Abstract
Objectives: Paraoxonase-1 (PON1) prevents oxidative stress by inhibiting the oxidation of cell membrane lipids by the reactive oxygen species that form during acute and chronic inflammation. The aim of this study was to investigate serum PON1 activity and oxidative stress in patients with chronic otitis media (COM).
Methods: Fifty consecutive patients with COM and 55 controls were enrolled in the present study. The patients were divided into two groups according to the presence of cholesteatoma. The serum PON1 arylesterase activities and lipid hydroperoxide (LOOH) levels were determined.
Results: Serum paraoxonase and arylesterase activities were significantly lower in the COM patients than in the controls (P < 0.001 for all comparisons), whereas the LOOH levels were significantly higher (P < 0.001).
Discussion: These results indicated that a lower level of PON1 activity was associated with an oxidant–antioxidant imbalance. In addition, decreased PON1 activity may play an important role in the pathophysiology of COM.
Introduction
Chronic otitis media (COM) is a disease that frequently requires surgical intervention by an otolaryngologist. COM typically causes conductive and cochlear type hearing loss. Despite its frequent occurrence, the pathophysiology of COM has not yet been explained. The current research attention is focused on the role of reactive oxygen species (ROS) produced by active leukocytes during the inflammatory response.Citation1–Citation3 Oxidative balance refers to the processes related to the production and elimination of ROS. Under pathological conditions, such as those that occur during inflammation, this balance is degraded as a result of ROS overproduction, which causes oxidative stress and related cell damage and lipid peroxidation.Citation3–Citation6 Lipid peroxidation is a well-known mechanism of cell detriment in humans and is used as an indicator of oxidative stress in cells and tissues.Citation3–Citation5
Paraoxonase-1 (PON1) is a serum esterase that is dependent on calcium related to the high-density lipoprotein (HDL) synthesized by the liver.Citation7 PON1 is known as an antioxidant enzyme because it hydrolyzes lipid peroxidase in oxidized lipoproteins.Citation8 Previous studies have shown that oxidative stress decreases PON1 activity and down regulates serum PON1 expression.Citation8,Citation9 Other studies have shown that PON1 suppresses oxidative stress by inhibiting the oxidation of the cell membrane lipids and ROS formed during acute and chronic inflammation.Citation10,Citation11
In some studies, PON1/arylesterase activity was investigated in patients with COM.Citation1 Thus, the purpose of this study is to investigate the serum PON1 and arylesterase activities in addition to serum LOOH levels in COM patients to examine whether these parameters are affected by cholesteatoma.
Materials and methods
Subjects
In this study, 50 consecutive patients with COM (27 males and 23 females) and 55 controls (29 males and 26 females) were enrolled between May 2013 and April 2014. The patients were divided into two groups according to the presence of cholesteatoma. Thirteen patients had cholesteatoma, and 37 did not (non-cholesteatoma). The patients were diagnosed according to their histories and routine otoscopic and otomicroscopic evaluations. Radiological examinations were performed on the patients who underwent the mastoid procedure.
The control group consisted of 55 healthy subjects without a history of chronic or recurrent disease. The subjects in the control group were asymptomatic with an unremarkable medical history and a normal physical examination. None of the control subjects were taking antioxidant vitamin supplements, including vitamins E and C.
The study protocol was conducted in accordance with the Helsinki Declaration (as revised in 1989) and approved by the local ethics committee. All subjects were informed of the study, and written consent was obtained from each participant.
Exclusion criteria
Patients with chronic diseases, such as cardiovascular, pulmonary, liver, renal, or haematological diseases or malignancies, and those who chronically used medication were excluded from the study. Subjects with a history of smoking were also excluded from the study. Additionally, patients with active infections according to history, physical examination, and complete blood count testing were not included in the study.
Blood samples
Blood samples were collected at 8:00 and 11:00 a.m. after an overnight fasting period. The blood samples were collected in empty tubes and immediately stored at 4°C. The serum was separated from the cells by centrifugation at 3000 rpm for 10 minutes, and the lipid parameters were measured immediately. The remaining serum was stored at −20°C until later analysis for PON1 activity and lipid hydroperoxide (LOOH) levels.
Measurement of paraoxonase and arylesterase activity
Paraoxonase and arylesterase activities were measured using commercial kits (Relassay, Gaziantep, Turkey). The PON1 activity was assayed using two different substrates and was provided as U/L, defined as 1 mmol p-nitrophenol generated per minute under well dried conditions.Citation12 Arylesterase activity was provided as kU/L and defined as 1 mmol phenol produced per minute under reaction conditions.Citation13
Measurement of lipid hydroxyperoxide levels
Serum LOOH levels were measured by the ferrous ion oxidation-xylenol orange measurement test.Citation14 This test is based on the conversion of ferrous ion to ferric ion via the oxidation of various oxidants, and the ferric ion concentration is measured using xylenol orange. LOOH is reduced by triphenyl phosphine (TPP), which is a specific reductant for lipids. The difference in the results of the presence or the absence of TPP pre examination process indicates the LOOH levels.
Statistical analysis
The results are expressed as the mean ± standard deviation. The comparisons of the parameters in the COM patients and those in the healthy controls were performed using Student's t- and Mann–Whitney U tests. The qualitative variables were assessed using the chi-square test. The differences were considered to be significant when the P value was less than 0.05. The data were analyzed using the SPSS® for Windows program (Version 11.0).
Results
The demographic characteristics of the COM patients and the controls are shown in Table . There were no significant differences between the groups with respect to age, gender, body mass index, blood pressure, blood lipid levels, and other parameters (P > 0.05) (Table ).
Table 1 Demographic characteristics of the two groups in this study
The serum paraoxonase and arylesterase activities were significantly lower in the COM patients than in the controls (for all, P < 0.001), whereas the LOOH levels were significantly higher (P < 0.001) (Table ).
Table 2 PON1 activity and lipid hydroperoxide levels in patients with chronic otitis media and controls subjects
The serum paraoxonase and arylesterase activities were lower in the patients with cholesteatoma than in those without cholesteatoma, whereas the LOOH levels were higher. However, these differences between groups were not statistically significant (P > 0.05 for all) (Table ).
Table 3 Paraoxonase and arylesterase activities and lipid hydroperoxide levels in patients with cholesteatoma and non-cholesteatoma
Discussion
COM with or without cholesteatoma is one of the most troublesome inflammatory diseases of the temporal bone and requires surgical operation by an otologic surgeon. The pathophysiology of this disease has not yet been explained. In the literature, various theories regarding bone damage and bone erosion in COM have been presented; however, many questions remain to be answered.Citation15 One of the most important mechanisms of tissue damage and bone erosion is imbalances in the oxidant–antioxidant system.Citation1,Citation3,Citation16
The oxidation products that are produced during inflammation may cause tissue damage. Antioxidants neutralize the destructive activities of these oxidation products. Because COM is a chronic inflammatory disease of the middle ear, the oxidation products formed during inflammation are neutralized by the antioxidants. It is possible that these oxidation products persist in middle ear diseases. Despite the relationship between the oxidant–antioxidant system and the otologic diseases, such as acute otitis media, otitis media with effusion and tympanosclerosis, the possible roles of oxidants and antioxidants in COM pathogenesis are not well understood.Citation2,Citation17,Citation18 Very little data exist regarding oxidant and antioxidant activities in COM patients.Citation1,Citation2
Lipids and lipoprotein peroxidation are important in the pathogenesis of inflammatory diseases.Citation19 When neutrophils, lymphocytes, and macrophages are activated, they release cytokines, growth factors, and ROS.Citation20 This is a significant pathogenic mechanism in various acute and chronic inflammatory diseases.Citation6,Citation21 Leukocyte-derived ROS may participate in tissue damage, especially that of endothelial tissue.Citation22 ROS have been shown to be significant mediators of various inflammatory processes, such as otitis media, maxillary sinusitis, adenotonsillitis, hepatitis, and Behçet's disease.Citation2,Citation17,Citation18,Citation23,Citation24
In COM patients, chronic inflammation in the middle ear is attributed to various mechanisms, such as the activation of macrophages and T-cells, the production of cytokines, and the presence of bacterial biofilms and endotoxins.Citation25,Citation26 It is well known that each of these factors increases ROS production and causes tissue damage that should be eliminated by antioxidants. Yılmaz et al. presented evidence of ROS damage in middle ear mucosa in an otitis media guinea pig model.Citation27 Takoudes et al. showed the presence of lipid hydroperoxidase in middle ear fluid in children with otitis media with effusion and that ROS may participate in inflammatory damage in human otitis media.Citation28 Shigemi et al. demonstrated a relationship between otitis media with persistent effusion (OME) and ROS.Citation29 In their study, the authors suggested that viral and bacterial pathogens are possible causal factors in the pathogenesis of COM. Additionally, they stated that infection causes excessive toxic oxygen production by inflammatory cells and thus leads to middle ear inflammation.Citation29 Throughout chronic infection, the antioxidant levels slowly decrease, and the level of oxidative stress gradually accumulates in the middle ear. The presence of oxidative stress and absence of antioxidants is one of the factors that cause persistent OME. In addition, the hypoxia and ischemia that result from inflammation in the middle ear in OME play a role in increasing the oxygen radical concentrations by inhibiting oxidative phosphorylation.Citation30
PON1 and arylesterase inhibit oxidative stress by hydrolyzing the lipid peroxidases in the oxidized lipoproteins.Citation31,Citation32 It is well known that PON1 activity can be reduced throughout the course of inflammation.Citation32,Citation33 Some studies have shown that PON1 prevents the oxidation of cell membrane lipids induced by ROS, which can result in acute and chronic inflammation.Citation23,Citation24 PON1 activity can be affected by inflammation. Serum PON1 activity is decreased in a series of pathological conditions, including coronary artery disease, hypercholesterolemia, type 2 diabetes, polycystic ovarian syndrome, and renal insufficiency.Citation32,Citation34–Citation37
Information regarding serum PON1 activity in COM patients is limited.Citation1 Baysal et al. reported decreased serum PON1 activity and increased oxidative stress levels in COM patients.Citation1 These authors suggested that oxidative stress and antioxidant enzyme imbalance are stronger in patients with COM with cholesteatoma than in patients with COM without cholesteatoma.Citation1
In this study, we found that the serum PON1 activity is affected by the oxidative stress levels. Thus, increased oxidative stress and decreased PON1 activity may have an important role in COM pathogenesis. Our results are in accordance with those obtained in previous publications.Citation1 In our study, serum PON1 and ARE activities were significantly lower in the patients with cholesteatoma than in the control groups, and these results were statistically significant. In addition, serum PON1 and arylesterase activities were significantly lower in the patients with cholesteatoma than in patients without cholesteatoma, whereas the LOOH levels were significantly higher. However, these differences were not significant, which was not in agreement with the results from previous publications.Citation1 In COM patients, decreased serum PON activity may be the result of increased PON1 inactivation that is dependent on excessive ROS production.
The mechanism of decreased serum PON1 activity observed in COM patients is still not clear. This decrease may be related to the increased lipid peroxidation because it has been shown that oxidized lipids inhibit PON1 activity. Increased PON1 inactivation depending on increased ROS production during inflammation may account for the decreased serum PON1 activity.Citation8 Another theory is that bacterial biofilm may play a role in cytokine production. Camps et al. demonstrated that the PON enzyme family can be an important defense mechanism against biofilms.Citation38
There are some limitations in our study. We have not ruled out differences in PON1 activity resulting from gene polymorphisms because we did not perform genotype analyses for the Q192R and L55M polymorphisms.
Conclusion
We determined that the serum PON1 and arylesterase activities were significantly lower in patients with COM than in the controls but that the LOOH levels were significantly higher. These results indicated that lower levels of PON1 and arylesterase activities were associated with an oxidant–antioxidant imbalance. Thus, serum PON1 activity measurements may be beneficial when evaluating the risk of developing COM during acute otitis media. The addition of antioxidant vitamins such as vitamins C and E may be useful in such patients. Further studies are required to investigate whether there is a strong relationship between decreased enzyme PON1 activity and the development of COM.
Disclaimer statements
Contributors Conception and design: MT, RU, MA, and HC; analysis and interpretation of the data: MT, FK, and AT; critical revision of the article for important intellectual content: MT, RU, and MFG; final approval of the article: MT, RU, and MA; collection and assembly of data: MT, MA, and MFG.
Funding None.
Conflicts of interest None.
Ethics approval Yes.
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