Helicobacter Pylori Infection, Lipid Profile, and Insulin Resistance in Obese Patients with Non-Alcoholic Fatty Liver Disease

Aim: Nonalcoholic fatty liver disease (NAFLD) is characterized by the presence of hepatic steatosis without any secondary causes contributing to the accumulation of fat in the liver. The influence of gut microbiota, including Helicobacter pylori (H. pylori), on liver injury has not been thoroughly investigated. Methods: This cross-sectional case-control study involved sixty patients with NAFLD (group 1) and sixty healthy individuals of matched age and sex as the control group (group 2). NAFLD patients were diagnosed by ultrasound, aged≥ 18 years old, and body mass index (BMI) ≥ 30 kg/m 2 . Investigations included fasting insulin level, fasting blood glucose (FBG), cholesterol, triglyceride (TG), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), liver profile, H. pylori antigen in stools, H. pylori IgG antibodies (IgG Abs) and homeostatic model assessment for insulin resistance (HOMA-IR). Results: Group 1 had significantly higher systolic and diastolic blood pressures (p < 0.001 and 0.015, respectively). Additionally, the NAFLD group exhibited significantly elevated levels of FBG (p = 0.004), HbA 1c (p < 0.001), cholesterol (p = 0.007), LDL-C (p = 0.001), alanine transferase (ALT) (p < 0.001), and aspartate transferase (AST) (p: 0.025). Furthermore, the NAFLD group had a significantly higher number of patients with positive H. pylori antibodies (p < 0.001), although the antibody titers were not statistically different between the two groups (p: 0.516). Conclusion


Introduction
Globally, non-alcoholic fatty liver disease (NAFLD) is a significant contributor to chronic liver diseases and is an essential indication for liver transplantation.[1] .Some estimates suggest that up to one-third of the population may be affected.[2].NAFLD spectrum includes nonalcoholic fatty liver (NAFL) and nonalcoholic steatohepatitis (NASH), depending on the presence or absence of substantial inflammation [3].
Non-alcoholic fatty liver disease is defined by the accumulation of excessive fat inside hepatocytes, with steatosis affecting more than 5% of these cells in the absence of alcohol consumption.[4] NAFLD can manifest as cirrhosis, inflammation, fibrosis, or simple steatosis [3].
Multiple factors contribute to the development of NAFLD, including lipid buildup, environmental factors, and genetics [5].Additionally, NAFLD is substantially linked to elements of metabolic syndrome, e.g., obesity, hypertension (HTN), and dyslipidemia.This suggests that insulin resistance (IR) is a significant contributing factor.[5,6] The gut microbiota is one of the additional variables that could influence the onset and course of the disease.There is evidence that Helicobacter pylori (H.pylori) infection may play a part in the onset and progression of NAFLD.[7].It is estimated that H. pylori infection impacts at least 50% of the global population.[8] Most infections start in childhood, especially in underdeveloped countries [9].Much research hasn't been done on how the gut microbiota, particularly H. pylori, produce liver damage.Potential mechanisms include the production of certain toxins.[10] and increased gut permeability, thus facilitating the entry of bacterial endotoxins into the liver through the portal vein [11].

Subjects and Methods
This cross-sectional case-control study included subjects attending outpatient clinics and wards of our hospital from June 2020 to May 2021.The study included sixty obese patients with NAFLD (group 1) and sixty healthy subjects of comparable age and sex as control (group 2).Patients in group 1 were further subdivided into three subgroups based on the severity of NAFLD by ultrasound (US): grade 1, grade 2, and grade 3 [12].
The diagnosis of NAFLD necessitates patients to exhibit evidence of steatosis through imaging or biopsy in the absence of significant alcohol consumption, other causes of steatosis, or concurrent chronic liver disease.[13]In our study, the US was the imaging modality of choice; no patients needed a biopsy.Patients in group 1 had an age ≥ 18 and a body mass index (BMI) ≥ 30 kg/m 2 .

Exclusion criteria
Patients with the following diseases/conditions were excluded from the study: • History of alcohol consumption [14].
• Active or past infection with hepatitis B or hepatitis C viral infection.
• Other causes of secondary liver diseases, e.g., Wilson disease.
• Recent rapid weight loss.
• History of bariatric surgery.
• Recent intake of hepatotoxic medications.
All participants underwent comprehensive clinical assessment, emphasizing blood pressure and anthropometric measurements, including BMI and waist-to-hip ratio (WHR).
Obesity was defined as a BMI ≥ 30 kg/m² [15].A WHR was considered normal if ≤ 0.85 for women and ≤ 0.9 for men.[16]Dietary intake was evaluated using a 24-hour food recall (24h-FR) method, known for its ease of application, cost-effectiveness, and independence from the respondent's literacy level.[17].Physical activity levels were evaluated using a self-report measure.[18].

Abdominal ultrasound
Abdominal ultrasound was conducted using consistent equipment and by the same operator.
Increased liver echogenicity served as the primary indicator of steatosis in the ultrasound scans.
Steatosis severity was classified as follows.[12]: • Grade 0: echogenicity of the right liver lobe is average compared to the right renal cortex.
• Grade 1: a slight, diffuse increase in delicate echoes in the liver parenchyma, with normal diaphragm and intrahepatic vessel borders visualization.
• Grade 2: moderate, diffuse increase in delicate echoes and slightly impaired visualization of the diaphragm and intrahepatic vessel borders.
• Grade 3: marked increase in delicate echoes, accompanied by poor or non-visualization of the intrahepatic vessel borders, diaphragm, and posterior right lobe of the liver.

Statistical analysis
The SPSS software version 22 (SPSS Inc., Chicago, IL, USA) was employed for data collection, presentation, and statistical analysis.Normally distributed data were presented as mean (M) (± standard deviation [SD]).Non-normally distributed data were analyzed using the Mann-Whitney test and Spearman's rank-order correlation and reported as the median and interquartile range (IQR), denoting the 25 th to 75 th percentiles.The Kolmogorov test for normality was applied to quantitative data.Pearson's Chi-square test was utilized to explore associations between two variables in qualitative data.A probability (p) value < 0.05 was considered significant for interpreting test results.[20].

Results
This study included 60 obese NAFLD patients diagnosed by ultrasound and 60 healthy apparent controls.Patients were subdivided into three subgroups (Grade I, Grade II, and Grade III) according to the severity of NAFLD by the US.No statistically significant difference was found between both groups regarding age and sex (p: 0.060 and 0.464, respectively).Around one-fifth of group 1 (13/60) had HTN.The control group showed statistically significantly higher values regarding physical activity levels than the NAFLD group (p: 0.039).No statistically significant difference in smoking status was found between both groups (p: 0.055).
The frequency of fast-food meals was significantly higher, while daily vegetable and fruit portions per week were significantly lower in the NAFLD group compared to the control.The most common clinical symptoms in the patient group were heartburn (76.7%), epigastric pain (75.0%), and reflux (70.0%).The patient group showed significantly higher BMI and WHR (p <0.001 for both) and considerably higher values for both SBP and DBP (p <0.001 and 0.015, respectively).There was no statistically significant difference between the three NAFLD subgroups regarding the number of patients with positive H. pylori Ag in the stool or the number of patients with positive H pylori Ab (p: 0.906 and 0.901, respectively).Laboratory parameters In the present study, no significant differences were observed between the NAFLD and control groups concerning age, sex, or smoking status (p=0.060,0.464, and 0.055, respectively) nor among NAFLD subgroups.The control group showed significantly higher values regarding physical activity (p=0.039).To ensure good health, some studies recommend that adults have at least 150 to 300 minutes per week of moderate-intensity exercise, 75 to 150 minutes per week of vigorous-intensity aerobic physical activity, or a combination of both.[21]The frequency of fast-food meals was statistically significantly higher in the NAFLD group.At the same time, the daily vegetable and fruit portion per week was statistically lower in the NAFLD group compared to the control.This was evaluated by asking about the daily intake of portions of vegetables and fruit per day and the frequency of fast-food meals per week for each group.
The most common clinical symptoms in the patient group were heartburn (76.7%), epigastric pain (75.0%), and reflux (70.0%).Werdmuller et al. [22] Almobarak et al. found similar results concerning gastrointestinal symptoms in H. pylori patients.Our study found a positive correlation between higher BMI, WHR, and the incidence of NAFLD.[23]Okushin et al. found that BMI is linked to NAFLD, with a significant relationship (p= 0.05).[24] They concluded that BMI was linked to NAFLD (their study included 13,737 participants).
The NAFLD group exhibited higher mean SBP and DBP than the control group.
Additionally, there was a notable discrepancy between NAFLD subgroups in terms of hypertension (HTN) prevalence, with an increasing incidence correlating with higher NAFLD grading.These results are consistent with those reported in a study conducted by Pardhe et al. [25].Our study revealed a significant difference between the NAFLD and control groups regarding HbA1c and FBG levels.While HOMA-IR and fasting insulin values were elevated in NAFLD patients compared to controls, this disparity did not reach statistical significance.
Other studies corroborated the observation of elevated HbA1c among NAFLD patients without diabetes mellitus.[26].
Tanaka et al. [27] illustrated that the prevalence of NAFLD rose with glycemia, reaching an HbA1c level of 8.0%.However, our study observed no significant difference between the two groups concerning insulin levels and HOMA-IR.In contrast, in their research, Novakovic et al. [28] identified an essential difference between the NAFLD group and controls regarding fasting insulin levels and HOMA-IR.Additionally, he noted a statistically significant difference between both groups concerning fasting insulin levels.Our study found that serum cholesterol and LDL-C were significantly higher in the NAFLD group (p= 0.007 and 0.001, respectively).At the same time, TG and HDL-C were not statistically different (p= 0.064 and 0.631, respectively).Cuenza et al. [29] found that mean LDL-C and cholesterol were higher in patients with NAFLD subjects.Pardhe et al. [25] found significant differences between the NAFLD group and the controls regarding serum cholesterol, TG, LDL-C, and HDL-C.Serum albumin had a significantly higher value in the control group than in the NAFLD group.Still, the two groups had no significant difference regarding bilirubin levels, PT, and INR.
ALT and AST were significantly higher in NAFLD patients than in the control group.
Also, there was a significant difference between NAFLD subgroups as regards both ALT and TG (p <0.001 and 0.009, respectively), with no significant differences regarding other laboratory parameters.Paschos et al [30] found that elevated serum AST and ALT values were the primary laboratory abnormalities in NAFLD patients.They also noticed that ALT levels are higher than AST levels.Elevated liver enzyme levels, particularly ALT, may serve as predictors for diabetes and NAFLD.[31].
H. pylori infection may contribute to the development of NAFLD.Our study observed that the NAFLD group had a significantly higher prevalence of H. pylori infection (p<0.001).
However, a cross-sectional study conducted in Japan found no association between H. pylori infection and NAFLD.Notably, this study included asymptomatic individuals and utilized ultrasonography to define NAFLD [24].
A meta-analysis (which included over 80.000 middle-aged individuals of Asian descent) concluded that H. pylori infection is linked to a slightly elevated risk of both existing and new cases of NAFLD.[32].The findings above were reaffirmed in another meta-analysis.[33].In a cross-sectional study conducted in Japan, a notable association was found between H. pylori seropositivity and metabolic syndrome.Subsequent regression analysis unveiled a significant relationship between H. pylori seropositivity and elevated LDL cholesterol, decreased HDL cholesterol, and higher systolic blood pressure levels.[34].
A study found no association between H. pylori infection and NAFLD.[35].The defined H. pylori infection is based on the positivity of both H. pylori Abs and H. pylori Ag in stool.
In addition, the patient and control group had no gastrointestinal symptoms; in contrast to our cohort, the patient group had gastrointestinal symptoms.

Study limitations
Further studies involving larger cohorts of patients and longer durations are necessary to establish the potential connections between NAFLD, H. pylori infection, dyslipidemia, and IR in NAFLD patients.

Conclusion
In the current study, we observed that obese patients with NAFLD had a significantly higher prevalence of positive H. pylori Abs and a substantially lower prevalence of positive H. pylori antigen in stool compared to the control group.Additionally, they exhibited higher levels of HbA1c and HOMA-IR compared to the control group, along with a higher incidence of dyslipidemia, which suggests a potential link between dyslipidemia and NAFLD.
Furthermore, the severity of steatosis detected by ultrasound was significantly and positively correlated with age, BMI, ALT, and AST levels.

Footnotes.
Ahmed Fathy (Assistant professor of internal medicine, gastroenterology, and hepatology unit) and Sara Salem (lecturer of internal medicine, gastroenterology, and hepatology unit) were the peer reviewers.E-Editor: Salem Youssef Mohamed, Osama Ahmed Khalil, Amany Mohammed.
Copyright ©.This open-access article is distributed under the Creative Commons Attribution License (CC BY).It may be used, distributed, or reproduced in other forums, provided the original author(s) and the copyright owner(s) are credited.The original publication in this journal must be cited according to accepted academic practice.

Disclaimer:
The authors' claims in this article are solely their own and do not necessarily represent their affiliated organizations or those of the publisher, the editors, and the reviewers.Any product evaluated in this article or its manufacturer's claim is not guaranteed or endorsed by the publisher.

Table 1 . Comparison of demographic characteristics of the studied groups.
* Significant at p-value <0.05

Table 2 . Main clinical symptoms of the patient group.
* Significant at p-value <0.05

Table 3 . Comparison of the primary clinical data of the studied groups.
* Significant at p-

value <0.05 ** FE: Fisher's exact test, X 2 ChS: Pearson's Chi-square test for independence of observations,
* ) and AST (p: 0.025), while group 2 shows statistically significant higher values for albumin level (p: 0.035).There was no statistically significant difference between the studied

Table 4 . Comparison of laboratory parameters of the studied groups.
* Significant at p-value <0.05Regarding H. pylori status, group 1 showed a significantly higher number of patients with positive H pylori antibodies (p <0.001).However, both groups had no statistical difference in antibody levels (p: 0.516).Group 2 revealed a considerably higher value of h pylori infection (positive H pylori Ag in stool) (p: 0.045).

Baeg et al. [26], found
that H. pylori infection is not a risk factor for NAFLD.NAFLD was diagnosed if hepatic steatosis index > 36 or ALD liver fat score > -0.640).There was no statistically significant difference among NAFLD subgroups regarding the number of patients with positive H pylori Ag in the stool or the number of patients with positive H pylori Abs.(p: 0.906 and 0.901, respectively).The study revealed a positive correlation between NAFLD severity and elevated BMI, ALT, and AST levels.