Antioxidant status & lipid peroxidation in patients with
F. Karatas, I. Ozates, H. Canatan*, I. Halifeoglu**, M. Karatepe & R. Colak†
Department of Chemistry, College of Science, Departments of *Medical Biology, **Biochemistry &
†Internal Medicine, College of Medicine, Firat University, Elazig 23119, Turkey
Received February 28, 2003
Background & objectives: Rheumatoid arthritis (RA) is a debilitating, chronic multisystem disease with
an unknown etiology. Recent findings indicate that increased oxidative stress and/or defective antioxidant
status contribute to the etiology of RA. The present study was undertaken to examine the oxidant and
antioxidant systems in patients with RA and healthy controls.
Methods: Twenty two patients with RA and 20 healthy volunteers were included in the study. Levels of
malondialdehyde (MDA) and antioxidant vitamins (A, E, C) in serum samples were determined by
high performance liquid chromatography (HPLC). Spectrophotometric methods were used to determine
activity levels of antioxidant enzymes, superoxide dismutase (SOD) and glutathione peroxidase (GSH-
Px), in erythrocytes.
Results: MDA levels in patients with RA were found to be significantly (P<0.005) higher than controls
whereas levels of vitamins A, E, C and activities of GSH-Px, SOD were lower in the patients compared
to controls (P<0.005 for SOD and antioxidant vitamins; P<0.05 for GSH-Px).
Interpretation & conclusion: There was an increased oxidative stress and a low antioxidant status in
patients with RA. These changes are probably due to efforts for reducing lipid peroxidation and hence
to lower tissue damage.
Key words Glutathione peroxidase - malondialdehyde - oxidative stress - rheumatoid arthritis - superoxide dismutase
Indian J Med Res 118, October 2003, pp 178-181
Rheumatoid arthritis (RA) is a chronic, multisystem
disease with an unknown etiology affecting about 1 per
cent of the world’s population1. RA is characterized by
persistent inflammation in the synovial membranes of
joints, associated with migration of activated phagocytes
and other leukocytes into synovial and periarticular
tissue2. During phagocytosis, monocytes, neutrophils and
macrophages generate superoxide radicals, hydrogen
peroxide and the highly reactive hydroxyl radicals3. These
cytotoxic reactive oxygen species (ROS) may cause
oxidative damage in the cells4. Activated oxygen
intermediates together with highly reactive radicals, such
as the hydroxyl radicals, are able to destroy membrane
lipids, proteins, deoxyribonucleic acid, hyaluronic acid, and
cartilage5. Enzymatic mechanisms include superoxide
dismutase (SOD), catalase and glutathione peroxidase
(GPX)1.Vitamins A, C, E and glutathione are some of the
major non-enzymic antioxidants in the body1,6. Plasma
concentrations of vitamin C, vitamin E and beta-carotene
were found to be decreased in patients with RA7,8.
Oxygen free radicals have been implicated as
mediators of tissue damage in patients with RA. Hence,
the aim of the present study was to assess the
lipid peroxidation and antioxidant status of patients
Material & Methods
Patients for the study were selected from individuals
attending the routine Rheumatology Clinic of Department
of Internal Medicine at Firat Medical Center, Turkey
during October 2001-April 2002. Criteria recommended
by the American Rheumatism Association9 were used
for the diagnosis of RA. The study protocol was approved
by the ethics committee of the Firat university. Informed
consent was obtained from all patients and controls.
A total of 22 patients with RA (10 males, 12 females,
mean age 48.9 ± 12.3 yr, mean disease duration 11.7 ±
7.6 yr) were included into the study group. Twenty
healthy volunteers (medical students, laboratory
personnel and officials) were included as controls
(8 males, 12 females, mean age 44.5±11.2 yr). None of
the patients and controls were smokers or consuming
alcohol or had any other chronic disease.
Blood samples collected from normal controls and
patients were centrifuged at 3000 g for 5 min at +40C
and the plasma separated. Levels of MDA and
antioxidant vitamins (A, E, C) were determined in
plasma samples. After separating the plasma,
erythrocytes were washed three times in 0.9 per cent
NaCl solution and were haemolysed by dilution in water
and stored at -200C until used for measurement of SOD
and GSH-Px activities. The haemoglobin (Hb) content
of erythrocytes was determined by the
SOD activity in erythrocytes was assayed using a kit
(Randox, UK) following the manufacturer's instructions.
In this method, xanthine and xanthine oxidase was used
to generate SOD radicals which react with 2-(4-
chloride to form a red formazan dye. SOD activity is
measured by the degree of inhibition of this reaction,
and expressed as U/g Hb.
GSH-Px activity levels were determined by the
method of Paglia and Valentine11 using a commercially
available kit (Randox, UK) and the activity levels were
expressed as U/g Hb.
The quantification of vitamin A and E was done by
the method of Catignani and Bieri12 and Miller et al13.
utilizing absorption spectra of 326 and 296 nm,
respectively. HPLC separations were done at room
temperature with a Cecil liquid chromatography system
(UK) (Series 1100) consisting a sample injection valve
(Cotati 7125) with a 20 µl sample loop, an ultra-violet
(UV) spectrophotometric detector (Cecil 68174),
integrator (HP 3395) and a Techsphere ODS-2 packed
(5 µm particle and 800A pore size) column
[250×4.6 internal diameter (ID)] with a methanol:
acetonitrile: chloroform (47: 42: 11, v/v) mobile phase at
a flow rate of 1 ml/min.
The extractions of vitamin C and MDA were done
following the method of Cerhata et al14. The supernatant
was filtered and vitamin C level was determined using
the method of Tavazzi et al15 and MDA level by the
method of Karatas et al16 by HPLC utilizing a column
(250×3.9 ID) packed with Tocnopak C18 reversed-phase
material (UK) (10µm particle size) For vitamin C 3.7
mM phosphate buffer, pH 4.0 was used as mobile phase
with a flow rate of 1 ml/min while for MDA the mobile
phase was 30 mM KH2PO4 buffer, pH 4 with H3PO4
and methanol at a flow rate of 1.5 ml/min.
All chemicals and reagents used were of analytical
grade and were purchased from Merck Chemical Co.,
The SPSS software (Chicago, IL, USA) was used
for statistical analyses. Differences in various parameters
between the two groups were analyzed for significance
using the Mann-Whitney U-test. Statistical significance
was defined as P<0.05.
Results & Discussion
The recovery rates were found to be 98.2 per cent
for vitamin A, 99.5 per cent for vitamin E, 97 per cent
for vitamin C and 98.8 per cent for MDA.
MDA levels were found to be significantly (P<0.005)
elevated in the patients with RA compared to the
controls (Table). This is in agreement with other studies
where higher MDA levels have been reported in
patients with RA17-19. Oliveri et al20, reported no
change in lipid peroxidation but generally increased lipid
peroxidation in serum17, plasma18 and erythrocytes19
has been reported.
KARATAS et al : OXIDANT & ANTIOXIDANT STATUS IN RHEUMATOID ARTHRITIS
Decreased SOD and GSH-Px activity levels in
patients with RA may indicate a degradation of these
antioxidant anzymes by free radicals during detoxification
processes. Decrease in antioxidant vitamin levels may
be related to their role in these antioxidant processes. It
is known that in rheumatic diseases, especially in RA,
Cu levels are increased whereas Zn levels are
decreased27-29. Decreased SOD activity levels may be
due to disturbance in Cu and/or Zn levels. It appears
that increased levels of superoxide and other radicals
are not detoxified in patients with RA due to decreased
efficiency of antioxidant enzymatic and non-enzymatic
mechanisms, and may act as mediators of tissue damage.
The authors acknowledge the financial support received from
the Firat University Research Fund, Elazig, Turkey.
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Antioxidant enzyme (SOD and GSH-Px) activities
were found to be significantly lower in patients
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respectively) in the present study. There are
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Akyol et al18 reported that SOD levels in erythrocytes
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and healthy controls.
Table. Antioxidant vitamins (A, E and C) and MDA in plasma and
erythrocyte antioxidant enzyme activities (SOD and GSH-Px) in
patients with RA and controls
Controls (n=20)Patients with RA (n=22)
Vitamin A (µg/dl)77.2±12.253.6±8.1**
Vitamin E (µg/ml)8.3±1.76.0±1.4**
Vitamin C (µg/ml)8.8±2.16.2±1.7**
MDA, malondialdehyde; SOD, superoxide dismutase; GSH-Px,
glutathione peroxidase; Data are mean±SD.
P*<0.05, **<0.005 compared to controls
INDIAN J MED RES, OCTOBER 2003
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Reprint requests : Dr Fikret Karatas, Assistant Professor, Department of Chemistry, College of Science,
Firat University, 23119 Elazig, Turkey
e-mail : firstname.lastname@example.org
KARATAS et al : OXIDANT & ANTIOXIDANT STATUS IN RHEUMATOID ARTHRITIS