A Study on Association Between Protein Carbonyl and Anti-cyclic Citrullinated Peptide Antibody in Rheumatoid Arthritis: Introducing a New Supplementary Biomarker

Abstract

Redox state and immune mechanisms are two major factors implicated in rheumatoid arthritis (RA). Regarding some limitations of anti-cyclic citrullinated peptide (anti-CCP) antibody in RA diagnosis, recruiting another strong marker of oxidative stress could lead to more definitive diagnosis. To evaluate the potential of protein carbonyl content as a supplementary biomarker for RA. Eighty patients with RA attending the Research Center from 2015 to 2016 were recruited in this study. Smoker and alcoholic subjects, or those with any other systemic illness were excluded from the study. Demographic information and clinical data were collected. Numbers of swollen and tender joints were determined and RA disease activity was assessed. Serum samples were used for assessing protein carbonyl level, platelet count, and anti-CCP antibody values. Statistical analyses for significant differences were performed according to parametric (Student t test) and nonparametric (Mann–Whitney test) tests. The correlation was determined by Pearson coefficient. There was a significant correlation between protein carbonyl levels and anti-CCP antibodies in active RA (p value = 0.01), but not in remission phase (p value = 0.28). A significant positive correlation was observed between protein carbonyl levels and platelets count in active RA (p value = 0.001), but not in remission phase (p value = 0.85). Protein carbonyl could be considered as a future cost-effective supplementary biomarker, alongside anti-CCP antibody, in active RA diagnosis as it showed a significant positive correlation with anti-CCP antibody and platelet, two major mediators in the disease pathogenesis.

Introduction

Rheumatoid arthritis (RA) is an inflammatory autoimmune disorder with unknown etiology, which affects diarthrodial joints [1]. It is marked by inflammatory cells infiltration into the synovium, synovial hyperplasia and, subsequently, erosions of articular cartilage as well as subchondral bone [2]. Increasing information in recent years has been proposed regarding the pathogenesis of RA, all of which have fostered the management of the disease [3]. Despite the latest advances in therapeutic strategies, the low survival rate is still partly unavoidable and sustained remission is hardly achieved [3]. Therefore, the current therapeutic strategy is to employ aggressive treatment soon after diagnosis [3]. This issue underscores the importance of discovering potential predictive biomarkers of disease prognosis and diagnosis. Although the exact etiological causes of RA are still unclear, studies aiming at better comprehending the pathogenesis and etiology of the disease have shed light on the participation of both autoimmunity and oxidative stress in the disease development [4].

It has been declared that anti-cyclic citrullinated peptide (anti-CCP) antibodies have a pivotal role in RA pathogenesis. These autoantibodies, directed against citrullinated proteins or peptides, have been observed in the majority of subjects with RA. Accordingly, accumulated evidence has signified that anti-CCP antibody, as a marker of autoimmunity, could be a useful prognostic tool for RA. However, research is still ongoing on its sensitivity, prognostic and diagnostic accuracy. Some studies believe that utilizing anti-CCP antibody as a prognostic marker in subjects with established RA has yet to be confirmed. Porto et al., for instance, claimed that their results showed no correlation between anti-CCP and the severity of disease. Thus, anti-CCP cannot be suggested as a prognostic marker in subjects with established RA [5]. Moreover, low sensitivity of this biomarker has been reported in a number of studies [6, 7]. Therefore, the concomitant use of anti-CCP with another diagnostic tools has been suggested to be useful in improving the diagnostic accuracy. For instance, Bizzaro et al. demonstrated that performing anti-CCP antibody testing concomitantly with RF could be of importance in diagnosis of subjects with suspected early RA [7].Thus, considering the fact that autoimmunity and oxidative stress are two main mechanisms involved in the disease pathogenesis, recruiting another strong marker of oxidative stress alongside the anti-CCP antibody could lead to a more definitive diagnosis. This fact necessitates us to investigate a reliable supplementary biomarker which represents the redox status.

Oxidative stress is a well-known phenomenon in which the balance between oxidants and antioxidants is disrupted mainly in favor of the oxidants [8]. Furthermore, it is known as a crucial mechanism underlying RA pathophysiology [9]. ROS production is believed to be one of the mediators implicated in tissue damage in this disease [9]. Although there has been a particular interest in assessing the correlation between oxidative stress and RA, limited information is available on introducing an oxidative biomarker being in line with anti-CCP antibody to validate the diagnosis. Among the various biomarkers of oxidative stress, protein carbonyl has been known as one of the most potential measures [10]. Studies have proven the elevation of protein carbonyl values in RA patients, and its positive correlation with Disease Activity Score in 28 joints (DAS28) [11]. Therefore, we aimed to investigate any correlation between protein carbonyl levels and anti-CCP antibody. Furthermore, as platelets seem to be implicated in both underlying redox and immune mechanisms [12], and since there has been a well-established association between anti-CCP antibody and platelets [13], we decided to assess any possible correlation between protein carbonyl levels and platelets to further validate the potency of protein carbonyl as a biomarker.

Methods

Study Population

The current study was conducted on 80 patients with established RA who fulfilled the revised American College of Rheumatology (ACR) criteria for RA [14]. All patients attending the Rheumatic Diseases Research Center for routine visits and follow-up of their condition from 2015 to 2016 were recruited in this study, and their demographic information was collected including gender, age, weight, and body mass index (BMI). Smokers and alcoholic subjects, or those with any other systemic illness were excluded from the study.

Clinical Variable

All individuals were interviewed, and, subsequently, physical examination was conducted by qualified rheumatologists. Numbers of swollen and tender joints were determined and RA disease activity was assessed by the DAS28 based on erythrocyte sedimentation rate (ESR).

The disease activity of RA patients is calculated according to the following formula: DAS28 = 0.56 × √ (tender 28 joint counts) + 0.28 × √ (swollen28 joint count) + 0.70 × ln (ESR), mm/hr.) + 0.014 × Visual Analog Scale (VAS), mm [15]. Subsequently, the patients fall into two categories based on DAS28-ESR score as follows: ≤ 2.6 (remission) and > 2.6 (active disease).

Furthermore, pain severity was determined by VAS using a 100 mm rating scale and asking the patients to determine their joint pain severity ranging from 0 (no pain) to 100 (the most severe pain).

Laboratory Measurements

Venous blood samples were collected from the brachial vein of all patients, centrifuged at 2000g for 10 min, and the sera were assessed for protein carbonyl level and anti-CCP antibody values. Platelet count was determined in whole blood samples.

Protein Carbonyl Assay

The assay procedure was done according to the manufacturer’s protocol (Protein carbonyl content assay kit, Sigma Aldrich, Germany—MAK094) which was based on the derivatization of protein carbonyl groups with 2,4-dinitrophenylhydrazine (DNPH). The final product is a stable dinitrophenyl (DNP) hydrazone adduct which can be detected by spectrophotometer at the absorbance of 375 nm. Finally, protein carbonyl concentration was calculated using a standard curve and expressed as nanomoles per milligram.

Anti-CCP Antibody Assessment

Anti-CCP antibodies were measured by enzyme-linked immunosorbent assay (ELISA) method in accordance with the manufacturer’s protocol (Immunoscan RA Anti-CCP test kit; EuroDiagnostica, Sweden—FCCP100). Briefly, sera were diluted 1:50 and incubated in the plate for 60 min at room temperature. Subsequently, they were incubated with the peroxidase-conjugated anti-human IgG antibody and, next, TMB solution each time for 30 min at room temperature. The reaction was terminated by the phosphoric acid solution. The optical density of the yellow color was read at 450 nm using an automated spectrophotometer. Pre-diluted anti-CCP standards and positive and negative controls were included in each plate. Finally, a standard curve was used to calculate anti-CCP antibody levels in the serum samples.

Statistical Analysis

All data were expressed as Mean ± SD. Statistical analyses for determination of correlation were performed according to nonparametric (Spearman rho) tests. The p value of 0.05 is used as the cutoff for significant difference.

Results

Demographic Information and Clinical Characteristics of Patients

A total of 80 individuals (70 females and 10 men) were included in this study with the mean age of 50.15 ± 1.51 years. The patients’ disease activity, measured by DAS28-ESR, was as follows: 71 patients (88.75%) had active disease, and 9 patients (11.25%) were in remission phase. Table 1 presents demographic information and clinical characteristics of the recruited patients.

Table 1.

Demographic and clinical characteristics of RA patients based on disease activity

	Active disease (DAS28 > 2.6, N = 71)	Remission DAS28 ≤ 2.6 (N = 9)
Age	50.73 ± 1.60	45.56 ± 4.69
BMI (kg/m2)	27.19 ± 0.65	26.31 ± 1.49
RA duration (year)	5.79 ± 0.72	4.33 ± 0.71
RA therapy (year)	4.84 ± 0.56	4.31 ± 0.71
VAS pain score	44.79 ± 2.77	14.44 ± 2.94
Tender joints number	7.97 ± 1.02	0.14 ± 0.14
Swollen joints number	6.39 ± 0.68	1.57 ± 1.41

DAS28 disease activity score in 28 joints, BMI body mass index, RA rheumatoid arthritis, VAS Visual Analog Scale

The Correlation Between Protein Carbonyl and Anti-CCP Antibody Levels

No significant correlation was observed between protein carbonyl level and anti-CCP antibody level in remission phase (p value = 0.28) (Table 2); however, this correlation was statistically significant in patients with active disease (p value = 0.01) (Table 3).

Table 2.

Correlation of serum protein carbonyl level with anti-CCP antibody level and platelets counts among the RA patients in remission phase

Remission (DAS28 ≤ 2.6, N = 9)		Spearman’s Rho (ρ)	p value
Serum protein carbonyl	Platelets	0.08	0.85
Serum protein carbonyl	Anti-CCP antibody	0.61	0.28

DAS28 disease activity score in 28 joints, anti-CCP anti-cyclic citrullinated peptide

Table 3.

Correlation of serum protein carbonyl level with anti-CCP antibody level and platelets counts among the RA patients with active disease

Active disease (DAS28 > 2.6, N = 71)		Spearman’s Rho (ρ)	p value
Serum protein carbonyl	Platelets	0.40	0.001
Serum protein carbonyl	Anti-CCP antibody	0.41	0.01

DAS28 disease activity score in 28 joints, anti-CCP anti-cyclic citrullinated peptide

The Correlation Between Protein Carbonyl Level and Platelets Counts

According to our result, there was not any significant correlation between protein carbonyl level and platelets counts in remission phase (p value = 0.85) (Table 2); while they were significantly correlated in patients with active disease (p value = 0.001) (Table 3).

Discussion

In the current study, we assessed the correlation between protein carbonyl levels and anti-CCP antibody, as well as platelet count in patients with RA. We found a positive correlation between protein carbonyl and anti-CCP antibody levels, as well as platelet count in patients with active disease (DAS28 > 2.6), but not those in remission phase (DAS ≤ 2.6).

RA is an inflammatory autoimmune disorder which can have severe consequences for multiple organs in our body [1, 16]. Despite the latest advances in therapeutic strategies, the low survival rate is still partly unavoidable and sustained remission is hardly achieved [3, 17, 18]. Studies have shown that therapeutic success is highly dependent on the early diagnosis so that the treatment can be initiated before the occurrence of permanent damages [3, 19, 20]. Accordingly, introducing potential biomarkers for diagnostic purposes have been of great importance. As the innate immunity and oxidative stress are two major mechanisms involved in the disease pathogenesis [1, 4], recruiting biomarkers presenting alteration in the immune system or redox status could be of value in disease diagnosis.

Anti-CCP antibodies are found to be implicated in RA pathogenesis. These autoantibodies, directed against citrullinated proteins or peptides, have been observed in the majority of subjects with RA. Hence, they have been used as a useful diagnostic tool for RA [21]. However, research is still ongoing on its accuracy. Some studies believe that utilizing anti-CCP antibody as a prognostic marker in subjects with established RA has yet to be confirmed. Despite the high specificity and early presence in the disease process, this biomarker presents low sensitivity, so that a negative result fails to exclude the disease [6, 22, 23]. However, some studies believe that not only anti-CCP antibody provides low sensitivity but also it shows lower specificity for RA diagnosis in comparison with other autoimmune diseases [22]. Accordingly, it has been reported that more than 30% of subjects with RA have negative tests for anti-CCP antibodies [24]. These facts necessitate us to evaluate a supplementary biomarker alongside the anti-CCP antibody to enhance its medical utility and improve the accuracy of diagnosis.

Oxidative stress is found to be a crucial phenomenon underlying RA pathophysiology. ROS production is believed to be one of the mediators implicated in tissue damage in this disease [1, 25]. Accordingly, a fivefold elevation in mitochondrial ROS generation has been observed in blood and monocytes of subjects with RA, suggesting oxidative stress as a pathogenic hallmark of the disease [1]. Moreover, investigation of numerous oxidative stress biomarkers, ranging from protein, lipid, and DNA oxidation markers to antioxidant enzymes and agents in RA patients, has been provided further strong evidence for the correlation between this phenomenon and the disease pathogenesis [1]. Studies have suggested that the assessment of oxidative biomarkers in RA, combined with currently used biomarkers, could provide better clinical control [1]. Considering the fact that autoimmunity and oxidative stress are two main mechanisms involved in the disease pathogenesis, recruiting another strong marker of oxidative stress alongside the anti-CCP antibody could lead to a more definitive diagnosis.

Although there has been a particular interest in assessing the correlation between oxidative stress and RA, little information is available regarding introducing an oxidative biomarker being in line with anti-CCP antibody in the disease diagnosis to validate the diagnosis. Among the various biomarkers of oxidative stress, protein carbonyl has been known as one of the most potential measures [26]. It is worth to note that elevated protein carbonyl levels in RA plasma and fluid synovial has been shown by different studies [27, 28]. Interestingly, protein carbonyl has also been found to be correlated positively with DAS28 [11]. Studies have shown that increased protein carbonyl values in RA patients, is a sign of strong redox imbalance since high levels of oxidative stress is required for carbonyl formation which is measurable in the rheumatoid synovium [1, 29]. Besides, among the other redox parameters, protein carbonyls form early and remain circulating in the blood for longer periods [29].

On the basis of these facts and the need for a more precise diagnosis, we decided to investigate any correlation between the anti-CCP antibody and protein carbonyl levels, as a possible supplementary biomarker. The obtained results clearly present a significant positive correlation between these two markers in patients with active disease but not those being in remission phase, thus, advancing notions of protein carbonyl possible potency to be recruited in future diagnostic strategies. The insignificant results in patients with remission phase might be due to the small population, as there were only 9 patients recruited with DAS28 ≤ 2.6.

To further validate the involvement of protein carbonyl in disease pathogenesis, we investigated the correlation between protein carbonyl levels and platelet count, since platelets have been shown to have a crucial role in RA pathogenesis [30]. For instance, studies have shown that platelet microparticles are abundantly present in arthritic joint fluid [30]. These microparticles induce production of inflammatory mediators in resident synovial fibroblasts, thus leading to arthritis progression [12]. Platelets are also implicated in synovial inflammation by generating prostaglandins. Moreover, serotonin released by platelets is involved in promoting vascular permeability in the inflamed vasculature in the synovium [12]. Interestingly, it has been suggested that anti-CCP antibodies are implicated in platelet activation in RA [13]. Thus, as platelets seem to be implicated in both underlying redox and immune pathogenesis mechanisms of the disease [12, 31], we decided to assess any possible correlation between protein carbonyl levels and platelets to further support the involvement of protein carbonyl in the disease pathogenesis and its candidacy as a biomarker. The obtained result presented a significant positive correlation between platelet count and protein carbonyl values. This correlation may be due to the antioxidant function of platelet as well. It has been demonstrated that platelet acts as an antioxidant by releasing serotonin. Also, the proved effect of antioxidants on platelet responses to stimuli could further evidence the correlation between oxidative stress and platelet activity [31]. In this view, the increased platelet count alongside the elevated protein carbonyl levels may serve as a compensatory mechanism to combat the elevated oxidant status. It should be noted that the positive correlation between protein carbonyl values and platelet count was only observed in patients with the active disease which might be due to the remission small sample size. Thus it is conceivable that, in our study, just patients with active disease presented the significant correlation both in terms of anti-CCP antibody and platelets count.

Conclusion

In conclusion, our obtained results signify that protein carbonyl, a well-known oxidative stress marker, could be considered as a future cost-effective supplementary biomarker, alongside anti-CCP antibody, in active RA diagnosis as it showed a significant positive correlation with anti-CCP antibody and platelet count, two major mediators in the pathogenesis of the disease. Elevated levels of protein carbonyl reported by previous studies further support its candidacy as a biomarker. Since anti-CCP antibody has shown some limitation in RA diagnosis, the combined determination of these two biomarkers involved in the pathogenesis of the RA could possibly improve diagnostic accuracy and, thus, their clinical utility. However, additional studies on larger population, particularly for remission phase, and for longer periods are required to confirm these results in clinical stage.

Compliance with Ethical Standards

Conflict of interest:

The authors declare no conflict of interest.

Ethical Approval

All procedures performed in the current study involving human participants were in accordance with the ethical standards of the institutional research committee and with the 1964 Helsinki declaration and its later amendments.

Informed consent

Awritten informed consent was obtained from each participating patient.