Comp Clin Pathol DOI 10.

1007/s00580-012-1407-8

REVIEW

Analysis of the parameters of oxidative stress in patients with Parkinsons disease

Galina Nikolova & Velina Mancheva

Received: 19 November 2011 / Accepted: 4 January 2012 # Springer-Verlag London Limited 2012

Abstract The increasing data provides enough evidences confirming the involvement of free radicals and other reactive oxygen species (ROS) superoxide radical (.O2), nitric oxide (NO.), hydrogen peroxide (H2O2) and hydroxyl radicals (.OH) in a number of physiological and pathological processes. Imbalance between levels of ROS resulting in the body and the capacity of antioxidant defense mechanisms occur oxidative stress (OS). OS is related to a number of structural and functional damages to cells and is involved in the pathogenesis of many diseases, including neurodegenerative diseases such as Alzheimers disease, Parkinsons disease (PD), amyotrophic lateral sclerosis, and Huntington disease. Defects in oxidative phosphorylation and oxidative damage play an important role in neurodegenerative diseases. The aim of this study was to investigate some biomarkers of OS such as the level of lipid peroxidation measured as malondialdehyde (MDA) reactive products and activity of antioxidant enzymes superoxide dismutase (SOD) and catalase (CAT) in the blood of PD patients compared with control group of healthy volunteers. By the present research we report higher levels of MDA products and an imbalance in SOD and CAT enzyme activities in PD patients compared to the control group.

Keywords Neurodegenerative diseases . SOD . CAT . MDA . Oxidative stress

Introduction Neurodegenerative disorders are diseases of the nervous systembrain, spinal cord and peripheral nerves, many of them resulting from infection, and some are hereditary or age-related disorders (Halliwell 1991). These neurodegenerative disorders are characterized by the deposition of abnormal forms of specific proteins in the brain (Ebadi et al. 1996). The etiology of neuronal death in the neurodegenerative diseases exemplified by Alzheimers disease (AD), Lewy body diseases such as Parkinsons disease (PD) and diffuses Lewy body disease, and amyotrophic lateral sclerosis (ALS), remains elusive (Sayre et al. 2001). A common feature of these diseases is the extensive evidence of oxidative stress, which might be responsible for the dysfunction or death of neuronal cells that contributes to disease pathogenesis (Barnham et al. 2004). Overproduction of free radicals can cause oxidative damage to biomolecules (lipids, proteins, DNA), eventually leading to many chronic diseases such as atherosclerosis, cancer, diabetics, rheumatoid arthritis, neurodegenerative disease (Barnham et al. 2004; Abraham et al. 2005). PD is a degenerative disease of the central nervous system characterized by paucity and slowing of normal human movements. It falls in the category of hypokinetic movement disorders, a group of diseases also known as Parkinsonism. The primary clinical manifestations of Parkinsonism are bradykinesia and akinesia. It is also known that injury to neurons in the basal ganglia and its connections from causes that include, but are not limited to, ischemic, toxic/metabolic, traumatic,

G. Nikolova (*) Department of Chemistry and Biochemistry, Medical Faculty, Trakia University, Armejska street 11, 6000 Stara Zagora, Bulgaria e-mail: gallina_nikollova@abv.bg V. Mancheva Department of Neurology and Psychiatry, Medical Faculty, Trakia University Hospital, Armeiska Street, 11, 6000 Stara Zagora, Bulgaria

Comp Clin Pathol

and infectious/inflammatory processes, can also manifest as Parkinsonism. Similarly, other neurodegenerative diseases of the brain can present with signs and symptoms of Parkinsonism. These are considered Parkinson plus syndromes and include multisystem atrophy, corticobasal ganglionic degeneration, and progressive supranuclear palsy. PD is associated with degeneration of dopaminergic neurons in the substantia nigra parts compacta. One of the pathological hallmarks of PD is the presence of intracellular inclusions called Lewy bodies that consist of aggregates of the presynaptic soluble called -syneclein (Gasser 2001; Beal 1995). It is clear that the underlying factor in the neurological disorders is increased oxidative stress substantiated by the findings that the protein side chains are modified either directly by reactive oxygen species (ROS) or indirectly by the products of lipid peroxidation (Halliwell 1994). In cases where the balance between ROS generation and antioxidant activity is disturbed, oxidative stress occurs and complicates the underlying disease. The main target substrates for free oxygen radical activity are polyunsaturated fatty acids in membrane phospholipids, the modification of which result in disorganization of cell framework and function. The end product of these reactions is malondialdehyde (MDA). It is excreted in urine, blood, and other fluids and therefore serves as a marker of lipid peroxidation and the presence of oxidative stress, respectively. Deactivation and removal of ROS depends on the activity of antioxidant defensive systems including the following enzymes: superoxide dismutase (SOD), catalase (CAT). CAT is a member of the peroxidases that contains heme at its active site. It is found in peroxisomes in most tissues and can cross membranes easily (Halliwell 2001; Gutteridge and Halliwell 2000). CAT reduced hydrogen peroxide (H2O2 H2O + O2), which is directly produced by some enzymes (e.g., monoamine, xanthine oxidase), to water and oxygen (Schulz et al. 2000). Under normal conditions, the continuous production of free radicals is compensated by the powerful action of protective enzymes. SOD, CAT and glutathione peroxidase (G-Px) are the major antioxidant enzymes presented in the human body that protect against the oxygen toxicity (Halliwell 1991; Yu 1994). Oxidative stress may be a consequence of reduced efficiency of these endogenous antioxidants that may render PD patients more vulnerable to oxidative stress (Abraham et al. 2005). Many authors supported the theory that ROS is a major determinant of life span and that it can be counteracted by pharmacological intervention (Gilgun-Sherki et al. 2001). The aim of the present study was to investigate some biomarkers of oxidative stress such as the concentration of lipid peroxidation measured as MDA reactive products and activity of antioxidant enzymes SOD and CAT in blood of

patients with PD compared with control group of healthy volunteers.

Materials and methods Patients and biological material Blood samples obtained from ten patients with PD (seven males and three females) all of them are patients of the Neurological Clinic of University Hospital, Stara Zagora, Bulgaria, and ten healthy volunteers (five males and five females) who had no family history of PD, were used as controls. To eliminate the factors that might affect parameters of oxidative stress, we excluded from PD patients and healthy controls all smoking and alcohol-drinking subjects, as well as individuals suffering from acute or chronic diseases. Informed consent was obtained from all participants in the study according to the ethical guidelines of the Helsinki Declaration. Laboratory analysis Fasting samples of venous blood were collected in the morning between 0800 and 1000 hours. Blood for determining the parameters of oxidative stress was collected in tubes containing 10%EDTA ethylenediamine tetraacetic acid, centrifuged at 3,000 rpm for 15 min, and plasma was carefully separated. The erythrocyte pellets were washed three times with saline, and 0.5 ml of the cell suspension was diluted with 2 ml cold water to lyse the erythrocytes. To precipitate hemoglobin, 0.1 ml lysate, 0.9 ml water and 0.5 ethanol/chloroform (5:3, v/v) were then added. The tubes were shaken on Vortex and centrifuged at 14,000 rpm for 5 min. The supernatant was used for determination of enzyme activities. Determination of the lipid peroxidation products The method of thiobarbituric acid (TBA), which measures MDA-reactive products, was used (Plaser et al. 1966). In brief, 1 ml plasma, 1 ml physiological solution, and 1 ml 25% trichloroacetic acid were mixed and centrifuged at 7,000 rpm for 20 min. Two milliliters protein-free supernatant was mixed with 0.5 ml 1% TBA and heated at 95C for 1 h. After cooling, the intensity of pink colour of the end fraction product was determined at 532 nm. The MDA concentration was calculated according to the following formula: 1 mol=l MDA OD532 1:75 0:156

where OD532 is the optic density in 0532 nm and extinction0 1.56105 M1 cm1

Comp Clin Pathol

Determination of SOD activity in erythrocytes The SOD activity was determined as described by Sun et al. (1988), with some modifications. The hypoxanthine/xanthinoxidase system was used for superoxide anion production. This anion reduces nitro blue tetrasole (NBT) to Formosan, which was monitored at 560 nm. SOD in the samples removes the superoxide anion and suppresses the reduction. The reduction rate was used to measure of SOD activity. The end concentrations of hypoxanthine, xanthinoxidase and NBT in the measurement were 50 mol, 10 U/ml, and 0.125 mM, respectively. One unit of SOD activity was determined as that enzyme quantity which causes 50% suppression of NBT reduction to Formosan. Determination of CAT activity in erythrocyte lysate CAT activity was estimated in erythrocyte lysate by the method of Beers and Sizer (1952). Hydrogen peroxide (30 mM) was used as a substrate and the decrease in its concentration at 22C in phosphate buffer (50 mM, pH07.0) was followed at 240 nm for 1 min. One unit CAT activity was determined as that quantity of enzyme which removes 1 mol H2O2 for 1 min. The results are presented as U/g Hb (units per gram hemoglobin). The hemoglobin concentration of the lysate was determined by the cyanmethaemoglobin method (Mahoney et al. 1993). Statistical analysis Statistical analysis was carried out using Statistica 7 for Windows. The results were reported as meansSD (SE). Students t-test was used to determine whether differences between means were significant. Correlations between the different parameters were calculated by linear regression analysis. A value p < 0.05 was considered statistically significant.

Fig. 2 SOD activity in PD patients and controls

Results and discussion The MDA levels in patients with PD are presented in Fig. 1. The values of MDA, measured in the group with PD, were significantly increased compared to the healthy controls (3.590.62 vs. 2.800.55 mol/l, p00.01). The results of enzyme activities for patients and controls are presented in Figs. 2 and 3. Erythrocyte SOD activity was statistically significantly reduced compared to the group of healthy individuals (mean 1,107.84669.5 vs. 1,622.13452. 97 U/g Hb, p00.005) (Fig. 2). Erythrocyte CAT activity in PD patients was significantly increased compared with that of controls (mean 42,178. 2310,245. 43 to 23,462. 91 123.86 U/g Hb, p00.001) (Fig. 3). Numerous hypotheses have been proposed to explain the neurodegenerative mechanisms that occur in PD and some of them show that oxidative stress plays a considerable role. Low activity of mitochondrial complex 1 in PD also results in generation of oxygen species (Schapira et al. 1990). 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 disease (Ischiropoulos and Beckman 2003). Mechanisms underlying neuronal death are poorly understood although several in vitro studies have suggested the involvement of oxidative stress. The

Fig. 1 Levels of MDA in PD patients and controls

Fig. 3 CAT activity in PD patients and controls

Comp Clin Pathol

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 ROS via autoxidation and the dopamine oxidation by the monoamine oxidase B (Cohen 1983; Sanya et al. 2009). In the brains of patients with PD, the amount of MDA (MDA is a product of lipid peroxidation in the substance nigra) is increased and the concentration of polyunsaturated fatty acids is reduced, which is seen as an indicator for the presence of lipid peroxidation. However, Molina et al. (1992) have shown that lipid peroxidation measured as MDA levels in serum are similar in both the PD patients and controls. Lipids are much more susceptible on storage to peroxidation than protein or DNA (Halliwall 1992). MDA is an intermediate compound and a major indicator of lipid peroxidation process (Halliwell et al. 1987). Results of the present work indicate that plasma MDA levels are significantly higher in PD patients than in controls and agree with those reported by Sanya et al. (2009). Although the significance of data is still unclear and problematic (Poirier and Barbeau 1987; Pall et al. 1986; Zhang et al. 2000), our study has speculated that the increased lipid peroxidation can be considered a risk factor for PD. Under normal conditions, the continuous production of free radicals is compensated by the powerful action of protective enzymes. SOD and CAT are the major antioxidant enzymes defense against harmful side effects of ROS in cells. Further, any reduction in antioxidant enzymes may result in ineffective removal of ROS. SOD showed significant reduction in activity in patients leading to an increase of superoxide radical. Superoxide radicals can also react with NO to generate peroxynitrite (ONOO), a putative neurotoxin (Beckman and Crow 1993). A significant reduction in both CAT activities in effect can increase the production of highly deleterious H2O2 (Abraham et al. 2005). There were reports by various groups about a significant decrease in the activities of SOD and CAT in erythrocytes of PD patients (Barthwal et al. 1999; Ihara et al. 1999; Urakami et al. 1992; Gatto et al. 1996). Abraham et al. (2005) reported that SOD and CAT activities were found to be significantly lower in patients with PD compared to the controls. Our results in accordance to the data show that levels of antioxidant enzymes in patients with PD observed statistically significant deviation (p<0.005) compared with the control group of healthy individuals. The results from our research show that patients with PD have increased oxidative stress due to elevated lipid peroxidation. Oxidative stress is accompanied by strong imbalance in antioxidant status decreased levels of antioxidant enzyme SOD and elevated antioxidant enzyme CAT. Interest is the extent to which treatment regimens influence the development of oxidative stress, and also comparisons of indicators of oxidative stress in other neurodegenerative diseases.

The exact cause of PD still is not known, but there is a growing body of evidence that nigral neurons may be damaged by cytotoxic substances known as free radicals. Free radicals are thought to be produced locally within the basal ganglia and to lead to progressive damage to and death of substantia nigra neurons in susceptible individuals. The increase in the oxidative stress due to the imbalance of the antioxidant enzymes and might be a reason for many secondary complications and may contribute to the neurodegeneration in PD (Ciccone 1998).

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