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Investigation of oxidative stress in patients with chronic tonsillitis
Auris Nasus Larynx 36 (2009) 340–344 www.elsevier.com/locate/anl
Investigation of oxidative stress in patients with chronic tonsillitis Tatjana Cvetkovic´ a, Predrag Vlahovic´ b,*, Miroljub Todorovic´ c, Milan Stankovic´ d a
b
Institute of Biochemistry, Faculty of Medicine, Nisˇ, Serbia Clinic of Nephrology & Hemodialysis, Clinical Center Nisˇ, Dr. Zorana Ðinpic´a 48, 18000 Nisˇ, Serbia c Clinic for Othorhinolaryngology, Cetinje, Montenegro d Clinic for Othorhinolaryngology, Clinical Center Nisˇ, Serbia Received 20 May 2008; accepted 17 October 2008 Available online 25 December 2008
Abstract Objectives: Oxidative stress has been implicated in numerous pathological conditions including chronic tonsillitis. The aim of this study was to assess levels of lipid peroxidation, evidenced by formation of thiobarbituric acid reactive substance (TBARS), level of total sulfhydryl groups (TSH), and carbonyl content in patients with tonsillar hypertrophy (TH) and recurrent tonsillitis (RT), before and after tonsillectomy. Methods: In this study the serum and tonsillar tissue levels of TBARS, TSH and carbonyl content were investigated in 35 patients with TH and RT, before and 1 month after the operation, compared to 30 age-matched controls. Results: In both TH and RT groups, a significantly higher serum TBARS levels were observed before and 4 weeks after tonsillectomy in comparison with healthy subjects. The serum level in TH patients after operation was even higher compared to the levels before. There was statistically significant difference in serum TSH levels between patients with RT before operation compared to the control group. After tonsillectomy the serum levels of TSH were higher compared to control groups and TH and RT patients before operation. Carbonyl content was attenuated only in TH patients after tonsillectomy. In tonsillar tissue, significantly lower level of glutathione content (GSH) has been observed in RT related to TH patients. Conclusions: Oxidative stress, in patients with tonsillar hypertrophy and recurrent tonsillitis, is still present 1 month after the removal of tonsillar tissue. Antioxidant therapy, during the recovery period after tonsillectomy, could be optional treatment. # 2008 Elsevier Ireland Ltd. All rights reserved. Keywords: Oxidative stress; Palatine tonsil; Tonsillitis; Recurrent tonsillitis; Tonsillar hypertrophy
1. Introduction Palatine tonsils are reactive lymphoepithelial organs which are considered the first place of contact with variety of antigenic substances present in air and food. They are located at the entrance of respiratory and alimentary tracts and during recurrent upper respiratory tract infections tonsillar humoral and cellular immunity is altered. Recurrent tonsillitis (RT) and idiopathic (obstructive) tonsillar hypertrophy (TH) represent two different pathogenic entities with different morphology [1] and patterns of local immune response [2,3]. Tonsillar hypertrophy is characterized histologically by an enlarge* Corresponding author. Fax: +381 18 53 01 27. E-mail address: [email protected] (P. Vlahovic´).
ment of lymphoid follicles and reduced extrafollicular tissue, indicating hyperplastic condition of lymphoid cells in the germinal centers. However, in the recurrent tonsillitis reduced number of follicles and fibrosis in extrafolicular tissue is demonstrable [4,5]. In addition, RT is caused by an immune response followed with strong inflammatory process compared to TH where environmental agents mediate local antigen stimulation without inflammation [6]. Although the pathophysiology of tonsillar disease processes remains still unknown, both TH and RT are the most common reasons for surgical tonsillectomy. Moreover, the benefit of tonsillar removal is still controversial. Oxidative stress has been implicated in numerous pathological conditions including cancer, cardiovascular diseases, rheumatoid arthritis, ischemia/reperfusion and
0385-8146/$ – see front matter # 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.anl.2008.10.004
T. Cvetkovic´ et al. / Auris Nasus Larynx 36 (2009) 340–344
341
2.2. Biochemical analyses
aging [7]. Considerably evidence indicated that reactive oxygen species, during chronic inflammation process, mediate the regulation of tonsillar pathophysiological functions [8,9]. The role of free radicals has been well elucidated in chronic tonsillitis [10,11]. The aim of this study was to investigate the effect of oxidative damage due to free radicals in patients with obstructive tonsillar hypertrophy and recurrent tonsillitis, in pre- and post-tonsillectomy period, and to compare the results with healthy control subjects.
2. Patients and methods
The biochemical determinations were carried out in serum and 10% homogenates of tonsillar tissue. Total sulfhydryl groups were determined in serum by the method of Ellman [13] as well as glutathione content (GSH) in tonsillar tissue using the method of Sedlak and Lindsay [14]. Lipid peroxidation, evidenced by formation of thiobarbituric acid reactive substance (TBARS), was determined in tonsillar tissue [15] and in serum [16]. The protein oxidation levels were monitored by determination of protein carbonyl content [17]. Protein content in tissue homogenates was determined according to the method of Lowry [18].
2.1. Subjects
2.3. Statistical analysis
The study was performed on 35 patients, 20 children (aged 5–12 years) with mean age of 7.5 years and clinical diagnosis of TH and 15 patients (aged 15–45 years) with mean age of 25.5 years and clinical diagnosis of RT who were planned to undergo tonsillectomy in the ENT clinic, at the Clinical Center Nisˇ. The patients in TH group were found to have no significant sleep apnea syndrome according to criteria of Pestalozza et al. [12]. The control group for TH consisted of 13 healthy children (aged 5–11 years) with mean age 7.9 years and from 17 adults (aged 13–27 years) with mean age 18.3 years for RT group. The investigation was performed after institutional ethical committee approval. Immediately after tonsillectomy, each tonsil was rinsed with 0.9%NaCl to eliminate any possible blood contamination and frozen at 20 8C until measurement of oxidative parameters. According to clinical history tonsils were classified in TH and RT, and diagnosis was confirmed by pathohistological examination of paraffin sections stained by the hematoxylin-eosin method [1,4]. Considering the age of patients, two healthy control groups were used, 13 children for TH group and 17 adults for RT group. Venous blood samples were taken from patients before tonsillectomy and 1 month postoperatively. After clotting, the serum was separated and stored at 20 8C. Tonsillar tissue samples were homogenized in Elvenjem– Potter homogenizer (10% homogenate) at a rate 800 g for 5 min.
Results were expressed as mean standard deviation. Statistical analyses were performed with Sigma Stat for Windows, using Student’s t-test and Mann Whitney Rank Sum test according to the distribution of data. Differences were considered significant at P < 0.05.
3. Results The differences between preoperative and postoperative blood levels of oxidative parameters are shown in Tables 1– 3. Lipid peroxidation was found to be significantly increased in TH and RT groups compared to control groups for TH and RT, respectively (Table 1). One month after tonsillectomy serum TBARS levels remained elevated in RT group and significantly increased in TH patients related to levels before tonsillectomy. Total SH group levels are shown in Table 2. Only patients with RT had elevated level of TSH compared to the control group for RT. Postoperatively statistically higher TSH levels were detected related to the values of control groups and both groups TH and RT before operation. Four weeks after tonsillectomy carbonyl content in TH group was decreased compared to the level before operation (Table 3). As shown in Table 4, there was no significant difference in TBARS and carbonyl content between TH and RT groups when parameters were tested in tonsillar tissue. GSH levels
Table 1 Serum TBARS levels in TH, RT and control groups. TBARS (mmol/l)
TH
Range
RT
Range
Control Before tonsillectomy After tonsillectomy
17.83 5.75 27.52 10.20b 44.52 16.02a,c
(12.61–35.96) (16.50–57.40) (20.00–71.80)
21.47 14.26 33.31 12.96b 48.99 23.47b
(9.17–64.60) (15.00–54.20) (21.00–88.10)
Values are means SD. a p < 0.001 vs. control. b p < 0.01 vs. control. c p < 0.01 vs. before tonsillectomy.
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342
Table 2 Serum TSH groups in TH, RT and control groups. TSH groups (mmol/l)
TH
Range
RT
Range
Control Before tonsillectomy After tonsillectomy
347.25 139.39 391.75 127.70 700.30 148.20a,b
(184–615) (168–719) (458–968)
257.43 80.19 422.73 122.74a 602.00 140.17a,c
(147–458) (183–652) (424–877)
Values are means SD. a p < 0.001 vs. control. b p < 0.001 vs. before tonsillectomy. c p < 0.01 vs. before tonsillectomy.
Table 3 Protein oxidation measured as protein carbonyl content in serum of TH, RT and control groups. Carbonyl content (mmol/g protein)
TH
Range
RT
Range
Control Before tonsillectomy After tonsillectomy
4.99 1.12 5.63 1.02 4.29 1.34a
(3.23–7.05) (4.26–8.18) (2.52–7.21)
4.91 2.11 5.33 1.23 4.79 0.49
(1.39–10.77) (3.30–7.67) (3.93–5.66)
Values are means SD. a p < 0.01 vs. before tonsillectomy.
were found statistically lower in the RT group compared with TH group.
4. Discussion There is a large number of distinct pathogens that can induce inflammatory reactions in the host. Palatine tonsils represent the first protective barrier against upper respiratory tract infection. Their constant exposure to external pathogens contribute to chronic inflammation which is, usually, in children characterized with tonsillar hypertrophy and in adults is associated with sclerosis of tonsillar structures. Several recent studies demonstrated that frequent inflammatory reactions are closely related to free radicals generation. It has been shown that oxidative metabolism of peripheral blood granulocytes is altered in 30% of patients with tonsillar hypertrophy and in 75–90% of patients with recurrent tonsillitis [19]. In the present study, elevated levels of oxidative stress markers with no improvement after tonsillectomy were noted. Lipid peroxidation, determined as TBARS level, is the most common indicator of free radicals mediated injury of the cellular membrane. In this investigation we demonstrated that serum TBARS levels in both TH and RT were significantly higher than those of the relevant Table 4 TBARS, GSH levels and carbonyl content in tonsillar tissue of patients with tonsillar hypertrophy and recurrent tonsillitis. Parameters
TH
RT
TBARS (nmol/mg protein) GSH (nmol/mg protein) Carbonyl content (nmol/mg protein)
9.94 4.82 4.51 2.47 10.87 4.16
9.05 4.57 2.84 1.0a 12.17 4.17
Values are means SD. a p < 0.01 vs. TH.
control groups. Furthermore, TBARS levels, 4 weeks after tonsillectomy, remained high in RT group and even increased in TH patients. Obtained results confirm previous findings where increased MDA content (malondialdehyde), an end product of membrane lipid peroxidation, in serum of patients with acute tonsillitis [9] and obstructive adenotonsillar hypertrophy [20] has been found. There are confusing data about MDA level after tonsillectomy. Our results are in contrast with previous findings where significantly decreased MDA content has been shown in obstructive adenotonsillar hypertrophy [20] and chronic tonsillitis [8]. Significant, but very moderate, attenuation in MDA levels, in chronic tonsillitis, after tonsillectomy has been also demonstrated by Kaygusuz et al. [21] and Yilmaz et al. [22]. On the other hand, Burduk et al. [23] reported decreased level of MDA in serum, 7 days after operation, with following elevation during the period of 1 and 6 months. Constant exposure of tonsillar tissue to variety of pathogens as well as repeated local infections, during the course of tonsillitis, is characterized with permanent activation of lymphoid cells and intensive hypoxia/reoxygenation episodes [20]. Moreover, the whole body is affected by oxidative stress which is generated in tonsillar tissue. As the consequence of this processes, imbalance between free radicals generation and activation of antioxidant defense mechanisms is present, so it seems reasonable to propose that the period of 1 month, after tonsillectomy, is too short for the restoring and maintenance of the balance between pro- and antioxidants. As Shukla et al. [24] pointed out, antioxidant therapy, including use of antioxidants such as vitamins A, E and C as well as food reach in polyphenols and other antioxidants, could be the treatment of choice. There is very limited number of studies reporting reduced glutathione blood levels (GSH) in tonsillitis before and after tonsillectomy [25]. In this study, total SH group levels in TH and RT patients were found to be significantly increased
T. Cvetkovic´ et al. / Auris Nasus Larynx 36 (2009) 340–344
after tonsillectomy compared to preoperative period and to the control groups. These findings are in agreement with the data of Yilmaz et al. [22] who also found moderate increase in GSH level after operation. Such elevation in blood GSH level could be explained as a result of defence mechanism activation against free radicals-induced oxidative stress in chronic tonsillitis. Our results showed that preoperative and postoperative blood levels of carbonyl content are different. They were higher in patients with TH and RT before operation compared to controls, However, after operation carbonyl content, even was not significantly changed, returned to the level of control groups. Despite these results, in tonsillar tissue, we could not find differences in TBARS and carbonyl content levels, only the level of GSH was lower in RT related to TH patients. Although in some studies, higher MDA level has been shown in tonsillar tissue of patients with adenotonsillar hypertrophy compared to chronic tonsillitis [26], our results are in agreement with the finding of Kaygusz et al. [21]. Furthermore, it has to be taken in consideration that RT is characterized with chronic inflammation and reduced levels of GSH could be attributed to permanent involvement of glutathione in diminishing of oxidative stress. Recently, long-term study of superoxide dismutase (SOD) activity was made, in 46 patients with tonsillar hypertrophy and 90 patients with recurrent tonsillitis who underwent tonsillectomy. This study revealed that, during 3year follow-up, SOD concentration reached stabilization 6 months after tonsillectomy in the group with TH, and almost 2 years in RT group [11]. Authors found that tonsillectomy reduces SOD level, as the result of oxidative stress, but these do not return to the normal level. Considering parameters of oxidative stress are elevated before tonsillectomy, it is clear that the generation of free radicals is the result of frequent exposure to external pathogens and changes in humoral and cellular immunity of patients with chronic tonsillitis. However, 1 month after tonsillectomy, the level of these markers remained high and become even higher. We may speculate that it could be the consequence of anesthesia and surgical treatment-operational stress, but it is well known that the effects of anesthesia on the level of oxidative markers are short-term effects [27]. It is also certain that the direct effects of surgical procedure, together with the effects of tissue repair after tonsillectomy, are followed by an acute and long-lasting systemic oxidative stress [28,29]. However, the influence of this treatment could not be estimated while oxidative parameters were not measured immediately after tonsillectomy.
5. Conclusions From the present data, we found that, both tonsillar hypertrophy and recurrent tonsillitis, are characterized with elevated serum parameters of oxidative stress, without any
343
improvement, 1 month after tonsillectomy. This study suggests that capacity of antioxidant defence system could be overhelmed with recurrent infection during the course of chronic tonsillitis. The removal of tonsils is not enough condition for restoring the balance between pro- and antioxidants during the short period of time. Antioxidant treatment during the recovery period after tonsillectomy could be optional therapy.
References [1] Zhang PC, Pang YT, Loh KS, Wang DY. Comparison of histology between recurrent tonsillitis and tonsillar hypertrophy. Clin Otolaryngol 2003;28:235–9. [2] Agren K, Andersson U, Litton M, Funa K, Nordlander B, Andersson J. The production of immunoregulatory cytokines is localized to the extrafollicular area of human tonsils. Acta Otolaryngol 1996;116:477– 85. [3] Perry M, Whyte A. Immunology of the tonsils. Immunol Today 1998;19:414–21. [4] Passali D, Damiani V, Passali GC, Passali FM, Boccazzi A, Bellussi L. Structural and immunological characteristics of chronically inflamed adenotonsillar tissue in childhood. Clin Diagn Lab Immunol 2004;11:1154–7. [5] Surjan L, Brantdzaeg P, Beral P. Immunoglobulin systems of human tonsils. II. Patients with chronic tonsillitis or tonsillar hyperplasia: quantification of Ig-producing cells, tonsillar morphometry and serum Ig concentrations. Clin Exp Immunol 1978;31:382–90. [6] Sugiyama M, Sakashita T, Cho JS, Nakai Y. Immunological and biochemical properties of tonsillar lymphocytes. Acta Otolaryngol (Stockh) 1984;(Suppl. 416):45–55. [7] Valko M, Leibfritz D, Moncol J, Cronin MT, Mazur M, Telser J. Free radicals and antioxidants in normal physiological functions and human disease. Int J Biochem Cell Biol 2007;39:44–84. [8] Shukla GK, Bhatia N, Pandey S, Kaur G, Shukla RN, Shar K. Significance of free radicals in chronic tonsillitis. Boll Chim Farm 2000;139:103–5. [9] Cemek M, Dede S, Bayiroglu F, Caksen H, Cemek F, Yuca K. Oxidant and antioxidant levels in children with acute otitis media and tonsillitis: a comparative study. Int J Pediatr Otorhinolaryngol 2005;69:823–7. [10] Garcı´a Callejo FJ, Orts Alborch MH, Velert Vila MM, Fernandez Julian EN, Blay Galaud L, Marco Algarra J. Oxidative profile in tonsillar infection. A study of the antioxidative enzymes in tonsil and in blood. Acta Otorrinolaringol Esp 2002;53:398–404. [11] Garcı´a Callejo FJ, Orts Alborch MH, Morant Ventura A, Costa Alcacer I, Velert Vila MM, Blay Galaud L. Tonsillar and erythrocyte levels of superoxid dismutase in children with and without recurrent tonsillitis. An Esp Pediatr 2002;56:310–7. [12] Pestalozza G, Tessitore E, Ballott R, Zucconi M. Tonsil surgery in heavy snoring young children. Adv Otorhinolaryngol 1992;47:251–9. [13] Ellman LG. Tissue sulphydryl groups. Arch Biochem Biophys 1959;82:70–7. [14] Sedlak J, Lindsday R. Estimation of total protein bound and nonprotein sulphydryl groups in tissue with Ellman’s reagent. Anal Biochem 1968;25:192–205. [15] Ohkava H, Ohishi N, Tagi K. Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Anal Biochem 1979;95:351–8. [16] Andreeva LI, Kozhemiakin LA, Kishkun AA. Modification of the method of determining lipid peroxidation in a test using thiobarbituric acid. Lab Delo 1988;11:41–3. [17] Levine RL, Wiliams JA, Stadtman ER, Shacter E. Carbonil assay for determination of oxidative modified proteins. Meth Enzymol 1994;233:346–57.
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[18] Lowry OH, Rosenbrough NJ, Farr AL, Randall T. Protein measurement with the Folin phenol reagent. J Biol Chem 1951; 193:265–75. [19] Kowalska M, Kowalska H, Zawadzka-Glos L, Debska M, Szerszen E, Chmielik M, et al. Dysfunction of peripheral blood granulocyte oxidative metabolism in children with recurrent upper respiratory tract infections. Int J Pediatr Otorhinolaryngol 2003; 67:365–71. [20] Dogruer ZN, Unal M, Eskandari G, Pata YS, Akbas Y, Cevik T, et al. Malondialdehyde and antioxidant enzymes in children with obstructive adenotonsillar hypertrophy. Clin Biochem 2004;37:718–21. [21] Kaygusuz I, Ilhan N, Karlidag T, Keles E, Yalcin S, Cetiner H. Free radicals and scavenging enzymes in chronic tonsillitis. Otolaryngol Head Neck Surg 2003;129:265–8. [22] Yilmaz T, Kocan EG, Besler HT. The role of oxidants and antioxidants in chronic tonsillitis and adenoid hypertrophy in children. Int J Pediatr Otorhinolaryngol 2004;68:1053–8. [23] Burduk PK, Betlejewski S, Drewa G. Antioxidants enzymes activity and concentration of lipid peroxidation products in chronic tonsillitis before and after surgery. Otolaryngol Pol 2005;59:693–7.
[24] Shukla GK, Sharma S, Shukla A, Pandey S, Mishra SC, Chandra M, et al. Comparative status of oxidative damage and antioxidant enzymes in chronic tonsillitis patients. Boll Chim Farm 1998;137:206–9. [25] Cemek M, Caksen H, Cemek F, Bayiroglu F, Dede S, Dulger H, et al. Investigation of antioxidant status in children with acute otitis media and tonsillitis. Int J Pediatr Otorhinolaryngol 2004;68:1381–5. [26] Kiroglu AF, Noyan T, Oger M, Kara T. Oxidants and antioxidants in tonsillar and adenoidal tissue in chronic adenotonsillitis and adenotonsillar hypertrophy in children. Int J Pediatr Otorhinolaryngol 2006;70:35–8. [27] Allaouchiche B, Debon R, Goudable J, Chassard D, Duflo F. Oxidative stress status during exposure to propofol, sevoflurane and desflurane. Anesth Analg 2001;93:981–5. [28] Mezzetti A, Guglielmi MD, Pierdomenico SD, Costantini F, Cipollone F, De Cesare D, et al. Increased systemic oxidative stress after elective endarterectomy relation to vascular healing and remodeling. Arterioscler Thromb Vasc Biol 1999;19:2659–65. [29] Zhou JF, Chen P, Yang JL, Zhu YG, Peng CH, Wu YL. Oxidative stress before and after operation in patients with chronic cholecystitis containing gallstone. Biomed Environ Sci 2000;13(4):254–62.
Investigation of oxidative stress in patients with chronic tonsillitis Tatjana Cvetkovic´ a, Predrag Vlahovic´ b,*, Miroljub Todorovic´ c, Milan Stankovic´ d a
b
Institute of Biochemistry, Faculty of Medicine, Nisˇ, Serbia Clinic of Nephrology & Hemodialysis, Clinical Center Nisˇ, Dr. Zorana Ðinpic´a 48, 18000 Nisˇ, Serbia c Clinic for Othorhinolaryngology, Cetinje, Montenegro d Clinic for Othorhinolaryngology, Clinical Center Nisˇ, Serbia Received 20 May 2008; accepted 17 October 2008 Available online 25 December 2008
Abstract Objectives: Oxidative stress has been implicated in numerous pathological conditions including chronic tonsillitis. The aim of this study was to assess levels of lipid peroxidation, evidenced by formation of thiobarbituric acid reactive substance (TBARS), level of total sulfhydryl groups (TSH), and carbonyl content in patients with tonsillar hypertrophy (TH) and recurrent tonsillitis (RT), before and after tonsillectomy. Methods: In this study the serum and tonsillar tissue levels of TBARS, TSH and carbonyl content were investigated in 35 patients with TH and RT, before and 1 month after the operation, compared to 30 age-matched controls. Results: In both TH and RT groups, a significantly higher serum TBARS levels were observed before and 4 weeks after tonsillectomy in comparison with healthy subjects. The serum level in TH patients after operation was even higher compared to the levels before. There was statistically significant difference in serum TSH levels between patients with RT before operation compared to the control group. After tonsillectomy the serum levels of TSH were higher compared to control groups and TH and RT patients before operation. Carbonyl content was attenuated only in TH patients after tonsillectomy. In tonsillar tissue, significantly lower level of glutathione content (GSH) has been observed in RT related to TH patients. Conclusions: Oxidative stress, in patients with tonsillar hypertrophy and recurrent tonsillitis, is still present 1 month after the removal of tonsillar tissue. Antioxidant therapy, during the recovery period after tonsillectomy, could be optional treatment. # 2008 Elsevier Ireland Ltd. All rights reserved. Keywords: Oxidative stress; Palatine tonsil; Tonsillitis; Recurrent tonsillitis; Tonsillar hypertrophy
1. Introduction Palatine tonsils are reactive lymphoepithelial organs which are considered the first place of contact with variety of antigenic substances present in air and food. They are located at the entrance of respiratory and alimentary tracts and during recurrent upper respiratory tract infections tonsillar humoral and cellular immunity is altered. Recurrent tonsillitis (RT) and idiopathic (obstructive) tonsillar hypertrophy (TH) represent two different pathogenic entities with different morphology [1] and patterns of local immune response [2,3]. Tonsillar hypertrophy is characterized histologically by an enlarge* Corresponding author. Fax: +381 18 53 01 27. E-mail address: [email protected] (P. Vlahovic´).
ment of lymphoid follicles and reduced extrafollicular tissue, indicating hyperplastic condition of lymphoid cells in the germinal centers. However, in the recurrent tonsillitis reduced number of follicles and fibrosis in extrafolicular tissue is demonstrable [4,5]. In addition, RT is caused by an immune response followed with strong inflammatory process compared to TH where environmental agents mediate local antigen stimulation without inflammation [6]. Although the pathophysiology of tonsillar disease processes remains still unknown, both TH and RT are the most common reasons for surgical tonsillectomy. Moreover, the benefit of tonsillar removal is still controversial. Oxidative stress has been implicated in numerous pathological conditions including cancer, cardiovascular diseases, rheumatoid arthritis, ischemia/reperfusion and
0385-8146/$ – see front matter # 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.anl.2008.10.004
T. Cvetkovic´ et al. / Auris Nasus Larynx 36 (2009) 340–344
341
2.2. Biochemical analyses
aging [7]. Considerably evidence indicated that reactive oxygen species, during chronic inflammation process, mediate the regulation of tonsillar pathophysiological functions [8,9]. The role of free radicals has been well elucidated in chronic tonsillitis [10,11]. The aim of this study was to investigate the effect of oxidative damage due to free radicals in patients with obstructive tonsillar hypertrophy and recurrent tonsillitis, in pre- and post-tonsillectomy period, and to compare the results with healthy control subjects.
2. Patients and methods
The biochemical determinations were carried out in serum and 10% homogenates of tonsillar tissue. Total sulfhydryl groups were determined in serum by the method of Ellman [13] as well as glutathione content (GSH) in tonsillar tissue using the method of Sedlak and Lindsay [14]. Lipid peroxidation, evidenced by formation of thiobarbituric acid reactive substance (TBARS), was determined in tonsillar tissue [15] and in serum [16]. The protein oxidation levels were monitored by determination of protein carbonyl content [17]. Protein content in tissue homogenates was determined according to the method of Lowry [18].
2.1. Subjects
2.3. Statistical analysis
The study was performed on 35 patients, 20 children (aged 5–12 years) with mean age of 7.5 years and clinical diagnosis of TH and 15 patients (aged 15–45 years) with mean age of 25.5 years and clinical diagnosis of RT who were planned to undergo tonsillectomy in the ENT clinic, at the Clinical Center Nisˇ. The patients in TH group were found to have no significant sleep apnea syndrome according to criteria of Pestalozza et al. [12]. The control group for TH consisted of 13 healthy children (aged 5–11 years) with mean age 7.9 years and from 17 adults (aged 13–27 years) with mean age 18.3 years for RT group. The investigation was performed after institutional ethical committee approval. Immediately after tonsillectomy, each tonsil was rinsed with 0.9%NaCl to eliminate any possible blood contamination and frozen at 20 8C until measurement of oxidative parameters. According to clinical history tonsils were classified in TH and RT, and diagnosis was confirmed by pathohistological examination of paraffin sections stained by the hematoxylin-eosin method [1,4]. Considering the age of patients, two healthy control groups were used, 13 children for TH group and 17 adults for RT group. Venous blood samples were taken from patients before tonsillectomy and 1 month postoperatively. After clotting, the serum was separated and stored at 20 8C. Tonsillar tissue samples were homogenized in Elvenjem– Potter homogenizer (10% homogenate) at a rate 800 g for 5 min.
Results were expressed as mean standard deviation. Statistical analyses were performed with Sigma Stat for Windows, using Student’s t-test and Mann Whitney Rank Sum test according to the distribution of data. Differences were considered significant at P < 0.05.
3. Results The differences between preoperative and postoperative blood levels of oxidative parameters are shown in Tables 1– 3. Lipid peroxidation was found to be significantly increased in TH and RT groups compared to control groups for TH and RT, respectively (Table 1). One month after tonsillectomy serum TBARS levels remained elevated in RT group and significantly increased in TH patients related to levels before tonsillectomy. Total SH group levels are shown in Table 2. Only patients with RT had elevated level of TSH compared to the control group for RT. Postoperatively statistically higher TSH levels were detected related to the values of control groups and both groups TH and RT before operation. Four weeks after tonsillectomy carbonyl content in TH group was decreased compared to the level before operation (Table 3). As shown in Table 4, there was no significant difference in TBARS and carbonyl content between TH and RT groups when parameters were tested in tonsillar tissue. GSH levels
Table 1 Serum TBARS levels in TH, RT and control groups. TBARS (mmol/l)
TH
Range
RT
Range
Control Before tonsillectomy After tonsillectomy
17.83 5.75 27.52 10.20b 44.52 16.02a,c
(12.61–35.96) (16.50–57.40) (20.00–71.80)
21.47 14.26 33.31 12.96b 48.99 23.47b
(9.17–64.60) (15.00–54.20) (21.00–88.10)
Values are means SD. a p < 0.001 vs. control. b p < 0.01 vs. control. c p < 0.01 vs. before tonsillectomy.
T. Cvetkovic´ et al. / Auris Nasus Larynx 36 (2009) 340–344
342
Table 2 Serum TSH groups in TH, RT and control groups. TSH groups (mmol/l)
TH
Range
RT
Range
Control Before tonsillectomy After tonsillectomy
347.25 139.39 391.75 127.70 700.30 148.20a,b
(184–615) (168–719) (458–968)
257.43 80.19 422.73 122.74a 602.00 140.17a,c
(147–458) (183–652) (424–877)
Values are means SD. a p < 0.001 vs. control. b p < 0.001 vs. before tonsillectomy. c p < 0.01 vs. before tonsillectomy.
Table 3 Protein oxidation measured as protein carbonyl content in serum of TH, RT and control groups. Carbonyl content (mmol/g protein)
TH
Range
RT
Range
Control Before tonsillectomy After tonsillectomy
4.99 1.12 5.63 1.02 4.29 1.34a
(3.23–7.05) (4.26–8.18) (2.52–7.21)
4.91 2.11 5.33 1.23 4.79 0.49
(1.39–10.77) (3.30–7.67) (3.93–5.66)
Values are means SD. a p < 0.01 vs. before tonsillectomy.
were found statistically lower in the RT group compared with TH group.
4. Discussion There is a large number of distinct pathogens that can induce inflammatory reactions in the host. Palatine tonsils represent the first protective barrier against upper respiratory tract infection. Their constant exposure to external pathogens contribute to chronic inflammation which is, usually, in children characterized with tonsillar hypertrophy and in adults is associated with sclerosis of tonsillar structures. Several recent studies demonstrated that frequent inflammatory reactions are closely related to free radicals generation. It has been shown that oxidative metabolism of peripheral blood granulocytes is altered in 30% of patients with tonsillar hypertrophy and in 75–90% of patients with recurrent tonsillitis [19]. In the present study, elevated levels of oxidative stress markers with no improvement after tonsillectomy were noted. Lipid peroxidation, determined as TBARS level, is the most common indicator of free radicals mediated injury of the cellular membrane. In this investigation we demonstrated that serum TBARS levels in both TH and RT were significantly higher than those of the relevant Table 4 TBARS, GSH levels and carbonyl content in tonsillar tissue of patients with tonsillar hypertrophy and recurrent tonsillitis. Parameters
TH
RT
TBARS (nmol/mg protein) GSH (nmol/mg protein) Carbonyl content (nmol/mg protein)
9.94 4.82 4.51 2.47 10.87 4.16
9.05 4.57 2.84 1.0a 12.17 4.17
Values are means SD. a p < 0.01 vs. TH.
control groups. Furthermore, TBARS levels, 4 weeks after tonsillectomy, remained high in RT group and even increased in TH patients. Obtained results confirm previous findings where increased MDA content (malondialdehyde), an end product of membrane lipid peroxidation, in serum of patients with acute tonsillitis [9] and obstructive adenotonsillar hypertrophy [20] has been found. There are confusing data about MDA level after tonsillectomy. Our results are in contrast with previous findings where significantly decreased MDA content has been shown in obstructive adenotonsillar hypertrophy [20] and chronic tonsillitis [8]. Significant, but very moderate, attenuation in MDA levels, in chronic tonsillitis, after tonsillectomy has been also demonstrated by Kaygusuz et al. [21] and Yilmaz et al. [22]. On the other hand, Burduk et al. [23] reported decreased level of MDA in serum, 7 days after operation, with following elevation during the period of 1 and 6 months. Constant exposure of tonsillar tissue to variety of pathogens as well as repeated local infections, during the course of tonsillitis, is characterized with permanent activation of lymphoid cells and intensive hypoxia/reoxygenation episodes [20]. Moreover, the whole body is affected by oxidative stress which is generated in tonsillar tissue. As the consequence of this processes, imbalance between free radicals generation and activation of antioxidant defense mechanisms is present, so it seems reasonable to propose that the period of 1 month, after tonsillectomy, is too short for the restoring and maintenance of the balance between pro- and antioxidants. As Shukla et al. [24] pointed out, antioxidant therapy, including use of antioxidants such as vitamins A, E and C as well as food reach in polyphenols and other antioxidants, could be the treatment of choice. There is very limited number of studies reporting reduced glutathione blood levels (GSH) in tonsillitis before and after tonsillectomy [25]. In this study, total SH group levels in TH and RT patients were found to be significantly increased
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after tonsillectomy compared to preoperative period and to the control groups. These findings are in agreement with the data of Yilmaz et al. [22] who also found moderate increase in GSH level after operation. Such elevation in blood GSH level could be explained as a result of defence mechanism activation against free radicals-induced oxidative stress in chronic tonsillitis. Our results showed that preoperative and postoperative blood levels of carbonyl content are different. They were higher in patients with TH and RT before operation compared to controls, However, after operation carbonyl content, even was not significantly changed, returned to the level of control groups. Despite these results, in tonsillar tissue, we could not find differences in TBARS and carbonyl content levels, only the level of GSH was lower in RT related to TH patients. Although in some studies, higher MDA level has been shown in tonsillar tissue of patients with adenotonsillar hypertrophy compared to chronic tonsillitis [26], our results are in agreement with the finding of Kaygusz et al. [21]. Furthermore, it has to be taken in consideration that RT is characterized with chronic inflammation and reduced levels of GSH could be attributed to permanent involvement of glutathione in diminishing of oxidative stress. Recently, long-term study of superoxide dismutase (SOD) activity was made, in 46 patients with tonsillar hypertrophy and 90 patients with recurrent tonsillitis who underwent tonsillectomy. This study revealed that, during 3year follow-up, SOD concentration reached stabilization 6 months after tonsillectomy in the group with TH, and almost 2 years in RT group [11]. Authors found that tonsillectomy reduces SOD level, as the result of oxidative stress, but these do not return to the normal level. Considering parameters of oxidative stress are elevated before tonsillectomy, it is clear that the generation of free radicals is the result of frequent exposure to external pathogens and changes in humoral and cellular immunity of patients with chronic tonsillitis. However, 1 month after tonsillectomy, the level of these markers remained high and become even higher. We may speculate that it could be the consequence of anesthesia and surgical treatment-operational stress, but it is well known that the effects of anesthesia on the level of oxidative markers are short-term effects [27]. It is also certain that the direct effects of surgical procedure, together with the effects of tissue repair after tonsillectomy, are followed by an acute and long-lasting systemic oxidative stress [28,29]. However, the influence of this treatment could not be estimated while oxidative parameters were not measured immediately after tonsillectomy.
5. Conclusions From the present data, we found that, both tonsillar hypertrophy and recurrent tonsillitis, are characterized with elevated serum parameters of oxidative stress, without any
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improvement, 1 month after tonsillectomy. This study suggests that capacity of antioxidant defence system could be overhelmed with recurrent infection during the course of chronic tonsillitis. The removal of tonsils is not enough condition for restoring the balance between pro- and antioxidants during the short period of time. Antioxidant treatment during the recovery period after tonsillectomy could be optional therapy.
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