Proton MR Spectroscopy for Diagnosis and Evaluation of Treatment Efficacy in Parkinson Disease 1

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1 Note: This copy is for your personal non-commercial use only. To order presentation-ready copies for distribution to your colleagues or clients, contact us at Leslie Mazuel, PhD Carine Chassain, PhD Betty Jean, MD Bruno Pereira, PhD Aurélie Cladière Claudine Speziale Franck Durif, MD, PhD Proton MR Spectroscopy for Diagnosis and Evaluation of Treatment Efficacy in Parkinson Disease Purpose: Materials and Methods: To assess the neurochemical profile in the putamen of patients with parkinsonian syndromes undergoing L-3,4- dihydroxyphenylalanine (L-DOPA) treatment (drug-on) or after withdrawal of L-DOPA medication (drug-off) compared with healthy volunteers to identify dopaminergic therapy sensitive biomarkers of Parkinson disease. The local institutional review board approved the study, and all participants gave informed consent. A short echotime (29 msec) single-voxel (-cm 3 ) proton (hydrogen [ H]) magnetic resonance (MR) spectroscopic approach was used at 3 T to explore the metabolic profile in the putamen of patients with Parkinson disease. Spectra obtained from 20 healthy volunteers were blindly compared with spectra obtained from 20 patients with parkinsonian syndromes in drug-on and drug-off conditions in a randomized permuted block study to assess the accuracy of diagnostic biomarkers for Parkinson disease and efficacy of L-DOPA therapy. The statistical tests were two sided, with a type-i error set at a of.05. Random-effects models were used to compare healthy subjects and patients with parkinsonian syndromes in drug-on or drug-off conditions. Original Research n Neuroradiology From the Department of UFR Medicine, Auvergne University, EA7280, Clermont-Ferrand, France (L.M., F.D.); Center for Magnetic Resonance Imaging (C.C., B.J., A.C., C.S.) and Department of Neurology (B.P., F.D.), CHU Gabriel Montpied, 58 rue Montalembert, Clermont-Ferrand, France. Received December, 204; revision requested January 26, 205; final revision received March 25; accepted April 9; final version accepted June 4. Address correspondence to F.D. ( fdurif@chu-clermontferrand.fr). Results: Conclusion: Measured concentrations of putaminal total N-acetylaspartate (tnaa) ( vs ; P,.0), total creatine (tcr) ( vs ; P,.0), and myo-inositol (m-ins) ( vs ; P,.00) were significantly lower in patients with parkinsonian syndromes in drug-off condition than in healthy volunteers. Moreover, L-DOPA therapy restored tnaa ( vs ; P,.0) and tcr ( vs ; P,.0) levels, whereas m-ins levels remained unchanged. The combined glutamate and glutamine and choline showed no changes in drug-off or drug-on condition compared with those in control subjects. tnaa, tcr, and m-ins were identified as putative biomarkers of Parkinson disease in the putamen of patients. tnaa and tcr levels are responsive to L-DOPA therapy. q RSNA, 205 q RSNA, 205 Radiology: Volume 000: Number n radiology.rsna.org

2 Parkinson disease (PD) is a neurodegenerative disorder characterized by the degeneration of dopaminergic neurons inside the substantia nigra pars compacta, leading to the appearance of motor disorders (). Diagnosis of PD is still reliant on clinical signs, and the accuracy of clinical diagnosis is less than 90% (2). In some cases even when there are patent signs of motor symptoms, PD often gets clinical misdiagnosis as other movement disorders. Furthermore, diagnosis is generally not confirmed until advanced stages of neurodegeneration, as clinical symptoms do not appear until 60% 80% of dopaminergic neurons are already lost. It is therefore crucial to identify markers that enable accurate early diagnosis of PD, assessment of progression rate, and evaluation of responsiveness to treatment. In this context, imaging techniques hold great promise, but there is still no fully validated marker (3). Magnetic resonance (MR) spectroscopy can track neurochemical alterations in a disease context. In PD, proton (hydrogen [ H]) MR spectroscopic studies have reported a number of metabolic changes in different brain Advances in Knowledge nn H MR spectroscopic assessment of neurochemical profile in the putamen of Parkinson disease (PD) patients after withdrawal of L-3,4-dihydroxyphenylalanine (L-DOPA) medication compared with healthy volunteers highlighted reduced total N-acetylaspartate (tnaa) ( vs ; P,.0), total creatine (tcr) ( vs ; P,.0), and myo-inositol ( vs ; P,.00) levels as potential markers of PD. nn PD patients undergoing L-DOPA therapy showed recovery of tnaa ( vs ; P,.0) and tcr ( vs ; P,.0) to levels approaching those found in healthy volunteers ( and , respectively). structures ranging from motor cortex (4) to temporoparietal cortex (5), specifically posterior cingulate gyrus (6) and occipital lobe (7,8), reduced total N-acetylaspartate (tnaa)-to-total creatine (tcr) ratio (4 6,8), and higher total choline (tcho)-to-tcr ratio (7). MR spectroscopic studies performed on the substantia nigra of patients with parkinsonian syndromes and control subjects have reported contradictory results on the tnaa/tcr ratio (7 9), possibly due to the poor signal-to-noise ratio (SNR) in this structure. Efforts to identify markers of PD have consequently turned to structures such as the putamen, to which the substantia nigra sends dense dopaminergic afferent projections (0). In the putamen, H MR spectroscopic studies have shown a lower tnaa/tcr ratio in PD patients compared with control subjects (), whereas other studies observed no change (2,3). Absolute quantitations using short echo-time H MR spectroscopy have shown no change in glutamate and glutamine levels (4,5), whereas 7-T H MR spectroscopy depicted higher g-aminobutyric acid levels in the putamen of PD patients than in control subjects (5). H MR spectroscopy was also used in a 997 study to assess L-3,4-dihydroxyphenylalanine (L-DOPA) effect on metabolism inside the putamen of PD patients (6) and showed that the reduced tnaa/tcho ratios observed in untreated patients may be reversed with L-DOPA therapy and may thus provide a reversible marker of neuronal dysfunction in the striatum. Higher magnetic field MR spectroscopic studies in rodent models of PD found changes in g-aminobutyric acid, glutamate, and glutamine in the putamen (7 9), and animal studies have Implication for Patient Care nn H MR spectroscopy can be used to assess metabolic changes as diagnostic markers of PD and ultimately to assess treatment efficacy in PD patients. shown that acute L-DOPA normalizes these alterations (9,20). Against this background, the current study used a short echo-time single-voxel H MR spectroscopic approach to track neurochemical profile changes in the putamen of PD patients compared with matched healthy volunteers in a randomized permuted block study design. The purpose was to assess the neurochemical profile in the putamen of PD patients undergoing L-DOPA treatment (drug-on) or after withdrawal of L-DOPA medication (drug-off) and compare them with those of healthy volunteers to identify dopaminergic therapy-sensitive biomarkers of PD. Materials and Methods Subjects This prospective imaging study included 20 patients with PD who were compared with 20 healthy age-matched and sex-matched subjects with no history of neurologic disorders. Between January 202 and March 204, PD patients were recruited by movement Published online before print 0.48/radiol Content codes: Radiology 206; 000: 9 Abbreviations: Glx = glutamate and glutamine L-DOPA = L-3,4-dihydroxyphenylalanine m-ins = myo-inositol PD = Parkinson disease SNR = signal-to-noise ratio tcho = total choline tcr = total creatine tnaa = total N-acetylaspartate UPDRS = Unified Parkinson s Disease Rating Scale Author contributions: Guarantors of integrity of entire study, L.M., C.C., F.D.; study concepts/study design or data acquisition or data analysis/interpretation, all authors; manuscript drafting or manuscript revision for important intellectual content, all authors; approval of final version of submitted manuscript, all authors; agrees to ensure any questions related to the work are appropriately resolved, all authors; literature research, L.M., C.C., B.J.; clinical studies, L.M., C.C., B.J., A.C., C.S., F.D.; experimental studies, L.M., C.C., A.C., C.S.; statistical analysis, L.M., C.C., B.P.; and manuscript editing, L.M., B.J. Conflicts of interest are listed at the end of this article. 2 radiology.rsna.org n Radiology: Volume 000: Number 0 206

3 disorder specialists after fully fulfilling the U.K. PD Brain Bank criteria for idiopathic PD (2). Inclusion criteria were idiopathic PD of at least 5-year duration, total Unified Parkinson s Disease Rating Scale (UPDRS) level in off-condition of 20/80 or greater, Hoehn and Yahr disease stage or greater, and UPDRS motor scale improvement to L-DOPA challenge test of 30% or greater. PD patients with deep brain stimulation, rest tremor (. 3 on one subitem of UPDRS), levodopa-induced dyskinesia in on state (motion incompatible with MR spectroscopic examination), and dementia (as assessed according to the fourth edition of the Diagnostic and Statistical Manual of Mental Disorders criteria) were not recruited. Healthy volunteers were recruited from healthy volunteers list owned and maintained by the Clinical Investigation Centre of Clermont- Ferrand. For each group, subjects who presented with brain disease, had MR imaging contraindications, or were on any medication that could interact with brain neurotransmitters (antidepressants, neuroleptics) were not recruited. All recruited participants had normal panel blood tests, excluding liver failure. At this stage of the study, no subject was excluded as the patients were carefully selected by a specialized medical doctor during the inclusion visit. The protocol was approved by the Regional Medical School Ethics Committee and was registered under number AU85. All healthy volunteers and PD patients gave informed consent according to the French national health authorities and the Declaration of Helsinki guidelines. Patients with parkinsonian syndromes in drug-off condition, with any antiparkinsonian medication withdrawn at least 2 hours prior to MR spectroscopy, arrived at the MR imaging department in the morning (8 am). MR spectroscopy was performed twice at -week intervals, alternately in drugoff condition and in drug-on condition (Fig ). Assignment to drug-off or drugon condition at the first MR spectroscopic examination was performed by Figure Figure : Diagram of the study design. using permuted-block randomization. For drug-on condition, patients received an acute 200 mg L-DOPA dose (Modopar Dispersible, Roche, France) while in drug-off condition. MR spectroscopy was performed hour later when parkinsonian symptoms improved. Clinical symptoms were assessed in both drug-on and drug-off condition. L-DOPA-equivalent daily dose was estimated according to Tomlinson et al (22). Healthy volunteers underwent only one H MR spectroscopic examination. MR Imaging and MR Spectroscopy Imaging and H MR spectroscopic experiments were performed with a 3-T imager (MR750; GE Medical Systems, Milwaukee, Wis) equipped with a 32-channel head coil. Foam padding and paper tape were used to restrict motion within the imager. To define voxel location, conventional anatomic MR images were obtained by using a three-dimensional T-weighted inversion recovery sequence: repetition time msec/echo time msec/inversion time msec, 8.8/3.5/400; section thickness,.2 mm; field of view, 24 cm; flip angle, 2 ; number of signals acquired, one; resolution, (Fig 2). Sections were aligned to anterior commissureposterior commissure line. The selected -cm 3 voxel position was determined by examining the T- weighted MR images rebuilt in all three dimensions. The putamen was located by the varying contrast between white matter and gray matter and the globus pallidus. To validate that voxel positioning was reproducible in all participants and between the two MR spectroscopic sessions, the percentage of voxel coverage was estimated by using Gimp 2.6. ( Briefly, for the Radiology: Volume 000: Number n radiology.rsna.org 3

4 Figure 2 three acquisition plans, one image acquired in drug-off condition was opened and the image acquired in the drug-on condition was opened as a layer. The two images were normalized according to gray levels by using the Gimp image registration toolbox (transformation model, shift and rotate). Then, by transparency, the two-voxel overlay was used to estimate the percentage of voxel coverage. H MR spectroscopy was performed inside the right and left putamen (Fig 2) by using a point-resolved spectroscopic sequence (repetition time msec/echo time msec,.5/29; 52 scans; volume of interest, mm; spectral bandwidth, 5000 Hz; 4096 complex data points). This volume-localized sequence was used with chemical-shift selective saturation water suppression Figure 2: Voxel location on H anatomic images. Three-plane T-weighted MR images in a healthy control subject (42-yearold woman) show a spectroscopic volume of interest located (white box) exactly on the left putamen in (a) axial, (b) coronal, and (c) sagittal sections. Four outer-volume suppressions were located at the bonebrain interface. and outer-volume suppression contiguous to the single volume of interest in the three dimensions. Four other outer-volume suppressions were placed far from the volume of interest at the brain-bone interface. Automatic shimming was conducted before the spectra were obtained. After shimming, a water suppression value at 95% and a spectral line width of less than 2.77 Hz on the water signal were considered as acceptable values. MR Spectroscopic Analysis All spectra were processed by using LCModel software version 6.3 (Steven Provencher, Oakville, Ontario, Canada) (23), which uses Bayesian analysis starting with solution spectra basis sets to provide estimates of metabolite and macromolecules concentrations without operator bias. Analyses were performed blindly by L.M. and C.C., who have 2 and 0 years of experience in spectra assessment, respectively. Quantitative analysis was performed for each spectrum set coming from the right and left putamen for each patient. Since the data were not significantly different between the left and right putamen (Fig 3), the values obtained for each side were averaged. Because the contribution of cerebrospinal fluid to the voxel and head motion relative to voxel size was low, neurochemical concentrations were not corrected for cerebrospinal fluid content. The LCModel reference for metabolite quantification used the unsuppressed water signal automatically collected. Spectra were included in the final analysis based on quality criteria defined by objective output parameters from the LCModel analysis, that is, sufficient spectral resolution (line width, 0. ppm, thus, 2.77 Hz), and residuals that were randomly scattered about zero to indicate a reasonable fit. Mean SNR was and ranged from 4 to 8. Assessment evaluated the following neurochemicals: tnaa, tcr, tcho, myo-inositol (m-ins), and combined glutamate and glutamine (Glx). In this study, quantification estimates for tnaa, tcr, tcho, and m-ins were included for Cramér-Rao Lower Band (CRLB) (percentage of standard deviation) of 20% or less. Glx was quantified by using CRLB of 25% or less. Values are expressed as means 6 standard error of the mean. The T relaxation times of water and the metabolites were similar and would therefore approximately cancel. Furthermore, the concentrations were not corrected for the significant T2 relaxation of the metabolites and are thus expressed in arbitrary units. Statistical Analysis Subjects. Statistical analysis on the clinical data was performed by using a paired Student t test comparing data for each group. Values are expressed as means 6 standard deviation. H MR spectroscopy. Statistical analysis was performed with Stata 3 software (StataCorp, College Station, 4 radiology.rsna.org n Radiology: Volume 000: Number 0 206

5 Figure 3 tests) and as number of patients and associated percentages for categorical parameters. For omnibus P values at,.05, log transformation was proposed to achieve normality of parameters. Comparisons between healthy subjects and PD patients assessed in drug-on or drug-off condition were performed by using random-effects models by (a) taking into account the within- and between-subject variability, (b) studying the (fixed) effect group, and (c) adjusting for relevant clinical parameters such as age, duration of disease, UPDRS III score in drug-on and drug-off conditions. Relations between quantitative data were studied by computing correlation coefficients (Pearson or Spearman according to statistical distribution). Values are expressed as means 6 standard deviation. P,.05 indicates statistically significant difference. Multiple comparisons integrated Šidák correction for inflation of type-i error. Results Participants Nineteen of the 20 patients received L- DOPA, while the remaining PD patient received dopaminergic agonists. Fourteen of the 20 patients also received a monoamine oxidase inhibitor B, and nine received a catechol-o-methyl transferase inhibitor (Table). PD patients had significantly lower UPDRS III scores in drug-on than drug-off condition ( vs , t = 6.96; P,.00). Average all-pd patient voxel coverage was 83.9% H MR Spectroscopy Figure 3: Quantification inside each putamen for the three groups by using LCModel software. A, Values obtained for healthy volunteers in right and left putamen. B, Values obtained for PD patients in drug-off condition. C, Values obtained for PD patients in drug-on condition. In patients, the analysis of the putamen contralateral (left putamen) or ipsilateral (right putamen) to clinical sign was chosen to prevent bias in the analysis as early stage, PD is mainly unilateral. Values are means 6 standard error of the mean. There were no significant changes between metabolite quantitations of the two putamen in each patient group. a.u. = arbitrary unit. Tex) by using two-sided tests with a type-i error set at a of.05 (except for multiple comparisons). Quantitative data are presented as means 6 standard deviation or medians (interquartile range) for continuous data (assumption of normality was assessed by using the Shapiro-Wilk and Agostino Analysis with the LCModel (Fig 4) showed that tnaa concentrations in the putamen of PD patients were lower in drug-off condition compared with those in healthy volunteers ( vs ; P,.0) and increased significantly after drug-on condition ( vs ; P,.0) to a level not significantly different from that in healthy volunteers ( vs ) (Fig 5). Similarly, tcr concentrations were also lower in PD patients in drug-off condition than in Radiology: Volume 000: Number n radiology.rsna.org 5

6 Demographic and Clinical Characteristics of the Subjects Variable Control Subjects (n = 20) PD Patients (n = 20) P Value Age (y) No. of women/men 9/ 9/ History of PD (y) Age at PD onset (y) Side of PD onset 4 right/6 left UPDRS III score,.00 Drug-off condition Drug-on condition Percentage improvement in motor signs UPDRS IV Hoehn and Yahr score.039 Drug-off condition Drug-on condition LEDD (mg/d)* Note. Unless otherwise indicated, data are means 6 standard deviation. * LEDD = L-DOPA equivalent daily dose healthy volunteers ( vs ; P,.0) and increased significantly after drug-on compared with drug-off ( vs ; P,.0) condition to a level not significantly different from that of healthy volunteers ( vs ). No changes in tcho were observed between healthy volunteers and PD patients in drug-off or drug-on condition ( , and , respectively). Interestingly, PD patients in drug-off condition showed significantly lower m-ins than did healthy volunteers ( vs ; P,.00), and this decrease was not reversed in drug-on condition ( vs ). We observed no changes in Glx between healthy volunteers and PD patients in drug-off or drug-on condition ( , and , respectively; Fig 5). There was no significant relationship between each metabolite, UPDRS III score assessed in drug-off or drugon conditions, and disease duration (all coefficients of correlation, r, were less than 0.304, which was the critical Pearson correlation value at two degrees of freedom). However, there was a weak correlation between Glx levels and UP- DRS III score in PD patients in drug-off condition (r = 0.337; P =.05). Discussion In this current study, we have demonstrated using H MR spectroscopy that patients with moderate PD had lower putamen m-ins, tcr, and tnaa levels in drug-off condition compared with healthy control subjects, making m-ins, tcr, and tnaa potential diagnostic biomarkers of PD. Moreover, this work showed that after L-DOPA therapy, tcr and tnaa recovered to levels not significantly different from those in control subjects. These neurochemical changes under L-DOPA treatment could be used as a way to monitor the efficacy of therapy in PD patients. H MR spectroscopy showed reduced putaminal tnaa levels in PD patients in drug-off condition. This decrease in tnaa in drug-off condition could point to mitochondrial dysfunction or neuronal loss inside the putamen (24,25). Furthermore, reduced putaminal tcr levels could indicate a restricted capacity of neurons and/or glia to supply energy demand. Thus, taken together, the codecrease in tnaa and tcr observed in PD patients in drug-off condition could indicate mitochondrial dysfunction in the putamen of PD patients rather than neuronal loss (26,27). Moreover, m-ins pools were profoundly altered in the putamen of PD patients compared with control subjects. The decrease in m-ins observed here could highlight a change in glial microenvironment in the putamen of PD patients (27,28). In PD patients given L-DOPA therapy (drug-on), tnaa and tcr concentrations increased back to near control levels, which provides indirect evidence of an effect of acute administration of L-DOPA in increasing neuronal function and thus might suggest that neural mitochondrial dysfunction in the putamen is reversible through dopaminergic therapy. Despite the neurochemical changes observed in PD patients between drugoff and drug-on conditions, we failed to find any correlation between disease stage and metabolites levels. The fact that our patient group was fairly homogeneous could explain this absence of correlation. The absence of significant correlations between MR spectroscopy derived data and disease duration or severity is also consistent with previous studies (6, 29). In our study, the quantitation of absolute tnaa revealed lower putaminal tnaa levels in PD patients in drug-off condition compared with control subjects, in agreement with a previous study (). Furthermore, a study in rat and primate models of progressive striatal neurodegeneration induced by the mitochondrial toxin 3-nitropropionic acid showed a decrease of tnaa in the striatum that was reversible in time (30). The observed increase in tnaa levels under dopaminergic therapy raises prospects for using tnaa to assess and monitor therapeutic efficacy in PD. Previous MR spectroscopic studies quantifying tnaa relative to tcr ( 3) in PD patients have reported conflicting results (7 9): Some studies found no changes in basal ganglia (7,8), while others showed differences in rostral to caudal substantia nigra NAA/Cr ratios between PD patients and control subjects (9). Indeed, use of tcr as reference warrants care, especially due to regional and individual variability. The decrease in putaminal tcr levels observed in PD patients in drug-off 6 radiology.rsna.org n Radiology: Volume 000: Number 0 206

7 Figure 4 Figure 4: H NMR spectra obtained from a -cm 3 voxel located on the putamen. Individual spectrum representative of mean spectral quality for (a) a healthy 42-year-old female volunteer (linewidth = ppm; SNR = 6) and a 46-year-old female PD patient in (b) drug-off condition (linewidth = ppm; SNR = 5) and (c) drug-on condition (linewidth = ppm; SNR = 6). Resonances of selected metabolites were assigned as follows: tnaa = ppm, tcr = 3.02 ppm, tcho = 3.2 ppm, m-ins = 3.56 ppm, Glx = ppm, and macromolecules (MM ) = ppm. Per our group findings, tnaa, tcr, and m-ins levels are lower in PD patients in drug-off condition than in healthy volunteers. In PD patients in drug-on condition, tnaa and tcr levels are not significantly different from those in healthy volunteers. For each condition, in vivo spectrum (black spectrum) was estimated with LCModel output (red spectrum). The difference between spectra is plotted at the top and represented the residual. The thin signal under the experimental spectrum is the baseline spline estimate determined with LCModel software. condition is consistent with previous H MR spectroscopic and phosphorus 3 MR spectroscopic studies in the same structure, where a decrease in tcr was associated with decrease in phosphocreatine (PCr) and adenosine triphosphate (ATP)levels (3). No changes in PCr or ATP were observed in other parts of basal ganglia in PD (4,32), suggesting that the putamen may be a key focal structure for MR spectroscopic assessment of disease state and treatment efficacy in PD patients. This current H MR spectroscopic study did not observe changes in Glx pool, which converges with previous studies in PD patients (5,32) but diverges from data obtained in animal models. Indeed, MR spectroscopic studies in animal models of PD suggest that dopaminergic denervation induces hyperactivity of the glutamatergic corticostriatal pathway, where the elevated glutamate concentrations observed inside the striatum were associated with increased glutamine and g-aminobutyric acid levels, which points to modified neuron-glia interactions inside this structure after dopaminergic denervation (8,20,33). As the modifications observed are associated with an acute lesion of the nigrostriatal pathway, changes in glutamate and glutamine in animal studies might reflect a temporary glial response in the striatum in response to dopaminergic denervation (8). An explanation for the unaltered Glx pools observed in our patients could be the fact that lesions in PD are progressive and may thus induce compensatory mechanisms masking the changes in Glx in drug-off condition. Furthermore, long-term chronic dopaminergic therapy could have blunted the change in Glx levels. Basal ganglia nuclei form a deep brain structure that hampers the accuracy of H MR spectroscopy for precise metabolic analysis. We addressed this issue by performing MR spectroscopic acquisitions at high magnetic field (3 T) on a -cm 3 size single voxel located inside the putamen. Single-voxel spectroscopy offers advantages in terms of accuracy of signal localization, shorter echo-time options, better magnetic field homogeneity after shimming, and Radiology: Volume 000: Number n radiology.rsna.org 7

8 Figure 5 Figure 5: LCModel analysis of metabolite concentrations for the two groups of participants. PD patients in drug-off or drug-on conditions were compared with healthy volunteers. Values are means 6 standard error of the mean. Metabolites that were significantly different between groups are flagged. n.s. = not significant, = P,.05, = P,.0, and 5 P,.00. thus more effective water signal suppression to enable a better absolute quantification of metabolite concentrations. This approach makes it possible to obtain a relatively good SNR. Furthermore, to reduce potential SNR bias, a Cramér-Rao Lower Band cut-off of 20% or less was used to select the reliable concentrations of tnaa, tcr, tcho, and m-ins. Previous.5-T MR spectroscopic studies in different brain structures of PD patients have reported conflicting results, likely explained by nonhomogenous patient groups (8). This prompted us to selectively include patients to get a patient group reflecting moderate disease state and to assess patients according to their medication conditions in a crossover design. This design made it possible to assess metabolic changes in response to acute L-DOPA administration to evaluate the therapeutic response. Given the combination of blinded randomization of patients with highly-informative short echo-time single-voxel spectroscopy and automated spectra quantization, we assume that our results are reliable under these experimental conditions. However, disease progression and the chronic dopaminergic therapy may have triggered compensatory mechanisms masking certain changes. Further H MR spectroscopic studies at early stages of PD (before dopaminergic therapy) could help define early markers. Here we observed changes in tnaa and tcr levels that could reflect impairment in mitochondrial energy metabolism in the putamen of PD patients in drug-off condition that is reversed under dopaminergic treatment, which suggests that tnaa and tcr might be used to assess responsiveness to therapy. Moreover, the significant decrease in m-ins levels that could reflect osmotic changes inside astrocytes at early-stage disease was not previously described in the literature. Further H MR spectroscopic studies at different stages of PD to assess the correlation between disease severity and metabolic profile or under different antiparkinsonian medications could pave the way to validate these in-putamen changes as markers of PD. Disclosures of Conflicts of Interest: L.M. disclosed no relevant relationships. C.C. disclosed no relevant relationships. B.J. disclosed no relevant relationships. B.P. disclosed no relevant relationships. A.C. disclosed no relevant relationships. C.S. disclosed no relevant relationships. F.D. disclosed no relevant relationships. References. Chaudhuri KR, Healy DG, Schapira AH; National Institute for Clinical Excellence. Nonmotor symptoms of Parkinson s disease: diagnosis and management. Lancet Neurol 2006;5(3): Hughes AJ, Daniel SE, Lees AJ. Improved accuracy of clinical diagnosis of Lewy body Parkinson s disease. Neurology 200;57(8): Jankovic J. Parkinson s disease: clinical features and diagnosis. J Neurol Neurosurg Psychiatry 2008;79(4): Lucetti C, Del Dotto P, Gambaccini G, et al. Proton magnetic resonance spectroscopy (H-MRS) of motor cortex and basal ganglia in de novo Parkinson s disease patients. Neurol Sci 200;22(): Hu MTM, Taylor-Robinson SD, Chaudhuri KR, et al. Evidence for cortical dysfunction in clinically non-demented patients with Parkinson s disease: a proton MR spectros- 8 radiology.rsna.org n Radiology: Volume 000: Number 0 206

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