Minerals composition of different types of gallstones in Sudanese population

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1 Open Science Journal of Analytical Chemistry 2014; 1(1): 1-5 Published online January 30, 2014 ( Minerals composition of different types of gallstones in Sudanese population Saadeldin Ahmed Idris 1, *, Kamal Elzaki Elsiddig 2, Mohamed Mahmoud Hafiz 1, Aamir Abdullahi Hamza 3, Mohammed H. F. Shalayel 4 1 Associate professor of Surgery, Alzaeim Alazhari University, Faculty of Medicine, Khartoum, Sudan 2 Associate professor of Surgery, University of Khartoum., Faculty of Medicine, Khartoum, Sudan 3 Associate professor of Surgery, Bahri University, Faculty of Medicine, Khartoum, Sudan 4 Professor of Biochemistry, National College for Medical and Technical Studies, Khartoum, Sudan address saadeldinahmed@hotmail.com (S. A. Idris), kamalelzaki@yahoo.com (K. E. Elsiddig), mohamedhafiz100@yahoo.com (M. M. Hafiz), aamirhamzza@yahoo.co.uk (A. A. Hamza), drmhfs@hotmail.com (M. H. F. Shalayel) To cite this article Saadeldin Ahmed Idris, Kamal Elzaki Elsiddig, Mohamed Mahmoud Hafiz, Aamir Abdullahi Hamza, Mohammed H. F. Shalayel. Minerals Composition of Different Types of Gallstones in Sudanese Population. Open Science Journal of Analytical Chemistry. Vol. 1, No. 1, 2014, pp Abstract Background: Gallstone disease is one of the major surgical problems in Sudanese population. Aim: The study was conducted to determine the varying composition of cations and anions of gallstones in a Sudanese population. Materials and methods: A cross-sectional study included all patients with symptomatic calcular cholecystitis that confirmed ultrasonographically and treated by cholecystectomy after acceptance of the informed consent. It was conducted in Khartoum teaching hospital in the period between January 2010 and December The stones were powdered and dissolved in different appropriate solvents depending upon the type of chemical constituent to be analyzed. Collected data was analyzed statistically using SPSS version 21. Results: The study included 94 patients (six (6.4%) males and 88 (93.6%) females) with female to male ratio of 14.7:1. Their age ranged between 27 and 80 years, with mean age 0f 45.9 years (SD ± 10.3). The stones were multiple in 61 (64.9%), with predominance of pigment calculi as found in 48 (51.07%). The sodium, potassium, calcium, magnesium, copper, iron, inorganic phosphate, oxalate and chloride were found in all gallstones in different concentrations in each different type of stones, collectively these minerals constituted about 9.36% of the stones composition. Conclusion: Calcium, sodium, potassium, magnesium, and chloride had the highest concentrations in pigment stones. Inorganic phosphate, copper, and oxalate recorded the highest levels in cholesterol calculi whereas; iron content was the highest in mixed stones. Keywords Gallstones (GS), Cholesterol Stone (CS), Pigment Stone (PS), Mixed Stone (MS), Cations, Anions 1. Introduction Gallstone disease (GSD) is a common digestive disorder with multifactorial origins [1]. Studies indicate its varying prevalence in different study populations [2]. It is incidence ranging from 10% to 20% in the world population. The incidence is four times higher in women than in men with high prevalence among younger age group. However, the true prevalence of the disease remains hard to derive as the majority of patients remain asymptomatic [3]. Gallstones, complex biomineralized deposits formed in the gallbladder and are frequently composed of more than one crystalline compound, although the exact mechanism of formation of multicomponent stones is not clearly understood [4]. Over the past several decades, there have been continuous efforts aimed at elucidating the pathogenesis of gallstones, and the rapid proliferation of novel methods for

2 2 Saadeldin Ahmed Idris et al.: Minerals Composition of Different Types of Gallstones in Sudanese Population analyzing the composition and the morphology of gallstones have been reported. With progress in scientific technology, new classes of gallstones can be added to the existing classification systems. Moreover, the incidence and composition of gallstones can change over time. Therefore, the compositions need to be revised from time to time [5]. Chemical composition of gallstones is essential for aetiopathogensis of gallstone disease [6]. They are generally classified into three major types: pure cholesterol, pigment and mixed gallstone [4]. Chemical classification and location of biliary calculi differ in various parts of the world and change over time because of nutritional, socio-economic and demographic constituents of different types of gallstones [6]. This study describes an extensive ionic cations and anions analysis of gallstones in Sudanese patients, including; calcium, phosphorus, sodium, potassium, magnesium, oxalates, and chlorides. 2. Materials and Methods A cross-sectional study was conducted in Khartoum teaching hospital in the period between January 2010 and December All patients with symptomatic calcular cholecystitis that confirmed ultrasonographically and treated by cholecystectomy were involved into our study after acceptance of the informed consent. 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 2-8 C, when not in use. 3. Results The study included 94 patients (six (6.4%) males and 88 (93.6%) females) with female to male ratio of 14.7:1. Their age ranged between 27 and 80 years, with mean age of 45.9 years (SD ± 10.3). The stones were multiple in 61 (64.9%). The highest incidence of gallstones cases were found in the age group years Stones Analysis The stones were divided into 3 groups based on their colour: cholesterol calculi, mixed calculi and pigment calculi in 16 (17.02%), 30 (31.91%) and 48 (51.07%) respectively. The collected gallstone specimens were of variable sizes, their size varied from as small as less than 0.5 x 0.6 cm to 0.9 x 0.9 cm. The sodium, potassium, calcium, magnesium, copper, iron, inorganic phosphate, oxalate and chloride were found in all gallstones in different concentrations in each different type of stones, collectively these minerals constitutes about 9.36% of stones composition (Table 1) Calcium Content 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 difference between calcium content of pigment calculi as compared to mixed calculi (p=0.0805) Inorganic Phosphate Content The inorganic phosphate content was highest in cholesterol calculi (16 mg/gm) and lowest in pigment calculi (10.2 mg/gm). 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) Sodium Content 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) Potassium Content 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 Magnesium Content 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

3 Open Science Journal of Analytical Chemistry 2014; 1(1): magnesium content of three calculi (p>0.05) Chloride Content 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) Copper Content 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) Iron Content 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 Oxalate Content 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), (Table 2 and 3). In female patients stones were found as follows; 45 (51.1 %) PS, 27 (30.7%) MS, 16 (18.2%) CS. Whereas, in male patients Cholesterol stones were not found, only they had PS in 3 (50%) and MS in 3 (50%) of patients. PS and MS In PS when mineral composition compared between genders, there was statistical difference in their composition of sodium, potassium, magnesium, copper, inorganic phosphate and chloride which were higher in male patients p values were p=0.03, p=0.01, p=0.02, p=0.02, p=0.01 and p=0.04 respectively. But there was no statistical difference in their composition of calcium, iron, and oxalate as p> In MS when mineral composition compared between genders, there was statistical difference in their composition of sodium and magnesium, which were higher in female patients p values were p=0.001, and p=0.009 respectively. But there was no statistical difference in their composition of calcium, potassium, iron, copper, chloride, inorganic phosphate and oxalate as p> Table 1. Percentage of mineral composition of gallstones of patients with symptomatic calcular cholecystitis in the study group (n=94). * The highest anion and cation Minerals Mg/gm Percent Ca 52.3* 2.16 PO Na K Mg Cl * 3.15 Copper Iron Oxalates Total Table 2. Quantitative analysis of cations and anions in different types of biliary calculi expressed as mg/gm dry stone powder (Data are mean±se) (n=94). Stone type Ca PO Na+ K+ 4 Mg Cl- Copper Iron Oxalate CS 9.6±2.7 16± ± ± ± ± ± ± ±1.3 MS 19.9± ± ± ± ± ± ± ± ±1.4 PS 22.8± ± ± ± ± ± ± ± ±0.9 *Note: CS, cholesterol stones; MS, mixed stones; PS, pigment stones. Values are the mean and standard error of mean. Comparison Table 3. P-values for differences in composition of various ions (n=94). Ca PO Na + K + 4 Mg Cl - Copper Iron Oxalate CS vs. MS CS vs. PS MS vs. PS

4 4 Saadeldin Ahmed Idris et al.: Minerals Composition of Different Types of Gallstones in Sudanese Population 4. Discussion Gallstones remain a serious health concern affecting millions of people throughout the world. An interesting finding in this study is that the females are at higher risk of cholelithiasis development than males, in a ratio of female: male (14.7:1). This finding might be attributed to high percentage of patients with high risk factors such as multiparity, use of oral contraceptives, obesity and family [7]. Study showed that gallstones were multiple in 61 (64.9%) patients and single in 33 (35.1%), this is in agreement with studies by Chandran P. et al. [8] in India and Banchob Sripa et al. [9] in Thailand where the percentages of multiple stones were 63% and 76% respectively. Whereas, Jaraari et al. [10] in Libya studied 41 patients and all had multiple gallstones. 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 agreement with studies by Jaraari et al. [10] and Pundir CS et al. [11]. It is known that bilirubin combines with calcium to form a precipitate of calcium bilirubinate [12]. Since PS had excess bilirubin, hence calcium forms calcium bilirubinate [13]. 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. [10] in their study 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 [5, 8]. The oxalate content was higher in CS (7.2 mg/gm) and lowest in MS (5.9 mg/gm). This disagrees with others [8, 10], where it was higher in PS. Whereas, Raha et al. [14] 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 [15]. The mean chloride content was highest in PS (36.1 mg/gm) and lowest in CS (17.8 mg/gm). Similar result was obtained by others in their series [8, 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 study [8]. The role of copper in pathogenesis of calcium bilirubinate in gallstones was suggested in literature [16]. 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 [8, 11]. Been et al. [16] 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 [5, 17]. Verma et al. [18] 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. Interestingly in this study we found difference in the minerals composition between genders, in literature search we could not find any study evaluated the mineral composition of gallstones in different genders. By finding of such difference might be a nidus for future researches. Starting from these particular observations, a better understanding of mechanisms of gallstone formation may see the light. Comparison of the ionic composition of gallstones in Sudanese individuals with those in other countries is summarized in Table 4. Table 4. Percentage of mineral composition of gallstones from various countries Country No. of patients Libya [10] Kuwait [18] India [19] England [18] South Africa [18] USA [18] Sweden [18] 27 6 Sudan (present study) Ca +2, PO - 4, Mg, Oxalates, Na +, K +, and Cl - (%) 5. Conclusion and Recommendation The sodium, potassium, calcium, magnesium, copper, iron, inorganic phosphate, oxalate and chloride were found in all gallstones in different concentrations in each different type of stones, collectively these minerals constitutes about 9.36% of stones composition. Calcium, sodium, potassium, magnesium, and chloride had the highest concentrations in pigment stones. Inorganic phosphate, copper, and oxalate recorded the highest levels in cholesterol calculi whereas; iron content was the highest in mixed stones.

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