Study of serum traditional and nontraditional biomarkers in Rheumatoid arthritis patients

The study of biomarkers in Rheumatoid arthritis (RA) is highly indispensable to understand mechanisms of pathogenesis, diagnosis, prognosis, and treatment of disease. The role of traditional biomarkers like Anti-CCP, RF, and in lammatory markers like ESR and CRP is well established. In this study, we aimed to measure nontraditional biomarkers like Hyaluronic acid (HA), Cartilage oligomeric matrix protein (COMP), and Osteocalcin in the serum of RA patients and also to establish an associationwith traditional markers. It was a cross-sectional study involving 152 RA patients based on the 1987 ACR criteria for the diagnosis of RA and 68 ageand sex-matched healthy controls. After the clinical examination, traditional markers were assessed to measure the disease activity along with non traditional markers in RA patients. All the valueswere expressed asmedian (25th–75th percentile). In our study, therewas a signi icant increase in serum HA levels in RA patients compared to healthy controls (p < 0.03), whereas no signi icant difference in serum COMP and osteocalcin levels. The traditional in lammatory markers were signi icantly increased in RA patients than controls with (p < 0.001). The serum HA levels were signi icantly correlated with traditional markers in RA patients. Conclusion: Signi icant increase in serum HA level in RA patients indicating synovial in lammation, but there were no notable changes in COMP and osteocalcin level in serum presuming the combination of these markers may be useful along with traditional markers in the different stages of RA.


INTRODUCTION
Rheumatoid arthritis (RA) is a chronic in lammatory systemic disease of unknown etiopathogenesis.
The course of the disease is variable among patients with RA. A substantial population of RA patients exhibits symptoms like poly-articular in lammation associated with synovitis, osteitis, periarticular osteopenia, and erosion of subchondral bone eventually leads to narrowing of joint space. These features commonly lead to progressive joint damage, impaired function, and permanent disability in these patients (Diarra et al., 2007). Arthritis is established as a devastating disorder resulting in excruciating pain, impairment, and dysfunction among patients having an injury to the synovium, cartilage, and bone of the affected joints. Based on a clinical presentation at the late stages of arthritis, many methods to diagnose and monitor the progression of the disease. So pertinent interventions in the early stages with effective treatment modalities can change the course and progression of the disease, minimize functional disability that can improve the standard of living. Therefore, ef icient biomarkers are needed for the early diagnosis and to monitor the prognosis of rheumatoid arthritis (Schett et al., 2005).
However, the majority of the chronic diseases have a standard test for diagnosis. On the contrary, quality, trustworthy laboratory test for RA diagnosis still elusive. The American College of Rheumatology (ACR) in 1987 and New Rheumatology criteria in 2010 used to de ine and classify / diagnosis RA internationally (Aletaha et al., 2010). By estimating the biomarkers of cartilage damage and repair, it is possible to better understand the variations in joint remodelling and monitor RA disease progress. COMP is one such biomarker, which is also known as thrombospondin 5, belongs to the family of pentameric calcium-binding proteins. Even though the exact function is not known, it plays an important structural role by interacting with collagen ibrils and matrix components in endochondral ossi ication and stabilization of the extracellular matrix, (Posey et al., 2018).Studies proved that COMP is detected in serum as well as synovial luid of RA and osteoarthritis (OA) patients (Morozzi et al., 2007).
Hyaluronic acid (HA) is a glycosaminoglycan made up of repeated units of N-acetyl glucosamine and glucuronic acid. It is an important constituent of synovium and cartilage, rendering the lubricating mechanism of synovial luid (Engstrom-Laurent and Hallgren, 1985). Many studies in the past proved that high volume of HA are produced locally from in lamed synovium of patients with different rheumatic diseases, including rheumatoid arthritis (Al-Dalaen et al., 2016).
One increasingly apparent thing is the bone loss at an accelerated rate in RA patients. Though the exact mechanisms are poorly understood, osteoporosis is part of clinical symptoms in the diagnosis of RA (Gevers et al., 1986). Bone formation and resorption is a tightly coupled continuous process happening all through life for remodelling of bone. Osteocalcin, also called bone Gla protein, is present in non-collagenous bone matrix. It is produced by osteoblasts re lecting the bone formation status. It is an established biochemical marker that is speci ic for bone metabolism most likely speculate bone formation (Nagaya et al., 1999).

Aim And Objectives
Thus, the aim of the study is to 1. To measure the serum concentrations of nontra-ditional biomarkers like HA, COMP, and Osteocalcin as cartilage and bone markers along with traditional in RA patients 2. To compare the serum traditional and nontraditional biomarkers in RA and healthy individuals and assess the relationship between these markers.

MATERIALS AND METHODS
It is a case-control study, including a total of 152 RA patients (75 early RA with the duration of disease less than two years and 77 late RA with more than two years from the onset of initial symptoms), with ages between 23 and 70 years attending the Rheumatology Clinic at Sri Ramachandra Medical College and Research Institute, Chennai from October to December 2010. The primary inclusion criterion was de inite RA ful illing the 1987 American College of Rheumatology criteria and disease duration of 6 months onward. The control group comprised of 68 age and sex-matched healthy volunteers with no other forms of in lammatory rheumatic disease. The exclusion criteria for both the groups included physiological statuses such as pregnancy, lactation, and individuals with habitual smoking and alcohol consumption excluded from our study. We also excluded individuals with known infectious diseases and other diseases such as diabetes mellitus, hypertension, thyroid dysfunction, neurological disorders, cancer, secondary osteoarthritis, any other forms of autoimmune diseases, and types of arthritis from the study.
All these RA patients met the new 2010 RA classi ication criteria retrospectively. Under the guidance of a rheumatologist, we recorded the values of physical activity, Visual Analog Score (VAS), disease duration, number of swollen and tender joints obtained by the personal interview of all the patients along with relevant clinical data, and the treatment history was collected using the Health Assessment Questionnaire (HAQ). We made sure that by selecting patients, who were under uniform dosage of nonsteroidal anti-in lammatory drugs and diseasemodifying antirheumatic drugs as per treatment protocol without in luencing our objective of the study. For the biochemical markers, 5 mL of fasting blood sample with and without anticoagulant were collected. The serum sample was collected and kept at a temperature of −70 • C for further biochemical analysis. The patients and controls were similar in ethnicity and nutritional habits. Other than Anti-CCP and RF autoantibodies, the conventional markers in RA consisted of the various Disease Activity Scores (DAS) which include compos-ite parameters such as counts of tender and swollen joints and levels of serum CRP or ESR were included in our study. Traditional markers of in lammation, like ESR and CRP, were analyzed using standard laboratory methods. The biochemical markers analyzed were anti-CCP, RF, and HA, COMP, and Osteocalcin in both patients and controls by in vitro quantitative ELISA method. There is no involvement of any kind of in vivo experiments or clinical trials on humans or animals in our study. Our study protocol (MEC/06/51/23) was approved by the Research ethics committee of Sri Ramachandra medical college and research institute, Chennai. All patients gave written informed consent before their enrolment in this study.

Statistical analysis
Statistical analysis in this study was performed using the SPSS ® statistical program version 16.0. Because most of the variables did not follow a normal distribution, all the statistical analyses had done using nonparametric tests. In the study group, non normally distributed quantitative variables were presented as median (interquartile range). Comparison of biochemical marker concentration between groups was calculated by nonparametric signi icant Kruskal-Wallis test, followed by Bonferroniadjusted Mann-Whitney test for unpaired differences. Spearman's Rho test and P ≤ 0.05 were considered statistically signi icant when we calculated the correlation between different parameters.

RESULTS AND DISCUSSION
The demographic data, including disease activity measures and serum biomarker levels, were compared between the rheumatoid arthritis patient group and the control, as shown in Table 1, and comparison of serum levels of biochemical markers between RA cases and controls (Table 2 ). Correlation analysis was performed between serum nontraditional markers like HA, COMP, and Osteocalcin with traditional markers like RF and Anti-CCP, as shown in Table 3. Figures 1 and 2 shows a signi icant increase of (p<0.001) Anti-CCP and RF between RA patients and normal control group respectively. A signi icant increase in HA between RA and normal is explained in Figure 3. We observed a significant positive correlation (p<0.01) between serum HA and anti-CCP levels in RA patients (Figure 4), and another Figure 5 shows a signi icant negative correlation (p<0.05) between COMP and anti-CCP level in the serum of RA patients.
The synovial proliferation of cartilage and bone is a characteristic feature of chronic in lammatory rheumatoid arthritis (Harris, 1990). When RA is clinically suspected, immunological studies such as testing for the presence of traditional markers such as RF and Anti-CCP along with disease activity measures like ESR, CRP, and joint count should be performed. Based on these tests and clinical examinations, the physician would rule out the possibility of other autoimmune diseases (Narayan et al., 2019). We have observed all these traditional markers of disease activity levels increased in RA patients compared with controls (Table 1). All the values are calculated as the median (25th-75th percentile). The age of the study participants for RA patients and healthy controls was 47 (39-53) and 50.5 (39-59.25) years, respectively, and patients had a disease duration of 2.6 (1-6.25) years. The in lammatory markers like ESR and CRP levels were signi icantly elevated (p < 0.001) in RA patients than healthy controls. The cartilage and bone tissue turnover biomarkers, so-called non-traditional, re lected in the systemic circulation due to the changes in the extracellular matrix of these tissues in RA patients. On evaluation, we also noticed that synovial in lammation traditional markers like AntiCCP and RF levels are signi icantly increased in RA patients than normal control group whereas no such signi icant changes noticed between COMP and Osteocalcin levels ( Table 2).

Hyaluronic acid
Synovial in lammation is a characteristic feature in rheumatoid arthritis, and HA is produced by synovial tissue and escapes out from in lamed synovium and raises its level in the blood (Yap et al.,

Figure 5: Correlation between serum COMP and anti-CCP levels in RA patients
2018). In results obtained from our study showed an increase in serum HA levels in RA patients signi icantly compared to the control group (Figure 3) suggesting an early indicator of structural damage and hence can be a useful prognostic marker of RA pathogenesis (Majeed et al., 2004).A study proved that serum hyaluronic acid level correlates with clinical and laboratory measures of disease activity like ESR, CRP in RA patients (Al-Dalaen et al., 2016). In agreement with this study, we have also proved a signi icant correlation between ESR, CRP with levels of hyaluronic acid in serum (Table 3) in RA patients suggesting synovial in lammation leads to RA patho-genesis. The release of HA and its fragments into the systemic circulation is due to the degeneration and turnover of the extracellular matrix. It is conirmed that localized in lammation of the synovial arthritic joints is the root cause of increased production and release of HA than cartilage degradation (Gupta et al., 2019).
A study demonstrated a signi icant positive association between CRP and HA level in RA patients suggesting increased synthesis of HA may be linked to the intensity of the in lammation process (Petrey and de la Motte, 2014). In our study, we have observed a signi icant positive correlation between HA with the traditional markers of RA such as Anti-CCP suggesting the involvement of synovium in RA pathogenesis (Figure 4). From rheumatoid synovium, a substantial amount of Hyaluronic acid from the synovial joint enters systemic circulation by lymphatic drainage as demonstrated by different stages of cell culture studies (Sokolove and Lepus, 2013). We were able to establish a reasonable correlation between HA with ESR, CRP, and Anti-CCP, all of which are directly involved in RA pathogenesis. According to conventional thinking, HA and Anti-CCP are more associated with synovial degradation and ESR and CRP with in lammation it is clear that synovial in lammation is a distinguishing feature of RA pathogenesis suggesting the role of the abovementioned markers in RA pathogenesis.

Cartilage oligomeric matrix protein ( COMP)
The serum COMP is one of the most extensively studied cartilage markers so far and researchers have found an association between increased COMP & progression in rheumatoid arthritis. Increased cartilage turnover is re lected upon as raised serum COMP levels, therefore used as a prognostic marker of cartilage breakdown in con irmed RA patients (Bi, 2018). But different from these observations in our study, there is not much signi icant change in the amount of COMP in RA patients when compared with controls, shows the degree of uncertainty on the role of COMP in RA pathogenesis. Several previous studies provide contradicting reports about serum COMP-either decrease or increase or no change in RA patients. Although COMP being a biomarker of cartilage wear and tear in RA and rheumatic / joint diseases, we have found that it is less signi icant unlike other biomarkers in predicting the disease progression, and from our observations, it is clear that serum COMP values are synonymous in RA patients and normal healthy controls.
In agreement with one such study, we also propose that there are no changes in serum COMP levels in RA patients signifying its limited role in RA patho-genesis (Tseng et al., 2009). Simultaneously both COMP and Osteocalcin showing a weak correlation with traditional markers of disease activity, in the study group.
From our study, we were unable to establish any kind of signi icant relationship between COMP with traditional disease activity markers of RA such as ESR, CRP, swollen and tender joint count. The same observation was reported in another study that, serum COMP showed a weak correlation with ESR, CRP in RA patients and increased serum COMP levels might occur early in the course of rheumatoid arthritis as a sign of cartilage involvement (Lindqvist, 2005). We have observed, according to Figure 5, the serum COMP and AntiCCP showed a signi icant negative correlation in RA patients. At the same time, we have found no signi icant correlation between serum levels of COMP with RF in RA patients. This biomarker strongly correlates with joint cartilage degradation; however, the results of all studies are not consistent. Besides, some studies have indicated that in advanced stages of rheumatoid arthritis there is a decline in serum COMP level (Wislowska and Jabloska, 2005), whereas synovial in lammation may persist, indicating normal or slightly elevated Anti-CCP autoantibodies. The main in luence of COMP in the RA pathogenesis is ambiguous, although there is proof about the role of COMP in turning on the complement system which contributes to disease development. In RA, there is a high turnover of cartilage matrix, eventually releasing COMP due to catabolic reaction, especially in late stages where repair mechanism cannot recompense joint destruction (Sagha i et al., 2017).

Osteocalcin
Rheumatoid arthritis is associated with severe progressive joint destruction and erosion of bone. So, we have studied serum osteocalcin, a bonespeci ic protein and marker of bone formation in rheumatoid arthritis patients and control. In our study, there was no statistical signi icance in serum osteocalcin level between RA patients with normal controls, which we had discussed in our earlier study (Vinod et al., 2011). Studies reported both increased and decreased serum osteocalcin concentrations in patients with RA (Shoji et al., 2017;Liu et al., 2018). Meanwhile, one study reported that there is inconsistency about the levels of markers of bone formation in RA patients and as we evaluated osteocalcin level found there is no signi icant difference between RA and controls suggesting normal bone turnover rate in RA patients (Riaz et al., 2020).
Normal osteocalcin levels in rheumatoid arthritis patients could be due to various reasons. A study reported that a higher amount of inactive osteocalcin and a low amount of active osteocalcin produced in the serum of chronic RA patients (Angela et al., 1990). It is reported that improper sample collection, specimen handling, and using different laboratories for successive measurements can seriously affect the precision of serum osteocalcin assay (Singer and Eyre, 2008). In our study, these possibilities are minimal since we have strictly followed the protocol and procedures as per the instruction manual, despite we have observed no signi icant changes in the serum osteocalcin levels between RA and controls. From the results of our study, we are suggesting a conventional bone resorption activity in RA, more probably due to imbalance between osteoblastic and osteoclastic activity in these patients.
In our study, there was no correlation between osteocalcin with traditional markers of RA. A study reported that OC concentrations were signi icantly decreased in RA patients compared with the controls and not correlated with ESR and CRP, suggesting reduced bone formation and bone remodelling in RA (Kroger et al., 1993). This is an important observation from our study on Indian patients with RA. In a broad and comprehensive nontraditional biomarkers are used to measure the disease severity in rheumatoid arthritis patients there was no single biomarker signi ied the biology of disease and helped in predicting the clinical course of the illness. Therefore, a blend of non-traditional biomarkers can help to evaluate disease activity better in the early stage of rheumatoid arthritis.

CONCLUSIONS
Based on all the indings it can be concluded that the serum non-traditional markers like COMP and osteocalcin, no doubt are good markers for RA disease activity but it would be premature to establish their involvement in RA pathogenesis as more future studies are required. However, Hyaluronic acid can be considered as a potential biomarker which in combination with traditional markers of RA can be useful for early diagnosis of rheumatoid arthritis.
interest in this study.

Funding Support
The result of this study was derived from the PhD (Faculty of Medicine) thesis of Dr Vinod Narayan registered in Sri Ramachandra medical college and research institute (SRU) University, Chennai, Tamil Nadu, India, and this was a self-funded project.