Serum malondialdehyde as a marker of oxidative stress in rheumatoid arthritis

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Background
Rheumatoid Arthritis (RA) is a chronic autoimmune inflammatory illness that mostly affects the small joints and leads to chronic progressive joint destruction [1].Although the cause of RA disease is unknown, reactive oxygen species (ROS) are considered a major factor in the disease's pathogenesis [2].Aging accelerates the course of RA, mostly because of systemic inflammation, and overproduction of ROS can exacerbate cell damage and a loss in antioxidant capacity [3].In RA patients, the oxidative stress mechanism appears to be highly correlated with inflammation and joint destruction [4].
In oxidative stress, the amount of ROS in the body rises gradually due to increased production, lowered antioxidant defenses, or a combination of the two.This impairs redox signaling and compromises the regulation of molecular damage [5].
The main attack of ROS is the unsaturated fatty acids.They damage nucleic acid, connective tissue, and the cell membrane's lipids [6].
The inflammatory response's fast cell division in RA results in a hypoxic synovial milieu and elevated intraarticular pressure.This results in repeated cycles of hypoxia and reoxygenation, which raises the formation of ROS through cellular oxidative phosphorylation.Furthermore, this could activate transcription factors like NF-κB and hypoxia-inducible factor-1α (HIF-1α), which respond to oxygenation.This will result in a range of gene expression that continues to cause synovitis [7].Increased production of IL-1 and TNF-α may be implied by the ROS-induced NF-κB pathway activation, which would provide positive feedback in a self-activation loop between the proinflammatory cytokines [8].
Oxidative stress-induced somatic mutations in fibroblast-like synoviocytes (FLS) may be involved in synovial hyperplasia and pannus formation, an overly proliferating synovial membrane with invasive behavior, together reinforce the oxidative damage in RA and directly contribute to the erosion of bone and cartilage destruction [3].
Reactive aldehydes are produced by ROS-triggered lipid peroxidation, an oxidative degradation process of polyunsaturated fatty acids [9].Lipid peroxidation's primary aldehyde byproduct is the 3-carbon dialdehyde species malondialdehyde (MDA) which can be processed by enzymes or react with proteins and tissues to generate adducts (MDA-modified macromolecules) that cause biomolecular damage [10].MDA, a reactive aldehyde, is one of the numerous reactive electrophile species that induce toxic stress in cells [11].
Thus, new complementary treatment options for controlling RA-disease activity may be provided by antioxidant therapy [12].
The utilization of oxidative stress markers has emerged and has recently received greater interest.It is a promising additional means of assessing RA patients' prognosis and extent of activity [13].
The study aimed to assess serum MDA levels among RA patients concerning disease activity as an indicator of oxidative stress.

Patients and methods
Twenty active RA patients achieved the 2010 American College of Rheumatology/ European League Against Rheumatism (ACR/EULAR) classification criteria included in this cross-sectional case-control study [14].The study also included 20 age and gender-matched controls.
Exclusion criteria: Diabetes mellitus, history of liver diseases, renal dysfunction, coronary artery disease, history of malignancy, pregnancy, smoking, and hyperlipidemia.Every participant gave their written consent.The study received ethics committee approval.Each participant completed the following: 1) Complete medical history including disease duration, morning stiffness duration, articular symptoms (swelling and painful joints), and extra-articular manifestations (as; Rheumatoid nodules, vasculitis, dyspnea, cough, expectoration, hemoptysis, chest pain).2) Clinical assessment with particular attention to joint examination for tenderness, warmth, effusion, edema, crepitus, range of motion, and joint deformities.3) Disease Activity Score (DAS28) is applied to determine disease activity [15].4) Measurement of complete blood counts (CBC), erythrocyte sedimentation rates (ESR), C-reactive protein (CRP), Rheumatoid Factor qualitative and quantitative, and Anti-Cyclic Citrullinated (Anti-CCP).

5) Quantitative Assessment of serum MDA by Double
Antibody Sandwich ELISA technique (Bioassay Technology Laboratory Co., Zhejiang, China) according to the manufacturer's protocol.Five milliliters of blood were drawn, centrifuged, and taken supernatant placed in aliquots at -20℃/-80℃ (1-3 months) for storage till measurement.

Statistical evaluation
Statistical Package for Social Science (IBM SPSS) version 20 was used to gather and analyze data.Quantitative data was shown as means, standard deviations, and ranges when their distribution was parametric, whereas qualitative data were expressed as numbers and percentages.The Chi-square test was employed to compare two groups' qualitative data, and the independent t-test was used to compare two independent groups utilizing quantitative data and a parametric distribution.The one-way ANOVA test compared more than two independent groups utilizing quantitative data and a parametric distribution.Total predictivity and the ideal cut-off value for detecting sensitivity and specificity at this cut-off value were determined by receiver operating characteristic (ROC) curve analysis.P-values less than 0.05 were considered significant, whereas P-values more than 0.05 were considered insignificant.

Results
Twenty active RA patients, ages 21 to 57, participated in our case-control study, with a median illness duration of 7 (4-15) years and a mean age of 39.25 ± 10.96.Furthermore, twenty controls were enrolled, ages 30 to 56, with a mean age of 44.10 ± 8.54.In most cases, the disease activity was high (85%), with a mean ± SD of 6.67 ± 1.09.
Comparing the CRP and ESR of the investigated cases to the control group, the RA patients showed statistical differences (Tables 1 and 2).Table 3 indicates a highly significant increase in the serum concentrations of MDA in RA patients.Higher sensitivity (100%) and specificity (100%) were observed for serum levels > 16.21 nmol/ml in sera's RA patients by the ROC curve (Fig. 1).
Table 4 illustrates the relationship between serum MDA levels with morning stiffness, clinical examination, laboratory investigations, and disease activity from RA patients.The morning stiffness (p = 0.016), ESR (p = 0.008), CRP (p = 0.021), and DAS28 (p < 0.001) all exhibited statistically significant positive correlation with serum MDA.We didn't find any statistical significance with disease duration (Years), tender joint count, swollen joint count, or visual analog scale (VAS).
Table 5 demonstrated a significant positive correlation between MDA and morning stiffness.There was no statistically significant correlation between MDA and family history, ocular manifestation, oral manifestation, pulmonary manifestation, neurological manifestation, deformities, DMARDS, and biologics.

Discussion
In RA patients, numerous indicators unique to joint metabolism have been studied.The most popular longterm biomarkers for assessment of RA severity and disease activity nowadays are the acute phase protein (ESR, and CRP).Still, there are differences in the relationship between CRP and ESR levels and clinical inflammation [16].
Targets of oxidation include lipids.Lipid peroxidation produces a range of somewhat stable breakdown products which may serve as indicators of oxidative stress [17].Consequently, the proinflammatory cytokines in RA positively correlate with blood levels of MDA, a measure of lipid peroxidation [4].
The study aimed to assess serum MDA level as a marker of oxidative stress in RA patients concerning disease activity.This case-control study was conducted on 20 RA patients and 20 healthy age and sex-matched controls.
The morning stiffness mean in the RA group was 48.75 ± 12.76 min and the patients having morning stiffness ≥ 1 h were 50%.The extra-articular manifestations (EAMs) presented as 35% of cases suffered from ocular manifestation, 30% suffered from oral manifestation, 5.0% suffered from pulmonary manifestation, 5% suffered from neurological manifestation, and 40% suffered from deformities.
Diabetes mellitus, history of liver diseases, renal dysfunction, coronary artery disease, history of malignancy, pregnancy, smoking, and hyperlipidemia.All of these are risk factors associated with increased levels of malondialdehyde.We excluded all of them to find the role of   increased oxidative stress in RA pathogenesis without any comorbidities.Patients in the current study showed disease activity with a DAS28 score's mean of 6.673 ± 1.09, where 17 patients (85%) had high disease activity and 3 patients (15%) had moderate disease activity.In another study by Azzeh and Kensara, the mean of DAS28 was 3.79 ± 0.87 with 16.7% having high disease activity and around 54.9% and 28.4% having moderate and low disease activity, respectively, this may be due to the greater number of patients included in this study which was 102 patients while our study was 20 patients only [19].
The current study showed that inflammatory markers, ESR and CRP, demonstrated a notable degree of variation, with a wide range suggesting ongoing inflammation Table 5 Relation between MDA and Morning stiffness, Family history, Extra-articular manifestation, Deformities, and treatment P-value > 0.05: Non-significant (NS); P-value < 0.05: Significant(S); P-value < 0.01: highly significant (HS)   where ESR ranged from 10-113 while the CRP ranged from 0.40-128.In the study of Ozkan et al. [20], the RA patients had elevated both ESR and CRP where the ESR mean was 38.8 ± 23.5 and the CRP mean was 15.5 ± 12.5.
Although CRP and ESR are frequently used to assess disease activity and direct treatment, both have drawbacks.According to a prior study, the sensitivity of.
CRP and ESR in detecting synovial inflammation in correlation to histology are 71.3% and 64.1%, respectively [21].Therefore, finding different markers of inflammation might enhance the treatment of RA patients and control their disease activity.
The current study showed a higher increase of serum MDA (nmol/ml) among the RA group than the control group with a mean of 60.81 ± 16.00 nmol/ml vs 9.29 ± 2.98 nmol/ml respectively, which indicates high oxidative stress in the RA.Previous studies suggested that serum MDA levels in RA significantly increase [22][23][24].So, we recommended adding antioxidant therapy as an adjuvant/complementary treatment option to control disease activity in RA.
The current study showed a statistically significant positive correlation between MDA and morning stiffness, DAS28 Score, ESR, and CRP which agrees with the results of El-barbary et al. [25].
The current study showed no statistical significance between MDA levels and DMARDs or biologics.This agrees with Herman et al. [26] who stated that methotrexate most likely exerts its immunosuppressive effects by induction of apoptosis through oxidative stress, and the serum MDA was not affected by anti-TNF-α biologic therapy.
The receiver operating characteristic curve (ROC) shows that the best cut-off point of MDA to detect the RA group was > 16.21 nmoles/ml with a sensitivity of 100%, specificity of 100.0%, and total accuracy of 100.0%.

Limitation of the study
The limitation of our study is that it runs a high risk of research biases, and the small number of patients in the study; so, a wide scale of patients is important to confirm the sensitivity and specificity of MDA, and lack of followup which is necessary to confirm the correlation of MDA to disease activity and the effect of treatment whether traditional or biological.

Conclusion
Statistically higher MDA among RA patients and a significant positive correlation with ESR, CRP, DAS-28 score, and morning stiffness.So, MDA measurement may be a good marker for RA activity and oxidative stress in RA patients.

Fig. 1
Fig. 1 Receiver operating characteristic (ROC) curve for serum levels of MDA in RA patients (N = 20) and in healthy controls (n = 20)

Table 2
Comparison between studied groups regarding demographic data

Table 3
Serum MDA levels in RA patients and controls Using: t-Independent Sample t-test for Mean ± SD; **p-value < 0.001 is highly significant (HS)

test MDA Test value P-value Sig Mean ± SD Range
a One Way ANOVA Test, b Independent t-

Table 4
Correlation between serum MDA levels and clinical, laboratory, and disease activity in RA patients CRP C-reactive protein, ESR Erythrocyte sedimentation rate, Anti-CCP Anti-Cyclic Citrullinated peptide, HDL High density lipoprotein, LDL Low-density lipoprotein,