Background & objectives: Oxidative stress is incriminated to play a central role in the pathogenesis of Parkinson's disease (PD). Oxidative stress, to which neurons are highly susceptible, is also known to induce oxidative changes in human red blood cells (RBCs), in vivo and in vitro. Earlier studies on oxidative stress in RBCs in patients with PD have yielded controversial results claiming unaltered activity to reduced activity. Using RBC as a model, we have undertaken this study to ascertain the possibility of oxidative damage to the RBCs in PD by measuring the cytosolic antioxidant enzymes viz., superoxide dismutase (SOD), catalase (CAT) glutathione peroxidase (G-Px) and glucose-6-phosphate dehydrogenase (G6PD).
Methods: Activities of antioxidant enzymes were measured in erythrocytes of 115 PD patients and 37 normal age-matched healthy persons as controls. 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.
Results: The SOD, CAT, G-Px and G6PD activities were significantly lower in patients with PD compared to the control. A significant (P
Interpretation & conclusion: Results of the present study showed involvement of oxidative stress as one of the risk factors, which can initiate and/or promote neurodegeneration in PD and was correlated to the severity of the disease.
Key words Antioxidant enzymes - glucose 6-phosphate dehydrogenase - oxidative stress - Parkinson's disease superoxide dismutase
Parkinsorf s disease (PD) is the second most common neurodegenerative disorder after Alzheimer's disease, affecting approximately 1 per cent of the population older than 50 yr1. It is characterized clinically by resting tremor, bradykinesia, rigidity and postural imbalance, and pathologically by the death of dopaminergic neurons in the substantia nigra (SN) with Lewy bodies in surviving neurons. Free radical theory was thought to be as one of the mechanisms involved in the pathogenesis in Parkinsoirs disease2. Oxidative stress, produced when there is an increased formation or defective inactivation of cytotoxic reactive oxygen species (ROS) can initiate and/or promote degeneration of dopaminergic (DA) neurons in PD. Cytotoxicity of ROS is related to the ability of these molecules to oxidize cell constituents, particularly lipids and nucleic acids which leads to deterioration of cellular structural architecture and signaling and ultimately death. ROS is also able to trigger both necrotic and apoptotic cell death1.
Under normal conditions, the continuous production of free radicals is compensated by the powerful action of protective enzymes. Superoxide dismutase (SOD), catalase (CAT) and gluthathione peroxidase (G-Px) are the major antioxidant enzymes present in the human body that protect against the oxygen toxicity3,4. Oxidative stress may be a consequence of reduced efficiency of these endogenous antioxidants that may render PD patients more vulnerable to oxidative stress. Glucose 6phosphate dehydrogenase (G6PD) is essential for keeping glutathione in the reduced state5.
There are reports suggesting a decrease in SOD and other antioxidant enzyme activities and increase in various markers of lipid peroxidation in SN of PD patients6,7. Based on the hypothesis that these deficits may not be organ specific, various groups have investigated oxidative stress in red blood cells (RBCs) and yielded controversial results claiming unaltered to reduced activities8-11. The studies by Urakami et al12 and Gatto et al13 showed that the erythrocyte SOD values were decreased only in treated patients and not in untreated PD group. To ascertain the possibility of oxidative damage to the RBCs in PD, the present study was undertaken in treated PD patients by evaluating the changes in erythrocyte activities of SOD, CAT, G-Px and G6PD and their possible correlation with age. age of onset, duration and stage of the disease was also investigated.
Material & Methods
Samples: Patients with PD (n=115, 73 males and 42 females) attending movement disorder clinic of Department of Neurology, All India Institute of Medical Sciences. New Delhi between 1999 to 2002 were included in the study. The patients fulfilling our inclusion and exclusion criteria were selected consecutively during the study period. The diagnosis of idiopathic PD was based on the presence of bradykinesia, 4-6Hz resting tremor, rigidity and postural instability not caused by cerebellar or proprioceptive dysfunction. The exclusion criteria for the PD 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, cerebellar signs, early severe autonomie disturbance, sustained remission, Balbinski sign, presence of brain tumour and hydrocephalous.
The age (58.2 ± 10.66 yr), disease onset age (54.85 ± 1 1.55 yr), duration of the disease (4.5 ± 5.19), United Parkinson's Disease Rating Scale (UPDRS) score14 (36.46 ± 18.6), modified Hoehn and Yahr staging15 (1.99 ± 0.89) and England and Schwab activities of daily living (ADL) (80.79 ± 15.53) were collected. All the patients were receiving treatment, spanning from 6 months to 12 yr. Most of the (90%) patients were receiving levodopa treatment either alone or in combination with other drugs. None of the patients were on vitamin E supplement and DA agonist monotherapy. Only two patients were receiving selegeline monotherapy since most of the patients coming here were not in the early stages of the disease. Untreated PD patients could not be included since most of the patients coming to our movement disorder clinic were referred cases. For comparison 37 age-matched normal healthy persons (24 males, 13 females), with mean age 57.1 7 ± 11.21 yr, were also included. The exclusion criteria were smoking, consumption of alcohol and any medications and vitamins in the previous six months. Since PD is an age-associated disease, we included persons above 45 yr of age and this combined with the exclusion criteria resulted in less number of controls. Blood was collected between 1000 to 1200 in EDTA vacutainers and plasma was removed after spinning the sample at 1200 g. RBCs were washed with normal saline thrice to remove white blood cells and kept at -20°C till analysis. All analysis were carried out within 2 days of blood collection.
The study protocol was approved by the ethics committee of the institute, and written consent was obtained from all patients and controls.
Assays of antioxidant enzymes: SOD was extracted by the method of Winterbourn et al16 from RBCs. The enzyme present in the clear top layer was assayed by the method of Nishikimi et al17 modified by Kakkar et al18. CAT was measured by the method of Aebi19. G-Px by the method of Flohe and Gunzler20, and G6PD by a commercially available kit (Sentinel, Italy).
Statistical analysis: The difference between PD and control groups was analyzed using unpaired Student t-test with log transformation whenever necessary. For PD patients, correlation coefficients were determined for relationship between values of different enzymes with age, age of onset, duration of the disease, UPDRS score and Hoehn & Yahr stage.
Results & Discussion
The SOD, CAT, G-Px and G6PD activities were found to be significantly lower (P
PD may serve as an excellent example to discuss the significance of oxidative processes as a central but not an initiating event for the development of clinical disease21. The concept that oxidative stress occurs in PD derives primarily from the realization that the metabolism of dopamine, by chemical or enzymatic means, can generate free radicals and other reactive oxygen species via autoxidation and dopamine oxidation by monoamine oxidase B22. The loss of dopaminergic neuron in PD results in enhanced metabolism of dopamine, augmenting the formation of H^sub 2^O^sub 2^ thus leading to the generation of highly neurotoxic hydroxyl radicals23. Low activity of mitochondrial complex 1 in PD also results in generation of oxygen species24. Further, any reduction in antioxidant enzymes may result in ineffective removal of ROS.
Our study showed a significant decrease in the activities of SOD, CAT, G-Px and G6PD in erythrocytes of PD patients as reported earlier by various groups9,10,12,13. The increase in the oxidative stress due to the low activity of antioxidant enzymes might pave way for many secondary complications and may contribute to the neurodegeneration in PD.
SOD showed a significant reduction in activity in patients leading to an increase of superoxide radical. Zhang et al25 found that some of the deleterious effects of N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) on striatal dopaminergic nerve terminals are mediated by both superoxide and hydroperoxides and they occur prior to dopaminergic neurodegeneration in the SN. Superoxide radicals can also react with NO to generate peroxynitrite (ONOO^sup -^), a putative neurotoxin26. A significant reduction in both CAT and G-Px activities in effect can increase the production of highly deleterious H^sub 2^O^sub 2^. G6PD is a key enzyme involved in the synthesis of NADPH, which is essential for keeping the normal level of reduced glutathione5. G6PD deficient erythrocytes are particularly sensitive to oxidative stress27 and reduction in G6PD activity may result in reduction of GSH level. The reduction in GSH level has already been reported in PD patients23. Sian et al28 measured the reduced and oxidised glutathione levels in various brain areas in PD and in few related disorders and found that GSH level in SN was significantly reduced only in PD.
Our results showed no correlation between enzyme activities and age of patients or age of onset of PD. In some studies, a negative correlation was reported between the SOD activity and the duration of illness12,29. We could not establish this in our study. We have found a significant negative correlation between the Hoehn and Yahr stage and enzyme activity. UPDRS score was also found to be negatively correlated to the activity of SOD. Bostantjopoulou el al29 reported a significant decrease of SOD activity in whole blood and in RBCs in stage III and IV PD patients while there was no relationship between L-Dopa treatment and SOD activity. Sudha et al11 reported that erythrocyte antioxidants in initial stage PD patients without any drug therapy were not significantly different from the controls.
The findings of our study showed the involvement of oxidative stress in PD. The neuronal degeneration may result from an increased exposure to free radicals coupled with a deficit of antioxidant mechanisms. The antioxidant enzyme levels are negatively correlated to the severity of the disease but independent of age and age of onset.
Authors acknowledge the Indian Council of Medieal Research and Institute Research Grant of All India Institute of Medical Sciences (AIIMS). New Delhi for financial support, and thank Dr R.M. Pandey. Department of Biostatistics, AIIMS, for the statistical analysis and interpretations.
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S. Abraham, C.C. Soundararajan, S. Vivekanandhan & M. Behari
Department of Neurology, All India Institute of Medical Sciences, New Delhi, India
Received September 16, 2003
Reprint requests: Dr S. Vivekanandhan. Associate Professor. Department of Neurobiochemistry, Neuroscience Centre All India Institute of Medical Sciences. Ansari Nagar. New Delhi 110029. India e-mail: email@example.com
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