Neurochem Res (2010) 35:15301537 DOI 10.

1007/s11064-010-0212-5

ORIGINAL PAPER

The Role of Oxidative Stress in Amyotrophic Lateral Sclerosis and Parkinsons Disease
Athan Baillet Vanessa Chanteperdrix Candice Trocme Pierre Casez Catherine Garrel Gerard Besson

Accepted: 1 June 2010 / Published online: 10 June 2010 Springer Science+Business Media, LLC 2010

Abstract We examined oxidative stress markers of 31 patients suffering from ALS, 24 patients suffering from PD and 30 healthy subjects were included. We determined the plasma levels of lipid peroxidation (malondialdehyde, MDA), of protein oxidative lesions (plasma glutathione, carbonyls and thiols) and the activity of antioxidant enzymes i.e. erythrocyte Cu,Zn-Superoxide dismutase (SOD), Glutathione peroxidase (GSH-Px) and catalase. MDA and thiols were signicantly different in both neurodegenerative diseases versus control population. A trend for an enhancement of oxidized glutathione was noted in ALS patients. Univariate analysis showed that SOD activity was signicantly decreased in ALS and GSH-Px activity was decreased in PD. After adjusting for demographic parameters and enzyme cofactors, we could emphasize a compensatory increase of SOD activity in PD. Different antioxidant systems were not involved in the same way in ALS and PD, suggesting that

oxidative stress may be a cause rather than a consequence of the neuronal death. Keywords Oxidative stress Reactive oxygen species Neurodegeneration Trace element Amyotrophic lateral sclerosis Parkinsons disease Glutathione peroxidase Superoxide dismutase Abbreviations ALS Amyotrophic lateral sclerosis EDTA Ethylenediaminetetraacetic acid GSH-Px Glutathione peroxidase MDA Malondialdehyde MOPS 3-N-morpholinopropaneslfonic acid PD Parkinsons disease SOD Cu,Zn-Superoxide dismutase

A. Baillet G. Besson Neurology Department, Hopital A. Michallon, 38043 Grenoble Cedex 9, France V. Chanteperdrix C. Garrel Biology Department, Hopital A Michallon, 38043 Grenoble Cedex 9, France C. Trocme Laboratory of Enzymology/DBPC, CHU Hopital Michallon, 38043 Grenoble Cedex 9, France P. Casez Medical Information Unit, A. Michallon, 38043 Grenoble Cedex 9, France A. Baillet (&) Grenoble University, Hopital A. Michallon, 38043 Grenoble Cedex 9, France e-mail: abaillet@chu-grenoble.fr

Introduction Due to its high rate of oxygen utilization, high content of unsaturated lipids and relative lack of antioxidant enzymes, the brain is very vulnerable to free radical damage [1, 2]. Indeed free radicals are very toxic for motor and dopaminergic neurons. Amyotrophic lateral sclerosis (ALS) and Parkinsons disease (PD) are threatening neurodegenerative diseases that affect patients with a critical impact on their quality of life and on the health-care resources. Several studies suggested that oxidative stress plays a role in neurodegeneration [3, 4]. Post-mortem studies found a higher level of oxidative stress in the substantia nigra of PD patients [5, 6]. Moreover a signicant decrease in the activity of the complex I of the mitochondrial electron

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transport chain was reported in the substantia nigra [7, 8] and in platelets [9] of patients suffering from Parkinsons disease. The neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine can cause parkinsonism by an inhibition of complex I [1012] and enhances lipid peroxidation in the substantia nigra. Finally a previous study proved that plasma from PD patients is more prone to peroxidation and contains higher levels of oxidative stress markers than in the control population [13]. Mutations in the Cu,Zn-Superoxide dismutase 1 (SOD1) gene account for 20% of familial ALS. Enhancement of oxidative damage markers was reported in ALS patients [3, 4, 1418]. Furthermore signs of increased compensatory response to oxidative stress were found in patients with sporadic ALS [19]. Although there is increasing evidence of oxidative stress involvement in these two degenerative diseases, the lack of efciency of antioxidant drugs raises the question of the role of oxidative stress in ALS and PD pathogenesis [2025]. Oxidative damage may only be the result of the neurodegenerative process. This study was designed to compare oxidative lesions and anti-oxidative systems in ALS, PD and healthy populations in order to clarify the specic role of oxidative stress in these two diseases.

Measurement of Oxidative Markers in PD and ALS MDA evaluates lipid peroxidation. The quantication method is based on the reaction of MDA with thiobarbituric acid followed by a reversed-phase high-performance liquid chromatography separation with uorescence detection. A standard curve was performed with increasing concentrations of 1,1,3,3-tetraethoxypropane diluted in ethanol, in order to calibrate the uorescence detector and to correlate uorescence intensity to the MDA rate as previously described [26]. The concentration of plasma free thiols, which reects the oxidative status of sulfhydril aminoacids, was evaluated according to the Ellmans test. This technic is based on the property of Ellmans reagent 5,50 -dithiobis (2-nitrobenzoic) acid to be reduced by free thiols in a yellow compound whose concentration was measured at 412 nm [27]. Carbonyls, which measure protein damage by free radicals, were evaluated through their reactivity with the 2,4-dinitrophenylhydrazine by forming a diphenylhydrazone compound that absorbs light at 380 nm. The carbonyl group content was calculated using an albumin standard curve and was referred to the protein concentration as previously described [28]. Evaluation of the Enzymatic Activity of SOD, GSH-Px and Catalase

Experimental Procedure Study Population and Sample Preparation This study enrolled a total of 85 patients. Twenty four patients were diagnosed with sporadic PD. All of them underwent bilateral subthalamus stimulation. Thirty one denite sporadic ALS patients (according to the El Escorial criteria) were included in this study. Venous blood samples were collected very soon after diagnosis and before any treatment in the ALS group or during a control consultation at least 3 months after surgery in the PD group. The control group consisted of 30 healthy individuals. All of them were questioned and examined by an experienced neurologist to rule out a possible beginning of neurodegenerative disease. All participants gave their written consent prior to inclusion, according to the Declaration of Helsinki. The study design was approved by the local medical ethic committee. Every patient in the ALS and PD groups was treated in the Neurology Department of Grenoble Hospital, France. Red blood cells, obtained from blood samples, were evaluated for their antioxidant capacity by measuring the erythrocyte SOD, catalase, glutathione peroxidase (GSH-Px) activity and both oxidized and reduced glutathione in each group. Plasmas were frozen at -80C for further measurement of oxidative stress markers (thiols, carbonyls, malondialdehyde (MDA)) and antioxidant enzyme cofactors (copper, zinc, selenium). Erythrocytes were rst lyzed by a fourfold dilution in H2O. SOD was extracted by a chloroform/ethanol solution (1:1, v/v) solution and after centrifugation (4,000 rpm, 25 min at ?4C), 50 ll of supernatant were added to 1,870 ll of the reaction buffer (50 mM cacodylic acid, 1 mM diethylenetriaminepentaacetic acid, 0.05 M Tris, pH 8.1) and 80 ll of 10 mM pyrogallol diluted in 36% HCl. Absorbance was measured at 420 nm. Values were converted to enzymatic units by reference to a SOD standard curve [29]. Specic Mn-SOD activity was obtained after inhibiting Cu,Zn-SOD activity with KCN. Specic Cu,Zn-SOD activity results from the difference between total-SOD activity and Mn-SOD activity. Erythrocyte GSH-Px activity was determined by oxidation of reduced glutathione in presence of terbutyl hydroperoxyde. NADPH2 was reduced into NADP? in presence of oxidized glutathione and glutathione reductase. Therefore GSH-Px activity was evaluated by the decrease of NADPH absorbance at 340 nm. Enzymatic reaction was performed at 25C in a tube containing 900 ll of reaction buffer (1 mM ethylenediaminetetraacetic acid (EDTA), 4 mM NaN3, 50 mM Tris, pH 7.6), 25 ll of a tenfold diluted hemolyzed erythrocytes, 20 ll of 0.15 M reduced glutathione, 20 ll of glutathione reductase (208 U/ml), 20 ll of 8.4 mM NADPH2 and 20 ll of terbutyl hydroperoxyde 70% [30].

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Measurement of catalase activity was performed by direct reading of H2O2 disappearance at 240 nm. 100 ll of plasma were diluted in 2.9 ml of reaction buffer (0.05 M Na2HPO4, 0.05 M NaH2PO4, pH 7) containing 100 ll of 33% H2O2. All enzymatic activities were expressed in units reported to the haemoglobin concentration [31]. Determination of Antioxidant Enzyme Cofactors: Trace Element and Glutathione Concentration Trace element concentration was determined by measurement of the atomic absorption spectrometry, at 324.8 nm for copper, 213.4 nm for zinc, and 196 nm for selenium. Given that glutathione is the most abundant nonprotein thiol, we used nonprotein thiols as a proxy of glutathione concentration. For the evaluation of the nonprotein thiols concentration, blood samples were rst deproteinized with 3.6 ml of 6% metaphosphoric acid and diluted (1:5, v/v) in 0.4 M MOPS (3-N-morpholinopropaneslfonic acid), 2 mM EDTA, pH 6.75. After centrifugation, total glutathione concentration was evaluated on the supernatant by the Ellmans test. For oxidized glutathione measurement supernatants were previously incubated during 1 h with 40 ll triethanolamine and 10 ll 2-vinylpyridine. Absorbances were read within 20 min at 412 nm and compared to a glutathione standard curve. Statistical Analysis Descriptive statistics were presented as numbers and percentages for categorical variables and means standard deviation (SD) for continuous variables. We assessed heterogeneity in group means using ANOVA followed by the Scheffe post hoc test to investigate the cause of rejection of the null hypothesis. We evaluated the correlation between trace elements and oxidative markers by the calculation of the regression coefcient. We assessed in a multivariate analysis the inuence of age and gender on SOD and GSHPx activities (MODEL1). Given that trace elements and cofactors rate may also inuence SOD or GSH-Px activity, we also introduce them in a second multivariate analysis (MODEL 2). We also compared oxidative stress markers of 45 age-matched patients suffering from ALS, PD and healthy subjects. Statistical analysis was performed using Stata 9.0.

younger than PD (39.4 11.3 vs. 57.8 8.5 years, P \ 0.01) and ALS populations (63.0 10.9 years, P \ 0.01). Mean ages in PD and ALS groups were not statistically different (P = 0.20). The male:female ratio in the healthy population (11:19) was statistically different from the PD group sex ratio (17:7, P \ 0.01) and from ALS group sex ratio (17:14, P \ 0.01). Sex ratio was not statistically different between ALS and PD groups. Evidences for Oxidative Stress in PD and ALS Lipid peroxidation was statistically different between groups (Table 2). The MDA plasmatic concentration in the control group was 1.41 0.22 lmol/l and was signicantly enhanced in PD (1.74 0.18 lmol/l, P \ 0.01) and ALS (1.78 0.31 lmol/l, P = 0.03) populations. Similarly a signicant difference in the plasmatic thiol rate was found between groups. Free thiols were decreased in both ALS (6.06 0.87 lmol/g prot, P = 0.02) and PD (6.15 0.69 lmol/g prot, P = 0.07) populations in comparison to control patients (6.64 0.69 lmol/g prot), but the difference between PD and ALS groups was not signicant (P = 0.91). We did not nd any statistical difference in the plasmatic carbonyl concentration between control (0.40 0.03 lmol/g prot), ALS (0.38 0.19 lmol/g prot, P = 0.87) and PD (0.45 0.09 lmol/g prot, P = 0.40) groups. Total and reduced glutathione plasmatic rates were not statistically different in the three groups (respectively, P = 0.97 and P = 0.99). A trend toward an increase of the oxidized glutathione in ALS and PD patients in comparison to controls was observed, but failed to reach statistical signicance (P = 0.08). Decreased Rate of Trace Elements and Antioxidant Enzyme Cofactors in PD and ALS Contrary to copper plasmatic rate which was slightly the same in the three populations (Table 3), selenium and zinc plasmatic concentrations were statistically different between groups (respectively, P = 0.02 and P \ 0.01).
Table 1 Demographic parameters in control group, amyotrophic lateral sclerosis (ALS) group and Parkinsons disease (PD) group Control Number of patients Age (years) 30 39.4 39.0 11.3 11/19 PD 24 57.8 59.0 8.5 17/7 ALS 31 63.0 64.0 10.9 17/14 0.04 P value NA \0.01

Results Study Population Mean age and sex ratio were statistically different in the 3 populations (Table 1). The control group was statistically

Mean Median SD Male:Female ratio

Statistical signicance was determined by ANOVA. SD, standard deviation

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Neurochem Res (2010) 35:15301537 Table 2 Oxidative stress in control group, amyotrophic lateral sclerosis group and in the Parkinsons disease group Oxidative stress markers Control Mean MDA (lmol/l) Thiols (lmol/g prot) Carbonyls (lmol/g prot) Total glutathione (lmol/l) Reduced glutathione (lmol/l) Oxidized glutathione (lmol/l) Reduced/oxidized glutathione ratio 1.41 6.64 0.40 974.67 949.00 11.88 89.49 SD 0.22 0.69 0.03 145.34 143.20 4.10 34.46 PD Mean 1.74 6.15 0.45 976.52 946.74 14.21 78.62 SD 0.18 0.69 0.09 178.72 176.46 6.03 42.59 ALS Mean 1.78 6.06 0.38 985.17 951.03 15.08 72.97 SD 0.31 0.87 0.19 176.01 175.02 5.41 37.05

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P value

0.02 0.01 0.40 0.97 0.99 0.08 0.14

Statistical signicance was determined by ANOVA. SD, standard deviation; MDA, malondialdehyde

Table 3 Trace element plasmatic rates in control group, amyotrophic lateral sclerosis group and in the Parkinsons disease group Trace elements Control Mean SD Selenium (lmol/l) Copper (lmol/l) Zinc (lmol/l) 1.10 0.21 PD Mean SD 1.01 0.30 ALS Mean SD 0.88 0.20 0.02 0.30 P value

17.96 5.35 16.14 2.74 17.09 3.98

13.84 2.28 13.03 1.85 12.24 1.61 \0.01

Statistical signicance was determined by ANOVA. SD, standard deviation

Selenium concentration was signicantly decreased in ALS patients (0.88 0.20 lmol/l) compared to the control group (1.10 0.21 lmol/l, P \ 0.01) whereas its rate in the PD group (1.01 0.30 lmol/l) was not statistically different from the control group (P = 0.36) or ALS group (P = 0.13). Zinc plasmatic rate was diminished in the ALS group (12.24 1.61 lmol/l) in comparison to the control population (13.84 2.28 lmol/l, P \ 0.01). The difference was not statistically signicant between control and PD groups (13.03 1.85 lmol/l, P = 0.31) and between PD and ALS populations (P = 0.33). Differential Regulation of Antioxidant Enzymes in PD and ALS While catalase activity was found similar in the three groups (Table 4), mean SOD and GSH-Px activities were

statistically different between groups (respectively, P = 0.01 and 0.048). SOD activity was lower in the ALS group (1.32 0.09 U/mg haemoglobin) than in the PD population (1.40 0.14 U/mg haemoglobin, P = 0.01). The difference between the control group (1.37 0.07 U/ mg haemoglobin) and PD or ALS group was not statistically signicant (respectively, P = 0.51 and 0.13). Although a signicant difference in GSH-Px activity was observed between the three populations, the difference between any pairs of groups did not reach statistical signicance in univariate analysis (respectively, P = 0.14, 0.08 and 0.91). Inuence of Demographic Parameters and Trace Element Rates on Oxidative Markers Gender did not signicantly inuence thiol (P = 0.91), carbonyl (P = 0.48), glutathione (P = 0.60) and MDA (P = 0.64) plasmatic rates. Linear regression analysis showed a negative correlation between age and thiol concentration. No correlation was found between age and carbonyl, oxidized gluthatione and MDA plasmatic rates (data not shown). Contrary to selenium which was not related to MDA, thiol, carbonyl and oxidized glutathione concentration, copper level was positively correlated with MDA plasmatic rate and negatively correlated with thiol level (Table 5). On the other side, zinc concentration positively correlated with thiol plasmatic rate.

Table 4 Antioxidant enzyme activities in control group, amyotrophic lateral sclerosis group and Parkinsons disease group Antioxidant enzymes Control Mean SOD (U/mg haemoglobin) GSH-Px (U/mg haemoglobin) Catalase (U/mg haemoglobin) 1.37 44.15 187.17 SD 0.07 8.34 24.46 PD Mean 1.40 38.68 186.38 SD 0.14 7.92 27.78 ALS Mean 1.32 39.69 181.39 SD 0.09 9.61 33.50 0.01 0.05 0.74 P value

Statistical signicance was determined by ANOVA. SD, standard deviation; GSH-Px, Glutathione peroxidase; SOD, superoxide dismutase

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1534 Table 5 Correlation between trace element plasmatic rates and plasmatic oxidative stress MDA Selenium Coefcient 95% CI P value Copper Coefcient 95% CI P value Zinc Coefcient 95% CI P value -0.005 [-0.39, 0.28] 0.798 0.095 [0.012, 0.177] 0.008 0.008 0.018 [0.002, 0.033] 0.04 -0.067 [-0.105, -0.29] \0.001 -0.005 -0.270 [-0.594, 0.053] 0.124 0.257 [-0.437, 0.951] 0.547 0.042 Thiols Carbonyls

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Oxidized glutathione

0.160 [-4.783, 5.102] 0.949 -0.225 [-0.508, 0.058] 0.118 -0.297 [-0.920, 0.326] 0.345

[-0.080, 0.164] 0.859

[-0.011, 0.002] 0.113

[-0.006, 0.022] 0.276

Statistical signicance was determined by regression analysis. MDA, malondialdehyde; 95% CI, 95% condence interval

Table 6 Multivariate analysis of Glutathione peroxidiase (GSH-Px) and Superoxide dismutase (SOD) activities according to demographic parameter (MODEL 1) or according to demographic parameters and anti-oxidant enzyme cofactors (MODEL 2) SOD activity Coefcient MODEL 1 Disease PD ALS Demographic characteristics Sex ratio Age MODEL 2 Disease PD ALS Demographic characteristics Sex ratio Age Copper Zinc Selenium Reduced glutathione -0.006 -0.003 0.004 -0.006 NA NA [-0.06, 0.05] [-0.01, 0.00] [-0.002, 0.01] [-0.02, 0.01] 0.82 \0.01 0.24 0.31 8.213 -0.001 -1.613 0.015 NA NA [-0.20, 16.63] [-0.01, 0.01] 0.06 0.90 [-5.65, 2.42] [-0.17, 0.20] 0.43 0.88 0.096 0.021 [0.03, 0.16] [-0.05, 0.09] \0.01 0.55 -5.853 -3.383 [-12.00, 0.29] [-10.02, 3.25] 0.06 0.31 0.010 -0.003 [-0.03, 0.06] [-0.01, 0.00] 0.65 \0.01 -2.214 0.019 [-6.15, 1.72] [-0.17, 0.20] 0.27 0.84 0.095 0.025 [0.03, 0.16] [-0.05, 0.10] \0.01 0.49 -6.568 -5.303 [-12.56, -0.58] [-11.57, 0.97] 0.03 0.10 95% CI P value GSH-Px activity Coefcient 95% CI P value

Antioxidant enzyme cofactors

Statistical signicance was determined by multivariate analysis. SD, standard deviation; PD, Parkinsons disease; ALS, amyotrophic lateral sclerosis; 95% CI, 95% condence interval: NA, not applicable; GSH-Px, Glutathione peroxidase; SOD, superoxide dismutase

Multivariate Analysis of SOD and GSH-Px Activity When adjusted for age and sex ratio, a signicant decrease of GSH-Px activity was shown in the PD population (P = 0.03, Table 6 MODEL 1). Since GSH-Px activity depends on selenium and reduced glutathione rate we included these parameters in the multivariate analysis.

When GSH-Px level was adjusted for selenium concentration and glutathione concentration the decrease of GSH-Px activity in PD group was no longer statistically signicant (P = 0.062, Table 6 MODEL 2). Linear regression analysis showed that SOD activity was signicantly enhanced in PD group (P \ 0.01) but not in ALS group (P = 0.49) when adjusted for age and sex

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ratio. Moreover SOD activity was statistically decreased when age increased (P \ 0.01). Although SOD activity depends on copper and zinc ions, the enzyme activity was independent from the plasmatic concentration of these cofactors (P = 0.24 and 0.31, respectively). Subgroup analysis of age-matched patients conrmed the signicant decrease of GSH-Px and the signicant increase SOD in PD group in comparison to the control group (data not shown).

Discussion We demonstrated in this study that patients suffering from ALS or PD displayed lipid and protein oxidative lesions. We found here an increased plasmatic rate of MDA which conrms the existence of lipid peroxidation, but also a decreased of plasmatic thiols in both populations and a trend for an enhancement of oxidized glutathione providing evidence of an alteration of the redox status. These results are consistent with previous studies which reported enhanced oxidative stress markers in plasma [3, 22] and spine [32, 33] in ALS and in plasma [34], cerebrospinal uid and the substantia nigra in PD [35]. As some studies showed an effect of age on SOD or GSH-Px activity [36, 37] we also performed multivariate analysis in order to identify possible confounders. Contrary to most of the studies evaluating oxidative stress in neurodegenerative diseases, we took into account enzyme cofactors and demographic parameters which may inuence the antioxidant enzyme activity. Although univariate analysis ans age-matched patients subgroup analysis suggested that SOD activity was diminished in the ALS group, this decrease was no longer signicant when adjusted for age and gender. Thus, the drop of SOD activity may be explained by the higher mean age in this specic ALS group which does not include familial forms of the disease. On the other side, SOD activity was enhanced in the PD group, strongly suggesting an upregulation of this antioxidant enzyme in order to maintain the balance of the redox status. Previous studies showed a reduction of SOD activity in erythrocyte [21] and cerebrospinal uid [38] in ALS whereas its activity was normal in PD [39, 40]. However in these studies a potential confounder cannot be ruled out since the enzymatic rates were not age-matched. Moreover SOD activity depends on the presence of the prosthetic group containing copper and zinc that stabilizes the apoenzyme in the native conguration. We have shown that zinc plasmatic rate was decreased in the ALS group. Although zinc depletion could cause a decrease of SOD activity, multivariate analysis proved that the decrease of this trace element was not correlated with the enzyme activity. However the

decreased level of zinc in ALS may still be related to the increase of some protein oxidative lesions. Indeed previous studies showed that zinc supplementation was associated with a decrease of lipid peroxidation in zinc-decient patients [4042] whereas zinc deciency caused an increase of oxidative stress in rats [43, 44]. Thus zinc depletion could explain, at least partly, the oxidative lesions observed in the ALS group. Such a decrease of zinc plasmatic rate may result from a trapping of this trace element by specic zinc binding proteins. Moreover ALS patients usually show signicant dysphagia and malnutrition which can cause a drop of this trace element intake. Contrary to zinc that has antioxidant properties, copper may be implicated in the outbreak of oxidative lesions, as shown by the positive correlation between its plasmatic rate and the increase of MDA level or the decreased concentration of free thiols. A previous study also suggested toxic effects of copper leading to lipid peroxidation, protein oxidation and oxidative DNA damage [45]. We also showed that the activity of another antioxidant enzyme, the GSH-Px, was decreased in PD patients whereas it remained normal in the ALS group. These results are in agreement with previous studies showing that this enzyme activity was normal in erythrocytes of ALS patients [3] but was decreased in the substantia nigra of PD patients [46]. Moreover our study demonstrated that this decline was dependent on the enzyme substrate, reduced glutathione, and selenium, whose four atoms bind to the catalytic site [30]. Although the rates of both parameters were in the normal range, the GSH-Px decrease in the PD group was not signicant anymore when adjusted to these two factors. This diminution of erythrocyte GSH-Px activity in PD patients could lead to the accumulation of hydrogen peroxide (H2O2) in the substantia nigra. This brain part also contains a high concentration of iron. All the conditions are gathered for the transformation of H2O2 into HO by the Fenton reaction. This very reactive free radical can attack both lipids and proteins and then participate to the neuronal oxidative damage. The preservation of catalase activity in neurodegenerative diseases has been noticed yet [40, 47]. In the brain H2O2 is mainly catabolyzed by GSH-Px rather than by catalase. This enzyme may not play an important role in neurodegenerative diseases. Although our study emphasized different patterns of oxidative stress-related substances among healthy, ALS and PD patients, few limitations should be emphasized. Firstly, these three populations were not age-matched. The healthy population was younger than ALS and PD populations. Previous studies emphasized that age impacts on both MDA and thiols [4850]. We performed a multivariate analysis and age-matched patients subgroup analysis in order to examine the inuence of demographic parameters

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Neurochem Res (2010) 35:15301537 7. Schapira AH, Mann VM, Cooper JM et al (1990) Anatomic and disease specicity of NADH CoQ1 reductase (complex I) deciency in Parkinsons disease. J Neurochem 55:21422145 8. Janetzky B, Hauck S, Youdim MB et al (1994) Unaltered aconitase activity, but decreased complex I activity in substantia nigra pars compacta of patients with Parkinsons disease. Neurosci Lett 169:126128 9. Parker WD Jr, Boyson SJ, Parks JK (1989) Abnormalities of the electron transport chain in idiopathic Parkinsons disease. Ann Neurol 26:719723 10. Singer TP, Castagnoli N Jr, Ramsay RR et al (1987) Biochemical events in the development of parkinsonism induced by 1-methyl4-phenyl-1, 2, 3, 6-tetrahydropyridine. J Neurochem 49:18 11. Przedborski S, Jackson-Lewis V, Djaldetti R et al (2000) The parkinsonian toxin MPTP: action and mechanism. Restor Neurol Neurosci 16:135142 12. Greenamyre JT, Sherer TB, Betarbet R et al (2001) Complex I and Parkinsons disease. IUBMB Life 52:135141 13. Agil A, Duran R, Barrero F et al (2006) Plasma lipid peroxidation in sporadic Parkinsons disease. Role of the L-dopa. J Neurol Sci 240:3136 14. Rosen DR, Siddique T, Patterson D et al (1993) Mutations in Cu/ Zn superoxide dismutase gene are associated with familial amyotrophic lateral sclerosis. Nature 362:5962 15. Oteiza PI, Uchitel OD, Carrasquedo F et al (1997) Evaluation of antioxidants, protein, and lipid oxidation products in blood from sporadic amyotrophic lateral sclerosis patients. Neurochem Res 22:535539 16. Bogdanov M, Brown RH, Matson W et al (2000) Increased oxidative damage to DNA in ALS patients. Free Radic Biol Med 29:652658 17. Beal MF, Ferrante RJ, Browne SE et al (1997) Increased 3-nitrotyrosine in both sporadic and familial amyotrophic lateral sclerosis. Ann Neurol 42:644654 18. Shibata N, Nagai R, Uchida K et al (2001) Morphological evidence for lipid peroxidation and protein glycoxidation in spinal cords from sporadic amyotrophic lateral sclerosis patients. Brain Res 917:97104 19. Cookson MR, Shaw PJ (1999) Oxidative stress and motor neurone disease. Brain Pathol 9:165186 20. Grundman M (2000) Vitamin E and Alzheimer disease: the basis for additional clinical trials. Am J Clin Nutr 71:630S636S 21. Sano M, Ernesto C, Thomas RG et al (1997) A controlled trial of selegiline, alpha-tocopherol, or both as treatment for Alzheimers disease. The Alzheimers Disease Cooperative Study. N Engl J Med 336:12161222 22. Ascherio A, Weisskopf MG, OReilly EJ et al (2005) Vitamin E intake and risk of amyotrophic lateral sclerosis. Ann Neurol 57:104110 23. Desnuelle C, Dib M, Garrel C et al (2001) A double-blind, placebo-controlled randomized clinical trial of alpha-tocopherol (vitamin E) in the treatment of amyotrophic lateral sclerosis. ALS riluzole-tocopherol Study Group. Amyotroph Lateral Scler Other Motor Neuron Disord 2:918 24. Group TPS (1996) Impact of deprenyl and tocopherol treatment on Parkinsons disease in DATATOP patients requiring levodopa. Parkinson Study Group. Ann Neurol 39:3745 25. Group PS (1993) Effects of tocopherol and deprenyl on the progression of disability in early Parkinsons disease. The Parkinson Study Group. N Engl J Med 328:176183 26. Fukunaga K, Yoshida M, Nakazono N (1998) A simple, rapid, highly sensitive and reproducible quantication method for plasma malondialdehyde by high-performance liquid chromatography. Biomed Chromatogr 12:300303 27. Ellman GL (1959) Tissue sulfhydryl groups. Arch Biochem Biophys 82:7077

on the results. Both thiol plasmatic rate and SOD activity were inuenced by the age whereas this potential confounder did not impact on the other oxidative stress markers. Signicant differences of anti-oxidant status could be shown between ALS and PD groups though the mean age in these two populations are not signicantly different. Therefore, misregulated oxidative stress markers in PD and ALS are more likely disease-specic rather than only age-dependant. Second, a limited number of healthy individuals and patients were included in this study. Additional studies with a higher number of aged-matched patients and are mandatory in order to conrm our results. Finally, cerebrospinal uid analysis on the same oxidative stress-related substances should be performed to further explore the origin of the oxidative stress. In conclusion this study demonstrated that the different antioxidant systems are not involved in the same way in ALS and in PD: increase of SOD and decrease of GSH-Px characterized in PD patients may contribute to the accumulation of H2O2 and then increase the oxidative protein lesions. On the other hand no difference in the level of the enzymes that we evaluated in this study was shown in ALS patients, suggesting that the oxidative lesions observed in this disease result from other mechanisms. It appears that each neurodegenerative disease is characterized by a specic oxidative ngerprint explaining the specic neuron population loss. However the relatively low efcacy of antioxidant supplementation in ALS [16, 17] and PD [18, 19, 51] suggests that oxidant damage in the brain are already considerable and irreversible hampering the efciency of such treatments.

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