Frequency of non alcoholic fatty liver in helicobacter pylori infected dyspeptic patients, is it a far reaching implication?

Background: Fatty inltration of the liver represents the intracytoplasmatic accumulation of triglycerides in the hepatocytes. We determined if H. pylori infection contributes to fatty liver inltration and if it was related to gastric mucosal changes. As fatty liver disease is seen prevalent in non obese non alcohol consuming population of some asian countries. Methods: This cross-sectional study conducted between 22/11/2016 to 30/06/2018 in the Department of Medicine Aga Khan University Hospital. Eligible patients were enrolled after taking informed consent. Sociodemographic data were collected on age, gender, smoking and alcohol use, along with medical history related to dyslipidemia, hypertension, Type 2 diabetes mellitus, and ischemic heart disease. Fatty liver inltrations were assessed using ultrasonography. H.pylori infection was detected by using carbon urea breath test (14C-UBT). Results: A total of 698 patients were enrolled during the study period with a mean age of 44.01±16.03 years and male patients were 373 (53%). A total of x patients were present with dyspepsia of the 299 (57%) of the patients were found to have positive H. pylori test. Of 299 patients with positive H. pylori test, 153 (20%) of the patients were diagnosed as having fatty liver inltrations. H. pylori infection induced chronic active gastritis was associated with fatty liver inltration in 62(71%) and absent in 200(64%) (P= 0.264). Conclusion: The frequency of fatty liver inltration in Helicobacter Pylori infected dyspeptic patients was 20%. Age and BMI are associated with fatty liver among dyspeptic H. pylori patients. Keywords: H. pylori, Fatty liver, BMI > 23; Type 2 diabetes. This is due to the incorporation of proinammatory cytokines by the helicobacter pylori and its impact on gut microbiota leading to leaky gut and GUT-LIVER-AXIS interaction at different levels. Despite low BMI in our population, fatty liver is seen in connection with helicobacter pylori


Background
Fatty in ltration of the liver is a frequently reported nding on liver ultrasonography, a re ection of increased hepatic parenchymal echogenicity. Fatty change is the result of increased accumulation of small vacuoles of triglycerides around perinuclear location of liver parenchymal cells.It can be alcoholic or nonalcoholic in etiology in the background of obesity or metabolic syndrome, commonly known as non-alcoholic fatty liver disease (NAFLD).Epidemiological studies showed aNAFLDprevalencein general population of 9-37% worldwide [1]. This has given an ever-increasing prevalence of "Fatty Liver" in Asian countries, ranging from 12-24% in the general population [2]. Non-alcoholic fatty liver is strongly associated with several factors, notably type II diabetes, obesity, low HDL and high triglycerides.
Premenopausal women possess a certain degree of safety which is lost in the post menopausal women.
Male gender is more prone to get fatty in ltration of the liver even at a lean body mass, and the risk increases with the advancing age [3]. The situation is worse in South Asia, where there are the highest reported rates of overweight/obesity are seen (25% among men and 37% among women) [4][5]. In sharp contrast to other ethnic/racial groups, South Asians are more prone to show insulin resistance at a lower body mass index (BMI[kg/m 2 ]; which is translated into their higher risk pro le of metabolic disorders, they have a higher cardiometabolic disease prevalence [6][7][8]. This has prompted World Health Organization (WHO) to establish new cutoffs for Asian population that are 18.5-<23.0, 23.0-27.5 and >27.5 kg/m 2 as normal, moderate and high respectively [9]. Susceptibility of weight gain related health risks and their deleterious effects are more pronounced in South Asians than most other ethnic/racial groups [6][7][8]. This has been taken into consideration by Indian health ministry by setting up more strict cutoffs of 23.0-25.0 and >25.0 kg/m 2 as overweight and obese respectively [10].
Signi cant metabolic impact posed by the non-alcoholic fatty liver disease (NAFLD) has spillover effects outside the liver. Our studied population with overweight and obesity have a basic lack of insight into the consequences of being overweight, with severe underestimation of metabolic disease burden.The group having a BMI between 23 and 27.5, have particularly common health-related consequences. Studies from Pakistan had shown a frequency of NAFLD of 14-15% however [11].It is becoming a big health-related concern for South Asian population who are prone to insulin resistance, cardiovascular disease, and diabetes at a BMI lower than other ethnic/racial groups [12][13].
Helicobacter pylori (H. pylori) are Gram-negative, ubiquitous bacteria that colonize gastric mucosal epithelium of humans [14]. The percentage of H. pylori infection in a the previous investigation conducted on dyspeptic patients in the Southern region of Pakistan showed 54% of seroprevalence [15].Low-grade in ammatory response initiated by the H. pylori,induces speci c mechanisms which express virulence peptides that resemble host antigens. Proin ammatory cytokine release is the main driver of host immune response with protean manifestation in the form of elevated tumour necrosis factor-alpha (TNFα) and other cytokines [16].H. pylori becomes a horseman of this whole equation and develop an environment for its prolonged stay causing a chronic in ammatory state. This study aimed at exploring the association between H.pylori infection and fatty liver in ltration among patients with dyspepsia.
Furthermore, the magnitude of gastric mucosal changes in these patients.
To our knowledge, ours is the rst research study describing fatty in ltration of the liver in our dyspeptic patients with H. pylori infection population who are at a risk for obesity, type 2 diabetes, and cardiovascular diseases.

Study population
The study was approved by the institutional ethics board of Aga Khan University Hospital on 21st of November 2016, informed consent was taken. The study was conducted among adults who attended the clinic for symptoms of dyspepsia in the form of abdominal discomfort or pain, fullness and bloating and underwent an endoscopic examination of upper gastrointestinal tract from 22nd November 2016 to 30th June 2018. There were 325(47%) females and 373 (53%) males with their age ranged from 18 to 90 year with a mean age of 44 ± 16 years.The age range of male was 18-85 year and female 18-90 year. A history, physical examination, and baseline tests were done. Data was collected regarding anthropometric, complete blood count, fasting blood, glycated hemoglobin A1C (HbA1c), serum cholesterol, triglyceride and result of H. pylori test. Body mass index (BMI) was calculated as weight in kilogram divided by the square of height in millimeter. The waist was measured between the iliac crest and rib cage. Modi ed criteria for South Asians was used for BMI calculation [17]. BMI was de ned as "Healthy" to "23" and "Overweight" when greater than 23. There were 204(29%) patients with BMI up to 23 while 494(71%) had a BMI greater than 23. The fasting plasma glucose (FPG) level of 7.0 mmol/ l was the criteria used for Type 2 diabetesmellitusdiagnosis [18]. The criteria used for labeling Hypertension was a blood pressure > than 140/90 mm of Hg as de ned by the JNC 7 (Joint National Committee on Prevention, Detection, Evaluation and Treatment of High Blood Pressure) [19]. Dyslipidaemia was de ned as a disorder of lipid metabolism re ected in abnormal levels of cholesterol or triglycerides in the blood. Patient's inclusion criteria included adults 18 and older with dyspepsia for more than six months. Celiac disease, in ammatory bowel disease, pregnant and lactating females, and those not giving consent or willing to participate in the study were excluded. Written and verbal information about the study was received by all the participants. Patients with dyspeptic symptoms were investigated for H. pylori infection using either gastroscopy with biopsy and histology examination or 14 C-urea breath test ( 14 C-UBT) [20]. H. pylori infection was detected in 399 (57%). The mean age of the two groups of patients was similar.

Diagnosis of H. pylori infection
H. pyloriinfection is diagnosed with the help carbon urea breath test (14C-UBT) commonly known as a urea breath test. For this, patients were asked to fast for six hours and wash their mouths before UBT.Patients are asked to take the test in sitting position and swallow a capsule containing 14C-urea (Helicap, Noster System AB Stockholm, Sweden) with water. This is followed by the collection of breath samples in a cartridge system (Heliprobe Breath Card, Noster System AB Stockholm, Sweden) after 10 min. The patients exhaled breath into the cartridge system which changed color to yellow from orange. Breath card was inserted into a β-scintillation counter (Heliprobe-analyser, Noster System AB Stockholm, Sweden) and activity was measured for 250 s. Results were expressed both as counts per minute (HCPM) and graded as (0: not infected, CPM < 25; 1: equivocal, CPM 25-50; 2: infected, CPM > 50) [21]. UBT is considered negative when grades 0 & 1 are the test result, so H. pylori-negative is the consideration given to test result.

Histological analysis
Gastric biopsies obtained at upper gastrointestinal endoscopy are xed with formalin and embedded in para n were stained with eosin and hematoxylin stains for histological assessment and examination. The extent and degree of in ammatory activity were referred to as acute and chronic in ammation and scored as per the updated Sydney classi cation system [22]. The gastric mucosal in ltration by mononuclear cells and polymorphonuclear leucocytes, intestinal metaplasia and atrophy were graded as follows: 0, none; 1, mild; 2, moderate; 3, marked [22]. In ammatory activity is said to be chronic, de ned by increased in ltration of lymphocytes and plasma cells in the lamina propria and is graded into mild, moderate or marked based on the density of cellular in ltrate. The activity of chronic gastric in ammation commonly reported as chronic active gastritis said to be present when neutrophilic in ltration of the lamina propria, pits or surface epithelium is encountered and graded as 0, nil; mild, <1/3 of pits and surface in ltrated; moderate, 1/3-2/3; and marked, >2/3. Gastritis as a whole,is the score calculated by total sum of grade of gastritis (mild, 1; moderate, 2; marked, 3 in ltrations with lymphocytes and plasma cells) and activity of gastritis (mild, 1; moderate, 2; marked, 3 in ltrations with neutrophilic leukocytes) either in the in the corpus or antrum [22].

Diagnosis of fatty liver disease (FLD)
Abdominal ultrasonography is the routine testing evaluation carried out for fatty liver in ltration. It routinely evaluates.g., liver, gallbladder, pancreas, kidneys, spleen, and abdominal aorta. Main determinants of fatty liver were described as a) an increase in the brightness of liver, b) an increase in the hepato-renal echo contrast pattern, c) existence of vascular blurring in the hepatic parenchyma, d)deep attenuation of hepatic echo, e) borderline blurring existing between liver and gallbladder, or right kidney and f) existence of focal hypoechoic lesion. For labeling a liver as fatty liver, ultrasonographic ndings satisfy both a) and b) in addition to at least one of the ndings between c) to f) [23]. The diagnosis was double-checked by the ultrasonographer and gastroenterologists.

Statistical Analysis
Statistical analysis was performed with SPSS 17.0 for Windows (SPSS, Chicago, IL). Continuous data are presented as mean ± standard error of the mean (SE). Categorical data are presented as numbers.
Independent samples t-test or Mann-Whitney test was used for comparison of continuous variables. Chisquare test or Fisher's exact test was used to compare categorical variables. Signi cance was set at P < 0 .05.

Results
A total of 698 patients were enrolled during the study period with a mean age of 44.01±16.03 years and male patients were 373 (53%). The baseline characteristics of the two study groups are similar. The H. pylori infection increased with age and plateaued in the 3 rd to the 6 th decade (Table 1). BMI was greater than 23 in the majority of patients. H. pylori infection was positive in 399(57%). Majority of patients had mild chronic active gastritis demonstrated on histological examination of the gastric biopsy specimen ( Table 1). The comorbidity factors associated with H. pylori infection included dyslipidemia 15%, type2 diabetes in 14%, hypertension in 13% and ischemic heart disease in 12% (Table 1). Fatty change of the liver was documented in 22% ( Table 1).

Association of age with liver fatty in ltration in association with H. pylori
The fatty change was present in 88 out of 399 (22.1%) H. pylori positive patients compared to 65/299 (21.7%) H. pylori negative patients (p=0.920).In the age group of 31-50 years, the change was present;in31/399 (8.0%) of H. pylori positive patients compared to 15/299 (5.0%) in H. pylori negative patients and the difference again was not statistically signi cant (p=0.147)( Table 2). ( Table 2).
Association of BMI with liver fatty in ltration in association with H. pyloriBMI>23 was seen in 319/399 (79.9%) of H. pylori positive patients compared with 175/299 (58.5%) H. pylori negative patients (p=0.000). However, this relation could not be found with fatty change in the liver. In patients with a BMI of up to 23,fatty change was seen in 4/80 (5%) patients with positive H. pylori compared to 10/124 (8%) H. pylori negative patients (p=0.572). In patients with BMI > 23, the fatty change was seen in 84/319(26.3%) patients who were positive for H. pylori compared to 55/175(31.5%) in H. pylori negative patients (p=0.572).
Association of comorbid with liver fatty in ltration in association with H. pylori infection Dyslipidaemia, type 2 diabetes, hypertension and ischemic heart disease were associated with liver fatty in ltration irrespective of H. pylori infection status, with p-value of < 0.001 in each case ( Table 2).

Association of the grade of gastritis with fatty in ltration with H. pylori infection
Moderate gastritis was associated with fatty liver change irrespective of H. pylori status. (Table 2).

Discussion
This study demonstrated that BMI >23 was associated with H. pylori infection. However, this study could not demonstrate the association of fatty change in the liver with H. pylori infection though there was an association of moderate to severe gastritis with the fatty change in the liver irrespective of H. pylori status.
Pathogenesis of fatty liver in ltration has been linked to an increase in free fatty acids. It occurred at an early age in the presence of the H. pylori infection whereas in the absence of H.pylori infection, fatty in ltration of the liver was demonstrated in the 5 th decade of life (Table 2). This is in keeping with the local prevalence of the H. pylori infection [15] that showed the seroprevalence of the H. pylori increased with age [15]. Fatty in ltration of the liver was not related to the gender of the patients as was the seroprevalence of H. pylori infection that was not associated with the gender distribution [15]. BMI greater than '23' with. pylori infection were associated with a higher incidence of the liver fatty in ltration in 84(96%) as compared to the 55(85%)in patients without H. pylori infection ( Table 2).This is in agreement with a previous study showing a high BMI was associated with an increased risk of theH. pylori infection [24].
In this study, dyslipidemia and type2diabetes were associated with H. pylori infection and a signi cant liver fatty in ltration ( Table 2). Both dyslipidemia and type 2 diabetes are individually known to be associated with fatty liver in ltration.H. pylori infection was signi cantly associated with dyslipidemia (Table 2). This is consistent with the results of an earlier study that showed H. pylori infection was signi cantly associated with higher total cholesterol level (coe cient = 2.114, P < 0.001), higher LDL-C level (coe cient = 3.339, P < 0.001) and lower HDL-C level coe cient = −1.237, P < 0.001) [25].A higher prevalence of H. pylori in patients with type2 diabetes has also been described. The prevalence of H. pylori infection was greater and described as 61.7% and 58.5 %, among type2 diabetes and nondiabetics, respectively [26].In our study, the moderate grade of gastritis and chronic active gastritis was associated with with fatty liver in ltration with and without the H. pylori infection. H. pylori inducesup regulation of several cytokines such as TNF-α, C-reactive protein and interleukin (IL)-1β, due to chronic low-grade in ammatory activity, which may in uence pancreatic β cell secretion and brings about a state where insulin action is altered.The milieu of autocrine and paracrine effects induced by H. pylori-induced gastritis have some indirect effects in the form of alteration of gastric hormones secretion, including gastrin, somatostatin,leptin, and ghrelin which could affect glucose homeostasis, insulin action and sensitivity.
H. pyloriare the culprit of peptic ulcer disease, several gastric and extragastric manifestation other than gastritis. Above all, the gastric malignancies are the most fearsome of all. DNA of gastric mucosal cells are vulnerable to the oxidative damage induced by the reach and free oxygen species by the H. pylori infection generated in ammation and accumulation of these toxic free radicals. H.pylori induced in ltration of the gastric mucosa and activation of neutrophils and macrophages leads to the generation of large amounts of reactive oxygen species(ROS) produces in ammation [28,29].The association of H. pylori infection with severe ongoing oxidative stress modulate many processes in gastric mucosa.Whether due to resistance to its action or de cient levels, alteration in the leptin action, is a coproducer of steatosis. There is a strong role of TNF-alpha in the early stages of fatty liver as evidenced by several animal studies and data from clinical models as well. More the 50% of the patients have derangement of liver enzymes and found to have simple steatosis as the cause for it. This is because of the asymptomatic course of fatty liver disease, most of them discovered incidentally on the workup of hepatomegaly and/or abnormal liver biochemical pro le [30]. In the nonalcoholic variant, the predominant elevation of liver biochemical tests is seen in alanine transaminase levels [31]. In conclusion, in our population, there was a strong association seen in 30-50 years age group which is an early onset of fatty liver in ltration in H. pylori-infected dyspeptic patients.A BMI > than 23, dyslipidemia and type 2 diabetes in the context of H. pylori infection have a greater predisposition to fatty liver. H. pylori bring about an intense milieu of proin ammatory cytokines and it is supposed to be having a close interaction with gut microbiota, further prospective studies in this regards need to be done to understand this situation more clearly.

Declarations
Ethical approval and consent to participate: This study was approved by the Ethical Review Committee of Aga Khan University Hospital, Karachi, Pakistan, on 21st of November 2016, ethical approval number granted was 4532-Med-ERC-16.
All procedures performed in this study were in accordance with the ethical standards of our institutional research committee (Ethical Review Committee, Aga Khan University Hosptial, Karachi, bearing reference number 4532-MED-ERC-6) and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.
Informed consent was obtained from all individual participants included in the study.
Consent for publication: Authors obtained consent for publication by using the data collection form approved by the Ethical Review Committee of our University Hospital.
Availability of data and materials: Data set used during the current study are available from the corresponding author on reasonable request.
Competing interest: Authors declare that they have no competing interests.
Funding: No funds were received for this study in any form by any of the authors.

Authors contributions:
Basit Siddiqui has made contributions to conception and design, interpretation of data, drafting the manuscript and revising it critically for important intellectual content. Rabeea Azmat has made contributions to conception and design, interpretation of data, drafting the manuscript and revising it critically for important intellectual content. Javed Yakoob has made a contribution to conception, acquisition, and interpretation of data and drafting the manuscript. Zaigham Abbas has made contributions to critically analyzing the data and manuscript. Shiyam Sundar Tikmani has made a contribution in acquisition and interpretation of data and nal revision. Zain Mushtaq has made a contribution in data collection. Saad Bin Zafar has made a contribution in data collection. Muhammad Tahir Khan has made contributions to design, drafting of the manuscript and revising it critically for important content.