**1. Introduction**

The gallstones (**Figure 1**) are hard, pebble-like pieces of material, usually made of cholesterol or bilirubin, that develop in the gallbladder [1]. These stones are formed due to various disorders. Five defects primarily play critical role in pathogenesis of cholesterol stones Viz lithogenes and genetic factors; hepatic hypersecretion of cholesterol; rapid phase transition of cholesterol in bile; impaired gallbladder motility; intestinal factors involving absorption of cholesterol, slow intestinal motility and altered gut microbiota.

Gallstones affect about 15% of United States population. About 10–20% of the United States population will get gallstones at some point in their life time and females are more affected than men [2].

**Figure 1.** *Cholesterol gallstones and gallbladder after cholecystectomy.*

Gallstones can cause biliary colic, cholecystitis, pancreatitis, empyema Gallbladder, perforation of gallbladder, cholangitis, bile duct obstruction [3], and cancers (gallbladder [4], colorectal [5, 6], pancreatic [7]). In 85% of patients with gallbladder cancer, gall stones are present [8]. Gallbladder cancer is a very fatal cancer with average survival of 6 months [9] and 5-year survival rate of 5% [8]. This bad prognosis is because of silent features. An increased mortality from cardiovascular disease and cancer was found to be associated with gallstones by an 18 year national study [10]. Asymptomatic cholelithiasis are present in 80% of cases and most of these are incidentally found when the patients are under investigation for other ailments [11]. In general, 10–25% of asymptomatic cases may develop symptoms during the patient's lifetime [12]. The estimated cost for gallbladder diseases was 6.5 billion dollar annually in United States only in 2002 [13].

There are three types of gallstones; first and most common type is cholesterol stone. Black and brown pigmented stones are the other two types of gallstones. The prevalence of various types of gallstones in the western world is: 75% of gallstones are cholesterol stones, 20% are black pigment stones, and 5% or less are brown pigment stones. Cholesterol and black pigment stones are formed exclusively in gallbladder in a sterile medium, while as brown pigment stones are formed everywhere in the biliary tree owing to the anaerobic bacterial infection. Stasis of bile is an essential component in the formation of gallstone or bile duct stone formation [14]. In underdeveloped countries parasitic infection of biliary tree is associated with brown pigment stones [15].

Physical-chemical origin is the basis of pathogenesis of sterile stones (cholesterol and black pigment stones) [16]. There is an alteration in lipid and lipo-pigment composition which results in the formation of stones. In cholesterol and black pigment stones the major component is cholesterol monohydrate crystals and calcium hydrogen bilirubinate respectively. During the long stay in gallbladder, the black pigment gets degraded and polymerized by free radicals and helps in the formation of black pigment stones while as in the brown pigment stones the main mechanism is infectious where the enzymatic hydrolysis of biliary lipids by anaerobic bacterial enzymes produces supersaturated long chain fatty and deconjugated bile acids [14, 17].

#### **2. Risk factors**

Gallstone formation occurs as a result of interaction between genetic and environmental factors in which few are unmodifiable and unaltered as age and genetic makeup. The traditional risk factors for gallstones are four "F" female, fertile, fat, forty and some have added fifth "F" for fair skin [18].

Various intrinsic and extrinsic risk factors for the causation of different types of gallstones are genetic factors, advanced age, female gender, parity, ethnicity, rapid weight loss, different medications (oestrogen replacement therapy, oral contraceptive pills), total parenteral nutrition [19], obesity, westernized diet, Type 2 diabetes mellitus, metabolic syndrome [20], dyslipidaemia, hyperinsulinemia [21], increased enterohepatic circulation of bilirubin [22], defective gall bladder motility [23] as shown in **Table 1**.

#### **2.1 Age and genetics**

Age is an important factor for the stone formation, with increase in age enzyme 7 α hydroxylase (Limiting enzyme for bile acid synthesis) activity decreases which increases the saturation of cholesterol in bile and hence increasing the chances of stone formation [24].


**Table 1.**

*Risk factors for gallbladder stone diseases.*

Various genes are linked with increased susceptibility of gallstones like Lith genes identified in mouse models [25]. Two variants of ATP binding Cassette transporters ABCG5-R50C and ABCG8-D19H [26]; 3 variants of Farnesoid X Receptor gene (FXR) (rs 35724, rs 11110358, rs 11110386) [27]; polymorphism of apolipoprotein E4 allele [28]; mucin genes [29]; fibroblast growth factor receptor 4 (FGFR4) [30], polymorphism in CCK 1-R gene [31] are linked with cholelithiasis. The genetic factors are responsible for 25–30% of symptomatic gallstones as inferred from twin studies [32]. Gene expressions are affected by environmental factors and geneenvironmental interactions through epigenetic mechanisms [33] also involving fat storage and insulin resistance [34]. These factors primarily include micro RNAs [35] Viz. 114 miRNA [36], miR-122 [37].

### **2.2 Gender and ethnicity**

Females are having a higher risk factors due to various reasons for cholelithiasis such as higher oestrogen levels naturally [38], multiparity [39] and ingestion of oral contraceptives containing oestrogens [38]. Females also tend to undergo cholecystectomy procedure more than males [40].

Ethnicity plays an important non-modifiable role in cholelithiasis as few ethnic groups are having higher incidence of having gall stones as in North American Indians (73% among women more than 30 years old within Prima Tribe) [14], American Indians (men 29.5%, women 64.1%) [41]. Prevalence of Gallstone and Gallbladder Cancer are co-related and effects certain indigenous population like South America & North India, especially younger population [42].

Risk of deaths from gallbladder cancer and other malignancies has been observed to be seven times more in tribal members with gallstones [43].

#### **2.3 Lipid profile and diet**

The co-relation between lipids and cholesterol stone formation is complex, multi factorial and is dependent on other factors also as few studies are in favour and few showing inverse relationship [14]. Few studies show high LDL [44], high cholesterol [45], low HDL [46] are associated with higher gall stone formation which seems to be evident but other studies show there are having inverse relationship with gallstone formation like low HDL, high cholesterol and high LDL [14].

Cholesterol gallstones are associated with western diet [47]. Multiple studies have shown high carbohydrate intake couples with high glycemic load [48], chronic hypernutrition [49] fibre depleted diet [50] are associated with higher risk for cholelithiasis in process which are associated with decreased risk are intake of in saturated fat [51], coffee [52], fibre [53], fish oil [54], calcium [55], ascorbic acid [56], fresh fruits and vegetables [57] and nuts [51].

#### **2.4 Obesity and weight loss**

Obesity is itself risk factor for cholelithiasis. It has been shown that an obese women (BMI ≥ 30 kg/m2 ) has two fold and morbidly obese women (BMI ≥ 45 kg/m<sup>2</sup> ) has seven fold higher risk for cholelithiasis as compared to lean women (BMI < 25 kg/m<sup>2</sup> ) [58].

Rapid weight loss i.e., >1.5 kg/week and or loss of body weight >25% [59] are risk factors for cholelithiasis which usually occur after bariatric surgery. Weight loss has also been found to reduce risk of gallstones except when rapid loss occurs.

### **2.5 Physical activity**

Physical activity decreases risk for cholelithiasis [60] as it improves hepatobiliary funtions, gut motility [61], increasing HDL [62], improves insulin release and plasma triglyceride levels [63]. An endurance exercise (cycling and running) 5 times per week for 30 minutes daily decreases risk by 34% for cholelithiasis [64] or 2–3 h/ week of recreational exercise decrease risk by 20% for cholecystectomy [60]. While as reduced physical activity increases risk for cholelithiasis [64]**.**

#### **2.6 Diseases**

Various diseases increase the risk for cholelithiasis as these work by different mechanisms like causing abnormal gallbladder motility, malabsorption of bile salts, decreased bile salt synthesis [65], increasing bile cholesterol saturation [66], supersaturation of bile and increased hepatic cholesterol secretion [67]. Different diseases increasing risk for cholelithiasis viz. metabolic syndrome, dyslipidaemia, diabetes (2–3 fold [68]), insulin resistance or hyperinsulinemia [69], chronic hepatitis C virus [66], liver cirrhosis (25–30% increased risk [65] and Chron's disease [70].

#### **2.7 Alcohol and smoking**

Both these habits have controversial results from various studies some favouring and others refuting association with cholelithiasis.

Men consuming alcohol 0–20 g per day have higher risk than those who consume 20–60 g per day [24]. But few studies show severe alcohol abuse in itself and also led to chronic cirrhosis (pigment stones), which is an independent risk factor for cholelithiasis [71].

Cigarette smoking is another risk factor for gallstones among woman [72], while as few studies refute these claims show no association with gallstones [73].

#### **2.8 Intestinal absorption of cholesterol**

An imbalance between absorption and synthesis of cholesterol i.e., increased biliary cholesterol secretion from high dietary cholesterol and decreased bile acid synthesis and pool, all driving bile supersaturation [22]. High cholesterol diet and high intestinal cholesterol absorption are two independent risk factors for gallstones [74], regulated by many factors like expression of sterol transport protein [75].
