Effect of Helicobacter pylori Infection on Insulin Resistance in non-Obese, ‎non-Diabetic Patients ‎ ‎

INTRODUCTION

Helicobacter pylori (H. Pylori) is one of the Gram-negative bacteria, which was previously ‎known as Campylobacter pylori, a spiral macrophile which attacks and lives in the human stomach ‎‎[1]. H. Pylori was first identified in 1982 by Barry Marshall and Robin Warren as the cause of ‎active chronic gastritis in the gastric epithelium [2]. H. Pylori is associated with the inflammation of ‎the mucosa-associated lymphoid tissues through the digestive tract [3], which might participate in ‎the prognosis of other serious diseases such as gastric cancer [4]. H. Pylori infection is usually ‎asymptomatic, however in severe infection, it might cause gastritis or ulcers in the stomach or even ‎in the duodenum [3]. Sometimes, it can cause an inflammation of the lymphoid tissue surrounding ‎the eye, called ocular adnexal Mucosa-Associated Lymphoid Tissue (MALT) lymphoma [5]. ‎Furthermore, the infection by H. pylori might cause sharp deficiencies in the levels of some ‎vitamins (A, C, E, and B12) and minerals (Iron and ‎Copper), which is a dangerous risk factor in ‎some medical situations such as pregnancy and breastfeeding [6]. H. pylori affect the melatonin ‎secretion and functions, which further disturb the sleep-wake cycles, as well [5]‎.

The infection by H. pylori is related negatively to the serum levels of leptin and positively to the tumor necrosis factor α (TNF-α), which commonly induces insulin resistance (IR) and the development of metabolic syndrome (MS) [7-9]. IR is a pathologic condition in which the normal insulin levels are not sufficient to function perfectly in the peripheral tissues to allow glycogen storage [10]. In humans, IR was found not to be associated with impaired insulin-stimulated glucose disposal [11], but with atherosclerosis and other cardiovascular diseases, as well [12]. The prognosis of IR is commonly associated with the excessive production of inflammatory cytokines such as (IL-8, IL-10, IL-12) and acute-phase proteins such as CRP [13]. The prevalence of IR increased all over the past years (15.5 to 46.5%, among adults) with associated metabolic disorders, particularly with older ages [14]. IR was found to be affected with different variations in the hormones that regulate insulin metabolism, such as gastrointestinal hormone, which deficiency is a common symptom of H. pylori infection, as well [15].

The Homeostatic model assessment (HOMA) is a reliable algorithm to evaluate the β-cell function and IR by basal (fasting) glucose and insulin levels in humans [16]. It was defined and developed in 1985 by Matthews and colleagues from the Diabetes Research Laboratories, Radcliffe Infirmary, Oxford, UK [16]. The combinational term (HOMA-IR) was used to describe a simple and convenient method to evaluate the level of IR and its correlation to the clamp-measured total glucose disposal [17-18].

To our knowledge, there is a limited number of reports related to the usage of the HOMA-IR model to study the relationship between H. pylori infection and the abnormal IR, however, there few studies were conducted in Egypt. So, the current study aimed to evaluate the effect of H. pylori on insulin resistance in non-obese and non-diabetic patients, registered in the outpatient virology clinics, Department of Gastroenterology and Hepatology, Sohag University Hospital, Egypt, using the HOMA-IR model.

PATIENTS AND METHODS

This study was conducted in outpatient clinic of Sohag University Hospital, Sohag, Egypt, in the period between February 2019 and December 2020. 62 patients were recruited in the study where all of them were complaining of various gastrointestinal manifestations and fulfilling the inclusion and exclusion criteria. Patients older than 30 years with various gastrointestinal symptoms with positive test for H. pylori infection (positive H. pylori stool Antigen test) were included. On the other hand, patients with liver cirrhosis, chronic renal failure (with or without dialysis), polycystic ovary, obese (BMI ≥ 30 kg/m2), treated with non-steroidal anti-inflammatory drugs (NSAIDS), and smokers were excluded from the current study. Besides, patients with local or systemic infection, diabetic, hypertensive, or those received eradication therapy for H. pylori in the last four weeks were excluded, as well. Patients were divided into two groups according to the positivity of H. pylori test; control group (1) with H. pylori negative and group (2) were H. pylori positive by stool antigen test for H. pylori infection. HOMA-IR index was used to assess insulin resistance.

All patients and controls were equally tested to the following variables: full medical history report, complete clinical examination, and routine laboratory investigations including complete blood count, serum total cholesterol, high-density lipoprotein, low-density lipoprotein, and triglycerides, liver and kidney functions tests, CRP by latex agglutination, fasting blood glucose, and fasting insulin levels.  BMI was calculated as body weight (kg)/height² (m).

Laboratory investigations:

To assess the H. pylori infection, a fresh stool sample is collected in sterile container and used for assessment of H. pylori Antigen. H. pylori infection was detected serologically by using the commercially available enzyme-linked immune-sorbent assay (ELISA) kits (H. pylori IgG ELISA kit, Padtan Elm Co., Tahran, Iran) and (H. pylori Qualitative ELISA kit; Eagle Biosciences Inc., Amherst, NH, USA) following the manufacturer guidelines.

To investigate the above-mentioned laboratory examinations; an amount of 5ml of venous blood was withdrawn from each participant, under aseptic conditions. The blood samples were allowed to clot at room temperature for 30 minutes and then were centrifuged at 2000-3000 rpm for 15 minutes. The sera supernatants were collected and divided into three aliquots:

a)    The portion was used for measurement of fasting blood glucose level (Pars Azmoon commercial kit, Pars Azmoon, Tehran, Iran), ALT (alanine aminotransferase), AST (aspartate aminotransferase), blood urea, and sCr) (serum creatinine) levels. A dedicated spectrophotometer (Cobas C 311 Chemistry Analyser System, Roch Diagnostic GmbH, Indianapolis, IN, USA) was used for OD measurements.

b)   The second portion was used to measure the levels of fasting serum insulin level by using by a suitable Immunenzymometric assay (IEMA) (ST AIA-PACK IRI, Tosoh Bioscience Inc., South San Francisco, CA, USA) and a dedicated ELIZA plate reader (ARCHITECT i1000SR immunoassay analyzer, Abbott Laboratories, Chicago, IL, USA).

c)    The third portion was used for the assessment of CRP by using Synchron Lx 20 Autoanalyser (Beckman Coulter, Fullertron, CA, USA).

To calculate the HOMA-IR, we used the following equation as described before [18]:

15HOMA-IR=Fastingglycemiamgdlأ—Fastinginsulinemia (آµUml) 405">

Statistical analysis:

The results and data from the above-mentioned observations and tests were analysed by the Statistical Package for Social Science (SPSS) (SPSS version 25, IBM, Armonk, NY, USA). Pearson Chi-Square was used to assess the significance of different variables between the two groups.  The correlations between homeostasis model assessment of insulin resistance and other variables of the study were assessed by Pearson product-moment correlation coefficient (PPMCC) to calculate the Pearson's r values and detect the significance of variables at P value

RESULTS:

In the current study, the participants were divided into two groups according to the results of stool antigen tests for H. pylori infection. As shown in Table 1, 35 males versus 27 females were recruited in the study. The results of stool antigen test showed that H. pylori infections were most common among male patients (73.5% of positive cases) compared to females (26.5%) with a significant P=0.003. All participants had an overall BMI of 26.59±1.48 %, which indicated that most of the participants had normal or little higher weights, but were definitely, not obese. However, the results indicated that patients with H. pylori infection had significant increases in BMI values, but were still not obese, P=0.001. All participants had an overall mean age of 47.24±5.43 years without any significant variation among patients and control groups.

The laboratory investigations showed significant variation in the results of CRP levels between patients and control groups, P=0.039. Despite the results of the other blood tests didn’t show any significance, the levels of AST, Urea, and sCr were slightly higher in H. pylori-infected patients, compared to the control group. By contrast, the levels of ALT were lower in H. pylori-infected group, despite insignificancy, as well. Most of the participants (81%) had different types of symptoms, despite none of them were significant to H. pylori infection (Table 1).

As shown in Table 2, all participants had normal fasting blood glucose level, as they are non-diabetic, however no significant difference were found between H. pylori-infected and non-infected individuals. On the contrary, the serum levels of fasting Insulin were significantly higher in H. pylori-infected group (16.95±4.67 µU/ml) compared to control group (10.49 ±6.5 µU/ml), P=0.001. The values of HOMA-IR index were significantly higher (3.8 ±1.16) in H. pylori-infected as compared to the control group (2.07 ±1.01), P=0.001 (Table 2).

To study the factors that might affect the results of the HOMA-IR model, we statically analyzed the correlations between different study variables and HOMA-IR values in both groups. The analysis showed that the level of fasting Insulin is directly proportional to H. pylori infection, where the higher levels of fasting Insulin were significantly correlated to positive H. pylori results of ELIZA, in contrast to the negative results with lower serum levels of Insulin (Table 3).

* P-value <0.05, ** P-value <0.01.

SD: Standard deviation; sCr: serum creatinine; ALT: alanine transferase; AST: aspartate transaminase; CRP: C reactive protein; BMI: Body Mass Index.

Table (2):    Correlations between levels of fasting blood glucose, fasting insulin, and homeostasis model assessment of insulin resistance in the studied groups:

** P-value <0.01.

SD: Standard deviation; HOMA-IR: Homeostasis Model Assessment of Insulin Resistance.

Table (3): Correlations between homeostasis model assessment of insulin resistance and other variables in the studied groups:

* P-value <0.05, ** P-value <0.01.

sCr: serum creatinine; CRP: C reactive protein; BMI: Body Mass Index.

DISCUSSION