НЕИРОХИМИЯ, 2011, том 28, № 2, с. 164-168
КЛИНИЧЕСКАЯ ^^^^^^^^^^^^^^ НЕЙРОХИМИЯ
УДК 577.1
PERIPHERAL MARKERS FOR OXIDATIVE STRESS IN PARKINSON'S DISEASE PATIENTS OF EASTERN INDIA
© 2011 J. Sanyal", B. Sarkar", T. K. Banerjee4, S. C. Mukherjeec, B. C. Rayd, V. R. Rao", *
aAnthropological Survey of India, Kolkata, India bDepartment of Neurology, NationalNeurosciences Center, Kolkata, India cDepartment of Neurology, Calcutta Medical College and Hospital, Kolkata, India dDepartment of Chemistry, Jadavpur University, Kolkata, India
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 cy-tosolic 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 age-matched 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.
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 60yrs. It is characterized clinically by resting tremor, bradykinesia, rigidity and postural imbalance, and pathologically by the degeneration of dopaminer-gic 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 neurode-generative diseases, oxidative stress is a primary 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 for-
* Corresponding Author; address: Anthropological Survey of India, 27 Jawaharlal Nehru Road, Kolkata, India; tel.: +91-3322861781; e-mail: parkinsons_research@rediffmail.com.
mation 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 their possible correlation with age, age of onset, disease duration and stage of the disease.
MATERIALS AND METHODS
The base population recruited for this study is idio-pathic 80 PD patients (61 males, 19 females), without a family history, visiting the Movement Disorder Clinic of Calcutta Medical College and Hospital and Na-
tional 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 diag-nosable 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, ocu-logyric 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 hy-drocephalous.
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 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
Table 1. Clinical characteristics of Parkinson disease patients
UPDRS scores
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
Student's t-test to compare PD patients with controls, one-way analysis of variance (ANOVA) and Pearson's correlation coefficient (r), using SPSS v 11.5 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 yrs, 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 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 cor-
Table 2. Enzyme activities in blood of Parkinson disease patients and control subjects
Patients (n = 80) Controls (n = 80) P value
SOD (U/mg Hb) 2.45 ± 0.57 2.02 ± 0.42 P = 0.0001
SOD (U/mg Hb) Disease duration <2 yrs with HY stages from I—II 2.91 ± 0.41 F = 159.06, P < 0.0001
SOD (U/mg Hb) Disease duratio
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