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Keywords:

  • malondialdehyde;
  • nitric oxide;
  • oxidative stress;
  • rheumatoid arthritis;
  • vitamin E

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. METHODS
  5. RESULTS
  6. DISCUSSION
  7. REFERENCES

Aim:  To assess the oxidative stress status in rheumatoid arthritis by measuring markers of free radical production, systemic activity of disease, free radical mediated tissue destruction and levels of antioxidant.

Methods:  Peripheral blood samples were used for all the assays. Total nitric oxide (NO) was quantitatively measured using immunoassay kit. Malondialdehyde (MDA) and vitamin E were measured by spectrophotometric methods.

Results:  Statistically significant changes were observed in the levels of MDA, vitamin E, total NO and erythrocyte sedimentation rate (ESR) in the patient group. Significant differences were also observed in ESR and vitamin E levels in patients with active disease.

Conclusions:  Increased oxidative stress status exists, which may lead to connective tissue degradation leading to joint and periarticular deformities in rheumatoid arthritis.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. METHODS
  5. RESULTS
  6. DISCUSSION
  7. REFERENCES

Rheumatoid arthritis (RA) is a systemic disease manifesting itself as a progressive, erosive chronic polyarthritis with intermittent acute inflammatory episodes. Oxidant stress generated within an inflammatory joint can produce autoimmune phenomena and connective tissue destruction in rheumatoid synovitis.1

Radical species with oxidative activity, which include reactive nitrogen species (RNS) and reactive oxygen species (ROS) represent the mediators and effectors of cartilage damage.2,3 Reactive oxygen species are involved in extracellular matrix degrading activity.4 RNS are derived from the oxidation of the guanido nitrogen of L-arginine, with production of a nitrogen-centred radical, nitric oxide (NO), by the enzyme nitric oxide synthase (NOS). Nitric oxide is known to have several physiological roles, including the regulation of platelet function, neurotransmission and the killing of intracellular pathogens.5–8 The oxidative stress related to reactive nitrogen and oxygen intermediates was evaluated in this study.

Growing evidence implicates NO in immune regulation, inflammation, autoimmunity and arthritis. Several studies suggest that tissue injury in inflammation involves NO production.9,10 Increased levels of NO in serum and synovial fluid have been reported in patients with rheumatoid arthritis (RA), ankylosing spondylitis (AS) and osteoarthritis (OA).11 It has been suggested earlier that ROS produced inside the joints may also contribute significantly to the pathogenesis of arthritis, since these inorganic oxidants are able to degrade matrix components by direct action or by indirect activation of latent collagenases.12

Because of the highly reactive nature of ROS and RNS, it is difficult to directly demonstrate their presence in vivo. It is considerably more practical to measure the ‘footprints’ of ROS and RNS, such as their effects on various lipids, proteins and nucleic acids. Under aqueous, aerobic conditions, NO spontaneously oxidizes to its inactive, stable end products nitrite and nitrate.5–8,13 The total nitrite and nitrate levels in plasma were determined by the Griess assay as an estimate of total nitric oxide radical formation.14 Previous studies have demonstrated oxidative damage to hyaluronic acid,15 lipid peroxidation products,16,17 oxidized low-density lipid proteins (LDL)18 and increased carbonyl groups reflective of oxidative damage to proteins.18,19

Lipid peroxidation has been implicated in the pathogenesis of cancer, atherosclerosis, degenerative diseases and inflammatory arthritis. During lipid peroxidation, polyunsaturated fatty acids (PUFAs) are oxidized to produce lipid peroxyl radicals that in turn lead to further oxidation of PUFA in a perpetuating chain reaction that can lead to cell membrane damage. In recent years, the measurement of isoprostanes in plasma and urine has emerged as a new marker of oxidative stress. Isoprostanes are stable end products of lipid peroxidation derived from arachidonic acid.20 In this study, a thiobarbituric acid reactive substance (TBARS), malondialdehyde (MDA) which is a byproduct of lipid peroxidation is measured as an estimate of the level of reactive oxygen intermediate radicals. Matrix degradation arising from cytokine-stimulated chondrocytes was shown to be primarily due to lipid peroxidation and to be preventable by vitamin E, the primary anti-oxidant for lipids.21 Endogenous antioxidants protect cellular systems from the damaging effects of ROS and reactive nitrogen species. Vitamin E (α-tocopherol) has lipid-soluble properties that allow it to act as a chain-breaking reagent in lipid peroxidation.22 Vitamin E, intercalated in cellular membranes plays an important role in reducing lipid hydroperoxides and is probably the best hydrophobic scavenger known.23

Rheumatoid arthritis is a condition known to be dependent on environmental factors and highly influenced by genetic composition. The data available about this disease condition and the biochemical aspects in our population is very minimal. Hence to establish baseline characteristics for our population, we formulated this study.

METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. METHODS
  5. RESULTS
  6. DISCUSSION
  7. REFERENCES

Study samples

One hundred and twenty patients with RA who attended the Rheumatology Clinic at Sri Ramachandra Hospital, Porur, Chennai, India were chosen for the study. Selection of patients was based on the revised American College of Rheumatology classification criteria.24 In addition 50 healthy volunteers served as a control group. The sample size considered in the study was obtained with a statistical power of 100%.

Study protocol

Informed consent was obtained from all the study subjects. The study was approved by the institutional ethics committee. Exclusion criteria were smoking, alcohol intake, narcotic drugs, hypertension, diabetes mellitus, hypothyroidism, hyperthyroidism and any other form of arthritis except RA. Patients on other lines of treatment such as ayurveda, homeopathy and siddha were also excluded from this study.

The number of swollen joints, tender joints and visual analogue score (VAS) of the selected patients were recorded with the help of a rheumatologist. Disease activity score (DAS28) was calculated using a universally accepted formula (Table 1).

Table 1.   Baseline characteristics of study samples
 Rheumatoid arthritis patientsControls
Total number of samples120 (12/108)50 (20/30)
Age in years
 Mean ± SD42.4 ± 11.538.4 ± 9.0
 Range 20–7325–60
Sex
 Male (n, %) 12, 10%20, 40%
 Female (n, %)108, 90%30, 60%
Duration of disease (years) 4.91 ± 5.47 –
Visual Analog Score (mm) 55.09 ± 17.29 –
Number of tender joints 4.61 ± 3.84 –
Number of swollen joints 2.44 ± 2.98 –
Disease Activity Score (DAS28) 4.82 ± 1.03 –

Five millilitres of peripheral blood was collected in lithium–heparin vacutainers. Plasma was separated by centrifugation and biochemical parameters. MDA and vitamin E were assayed within 24 h. Plasma samples for total NO were stored at –70°C until the analysis.

Two millilitres of peripheral blood was collected in potassium–edetic acid (EDTA) vacutainers and used for erythrocyte sedimentation rate (ESR) estimation.

The patient and control samples were coded during collection and blinded during the actual investigation procedures.

Laboratory analyses

Erythrocyte sedimentation rate was measured using disposable EaSeR tubes by Westergren method. For men, an ESR > age/2 and for women, an ESR > (age + 10)/2 has been accepted as criteria for the systemic activity of disease (SAD).11 Quantitative determination of total NO in samples was performed using a total nitric oxide assay kit (Assay Designs, Anne Arbor, MI, USA). Plasma MDA and vitamin E were estimated by methods described earlier.25

Statistical analyses

Statistical analyses were done using standard statistical procedures. Data in tables are expressed as mean ± standard deviation (SD). Student's t-test and Pearson correlation tests were used as appropriate statistical methods for analyses. Differences were considered significant at P ≤ 0.05.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. METHODS
  5. RESULTS
  6. DISCUSSION
  7. REFERENCES

The levels of MDA were found to be significantly increased in the patient group, whereas the levels of vitamin E were found to be significantly decreased in the patient group in comparison with the controls (P < 0.001). A negative correlation was observed between MDA and vitamin E levels in the control group (r2 = 0.268). A statistically significant increase in the levels of total NO was also observed in the patient group when compared to the controls (P < 0.01). The increase in the levels of ESR in the patient group was found to be highly significant (P < 0.001) (Table 2).

Table 2.   Comparison of the status of free radicals and antioxidant in rheumatoid arthritis (RA) patients and controls
ParameterRA patientsControlsP-value
  1. Values are represented as mean ± SD. ESR, erythrocyte sedimentation rate; NO, nitric oxide; MDA, malondialdehyde.

ESR (mm, 1 h)59.66 ± 31.3123.35 ± 16.78P < 0.001
Total NO (µmole/L)105.11 ± 89.5575.97 ± 54.73P < 0.01
MDA (nmol%)169.62 ± 56.53124.17 ± 49.12P < 0.001
Vitamin E (mg%)0.46 ± 0.240.85 ± 0.32P < 0.001

A statistically significant increase was observed in ESR in RA patients with active disease in comparison with those with inactive disease (P < 0.001). A significant decrease was recorded in the levels of vitamin E in the active disease group compared to the inactive disease patients (P < 0.001). The levels of total NO were found to be increased in the active disease group (Table 3). No correlations were observed among these parameters in both the groups. DAS28 was correlated with the levels of total NO, MDA and vitamin E levels. However, no correlation was obtained between DAS28 and the measured parameters.

Table 3.   Status of free radicals and antioxidant in patients with active and inactive disease
ParameterActive RAInactive RAP-value
  1. Values are represented as mean ± SD; RA, rheumatoid arthritis; M, ale; F, female; ESR, erythrocyte sedimentation rate; NO, nitric oxide; MDA, malondialdehyde.

Number (M/F) 101 (7/94)     19 (5/14) 
ESR (mm, 1 h)67.32 ± 28.0018.95 ± 6.56P < 0.001
Total NO (µmole/L)102.86 ± 85.7258.91 ± 48.25P > 0.05
MDA (nmol%)167.93 ± 53.96178.58 ± 69.65P > 0.01
Vitamin E (mg%)0.43 ± 0.190.64 ± 0.34P < 0.001

DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. METHODS
  5. RESULTS
  6. DISCUSSION
  7. REFERENCES

In this study, parameters such as total NO as a marker of free radical production, plasma MDA as a marker of free radical mediated tissue destruction, vitamin E, a lipid soluble antioxidant and ESR as a marker of systemic activity of disease, were assayed to gain insight into the oxidative stress status in RA.

Nitric oxide is known to play an important role in autoimmunity and inflammation. The role of NO in the cytotoxic mechanism of activated macrophages, inhibition of iron-sulphur-centred enzymes and their antiproliferative effects were hypothesized and studied by Clancy et al.8 Another substantial source of NO in RA was found to be the articular chondrocytes and synovial fibroblasts.26 In inflamed joints, cells such as neutrophils, lymphocytes, mast cells and macrophages could result in production of NO. These findings clearly suggest increased endogenous NO synthesis in RA, RNS and ROS production by the synovium and also by other tissues. Our results of increased NO levels corroborate with the existing published data.

The possible cause for the increased levels of NO is not very clear. Enhanced synovial inflammation may result in increased serum levels of NO when the synovial fluid enters the systemic circulation and equilibrates with the vascular compartment within the synovium. Another possible source of increased NO is the systemic vasculature and other cells in which the induction of NO has been shown.27

Positive correlations between serum nitrate/nitrite concentrations and SAD variables, parameters of clinical presentation and severity of disease have been shown in patients with RA.28,29 In our study, we did not observe any correlation between NO and ESR, indicating that ESR may not be a good SD variable when measured as a stand-alone parameter.26

Oxidative damage of lipids, especially PUFAs result in formation of lipid hydroperoxides and conjugated dienes which further decompose to form several byproducts including alkanols, alkenals, hydroxyalkenal, volatile hydrocarbons and MDA. In this study, plasma MDA was found to be significantly increased (P < 0.001) in RA patients in comparison to the controls. Several researchers have reported similar findings and a probable mechanism for such elevation has also been suggested. In the inflamed joint, hypoxia-reperfusion occurs which may result in increased lipolysis. During this process, some lipids may get oxidatively modified by free radicals and removed by macrophages.30 MDA may also be produced as a byproduct of thromboxane synthesis, degradation of endoperoxides and cycloxgenase reactions.31 These processes involving MDA production could form the basis of elevation of MDA observed in this study.

Patients with inflammation of the synovial membrane respond relatively frequently by a reduction of indicators of antioxidants and antioxidant enzymes.32 Researchers suggest that a defense reaction of the organism is obviously involved against the increased formation and action of free radicals in the inflamed articular lining.

Several studies have clearly indicated the possibility that these changes observed in the oxidative stress parameters in RA patients may be considered to be an accurate reflection of the synovial and articular changes.32–35 In a study conducted by Chaturvedi et al. MDA was suggested to be a valid biomarker of free radical-mediated tissue destruction in RA.36

Our results are in agreement with other recent studies that indicate that oxidative stress generated within an inflamed joint can produce connective tissue destruction leading to joint and periarticular deformities in RA.27,30,37–39 The level of oxidative stress is found to be much higher in patients with active disease. Further indepth knowledge about this aspect of RA may lead to therapeutic protocols involving correction of oxidative stress levels.

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. METHODS
  5. RESULTS
  6. DISCUSSION
  7. REFERENCES
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