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Nuclear Instruments and Methods in Physics Research B 255 (2007) 409–415

NIMBBeam Interactions

with Materials & Atoms

Analysis of Indian pigment gallstones

T.R. Rautray a,*, V. Vijayan b, S. Panigrahi a

a Department of Physics, National Institute of Technology, Rourkela 769 008, Orissa, Indiab Institute of Physics, Bhubaneswar 751 005, Orissa, India

Received 16 August 2006; received in revised form 17 November 2006Available online 5 January 2007

Abstract

Particle induced X-ray emission and particle induced c-ray emission spectroscopic techniques have been carried out to analyse theelemental 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 presentin the pigment gallstone samples of both the regions. Our study reveals that average concentration of all elements except Ni in southIndian pigment gallstone samples is higher than that of corresponding values in east Indian pigment gallstone samples whereas elementslike Al, P, S, Cl and V did not show much variation between these two regions. Fourier transform infra-red analysis was carried out toidentify the functional groups and the classification of the pigment type gallstones of both the regions. The thermal behaviour of pigmentgallstones was carried out by thermogravimetry-derivative thermogravimetry analysis.� 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 commondigestive diseases in the world. Since the pathogenesis ofgallstones is not yet clearly understood, its analysis-usingchemical and spectroscopic techniques have provided someclues. In gallstone disease, trace elements are believed toplay an important role in stone formation [1,2]. They arebroadly classified into three categories according to theircomposition namely cholesterol type (cholesterol rich), pig-ment type (bilirubin and calcium bilirubinate rich) andmixed type (combination of cholesterol, calcium carbonate,calcium bilirubinate) [3]. In particular, septic complicationsare much more common in patients with pigment gall-stones than in patients with cholesterol gallstones. Whilecholesterol gallstones are due to supersaturated bile, the

0168-583X/$ - see front matter � 2006 Elsevier B.V. All rights reserved.

doi:10.1016/j.nimb.2006.12.147

* Corresponding author. Tel.: +91 9853262167; fax: +91 674 2300142.E-mail address: tapash77@hotmail.com (T.R. Rautray).

black pigment gallstones are common in haemolytic ane-mia (secondary to excessive degradation of red blood cells)or in the presence of infected bile. In the absence of theseknown precipitating factors, the pathogenesis of pigmentor mixed gallstone formation in Indian patients remainsunclear [4]. Trace elements play a significant role in, or pro-vide an indication of possible mechanisms of stone forma-tion. The relative importance of the elements in stoneformation was subsequently evaluated based on the statis-tical analysis of the data obtained. In general, the variationof chemical composition in different gallstones varies sig-nificantly in different parts of India. Food habits may beone of the main reasons. Cholesterol gallstones are pre-dominant in the northern, eastern and western parts ofIndia, while pigment gallstones are common in the south-ern region [5]. Despite decades of research, the formationmechanism of gallstones remains incompletely understood.The study of stone formation inside the gall bladderbecomes one of the major problems for the contemporarymedicine. It was found that the principal components of

410 T.R. Rautray et al. / Nucl. Instr. and Meth. in Phys. Res. B 255 (2007) 409–415

human gallstone are cholesterol and bilirubin in the formof various bilirubinate salts. Other compounds that havebeen found in gallstones include calcium carbonate, cal-cium phosphate salts, calcium bilirubinate, fatty salts, var-ious cholic acid derivatives, polysaccharide and proteins[3,6]. Metals in the human body are metabolized mainlyvia the kidney and liver and excreted through urine andpartly by bile. Changes in the composition of bile mayinduce cholecystitis and cholelithiasis [7]. Pigment gall-stones can be further classified into brown and blackstones. Brown stones have a characteristic appearanceand are typical in Asia. These stones occur as a result ofinfection. Black stones generally are not associated withinfected bile. These stones are found in patients with hemo-lytic disorders or cirrhosis [8]. The toxic elements areknown to be very harmful even at extremely low concentra-tions. For this reason, reliable analyses will help to clarifyand define effective treatment. Factors responsible for theformation of stones include altered hepatic bile composi-tion, biliary glycoprotein, infection, age, genetic, sex, estro-gens, dietary factors, geographical prevalence and cirrhosisof the liver [9]. Since particle induced X-ray emission(PIXE) and particle induced gamma-ray emission (PIGE)techniques are simultaneous, reliable, rapid, multi-elemen-tal, sensitive and non-destructive in nature to analyse evenfew ppm level elements [10], the present investigation wascarried out for the pigment gallstone samples. The presenceof functional groups was confirmed by Fourier transforminfra-red (FTIR) analysis and the thermal behaviour wasstudied by thermogravimetry-derivative thermogravimetry(TG-DTG) analysis for the pigment gallstones of boththe eastern and southern regions of India.

2. Experimental

2.1. Sample preparation

The east Indian and the south Indian pigment gallstonesamples of both sexes were collected from Kalinga Hospi-tal, Bhubaneswar, Orissa and Stanley Medical College,Chennai, respectively, in the age group of 33–68 years. Fif-teen pigment gallstone samples of different patients weretaken from various places of a south Indian state and 12pigment gallstone samples of different patients were takenfrom different places of an east Indian state. The pigmentgallstones samples were washed with deionised water 5–6times and heated at 40 �C in an oven for 8 h. After drying,the gallstone samples were crushed to make pellets. ForPIXE and PIGE analysis, 150 mg of powdered sampleswere mixed with 150 mg of high pure graphite powder in1:1 ratio by mass to make pellets of size 13 mm diameterin a hydraulic pressure. The aim of adding graphite powderis that it can be used as a binder of the homogenized pow-dered sample as well as to monitor the beam current sincegraphite is a good conductor of electricity and another pur-pose of adding graphite is to reduce bremsstrahlung back-ground 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 animalblood (A-13) were also prepared by the same procedurefor calibration purposes.

For FTIR measurements, 100 mg of KBr powder wasthoroughly mixed with 10 mg of pigment gallstones tomake a pellet of size 13-mm diameter and 250 mg of driedpure powdered gallstones were taken for the TG-DTGmeasurements.

2.2. PIXE, PIGE, FTIR and TGDTG measurements

Simultaneous PIXE–PIGE analysis was carried outusing the 3 MV horizontal Tandem Pelletron accelerator(9SDH-2, NEC, USA) at Institute of Physics, Bhubane-swar, India. The proton beam was collimated to a diameterof 2 mm on the target in vacuum (10�6 Torr) inside a PIXEchamber. The multiple-target holder was placed at 45� tothe beam direction. The target holder was mounted on aninsulated stand and is surrounded by a cylindrical electronsuppressor held at negative potential with respect to thetarget. Integrated charge on the thick sample was measuredusing a current integrator, which was connected to the tar-get holder. The targets were bombarded with 3 MeV pro-ton beams with 20 nA beam current. A Si (Li) detector(active area 30 mm2) with a resolution of 170 eV at5.9 keV with beryllium window placed at 90� to the beamdirection was used to detect the characteristic X-rays emit-ted from the targets. The X-rays exit the scattering cham-ber through a 95 lm Mylar window before entering thedetector. Spectra were recorded by using a multi-channelanalyser calibrated with 241Am X-ray source [11]. No X-ray absorbers were used between the detector and targetduring data collection. For PIGE analysis, a high puritygermanium (HPGe) detector with a resolution of 1.9 keVat 1.33 MeV placed at an angle 135� to the beam directionwas also simultaneously used in the scattering chamber forthe gamma ray measurements. A 60Co c-ray source wasused for the c-energy calibration. In PIGE, inelastic scat-tering of protons follows de-excitation of low-lying nuclearexcited states resulting in the emission of characteristicc-rays of a particular nuclide that serves as a basis for qual-itative as well as quantitative assessment of a particular ele-ment [12]. The concentration of low Z elements like Na,Mg and Al were analysed by PIGE whereas other elementswere analysed by PIXE technique.

The PIXE spectral analyses were performed usingGUPIX-2000 software [13]. This provides a non-linearleast square fitting of the spectrum, together with subse-quent conversion of the fitted X-ray peak intensities intoelemental concentrations, utilising the fundamental param-eter method (FPM) for quantitative analysis. The uncer-tainty in the concentration estimation for the minorelements such as Na, Mg, P, S, Cl, K, Ca, Mn, Fe andCu is found to be between 8% and 10%, whereas for thetrace elements it is found to be between 17% and 20%.

T.R. Rautray et al. / Nucl. Instr. and Meth. in Phys. Res. B 255 (2007) 409–415 411

The PIGE spectral analysis was done using the ANGES(IAEA, Vienna) software [14].

The chemical analysis of the pigment gallstones, innitrogen gas atmosphere, was carried out by a FTIR spec-trophotometer [15] (Thermo Nicolet Avatar 370, USA) toidentify the functional groups and the measurements werecarried out in the mid-infrared range (4000–400 cm�1) atthe resolution of 4 cm�1 in the transmission mode. The col-limator through which the infrared beam passes before fall-ing on the sample is 0.5-mm in breadth and 10-mm inlength.

TG-DTG analysis was performed using the powderedsamples in a Shimadzu thermal analyser. The weight lossof the sample was monitored under air atmosphere. Eachsample was heated under air atmosphere [16] at a linearheating rate of 10 �C min�1 from 25 �C to 525 �C.

Fig. 2. PIXE spectrum of a south indian pigment gallstone.

3. Results and discussion

Two representative PIXE spectra of pigment gallstonesamples of eastern India and southern India are shown inFigs. 1 and 2, respectively, and two representative PIGEspectra of pigment gallstones of eastern and southern Indiaare shown in Figs. 3 and 4, respectively. The spectraobtained from all the pigment gallstone samples were ana-lysed. The elemental concentrations of the east and southIndian pigment gallstones are shown in Table 1, which iscompared with the literature values of south Indian gall-stones 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 inthe pigment gallstone samples.

Average concentration of Mg in southern region pig-ment gallstones are about 2.3 times more than that of theeast Indian pigment gallstones. Since high intakes of die-tary fibre (40–50 g/day) leads to lower Mg absorption, thismay be the reason for the low Mg concentration in the east

Fig. 1. PIXE spectrum of an east indian pigment gallstone.

Fig. 3. PIGE spectrum of an east indian pigment gallstone.

Indian gallstones. This is probably attributable to the Mg-binding action of phytate phosphorus associated with thefibre [17–19]. Rice is a regular and primary food consumedin both the eastern and southern regions. Since, the Mgcontent of Indian milled rice is 380 ppm, the higher valuesof Mg in the pigment gallstones of both the regions may bedue to rice consumption. However, just like rice, tamarindis consumed in higher amount as a regular basis in southIndia. Since the concentration of Mg in Indian tamarindis 410 ppm [20], higher intake of tamarind gives rise tohigher concentration of Mg in the south Indian pig-ment gallstones. Even though the concentration of P(1100 ppm) is also high in tamarind, concentration of Pdoes not show much variation between the two regionswhich may be ascribed due to the presence of calcium phos-phate or hydroxyapatite in both types of pigment gall-stones as evidenced by FTIR.

Fig. 4. PIGE spectrum of a south indian pigment gallstone.

412 T.R. Rautray et al. / Nucl. Instr. and Meth. in Phys. Res. B 255 (2007) 409–415

However, consumption of phytate- and cellulose-richproducts (usually containing high concentrations of Mg)increases Mg intake, which often compensates for thedecrease in gastrointestinal absorption. The effects of die-tary components such as phytate on Mg absorption areprobably critically important only at low magnesiumintake. There is no consistent evidence that modestincreases in the intake of Ca [21–23], Fe, or Mn [24] affectMg balance. In contrast, high intakes of zinc (142 mg/day)decrease Mg absorption and contribute to a shift towardnegative balance [25].

Concentration of Ca in southern region pigment gall-stones is more than one and half times than that of easternregion, but it is less than half than that reported by Ashoket al. From among the detected elements, Ca has the high-

Table 1Average concentration (in ppm by weight) of various elements in Indian pigm

Elements Eastern India (n = 12)(current study)

Southe(curren

By PIGE Na 611 1122Mg 5567 16,653Al 277 252

Estimated byPIXE

P 3433 3621S 15,221 14,702Cl 5593 5551K 2022 3417Ca 11,898 19,434Ti 26 98V 35 32Cr 96 179Mn 138 304Fe 368 898Ni 18 12Cu 1448 4581Zn 70 141Br 15 36Pb 6 34

est average concentration which is because of presence ofcalcium bilirubinate in the pigment gallstones. Ca ions playa vital role in many if not most metabolic processes. Caleaves 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 thetotal Ca absorbed from the calcium pool in the intestinesand therefore contains both dietary and digestive juicecomponents. In adults, the rate of Ca absorption fromthe gastrointestinal tract needs to match the rate of alllosses from the body if the skeleton is to be preserved; inchildren and adolescents, an extra input is needed to coverthe requirements of skeletal growth.

Average concentration of Fe in south Indian pigmentgallstones is about 2.5 times more than that of the eastIndian gallstones whereas concentration of Fe is still higherin other parts of south India as analysed by Ashok et al.Absorption from the gastrointestinal tract is the primaryhomeostatic mechanism for iron. With respect to the mech-anism of absorption, there are two kinds of dietary iron:heme iron and non-heme iron [27]. In the human diet theprimary sources of heme iron are the haemoglobin andmyoglobin from consumption of meat, poultry, and fishwhereas non-heme iron is obtained from cereals, pulses,legumes, fruits, and vegetables [28]. The absorption ofheme iron can vary from about 40% during iron deficiencyto about 10% during iron repletion [29]. Fe absorptiondecreases until an equilibrium is established betweenabsorption and requirements. For a given diet this regula-tion of Fe absorption, however, can only balance losses upto a certain critical point beyond which iron deficiency willdevelop [30]. About half of the basal Fe losses are fromblood, primarily in the gastrointestinal tract. Absorptionfrom the gastrointestinal tract is the primary homeostaticmechanism for iron. Since the concentration of Fe is

ent gallstone samples by PIGE and PIXE

rn (Chennai region) India (n = 15)t study)

Southern India (n = 10)(Ashok et al.) [4]

–––

–––333950,70117–297372502–77052651085129

Fig. 5. Concentration of elements in different regions.

T.R. Rautray et al. / Nucl. Instr. and Meth. in Phys. Res. B 255 (2007) 409–415 413

170 ppm in tamarind [20], higher intake of tamarind givesrise to higher concentration of Fe in the south Indian pig-ment gallstones.

In a hospital based case controlled study [31] of 346patients, it was also reported that dietary parameters didnot differ between patients and controls, except for theuse of tamarind (Garcinia camborginia) more than equalto 3 times a week, as determined by univariate (v2

trend = 4.73, odds ratio 1.76; 95% confidence interval1.05, 2.96; p = 0.03) and multivariate (p = 0.02) analysis.Most of the south Indian states consume tamarind as acommon ingredient in their regular food which may bethe primary cause leading to the higher concentration ofFe in their gallstones by gastrointestinal absorption fromsuch diets.

Average concentration of Cu in south Indian pigmentgallstones is about three times more than that of the eastIndian gallstones whereas Cu concentration in other partsof south India as analysed by Ashok et al. is about 1.7times higher than that of southern region gallstone in thepresent study. Higher amount of Cu in the pigment gall-stones may be due to presence of copper bilirubinate whichgives the pigment gallstones the black colour. Transport ofCu from the liver into the bile is the primary route forexcretion of endogenous copper. Copper of biliary originand non-absorbed dietary Cu are eliminated from the bodyvia the feces. The absorption and retention of Cu varieswith dietary intake and status [32,33]. Intake of beverageswith elevated Cu concentrations can induce acute gastroin-testinal symptoms, such as epigastric pain, nausea, vomit-ing, and diarrhea. However, Cu concentrations at whichthese symptoms appear and the scope of responsesobserved are not clear [34]. For this reason, the thresholdfor gastrointestinal symptoms of Cu in beverages has notbeen precisely established in controlled prospective studies.Both copper sulfate (a soluble compound) and copperoxide (an insoluble compound) have comparable effects,implying that the ionic Cu present in the stomach is respon-sible for the induction of gastrointestinal manifestations[35].

Average concentration of Zn in southern region pigmentgallstones is about twice more than the average concentra-tion of east Indian stones and is about 0.5 times as reportedby Ashok et al. Zn is lost from the body through the kid-neys, skin, and intestine. The endogenous intestinal lossescan vary from 7 lmol/day (0.5 mg/day) to more than45 lmol/day (3 mg/day), depending on Zn intake [36].The utilisation of Zn depends on the overall compositionof the diet. Experimental studies have identified a numberof dietary factors as potential promoters or antagonistsof gastrointestinal Zn absorption. The risk for competitiveinteractions seems mainly to be related to high doses in theform of supplements or in aqueous solutions. However, atlevels present in food and at realistic fortification levels, Znabsorption appears not to be affected, for example, by Feand Cu [37]. Long-term Zn intakes higher than the require-ments could, however, interact with the metabolism of

other trace elements. Cu seems to be especially sensitiveto high Zn doses. A Zn intake of 50 mg/day (760 lmol)affects Cu status indexes, such as CuZn superoxide dismu-tase in erythrocytes [38,39]. Because Cu also has a centralrole in immune defence, these observations call for cautionbefore large-scale Zn supplementation programmes areundertaken. Any positive effects of Zn supplementationon growth or infectious diseases could be disguised orcounterbalanced by negative effects on copper relatedfunctions.

The average concentrations of Cr in south Indian gall-stones is about twice more than that of eastern region gall-stones, whereas it is about six times more than as reportedby Ashok et al. So, the reason for less amount of Cr insouth Indian gallstones is yet to be fully understood. Thecomparison of average concentrations of various elementsis depicted in Fig. 5. In the present investigation, averageconcentration of Br in southern region gallstones is morethan twice than eastern region. However, average Br con-centration in other parts of south India is about 30 timesthan southern region pigment gallstones which is notunderstood properly.

Figs. 6 and 7 show the FTIR spectrum of both theregions. The characteristic bands namely 2932, 2865,1459, 1378 and 1054 cm�1 are due to cholesterol in the pig-ment gallstones of both the eastern and southern regions.The calcium bilirubinate has characteristic bands at 1660,1626, 1559 and 1254 cm�1 which are assigned to ( C@C,C–N, C@O) stretching vibration of lactam, (C@O) stretch-ing of COOH; (C@O, C–N, C@C) stretching, asymmetricstretching tas (COO�) and (C–O) stretching or C–Nstretching coupled with NH deformation t(C–N) +d(NH), respectively [40]. The band at 3409 cm�1 is due toNH stretching vibration of pyrrole of bilirubin. Calciumbilirubinate is the main component of pigment gallstones.At present no crystal structure of Calcium bilirubinate isavailable.

Fig. 6. FTIR spectrum of an east indian pigment gallstone.

Fig. 7. FTIR spectrum of a south indian pigment gallstone.

Fig. 8. TG-DTG spectrum of an east indian pigment gallstone.

Fig. 9. TG-DTG spectrum of a south east indian pigment gallstone.

414 T.R. Rautray et al. / Nucl. Instr. and Meth. in Phys. Res. B 255 (2007) 409–415

The bands due to phosphate ðPO3�4 Þ ions are 1105, 1037,

963, 605 and 563 cm�1, which may be due to presence ofcalcium phosphate in the pigment gallstones. The band vis-ible at 1251 cm�1 may be due to amide III which overlapswith the calcium bilirubinate band. The very broad bandsin the spectra of the pigment gallstones are said to be indic-ative of multiple metal ions (Mg, Ca, Fe, Cu) present inhigh concentrations. Longitudinal optic mode frequencyof MgO is observed at 796 cm�1.

The TG and DTG curves, (Figs. 8 and 9), from easternand southern India, respectively, show that the pigmentgallstone samples are thermally unstable early in thecurve i.e. between 25 �C and 70 �C. After this temperature,the gallstone samples are stable up to 210 �C. Althoughall samples were dried during sample preparation, the sam-

ples still contain a little inherent water, which is evapo-rated below around 150 �C. The thermal decompositionobserved in the TG and DTG curves occurs in two con-secutive steps, between 210 �C and 446 �C (east Indiangallstones) and 210 �C and 498 �C (in southern region gall-stones). In the south Indian gallstones, the first weight lossup to 336 �C occurs through a fast process with weight lossof 42%. This region is related to primary devolatilization,during which carbon, hydrogen and oxygen compoundsare released. The observed weight loss in this region maybe suggested that this residue is composed mainly of carbo-naceous material formed only in the presence of oxygen.The second weight loss begins with a very slow process withweight loss of 48%. But, in the east Indian gallstones, theweight loss up to 362 �C occurs in a very fast process withweight loss of 78%, whereas the weight loss between 362 �Cand 446 �C occurs very slowly with a weight loss of 88%.After weight loss of 88% it was observed that there wasno more thermal degradation, which may be ascribed dueto stability of chemical substances like calcium bilirubinate,

T.R. Rautray et al. / Nucl. Instr. and Meth. in Phys. Res. B 255 (2007) 409–415 415

calcium phosphate or hydroxyapatite present in the pig-ment gallstones in this range of temperature. The weightloss 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 Cain south Indian pigment gallstones is more than 1.6 timesthan that of east Indian pigment gallstones, it is expectedthat the concentrations of different Ca compounds maybe higher in the south Indian pigment stones. Moreover,FTIR results suggests that calcium phosphate may be pres-ent in the pigment gallstones. Higher amount of calciumphosphate will give rise to more thermal stability of the pig-ment gallstone. So, the thermal degradation of southIndian pigment gallstones does not fall below 48% due topresence of higher amount of calcium phosphate orhydroxyapatite present in it. This shows that the southIndian pigment stones contains compounds which are notdegradable even at 525 �C.

4. Conclusion

Twenty-seven Indian pigment gallstone samples wereanalysed by PIXE and PIGE and minor/trace elementsnamely Na, Mg, Al, P, S, Cl, K, Ca, Ti, V, Cr, Mn, Fe,Ni, Cu, Zn, Br and Pb were quantified. Our study revealsthat average concentration of all elements except Ni insouth Indian pigment gallstone samples is higher than thatof corresponding values in east Indian pigment gallstonesamples whereas elements like Al, P, S, Cl and V did notshow much variation between these two regions. The rea-son for this may be due to different types of food habitsof the patients in these two regions. The higher concentra-tion of Fe in south Indian pigment gallstone samples maybe due to intake of tamarind as their regular food. Whilethe presence of different functional groups was detectedby FTIR analysis, TG and DTG curves provided informa-tion on the thermal decompositions of these compounds.

Acknowledgements

The help extended by Dr. Md. Ibrarullah, Dept. ofGastroenterology, Kalinga Hospital, Bhubaneswar andDr. V. Jayanthi, Dept. of Gastroenterology, Stanley Med-ical College Hospital, Chennai for providing samples areacknowledged and thanks to Prof. S.N. Sahu and Mr.S.N. Sarangi, Institute of Physics, Bhubaneswar for usingFTIR facility. We are grateful to Prof. S. Jena and Mr.D.K. Ray, Dept. of Chemistry, Utkal University, Bhu-baneswar for their help in carrying out thermogravimetricanalysis.

References

[1] T. Maki, Arch. Surg. 82 (1961) 599.[2] T. Markkanen, A.J. Aho, Acta Chir. Scand. 138 (1972) 30.

[3] T.R. Rautray, V. Vijayan, S. Panigrahi, Eur. J. Gastroen. Hepat. 18(2006) 999.

[4] M. Ashok, S.N. Kalkura, V.J. Kennedy, A. Markwitz, V. Jayanthi,K.G.M. Nair, V. Vijayan, Int. J. PIXE 12 (2002) 137.

[5] M. Ashok, T.R. Rautray, P.K. Nayak, V. Vijayan, V. Jayanthi, S.N.Kalkura, J. Radioanal. Nucl. Chem. 257 (2003) 333.

[6] G. Liu, D. Xing, H. Wang, J. Wu, J. Mol. Struct. 616 (2002) 187.[7] K. Szentmihalyi, P. Sipos, A. Blazovics, P. Vinkler, M. Szilagyi, Trace

Elem. Electroly. 19 (160) (2002) 3.[8] S. Vatankhah, K. Moosavi, H. Peroval, J. Salimi, Int. J. PIXE 13

(2003) 97.[9] N. Ekinci, Y. Sahin, Spectrochim. Acta B 57 (2002) 167.

[10] V. Vijayan, S.N. Behera, V.S. Ramamurthy, S. Puri, J.S. Shahi, N.Singh, X-ray Spectrom. 26 (1997) 65.

[11] V. Vijayan, T.R. Rautray, P.K. Nayak, D.K. Basa, X-ray Spectrom.34 (2005) 128.

[12] P.K. Nayak, V. Vijayan, Nucl. Instr. and Meth. B 245 (2006) 505.[13] J.L. Campbell, T.L. Hopmann, J.A. Maxwell, Z. Nezedly, Nucl.

Instr. and Meth. B 170 (2000) 193.[14] ANGES software package, IAEA, Vienna. <www.iaea.org>.[15] A. Rapacz-Kmita, C. Paluszkiewicz, A. Slosarczyk, Z. Paszkiewicz, J.

Mol. Struct. 744–747 (2005) 653.[16] N.S. Fernandes, M.A. da Silva, C. Filho, R.A. Mendes, M.

Ionashiro, J. Braz. Chem. Soc. 10 (1999) 459.[17] R.A. McCance, E.M. Widdowson, J. Physiol. 101 (1942) 304.[18] R.A. McCance, E.M. Widdowson, J. Physiol. 101 (1942) 44.[19] J.L. Kelsay, K.M. Bahall, E.S. Prather, Am. J. Clin. Nutr. 32 (1979)

1876.[20] C.G. Gopalan, B.V. Rama Sastri, S.C. Balasubramanian, National

Institute of Nutrition, Hyderabad, India (2000).[21] M.B. Andon, J.Z. Ilich, Tzagornnis, V. Matkovic, Am. J. Clin. Nutr.

63 (1996) 950.[22] S.A. Abrams, M.A. Grusak, J. Stuff, K.O. O’Brien, Am. J. Clin. Nutr.

66 (1997) 1172.[23] J. Sojka, M. Wastney, S. Abrams, S.F. Lewis, B. Martin, C. Weaver,

M. Peacock, Am. J. Physiol. 273–42 (1997) R710.[24] E. Wisker, R. Nagel, T.K. Tamudjaja, W. Feldheim, Am. J. Clin.

Nutr. 54 (1991) 533.[25] H. Spencer, C. Norris, D. Williams, J. Am. Coll. Nutr. 13 (1994) 479.[26] E.M. Brown, S.C. Hebert, Bone 20 (1997) 303.[27] L. Hallberg, Ann. Rev. Nutr. 1 (1981) 123.[28] L. Hallberg, Scand. J. Gastroentero. 14 (1979) 769.[29] L. Hallberg, L. Hulthen, E. Gramatkovski, Am. J. Clin. Nutr. 66

(1997) 347.[30] L. Hallberg, Eur. J. Clin. Nutr. 49 (1995) 200.[31] V. Jayanthi, L. Anand, L. Ashok, S. Vijaya, Ind. J. Gastroentero. 24

(2005) 97.[32] M.A. Silva, T. Wong, J. Gastrointest. Surg. 9 (2005) 739.[33] M. Acalovschi, D. Blendea, C. Feier, A.I. Letia, N. Ratiu, D.L.

Dumitrascu, A. Veres, Am. J. Gastroentero. 98 (2003) 1856.[34] F. Pizarro, M. Olivares, V. Gidi, M. Araya, Rev. Environ. Health 14

(1999) 231.[35] F. Pizarro, M. Olivares, M. Araya, V. Gidi, R. Uauy, Environ.

Health Persp. 109 (9) (2001) 949.[36] J.C. King, J.R. Turnlund, Human zinc requirements, in: C.F. Mills

(Ed.), Zinc in Human Biology, Springer-Verlag, Devon, U.K., 1989,p. 335.

[37] B. Sandstrom, B. Lonnerdal, Promoters and antagonists of zincabsorption, in: C.F. Mills (Ed.), Zinc in Human Biology, Springer-Verlag, Devon, U.K., 1989, p. 57.

[38] P.W.F. Fischer, A. Giroux, M.R. L’Abbe, Am. J. Clin. Nutr. 40(1984) 743.

[39] M.K. Yadrick, M.A. Kenney, E.A. Winterfeldt, Am. J. Clin. Nutr. 49(1989) 145.

[40] G. Liu, Da Xing, H. Yang, J. Wu, J. Mol. Struct. 616 (2002) 187.