ORIGINAL ARTICLE

Cross-sectional study of kidney stones by laser-inducedbreakdown spectroscopy

V. K. Singh & A. K. Rai & P. K. Rai & P. K. Jindal

Received: 5 July 2008 /Accepted: 25 November 2008 /Published online: 23 December 2008# Springer-Verlag London Limited 2008

Abstract We performed laser-induced breakdown spec-troscopy (LIBS) for the in situ quantitative estimation ofelemental constituents distributed in different parts ofkidney stones obtained directly from patients by surgery.We did this by focusing the laser light directly on thecenter, shell, and surface of the stones to find the spatialdistribution of the elements inside the stone. The elementsdetected in the stones were calcium, magnesium, manga-nese, copper, iron, zinc, strontium, sodium, potassium,carbon, hydrogen, nitrogen, oxygen, phosphorus, sulfur,and chlorine (Cl), etc. We optimized the LIBS signals byvarying the laser energy from 10 mJ to 40 mJ to obtain thebest signal-to-background and signal-to-noise ratios. Weestimated the quantities of different elements in the stones bydrawing calibration curves, plotting graphs of the analytesignal versus the absolute concentration of the elements instandard samples. The detection limits of the calibrationcurves were discussed. The concentrations of the differentelements were found to be widely different in differentstones found in different age groups of patients. It wasobserved that stones containing higher amounts of copperalso possessed higher amounts of zinc. In general, theconcentrations of trace elements present in the kidney stonesdecreased as we moved from center to shell and surface. Our

results also revealed that the concentrations of elementspresent in the stones increased with the age of the patients.The results obtained from the calibration curves werecompared with results from inductively coupled plasmamass spectrometry (ICP-MS). We also used the intensityratios of different elemental lines to find the spatialdistribution of different elements inside the kidney stones.

Keywords Laser-induced breakdown spectroscopy (LIBS) .

Kidney stones . Calibration curve . Trace elements

Introduction

The major aim of research in the field of biomedical scienceover the past few decades has been to determine theconcentrations of various elements in human tissues [1]. Inparticular, much research has been done to explain the effectsof the accumulation of certain elements by specific organswhich results in disease and disorders of these organs.

Kidney stone formation in the urinary tract of the humanbody is one of the most painful urological disordersthroughout the world [2–5]. Kidney stones are classified,according to their location, as renal/kidney stones, ureteralstones, bladder stones, and urethral stones. After removalby surgery, treatment with extracorporeal shock wavelithotripsy (ESWL), or drugs, the stones can be subjectedto further analysis and studies by several techniques todetermine their constituents [2, 3].

It has been reported that approximately 75% of kidneystones are composed mainly of calcium oxalate (CaC2O4.H2O) [4], and they are known as calcium oxalate stones. Inaddition to CaC2O4.H2O, other calcium (Ca) compoundsare also present in human urinary stones: calcium phos-phate [Ca10(PO4)6.2H2O], calcium carbonate (CaCO3),

Lasers Med Sci (2009) 24:749–759DOI 10.1007/s10103-008-0635-2

V. K. Singh :A. K. Rai (*)Laser Spectroscopy Research Laboratory, Department of Physics,University of Allahabad,Allahabad 211002, Indiae-mail: awadheshkrai@rediffmail.com

V. K. Singhe-mail: singh_vivekkumar2005@yahoo.com

P. K. Rai : P. K. JindalDepartment of Nephrology and Urology, Opal Hospital,Varanasi, India

brushite (CaHPO4.2H2O), gypsum (CaSO4.2H2O), anddolomite [CaMg(CO3)2] [5], but a much larger percentageof kidney stones is reported to be mostly calcium oxalate[6]. Those authors reported that 63% of the compositions ofkidney stones were calcium oxalate stones, which were inthe form of mineral whewellite (CaC2O4.H2O) (43%) andweddellite (CaC2O4.2H2O) (20%). On the basis of theirmajor constituents, the other types of kidney stones aremagnesium ammonium phosphate (MgNH4.PO4.6H2O) orstruvite stones, uric acid (C5H4N4O3) stones, and cysteine(C6H12N2O4S2) stones, but the percentages of these stonesare 15%, 5–10%, and 1%, respectively of the total kidneystones [5].

The diagnostic information regarding the chemical com-position of kidney stones has been recognized since the 1950sand has significantly improved during past few years. In thepast, several techniques, such as chemical analysis [7, 8],infra-red spectroscopy [9], and X-ray powder diffraction [10,11] have been used for the routine analysis of kidney stones.

There is a great need for the etiological factors in urinarystone disease to be determined, so that the cause of thisproblem can be explained. The bulk elemental compositionof kidney stones has been investigated previously byseveral authors [12–17], but there are very few reportsavailable for the spatial distribution of the elements withinthese stones [18]. Knowledge of the spatially distributedelemental constituents of kidney stones is essential if we areto know about their initiation and formation, which mayprovide a precise diagnosis and mechanism to help usunderstand the molecular growth in kidney stones. Thisknowledge helps the nephrologist to elucidate the initiationand growth controlling factors associated with the struc-tures of this painful disease and may also help to facilitatethe treatment protocols.

In order to overcome the aforesaid problems, laser-induced breakdown spectroscopy (LIBS) has been utilizedfor the detection and quantification of spatially distributedmajor and trace elements present in kidney stones. In LIBSa pulsed laser beam is focused on the surface of the sampleunder investigation. This generates high-density plasma thatexcites various atomic elements and induces elementaltransitions in the focal volume. The light emitted by theplasma is collected and subsequently analyzed to determineelemental concentrations in the sample. Element identifica-tion is based on the emission wavelength, and the relativeabundance is related to the calibrated intensity of theemission peaks present in a LIBS spectrum. LIBS permitsreal-time qualitative and quantitative identification of thetraces in solids, liquids and gases. In recent years, LIBS hasbeen successfully applied to fast multi-elemental pollutantanalysis in the atmosphere and in the solid matrix. LIBScan be useful for analysis of liquid and solid samples for avariety of applications [19, 20].

Fang et al. [21] used laser-induced plasma spectroscopy(LIPS) to analyze and identify the elemental constituents ofurinary stones without giving any specification for spatialdistribution, and they quantified some elements withoutgiving any more idea about calibration related to thoseparticular elements. We have already demonstrated theusefulness of the LIBS technique for variational study ofthe constituents of cholesterol stones and other kinds ofgallstones [22, 23]. Further, our group has alreadydemonstrated the ability of LIBS to detect and quantifytraces in environmental samples like soils [24, 25] andindustrial wastes [26], biomedical applications such asdiabetes management [27], and also for the detection ofnitro-compounds [28]. The unique features of LIBS are: itis a simple, rapid, remote, real-time analysis withoutsampling requirements. The point detection capability ofLIBS is a motivation for the study of the relativedistribution (from center to surface) of major and minorconstituents present inside gallbladder and kidney stones,and such type of study is very difficult using othertechniques, even X-ray fluorescence (XRF), inductivelycoupled plasma atomic emission spectrometry (ICP-AES),laser ablation-inductively coupled plasma mass spectrome-try (LA-ICP-MS), atomic absorption spectroscopy (AAS),etc. Thus, by using LIBS, it is possible to study theelemental distribution of numerous elements across thewidth of the stones which will elucidate the variations inthe element accumulation profiles in these stones.

In this work we estimated the concentrations of the traceelements copper (Cu), magnesium (Mg), zinc (Zn), andstrontium (Sr) in the different parts (center, shell and surface)of the kidney stones by using calibration curves. Variationsin the relative concentrations of different constituents acrossthe width of the stones were also examined.

This study is important, because if we look at theetiology of kidney stone formation, then many factors areresponsible for stone formation. To carry out numerousinvestigations on patients is time consuming and expensive,and it can also cause discomfort to the patients. Elementalanalyses of kidney stones by LIBS may help us tounderstand the process of stone formation inside the humanbody and, thus, restrict investigation, expense, and discom-fort to patients. For this reason, we intend to explain theelemental content of the different parts of stones that areresponsible for the pathogenesis of the stones.

Material and methods

Collection and preparation of samples

The kidney stones were surgically removed from thepatients by the expert surgeon at the Opal Hospital,

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Varanasi, India. The specifications of the kidney stonesamples that we used are tabulated in Table 1. The stoneswere washed with deionized water to remove traces ofurine, blood and other possible contaminants, dried, storedin sealed pots and sent to the Laser Spectroscopy ResearchLaboratory, Department of Physics, University of Allaha-bad, Allahabad, India. The samples (Fig. 1a–e) supplied bythe surgeon at the Opal Hospital were used withoutundergoing any pre-treatment. The stones were cut intohalves with a blade so that the laser beam could be focusedon the cross-section at different points, from the center tothe shell and surface of the stone. Minute amounts of thesamples from the center, shell and surface of the stone werecarefully removed and sent to the Indian Institute ofTechnology Roorkee, India for ICP-MS analysis.

For the calibration curve (a graph between intensity ofthe analyte line and its concentration in the matrix),certified reference materials (CRMs) having a knownconcentration of analyte elements and having a similarmatrix to that of the sample which has to be analyzed arerequired. In the case of biological materials, it is difficult tofind a CRM with a similar matrix; therefore, such samplesare prepared by mixing the metal (powder or aqueousstandard solution) with a calcium oxalate monohydratedcompound, which is the major matrix compound of thekidney stone. The calibration curve is very sensitive to thehomogeneity of the artificially prepared CRM, due tothe very small size of the focused laser spot and smallmass (order of micrograms) of the sample vaporized by thelaser spark. In this work, a standard solution method waspreferred for the preparation of the CRM, to maintain thehomogeneity in the samples. The 5,000 μg/ml standardsolution of Cu, Mg Zn, and Sr was diluted with double-distilled water, and standard solutions with concentrationsranging from 50 p.p.m. (parts per million) to 5,000 p.p.m.of Cu, Zn, Sr, and Mg by weight were prepared. Thesestandard solutions were used for spiking in the standardsample, and the wet paste was rigorously shaken in a plasticvial and eventually dried at room temperature for severalhours. The dried sample was placed into a 13 mm diameterEvacuable KBr Die Set (Kimaya Engineers, India) andpressed with 10 tons of force for 2 min by a hydraulicmachine to form pellets, which were finally used to recordthe LIBS spectra of the standard samples.

Experimental setup

The schematic experimental setup used for the analysis ofthe kidney stone samples is presented in Fig. 2. Aneodymium:yttrium–aluminum–garnet (Nd:YAG) laser(Continuum Surelite III-10) was employed for the produc-tion of plasma on the surface of the kidney stone sample.The laser can deliver a maximum pulse energy of 425 mJ,with a pulse width of 3–4 ns at a wavelength of 532 nm,and can operate at a 10 Hz pulse repetition rate in Q-switched mode. The laser pulse at the second harmonicwavelength of 532 nm was focused by a convex lenshaving a focal length of 15 cm onto the surface of thesample to create plasma for the LIBS studies. The pulseenergy utilized was in the range of 10–40 mJ. The laserenergy was monitored by direct measurement on acalibrated energy meter (Gentec-e model: UP19K-30H-VM-DO) placed in the path of the main laser beam.

The fiber bundle with a small lens at one end was usedfor the efficient collection of emissions from the plasmagenerated on the sample surface. The other end of the fiberswas connected to a broadband spectrometer (Ocean OpticsLIBS 2000+), which had four spectrometer modules toprovide high resolution of 0.1 nm in the 200 nm to 510 nmwavelength regions and low resolution of 0.75 nm in thespectral range of 200 nm to 900 nm. The detector had agated charge-coupled device (CCD) camera having 2,048pixels. This made it possible for us to measure a LIBSspectrum over a broad spectral range of 200–510 nm, withspectral resolution (0.1 nm), and 200–900 nm, with spectralresolution (0.75 nm), simultaneously. Software (OOILIBSsoftware, working in Windows 2000 professional mode)built into the spectrometer read the data from the chip andreconstructed the spectrum. The software provided elemen-tal identification through a spectral database for qualitativeanalysis. To record the LIBS spectra, the sample was placedon a platform mounted on a rotary table so that, every time,a new portion of the sample was irradiated to avoid craterformation on the sample surface. Fifty laser pulses weredirected onto the target sample to record one LIBSspectrum for analysis. The concentrations of different tracemetals present in the kidney stone samples were alsomeasured by ICP-MS to verify the results achieved with theLIBS technique.

Table 1 Specifications of the kidney stone samples used in our study

Sample Patients age (years) Gender Number of stones, shape and size

First (Fig. 1a) 24 Male Two; oval; ranging from 2.5–5 mm along the diameterSecond (Fig. 1b) 30 Male One; irregular shape; 2.5 mm×8 mmThird (Fig. 1c) 40 Male Two; oval, distorted shape; ranging from 2.5 mm–7.5 mm along the diameterFourth (Fig. 1d) 45 Male One; oval; approximately 10 mm in diameterFifth (Fig. 1e) 55 Male One; oval; approximately 12 mm in diameter

Lasers Med Sci (2009) 24:749–759 751

Results and discussion

Optimization of laser energy

To study the effect of the laser energy on the intensity of theatomic line present in the LIBS spectra of any sample, werecorded the LIBS spectra at different laser energies. Allother parameters, such as target rotation speed, or the sizeof the focus spot on the sample surface, were kept constant.The LIBS spectra of the standard samples (CRMs) wererecorded in the 200–510 nm spectral range by varying laser

energies from 10mJ per pulse to 40mJ per pulse in the steps of5 mJ (Fig. 3). Figure 4 shows a typical trend of energy-dependent intensity of the atomic line of Sr I (460.7 nm) inthe LIBS spectra of the sample containing Sr. It can be seenfrom Fig. 4 that the intensity of the atomic line increaseslinearly with increase in laser energy up to 30 mJ, but, athigher laser energies, the rate of increase becomes slower dueto saturation in signal intensity at higher laser pulse energies.Thus, it was concluded that a laser energy of 30 mJ wasenough for us to obtain reasonable line intensity for detectionof the elements present in the kidney stone samples.

(a) (b)

(c) (d)

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Fig. 1 (a)–(e) Photographs ofthe kidney stone samples exam-ined by laser-induced break-down spectroscopy (LIBS)

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Laser-induced breakdown spectra of kidney stone samples

The LIBS spectra of several kidney stones at differentlocations were recorded in the spectral range 200–510 nmwith spectral resolution 0.1 nm and in the spectral range200–900 nm with spectral resolution 0.75 nm, and a typicalLIBS spectrum of one kidney stone is shown in Fig. 5. Thespectra collected from the samples were representative ofvarious elements corresponding to the composition of the

samples. All the elements present in the sample wereidentified from the spectral analysis using the NationalInstitute of Standards and Technology (NIST) data base andspectra reference data book [29]. The elements detected inthe kidney stones were: calcium (Ca), magnesium (Mg),manganese (Mn), copper (Cu), iron (Fe), zinc (Zn),strontium (Sr), sodium (Na), potassium (K), phosphorus(P), sulfur (S), and chlorine (Cl), etc. The light organicelements such as carbon (C), hydrogen (H), nitrogen (N)and oxygen (O) were also detected in the kidney stones andare difficult to detect by other conventional techniques like

Fig. 2 A schematic diagram ofthe experimental setup of laser-induced breakdown spectrosco-py (LIBS) for the analysis ofkidney stone samples

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Fig. 3 LIBS spectra of a standard sample (matrix of calcium oxalatecontaining 400 p.p.m. Sr) at different laser energies. The emissionpeaks due to trace metals (Sr) present in the samples are indicated inthe spectra

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Lasers Med Sci (2009) 24:749–759 753

X-ray fluorescence and ICP-MS. The presence of theseelements clearly shows that the constituents of kidney stonesare inorganic and organic compounds. The presence of theatomic lines of Ca and P indicate that the one of the probableconstituents of kidney stones is calcium phosphate. Thepresence of S and Ca, Mg and Ca, suggests that the otherprobable constituents of the kidney stones were gypsum(CaSO4.2H2O) and dolomite [CaMg (CO3)2], respectively.The presence of Cl, Na and K in the stones can be attributedto the formation of the halides, NaCl and KCl, in ionic formduring the initial precipitation stage of the stone formation.

To determine the type of the stone, it is very important toknow its major constituents. Thus, the stone samples wereanalyzed by ICP-MS, which showed that the percentageconcentration of Ca in the kidney stones was up to 43%.The relatively high concentrations of Ca present in all thestones showed that the major component of the kidneystones utilized in our study was calcium oxalate (CaC2O4.H2O). Therefore, we chose this compound as the matrixmaterial to make the standard sample for drawing thecalibration curve, because the LIBS signals are verysensitive to the matrix of the materials.

Quantitative analysis of trace elements in the kidney stonesby using the calibration curve

For quantitative analysis of Cu, Mg, Zn, and Sr present inthe kidney stone samples, we required different CRMshaving varying concentrations of these elements to obtainproper calibration curves. Thus, for the construction of thecalibrations curves, different stoichiometric samples (hav-ing 5,000 p.p.m., 1,000 p.p.m., 800 p.p.m., 600 p.p.m.,400 p.p.m., 200 p.p.m., 100 p.p.m., and 50 p.p.m. of Cu,Mg, Zn, and Sr in the matrix of calcium oxalate (CaC2O4.

H2O) were prepared in pellet form by the standard solutionmethod described earlier. In order to test the homogeneityof our samples, we performed several LIBS measurementsat different locations on the surface of the pellets, and weobserved that the ratio of the intensity of the atomic line oftrace elements (Cu, Mg, Zn, and Sr) to the intensity of theatomic line of the major element (Ca) was same in eachspectrum obtained at the different locations. Each spectrumwas obtained from an average of 50 laser shots, at differentlocations on the sample surface, and five such spectra wereaveraged to obtain better signal-to-background and signal-to-noise ratios. This averaging of the spectra reduced thebackground noise to a great extent when compared with thesingle-shot spectrum of the sample. For drawing the calibra-tion curve we chose those atomic lines of the elements thatwere persistent, i.e., lines must appear with the lowestconcentration of the particular element present in the sample.The atomic lines chosen for this purpose were based on thestrongest transitions of Cu [3d10(1S) 4p (2P3/2) → 3d10(1S) 4s(2S1/2)] at 324.7 nm, Mg [3s3p (1P3) → 3s2 (1S0)] at285.2 nm, Zn [3d104p (2P3/2) → 3d104s (2S1/2)] at 202.5 nm,and Sr [5s5p (1P1) → 5s2 (1S0)] at 460.7 nm, with the leastpossible laser irradiation of the samples.

We drew the calibration curves for these lines by plottingelemental concentration vs emission intensity of theseelements, using ORIGIN software. These calibration curvesare shown in Figs. 6, 7, 8, and 9. We obtained the slopes ofthese calibration curves by fitting data in the linearregression functions for these spectral lines:

Cu I 324:7 nm; y ¼ 0:2xþ 2:9;R ¼ 0:999Zn II 202:5 nm; y ¼ 0:101x� 1:83;R ¼ 0:994Sr I 460:7 nm; y ¼ 0:342xþ 148:6;R ¼ 0:997Mg II 285:2 nm; y ¼ 0:198xþ 169:25;R ¼ 0:999

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Fig. 6 Calibration curve for Cu I at 324.7 nm

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The non-zero intercept in the calibration curves is due tobackground signal [26, 30].

The limit of detection (LOD) is one of the importantparameters for any analytical technique, and it is basicallythe lowest concentration that can be detected. In our workthe LOD was estimated by the 3σ rule, which is describedin detail elsewhere [26].

The LOD was calculated by the formula

Limit of detection ¼ 3sB

S

where σB is the standard deviation of the background and Sis the calibration sensitivity (slope of the calibration curve).

For the calculation of the standard deviation of thebackground (σB), five pixel-to-pixel measurements ofcontinuum background signal, on either side (left and right)of the chosen atomic lines, were performed. These measure-ments were made for five spectra, and, finally, the meanrelative standard deviation (RSD) of the background signalwas calculated for each element separately. The LODs forthe elements Cu, Mg, Zn, and Sr, estimated from ourexperimental setup, were 9 p.p.m., 8.5 p.p.m., 10 p.p.m.,and 17 p.p.m., respectively.

We recorded the LIBS spectra of several kidney stonesamples under the same experimental conditions as used forthe standard samples. The intensity of the atomic lines forCu at 324.7 nm, Mg at 285.2 nm, Zn at 202.5 nm, and Sr at460.7 nm observed in the LIBS spectra of each kidneystone were measured, and the concentrations of theelements were calculated from the calibration curves. Theresults are tabulated in Table 2. To validate our results, wealso measured the concentration of these elements in a fewsamples by using the ICP technique, and the results are alsotabulated in Table 2. From Table 2 it is clearly seen that theLIBS measurements are in good agreement with the ICPspectrometry measurements, except the concentration of Cuand Mg in the center and shell of the first stone. ICPanalyses did not detect the elements Cu and Mg in thecenter and shell of this stone, which seems to be not correct,because we observed the persistent lines of Cu [3d10(1S) 4p(2P3/2) → 3d10(1S) 4s (2S1/2)] at 324.7 nm and Mg [3s3p(1P3) → 3s2 (1S0)] at 285.2 nm in their LIBS spectra[Fig. 5]. Further, our results reveal that the concentrationsof Cu and Zn were higher in the center than in the shell andsurface parts of the kidney stone (Table 2), which was inagreement with the results obtained by Durak et al. [31].However, according to ICP analysis, the concentration of

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Fig. 7 Calibration curve for Mg I at 285.2 nm

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Fig. 8 Calibration curve for Zn II at 202.5 nm

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Fig. 9 Calibration curve for Sr I at 460.7 nm

Lasers Med Sci (2009) 24:749–759 755

Zn in the center was less than at the surface, and even Znwas absent in the shell of the first kidney stone, whichcontradicted previous results [22, 31] as well as thepresence of the persistent atomic lines of Zn [3d104p(2P3/2) → 3d104s (2S1/2)] at 202.5 nm in the LIBS spectraof the shell of this kidney stone.

It was clear from ICP-MS analysis that the majorconstituents/main matrix of the kidney stones was calcium,and this fact was supported by the LIBS spectra of thekidney stones [Fig. 10]. It is clear from Fig. 10 that theintensity of the Ca lines at 714.8 nm, 720.2 nm, and732.6 nm in the LIBS spectra of the center, shell, andsurface of the stone is nearly same. This is because; theconcentration of the main matrix of the stones did not varyby an appreciable amount throughout the whole stone.Thus, according to our analysis, the majority of the stoneswere rich in calcium. It is probable that hypercalciuria wasone of the important risk factors in the formation of calciumstones in this study. The high content of calcium in kidneystones also promotes the idea that compositional analysisshould be an integral part of metabolic evaluation inpatients with nephrolithiasis.

There has been a lot of interest in the role and effects ofinorganic Zn ions and Cu ions in urolithiasis because theyare found in both urine and urinary stones at the trace level[32]. It is clear from Table 2 that the stones containing asignificant higher amount of Zn also contained higheramounts of Cu. Therefore, Zn and Cu may have a triggereffect on stone formation and may finally cause urolithiasis.As a result, the higher concentration of Zn and Cu in thecenter than in the shell and surface should still beinvestigated to find a possible connection between inor-

ganic ions and kidney stone formation, to clarify thepathological mechanisms of stone formation.

Mg is an important element in the biological calcifica-tion process, but the exact role of Mg in kidney stoneformation still remains undefined. In vitro studies havedemonstrated decreased nucleation and growth of calciumoxalate crystals in the presence of super-physiologicconcentrations of Mg [33, 34]. Several animal studieshave suggested hypomagnesuria to be a risk factor forstone formation. Most trials have shown that supplemen-tation with Mg and/or vitamin B6 significantly lowers therisk of kidney stone formation [33, 34]. Similar to other

Table 2 The absolute concentrations of the elements detected in the kidney stones by LIBS and ICP spectrometry

Sample Location Cu Mg Zn Sr

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ICP(μg/g)

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ICP(μg/g)

LIBS(μg/g)

ICP(μg/g)

LIBS(μg/g)

ICP(μg/g)

First stone Center 24.24 0 593 0 52 12 25 22.4Shell 18.76 0 227 0 44.95 0 28.5 24Surface 12.34 7.2 1,019 1,100 29.73 20 38.13 41.5

Second stone Center 28.52 4.86 1,236 1,460 76.5 88.8 51.35 55.2Shell < 9 2.73 1,205 1,130 72.97 80.4 65.7 65.3Surface < 9 0.676 977 932 52.55 47.9 56.14 59.4

Third stone Center 153 – 1,067 – 92.2 – 101 –Shell 93 – 945 – 89.7 – 88 –Surface 88 – 767 – 57.3 – 79 –

Fourth stone Center 225 – 1,081 – 109 – 106 –Shell 120.4 – 789 – 91 – 99 –Surface 108 – 991 – 75.3 – 88.5 –

Fifth stone Center 3,682 4,598 3,121 3,900 1,100 1,359 913 –Shell 840 – 2,250 – 576 – 423 –Surface 448 – 1,469 – 191 – 267 –

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Fig. 10 LIBS spectra from the center, shell and surface of the firstkidney stone sample in the spectral range 700–800 nm

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studies [32–34], we also detected Mg in appreciableamounts in every part of all the stones in our study. Theconcentration of Mg varied across the width of the stone,and, in most of the stones, the concentration was higher inthe center than in the shell and surface, except in firststone, which did not follow this trend (Table 2). A furtherstudy with a large sample size is required to elucidate thiscontradictory concentration of Mg across the width of thestone.

The variation in concentration of Sr across the width ofthe stones was very small, therefore no regular pattern inthe variation of spatial distribution of Sr inside the stonewas observed, which is in agreement with the resultreported by Chaudhri et al. [18]. The concentration of Srwas higher in the center than in the shell and surface of thekidney stone, except for in the first two stones. Theconcentration of Sr across the width of the first two stoneswas not uniform, which indicated a difference in chemicalcomposition between the nucleus and outer parts of thesetwo stones. Perhaps these two stones had grown underrelatively changing conditions that had given rise to a non-uniform chemical deposition across the stone. Strontiumconcentrations in water and food contribute to the strontiumconcentrations in the human body and, consequently, inpatients with kidney stones, which may vary in differentgeographical areas. It is also clear from Tables 1 and 2 thatthe strontium content in stones found in older patients washigher than in the stones found in younger patients. If weconsider the chemical composition in the central part of thestones, then it was observed that the content of the entireelement in the stone found in older patients was higher thanin the stone found in younger patients. It is not an easytask to correlate the higher concentration of elementspresent in the samples of the older patients. However, itwas observed that the concentrations of elements in thestones in the older age group were higher than in those inthe younger group. This can be clearly seen from Table 2,and demonstrates that the chemical mechanism of deposi-

tion of minor elements in the stone is dependent on the ageof the patient.

We also estimated relative spatial distribution of ele-ments potassium (K) and phosphorous (P) across the widthof the stone by taking the line intensity ratio (intensity ofminor element/intensity of major element). The intensityratios of the elements present in the different part of thestones are tabulated in Table 3. As with the elements Cu,Mg, Zn, and Sr, the concentration of K and P was higher inthe central part than in the shell and surface parts (Table 3).The LIBS spectra of center, shell and surface of a kidneystone also revealed that the atomic lines of K at 766.9 nmdecrease as we go from center to surface (Fig. 10). Thehigher amount of phosphorous in the central part of thestones may be explained by the anchoring of crystals to therenal epithelium, which allows the crystal to grow into aclinically significant calculus or stone. Calcium oxalatecrystals have been shown to anchor onto areas of calciumphosphate deposits termed Randall’s plaques, which aremostly composed of apatite crystals [35]. During nucle-ation, calcium oxalate crystals attach to these plaques,allowing significant stone growth, and, hence, phosphorousis deposited in the form of apatite in different parts of thestones.

The elements Cu, Zn, P, and K had a similar distributionpattern across the width of the stones, although there weresignificant differences between the absolute concentrations.The higher concentration of these elements in the centerpart of the stones than in the shell and surface suggestedthat a completely different metabolic process is involved forthe nucleation of the center than the deposition process inthe shell and surface. It is probable that the higherconcentration of the elements Cu, Zn, P, and K along withCa as well as oxalic acid in urine may become an initiatingfactor in the nucleation process for rapid stone precipitationwithin the renal system and, consequently, may be thecause of the higher level of these elements in the center ornucleus of the stones. However, a more detailed study of

Table 3 Intensity ratios of analyte line and reference line measured from LIBS spectra of the center, shell and surface of the kidney stones

Sample Analyte line (nm)/reference line (nm) Intensity ratios

Center Shell Surface

First stone K (766.9)/Ca (732.6) 0.75 (±7.6%) 0.45 (±11%) 0.23 (±8.6%)P (649.6)/Ca (732.6) 3.62 (±7.4%) 3.5 (±1.4%) 3.42 (±8%)

Second stone K (766.9)/Ca (732.6) 0.75 (±19.2%) 0.35 (±5%) 0.23 (±13.1%)P (649.6)/Ca (732.6) 3.59 (±5.6%) 3.24 (±1.03%) 2.93 (±1.6%)

Third stone K (766.9)/Ca (732.6) 0.63 (±17.9%) 0.38 (±12.6%) 0.25 (±12.2%)P (649.6)/Ca (732.6) 3.49 (±11%) 3.4 (±2.9%) 2.8 (±1.76%)

Fourth stone K (766.9)/Ca (732.6) 0.505 (±7.6%) 0.29 (±3.4%) 0.25 (±12.8%)P (649.6)/Ca (732.6) 3.01 (±10.5%) 2.76 (±22%) 2.55 (±18%)

Fifth stone K (766.9)/Ca (732.6) 0.72 (±18.7%) 0.406 (±9.8%) 0.25 (±9.3%)P (649.6)/Ca (732.6) 3.01 (±18%) 2.7 (±6.3%) 2.1 (±20%)

Lasers Med Sci (2009) 24:749–759 757

the nucleation process must be undertaken, as this may givefundamental information on nucleation, and, possibly, itwould lead to more concrete concepts for a preventivemechanism.

Conclusion

For the first time, the in situ spatial distribution of kidneystones has been studied successfully by the use of LIBS.Quantitative estimation of the concentrations of the traceelements Cu, Mg, Zn, and Sr in different parts of the stoneswill help us to understand the process of urinary stoneformation, and, accordingly, nephrologists may plan safermethods to prevent the formation of stones in the body. Ourresult reveals that LIBS offers a suitable method to obtaininformation about the spatial distribution of elements indifferent kinds of stones found in the human body, withoutdestroying the stones.

Acknowledgments The authors would like to thank Dr. A.K.Chawdhary, of the Indian Institute of Technology Roorkee (IIT-Roorkee)for providing the facility of ICP-MS analysis of the kidney stone samples.Financial assistance from the Defence Research & DevelopmentOrganization (DRDO) project (no. ERIP/ER/04303481/M/01/787) isduly acknowledged. V.K. Singh thanks Allahabad University, Allahabad,India, for financial support in the form of a D. Phil. Scholarship under theUniversity Grants Commission (UGC) scheme.

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