Lipid peroxidation and antioxidant status in patients with rheumatoid arthritis
ABSTRACT The changes in the erythrocyte lipid peroxidation products (MDA), levels of glutathione (GSH), ascorbic acid and plasma vitamin
E (non enzymatic antioxidant parameters) and activities of antioxidant enzymes superoxide dismutase (SOD), glutathione peroxidase
(GPX), catalase in erythrocytes and plasma glutathione - S - transferase (GST) activity were estimated in patients with rheumatoid
arthritis. This work was undertaken to assess oxidative stress and anti oxidant status in patients with rheumatoid arthritis.
It was observed that there was a significant increase in erythrocyte MDA levels, activities of SOD, GPX, plasma GST and a significant decrease in erythrocyte GSH, ascorbic acid, plasma vitamin E levels and catalse activity in
patients with rheumatoid arthritis when compared to controls. The results of our study suggests higher oxygen free radical
production, evidenced by increased MDA and decreased GSH, ascorbic acid, vitamin E and Catalase activity, support to the oxidative
stress in rheumatoid arthritis. The increased activities of antioxidant enzymes may be a compensatory regulation in response
to increased oxidative stress.
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ABSTRACT: Our study goals to investigate the anti-arthritic potential of Salix nigra bark methanol extract (SNME) against both inflammation and oxidative stress in the collagen-induced arthritis (CIA) rat model. Results showed that SNME exhibited maximum scavenging activity against superoxide, hypochlorous acid and hydrogen peroxide radicals along with the suppression of lipid peroxidation. Female wistar rats were immunized with porcine type II collagen and treated with SNME (100 mg/kg body weight) for 15 days starting on day 20. SNME significantly inhibited the paw swelling and arthritic score; exhibited maximum CIA inhibition of 93.7% by the end of the experimental period. Administration of SNME to arthritic rats significantly improved the histological findings in joints as evident by reduced infiltration of polymorphonuclear cells and smooth synovial lining. Roentgenograms of tibiotarsal joints of both SNME and indomethacin-treated rats showed protection against osteophyte formation, soft tissue swelling and bone resorption. Furthermore, levels of inflammatory mediators (nitric oxide, TNF-α, IL-1β, IL-6) measured in both plasma and joint exudates were significantly reduced by SNME treatment. Increased oxidative stress observed in the arthritic animals was also found to be significantly restored in SNME- treated rats. Taken together, our studies clearly indicate the potential of S. nigra as an anti-arthritic agent.Food and chemical toxicology: an international journal published for the British Industrial Biological Research Association 12/2011; 49(12):3395-406. · 2.99 Impact Factor
Indian Journal of Clinical Biochemistry, 2008 / 23 (1) 41-44
LIPID PEROXIDATION AND ANTIOXIDANT STATUS IN PATIENTS WITH RHEUMATOID
Krishna Mohan Surapneni and V S Chandrasada Gopan*
Department of Biochemistry, Dr. Pinnamaneni Siddhartha Institute of Medical Sciences & Research Foundation,
Chinoutpally, Gannavaram (Mandal) – 521286, A.P, INDIA.
* Department of Biochemistry, Katuri Medical College & Hospital, Chinakondrupadu, Guntur – 522019, A.P, INDIA.
The changes in the erythrocyte lipid peroxidation products (MDA), levels of glutathione (GSH), ascorbic acid
and plasma vitamin E (non enzymatic antioxidant parameters) and activities of antioxidant enzymes superoxide
dismutase (SOD), glutathione peroxidase (GPX), catalase in erythrocytes and plasma glutathione – S –
transferase (GST) activity were estimated in patients with rheumatoid arthritis. This work was undertaken to
assess oxidative stress and anti oxidant status in patients with rheumatoid arthritis. It was observed that there
was a significant increase in erythrocyte MDA levels, activities of SOD, GPX, plasma GST and a significant
decrease in erythrocyte GSH, ascorbic acid, plasma vitamin E levels and catalse activity in patients with
rheumatoid arthritis when compared to controls. The results of our study suggests higher oxygen free radical
production, evidenced by increased MDA and decreased GSH, ascorbic acid, vitamin E and Catalase activity,
support to the oxidative stress in rheumatoid arthritis. The increased activities of antioxidant enzymes may be
a compensatory regulation in response to increased oxidative stress.
Malondialdehyde, Glutathione, Ascorbic acid, Vitamin E, Superoxide dismutase, Catalase, Glutathione
peroxidase, Glutathione -S - transferase, Rheumatoid arthritis.
Address for Correspondence :
Dr. Krishna Mohan Surapaneni
Department of Biochemistry,
Dr. Pinnamaneni Siddhartha Institute of Medical Sciences &
Research Foundation, Chinoutpally,
Gannavaram (Mandal)-521286, A.P, INDIA.
Arthritis, the joint inflammation, refers to a group of diseases
that cause pain, swelling, stiffness and loss of motion in the
joints. Rheumatoid Arthritis (RA) is a chronic, systemic disease,
in which various joints in the body are inflamed, leading to
swelling, pain, stiffness, and the possible loss of function. It is
an autoimmune disease in which the body’s immune system
attacks itself. Rheumatoid Arthritis affects approximately 1-
2% of the total world’s population (1). Annual incidence rate
of rheumatoid arthritis between 0.5%-1% of total population
is reported every year in both developed and developing
countries (2). Lower incidences of rheumatoid arthritis are
reported every year in East Asia (3). Rheumatoid Arthritis
affects around 1 in 50 people and is more common in women
than men. It is most common after the age of 40, but can
happen at any age. Lipid peroxidation mediated by free radicals
is considered to be the major mechanism of cell membrane
destruction and cell damage. Free radicals are formed in both
physiological and pathological conditions in mammalian
tissues (4). The uncontrolled production of free radicals is
considered as an important factor in the tissue damage
induced by several pathophysiologies (5, 6). Alteration in the
oxidant – antioxidant profile is known to occur in rheumatic
diseases (7, 8). Oxidative stress due to damage brought about
by free radicals is also known to influence the response of
these patients to therapy. Moreover the body’s defense
mechanisms would play an important role in the form of
antioxidants and try to minimize the damage, adapting itself
to the above stressful situation. Antioxidants are compounds
that dispose, scavenge, and suppress the formation of free
radicals, or oppose their actions (9) and two main categories
of antioxidants are those whose role is to prevent the
Indian Journal of Clinical Biochemistry, 2008 / 23 (1)
generation of free radicals and those that intercept any free
radicals that are generated (10). They exist in both the aqueous
and membrane compartment of cells and can be enzymes or
non-enzymes. The aim of our study was to investigate the
changes in oxidant and antioxidant status in patients with
MATERIALS AND METHODS
The study was conducted in Department of Biochemistry and
Orthopaedics, Dr. Pinnamaneni Siddhartha Institute of Medical
Sciences & Research Foundation, Chinoutpally, Gannavaram
(Mandal), A.P, India. Thirty clinically diagnosed patients from
orthopaedics OPD, who had not undergone any previous
treatment for their arthritis, were chosen for the study. Out of
the thirty patients 12 were females. An equal number of age &
sex matched healthy subjects with similar socio economic
status were also investigated. Due permission was obtained
from the ethical committee of the Dr.PSIMS&RF General
Hospital, Chinoutpally before the start of the work. The written
consents were also taken from the patients prior to study and
the objectives of the study were fully explained. Nine of the
participants were dropped out at the end of the selection, as
they did not like the idea of giving blood.
The complete clinical and personal history of the subjects was
recorded. The subjects were ranging in age 35 – 60 years. All
the patients in the study were clinically diagnosed as patients
with rheumatoid arthritis. The presence of rheumatoid arthritis
in patients was diagnosed by carrying out X – ray analysis of
joint destruction, rheumatoid factor test, C – reactive protein
and antinuclear anti bodies test. Subjects with diabetes/
systemic diseases like hypertension/diseases of any origin
other than osteoarthritis which could cause oxidative stress
or those receiving anti-inflammatory drugs in last 6 months
were excluded. None of the participants was alcoholic or
chronic smoker. Subjects with normal nutritional habits without
supplementing any vitamins during last 6 months were
The heparinised venous blood samples obtained from these
subjects were used for the analysis. Plasma was separated
by centrifugation at 1,000 g for 15 minutes. Separated plasma
was used for the estimation of vitamin E and GST. The buffy
coat was removed and the packed cells were washed three
times with physiological saline. The erythrocyte suspension
was prepared by the method of Dodge et al (11), modified by
Quist (12). The packed cells were used for the analysis of
GSH, ascorbic acid, MDA, SOD, catalase, GPX. Erythrocyte
GSH was estimated by the method of Beutler et al (13) using
di thio bis nitro benzoic acid (DTNB). Ascorbic acid levels were
estimated by the method of Tietz (14). Plasma vitamin E levels
were estimated by the method of Baker H et al (15). MDA was
determined as the measure of thio barbituric acid reactive
substances (TBARS) (16). SOD (EC 184.108.40.206) activity was
determined in the hemolysate by the method of Misra &
Fridovich based on the inhibition of auto oxidation of
epinephrine to adenochrome at pH 10.2 (17). Catalase (EC
220.127.116.11) activity was measured by the method of Beers and
Sizer (16). The activity of Glutathione Peroxidase (GPx, EC
18.104.22.168) was measured as described by Paglia and Valentine
(19) in erythrocytes and GST (EC 22.214.171.124) was measured by
using 1-chloro-2, 4-dinitro benzene (CDNB) (20). Necessary
care was taken during sample collection, storage and assay.
All reagents used were of analytical reagent grade. DTNB,
CDNB and thio barbituric acid were obtained from sigma
chemicals, St.Louis, MO. Statistical analysis between group
1 (controls) and group 2 (patients) was performed by the
student’s ‘t’ – test using the stat -view package. The data were
expressed as Mean±SD. P < 0.05 was considered as
RESULTS AND DISCUSSION
The Mean ± SD of erythrocyte GSH, ascorbic acid, MDA, SOD,
Catalase, GPx, plasma vitamin E and GST are described in
the Table1. There was a statistically significant increase in
the erythrocyte MDA levels in patients with rheumatoid arthritis
compared to controls. The activities of erythrocyte antioxidant
Table1: Malondialdehyde (MDA), glutathione, ascorbic acid,
vitamin E, superoxide dismutase (SOD), catalase, glutathione
peroxidase (GPX) and glutathione – S – transferase in controls
and patients with rheumatoid arthritis.
ParameterGroup1 (controls) Group2 (Patients)
Glutathione ( mg/gm of Hb)18.6±3.01 14.35±2.35 *
Ascorbic acid (mg/dl) 4.86±0.333.16±0.28 **
Vitamin E (µmoles/liter) 7.55±2.745.68±1.42 **
MDA (nmoles/gm of Hb)4.62±0.585.63±0.82 ***
SOD (EU/gm of Hb)
1877.90 ± 532.672712.57± 497.79* * *
9.54 ± 0.13 6.91 ± 0.15 **
GPX (u/gm of Hb)
GST(micromoles/dl of plasma) 13.65 ± 6.46
16.91 ± 1.6739.7 ± 2.01 **
21.43 ± 6.97 ****
Values are mean ± SD
*P < 0.0001 compared to controls; ** P < 0.05 compared to controls
***P< 0.001 compared to controls; ****P < 0.01 compared to controls
enzymes SOD, GPX and plasma GST were significantly
increased in group2 compared to group1. The levels of
erythrocyte GSH, ascorbic acid, plasma vitamin E and
Catalase activity were significantly decreased in patients with
rheumatoid arthritis compared to controls.
In the present study the lipid peroxidation product i.e. MDA
levels have been increased significantly in erythrocytes of the
patients with rheumatoid arthritis. Rise in MDA could be due
to increased generation of reactive oxygen species (ROS) due
to the excessive oxidative damage generated in these patients.
These oxygen species in turn can oxidize many other important
biomolecules including membrane lipids. Similar reports of
elevated MDA levels have been reported in patients with
rheumatic disease (7, 8). In contrast to our study,
Kajanachumpol et al reported no significant change in MDA
levels in patients with rheumatoid arthritis compared to
We observed a significant decrease in the levels of erythrocyte
GSH, ascorbic acid and plasma vitamin E (non enzymatic
antioxidant defense system) in patients with rheumatoid
arthritis when compared to controls. The decrease in the levels
of these non-enzymatic antioxidant parameters may be due
to the increased turnover, for preventing oxidative damage in
these patients suggesting an increased defense against
oxidant damage in Rheumatoid Arthritis. Similar reports of
elevated MDA levels have been reported in patients with
rheumatoid arthritis (22, 23).
In our study the erythrocyte antioxidant enzymes, i.e. SOD &
GPX activities have been increased significantly in patients
with rheumatoid arthritis. Similar reports of raised SOD & GPX
activities have been reported in patients with rheumatoid
arthritis (7, 24). SOD is the important antioxidant enzyme
having an antitoxic effect against super oxide anion. The over
expression of SOD might be an adaptive response and it
results in increased dismutation of superoxide to hydrogen
peroxide. GPX, an oxidative stress inducible enzyme plays a
significant role in the peroxyl scavenging mechanism and in
maintaining functional integration of the cell membranes (25).
The rise in the activity of GPX could be due to its induction to
counter the effect of increased oxidative stress. Ostalowska
et al have reported increased activities of SOD, glutathione
peroxidase and glutathione reductase in synovial fluid of
patients with primary and secondary rheumatoid arthritis of
the knee joint (6).
The glutathione-S-transferases are a group of multifunctional
proteins, which play a central role in detoxification of
electrophilic chemicals & the hepatic removal of potentially
harmful hydrophobic compounds from blood (26). We have
observed a significant rise in the activity of GST in patients
with rheumatoid arthritis compared to controls. Similar reports
of raised GST activity were observed in rheumatic diseases
(27). The rise in the activity of GST could be due to its induction
to counter the effect against increased oxidative stress.
In the present study, we have observed a significant decrease
in the catalase activity in patients with rheumatoid arthritis
compared to controls. Catalase is the enzyme, which protects
the cells from the accumulation of hydrogen peroxide by
dismutating it to form water and oxygen or by using it as an
oxidant in which it works as a peroxidase (28). Similar reports
of decreased catalase activity were observed in rheumatoid
arthritis by Kerimova et al (29). However others have reported
an increase in plasma catalase activity in patients with
rheumatoid arthritis when compared to controls (7). In
conclusion, Oxidative stress may be involved in rheumatoid
arthritis. There is a shift in the oxidant – antioxidant balance
in favour of lipid peroxidation, which could lead to the tissue
damage observed in this disease.
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