Title: Serum and synovial fluid adenosine deaminase activity in patients with rheumatoid arthritis and osteoarthritis
Author(s): Mehrnoush Hassas Yeganeh MD, Seyyed Mohammad Qoreishy MD*
Affiliattion(s): Shahid Beheshti University of Medical Sciences, Tehran, Iran
* Corresponding Author
Vol 1, Num 1, July 2014
Introduction: Adenosine deaminase (ADA) is an enzyme involved in purine metabolism and plays a significant role in the immune system. In this study, we try to assess the reliability of the serum and synovial levels of adenosine deaminase (ADA) in differentiating rheumatoid arthritis from osteoarthritis.
Materials and Methods: We selected 15 patients with rheumatoid arthritis and 15 osteoarthritis patients to enter the study. Adenosine deaminase activity was determined in paired samples of serum and synovial fluid taken from patients with rheumatoid arthritis and osteoarthritis.
Results: The results showed a statistically significant difference between ADA levels in joint effusion of patients with rheumatoid arthritis and osteoarthritis (p<0.001). Increased activity was found in the synovial fluid taken from patients with rheumatoid disease. Synovial fluid taken from patients with osteoarthritis did not show significantly raised adenosine deaminase activity as compared with that of normal controls.
Conclusion: We concluded that synovial total ADA assay could be a sensitive and specific test, being suitable for rapid diagnosis of rheumatoid effusions.
Keywords: Adenosine deaminase (ADA), Purine metabolism, Rheumatoid arthritis, Serum, Synovial fluid, Osteoarthritis
Adenosine deaminase (ADA) is one of the main enzymes of the purine metabolic pathway, catalyzing the deamination of adenosine to inosine and deoxyadenosine to deoxyinosine. Adenosine deaminase (ADA) is released into the serum in patients with different types of malignancies and infections, including viral hepatitis, infectious mononucleosis and tuberculosis.
During inflammatory reactions the enzyme is released into the extracellular fluid, and in serous effusions of different pathologic conditions, the levels of ADA activity increase considerably. This increase has been shown to correlate with the number of nucleated cells, particularly T lymphocytes and macrophages, present in the effusions.(1,2)
It has also been shown that pleural effusions due to rheumatoid arthritis (RA) or tuberculosis contain significantly higher levels of ADA than effusions caused by systemic lupus erythematosus or malignant diseases.(3-5)
According to different studies, increases in ADA activity in synovial fluid have a close relationship with activity of underlying disease and can show the degree of inflammation in joints. This has led to the proposal that determination of ADA activity may be a useful technique to differentiate between RA and less inflammatory conditions such as osteoarthritis.
Thus, we designed a study to evaluate ADA levels in synovial fluids in patients with RA or OA ad comparing them to normal controls.
Materials and Methods
After hospital ethical committee approval and obtaining the informed consent from all the patients, fifteen patients with rheumatoid arthritis and 15 patients with osteoarthritis entered the study as the study groups. The 15 patients with RA had definite diagnosis according to the criteria of the American College of Rheumatology in 1987 and all of them had signs of active arthritis in one or both knee joints. Active disease was defined by the presence of swelling and or limitation of movement or tenderness in at least one joint, and elevated C-reactive protein (CRP) level and erythrocyte sedimentation rate (ESR). The diagnosis of OA was based on the criteria of the American College of Rheumatology 1987 and all of them had these criteria in one or both knee joints.
All these patients were candidate for knee joint arthroplasty. We selected 15 patients with carpal tunnel syndrome who needed a surgery on their wrist and without any symptoms in their knee joints and any radiographic sign of OA as the control group. Synovial fluid specimens were collected from knee joint effusion at the time of surgery. We collected 10cc of synovial fluid and concurrently 10cc of patient's blood in clot form. Synovial fluid was examined for infectious causes according to its cell count and smear results. In addition to the routine laboratory investigations, which were performed on all samples, synovial fluids were examined for the presence of crystals. After disproving the infection and crystal induced arthropathies, ADA test was performed.
In laboratory, after centrifuging, the samples were kept in -25 degrees centigrade for one night. The ADA assay is based on the enzymatic deamination of adenosine to inosine. ADA activity was accountable at least up to 100 IU/L (international unite per liter).
Descriptive statistical exams, Chi Square, Fisher test and independent t-test for binary and continuous variants were used and also odds ratio was estimated as appropriate (using SPSS software). Standard deviation and mean values were also calculated for each group. For all statistical parameters, the confidence interval of 95% and p-values less than 0.001 were considered significant.
The mean age of patients was 45 ± 5.6 years in RA group and was 58 ± 10.7 years in OA group and 34±4.9 years in control group. Cell counts of all synovial samples were examined to rule out the presence of infectious causes. Cell counts of synovial fluids were 100-1800 WBC/ml and 1600-35000 WBC/ml in OA an RA groups, respectively. The average level of synovial ADA in RA patients was 29.1 ± 6.9 IU/L and in OA group was 17.5 ± 6.8 IU/L and undetectable in control group.
Patients with rheumatoid arthritis had significantly increased ADA activity in their synovial fluids as compared with patients having from osteoarthritis and normal controls (p<001). Although ADA activity in the synovial fluid of the osteoarthritic group was significantly less than that present in the synovial fluid of patients with rheumatoid arthritis, the mean value was higher than that of normal control subjects.
The average level of serum ADA in RA patients was 18.9±3.7 IU/L and in OA group was 14.3 ± 6.8 IU/L and 13.39 ±4.2 in control group.
Serum ADA activity of patients with arthritis did not differ significantly from that of the normal controls (p<001). No significant differences between the groups could be shown. As expected, synovial fluids obtained from patients with RA contained elevated concentrations of protein and increased numbers of polymorphs and lymphocytes as compared to patients with osteoarthritis. There was, however, no significant association between the number of white cells and lymphocytes present in the synovial fluids and the synovial ADA activities.
According to multivariate Logistic Regression assay, the odd ratios for ADA in synovial fluid were 1.6. This showed that for every elevation in ADA amount in synovial fluid, there was 60% raise in the probability of being in RA group.
All nucleated cells contain adenosine deaminase. Distribution of this enzyme in different tissues, however, varies considerably, and it has been shown that lymphoid organs, in particular the thymus, contain the highest level of enzyme activity.(6) Peripheral T lymphocytes have been shown to contain five to 20 times more ADA activity than B lymphocytes or non-T and non-B cells.(7) It is possible, therefore, that determination of serum ADA activity, or activity in different tissues, and particularly activity in serous fluids, may be a useful indicator of the presence of increased lymphoreticular activities. The most useful application of the ADA determination, in terms of differential diagnosis, is in patients with tuberculosis.(8)
Pettersson et al and Ocana et al also showed that pleural effusions caused by rheumatoid arthritis had significantly raised ADA activity.(9-11) A raised level of ADA activity in the synovial fluid of patients with rheumatoid arthritis, therefore, is not an unexpected finding and might have been expected to be a discriminative test for identifying rheumatoid joint swellings.
Considering the chronic course of RA and not completely known pathology of the disease and the destructive debilitating nature of RA, it is important to determine the level of inflammation and response to therapy. Therefore, due to non-specific clinical features and insufficient diagnostic tests, competency of synovial fluid ADA for differentiating RA from OA was evaluated in this study.
All previous studies have shown a significant relation between the average level of ADA in synovial fluid and/or serum and activity of RA. (12) In 2001, Hitogloue et al. evaluated activity of total ADA in juvenile rheumatoid arthritis and systemic lupus erythematous patients in different stages of the diseases. They showed that higher levels of serum ADA had close association with activity and relapse of these diseases.(13) In another study performed by Sari et al. in 2003, the correlation of serum activity level of ADA and its isoenzymes with disease activity of RA was assessed. Results showed that activity of total ADA and ADA2 had correlation with RA activity. They concluded that this noninvasive test in concordance with CRP and ESR could be used as a biomarker to diagnose inflammation.(14)
Our results showed that the average level of synovial fluid ADA has statistically significant difference between RA and OA and normal controls. Furthermore, synovial fluid ADA can be used as an appropriate screening test for diagnosing rheumatoid joint effusions.
In this study, we found high levels of ADA activity in rheumatoid synovial effusions. Furthermore, although osteoarthritic patients as a group had lower levels of ADA activity in their synovial fluids, this does not hold true for individual cases, and this decreases the value of ADA determination in terms of differential diagnosis. Although measurement of synovial fluid ADA activity levels does not discriminate absolutely between joint swellings of different etiologies, we feel that in certain instances it may be of some value in differentiating between osteoarthritis and rheumatoid arthritis.
CRP is released by liver cells and under the influence of cytokines secreted from Macrophages. Recent studies have shown that CRP is a pre-inflammatory factor that causes Monocytes stimulation to secret IL-6 and TNFα, which all directly activate macrophages.(15) According to our study, CRP level of RA patients was significantly higher than that of the control group. In 2003, Kilmanuiuk et al found that high levels of CRP related with active inflammation of RA.(16)
IN 2004, Yildirim et al. studied the relationship between acute phase reactants and RA activity level. They found that CRP had the most diagnostic value among other acute phase reactants.(16)
By taking into account the ESR and CRP to ADA synovial level, the physician can distinguish between RA and OA more accurately.
Mehrnoush Hassas Yeganeh MD Paediatric rheumatologist, Assistant Professor, Shahid Beheshti Medical University, Tehran, Iran firstname.lastname@example.org
Mohammad Qoreishi MD Orthopaedic surgeon, Assistant professor, Shahid Beheshti Medical University, Tehran, Iran email@example.com Corresponding author
Acknowledgements: None declared.
Financial disclosure: None declared.
1. Lee DM, Weinblatt ME. Rheumatoid arthritis. Lancet 2001; 358: 903-911.
2. Rothschild BM, Turner KR, DeLuca MA. Symmetrical erosive peripheral polyarthritis in the Late Archaic Period of Alabama. Science 1988; 241: 1498-1501.
3. Iwaki-Egawa S, WatanabeY, Matsuno H. Correlations between matrix metalloproteinase-9 and adenosine deaminase isozymes in synovial fluid from patients with rheumatoid arthritis. J Rheumatol 2001; 28: 485-489.
4. Chan ES, Fernandez P, Cronstein BN. Adenosine in inflammatory joint diseases. Purinergic Signal 2007; 3: 145-152.
5. Wortmann RL, Veum JA, Rachow JW. Purine catabolic enzymes in human synovial fluids. Adv Exp Med Biol 1989; 253A: 393-398.
6. Chechic B E, Schrader W P, Minowado J. An immunomorphologic study of adenosine deaminase distribution in human thymus tissue, normal lymphocytes and haemopoietic cell lines. J Immunol 1981; 126: 1003-7.
7. Shohat B, Agam G, Brosh S. Adenosine deaminase activity in lymphocyte subpopulations of B-16 melanoma and normal C57BC bearing mice. Immunol Lett 1984; 8: 307-10.
8. Yuksel H, Akoolu TF. Serum and synovial fluid adenosine deaminase activity in patients with rheumatoid arthritis, osteoarthritis, and reactive arthritis .Annals of the Rheumatic Diseases, 1988; 47, 492-495
9. Pettersson T, Ojala K, Weber T H. Adenosine deaminase in the diagnosis of pleural effusions. Acta Med Scand 1984; 215: 299-304.
10. Pettersson T, Klockars M, Weber T. Pleural fluid adenosine deaminase in rheumatoid arthritis and systemic lupus erythematosus. Chest 1984; 86: 273.
11. Ocana I, Martinez-Vazquez J M, Ribera E, Capdevila J A, Fernandez de Sevilla T. Pleural fluid adenosine deaminase activity in rheumatoid arthritis and systemic lupus erythematosus. Chest 1984: 86: 273-4.
12. Yagawa K, Okamura J. Role of adenosine deaminase in activation of macrophages. Infect Immun. 1981; 32: 394-397.
13. Hitoglou S, Hatzistilianou M, Gougoustamou D, Athanassiadou F, Kotsis A, Catriu D. Adenosine deaminase activity and its isoenzyme pattern in patients with juvenile rheumatoid arthritis and systemic lupus erythematosus. Clin Rheumatol 2001; 20: 411-416.
14. Sari RA, Taysi S, Yilmaz O, Bakan N. Correlation of serum levels of adenosine deaminase activity and its isoenzymes with disease activity in rheumatoid arthritis. Clin Exp Rheumatol 2003; 21: 87-90.
15. Ballou SP, Lozanski G. Induction of inflammatory cytokine release from cultured human monocytes by C-reactive protein. Cytokine 1992; 4: 361-368.
16. Klimiuk PA, Sierakowski S, Chwiecko J. Serum interleukin 6 (il-6A) concentration correlates with matrix metalloproteinases and their tissue inhibitors in rheumatoid arthritis. Pol Arch Med Wewn 2003; 109: 119-123.
17. Yildirim K, Karatay S, Melikoglu MA, Gureser G, Ugur M, Senel K. Associations between acute phase reactant levels and disease activity score (DAS28) in patients with rheumatoid arthritis. Ann Clin Lab Sci 2004; 34: 423-426.