PERIPHERAL MARKERS FOR OXIDATIVE STRESS IN PARKINSON’S DISEASE PATIENTS OF EASTERN INDIA

ISSN 1819-7124, Neurochemical Journal, 2011, Vol. 5, No. 2, pp. 146–149. © Pleiades Publishing, Ltd., 2011. CLINICAL NEUROCHEMISTRY Peripheral Markers for Oxidative Stress in Parkinson’s Disease Patients of Eastern India1 J. Sanyala, B. Sarkara, T. K. Banerjeeb, S. C. Mukherjeec, B. C. Rayd, and V. R. Raoa, 2 a Anthropological Survey of India, Kolkata, India Department of Neurology, National Neurosciences Center, Kolkata, India c Department of Neurology, Calcutta Medical College and Hospital, Kolkata, India d Department of Chemistry, Jadavpur University, Kolkata, India e-mail: parkinsons_research@rediffmail.com b Received November 18, 2010 Abstract—Oxidative stress is thought to play a major role in the pathogenesis of Parkinson’s disease (PD). Neurons are highly susceptible to a defective antioxidant scavenging system, thus inducing oxidative changes in human red blood cells (RBCs), in vivo and in vitro. Previous studies on oxidative stress in RBCs in patients with PD have yielded controversial results claiming unaltered activity to reduced activity. We have thus undertaken this study to investigate the possibility of oxidative damage to the RBCs in PD by measuring the cytosolic antioxidant enzymes viz., catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (G-Px). The biochemical parameters were measured in erythrocytes of 80 PD patients and 80 normal agematched healthy controls. The enzymes activities were correlated with age of patients, age of onset of disease, duration of disease, United Parkinson’s Disease Rating Scale (UPDRS) and Hoehn and Yahr stage. Patients with PD had higher red blood corpuscle (RBC) activity of SOD. The CAT, and G-Px activities were significantly lower in patients with PD compared to the controls. Erythrocyte SOD, CAT and G-Px were markedly lower in those PD patients who were suffering for a greater duration of the disease and in advanced cases of PD. A significant (P < 0.05) negative correlation of enzyme activities with disease duration, UPDRS score and Hoehn and Yahr stage of the disease was found. Results of our present study concludes the implication of oxidative stress as one of the risk factors, which can initiate or promote neurodegeneration in PD by playing a role in dopaminergic neuronal loss and was correlated to the severity of the disease. Keywords: Antioxidant enzymes, Catalase, glutathione peroxidase, superoxide dismutase, oxidative stress, Parkinson’s disease DOI: 10.1134/S1819712411020073 21 INTRODUCTION Parkinson’s disease (PD) is the second most common neurodegenerative disorder after Alzheimer’s disease, affecting more than 1% of the population after the age of 60 yr. It is characterized clinically by resting tremor, bradykinesia, rigidity and postural imbalance, and pathologically by the degeneration of dopaminergic neurons in the substantia nigra pars compacta (SN) with Lewy bodies in surviving neurons. The exact cause of nigral neuronal death in Parkinson’s disease is still unknown; however, oxidative stress [1] and mitochondria respiratory failure [2–4] have been implicated as major contributors. Due to increase in free radicals and other reactive oxygen species, which play an important part in neuronal death in major neurodegenerative diseases, oxidative stress is a primary 1 The article is published in the original. 2 Corresponding Author; address: Anthropological Survey of India, 27 Jawaharlal Nehru Road, Kolkata, India; tel.: +91-3322861781; e-mail: parkinsons_research@rediffmail.com. causal event in the etiology of PD. Under normal conditions, the actions of reactive species are opposed by a balanced and coordinated system of antioxidant defenses like Superoxide dismutase (SOD), catalase (CAT) and Glutathione peroxidase (G-Px). The free radical formation is a result of MAO-B in the SN, and of the oxidation of dopamine via 6-hydroxydopamine [5, 6]. Oxidative stress might be a consequence of reduced efficiency of these endogenous antioxidants that render PD patients more vulnerable to oxidative stress. Several previous reports have suggested a decrease in these antioxidant enzymes with PD suggesting the possible contribution of a metabolic failure in antioxidant mechanisms [7–10]. A single report from Eastern India depicting an increase in lipid peroxidation [11] and nitrates [12] in plasma of PD patients further lead us to investigate and ascertain the possibility of oxidative damage influencing the pathogenesis of PD in these patient groups. To the best of our knowledge, this is the first report from Eastern India to determine SOD, CAT and G-Px activity and 146 PERIPHERAL MARKERS FOR OXIDATIVE STRESS their possible correlation with age, age of onset, disease duration and stage of the disease. 147 Table 1. Clinical characteristics of Parkinson disease patients UPDRS scores MATERIALS AND METHODS The base population recruited for this study is idiopathic 80 PD patients (61 males, 19 females), without a family history, visiting the Movement Disorder Clinic of Calcutta Medical College and Hospital and National Neurosciences Centre (NNC), Kolkata, India from August 26th, 2007 to July 31st, 2008. The Ethics Committee of the Institute and collaborating hospitals approved the study protocol. Clinical data and detailed family history of each patient was collected with the help of collaborating clinicians. The Unified Parkinson’s Disease Rating Scale (UPDRS) [13] and Hoehn and Yahr staging (HY) [14] were performed to quantify disease severity. The control group consisted of 80 healthy community-based, age and sex-matched volunteers (68 males, 12 females), residing in the same areas and from the same ethnic background as the PD patients (P > 0.05). None of the controls had any diagnosable neurological disorders. OPERATIONAL DEFINITIONS All PD patients recruited met the following criteria (at the time of diagnosis and within the study period): (1) The presence of at least three of the following signs: resting tremor, cogwheel rigidity, bradykinesia and postural reflex impairment, atleast one of which must be either rest tremor or bradykinesia14; (2) No suggestion of secondary parkinsonism due to drugs, trauma, brain tumor or treatment within the last 12 months with dopamine-blocking or dopamine-depleting agents; and (3) No atypical features such as prominent oculomotor palsy, cerebellar signs, severe orthostatic hypotension, pyramidal signs, amyotrophy or limb apraxia. The exclusion criteria applied were history of repeated strokes and head trauma, encephalitis, oculogyric crises, neuroleptic treatment within one year of onset of symptoms, more than one affected relatives, early severe autonomic disturbance, sustained remission, Balbinski sign, presence of brain tumour and hydrocephalous. COLLECTION OF BLOOD SAMPLES AND PLASMA SEPARATION Approximately 5 ml of peripheral blood was collected after an overnight fast in a Vacutainer containing K2 EDTA (Becton Dickinson Vacutainer system), with written informed consent from all subjects. Blood was centrifuged at 3000 rpm for 8 minutes. Plasma was separated from the buffy coat carefully and stored at 4°C until analysis. RBCs were washed thrice with normal saline to remove white blood cells. All analysis was carried out within 72 hours of blood collection. SOD activity was determined by the method of Marklund NEUROCHEMICAL JOURNAL Vol. 5 No. 2 2011 Total for parts I–III (items 1–31) 31.2 ± 5.2 ADL scale (items 5–17) 13.8 ± 1.8 Motor scale (items 18–31) 14.9 ± 2.1 Hoehn and Yahr stage 2.6 ± 1.1 and Marklund [15]. Catalase was estimated in erythrocytes hemosylate, according to method of Aebi [16]. Flohe and Gunzler’s [17] method was used to measure G-Px. STATISTICAL ANALYSIS The results are expressed as mean ± standard deviation (SD). Statistical analysis included the two-tailed Student’s t-test to compare PD patients with controls, one-way analysis of variance (ANOVA) and Pearson’s correlation coefficient (r), using SPSS v11.5 software. A P value of less than 0.05 was considered statistically significant. RESULTS One hundred and sixty subjects were included in this study comprising of 80 PD patients and 80 healthy controls. Clinical data and parameters of oxidative stress presented as mean values, ranges and SD of PD and control subjects are summarized in Table 1 and Table 2 respectively. The age (P = 0.74) and sex (P = 0.23) distribution of patients (57.2 ± 12.2 years) and controls (57.6 ± 9.1 years) was similar. More than three-fourths of each group comprised of males. The mean age of onset for PD was 55.3 ± 5.2 years with an average of duration of illness to be 3.6 ± 1.6. All patients, except four were receiving levodopa medications either alone or in combination with other drugs. Compared to the control groups, PD patients had a significant higher RBC SOD activity (t = 12.1069, P = 0.0001). The mean RBC activity of CAT (t = 105.4655, P = 0.0001) and G-Px (t = 30.2015, P = 0.0001) were found to be lowered in patients compared to the controls (Table 2). No correlation was observed between the age, age of onset and enzyme values (r = 0.025, and 0.146 respectively). 41 patients were included in the first group with disease duration less than 2 yr, HY stages from I-II, and lower UPDRS scores (<30), another group of patients (n = 39) were in the advanced stage of disease suffering for a longer time (greater than 5 years) with HY stages III-IV, UPDRS scores greater than 30. PD patients of the first group had a significantly higher CAT and G-Px activity than PD patients with HY stages III 148 SANYAL et al. Table 2. Enzyme activities in blood of Parkinson disease patients and control subjects Patients (n = 80) Controls (n = 80) SOD (U/mg Hb) 2.45 ± 0.57 2.02 ± 0.42 SOD (U/mg Hb) Disease duration <2 yrs with HY stages from I–II 2.91 ± 0.41 SOD (U/mg Hb) Disease duration >5 yrs with HY stages from III–V 1.97 ± 0.21 154.83 ± 6.24 CAT (U/g Hb) 160.03 ± 2.29 Disease duration >5 yrs with HY stages from III–V 149.88 ± 4.52 36.64 ± 5.06 G-Px (U/g Hb) 40.90 ± 2.19 G-Px (U/g Hb) Disease duration >5 yrs with HY stages from III–V 32.17 ± 2.85 and IV (160.03 ± 2.29 vs. 149.88 ± 4.52 U/g Hb; 40.90 ± 2.19 vs. 32.17 ± 2.85 U/g Hb). An ANOVA showed that SOD, CAT and G-Px levels differed significantly with the increase in disease duration and clinically estimated stages of disease progression (F = 159.06, P < 0.0001; F = 158, P < 0.0001; F = 166.859, P < 0.0001 respectively). A negative correlation was observed between HY stages, UPDRS and enzyme activities (Table 2). DISCUSSION Findings of our previous study [11, 12] inspired us to aim whether there is any difference in antioxidant activity between PD patients at the early stage of their disease (duration of illness less than 2 yr) than those suffering for a longer time from advanced PD. Parkinsonian patients in the late phase of the disease have a lower antioxidant activity as expressed by a significantly decreased SOD, CAT and G-Px activity. These enzymes do not decrease with age or age of onset, but the correlation with duration of disease, UPDRS and HY stages was significant for those patients suffering for a greater duration with later stages of PD progression; thus the lowering of activity of these biochemical parameters. In the initial stages of the disease, increased SOD, CAT, G-Px activity in the nervous system of PD patients may be a protective response to the increased production of the anions. Mechanisms underlying neuronal death in PD are poorly understood, although several in vitro studies have suggested P = 0.0001 F = 158, P < 0.0001 47.78 ± 4.69 G-Px (U/g Hb) Disease duration <2 yrs with HY stages from I–II P = 0.0001 F = 159.06, P < 0.0001 216.43 ± 7.35 CAT (U/g Hb) Disease duration <2 yrs with HY stages from I–II P value P = 0.0001 F = 166.859, P < 0.0001 the involvement of oxidative stress [18]. SOD is the first enzymatic line of defense against superoxide anion. Our study pointed its activity to be increased with the onset of disease in patients than controls, which gradually decrease as the disease progress over the years. Studies indicated a lowering of SOD activity in blood of PD patients [8, 9, 19–21] has been supportive of our results. Although few previous studies indicated no significant change in erythrocyte CAT activity of PD patients [22, 23] or unaltered CAT levels in post mortem parkinsonian brains [24], our results depicted a deficit of CAT when compared to controls; which is at par with the findings of Torre et al. [7], Nikam et al. [8] and Abraham et al. [9]. However, this study points to a significantly lower RBC CAT activity in severe PD (HY stage III/IV) with greater duration than early PD with lesser duration of illness (HY stages I/II). The present study depicts a significantly lowering of G-Px activity in PD patients and a negative correlation of G-Px with duration of illness and clinical stages. Nikam et al. [8], Johannsen et al. [10], Abraham et al. [9], Kilinc et al. [22], previously reported such reduced activity of GPx in PD patients. Johansen et al. [10] showed that PD patients in the late phase of disease had a lowered antioxidant activity that confirms our findings; although this decrease could not be explained by differences in dietary habits, energy intake, and lack of trace element supplementation. With respect to the Indian scenario, three studies have shown contrasting results; Sudha et al. [23] conNEUROCHEMICAL JOURNAL Vol. 5 No. 2 2011 PERIPHERAL MARKERS FOR OXIDATIVE STRESS cluded no significance of erythrocyte antioxidants in PD patients whereas Nikam et al. [8] and Abraham et al. [9] clearly demonstrated that these enzymes decreased in PD patients when compared to controls. With such contrasting results, we thus initiated this study in Eastern Indian PD patients and our analysis concluded that our patient cohort is subjected to oxidative stress. CONCLUSIONS High MDA, nitrate levels, excessive SOD activity in patients with less duration, early stages, decreased CAT, G-Px may indicate a systematic reaction related to chronic oxidative stress in brain. These can be viewed as peripheral markers for PD although such markers might not be of any diagnostic value. We find the difference between early and late PD patients convincing. We therefore, conclude that some defect in the free radical protecting enzymes does not develop over the years of Parkinson’s disease. This is supported by the observation that early patients, even those that are elderly, have high levels of these enzymes, while late patients, even young ones, seem to have lost the ability to increase their antioxidant levels and thus have less free radical protecting capacity. It is possible that this loss is due to increased oxygen stress induced by the levadopa therapy. On the whole, it can be concluded that erythrocytes of Eastern Indian PD patients are under oxidative stress as is evidenced by reduced SOD, CAT, G-Px with greater duration and later phase of PD. ACKNOWLEDGMENTS This research work was supported by a grant from the Anthropological Survey of India, Ministry of Culture, Government of India (to J. Sanyal). We are thankful to the PD patients and control subjects for voluntarily taking part in this research work and donating their blood samples. Mitali Maity (nurse in NNC) helped in the collection of blood samples from the outpatient Department of NNC. REFERENCES 1. Jenner, P. and Olanow, C.W., Neurol., 1996, vol. 47, pp. 161–170. 1 2. Mizuno, Y., Ohta, S., Tanaka, M., Takamiya, S., Suzuki, K., Sato, T., Oya, H., Ozawa, T., and Kagawa, Y., Biochem. Biophys. Res. Commun., 1989, vol. 163, pp. 1450–1455. 3. Schapira, A.H., Cooper, J.M., Dexter, D., Dexter, D., Clark, J.B., Jenner P., and Marsden, C.D., Lancet, 1989, vol. 1, pp. 1269–1269. NEUROCHEMICAL JOURNAL SPELL: 1. ok Vol. 5 No. 2 2011 149 4. Hattori, N., Tanaka, M., Ozawa, T., and Mizuno, Y., 1 Ann. Neurol., 1991, vol. 30, pp. 563–571. 5. Cohen, G. and Heikkila, R., J. Biol. Chem., 1974, vol. 249, pp. 2447–2452. 6. Graham, D.G., Tiffany, S.M., Bell, W.R., and Gutknecht, W.F., Mol. Pharmacol., 1978, vol. 14, pp. 644– 653. 7. De la Torre, R., Casado, A., Lopez-Fernandez, E., Carrascosa, D., Casado, C, Venarucci, D., and Venaruccil, V., Neurochem. Res., 1996, vol. 21, pp. 885–888. 8. Abraham, S., Soundararajan, C.C., Vivekanandhan, S., and Behari, M., Indian J. Med. Res., 2005, vol. 121, pp. 111–115. 9. Shashikant, N., Padmaja, N., Ahaley, S.K., and Sontakke, A.V., Indian J. Clin. Biochem., 2009, vol. 24, pp. 98–101. 10. Johannsen, P., Velander, G., Mai, J., Thorling, E.B., and Dupont, E., J. Neurol. Neurosurg. Psychiatry, 1991, vol. 54, pp. 679–682. 11. Sanyal, J., Bandyopadhyay, S.K., Banerjee, T.K., Mukherjee, S.C., Chakraborty, D.P., Ray, B.C., and Rao, V.R., Eur. Rev. Med. Pharmacol. Sci., 2009, vol. 13, pp. 129–132. 12. Sanyal, J., Sarkar, B.N., Banerjee, T.K., Ojha, S., Ray, B.C., and Rao, V.R., Neurol. Asia, 2010, vol. 15, pp. 55–59. 13. Fahn, S., Elton, R.L., Recent Developments in Parkinson’s Disease, Vol. II, Fahn S., Marsden, C.D., Goldstein, M., and Calne, D.B., Eds., UPDRS Development Committee, Unified Parkinson’s Disease Rating Scale, New Jersey, Florham Park, 1987, pp. 153–163. 14. Hoehn, M.M. and Yahr, M.D., Neurol., 1967, vol. 17, pp. 427–442. 15. Marklund, S. and Marklund, G., Eur J. Biochem., 1974, vol. 47, pp. 469–474. 16. Aebi and Catalase, H., In Methods, in Enzymatic Analysis, Bergmeryer, H.U., Ed., N.Y.: Academic Press, 1983, vol. 3, no. 276–286. 17. Flohe, L. and Gunzler, W.A., Methods Enzymol., 1984, vol. 105, pp. 114–121. 18. Gotz, M.R.E., Freyberger, A., and Riederer, P., J. Neural. Transm., 1990, vol. 29, pp. 241–249. 19. Bostantjopoulou, S., Kyriaz, G., Katasarou, Z., Kiosseoglou, G., Kazis, A., and Mentenopoulos, G., Funct. Neurol., 1997, vol. 12, pp. 63–68. 20. Ihara, Y., Chuda, M., Kuroda, S., and Hayabara, T., J. Neurol. Sci., 1999, vol. 170, pp. 90–95. 21. Torsdottir, G., Kristinsson, J., Sveinbjornsdottir, S., Snaedal, J., and Johanhesson, T., Pharmacol. Toxicol., 1999, vol. 85, pp. 239–243. 22. Kilinc, A., Yalcin, A.S., Yalcin, D., Taga, Y., and Emerk, K., Neurosci. Lett., 1988, vol. 87, pp. 307–310. 23. Sudha, K., Rao, A.V., Rao, S., and Rao, A., Neurol. India, 2003, vol. 51, pp. 60–62. 24. Marttila, R.J., Lorentz, H., and Rinne, U.K., J. Neurol. Sci., 1988, vol. 86, pp. 321–331.