Abstract
The aim of this study is to investigate serum selenium (Se) and glutathione peroxidase (GSH-Px) levels in patients with recurrent tonsillitis. The study included 53 patients with recurrent tonsillitis and 51 healthy children. The measurement of serum Se levels were done in graphite furnace atomic absorption spectrophotometer using Zeeman background correction. Erythrocyte GSH-Px levels were indirectly measured by using the spectrophotometry. The ages of children in both groups ranged between 3 and 13 years, with a mean age of 7.60 years for patient group and 7.22 years for control group. Mean serum Se levels in patient and control groups were 60.4 and 78.7 µg/dL respectively. Mean erythrocyte GSH-Px levels in patient and control groups were 7.0 and 23.1 U/g hb, respectively. The mean blood Se and GSH-Px levels in patients with recurrent tonsillitis were significantly (P < 0.001) lower than control group. In our study, we found that serum Se and erythrocyte GSH-Px levels of cases with recurrent tonsillitis were significantly lower than healthy controls. The decline in serum Se and erythrocyte GSH-Px enzyme levels may predispose a chronic disease state but this issue needs further investigation.
Keywords: Selenium, Glutathione peroxidase, Recurrent tonsillitis, Trace element, Oxidant, Antioxidant
Introduction
Trace elements like selenium (Se) are actually very important for human health (1). Antioxidant enzymes need trace metal co-factors for optimum efficiency, including selenium for glutathione peroxidase (GSH-Px), copper, zinc or manganese for superoxide dismutase (SOD) and iron for catalase (2). GSH-Px is the general name of an enzyme family with peroxidase activity whose main biological role is to protect the organism from oxidative damage. The enzyme system, GSH-Px, reduces many organic peroxides (like lipid and DNA hydroperoxides) and hydrogen peroxide. In this reaction, glutathione takes part as a reducing agent [1, 2]. It also provides stability to cell membranes which are composed of lipids from probable oxidant damage. The concentration of GSH-Px is selenium dependent. Therefore, a decrease in GSH-Px activity can be a good indicator of selenium status. GSH-Px has at least 4 different isoenzymes. GSH-Px-1 and GSH-Px-4 exist in most of the tissues. GSH-Px-1 is mainly located in erythrocyte, liver and kidneys. GSH-Px isoenzymes account for about 36% of total body selenium [2, 3].
Se is not only a constituent of the structure of antioxidant enzymes, but plays an important role in antibody and complement formation. The integrity of the cellular membranes is largely dependant on GSH-Px, on the other hand, the antioxidative protective system of GSH-Px itself depends heavily on the presence of selenium [2].
GSH-Px and Se are present in many tissues and fluids of the body, and abnormal level of those increases the tendency of cellular damage which may contribute to the pathogenesis of some diseases including inflammatory diseases of the upper airways like tonsillitis.
The aim of our study is to evaluate the status of Se and GSH-Px levels in patients with recurrent tonsillitis.
Materials and methods
This study contained 53 patients with recurrent tonsillitis applied to Kahramanmaras Sutcuimam University otorhinolaryngology clinic and 51 healthy children as control. Patient group had complaints of recurrent throat infection with a frequency of at least 7 episodes in the past year or at least 5 episodes per year for 2 years or at least 3 episodes per year for 3 years. According to history and medical examination of the patients were diagnosed with recurrent tonsillitis. Control group composed of 51 healthy children admitted to Healthy child outpatient clinic had no recurrent tonsillitis or any other disease.
Blood Sampling
Fasting venous blood specimens were drawn from the antecubital vein and collected into EDTA tubes (Becton-Dickinson, Franklin Lakes, NJ, USA) and plain tubes without anticoagulant. The blood samples in EDTA tubes were used for determination of blood hemoglobin concentration and for measurements of the red blood cell GSH-Px enzyme activity. The plain tubes without any anticoagulant were allowed time for clot retraction, the tubes were then centrifuged for 10 min at 3000×g and serum was then separated. Serum samples were stored at − 70 °C.
Measurement of Serum Se Level
Measurements of serum Se levels were done in graphite furnace atomic absorption spectrophotometer (Perkin Elmer Analyst 800) using Zeeman background correction. Matrix modifiers were palladium (4 mg in 20-mL sample) and Mg sulfate (3 mg in 20-mL sample). Samples and calibration standards were diluted in 1:3 with 0.05% Triton X-100 to improve the sample viscosity and reproducibility of the results. Se calibration standards were prepared from the commercial Se standard (1000 mg/L) by serial dilutions. Se levels in all groups were evaluated according to a standard curve [4].
Preparation of Hemolysate
EDTA tubes containing blood samples were used for preparation of hemolysate. The red blood cells from each sample was washed three times with 2 mL saline (0.9% NaCl), after each wash the samples were centrifuged at 3000 rpm. Thereafter 50 μL blood cells were placed in another tube and hemolysed with 2 mL double distilled cold water. These hemolysate was used for measurement of erythrocyte GSH-Px enzyme activity and hemoglobin (Hb) analysis.
Measurement of erythrocyte GSH-Px enzyme activity: Hemolysate were used for determination for GSH-Px enzyme activity. GSH-Px in the presence t-butyl hydroperoxide reduced glutathione (GSH) to oxidized glutathione (GSSG). At the and of this reaction oxidized glutathione again convert to reduced glutathione form by GSH-Px enzyme. During this reaction nicotine amide adenine dinucleotide phosphate (NADPH) is used as cofactor. Erythrocyte GSH-Px levels were indirect measured as reduction of NADPH to NADP at 340 nm using the spectrophotometry [5, 6].
Hemoglobin Concentration
The concentration of hemoglobin in the hemolysate samples were determined using Drabkin’s solution and read at 546 nm with spectrophotometer. Optical density of hemolysate samples were calculated according to hemoglobin standard curve [7].
Statistical Analysis
Statistical analysis was carried out by SPSS version 15.0 (SPSS Inc, Chicago, IL, USA) Chi-square test and Student’s t test were used to compare the results. Data were expressed as mean ± standard deviation (SD). The significance was set at P < 0.05.
Results
The demographic data of patient and control groups are shown in Table 1. There were no statistical difference between control group and patients with recurrent tonsillitis group in the number of girls and boys, and the ages. Mean serum Se levels in patient and control groups were 60.4 and 78.7 µg/dL respectively. Mean erythrocyte GSH-Px levels in patient and control groups were 7.0 and 23.1 U/g hb, respectively. Patients with recurrent tonsillitis, the mean blood Se and GSH-Px levels were significantly (P < 0.001) lower than control group’s (Table 2). Figure 1 shows as column the concentrations of Se and GSH-Px in patients with recurrent tonsillitis and control groups.
Table 1.
Patients and control group’s subject number, mean ± standard deviation (SD), min–max, male and female number and percents
| Patient group | Control group | P value | |
|---|---|---|---|
| Age | |||
| Mean ± SD | 7.60 ± 2.71 | 7.22 ± 2.17 | * |
| Min–max | 3.0–13.0 | 4.0–13.0 | * |
| Female | |||
| n(%) | 24 (45.3%) | 22 (43.1%) | * |
| Male | |||
| n(%) | 29 (54.7%) | 29 (56.9%) | * |
N, subject number; P, statistically significance, min, minimum; max, maximum; age, year; *, > 0.05
Table 2.
Serum Se, erythrocyte GSH-Px and haemoglobin levels in patient and control groups
| Patient (mean ± SD) | Control (mean ± SD) | P | |
|---|---|---|---|
| Serum Se (µg/L) | 60.4 ± 15.21 | 78.7 ± 13.18 | <0.001* |
| Erythrocyte GSH-Px (U/g hb) | 7.0 ± 2.09 | 23.1 ± 4.68 | <0.001* |
Hb, hemoglobin; *, P < 0.001
Fig. 1.
Patients and control group’s Se and GSH-Px levels
Discussion
Bacterial and viral agents are involved in most of the infections. In addition to both agents, the immune system is also involved in the process of inflammation. Immune system cells, T and B lymphocytes, and neutrophils are in a constant struggle against viruses and bacteria. The neutrophils use reactive oxygen species formed during microbicidal activity at burst reactions [8]. The reactive oxygen derivatives kill microbial invaders but at the same time these may also kill normal cells and lead to inflammation and swelling of the tonsils, and this condition may harm the host if not balanced by antioxidant enzymes. Antioxidant enzymes, especially erythrocyte GSH-Px’s play a major role in chronic infections to reduce these oxidants. Selenium is an active component of GSH-Px system, therefore reduction in selenium concentration could reduce the level of the enzyme system. Reimund et al. [9] reported that, Se and GSH-Px levels were lower in patients with chronic diseases compared to normal people with adequate intake of anti-oxidant enzymes.
Kaygusuz et al. [10] found that in patients with chronic tonsillitis, preoperative plasma malondialdehyde (MDA) levels, an indicator of oxidative stress, were higher than postoperative MDA levels; but preoperative SOD levels were lower than postoperatively. In their study, they indicated that oxidative stress was increased and antioxidant mechanisms were decreased in patients with chronic tonsillitis. Yilmaz et al. [11] found that lowered levels of antioxidant vitamins (such as ascorbic acid) and antioxidant enzymes including GSH-Px before the surgery were elevated, and oxidation product MDA was decreased after the surgery in patients with chronic tonsillitis and adenoid hypertrophy. However they did not measure Se levels in their patients.
In this study, we showed that the mean blood Se and GSH-Px levels in patients with recurrent tonsillitis were significantly lower than control group. In children, tonsils are an important part of the immune system, and its inflammation (tonsillitis) can cause obstruction of respiratory tract. It is known that recurrent tonsillitis is a frequent and important problem during childhood, and it is possible that the condition could be related to reduction on the GSH-Px anti-oxidant system. Since selenium is an active component of GSH-Px system, reduction in selenium concentration could reduce the level of the enzyme system.
Selenium is an important trace element. It not only a constituent of the structure of antioxidant enzymes, but plays an important role in antibody and complement formations [12]. Investigations showed that in Se deficient individuals, decreased neutrophil functions, increased H2O2 formation during neutrophil phagocytosis, decreased NADPH dependent superoxide production in neutrophils, decreased T lymphocyte activity, and increased bacterial and mycotic antigen levels are observed (15–17). When given oral Se, antibody production by B lymphocytes, T cell (T helper) dependent antibody production, Se content in lymph nodes and immune tissues, neutrophils and lymphocyte GSH-Px levels were increased [13–15].
In addition to GSH-Px, selenomethionine and selenocysteine have similar protective actions against these oxidant agents. Additionally, thioredoxin reductase, a selenium containing molecule, along with GSH-Px participate in the removal of peroxynitrites [16, 17]. Selenium also prevents the release of inflammatory mediators from taking place during the reduction of organoperoxides which participate in the production of active leukotrienes, in this way it, exerts anti-inflammatory actions. Selenium is also important in thrombocyte GSH-Px activities. Its deficiency leads to thrombocyte aggregation, and increased thromboxane B2 and lipoxygenase production. Selenium plays a role in production of certain molecules such as tumor necrosis factor (TNF-α) and interleukin 1 and thus prevents inflammation. Additionally, T and B lymphocytes efficiency are increased through increasing the number and sensitivity of interleukin 2 receptors, α and p subunits in the presence of selenium. Consequently Se and Se containing molecules (especially GSH-Px) may play an important role in the body’s immune functions, antioxidant system and normal metabolism. Selenium has been implicated in the protection of body tissues against oxidative stress, maintenance of defences against infection, and modulation of growth and development [2].
Conclusion
In this study, we found that serum Se and erythrocyte GSH-Px levels of cases with recurrent tonsillitis were significantly lower than healthy controls. The decline in serum Se and erythrocyte GSH-Px enzyme levels in the case of recurrent tonsillitis support the thought that selenium has been implicated in the protection of body tissues against oxidative stress and maintenance of defences against infection. We therefore believe that decreased Se and GSH-Px levels in tonsillitis patients may constitute or predispose to a chronic disease state although this issue needs further clarification.
Acknowledgement
We thank Prof. Dr. Prem Gathiram (Ph.D.) for grammatical corrections.
Compliance with Ethical Standards
Conflict of interest
All authors declare that they have no conflict of interest.
Contributor Information
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