Extensive Quantitative Analysis of Gallstones

Background: The chemical composition of gallstones is essential to study aetiopathogenesis of gallstone disease. Objective: To determine the composition of gallstones in a Sudanese population. Patients and methods: It describes an extensive quantitative analysis of gallstones from patients presented with symptomatic gall stone disease and treated by cholecystectomy after the acceptance of the pre-given informed consent in Khartoum teaching hospital in the period between Jan 2010 and Dec 2010. Using a pretested questionnaire data collected from and analyzed statistically by SPSS computer program version 21. Results: Data are analyzed from 94 patients (six males and 88 females). Cholesterol stones showed a significantly higher cholesterol content than pigment stones (p = 0.0042), though not significantly higher than mixed stones. Their phospholipids content and inorganic phosphates were higher than in the other types of stones and oxalate content was significantly elevated in comparison with mixed stones (p = 0.0402). In mixed stones, the cholesterol, bile acids, and bilirubin were intermediate between cholesterol and pigment stones, whereas triglycerides were significantly more than pigment stones (p = 0.0007). Bilirubin (p = 0.0021) and bile acids (p = 0.0016) were significantly higher than cholesterol stones (p = 0.0001) and (p = 0.0001) respectively. However, they contained the lowest amounts of sodium, potassium, magnesium, and oxalate. In pigment stones, bilirubin was significantly higher (p = 0.0001) than both groups. Conclusion: Collaborations between surgeons, nutritionists, biochemists, and physicians should be stimulated in future studies to define the different types of gall stones in different areas in Sudan and the relevancy of such types with diets’ traditions.


Introduction
Gallstones, complex biomineralized deposits formed in the gallbladder, are still a major health problem all over the world [1].When symptoms arise, cholecystectomy is recommended [2][3][4].
Cholelithiasis is common with the incidence ranging from 10% to 20% of the world population, 11% of the general population of the United States (US) [5].The incidence is four times higher in women than in men with the high prevalence among younger age group [6].
On the basis of their composition, gallstones can be divided into the three types: Cholesterol stones (CS) that vary in colour from light-yellow to dark-green or brown and are oval, and they must have at least 80% cholesterol by weight (or 70%, according to the Japanese classification system); Pigment stones (PS) which are small, dark stones made of bilirubin and calcium salts that are found in bile, and they contain less than 20% of cholesterol (or 30%, according to the Japanese classification system); and Mixed stones (MS) which typically contain 20% -80% cholesterol (or 30% -70%, according to the Japanese classification system) [7].By defining the pattern and type of the gallstone, we will open new windows for further investigations in the future helping in implementing the non-surgical interventions measures.The aim of the current study is to determine the prevalence of different types of gallstone, and to explore the composition of the stones in symptomatic gallstone disease (GSD) in Sudan.

Patients and Methods
A cross-sectional study was conducted in Khartoum teaching hospital, Sudan (January 2010 and December 2011).Khartoum is a capital city of the Sudan with more than six million inhabitants [8].All patients with symptomatic calcular cholecystitis that confirmed ultrasonographically and treated by cholecystectomy were involved into our study after acceptance of the informed consent.Before commencing the study ethical approval was obtained from ethical committee Sudan Medical Specialization Board.Using predesigned questionnaire, general physical examination was performed to participants and then enrolled into an appropriate investigation.
The stones were divided into 3 groups depending upon their colors: pale yellow and whitish stones as cholesterol calculi, black and blackish brown as pigment calculi and brownish yellow or greenish with laminated features as mixed calculi.Then, they were powdered in a pestle and mortar and dissolved in different solvents depending upon the type of ionic constituents that would be analyzed.To determine calcium, oxalate, inorganic phosphate, magnesium, chloride, soluble proteins, triglycerides, iron, copper, sodium and potassium, 30 mg stone powder was dissolved in 3 ml I N HCl (one normal HCl) in a graduated 10 ml tube and its final volume was made up to 10 ml with distilled water.The tube was kept in boiling water bath for 1 hour.Total oxalate, inorganic phosphate, magnesium, and chloride all were estimated by a suitable colorimetric method, while iron and copper were estimated by atomic absorption spectrophotometer, sodium and potassium by flame photometer.The dissolved stone solutions were stored at 2˚C -8˚C, when not in use.
The collected data was spread on master sheet, entered computer and managed statistically using SPSS computer program version 21.Numerical data was expressed as a mean ± SD.The stone-types were compared between groups using the Chi-square test.The confidence level was set at 95% CI and p values less than 0.05 were statistically considered significant.
Cholesterol stones (CS) were bigger in size as compared to mixed stones (MS) and pigment stones (PS) Table 1.The incidence of gallstones was highest in age group 41 -50 years (43.6%),Table 2.

Determination of Metabolites
The cholesterol, bilirubin, bile acids, fatty acids, triglycerides, phospholipids and soluble protein were found in all 94 gallstones.
Cholesterol stones (CS) had the highest composition of cholesterol, while mixed stones (MS) had a high content of triglycerides and pigment stones (PS) were composed mostly of bilirubin (Tables 3 and 4).
The mean total cholesterol concentration was significantly higher in cholesterol calculi (713 mg/gm), compared to pigment (421 mg/gm) (p = 0.0042) and in mixed stones (637 mg/gm) as compared to pigment stones (p = 0.0261).However, there was an insignificant difference between total cholesterol content of cholesterol Size (cm, mean) 0.9 × 0.9 0.5 × 0.6 0.7 × 0.8 stones and mixed stones (p = 0.1032).The mean triglyceride content was highest in cholesterol stones (69.3 mg/gm) and lowest in pigment stones (32.8 mg/gm).The triglyceride content was significantly higher in cholesterol calculi (p = 0.0371) and mixed calculi (p = 0.0183) as compared to pigment calculi.However, the difference was insignificant between cholesterol calculi and mixed calculi (p = 0.1303).
The mean fatty acid content was highest in cholesterol calculi (21.71 mg/gm) and lowest in mixed calculi (13.82 mg/gm).The difference of fatty acid content was significant between cholesterol calculi and mixed calculi (p = 0.0216) and between cholesterol calculi and pigment calculi (p = 0.0093) but insignificant between mixed and pigment calculi (p = 0.1017).
The mean total bilirubin concentration was highest in pigment stones (4 mg/gm) and lowest in cholesterol stones (0.3 mg/gm).It was significantly higher in pigment stones compared to cholesterol stones and mixed stones (p = 0.0001) and insignificantly higher in mixed stones as compared to cholesterol stones (p = 0.0021).
The mean bile acid content in pigment stones (25 mg/ gm) was comparatively higher than that in cholesterol calculi (7.2 mg/gm) and mixed calculi (23 mg/gm).There was a significant difference between bile acid content of pigment calculi and cholesterol calculi (p = 0.0001), and between mixed calculi and cholesterol calculi (p = 0.0012).However, the difference was insignificant between pigment calculi and mixed calculi (p = 0.7359).
The mean phospholipids content was highest in cholesterol stones (6.9 mg/gm) and lowest in mixed stones (5.8 mg/gm).There was a significant difference between phospholipid content of cholesterol calculi and mixed calculi (p = 0.0095), and between cholesterol calculi and pigment calculi (p = 0.0201).However, the difference was insignificant between mixed calculi and pigment calculi (p = 0.3978).
The soluble protein content was highest in pigment calculi (98.62 mg/gm) and lowest in mixed calculi (10.03 mg/gm).The protein content was significantly higher in pigment calculi as compared to mixed calculi (p = 0.0001) and cholesterol calculi (p = 0.0091).However, the difference was insignificant between cholesterol calculi and mixed calculi (p = 0.0983).

Determination of Cations and Anions
The sodium, potassium, calcium, magnesium, copper, iron, inorganic phosphate, oxalate and chloride were found in all 94 gallstones Tables 5 and 6.
The concentrations of different ions also varied with the type of stone.The mean calcium content was highest in pigment calculi (22.8 mg/gm) and lowest in cholesterol calculi (9.6 mg/gm).It was significantly higher in pigment calculi as compared to cholesterol calculi (p = 0.0001) and in mixed calculi as compared to cholesterol calculi (p = 0.0018).However, there was an insignificant    There was a significant difference between cholesterol calculi and pigment calculi (p = 0.0001) and cholesterol calculi and mixed calculi (p = 0.0081) but no significant difference between mixed and pigment calculi (p = 0.1073).
The mean sodium content was highest in pigment calculi (3.8 mg/gm) and lowest in mixed calculi (1.3 mg/ gm).The difference in sodium content was highly significant in pigment calculi as compared to mixed calculi (p = 0.0010) and cholesterol calculi (p = 0.0027) but insignificant between cholesterol calculi and mixed calculi (p = 0.9101).
The mean potassium content was highest in pigment calculi (0.73 mg/gm) and lowest in mixed calculi (0.38 mg/gm).The potassium content was significantly higher in pigment calculi as compared to mixed (p = 0.0391).The difference of potassium content of cholesterol calculi and mixed calculi (p = 0.0520) and between pigment and cholesterol calculi (p = 0 .0975)was however insignificant.
The magnesium content was highest in pigment calculi (10.2 mg/gm) and lowest in mixed calculi (9.1 mg/gm).However, there was no significant difference between magnesium content of three calculi (p > 0.05).
The mean chloride content was highest in pigment calculi (36.1 mg/gm) and lowest in cholesterol calculi (17.8 mg/gm).The chloride content was significantly higher in pigment calculi as compared to cholesterol (p = 0.0012) and mixed calculi (p = 0.0424) but insignificant between cholesterol calculi and mixed calculi (p = 0.0847).
The copper content was highest in cholesterol calculi (0.72 mg/gm) and lowest in pigment calculi (0.348 mg/gm).However, there was no significant difference between copper content of three calculi (p > 0.05).
The iron content was highest in mixed calculi (1.09 mg/gm) and lowest in cholesterol calculi (0.64 mg/gm).The content of iron was significantly higher in mixed calculi as compared to cholesterol calculi (p = 0.0164) and pigment calculi (p = 0.0403).However, there was an insignificant difference (p = 0.5718) between iron content of cholesterol calculi and pigment calculi.
The oxalate content was higher in cholesterol stone (7.2 mg/gm) and lowest in mixed stone (5.9 mg/gm).However, there was no significant difference between oxalate content of the three calculi (p > 0.05).

Discussion
Gallstone disease known as cholelithiasis is the most common digestive surgical disorder and account for an important part of health care expenditure.The most commonly involved age group for gallstone disease (43.6%) is found to be 41 -50 years with a female predominance (F:M = 14.7:1), this is in concordance with Jaraari et al. [9] in Libya and P. Chandran et al. [10] in India, and differ from others [6,11,12].
In this study Pigment stone was found to be the commonest comprising 51.06%, followed by mixed stone 31.91% and cholesterol stone 17.02%.The findings are similar with the findings in the study done in Libya and San Francisco [9,13].Where as in the study in Nepal (India) [14] mixed stone was found to be the commonest comprising 78.75%, followed by cholesterol 12.5%, pigment 8.75% and black pigment stone 1.25%.In other study in Haryana, India mixed calculi (38%) more than pigment calculi (36%) more than cholesterol calculi (26%) [10].Study showed that gallstones were multiple in 64.9% patients; this is in agreement with P. Chandran et al. and Banchob Sripa et al. [10,12].Whereas, Jaraari et al. [9] in Libya studied 41 patients all had multiple gallstones.Study showed that there is difference in the number of stone detected by ultrasound and that found intraoperatively (p < 0.0001), this might be due to the experience ultrasonographists.
CSs were bigger in size as compared to mixed and pigment stones.The size of cholesterol calculi was in the range 0.3 -2.7 cm and 0.3 -3.1 cm with an average of 0.9 cm and 0.9 cm, this is in agreement with others [9,10].
Determination of metabolites showed that the mean total cholesterol was higher in cholesterol calculi (713 mg/gm), compared to mixed stones (637 mg/gm) and pigment stone (421 mg/gm).
There was an insignificant difference between total cholesterol content of cholesterol and mixed stones (p > 0.05), others found similar result [9,10].Gallstones are believed to form when the concentration of cholesterol exceeded that which can be held in mixed micellar solution with bile acids and phospholipids.Supersaturation of cholesterol is believed to be due to abnormal production of bile from liver.The concept of cholesterol supersaturation as a basis for gallstone formation has been emphasized for cholesterol stones, which are composed mainly of cholesterol [12].The co-existence of nucleating factors, gallbladder hypomobility, and mucus hypersecretion also contribute to cholesterol precipitation leading to the development of gallstones [9].
The triglyceride content was higher in cholesterol calculi (69.3 mg/gm) and mixed calculi (51 mg/gm) as compared to pigment calculi (32.8 mg/gm).The triglyceride content was significantly higher (p < 0.05) in cholesterol and mixed calculi as compared to pigment calculi.However, the difference was insignificant between cholesterol and mixed calculi (p > 0.05).The result is similar to study by Chandran et al. [10] but is comparable to study by Jaraari et al. [9] in Libya where triglyceride content was higher in mixed stones than in the other two types of stones.The higher content of triglycerides in MS or CS compared to PS might be due to a higher deposition of calcium salts of cholesterol and esters of fatty acids in MS and CS when compared to PS in which calcium bilirubinate is the major salt [15].
The mean fatty acid content was highest in cholesterol calculi (21.71 mg/gm) and lowest in mixed calculi (13.82 mg/gm).The difference of fatty acid content was significant between cholesterol and mixed calculi (p < 0.05) and between cholesterol and pigment calculi (p < 0.05) but insignificant between mixed and pigment calculi (p > 0.05).
The esterified fatty acid content was highest in cholesterol and lowest in mixed calculi.Similar result also found by Chandran et al. [10].The high content of esterified fatty acids in cholesterol stones might be due to interaction between excessive cholesterol and fatty acids.
The mean total bilirubin concentration was highest in pigment stones (4 mg/gm) and lowest in cholesterol stones (0.3 mg/gm).
The total bilirubin concentration was highest in pigment and lowest in mixed calculi.It was significantly higher in pigment calculi compared to mixed and cholesterol calculi (p < 0.009) and in cholesterol calculi as compared to mixed calculi (p < 0.02).
These observations are in agreement with other reports by Chandran et al. [10], Kumar D et al. [16] and Pundir CS et al. [17].The colour of the pigment stones could be attributed to colour of bilirubin, which form salt with calcium to form calcium bilirubinate.It is known that β-glucuronidase of bacterial origin hydrolyses conjugated bilirubin into free bilirubin, which form salt with calcium as calcium bilirubinate [18].
The mean bile acid content in pigment stones (25 mg/ gm) was comparatively higher than that in cholesterol calculi (7.2 mg/gm) and mixed calculi (23 mg/gm).The bile acid content in pigment stone was comparatively higher than that in cholesterol and mixed calculi.The difference was significant between PS and CS and between MS and CS (p < 0.05), but the difference was insignificant between PS and MS (p > 0.05).The result was similar to that achieved by both Jaraari et al. [9] and Chandran et al. [10].But it was five times higher than that reported in Japan [19].
Bile acids were significantly lower in CS than MS and PS, while it were significantly higher in PS.Supersaturation of bile with calcium bilirubinate is inhibited by bile salts, which bind calcium, reducing the activity of free calcium ions.When supersaturation occurs, usually due to increased concentrations of bilirubinate anion, nucleation may be initiated by binding of calcium bilirubinate to mucin glycoproteins in bile [20].
Similarly, the phospholipid content was marginally higher in CS than PS and MS.As the mean phospholipids content was highest in CS (6.9 mg/gm) and lowest in MS (5.8 mg/gm).
Cholesterol has to be in relative proportion with the bile salts and phosphatidyl choline to remain soluble in the bile and thereby stone formation [21].
About 5% of cholesterol is barely soluble in 20% phosphatidyl choline and this should require approximately 60% of bile salts.Any concentration above this level would cause precipitation of cholesterol, as shown by the presence of the highest amount of cholesterol in CS is an indicator of this fact [22].PS contained less cholesterol than the other two types of stones.Cholesterol in PS is mostly because of co-precipitation with bilirubin and other compounds but not due to its supersaturation in the bile [23].In contrast to this, MS have less cholesterol than CS but more than PS.This might be due to the presence of both cholesterol and bile pigment in these stone.
The protein content was significantly higher in PS (98.62) as compared to MS (10.03) and CS (27.37).Similarly Chandran et al. [10] was achieved nearer results.
Binette et al. [24] suggested that the proteins to be the candidates either to facilitate or hinder the formation of stones.Maki [25] proposed the ability of bacterial βglucuronidase enzymes (protein) to hydrolyse the bilirubin glucuronide complex thus releasing a poorly soluble bilirubin to explain the formation of PS as well as CS.
Determination of cations and anions showed that the mean calcium content was highest in pigment calculi (22.8 mg/gm) and lowest in cholesterol calculi (9.6 mg/gm).The inorganic phosphate content was highest in cholesterol calculi (16 mg/gm) and lowest in pigment calculi (10.2 mg/gm).
So in this study calcium content was highest in PS, while phosphorus was lowest.This is in agrees with studies by Jaraari et al. [9] and Pundir CS et al. [17].It is known that bilirubin combines with calcium to form a precipitate of calcium bilirubinate [26].Since PS has excess bilirubin, calcium forms calcium bilirubinate [27].
It was not surprising that bile acids were more in PS because an increase in sodium content facilitates excessive formation of bile salts.Potassium was also higher in PS.Jaraari et al. [9] in their study were concluded similar results.It is presumed that the sodium to potassium ratio will be maintained in the bile.Hence, higher sodium content is associated with higher potassium content, although the increase in the latter was not as much as that of the sodium content.
The magnesium content was highest in PC (10.2 mg/gm) and lowest in MS (9.1 mg/gm).This is in conformity with observations made by others [9,10].
The oxalate content was higher in CS (7.2 mg/gm) and lowest in MS (5.9 mg/gm).This disagrees with others [9,10], where it was higher in PS.Whereas, Raha et al. [28] were found calcium oxalate to be one of the major component of MS.Both magnesium and oxalate form insoluble salt which might be crystallized to strengthen the texture of PS [16].
The mean chloride content was highest in PS (36.1 mg/gm) and lowest in CS (17.8 mg/gm).Similar result also obtained by others in their series [9,10].Same authors proposed that the chloride ions are always present in biological fluids in human beings including bile, which might get deposited in the form of sodium chloride salt along with major salts of the gallstones.
The copper content was highest in CS (0.72 mg/gm) and lowest in PS (0.348 mg/gm).This is also observed in another one study [10].
The role of copper in pathogenesis of calcium bilirubinate in gallstones was suggested in literature [29].
The iron content in this study was highest in MS (1.09 mg/gm) and lowest in CS (0.64 mg/gm).These observations are in agreement with others [10,17].Been et al. [29] reported the presence of small amount of iron in a thick black shell around the central dark inclusion of gallstones.
The high iron content in MS might be promoting aggregation of calcium bilirubinate particles which are the major constituents especially due to the polyelectrolytic nature of iron [7,30].
Verma et al. [31] suggested that the central aggregates of calcium salts may lead to ulceration of gallbladder mucosa and microscopic haemorrhage.The iron released by this process might be another source of its deposition in gallstones.
Comparison of the chemical composition of gallstones in Sudanese individuals with those in other countries is summarized in Table 8.Though cholesterol is a major component of gallstones, the composition of gallstones varies from country to country and region to region.
A major limitation of the present study is the potential self-selection bias due to the hospital-based study design, which is not being exactly representative of the whole general population.Second, it is well established that a large proportion of GSD patients remain asymptomatic and they are therefore often ignored for many years.

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Thus the present study may be representative of the clinical and not of the true prevalence.The solution to such study is to conduct a number of prospective longitudinal analogous studies, the results of which would be expected to complement the cross-sectional findings of this study.

Conclusions
In conclusion, CS showed significantly higher cholesterol content than pigment stones though not significantly higher than MS.In MS, the cholesterol, bile acids, and bilirubin were intermediate between CS and PS, whereas triglycerides were significantly higher than PS.Bilirubin and bile acids were significantly higher than CS.In PS, bilirubin was significantly higher than both groups.Bile acid content was significantly higher than CS but not significantly more than MS.Collaborations between surgeons, nutritionists, biochemists, and physicians should be stimulated in future studies to define the different types of gall stones in different areas in Sudan and the relevancy of such types with diets' traditions.

Table 3 . Concentrations of metabolites in the different types of biliary calculi in patients with symptomatic calcular cholecys- titis in the study group (n = 94).
a PL, phospholipids; b CS, cholesterol stones; c MS, mixed stones; d PS, pigment stones.Values are the mean and standard deviation.