1. Background
One of the common causes of chronic inflammatory disorders is arthritis which affects cartilage and joints (1-4). Synovial inflammation is often progressive and leads to joint destruction (5, 6). Juvenile idiopathic arthritis (JIA) is the common form of arthritis among children and adolescents from the age of 7 to 12. Characteristics for JIA is the appearance of immunological abnormalities such as the presence of circulating immune complexes, the production of a variety of autoantibodies, and dysregulation of T-cell/B-cell interactions along with the involvement of multiple systems or organs (7, 8).
Clinical data (such as number of tender or swelling joints), global pain reported by the patients, and serological markers have been widely used to extract different indices in order to diagnose the disease activity. Inflammatory biomarkers and acute-phase reactants such as C-reactive protein (CRP) and erythrocyte sedimentation rate (ESR) are related to progression of RA and are useful for evaluation of disease activity and its response to treatment (9-11).
Cell-mediated immune response, manifests as secretion of different inflammatory products from activated lymphocytes, macrophages, fibroblasts and leukocytes in synovial joints. Endogenous adenosine remarkably suppresses this process and actually adenosine pathway mediates the inflammatory cascade (12). The level of extracellular adenosine is important in determining its ability to regulate various biological processes (13, 14). Adenosine deaminase (ADA) is one of the main enzymes of purine metabolic pathway, and is responsible to metabolize adenosine. ADA represents an exclusive checkpoint in the extracellular adenosine regulation and, consequently, in the control of immune system activity through its modulation of adenosine pathways (15-20).
Studies have shown that level of serum ADA is higher in RA patients compared with the healthy controls (19, 21, 22). Also, serum ADA is widely used to differentiate between various infectious and malignant conditions in body fluids (23-25). Moreover, ADA has been suggested as an alternative parameter representing disease activity in systemic lupus erythematous, Juvenile Idiopathic arthritis, and Behcet’s Disease (18, 20, 26). Synovial fluid ADA can also differentiate between RA and non-inflammatory arthritis (27). Thus, determination of serum and synovial fluid adenosine level can be a suitable way to assess disease activity.
Currently, there is not an exact clinical finding or laboratory test for diagnosis of JIA. Thus, the detection and control of disease recurrence is difficult. The aim of this study was, comparing the level of serum ADA with synovial fluid ADA in JIA patients, to find a correlation with oligoarticular and polyarticular subtypes of the disease and its blood acute-phase responses.
2. Methods
This cross-sectional study was conducted on 55 children who were diagnosed with juvenile idiopathic arthritis (JIA) based on International League of Associations for Rheumatology (ILAR) diagnosis criteria, referred to division of rheumatology of children’s medical center, pediatrics center of excellence (Tehran, Iran) during February 2013 - May 2014.
2.1. Inclusion and Exclusion Criteria
Synovial fluid aspiration and synovial fluid ADA measure were performed routinely. Children with joint diseases other than JIA such as septic arthritis, brucellosis, and other rheumatoid diseases like as systemic lupus erythematosus (SLE) were excluded from the study.
2.2. Performance
Information of patients were extracted from their files and recorded in questionnaires. Clinical and paraclinical data, including number of involved joints, systemic symptoms, and results of blood and synovial fluid tests in addition to patients’ demographical data such as age and gender were recorded. Blood laboratory finings like Coomb’s test, Wright test, anti-cyclic citrullinated peptide (anti-CCP), anti-nuclear antibodies (ANA), rheumatoid factor (RF), CRP, ESR, complete blood count (CBC), ADA, 2-mercaptoethanol (2ME) were recorded. If there was more than one CBC vakue, the one with highest measure was chosen. In this study, correlation between synovial fluid ADA and blood ADA, and its relation to acute-phase reaction, duration of the disease and number of involved joints were assessed. Amounts of both blood and synovial fluid ADA were measured using special kits, and the normal range of serum ADA was considered 0-15 IU/L.
2.3. Ethics
Personal identity of patients was confidentially recorded in questionnaires and only analysis of results was reported. Moreover, the researchers regarded all contents of the Declaration of Helsinki (DoH) throughout the study.
2.4. Data Analysis
Quantitative and qualitative data were analyzed and reported using SPSS software (version 18) as average±standard deviation, number and percent. For all patients, average number of platelet, ESR, and CRP of blood serum, and glucose, protein, and LDH of synovial fluid have been reported. Statistical analyses were performed using t-test, Mann-Whitney U test, ANOVA, and chi-square test. Correlation study was done by Pearson correlation test. P value < 0.05 was considered significant.
3. Results
Of 55 patients (24 females and 24 31 males), 44 (80%) cases had oligoarticular JIA. Some of the serum and synovial fluid laboratory findings are presented in Table 1. It can be seen that there is no significant difference between number of white blood cells (WBC), and polymorphonuclear leukocytes of serum with synovial fluid.
Table 1.
Laboratory Findings of Blood Serum and Synovial Fluid in Studied Patientsa
| Variable | Mean (SD) | Range |
|---|---|---|
| Serum content | ||
| Average number of white blood cells (WBC), mm-3 | 10379 ± 4535 | 4,000 - 34,000 |
| Polymorphonuclear leukocytes (PMN), % | 53.2 ± 17.2 | 15.4 - 91.4 |
| Average number of platelets, mm-3 | 400910 ± 126840 | 196,000 - 677,000 |
| Erythrocyte sedimentation rate (ESR) | 34.0 ± 11.9 | 7 - 101 |
| C-reactive protein (CRP), mg/mL | 35.5 ± 5.69 | 5 - 260 |
| Synovial fluid content | ||
| Average number of WBC, mm-3 | 10604.5 ± 13129.6 | 60 - 64,000 |
| PMN, % | 62.6 ± 29.6 | 8 - 98 |
| Glucose, mg/dL | 79.7 ± 22.2 | 9 - 210 |
| Protein, mg/dL | 4501.7 ± 1683.9 | 1030 - 15,500 |
| LDH, U/L | 981.3 ± 750.8 | 232.7 - 3,599 |
aValues are expressed as mean ± standard deviation (range of alteration).
As shown in Table 2, most of the patients had oligo-articular than poly-articular JIA subtype (44 vs 11). However, there was no significant difference of JIA subtype distribution between males and females. Also, the age of patients of the two subtypes did not show any significant difference. Similarly, the average number of affected joints, serum ADA levels in average, and synovial fluid ADA levels in average do not significantly differ in relation to patient’s gender.
Table 2.
Distribution of Patients’ Characteristics in Relation to Gendera
| Variable | Value | P Value |
|---|---|---|
| Gender | 0.6 | |
| Female | 24 (43.6) | |
| Male | 31 (56.4) | |
| Average age, y | 8.42 ± 4.67 (1.5 - 15.8) | 0.4 |
| Female | 7.7 | |
| Male | 8.7 | |
| JIA typeb | 0.6 | |
| Oligoarticular | 44 (80) | 0.6 |
| Female | 20 | |
| Male | 24 | |
| Polyarticular | 11 (20) | 0.7 |
| Female | 4 | |
| Male | 7 | |
| Average age according to JIA type, yc | 0.2 | |
| Oligo-articular | 8.2 ± 4.7 | 0.3 |
| Female | 7.9 | |
| Male | 8.7 | |
| Poly-articular | 9.0 ± 2.7 | 0.04 |
| Female | 7.9 | |
| Male | 10.1 | |
| Average level of serum ADA, UI/Ld | 16.2 ± 5.5 | 0.02 |
| Average level of synovial fluid ADA, UI/Ld | 26.9 ± 11.5 | |
| Average level of serum ADA, UI/Le | 16.2 ± 5.5 | 0.5 |
| Female | 13.9 ± 2.8 | |
| Male | 15.2 ± 6.8 | |
| Average l evel of synovial fluid ADA, UI/Le | 26.9 ± 13.5 | 0.2 |
| Female | 24.9 ± 14.8 | |
| Male | 28.4 ± 12.6 |
Abbreviations: ADA, adenosine deaminase; JIA, juvenile idiopathic arthritis.
aValues are expressed as No. (%) and mean ± standard deviation (range of alteration).
bChi2 was 0.002.
cIndependent t-test was -1.361.
dIndependent t-test was 0.652.
eIndependent t-test was 1.346.
Level of ADA in serum and synovial fluid, JIA subtype, disease duration, and blood acute phase reactants (WBC, CRP, platelets, and ESR) were recorded separately for both genders (Table 3). Then, amount of ADA in serum and synovial fluid was compared regarding each criterion to find a correlation between them. Results showed that there was no significant correlation between these criteria and level of ADA or for serum and synovial fluid. It should be noted that the difference of synovial fluid ADA in relation to low and high levels of CRP was not significant (P value = 0.1), although it might be significant in larger populations.
Table 3.
Level of Blood Serum ADA and Synovial Fluid ADA in Relation to Blood Acute-Phase Reactants, JIA Subtypes, and Gendera
| Average Serum ADA | Average Synovial Fluid ADA | P Value | |
|---|---|---|---|
| Total | 15.2 ± 5.5 | 27.9 ± 11.5 | 0.03 |
| JIA subtype | |||
| Oligoarticular | 13.8 ± 3.2 | 24.5 ± 12.7 | 0.2 |
| Poly-articular | 18.3 ± 7.7 | 37.0 ± 9.4 | 0.04 |
| P Value | 0.3 | 0.2 | |
| Negative rheumatoid factor | 14.8 ± 5.6 | 30.5 ± 15.7 | 0.1 |
| WBC, mm-3 | |||
| Normal, < 12,000 | 14.7 ± 6.1 | 31.06 ± 12.5 | 0.09 |
| High, > 12,000 | 12.2 ± 1.9 | 30.87 ± 12.4 | 0.04 |
| P Value | 0.5 | 0.9 | |
| CRP, mg/mL | |||
| Low, < 10 | 13.8 ± 2.4 | 26.4 ± 16.2 | 0.2 |
| High, > 10 | 15.9 ± 7.2 | 35.6 ± 10.0 | 0.03 |
| P Value | 0.3 | 0.1 | |
| Platelets, mm-3 | |||
| Normal, < 400,000 | 14.5 ± 2.9 | 24.3 ± 14.5 | 0.3 |
| High, > 400,000 | 15.2 ± 7.5 | 33.5 ± 9.5 | 0.03 |
| P Value | 0.7 | 0.09 | |
| ESR, mm/h | |||
| Low, < 20 | 13.2 ± 2.5 | 24.0 ± 1.1 | 0.1 |
| Moderate, 20 < ESR < 40 | 18.1 ± 6.4 | 27.5 ± 1.2 | 0.03 |
| High, > 40 | 13.1 ± 3.2 | 38.1 ± 1.5 | 0.001 |
| P Value | 0.1 | 0.2 |
Abbreviations: ADA, adenosine deaminase; CRP, C-reactive protein; ESR, Erythrocyte sedimentation rate; JIA, juvenile idiopathic arthritis; WBC, white blood cells.
aValues are expressed as mean ± standard deviation.
Overall, synovial ADA was significantly higher than serum ADA (P = 0.03). Although synovial ADA was higher in both subtypes of JIA, this difference was statistically only significant in polyarticular subtype (P = 004). In patients with evidence of laboratory inflammation such as higher ESR, CRP and peripheral leukocytosis and thrombocytosis, synovial ADA was significantly higher than in serum ADA level.
Also there was no significant difference between acute phase responses, including WBC, platelet, and CRP at low and high ADA level of synovial fluid (Table 4). On the other hand, difference of ESR at low and high level synovial fluid ADA was statistically significant. This result demonstrates ESR as a remarkable indicator for the high level of ADA and presence of high inflammation.
Table 4.
Blood Acute-Phase Reactants in Relation to Low and High Levels of ADA in Synovial Fluida
| Synovial Fluid ADA < 20 | Synovial Fluid ADA > 20 | P Value | |
|---|---|---|---|
| Average of WBC, mm3 | 10104.5 ± 4573.94 | 10830.3 ± 5962.53 | 0.7 |
| Average platelet count, mm3 | 363181 ± 130927 | 447666 ± 110599 | 0.5 |
| Average ESR, mm/h | 22.72 ± 15.58 | 48.20 ± 33.73 | 0.004 |
| Average CRP, mg/L | 21.99 ± 39.02 | 53.05 ± 61.54 | 0.1 |
Abbreviations: ADA, adenosine deaminase; CRP, C-reactive protein; ESR, erythrocyte sedimentation rate; WBC, white blood cells.
aValues are expressed as mean ± standard deviation.
Correlation of serum ADA with synovial fluid ADA was assessed to find whether these variables have any impact on each other. There was no correlation between serum ADA and synovial fluid ADA (P value = 0.2) indicating that alteration in ADA level of serum does not affect the ADA level of synovial fluid, and vice versa.
4. Discussion
In spite of investigation of many possible causes, including infectious, genetic, immunologic etc., the etiology of rheumatic diseases such as JIA is a mystery (28). Adenosine is a purine nucleoside and has anti-inflammatory functions by decreasing proinflammatory cytokines, increasing anti-inflammatory cytokines, cytokine modulation of macrophages and monocytes and regulation of endothelial cell inflammatory functions (29-31). It has been suggested that ADA concentration may help in identification of immune system activity and differentiation of inflammatory from non-inflammatory conditions (32).
In this study, the average level of serum ADA in female and male groups was normal (i.e. < 15 IU/L), and no significant difference was observed between them. This result was compatible with the previous studies (32, 33). The average of synovial fluid ADA was 30.73 ± 15.55 UI/L which was higher than the differentiation cut-off of inflammatory and non-inflammatory arthritis (16). This result indicates the direct relation of synovial fluid ADA with arthritis in comparison with serum ADA, even though there was no significant difference between female and male groups. Same relation was observed between synovial fluid ADA and oligo-articular and poly-articular JIA subtypes which demonstrates higher diagnostic precision of synovial fluid ADA.
There was no significant relation between level of serum ADA and blood acute-phase reactants such as WBC, platelet, CRP, and ESR at high and low levels. In our previous study, we found inverse correlation between serum ADA level and ESR during acute phase of JIA and between serum ADA level and WBC counts after disease control (25). Salesi et al reported significant correlation between serum ADA and ESR (15), but the result of our recent study was compatible with that of Sari et al. (19). Vinapamula et al found significant positive correlation between serum ADA and CRP in RA patients. They reported cut-off value of 25.3 IU/L for serum ADA level in RA with 82.6% sensitivity and 65.2% specificity (34). It seems that cut off point for serum and synovial level of ADA is an important factor that influences various results in different studies. On the contrary, level of synovial fluid ADA results in higher levels of acute-phase responses (except for WBC); however, there was no significant difference between synovial fluid ADA at higher and lower levels of WBC, platelets, ESR, and CRP. This result depicts the close relation of synovial fluid ADA with inflammatory arthritis than serum ADA does.
Unlike the results of Yuksel et al. (32), in our study, there was no correlation between serum ADA and synovial fluid ADA which means that increases in serum and synovial ADA do not affect each other. Also, there was no relation between duration of disease and the level of synovial fluid ADA.
Comparison of blood acute-phase reactions between two groups of synovial fluid ADA higher and lower than 20 showed no significant difference for WBC, CRP and platelets; although, the amount of CRP and number of platelets increased in higher level of ADA. On the other hand, significant difference was observed in average ESR in two groups of synovial fluid ADA. This result shows direct relation of ESR with synovial fluid ADA in arthritis activity. Entirely, it seems that ESR, platelet and CRP are predicting indices of high levels of synovial fluid ADA, and consequently, inflammatory arthritis, and their simultaneous measurement would represent the active inflammation.
There were some limitations in this study which should be removed in future studies. First there were only 38 cases which is not a suitable number for statistical analysis. Larger numbers of patients could alter both negative and positive rheumatoid factors. Second, unavailable measurement of subjective pain and joint inflammation was an obstacle to determine the intensity of symptoms in each patient and at different times of hospitalization. Thus, there is a need to record information more precisely.
In conclusion, our results demonstrated that the level of synovial fluid ADA is a more precise index than level of serum ADA to assess the activity of arthritis. Also, synovial fluid ADA is in relation with blood acute phase responses including platelets, CRP, and ESR which could be used to diagnose inflammatory from non-inflammatory arthritis.
