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ORIGINAL ARTICLE
Year : 2013  |  Volume : 6  |  Issue : 1  |  Page : 55-59  

The effect of Helicobacter pylori infection on oxidative stress status in erosive reflux disease


1 Department of Gastroenterology, Faculty of Medicine, Urmia University of Medical Sciences, Urmia, Iran
2 Department of Biochemistry, Faculty of Medicine, Urmia University of Medical Sciences, Urmia, Iran
3 Department of Biology, Payame Noor University, P.O. Box 19395-3697, Tehran, Iran
4 Department of Biochemistry, Faculty of Medicine, Tabriz University of Medical Sciences, Tabriz, Iran

Date of Web Publication14-Mar-2013

Correspondence Address:
Yousef Rasmi
Department of Biochemistry, Faculty of Medicine, Urmia University of Medical Sciences, Urmia
Iran
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Source of Support: Urmia University of Medical Sciences, Urmia, Iran, Conflict of Interest: None


DOI: 10.4103/0975-2870.108643

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  Abstract 

Background and Objectives: Helicobacter pylori (H. pylori) infection has been associated with increased production of reactive oxygen species, which leads to oxidative stress in the gastrointestinal mucosa. Nevertheless, the association of H. pylori infection and oxidative stress in erosive reflux disease (ERD) is still unclear. We sought to investigate the association between oxidative stress status and H. pylori infection in ERD. Materials and Methods: Eighty-three ERD patients (45 male/ 38 female; mean age: 40.4 ± 14.3 years) who had heartburn and/or regurgitation and erosion(s)-confirmed by endoscopy-in the distal esophagus were enrolled. Two antral biopsies were taken from the patients for rapid urease test. Blood samples were drawn for measurement of oxidative stress parameters, including malondialdehyde (MDA), ferric reducing antioxidant power (FRAP), superoxide dismutase (SOD) activity, and glutathione peroxidase (GPX) activity. Data were compared among H. pylori(+) and H. pylori(-) patients. Results : The overall prevalence of H. pylori infection was 71% (59/83). There was a significant increase in the levels of MDA in H. pylori(+) patients (0.98 ± 0.28 μmol/l) when compared with H. pylori(-) patients (0.84 ± 0.33 μmol/l; P = 0.048). However, the levels of FRAP in the H. pylori-infected patients were significantly lower than in those without infection (941 ± 211.8 vs. 1060.3 ± 216.6 μmol/l, respectively; P = 0.028). There were no significant differences in SOD activity, GPX activity, age, sex, and body mass index (BMI) of H. pylori(+) vs. H. pylori(-) patients (P > 0.05). Conclusion: Increased levels of MDA in H. pylori(+) patients showed association between oxidative stress and H. pylori infection in EDR patients. Also, considerable changes of antioxidant concentrations indicate a compensatory mechanism to cope with the increased oxidant status in H. pylori(+) patients. It may be concluded that oxidative stress increases in the presence of H. pylori in ERD patients, and antioxidant defense mechanisms, try to minimize oxidative stress damage.

Keywords: Erosive reflux disease, GERD, Helicobacter pylori, oxidative stress


How to cite this article:
Sadreddini M, Rasmi Y, Shahsavari Z, Khosravifar F, Rahmati M. The effect of Helicobacter pylori infection on oxidative stress status in erosive reflux disease. Med J DY Patil Univ 2013;6:55-9

How to cite this URL:
Sadreddini M, Rasmi Y, Shahsavari Z, Khosravifar F, Rahmati M. The effect of Helicobacter pylori infection on oxidative stress status in erosive reflux disease. Med J DY Patil Univ [serial online] 2013 [cited 2024 Mar 29];6:55-9. Available from: https://journals.lww.com/mjdy/pages/default.aspx/text.asp?2013/6/1/55/108643


  Introduction Top


Helicobacter pylori (H. pylori) infection is probably one of the most common chronic bacterial infections worldwide, [1] which is associated with gastrointestinal complications, including ulceration, dyspepsia, adenocarcinoma, and gastroesophageal reflux disease (GERD). [2] GERD, which is induced by reflux of the gastric and duodenal contents into the esophagus, has recently come to be recognized as a serious clinical problem. [3] In recent decades, while the prevalence of H. pylori infection has been declining, the incidence of GERD has been increasing in the developed countries, [4] and we proposed that a neuroimmunological mechanism is responsible for the protective effect of H. pylori on GERD. [5] It is noteworthy that GERD is heterogeneous in terms of endoscopic features and etiologic factors. On the basis of whether mucosal breaks or ulcerations were noted in endoscopic examination, GERD can be classified into erosive reflux disease (ERD) and non-erosive negative reflux disease (NERD).

Studies of GERD that have focused on parameters related to inflammation, such as oxidative stress, chemokines, inflammatory cells, and growth factors, have increasingly drawn attention to a new approach to GERD as an inflammatory disease. [3] Some authors have reported that oxidative stress is involved in the esophageal mucosal damage induced by ERD. [3],[6]

On the other hand, H. pylori infection has been associated with generation of reactive oxygen species (ROS), with leads to oxidative stress in the gastrointestinal mucosa. [7] Oxidative stress causes a variety of conditions that stimulate either additional ROS production or a decline in antioxidant defenses. [8] Oxidative stress is not only involved in the pathogenesis of gastric inflammation, ulcers, and carcinogenesis in H. pylori infection, but also in that of lifestyle-related diseases, including atherosclerosis, hypertension, ischemic heart diseases, diabetes mellitus, and cancers. [9] H. pylori induces infiltration and activation of neutrophils, which produce inflammatory mediators that include oxidative stress parameters. [10] The extent of ROS-induced oxidative damage can be exacerbated by a decreased efficiency of antioxidant defense systems. The deleterious influences of the free radicals are kept under check by a delicate balance between the rate of their production and the rate of their elimination by these defense systems. [11],[12]

Malondialdehyde (MDA) is a decomposition product of auto-oxidation of polyunsaturated fatty acids, which is used as an index of oxidative damage. [11],[13] Increased MDA concentration indicates increased membrane lipid peroxidation.

On the other hand, activity of free radical scavenger enzymes namely superoxide dismutase (SOD) and glutathione peroxidase (GPX) represents the enzymatic part. The non-enzymatic part includes a large number of natural and synthetic antioxidant compounds such as glutathione that have the ability to inhibit oxidative stress by scavenging the highly destructive free radicals. [14]

According to the literature, the mechanism of esophageal mucosal injury has been understood at the molecular biological level, but the profile and relation between oxidant and antioxidant systems in H. pylori(+) and H. pylori(-) is not fully understood in ERD patients. Therefore, the study was designed to investigate the association between oxidative stress status and H. pylori infection in ERD patients.


  Materials and Methods Top


Study population

Between September 2007 and February 2008, 83 consecutive patients (45 male/38 female) who underwent endoscopy at the Gastroenterology Center at Urmia University of Medical Sciences, Urmia, Iran, and diagnosed with ERD were considered for inclusion into our study. ERD patients were those who had heartburn and/or regurgitation with erosions in the distal esophagus. Demographics were recorded and all patients were required to give written informed consent before participating in the study. This study was conducted in accordance with the Declaration of Helsinki and its revisions, and was approved by the ethics committee of our university.

Rapid urease test

For diagnosis of H. pylori, two antral biopsy materials obtained during endoscopy for rapid urease test.

Exclusion criteria

Patients with a history of pregnancy, antioxidant usage, taking drugs interfering with free radical production such as non-steroidal anti-inflammatory drugs (NSAIDs) or vitamin supplements, receiving H. pylori eradication therapy, H 2 receptor antagonist or proton pump inhibitor within the last four weeks, having active infection, hypertension, diabetes mellitus, hyper-lipidemia, respiratory insufficiency, rheumatoid arthritis, cirrhosis, renal disease, vascular disease, and malignancy were excluded from the study.

Blood collection

An 8-ml heparinized blood sample was obtained from all subjects. A 2-ml volume of whole blood was transferred into a labeled vial and 6 ml of blood was centrifuged at 2000g for 15 min. Plasma was aliquoted and stored at -80°C until analysis.

Estimation of MDA

MDA was estimated in plasma by a standard technique. [15] Briefly, the pink chromogen produced by the reaction between thiobarbituric acid and MDA, a secondary product of lipid peroxidation, was estimated. Absorbance of the clear supernatant was measured against a reference blank at 535 nm. Results were expressed as mmol/ml for plasma.

Estimation of total antioxidant capacity

Total antioxidant capacity was determined by the FRAP method. [16] Briefly, 300 mmol/l of acetate buffer (pH 3.6) and 10 mmol/l 2, 4, 6-tri-pyridyl-s-triazine (TPTZ) in 40 mmol/l HCl and 20 mmol/l FeCl 3 ×6H 2 O in the ratio of 10:1:1 gave the working FRAP reagent. Seven hundred and fifty microliters of the working FRAP reagent were mixed with 25 μl of plasma or a standard in a test tube. After exactly 10 min at room temperature, absorbance was read at 593 nm against a reagent blank. Fe(II) standards were used. Fe(II) (1000 μmol/l) is equivalent to 1000 μmol/l of FRAP. Change in absorbance is directly related to the "total" reducing power of the electron-donating antioxidants present in the reaction mixture.

Determination of SOD and GPX activity

SOD and GPX activities were spectrophotometrically assayed in whole blood using "Ransod" and "Ransel" kits, respectively (Randox Laboratories Ltd, UK). Hemoglobin (Hb) level was measured by routine methods. Enzymes activities were expressed as U/mg Hb.

Statistical analysis

For statistical analysis Chi square and Student's t-tests were carried out using the SPSS software (version 16.0). P-values less than 0.05 were considered statistically significant. Results are expressed as mean ± SD.


  Results Top


The overall prevalence of H. pylori infection in ERD patients was 71% (59 out of 83 patients). The demographic and clinical data of H. pylori (+) and H. pylori (-) patients with ERD are shown in [Table 1]. No statistically significant difference was observed between H. pylori(+) and H. pylori(-) ERD patients regarding age, sex, body mass index (BMI), systolic blood pressure, diastolic blood pressure, duodenitis, and gastritis (P > 0.05) [Table 1].
Table 1: Demographic and clinical characteristics in H. pylori (+) or H. pylori (– ) patients with ERD

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Comparison of oxidative stress parameters between H. pylori(+) and H. pylori(-) patients with ERD is shown in [Table 2]. As expected, the H. pylori(+) subjects had a significantly higher level of MDA than the H. pylori(-) subjects with ERD (P = 0.048), whereas mean plasma total antioxidant capacity levels were significantly lower than in the infected patients (P = 0.026). Mean SOD and GPX activities in the H. pylori(+) subjects were not significantly different than those in the H. pylori(-) ERD patients (P = 0.113 and 0.803, respectively) [Table 2].
Table 2: Oxidative stress status in H. pylori (+) or H. pylori (– ) patients with ERD

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  Discussion Top


ERD is a common disease entity in which gastric juice gains access to the esophagus via an incompetent lower esophageal sphincter. The presence of refluxed materials induces different grades of esophageal damage ranging from low- to high-grade esophagitis. [17] It is generally believed that reflux of gastric juices causes inflammation, ulceration, and destruction of the normal squamous epithelium of the esophagus. [18] However, the exact pathophysiological mechanisms of esophageal cell damage during reflux cannot be fully explained by acid reflux alone. In some studies, it has been shown that mucosal damage in ERD is mediated primarily by oxygen-derived free radicals. [19],[20] The role of ROS has been studied in acute gastric and esophageal mucosal injury caused by ischemia, anti-inflammatory drugs such as NSAIDs, and ethanol. [20] It is particularly important that pro-inflammatory factors, such as inflammatory cytokines, and oxidative stress have been demonstrated to be involved in the development of ERD.

On the other hand, more than 70% of the patients, either NERD or ERD, acquire H. pylori infection. [21] and several studies suggest that H. pylori-induced chronic gastritis correlates inversely with ERD, Barrett's esophagus, and a higher risk of distal esophageal and cardiac adenocarcinoma. [22],[23],[24] Also, recently published results showed that mucosal oxidative damage in H. pylori infection is associated with increased inflammatory cell infiltration, enhanced apoptosis, and cell proliferation. [7],[25],[26]

Oxidative stress is well documented in chronic gastric inflammatory diseases. For example, Turkkan et al. [27] demonstrated that there may be an inverse relation between severity of symptoms and level of H. pylori-induced gastric inflammation or oxidative stress in patients with functional dyspepsia. Felley et al. [28] suggested that oxidative stress occurs in asymptomatic patients and can be modulated by H. pylori eradication.

As stated above, oxidative stress is characterized by an increased concentration of oxygen-derived products that arouse critical, even irreversible, cell injury. Oxygen reduction leads to the synthesis of reactive intermediate compounds such as the superoxide anion, hydroxyl radical, hydrogen peroxide, and MDA. [29],[30] Elevated plasma MDA in H. pylori (+) patients reveals a possible role of infection in the development of ERD. Increased MDA is thought to be a consequence of oxidative stress, which occurs when the dynamic balance between the pro-oxidant and antioxidant mechanism is impaired. A reason for increased MDA in the plasma of H. pylori(+) patients may be a poor enzymatic and non-enzymatic antioxidant defense system.

Several phenotypes of gastrointestinal diseases are known to be related to antioxidant property dysfunction. [8] Antioxidants constitute the foremost defense system that limits toxicity associated with free radicals. The deleterious effects of the free radicals are kept under check by a delicate balance between the rate of their production and elimination by these defense systems. [31] Considerable changes of FRAP indicates a compensatory mechanism(s) to cope with increased oxidant parameters in H. pylori(-) patients.

When there is excessive addition of free radicals from exogenous sources to endogenous production, the available tissue defense system becomes overwhelmed, resulting in oxidative damage to the tissues. [12] SOD along with GPX, the preventive antioxidants, plays a very important role in protection against lipid peroxidation. Although mean SOD and GPX activities, as the enzymatic antioxidant defense system, in the H. pylori-infected ERD patients were not significantly different than those in the H. pylori(-) patients, they tended to decrease.

A limitation of the study was that the study population was relatively small (n = 83). In addition, it would have been preferable to compare the results with other conditions such as NERD and severity of symptoms or ERD grade.


  Conclusion Top


As for the distal esophagus, a lot of data have been published thus far and the overall feeling is that H. pylori eradication may trigger a more severe ERD in patients. [32]

But we could not find data in the literature about the influence of H. pylori infection on oxidative stress parameters in ERD patients, and this is the first demonstration-to our knowledge-of oxidative stress status in H. pylori(+) vs. H. pylori(-) patients with ERD.

Our results show there may be an association between H. pylori infection and oxidative stress status in ERD patients, as reflected by the increased MDA and decreased total antioxidant capacity, SOD, and GPX activity. We suggest that H. pylori caused considerable levels of oxidative stress in the esophageal mucosa and the antioxidant defense mechanisms try to minimize this damage.

Thus antioxidant treatment should be considered as supplementary therapy in the prevention or treatment of H. pylori-dependent oxidative damage in ERD patients. Further studies are needed to confirm these results in humans.


  Acknowledgements Top


This work is supported by research grant from the Urmia University of Medical Sciences, Urmia, Iran.

 
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    Tables

  [Table 1], [Table 2]


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