J

A

Nb

DASa

b

c

d

e

f

g

h

i

j

a

ARA

KBGCMI

aS(x((

M

A

0h

ARTICLE IN PRESSG ModelTEMB-25416; No. of Pages 6

Journal of Trace Elements in Medicine and Biology xxx (2012) xxx–xxx

Contents lists available at SciVerse ScienceDirect

Journal of Trace Elements in Medicine and Biology

jou rn al h omepage: www.elsev ier .de / j temb

nalytical methodology

ew insights into the chemical and isotopic composition of human-bodyiominerals. I: Cholesterol gallstones from England and Greece

imitra Athanasiadoua,1, Athanasios Godelitsasa,∗, Dimosthenis Sokarasb,2,ndreas-Germanos Karydasc,d, Elisavet Dotsikae, Constantinos Potamitis f, Maria Zervouf,telios Xanthosg, Elias Chatzitheodoridish, Hock Chye Gooi i, Udo Becker j

School of Sciences, University of Athens, Panepistimioupoli Zographou, 15784 Athens, GreeceInstitute of Nuclear Physics, National Center for Scientific Research “Demokritos”, Aghia Paraskevi, 15310 Athens, GreeceInstitute of Nuclear Physics, National Center for Scientific Research “Demokritos”, Aghia Paraskevi, 15310 Athens, GreeceInternational Atomic Energy Agency (IAEA), Nuclear Spectrometry and Applications Laboratory, Seibersdorf Laboratories, AustriaNational Center for Scientific Research “Demokritos”, I.M.S., 15310 Aghia Paraskevi Attikis, GreeceNational Hellenic Research Foundation, Institute of Biology, Medicinal Chemistry and Biotechnology, Vas. Constantinou 48, 11635 Athens, GreeceDepartment of Electrical and Computer Engineering, Aristotle University of Thessaloniki, GR-54124 Thessaloniki, GreeceSchool of Mining and Metallurgical Engineering, National Technical University of Athens, 15780 Zographou, Athens, GreeceBeaumont Hospital, Dublin, IrelandDepartment of Geological Sciences, University of Michigan, 1100 North University Ave., Ann Arbor, MI 48109, USA

r t i c l e i n f o

rticle history:eceived 6 August 2011ccepted 27 August 2012

eywords:iomineralsallstonesholesteroletals

a b s t r a c t

We have analyzed gallstones from four patients of Europe and particularly from England (includingsamples from a mother and a daughter) and Greece. According to the XRD, FTIR, NMR and laser micro-Raman results the studied materials correspond to typical cholesterol monohydrate (ChM). The micro-morphology of cholesterol microcrystals was investigated by means of SEM–EDS. The XRF results revealedthat Ca is the dominant non-organic metal in all gallstones (up to ∼1.95 wt.%) together with Fe, Cu, Pband Ni (up to ∼19 ppm for each metal). Gallstones from England contain additional Mn (up to ∼87 ppm)and Zn (up to ∼6 ppm) while the sample of the mother contains negligible Zn and Mn, compared to thatof her daughter, but significant As (∼4.5 ppm). All cholesterol gallstones examined are well enriched in

sotopes potentially toxic metals (Pb, as well as Ni in one case) and metalloids (As also in one case) as compared tothe global average. The position of Zn, which is a characteristic biometal, in the structure of cholesterol,was investigated by molecular simulation using the Accelrys Materials Studio® software. On the basisof IRMS results, all gallstones examined exhibit a very light �13C signature (average �13C ∼−24‰ PDB).Gamma-ray spectrometry measurements indicate the presence of 214Pb and 214Bi natural radionuclidesdue to the 238U series as well as an additional amount of 40K.

Please cite this article in press as: Athanasiadou D, et al. New insights inerals. I: Cholesterol gallstones from England and Greece. J Trace Elem

∗ Corresponding author. Tel.: +30 2107274689.E-mail addresses: dimitra.athanasiadou@mail.mcgill.ca (D. Athanasi-

dou), agodel@geol.uoa.gr (A. Godelitsas), dsokaras@slac.stanford.edu (D.okaras), A.Karydas@iaea.org (A.-G. Karydas), edotsika@ims.demokritos.grE. Dotsika), potamitis@eie.gr (C. Potamitis), mzervou@eie.gr (M. Zervou),anthos@nestoras.ee.auth.gr (S. Xanthos), e.chatzitheodoridis@metal.ntua.grE. Chatzitheodoridis), jimmygooi@beaumont.ie (H.C. Gooi), ubecker@umich.eduU. Becker).

1 Present Address: Faculty of Dentistry, McGill University, 3640 University Street,ontreal, QC, Canada H3A 0C7.2 Present Address: Stanford Synchrotron Radiation Lightsource, SLAC Nationalccelerator Laboratory, 2575 Sand Hill Rd, Menlo Park, 94025 CA, USA.

946-672X/$ – see front matter © 2012 Elsevier GmbH. All rights reserved.ttp://dx.doi.org/10.1016/j.jtemb.2012.08.004

© 2012 Elsevier GmbH. All rights reserved.

Introduction

Gallstone disease is a common and crucial health problemaround the world. Many theories have been postulated for itspathogenesis but the formation and growth of gallstones are notfully understood. Bile is a digestive fluid that is extracted by liverand stored in the gallbladder, a small pear-shaped organ on theunderside of the liver. It consists of a mixture of water (>90%), lipids,electrolytes (alkalis and alkaline earth metals such as Na, K, and Ca),and proteins. The primary lipid components of bile are bile acids,phospholipids and cholesterol. The hepatocyte is the major site forcholesterol synthesis and peripheral uptake, and excess cholesterol

to the chemical and isotopic composition of human-body biomin-Med Biol (2012), http://dx.doi.org/10.1016/j.jtemb.2012.08.004

is directly secreted into bile or converted into bile salts. As a resultof the lipid imbalance, the micelles become supersaturated withcholesterol, giving rise to the nucleation of crystals. As the conditionprogresses, the gallbladder secretes excess mucus, which results

IN PRESSG ModelJ

2 ents in Medicine and Biology xxx (2012) xxx–xxx

iicTcttge

sawgmmaioaXtaASwtTpkcs

ssIsoEXtTetc[

cth

M

fEawtaswl

m

ARTICLETEMB-25416; No. of Pages 6

D. Athanasiadou et al. / Journal of Trace Elem

n the formation of insoluble solid cholesterol crystals constitut-ng the gallstones. Gallstones themselves consist mainly of organicompounds such as cholesterol, bilirubin, bile acid and fatty acid.hey are broadly classified into three categories according to theirhemical composition: cholesterol type (cholesterol rich), pigmentype (bilirubin and Ca-bilirubinate rich) and mixed type (combina-ion of cholesterol, Ca-bilirubinate and Ca-carbonate). Cholesterolallstone disease results from a complex interaction of genetic andnvironmental risk factors [e.g. 1–5].

Therefore, the elemental and isotopic composition of gall-tones is considered to play a significant role in the formationnd growth of cholesterol gallstones. In addition to the majorell-known constituents (cholesterol, bilirubin and phospholipid)

allstones also contain a number of other elements with Ca as theajor biometal [e.g. 6–9]. The role of microelements, includingetals, in cholelithiasis has not yet been understood completely,

lthough it is known that many of these elements play a signif-cant role in metabolism and sustain permanency of the internalsmotic stability of tissue liquids [7]. In previous works, X-raynalytical techniques (X-ray Fluorescence/XRF and Proton Induced-ray Emission/PIXE) were used to determine the concentra-

ions of trace elements in cholesterol gallstones [7–17]. Additionalnalytical techniques, such as Instrumental Neutron Activationnalysis/INAA, Inductively Coupled Plasma – Optical Emissionpectrometry/ICP-OES and Atomic Absorption Spectrometry/AAS,ere also used [10,18–21]. Notably, most of the existing litera-

ure concerns the study of gallstones from Asia (India, Iran, Japan,urkey, Pakistan) and Africa whereas the data for Europe are sur-risingly rather limited [10,12,19]. Moreover, to the best of ournowledge, there are no particular papers on the precise chemi-al composition of cholesterol gallstones from the Americas, exceptome data from Canada [18].

One of the goals of the present work is to contribute to thetudy of European gallstones, and specifically to compare gall-tones from northern and southern Europe (England and Greece).n our case, XRF was also used for analysis of selected gall-tones which were previously characterized using a combinationf microscopic, diffraction and spectroscopic techniques (Scanninglectron Microscopy–Energy Dispersive Spectrometry/SEM–EDS,-ray Diffraction/XRD, Fourier-Transform Infrared Spectrome-

ry/FTIR, Nuclear Magnetic Resonance/NMR, laser micro-Raman).he position of divalent biometals (e.g. Zn) into cholesterol wasxplored using molecular simulation software. It should be noted,hat crystal structures of anhydrous cholesterol (C27H46O) andholesterol monohydrate (C27H46O·H2O) are known since the 70s22–24], however, the data for occluded metals are very limited.

Finally, for the first time in literature, stable carbon isotopes andarbon radionuclides were measured in cholesterol gallstones fromhe human body, using Isotope Ratio Mass Spectrometry/IRMS andigh-resolution Gamma (�)-ray spectrometry.

aterials and methods

A total of 20 gallstones were removed from four patients (threeemales and one male) during surgical operation at hospitals inngland (Leeds) and Greece (Athens). From the above collection,fter optical examination, only 4 typical and homogeneous samplesere finally subjected to further detailed study. It should be noted

hat 2 of the examined gallstones, chosen from England, belong to mother and a daughter, respectively. Both mother and daughteruffer from the autosomal disorder C1 esterase inhibitor deficiency,

Please cite this article in press as: Athanasiadou D, et al. New insights inerals. I: Cholesterol gallstones from England and Greece. J Trace Elem

hereas the other patient from England has chronic chest prob-ems.

SEM–EDS investigation was carried out using a Jeol JSM-5600icroscope equipped with an Oxford EDS. The XRD patterns were

Fig. 1. SEM image of human gallstone from the present study.

recorded by means of a Siemens D5005 (Bruker AXS) using Cu K�

radiation. The FTIR spectroscopic measurements were performedusing a Perkin Elmer Spectrum One spectrometer in the frequencyrange 4000–450 cm−1 with a nominal energy resolution of 2 cm−1.For each measurement 1.3 mg of powdered sample was used toprepare KBr discs. The 1H NMR measurements were performed ona Varian 600 MHz spectrometer. In each case, 5 mg of each samplewere dissolved in 0.7 mL chloroform-d. Chemical shifts are reportedin ppm while spectra were referenced by the standard experimen-tal setup. Laser micro-Raman spectra were acquired by a RenishawRamascope RM1000 Raman micro-spectrometer. Typical spectrawith a resolution of 0.4 cm−1 measured on the Rayleigh line of theHe–Ne laser, were acquired with a laser power of 4 mW energy anda laser of 632.8 nm. All the spectra were acquired and processedwith the GRAMS of Thermo Scientific software.

The XRF analysis was performed using an in-house developedportable milli-beam XRF spectrometer. It comprises a Rh-anodeside-window low power X-ray tube (50 W, 40 kV, 75 �m Be win-dow) and a Si–PiN diode X-ray detector (500 �m nominal crystalthickness, energy resolution of about 165 eV (FWHM) at Mn K�).For the present study, the X-ray tube was operated at 40 kV withhard filtering (i.e., Ni: 42.5 mg/cm2, V: 33.0 mg/cm2) to optimizepeak-to-background ratio for the metallic trace elements. Thequantitative analysis was based on elemental calibration factorsexperimentally obtained by means of a set of reference thin mono-elemental targets, whereas pure cholesterol was taken as the darkmatrix of the measured samples. A 5 tonnes hydraulic press wasused to compress the sample powder into a 1.3-cm diameter thinpellet.

Moreover, the Accelrys Materials Studio® software was used tosimulate divalent biometals, and specifically Zn, in the structure ofcholesterol monohydrate.

Stable carbon isotopes were measured in a Thermo ScientificDelta V Plus IRMS equipped with a FlashEA 1112 elemental anal-yser. 150–200 mg of each sample was used for the determinationof the �13C values of the studied gallstones. Finally, radioactiveisotopes were determined by high-resolution �-ray spectrometryusing a Canberra high-purity Ge detector (HPGe).

Results and discussion

The morphology and the aggregations of organic microcrys-

to the chemical and isotopic composition of human-body biomin-Med Biol (2012), http://dx.doi.org/10.1016/j.jtemb.2012.08.004

tals into human gallstones from England and Greece, as recordedby SEM–EDS, are shown in Fig. 1. The powder XRD patternsof the samples disclosed two characteristic peaks at d = 17.1 Aand d = 5.7 A, corresponding to cholesterol [25], whereas the

Please cite this article in press as: Athanasiadou D, et al. New insights into the chemical and isotopic composition of human-body biomin-erals. I: Cholesterol gallstones from England and Greece. J Trace Elem Med Biol (2012), http://dx.doi.org/10.1016/j.jtemb.2012.08.004

ARTICLE IN PRESSG ModelJTEMB-25416; No. of Pages 6

D. Athanasiadou et al. / Journal of Trace Elements in Medicine and Biology xxx (2012) xxx–xxx 3

Fig. 2. 1H NMR spectrum of a human cholesterol gallstone from the present study (acquired in CDCl3 at 600 MHz).

Fig. 3. XRF spectra of human cholesterol gallstones from the present study (a: female patient from England/mother; b: female patient from England/daughter; c: male patientfrom England; d: female patient from Greece). The presence of V K- and Au L-characteristic X-rays (indicated with an asterisk) in the XRF spectra is due to contamination bythe XRF spectrometer materials, namely the filter and X-ray detector (contact electrode), respectively.

ARTICLE ING ModelJTEMB-25416; No. of Pages 6

4 D. Athanasiadou et al. / Journal of Trace Elements i

Foc

F4af

ig. 4. Average concentration of elements in human cholesterol gallstones (averagef global values from the literature [9–21] and present study, Ni and As refer to onease).

Please cite this article in press as: Athanasiadou D, et al. New insights inerals. I: Cholesterol gallstones from England and Greece. J Trace Elem

TIR spectra included characteristic bands in the region between000–400 cm−1 due to the stretching vibrations of C C, C Hnd OH at 1056, 1365, 1466, 2932 and 3400 cm−1 also arisingrom cholesterol [26,27]. A representative 1H NMR spectrum of

Fig. 5. Triangular diagrams showing the main heavy metals in h

PRESSn Medicine and Biology xxx (2012) xxx–xxx

the studied gallstones is shown in Fig. 2. The 1H NMR spectrumidentified the presence of cholesterol resonance peaks accordingto literature [28]. Notations to distinct non overlapping signalsof H3-18, H-3 and H-6 protons are highlighted. All the abovebulk data confirmed that the examined biominerals belong tocholesterol-type gallstones. The subsequent examination by lasermicro-Raman, in accordance to SEM–EDS, proved the homogeneityand purity of the cholesterol in microscale and the absence of asso-ciated phases, including proteins, bilirubin metal complexes andsalts of Ca such as Ca-carbonate and Ca-bilirubinate [29].

The XRF analyses (Fig. 3) revealed the presence of the essentialfor the normal functioning of the body biometals (Ca, Fe, Mn, Cu, Zn)and, in addition, of potentially toxic metals and metalloids (Ni, Pb,As). The presence of V K- and Au L-characteristic X-rays in the XRFspectra is due to contamination by the XRF spectrometer materials,namely the filter and X-ray detector (contact electrode), respec-tively. Calcium was found to be the predominant non-organicconstituent in all cholesterol gallstones (max. 1.95 wt.%). It hasbeen reported that calcium behaved as a nidus for the gallstone

to the chemical and isotopic composition of human-body biomin-Med Biol (2012), http://dx.doi.org/10.1016/j.jtemb.2012.08.004

formation [30,31]. It is obvious that Ca content is affected by thetype of food and drinks the patients take, including diary and milkproducts, eggs, tea, and hard water [32]. Trace element Sr (max.∼13 ppm) might also be related to Ca, due to same valence and

uman cholesterol gallstones ([9–21] and present study).

ARTICLE ING ModelJTEMB-25416; No. of Pages 6

D. Athanasiadou et al. / Journal of Trace Elements i

Fig. 6. Position of Zn (magenta color) in the structure of cholesterol as simulatedbfi

cfwtMiFmuoFwcotao∼twoZgitictM∼aIM

Gea

topes, �-ray spectrometry measurements revealed the existence of

y Accelrys Materials Studio® . (For interpretation of the references to color in thisgure legend, the reader is referred to the web version of the article.)

lose ionic radii as well as similar metabolism [33], whereas theunction of Br (max. ∼25 ppm) is rather unknown. Iron and Cuere present in all examined samples, with a maximum concentra-

ion of ∼19 ppm, in the gallstones from both England and Greece.uch higher concentrations of Cu were found by Salimi et al. [13]

n cholesterol gallstones from Iran while higher concentrations ofe were found by Ashok et al. [9] and Alatise et al. [17] in relevantaterials from India and Nigeria (Figs. 4 and 5). The kind of crockery

sed in India and Iran may be responsible for the high concentrationf Cu. Johnston et al. [34] and Kumar et al. [35] suggested that ane-deficient diet might alter the metabolism of the hepatic enzyme,hich later increases gallbladder bile cholesterol and promotes

holesterol crystal formation. Furthermore, the central aggregatesf calcium salts constitute hard foreign bodies which may leado ulceration of the gall bladder mucosa, microscopic hemorrhagend inflammation. This releasing process may be another sourcef Fe deposition in gallstones [36]. On the other hand, Zn (max.6 ppm), which is considered to be a critical biometal, was found

o occur only in the samples coming from England (Fig. 5), whichere reported to be richer in all metals. In addition, the gallstones

f the English young lady (daughter) contained interestingly moren in contrast to the gallstones of her mother containing negli-ible (almost zero) amounts. However, the global average of Znn cholesterol gallstones was greater compared to the average ofhe European samples studied herein (Fig. 4). The presence of Znn gallstones could be explained as simple co-precipitation withholesterol [37]. The position of Zn in the structure of choles-erol, as suggested by molecular simulation, is shown in Fig. 6.

anganese was also significant in the studied gallstones (max.87 ppm), though lower than the global average. Manganese forms

salt with bile acid which gets accumulated during cholelithiasis.t should be emphasized that, as in the case of Zn, the presence of

n was proved only for the case of gallstones from England (Fig. 5).It is evident that the cholesterol gallstones from England and

Please cite this article in press as: Athanasiadou D, et al. New insights inerals. I: Cholesterol gallstones from England and Greece. J Trace Elem

reece examined in the frame of the present paper are wellnriched in potentially toxic metals (Pb, Ni) and metalloids (As),s compared to the global average (Fig. 4). However, the highest

PRESSn Medicine and Biology xxx (2012) xxx–xxx 5

concentration of Pb (∼19 ppm) is observed in the samples fromEngland. Lead is known to be hepatotoxic and the sources of thisharmful heavy metal might be natural, industrial, or even unex-pected (e.g. home made wine [38]). It is notable that Ni and Aswere also concentrated in individual samples from England (∼19and ∼4.5 ppm, in one case, respectively) but not in the samplesfrom Greece. Thus, gallstones from England are mostly rich in toxicelements. This may be related to possible toxic diet and environ-mental pollution issues. At this point it should be noted that, exceptPb, the elements Ni and As (as well as Zn) could also be associated toatmospheric pollution [39]. No significant Cd concentrations, whichhas been reported for patients from India having cholelithiasis andcancer [40], were detected in the studied European cholesterolgallstones (also considering the low analytical sensitivity of XRFanalysis for this metal).

The stable carbon isotopic composition of the gallstones wasfound to occur in the expected range. The average of �13C values ofthe samples was calculated at −24‰VPDB (Vienna Pee Dee Belem-nite; international reference standard for carbon isotopes). Thesevalues are generally in agreement with the findings on other studies[41,42]. The isotopic composition of carbon reflects the diet habitsof the patients. From the �13C values, it is obvious that the gall-stones are enriched in the light isotope 12C. DeNiro and Epstein[41] and Jim et al. [42] were able to determine the influence ofvarious food animals (pigs and rats) in the isotopic composition ofcarbon. However, the accurate determination of food influence inthe isotopic composition of humans is difficult because of the vari-ety of their diet habits. The high-resolution �-ray spectrometricinvestigation revealed, for the first time in the literature, that thestudied biominerals contain radioactive isotopes. Natural radionu-clides such as 214Pb (196 Bq/kg) and 214Bi (170 Bq/kg) due to 238Useries and 40K (54 Bq/kg) were determined. This also reveals thattraces of U are present in the gallstones. Finally, it is noteworthythat artificial radionuclides, such as 137Cs (due to nuclear accidentsincluding Chernobyl) were not observed.

Conclusions

The present study concerns the characterization of human gall-stones by means of microscopic, diffraction and spectroscopictechniques (SEM–EDS, XRD, FTIR, NMR, laser micro-Raman) as wellas the elucidation of their chemical and isotopic composition bymeans of XRF, IRMS and high-resolution �-ray spectrometry. Wehave analyzed gallstones from northern and southern Europe andparticularly from England and Greece. The characterization studyshowed that all gallstones selected for detailed analyses (includ-ing samples from mother and daughter from England) correspondto typical cholesterol monohydrate. The analyses by XRF indicatedthat Ca is the main foreign element (up to ∼1.95 wt.%) into choles-terol crystal aggregates together with Fe, Cu, Pb and Ni (up to∼19 ppm for all metals). Gallstones from England contain additionalMn (up to ∼87 ppm) and Zn (up to ∼6 ppm) whereas the sampleof mother contain negligible Zn and Mn, compared to that of herdaughter, and considerable amount of As (∼4.5 ppm). The positionof Zn, as representative biometal, in the structure of cholesterol,was investigated, for the first time in the literature, by molecularsimulation using the Accelrys Materials Studio® software. On thebasis of IRMS results, all examined gallstones show very light �13Csignature (average �13C ∼−24‰ PDB), in accordance to recent dataabout cholesterol from humans as well as from rats raised on avariety of isotopically controlled diets. Concerning radioactive iso-

to the chemical and isotopic composition of human-body biomin-Med Biol (2012), http://dx.doi.org/10.1016/j.jtemb.2012.08.004

214Pb and 214Bi, due to 238U series, and also a detectable amountof 40K. We conclude that the examined European cholesterol gall-stones, though they are enriched in metals essential for the normal

ING ModelJ

6 ents i

ftat

A

s

R

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

ARTICLETEMB-25416; No. of Pages 6

D. Athanasiadou et al. / Journal of Trace Elem

unctioning of the body, such as Ca (related also to increased Sr),hey contain excess harmful metals and metalloids, such as Pb, Ni,nd As, as well as traces of U. This might be related to potentialoxic diet and environmental pollution issues.

cknowledgments

We would like to thank Profs. E. Iatrou and A. Argyraki (Univer-ity of Athens) for their collaboration.

eferences

[1] Shiff ER, Sorrell MF, Maddrey WC. Shiff’s diseases of the liver. 10th ed. Philadel-phia: Lippincott Williams & Wilkins; 2007.

[2] Portincasa P, Moschetta A, Mazzone A, Palasciano G, Svelto M, Calamita G.Water handling and aquaporins in bile formation: recent advances and researchtrends. J Hepatol 2003;39:864–74.

[3] van Erpecum KJ. Biliary lipids, water and colesterol gallstones. Biol Cell2005;97:815–22.

[4] Russell DW, Setchell KD. Bile acid biosynthesis. Biochemistry1992;31:4737–49.

[5] Portincasa P, Moschetta A, Palasciano G. Cholesterol gallstone disease. Lancet2006;368:230–9.

[6] Gleeson D, Murphy GM, Dowling RH. Calcium binding by bile acids: in vitrostudies using a calcium ion electrode. J Lipid Res 1990;31(5):781–91.

[7] Golovanova OA, Palchic NA, Berezina NYU, Yudina LN. Comparative character-istics of the mineral and microelement composition of gallstones extractedfrom patients in the Novosibirsk and Omsk regions. Chem Sustain Dev2006;14:125–31.

[8] Rautray TR, Dutta K, Das SL, Rautray AC. In situ analyses of gallstone inner layersby external PIXE. Nucl Instrum Methods B 2010;268:2773–6.

[9] Ashok M, Rautray TR, Nayak PK, Vijayan V, Jayanthi V, Kalkura SN. Energydispersive X-ray fluorescence analysis of gallstones. J Radioanal Nucl Chem2003;257:333–5.

10] Al-Kinani AT, Harris IA, Watt DE. Analysis of minor and trace element analysisin gallstones by induction of characteristic ionising radiation. Phys Med Biol1984;29(2):175–84.

11] Shizuma K, Iwatani K, Hasai H, Hamanaka S, Wen XQ, Horiuchi I, et al. PIXEanalysis of gallstones: trace element analysis and millibeam scanning of stonesections. Nucl Instrum Methods B 1997;129:401–9.

12] Paluszkiewicz C, Kwiatek WM, Galka M, Sobieraj D, Wentrup-Byrne E. FT-Raman, FT-IR spectroscopy and PIXE analysis applied to gallstones specimens.Cell Mol Biol 1998;44(1):65–73.

13] Salimi J, Moosavi K, Vatankhah S. The concentration of heavy trace elementsin pigment and cholesterol human gallstones: comparative studies by PIXEanalysis. Iran J Radiat Res 2003;1:93–7.

14] Rautray TR, Vijayan V, Ashok M, Kennedy JV, Jayanthi V, Ibrarullah MD, et al.Int J PIXE 2005;15(3&4):147–52.

15] Rautray TR, Vijayan V, Panigrahi S. Analysis of Indian cholesterol gallstonesby particle induced X-ray emission and thermogravimetry–derivative thermo-gravimetry. Eur J Gastroenterol Hepatol 2006;18:999–1003.

16] Okumus oglu NT, Korkmaz F, Birchall J. PIXE analysis of gallstones from Turkishpatients. Nucl Instrum Methods A 2006;562:1054–6.

17] Alatise OI, Obiajunwa EI, Lawal OO, Adesunkanmi ARK. Particle-induced X-ray

Please cite this article in press as: Athanasiadou D, et al. New insights inerals. I: Cholesterol gallstones from England and Greece. J Trace Elem

emission (PIXE) analysis of minor and trace elements in gallstones of Nigerianpatients. Biol Trace Elem Res 2010;134(1):13–24.

18] Harvey RC, Taylor D, Petrunka CN, Murray AD, Strasberg SM. Quantitative anal-ysis of major, minor and trace elements in gallbladder bile of patients with andwithout gallstones. Hepatology 1985;5(1):129–32.

[

PRESSn Medicine and Biology xxx (2012) xxx–xxx

19] Sahuquillo A, Rubio R, Ribó JM, Ros E, Vela M. Application of focused-microwavewet digestion to the determination of trace elements in human gallstones byICP/AES. J Trace Elem Med Biol 2000;14:96–9.

20] Channa NA, Khand FD, Khuhawar MY, Bhanger MI. Study of some metal ele-ments in four chemically different types of human gallstones. J Chem Soc Pak2004;26(2):123–6.

21] Chandran P, Kuchhal NK, Garg P, Pundir CS. An extended chemical analysis ofgallstone. Ind J Clin Biochem 2007;22(2):145–50.

22] Craven BM. Crystal structure of cholesterol monohydrate. Nature1976;260:727–9.

23] Craven BM. Pseudosymmetry in cholesterol monohydrate. Acta Cryst1979;B35:1123–8.

24] Shieh HS, Hoard LG, Nordman CE. The structure of cholesterol. Acta Cryst1981;B37:1538–43.

25] Konikoff FM, Cohen DE, Carey MC. Filamentous crystallization of choles-terol and its dependence on lecithin species in bile. Mol Cryst Liq Cryst1994;248:291–6.

26] Kleiner O, Ramesh J, Huleihel3, Cohen B, Kantarovich K, Levi C, et al. A compar-ative study of gallstones from children and adults using FTIR spectroscopy andfluorescence microscopy. BMC Gastroenterol 2002;2(3):1–14.

27] Sikkandar S, Jayakumar S, Gunasekaran S, Renugadevi TS, Alwar B. Study on theanalysis of human gallstones using Fourier transform infrared spectroscopictechnique. Int J ChemTech Res 2011;3(1):149–54.

28] Castagne D, Dive G, Evrard B, Frédérich M, Piel G. Spectroscopic Studies andMolecular Modeling for Understanding the Interactions Between Cholesteroland Cyclodextrins. J Pharm Pharmaceut Sci 2010;13(2):362–77.

29] Zhou XS, Shen GR, Wu JG, Li WH, Xu YZ, Weng SF, et al. A spectroscopic studyof pigment gallstones in China. Biospectroscopy 1997;3(5):371–80.

30] Murphy GM. Facets of cholesterol crystallization in human bile. J Phys D ApplPhys 1991;24:119–22.

31] Soloway RD, Trotman BW, Ostrow JD. Pigment gallstones. Gastroenterology1977;72:167–82.

32] Abboud IA. Concentration effect of trace metals in Jordanian patients of urinarycalculi. J Soc Environ Geochem Health 2008;30(1):11–20.

33] Lazeeza Sattar Omer. Quantitative analysis in (33) traces metals in humangallstones by ICP-AES. Int J Chem 2011;3(2):105–10.

34] Johnston SM, Murray KP, Martin SA, Fox-Talbot K, Lipsett PA, LillemoeKD, et al. Iron deficiency enhances cholesterol gallstone formation. Surgery1997;122(2):354–61.

35] Kumar M, Goyal BB, Mahajan M, Singh S. Role of iron deficiency in the formationof gallstones. Indian J Surg 2006;68(2):80–3.

36] Verma GR, Pandey AK, Bose SM, Prasad R. Study of serum calcium and traceelements in chronic cholelithiasis. ANZ J Surg 2002;72:596–9.

37] Abu-Farsakh F. Correlation between copper, zinc and some lipids in serum,bile and stones of patients with gallstone disease. Dirasat Med Biol Sci1997;24(1):54–9.

38] Sipos P, Szentmihályi K, Fehér E, Abaza M, Szilágyi M, Blázovics A. Someeffects of lead contamination on liver and gallbladder bile. Acta Biol Szeged2003;47(1–4):139–42.

39] Godelitsas A, Nastos P, Mertzimekis TJ, Toli K, Simon R, Göttlicher J. A micro-scopic and Synchrotron-based characterization of urban particulate matter(PM10–PM2.5 and PM2.5) from Athens atmosphere, Greece. Nucl Instrum Meth-ods B 2011;269(24):3077–81.

40] Shukla VK, Prakash A, Tripathi BD, Reddy DCS, Singh S. Biliary heavy metalconcentrations in carcinoma of the gall bladder: case–control study. BMJ1998;317(7168):1288–9.

41] DeNiro MJ, Epstein S. Influence of diet on the distribution of carbon isotopes in

to the chemical and isotopic composition of human-body biomin-Med Biol (2012), http://dx.doi.org/10.1016/j.jtemb.2012.08.004

animals. Geochim Cosmochim Acta 1978;42:495–506.42] Jim S, Ambrose SH, Evershed RP. Stable carbon isotopic evidence for differences

in the dietary origin of bone cholesterol, collagen and apatite: implica-tions for their use in palaeodietary reconstruction. Geochim Cosmochim Acta2004;68(1):61–72.