Eur Arch Otorhinolaryngol (2014) 271:1797–1802 DOI 10.1007/s00405-013-2843-z
MISCELLANEOUS
Assessment of adenosine deaminase (ADA) activity and oxidative stress in patients with chronic tonsillitis Mehmet Fatih Garca • Halit Demir • Mahfuz Turan • Nazım Bozan • Ahmet Kozan • S¸ eyda Bayel Belli • Ays¸ e Arslan • Hakan Cankaya
Received: 9 July 2013 / Accepted: 5 November 2013 / Published online: 4 December 2013 Ó Springer-Verlag Berlin Heidelberg 2013
Abstract To emphasize the effectiveness of adenosine deaminase (ADA) enzyme, which has important roles in the differentiation of lymphoid cells, and oxidative stress in patients with chronic tonsillitis. Serum and tissue samples were obtained from 25 patients who underwent tonsillectomy due to recurrent episodes of acute tonsillitis. In the control group, which also had 25 subjects, only serum samples were taken as obtaining tissue samples would not have been ethically appropriate. ADA enzyme activity, catalase (CAT), carbonic anhydrase (CA), nitric oxide (NO) and malondialdehyde (MDA) were measured in the serum and tissue samples of patients and control group subjects. The serum values of both groups were compared. In addition, the tissue and serum values of patients were compared. Serum ADA activity and the oxidant enzymes MDA and NO values of the patient group were
significantly higher than those of the control group (p \ 0.001), the antioxidant enzymes CA and CAT values of the patient group were significantly lower than those of the control group (p \ 0.001). In addition, while CA, CAT and NO enzyme levels were found to be significantly higher in the tonsil tissue of the patient group when compared to serum levels (p \ 0.05), there was no difference between tissue and serum MDA and ADA activity (p [ 0.05). Elevated ADA activity may be effective in the pathogenesis of chronic tonsillitis both by impairing tissue structure and contributing to SOR formation. Keywords Chronic tonsillitis Adenosine deaminase (ADA) Oxidative stress Malondialdehyde (MDA)
Introduction M. F. Garca (&) M. Turan N. Bozan H. Cankaya Department of Otorhinolaryngology, Medical Faculty, Yuzuncu Yıl University, 65400 Van, Turkey e-mail: [email protected] H. Demir Division of Biochemistry, Department of Chemistry, Yuzuncu Yıl University, Van, Turkey A. Kozan Department of Clinical Biochemistry, Gazi State Hospital, Samsun, Turkey S¸ . B. Belli Department of Otorhinolaryngology, Bag˘cılar Training and Research Hospital, Istanbul, Turkey A. Arslan Yuzuncu Yıl University, University of Health Sciences, Van, Turkey
Tonsil tissue has a lymphoepithelial structure and is part of the Waldeyer ring [1]. It has strategic importance in the defense against antigens which enter the body via the respiratory route or by digestion [2]. On the other hand, tonsillectomy is among the most commonly performed operations by ENT specialists alone or with adenoidectomy due to the recurrent or chronic infection of tonsil tissue. Better understanding of the active role of tonsil tissue in the immune system has put forth the necessity to more carefully assess tonsillectomy indications and limit its frequency [2]. The free oxygen radicals (FORs) are effective in the pathogenesis of chronic tonsillitis has been emphasized with studies [3–5]. They may react with proteins, nucleic acids and membrane phospholipids in the cell and may impair cellular functions [6, 7].
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Adenosine deaminase (ADA) enzyme directly or indirectly contributes to FORs development [8, 9]. This enzyme has an important role in the differentiation and proliferation of lymphoid cells in lymphoid tissue [10, 11]. Therefore, ADA enzyme activity is used as an important immunoenzyme marker in biochemical and immunomorphologic studies [12]. Although the importance of ADA for various infectious or inflammatory diseases has been demonstrated in literature [13–19], its role in chronic tonsillitis has not yet been identified. In the present study, ADA activity and stress oxidation markers values including malondialdehyde (MDA), nitric oxide (NO) and the antioxidant markers, catalase (CAT) and carbonic anhydrase (CA) which had not been previously studied were measured.
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head and neck region and without any infection or other systemic diseases. CAT and CA activity as antioxidants, MDA and NO product as peroxidation and ADA activity were determined in the plasma of both patients with chronic tonsillitis group and control group. The same measurements were also made in the patient’s tonsil tissue. Because it was not ethically acceptable to create a control group for the tonsillar tissue measurements to statistically compare these values with any corresponding value, only blood samples were obtained from the control group. ADA activities, antioxidant and oxidation product levels in the tonsil tissues of patients with chronic tonsillitis were compared with those of the serum.
Biochemical measurements Methods and materials Measurement of adenosine deaminase activity The study was performed in the tertiary referral center after institutional ethical committee approval and informed consent was obtained from all the patients. The study group consisted of 25 patients (aged 9–28 years) with a diagnosis of chronic tonsillitis who were planned to undergo tonsillectomy in the otolaryngology clinic. The examination of the patients included recording of current medical conditions, a physical and otoscopic examination. Chronic tonsillitis was diagnosed by history and ENT examination. Frequent attacks of tonsillitis and chronically inflamed tonsils with white debris originating from the crypts of the tonsils were accepted as indications for tonsillectomy. Indications other than recurrent attacks of acute tonsillitis such as chronic tonsillitis with no response to medical treatment associated with halitosis, persistent sore throat or painful cervical adenitis were not accepted. Patients with complications were not included in the study. Preoperatively in the study group, after overnight fasting, a total of 10 ml venous blood samples were obtained from each person in addition to the routine preoperative blood tests. Serum was obtained by centrifugation at 2,0009g for 10 min of blood samples taken without anticoagulant and stored at -40 °C until the analysis date. Tonsillectomy was performed on all the patients under general anesthesia. Immediately after tonsillectomy, each tonsil was rinsed with 0.9 % NaCl to eliminate any possible blood contamination. They were stored individually in plastic bottles at -40 °C until biochemical analyses were carried out. At the time of analysis, tonsillar tissue samples were weighed, placed in 1.15 % KCl solution, and homogenized for 30 min at 14,000 speed. The homogenates were centrifuged at 10,0009g for 20 min at 4 °C. The control group consisted of 25 healthy people with normal physical examinations with no complaints in the
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Serum and homogenates of tonsil tissue ADA activity was estimated spectrophotometrically by the Giusti method [5], which is based on the direct measurement of the ammonia, produced when ADA acts in excess of adenosine. The results were expressed as units per liter (U/L) for serum and units per milligram-tissue (U/mg-tissue) for tonsil tissue [20]. Measurement of lipid peroxidation Serum and homogenates of tonsil tissue lipid peroxidation was measured by estimating MDA levels as described by Yoshioka et al. [21]. The results were expressed as nanomoles per milliliter (nmol/ml) for serum and nanomoles per milligram-tissue (nmol/mg-tissue) for tonsil tissue. Measurement of nitric oxide NO measurement is very difficult in biological specimens because it is easily oxidized to nitrite (NO-) and subsequently to nitrate (NO3-) which serve as index parameters of NO production. Serum NO2-/NO3- concentrations were determined using the Griess reaction according to Tracey et al. [22]. The results were expressed as micromole per liter (lmol/l) for serum and micromole per gram tissue (lmol/g-tissue) for tonsil tissue. Measurement of catalase activity Serum and homogenate CAT activity was measured using H2O2 as a substrate [23]. The degradation of H2O2 was monitored at 240 nm for 5 min using a spectrophotometer, and enzyme activity was expressed in units per liter (U/L)
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Table 1 Serum MDA, NO, CA and CAT levels and ADA activity of the chronic tonsillitis patients and control group (p \ 0.001)
CAT
CA
Serum
Control group
Patients
p value
Mean ± SD
3.73 ± 0.754
1.139 ± 1.119
0.000
Median
3.80
1.12
Min–max
2.67–5.11
0.99–1.35
Mean ± SD
2.14 ± 0.187
1.03 ± 0.071
Median
2.22
1.04
Min–max
1.80–2.56
0.891–1.20
NO
Mean ± SD Median
4.93 ± 0.967 4.98
11.01 ± 0.642 11.02
Min–max
3.34–6.32
10.02–12.09
MDA
Mean ± SD
5.51 ± 1.033
11.98 ± 0.864
Median
5.22
11.98
ADA
Min–max
3.98–6.98
10–13.2
Mean ± SD
7.07 ± 1.94
17.62 ± 2.39
Median
7.00
17.32
Min–max
3.98–10.34
13.87–22.89
0.000
0.000
0.000
0.000
CAT Catalase, CA carbonic anhydrase, NO nitric oxide, MDA malondialdehyde, ADA adenosine deaminase
Fig. 1 Serum ADA activity, MDA and NO levels of patients were found to be significantly higher than those of control group (p \ 0.001). NO Nitric oxide, MDA malondialdehyde, ADA adenosine deaminase
for serum and units per gram tissue (U/g-tissue) for tonsil tissue. Measurement of carbonic anhydrase enzyme activity Serum and homogenate CA activity was measured by CO2 hydration. The hydration of CO2 was measured using the method of Rickli and Wilbur-Anderson, with bromothymol blue as the indicator [24]. The results were expressed as units per liter (U/L) for serum and units per gram tissue (U/ g-tissue) for tonsil tissue. Statistical analysis Levels of all the parameters both in blood and tissue were compared. The results were expressed as means and standard deviation (mean ± SD). Non-parametric continuous variables were compared by the Mann–Whitney U test. Parametric variables were compared using the Student’s t test. A p value \0.05 was considered to be statistically significant. Statistical evaluation was carried out with the SPSS 11.0.
Results Serum MDA, NO, CA and CAT levels and ADA activity of the chronic tonsillitis patients and control groups are given in Table 1. Serum ADA activity, MDA and NO levels of patients were found to be significantly higher than those of
Fig. 2 Serum CA and CAT levels were found to be significantly lower in control group statistically (p \ 0.001). CAT Catalase, CA carbonic anhydrase
the control group statistically (p \ 0.001) (Fig. 1). However, serum CA and CAT levels were found to be significantly lower in the control group statistically (p \ 0.001) (Fig. 2). Serum and tonsil tissue values of the patient group are given in Table 2. There was no statistically significant difference between ADA activity and MDA levels of serum and tonsil tissues (p [ 0.05) (Table 2). However, tonsil tissue levels of NO, CAT and CA were found to be
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Table 2 Serum and tonsil tissue parameter values in the patient group Patient
significantly higher than (p \ 0.05) (Table 2).
serum
levels
statistically
p value
CAT
Discussion
Serum Mean ± SD
1.139 ± 1.119
Median
1.12
Min–max
0.99–1.35
0.000
Tissue Mean ± SD Median
1.78 ± 0.364 1.82
Min–max
1.20–2.34
CA Serum Mean ± SD
1.03 ± 0.071
Median
1.04
Min–max
0.891–1.20
0.013
Tissue Mean ± SD
1.11 ± 0.125
Median
1.06
Min–max
1.02–1.53
NO Serum Mean ± SD
11.01 ± 0.642
Median Min–max
11.02 10.02–12.09
0.001
Tissue Mean ± SD
11.85 ± 11.82
Median
11.82
Min–max
10.53–13.77
MDA Serum Mean ± SD
11.98 ± 0.864
Median
11.98
Min–max
10–13.2
0.222
Tissue Mean ± SD
12.28 ± 0.860
Median
12.44
Min–max
10.53–14.11
ADA Serum Mean ± SD
17.62 ± 2.39
Median
17.32
Min–max
13.87–22.89
0.126
Tissue Mean ± SD
18.5 ± 1.96
Median
18.54
Min–max
15.44–22.89
CAT Catalase, CA carbonic anhydrase, NO nitric oxide, MDA malondialdehyde, ADA adenosine deaminase
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Inflammation developing as a result of chronic antigenic attacks in tonsil tissue affects the structure of the tonsils and leads to hypertrophy or fibrosis. As a result, although tonsillectomy for recurrent acute tonsillitis episodes is one of the most commonly performed operations, the process of the disease and the indications for tonsillectomy are still debated [2]. In recent years, the effects of oxidative stress in tonsil hypertrophy and chronic tonsillitis have also been emphasized [3–5, 25]. Oxidative stress has been shown to be effective in many pathologic conditions including cancer, cardio-vascular diseases, rheumatoid arthritis, ischemia/reperfusion and aging [6]. In addition, a close relationship has been demonstrated between inflammatory reactions and FORs formation in chronic diseases [6]. In the study by Kowalska et al. [26] demonstrated that FORs play a role in the pathogenesis of inflammation, while a FORs-related increase in oxidative stress was shown in 30 % of patients with tonsil hypertrophy, this increase was shown in approximately 90 % of the patients who experienced recurrent tonsillitis. Oxidative stress was found to be significantly high in both groups in the study by Cvetkovic et al. [4]. In chronic tonsillitis patients, FORs arise from the frequent interaction of tonsil tissue with pathogens which activates cellular and humoral immunity [4]. FORs affect the cellular membrane and react with lipids, proteins and nucleotides that lead to cellular destruction [6, 7]. Under normal conditions, elevated FORs are balanced with the clearing affect of antioxidants. Cellular damage and even death occur when this balance is impaired in favor of oxidants [3]. This effect is important in the development of many chronic diseases [6]. Antioxidants are used during the neutralization of oxidant products and this decrease in antioxidants results in oxidative stress. This condition contributes to the chronicity of inflammation and thereby the disease. In previous studies, the role of free radicals, which increase due to the constant inflammation developing with leucocytes and lymphocytes, was emphasized in the chronicity of tonsillitis episodes [3, 4]. FORs lead to lipid peroxidation by degrading polyunsaturated fatty acids in the cellular wall. MDA and NO are the most important markers for indicating FORs-related tissue injury [6, 7]. On the other hand, the most important enzymes for oxidative balance are superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), CAT and CA. In
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addition, non-enzymatic antioxidants include glutathione, tocopherol (vit E), ascorbic acid (vit C), carotene (vit A), albumin, bilirubin and uric acid [3, 4, 6, 7]. The human body balances its oxidative state with these enzymatic and non-enzymatic antioxidants. Chronic tonsillitis is characterized by local infection episodes with various pathogens, permanent activation of the lymphoid cells and intensive hypoxia/reoxygenation episodes [27]. In the inflammation process of the tonsils, FORs, which are one part of the defense mechanism, are abundantly produced by neutrophils, monocytes, eosinophils and macrophages to kill bacteria [3, 4]. Yılmaz et al. [3] claimed that the overall oxidative stress is reduced through tonsillectomy as microbial sources are eliminated and thereby the immunologic system is supported. The influence of FORs on the immune system is as follows: it negatively affects the immune system by reducing the reproduction capacity of defensive cells through DNA damage, by reducing synthesis of some critical factors and leading to decreases in antioxidant levels, thus tendency of upper respiratory tract infections occurs. Cvetkovic et al. [4] emphasized that the antioxidant defense system may be protective against tonsillitis recurrence, but that tonsillectomy alone cannot provide this protection. The ADA enzyme is present in all cells of the body [13]. It contributes to lymphoid tissue development and lymphoid maturation [11]. It is a purine metabolism enzyme which regulates adenosine levels by irreversibly catalyzing adenosine to inosine and 2-deoxyadenosine to 2-deoxyinosine [11, 16, 17]. Adenosine is a metabolic precursor for nucleic acids and an important signal molecule for regulation of various physiologic mechanisms [11, 16, 17]. Its physiologic role in different tissues has not been clearly understood, although it plays a central role in the immune system. The ADA activity of leucocytes increases in pathologies where the cell-mediated immune system is effective [11]. This indicates that ADA enzyme activity may play a role in the pathogenesis of some diseases. It is even used in the diagnosis and follow-up of certain diseases such as rheumatoid arthritis, systemic lupus erythematosus, infectious mononucleosis, brucellosis, Behcet’s disease and tuberculosis as an indicator of cellular immunity [13–16]. In the studies by Okur et al. [8, 28], ADA activity was also demonstrated in the inferior concha, nasal mucosa and adenoid tissue. Although tonsil tissue is a part of the lymphoid system, we did not encounter any studies showing ADA enzyme activity in this tissue. In our study, the aim was to determine the differences in ADA enzyme activity in relation to chronic tonsillitis. The main role of tonsils in the immune system is to form antigen-specific memory and effector B cells and other immune globulins, mainly secretory IgA [2, 29]. Activated
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T cells are present in the extrafollicular field and B cells, which recognize specific antigens and antibody-producing plasma cells, are present in the germinal center. To differentiate, B lymphocytes and plasma cells need to be activated by T lymphocytes. T lymphocytes can cause this activation by differentiating into antigen-specific T cells. This differentiation of T lymphocytes is accomplished by contact with the surface of many antigen-presenting cells while migrating along the interfollicular field. Differentiated and activated T cells activate B cells. Consequently, the proliferation of activated B cells leads to antigen-specific Ig production [2]. The ADA enzyme has a central role in the differentiation and proliferation of T cell which is effective in the formation of the defense system of tonsil tissue against antigens and it is a marker of T cell activation [10, 11]. Okur et al. [28] found ADA activity to be high in patients with adenoid hypertrophy and emphasized that T lymphocyte activation could have a potential role in adenoid hypertrophy development. So, ADA enzyme activity may both be used in the diagnosis and follow-up of diseases where cellular immunity plays a role as is the case in Tbc, H. pylori and Behcet’s disease and it may also be useful for the prediction of disease processes in tonsil tissue [13–16]. Increases in ADA enzyme activity have effects on the increase of FORs which plays an important role in tissue damage [8, 9, 28]. It does this by increasing hypoxanthine and xanthine production, which is necessary for xanthine oxidase production, which has been shown as the central mechanism in FORs formation. In addition, ADA enzyme activity irreversibly converts adenosine, which takes part in the structure of adenosine triphosphate, which is a multifunctional intracellular nucleotide to inosine and leads to its reduction [8, 9]. Adenosine is a protein which forms cellular function and hinders lipopolysaccharide-dependent formation of reactive oxygen radicals with its potent anti-inflammatory effect [8, 9, 30]. Consequently, an increase in ADA enzyme activity may contribute to increases in FOR through reducing adenosine and thereby tissue injury. In our study, ADA enzyme activity and the oxidative stress reflectors MDA and NO, were found to be significantly high in chronic tonsillitis patients when compared to the control group (p \ 0.001). In addition, serum levels of antioxidant enzymes, CA and CAT were found to be significantly lower than the control group (p \ 0.001). These findings were similar with those of Okur et al. [8], who investigated patients with adenoid hypertrophy. So it can be stated that the ADA enzyme impairs the main structure of tonsil tissue and contributes to FORs formation and thereby leads to chronic tonsillitis. In addition, while tissue CA, CAT and NO levels of patients were
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found to be higher than those of serum levels (p \ 0.05), there was no difference between ADA and MDA levels of serum and tissue (p [ 0.05). In our study, tissue levels could not be studied in the control group as removal of healthy tonsil tissue is not ethically appropriate. However, given the similarity of tissue and serum ADA levels, serum levels may be sufficient to reflect ADA enzyme activity. In conclusion, tissue and serum ADA enzyme activity was found to be elevated in patients with chronic tonsillitis, so activated T lymphocytes may be considered to have an important role in the pathogenesis of chronic tonsillitis. In addition, measurements of serum ADA activity may provide valuable information about the disease as serum values are similar to tissue values.
References 1. Boyaka PN, Wright PF, Marinaro M, Kiyono H, Johnson JE, Gonzales RA, Ikizler MR, Werkhaven JA, Jackson RJ, Fujihashi K, Di Fabio S, Staats HF, McGhee JR (2000) Human nasopharyngeal-associated lymphoreticular tissues. Am J Pathol 157(6):2023–2035 2. Nasrin M, Miah MR, Datta PG, Saleh AA, Anwar S, Saha KL (2012) Effect of tonsillectomy on humoral immunity. Bangladesh Med Res Counc Bull 38(2):59–61 3. Yilmaz T, Koc¸an EG, Besler HT (2004) The role of oxidants and antioxidants in chronic tonsillitis and adenoid hypertrophy in children. Int J Pediatr Otorhinolaryngol 68(8):1053–1058 4. Cvetkovic´ T, Vlahovic´ P, Todorovic´ M, Stankovic´ M (2009) Investigation of oxidative stress in patients with chronic tonsillitis. Auris Nasus Larynx 36(3):340–344 5. Passali D, Damiani V, Passali GC, Passali FM, Boccazzi A, Bellussi L (2004) Structural and immunological characteristics of chronically inflamed adenotonsillar tissue in childhood. Clin Diagn Lab Immunol 11:1154–1157 6. Valko M, Leibfritz D, Moncol J, Cronin MT, Mazur M, Telser J (2007) Free radicals and antioxidants in normal physiological functions and human disease. Int J Biochem Cell Biol 39:44–84 7. Kavak S, Garca MF, Gecit I, Meral I, Cengiz N, Demir H (2012) Effects of extracorporeal shock-wave lithotripsy directed at the parotid gland on oxidative stress parameters and some trace element levels in facial nerve of rats. Muscle Nerve 45(4):562–566 8. Okur E, Inanc F, Yildirim I, Kilinc M, Kilic MA (2006) Malondialdehyde level and adenosine deaminase activity in nasal polyps. Otolaryngol Head Neck Surg 134(1):37–40 9. Xia Y, Khatchikian G, Zweier JL (1996) Adenosine deaminase inhibition prevents free radical-mediated injury in the postischemic heart. J Biol Chem 271(17):10096–10102 10. Aldrich MB, Chen W, Blackburn MR, Martinez-Valdez H, Datta SK, Kellems RE (2003) Impaired germinal center maturation in adenosine deaminase deficiency. J Immunol 171(10):5562–5570 11. Kameoka J, Tanaka T, Nojima Y, Schlossman SF, Morimoto C (1993) Direct association of adenosine deaminase with a T cell activation antigen, CD26. Science 261(5120):466–469 12. Franco R, Valenzuela A, Lluis C, Blanco J (1998) Enzymatic and extraenzymatic role of ecto-adenosine deaminase in lymphocytes. Immunol Rev 161:27–42
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Eur Arch Otorhinolaryngol (2014) 271:1797–1802 13. Saghiri R, Ghashghai N, Movaseghi S, Poursharifi P, Jalilfar S, Bidhendi MA, Ghazizadeh L, Ebrahimi-Rad M (2012) Serum adenosine deaminase activity in patients with systemic lupus erythematosus: a study based on ADA1 and ADA2 isoenzymes pattern. Rheumatol Int 32(6):1633–1638 14. Ungerer JP, Oosthuizen HM, Bissbort SH, Vermaak WJ (1992) Serum adenosine deaminase: isoenzymes and diagnostic application. Clin Chem 38(7):1322–1326 15. Dikensoy O, Namiduru M, Hocaoglu S, Ikidag B, Filiz A (2002) Increased pleural fluid adenosine deaminase in brucellosis is difficult to differentiate from tuberculosis. Respiration 69(6):556–559 16. Canpolat F, Unver M, Eskiog˘lu F, Ko¨sebalaban S, Durmazlar SP (2006) Serum and erythrocyte adenosine deaminase activities in patients with Behcet’s disease. Int J Dermatol 45(9):1053–1056 17. Liao YJ, Wu CY, Lee SW, Lee CL, Yang SS, Chang CS, Lee TY (2012) Adenosine deaminase activity in tuberculous peritonitis among patients with underlying liver cirrhosis. World J Gastroenterol 18(37):5260–5265 18. Erel O, Kocyigit A, Gurel MS, Bulut V, Seyrek A, Ozdemir Y (1998) Adenosine deaminase activities in sera, lymphocytes and granulocytes in patients with cutaneous leishmaniasis. Mem Inst Oswaldo Cruz 93:491–494 19. Lee SJ, Hwang HS, Kim BNR, Kim MA, Lee JW, Park YW, Kim YH (2007) Changes in serum adenosine deaminase activity during normal pregnancy. J Korean Med Sci 22:718–721 20. Giusti G (1974) Adenosine deaminase. In: Bergmeyer HI (ed) Methods of enzymatic analyses. Academic Press, New York, pp 1092–1098 21. Yoshioka T, Kawada K, Shimada T, Mori M (1979) Lipid peroxidation in maternal and cord blood and protective mechanisms against activated oxygen toxicity in the blood. Am J Obster Gynecol 135:372–376 22. Tracey WR, Tse J, Carter G (1995) Lipopolysaccharide-induced changes in plasma nitrite and nitrate concentrations in rats and mice: pharmacological evaluation of nitric oxide synthase inhibitors. J Pharmacol Exp Ther 272:1011–1015 23. Aebi H, Bergmayer HU (1974) Catalase methods of enzymatic analysis. Academic Press Inc, New York, pp 673–677 24. Rickli EE, Ghazanfar SAS, Gibbons BH, Edsall JT (1964) Carbonic anhydrase from human erythrocytes. Preparation and properties of two enzymes. J Biol Chem 239:1065–1078 ¨ , Ekinci A (2011) 25. Koc S, Aksoy N, Bilinc H, Duygu F, Uysal IO Paraoxonase and arylesterase activity and total oxidative/antioxidative status in patients with chronic adenotonsillitis. Int J Pediatr Otorhinolaryngol 75(11):1364–1367 26. Kowalska M, Kowalska H, Zawadzka-Głos L, Debska M, Szerszen´ E, Chmielik M, Wasik M (2003) Dysfunction of peripheral blood granulocyte oxidative metabolism in children with recurrent upper respiratory tract infections. Int J Pediatr Otorhinolaryngol 67:365–371 27. Kutluhan A, Ugras S, Kiris M, Cankaya H, Kiroglu AF, Yurttas V (2003) Differences in clinical and histopathologic features between chronic adenotonsillitis and chronic adenotonsillar hypertrophy. Kulak Burun Bogaz Ihtis Derg 10(2):61–67 28. Okur E, Tolun FI, Yildirim I, Kilinc M, Kilic MA (2012) Malondialdehyde level and adenosine deaminase activity in adenoid tissue of patients with OME and obstructive adenoid hypertrophy. Int J Pediatr Otorhinolaryngol 76(4):579–582 29. Van Kempen MJP, Rijkers GT, Van Cauwenberge PB (2000) The immune response in adenoids and tonsils. Int Arch Allergy Immunol 122:8–19 30. Bor MV, Durmus O, Bilgihan A, Cevik C, Turkozkan N (1999) The beneficial effect of 20-deoxycoformycin in renal ischemiareperfusion is mediated both by preservation of tissue ATP and inhibition of lipid peroxidation. Int J Clin Lab Res 29(2):75–79
MISCELLANEOUS
Assessment of adenosine deaminase (ADA) activity and oxidative stress in patients with chronic tonsillitis Mehmet Fatih Garca • Halit Demir • Mahfuz Turan • Nazım Bozan • Ahmet Kozan • S¸ eyda Bayel Belli • Ays¸ e Arslan • Hakan Cankaya
Received: 9 July 2013 / Accepted: 5 November 2013 / Published online: 4 December 2013 Ó Springer-Verlag Berlin Heidelberg 2013
Abstract To emphasize the effectiveness of adenosine deaminase (ADA) enzyme, which has important roles in the differentiation of lymphoid cells, and oxidative stress in patients with chronic tonsillitis. Serum and tissue samples were obtained from 25 patients who underwent tonsillectomy due to recurrent episodes of acute tonsillitis. In the control group, which also had 25 subjects, only serum samples were taken as obtaining tissue samples would not have been ethically appropriate. ADA enzyme activity, catalase (CAT), carbonic anhydrase (CA), nitric oxide (NO) and malondialdehyde (MDA) were measured in the serum and tissue samples of patients and control group subjects. The serum values of both groups were compared. In addition, the tissue and serum values of patients were compared. Serum ADA activity and the oxidant enzymes MDA and NO values of the patient group were
significantly higher than those of the control group (p \ 0.001), the antioxidant enzymes CA and CAT values of the patient group were significantly lower than those of the control group (p \ 0.001). In addition, while CA, CAT and NO enzyme levels were found to be significantly higher in the tonsil tissue of the patient group when compared to serum levels (p \ 0.05), there was no difference between tissue and serum MDA and ADA activity (p [ 0.05). Elevated ADA activity may be effective in the pathogenesis of chronic tonsillitis both by impairing tissue structure and contributing to SOR formation. Keywords Chronic tonsillitis Adenosine deaminase (ADA) Oxidative stress Malondialdehyde (MDA)
Introduction M. F. Garca (&) M. Turan N. Bozan H. Cankaya Department of Otorhinolaryngology, Medical Faculty, Yuzuncu Yıl University, 65400 Van, Turkey e-mail: [email protected] H. Demir Division of Biochemistry, Department of Chemistry, Yuzuncu Yıl University, Van, Turkey A. Kozan Department of Clinical Biochemistry, Gazi State Hospital, Samsun, Turkey S¸ . B. Belli Department of Otorhinolaryngology, Bag˘cılar Training and Research Hospital, Istanbul, Turkey A. Arslan Yuzuncu Yıl University, University of Health Sciences, Van, Turkey
Tonsil tissue has a lymphoepithelial structure and is part of the Waldeyer ring [1]. It has strategic importance in the defense against antigens which enter the body via the respiratory route or by digestion [2]. On the other hand, tonsillectomy is among the most commonly performed operations by ENT specialists alone or with adenoidectomy due to the recurrent or chronic infection of tonsil tissue. Better understanding of the active role of tonsil tissue in the immune system has put forth the necessity to more carefully assess tonsillectomy indications and limit its frequency [2]. The free oxygen radicals (FORs) are effective in the pathogenesis of chronic tonsillitis has been emphasized with studies [3–5]. They may react with proteins, nucleic acids and membrane phospholipids in the cell and may impair cellular functions [6, 7].
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Adenosine deaminase (ADA) enzyme directly or indirectly contributes to FORs development [8, 9]. This enzyme has an important role in the differentiation and proliferation of lymphoid cells in lymphoid tissue [10, 11]. Therefore, ADA enzyme activity is used as an important immunoenzyme marker in biochemical and immunomorphologic studies [12]. Although the importance of ADA for various infectious or inflammatory diseases has been demonstrated in literature [13–19], its role in chronic tonsillitis has not yet been identified. In the present study, ADA activity and stress oxidation markers values including malondialdehyde (MDA), nitric oxide (NO) and the antioxidant markers, catalase (CAT) and carbonic anhydrase (CA) which had not been previously studied were measured.
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head and neck region and without any infection or other systemic diseases. CAT and CA activity as antioxidants, MDA and NO product as peroxidation and ADA activity were determined in the plasma of both patients with chronic tonsillitis group and control group. The same measurements were also made in the patient’s tonsil tissue. Because it was not ethically acceptable to create a control group for the tonsillar tissue measurements to statistically compare these values with any corresponding value, only blood samples were obtained from the control group. ADA activities, antioxidant and oxidation product levels in the tonsil tissues of patients with chronic tonsillitis were compared with those of the serum.
Biochemical measurements Methods and materials Measurement of adenosine deaminase activity The study was performed in the tertiary referral center after institutional ethical committee approval and informed consent was obtained from all the patients. The study group consisted of 25 patients (aged 9–28 years) with a diagnosis of chronic tonsillitis who were planned to undergo tonsillectomy in the otolaryngology clinic. The examination of the patients included recording of current medical conditions, a physical and otoscopic examination. Chronic tonsillitis was diagnosed by history and ENT examination. Frequent attacks of tonsillitis and chronically inflamed tonsils with white debris originating from the crypts of the tonsils were accepted as indications for tonsillectomy. Indications other than recurrent attacks of acute tonsillitis such as chronic tonsillitis with no response to medical treatment associated with halitosis, persistent sore throat or painful cervical adenitis were not accepted. Patients with complications were not included in the study. Preoperatively in the study group, after overnight fasting, a total of 10 ml venous blood samples were obtained from each person in addition to the routine preoperative blood tests. Serum was obtained by centrifugation at 2,0009g for 10 min of blood samples taken without anticoagulant and stored at -40 °C until the analysis date. Tonsillectomy was performed on all the patients under general anesthesia. Immediately after tonsillectomy, each tonsil was rinsed with 0.9 % NaCl to eliminate any possible blood contamination. They were stored individually in plastic bottles at -40 °C until biochemical analyses were carried out. At the time of analysis, tonsillar tissue samples were weighed, placed in 1.15 % KCl solution, and homogenized for 30 min at 14,000 speed. The homogenates were centrifuged at 10,0009g for 20 min at 4 °C. The control group consisted of 25 healthy people with normal physical examinations with no complaints in the
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Serum and homogenates of tonsil tissue ADA activity was estimated spectrophotometrically by the Giusti method [5], which is based on the direct measurement of the ammonia, produced when ADA acts in excess of adenosine. The results were expressed as units per liter (U/L) for serum and units per milligram-tissue (U/mg-tissue) for tonsil tissue [20]. Measurement of lipid peroxidation Serum and homogenates of tonsil tissue lipid peroxidation was measured by estimating MDA levels as described by Yoshioka et al. [21]. The results were expressed as nanomoles per milliliter (nmol/ml) for serum and nanomoles per milligram-tissue (nmol/mg-tissue) for tonsil tissue. Measurement of nitric oxide NO measurement is very difficult in biological specimens because it is easily oxidized to nitrite (NO-) and subsequently to nitrate (NO3-) which serve as index parameters of NO production. Serum NO2-/NO3- concentrations were determined using the Griess reaction according to Tracey et al. [22]. The results were expressed as micromole per liter (lmol/l) for serum and micromole per gram tissue (lmol/g-tissue) for tonsil tissue. Measurement of catalase activity Serum and homogenate CAT activity was measured using H2O2 as a substrate [23]. The degradation of H2O2 was monitored at 240 nm for 5 min using a spectrophotometer, and enzyme activity was expressed in units per liter (U/L)
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Table 1 Serum MDA, NO, CA and CAT levels and ADA activity of the chronic tonsillitis patients and control group (p \ 0.001)
CAT
CA
Serum
Control group
Patients
p value
Mean ± SD
3.73 ± 0.754
1.139 ± 1.119
0.000
Median
3.80
1.12
Min–max
2.67–5.11
0.99–1.35
Mean ± SD
2.14 ± 0.187
1.03 ± 0.071
Median
2.22
1.04
Min–max
1.80–2.56
0.891–1.20
NO
Mean ± SD Median
4.93 ± 0.967 4.98
11.01 ± 0.642 11.02
Min–max
3.34–6.32
10.02–12.09
MDA
Mean ± SD
5.51 ± 1.033
11.98 ± 0.864
Median
5.22
11.98
ADA
Min–max
3.98–6.98
10–13.2
Mean ± SD
7.07 ± 1.94
17.62 ± 2.39
Median
7.00
17.32
Min–max
3.98–10.34
13.87–22.89
0.000
0.000
0.000
0.000
CAT Catalase, CA carbonic anhydrase, NO nitric oxide, MDA malondialdehyde, ADA adenosine deaminase
Fig. 1 Serum ADA activity, MDA and NO levels of patients were found to be significantly higher than those of control group (p \ 0.001). NO Nitric oxide, MDA malondialdehyde, ADA adenosine deaminase
for serum and units per gram tissue (U/g-tissue) for tonsil tissue. Measurement of carbonic anhydrase enzyme activity Serum and homogenate CA activity was measured by CO2 hydration. The hydration of CO2 was measured using the method of Rickli and Wilbur-Anderson, with bromothymol blue as the indicator [24]. The results were expressed as units per liter (U/L) for serum and units per gram tissue (U/ g-tissue) for tonsil tissue. Statistical analysis Levels of all the parameters both in blood and tissue were compared. The results were expressed as means and standard deviation (mean ± SD). Non-parametric continuous variables were compared by the Mann–Whitney U test. Parametric variables were compared using the Student’s t test. A p value \0.05 was considered to be statistically significant. Statistical evaluation was carried out with the SPSS 11.0.
Results Serum MDA, NO, CA and CAT levels and ADA activity of the chronic tonsillitis patients and control groups are given in Table 1. Serum ADA activity, MDA and NO levels of patients were found to be significantly higher than those of
Fig. 2 Serum CA and CAT levels were found to be significantly lower in control group statistically (p \ 0.001). CAT Catalase, CA carbonic anhydrase
the control group statistically (p \ 0.001) (Fig. 1). However, serum CA and CAT levels were found to be significantly lower in the control group statistically (p \ 0.001) (Fig. 2). Serum and tonsil tissue values of the patient group are given in Table 2. There was no statistically significant difference between ADA activity and MDA levels of serum and tonsil tissues (p [ 0.05) (Table 2). However, tonsil tissue levels of NO, CAT and CA were found to be
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1800
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Table 2 Serum and tonsil tissue parameter values in the patient group Patient
significantly higher than (p \ 0.05) (Table 2).
serum
levels
statistically
p value
CAT
Discussion
Serum Mean ± SD
1.139 ± 1.119
Median
1.12
Min–max
0.99–1.35
0.000
Tissue Mean ± SD Median
1.78 ± 0.364 1.82
Min–max
1.20–2.34
CA Serum Mean ± SD
1.03 ± 0.071
Median
1.04
Min–max
0.891–1.20
0.013
Tissue Mean ± SD
1.11 ± 0.125
Median
1.06
Min–max
1.02–1.53
NO Serum Mean ± SD
11.01 ± 0.642
Median Min–max
11.02 10.02–12.09
0.001
Tissue Mean ± SD
11.85 ± 11.82
Median
11.82
Min–max
10.53–13.77
MDA Serum Mean ± SD
11.98 ± 0.864
Median
11.98
Min–max
10–13.2
0.222
Tissue Mean ± SD
12.28 ± 0.860
Median
12.44
Min–max
10.53–14.11
ADA Serum Mean ± SD
17.62 ± 2.39
Median
17.32
Min–max
13.87–22.89
0.126
Tissue Mean ± SD
18.5 ± 1.96
Median
18.54
Min–max
15.44–22.89
CAT Catalase, CA carbonic anhydrase, NO nitric oxide, MDA malondialdehyde, ADA adenosine deaminase
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Inflammation developing as a result of chronic antigenic attacks in tonsil tissue affects the structure of the tonsils and leads to hypertrophy or fibrosis. As a result, although tonsillectomy for recurrent acute tonsillitis episodes is one of the most commonly performed operations, the process of the disease and the indications for tonsillectomy are still debated [2]. In recent years, the effects of oxidative stress in tonsil hypertrophy and chronic tonsillitis have also been emphasized [3–5, 25]. Oxidative stress has been shown to be effective in many pathologic conditions including cancer, cardio-vascular diseases, rheumatoid arthritis, ischemia/reperfusion and aging [6]. In addition, a close relationship has been demonstrated between inflammatory reactions and FORs formation in chronic diseases [6]. In the study by Kowalska et al. [26] demonstrated that FORs play a role in the pathogenesis of inflammation, while a FORs-related increase in oxidative stress was shown in 30 % of patients with tonsil hypertrophy, this increase was shown in approximately 90 % of the patients who experienced recurrent tonsillitis. Oxidative stress was found to be significantly high in both groups in the study by Cvetkovic et al. [4]. In chronic tonsillitis patients, FORs arise from the frequent interaction of tonsil tissue with pathogens which activates cellular and humoral immunity [4]. FORs affect the cellular membrane and react with lipids, proteins and nucleotides that lead to cellular destruction [6, 7]. Under normal conditions, elevated FORs are balanced with the clearing affect of antioxidants. Cellular damage and even death occur when this balance is impaired in favor of oxidants [3]. This effect is important in the development of many chronic diseases [6]. Antioxidants are used during the neutralization of oxidant products and this decrease in antioxidants results in oxidative stress. This condition contributes to the chronicity of inflammation and thereby the disease. In previous studies, the role of free radicals, which increase due to the constant inflammation developing with leucocytes and lymphocytes, was emphasized in the chronicity of tonsillitis episodes [3, 4]. FORs lead to lipid peroxidation by degrading polyunsaturated fatty acids in the cellular wall. MDA and NO are the most important markers for indicating FORs-related tissue injury [6, 7]. On the other hand, the most important enzymes for oxidative balance are superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), CAT and CA. In
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addition, non-enzymatic antioxidants include glutathione, tocopherol (vit E), ascorbic acid (vit C), carotene (vit A), albumin, bilirubin and uric acid [3, 4, 6, 7]. The human body balances its oxidative state with these enzymatic and non-enzymatic antioxidants. Chronic tonsillitis is characterized by local infection episodes with various pathogens, permanent activation of the lymphoid cells and intensive hypoxia/reoxygenation episodes [27]. In the inflammation process of the tonsils, FORs, which are one part of the defense mechanism, are abundantly produced by neutrophils, monocytes, eosinophils and macrophages to kill bacteria [3, 4]. Yılmaz et al. [3] claimed that the overall oxidative stress is reduced through tonsillectomy as microbial sources are eliminated and thereby the immunologic system is supported. The influence of FORs on the immune system is as follows: it negatively affects the immune system by reducing the reproduction capacity of defensive cells through DNA damage, by reducing synthesis of some critical factors and leading to decreases in antioxidant levels, thus tendency of upper respiratory tract infections occurs. Cvetkovic et al. [4] emphasized that the antioxidant defense system may be protective against tonsillitis recurrence, but that tonsillectomy alone cannot provide this protection. The ADA enzyme is present in all cells of the body [13]. It contributes to lymphoid tissue development and lymphoid maturation [11]. It is a purine metabolism enzyme which regulates adenosine levels by irreversibly catalyzing adenosine to inosine and 2-deoxyadenosine to 2-deoxyinosine [11, 16, 17]. Adenosine is a metabolic precursor for nucleic acids and an important signal molecule for regulation of various physiologic mechanisms [11, 16, 17]. Its physiologic role in different tissues has not been clearly understood, although it plays a central role in the immune system. The ADA activity of leucocytes increases in pathologies where the cell-mediated immune system is effective [11]. This indicates that ADA enzyme activity may play a role in the pathogenesis of some diseases. It is even used in the diagnosis and follow-up of certain diseases such as rheumatoid arthritis, systemic lupus erythematosus, infectious mononucleosis, brucellosis, Behcet’s disease and tuberculosis as an indicator of cellular immunity [13–16]. In the studies by Okur et al. [8, 28], ADA activity was also demonstrated in the inferior concha, nasal mucosa and adenoid tissue. Although tonsil tissue is a part of the lymphoid system, we did not encounter any studies showing ADA enzyme activity in this tissue. In our study, the aim was to determine the differences in ADA enzyme activity in relation to chronic tonsillitis. The main role of tonsils in the immune system is to form antigen-specific memory and effector B cells and other immune globulins, mainly secretory IgA [2, 29]. Activated
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T cells are present in the extrafollicular field and B cells, which recognize specific antigens and antibody-producing plasma cells, are present in the germinal center. To differentiate, B lymphocytes and plasma cells need to be activated by T lymphocytes. T lymphocytes can cause this activation by differentiating into antigen-specific T cells. This differentiation of T lymphocytes is accomplished by contact with the surface of many antigen-presenting cells while migrating along the interfollicular field. Differentiated and activated T cells activate B cells. Consequently, the proliferation of activated B cells leads to antigen-specific Ig production [2]. The ADA enzyme has a central role in the differentiation and proliferation of T cell which is effective in the formation of the defense system of tonsil tissue against antigens and it is a marker of T cell activation [10, 11]. Okur et al. [28] found ADA activity to be high in patients with adenoid hypertrophy and emphasized that T lymphocyte activation could have a potential role in adenoid hypertrophy development. So, ADA enzyme activity may both be used in the diagnosis and follow-up of diseases where cellular immunity plays a role as is the case in Tbc, H. pylori and Behcet’s disease and it may also be useful for the prediction of disease processes in tonsil tissue [13–16]. Increases in ADA enzyme activity have effects on the increase of FORs which plays an important role in tissue damage [8, 9, 28]. It does this by increasing hypoxanthine and xanthine production, which is necessary for xanthine oxidase production, which has been shown as the central mechanism in FORs formation. In addition, ADA enzyme activity irreversibly converts adenosine, which takes part in the structure of adenosine triphosphate, which is a multifunctional intracellular nucleotide to inosine and leads to its reduction [8, 9]. Adenosine is a protein which forms cellular function and hinders lipopolysaccharide-dependent formation of reactive oxygen radicals with its potent anti-inflammatory effect [8, 9, 30]. Consequently, an increase in ADA enzyme activity may contribute to increases in FOR through reducing adenosine and thereby tissue injury. In our study, ADA enzyme activity and the oxidative stress reflectors MDA and NO, were found to be significantly high in chronic tonsillitis patients when compared to the control group (p \ 0.001). In addition, serum levels of antioxidant enzymes, CA and CAT were found to be significantly lower than the control group (p \ 0.001). These findings were similar with those of Okur et al. [8], who investigated patients with adenoid hypertrophy. So it can be stated that the ADA enzyme impairs the main structure of tonsil tissue and contributes to FORs formation and thereby leads to chronic tonsillitis. In addition, while tissue CA, CAT and NO levels of patients were
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found to be higher than those of serum levels (p \ 0.05), there was no difference between ADA and MDA levels of serum and tissue (p [ 0.05). In our study, tissue levels could not be studied in the control group as removal of healthy tonsil tissue is not ethically appropriate. However, given the similarity of tissue and serum ADA levels, serum levels may be sufficient to reflect ADA enzyme activity. In conclusion, tissue and serum ADA enzyme activity was found to be elevated in patients with chronic tonsillitis, so activated T lymphocytes may be considered to have an important role in the pathogenesis of chronic tonsillitis. In addition, measurements of serum ADA activity may provide valuable information about the disease as serum values are similar to tissue values.
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