Animal and Vegetarian Protein Sources in Relationship with the Risk of Biliary Stone; A Case-Control Study from Iranian Adults

Previous studies suggested that protein has a protective effect on the formation of biliary stones. The aim of the current study is the comparison between the effect of animal protein and plant protein on the formation of biliary stone. One hundred and ten cases who had the history of biliary stone disease and 230 controls who were normal in terms of biliary diseases enrolled in this study. Food frequency questionnaire was used for the nutritional assessment; moreover, demographic and anthropometric data, in addition to habitual history and comorbidities were collected by a questionnaire. Results have a signicant negative on


Introduction
Biliary stones are one of the most common disorders of the biliary system in the world with a 15-20% prevalence rate in western countries 1 − 4 . It is also one of the most common diseases affecting emergency-room patients with epigastric pain, nausea, vomiting, abdominal pain, and loss of appetite 5 . The pathogenesis of biliary stones is believed to be multifactorial and probably develops from interactions between several genetic and environmental factors such as age, gender, ethnicity, family history, obesity rapid weight loss and, pregnancy 6 − 8 .
One of the modi able risk factors for biliary stones is nutritional intake 9−11 .Dietary proteins have an in uence on biliary cholesterol density and biliary stone formation 12 − 14 . Some of the studies suggest that plant protein intake is associated with lower risk of biliary stones in comparison to animal protein intake 15 16 . In addition, some of studies have observed no signi cant association between protein intake and biliary stones 17 18 .
Assessing the dietary habit in biliary stones patient can determine the relationship between protein intake and biliary stones 19 . The purpose of the current study was to determine the association between plant and animal protein intake with the risk of biliary stone.

Study Design
This case-control study was conductedin Research Institute for Gastroenterology and Liver Diseases of Taleghani hospital (Tehran, Iran) from November 2017 to October 2018. Our samples consisted of 110 cases (aged 21-91 years) who had the history of gallstone or common bile duct (CBD) stone which was con rmed by ultrasonography in a 6 months' period before the presentation to gastrointestinal disease clinic. Furthermore, the control group consist of 230 subjects (aged 23-84 years) who admitted to the other wards of the hospital and did not have the history of biliary stones. Moreover, controls were matched to cases based on age (± 5 years) and sex.

Nutritional Assessment
In order to assess the dietary consumption of samples during 1 year before biliary stone determination in the case group or hospital admission in the control group, a validated food frequency questionnaire (FFQ) was used 20 . Researchers asked participantsto explain the frequency of their dietary consumption on a daily, weekly, monthly or yearly manner. After that, dietaryintakeswereconverted to daily frequency for each group of protein consumption.

Data Collection
Demographic data, anthropometric measurements, physical activity, history of smoking, history of alcohol consumption and comorbidities were collected by a questionnaire. Physical activity was assessed by using a validated questionnaire as metabolic equivalents hour/day (METs h/d) 21 .
Furthermore, weight measurement was done in standing position by digital scales (Soehnle®, Berlin, Germany) with an accuracy of 100 g. In addition, height was measured using a tape-meter which xed to a wall with an accuracy of 0.5 cm. BMI was calculated by dividing the weight by the square of height in meter.

Statistical Analysis
Statistical analysis was conducted on the data by using SPSS software version 16 (SPSS Inc., Chicago, Illinois). Baseline characteristics, biochemical parameters and dietary intakes of study participants between case and control group were compared t-test or Mann-Whitney for quantitative variables and Chisquare test for qualitative variables. Logistic regression was used to calculate the odds ratio of biliary stone disease as the dependent variable in relation to the animal and plant protein as independent variables in 4 models: Crudemodel, model2(adjusted for energy intake and physical activity), model 3 (further controlled for body mass index and history of diabetes) and, model4(additionally adjusted for dietary cholesterol and dietary ber). The odds ratios of the outcomes were determined with 95% con dence interval.

Ethical Consideration
The protocol of study was approved by Research Institute of Gastroenterology and Liver Diseases Ethics Committee (IR.SBMU.RIGLD.REC.1396.159).

Results
Baseline characteristics, biochemical parameters and dietary intakes of study participants were shown based on case and control group (Table 1). Individuals in the case group as compared to those in the control group were higher in total energy intake and prevalence of type-2 diabetes and lower in physical activity, protein intake, dietary cholesterol, and dietary ber (p-value < 0.05). Dietary intake of animal and plant protein base on case and control group for men, women and both sex were shown in Table 2. Men in the case group as compared to men in control group consumed higher animal protein and lower plant protein (p-value < 0.05). for women there was no signi cant differences between case and control groups. When both sex considered, case group as compared to control group consumed lower plant protein (p-value < 0.05).  Table 3 shows the odds ratios for the biliary stone disease for male, female and both sex together in four models. In the crude model, there was a signi cant relationship between biliary stone disease and animal protein intake for male (OR: 1.03, 95% CI = 1.01-1.05) and both sex together (OR: 1.01, 95% CI = 1.00 -1.01). In fact, patients with higher consumption of animal protein intake were signi cantly more likely to have the biliary stone disease. In model 2 (adjusted for energy intake and physical activity), model 3 (further controlled for, body mass index and history of diabetes), and model 4 which were adjusted for (additionally adjusted for dietary cholesterol and dietary ber), these relationships were signi cant for men, women, and both sex (Table 3).   Table 3).

Discussion
In this study, nutritional habits, physical activity and protein consumption were investigated on the incidence of biliary stones. Lower rate of physical activity, lower protein intake, ber intake and dietary cholesterol were associated with higher risk of biliary stone. In addition, energy intake, and the rate of type 2 diabetes were higher in biliary stone patient.
Many studies have addressed the association of dietary patterns with biliary stones; in the study of Jessri et al, the rate of biliary stone was signi cantly lower in the patient with healthy nutrition in comparison to unhealthy nutrition 9 . In addition, in the study of Goktas et al, the rate of biliary stone was signi cantly higher in patients who consumed liquid oil and did not consume milk 22 . Furthermore, Ortega et al suggested that the prevalence of biliary stone is signi cantly higher in patients with high energy intake, high intake of fat, low ber intake and low rate of physical activity 23 . Moreover, in the study of Park et al, the rate of cholesterol biliary stone had a direct association with the high intake of lipid, meat and fried food 24 . These results indicate the role of dietary pattern in the formation of biliary stone which is in line with the current study.
In clinical investigations, high plasma level of triglyceride and low plasma level of HDL-C are correlated with higher risk of biliary stones 25  Our study results suggest that the vegetable proteins are associated with lower risk of biliary stone compared to animal proteins. These results are associated with previous studies. Lander et al suggested that plant proteins are signi cantly related to lower risk of gallstone disease in post-menopausal female 11 ; moreover, in the study of Tsai et al the risk of cholecystectomy due to biliary stone was inversely associated with vegetable proteins 13 .

Conclusion
Dietary habit is associated with biliary stone formation. The proteins have a negative effect on the formation of biliary stones. In addition, vegetable proteins have a signi cant effect on biliary stone disease compared to animal proteins.

Limitation
The main limitation of this study was the geographical issue; we evaluate the patients who were in Tehran, the capital of Iran. It will be better to conduct feature study as an epidemiologic evaluation with larger samples. Abbreviations