Gallstones are a common digestive disorder, yet the association between the fibrosis-4 index (FIB-4) and gallstone formation remains poorly understood. This study explores the link between FIB-4 levels and gallstone prevalence among US adults.

This study was based on data from the 2017–2020 National Health and Nutrition Examination Survey (NHANES), which included 7,771 participants. The association between FIB-4 and gallstone risk was analyzed using multivariate logistic regression with restricted cubic spline (RCS) analysis to assess nonlinear correlations, and threshold effects analysis to identify inflection points. Robustness checks included subgroup analysis.

The weighted prevalence of gallstones in this study was 11%. Multiple logistic regression analysis showed that FIB-4 levels were significantly and positively associated with the risk of gallstones. In the fully adjusted model, each unit increase in FIB-4 as a continuous variable increased the risk of gallstones by 19% (OR = 1.19, 95% CI: 1.10, 1.29). When FIB-4 was grouped by quartiles, the risk of gallstones was increased by 60% (OR = 1.60, 95% CI: 1.25, 2.03) in the Q4 group compared to the Q1 group. RCS analysis further revealed a nonlinear positive correlation between FIB-4 and gallstones (P for nonlinear = 0.015) with an inflection point at 2.43, (P for log likelihood ratio test = 0.001). Bonferroni-corrected subgroup analyses showed that the association of FIB-4 with gallstones was statistically significant among non-Hispanic whites, those without heart failure, those without coronary heart disease, alcohol drinkers, and smokers (P < 0.00217).

In this study, we found that elevated levels of FIB-4 were significantly and positively association with the risk of gallstones, and showed a non-linear trend. FIB-4 may have a potential application in the risk assessment of gallstones.

Gallstones are a common biliary disorder worldwide, usually manifesting as stone formation in the gallbladder [1]. The occurrence of gallstones is closely related to a number of factors, including genetics, diet, obesity, and metabolic abnormalities [2,3,4]. The prevalence of gallstones is relatively high in Europe and the United States, with approximately 10–15% of the population affected by gallstones [5]. In addition, obese and diabetic patients are often accompanied by gallstones due to disturbed lipid metabolism in the body [6]. Although gallstones can exist asymptomatically, they can lead to a range of clinical symptoms and, in severe cases, can cause biliary tract infections or pancreatitis [7, 8]. Although previous studies have identified a number of risk factors for gallstones, there is still a lack of more reliable clinical indicators to further explore the association with gallstones [9].

The Fibrosis 4 Index (FIB-4) was originally identified as a prognostic indicator of liver fibrosis in patients with viral infections [10]. With ongoing research, the FIB-4 index is widely recognized as a simple and cost-effective indicator of liver disease [11]. In addition, it is a valuable prognostic marker for individuals with cardiovascular disease (CVD), and liver fibrosis has been associated with CVD even in subclinical stages [12]. Recent studies have revealed that FIB-4 is substantially linked with all-cause mortality and CVD mortality, highlighting the fact that liver fibrosis may contribute to greater mortality in diabetes patients [13].

However, there is a lack of studies on the association between the FIB-4 index and gallstones. Therefore, we hypothesized that there is an association between FIB-4 and the occurrence of gallstones, and we therefore evaluated the value of FIB-4 in the occurrence of gallstones in the U.S. population, with the objective of presenting a population-level epidemiologic explanation for the avoidance of gallstones.

NHANES is a nationwide study that evaluates the nutritional status and health of adults and children in the United States. The survey has been conducted every two years since 1999 and uses a complex multi-stage stratified probability design to ensure representativeness. It looks at the association between environmental factors, lifestyle, nutrition, and health. The study methodology was approved by the Ethical Review Committee of the National Center for Health Statistics, and each participant filled out an informed consent form. The participant screening process for this study is shown in (Fig. 1). Initially 15,560 participants were selected from the NHANES 2017–2020 cycle. Subsequently, We did not include 6,328 participants who were under 20. We further excluded 1388 participants without FIB-4 assessment data. In addition, we excluded 19 subjects without data on gallstones, and on this basis we excluded 54 subjects without complete covariates. This study involved the recruitment of 7,771 eligible volunteers.

NHANES 2017–2020 participant selection flowchart

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Fibrosis-4 index (FIB-4) was the exposure variable in this study. Age, serum aspartate transaminase (AST), alanine transaminase (ALT), and platelets (PLT) were all included in the FIB-4 formula. In calculating FIB-4, we followed the accepted calculation methods of previous studies [14]:

Gallstones were the exposure variable in this study. The “MCQ” section of the NHANES questionnaire: “ Have you ever been told you have gallstones? Participants who answered yes were defined as having gallstones [15].

To control for potential confounders, a number of covariates were included in this study: gender, race, education level, marital status, poverty-to-income ratio (PIR), body mass index (BMI), smoking status, alcohol, diabetes, hypertension, heart failure, coronary heart disease, angina pectoris, stroke, physical activity, and total cholesterol.

Educational attainment is categorized as less than high school, high school or GED, and more than high school. BMI was categorized as < 25 kg/m², 25–30 kg/m² and ≥ 30 kg/m². PIR is categorized into three categories: <1.3, 1.3–3.5, and ≥ 3.5.Smoke at least 100 cigarettes per year? “Smoke at least 100 cigarettes per year?” Individuals were classified as smokers if they answered ‘Yes’ to the question and had smoked at least 100 cigarettes in their lifetime. Individuals were classified as drinkers if they answered ‘Yes’ and had consumed at least 12 alcoholic drinks in their lifetime. Marital status was categorized into three groups: never married, married or living with a partner, and widowed, divorced, or separated. For the question, “Has a doctor ever told you that you have diabetes, except during pregnancy?” question, those who answered “yes” and “borderline” were categorized as having a history of diabetes. Those who answered “yes” to the question. Hypertension was defined as a previous diagnosis, current use of antihypertensive medications, or a blood pressure reading ≥ for 140/90 mm Hg. Participants were categorized into three classes, based on standardized scores from the International Physical Activity Questionnaire (IPAQ). low (< 600MET min/week), moderate (300–3000 MET min/week), High (≥ 3000 MET min/week). Heart failure, coronary heart disease, and angina pectoris were identified through the Cardiovascular Health Questionnaire [16, 17]. All research variable measurements are available on the NHANES website (www.cdc.gov/nchs/nhanes/).

To better represent the entire U.S. population, all analyses in this study were conducted using complex sampling. The study examined the association between FIB-4 and gallstone prevalence in the U.S. population, with FIB-4 assessed as both a continuous variable and in quantiles. Odds ratios and 95% confidence intervals are presented for both analyses. For each individual, we provided baseline characteristics. We evaluated the association between FIB-4 and gallstones using multivariate logistic regression models. We evaluated nonlinear interactions using restricted cubic spline plots, and a threshold effects analysis was conducted. We evaluated the stability of the association between FIB-4 and gallstones using subgroup analysis and interaction testing. Because multiple testing may increase the risk of Type I error, we applied a Bonferroni correction to the results of the subgroup analyses.The Bonferroni correction has a significance level of α’ = 0.05/n, where n is the number of subgroups to be compared (n = 23 in this study), resulting in an adjusted significance level of α’ = 0.00217. We considered the result still statistically significant only if the p-value was below 0.00217. In addition, we recognize that the Bonferroni correction method is more conservative and may increase the risk of Type II errors. Therefore, we exercise caution in the interpretation of the results and recommend that future studies further validate these associations. We used R4.4.2 (R Foundation, http://www.R-project.org) and Empower software (www.empowerstatas.com) to conduct heterogeneity analyses. To ensure reliable findings, the analytical algorithm kept three decimal places. A two-sided p-value of less than 0.05 was considered statistically significant.

Table 1 shows the weighted baseline characteristics of the 7,771 participants in this study. The quartiles of FIB-4 were 0.12 ≤ Q1 < 0.61; 0.61 ≤ Q2 < 0.94; 0.94 ≤ Q3 < 1.40; 1.40 ≤ Q4 ≤ 37.95. The weighted mean age of the participants was 48.37 years, with 48.33% males and 51.67% females. Compared to the lowest FIB-4 quartile, participants in the highest FIB-4 quartile were older, and had a higher proportion of males, were less educated, had lower rates of being unmarried, but higher rates of being divorced, widowed, or separated. They tended to have normal or obese BMIs and had higher rates of heart failure, coronary heart disease, angina, stroke, hypertension, and diabetes. In addition, the high FIB-4 group had higher rates of smoking, lower rates of alcohol consumption, and lower levels of physical activity. FIB-4 levels were positively correlated with gallstone prevalence, with the highest quartile group having the highest prevalence of gallstones (P < 0.05).

Multiple logistic regression analyses showed that FIB-4 as a continuous variable, FIB-4 levels were positively associated with gallstone occurrence in all models. In the model without adjustment for covariates (model 1), the risk of gallstones increased by 22% per unit increase (OR = 1.22, 95% CI: 1.14–1.32). In the model adjusted for sex and race (Model 2), the OR further increased to 1.29 (95% CI: 1.19–1.39). However, in the fully adjusted model (Model 3), although FIB-4 was still significantly positively associated with gallstone risk, the OR decreased slightly to 1.19 (95% CI: 1.10–1.29). When FIB-4 was analyzed in quartiles of subgroups, the Q4 group demonstrated a significantly higher risk of gallstones in all models. In the fully adjusted model, the Q4 group had a 60% increased risk of gallstones compared with the Q1 group (OR = 1.60, 95% CI: 1.25, 2.03). However, in groups Q2 and Q3, the OR showed some increasing trend in Model 1 and Model 2, but failed to maintain statistical significance in Model 3. (Table 2)

To further explore whether there was a nonlinear association between FIB-4 and gallstones, we used a restricted cubic spline plot for fitting (Fig. 2). The RCS results showed a positively nonlinear correlation between FIB-4 and the prevalence of gallstones (P for nonlinear = 0.015). The analysis showed a nonlinear positive correlation between FIB-4 and gallstones, (log-likelihood ratio test p-value = 0.001). (Table 3).

The RCS curve of the association between FIB-4 and gallstones odds ratio among all the study participants. CI: confidence interval. RCS, restricted cubic spline. FIB-4, fibrosis-4 index

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Figure 3 displays the outcomes of interaction tests and subgroup analysis. Bonferroni corrections were applied to the results of the subgroup analyses to minimize the effect of multiple testing. After correction, the subgroups of non-Hispanic whites, those without heart failure, those without coronary artery disease, alcohol drinkers, and smokers were statistically significant (P < 0.00217). Other subgroups were no longer statistically significant after correction.The correlation between FIB-4 and gallstones was not significantly different in the subgroups analyzed (p for interaction > 0.05). (Fig. 3).

Investigation of the association between gallstones and FIB-4 by subgroup. Note 1: The above model adjusted for gender, race, education level, marital status, PIR, BMI, smoking status, alcohol, diabetes, hypertension, heart failure, coronary heart disease, angina pectoris, stroke, physical activity, and total cholesterol. Note 2: In each case, the model is not adjusted for the stratification variable. Note 3: Given the multiple subgroup analyses, we acknowledge the potential for Type I errors. After Bonferroni correction, significant associations remained in Non-Hispanic white, no heart failure, no coronary heart disease, alcohol drinker, and smoker but not in other subgroups

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We compared the baseline characteristics of the study population with those of the FIB-4 deficient population (Table S1). The results showed that there were no differences between the two groups on several key covariates, such as gender, education, and marital status. However, there were still a few covariates that differed. To further validate the robustness of the findings, we performed a multifactor logistic regression analysis incorporating the adjusted covariates. The results showed that, using FIB-4 as a continuous variable, FIB-4 was positively associated with the occurrence of gallstones in all models. In Model 3, each one-unit increase in FIB-4 was associated with a 13% increased risk of gallstones (OR = 1.13, 95% CI: 1.05–1.22). (Table 4)

Based on information from a sizable population-based survey conducted in the US, this study is the first to investigate the connection between FIB-4 and gallstones. Our results showed a positive association between FIB-4 and gallstones. Positive associations of FIB-4 and gallstones differed significantly among non-Hispanic whites, those without heart failure, those without coronary artery disease, alcohol drinkers, and smokers. Bonferroni corrections assessed the robustness of our conclusions.

In this study, the diagnosis of gallstones relied on self-reporting by the subjects rather than imaging such as ultrasound. This approach may introduce classification bias, and if misclassification of gallstones is uniformly distributed among study subjects with different FIB-4 levels, then this random misclassification may lead to bias of the results toward a null effect. This means that the association between FIB-4 and gallstones may be underestimated. If the degree of misclassification of gallstones is correlated with the level of FIB-4, and if subjects in the high FIB-4 group are more health-conscious and more likely to recall and report gallstones, or if subjects in the low FIB-4 group are more likely to underreport gallstones, then this systematic misclassification may lead to an unpredictable direction of bias. However, because symptoms of gallstones, such as abdominal pain, are usually more pronounced and because NHANES uses a standardized survey, we believe that misclassification is more likely to be nondifferential, and therefore the study is not a spurious finding.

In the United States, the incidence of gallstones is relatively high, with approximately 10–15% of the population developing gallstones during their lifetime [18]. In our study, the prevalence of gallstone disease was 11%, which is close to the reported incidence of in U.S. adults [19]. Studies have shown that gallstones in young individuals tend to be associated with genetic disorders, choledochal obstruction and a high incidence of pancreatitis, among others [20, 21]. Gallstones are relatively rare in this age group and their incidence is much lower than in the adult population [22]. Therefore, participants younger than 20 years of age were excluded from this study. Studies have founded that the association between age and gallstones was significantly weakened by adjusting for metabolic-related factors [23, 24]. This suggests that metabolic factors are mediating variables in the association between age and gallstones. Several other studies have reported age as one of the major factors in the incidence of gallstones, which is consistent with our results [25,26,27]. Previous studies have shown that gallstones are more prevalent in women. In our study, we analyzed baseline characteristics stratified by FIB-4 quartiles, in which the proportion of female participants varied between quartiles [28].

The specific mechanism linking elevated FIB-4 to increased prevalence of gallstones is not known, but many potential mechanisms may exist based on previous studies. As mentioned earlier, FIB-4 is an important indicator for assessing liver fibrosis [29]. Hepatic fibrosis leads to obstruction of bile flow and cholestasis is an important factor in gallstone formation [30]. Cholestasis increases the risk of oversaturation of cholesterol in bile, which promotes stone formation [31]. The FIB-4 score is strongly associated with metabolic syndrome, which has been widely recognized as a risk factor for gallstones [32, 33]. Elevated FIB-4 may reflect a systemic chronic inflammatory state [34]. Chronic inflammation may lead to impaired contractile function of the gallbladder, slowing down bile emptying and increasing the retention time of cholesterol in the gallbladder, thus promoting stone formation [35, 36]. In addition, liver disease and metabolic syndrome are often accompanied by oxidative stress, which can further promote gallstone production by damaging gallbladder wall cells and altering the physicochemical properties of bile [37]. Elevated FIB-4 scores are usually indicative of nonalcoholic fatty liver disease (NAFLD) [38], and the association between NAFLD and gallstones has been well established NAFLD patients tend to have higher cholesterol levels in the bile with concomitant gallbladder dysfunction [39, 40]. Thus, chronic inflammation and metabolic disorders triggered by fatty liver may be intermediate in gallstone formation [41]. In summary, we hypothesize that FIB-4 may be associated with bile metabolism, metabolic syndrome, inflammation and oxidative stress, leading to potential gallstone prevalence. Therefore, detection of FIB-4 levels could help in the prevention and management of gallstones.

To our knowledge, this is the first study to investigate the association between FIB-4 and gallstones in a large population. Strengths of this study include the fact that we included a nationally representative sample of U.S. adults and conducted sensitivity analyses for different metrics and subgroups as well as dose-response association.

However, we must recognize certain limitations of this study. First, as an observational study, our results cannot establish a causal association between FIB-4 and gallstones. Second, although we adjusted for a variety of possible confounders in our model, there may still be unadjusted residual confounders that affect the accuracy of the study conclusions. Third, the gallstone diagnoses in this study were based on self-reported data collected by trained clinical staff through questionnaires, which may have been affected by recall bias, resulting in the study primarily capturing associations of clinically evident gallstones, with higher reporting rates in more symptomatic individuals and possible underreporting of asymptomatic gallstones. Fourth, as the measurement limitations of dietary factors kept them from being included in the final model, future studies could further incorporate dietary data to more fully assess the association between FIB-4 and gallstones.

NHANES:

National Health and Nutrition Examination Survey

NCHS:

National Center for Health Statistics

BMI:

Body mass index

PIR:

Poverty-to-income ratio

FIB-4:

Fibrosis-4 index

RCS:

Restricted cubic spline

CVD:

Cardiovascular disease

AST:

Transaminase

ALT:

Alanine transaminase

PLT:

Platelets

NAFLD:

Nonalcoholic fatty liver disease

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Author notes

FC and GM designed the research. ZZ, DH, and ZH collected and analyzed the data, and drafted the manuscript. CX and CH revised the manuscript. All authors contributed to the article and approved the submitted version.

Correspondence to Xintian Cai or Hongping Cheng.

The authors declare no competing interests.

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