Association between nonalcoholic fatty liver disease and Helicobacter pylori infection in Dali City, China
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* Ping Yan
* Bocheng Yu
* Min Li
* Weidong Zhao

## Abstract

**Objectives:** To investigate whether gastric *Helicobacter pylori (H. pylori*) infection is associated with the progress of nonalcoholic fatty liver disease (NAFLD) in Dali city, China.

**Methods:** One thousand and one hundred eighty-five adults who had visited a health examination center were enrolled. Nonalcoholic fatty liver disease and *H. pylori* infection were assessed by abdominal color ultrasound and 13C-urea breath test, respectively. Serum lipid metabolic indices were compared between NAFLD subjects without and with *H. pylori* infection.

**Results:** The prevalence rate of NAFLD for the entire study population (n=1185) was 44.6% (n=529), including 167 women and 362 men. Among 529 NAFLD subjects, *H. pylori* was confirmed as a significant and independent risk factor for NAFLD (95% CI 1.02-1.79, *p*=0.036, OR =1.35). Compared with *H. pylori* non-infected individuals (n=721), higher contents of LDL-C (*p*=0.012) along with TC (*p*=0.014) were also observed in *H. pylor*i-infected individuals (n=464).

**Conclusion:** A positive correlation is identified between *H. pylori* infection and NAFLD.

*   *H. pylori* infection
*   nonalcoholic fatty liver disease
*   lipid metabolic dysfunction
*   Dali city

**N**onalcoholic fatty liver disease (NAFLD) is a frequent metabolic condition in humans, creating a global public health concern.1 An estimated 400 million people have NAFLD in China, and it has been linked to more than 100 thousand deaths.2,3 Research evidence shows that NAFLD is strongly related to insulin resistance, metabolic syndrome, as well as obesity.4 However, the association of gastrointestinal microbes to the pathogenesis of NAFLD remains unclear.5

*Helicobacter pylori (H. pylori*) pose a threat to nearly half of the world’s population.6,7 Infection of *H. pylori* is commonly responsible for gastritis, gastric cancer, as well as peptic ulcer disease. In particular, novel research findings have opined a strong link between *H. pylori* infection and extra-digestive manifestations, namely, diabetes, obesity, liver tumors, cardiovascular disease, and lipid metabolism disorders.8 Notably, in recent years, *H. pylori* infection has been viewed as a potentially important causative agent associated with the development of NAFLD.

However, connection linking *H. pylori* infection to NAFLD remains debated. Positive associations of NAFLD with *H. pylori* infection have been investigated in numerous studies,9-11 whereas some studies have reported conflicting results.12-15 Under these circumstance, more clinical data are needed to address the connection of NAFLD with *H. pylori* infection. Locally, no studies regarding NAFLD and *H. pylori* association are available. Therefore, herein, we primarily purposed to explore the association of NAFLD with *H. pylori* infection in medical check-ups in Dali city, a Bai ethnic minority area in western Yunnan province of China.

## Methods

We conducted this retrospective research using electronic medical data from participants (age, >18 years) who underwent comprehensive health check-ups at a tertiary hospital in Dali city between January 2017 and June 2019 (Figure 1). We included data from participants consisting of anthropometric parameters and results of 13C-urea breath test (UBT), serum biochemistry, ultrasound color imaging of the abdomen, and ultrasound color imaging of neck. We excluded subjects who had (i) a history of significant alcohol intake: females (≥140 g/week), and males (≥210 g/week); (ii) a treatment history with amiodarone, methotrexate, glucocorticoid and tamoxifen; (iii) individuals with celiac disease, Wilson’s disease, viral hepatitis, lack of beta lipoprotein, autoimmune hepatitis, total parenteral nutrition, and congenital lipid atrophy; (iv) those under treatment with proton pump inhibitors (PPI) for 2 weeks or under treatment with bismuth salts and antibiotics for 4 weeks prior to the 13C-UBT; (v) individuals with upper gastrointestinal disorders’ history; (vi) participants with cancer or mental conditions; and (vii) previously underwent gastric surgery. The ethical committee of Dali University Hospital granted the study protocol and ethical approval (No: 20200118).

![Figure 1](http://smj.org.sa/https://smj.org.sa/content/smj/42/7/735/F1.medium.gif)

[Figure 1](http://smj.org.sa/content/42/7/735/F1)

Figure 1 
- Flowchart of study population selection flowchart. NAFLD: nonalcoholic fatty liver disease, *H. pylori: Helicobacter pylori*

### Physical examinations and serum biochemical examinations

Anthropometric parameters (weight along with height) were also assessed. And body mass index (BMI) was determined as kg/m2. We recorded the systolic, as well as diastolic blood pressure during the physical examination. A fully automatic biochemical system (Hitachi 7600, Tokyo, Japan) was used to test the serum biochemical indices: alanine aminotransferase (ALT), total cholesterol (TC), aspartate aminotransferase (AST), low-density lipoprotein cholesterol (LDL-C), fasting plasma glucose (FPG), urea, albumin (ALB), triglyceride (TG), high-density lipoprotein cholesterol (HDL-C), creatinine, and uric acid.

### Assessment of NAFLD

Each of the participant was subjected to Color Doppler ultrasound imaging of the abdomen by an independent radiologist with a Logiq E8 radiology ultrasound equipment (GE Corp., Chicago, IL, USA). We diagnosed NAFLD on the basis of the results of the ultrasound photographs and the current diagnostic reference criteria.16

### Establishment of H. pylori infection

Each subject was assessed for *H. pylori* infection using 13C-UBT kit (Headway, Zhonghe, Shenzhen, China). After ≥8 hours of fasting overnight, we orally administered a 75 mg 13C-urea capsule to all subjects and collected 2 samples of exhaled breath at baseline, as well as 30 mins after the capsule was consumed. We analyzed the samples using a HCBT-01 13C detection system (Headway, Shenzhen, China). If the results of 2 samples differed by >4/thousand (δ‰) meant a participant was positive for *H. pylori* infection, as previously described.17

### Statistical statistics

The characteristics of the subjects are presented as mean ± SD for normally distributed continuous variables and as percentages for categorical data. We compared the characteristics of subjects across the NAFLD status with the Chi-square test or t-test. Then logistic regression was employed to investigate the effect of various parameters on NAFLD and *H. pylori* infection association by adjusted odds ratios (ORs). We also constructed 4 different models. Model 1 included only *H. pylori* infection. Age and carotid plaque status were added to Model 2. Model 3 was Model 2 with further adjustment for ALT, AST, and UA levels. Finally, more potential indicators and confounders, including FPG, TC, TG, SBP, DBP, LDL-C, and BMI were added to Model 4. Point-biserial correlation was employed to evaluate the correlation between *H. pylori* infection and TC, as well as LDL-C.

Statistical analysis was implemented in the SPSS Statistics 23.0 software (for windows, IBM, Armonk, NY, USA). P-value of <0.05 signified statistical significance.

## Results

Overall, 1185 participants (407 women and 778 men) were enrolled. The prevalence rate of NAFLD for the entire study population (n=1185) was 44.6% (n=529), including 167 women and 362 men. Participants with NAFLD were significantly older, with lower levels of HDL-C and higher weight, SBP, and DBP, as well as elevated contents of TC, AST, TG, FPG, LDL-C, and ALT (all *p*<0.05). Differences in ALB contents and height between the NAFLD and non-NAFLD subjects were not significant (*p*>0.05). A higher prevalence rate of *H. pylori* infection was reported in the individuals with NAFLD (43.5%) relative to that of in the individuals without NAFLD (35.7%, *p*=0.007) (Table 1).

View this table:
[Table 1](http://smj.org.sa/content/42/7/735/T1)

Table 1 
- Comparison of study participants demographics and biochemical profiles between cgroup and non-NAFLD group.

Table 2 summarizes several risk factors related to NAFLD after logistic regression analysis. The levels of ALT (OR =1.02, 95% CI 1.01-1.03), BMI (OR=4.38, 95% CI 3.29-5.84), FPG (OR=2.29, 95% CI 1.31-3.40), TG (OR =2.33, 95% CI 1.73-3.14), and DBP (OR =1.73, 95% CI 1.17-2.56) were independent risk factor of NAFLD (all *p*<0.05). Notably, *H. pylori* was similarly a strong independent risk factor for NAFLD (95% CI 1.02-1.79, OR=1.35, *p*=0.036). These findings illustrate that *H. pylori* functions as a mediator of NAFLD.

View this table:
[Table 2](http://smj.org.sa/content/42/7/735/T2)

Table 2 
- Univariate regression analysis for risk factors associated with nonalcoholic fatty liver disease (NAFLD).

Furthermore, to clarify the link between NAFLD and *H. pylori* infection, we constructed 4 different models. Consequently, *H. pylori* infection (unadjusted OR=1.38; for 95% CI 1.09-1.75, *p*=0.007) was remarkably linked to NAFLD as indicated in Table 3. Additional adjustment for carotid plaque and age attenuated the result was 95% CI 1.05-1.68, OR =1.33, *p*=0.020. However, after adjusting the model for AST, UA, and ALT, the OR for NAFLD was escalated (95% CI 1.10-1.83, OR=1.42, *p*=0.008). In the fully adjusted model (Model 4), infection of *H. pylori* correlated with an elevated risk of NAFLD (95% CI 1.02-1.79, OR 1.35, *p*=0.036). To elucidate the potential underlying mechanisms of *H. pylori* infection in NAFLD, we conducted an analysis to determine the correlation of *H. pylori* infection with several metabolic syndrome constituents. Compared to the *H. pylori* (-) individuals, the *H. pylori* (+) individuals had elevated TC and LDL-C contents (all *p*<0.05, Table 4). However, the differences in FPG (*p*=0.492), TG (*p*=0.409), DBP (*p*=0.614), BMI (*p*=0.142), SBP (*p*=0.776), and HDL-C (*p*=0.087) between these 2 groups were not significant. Furthermore, point-biserial correlation was employed to evaluate the correlation between *H. pylori* infection and TC, as well as LDL-C. As presented in Figure 1, there was a positive correlation between *H. pylori* infection and TC (*p*=0.015, r=0.071, Figure 2A). And *H. pylori* infection was also positively correlated with LDL-C (*p*=0.013, r=0.072, Figure 2B). Collectively, our results provide evidence that infection of *H. pylori* may be involved in the pathogenesis of NAFLD by influencing the metabolism of cholesterol.

![Figure 2](http://smj.org.sa/https://smj.org.sa/content/smj/42/7/735/F2.medium.gif)

[Figure 2](http://smj.org.sa/content/42/7/735/F2)

Figure 2 
- *Helicobacter pylori (H. pylori)* infection rates in nonalcoholic fatty liver disease (NAFLD) individuals categorized by A) total cholesterol (TC) and B) low-density lipoprotein cholesterol (LDL-C).

View this table:
[Table 3](http://smj.org.sa/content/42/7/735/T3)

Table 3 
- Multivariate logistic regression analysis for nonalcoholic fatty liver disease (NAFLD) after adjusting effects of confounders.

View this table:
[Table 4](http://smj.org.sa/content/42/7/735/T4)

Table 4 
- Effect of *Helicobacter pylori (H. pylori)* infection on metabolic syndrome indicators in nonalcoholic fatty liver disease (NAFLD) individuals.

## Discussion

Nonalcoholic fatty liver disease is a frequent metabolic affecting nearly one-third of the world’s population, but the underlying mechanism remains poorly understood. Many factors, namely, metabolic, behavioral, genetic, and environmental, may be responsible for NAFLD pathogenesis.18 Findings over recent years have established possible associations of *H. pylori* infection with NAFLD.19 Herein, a positive influence of *H. pylori* infection on NAFLD was demonstrated. Remarkably, after ruling out additional confounders, consisting of age, blood lipids, carotid plaque, blood glucose, liver enzymes, and blood pressure, *H. pylori* infection (95% CI 1.02-1.79, OR 1.35, *p*=0.036) remained the only independent factor for NAFLD. This finding is in accordance with that of a previous report which documented a positive correlation between NAFLD and *H. pylori* infection.20

The initial view of NAFLD is a hepatic indication of the metabolic disorder has been changing, and it is currently regarded that the occurrence of NAFLD may precede metabolic syndrome.21 Thus, when combined with the analysis of several metabolic indices, we obtained higher weight, DBP, and SBP and elevated contents of TG, FPG, TC, BMI, and LDL-C (all *p*<0.05) in individuals with NAFLD in contrast with those without NAFLD. We also identified DBP, BMI, FPG, and TG, as remarkable risk factors of NAFLD. Therefore, we speculated that a potential interaction exists between the NAFLD and metabolic syndrome constituents. As noted in another study, metabolic syndrome itself may contribute to the onset and progress of NAFLD, and vice versa.22

Abundant published research has demonstrated that infection of *H. pylori* is strongly associated with NAFLD, as well as metabolic syndrome.23-26 Herein, we also evaluated whether *H. pylori* infection affected various metabolic syndrome indicators (namely, HDL-C, SBP, FPG, TC, DBP, BMI, TG, and LDL-C). Notably, both LDL-C and TC contents were elevated in the *H. pylori* (+) group (*p*<0.05). Consequently, *H. pylori* infection may contribute to an escalation in LDL-C and TC contents. This finding is in accordance with a previous meta-analyses report that confirmed the influence of *H. pylori* infection on the lipid profile in serum.27 However, herein, the effects of *H. pylori* infection on other metabolic indicators were not verified. Here, we enumerate some possible reasons for our results. First, some guidelines before blood collection were provided to the participants. Some biochemical and physiological indicators of the participants in their original living environment (namely, kidney function, blood lipid, liver function, blood pressure, and blood glucose,) may have been underestimated. Second, the study included only subjects with higher education level working in government-affiliated institutions in Dali City. They are likely to have a higher level of health awareness than rural populations. Therefore, these study findings may be biased toward participants with better living standards, better dietary profiles, as well as good health awareness.

### Study limitations

Our findings merit consideration, along with several potential limitations. First, this was a retrospective, single-center research. Another limitation of this study is the possible sample bias. Only subjects who underwent health check-ups were enrolled in the analysis. Thus, generalizing the findings to other populations is a challenge. Our present results require further verification using larger samples with different socioeconomic backgrounds. Third, no follow-up of the participants was performed. Fourth, we only confirmed that the infection of *H. pylori* was an independent risk factor for NAFLD in present study. The underlying mechanism of *H. pylori* infection evoking NAFLD was mere speculation. More in-depth prospective studies are required for the precise mechanistic association between *H. pylori* infection and NAFLD.

In summary, *H. pylori* infection may be a potential important contributor of NAFLD. However, further studies are needed to elucidate the precise mechanism of the relationship between *H. pylori* infection and NAFLD.

## Acknowledgment

*This work was funded by Yunnan Health Training Project of High Level Talents (#H-2019045), the Special Basic Cooperative Research Programs of Yunnan Provincial Undergraduate Universities’ Association (#202001BA070001-040 & 202001BA070001-055), and the National Natural Science Foundation, China (#81960363). We thank the Editage company in Shanghai ([www.editage.cn](http://www.editage.cn)) for their English language editing services of this manuscript.*

## Footnotes

*   **Disclosure.** Authors have no conflict of interests, and the work was not supported or funded by any drug company.

*   Received January 18, 2021.
*   Accepted May 11, 2021.


*   Copyright: © Saudi Medical Journal

This is an Open Access journal and articles published are distributed under the terms of the Creative Commons Attribution-NonCommercial License (CC BY-NC). Readers may copy, distribute, and display the work for non-commercial purposes with the proper citation of the original work.

## References

1.  1.Loomba R, Lim JK, Patton H, El-Serag HB. AGA Clinical practice update on screening and surveillance for hepatocellular carcinoma in patients with nonalcoholic fatty liver disease: Expert review. Gastroenterology 2020; 158: 1822–1830.
    
    [PubMed](http://smj.org.sa/lookup/external-ref?access_num=http://www.n&link_type=MED&atom=%2Fsmj%2F42%2F7%2F735.atom) 

2.  2.Zhou F, Zhou J, Wang W, Zhang XJ, Ji YX, Zhang P, et al. Unexpected Rapid Increase in the Burden of NAFLD in China from 2008 to 2018: A systematic review and meta-analysis. Hepatology 2019; 70: 1119–1133.
    
    [CrossRef](http://smj.org.sa/lookup/external-ref?access_num=10.1002/hep.30702&link_type=DOI) 
    
    [PubMed](http://smj.org.sa/lookup/external-ref?access_num=31070259&link_type=MED&atom=%2Fsmj%2F42%2F7%2F735.atom) 

3.  3.Estes C, Anstee QM, Arias-Loste MT, Bantel H, Bellentani S, Caballeria J, et al. Modeling NAFLD disease burden in China, France, Germany, Italy, Japan, Spain, United Kingdom, and United States for the period 2016-2030. J Hepatol 2018; 69: 896-904.
    
    

4.  4.Younossi ZM. Non-alcoholic fatty liver disease - A global public health perspective. J Hepatol 2019; 70: 531-544.
    
    

5.  5.Safari Z, Gerard P. The links between the gut microbiome and non-alcoholic fatty liver disease (NAFLD). Cell Mol Life Sci 2019; 76: 1541–1558.
    
    [CrossRef](http://smj.org.sa/lookup/external-ref?access_num=10.1007/s00018-019-03011-w&link_type=DOI) 

6.  6.Mansori K, Moradi Y, Naderpour S, Rashti R, Moghaddam AB, Saed L, et al. Helicobacter pylori infection as a risk factor for diabetes: a meta-analysis of case-control studies. BMC Gastroenterol 2020; 20: 77.
    
    [CrossRef](http://smj.org.sa/lookup/external-ref?access_num=10.1186/s12876-020-01223-0&link_type=DOI) 

7.  7.Crowe SE. Helicobacter pylori Infection. N Engl J Med 2019; 380: 1158–1165.
    
    [CrossRef](http://smj.org.sa/lookup/external-ref?access_num=10.1056/NEJMcp1710945&link_type=DOI) 

8.  8.Razuka-Ebela D, Giupponi B, Franceschi F. Helicobacter pylori and extragastric diseases. Helicobacter 2018; 23 Suppl 1: e12520.
    
    

9.  9.Zhou BG, Yang HJ, Xu W, Wang K, Guo P, Ai YW. Association between Helicobacter pylori infection and nonalcoholic fatty liver disease: A systematic review and meta-analysis of observational studies. Helicobacter 2019; 24: e12576.
    
    

10. 10.Yu YY, Cai JT, Song ZY, Tong YL, Wang JH. The associations among Helicobacter pylori infection, white blood cell count and nonalcoholic fatty liver disease in a large Chinese population. Medicine (Baltimore) 2018; 97: e13271.
    
    

11. 11.Jiang T, Chen X, Xia C, Liu H, Yan H, Wang G, et al. Association between Helicobacter pylori infection and non-alcoholic fatty liver disease in North Chinese: a cross-sectional study. Sci Rep 2019; 9: 4874.
    
    

12. 12.Okushin K, Takahashi Y, Yamamichi N, Shimamoto T, Enooku K, Fujinaga H, et al. Helicobacter pylori infection is not associated with fatty liver disease including non-alcoholic fatty liver disease: a large-scale cross-sectional study in Japan. BMC Gastroenterol 2015; 15: 25.
    
    

13. 13.Baeg MK, Yoon SK, Ko SH, Noh YS, Lee IS, Choi MG. Helicobacter pylori infection is not associated with nonalcoholic fatty liver disease. World J Gastroenterol 2016; 22: 2592–2600.
    
    

14. 14.Fan N, Peng L, Xia Z, Zhang L, Wang Y, Peng Y. Helicobacter pylori infection is not associated with non-alcoholic fatty liver disease: A cross-sectional study in china. front microbiol 2018; 9: 73.
    
    

15. 15.Cai O, Huang Z, Li M, Zhang C, Xi F, Tan S. Association between Helicobacter pylori infection and nonalcoholic fatty liver disease: A single-center clinical study. Gastroenterol Res Pract 2018; 2018: 8040262.
    
    

16. 16.Zou B, Sun L, Wang X, Chen Z. The prevalence of single and multiple thyroid nodules and its association with metabolic diseases in Chinese: A cross-sectional study. Int J Endocrinol 2020; 2020: 5381012.
    
    

17. 17.Xue Y, Zhou LY, Lu HP, Liu JZ. Recurrence of Helicobacter pylori infection: incidence and influential factors. Chin Med J (Engl) 2019; 132: 765–771.
    
    

18. 18.Friedman SL, Neuschwander-Tetri BA, Rinella M, Sanyal AJ. Mechanisms of NAFLD development and therapeutic strategies. Nat Med 2018; 24: 908–922.
    
    [CrossRef](http://smj.org.sa/lookup/external-ref?access_num=10.1038/s41591-018-0104-9&link_type=DOI) 
    
    [PubMed](http://smj.org.sa/lookup/external-ref?access_num=http://www.n&link_type=MED&atom=%2Fsmj%2F42%2F7%2F735.atom) 

19. 19.Okushin K, Tsutsumi T, Ikeuchi K, Kado A, Enooku K, Fujinaga H, et al. Helicobacter pylori infection and liver diseases: Epidemiology and insights into pathogenesis. World J Gastroenterol 2018; 24: 3617–3625.
    
    

20. 20.Liu R, Liu Q, He Y, Shi W, Xu Q, Yuan Q, et al. Association between Helicobacter pylori infection and nonalcoholic fatty liver: A meta-analysis. Medicine (Baltimore) 2019; 98: e17781.
    
    [CrossRef](http://smj.org.sa/lookup/external-ref?access_num=10.1097/md.0000000000017781&link_type=DOI) 

21. 21.Neves C, Rodrigues T, Sereno J, Simoes C, Castelhano J, Goncalves J, et al. Dietary Glycotoxins Impair Hepatic Lipidemic Profile in Diet-Induced Obese Rats Causing Hepatic Oxidative Stress and Insulin Resistance. Oxid Med Cell Longev 2019; 2019: 6362910.
    
    

22. 22.Federico A, Zulli C, de Sio I, Del Prete A, Dallio M, Masarone M, et al. Focus on emerging drugs for the treatment of patients with non-alcoholic fatty liver disease. World J Gastroenterol 2014; 20: 16841–16857.
    
    [CrossRef](http://smj.org.sa/lookup/external-ref?access_num=10.3748/wjg.v20.i45.16841&link_type=DOI) 
    
    [PubMed](http://smj.org.sa/lookup/external-ref?access_num=25492998&link_type=MED&atom=%2Fsmj%2F42%2F7%2F735.atom) 

23. 23.Chen C, Zhang C, Wang X, Zhang F, Zhang Z, Ma P, et al. Helicobacter pylori infection may increase the severity of nonalcoholic fatty liver disease via promoting liver function damage, glycometabolism, lipid metabolism, inflammatory reaction and metabolic syndrome. Eur J Gastroenterol Hepatol 2020; 32: 857–866.
    
    

24. 24.Alvarez CS, Florio AA, Butt J, Rivera-Andrade A, Kroker-Lobos MF, Waterboer T, et al. Associations between Helicobacter pylori with nonalcoholic fatty liver disease and other metabolic conditions in Guatemala. Helicobacter 2020: e12756.
    
    

25. 25.Lim SH, Kim N, Kwon JW, Kim SE, Baik GH, Lee JY, et al. Positive association between Helicobacter pylori infection and metabolic syndrome in a Korean population: A multicenter nationwide study. Dig Dis Sci 2019; 64: 2219–2230.
    
    

26. 26.Liou JM, Chen CC, Chang CM, Fang YJ, Bair MJ, Chen PY, et al. Long-term changes of gut microbiota, antibiotic resistance, and metabolic parameters after Helicobacter pylori eradication: a multicentre, open-label, randomised trial. Lancet Infect Dis 2019; 19: 1109-1120.
    
    

27. 27.Shimamoto T, Yamamichi N, Gondo K, Takahashi Y, Takeuchi C, Wada R, et al. The association of Helicobacter pylori infection with serum lipid profiles: An evaluation based on a combination of meta-analysis and a propensity score-based observational approach. PLoS One 2020; 15: e0234433.