Vol. 58, No.

2/2011
165–169
on-line at: www.actabp.pl
Regular paper

Altered oxidative stress levels in Indian Parkinson’s disease

patients with PARK2 mutations
Monika Vinish*, Akshay Anand* and Sudesh Prabhakar*
Department of Neurology, PostGraduate Institute of Medical Education and Research (PGIMER), Sector-12, Chandigarh, India

The aim of this pilot study was to determine the base- factors appear to play an important role in its develop-
line state of oxidative stress indices in patients with ment. Parkin is a Parkinson disease-related E3 ubiqui-
Parkinson’s disease (PD). Peripheral blood samples of tin ligase; parkin-deficient animals exhibit mitochondrial
15 PD subjects were analyzed and compared with ten degeneration and increased oxidative stress vulnerability,
age matched healthy controls. Patients with PARK2 mu- and both mice and flies lacking DJ-1 are hypersensitive
tations were also compared with PD patients without to environmental toxins associated with PD (Palacino et
mutations. There was significant increase in malondial- al., 2004; Shen & Cookson, 2004). Currently, accumu-
dehyde content and superoxide-dismutase (SOD) activity lating evidence indicates that parkin may play a role in
in peripheral blood parameters in PD patients (p < 0.05) maintaining mitochondrial function and preventing oxi-
in comparison to controls. These findings suggest an dative stress (Hyun et al., 2005). We therefore examined
important role of oxidative stress in Parkinson’s disease if PARK2 deletions alter the antioxidant profile of In-
evolution and progress. No changes were observed in dian PD patients.
glutathione peroxidase and nitric oxide levels. We found
significant correlation between SOD activity and lipid
Material and Methods
peroxidation when the biochemical data was further
analyzed. In addition, significant increase in the levels of
SOD among the PD patients with PARK2 mutations was Patients. The study group included 15 sporadic or
observed, which can be ascribed to chronic oxidative non-consanguineous PD patients visiting the Neurology
stress induced by PARK2 mutations. Clinic at the PostGraduate Institute of Medical Educa-
Key words: oxidative stress, mutations, Parkinson’s disease, PARK2
tion and Research (Chandigarh, India) and ten healthy
controls. The diagnosis of Parkinson’s disease was made
Received: 19 November, 2009; revised: 24 January, 2011; accepted: on the basis of the UK Parkinson’s Disease Society Brain
08 May, 2011; available on-line: 17 May, 2011
Bank Research criteria, London (Hughes et al., 1992).
Clinical diagnosis was established with the presence of at
least two of the cardinal symptoms, i.e., tremors, muscu-
Introduction lar rigidity, bradykinesia and postural instability while pa-
tients with vertical gauge impairment, marked autonomic
disturbances, atypical Parkinsonism and those on antip-
Parkinson’s disease (PD) is characterized by a loss of sychotic drugs were excluded from the study (Lang &
dopaminergic neurons in the substantia nigra, leading Lozano, 1998). Written informed consent was obtained
to the major clinical and pharmacological abnormalities from all patients and controls as per the Institute Eth-
that characterize the disease. Although the pathogen- ics Committee guidelines. Genetically unrelated controls
esis of PD remains ambiguous, oxidative stress (OS) to were also examined for the absence of extra-pyramidal
dopaminergic neurons in the substantia nigra pars com- signs, which included spouse of the patient and other
pacta (SNpc) has been reported to be one of the lead- age, sex and ethnicity matched healthy individuals. The
ing causes of neurodegeneration in PD (Bahmann et al., mean age of onset for patients recruited for the study
2004). The human body has evolved several defense was 46.5 ± 2.1 years while that for healthy volunteers was
mechanisms to counteract OS such as vitamin E, vita- 43.4 ± 2.2. About 10 mL of venous blood was drawn for
min C, vitamin A, glutathione and various antioxidant genetic analysis from these patients and controls. All the
enzymes, but the brain appears to be more susceptible biochemical assays were performed in duplicates.
to these assaults than other organs because of its low Superoxide dismutase (SOD) assay. Determination
antioxidant capacity. Being highly metabolic, brain tissue of Cu,Zn-SOD activity was performed using a commer-
generates more oxyradicals. Alteration in the oxidative cial kit (Ransod; Randox, CrumLin, UK) based on the
stress has been proposed to cause the loss of dopamin- method developed by McCord and Fridovich (1988).
ergic neurons in PD patients (Hung & Lee, 1998). Al- Coupling of O2–• generators (xanthine and xanthine oxi-
though the changes in lipid peroxidation and antioxidant
defenses are documented in the SNpc of PD patients *
but there is difficulty in obtaining a brain biopsy, until e-mail: pgineurology@gmail.com
*
These authors contributed equally to this work
after the death of the afflicted individual. It is therefore Abbreviations: Gpx, glutathione peroxidase; INT, (2-(4-iodophenyl)-
crucial to develop suitable peripheral markers, which can 3-(4-nitrophenol)-5-phenyltetrazolium chloride LRRK2, leucine rich
help in the diagnosis of PD during life. repeat kinase; MDA, malondialdehyde; PD, Parkinson’s disease;
The etiology of Parkinson’s disease is unknown al- PARK2, Parkin gene; SNc, substantia nigra; SOD, superoxide dis-
mutase; PINK-1, PTEN-induced putative kinase 1
though both genetic susceptibility and environmental
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M. Vinish and others 2011

dase) with an O2• detector INT (2-(4-iodophenyl)-3-(4- Results

nitrophenol)-5-phenyltetrazolium chloride) leads to for-
mation of red formazan dye. One unit of SOD activ-
ity was defined as the amount of protein that inhibits Central Nervous System related studies have been
the rate of INT reduction by 50 %. Enzyme activity was confronted with the complexity of direct investigation
measured in SOD units/mL of whole blood. because of difficulty in obtaining biopsies. It is therefore
crucial to investigate peripheral markers, which can help
Glutathione peroxidase (Gpx) assay. Measurement in the early diagnosis of Parkinson’s disease (PD). Our
of glutathione peroxidase (Gpx) activity was performed recent study has revealed upregulated levels of SOD and
using Ransel reagents (Randox Laboratories, UK) and lipid peroxidation in PD patients (Sharma et al., 2008).
is based on the method of Paglia and Valentine (1967). Molecular studies in familial forms of the disease have
Gpx catalyzes the oxidation of glutathione (GSH) by identified genes: α-synuclein, parkin, DJ-1 (PARK7),
cumene hydroperoxide. In the presence of glutathione PINK-1 (PTEN-induced putative kinase 1) and LRRK2
reductase (GR) and NADPH the oxidized glutathione (leucine rich repeat kinase) encoding key proteins in-
(GSSG) is immediately converted into the reduced form volved in PD pathogenesis, and support a major role for
with a simultaneous oxidation of NADPH to NADP+. mitochondrial dysfunction and oxidative stress (Thomas
For the Gpx assay, hemolysates (50 μL) were diluted & Beal, 2007). Here we studied the antioxidant profile
with 1.0 mL of Ransel diluting agent and incubated for (SOD, Gpx, nitric oxide and lipid peroxidation (MDA) in
5 minutes, followed by the addition of 1.0 mL of the blood of PD patients and controls using spectrophoto-
Drabkin reagent. The decrease in absorbance at 340 nm metric analysis.
was measured.
Nitric oxide estimation. Nitric oxide estimation Superoxide dismutase (SOD)
was done by the method of Titheradge (1998). About
100 μL of the sample (plasma) was added to 400 μL SOD is a Cu,Zn containing enzyme responsible for
of distilled water, 500 μL of freshly prepared solution catalytic dismutation of highly reactive and potential-
C (Griess reagent) was then added to the vials con- ly toxic superoxide radicals (O2–·) to H2O2 (McCord
taining sample. The reaction mixture was incubated at and Fridovich, 1988). O2–· is constantly generated in
room temperature for 10 minutes and absorbance was the body tissues and failure in its removal can initi-
read at 546 nm. ate a damaging effect on polyunsaturated fatty acids
Lipid peroxidation. Brain tissues are rich in phos- and structural proteins of plasma membranes. We es-
pholipids and vulnerable to attack by oxygen-derived timated SOD levels among PD patients and controls
free radicals to initiate lipid peroxidation. Lipid perox- using spectrophotometric analysis. The results showed
idation was evaluated as the concentration of malond- significantly increased SOD levels (P = 0.026) in PD
ialdehyde (MDA), a lipid peroxidation end product patients as compared to controls (n = 10) (Fig. 1).
that reacts with TBA (thiobarbituric acid) to form a
conjugate. MDA levels thus provide valuable infor- Glutathione peroxidase (Gpx)
mation for evaluation of oxygen radical-induced oxi-
dative stress. In our study, MDA was assayed by the Gpx has been previously reported as an important
method of Buege and Aust (1978). About 1.0 mL of hydroperoxide-degrading enzyme and the importance
plasma was mixed with 2 mL of mildly heated reagent of glutathione reductase (GR) lies in its ability to keep
(15 % (w/v) trichloroacetic acid, 0.375 % (w/v) TBA, glutathione in its reduced (biologically active) form.
0.25 M HCl). The solution containing plasma and rea- Numerous chemical processes in aerobic cells lead
gent was heated in boiling water bath for 15 min and to the production of peroxides by activated forms
then cooled. The flocculation precipitate was removed of oxygen. These peroxides by their decomposition
by centrifugation at 1 000 r.p.m. for 10 min. The su- to free radicals and other reactive chemical species
pernatant was collected and the absorbance was read cause oxidative damage in biological tissues. The sim-
at 535 nm against appropriate blank. The amount of plest hydroperoxides such as H2O2 and lipid perox-
MDA was calculated using molar absorption coeffi- ides can be detoxified by the selenium-dependent Gpx.
cient of MDA (1.56 × 105 M–1 · cm–1). The results were The activity was estimated by following the oxidation
expressed as mmol of MDA/L. of NADPH, required for the reduction of GSSG to
PARK2 analysis. Genomic DNA was extracted from GSH. We only found an insignificant decrease in the
the sporadic PD patients and controls. All exons of PARK2 Gpx levels (699 ± 59.4) in patients as compared to
were amplified by PCR using parkin specific primers (Ki- controls (730 ± 79.0) (Fig. 2).
tada et al., 1998). PCR products were visualized on 2 %
agarose gel for absence or presence of amplified product.
Gene dosage analysis was not carried out because lack of
real-time PCR device in our institute. Therefore this study
was restricted to qualitative analysis alone.
In order to determine the mutations in PARK2,
PCR amplified exons were subjected to SSCP and
band pattern was obtained by silver staining. Sequenc-
ing was performed for all those PCR products of
patients and controls that showed mobility shift by
SSCP analysis.
Statistical analysis. The mean values of various anti-
oxidants were compared using Student’s t-test. Correlation
between different variables was tested using nonparamet-
ric Spearman’s coefficient and statistical significance was
Figure 1. SOD levels in PD patients and controls.
considered at P < 0.05. (SPSS 17.0, Chicago, IL, USA).
Vol. 58
Antioxidant profile of Indian PD patients 167

Figure 2. Glutathione peroxidase (Gpx) levels in PD patients and

controls Figure 5. SOD levels in PD patients with and without PARK2 mu-
tations

Figure 3. Nitric oxide levels in PD patients and controls Figure 6. Glutathione peroxidase (Gpx) levels in PD patients with
and without PARK2 mutations

Figure 4. Lipid peroxidation (MDA) levels in PD patients and

controls
Figure 7. Nitric oxide levels in PD patients with and without
PARK2 mutations
Nitric oxide estimation

Nitric oxide is a unique biological messenger mol-

ecule which plays diverse physiologic roles. NO me-
diates blood vessel relaxation, immune activity of
macrophages and neurotransmission of central and
peripheral neurons. Our results revealed a slight in-
crease in NO levels in PD patients (32.0 ± 3.3) vs con-
trols (26.9 ± 2.4) but this was statistically insignificant
(Fig.  3).
Lipid peroxidation

Lipid peroxidation reflects oxidative deterioration of Figure 8. Lipid Peroxidation (MDA) levels in PD patients with
polyunsaturated fatty acids, important constituents of and without PARK2 mutations.
biological membranes and is measured in terms of nmols
of MDA formed/mg protein. Higher levels of MDA, tant role to play in the pathogenesis of neurodegenera-
a marker of oxidative stress, have been reported in the tive diseases.
SNpc of PD patients (Dexter et al., 1989). Increased lipid
peroxidation is well reported in neurodegenerative dis- PARK2 analysis
eases (Dei et al., 2002). Similarly, we found a significant
increase in the plasma MDA levels of PD patients as All the 12 exons of PARK2 were amplified in PD pa-
compared to controls (Fig. 4). Thus these increased lipid tients and healthy controls. Absence of a band was con-
peroxidation products suggest that ROS have an impor- firmed by repeating PCR and revalidated by GAPDH
168
M. Vinish and others 2011

a potential peripheral oxidative stress marker in

Table 1. Correlation among the various biochemical variables
plasma of PD patients (Llic et al., 1999; Serra et
Variables Sex Age SOD Gpx Lpx NO al., 2001; Younes-Mhenni et al., 2007). Our study
Sex 1.00 0.175 0.456 0.171 –0.116 0.463 also revealed increase in plasma MDA levels in
PD patients as compared to healthy controls,
Age 0.175 1.0 –0.378 –0.113 –0.507 0.031 which reflects a state of OS in these patients.
SOD –0.456 –0.378 1.0 –0.093 0.797** –0.187 Therefore, the increased SOD and lipid peroxi-
dation levels in PD patients appear to play an
Gpx 0.171 –0.113 –0.093 1.0 –0.225 0.341
important role in PD pathogenesis. The elevated
Lpx –0.116 –0.507 0.797** –0.225 1.0 –0.279 SOD levels have been earlier reported in blood
NO 0.463 0.031 –0.187 0.341 –0.279 1.0 of PD patients (Llic et al., 1999; Serra et al., 2001;
Younes-Mhenni et al., 2007; Sharma et al., 2008).
**Correlation is significant at p < 0.01 level (2-tailed) Besides, we also found excellent correlation be-
tween SOD and lipid peroxidation levels in the
(positive control) amplification of the same template. blood of these patients which can serve as a bi-
PARK2 analysis in these patients revealed exonic dele- omarker in the blood (Table 1).
tions in exons 1, 2, 3 and 12 by PCR and SSCP analysis.
The exons were amplified thrice under same set of PCR PARK2, DJ-1 and PINK-1 gene mutations have been
conditions. In addition, when the biochemical values of previously reported (Thomas & Beal, 2007) in the eleva-
these PD patients carrying PARK2 mutations and those tion of oxidative stress. Our results partly match such
without mutations were compared (Figs. 5–8), a signifi- antioxidant profile. Although the antioxidant profile was
cant increase in the levels of SOD among the PD pa- altered in PD patients with mutations when compared to
tients with PARK2 mutations was observed, which can those without mutations a larger study will determine the
be attributed to chronic oxidative stress induced by importance of these results. Nevertheless, this pilot study
PARK2 mutations. provides preliminary information which forms the basis
of the investigations to follow. Based on the analysis,
Correlation between biochemical parameters we conclude that alterations in SOD and lipid peroxida-
tion levels are possibly due to increased oxidative stress
Spearman correlation analysis was performed to study in these patients or as a general compensatory response.
the association between the biochemical parameters. A Mutations in PARK2 might affect the oxidative machin-
significant positive correlation between superoxide dis- ery of PD patients but a larger study can establish this.
mutase and MDA levels was found (P = 0.001) (Table 1). Our study supports the involvement of oxidative stress
that is implicated in the pathogenesis of PD. Perspec-
tives for treatment of PD in the future should investi-
Discussion
gate the role of antioxidant therapy.

Oxidative stress represents one of the risk factors Acknowledgements

that can promote neurodegeneration in PD. Recent evi-
dence suggests that several known mutations cause fa- We acknowledge the Council of Scientific and Indus-
milial Alzheimer disease (AD) (amyloid β protein pre- trial Research (CSIR), New Delhi, for financial support to
cursor, presenilin-1, or presenilin-2 gene) while familial PhD scholar (Monika Vinish). We are also thankful to the
PD genes such as Parkin, PINK-1, or DJ-1 are associated valuable inputs of Mr. R.C. Goyal to statistical analysis.
with increased oxidative stress. Also, several known ge-
netic (e.g., apolipoprotein Eε4 variant) and environmen- References
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