Elemental Analysis as a Strong Tool for the Determination of Gallstones Composition

Elemental analysis of C, H, N, S and Ca was used for the classification of gallstones from 94 patients from the Western Region of Saudi Arabia. Fourier transform infrared spectroscopy (FT-IR) was used to provide rapid qualitative information about the gallstones composition. The density, color and surface of the stones were found to give a good indication of stones composition. From the studies made, it was found that 64.9% of the stones analyzed were cholesterol stones, 25.5% were mixed stones, which contain cholesterol and some calcium salt. The rest of the stones were rare stones which contain either calcium carbonate or a mixture of calcium carbonate and ammonium and magnesium salt. Introduction Gallstones are formed in the biliary tree, usually in the gall bladder. They occur when certain biliary solutes such as cholesterol or calcium salt precipitate as solid crystals that subsequently grow and aggregate within the mucine layer lining the gall bladder. There are two major categories of gallstones; cholesterol stones and calcium stones. Cholesterol stones consist of crystalline cholesterol (C27 H45 OH-M.W. 386.67) with variable amounts of other components including mucine glycoprotein, and calcium bilirubin. The predominant components of calcium stones are calcium salts of organic and inorganic anions, especially bilirubin (C33 H36 N4O6-M.W. 584.67). Black gallstones are mostly hard, dense and brittle. They are composed of calcium bilirubinate with inorganic calcium salt of carbonate and phosphate. Some of these stones contain substantial proportion of calcium soaps of fatty acids. A.A. Al-Sibaai, et al. 34 Cholesterol stones represent about 80% of all gallstones in United States[1], 46% in Southeast Asia[2], 100% in Himalayas (Sikkim and north Bengal)[3], less than 2% in Coimbatore District of Tamil Nadu State[4], 34% in Ghana[5], 56% in Sanata Cruz, Bolivia[6], 94% in Northern India[7]. In Siena, Italy[8], out of 1226 gall bladder stones 94 were black pigment gallstones Out of these, 13 patients were found to have black pigment gallstones and cholesterol gallstones in their gall bladders. The knowledge of the chemical composition of gallstones is very important from the viewpoint of litho-genesis and possible medical therapy of stone dissolution. Different techniques have been used for the determination of the components of the gallstones. Thermogravimetry has been used for the determination of cholesterol, calcium carbonate and calcium oxalate[9]. Enzymes method was used for the determination of total cholesterol in gallstones[10]. FT-IR was used as the main analytical technique for the determination of human gallstone structural composition[11]. Computed tomography (CT) was used for the analysis of gallstone composition in vivo[12]. Correlation between chemical analysis and in vitro CT scan was investigated[13]. The analysis of minor and trace elements in the gallstones was made using neutron activation analysis (NAA), proton induced X-ray emission (PIXE) and X-ray fluorescence spectroscopy (XRF)[14]. Also inductive-coupled-plasma atomic emission spectroscopy (ICP-AES) and atomic absorption spectroscopy (AAS) were used for the determination of Co, Cr, Cu, Fe, Mn, Ni and Zn in gallstones after digestion with HNO3 and H2O2 [15]. Experimental Gallstones were received from the Faculty of Medicine at King Abdulaziz University Hospital, Jeddah, Saudi Arabia. Any possible superficial contamination of the stones was removed by washing with distilled water after which the stones were dried in oven at 50oC for 24 h. The physical properties were recorded including the weight, number, color, shape and surface. Photographs of the stones were taken to document any special feature. In all cases only one stone was taken for analysis. Half of the dried stone was ground carefully to a fine powder in porcelain mortar. The resulting powder was analyzed by FT-IR and elemental analyzer. The density of each stone was measured on the other half of the stone using density bottle. Physical Measurements 1 – FT-IR Analysis: About 5 mg of the dry stone powder was ground with 25 mg of pure KBr (Analytical grade) in an agate mortar. The resulting powder was put in the sample cavity of Perkin Elmer Spectrum RXI-FT-IR System. Background correction was made with pure KBr. Elemental Analysis as a Strong Tool... 35 2 – C,H,N and S Analysis: was made using Perkin Elmer Elemental Analyzer (model 2400). 3 – Al, Ba, Ca, Cu, K, Mg, Mn, Na, Sr and Zn Analysis: Exact weight of the stone powder was heated in a porcelain crucible at 700oC using muffle furnace. The residue was digested with 5 ml concentrated HNO3 (Analytical grade) for one hour on a water bath, then evaporated to dryness and dissolved in dilute HNO3 (0.1 M). The solution was transferred to a 25 ml volumetric flask and the volume was completed to the mark using 0.1 M HNO3. The blank was prepared in the same way. Standard solutions of Al, Ba, Ca, Cu, K, Mg, Mn, Na, Sr and Zn were prepared using the nitrate salt of these metals (Analytical grade). Na and K were determined using a Perkin Elmer, model 5000, atomic absorption spectrometer. The rest of metals were determined using a Perkin Elmer inductively coupled plasma spectrometer, model Optima 4100-DV. Results and Discussion The stones from 94 patients from the Western Region of Saudi Arabia have been studied. 86.17% of the patients were women and 13.83% were men. 40.43% of the cases have single stone only, 8.51% of the cases have two stones, 25.53% of the cases have between 3-10 stones and more than 10 stones in the rest of the cases. In one case 40 stones were found. Different shapes of the stones were observed. 44% of the stones were spherical, 18% egg shape, 11% almost cubic, 11% pyramidal shape, 8% different shapes and the rest were crushed pieces. The color of the stones was observed too. 67% of the stones were brown, 13% were black and the rest of the stones were either white or light gray. The surface of the stones was smooth in 80% of the cases, rough in 17% of the cases and needle like in three stones. The photographs of some of these stones are shown in Fig. (1). The figure shows the different shapes and different colors of some studied stones. FT-IR Spectra FT-IR spectra provide a rapid qualitative information about the chemical composition of gallstone[11]. Figure (2) is the spectrum of stone No. 44 which shows bands at 3372, 2936, 1465, 1377 and 1057 cm–1 is almost similar to the spectra of pure cholesterol. Figure (3) is the spectrum of stone No. 15 which exhibits bands at 3286, 2929, 2517, 1793, 1487, 1082, 876 and 713 cm–1 is similar to the spectrum of pure CaCO3. Fig. (4) is the spectrum of stone No. 70 which gives bands at 3282, 1665, 1250, 1036 and 564 cm–1, is an indicative of the presence of N-H group. A.A. Al-Sibaai, et al. 36 Fig. 1. Photographs of some gallstones and their corresponding numbers. Elemental Analysis as a Strong Tool... 37