Neurol India Home 

Year : 2003  |  Volume : 51  |  Issue : 1  |  Page : 60--62

Free radical toxicity and antioxidants in Parkinsonís disease

K Sudha1, A Rao2, S Rao3, A Rao4,  
1 Departments of Biochemistry, Kasturba Medical College, Mangalore, India
2 Department of Biochemistry, Father Mullers Medical College, Mangalore, India
3 Departments of Neurology, Kasturba Medical College, Mangalore, India
4 Department of Biochemistry, Kasturba Medical College, Manipal-576119, India

Correspondence Address:
A Rao
Kasturba Medical College, Manipal-576119


Erythrocyte lipid peroxidation, oxidative hemolysis, erythrocyte antioxidant enzymes, viz. superoxide dismutase, glutathione reductase, glutathione peroxidase, catalase and plasma antioxidants, viz. vitamin A, vitamin E, vitamin C and ceruloplasmin have been determined by spectrophotometric methods in 15 patients with Parkinsonís disease (PD) and in 50 controls. Lipid peroxidation, oxidative hemolysis and plasma ceruloplasmin were significantly higher in PD patients as compared to normals. Erythrocyte antioxidants in PD patients were not significantly different from the controls. However, plasma vitamin C in PD patients was significantly lower than the controls. It is concluded that these patients are under oxidative stress which points to a possible involvement of free radicals in PD.

How to cite this article:
Sudha K, Rao A, Rao S, Rao A. Free radical toxicity and antioxidants in Parkinsonís disease .Neurol India 2003;51:60-62

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Sudha K, Rao A, Rao S, Rao A. Free radical toxicity and antioxidants in Parkinsonís disease . Neurol India [serial online] 2003 [cited 2023 Mar 29 ];51:60-62
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Oxygen-derived free radicals have recently been implicated in pathogenesis of various diseases including atherosclerosis, diabetes mellitus, epilepsy, inflammatory diseases and cancer.[1],[2],[3],[4],[5] Lipid peroxidation induced by free radicals is believed to be one of the major causes of cell membrane damage leading to lysis of cell.[6]

The body possesses a complex protective antioxidant system against these potentially toxic products such as vitamin E, vitamin C, vitamin A, glutathione and antioxidant enzymes. These enzymes include glutathione reductase (GR), glutathione peroxidase (GP), superoxide dismutase (SOD) and catalase (CT).[7]

In Parkinson's disease (PD), there is progressive death of substantia nigral cells leading to less availability of dopamine to the striatum which controls movement. Neurons of substantia nigra (SN) may be particularly vulnerable to oxidant stress, because the oxidative metabolism of dopamine has the potential to generate cytotoxic free radicals. Dopamine can be oxidized by either monoamine oxidase or undergo autooxidation to generate hydrogen peroxide (H2O2). H2O2 can damage the neuron directly or indirectly through the formation of hydroxyl radicals in presence of ferrous ions.[8] Neuromelanin present within the SN neurons has the potential to promote site-specific accumulation and reduction of iron thereby potentiating iron-induced lipid peroxidation and consequent cell death.[9],[10] H2O2 is normally detoxified by reduced glutathione (GSH) in the reaction catalyzed by GP, thus an increased rate of dopamine turnover or a deficiency of GSH could lead to oxidative stress. Thus, it appears that free radicals may be one of the important agents responsible for destruction of SN neurons, thereby leading to PD.

However, at present, very few reports[11],[12],[13],[14],[15] are available on antioxidants of blood in patients with PD. Hence, the aim of the study is to evaluate various blood antioxidants and establish the possibility of oxidant damage to the RBC in PD.


   Materials and Methods

The study population consisted of 15 patients who had PD and 50 age and sex matched healthy controls. Diagnosis of PD was mainly by taking a detailed history and studying the symptoms of the disease. The diagnostic criteria for PD were based on those given by Hughes et al.[16] Most of the patients showed bradykinesia and resting tremor. One patient suffered from mild hypertension and another from early dementia. The patients were in the age group of 40-60 years and in the initial stage of the disease (1-2 years) without any drug therapy.

Random blood samples were collected in EDTA bottles from normal subjects and patients. Blood was centrifuged at 3000 g for 10 minutes. Plasma was separated, buffy coat was carefully removed and separated erythrocytes washed thrice with 0.01M saline phosphate buffer pH 7.4 (containing 0.15 M NaCl), then diluted 1:2 with the same buffer and stored at 4-50C. The hemoglobin content of the erythrocytes was determined by cyanmethemoglobin method. Erythrocyte enzymes were estimated in appropriately diluted hemolysates prepared by the addition of distilled water. Erythrocyte GR and GP activity was determined by recording the decrease in absorbance due to depletion of NADPH at 340 nm for a period of 5 minutes.[17],[18] SOD was determined according to the method of Beauchamp and Fridovich[19] based on inhibition of nitroblue tetrazolium reduction. CT activity in the hemolysate was determined by the method of Brannan et al.[20] The assay is based on the disappearance of H2O2 in the presence of the enzyme source at 260C. The lipid peroxidation and oxidative hemolysis of RBC were determined by incubating RBC suspension in saline phosphate buffer containing 0.44M H2O2 at 370C for a period of 2 hours.[21],[22] Aliquots were withdrawn from the above mixture at 0 hour and 2 hours. Lipid peroxidation in RBC was determined by estimating malondialdehyde (MDA) produced using thiobarbituric acid (TBA).

Plasma ceruloplasmin was determined by its p-phenylene diamine oxidase activity.[23] Plasma alpha tocopherol was measured by the Emmorie Engel reaction given by Bieri et al.[24] Vitamin A was determined by reading the extinction at 327 nm before and after exposure to UV light.[25] Plasma vitamin C was determined chemically using dintrophenyl hydrazine as a color compound.[26]

Data was analyzed statistically by Mann Whitney U test. The difference of P   


The erythrocyte lipid peroxidation in PD patients was significantly high at 0 hour compared to the healthy controls. However after 2 hours of incubation of RBC with H2O2 the increase in lipid peroxide levels of PD patients was not statistically significant. Oxidative hemolysis was significantly high both at 0 hour and 2 hours in PD patients as compared to controls [Table:1].

A comparison of erythrocyte GR, GP, SOD and CT activities in PD patients to that of controls showed no significant change [Table:2]. However plasma ceruloplasmin concentration was significantly higher in PD patients than in normal subjects. The mean plasma vitamin C in PD patients was significantly lower compared to that of controls but vitamin A and E levels were within normal range [Table:3].



In PD, the environment within SN is conducive to the formation of cytotoxic free radicals. These free radicals react instantaneously with membrane lipids and cause lipid peroxidation and cell death.[27] In the present study, there is significantly high erythrocyte lipid peroxidation at 0 hour which supports the notion of increased lipid peroxidation in the PD brain as well.[28] Lipid peroxidation of RBC membrane causes them to lose their ability to change shape and squeeze through the smallest capillaries, thus eventually leading to hemolysis. Jenner et al[28] have shown an enhanced basal lipid peroxidation in SN of post mortem brain in PD. In MPTP (a neurotoxin producing PD) treated rats the basal lipid peroxide concentration is significantly high in SN.[29] Brain tissue extracts of PD patients showed a tenfold increase in lipid hydroperoxides in SN compared to control subjects.[30] This evidence indicates that increased lipid peroxidation could possibly play a role in neurodegeneration leading to PD. The present study also shows similar changes in blood where increased lipid peroxidation has lead to lysis of erythrocytes. This finding is also supported by the work on lipid peroxides in RBC elsewhere.[15]

Moreover, in this study, there has been no change observed in the antioxidant enzyme levels of RBC in PD. Though the mean values of these enzymes were lower in PD patients compared to normal, none of them were statistically significant. This observation is supported by the reports made on post mortem brains and in MPTP treated animals, where activities of CT, GP and GSH concentrations in SN remained unchanged compared to controls.[28],[29] Further, several studies indicated a decreased SOD activity in blood of PD patients.[12],[13],[14] In contrast, increase in erythrocyte SOD level in PD has also been reported.[15]

Significantly high plasma ceruloplasmin level in PD patients observed in the present study may be attributed to its ferroxidase activity. The ferroxidase activity inhibits iron dependent lipid peroxidation[31] and also formation of hydroxyl radicals. Good et al[32] have demonstrated that iron-induced oxidant stress contributes to cell death in PD. However, significantly decreased serum ceruloplasmin levels have also been observed in PD.[14] A significant decrease in plasma vitamin C is seen in PD patients compared to controls in this study. A similar observation is made by Youdim et al[10] who have shown low vitamin C levels in SN of PD patients. Addition of vitamin C to an incubation mixture containing tissue from different brain areas and ferrous sulfate, decreased lipid peroxidation in SN of MPTP treated monkeys.[29] Low plasma vitamin C level in PD patients as observed here is probably due to increased utilization of the vitamin to mitigate the toxicity of free radicals.

On the whole, it can be concluded that erythrocytes of PD patients are under oxidative stress as evidenced by increased lipid peroxidation and oxidative hemolysis. These findings are in keeping with the possible role of free radical damage in PD.


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