Original Research. Vahid Ziaee 1-2-3, Ali Amiran 3, Mohammad-Hassan Moradinejad 1-3-4, Mohammad-Taghi Haghi-Ashtiani 4-5. Department of Pediatrics; 2

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1 Original Research Evaluation of Serum Adenosine Deaminase Changes Before and After Treatment in Patients with Systemic Lupus Erythematosus, Henoch-Schonlein Purpura and Juvenile Idiopathic Arthritis Vahid Ziaee 1-2-3, Ali Amiran 3, Mohammad-Hassan Moradinejad 1-3-4, Mohammad-Taghi Haghi-Ashtiani Department of Pediatrics; 2 Pediatric Rheumatology Research Group, Rheumatology Research Center; 3 Children s Medical Center, Pediatrics Center of Excellence; 4 Growth and Development Research Center; 5 Department of Pathology, Tehran University of Medical Sciences, Tehran, Iran Corresponding Author: Vahid Ziaee, Division of Pediatric Rheumatology, Children s Medical Center, No 62, Dr Gharib St, Keshavarz Blvd, Tehran, Iran ziaee@tums.ac.ir Received: Dec 21, 2012 Accepted: Feb 18, 2013 Ann Paediatr Rheum 2013; 2:21-26 DOI: /apr Abstract Background: Adenosine deaminase (ADA) is a purine metabolism enzyme that can be used as an indicator of cellular immunity for monitoring of inflammatory diseases. The aim of this study was to investigate the changes of serum adenosine deaminase before and after treatment in patients with systemic lupus erythematosus (SLE), Henoch-Schonlein purpura (HSP) and juvenile idiopathic Arthritis ( JIA). Methods: The study comprised 103 patients with JIA, 17 with SLE and 30 HSP. The serum ADA was measured of all patients before and after treatment or remission. Serum ADA was measured by an ultraviolet kinetic method with NADH as substrate. Normal value for serum ADA level was less than 15 IU/L. Results: Positive serum ADA was found in 38 patients with JIA (38.9%), 9 patients (52.9%) with SLE and 6 patients (20%) with HSP. During the disease activity (before treatment), the mean serum ADA in patients with JRA was 15.1±10.9 IU/l, vs. 12.6±4.5 IU/l in patients with HSP and 18.8±11.95 IU/l in patients with SLE (P=0.097). There was no significant differences in changes of ADA levels between disease activity and after control (p= 0.1) in SLE. In patients with JIA, there was significant differences in changes of ADA levels between disease activity and control (p= 0.01). There was no significant differences in changes of ADA levels between disease activity and control (p= 0.2) in HSP. Conclusion: Determination of ADA serum levels is as noninvasive method, reliable and easy for diagnosis of JIA and can be used as alternative parameters representing disease activity in SLE patients. Key words: Adenosine deaminase, systemic lupus erythematosus, Henoch-Schonlein purpura, Juvenile idiopathic arthritis, biomarker Introduction Adenosine deaminase (ADA) is a purine metabolism enzyme that playing an significant role on differentiation of lymphoid cells, and it can be used as an indicator of cellular immunity for monitoring of inflammatory diseases[1]. The changes of the serum level of ADA has been expected in some rheumatologic disorders

2 ADA in rheumatologic disorders 22 such as juvenile idiopathic arthritis (JIA), systemic lupus erythematosus (SLE), and Henoch-Schonlein purpura (HSP) in which cell-mediated immunity has been altered [2-3]. Multiple organ involvement is a common characteristic of these rheumatic disorders. Immunological charcthristic of these diseases are production of autoantibodies due to disregulation of T-cell/B-cell interactions and presence of circulating immune complexes[4-6]. Activated macrophages and T cells has an important role in in inflammatory process in JIA and SLE. Increasing of percentages of these cells have been found in peripheral blood of patients with rheumatic diseases[2]. So it has been suggested that these cells are responsible for immunoregulation and inflammatory response. Serum ADA activity has closely relation with active lupus disease activity and it can be used as diagnostic factor [3,7]. There is a few study on ADA in JIA patients and it is suggested serum ADA is increased in chronic inflammatory arthritis [2,8]. HSP is multisystem inflammatory disease with abnormal inflammatory response and it seems cellular immunity was involved in this vasculitis [9]. So increase of ADA in this disorder is expected. SLE, HSP and JIA are recurrent chronic inflammatory diseases and there is a not definite clinical finding or laboratory test for diagnosis or flare up of these disorders. Therefore, the detection and control of disease recurrence is about doubt. The aim of this study was to investigate the changes of serum ADA in acute phase and after treatment in patients with SLE, HSP and JIA. Materials and Methods The study comprised 150 patients with rheumatic diseases, 103 with JIA (45 males and 58 females), 17 with SLE (3 males and 14 females) and 30 HSP (19 males and 11 females). After parent s patients gave written informed consent for participation in the study, approved by Tehran University Medical of Sciences, patients with SLE, JIA and HSP included the study. All patients in the acute phase of disease without tuberculosis and other infection concomitant included the study. Patients who were diagnosed during the follow-up to change, death, no follow-up care after leaving the disease activity, patients with TB disease or other infection during the study, excluded. The diagnosis of JIA was based on international league of associations for rheumatology classification of juvenile idiopathic arthritis, SLE based on the criteria of the American College of Rheumatology and HSP based on EULAR/PreS Criteria[10-12]. At the time of admission, in all patients serum ADA level, white blood cells (WBC), platletes (PLT), hemoglobin (Hgb), erythrocyte sedimentation rate (ESR), C-reactive protein (CRP) have been measured. In patients with JIA, rheumatoid factors, AntiCCP and for SLE complement components (C3, C4), CH50 and Anti dsdna have been measured. Also, in patients with Henoch-Schonlein purpura involvement joint recorded. After treatment in SLE and JIA and disease control, the drug dosage was reduced and serum ADA level was checked again. In HSP serum ADA level was checked after 2 month or reduces all symptoms of disease. Serum ADA level was checked again in patients with ADA>15 IU/L before treatment. Serum samples Fasting blood samples were collected without using any anticoagulant and were subjected to centrifugation at 3,000 rpm for 10 min at 4 C to obtain serum. All serum samples were stored at -80 C immediately after separation from peripheral blood prior to analysis. ADA assay Serum ADA was measured by an ultraviolet kinetic method with NADH as substrate [13], adapted to a RA-1000 Analyzer (Technicon Ireland LTD., Dublin, Ireland). One unit of ADA is dewned as the amount of enzyme that converts 1 mm adenosine to inosine and ammonia per minute under standard assay conditions and is expressed as IU/l. The enzyme is stable for at least 24 h at 25 C, 7 days at 4 C, and 3 months at -20 C [13,14]. To minimize the measurement errors, all the assays were carried out three times and the mean value is presented. Normal value for serum ADA level was less than 15 IU/L. Statistical analysis All results were expressed in terms of mean ± SD, Statistical significance of differences between mean values was tested by one-way ANOVA test, paired t test. Correlations analysis DOI: /apr

3 23 Ziaee V et al. was performed by Pearson s correlation coeycient (r). All analyses were performed using the Statistical Package for the Social Sciences (SPSS, version 19.0). Statistically was considered as a significant p < Results The mean age of patients with SLE was 12.4±2.9 years (ranging from 2-16 years), in patients with JIA was 6.6±4.0 years (ranging from 1-16 years), and in patients with HSP was 6.2±3.1 years (ranging from 2-14 years). During the disease activity (before treatment), the mean serum ADA in patients with JIA was 15.1±10.9 IU/l, vs. 12.6±4.5 U/l in patients with Henoch-Schonlein purpura and 18.8±11.95 IU/l in patients with SLE (P=0.097). In patients with SLE, 9 patients (52.9%) had ADA posi- tive. We found joint involvement in 10 patients (58.8%) and renal involvement 5 patients (29.4%). Compare of ADA levels and other laboratory findings between disease activity and control in patients with SLE have been shown in table 1. There was no significant differences in changes of ADA levels between disease activity and control (p= 0.1). Before treatment, we found significant inverse correlation with ADA levels and WBC counts (p=0.027, r= ), during disease control, a significant positive correlation with ADA levels and Anti ds- DNA (r=0.925, p=0.003). During disease activity and control there was no significant correlation with ADA levels and other laboratory findings, also joint, renal and skin involvements. In patients with JIA, ADA was positive in 38 patients (38.9%). There was significant differences in changes of ADA Table 1. Comparison ADA levels and other laboratory findings between disease activity and control in patients with SLE Parameter Disease activity Disease control P Value ADA (U/l) 28.2± ± WBC * 10 3 (/mm 3 ) 8.4±6 5.8± Hgb (mg/dl) 11.3± ± PLT*10 3 (/mm 3 ) 265.5± ± ESR (mm/hr) 36.7± ± CRP (mg/l) 12.8±12.2 3± Anti Ds DNA 237.2± ± CH ± ± C3 67.5± ± C4 15.9± ± ADA: Adenosine deaminase; SLE: systemic lupus erythematosus; WBC: white blood cells; PLT: platelets; Hgb: hemoglobin; ESR: erythrocyte sedimentation rate; CRP: C-reactive protein Table 2. Comparison ADA levels and other laboratory findings between disease activity and control in patients with JIA Parameter Disease activity Disease control P Value ADA (U/l) 24.0± ± WBC * 10 3 (/mm 3 ) 10.6± ± Hgb (mg/dl) 11.2± ± PLT*10 3 (/mm 3 ) 423.4± ± ESR (mm/hr) 39.3± ± CRP (mg/l) 20.1± ± ADA: Adenosine deaminase; JIA: Jevenile Idiopathic Arthritis; WBC: white blood cells; PLT: platelets; Hgb: hemoglobin; ESR: erythrocyte sedimentation rate; CRP: C-reactive protein Annals of Paediatric Rheumatology Year 2013 Volume:2 Issue:

4 ADA in rheumatologic disorders 24 Table 3. Comparison ADA levels and other laboratory findings between disease activity and control in patients with HSP Parameter Disease activity Disease control P Value ADA (U/l) 21.3± ± WBC * 10 3 (/mm 3 ) 12.9± ± Hgb (mg/dl) 12.2± ± PLT*10 3 (/mm 3 ) 356.0± ± ESR (mm/hr) 16.3± ± CRP (mg/l) 6.3± ± ADA: Adenosine deaminase; HSP: Henoch-Schonlein purpura; WBC: white blood cells; PLT: platelets; Hgb: hemoglobin; ESR: erythrocyte sedimentation rate; CRP: C-reactive protein levels between disease activity and control (p= 0.01). Compare of ADA levels and laboratory findings between disease activity and control in patients with JIA have been shown in table 2. During disease activity, there was no significant correlation between ANA and/or Anti-ccp with ADA levels. In patient with JIA rheumatoid factors was in 100 patients (97.1%) negative, 3 (2.9%) positive. Also, we found during disease control significant inverse correlation with ADA levels and WBC counts (p=0.03, r= ), during disease activity significant inverse correlation with ADA levels and hemoglobin (p=0.03, r= ), and significant positive correlation with ESR (r=0.297, p=0.002). There was no significant correlation with ADA levels and other laboratory findings. Compare of ADA levels and other laboratory findings between disease activity and control in patients with HSP have been shown in table 3. The ADA level was higher than normal in 6 HSP patients (20%), and there was no significant differences in changes of ADA levels between disease activity and control (p= 0.2). In patients with HSP joint involvement was found in 23 patients (76.6%). There was renal involvement in 6 (20%), gastrointestinal involvement 12 (40%). There was no significant correlation with ADA levels and organ involvements or laboratory findings. Discussion The etiology of rheumatic diseases such as JRA and SLE is still unknown, although many possible causes, including infectious, genetic, immunologic, have been investigated [15]. Treatment of the rheumatic diseases is expected after control of inflammation and decreasing the level of autoimmune activity [5,6,16]. The ubiquitous enzyme, ADA, has been found to play a critical role in the diverentiation and maturation of the immune cells including lymphocytes and monocyte macrophage cell lines [17]. Obviously, it seems to maintain the proper function of human immune system. The vital and putative role of ADA on immune system s function was first described in patients with severe combined immunodeficiency (SCID) [18]. Moreover, elevated serum concentrations of ADA have been reported in many autoimmune and inflammatory diseases such as rheumatoid arthritis, systemic sclerosis, and psoriasis [19-22]. In this study, there was significant reduction in ADA levels after disease control in patients with JIA. Possibly, the increased serum ADA levels in disease activity can be correlated with the immune response [2,23,24]. In other study, investigators demonstrated that ADA serum levels increased, significantly, before treatment [2]. This cause might originate from both monocyte macrophage and T cell stimulations, the compartments of cellular immunity with specific responsibility for immune regulation and inflammatory responses [6,10,25]. In our study, mean ADA level was very high in SLE patients (28.2) and it decrease to 18.3 after treatment, but this change was not statistically significant. Some studies have been reported significantly higher level of ADA in disease activity of SLE patients [2,10,26]. Our finding is not compatible with these studies. It can be due to low sample size or it means ADA level return to normal value with delay after specific treatment. It seems, ADA activity is correlated with the increased ADA2 DOI: /apr

5 25 Ziaee V et al. level in SLE patients [26]. Another study with higher sample size and specific organ involvement and serum ADA level in SLE patients was recommended. In our study, the ADA level was slightly higher than normal in HSP patients in acute phase, but there is no significant association between disease activity and ADA level in HSP. We didn t find similar study in the literature, so more studies in this subject is recommended. The study on Behcet s disease showed that the increased serum ADA level was the result of erythrocyte membrane damage induced by lipid peroxidation, which leads to the releasing of ADA into the plasma [27]. Some studies have found the increased level of ADA in serum or other body fluids as a marker to specify the disease states [23,28,29] and the other studies have proposed it as a diagnostic procedure [30,31]. It seems, assessment of the use of ADA serum levels could be used for diagnose and follow up in rheumatologic disease with systemic involvements. Conclusion According to the findings of the present study, determination of ADA serum levels is as noninvasive biomarker, reliable and easy for diagnosis of JIA and can be used as alternative parameters representing disease activity. Also, determination of ADA serum levels can be helpful as an indicator during the follow up course of treatment for SLE patients. Acknowledgment: This study was part of a post graduated dissertation (of Dr A. Amiran) and was approved by Vicechancellor for Research of School of Medicine, Tehran University of Medical Sciences. The authors would like to thank parents and children who participated in the study. Competing interests: The authors declared no competing interest. Funding: None. Provenance and peer review: Not commissioned; externally peer reviewed. References 1. Antonioli L, Colucci R, La Motta C, Tuccori M, Awwad O, Da Settimo F, et al. Adenosine deaminase in the modulation of immune system and its potential as a novel target for treatment of inflammatory disorders. Curr Drug Targets ;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: Moon J, Han C, Kang S, Park M, Hwang S, Byun M, et al. The relationship between age and pleural fluid adenosine deaminase activity in pleural tuberculosis. Tuberc Respir Dis 2005; 58: Appelboom T, Manelbaum I, Vertongen F. Purine enzyme levels in rheumatoid arthritis. J Rheumatol 1985;12: Samsonov MY, Tilz GP, Egorova O. Serum soluble markers of immune activation and disease activity in systemic lupus erythematosus. Lupus 1995; 4: Steinberg AD, Klinman DM. Pathogenesis of systemic lupus erythematosus. Rheum Dis Clin North Am 1989;14: Stancikova M, Lukac J, Istok R, Cristalli G, Rovensky J. Serum adenosine deaminase activity and its isoenzyme pattern in patients with systemic lupus erythematosus. Clin Exp Rheumatol. 1998; 16: Pettersson T, Klockars M, Weber TH, von Essen R. Adenosine deaminase activity in joint effusions. Scand J Rheumatol 1988; 17: Jen HY, Chuang YH, Lin SC, Chiang BL, Yang YH. Increased serum interleukin-17 and peripheral Th17 cells in children with acute Henoch-Schönlein purpura. Pediatr Allergy Immunol ;22: Petty RE, Southwood TR, Manners P, Baum J, Glass DN, Goldenberg J, et al. International League of Associations for Rheumatology. International League of Associations for Rheumatology classification of juvenile idiopathic arthritis: second revision, Edmonton, J Rheumatol. 2004; 31: Hochberg MC. Updating the American College of Rheumatology revised criteria for the classification of systemic lupus erythematosus. Arthritis Rheum. 1997; 40: Ozen S, Ruperto N, Dillon MJ, Bagga A, Barron K, Davin JC, et al. EULAR/PReS endorsed consensus criteria for the classification of childhood vasculitides. Ann Rheum Dis 2006; 65: Annals of Paediatric Rheumatology Year 2013 Volume:2 Issue:

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