The major clinical disturbances in Parkinson’s disease (PD) are consequence of dopamine depletion in the neostriatum, due to degeneration of dopaminergic neurons. The aim of the present study was to determine whether oxidative stress (OS) occurs during the clinical course of Parkinson’s disease and to evaluate the influence of therapy on the levels of some important final products of oxidation of lipids, proteins and nucleic acids in PD patients with drug therapy. For this purpose, we investigated the levels of malondialdehid (MDA), protein carbonyl content (PCC) and 8-hydroxy-2’-deoxyguanosine quantity (8-OHdG) in PD patients with and without drug therapy. The observed changes in MDA levels, PCC and 8-OHdG quantity in blood of untreated PD patients, suggested impaired antioxidant status and presence of oxidative stress in Parkinson disease. After treatment with Madopar, the elevation in by-products significantly progresses. Our results demonstrate that administration of Madopar causes in greater degree oxidative stress than that induced by Parkinson disease, by itself.
Parkinson’s disease (PD) takes a leading place among contemporary frequent diseases of the central nervous system (CNS). This is a slow, progressive disease, characterized by loss of dopaminergic neurons in the Subs- tantia Nigra (SN) pars compacta [
The most commonly used drug in the PD treatment is levodopa (L-dopa). It is a precursor of dopamine and thus complete deficiency of dopamine in the CNS. After oral intake L-dopa undergoes metabolism, including oxidative dopamine metabolism, auto-oxidation and across the blood-brain barrier (BBB). Only less than 5% of an oral dose of L-dopa is delivered to the brain. Remaining plasmatic levels of L-dopa undergoes peripheral oxidative metabolism and may generate ROS. Likely peripheral oxidative status in PD patients might be af- fected by L-dopa therapy [
The present study was aimed to determine whether OS occurs during the clinical course of PD and to evaluate the influence of drug therapy with Madopar on the levels of some important final products of oxidation of lipids, proteins and nucleic acids, such as plasma concentrations of MDA, PCC and quantity of 8-OHdG.
The Thiobarbituric acid and Protein Carbonyl and Oxidative DNA Damage ELISA kits were purchased from Sigma-Aldrich Chemie GmbH (Germany). All other chemicals used in this study were analytical grade. De- ionized and distillated water was used for all experiments.
The study population consisted 20 PD patientstreated with Madopar, 18 PD patients without administration. All of patients were in the age group of 59 - 78 years old and were patients of the Neurological Clinic of University Hospital, Stara Zagora, Bulgaria. The PD diagnoses were made by taking a detailed history and studying the disease symptoms. For controls were used 20 age-matched healthy volunteers (
No obvious differences were found in the severity of disease and the dose of antiparkinsonian drugs between female and male cases. To eliminate other factors that might affect the oxidative stress parameters, we have chosen PD patients and healthy volunteers all were neither smokers nor 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.
Fasting samples of venous blood were collected in the morning in tubes with anticoagulant and was used to
. Clinical characteristics of PD patients control subjects
Group | Therapy | Male/Female | Age | Disease Duration | Therapy Duration |
---|---|---|---|---|---|
Parkinson Disease | Therapy —Madopar 250 mg 1 + 1 + 1 + 1/2 | Men - 8 women - 10 | 55 - 70 | from 6 to 8 years | from 2 to 4 years |
Parkinson Disease | Without therapy | men - 10 women - 10 | 55 - 70 | from 1 to 2years | without therapy |
Healthy Volunteers | Controls | men - 8 women - 12 | 50 - 65 | healthy volunteers | healthy volunteers |
determinated the product of lipid peroxidation. For PCC and 8-OHdG was collected whole blood in a covered test tube (without anticoagulant). All samples from each subject were split and run in triplicate.
Total amount of lipid peroxidation products in the plasma of healthy volunteers and patients was estimated using the thiobarbituric acid (TBA) method modified by Gadjeva et al., and measurement of MDA at 532 nm [
Haemoglobin lysates concentrations were analyzed by cyanmethaemoglobin method [
PCC was measured by using a commercial ELISA kit followed manufacturer’s instructions.
The quantitative measurement of 8-OHdG were carried out using commercial ELISA kit, followed manufacturer’s instructions.
Statistical analysis was carried out using Statistics 7 for Windows. The results were reported as means ± SD (SE). Student’s t-test was used to determine the statistical difference between groups. P ≤ 0.001 was considered statistically significant.
Lipids are the most easily oxidizable which makes them the most preferred object for the study of free radical injuries via lipid peroxidation. Literature data show that in the brain of PD patients the amount of products of lipid peroxidation (LPO) (measured as MDA) is increased and the concentration of polyunsaturated fatty acids decreased, which is considered as an indicator of increased LPO in the SN.
Sanyal et al. reported that PD patients levels of plasma MDA is significantly higher than in controls [
It is well known that lipid peroxidation of plasma and intracellular membranes leads to distortion of the overall cell metabolism, and has a cytotoxic effect. MDA can react with proteins and nucleic acids, and so impair certain physiological mechanisms in the human body. 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. The concept that OS occurs in PD derives primarily from the realization of dopamine metabolism, by chemical or enzymatic means that can generate free radicals and other ROS via autoxidation and dopamine oxidation. Biochemical evidence of oxidative injury can be suggested through the detection of products of oxidation of lipids, proteins and nucleic acids, such as MDA, PCC and quantity of 8-OHdG [
Therefore the results of our study indicate that oxidative stress increased in PD patients without therapy which is due to the increased levels of LPO and are in agreement with studies of Jenner & Olanov, Sudha et al. and Barnham et al. [
MDA plasma levels in controls, PD patients with therapy Madopar and PD patients without drug therapy. Results are presented as mean values ±SE p < 0.05; (*)—statistical significance relative to the controls; (++)— statistical significance compared to PD patients without drugtreatment
comparing with controls, which is consistent with our results [
The oxidative modification of proteins leads to increased recognition and degradation by proteases and loss of enzymatic activity [
In comparison between the group of PDpatients without therapy with controls also had a statistically significant increase (mean 5.17 nmol/mg ± 0.1, vs mean 1.25 nmol/mg ± 0.2, р = 0.00, t-test), which indicates that impacts of disease may also damaged the proteins and confirmed the role of oxidative stress in disease path- ology.
There are significant data [
Protein Carbonyl Content (PCC) in PD patients with Madopar therapy, patients without therapy and controls; p < 0.05; (*) relative to con- trols, (++) compared with PD patients without drug treatment
DNA damage, measured as 8-OHdG (ng/ml) in controls, PD pa- tients with therapy Madopar and PD patients without drug therapy. p < 0.05; (*)—relative to the controls, (++)—compared with PD patients without drug treatment
The results suggested that in PD patients without drug therapy OS increased, this is demonstrated by elevated levels of the end products of oxidation of lipids (measured as MDA), proteins (PCC) and nucleic acids (8-OHdG). In the group of PD patients with drug therapy our results coincide with Stadman et al., Alam et al., Cornetta et al., Prigione et al., Floor et al., [
There is a lot of evidence that PD brain is oxidatively damaged, which is observed within L-dopa therapy [
In view of these facts we can conclude that presented results from our study confirm the concept that oxidative stress is increased in PD patients by elevated plasma levels of MDA, PCC modified proteins and the amount of 8-OHdG. The long-term drug administration can cause further oxidative stress and imbalance between the production and elimination of ROS could contribute to the pathogenesis of PD and other neurodegenerative disorders. In conclusion, we believe that the Levodopa (Madopar) administration results in a greater degree of oxidative stress, than is induced by PD itself. Further studies are needed to clarify the effect of the combined therapy with antioxidants, which would have a protective effect on Madopar-induced oxidative toxicity
This work was supported by the scientific projects No 12/2012 and No 4/2013, Medical faculty, Trakia University. Authors thank to the European Social Fund, Structural Funds, and Operational Programme “Human Resources Development” for financial support in frame of Grant BG051PO001-3.3.06-0006.