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Analysis of Indian pigment gallstones
NIM B Beam Interactions with Materials & Atoms
Nuclear Instruments and Methods in Physics Research B 255 (2007) 409–415 www.elsevier.com/locate/nimb
Analysis of Indian pigment gallstones T.R. Rautray a
a,*
, V. Vijayan b, S. Panigrahi
a
Department of Physics, National Institute of Technology, Rourkela 769 008, Orissa, India b Institute of Physics, Bhubaneswar 751 005, Orissa, India Received 16 August 2006; received in revised form 17 November 2006 Available online 5 January 2007
Abstract Particle induced X-ray emission and particle induced c-ray emission spectroscopic techniques have been carried out to analyse the elemental concentrations of human pigment gallstone samples from eastern region (Orissa) and southern region (Chennai) of India. It was observed that 18 minor/trace elements namely Na, Mg, Al, P, S, Cl, K, Ca, Ti, V, Cr, Mn, Fe, Ni, Cu, Zn, Br and Pb were present in the pigment gallstone samples of both the regions. Our study reveals that average concentration of all elements except Ni in south Indian pigment gallstone samples is higher than that of corresponding values in east Indian pigment gallstone samples whereas elements like Al, P, S, Cl and V did not show much variation between these two regions. Fourier transform infra-red analysis was carried out to identify the functional groups and the classification of the pigment type gallstones of both the regions. The thermal behaviour of pigment gallstones was carried out by thermogravimetry-derivative thermogravimetry analysis. Ó 2006 Elsevier B.V. All rights reserved. PACS: 87.64.Ni; 87.68.+z; 87.64.Gb; 78.30.Er; 65.60.+a Keywords: PIXE; PIGE; FTIR; TG-DTG; Calcium bilirubinate
1. Introduction Formation of gallstones is still one of the most common digestive diseases in the world. Since the pathogenesis of gallstones is not yet clearly understood, its analysis-using chemical and spectroscopic techniques have provided some clues. In gallstone disease, trace elements are believed to play an important role in stone formation [1,2]. They are broadly classified into three categories according to their composition namely cholesterol type (cholesterol rich), pigment type (bilirubin and calcium bilirubinate rich) and mixed type (combination of cholesterol, calcium carbonate, calcium bilirubinate) [3]. In particular, septic complications are much more common in patients with pigment gallstones than in patients with cholesterol gallstones. While cholesterol gallstones are due to supersaturated bile, the
*
Corresponding author. Tel.: +91 9853262167; fax: +91 674 2300142. E-mail address: [email protected] (T.R. Rautray).
0168-583X/$ - see front matter Ó 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.nimb.2006.12.147
black pigment gallstones are common in haemolytic anemia (secondary to excessive degradation of red blood cells) or in the presence of infected bile. In the absence of these known precipitating factors, the pathogenesis of pigment or mixed gallstone formation in Indian patients remains unclear [4]. Trace elements play a significant role in, or provide an indication of possible mechanisms of stone formation. The relative importance of the elements in stone formation was subsequently evaluated based on the statistical analysis of the data obtained. In general, the variation of chemical composition in different gallstones varies significantly in different parts of India. Food habits may be one of the main reasons. Cholesterol gallstones are predominant in the northern, eastern and western parts of India, while pigment gallstones are common in the southern region [5]. Despite decades of research, the formation mechanism of gallstones remains incompletely understood. The study of stone formation inside the gall bladder becomes one of the major problems for the contemporary medicine. It was found that the principal components of
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human gallstone are cholesterol and bilirubin in the form of various bilirubinate salts. Other compounds that have been found in gallstones include calcium carbonate, calcium phosphate salts, calcium bilirubinate, fatty salts, various cholic acid derivatives, polysaccharide and proteins [3,6]. Metals in the human body are metabolized mainly via the kidney and liver and excreted through urine and partly by bile. Changes in the composition of bile may induce cholecystitis and cholelithiasis [7]. Pigment gallstones can be further classified into brown and black stones. Brown stones have a characteristic appearance and are typical in Asia. These stones occur as a result of infection. Black stones generally are not associated with infected bile. These stones are found in patients with hemolytic disorders or cirrhosis [8]. The toxic elements are known to be very harmful even at extremely low concentrations. For this reason, reliable analyses will help to clarify and define effective treatment. Factors responsible for the formation of stones include altered hepatic bile composition, biliary glycoprotein, infection, age, genetic, sex, estrogens, dietary factors, geographical prevalence and cirrhosis of the liver [9]. Since particle induced X-ray emission (PIXE) and particle induced gamma-ray emission (PIGE) techniques are simultaneous, reliable, rapid, multi-elemental, sensitive and non-destructive in nature to analyse even few ppm level elements [10], the present investigation was carried out for the pigment gallstone samples. The presence of functional groups was confirmed by Fourier transform infra-red (FTIR) analysis and the thermal behaviour was studied by thermogravimetry-derivative thermogravimetry (TG-DTG) analysis for the pigment gallstones of both the eastern and southern regions of India. 2. Experimental 2.1. Sample preparation The east Indian and the south Indian pigment gallstone samples of both sexes were collected from Kalinga Hospital, Bhubaneswar, Orissa and Stanley Medical College, Chennai, respectively, in the age group of 33–68 years. Fifteen pigment gallstone samples of different patients were taken from various places of a south Indian state and 12 pigment gallstone samples of different patients were taken from different places of an east Indian state. The pigment gallstones samples were washed with deionised water 5–6 times and heated at 40 °C in an oven for 8 h. After drying, the gallstone samples were crushed to make pellets. For PIXE and PIGE analysis, 150 mg of powdered samples were mixed with 150 mg of high pure graphite powder in 1:1 ratio by mass to make pellets of size 13 mm diameter in a hydraulic pressure. The aim of adding graphite powder is that it can be used as a binder of the homogenized powdered sample as well as to monitor the beam current since graphite is a good conductor of electricity and another purpose of adding graphite is to reduce bremsstrahlung background of PIXE spectrum [10]. Thick targets have been
prepared by pressing the above mixture with a pelletizer. Pellets of NIST bovine liver (1577b) and IAEA animal blood (A-13) were also prepared by the same procedure for calibration purposes. For FTIR measurements, 100 mg of KBr powder was thoroughly mixed with 10 mg of pigment gallstones to make a pellet of size 13-mm diameter and 250 mg of dried pure powdered gallstones were taken for the TG-DTG measurements.
2.2. PIXE, PIGE, FTIR and TGDTG measurements Simultaneous PIXE–PIGE analysis was carried out using the 3 MV horizontal Tandem Pelletron accelerator (9SDH-2, NEC, USA) at Institute of Physics, Bhubaneswar, India. The proton beam was collimated to a diameter of 2 mm on the target in vacuum (106 Torr) inside a PIXE chamber. The multiple-target holder was placed at 45° to the beam direction. The target holder was mounted on an insulated stand and is surrounded by a cylindrical electron suppressor held at negative potential with respect to the target. Integrated charge on the thick sample was measured using a current integrator, which was connected to the target holder. The targets were bombarded with 3 MeV proton beams with 20 nA beam current. A Si (Li) detector (active area 30 mm2) with a resolution of 170 eV at 5.9 keV with beryllium window placed at 90° to the beam direction was used to detect the characteristic X-rays emitted from the targets. The X-rays exit the scattering chamber through a 95 lm Mylar window before entering the detector. Spectra were recorded by using a multi-channel analyser calibrated with 241Am X-ray source [11]. No Xray absorbers were used between the detector and target during data collection. For PIGE analysis, a high purity germanium (HPGe) detector with a resolution of 1.9 keV at 1.33 MeV placed at an angle 135° to the beam direction was also simultaneously used in the scattering chamber for the gamma ray measurements. A 60Co c-ray source was used for the c-energy calibration. In PIGE, inelastic scattering of protons follows de-excitation of low-lying nuclear excited states resulting in the emission of characteristic c-rays of a particular nuclide that serves as a basis for qualitative as well as quantitative assessment of a particular element [12]. The concentration of low Z elements like Na, Mg and Al were analysed by PIGE whereas other elements were analysed by PIXE technique. The PIXE spectral analyses were performed using GUPIX-2000 software [13]. This provides a non-linear least square fitting of the spectrum, together with subsequent conversion of the fitted X-ray peak intensities into elemental concentrations, utilising the fundamental parameter method (FPM) for quantitative analysis. The uncertainty in the concentration estimation for the minor elements such as Na, Mg, P, S, Cl, K, Ca, Mn, Fe and Cu is found to be between 8% and 10%, whereas for the trace elements it is found to be between 17% and 20%.
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The PIGE spectral analysis was done using the ANGES (IAEA, Vienna) software [14]. The chemical analysis of the pigment gallstones, in nitrogen gas atmosphere, was carried out by a FTIR spectrophotometer [15] (Thermo Nicolet Avatar 370, USA) to identify the functional groups and the measurements were carried out in the mid-infrared range (4000–400 cm1) at the resolution of 4 cm1 in the transmission mode. The collimator through which the infrared beam passes before falling on the sample is 0.5-mm in breadth and 10-mm in length. TG-DTG analysis was performed using the powdered samples in a Shimadzu thermal analyser. The weight loss of the sample was monitored under air atmosphere. Each sample was heated under air atmosphere [16] at a linear heating rate of 10 °C min1 from 25 °C to 525 °C. 3. Results and discussion
Fig. 2. PIXE spectrum of a south indian pigment gallstone.
Two representative PIXE spectra of pigment gallstone samples of eastern India and southern India are shown in Figs. 1 and 2, respectively, and two representative PIGE spectra of pigment gallstones of eastern and southern India are shown in Figs. 3 and 4, respectively. The spectra obtained from all the pigment gallstone samples were analysed. The elemental concentrations of the east and south Indian pigment gallstones are shown in Table 1, which is compared with the literature values of south Indian gallstones by Ashok et al. [4]. It was observed that 18 minor/ trace elements namely Na, Mg, Al, P, S, Cl, K, Ca, Ti, V, Cr, Mn, Fe, Ni, Cu, Zn, Br and Pb were present in the pigment gallstone samples. Average concentration of Mg in southern region pigment gallstones are about 2.3 times more than that of the east Indian pigment gallstones. Since high intakes of dietary fibre (40–50 g/day) leads to lower Mg absorption, this may be the reason for the low Mg concentration in the east Fig. 3. PIGE spectrum of an east indian pigment gallstone.
Fig. 1. PIXE spectrum of an east indian pigment gallstone.
Indian gallstones. This is probably attributable to the Mgbinding action of phytate phosphorus associated with the fibre [17–19]. Rice is a regular and primary food consumed in both the eastern and southern regions. Since, the Mg content of Indian milled rice is 380 ppm, the higher values of Mg in the pigment gallstones of both the regions may be due to rice consumption. However, just like rice, tamarind is consumed in higher amount as a regular basis in south India. Since the concentration of Mg in Indian tamarind is 410 ppm [20], higher intake of tamarind gives rise to higher concentration of Mg in the south Indian pigment gallstones. Even though the concentration of P (1100 ppm) is also high in tamarind, concentration of P does not show much variation between the two regions which may be ascribed due to the presence of calcium phosphate or hydroxyapatite in both types of pigment gallstones as evidenced by FTIR.
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Fig. 4. PIGE spectrum of a south indian pigment gallstone.
However, consumption of phytate- and cellulose-rich products (usually containing high concentrations of Mg) increases Mg intake, which often compensates for the decrease in gastrointestinal absorption. The effects of dietary components such as phytate on Mg absorption are probably critically important only at low magnesium intake. There is no consistent evidence that modest increases in the intake of Ca [21–23], Fe, or Mn [24] affect Mg balance. In contrast, high intakes of zinc (142 mg/day) decrease Mg absorption and contribute to a shift toward negative balance [25]. Concentration of Ca in southern region pigment gallstones is more than one and half times than that of eastern region, but it is less than half than that reported by Ashok et al. From among the detected elements, Ca has the high-
est average concentration which is because of presence of calcium bilirubinate in the pigment gallstones. Ca ions play a vital role in many if not most metabolic processes. Ca leaves the extra-cellular fluid via the gastrointestinal tract, kidneys, and skin and enters into bone via bone formation [26]. True absorbed Ca by the gastrointestinal tract is the total Ca absorbed from the calcium pool in the intestines and therefore contains both dietary and digestive juice components. In adults, the rate of Ca absorption from the gastrointestinal tract needs to match the rate of all losses from the body if the skeleton is to be preserved; in children and adolescents, an extra input is needed to cover the requirements of skeletal growth. Average concentration of Fe in south Indian pigment gallstones is about 2.5 times more than that of the east Indian gallstones whereas concentration of Fe is still higher in other parts of south India as analysed by Ashok et al. Absorption from the gastrointestinal tract is the primary homeostatic mechanism for iron. With respect to the mechanism of absorption, there are two kinds of dietary iron: heme iron and non-heme iron [27]. In the human diet the primary sources of heme iron are the haemoglobin and myoglobin from consumption of meat, poultry, and fish whereas non-heme iron is obtained from cereals, pulses, legumes, fruits, and vegetables [28]. The absorption of heme iron can vary from about 40% during iron deficiency to about 10% during iron repletion [29]. Fe absorption decreases until an equilibrium is established between absorption and requirements. For a given diet this regulation of Fe absorption, however, can only balance losses up to a certain critical point beyond which iron deficiency will develop [30]. About half of the basal Fe losses are from blood, primarily in the gastrointestinal tract. Absorption from the gastrointestinal tract is the primary homeostatic mechanism for iron. Since the concentration of Fe is
Table 1 Average concentration (in ppm by weight) of various elements in Indian pigment gallstone samples by PIGE and PIXE Elements
Eastern India (n = 12) (current study)
Southern (Chennai region) India (n = 15) (current study)
Southern India (n = 10) (Ashok et al.) [4]
By PIGE
Na Mg Al
611 5567 277
1122 16,653 252
– – –
Estimated by PIXE
P S Cl K Ca Ti V Cr Mn Fe Ni Cu Zn Br Pb
3433 15,221 5593 2022 11,898 26 35 96 138 368 18 1448 70 15 6
3621 14,702 5551 3417 19,434 98 32 179 304 898 12 4581 141 36 34
– – – 3339 50,701 17 – 29 737 2502 – 7705 265 1085 129
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170 ppm in tamarind [20], higher intake of tamarind gives rise to higher concentration of Fe in the south Indian pigment gallstones. In a hospital based case controlled study [31] of 346 patients, it was also reported that dietary parameters did not differ between patients and controls, except for the use of tamarind (Garcinia camborginia) more than equal to 3 times a week, as determined by univariate (v2 trend = 4.73, odds ratio 1.76; 95% confidence interval 1.05, 2.96; p = 0.03) and multivariate (p = 0.02) analysis. Most of the south Indian states consume tamarind as a common ingredient in their regular food which may be the primary cause leading to the higher concentration of Fe in their gallstones by gastrointestinal absorption from such diets. Average concentration of Cu in south Indian pigment gallstones is about three times more than that of the east Indian gallstones whereas Cu concentration in other parts of south India as analysed by Ashok et al. is about 1.7 times higher than that of southern region gallstone in the present study. Higher amount of Cu in the pigment gallstones may be due to presence of copper bilirubinate which gives the pigment gallstones the black colour. Transport of Cu from the liver into the bile is the primary route for excretion of endogenous copper. Copper of biliary origin and non-absorbed dietary Cu are eliminated from the body via the feces. The absorption and retention of Cu varies with dietary intake and status [32,33]. Intake of beverages with elevated Cu concentrations can induce acute gastrointestinal symptoms, such as epigastric pain, nausea, vomiting, and diarrhea. However, Cu concentrations at which these symptoms appear and the scope of responses observed are not clear [34]. For this reason, the threshold for gastrointestinal symptoms of Cu in beverages has not been precisely established in controlled prospective studies. Both copper sulfate (a soluble compound) and copper oxide (an insoluble compound) have comparable effects, implying that the ionic Cu present in the stomach is responsible for the induction of gastrointestinal manifestations [35]. Average concentration of Zn in southern region pigment gallstones is about twice more than the average concentration of east Indian stones and is about 0.5 times as reported by Ashok et al. Zn is lost from the body through the kidneys, skin, and intestine. The endogenous intestinal losses can vary from 7 lmol/day (0.5 mg/day) to more than 45 lmol/day (3 mg/day), depending on Zn intake [36]. The utilisation of Zn depends on the overall composition of the diet. Experimental studies have identified a number of dietary factors as potential promoters or antagonists of gastrointestinal Zn absorption. The risk for competitive interactions seems mainly to be related to high doses in the form of supplements or in aqueous solutions. However, at levels present in food and at realistic fortification levels, Zn absorption appears not to be affected, for example, by Fe and Cu [37]. Long-term Zn intakes higher than the requirements could, however, interact with the metabolism of
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other trace elements. Cu seems to be especially sensitive to high Zn doses. A Zn intake of 50 mg/day (760 lmol) affects Cu status indexes, such as CuZn superoxide dismutase in erythrocytes [38,39]. Because Cu also has a central role in immune defence, these observations call for caution before large-scale Zn supplementation programmes are undertaken. Any positive effects of Zn supplementation on growth or infectious diseases could be disguised or counterbalanced by negative effects on copper related functions. The average concentrations of Cr in south Indian gallstones is about twice more than that of eastern region gallstones, whereas it is about six times more than as reported by Ashok et al. So, the reason for less amount of Cr in south Indian gallstones is yet to be fully understood. The comparison of average concentrations of various elements is depicted in Fig. 5. In the present investigation, average concentration of Br in southern region gallstones is more than twice than eastern region. However, average Br concentration in other parts of south India is about 30 times than southern region pigment gallstones which is not understood properly. Figs. 6 and 7 show the FTIR spectrum of both the regions. The characteristic bands namely 2932, 2865, 1459, 1378 and 1054 cm1 are due to cholesterol in the pigment gallstones of both the eastern and southern regions. The calcium bilirubinate has characteristic bands at 1660, 1626, 1559 and 1254 cm1 which are assigned to ( C@C, C–N, C@O) stretching vibration of lactam, (C@O) stretching of COOH; (C@O, C–N, C@C) stretching, asymmetric stretching tas (COO) and (C–O) stretching or C–N stretching coupled with NH deformation t(C–N) + d(NH), respectively [40]. The band at 3409 cm1 is due to NH stretching vibration of pyrrole of bilirubin. Calcium bilirubinate is the main component of pigment gallstones. At present no crystal structure of Calcium bilirubinate is available.
Fig. 5. Concentration of elements in different regions.
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Fig. 8. TG-DTG spectrum of an east indian pigment gallstone.
Fig. 6. FTIR spectrum of an east indian pigment gallstone.
Fig. 9. TG-DTG spectrum of a south east indian pigment gallstone.
Fig. 7. FTIR spectrum of a south indian pigment gallstone.
The bands due to phosphate ðPO3 4 Þ ions are 1105, 1037, 963, 605 and 563 cm1, which may be due to presence of calcium phosphate in the pigment gallstones. The band visible at 1251 cm1 may be due to amide III which overlaps with the calcium bilirubinate band. The very broad bands in the spectra of the pigment gallstones are said to be indicative of multiple metal ions (Mg, Ca, Fe, Cu) present in high concentrations. Longitudinal optic mode frequency of MgO is observed at 796 cm1. The TG and DTG curves, (Figs. 8 and 9), from eastern and southern India, respectively, show that the pigment gallstone samples are thermally unstable early in the curve i.e. between 25 °C and 70 °C. After this temperature, the gallstone samples are stable up to 210 °C. Although all samples were dried during sample preparation, the sam-
ples still contain a little inherent water, which is evaporated below around 150 °C. The thermal decomposition observed in the TG and DTG curves occurs in two consecutive steps, between 210 °C and 446 °C (east Indian gallstones) and 210 °C and 498 °C (in southern region gallstones). In the south Indian gallstones, the first weight loss up to 336 °C occurs through a fast process with weight loss of 42%. This region is related to primary devolatilization, during which carbon, hydrogen and oxygen compounds are released. The observed weight loss in this region may be suggested that this residue is composed mainly of carbonaceous material formed only in the presence of oxygen. The second weight loss begins with a very slow process with weight loss of 48%. But, in the east Indian gallstones, the weight loss up to 362 °C occurs in a very fast process with weight loss of 78%, whereas the weight loss between 362 °C and 446 °C occurs very slowly with a weight loss of 88%. After weight loss of 88% it was observed that there was no more thermal degradation, which may be ascribed due to stability of chemical substances like calcium bilirubinate,
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calcium phosphate or hydroxyapatite present in the pigment gallstones in this range of temperature. The weight loss of pigment stones in east Indian gallstones was 88%, whereas it was only 48% incase of south Indian gallstones. As evidenced by PIXE technique, the concentration of Ca in south Indian pigment gallstones is more than 1.6 times than that of east Indian pigment gallstones, it is expected that the concentrations of different Ca compounds may be higher in the south Indian pigment stones. Moreover, FTIR results suggests that calcium phosphate may be present in the pigment gallstones. Higher amount of calcium phosphate will give rise to more thermal stability of the pigment gallstone. So, the thermal degradation of south Indian pigment gallstones does not fall below 48% due to presence of higher amount of calcium phosphate or hydroxyapatite present in it. This shows that the south Indian pigment stones contains compounds which are not degradable even at 525 °C. 4. Conclusion Twenty-seven Indian pigment gallstone samples were analysed by PIXE and PIGE and minor/trace elements namely Na, Mg, Al, P, S, Cl, K, Ca, Ti, V, Cr, Mn, Fe, Ni, Cu, Zn, Br and Pb were quantified. Our study reveals that average concentration of all elements except Ni in south Indian pigment gallstone samples is higher than that of corresponding values in east Indian pigment gallstone samples whereas elements like Al, P, S, Cl and V did not show much variation between these two regions. The reason for this may be due to different types of food habits of the patients in these two regions. The higher concentration of Fe in south Indian pigment gallstone samples may be due to intake of tamarind as their regular food. While the presence of different functional groups was detected by FTIR analysis, TG and DTG curves provided information on the thermal decompositions of these compounds. Acknowledgements The help extended by Dr. Md. Ibrarullah, Dept. of Gastroenterology, Kalinga Hospital, Bhubaneswar and Dr. V. Jayanthi, Dept. of Gastroenterology, Stanley Medical College Hospital, Chennai for providing samples are acknowledged and thanks to Prof. S.N. Sahu and Mr. S.N. Sarangi, Institute of Physics, Bhubaneswar for using FTIR facility. We are grateful to Prof. S. Jena and Mr. D.K. Ray, Dept. of Chemistry, Utkal University, Bhubaneswar for their help in carrying out thermogravimetric analysis. References [1] T. Maki, Arch. Surg. 82 (1961) 599. [2] T. Markkanen, A.J. Aho, Acta Chir. Scand. 138 (1972) 30.
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Nuclear Instruments and Methods in Physics Research B 255 (2007) 409–415 www.elsevier.com/locate/nimb
Analysis of Indian pigment gallstones T.R. Rautray a
a,*
, V. Vijayan b, S. Panigrahi
a
Department of Physics, National Institute of Technology, Rourkela 769 008, Orissa, India b Institute of Physics, Bhubaneswar 751 005, Orissa, India Received 16 August 2006; received in revised form 17 November 2006 Available online 5 January 2007
Abstract Particle induced X-ray emission and particle induced c-ray emission spectroscopic techniques have been carried out to analyse the elemental concentrations of human pigment gallstone samples from eastern region (Orissa) and southern region (Chennai) of India. It was observed that 18 minor/trace elements namely Na, Mg, Al, P, S, Cl, K, Ca, Ti, V, Cr, Mn, Fe, Ni, Cu, Zn, Br and Pb were present in the pigment gallstone samples of both the regions. Our study reveals that average concentration of all elements except Ni in south Indian pigment gallstone samples is higher than that of corresponding values in east Indian pigment gallstone samples whereas elements like Al, P, S, Cl and V did not show much variation between these two regions. Fourier transform infra-red analysis was carried out to identify the functional groups and the classification of the pigment type gallstones of both the regions. The thermal behaviour of pigment gallstones was carried out by thermogravimetry-derivative thermogravimetry analysis. Ó 2006 Elsevier B.V. All rights reserved. PACS: 87.64.Ni; 87.68.+z; 87.64.Gb; 78.30.Er; 65.60.+a Keywords: PIXE; PIGE; FTIR; TG-DTG; Calcium bilirubinate
1. Introduction Formation of gallstones is still one of the most common digestive diseases in the world. Since the pathogenesis of gallstones is not yet clearly understood, its analysis-using chemical and spectroscopic techniques have provided some clues. In gallstone disease, trace elements are believed to play an important role in stone formation [1,2]. They are broadly classified into three categories according to their composition namely cholesterol type (cholesterol rich), pigment type (bilirubin and calcium bilirubinate rich) and mixed type (combination of cholesterol, calcium carbonate, calcium bilirubinate) [3]. In particular, septic complications are much more common in patients with pigment gallstones than in patients with cholesterol gallstones. While cholesterol gallstones are due to supersaturated bile, the
*
Corresponding author. Tel.: +91 9853262167; fax: +91 674 2300142. E-mail address: [email protected] (T.R. Rautray).
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black pigment gallstones are common in haemolytic anemia (secondary to excessive degradation of red blood cells) or in the presence of infected bile. In the absence of these known precipitating factors, the pathogenesis of pigment or mixed gallstone formation in Indian patients remains unclear [4]. Trace elements play a significant role in, or provide an indication of possible mechanisms of stone formation. The relative importance of the elements in stone formation was subsequently evaluated based on the statistical analysis of the data obtained. In general, the variation of chemical composition in different gallstones varies significantly in different parts of India. Food habits may be one of the main reasons. Cholesterol gallstones are predominant in the northern, eastern and western parts of India, while pigment gallstones are common in the southern region [5]. Despite decades of research, the formation mechanism of gallstones remains incompletely understood. The study of stone formation inside the gall bladder becomes one of the major problems for the contemporary medicine. It was found that the principal components of
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human gallstone are cholesterol and bilirubin in the form of various bilirubinate salts. Other compounds that have been found in gallstones include calcium carbonate, calcium phosphate salts, calcium bilirubinate, fatty salts, various cholic acid derivatives, polysaccharide and proteins [3,6]. Metals in the human body are metabolized mainly via the kidney and liver and excreted through urine and partly by bile. Changes in the composition of bile may induce cholecystitis and cholelithiasis [7]. Pigment gallstones can be further classified into brown and black stones. Brown stones have a characteristic appearance and are typical in Asia. These stones occur as a result of infection. Black stones generally are not associated with infected bile. These stones are found in patients with hemolytic disorders or cirrhosis [8]. The toxic elements are known to be very harmful even at extremely low concentrations. For this reason, reliable analyses will help to clarify and define effective treatment. Factors responsible for the formation of stones include altered hepatic bile composition, biliary glycoprotein, infection, age, genetic, sex, estrogens, dietary factors, geographical prevalence and cirrhosis of the liver [9]. Since particle induced X-ray emission (PIXE) and particle induced gamma-ray emission (PIGE) techniques are simultaneous, reliable, rapid, multi-elemental, sensitive and non-destructive in nature to analyse even few ppm level elements [10], the present investigation was carried out for the pigment gallstone samples. The presence of functional groups was confirmed by Fourier transform infra-red (FTIR) analysis and the thermal behaviour was studied by thermogravimetry-derivative thermogravimetry (TG-DTG) analysis for the pigment gallstones of both the eastern and southern regions of India. 2. Experimental 2.1. Sample preparation The east Indian and the south Indian pigment gallstone samples of both sexes were collected from Kalinga Hospital, Bhubaneswar, Orissa and Stanley Medical College, Chennai, respectively, in the age group of 33–68 years. Fifteen pigment gallstone samples of different patients were taken from various places of a south Indian state and 12 pigment gallstone samples of different patients were taken from different places of an east Indian state. The pigment gallstones samples were washed with deionised water 5–6 times and heated at 40 °C in an oven for 8 h. After drying, the gallstone samples were crushed to make pellets. For PIXE and PIGE analysis, 150 mg of powdered samples were mixed with 150 mg of high pure graphite powder in 1:1 ratio by mass to make pellets of size 13 mm diameter in a hydraulic pressure. The aim of adding graphite powder is that it can be used as a binder of the homogenized powdered sample as well as to monitor the beam current since graphite is a good conductor of electricity and another purpose of adding graphite is to reduce bremsstrahlung background of PIXE spectrum [10]. Thick targets have been
prepared by pressing the above mixture with a pelletizer. Pellets of NIST bovine liver (1577b) and IAEA animal blood (A-13) were also prepared by the same procedure for calibration purposes. For FTIR measurements, 100 mg of KBr powder was thoroughly mixed with 10 mg of pigment gallstones to make a pellet of size 13-mm diameter and 250 mg of dried pure powdered gallstones were taken for the TG-DTG measurements.
2.2. PIXE, PIGE, FTIR and TGDTG measurements Simultaneous PIXE–PIGE analysis was carried out using the 3 MV horizontal Tandem Pelletron accelerator (9SDH-2, NEC, USA) at Institute of Physics, Bhubaneswar, India. The proton beam was collimated to a diameter of 2 mm on the target in vacuum (106 Torr) inside a PIXE chamber. The multiple-target holder was placed at 45° to the beam direction. The target holder was mounted on an insulated stand and is surrounded by a cylindrical electron suppressor held at negative potential with respect to the target. Integrated charge on the thick sample was measured using a current integrator, which was connected to the target holder. The targets were bombarded with 3 MeV proton beams with 20 nA beam current. A Si (Li) detector (active area 30 mm2) with a resolution of 170 eV at 5.9 keV with beryllium window placed at 90° to the beam direction was used to detect the characteristic X-rays emitted from the targets. The X-rays exit the scattering chamber through a 95 lm Mylar window before entering the detector. Spectra were recorded by using a multi-channel analyser calibrated with 241Am X-ray source [11]. No Xray absorbers were used between the detector and target during data collection. For PIGE analysis, a high purity germanium (HPGe) detector with a resolution of 1.9 keV at 1.33 MeV placed at an angle 135° to the beam direction was also simultaneously used in the scattering chamber for the gamma ray measurements. A 60Co c-ray source was used for the c-energy calibration. In PIGE, inelastic scattering of protons follows de-excitation of low-lying nuclear excited states resulting in the emission of characteristic c-rays of a particular nuclide that serves as a basis for qualitative as well as quantitative assessment of a particular element [12]. The concentration of low Z elements like Na, Mg and Al were analysed by PIGE whereas other elements were analysed by PIXE technique. The PIXE spectral analyses were performed using GUPIX-2000 software [13]. This provides a non-linear least square fitting of the spectrum, together with subsequent conversion of the fitted X-ray peak intensities into elemental concentrations, utilising the fundamental parameter method (FPM) for quantitative analysis. The uncertainty in the concentration estimation for the minor elements such as Na, Mg, P, S, Cl, K, Ca, Mn, Fe and Cu is found to be between 8% and 10%, whereas for the trace elements it is found to be between 17% and 20%.
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The PIGE spectral analysis was done using the ANGES (IAEA, Vienna) software [14]. The chemical analysis of the pigment gallstones, in nitrogen gas atmosphere, was carried out by a FTIR spectrophotometer [15] (Thermo Nicolet Avatar 370, USA) to identify the functional groups and the measurements were carried out in the mid-infrared range (4000–400 cm1) at the resolution of 4 cm1 in the transmission mode. The collimator through which the infrared beam passes before falling on the sample is 0.5-mm in breadth and 10-mm in length. TG-DTG analysis was performed using the powdered samples in a Shimadzu thermal analyser. The weight loss of the sample was monitored under air atmosphere. Each sample was heated under air atmosphere [16] at a linear heating rate of 10 °C min1 from 25 °C to 525 °C. 3. Results and discussion
Fig. 2. PIXE spectrum of a south indian pigment gallstone.
Two representative PIXE spectra of pigment gallstone samples of eastern India and southern India are shown in Figs. 1 and 2, respectively, and two representative PIGE spectra of pigment gallstones of eastern and southern India are shown in Figs. 3 and 4, respectively. The spectra obtained from all the pigment gallstone samples were analysed. The elemental concentrations of the east and south Indian pigment gallstones are shown in Table 1, which is compared with the literature values of south Indian gallstones by Ashok et al. [4]. It was observed that 18 minor/ trace elements namely Na, Mg, Al, P, S, Cl, K, Ca, Ti, V, Cr, Mn, Fe, Ni, Cu, Zn, Br and Pb were present in the pigment gallstone samples. Average concentration of Mg in southern region pigment gallstones are about 2.3 times more than that of the east Indian pigment gallstones. Since high intakes of dietary fibre (40–50 g/day) leads to lower Mg absorption, this may be the reason for the low Mg concentration in the east Fig. 3. PIGE spectrum of an east indian pigment gallstone.
Fig. 1. PIXE spectrum of an east indian pigment gallstone.
Indian gallstones. This is probably attributable to the Mgbinding action of phytate phosphorus associated with the fibre [17–19]. Rice is a regular and primary food consumed in both the eastern and southern regions. Since, the Mg content of Indian milled rice is 380 ppm, the higher values of Mg in the pigment gallstones of both the regions may be due to rice consumption. However, just like rice, tamarind is consumed in higher amount as a regular basis in south India. Since the concentration of Mg in Indian tamarind is 410 ppm [20], higher intake of tamarind gives rise to higher concentration of Mg in the south Indian pigment gallstones. Even though the concentration of P (1100 ppm) is also high in tamarind, concentration of P does not show much variation between the two regions which may be ascribed due to the presence of calcium phosphate or hydroxyapatite in both types of pigment gallstones as evidenced by FTIR.
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Fig. 4. PIGE spectrum of a south indian pigment gallstone.
However, consumption of phytate- and cellulose-rich products (usually containing high concentrations of Mg) increases Mg intake, which often compensates for the decrease in gastrointestinal absorption. The effects of dietary components such as phytate on Mg absorption are probably critically important only at low magnesium intake. There is no consistent evidence that modest increases in the intake of Ca [21–23], Fe, or Mn [24] affect Mg balance. In contrast, high intakes of zinc (142 mg/day) decrease Mg absorption and contribute to a shift toward negative balance [25]. Concentration of Ca in southern region pigment gallstones is more than one and half times than that of eastern region, but it is less than half than that reported by Ashok et al. From among the detected elements, Ca has the high-
est average concentration which is because of presence of calcium bilirubinate in the pigment gallstones. Ca ions play a vital role in many if not most metabolic processes. Ca leaves the extra-cellular fluid via the gastrointestinal tract, kidneys, and skin and enters into bone via bone formation [26]. True absorbed Ca by the gastrointestinal tract is the total Ca absorbed from the calcium pool in the intestines and therefore contains both dietary and digestive juice components. In adults, the rate of Ca absorption from the gastrointestinal tract needs to match the rate of all losses from the body if the skeleton is to be preserved; in children and adolescents, an extra input is needed to cover the requirements of skeletal growth. Average concentration of Fe in south Indian pigment gallstones is about 2.5 times more than that of the east Indian gallstones whereas concentration of Fe is still higher in other parts of south India as analysed by Ashok et al. Absorption from the gastrointestinal tract is the primary homeostatic mechanism for iron. With respect to the mechanism of absorption, there are two kinds of dietary iron: heme iron and non-heme iron [27]. In the human diet the primary sources of heme iron are the haemoglobin and myoglobin from consumption of meat, poultry, and fish whereas non-heme iron is obtained from cereals, pulses, legumes, fruits, and vegetables [28]. The absorption of heme iron can vary from about 40% during iron deficiency to about 10% during iron repletion [29]. Fe absorption decreases until an equilibrium is established between absorption and requirements. For a given diet this regulation of Fe absorption, however, can only balance losses up to a certain critical point beyond which iron deficiency will develop [30]. About half of the basal Fe losses are from blood, primarily in the gastrointestinal tract. Absorption from the gastrointestinal tract is the primary homeostatic mechanism for iron. Since the concentration of Fe is
Table 1 Average concentration (in ppm by weight) of various elements in Indian pigment gallstone samples by PIGE and PIXE Elements
Eastern India (n = 12) (current study)
Southern (Chennai region) India (n = 15) (current study)
Southern India (n = 10) (Ashok et al.) [4]
By PIGE
Na Mg Al
611 5567 277
1122 16,653 252
– – –
Estimated by PIXE
P S Cl K Ca Ti V Cr Mn Fe Ni Cu Zn Br Pb
3433 15,221 5593 2022 11,898 26 35 96 138 368 18 1448 70 15 6
3621 14,702 5551 3417 19,434 98 32 179 304 898 12 4581 141 36 34
– – – 3339 50,701 17 – 29 737 2502 – 7705 265 1085 129
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170 ppm in tamarind [20], higher intake of tamarind gives rise to higher concentration of Fe in the south Indian pigment gallstones. In a hospital based case controlled study [31] of 346 patients, it was also reported that dietary parameters did not differ between patients and controls, except for the use of tamarind (Garcinia camborginia) more than equal to 3 times a week, as determined by univariate (v2 trend = 4.73, odds ratio 1.76; 95% confidence interval 1.05, 2.96; p = 0.03) and multivariate (p = 0.02) analysis. Most of the south Indian states consume tamarind as a common ingredient in their regular food which may be the primary cause leading to the higher concentration of Fe in their gallstones by gastrointestinal absorption from such diets. Average concentration of Cu in south Indian pigment gallstones is about three times more than that of the east Indian gallstones whereas Cu concentration in other parts of south India as analysed by Ashok et al. is about 1.7 times higher than that of southern region gallstone in the present study. Higher amount of Cu in the pigment gallstones may be due to presence of copper bilirubinate which gives the pigment gallstones the black colour. Transport of Cu from the liver into the bile is the primary route for excretion of endogenous copper. Copper of biliary origin and non-absorbed dietary Cu are eliminated from the body via the feces. The absorption and retention of Cu varies with dietary intake and status [32,33]. Intake of beverages with elevated Cu concentrations can induce acute gastrointestinal symptoms, such as epigastric pain, nausea, vomiting, and diarrhea. However, Cu concentrations at which these symptoms appear and the scope of responses observed are not clear [34]. For this reason, the threshold for gastrointestinal symptoms of Cu in beverages has not been precisely established in controlled prospective studies. Both copper sulfate (a soluble compound) and copper oxide (an insoluble compound) have comparable effects, implying that the ionic Cu present in the stomach is responsible for the induction of gastrointestinal manifestations [35]. Average concentration of Zn in southern region pigment gallstones is about twice more than the average concentration of east Indian stones and is about 0.5 times as reported by Ashok et al. Zn is lost from the body through the kidneys, skin, and intestine. The endogenous intestinal losses can vary from 7 lmol/day (0.5 mg/day) to more than 45 lmol/day (3 mg/day), depending on Zn intake [36]. The utilisation of Zn depends on the overall composition of the diet. Experimental studies have identified a number of dietary factors as potential promoters or antagonists of gastrointestinal Zn absorption. The risk for competitive interactions seems mainly to be related to high doses in the form of supplements or in aqueous solutions. However, at levels present in food and at realistic fortification levels, Zn absorption appears not to be affected, for example, by Fe and Cu [37]. Long-term Zn intakes higher than the requirements could, however, interact with the metabolism of
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other trace elements. Cu seems to be especially sensitive to high Zn doses. A Zn intake of 50 mg/day (760 lmol) affects Cu status indexes, such as CuZn superoxide dismutase in erythrocytes [38,39]. Because Cu also has a central role in immune defence, these observations call for caution before large-scale Zn supplementation programmes are undertaken. Any positive effects of Zn supplementation on growth or infectious diseases could be disguised or counterbalanced by negative effects on copper related functions. The average concentrations of Cr in south Indian gallstones is about twice more than that of eastern region gallstones, whereas it is about six times more than as reported by Ashok et al. So, the reason for less amount of Cr in south Indian gallstones is yet to be fully understood. The comparison of average concentrations of various elements is depicted in Fig. 5. In the present investigation, average concentration of Br in southern region gallstones is more than twice than eastern region. However, average Br concentration in other parts of south India is about 30 times than southern region pigment gallstones which is not understood properly. Figs. 6 and 7 show the FTIR spectrum of both the regions. The characteristic bands namely 2932, 2865, 1459, 1378 and 1054 cm1 are due to cholesterol in the pigment gallstones of both the eastern and southern regions. The calcium bilirubinate has characteristic bands at 1660, 1626, 1559 and 1254 cm1 which are assigned to ( C@C, C–N, C@O) stretching vibration of lactam, (C@O) stretching of COOH; (C@O, C–N, C@C) stretching, asymmetric stretching tas (COO) and (C–O) stretching or C–N stretching coupled with NH deformation t(C–N) + d(NH), respectively [40]. The band at 3409 cm1 is due to NH stretching vibration of pyrrole of bilirubin. Calcium bilirubinate is the main component of pigment gallstones. At present no crystal structure of Calcium bilirubinate is available.
Fig. 5. Concentration of elements in different regions.
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Fig. 8. TG-DTG spectrum of an east indian pigment gallstone.
Fig. 6. FTIR spectrum of an east indian pigment gallstone.
Fig. 9. TG-DTG spectrum of a south east indian pigment gallstone.
Fig. 7. FTIR spectrum of a south indian pigment gallstone.
The bands due to phosphate ðPO3 4 Þ ions are 1105, 1037, 963, 605 and 563 cm1, which may be due to presence of calcium phosphate in the pigment gallstones. The band visible at 1251 cm1 may be due to amide III which overlaps with the calcium bilirubinate band. The very broad bands in the spectra of the pigment gallstones are said to be indicative of multiple metal ions (Mg, Ca, Fe, Cu) present in high concentrations. Longitudinal optic mode frequency of MgO is observed at 796 cm1. The TG and DTG curves, (Figs. 8 and 9), from eastern and southern India, respectively, show that the pigment gallstone samples are thermally unstable early in the curve i.e. between 25 °C and 70 °C. After this temperature, the gallstone samples are stable up to 210 °C. Although all samples were dried during sample preparation, the sam-
ples still contain a little inherent water, which is evaporated below around 150 °C. The thermal decomposition observed in the TG and DTG curves occurs in two consecutive steps, between 210 °C and 446 °C (east Indian gallstones) and 210 °C and 498 °C (in southern region gallstones). In the south Indian gallstones, the first weight loss up to 336 °C occurs through a fast process with weight loss of 42%. This region is related to primary devolatilization, during which carbon, hydrogen and oxygen compounds are released. The observed weight loss in this region may be suggested that this residue is composed mainly of carbonaceous material formed only in the presence of oxygen. The second weight loss begins with a very slow process with weight loss of 48%. But, in the east Indian gallstones, the weight loss up to 362 °C occurs in a very fast process with weight loss of 78%, whereas the weight loss between 362 °C and 446 °C occurs very slowly with a weight loss of 88%. After weight loss of 88% it was observed that there was no more thermal degradation, which may be ascribed due to stability of chemical substances like calcium bilirubinate,
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calcium phosphate or hydroxyapatite present in the pigment gallstones in this range of temperature. The weight loss of pigment stones in east Indian gallstones was 88%, whereas it was only 48% incase of south Indian gallstones. As evidenced by PIXE technique, the concentration of Ca in south Indian pigment gallstones is more than 1.6 times than that of east Indian pigment gallstones, it is expected that the concentrations of different Ca compounds may be higher in the south Indian pigment stones. Moreover, FTIR results suggests that calcium phosphate may be present in the pigment gallstones. Higher amount of calcium phosphate will give rise to more thermal stability of the pigment gallstone. So, the thermal degradation of south Indian pigment gallstones does not fall below 48% due to presence of higher amount of calcium phosphate or hydroxyapatite present in it. This shows that the south Indian pigment stones contains compounds which are not degradable even at 525 °C. 4. Conclusion Twenty-seven Indian pigment gallstone samples were analysed by PIXE and PIGE and minor/trace elements namely Na, Mg, Al, P, S, Cl, K, Ca, Ti, V, Cr, Mn, Fe, Ni, Cu, Zn, Br and Pb were quantified. Our study reveals that average concentration of all elements except Ni in south Indian pigment gallstone samples is higher than that of corresponding values in east Indian pigment gallstone samples whereas elements like Al, P, S, Cl and V did not show much variation between these two regions. The reason for this may be due to different types of food habits of the patients in these two regions. The higher concentration of Fe in south Indian pigment gallstone samples may be due to intake of tamarind as their regular food. While the presence of different functional groups was detected by FTIR analysis, TG and DTG curves provided information on the thermal decompositions of these compounds. Acknowledgements The help extended by Dr. Md. Ibrarullah, Dept. of Gastroenterology, Kalinga Hospital, Bhubaneswar and Dr. V. Jayanthi, Dept. of Gastroenterology, Stanley Medical College Hospital, Chennai for providing samples are acknowledged and thanks to Prof. S.N. Sahu and Mr. S.N. Sarangi, Institute of Physics, Bhubaneswar for using FTIR facility. We are grateful to Prof. S. Jena and Mr. D.K. Ray, Dept. of Chemistry, Utkal University, Bhubaneswar for their help in carrying out thermogravimetric analysis. References [1] T. Maki, Arch. Surg. 82 (1961) 599. [2] T. Markkanen, A.J. Aho, Acta Chir. Scand. 138 (1972) 30.
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