Consumption of heme iron: A major factor in pigment gallstone formation

Objective: To study the various risk factors responsible for pigment gallstone formation. As there is not any reported medicine which can dissolve pigmented gallstone. Design: Study of 526 cases of cholelithiasis and 526 control cases was done. Query forms were filled with various risk factors like age, gender, BMI, diabetes, genetics, diet, smoking, etc. Collected data and hypothesis were statistically analysed. ICP-OES was done to know the elementary composition of gallstone. Statistical Analysis: Univariate and multivariate were done using SPSS to conclude the risk factors for gallstone disease. Chi-square was applied for the relation between types of stone and age while ANOVA and post hoc ANNOVA, Least significant difference (LSD) were use for the analysis of variance between prevalence of different types of stone with risk factors. Results: In our study, non-veg diet and ground water rich in iron beyond the permissible limit, as a drinking source were at higher risk factor for the gallstone disease (p<0.0001). Non-veg diet as a rich source of heme iron and supplementation of iron from drinking water source may led to pigmented gallstone formation. Conclusion: Controlled dietary habit and use of supply water may reduce the chance for pigmented gallstones.


Sample Collection
Case control studies of 1052 cases were done. Of these 526 cases were of cholelithiasis and 526 (age-25-55yrs) non-gallstones, who were taken as control. Gallstones of the patients were collected, washed with normal saline and air dried. Gallstones were classified on the basis of their cross section and morphology 34 into three categories: Cholesterol stones, pigmented stones and mixed stones.

Measures for probing risk factors for gallstone formation:
Query form of the gallstone patients were filled which included age, gender, weight, height, source of drinking water, dietary habit, genetic relation, sugar level or diabetic status and smoking habit etc. BMI (Body Mass Index) were calculated as weight and height ratio.

Collection of data of Drinking Water
Data of elementary constituents of ground water of 21 blocks of Jharkhand, India were collected from State Level Water Laboratory, Drinking Water and Sanitation Department, Ranchi, Jharkhand.

Elementary analysis of Gallstone
Elementary analysis of human gallstones was determined by ICP-OES (Inductively Coupled Plasma-Optical Emission Spectroscopy). This is an analytical technique used for the detection of trace metals. It is a type of emission spectroscopy that uses the inductively coupled plasma to produce excited atoms and ions that emit electromagnetic radiation at wavelengths characteristic of a particular element. The intensity of this emission is indicative of the concentration of the element within the samples. For the elementary analysis, 0.2 gm of gallstones were digested with a mixture of HNO3 (5 ml) and H2O 2(3 ml) in teflon stub 35 . After digestion the samples were diluted to 100 ml with deionised water. Then the samples were subjected to ICP-OES.

Statistical analysis
Univariate analysis as well as multivariate analysis was used to correlate between independent variants with the prevalence of gallstone disease. Chi-square applied for the relation between types of stone and age while ANOVA and post hoc ANNOVA Least significant difference (LSD) is use for the analysis of variance between prevalence of different types of stone with non-veg diet and source of drinking water. For all the analysis SPSS 16 software was used.

Results
The study included 526 gallstone patients and 526 non-gallstone persons. Out of 526 patients 384 were female and 142 were male. Females were 2.7 times more prone to gallstone. Out of 526 cases dealt, only 459 gallstones were collected. Rest of the 67 stone were either sucked along the suction pressure during surgery or fully crushed. Out of these, pigmented gallstones (238/459) were predominant followed by Cholesterol (104/459) and Mixed gallstones (117/459). Though pure cholesterol gallstones were not seen, as some pigmentation were observed in their inner surface as shown in Figure: 1. Like others study dietary factors were found dominating one in Jharkhand region also. Individuals having non-veg diet (O.R=2.3204-3.8717; p<0.0001) and ground water (O.R=2.1345;1.6677-2.7320; p<0.0001) as a drinking water source were at higher risk. The Sources of drinking water were also seen to be related with the chemical constituent of gallstones. Accor ding to the data from Level Water Laboratory, Drinking Water and Sanitation Department, Ranchi, Jharkhand of 21 blocks of Jharkhand, shows that the iron content of ground water of Jharkhand is very high beyond the permissible limit (0.3ppm-1ppm). Mean±SD iron content of ground water of these 21 blocks were found to be 2.831±0.5 ppm and the ICP-OES (Perkin Elmer, USA; Optical 2100DV) results of pigmented and Cholesterol gallstones shows the mean±SD iron content of 0.83±0.12 ppm and 0.514±0.09 respectively. Pigmentations in cholesterol ( Figure: 1) gallstone may be due to presence of iron which may get absorbed from drinking water source (F= 22.29; p<0.0001; Chol vs Pigmented and mixed; p<0.0001 ). Average BMI for diseased and control were 22.34 and 21.57 respectively, which is a normal range as per WHO (World Health Organization) Expert Committee. Thus BMI was not an influencing factor for gallstone formation in our study. Consumption of beverages like tea, coffee or combination of tea and coffee by gallstone patient and control cases were also compared. 293, 121 and 147 patients were having tea, coffee or combination of both respectively in their routine life while 302, 115 and 153 control cases were having tea, coffee or combination of both respectively.

Discussions
Previous studies stated BMI 36 , ageing 17 , diet 7 , Gender 19 are the major risk factors for gallstone formation. Whereas diabetes, genetics, smoking, beverages like tea and coffee are still controversial 37 . Of these factors, diets have major effect in influencing the types of gallstones as well as renal gallstone 38 formation. Additionally the composition of gallstones is greatly affected by the elementary constituents of the sources of IJBR (2014) 05 (01) www.ssjournals.com drinking water. Shareef et al, 2008 found higher concentration of calcium in gallstone as well as in the source of drinking water in Erbil (Iraq) 39 . In our case based study also, non-vegetarian taking ground water as drinking water sources are at higher risk factor with types of gallstones on univariate (Table: 1) as well as multivariate analysis (Table: 2). From our study we found that 72% of the cases were regularly having nonvegetarian diet. Non-vegetarian food (meat, poultry and fish) are the good source of heme form of iron 40,41 which is greatly associated with the gallstone disease in comparison to nonheme iron 42 . Heme iron breakdown to form bilirubin 43 which precipitates to initiate pigmented gallstone formation. The gallstone sample collected by us also shows the variance of pigmented gallstone with relation to non-veg diet as shown in Table 3.  Though patients were also taking non-heme iron in their diet but supplementation of iron abate the absorption of non-heme iron 44 . The source of drinking water was supplementing the iron in those patients. According to the data from Level Water Laboratory, Drinking Water and Sanitation Department, Ranchi, Jharkhand, mean iron content of ground water was 2.831±0.5 ppm which is far higher than the permissible limit (.3ppm-1ppm). Such a high content of body iron is regulated primarily by absorption, since human have no physiological mechanism by which excess iron is excreted. Excess of iron remains within shed intestinal cells, in bile, urine and even in small amount in sweat, nails and hairs 45 . Accumulation of iron in bile may expedite the iron content in gallstones. ICP-OES result also show the presence of iron in gallstones. The mean iron content was found to be 0.514±0.09ppm, 0.72±0.1and 0.83±0.12ppm in cholesterol, mixed and pigmented stones respectively. This proportion of iron in cholesterol stone is because of some pigmentation on their inner surface as shown in Figure: 1 which may be from the drinking water source (p<0.0001).
With increasing age absorption of iron decreases, the increased iron is accumulate in the bile, may expedite the pigmented gallstone in higher age group but no such relation with the types of stone were seen biostatisticaly in our study (χ 2 =6; p=0.199). This study is contradicted by Diehl et al, 1995, who found cholesterol stone in the patients with age below 40yrs and pigmented ston e in the age of more than 40yrs 46 . Also Kurtin et al, 2000 stated that the cholesterol content of stone steadily decreases after the age of 50 47 . Genetic is related with gallstone disease, as its frequency varies between ethnicities. But in this study we have not found such an association with gallstone formation (p=0.1333). Likewise, cigarette smokers have lower level of HDL in comparison to non-smokers, a risk factor for gallstone formation in some studied 48,49 but not in all 18,37 which is supporting our analysis (p=0.0732).

Conclusions
Use of heme iron rich diet and high concentration of iron in drinking water may predispose pigmented stone formation.