European Journal of Internal Medicine 20 (2009) 44 – 47 www.elsevier.com/locate/ejim
Original article
Serum adenosine deaminase activities during acute attacks and attack-free periods of familial Mediterranean fever Bunyamin Kisacik a,⁎, Ali Akdogan a , Gulsen Yilmaz b , Omer Karadag a , Fatma Meric Yilmaz b , Seyfettin Koklu c , Osman Yuksel c , A. Ihsan Ertenli a , Sedat Kiraz a a
Hacettepe University Faculty of Medicine Department of Internal Medicine Division of Rheumatology, Ankara, Turkey b Ministry of Health, Ankara Education and Research Hospital, Clinical Biochemistry Laboratory, Ankara, Turkey c Ministry of Health, Ankara Education and Research Hospital, Department of Gastroenterology, Ankara, Turkey Received 1 November 2007; received in revised form 20 March 2008; accepted 26 April 2008 Available online 20 June 2008
Abstract Background: Familial Mediterranean Fever (FMF) is a systemic relapsing autoinflammatory disorder. Adenosine deaminase (ADA) is an enzyme widely distribute in tissues and body fluids. Circulating levels of ADA have been shown to increase in several inflammatory conditions. This study was designed to evaluate the serum ADA in patients with FMF during acute attacks and attack-free periods. Methods: The study groups comprised 23 FMF patients in attack-free period (male/female: 11/12), 30 FMF patients in attack period (male/female: 11/19) and 20 healthy control (male/female:10/10). The groups were similar for age, gender and disease duration. Results: The mean age of FMF patients in attack-free period, patient with acute attack were 34.3 ± 11.7 and 29.4 ± 11.1 respectively. The disease durations were 13.1 ± 10.2 and 8.2 ± 7.6 years for patients in attack-free periods and patients with acute FMF attack, respectively. Patients with acute attack had significantly higher ADA levels than both patients with attack-free periods and healthy controls (for each, p b 0.001). Conclusion: In this study we demonstrated that FMF patients with acute attacks had higher serum ADA levels than attack-free periods and healthy controls. It is likely that ADA may have a role in the cytokine network of the inflammatory cascade of FMF. Also, elevated ADA levels may be a part of the activated Th1 response in the disease. ADA may be used as a supportive marker to differentiate FMF attacks from attack-free periods. Further larger-scale studies are needed to support this result. © 2008 European Federation of Internal Medicine. Published by Elsevier B.V. All rights reserved. Keywords: Familial Mediterranean Fever (FMF); Adenosin deaminase (ADA)
Familial Mediterranean Fever (FMF) is a systemic relapsing autoinflammatory disorder occurring in populations originating from the Mediterranean basin, mainly Turks, Levantine Arabs, Sephardic Jews, Druze and Armenians [1]. The gene causing FMF, designated MEFV, encodes a protein ⁎ Corresponding author. Hacettepe Universitesi Tip Fakültesi Romatoloji Unitesi, 06100 Sihhiye-Ankara, Turkiye. Tel.: +90 312 3100194; fax: +90 312 3100194. E-mail addresses:
[email protected] (B. Kisacik),
[email protected] (A. Akdogan),
[email protected] (G. Yilmaz),
[email protected] (O. Karadag),
[email protected] (F.M. Yilmaz),
[email protected] (S. Koklu),
[email protected] (O. Yuksel),
[email protected] (A.I. Ertenli),
[email protected] (S. Kiraz).
called pyrin or marenostrin that is expressed mainly in myeloid bone marrow precursors, neutrophils, and monocytes [2]. The most common clinical manifestations are peritonitis, articular disease, and pleurisy accompanied by fever. Several nonspecific immunological abnormalities and elevations in acute-phase reactant levels have been observed during the FMF attacks [3]. Adenosine deaminase (ADA) is an enzyme widely distributed in tissues and body fluids. The most important biological activity of ADA related to lymphoid tissue and is necessary for proliferation and differentiation of T lymphocytes. T lymphocytes have ADA levels 10 to 12 times higher than B lympocytes. ADA activity varies depending on proliferative status and maturity of cells [4].
0953-6205/$ - see front matter © 2008 European Federation of Internal Medicine. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.ejim.2008.04.020
B. Kisacik et al. / European Journal of Internal Medicine 20 (2009) 44–47
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Table 1 Demographic features and ADA levels in all groups
Age (year) Male/female Disease duration (year) ADA (N15 U/L)
FMF (attack-free) n = 23
FMF (attack) n = 30
Healthy control n = 20
p
34.3 ± 11.7 11/12 13.1 ± 10.2 0
29.4 ± 11.1 11/19 8.2 ± 7.6 22 (73.3%)
34.8 ± 11.1 10/10 – 1 (5%)
NS NS NS p b 0.001
NS: not significant (p N 0.05 compared to all group).
Circulating levels of ADA have been shown to increase in several inflammatory conditions including Behçet's disease, systemic lupus erythematosus and rheumatoid arthritis [5–8]. However, to our knowledge the role of ADA in FMF has not yet been assessed. The aim of this study was to investigate the diagnostic value of serum ADA levels in FMF patients with acute attacks and attack-free period. 1. Material and methods The study groups comprised 23 FMF patients in attack-free period (male/female: 11/ 12), 30 FMF patients in attack period (male/female: 11/19) and 20 healthy control (male/female:10/ 10). The diagnosis of FMF was established according to the TelHashomer criteria [9]. Out of 30 FMF patients in attack period, 26 had fever and signs of aseptic peritonitis, 2 cases had pleural involvement and 2 cases had joint inflammation. No case had dermatolologic finding and/or amyloidal accumulation. All FMF patients were on 1–1.5 mg per day of colchicine therapy and none of the patients was receiving any drug other than colchicine that could influence cytokine levels. The other causes of increased ADA were excluded for the patients in this study such as other rheumatologic diseases (rheumatoid arthritis, SLE, scleroderma, Behçet's disease), infectious diseases (especially tuberculosis) and other disease (pancreatic diseases, leukemia). Serum samples were stored at − 80 ° until analysis was performed. All serum samples were obtained during morning hours to avoid diurnal variations. Serum samples were obtained from venous blood without venous occlusion and frozen
immediately. Erthrocyte sedimentation rate (ESR), C-reactive protein (CRP) and white blood cell (WBC) count were evaluated concurrently. ADA activity was measured with an enzymatic spectrophotometric method [10] in CL-770 clinical spectrophotometer (Shimadzu, Japan). All participants were informed about the study, and the procedures were in accordance with Declaration of Helsinki and institutional guidelines. The study was approved by the Ankara Education and Research Hospital ethics committee. The Statistical Package for Social Sciences (SPSS) v10.0 for windows was used to analyze the data. Non-parametric Kruskal–Wallis test was used to analyze the variance groups. Statistically significant differences obtained from Kruskal– Wallis analysis were further tested by Mann–Whitney U-test for post hoc pairwise comparisons between groups. Correlation analysis between the parameters was performed by Pearson's correlation test. p values below 0.05 were considered as statistically significant. 2. Results The mean age of FMF patients in the attack-free group, patients with acute attack were 34.3 ± 11.7 and 29.4 ± 11.1 years respectively. The disease durations were 13.1 ± 10.2 and 8.2 ± 7.6 years for FMF patients in attack-free group and patients with acute attack, respectively. The mean age of healthy controls were 34.8 ± 11.1 years. The groups were similar for age, gender and disease duration (for each, p N 0.05). FMF patients with acute attacks had significantly higher ADA levels than attack-free group, healthy controls (for each, p b 0.001) (Graphic 1). ADA levels were similar between attack-free group and healthy control groups. ADA levels (N 15 U/L) in the FMF attack group were significantly higher than in the FMF attack free and control groups (p b 0.001). Demographic features and ADA levels were presented in Table 1(see Fig. 1). Acute-phase reactants levels of FMF patients were presented in Table 2. ESR levels were significantly higher in acute attack Table 2 Acute-phase reactant levels of FMF patients FMF (attack-free) n = 23 FMF (attack) n = 30 p ESR (mm/h)⁎ CRP(mg/l)⁎⁎ WBC (/mm3)⁎ Fibrinogen (mg/dl)⁎
Fig. 1. ADA levels of all groups.
11.7 ± 8.6 0.4 (0.1–3.2) 7570 ± 3530 307.7 ± 74.6
29.2 ± 24.4 4.8 (0.7–2.4) 9790 ± 4490 463.9 ± 149.8
b0.001 b0.001 0.053 b0.001
ESR = erthrocyte sedimentation rate, CRP = C-reactive protein, WBC = white blood cell. ⁎mean ± SD, ⁎⁎mean (range).
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B. Kisacik et al. / European Journal of Internal Medicine 20 (2009) 44–47
Fig. 2. Correlation of ADA and C-relative protein in FMF attack group.
than attack-free group and healthy controls (p = 0.001). CRP levels were significantly (p b 0.001) higher than attack-free group (p b 0.001). WBC levels were similar in acute attack and attack-free group. In FMF attack group, significant correlation were not found between ADA and CRP (r = − 0.180; p = 0.342), fibrinogen (r = − 0.002; p = 0.941) (Graphic 2–3) (Fig. 2)(Fig. 3). 3. Discussion In this study we demonstrated that FMF patients with acute attacks had higher serum ADA levels than attacks-free group and healthy controls. It is likely that ADA activity involved in the cytokine network of the inflammatory cascade of FMF and elevated ADA levels may be a part of the activated Th1 response in the disease. Moreover, it may be used as a supportive marker to differentiate FMF attacks from attack-free periods. The exact reason for the increased levels in acute FMF attacks remains unclear. ADA, a polymorphic enzyme that is involved in purine metabolism, catalyzed the deamination of adenosine to inosine and ammonium. ADA activity of the lymphocytes is ten-times higher than that of the erythrocytes and B-lymphocytes. ADA is related to the differentiation and proliferation of the lymphocytes, and its levels increase during mitogenic and antigenic responses of these cells. It occurs via the stimulation of cellular immunity, activation of macrophages and lymphocytes [4]. The immune system is regulated by T-helper cells (Th) which develop different immune responses according to different cell types, type 1 Th-cells (Th1) or type 2 Th-cells (Th2). The primary cytokines produced by human Th1 cells are interferon (IFN)-γ and tumour necrosis factor (TNF)-β, whereas Th2 cells produce interleukin (IL)-4, IL-5 and IL-9 [11,12]. FMF shows a Th1 polarization. Aypar et al. have recently shown that IFN-γ, which is a Th1 type cytokine with immunomodulatory properties, were higher during acute attacks compared to attack-free periods with FMF patients. IL-4, which is a Th2 type cytokine, was not significantly different between
acute attacks and attack-free-period [13]. Supporting the previous studies, Köklü et al.found increased plasma IFN-γ levels in FMF patients both with and without attack as compared to healthy controls [14]. FMF attacks are characterized by serosal inflammation rich in polymorphonuclear leukocytes (PMNL) [4]. Therefore we can propose that increased numbers of PMNL infiltrating the serosal membranes during acute attacks of FMF may be responsible for the higher ADA serum levels in the patients. Elevated concentrations of ADA degradation products, on the other hand, might contribute to increased chemotaxis of PMNL during FMF attacks, since proteolytic fragments of ADA were shown to enhance PMNL migration to the site of inflammation [15]. Several diseases are characterized by T-cell activation, and this indicates the importance of ADA enzyme activity in the aetiopathogenesis of such diseases. Stancikova et al have reported a relationship between ADA levels and disease activity in patients with systemic lupus erythematosus (SLE), suggesting its concentration as a useful activity parameter [6]. ADA and its isoenzymes may play a role during the pathophysiology of progressive systemic sclerosis, rheumatoid arthritis and SLE [7]. ADA activity has also been found to be higher in the synovial fluid of rheumatoid arthritis patients than in those suffering from gonarthrosis or trauma-related knee effusions [8]. Behçet's disease (BD) etiology and immune pathogenesis are not clear yet, but T-cell-mediated immune response has been shown as the major pathophysiological mechanism, with histopathologically demonstrated perivascular T-cell infiltrates in related tissues [16]. Calis et al. shown that ADA levels of patients with BD may be a valuable and supportive indicator of disease activity [17]. All our patients were on colchicine therapy during both attack and attack-free periods. Colchicine is known to inhibit upregulated PMNL functions; one might think that colchicine therapy would affect our results. However, it would be not only unethical to follow patients without colchicine once the diagnosis of FMF has been made, but colchicine therapy also
Fig. 3. Correlation of ADA and fibronogen in FMF attack group.
B. Kisacik et al. / European Journal of Internal Medicine 20 (2009) 44–47
seemed to have no significant effect on the results, as our patients were taking colchicine during both acute attacks and attack-free-periods. Moreover, Calis et al. shown that colchicine did not affect the levels of ADA activity [17]. This is the first report suggesting that the levels of ADA with FMF patients. We suggest that ADA levels may be used as a supportive marker to differentiate FMF attacks from attacksfree periods. Further larger-scale studies are needed to support this result. 4. Learning points • Plasma levels of ADA were significantly higher in FMF patients with acute attack compared to attacks-free period and healthy control group. • ADA may have a role in the ongoing inflammatory cascade in FMF. References [1] Ben-Chetrit E, Levy M. Familial Mediterranean fever. Lancet 1998;351: 659–64. [2] Centola M, Wood G, Frucht DM, Galon J, Aringer M, Farrell C, et al. The gene for familial Mediterranean fever, MEFV, is expressed in early leukocyte development and is regulated in response to inflammatory mediators. Blood 2000;95:3223–31. [3] Tunca M, Kirkali G, Soyturk M, Akar S, Pepys MB, Hawkins PN. Acute phase response and evolution of familial Mediterranean fever. Lancet 1999;353(9162):1415. [4] Van der Weyden MB, Kelley WN. Human adenosine deaminase distribution and properties. J Biol Chem 1976;251: 5448–56.
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