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ORIGINAL ARTICLE
Year : 2019  |  Volume : 22  |  Issue : 3  |  Page : 375-379

Evaluation of oxidative stress and antioxidants effect on turning process acute otitis media to chronic otitis media with effusion


1 Department of Otorhinolaringology, Faculty of Medicine, Kahramanmaras Sutcu Imam University, Kahramanmaras, Turkey
2 Department of Infectious Disease, Faculty of Medicine, Kahramanmaras Sutcu Imam University, Kahramanmaras, Turkey
3 Department of Biochemisty, Faculty of Medicine, Kahramanmaras Sutcu Imam University, Kahramanmaras, Turkey
4 Necip Fazıl Sehir Public Hospital, Department of Otorhinolaringology, Kahramanmaras, Turkey

Date of Acceptance12-Nov-2018
Date of Web Publication6-Mar-2019

Correspondence Address:
Dr. S Sagiroglu
Department of Otorhinolaringology, Faculty of Medicine, Kahramanmaras Sutcu Imam University, Avsar Campus, 46100, Kahramanmaras
Turkey
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/njcp.njcp_126_18

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   Abstract 


Objective: The aim of this study was to investigate the effect of oxidative stress and antioxidant situation on chronic otitis media with effusions (COME) and acute otitis media (AOM) in children. Methods: A total of 107 children aged 2 to 13 years were examined. The study included 31 patients with AOM, 39 with COME, and 37 as control subjects. Venous blood samples were collected from all patients and control group. Myeloperoxidase (MPO), glutathione peroxidase (GPx), catalase (CAT), nitric oxide (NO), malondialdehyde (MDA), and superoxide dismutase (SOD) activities were investigated in the blood samples. Results: The mean age was found as 7.3 ± 3.3 in the AOM group, 6.2 ± 3.0 in the COME group, and 6 ± 2.4 in the control group. MPO, NO, and CAT were found to be significantly higher in the AOM and COME groups than the control groups (P = 0.040, P = 0.001, and P = 0.044). Conclusion: In this study, we observed activity of antioxidant and oxidative stress in children with COME and AOM. These results may be important in the diagnosis of these diseases and may affect the theurapeutic approach to the patients with COME and AOM.

Keywords: Acute otitis media, chronic otitis media with effusions, antioxidants, oxidative stress


How to cite this article:
Sagiroglu S, Ates S, Tolun F I, Oztarakci H. Evaluation of oxidative stress and antioxidants effect on turning process acute otitis media to chronic otitis media with effusion. Niger J Clin Pract 2019;22:375-9

How to cite this URL:
Sagiroglu S, Ates S, Tolun F I, Oztarakci H. Evaluation of oxidative stress and antioxidants effect on turning process acute otitis media to chronic otitis media with effusion. Niger J Clin Pract [serial online] 2019 [cited 2019 May 21];22:375-9. Available from: http://www.njcponline.com/text.asp?2019/22/3/375/253442




   Introduction Top


Acute otitis media (AOM) is an extremely common infection in children and is the leading cause of hearing loss.[1] AOM symptoms include middle ear effusion and signs of acute infection such as fever, pain, and membrane redness and bulging. It is most typically seen in children, and it requires antimicrobial treatment and sometimes surgery.[2] Chronic otitis media with effusion (COME) and serous otitis media are characterized by an accumulation of fluid behind the tympanic membrane, while lacking the signs and symptoms of acute infection.

Reactive oxygen species (ROS) are important pathological mediators of many diseases under normal physiological conditions. In addition to being essential for human life, some reactive forms of oxygen have the potential to damage the body. ROS, which are formed during chemical and metabolic reactions that might include substances such as hydrogen peroxide, hydroxyl peroxide, hydroxyl free radicals, lipid peroxides, and many others derivatives, can cause toxicity in various tissues.[3],[4] Nitric oxide (NO) and myeloperoxidase (MPO) are oxidative enzymes.

Antioxidants are substances that inhibit the oxidation caused by free radicals by capturing and stabilizing them. Antioxidants lead to reactions converting these free radicals into their less harmful forms and also prevent the formation of new free radicals. The increase and accumulation of ROS in tissues that have been neutralized by antioxidants lead to damage to tissues, lipids, proteins, and DNA, causing cellular death.[3],[4],[5],[6] MPO is an oxidative enzyme and plays a role in chronic inflammation. To protect themselves against these toxic products, organisms have developed enzymatic antioxidant systems such as super oxide dismutase (SOD), erythrocyte malondialdehyde (MDA), catalase (CAT), and glutathione peroxidase (GPx).[7],[8] When ROSs are increased due to chronic inflammation, the antioxidant system is activated to eliminate them.[9]

The oxidative stress plays a role in the pathogenesis of chronic adenotonsillitis, COME, and tympanosclerosis.[6],[10],[11] According to our knowledge, no studies were reported in the literature investigating the levels of antioxidative/oxidative stress biomarkers in patients with both AOM and COME. In this study, we attempted to reveal whether any change takes place in the ROS and antioxidant balance in the peripheral blood, which reflects the reaction of the entire body in AOM and COME.


   Materials and Methods Top


Patients

The study included 107 children; 31 patients (17 males, 14 females) with AOM, 39 patients (24 males, 15 females) with COME, and 37 healthy subjects (23 males, 14 females) as the control group. The age of children ranged from 2 to 13 years. Patients presenting with ear pain, fever, and/or hearing loss and diagnosed as AOM were followed-up. Twenty-one patients had acute otitis accompanying upper respiratory tract infections. Ten patients had only otitis complaint. Full blood count, sedimentation, and C-reactive protein (CRP) were studied in the venous blood samples collected from the patients with AOM. We administered medical therapy for patients with AOM. We performed paracentesis only in 5 patients. Patients with AOM were followed regularly at 3-weeks intervals. Three weeks later, patients who were still suffering from otitis media at the first control visit were excluded from the AOM group.

Patients with a 3-months effusion were included in the COME group. COME was defined as persistent evidence of effusion by otoscopy and/or tympanogram with a persistent conductive hearing loss after 3 months of adequate medical therapy including antihistamines, decongestants, or both. Blood samples were collected after 3 months of follow-up from the patients with COME. COME patients showing no response to medical treatment also underwent adenoidectomy, and routine grommet insertion was performed to maintain the airflow in the middle ear. Adenoidectomy was not performed unless it was chronic adenoiditis with nasal obstruction. Patients with chronic tonsillitis in their past history also underwent tonsillectomy.

The control group was formed of age and gender-matched children who presented at the polyclinic with no AOM and COME. These completely healthy subjects were also evaluated by otoscopic, otomicroscopic, and tympanometric methods. Control group with acute and chronic underlying disease (including AOM, COME, upper-lower respiratory tract infection allergic rhinitis, asthma, or immunodeficiency) were excluded from study. The study was approved by the local ethics committee, and informed consent was received from the parents of each subject.

Blood study

Venous blood samples were collected from all participants after an overnight fasting period at two sessions (one at 8:00 AM and one at 9:00 AM). Within 1 hour of collection of blood samples, the blood was centrifuged (Eppendorf Centrifuge 5810R, Germany) at 4000 rpm for 10 min, and the serum was stored at −70°C until time of analysis. The serum was analyzed to measure MPO, GPx, CAT, NO, MDA, and SOD activity. The reagents used in this study were obtained from Sigma-Aldrich GmbH, Seelze-Germany, and Merck KGaA Darmstadt-Germany.

MPO activity was determined using a modification of the O-dianisidine method.[12] Specific activity was given in IU/L. The Beutler's method was used for GPx activity measurement.[13] CAT activity was assayed by measuring the degradation rate of H2O2 using Beutler's method.[14] CAT activity in the serum was expressed in U/ml. Serum NO levels were measured with the use of Griess reagent, as previously described.[15] NO results were reported in μM/L. The concentration of serum lipid peroxidation (total MDA) was determined using the Ohkawa method with slight modifications.[16] MDA results were expressed in nanomoles per milliliter (nmol/ml). SOD activity was determined as described by Fridovich.[17]

Statistical analysis

The data obtained were analyzed using SPSS software (version 16.0; SPSS, Inc., Chicago, IL, USA). The normality of the data was determined using the Shapiro–Wilk test. None of the variables for MDA and GPx showed normal distribution in any of the groups, whereas CAT, MPO, and SOD variables were normally distributed. Normally distributed variables were compared using a one-way ANOVA test, followed by Tukey's post-hoc test for paired comparison, whereas the Kruskal–Wallis H test was used to compare the non-normally distributed variables. The P values less than 0.05 were accepted as statistically significant.


   Results Top


The AOM study group consisted of 31 subjects (17 males, 14 females) between 2 and 13 years of age (mean age 7.3 ± 3.3). The COME study group consisted of 39 subjects (25 males, 14 females) between 2 and 13 years of age (mean age 6.2 ± 3.0). The control group consisted of 37 children (20 males, 17 females) between 2 and 11 years of age (mean age 6 ± 2.4). There were no statistically significant differences among three groups in terms of age and gender (P > 0.05). The mean, standard deviations, median, and minimum–maximum values for plasma MDA, GPx, NO, CAT, MPO, and SOD enzyme activities in the blood samples from all groups, as well as the statistical results, are shown in [Table 1].
Table 1: Comparison of statistics between patients with AOM and EOM with control group

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When the three groups were compared, NO, CAT, and MPO were found to be significantly and statistically different between groups. NO, CAT, and MPO were found significantly higher in COME patients than the controls and AOM patients (P = 0.001, P = 0.044, P = 0.040, respectively).

The distribution of NO was found significantly higher in COME and AOM groups [Figure 1]. The distribution of MPO and CAT were found significantly higher in COME group [Figure 2] and [Figure 3]. However, CAT was found to be lower in AOM patients than the control group and the COME groups. Distribution of MDA, GPx, and SOD were not statistically significant (P = 0.365, P = 0.285, P = 0.497, respectively).
Figure 1: Distribution of NO according to groups

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Figure 2: Distribution of MPO according to groups

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Figure 3: Distribution of CAT according to groups

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   Discussion Top


In this study, MPO, CAT, and NO were found higher in COME patients. The high level of MPO values in patients with COME is particularly noticeable. CAT was low in the acute phase and high in the chronic phase. In both AOM and COME, the most elevated oxidative stress marker was NO. Our study did not reveal a clear effect of SOD on otitis media. It may be thought that GPx is in a passive state in this process. According to these results, NO, CAT, and MPO from oxidants and antioxidants play an important role in otitis media.

There are a number of causative factors contributing to AOM and COME, initiated by dysfunction of the  Eustachian tube More Details. However, the three most important factors are infection, inflammation, and problems with airflow in the middle ear.[2],[18] The obstruction of the Eustachian tube causes an increase in middle ear pressure and fluid build-up, blockage of the veins, and local acidosis.[19] In AOM, leukocytes release MPO, which makes way for lipid peroxidases. In turn, lipid peroxidase leads to the death of bacteria and causes an increase of oxidative stress in the middle ear, leading to DNA and proteins damage within cells. For these reasons, tissue damage occurs in the ear and Eustachian tube. Leukocytes release ROS for the elimination of necrotic cells. These ROSs lead to lipid, protein, lipoprotein, and DNA damage causing chronic inflammation. MPO is an antimicrobial oxidative enzyme, and it plays a role at chronic inflammation (atherosclerosis and neurodegenerative diseases, etc.)[20],[21] In our study, the MPO value was significantly higher in patients with COME. This finding was consistent with the other studies in the literature.

Lipid peroxidation is a reaction started by free radicals, which oxidizes polyunsaturated fats found in membranes.[4] The degree of oxidative stress is affected by the number of lipid proteins found in the membrane, the number of phospholipids, fatty acid composition their degree of unsaturation, and membrane permeability. Lipid peroxidation reduces and affects membrane permeability. MDA is formed from membrane lipid peroxidation and is the most commonly used indicator for oxygen free radicals.[7],[20] The studies done by Parks et al.[22] on Guinea pigs revealed that there was a high MDA number and hydroperoxide in the infectious middle-ear mucosa. Similarly, Doner et al.[3] found that middle-ear infection leads to a high MDA number, which is a free radical found in local tissue. It was found in another study by Cemek et al.[7] that patients with AOM and acute tonsillitis had higher levels of MDA in their blood streams than the control group. Unlike these studies, MDA was not significant in our study (P = 0.365).

Erythrocytes are excellently equipped to handle intracellular oxidative stress through the combined activity of CAT, GPx, and SOD. Under normal conditions, it plays an important role in the acquisition of tolerance to oxidative stress in the adaptive response of cells.[23] Studies by Khakimov et al.,[24] have shown that children with supurative otitis media have low levels of antioxidants (SOD, CAT) in their serum samples, whereas the products of oxidation (MDA, lipid peroxidase) are high. In a study by Yariktas et al.[10] patients with COME showed statistically significant levels of CAT. In our study, patients with COME showed increased CAT levels. Interestingly, CAT was found to be lower in AOM patients than in the control patients.

ROS are known to play an important role in the intracellular killing of microorganisms. The challenge of polymorphonuclear cells by many activating agents, including immune complement, evokes a potent response that produces toxic oxygen species such as oxide ion and hydrogen peroxide. SOD specifically quenches aberrant oxide ion. The role of SOD in otitis media is not clearly understood. Although SOD formation is high in vivo, its intracellular levels are very low, and it provides the catalysis for superoxide ion break down. In a study, it was reported that plasma SOD levels in patients with otitis media were significantly elevated.[25] In our study, patients in the COME group had high levels of SOD, but the difference was not statistically significant. Shukla et al.[26] found that the levels of MDA in blood were very high, whereas SOD and CAT levels were low in patients with chronic tonsillitis. Following tonsillectomy, MDA levels were lower; SOD and catalase (antioxidant) levels were high.[26] A study by Yariktas et al.[10] revealed that patients with COME had lower levels of SOD compared to the controls. Different studies have revealed variable results.[10],[25],[27]

NO is produced in the respiratory tract and plays an important role in pathophysiology of the respiratory tract. NO is both pro-oxidative and an antioxidant, also found in high levels in inflammatory diseases.[19] Kasperska-Zajac et al.[28] found that the levels of NO were high in patients with recurrent chronic tonsillitis and chronic adenotonsillar hypertrophy. In many of the studies, patients with chronic otitis showed high levels of NO.[21],[24] In our study, the levels of NO were found to be high in the AOM and COME, and it proved statistically significant. Similarly, the elevated serum NO levels might be a result of increased oxidative stress in the present study.

High concentrations of reduced glutathione (GSH) and low levels of its oxidized form glutathione disulfide (GSSG) are essential for life. Therefore, high GSH and low GSSG levels are important. High levels of GSSG form reactions with protein sulfhydryl and lead to the production of GSH-protein sulfhydryl, which is involved in inactivating other proteins.[7] In a study of 59 children, Testa et al.[29] found that analysis of middle-ear fluid showed high levels of lipid peroxidase. They showed that the oxidative damage by COME had multifactorial etiology and that GSH treatment could have positive treatment effects. Cemek et al.[7] showed that patients with COME and acute tonsillitis had lower levels of GSH compared to the control group. However, in our study, GPx levels were not significant in all three groups (P = 0.285), and it was lower in the COME and AOM groups.


   Conclusion Top


In conclusion, we consider that serum levels of oxidative stress and antioxidant products play an active role in the pathogenesis of COME and AOM. We think that these results are important in the diagnosis and treatment of the patients.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
   References Top

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