5 −4.9% .54NAA/W (×10−2) 18.4 ± 1.7 19.7 ± 3.1 −6.6% .26tCho/W (×10−2) 7.6 ± 1.6 6.5 ± 2.3 +16.9% .20tCr/W (×10−2) 9.3 ± 1.1 8.9 ± 1.3 +4.5% .44Internal water (W; ×1011) 14.7 ± 1.3 14.9 ± 1.3 −1.3% .72GSH/tCr (×10−2) 1.2 ± 0.5 1.9 ± 0.8 −35.9% .02*Lactate/tCr (×10−2) 8.6 ± 1.9 9.4 ± 1.7 −8.0% .36NAA/tCr 2.0 ± 0.2 2.2 ± 0.2 −9.1% .013*

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ignificant group differences are marked with an asterisk.

p = .11) nor white matter (p = .73) volume within the voxel of inter-st differed between the groups.

Motor cortex GSH/W levels were 31.3% lower in ALSatients ([1.1 ± 0.3] × 10−3) than in HV ([1.6 ± 0.5] × 10−3)p = .005, t(20) = 3.11), and GSH/tCr levels were 35.9% lower inLS ([1.2±.5] × 10−2) than in HV ([1.9 ± 0.8] × 10−2) (p = .02,

(20) = 2.47) (Fig. 2). On the other hand, NAA levels relativeo W did not differ between ALS ([18.4 ± 1.7] × 10−2) and HV[19.7 ± 3.1] × 10−2) (p = .26, t(20) = 1.15), but were lower by 9% inLS (2.0 ± 0.2) than in HV (2.2 ± 0.2) (p = .013, t(20) = 2.74) whenxpressed as ratios relative to tCr (Fig. 2).

There were no significant group differences in any of the otheretabolites or ratios (Table 1), including a numerically 4.5% higher

Cr/W (p = .44) and a numerically 6.6% lower NAA/W (p = .26) in ALS

han in HV.

Lastly, there were no significant linear correlations between anyf the metabolite levels and disease duration, ALSFRS or FVC%.

ig. 2. Glutathione (GSH) and NAA levels in ALS patients and healthy controls. Scatter plotsealthy volunteer (HV) subjects expressed as ratios to intra-voxel tissue water (W) or to

xpressed as ratios to W [all values depicted are ×10−3] (panel A) or to tCr [×10−2] (panelxpress as ratios to W [×10−1] (panel C), but were lower in ALS than in HV when express

+10.8% .38

4. Discussion

To our knowledge, this is the first study to measure and reportGSH levels in the motor cortex of patients with ALS in vivo, findinga significant deficit of the antioxidant compared to age-matchedhealthy volunteers, whether expressed as ratios relative to voxeltissue water or to tCr. This finding is consistent with the resultsof prior ex vivo studies that reported decreased GSH levels and ele-vated markers of oxidative stress in blood, urine, cerebrospinal fluidand spinal tissue derived from patients with ALS [2,3,9,19,25,26]. Inpreclinical ALS models, GSH levels have been associated with bothanimal survival and cell pathology [30].

A number of factors may account for the lower GSH levels inALS. First, this could reflect increased consumption of antioxidant

reserves in neutralizing the excessive production of ROS/RNS. Sec-ond, glutamate excitotoxicity might impair glial GSH synthesis byinhibiting the gradient-driven glutamate-cystine antiporter [23],

of motor cortex GSH (left) and NAA (right) in patients with ALS and in age-matchedtotal creatine (tCr). GSH levels were significantly lower in ALS than in HV, whether

B). On the other hand, motor cortex NAA levels in ALS did not differ from HV whened as ratios to tCr (panel D).

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hus depleting the cells of cystine, which readily reduces to cys-eine, the rate-limiting substrate for GSH synthesis in vivo [10,11].hird, the availability of cysteine might be decreased due to oxida-ive stress-mediated or inherent dysfunction in ALS [1] of thexcitatory amino acid transporter (EAAT) [10,11], which is respon-ible for up to 90% of total cysteine uptake into neurons [17].

In addition, normal aging, tobacco smoking and a variety of neu-ological disorders have been associated with GSH deficits [20].owever, none of these factors adequately explains the presentbservation of a cortical GSH deficit, since the mean age did notiffer between the groups; all participants were non-smokers orad abstained from smoking for at least 4 weeks prior to theeuroimaging studies; and the presence of a significant comorbidisorder was exclusionary. Thus, the GSH decrease found in thistudy is likely the manifestation of pathophysiological processeshat may be specific for ALS. Further studies are needed to evaluatehe specificity of this GSH deficit relative to common differentialiagnoses.

While we replicated the widely reported finding of decreasedAA/tCr in ALS compared to HV, this apparent NAA deficit vanishedhen the levels were expressed as ratios to W. As a result, it wasnclear whether NAA levels were decreased or unchanged in ALSompared to HV. Since the levels of all metabolites were derivedrom spectra that had been acquired and processed identically, andhe internal unsuppressed voxel tissue water signal, W, did not dif-er between the groups, we postulated that the most likely sourcef this discrepancy in the two measures of NAA was that NAA/Was numerically 6.6% lower and tCr/W was numerically 4.5% higher

n the ALS group than in the HV group, so that the net effect of com-uting the NAA/tCr ratio was to magnify the numerically oppositehanges in NAA and tCr in ALS to the point of reaching a statisticallyignificant difference between the groups. We thus conclude thatur metabolite levels reported as ratios to W are more reliable thanhose expressed as ratios relative to tCr.

As motor neuron degeneration is the hallmark of ALS, decreasesn the levels of NAA – which is synthesized in neuronal mitochon-ria and thought to reflect neuronal damage, dysfunction or deaths well as mitochondrial dysfunction [6] – have long been pos-ulated. In vivo MRS evidence is generally consistent with thisypothesis, and nearly all prior studies found decreased motorortex NAA levels in the disorder, whether expressed as absoluteoncentrations [19] or as ratios to creatine [14,15,19,28,31,32], toholine [14,24,31,32], to creatine + choline [24] or to myoinositol14]. Our finding of a decrease in motor cortex NAA/tCr in ALSatients is in agreement with those prior results. However, as dis-ussed, our failure to find a significant group difference in meanAA relative to the more reliable water reference suggests that,epending on the stage and/or severity of the disease NAA abnor-alities may be missed. That most prior studies and the present

tudy found decreased NAA/tCr in ALS suggests that abnormalitiesoo subtle to detect by MRS can be magnified and uncovered byomparing ratios of metabolites that change in opposite directions.herefore, caution in the use and interpretation of metabolite ratiosppears warranted.

This study has potential limitations. First, it is unclear whetherhe duration of the medication washout period was adequate to

inimize or eliminate the potential effects of any drugs or supple-ents taken prior to enrollment on the metabolite levels, especiallySH. Riluzole has a half-life of 12 h so that our washout period oft least 3 days would have reduced its systemic levels to less than% of the baseline concentration. Second, our sample size was rela-ively small, which might have limited the statistical power to find

n association between clinical indices and MRS markers.

The novelty of this study is in our finding of a GSH decrease inLS, which suggests limited antioxidant capacity in the motor cor-

ex of affected patients and seems to corroborate prior laboratory

[

Letters 570 (2014) 102–107

and preclinical findings of increased peripheral markers of oxida-tive stress in ALS. In addition, this study has replicated the priorfinding of decreased NAA/tCr levels in ALS, while also suggestingcaution in the common practice of expressing metabolite levels asratios of peak areas, especially to tCr. With proper implementa-tion and confirmation in larger studies, in vivo 1H MRS measuresof cortical GSH could, potentially, serve as objective biomarkersof metabolic alterations that characterize upper motor neuroninvolvement in ALS.

Conflict of interest

The authors report no disclosures.

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