The association between dietary protein and gallstones in adults under 60 years old

Abstract

Although there is a clear association between dietary composition and risk factors such as obesity and insulin resistance, the link between protein intake and gallstones remains unclear. The aim of this study is to investigate the association between dietary protein and the development of gallstones in American adults under 60 years old. The study employed weighted multivariate logistic regression, and smoothed curve fitting to examine the association between dietary protein and gallstones using data from National Health And Nutrition Examination Survey from 2017 to 2020. To correct for the skewed distribution of protein intake, the data underwent Box–Cox transformation. Among the 3150 participants aged 20 years and older, there was a significant negative association between Box–Cox-transformed protein intake and gallstone incidence for the United States adults under 60 years old. The risk of gallstones was reduced by 30 percent for each unit of Box–Cox-transformed dietary protein intake. The group with the highest protein intake had a 76% lower risk of gallstones compared to the group with the lowest protein intake. Threshold effect analysis revealed that the optimal threshold identified by the model was 9.79; when protein intake was below this threshold, each unit increase in intake was significantly associated with a 27% reduction in the risk of gallstones; once this threshold was exceeded, this association was no longer significant. Among adults under the age of 60 in the United States, there is an inverse association between dietary protein intake and gallstones. After Box–Cox transformation, the optimal threshold for dietary protein intake was 9.79, when protein intake exceeded this threshold, its risk-reducing effect was no longer statistically significant.

1. Introduction

Gallstones and their complications are one of the common causes of hospitalization for digestive diseases.^[1] The prevalence of cholelithiasis and the rate of surgical treatment have both approximately doubled in the last 30 years,^[2] and the prevalence of gallstones in Western countries is 15 to 20%.^[3] Gallstones can be classified according to their composition as cholesterol stones, pigment stones, and mixed stones^[4,5] with more than 80% of gallstones consisting of cholesterol.^[6] Surveys have shown that more than 20% of people with gallstones will experience symptoms such as colic or infection during their lifetime^[7] and undergo cholecystectomy.^[8] In addition, gallstones are associated with a heightened risk of diabetes, neoplasia, and overall mortality, imposing a significant economic burden on patients.^[9] Therefore, exploring primary prevention strategies for gallstones, such as dietary and lifestyle changes, may help to reduce their incidence and associated healthcare costs.

The occurrence of gallstones is affected by numerous factors,^[10] including female sex, age, body mass index (BMI), pregnancy, physical inactivity, diabetes mellitus, fatty liver, hemolytic anemia, and medications.^[11–13] Among these factors, dietary factors have received widespread attention, and diet is considered a modifiable risk factor.^[14,15] Earlier research has indicated that the risk of gallstones is positively associated with meat consumption, high energy, high fat and saturated fatty acid intake, and negatively associated with vegetable and fiber consumption.^[16] It has been suggested that plant protein intake is protective against the development of gallstones,^[17] whereas dietary patterns high in animal protein and fatty foods are associated with cholesterol gallstone risk,^[16] and coincidentally, vegetarian diets are also associated with a reduction in the likelihood of symptomatic cholelithiasis by lowering cholesterol.^[18] It is worth noting that different sources of animal protein have varying effects on digestive health; whilst the intake of dairy proteins is associated with a reduced risk of inflammatory bowel disease, total meat intake may increase this risk.^[19] This differential effect may extend to the process of gallstone formation. Concurrently, higher milk intake is negatively correlated with nonalcoholic fatty liver disease indices; as nonalcoholic fatty liver disease and gallstones share core risk factors such as insulin resistance,^[20] this suggests that dairy proteins may indirectly reduce susceptibility to gallstones by protecting hepatic metabolism. Bioactive compounds in the diet are closely linked to the regulation of metabolic pathways; for example, chlorogenic acid in green coffee extract can improve fasting blood glucose, insulin and triglyceride levels,^[21] and abnormalities in these metabolic markers are key drivers of cholesterol stone formation. Furthermore, studies have indicated that an imbalance in the intake of specific protein sources may disrupt metabolic homeostasis,^[22] which further highlights the need to investigate the dose-response relationship between dietary protein intake and the risk of gallstones.

Although research has investigated the association between dietary protein type and gallstones, no study has concentrated on the link between dietary protein intake and gallstone risk. Because of the long period of gallstone formation and treatment modalities that rely primarily on surgical removal, and because previous studies have shown that non-elderly adults are more susceptible to cholesterol stones and diet-related diseases.^[23,24] Clinical studies show that the highest proportion of gallstone patients (44.3%) is found in the 41 to 60 age group, with the lowest prevalence among those under 20, while the incidence drops significantly in those over 60 (28.6%).^[25] Other studies have also indicated that patients aged 50 to 60 years account for approximately one-quarter of all cases.^[26] Given that early screening is crucial for gallstones, conducting research focused on middle-aged and young adults under 60 years old is more clinically targeted and practically meaningful. Therefore, this study aims to utilize data from the National Health and Nutrition Survey to explore the potential association between dietary protein intake and the incidence of gallstones in adults under 60 years old. We hypothesize that dietary protein intake is negatively associated with the risk of gallstone development in adults under 60 years old.

2. Methods

2.1. Study population

Data were obtained from the National Health and Nutrition Examination Survey (NHANES) database and authorization of the study protocol was granted by the National Center for Health Statistics Research Ethics Review Board. All participants provided written informed consent prior to recruitment. Surveys are typically conducted over a 2-year cycle, but a new coronavirus outbreak in 2019 caused the study to be paused midway through the study. Therefore, we used data from a nationally representative sample from 2017 to March 2020 before the NHANES outbreak. The study population was screened according to the study objectives; the detailed inclusion and exclusion criteria are shown in Figure 1. Ultimately, 3150 individuals were included in this study, of whom 246 reported a personal history of gallstones.

Figure 1.

Flow chart of participant selection. n = number of participants, BMI = body mass index.

2.2. Gallstones

The questionnaire was administered by skilled interviewers in a home environment using a computer-assisted personal interviewing system. The questionnaire was designed to determine whether a physician or another healthcare provider had informed the participants about the presence of gallstones. In this study, gallstone disease (GSD) were defined based on self-reported diagnoses from the NHANES questionnaire (Medical Conditions Questionnaire 550: “Has a doctor or other healthcare professional ever told you that you have gallstones?”). Participants who answered “Yes” were classified as cases of GSD, whilst those who answered “No” were classified as controls; participants with missing, refused, or unknown responses were excluded.^[27,28]

2.3. Dietary protein

The objective of the dietary interview section was to gather comprehensive information regarding the participants’ dietary habits. All participants in the NHANES were allowed to participate in a meal review 24 hours prior to the interviews.^[29,30] The initial dietary recall interview was conducted at a mobile examination center, followed by a second interview conducted via telephone 3 to 10 days later. For the purposes of this study, the average dietary protein value with complete data on both occasions was used as the independent variable.

2.4. Covariables

The covariates included age, race, sex, smoking, alcohol use, poverty-to-income ratio (PIR), education, marital status, waist circumference, BMI, high cholesterol, high blood pressure, coronary heart disease (CHD), heart failure, angina, diabetes, cancer, total dietary energy, dietary fiber, dietary fat, and dietary saturated fatty acids. High cholesterol, high blood pressure, CHD, angina pectoris, heart failure, diabetes, and cancer were identified by whether a physician or another healthcare provider had ever informed the participant that they had the disease, which was “yes” if answered “Yes,” or if answered “no,” which was “No.” The diabetes survey added the question of whether they were part of the critical diabetes option, where participants who answered the critical diabetes question were categorized as diabetic for the purposes of this survey. The doctor or other healthcare professional asked: “Have you ever had gallbladder surgery?.” Participants who answered “yes” were classified into the group with a history of cholecystectomy, whilst those who answered “no” were classified into the control group. There were 488 missing values for the covariate PIR; this study employed multiple imputation methods to impute the missing values for PIR.

2.5. Statistical analysis

The demographic characteristics of the subjects were assessed by the presence or absence of cholelithiasis using the chi-square test and t-test. Weighed logistic regression analysis was employed to examine the linear relationship between dietary protein and GSD. After converting dietary protein from a continuous variable to a categorical variable (3 categories) using Box–Cox transformation, trend analysis was performed to investigate the linear relationship pattern between dietary protein and GSD. Subgroup analyses were conducted to investigate the relationships between dietary protein and gallstones in populations of different sexes, races, educational levels, diabetes mellitus, hypertension, and CHD status. Interaction tests were used to examine the consistency of the relationships among different subgroups. The linear relationship between dietary protein and gallstones was explored using smoothing curve fitting and threshold effect analyses. Sensitivity analyses were performed to confirm the robustness of the results. All analyses were performed using R (version 4.2.0) or EmpowerStats (version 5.2; X&Y Solutions, Inc.). Statistical significance was defined as a 2-sided P < .05.

3. Results

3.1. Baseline characteristics

The mean age of the 3150 participants in the survey was 39.38 (± 11.48) years. Of these, 52.32% were female, and 33.31% were non-Hispanic White. Of all participants, 246 were diagnosed with cholelithiasis. The mean (standard deviation) dietary protein value was 82.44 (± 36.59) for all participants and 73.70 (± 35.81) for those with gallstones; the difference between the 2 groups was statistically significant (t = 3.86, df = 3148, P < .001). Individuals with gallstones were more likely to be older, female, non-Hispanic White, and more likely to have high cholesterol, hypertension, diabetes, heart disease, malignancy, and a history of cholecystectomy. In addition, patients with gallstones generally had a higher BMI (Table 1).

Table 1.

Basic characteristics of participants by gallstones among U.S. adults.

Characteristics	Total adults	No gallstones	Gallstones	P value
N	3150	2904	246
Age (± SD) (yrs)	39.38 ± 11.48	39.03 ± 11.53	43.46 ± 10.00	< .001
Gender (%)				< .001
Male	1502 (47.68)	1449 (49.90)	53 (21.54)
Female	1648 (52.32)	1455 (50.10)	193 (78.46)
Race (%)				< .001
Mexican American	401 (12.73)	364 (12.53)	37 (15.04)
Other Hispanic	313 (9.94)	287 (9.88)	26 (10.57)
Non-Hispanic White	1043 (33.11)	945 (32.54)	98 (39.84)
Non-Hispanic Black	889 (28.22)	834 (28.72)	55 (22.36)
Other race	504 (16.00)	474 (16.32)	30 (12.20)
PIR (± SD)	2.71 ± 1.66	2.72 ± 1.66	2.57 ± 1.62	.181
Education level (%)				.544
< High school	377 (11.97)	348 (11.98)	29 (11.79)
High school	713 (22.63)	664 (22.87)	49 (19.92)
> High school	2060 (65.40)	1892 (65.15)	168 (68.29)
Marital status (%)				.467
Cohabitation	1813 (57.56)	1666 (57.37)	147 (59.76)
Solitude	1337 (42.44)	1238 (42.63)	99 (40.24)
Alcohol (%)				.229
No	3133 (99.46)	2887 (99.41)	246 (100.00)
Yes	17 (0.54)	17 (0.59)	0 (0.00)
Smoked (%)				< .001
No	1878 (59.62)	1756 (60.47)	122 (49.59)
Yes	1272 (40.38)	1148 (39.53)	124 (50.41)
Diabetes (%)				< .001
No	2856 (90.67)	2653 (91.36)	203 (82.52)
Yes	294 (9.33)	251 (8.64)	43 (17.48)
High cholesterol (%)				< .001
No	2391 (75.90)	2229 (76.76)	162 (65.85)
Yes	2391 (75.90)	675 (23.24)	84 (34.15)
CHD (%)				.098
No	3118 (98.98)	2877 (99.0)	241 (97.97)
Yes	32 (1.02)	27 (0.93)	5 (2.03)
BMI (± SD) (kg/m2)	30.44 ± 8.20	30.03 ± 7.88	35.39 ± 10.02	< .001
Hypertension (%)				< .001
No	2365 (75.08)	2210 (76.10)	155 (63.01)
Yes	785 (24.92)	694 (23.90)	91 (36.99)
Dietary protein (± SD)	82.44 ± 36.59	83.18 ± 36.56	73.70 ± 35.81	< .001
Heart failure (%)				.002
No	3112 (98.79)	2874 (98.97%)	238 (96.75)
Yes	38 (1.21)	30 (1.03)	8 (3.25)
Angina pectoris (%)				< .001
No	3119 (99.02)	2881 (99.21)	238 (96.75)
Yes	31 (0.98)	23 (0.79)	8 (3.25)
Cancer (%)				.017
No	3023 (95.97)	2794 (96.21)	229 (93.09)
Yes	127 (4.03)	110 (3.79)	17 (6.91)
Total dietary energy	2159.00 ± 879.77	2168.48 ± 879.13	2047.08 ± 881.31	.038
Dietary fiber	15.94 ± 8.98	16.04 ± 9.06	14.72 ± 7.92	.027
Dietary fat	87.77 ± 41.24	88.08 ± 41.12	84.03 ± 42.57	.138
Dietary saturated fatty acids	27.98 ± 14.56	28.09 ± 14.41	26.73 ± 16.30	.161
History of cholecystectomy (%)				< .001
No	2916 (92.60)	2842 (97.90)	74 (30.08)
Yes	233 (7.40)	61 (2.10)	172 (69.92)

Mean ± SD for continuous variables: the P value was calculated using the linear regression model; (%) for categorical variables: the P value was calculated using the chi-square test. BMI = body mass index, CHD = coronary heart disease, N = number of participants, PIR = poverty-to-income ratio, SD = standard deviation, U.S. = United States.

3.2. Association between dietary protein and GSD

Table 2 shows the association between dietary protein intake and GSD. Since dietary protein values were found to be non-normally distributed in the study, we performed a skewed distribution transformation (Box–Cox transformation) of dietary protein values to minimize bias and fluctuations in the data. Therefore, we investigated the linear relationship between the Box–Cox transformation of dietary protein and GSD. The results demonstrated a negative correlation between dietary protein intake and GSD incidence across all models, with a particularly strong association observed in the unadjusted and fully adjusted models. After adjusting for all covariates, a 1-unit increase in dietary protein (as measured by the Box–Cox transformation) was associated with a 30% reduction in the incidence of gallstones (odds ratio = 0.70 [0.56, 0.88]) The correlation remained significant in the fully adjusted model (P < .05) after dividing the Box–Cox transformation value of dietary protein into tertiles, with a 76% lower prevalence of cholelithiasis in participants in the highest tertile of Box–Cox transformation values of dietary protein than in those in the lowest tertile of Box–Cox transformation values of dietary protein (odds ratio = 0.24 [0.13, 0.44]). Furthermore, analyses of smoothed curve fits confirmed a negative association between the logarithm of dietary protein and GSD (Fig. 2). Through threshold saturation effect analysis (Table 3), we identified the saturation point of this negative association. When the Box–Cox-transformed dietary protein value was < 9.79 (corresponding actual intake: 143 g/day), the association between dietary protein and gallstones was significantly negative; when the Box–Cox transformed dietary protein value was < 9.79, the association was not significant.

Table 2.

Associations between dietary protein and gallstones.

Exposure	Model 1, OR (95% CI)	Model 2, OR (95% CI)	Model 3, OR (95% CI)
Box–Cox transformed values of dietary protein (continuous)	0.80 (0.71–0.91)	0.90 (0.77–1.05)	0.70 (0.56–0.88)
Box–Cox transformed values of dietary protein (tertile)
T1	1.0 (reference)	1.0 (reference)	1.0 (reference)
T2	0.74 (0.48–1.13)	0.83 (0.53–1.30)	0.57 (0.36–0.91)
T3	0.38 (0.23–0.62)	0.55 (0.32–0.96)	0.24 (0.13–0.44)
P for trend	< .0001	.0083	< .0001

Model 1: no covariates were adjusted. Model 2: age, sex, and race were adjusted. Model 3: sex, age, race, education, marriage, PIR, smoking, drinking, BMI, high cholesterol, hypertension, diabetes, heart failure, coronary heart disease, angina pectoris, cancer, total dietary energy, dietary fiber, dietary fat, and dietary saturated fatty acids were adjusted.

BMI = body mass index, CHD = coronary heart disease, CI = confidence interval, OR = odds ratio, PIR = poverty-to-income ratio.

Figure 2.

Nonlinear correlation between dietary protein and gallstones. Sex, age, race, education, marriage, PIR, smoking, drinking, BMI, high cholesterol, hypertension, diabetes, heart failure, CHD, angina pectoris, cancer, total dietary energy, dietary fiber, dietary fat, and dietary saturated fatty acids were adjusted. The solid and red lines represent the smooth curve fitting between the variables. The blue band represents the 95% CI for the fit. BMI = body mass index, CHD = coronary heart disease, CI = confidence interval, PIR = poverty-to-income ratio.

Table 3.

Threshold effect analysis of dietary protein on gallstones.

Box–Cox transformed values of dietary protein	Adjusted OR (95% CI), P value
Model 1
Fitting by the standard linear model	0.76 (0.64–0.90), .0013
Model 2
Inflection point	9.79
< 9.79	0.73 (0.62–0.87), .0003
> 9.79	1.28 (0.81–2.03), .2957
Log likelihood ratio	0.032

Sex, age, race, education, marriage, PIR, smoking, drinking, BMI, high cholesterol, hypertension, diabetes, heart failure, CHD, angina pectoris, cancer, total dietary energy, dietary fiber, dietary fat, and dietary saturated fatty acids were adjusted.

BMI = body mass index, CHD = coronary heart disease, CI = confidence interval, OR = odds ratio, PIR = poverty-to-income ratio.

3.3. Subgroup analyses

We conducted subgroup analyses and interaction tests based on covariates such as age, sex, ethnicity, diabetes, hypertension, and cancer. The results showed no significant differences in the associations across these subgroups (P for interaction > .05; Table 4), indicating that the association between dietary protein intake and GSD is consistent across the general population.

Table 4.

Subgroup analysis of the association between dietary protein and gallstones.

Subgroup	Gallstones, OR (95% CI)	P for interaction
Gender		.8093
Male	0.74 (0.58–0.95)
Female	0.76 (0.64–0.91)
Age		.4785
≤ 40 yrs	0.73 (0.59–0.89)
> 40 yrs	0.79 (0.65–0.95)
Race		.3234
Mexican American	0.68 (0.50–0.93)
Other Hispanic	0.74 (0.54–1.01)
Non-Hispanic White	0.71 (0.57–0.87)
Hispanic Black	0.81 (0.64–1.04)
Other race	0.96 (0.71–1.31)
Diabetes		.5119
No	0.77 (0.65–0.92)
Yes	0.70 (0.52–0.94)
CHD		.1632
No	0.76 (0.65–0.90)
Yes	0.36 (0.11–1.16)
Hypertension		.2028
No	0.80 (0.66–0.96)
Yes	0.70 (0.56–0.86)

Sex, age, race, education, marriage, PIR, smoking, drinking, BMI, high cholesterol, hypertension, diabetes, heart failure, CHD, angina pectoris, cancer, total dietary energy, dietary fiber, dietary fat, and dietary saturated fatty acids were adjusted.

BMI = body mass index, CHD = coronary heart disease, CI = confidence interval, OR = odds ratio, PIR = poverty-to-income ratio.

3.4. Sensitivity analysis

To rule out the confounding effects of potential mediating variables such as BMI, diabetes, and hyperlipidemia, we conducted further sensitivity analyses after adjusting for these potential mediating variables as covariates. The results showed that the negative association between dietary protein and gallstones remained highly robust, indicating that the core findings were not influenced by potential mediating variables (Table 5).

Table 5.

Associations between dietary protein and gallstones.

Exposure	Model 3, OR (95% CI)]
Box–Cox transformed values of dietary protein (continuous)	0.72 (0.57–0.91)
Box–Cox transformed values of dietary protein (tertile)
T1	1.0 (reference)
T2	0.58 (0.36–0.94)
T3	0.28 (0.16, 0.50)
P for trend	< .0001

Model 3: sex, age, race, education, marriage, PIR, smoking, drinking, hypertension, heart failure, CHD, angina pectoris, cancer, total dietary energy, dietary fiber, dietary fat, and dietary saturated fatty acids were adjusted.

CHD = coronary heart disease, CI = confidence interval, OR = odds ratio, PIR = poverty-to-income ratio.

4. Discussion

The present study revealed a significant negative correlation between dietary protein intake and the prevalence of gallstones in individuals under 60 years old. In individuals under 60 years old, dietary protein intake was negatively correlated with gallstones. When the Box–Cox transformed value for dietary protein intake was < 9.79, there was a significant negative correlation between dietary protein intake and gallstones. Conversely, when this value was > 9.79, this association was not significant. To the best of our knowledge, this study is the first to explore the relationship between dietary protein intake and the occurrence of gallstones. In previous studies, dietary protein intake has been frequently used as an indicator for the assessment of digestive diseases. A randomized controlled trial of 130,859 postmenopausal women demonstrated that the consumption of plant-based protein was inversely associated with the likelihood of developing gallbladder disease,^[31] whereas another meta-analysis suggested that total dietary protein intake may not be associated with pancreatic cancer risk.^[32] Furthermore, many studies have shown that a protein-rich diet is advantageous for patients with cirrhosis in terms of their nutritional status^[33] and is not associated with hepatic encephalopathy. In addition, 1 study showed that high BMI, high dietary fat, and meat intake were strongly associated with GSD.^[34] Another study noted that meat consumption may increase the risk of gallstones in women,^[18] while a Korean case-control study also found that beef and pork consumption was associated with the risk of cholesterol gallstones.^[16]

Our research indicates a significant link between dietary protein and gallstone formation, as gallstones primarily develop in the gallbladder, which derives its components from dietary cholesterol intake.^[35] The dietary effects on gallstones are mediated through multiple pathways, including metabolic abnormalities,^[36,37] obesity,^[38,39] and insulin resistance.^[40,41] Healthy dietary patterns (characterized by skinless chicken, lean beef, and fish) have been shown to significantly reduce the risk of gallstone formation^[42]; whereas dietary patterns that increase the intake of plant proteins have also been shown to increase cholesterol excretion.^[43] Previous studies have confirmed that elevated cholesterol levels are a key factor in cholesterol stone formation.^[6,44] In summary, dietary protein intake has a significant effect on gallstone formation, and understanding these factors can help reduce the risk of gallstones. The results of our study indicate a notable correlation between dietary protein intake and the incidence of gallstones among individuals under 60 years old. This finding is supported by several other studies that have also identified an association between gallstones and age,^[45,46] which may be related to age-related changes in hormone levels and gallbladder function,^[47,48] that in turn affect cholesterol metabolism. In addition, dietary protein intake is inversely associated with the risk of GSD; the underlying mechanisms may involve various aspects of cholesterol and bile acid metabolism, as well as gallbladder motility. Adequate protein intake stimulates the secretion of cholecystokinin, thereby promoting gallbladder contractions and facilitating efficient bile emptying.^[49,50] Furthermore, moderate protein intake promotes the conversion of cholesterol into bile acids and enhances the fecal excretion of bile acids and cholesterol, thereby reducing systemic cholesterol levels. Specifically, high-quality protein may upregulate the expression of CYP7A1 whilst downregulating the activity of HMG-CoA reductase, thereby inhibiting hepatic cholesterol synthesis.^[51,52] Conversely, inadequate protein intake reduces endogenous bile acid production and bile flow, thereby promoting cholesterol precipitation and the formation of gallstones.^[53] It is worth noting that this inverse relationship exhibits a saturation effect; no further significant reduction in risk was observed when protein intake was ≥ 143 g/day. This phenomenon can be partly attributed to the fact that high protein intake is often associated with a Western-style diet characterized by high fat and low dietary fiber^[54–56]; this dietary pattern offsets the protective effects of protein and eliminates the additional statistical significance of the association between protein intake and gallstone risk.^[56–58] Furthermore, given that cholesterol metabolism involves multiple regulatory steps, the liver maintains a steady-state balance of cholesterol, which may limit the further stimulatory effect of excessive protein intake on bile acid synthesis and cholesterol excretion.

The diagnosis of gallstones relies on self-reporting, which may be subject to information bias. We identified a saturation point in the association between dietary protein and gallstones, and moderate protein intake may serve as a reference for the prevention of gallstones in adults under 60 years of age. Since the present study was a cross-sectional study, a causal association could not be determined. Further clinical cohort studies and studies on mechanisms related to gallstones are needed to determine whether adjusting dietary protein intake will reduce the prevalence of gallstones in future clinical practice. The NHANES database does not provide stratified data on dietary protein sources; consequently, it is not possible to analyze in greater detail the differing effects of plant-based and animal-based proteins on the risk of gallstones. Future research involving cohort studies that include information on protein sources is required to explore in greater depth the differences in the association between proteins from different sources and the incidence of gallstones. Twenty-four–hour dietary recalls reflect only recent intake rather than long-term dietary exposure throughout the entire period of gallstone formation. Although we used the average of 2 nonconsecutive 24-hour dietary recalls (spaced 3–10 days apart) to minimize diurnal fluctuations, and dietary protein intake patterns in adults are generally stable over the medium term, this measurement method still fails to fully reflect habitual intake over the multi-year timeframe of gallstone formation. This limitation precludes drawing causal inferences regarding the temporal relationship between protein intake and gallstone risk. Furthermore, the NHANES database does not contain detailed information on the causes of cholecystectomy, cholecystitis, disease severity, or specific indications for treatment. This limits our ability to further explore the association between dietary protein and the risk of cholecystitis, as well as the risk of symptomatic gallstones requiring treatment. Future analyses should be based on studies that include comprehensive clinical and imaging data. In addition, 24-hour dietary recalls are subject to certain measurement errors; future studies could employ longer-term dietary assessment methods to further validate these findings. Failure to include all covariates that may influence the association between dietary protein and gallstones is also a limitation. However, the use of a large nationally representative database to assess dietary quality was a major strength of this study.

5. Conclusion

In conclusion, a Box–Cox-converted dietary protein value below 9.79 (corresponding to a daily protein intake of 143 g/day) was significantly and negatively associated with the risk of gallstones; otherwise, the association was not significant. This finding may provide guidance for future dietary protein intake.

Acknowledgments

We would like to thank all participants in this study.

Author contributions

Conceptualization: Weihui Liu, Jian Wu.

Data curation: Weihui Liu, Lishan Bai, Jian Wu.

Formal analysis: Jing Tang, Jian Wu.

Funding acquisition: Lishan Bai.

Investigation: Jing Tang, Yi Zhou.

Methodology: Yuanming Li, Biqian Yang, Lishan Bai, Yi Zhou.

Project administration: Biqian Yang, Yi Zhou.

Resources: Weihui Liu, Jian Wu.

Software: Yuanming Li, Biqian Yang, Lishan Bai.

Supervision: Weihui Liu, Biqian Yang, Jian Wu.

Validation: Weihui Liu, Biqian Yang, Jian Wu.

Visualization: Weihui Liu, Jian Wu.

Writing – original draft: Yuanming Li, Biqian Yang, Jing Tang.

Writing – review & editing: Yuanming Li, Weihui Liu, Jian Wu.