SERUM MALONDIALDEHYDE AS PREDICTOR OF OXDATIVE STRESS IN EARLY RHEUMATOID ARTHRITIS.

Objective: To measure the concentration and role of malondialdehyde to determine oxidative stress in rheumatoid arthritis patients and comparing them with healthy individuals. Methodology: This cross sectional study was conducted on 180 patients of age 49 to 70 years and both gender. Study was completed in the department of physiology Sheikh Zaid Medical College, Quid e Azam Medical college and Lahore General Hospital, from October 2017 to October 2018. Study was started after obtaining ethical approval and informed consent from the patients. SPSS version 23 was used to analyze data related to study. Continuous variables were presented as mean and standard deviation like age and Serum MDA concentration, categorical variables were presented as numbers and percentages like gender. Post operative chi square test was applied and p value less than or equal to 0.05 was considered as significant. Results: Overall, there were 100% (n=180) patients were included, in this study. The study group was further divided into two equal groups, 50% (n=90) in each, i.e. Rheumatoid arthritis (RA) group and control group. RA group patients include those diagnosed as rheumatoid arthritis clinically and through lab investigations and control group consist of normal healthy adults. The serum MDA levels in the RA patients were 3.97±1.03 nmoles/ml and it was 1.59±0.32 nmol/ml in control patients. The difference was statistically significant (t=20.87, p=0.001). Conclusion: the observations of our study showed statistically significant raised serum Malondialdehyde level as compared to control group. Keywords: Malondialdehyde, Rheumatoid arthritis, Lipid per oxidation, Free radicals. DOI : 10.7176/JMPB/55-18 Publication date :May 31 st 2019

Malondialdehyde (MDA) is a main contributing factor of DNA mutation in human and bacterial cells, it produces as a result of lipid peroxidation and also from biosynthesis of prostaglandins. MDA also reported as carcinogenic agent as reported in some studies conducted on rats. It was also reported that in Salmonella typhimurium MDA responsible for their gene mutation by affecting base substances. In Escherichia coli replication was observed on single standard M13 genome mutation (G→T, A→ G, and C→ T). These three modifications are possible in DNA as a result of MDA involvement. It was separated from tissues of healthy humans in form of M1G which was produced as a result of reaction with guanine 2 .
Cytokinesand T cells in combination with oxygen radicals and hydrogen peroxide plays major role in progressive activity of macrophages and enhancement of Rheumatoid Arthritis. Nitrogen and oxygen species have both types of effects beneficial and harmful 3 . Increased level of malonaldehyde (oxidative stress) is stage when reactive nitrogen and oxygen reaches to a damaging range and harm the cells and other biological body markers. When body's mechanism of antioxidants and oxidative stress become imbalance it can cause serious autoimmune diseases like Rheumatoid Arthritis 4 . Oxygen species in reactive form plays an important role in RA, main attack of these reactive oxygen molecule is unsaturated fatty acids.
They damage nucleic acid, connective tissue and lipids of cell membrane, as a result of these damage free radicals and their result products cause inflammation by realizing mediators 5 . In synovial joints, synovial fluids have chemo attractant property; this property leads to the anaerobic glycolysis and enhancement the oxygen consumption. This whole activity generates hydroxyl, hypochloricradicals and superoxide. Among all these components neutrophils are found to be active in synovial fluid and cause erosion when Rheumatoid Arthritis develops 6 .
In lipid peroxidation malondialdehyde is an important factor and hallmark of Rheumatoid Arthritis diagnosis 7 . In recent studies it was observed and suggested that early and right management of malondialdehyde reduced and delayed the incidence of Rheumatoid Arthritis. Vyas S et al 8 conducted a study in 2016 and observed a significant increase in malondialdehyde level in serum of Rheumatoid Arthritis patients; p value was 0.0001 which is significant. Determine the serum malondialhyde level in Rheumatoid Arthritis patients and to compare with normal and healthy patients is objective of our study 9 .

Methodology:
This cross sectional study was conducted on 180 patients of age 49 to 70 years and both gender. Study was completed in the department of physiology Sheikh Zaid Medical College, Quid e Azam Medical college and Lahore General Hospital, from October 2017 to October 2018. Study was started after ethical approval from institutions committee of ethics. Patients were divided into two equal groups (90 patients in each group). Non probability consecutive sampling technique was used. Sample size was calculated by using openepi.com an online source for sample size calculation. Complete physical examination, routine laoratoryand radiological investigations was done. Rheumatoid arthritis was confirmed by X ray, c reactive protein, antinuclear antibody and rheumatoid arthritis factor. Patients of normal healthy diet, no history of nutritional supplement and post and pre menopausal women were included in the study. Patients with known history of diabetes, hypertension, smokers, alcoholic, history of anti inflammatory dugs and any trauma to joint were excluded from the study. With all antiseptic measures 5 ml blood was drawn from anticubetal vein and sent to laboratory for laboratory investigation. Sampling tube was opened with care to avoid hemolysis. Blood was allowed to clot at room temperature for some time than serum was drawn after centrifugation (centrifugation was done at 3000 rpm for 15-20 minutes in morning shift. Hemolytic samples were discarded. Thiobarbituric acid assay test was used to estimate MDA concentration. UV-VIS Spectrophotometer was used to calculate optical density. Thiobarbituric acid assay method measures Malondialdehyde by measuring aldehyde products of lipid peroxide. Exact mechanism behind this procedure is reaction of two molecules of thiobarbituric acid and one molecule of Malondialdehyde to make MDA-TBA red product which can be measured at 535 nm.
SPSS version 23 was used to analyze data related to study. Continuous variables were presented as mean and standard deviation like age and Serum MDA concentration, categorical variables were presented as numbers and percentages like gender. Post operative chi square test was applied and p value less than or equal to 0.05 was considered as significant.
The serum MDA levels in the RA patients were 3.97±1.03 nmoles /ml and it was 1.59±0.32 nmol/ml in control patients. The difference was statistically significant (t=20.87, p=0.000). (Table. 2). In our study we found that serum Malondialdehyde level was increased in patients of rheumatoid arthritis, similar results were reported by Lunec et al in his study, he revealed that serum uric acid concentration found to be increased in serum and synovial fluid of RA patients 19 . Another study was conducted by Gambriz et al in 1997 on topic of serum uric acid level in RA patients and reported that increase in serum uric acid concentration are the cause of RA specifically in aged patients 20 . Another similar study was conducted by Chaturvedi et al 1990 and reported similar finding that serum uric acid was increased in RA patient's blood. Findings of these studies are comparable with our findings and goes in favor of our results 21 .

Conclusion:
the observations of our study showed statistically significant raised serum Malondialdehyde level as compared to control group.