Received 9 June 2016; accepted 9 July 2016; published 12 July 2016
Helicobacter pylori is an spiral-shaped gram-negative pathogenic bacterium that specifically colonizes in the gastric epithelium and causes several acute and chronic gastro-duodenal diseases including chronic gastritis, peptic ulcer disease, and/or gastric malignancies  . It is estimated that half of the world population was infected with H. pylori, with more prevalence in developing countries   .
Eradication of H. pylori was reported to lead to a more healthy development in young population. In patients with unexplained iron deficiency anemia (IDA), successful eradication of H. pylori infection leads to improvement in hemoglobin, iron, total iron-binding capacity and ferritin values  .
Recent studies indicate a strong association between H. pylori infection and the incidence of Type 2 diabetes mellitus  , a growing body of evidence suggests that the infection with H. pylori may cause insulin resistance and chronic inflammation related to diabetes  . The mechanisms of pathogenesis of diabetes are complex, including insulin resistance and chronic inflammation, moreover, previous studies suggest a correlation between H. pylori infection and poor glycemic control  .
The extra-intestinal manifestations of H. pylori have been shown in many previous studies  , considerable evidence suggest the potential role of Helicobacter pylori (H. pylori) infections in the initiation, development or persistence of atherosclerosis and coronary heart disease (CHD)    .
There remains a critical need to better understand the underlying disease mechanisms responsible for diabetes complications, which may help in developing new and improved therapeutic strategies. Atherosclerosis and subsequent events are considered as the most prevalent and serious complications of diabetes mellitus  , dyslipidemia and oxidized low density lipoproteins (ox-LDL) play a major role in macrovascular complications of diabetes mellitus, and H. pylori was considered to be a reversible risk factor in Coronary Artery Disease (CAD)  .
The present study was conducted to investigate the possible relationship between H. pylori infection and disturbance in lipid profile, especially the levels of atherogenic ox-LDL in Type-2 diabetic patients.
2. Subjects and Methods
The study was conducted in Makka Al-Mukarama, in Saudi Arabia. One hundred and five participants who met the inclusion criteria were enrolled in the present study, ethical consent was obtained from each participant. The overall study methodology was approved by the Biomedical Ethics Committee, Faculty of Medicine, Umm Al- Qura University, Makkah, Kingdom of Saudi Arabia.
The study population was categorized into two groups of H. pylori positive cases and negative controls according to their results of Helicobacter pylori IgG antibodies. Subjects in both groups fill the structured questionnaire and subjected to anthropometric measurements and clinical investigations. Blood samples were drawn using standard collection procedures for evaluation of FBS, 2hr-PP blood sugar, HbA1c, Lipid profile and oxidized LDL.
Routine analysis was performed using a full automated chemistry analyzer (Humastar-80), from Human Diagnostics. The protocols were followed as manufacturer’s guidance using the kits and controls of Human.
H. pylori antibodies were assessed using Abcam’s Human Helicobacter pylori IgG ELISA Test kit. Briefly, 96-well plates have been pre-coated with Helicobacter pylori antigens, cognate antibodies were bound. Controls or test samples were added and incubated. Washing cycles then followed, a horseradish peroxidase (HRP) labeled anti-Human IgG conjugate was added which binds to the Helicobacter pylori-specific antibodies. TMB with HRP then produce a blue color product that changes to yellow after addition of an acid to stop the reaction. The density of the yellow color is directly proportional to the amount of Helicobacter pylori IgG in the samples.
Oxidized Low Density Lipoproteins was measured using Mercodia Oxidized LDL Competitive ELISA. The assay kit Manufactured by Mercodia AB, Sylveniusgatan 8A, SE-754 50 Uppsala, Sweden. This assay uses Holvoetet al monoclonal antibody, 4E6 specific for oxidatively modified LDL  . The 4E6 antibody is directed against a conformational change in the apoB-100 of LDL.
The principle is a competitive ELISA were the Oxidized LDL in the sample or standard competes with a fixed amount of oxidized LDL bound to the well for the binding to the specific antibodies 4E6. A washing step removes unreactive sample components, then the biotin-labeled antibody bound to the well is detected by HRP- conjugated streptavidin. The bound conjugate is detected by reaction with 3, 3´, 5, 5´-tetramethylbenzidine (TMB). The stop solution is added and a yellow color developed which can be read spectrophotometrically and the concentration of LDL determined using the curve generated from standards.
3. Statistical Analysis
The statistical analysis was done using Statistical Package for the Social Sciences (SPSS) for windows, version 20. Results were expressed as mean ± SD. Descriptive statistics, the mean procedures and independent sample t-test were used to compare the different studied parameters between groups of H. pylori positive cases and negative controls, P < 0.05 was considered as statistically significant.
Among the 105 Type-2 Diabetic patients, 48 cases (45.7%) were diagnosed as H. pylori seropositive. The negative controls were 57 patients (54.3%). The ages of participants in the present study ranges from 25 - 64 years, there is no significant difference in the mean age or mean BMI between the H. pylori negative and positive cases as shown in Table 1.
Glycemic control was similar in the two groups, HbA1c was higher in the H. pylori positive cases suggesting uncontrolled diabetes, but difference between the means was not statistically significant.
Total Cholesterol was found to be elevated in cases of positive H. pylori compared to negative controls (P < 0.001) and Triglycerides was found to be significantly elevated too in the same group (P < 0.005).
While there was no significant difference in the levels of HDL-Cholesterol between the two groups, the mean LDL-Cholesterol was found to be significantly increased in cases of H. pylori positive compared to negative controls (P < 0.001).
Oxidized LDL levels confirm the disturbance in lipid profile in the positive cases and found to be increased significantly in the H. pylori positive cases (P = 0.001) compared to negative controls as shown in Table 1. Lipid profile, when compared between the two groups shows marked dyslipidemia in cases of H. pylori, moreover, the increased atherogenic oxidized LDL in this group suggest a correlation between infection of H. pylori and disturbance in lipid metabolism.
Since the discovery of Helicobacter pylori by Barry J. Marshall and Robin Warren in 1984, where they found that the bacteria was present in almost all subjects with active chronic gastritis, duodenal ulcer, or gastric ulcer
Table 1. Age, BMI, glucose tolerance and lipid profile in the studied groups.
and concluded that it may be an important etiological factor of these diseases, since then and the bulk of research was focused on the effect of the bacteria on the gastro-intestinal region  .
Current data suggestan extra-gastric role of H. pylori, there is evidence that H. pylori infection is associated with increased incidence of Type-2 Diabetes mellitus  , and may be a major contributing factor for development of insulin resistance  , long-term diabetes complications, and cardiovascular risk factors  , however there is limited data supporting this controversial association  and some previous studies did not find it  -  . Moreover, while some studies look at H. pylori infection as a risk factor for diabetes an eradication can improve the mean glycated hemoglobin in diabetic patients  , others interpret the association as susceptibility of diabetic patients to the infection  .
The present study investigates the disturbance in lipid profile in Type-2 diabetic patients with and without H. pylori infection. The findings suggest marked dyslipidemia and a significant increase in Total Cholesterol levels in seropositive cases as shown in Figure 1, these findings are consistent with many previous studies that report elevated serum LDL cholesterol levels in elder population with H. pylori  . Cardiovascular complications of diabetes mellitus are caused by dyslipidemia, in particular the increased LDL and oxidized LDL  . H. pylori infection was reported to be more prevalent in CAD positive patients  , this may be due to the disturbance in lipid metabolism associated with H. pylori infection. Diabetic patients with positive H. pylori score high in triglyceride levels with a significant difference when compared with their negative peers in the present study. Although most of the previous studies focus on total cholesterol and LDL-C as parameters of cardio-metabolic disorders, but the significant increase in seropositive subjects may support the hypothesis that the dyslipidemia observed in this group have a strong association with the H. pylori infection.
Oxidized LDL is considered as a biomarker of cardiovascular diseases  . In the present study the levels of ox-LDL was significantly higher in H. pylori positive diabetic patients as shown in Figure 2, eradication of the infection was supposed to be a promising strategy to control lipid profile as well as other metabolic parameters  .
The gastric inflammation caused by H. pylori infection was proposed as a risk factor for insulin resistance and cardiovascular diseases in Type-2 Diabetes  . The disturbance in lipid profile which is associated with H. pylori infection seems to play a significant role in the mechanism of these diabetic complications, considering the different metabolic roles of H. pylori especially in diabetic patients may help in treatment and prevention of many of these complications.
Before Warren and Marshall announced their findings about the role of H. pylori in gastritis and duodenal ulcer, it was a common and long-standing belief in medical teaching and practice that the major causes of peptic ulcer disease were stress and lifestyle factors. Soon after, it was clear with strong evidences that H. pylori, causes more than 90% of duodenal ulcers and up to 80% of gastric ulcers  . Now a growing evidence suggests that there are many extra-intestinal manifestations of H. pylori infection, and if validated, this may provide a better understanding and monitoring of many of the pathologies associated with H. pylori.
Figure 1. Cholesterol, triglyceride, HDL-C, and LDL-C in H. pylori positive and H. pylori negative subjects.
Figure 2. Oxidized LDL in H. pylori positive band H. pylori negative subjects.
Infection with H. pylori is associated with increased levels of Total Cholesterol, Triglycerides, LDL-C and oxidized LDL in Type 2 Diabetic patients. And eradication of the pathogen may represent a useful tool for monitoring dyslipidemia in infected diabetic subjects.
This research project was supported and funded by a grant from the Institute of Scientific Research and Revival of Islamic Culture, Umm Al-Qura University, Makkah, Kingdom of Saudi Arabia.
Conflict of Interests
The authors declare that there is no conflict of interests regarding the publication of this paper.
MHM: design of the study, interpretation of biochemical laboratory investigations, and editing, styling, writing, and revising of the main manuscript text. WMN: design of the study, interpretation of biochemical laboratory investigations, preparation of tables and figures, and editing, styling, writing, and revising of the main manuscript text. ATB: biochemical laboratory investigations, statistical analysis, preparation of tables and figures, and editing, styling, writing, and revising of the main manuscript text. All authors read and approved the final manuscript.
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