ARTICLE pubs.acs.org/jpr
Metabolic Profiling of 3-Nitropropionic Acid Early-Stage Huntington’s Disease Rat Model Using Gas Chromatography Time-of-Flight Mass Spectrometry Kai Lun Chang,† Lee Sun New,† Mainak Mal,† Catherine W. Goh,§ Chiu Cheong Aw,§ Edward R. Browne,§ and Eric C. Y. Chan*,† †
Department of Pharmacy, Faculty of Science, National University of Singapore, 18 Science Drive 4, Singapore 117543, Singapore GlaxoSmithKline Centre for Cognitive and Neurodegenerative Disorders, Singapore, 11 Biopolis Way, The Helios Building #03-01/02, Singapore 138667, Singapore
§
bS Supporting Information ABSTRACT: 3-Nitropropionic acid (3-NP), a potent irreversible inhibitor of mitochondrial complex II enzyme, leads to mitochondrial dysfunction and oxidative stress in Huntington’s disease (HD) rat model. In this study, biochemical assays were used to demonstrate the presence of oxidative stress and mitochondrial dysfunction in 3-NP early stage HD rat models. Gas chromatography time-of-flight mass spectrometry (GC/ TOFMS) was applied to analyze metabolites in brain and plasma of 3-NP-treated and vehicle-dosed rats. The orthogonal partial least-squares discriminant analysis (OPLS-DA) model generated using brain metabolic profiles robustly differentiated the 3-NP early stage HD rat model from the control. Metabonomic characterization of the 3-NP HD rat model facilitated the detection of biomarkers that define the physiopathological phenotype of early stage HD and elucidated the treatment effect of galantamine. Brain marker metabolites that were identified based on the OPLS-DA model were associated with altered glutathione metabolism, oxidative stress, and impaired energy metabolism. The treatment effect of galantamine in early stage HD could not be concluded mechanistically using the brain metabotype. Our study confirmed that GC/TOFMS is a strategic and complementary platform for the metabonomic characterization of 3-NP induced neurotoxicity in the early stage HD rat model. KEYWORDS: 3-nitropropionic acid, Huntington’s disease, metabolic profiling, metabonomics, neurodegeneration, oxidative stress, mitochondrial dysfunction, galantamine
’ INTRODUCTION Huntington’s disease (HD) is a neurodegenerative disorder caused by mutation in gene encoding Huntingtin protein, which leads to production of neurotoxic form of mutated Huntingtin (m-Htt) protein.1 Typically, the disease is characterized by a progressive neurodegeneration from the putamen to the caudate nucleus,2 and manifests with cognitive disturbance, behavioral disorder, and movement incoordination.3 In HD patients, a marked decrease in the activity of mitochondrial Complex II enzyme (succinate dehydrogenase, SDH) was observed4 and ultrastructural abnormalities in mitochondria were reported in HD cortical tissue.5 Particularly, severe defects of mitochondrial Complexes II and III were found in the brain tissues of postmortem HD patients6,7 and experimental findings on mitochondrial dysfunction further supported that Complex II enzyme is the defective component in HD.8 Mitochondrial dysfunction in HD is also intimately associated with oxidative damage as leakage of electron from mitochondria can cause oxidative stress that leads to cell death.9,10 Mitochondrial Complex II dysfunction and r 2011 American Chemical Society
oxidative damage have therefore been proposed to play a major causative role in the pathogenesis of HD.11 The administration of 3-nitropropionic acid (3-NP), a potent irreversible inhibitor of SDH, leads to mitochondrial dysfunction in a biological system. A selective degeneration of the putamen and caudate in humans had been demonstrated following an accidental consumption of 3-NP in China.12 A majority of the patients who accidentally ingested 3-NP displayed persistent cognitive and behavioral impairments similar to the symptoms of HD.12 In rodents, chronic administration of 3-NP produced consistent neurodegenerative changes that were reminiscent of HD, specifically mitochondrial dysfunction in the neurones which was similarly observed in transgenic models of the disease.1316 The basis for considering the 3-NP HD rat model as a suitable disease model for the elucidation of the pathogenesis of HD had been thoroughly reviewed by Brouillet et al.17 Although this model may only be seen as “phenotypic” model Received: January 14, 2011 Published: February 28, 2011 2079
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Journal of Proteome Research of HD, its relevance to the disease is emphasized when pathological mechanisms common to both 3-NP and m-Htt toxicities were discussed.17 A recent literature by Tsang et al.18 documented the use of high-resolution magic-angle spinning nuclear magnetic resonance spectroscopy (HR-MAS NMR) in the metabolic profiling of the 3-NP rat model that mimics early stage HD. Remarkably, Tsang et al.18 demonstrated that HR-MAS NMR metabolic profiling technique is a more sensitive method in characterizing 3-NP-induced neurotoxicity than the standard histopathological criteria. In their study, HR-MAS NMR-based metabonomics was proven to be capable of detecting precursory signals prior to striatal neurodegeneration in 3-NP early stage HD rat model that displayed behavioral symptoms consistent with 3-NP-induced neurotoxicity.18 The study underscored that global metabolic profiling of animal disease model is a strategic approach to identify the early metabolic phenotypes of HD so as to gain a better insight into the pathogenesis and progression of the disease. While not investigated by Tsang et al.,18 such a metabolic profiling approach may also be explored for elucidating the pharmacological effects of potential pharmacotherapy targeted against early stage HD, where neuronal cell death has just started to occur.19 Galantamine is a reversible acetylcholinesterase (AChE) inhibitor that is currently being used for the treatment of Alzheimer’s disease (AD) and various memory impairments. More recently, galantamine has been shown to demonstrate in vitro antioxidative effect on neuronal damage induced by oxidative stress.20 It was also found to be able to attenuate 3-NPinduced striatal neurodegeneration in a 3-NP HD rat model, which was demonstrated to be associated with modulation of nicotinic acetylcholine receptor (nAChR).21 In addition, a case study involving a patient with HD reported that galantamine improved both motor and psychotic symptoms of HD.22 Considering the therapeutic effects observed with the administration of galantamine in HD, a metabolic profiling approach could potentially uncover novel pathway related to the treatment effect of galantamine. Apart from using brain tissue as sample in metabolic profiling technique, the usage of plasma as a nonterminal biological matrix would be valuable as it could be sampled for the longitudinal metabolic profiling of the 3-NP early stage HD rat model. Increased oxidative damage has been observed in plasma samples of HD patients,23,24 and such observations could translate into detectable plasma metabolic fluxes. In this study, a global analysis of metabolites in brain and plasma of 3-NP early stage HD rat model using the gas chromatography time-of-flight mass spectrometry (GC/TOFMS) analytical platform was performed for the first time. On the basis of the complementary nature of the different analytical platforms,25 a careful comparison drawn between our findings and the HR-MAS NMR study by Tsang et al.18 would enhance our understanding of HD pathology. The possible pharmacological roles of galantamine in mitigating 3-NP-induced HD would also be investigated. On the basis of the HR-MAS NMR metabolic profiling study performed by Tsang et al.,18 we propose a similar 14-day high-dose 3-NP dosing regimen to induce neurotoxicity in our 3-NP early stage HD rat model. As biochemical alterations in this model that mimics early stage HD have not been ascertained before, we intend to investigate in the brain and plasma the extent of mitochondrial Complex II dysfunction and oxidative stress. These two major causative factors of HD11 were suitably measured using
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biochemical assays to ensure that the 3-NP dosing regimen employed in our study could induce HD-related neurotoxicity effectively. In phase-1 of our study, biochemical assays were performed on selected brain regions, namely, the striatum, cortex, and hippocampus, of the 3-NP early stage HD rat model to validate the inhibition of mitochondrial Complex II enzyme and occurrence of oxidative damage as compared to vehicle-dosed rats. Plasma samples were also assessed to ascertain the level of oxidative stress following administration of 3-NP. Once the 3-NP dosage regimen was validated, we proceeded to phase-2 of our study and characterized the brain and plasma metabolic profiles of the 3-NP HD rat model, with and without galantamine intervention, using GC/TOFMS-based metabonomics.
’ MATERIALS AND METHODS Chemicals and Reagents
3-NP was purchased from Sigma-Aldrich (St. Louis, MO). Galantamine hydrobromide was obtained from J. INC (India). N-Methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA) with 1% trimethylchlorosilane (TMCS) and 2% methoxamine hydrochloride in pyridine (MOX reagent) were purchased from Pierce (Rockford, IL). Bradford protein assay kit was purchased from Bio-Rad Laboratories (Hercules, CA). Mitochondria isolation kit and Complex II enzyme activity microplate assay kit were purchased from MitoSciences (Eugene, OR). Nitrate/nitrate colorimetric (NOS) and superoxide dismutase (SOD) assay kits were purchased from Caymen Chemicals (Ann Arbor, MI). All other reagents used were of analytical grades. Animal Handling
Twenty 16-week-old male SpragueDawley (SD) rats (290350 g, Animal Resources Centre, WA, Australia) were used in the phase-1 study for the assessment of mitochondrial dysfunction and oxidative stress induced by the 14-day high-dose 3-NP dosing regimen reported by Tsang et al.18 Once the 3-NP dosing regimen was validated, 32 16-week-old male SD rats (290350 g) were dosed in our phase-2 study to generate the early stage HD rat model for GC/TOFMS-based metabolic profiling. All experiments were performed at the Biological Resource Centre (BRC) rodent facility, Biopolis, Singapore. All protocols were approved by the BRC Institutional Animal Care and Use Committee (IACUC), in accordance with the Singapore National Advisory Committee for Laboratory Animal Research (NACLAR) Guidelines. Food and water were available ad libitum and the animals were allowed to acclimate for a period of 7 days before treatment. During the study, the rats were housed in clear cages at 20 ( 1 °C and 45 ( 5% humidity and subjected to 12 h light/dark cycle. Dosage Regimen
3-NP was dissolved in normal saline (adjusted to pH 7.2) and administered intraperitoneally (ip) as a single bolus injection at a dosage of 7.5 mg/kg. Galantamine was dissolved (sonicated for 10 min at 30 °C) in 1% methyl cellulose solution and administered via oral gavage (po) at a dosage of 20 mg/kg. When animals were treated with both 3-NP and galantamine, galantamine doses were given 3045 min before 3-NP injections. Drugs were administered once daily at a consistent timing for 14 consecutive days. For phase-1 study, the SD rats were randomly divided into four groups (n = 5 per group). Group-1 (control) received normal saline (ip) and 1% methyl cellulose (po). Group-2 received 3-NP injections (ip) and 1% methyl cellulose (po). 2080
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Journal of Proteome Research Group 3 received both 3-NP injections (ip) and galantamine treatment (po). Group-4 received normal saline (ip) and galantamine treatment (po). The volumes of control and test solutions were adjusted daily according to the body weight of each individual rat. During phase-2 study, 32 SD rats were randomly allocated to the similar four treatment groups: group-1 (n = 5), group-2 (n = 11), group-3 (n = 11), and group-4 (n = 5). Similarly, the volumes of control and test solutions were adjusted daily according to the body weight of each individual rat. Biological Sampling for Biochemical Assays
Twenty-four hours after the last dosing for all four treatment groups, the rats were sacrificed by rapid decapitation. Sampling of brain and plasma was performed immediately. Each whole rat brain was extracted and placed on ice, followed by dissection and isolation of striatum, cortex, and hippocampus. The isolated brain parts were collected into one Eppendorf tube for each rat and stored at 80 °C until analysis. Blood was collected in ethylenediaminetetraacetic acid (EDTA) tubes and centrifuged at 4 °C for 10 min at 4000g. After centrifugation, 1 mL of plasma was transferred into a new Eppendorf tube and stored at 80 °C until analysis. On the day of biochemical assay, the brain parts were minced collectively and split into 3 portions for each biochemical assay. Superoxide Dismutase (SOD) Activity Assay
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nitrate and nitrite for all samples were determined using a nitrate standard curve. The results were expressed as concentration of nitrite after complete conversion in nanomoles per milligram of protein (nmol/mg protein). Mitochondrial Complex II Enzyme Activity
Complex II enzyme activities of brain samples collected from animal groups 1, 2, and 3 were determined using the complex II enzyme activity microplate assay kit. Extraction of mitochondria was first performed using the MitoProfile benchtop mitochondria isolation kit, where differential centrifugation was used to isolate intact mitochondria from brain tissue. Prechilled Dounce homogenizer was used to further homogenize each minced brain tissue, which was subsequently suspended in an isolation buffer and centrifuged at 1000g for 10 min at 4 °C. Supernatants were collected and centrifuged at 12 000g for 15 min at 4 °C to obtain pellets of intact mitochondria. The pellets were suspended to 5.5 mg/mL in PBS. A microplate, coated with anti-Complex II monoclonal antibody (mAb), was used to purify the Complex II enzyme from the samples. Once the enzyme was immobilized, an activity solution, containing ubiquinone, succinate, and 2,6-diclorophenolindophenol (DCPIP), was added to initiate the reduction of ubiquinone by the active Complex II enzyme. Subsequent reduction of DCPIP was measured as a decrease in absorbance at 600 nm. Complex II activities were reported as the rate of disappearance in absorbance (mAbs/min).
Brain and plasma samples were assayed for their total SOD activity using the SOD assay kit. The assay kit utilized a tetrazolium salt for the detection of superoxide radicals based on an absorbance at 440 nm measured using the Infinite M200 Tecan microplate reader (Tecan Group Ltd., M€annedorf, Switzerland). These radicals were diminished in the presence of SOD, thus, allowing the absorbance reading to reflect SOD activity. The minced brain tissues were further homogenized in 5 mL/g of cold 20 mM HEPES buffer, pH 7.2, containing 1 mM EGTA, 210 mM mannitol, and 70 mM sucrose. The homogenates were centrifuged at 4 °C for 5 min at 1500g and the supernatants were used for the assay. Plasma samples were diluted 1:5 with sample buffer (50 mM Tris-HCl, pH 8.0) before assaying for total SOD activity. Briefly, the reaction was initiated by the addition of xanthine oxidase to the mixture containing hypoxanthine, tetrazolium salt, and sample. The results were expressed as units per milligram of protein (unit/mg protein), where one unit of SOD was defined as the amount of enzyme needed to exhibit 50% dismutation of the superoxide radicals.
Statistical analyses were performed to identify significant differences between the four treatment groups with regards to SOD activity, NO production, and Complex II enzyme activity. All the result values were expressed as percentages of vehicledosed group and converted into mean ( standard deviation (SD). The data was analyzed using one way analysis of variance (ANOVA) followed by Tukey’s posthoc test. For all biochemical assays, a p-value of less than 0.05 was considered to be significantly different.
Estimation of Nitric Oxide (NO) Production
Biological Sampling for Metabolic Profiling
The sum of both nitrate and nitrite is the best index of total NO production and was measured in our study using the NOS assay kit. The assay kit utilized nitrate reductase to convert all nitrate into nitrite, and total nitrite concentration was then determined with a colorimetric assay using Griess reagents (sulfanilamide and N-(1-naphthyl)ethylenediamine). Each brain sample was first homogenized in phosphate buffered saline (PBS) and centrifuged at 4 °C for 20 min at 10 000g. Brain supernatants and plasma samples were ultrafiltered using prerinsed 30 kDa molecular weight cutoff filters and the filtrates were used for the assay. Nitrate reductase and cofactors were added to each assay sample and incubated for 3 h at room temperature (25.0 ( 0.5 °C) to ensure complete conversion of all nitrate to nitrite. An equal volume of Griess reagents was then added and the color was allowed to develop for 10 min at room temperature. Absorbance readings at 540 nm were measured and concentrations of total
Twenty-four hours after the last dosing for all four treatment groups in our phase-2 study, the rats were sacrificed by rapid decapitation and their brain and plasma were collected immediately as described previously in the phase-1 study. Prior to metabolic profiling, the brain parts were homogenized with water in the ratio of 1 part of tissue mass to 1 part of water. Then, 100 μL of each homogenate was transferred to a 15-mL glass centrifuge tube and 1 mL of monophasic extraction solvent of chloroformmethanolwater (2:5:2, v/v/v) was added to each sample. After 2 min of vortex-mixing, the samples were centrifuged at 3500 rpm for 3 min and 800 μL of supernatant from each sample was transferred into a new 15-mL glass tube. Likewise, plasma samples were prepared similarly but using acetonitrile as the extraction solvent instead. The supernatants of both brain and plasma samples were concentrated to complete dryness at a temperature of 50 °C for approximately 30 min using
Protein Measurement
Protein levels in all samples were assessed using the Bradford protein assay kit. Using bovine serum albumin as standard, concentrations of protein in samples (mg/mL) were quantified based on a differential color change of Coomassie Brilliant Blue G-250 dye and a shift in absorbance maximum from 465 to 595 nm. The measured protein concentrations were used to normalize the results of the respective biochemical assays. Statistical Analysis
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Table 1. Biochemical Changes in Rat Brain Parts Induced by 3-NP with and without Galantamine treatment Control
a
SOD activity (unit/mg)a
nitrate/nitrite level (nmol/mg)a
100.00 ( 15.99
Complex II enzyme activity (mAbs/min)a
100.00 ( 50.50
100.00 ( 10.63
278.51 ( 133.09b
23.69 ( 9.83c
3-NP and Galantamine
b
53.51 ( 8.89
86.77 ( 46.21
17.37 ( 3.98c
Galantamine
92.57 ( 25.32
71.89 ( 15.26
3-NP
41.86 ( 25.86b
d
Values are expressed as mean % of control ( standard deviation (SD). b p < 0.01 versus Control. c p < 0.001 versus Control. d p < 0.001 versus 3-NP.
the Turbovap nitrogen evaporator (Caliper Life Science, Hopkinton, MA). Afterward, 100 μL of anhydrous toluene (stored with sodium sulfate) was added to each of the dried tissue and plasma extracts. Following 1 min of vortex-mixing, the samples were evaporated to dryness using the evaporator to ensure the complete elimination of any traces of water which might interfere with the subsequent GC/TOFMS analysis. Then, 40 μL MOX reagent was added to the dried samples, vortex-mixed for 2 min, and incubated at room temperature (25.0 ( 0.5 °C) for at least 16 h as an overnight methoximation step. Derivatization reaction aimed to increase the volatility of polar metabolites was then initiated by adding 60 μL of MSTFA (with 1% TMCS) to each sample, vortex-mixed for 2 min, and incubated at 70 °C for 30 min. Following the incubation, each sample was vortex-mixed for 2 min and carefully transferred to the autosampler vials for GC/ TOFMS analysis. GC/TOFMS Conditions
GC/TOFMS analysis was performed using 7890A Gas Chromatography (Agilent Technologies, Santa Clara, CA) coupled to PEGASUS 4D Time-of-Flight Mass Spectrometer (LECO Corporation, St. Joseph, MI). A DB-1 GC column (Agilent Technologies) with a length of 23 m, internal diameter of 250 μm, and film thickness of 0.25 μm was used as the primary column. A Rxi17 GC column (Restek Corporation, Bellefonte, PA) with a length of 1 m, internal diameter of 100 μm, and film thickness of 0.1 μm was used as the secondary column. Helium was used as the carrier gas at a flow rate of 1 mL/min. An injection volume of 1 μL was used and the injector split ratio was set to 1:2. The front inlet and ion source temperatures were maintained at 250 and 200 °C, respectively. The primary column temperature was kept at 60 °C for 0.2 min, then increased by 5 °C/min to 125 °C and further increased by 15 °C/min to 270 °C where it was held for 25 and 7 min for analysis of brain and plasma samples, respectively. The secondary column temperature was set to an initial temperature of 70 °C for 0.2 min, then ramped at 5 °C/min to 135 °C and further increased by 15 °C/min to 280 °C with a 25 and 7 min hold time for analysis of brain and plasma samples, respectively. Modulator temperature offset of þ20 °C relative to secondary oven was used. The MS mass range was m/z 50650 with an acquisition rate of 50 Hz. The detector voltage was set at 1650 V with electron energy of 70 eV. Solvent cutoff time was determined to be 8.3 min. Chromatogram acquisition, peak deconvolution, analyte alignment, and preliminary analyte identification by the National Institute of Standards and Technology (NIST) library were performed using the LECO ChromaTOF software version 4.21 prior to conducting multivariate data analysis for all the sample groups. Peaks with similarity index of more than 70% were assigned putative metabolite identities based on the NIST mass spectral library. Identities of selected marker metabolites preliminarily identified by NIST mass spectral library were further confirmed based on comparison of their mass spectra and retention times with those obtained using
commercially available reference standards (glycerol, L-Threonine, succinic acid, L-Serine, glycine, L-Methionine, L-Aspartic acid, L-Glutamine, citric acid, L-Ascorbic acid, palmitic acid, arachidonic acid, oleamide, docosahexaenoic acid, D-fructose, L-tryptophan). Multivariate Data Analysis
The resulting data was first processed by normalizing peak area of each analyte based on total integral area calculation performed using an in-house script (Microsoft Office Excel). All processed data were then mean-centered and unit-variance scaled before it was subjected to principal component analysis Sweden) to iden(PCA) (SIMCA-P software, Umetrics, Umea, tify clustering trend, as well as detect and exclude outlier. Quality control (QC) samples for both brain and plasma samples were prepared by pooling randomly 5 μL from each of the five samples belonging to the four different test groups. QC samples were analyzed at constant intervals to ensure that the data acquisition for GC/TOFMS metabolic profiling was reproducible for all samples. After exclusion of sample outliers, brain and plasma samples of 3-NP-treated and vehicle-dosed rats were further subjected to orthogonal partial least-squares-discriminant analysis (OPLS-DA) for identification of discriminant metabolites that characterized the two sample groups. Variable importance in the projection (VIP) cutoff value was defined as 1.00. Independent t-test with Welch’s correction was then used for statistical comparison of discriminant metabolite levels between the two test groups. A p-value of less than 0.05 was considered to be significantly different for discriminant metabolites in both brain and plasma samples. In an attempt to elucidate the metabolic consequences due to pharmacological effects of galantamine in 3-NP HD rat model, we further subjected brain samples from group-1 (vehicle-dosed), group-2 (3-NP treatment only), and group-3 (3-NP and galantamine treatments) to PCA. Metabolic profiles of brain samples from group-3 were also evaluated to investigate effects of galantamine treatment on the levels of brain discriminant metabolites determined for 3-NP HD rat model.
’ RESULTS Biochemical Assays
Results of the biochemical assays for brain samples are summarized in Table 1. As compared to the vehicle-dosed group, significant elevation in nitrate/nitrite level (p < 0.01) in SD rat brain, specifically the striatum, cortex, and hippocampus, was observed in the 3-NP-treated rats, indicating an increased NO production. Systemic administration of 3-NP also caused significant reduction in SOD enzyme activity (p < 0.01) as well as complex II enzyme activity (p < 0.001) in all the examined brain parts of SD rats as compared to the control group. Galantamine co-administration prevented the increase in nitrate/nitrite level (p < 0.001) in 3-NP-treated rats, demonstrating its effectiveness in halting excess NO production induced by 3-NP. However, 2082
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Table 2. Biochemical Changes in Rat Plasma Samples Induced by 3-NP with and without Galantamine SOD activity
nitrate/nitrite level
(unit/mg)a
(nmol/mg)a
Control 3-NP
100.00 ( 12.32 95.14 ( 14.58
100.00 ( 47.49 77.80 ( 29.66
3-NP and Galantamine
112.55 ( 20.40
97.05 ( 47.90
94.13 ( 8.24
93.92 ( 46.52
treatment
Galantamine a
Values are expressed as mean % of control ( standard deviation (SD).
galantamine failed to restore the decreased SOD enzyme and complex II enzyme activities as induced by 3-NP treatment. Galantamine per se treatment, in the absence of concomitant 3-NP treatment, did not produce any significant effect on the SOD activity and nitrate/nitrite level as compared to the vehicledosed rats. In contrast to the biochemical findings in brain, both SOD enzyme activity and NO production level were not significantly altered in the plasma of rats dosed with 3-NP and/or galantamine, indicating little or no biochemical changes in the plasma following administration of 3-NP and/or galantamine. Results of the biochemical assays for plasma samples are summarized in Table 2. GC/TOFMS Metabolic Profiling
PCA of brain samples indicated that 3-NP-treated and vehicledosed rats formed two distinct clusters on the scores plot (Supporting Information, Figure 1A). The scores plot of the OPLS-DA model generated using the normalized, mean-centered and unit-variance scaled data of brain samples from five control rats and nine 3-NP-treated rats is presented in Figure 1A (3 LV, R2(Y) and Q2(cum) were 0.980 and 0.524, respectively). LV are the latent variables, R2(Y) is the fraction of the sum of squares of all Y-values explained by the current latent variables, and Q2(cum) is the cumulative Q2 for the extracted latent variables. Q2 is given by the expression Q2 = 1 ∑(Ypredicted Ytrue)2/∑Y2true. As shown in Figure 1A, 3-NP-treated rats were clearly separated from vehicletreated rats along LV 2. A list of discriminant brain metabolites that were responsible for defining the 3-NP rats distinctly from the control rats in the OPLS-DA model is summarized in Table 3. With the exception of succinic acid, all the other discriminant metabolites were found to be present at lower levels in the brain samples of 3-NP-treated rats. The results from independent t test with Welch’s correction indicated that all the discriminant metabolites were present at levels that were statistically different between the two sample groups (p < 0.05). The representative GC/TOFMS chromatogram of brain sample of 3-NP-treated rat depicting the peaks of discriminant metabolites is shown in Figure 1B. No observable clustering trend was determined in the PCA of plasma samples (Supporting Infromation, Figure 1B), suggesting little or no evidence of differences in the plasma metabolic phenotypes of the different test groups. The scores plot of the OPLS-DA model generated using the plasma samples from five control rats and eight 3-NP-treated rats is presented in Figure 2A (2 LV, R2(Y) and Q2(cum) were 0.954 and 0.302, respectively). A low Q2(cum) in this OPLS-DA model indicates weak separation between 3-NP-treated and vehicle-dosed rats. A few discriminant metabolites of plasma samples were identified and summarized in Table 4. The results from independent t test with
Figure 1. (A) OPLS-DA scores plot discriminating 3-NP-treated and vehicle-dosed rats based on GC/TOFMS brain metabolic profiles. (B) GC/TOFMS chromatogram of brain sample of 3-NP-treated rat depicting the peaks of discriminant metabolites.
Welch’s correction indicated that all the discriminant metabolites were present at levels that were statistically different between the two sample groups (p < 0.05). The representative GC/TOFMS chromatogram of plasma sample of 3-NP-treated rat depicting the peaks of discriminant metabolites is illustrated in Figure 2B. In the PCA of group-1, group-2 and group-3 treatment groups, rats co-administered with both 3-NP and galantamine were found to be clustered together with 3-NP-treated rats in the scores plot (Supporting Information, Figure 1C), suggesting that galantamine treatment did not alter the brain metabotype of 3-NP-treated rats significantly. Among the list of discriminant brain metabolites that were responsible for defining 3-NP early stage HD rat model (Table 3), administration of galantamine mitigated the accumulation of succinic acid, lowering the percentage change of disease model from control for succinic acid from 160.3 to 58.5% (p = 0.05).
’ DISCUSSION In this study, we employed a recently reported 3-NP rat model that mimics early stage HD, which allows for detection of precursory signals prior to striatal neurodegeneration as demonstrated by Tsang et al.18 To further evaluate this 3-NP early stage HD rat model, we investigated the biochemical effects of 3-NP administration on the oxidative status and mitochondrial complex II enzyme activity, the two main factors in HD pathogenesis,11 in the rat brain and plasma. Following a similar 14-day high-dose 3-NP dosing regimen as reported by Tsang et al.,18 oxidative stress and mitochondrial complex II enzyme dysfunction were clearly observed in the brain tissues of 3-NP-treated rats in our study. We documented significant reduction of SOD enzyme activity and increased production of NO in the brain of 3-NP-treated rats. Systemic administration of 3-NP also resulted in significant central 2083
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Table 3. Marker Metabolites Found in GC/TOFMS Analysis of Brain Samples of 3-NP-Treated and Vehicle-Dosed Rats metabolite Methylmalonic acid d
identified by a
Rt (min)
chemical class
14.00,
Dicarboxylic acid
NIST
% change of disease model from control b
p-value c
44.5
