Dietary fatty acids and gallstone risk: insights from NHANES and Mendelian randomization analysis

Background Prior research suggests polyunsaturated fatty acids (PUFA) may prevent gallstones, but evidence on saturated fatty acids (SFA) and monounsaturated fatty acids (MUFA) is limited. This study aims to explore the associations between fatty acids and gallstones using a large sample of American population and Mendelian randomization (MR) methods. Methods The cross-sectional study involved 6,629 participants from the National Health and Nutrition Examination Survey (NHANES) 2017–2020. Logistic regression and restricted cubic spline (RCS) analysis were conducted after stratifying by gender subgroups. Two-sample MR analysis was used to explore the causal relationship between fatty acids and gallstones without confounding factors. Results In females, higher SFA intake was positively associated with gallstone risk, while higher intake of n-3 and n-6 PUFA was negatively associated. No significant associations were found in males. No nonlinear correlations were found in any group by RCS analysis. MR analysis indicated that SFA, n-3, and n-6 PUFA could reduce gallstone risk. Conclusion The influence of dietary fatty acid composition on gallstone development differs by gender, providing insights into dietary prevention and treatment of gallstones.


Introduction
Gallstones are a globally prevalent digestive disease, affecting 10-20% of individuals in developed countries and often presenting without obvious symptoms in the early stages (1).However, as the disease progresses, serious and potentially life-threatening complications may occur, including acute cholangitis, cholecystitis, and biliary pancreatitis (2-4).The high incidence and complications of gallstones impose significant economic and health burdens, necessitating effective preventive and curative measures.
Gallstone formation is influenced by genetic predisposition, lipid metabolic disorders, and lifestyle habits, with genetic factors contributing only 25% to the risk, emphasizing the importance of environmental and lifestyle influences (1,5,6).Growing emphasis is placed on the influence of dietary fatty acids on gallstone formation.Prior research has explored the potential protective role of polyunsaturated fatty acids (PUFA) in preventing gallstones (7)(8)(9)(10).However, the evidence on saturated fatty acids (SFA) and monounsaturated fatty acids (MUFA) is limited by small sample sizes and the influence of confounding factors.

Study variables
The dietary fatty acids of interest in this study include SFA, MUFA, PUFA, n-3 PUFA, and n-6 PUFA.Each participant underwent two 24-h dietary recall interviews: the first was conducted in person at the Mobile Examination Center, and the second over the phone 3-10 days later.In order to maintain precision, the analysis utilized the average of the two dietary data, with "dietary two-day sample weight" serving as weight.
Gallstone cases were identified using the inquiry "Has DR ever said you have gallstones".Participants who responded affirmatively were classified as having gallstones.

Covariates
To address potential confounding variables, the study incorporated the following covariates.These covariates encompassed continuous variables such as age, body mass index (BMI), triglyceride, high-density lipoprotein-cholesterol (HDL-C), dietary energy intake, fat intake, and cholesterol intake.Additionally, categorical variables such as gender, race, education level, marital status, poverty income ratio (PIR), hypertension, diabetes, hepatic steatosis, smoking, and Flowchart of participant selection.drinking were also considered.Detailed definitions of covariates are available in Supplementary Table S1.

Statistical analysis
The sample weights from the NHANES stratified multistage sampling were taken into account in the baseline characteristics and correlation analysis.The baseline characteristics of the participants were determined depending on their gallstone status.As the continuous variables were all skewed, median and interquartile range (IQR) were used to describe them, and frequency and weighted percentage were used to describe the categorical variables.Group comparisons were carried out employing the weighted χ 2 test and the Wilcoxon rank-sum test.
Fully adjusted for all covariates, a weighted multivariate logistic regression model was used for analysis.We first analyzed fatty acid intake as a continuous variable for its association with gallstones.Subsequently, fatty acid intake was divided into quartiles to conduct a trend test.Given that gallstones are more prevalent in women and that there are gender disparities in fatty acid intake (11,12), subgroup logistic regression analysis was conducted by gender.Additionally, restricted cubic splines (RCS) were employed to examine the potential nonlinear relationship in different genders.

Data sources
The fatty acid genome-wide association studies (GWAS) data were acquired from the IEU OpenGWAS project, which included a total of 115,006 European participants.The gallstone GWAS data were acquired from the Finngen R10 Release (13), comprising 40,191 European cases and 361,641 European controls overall.For these original GWAS studies, corresponding ethical approvals have been obtained.The population samples of exposure and outcome were from different consortia, ensuring minimal overlap.Detailed GWAS data information is presented in Table 1.

Instrumental variables selection
Single nucleotide polymorphisms (SNPs) strongly associated with the exposure factors were employed as unconfounded instrumental variables (IVs) for the analysis.The MR analysis was based on three fundamental assumptions: (1) IVs are substantially linked with exposure; (2) IVs do not influence outcomes via confounders; (3) IVs impact outcomes only through their effect on exposure.Based on the above criteria, the candidate SNP must reach the genome-wide significance (5 × 10 −8 ).To guarantee the independence of candidate SNPs, the linkage disequilibrium threshold was set to be r 2 = 0.001 and clumping distance = 10,000 kb.The intensity of each SNP was calculated using the formula: F statistic = Beta 2 /SE 2 (14), and SNPs with F < 10 were discarded as weak IVs.To ensure adherence to the core assumptions of MR, we screened for and removed confounders using the GWAS Catalog (15).Detailed information on the confounding SNPs and traits is provided in Supplementary Table S2.

Mendelian randomization analysis
The main method for MR analysis was the inverse variance weighted (IVW) approach.It combines Wald ratios of each IV to conduct a meta-analysis.It is considered the most accurate statistical method when horizontal pleiotropy does not exist (16).Additionally, supplementary analytical methods such as weighted median, MR-Egger, and MR Robust Adjusted Profile Score (MR-RAPS) were also employed in this study (17)(18)(19).We employed MR-PRESSO and RadialMR to detect and eliminate outliers (20,21).Horizontal pleiotropy was evaluated using the MR Egger intercept and MR-PRESSO global test, while heterogeneity was assessed with Cochran's Q test (22).p values below 0.05 suggest the existence of pleiotropy or heterogeneity.The impact of individual outlier IVs was assessed using the funnel plot and the leave-one-out analysis.We applied STROBE-MR to design this study (Supplementary Table S3) (23).The R packages used for MR analysis were "TwoSampleMR" (version 0.6.3),"mr.raps" (version 0.2), "MRPRESSO" (version 1.0), and "RadialMR" (version 1.1).

Baseline characteristics
Table 2 presents the weighted baseline characteristics of the study participants grouped by gallstone status.The study involved 6,629 participants, comprising 5,894 individuals without gallstones and 735 individuals with gallstones.The analysis showed that individuals with gallstones were more likely to be older, female, obese, former smokers, and former drinkers, and exhibited a higher prevalence of hypertension, diabetes, and hepatic steatosis compared to those without gallstones (p < 0.001).Additionally, the two groups differed significantly in race, education level, marital status, and PIR.The

Association between dietary fatty acids and gallstone
After adjusting for all covariates, the results of the weighted logistic regression analysis are presented in Table 3.The results showed that in the female subgroup, higher consumption of SFA was positively associated with an increased risk of gallstones (OR = 1.054, 95% CI: 1.003-1.107),while higher n-3 PUFA (OR = 0.647, 95% CI: 0.420-0.996)and n-6 PUFA (OR = 0.926, 95% CI: 0.871-0.984)intake was linked to a reduced risk.When analyzing fatty acid intake by quartiles, compared to Q1, Q3 and Q4 groups of n-3 PUFA and Q3 group of n-6 PUFA were inversely associated with gallstone risk among females.However, no notable associations were found in the male subgroup.Figure 2 displays the RCS study illustrating the impact of dietary fatty acids on gallstone risk according to gender.After accounting for all confounding variables, no nonlinear correlations were discovered in any of the groups (P-nonlinear >0.05).

Mendelian randomization study
After applying the selection criteria for IVs and removing outliers, the SNPs utilized in the MR analysis are listed in Supplementary Tables S4-S9.All SNPs exhibited F-statistics over 10, indicating no weak IVs.
The IVW method showed that SFA (OR = 0.842, 95% CI: 0.781-0.908),n-3 PUFA (OR = 0.895, 95% CI: 0.841-0.952),and n-6 PUFA (OR = 0.887, 95% CI: 0.838-0.939)could reduce gallstone risk (all p < 0.001), as shown in Figure 3.All three supplementary MR methods demonstrated results consistent with the IVW method, enhancing the reliability of the findings.Table 4 shows that there is no indication of pleiotropy or heterogeneity.No potential outliers were observed that could affect the results, as shown by the leave-one-out results and funnel plots (Supplementary Figures S1, S2).

Discussion
This study is the first to utilize NHANES large sample data and the latest GWAS data to investigate the relationship between various dietary fatty acids and gallstones in the American population, and to explore the causal relationship without confounding factors using MR analysis.Results from the NHANES study indicated that for females, higher SFA intake was linked to higher gallstone risk, whereas higher n-3 and n-6 PUFA intake were linked to a reduced risk.No notable associations were found in the male group.MR analysis indicated that SFA, n-3 and n-6 PUFA could reduce gallstone risk.
Studies have demonstrated that the main causes contributing to the development of gallstones are supersaturation of bile components, promotion of crystal nucleation by mucin and similar substances, and reduced gallbladder motility (24).Multiple epidemiological studies have confirmed that PUFA can prevent gallstone formation (10,25,26).This may be attributed to the following mechanisms: firstly, the intake of fish oil, which contains high levels of n-3 PUFA, can lower cholesterol saturation in bile, inhibiting the formation of cholesterol crystals (8,27).Secondly, n-3 PUFA can also increase the secretion of bile acids and phospholipids, inhibit the formation of biliary mucin, and improve gallbladder motility to prevent gallstone formation (9,28,29).Thirdly, PUFA can reduce serum and liver cholesterol and triglyceride levels, thereby lowering the risk of gallstones (9,(30)(31)(32).Additionally, since inflammation plays a significant role in gallstone formation (33,34), PUFA may also prevent gallstones through their anti-inflammatory effects (35,36).
Regarding SFA, two clinical studies conducted in France and Italy found a positive correlation between dietary SFA and gallstones (37,38).An American clinical investigation found that consuming more long-chain SFA is linked to a greater likelihood of developing gallstones in men, whereas intake of medium-or shortchain SFA does not seem to affect this risk (39).The close correlation between SFA and metabolic syndrome has been highlighted (40).SFA has been found to be linked to several disorders connected to   Dose-response analysis of fatty acids intake and gallstone risk.Models grouped by gender were adjusted for age, race, education level, marital status, poverty income ratio, body mass index, triglyceride, high-density lipoprotein-cholesterol, hypertension, diabetes, hepatic steatosis, smoking, drinking, energy intake, total fat intake and cholesterol intake.Abbreviation: OR, odds ratio; CI, confidence interval; SFA, saturated fatty acid; MUFA, monounsaturated fatty acid; PUFA, polyunsaturated fatty acid.).Anyway, the precise impact of MUFA remains uncertain, with some clinical studies finding a negative correlation between MUFA and gallstone risk (10,38), while others have found the opposite result (47, 48).
Our study findings suggest that in females, consuming dietary PUFA can lower the likelihood of developing gallstones, while higher intake of SFA is associated with an increased risk.These findings align with prior research.However, no such correlation was observed in males.Differences in estrogen and lipid metabolism may lead to a higher prevalence of gallstones in females (49,50), and this disparity may influence the effects of fatty acids on different genders.Moreover, the cross-sectional study and the MR study yielded contradictory findings regarding the role of SFA.This discrepancy can be attributed to the inherent limitations of each study design.Despite adjusting for potential covariates as thoroughly as possible, the cross-sectional study remains susceptible to residual confounding.In contrast, the MR study indicates a causal relationship between lifetime exposure and outcome with minimal confounding, but it cannot account for gender differences and cannot fully eliminate the influence of pleiotropy.However,  further clinical and experimental studies are required to validate these gender differences and explore possible mechanisms.This study utilized a nationally representative NHANES sample for weighted analysis and conducted subgroup analysis by gender, making the results generalizable and instructive.Combining observational studies with MR analysis enhanced the reliability of the findings.However, there are some limitations to this study that should not be overlooked.Firstly, the dietary and gallstone data were selfreported, which introduces the possibility of recall bias and lack of precise imaging diagnosis.Secondly, the 24-h dietary recall interview data may not accurately reflect an individual's long-term dietary habits.Thirdly, this study only targeted adults in the United States, so caution is needed when generalizing the results to other populations.

Conclusion
Our study found that in females, dietary SFA was positively associated with gallstone risk, while higher intake of n-3 and n-6 PUFA was associated with a decreased risk.No significant associations were found in men.MR analysis supports that SFA, n-3 and n-6 PUFA could reduce gallstone risk.These findings provide new perspectives on dietary strategies for the prevention and treatment of gallstones.However, further prospective cohort studies and experimental research are required to validate these results and explore the underlying mechanisms.

FIGURE 3
FIGURE 3Mendelian randomization analysis of fatty acids and gallstone risk.Abbreviation: OR, odds ratio; CI, confidence interval; PUFA, polyunsaturated fatty acid; MR-RAPS, MR Robust adjusted profile score.

TABLE 1
GWAS data information in Mendelian randomization study.

TABLE 2
Weighted basic characteristics of participants by gallstone.
intake of energy, fat, cholesterol, MUFA, and n-6 PUFA was found to be lower in the gallstone group.

TABLE 3
Weighted logistic regression analysis of the association between fatty acid intake and gallstone.

TABLE 4
Assessment of pleiotropy and heterogeneity in Mendelian randomization study.