Pradeep K. Rai

n this study, we analyzed a tooth sample of a renal patient using wavelength-dispersive X-ray fluorescence (WDXRF) spectrometry and Fourier transform infrared (FT-IR) spectrometry to quantify major and trace elements, and heavy and toxic elements in particular. The elements detected and quantified in the tooth sample were calcium (Ca), phosphorus (P), carbon (C), oxygen (O), magnesium (Mg), sodium (Na), chlorine (Cl), sulfur (S), potassium (K), silicon (Si), iron (Fe), aluminum (Al), zinc (Zn), strontium (Sr), chromium (Cr), nickel (Ni), copper (Cu), and zirconium (Zr). FT-IR spectrometry has been used to extract relevant information about the molecular contents of the tooth sample. We have compared our results with those obtained in the laboratory for the healthy subjects, and also with the values reported in the literature as obtained using XRF spectrometry only. Elements found in the tooth sample were correlated with the chronic kidney disease (CKD) of the patients. An outline of the XRF apparatus and applications of XRF as a tool for analyzing to dental specimens are also briefly highlighted.

Vivek K. Singh, Shikha Sharma, Mahima Sharma, Neha Sharma, Jitendra Sharma, and Pradeep K. Rai

	
	Calcified tissues, such as bones and teeth, contain biological signatures from their living phase. The habitat and nutritional and environmental conditions of people can be determined by monitoring the trace elements present in these samples (1). This is not unexpected, as teeth have both an organic and inorganic phase. Teeth and bones consist of an inorganic calcium phosphate mineral approximated by hydroxylapatite and matrix proteins with a small amount of proteolglycans and lipids (2). In contrast, the inorganic phase is composed of well-packed nanocrystals of calcium apatite, together with trace elements (3). The trace elements in the tooth can vary, depending on the source of food and water, as well as from soil and dermal absorption (1–4). Accurate determination of trace elements in calcified tissues still remains a challenge for biomedical researchers. X-ray fluorescence (XRF) spectrometry offers a reliable approach to provide rapid multi-elemental analysis of biological samples in µg/g (ppm) range, and it is a non-destructive technique that allows samples to be preserved for further experimentation (5). Instrumentation, such as wavelength-dispersive XRF (WDXRF), can now be effectively used to accurately measure trace elements in the human tooth. Such information not only provides a useful assessment of the diet, but reflects specific environmental and disease conditions as well.

	Recent biochemical studies provide insight into the factors controlling the formation and growth of bioapatite crystals, and how alterations in the mineralization process can lead to diseases such as osteoporosis. The relationship between chronic kidney disease (CKD), specifically hypercalciuric nephrolithiasis, osteoporosis, and bone mineral density (BMD), is well established. A large number of epidemiologic studies linked to evaluating factors associated with BMD in men have amply demonstrated that history of renal disease is indeed associated with lower BMD (6). The kidneys play an important role in maintaining healthy bone mass and structure by balancing calcium (Ca) and phosphorus (P) levels in the blood. Mineral and bone disorders in CKD occur when damaged kidneys and abnormal hormone levels cause Ca and P levels in a patient’s blood to be out of balance. CKD causes mineral and bone disorders, and thus kidneys stop activating calcitriol. The low levels of calcitriol in the body in turn create an imbalance of Ca in the blood by not removing the P in the blood proportionately, so the P level rises in the blood. The extra P pulls Ca out of the bones, causing them to weaken. Consequently, bone losses from the jaw occur in the CKD patients. Such weaker bones can cause teeth to become loose, and eventually fall out.

	Therefore, in this paper, we present results from our investigation of major (Ca, P, magnesium [Mg], sodium [Na], chlorine [Cl], sulfur [S], potassium [K], silicon [Si], iron [Fe], aluminum [Al], zinc [Zn], strontium [Sr}, chromium [Cr], nickel [Ni], copper [Cu], and zirconium [Zr]), trace elements, and toxic metals using WDXRF spectrometry in a tooth sample obtained from a CKD patient. We have compared our results with literature reported values (using XRF spectrometry only) and laboratory values for healthy subjects. Fourier transform infrared (FT-IR) spectrometry has also been used to investigate the phosphate, carbonate, and amide contents of teeth samples. This paper presents a very crucial application of WDXRF spectrometry to quantify heavy and toxic elements of calcified tissue materials like tooth samples from renal patients with CKD.

	The tooth sample from the CKD patient was kindly provided by the Department of Nephrology at Opal Hospital in Varanasi, India. In the present work, the studied tooth sample was one that had become loose, and had eventually fallen out of the jaw of a CKD patient. The received tooth sample was dried and stored in a sealed pot after washing in deionized water to remove all traces of possible contaminants. The as-received tooth sample was examined visually, to check for caries. The tooth sample was not affected by caries, and therefore the present study was not influenced by caries formation. The tooth was then powdered using a mortar and pestle, and the powdered tooth sample was used subsequently for experimental measurements using FT-IR and WDXRF spectrometry.

	The ground tooth sample was used to record the FT-IR spectrum in a transmittance mode setting for the spectral region of 4000-400 cm

 on an IRTracer-100 Fourier transform infrared spectrophotometer (Shimadzu), using a single-reflection attenuated total reflection (ATR) accessory. In this spectrophotometer, the FT-IR spectrum is recorded in a very simple manner, as only few milligram (mg) of sample (solid or liquid ) are required to place on the surface of the ATR prism. However, solid samples must be held on the surface of the prism using the provided pressure clamp. The spectra can be recorded either in absorption mode or in transmission mode.

	XRF analysis provides elemental mapping and imaging information using emitted characteristic X-rays from the sample. Figure 1 shows the mechanism of characteristic X-ray generation. With the bombardment of high-energy electrons or high-energy X-rays, a bound electron is struck out of the target atom. After the electron is ejected, an outer shell electron falls into the vacant inner shell, resulting in the emission of characteristic X-rays with an energy equal to the difference of energy between the outer and inner shell energy levels. These X-rays generated through the bombardment of high energy X-ray irradiation are termed

. Each element with inherently different energy levels (for the set of electrons it possesses) emits X-rays of energies that uniquely identify this characteristic element. Therefore, such characteristic X-rays can easily be exploited to perform elemental analysis of a sample. Further details of the theory and applications of XRF spectrometry can be found in reference sources (7–11).

	High performance WDXRF spectrometry is now being used for the determination of traces in plant materials, biological samples, and geological samples. To fulfill today’s requirements for quality data, modern instruments must provide high elemental sensitivities to achieve low detection limits and optimal spectral resolution to minimize line overlays. In this work, we have used a WDXRF spectrometer S8 Tiger (Bruker) for the quantitative measurements of major, trace, and heavy elements in the sample. It provided the optimum quantitative results in terms of detection limits, resolution, and reliability for each element with the best excitation and highest intensity over the entire elemental range.

	In this WDXRF spectrometer, an end-window X-ray tube with 

Rh anode is adjusted to a voltage of 60 kV and a current of 170 mA, correlating to a maximum power supply of 4kW to generate the primary beam. To ensure more accurate and reproducible results, the tooth sample was analyzed three times. For the detection of lighter elements, a flow proportional counter is used, and, subsequently, a scintillation counter detector is used to detect heavier elements. The acquisition time taken to capture a single spectrum of the sample was 20−25 min. The analyzer crystal LiF(220) ensures that in combination with the 0.23° collimator, the best separation is achieved for adjacent elements. The mask size was 23 mm, and the spectrum was recorded in vacuum. Technical details of the spectrometer used in the present study can be found elsewhere (12). In the present work, while analyzing data, the maximum relative standard deviation (RSD) obtained was 5%. To measure the concentration of trace and heavy elements, the S8 Tiger WDXRF spectrometer uses a standardless semi-quantitative Spectraplus software (Quant Express) package. The technical details of this software can be found elsewhere (12). The unique feature of this software is that it corrects all the matrix effects based on theoretically fundamental physical parameters, such as absorption coefficients and secondary fluorescence enhancement, automatically. In this software, the elemental concentration has been determined using Sherman’s equation (13):

 is the measured intensity of the corresponding line; 

 are the concentrations of the other elements 

 are the coefficients of the calibration regression line, respectively, namely offset and slope. The advantage of this software is that it does not need any standard reference materials to check its reliability and reproducibility with respect to the given samples.

	Because of the limited amount of the tooth sample, we have used the sandwich method to create a pellet. The powdered tooth samples are pressed onto approximately two spatulas full of boric acid using a constant pressure of 15 tons for 15 s on the stainless steel die head using a hydraulic press to prepare a smooth and uniform surface. This method is generally used in cases of limited sample size, and, in such cases, the use of a non backscattering boric acid matrix is recommended (7–11).

Analysis of Tooth Sample Using FT-IR Spectrometry

	Unless otherwise noted, the literature citations for band assignments are given in references (14,15). The FT-IR spectra of the tooth sample are shown in Figures 2 and 3, and the functional groups found in the sample are indicated in their respective wide scan FT-IR spectra (Figures 2 and 3). A typical -OH stretching band is clearly visible at 3300 cm

. The characteristics peaks of phosphates appear at 579, 590, 608, and 614 cm

 asymmetric bending mode of ), 1023, 1043, 1052, and 1069 cm

 asymmetric stretching mode of ), 431, and 446 cm

 symmetric stretching mode of ). Phosphates in the sample are identified by the characteristic absorption band at 1035 cm

 asymmetric stretching of . Other phosphate bands (relatively broader) appeared around at 985 and 925 cm

 stretching in , is assigned to type B carbonate
	( ions substituting  sites), located at 870 cm

 mode presented two bands, one at 1412 cm

, respectively, while type B carbonate ν

 (shoulder peak) (8). Type A carbonate (substituting  sites) in the ν

	The four primary amide bands are amide I (1700−1600 cm

 represents amide III
	(N–H stretching vibration). The amide I band is representative of the secondary structure of proteins. It can be decomposed into three main components; the most intense being the central one, at about 1660 cm

, which has low- and high-frequency shoulders. Usually a shoulder peak is observed near 1620 cm

, which is attributed to the amide I feature of dentin and hydrated gel-like collagen. In the present study, the amide I vibrations arising from C=O stretching were centered in the broader 1627 cm

 band, with shoulders at 1604 and 1665 cm

, respectively. The amide A region, where the N–H stretching vibrations of the amide normally appear, had its major intensity peak near 3300 cm

 (overlapped with an OH stretching broadband around 3300 cm

). The amide B region, arising from C–H stretching, exhibited a band with higher intensities at 2857 and 2927 cm

, respectively. Absorption peaks centered at 1242 cm

 have been characterized as amide III (N–H stretching vibrations).

Analysis of Tooth Sample Using WDXRF Spectrometry

	The concentration of different elements present in the tooth sample was determined using WDXRF spectrometry measurements, and the values obtained are presented in Table I. The major and trace elements detected and quantified in the sample were calcium (Ca), phosphorus (P), carbon (C), oxygen (O), magnesium (Mg), sodium (Na), chlorine (Cl), sulfur (S), potassium (K), silicon (Si), iron (Fe), aluminum (Al), zinc (Zn), strontium (Sr), chromium (Cr), nickel (Ni), copper (Cu), and zirconium (Zr). Figure 4 depicts a typical WDXRF spectrum of the tooth sample obtained from the WDXRF spectrometer indicating the different elements present in it. In Table I, we have also summarized the concentration of major and trace elements found in the sample and compared them with those reported in the literature using only XRF techniques (16–21). Similarly, Table II presents the concentration of some major elements, including Ca, P, Mg, Na, K, and Cl, found in the studied sample, along with the average values for sound human enamel and dentin as reported by different laboratories (22–26). Here, to compare with our data, the concentration values from the healthy teeth were considered in the same way as we have analyzed the tooth sample from CKD patients. For the present study, we have analyzed the whole tooth, including enamel and dentine, by making them into a powder. As a constraint (refer to Table II), we are bound to compare our results with the literature-reported values of healthy subjects only, and not with the healthy teeth of the same CKD patient because we are unable to collect healthy teeth from the same patient.

	Organic matrix elements detected in the tooth sample were carbon (C) and oxygen (O). The elements Ca (14.73%), and P (7.98%) were the most abundant elements in the studied sample, as substantiated by other reports as well (16–26). It is clear from Table II that the concentrations of elements Ca, P, Mg, K, and Cl are lower in the tooth sample of the CKD patient than that from healthy teeth. Figure 5 clearly shows the relative concentration of such elements in the studied tooth sample. No such variation was observed for Na. The Ca to P ratio was also not consistent with the literature reported value. We noted that CKD apparently caused the imbalance of P in the blood, which in turn affects the Ca in tooth and bone, which is considered as being a major cause of imbalance of Ca in tooth and bone. Reduced amounts of Ca, P, and other major elements (necessary for healthy teeth) were indicated in the tooth sample and may have been caused by CKD and its consequences on the patient.

	Most of the elements detected in the tooth were those that accumulated within the structure of hydroxyapatite over time. Numerous substitution metals can be found in hydroxyapatite crystals; metal cations such as K

, whereas anionic complexes, including ,  or , can provide a substitute for , and anions such as Cl

 in the crystal structure (26–29). Therefore, it is not surprising to find these elements in the tooth sample we studied. However, our main objective was to present a comparative study (comparing to a healthy subject) to investigate the accumulation of elements in tooth samples of renal patients.

	In Table I, we have summarized the concentrations of major and other heavy and toxic elements found in the tooth sample of the present study along with the literature reported values (16–21). We have considered the recent studies of tooth samples using XRF spectrometry alone. Figures 6–11 show the relative concentrations of different elements and their variations. The Ca to P ratio (Ca/P) is an indicator of the relative strength of bones or teeth (30,31). From the Ca/P ratio plotted in Figure 6, one can easily see that the Ca and P values measured from the sample are relatively less as compared to the values reported by others like Buddhachat and associates (16), Oprea and colleagues (17), Baranowska and co-workers (18), Aadim and associates (19), Teruel and colleagues (20), and Bielecka and co-workers (14). Also, the Ca/P ratio reported by Buddhachat and associates (9), Oprea and colleagues. (10), Baranowska and co-workers. (18), Aadim and associates (19), and Teruel and colleagues (20) lie in the range of 1.94 to 2.44, which is also in good agreement with the reported values (average value ~2.07) for sound enamel and dentin, with the exception of Bielecka and co-workers (21) (value ~1.77). In the present study, the value obtained was ~1.84, which indicates different Ca and P levels due to the CKD affected tooth. The major elements Mg, Na, and Cl were also found to be lower in the tooth sample as compared to the reported values (16–21), which can be seen in Figures 7 and 8.

	In addition, the concentration of essential elements such as S, Si, and Zn was observed to be lower (as can be seen in Figure 9) than the literature reported values (16–21). Values obtained for potassium (K) were a little higher (Figure 8) than those in the literature. The other heavy and trace elements such as Fe, Al, Sr, Ni, Cu, and Zr were found in a different concentration range, as shown in Figures 8–11. The overall relative concentrations in log percentage value of major, trace, heavy, and toxic elements found in the tooth sample in the present study and reported values are plotted for comparison. The relative difference in the concentration of elements, particularly Fe, Al, Sr, Ni, Cu, and Zr, may be due to the different environmental and geographical regions to which the patient belongs, and also the patient’s dietary habits, which is a major factor that can influence the concentration of such elements in teeth. Refer to references (16–21) for these elemental values.

	Recently, Boskey (32) has discussed a variety of human diseases in which altered mineral properties occur in bones and teeth in relation to age and sex-matched healthy individuals, presenting some examples of the mineral changes that include frequently encountered diseases, such as osteoporosis and osteomalacia, and less-common diseases such as osteopetrosis, osteonecrosis, and osteogenesis imperfecta and odontogenesis imperfecta. Some of these diseases are due to genetic abnormalities, but they may also be attributed, to a varying degree, to environmental factors such as diet, exposure to sunlight, and exercise (33). Renal osteodystrophy was reported as a consequence of kidney malfunction, which leads to osteoporotic bone and decreased mineral contents (32−34). The decreased contents (as compared to healthy subjects) of essential elements, particularly Ca, P, Mg, Na, and Cl, in the tooth sample may also be attributed to the chronic kidney disease and hence may be related to renal osteodystrophy. The presence of other nonessential and toxic elements indicates their role in CKD and tooth mineralization. However, more elaborate experiments on multiple samples from different patients and their extensive studies will be needed.

	The present study presents an interesting aspect for further studies of tooth samples of renal patients. In this case, we were only able to analyze the tooth sample from one CKD patient, as such tooth samples from renal patients (of different history) scarcely become available. We, therefore, have presented a case study only, on the elemental composition of a tooth sample from a renal patient. However, to extract detailed scientific data, more extensive studies will be needed to draw definite conclusions on the effects of CKD in calcified tissue materials like teeth and bones.

	New spectroscopic and microscopic techniques are emerging every day, providing scientists with useful tools for characterizing even minute changes or transformations in crystal properties from diseases, providing potentially fruitful areas of collaboration between physicists and biologists and offering hope for the development of novel therapies. In the present study, we have successfully utilized WDXRF spectrometry for the detection and quantification of the elemental composition of a tooth sample from a renal patient, which indicates the potential usefulness of this technique toward the analysis of calcified tissue materials like bones and teeth. The elemental contents were compared with the literature values for a more fruitful discussion. The data demonstrated that the distribution of elements can be used to explain biological processes related to CKD. We have also utilized FT-IR as a powerful tool for detecting organic and inorganic constituents of biomaterials and successfully characterized phosphates, type A and B carbonates, amide, and water content in the tooth sample of the CKD patient. Therefore, our studies on the tooth sample demonstrate that FT-IR is a robust tool for detecting organic and inorganic constituents of biomaterials. The presence of variable contents of essential and nonessential elements indicates their role in CKD and renal osteodystrophy. However, more extensive spectroscopic investigations on tooth samples are required for more fruitful results using XRF spectrometry. 

	Both Ms. Shikha Sharma and Ms. Mahima Sharma both contributed equally in carrying out the experiments and drafting of the manuscript. We are thankful to the Sophisticated Analytical Instrumentation Facility (SAIF), at Panjab University, in Chandigarh, for providing WDXRF experimental facilities. Our special thanks go to Mr. Tejbir Singh at the WDXRF Lab SAIF, for his valuable suggestions and discussions related to WDXRF spectrometry and instrumentation. The authors are also thankful to staff members at the Department of Nephrology at Opal Hospital, in Varanasi, India, for their constant and timely support in planning the experiments on a sample of choice.
	
	

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Articles

In this study, WDXRF and FT-IR are used to analyze a tooth sample of a renal patient, and to compare the results to healthy patients. The quantities of multiple elements are reported using the XRF technique, and FT-IR spectroscopy is used to extract relevant information about the molecular contents of the sample with the important absorption bands identified.

Application of WDXRF and FT-IR for Human Tooth Analysis

Wavelength-dispersive X-ray fluorescence (WD-XRF) spectrometry involves the classification and quantification of several kinds of biological samples. Multiple applications of WD-XRF spectrometry are anticipated in the mining industries, nuclear power industry, medical diagnostics, forensics, environmental, and particularly in agricultural studies. Here, we present a concise review of the quantitative measurements performed to classify and quantify the traces of essential and toxic minerals in wheat seed gall nematodes (

 rhizomes, gallstones, and kidney stones. Based on the studies reported, WD-XRF was found to be a robust analytical tool that is useful for agricultural studies and for the diagnosis of urological and gastroenterological disorders. This review is aimed at analyzing major and trace levels of heavy and toxic minerals in biological specimens related to agricultural crops (wheat grains and 

) and human diseases (gallstones and kidney stones) and shows some interesting prospects for using WD-XRF spectrometry.

	Trace and heavy metal detection in agricultural and biological samples currently is of crucial importance because of the pollution occurring in the environment, particularly in soil and water, which constantly affect human health. Agricultural crops, medicinal herbs, and natural resources used for human benefits are being contaminated by the presence of toxic elements such as lead, arsenic, mercury, chromium, and nickel. These elements then move from soils to plants and then to human beings. They can cause serious diseases such as cancer or stone formation in the human body (gallstones and kidney stones). Hence, a holistic approach is needed to analyze agricultural and biological samples to find the source of these toxic elements and take necessary steps for prevention.

	Over the last few decades, the major aim of research in the field of agricultural and biomedical science has been to determine the concentrations of various toxic elements in many agricultural and biological specimens to find the cause of disease for its prevention. X-ray fluorescence (XRF) spectrometry is a versatile tool in many analytical problems. Major, minor, and trace elements can be qualitatively and quantitatively determined in various kinds of samples: metals, alloys, glasses, cements, minerals, rocks, ores, and polymers as well as environmental and biological materials (1). The application of wavelength-dispersive X-ray fluorescence (WD-XRF) spectrometry allows efficient determination of low-Z elements down to even beryllium (Be) without sample treatment with ensured high quality quantitative results. Because of the nondestructive character of X-ray measurement, XRF spectrometry is widely applied for the analysis of art, for example, museum and archeological objects such as manuscripts, paintings, icons, pottery, ancient glasses, ceramics, and coins.

	XRF is based on the principle that all elements produce secondary "fluorescent" X-rays of characteristic energy when they are exposed to X-rays of appropriate higher energy. The energy and intensity of the emitted X-rays can then be used to determine elemental composition. In principle, the higher the atomic weight of an element, the higher the energy needed to induce fluorescence and the fluorescence energy. It is also easier to detect fluorescence as the atomic weight of an element increases. Because XRF offers many unique capabilities, it has become the technique of choice for many applications requiring accurate elemental analysis for the purpose of meeting tight specifications at high concentration levels (2). High-performance WD-XRF spectrometry is now being used for the determination of traces in plant materials, biological samples, and geological samples. To fulfill today's requirements for quality data, modern instruments must provide high elemental sensitivities to achieve low detection limits and the best spectral resolution to minimize line overlays. In this article, we discuss the use of WD-XRF spectrometry to analyze trace and heavy metal contents in some agricultural and biological samples.

	The studied infected and uninfected wheat grain samples are shown in Figure 1. Figures 1a, 1b, and 1c are images of uninfected wheat grain, low infection wheat seed galls with 

, and high infection wheat seed galls with 

. Microscopic observations revealed the infection in wheat samples with 

 and details can be found in the literature (3). Also, the galls have a similar shape to the uninfected grains but are dark brown in color. The studied 

, which is widely distributed in many tropical and subtropical regions of the world) samples were collected and washed thoroughly to remove all the traces of soils and other possible contaminations. The biological applications and use of 

 can be found in the literature (4). The gallstones and kidney stone samples (5,6) studied using WD-XRF were collected from the Department of Nephrology at Opal Hospital in Varanasi, India.


	Figure 1: Photographs of (a) uninfected wheat grain, (b) wheat seed galls (low infection) with Anguina tritici, and (c) wheat seed galls (high infection) with Anguina tritici. Adapted with permission from reference 3.

, gallstones, and kidney stones, we used an S8 TIGER WD-XRF spectrometer (Bruker) for the determination of traces in these biological samples. The instrument was optimized to obtain better quantitative results, resolution, reproducibility, and reliability for each element with the best excitation and highest intensity over the whole elemental range. The quantitative measurements of the elemental constituents of kidney stones were performed using the same WD-XRF spectrometer. A 4-kW Rh anode X-ray tube (with a voltage of 60 kV max; and current 170 mA max) equipped with proportional flow counter and scintillation counter detectors was used as the energy source. Exposure time per sample was 20 min. The analyzer crystal LiF220 ensured that, in combination with the 0.23° collimator, the best separation was achieved for adjacent elements. The quantitative determination of major and trace elements was done using Quant-Express software (Bruker), which comprises a unique multipurpose preprepared calibration using certified standards. It automatically created the optimal measurement method to match each element and concentration range quickly, simply, and reliably.

Using WD-XRF to Analyze Uninfected and Infected Wheat Gall Nematodes

	Microscopic observations (3) have been performed to identify nematodes in the infected wheat grains that clearly indicated the presence of dead nematodes and fungal spores inside the wheat galls. Figures 2a and 2b depict the nematodes identified as 

 while Figures 2c and 2d show the silicified hairs on the galls. Further, Figures 2e and 2f show the features identified as the fungal spores helminthosporium and alternaria, which originated from air during storage of the wheat grains and caused further grain damage.


	Figure 2: Anguina tritici (a, b), silicified hairs (c, d), helminthosporium (e), and alternaria (f). Adapted with permission from reference 3.

	The typical overlapped WD-XRF spectra of uninfected wheat grain and seed gall nematode (low and high infection) wheat samples are shown in Figure 3 in different energy ranges. The relative intensities of the elements present in the uninfected, low infection, and high infection wheat samples are different, as can be seen in Figure 4, which indicates that their concentrations are different in different samples. The detected and quantified minerals in uninfected, low infection, and high infection wheat samples were potassium (K), sulfur (S), phosphorus (P), chlorine (Cl), calcium (Ca), magnesium (Mg), iron (Fe), silicon (Si), zinc (Zn), copper (Cu), sodium (Na), chromium (Cr), manganese (Mn), nickel (Ni), and aluminium (Al). The relative concentrations of these elements present in wheat samples are plotted in Figure 5. Among minerals, K, P, and S were predominant in uninfected grains as compared to Ca, Mg, Fe, Si, Cu, and Zn. The observed order of elemental concentration, particularly S, P, Ca, Mg, Fe, and Si, was found as follows: high infection wheat samples > low infected wheat samples > uninfected wheat samples. The concentration of K and Cl was highest in the low infection wheat sample. The concentration of Zn was higher for high infection wheat samples, a trend that may be correlated to the infection of grains caused by the nematodes. The elements Cr, Mn, and Ni were detected only in low infection and high infection wheat samples and they were completely absent in uninfected wheat samples which may be attributed to the activity of nematodes. Cu was only detected in uninfected and high infection wheat samples and was absent in low infection wheat samples. The mineral Na was only detected in low infection samples and Al was only detected in high infection wheat samples.


	Figure 3: Typical WD-XRF spectra of uninfected and infected wheat grains in energy ranges (a) 0.5–1.5 KeV indicating the presence of Mg and Al; (b) 1.7–3.3 KeV indicating the presence of Si, P, S, and Cl; (c) 3.3–5 KeV indicating the presence of K and Ca; and (d) 5–11 KeV indicating the presence of Cr, Mn, Fe, Ni, Cu, and Zn. Adapted with permission from reference 3.

	The toxic elements Al, Cr, and Ni were only detected in infected wheat samples and were not found in uninfected wheat samples. This result may be due to the presence of infected areas in grains because of the fungus and nematodes. The detected concentration of Al up to 600 ppm clearly indicated the toxic effect of Al in gall formation in wheat samples (7). Also, the presence of Cr and Ni at excessive levels in infected wheat samples were also the important reasons of gall formation in wheat grains. Thus, it was concluded that their presence was one of the factors leading to this disease and the formation of galls in wheat samples. The maximum concentration of Zn high infection wheat samples was responsible for the change in color of infected wheat samples from light brown to black. Along with WD-XRF, the authors also applied Fourier transform infrared (FT-IR) spectroscopy to study the molecular compositions of the infected and uninfected wheat grain samples. Diffuse reflectance measurements of uninfected and infected wheat samples were also used to identify spectral differences among wheat samples.


	Figure 4: Bar diagrams showing the concentration of elements present in uninfected, low infection, and high infection wheat samples as measured using WD-XRF. Adapted with permission from reference 3.

	The spectroscopic revolution has not only been regarded as the milestone for physical sciences, but also has opened the possibilities of new avenues in plant science and applied science. These findings underline the value of considerable supporting information using XRF spectrometry to avoid risks caused by microrganisms and are also helpful in avoiding damage to the environment, thus offering new investigation routes of agriculture- and disease-related issues.

	The high numbers of inorganic mineral elements present in nearly all plants ensure the successful growth and development of both vegetative and reproductive tissues. Currently, Fe, Zn, Mn, Cu, boron (B), Cl, molybdenum (Mo), and Ni have been recognized as micronutrients that are found at concentrations less than 0.01% of dry tissue weight (4). A few other minerals, such as cobalt (Co), sodium (Na), silicon (Si), selenium (Se), iodine (I), and vanadium (V), have also been shown to be essential and beneficial for certain plant species (4). Many other elements can be found in plants, but these are thought to enter plants nonselectively. Most of these nonessential elements confer no known benefit to the plant and many, such as cadmium (Cd) or Cr, are actually harmful to plant growth.

 family and is widely distributed in many tropical and subtropical regions of the world. It is also known as purple nut sedge or nut grass and is a common weed found mostly in the fields, agricultural land, and moist waste land of all over India (4). The tubers of 

 have a wide range of medicinal and pharmacological applications. They are generally used as anthelmintic, aphrodisiac, diuretic, sedative, carminative, stimulant, tonic, and stomachic. They are also used as a remedy for renal colic and dysentery. Furthermore, the plant possesses different biological activities such as antioxidant, cytotoxic, antiallergic, antipyretic, anti-inflammatory, antiemetic, hypotensive, anticonvulsant, antimalarial, antimicrobial, antidiarrhoeal, antidiabetic, hepatoprotective, and insecticide (4).

	The typical overlapped WD-XRF spectra of 

 samples are shown in Figure 5. The macro- and micronutrient minerals detected and quantified in 

 sample using WD-XRF were C, O, Na, Cl, Mg, Si, K, Ca, S, P, Al, Fe, Zn, Mn, Pd, Ru, Cr, Mo, Sr, Cu, and Ni. The concentrations of macronutrients Ca, Mg, K, P, and S were 0.36%, 0.46%, 0.38%, 0.20%, and 0.26%, respectively. The detected concentrations some micronutrients such as Al, Fe, Si, Na, and Cl were 0.06%, 0.05%, 0.42%, 0.8%, and 0.65%, respectively. Other micronutrients including some heavy minerals were detected at low levels ranging from 7 ppm to 44 ppm. The presence of elements found in 

 species were discussed in the literature (4). For the first-time, we used WD-XRF techniques to determine the macro- and micronutrient mineral contents of 

 rhizomes. Our results revealed that WD-XRF is a promising technique that is capable of analyzing plant samples with complex matrices used for medicinal purposes. Furthermore, the results obtained in the present paper show interesting prospects for WD-XRF to study plant samples. Other techniques, such as FT-IR and ultraviolet–visible (UV–vis) spectroscopy, have also been used for molecular analysis of c

 which revealed the capability of those techniques for the study of medicinal plants.


	Figure 5: Typical WD-XRF spectrum of Cyperus rotundus (snapshot shown in inset) in different energy regions indicating the presence of macro- and micronutrient minerals. Details about spectral information can be found in reference 4.

Using WD-XRF to Analyze Heterogeneous Gallstones

	Stone formation (gallstone and kidney stones) inside human organs are common diseases and constitute a major health problem worldwide (8–10). These stones are the result of a buildup of chemical particles in the gallbladder and kidneys, respectively. The chemical composition of stones is complex and varied. Trace elements are essential components of biological structures and are necessary for a multitude of vital functions in the human body. The precise role of trace constituents in the pathogenesis of stones is still unclear and under debate, but researchers are increasingly focusing on the role of trace elements in their pathogenesis (8–10). To understand the formation of these stones and their growth, it is important to study their chemical and elemental compositions as well as the function of the different elements as affected by the stone type. Determining and studying the chemical composition of stones is often among urologists' main means of shedding light on the pathogenesis of stones. Analytical techniques such as atomic absorption spectroscopy (AAS), scanning electron microscopy coupled with energy-dispersive X-ray spectroscopy (SEM-EDX), XRF, proton-induced X-ray emission (PIXE), X-ray diffractometry (XRD), Raman spectroscopy and microscopy, FT-IR spectroscopy, and laser-ablation inductively coupled plasma–mass spectrometry (LA-ICP-MS) are currently being used to answer questions about the composition of stones worldwide (8,9). Routine analysis in the field of medical science is still based on X-ray, ion-beam, and spectroscopic techniques. Each technique has its own merits and demerits. Most of the recently developed optical techniques such as laser-induced breakdown spectroscopy (LIBS) are based on the detection of the radiation emitted by the elements present in the biological samples (8–10).

	The WD-XRF spectra of these gallstone samples have been recorded and analyzed thoroughly (for more details see references 5 and 6). The major, trace, and heavy elements detected and quantified in gallstone (GS1, GS2, and GS3) were Ca, Na, K, P, Mg, Mn, Zn, Na, Cu, Fe, P, S, Si, Cl, Al, Br, Pd, and Ru. The relative concentrations of these elements are shown in the bar diagram in Figure 6. The observed concentrations of the elements Ca, Mg, Na, Cl, P, and S were higher in comparison to other elements. All other heavy elements were detected at trace amounts (parts-per-million range).

	The presence of Na and Cl in higher amounts is because of the daily dietary consumption of NaCl (in the form of salt) by the patients, which deposited in ionic form in the stones during the initiation precipitation stage of the stone formation. Ca was found to be the predominant nonorganic constituent in all gallstones, which behaves as a nidus for the gallstone formation. The presence of Ca in higher amounts is mainly because of the type of food and drinks the patients take, including diary and milk products, eggs, tea, and hard water (5,6). An iron-deficient diet might alter the metabolism of the hepatic enzyme, which later increases gallbladder bile cholesterol and promotes cholesterol crystal formation. Furthermore, the central aggregates of calcium salts constitute hard foreign bodies that may lead to ulceration of the gall bladder mucosa, microscopic hemorrhage, and inflammation. This releasing process may be another source of Fe deposition in gallstones (5,6). Manganese (Mn) was also significant in the studied gallstones. Manganese forms a salt with bile acid, which gets accumulated during cholelithiasis. Gallstones also show the presence of some elements like Pd, Ru, and other trace elements using WD-XRF which may be due to the different geographical locations of patients with gallstones, the dietary differences of the patients, and environmental pollution issues.


	Figure 6: Gallstone samples and heavy and trace elements quantified using WD-XRF. Details and spectral information can be found in reference 5.

	In addition, we have also applied FT-IR spectroscopy to study the major presence of cholesterol and bilirubin, as well as the minor presence of calcium carbonate and calcium phosphate in gallstones. Our results revealed that FT-IR and WD-XRF are promising techniques that are capable of analyzing stone samples present inside the human body. The results obtained with this study show interesting prospects of WD-XRF to study cholelithiasis better.

Using WD-XRF to Analyze Traces in Kidney Stones

	Kidney stones are a painful and prevalent disorder of the urinary system that is seen in patients throughout the world; its prevalence has drastically increased, particularly in industrialized countries. These stones are small, hard mineral deposits that build up on the inner surface of the kidney and grow rapidly because of the trace minerals in contact. Some minerals inhibit the growth of stones and others strengthen their growth rate depending upon the stone type. Hence, in this situation trace analysis of kidney stones becomes important to know its pathogenesis for prevention. Quantitative analysis of the stones removed from the body can not only be very valuable methods for the treatment of disease, but also to avoid conventional 24-h urine tests, which are generally complicated and imprecise. Despite several investigations about the elemental composition of kidney stones, there is insufficient information with regard to the distribution of trace elements in these stones, especially within the different stone types. Therefore, the present study (11) investigated various factors that play a regulatory role in the pathogenesis of two different kinds of kidney stones-oxalate stones and struvite stones-by using WD-XRF spectrometry.

	The images of the kidney stone samples (KS1, KS2, KS3, KS4, and KS5) analyzed are shown in Figure 7. Clinical details of the patients can be seen in reference 11. FT-IR spectroscopy was used to classify the stone samples. The FT-IR spectral data indicated calcium oxalate monohydrate (COM) as the major constituent and calcium phosphate as the minor constituents of the kidney stones and hence stones (KS1, KS3, KS4, and KS5) could be classified as oxalate stones. Stone sample KS2 was identified as a struvite stone with the major presence of magnesium ammonium phosphate (MAPH), calcium phosphate, and the minor presence of COM.


	Figure 7: Photos of kidney stone samples. Adapted with permission from reference 11.

	The WD-XRF studies were performed on these two kinds of samples. The elements determined in stone samples (KS1–KS5) were Ca, Mg, P, Na, K, Cl, S, Si, I, Ti, Fe, Ru, Zn, Al, Sr, Ni, Cu, and Br. The relative concentrations of these elements are shown as a bar diagram in Figure 8. Detailed analysis of FT-IR and WD-XRF spectral studies can be seen in reference 11. I (200 ppm) and Ti (69 ppm) were detected only in sample KS2 in low concentration. Fe was detected in all stone samples and its concentration was found to be nearly comparable in all stone samples. No measurable differences of the concentration of Fe in stone samples was observed. Strontium (Sr) was detected only in KS3 and KS4, and heavy elements such as Ni, Cu, and Br were detected only in the oxalate stone sample (KS3).


	Figure 8: Relative concentrations of heavy and trace elements detected in kidney stones using WD-XRF spectrometry. Adapted with permission from reference 11.

	The content of Mg and P was much higher in the struvite stone (KS2) than that of oxalate type stone samples (KS1, KS3–KS5). It is because of the stone matrix that magnesium ammonium phosphate (MgNH

O) is called struvite. Ca was detected in all stone samples and was the major constituent of all kidney stones. However, the concentrations of Ca (0.55–10.42%) in oxalate type stone samples were higher than that of struvite stone (~ 0.15%). The concentration of Na, K, and Cl were also higher in the struvite stone samples than that of oxalate type stones. This higher concentration may be because of the higher presence of NaCl and KCl in struvite stone samples and indicates their role in struvite stones. For the first time, we have detected ruthenium (Ru) in all stone samples (except sample KS3) in low concentrations (ranging from 22 to 30 ppm). No measurable difference has been observed for Ru among the stones. Zn was only observed in oxalate type stone samples (KS1 and KS3) in low concentration (up to 20 ppm). The presence of Ru and Zn only in some stones is probably due to the dietary differences of the patients. We have also compared our results from the reported literature value of kidney stones using the XRF technique and good agreements were found (11).

	We detected a wide range of elements -Ca, Mg, P, Na, K, Cl, S, Si, I, Ti, Fe, Ru, Zn, Al, Sr, Ni, Cu, and Br-in kidney stone samples. The concentration difference of the elements present in stones samples may be due to the different dietary habits of patients (vegetarian or nonvegetarian) as well as their differing environmental and geographical regions. Here, the stones samples collected were from patients from various regions or states of India with differing dietary habitats (11). Particularly, the studied stone samples were collected from the patients who were vegetarian and nonvegetarian. Also the patients have some habitats of nephrotoxins such as herbominerals, chewing tobacco products, and drinking alcohol.

	Meats, meat products, and eggs are good sources of essential minerals not limited to Fe, Cu, Mg, Zn, and toxic and heavy metal elements particularly Pb, Co, Ni, and Br (12). Also, tobacco is indigenous to many areas such as India, the Middle East, Southeast Asia, and the Americas (13). The tobacco samples (spit tobacco, gutkaas, and paan masalas) were found enriched with the presence of mineral elements such as Na, K, Ca, Cl, Mg, P, Cu, Fe, and Zn and traces of toxic and heavy elements, particularly As, Cr, Co, Al, Ni, and Br (13). In our study, some heavy and toxic elements such as Al, Cu, Ni, Br, and Sr were observed in the stone samples of patients with habits of chewing tobacco and meat products, which are potential sources of these heavy and toxic elements (13). The presence of toxic and heavy elements, particularly Ni, Br, and Cu, only in the KS3 stone sample may be due to the chewing habits because chewing samples were reported as enriched with these toxic elements (as mentioned earlier). Thus, it may be concluded that the presence of these elements in the samples may be due to chewing habits, alcoholic nature, and eating meat products. Along with these factors, the formation of stones in humans may also be influenced by the living style in a far more complex environment, drinking water, dietary differences, as well as genetic predisposition.

	The spectroscopic revolution has not only been regarded as a milestone for physical sciences, but also has opened the possibilities of new avenues in plant science and applied sciences. We demonstrated several possibilities of using WD-XRF spectrometry for rapid analysis of some varieties of biologically important specimens such as wheat seed gall nematodes (

, gallstones, and kidney stones. A significant advantage of this technique is that solid samples can be analyzed in their original unaltered condition, without undergoing pretreatment procedures. For the first time, we have successfully measured the concentrations of minerals such as K, S, P, Cl, Ca, Mg, Fe, Si, Zn, Cu, Na, and Mn in uninfected wheat grains along with some toxic elements such as Cr, Ni, and Al in infected wheat grains using WD-XRF. These findings underline the value of considerable supporting information through physical instruments to avoid risks caused by microorganisms and simultaneously help to avoid damage to the environment, thus offering new investigation routes of agriculture- and disease-related issues.

	Using WD-XRF spectroscopy we have both analyzed and quantified a wide range of major and trace elements, including toxic elements in gallstones and kidney stones. We found the major differences between the elemental contents of calcium oxalate type stones and struvite stones. This study showed that the distribution and presence of elements in kidney stones are not only associated with geochemical factors but include environmental conditions and biology also. Occupations with higher insensible fluid losses, such as a hot environment, increase the risk of stone formation (14). The risk may also be higher when individuals have restricted access to water or bathroom facilities, leading to lower fluid intake and lower urine volume. In our study, the patients belong to different regions and different environments. In addition, dietary intake influences urine composition, thereby modifying the risk of nephrolithiasis. Heavy and toxic elements such as Al, Cu, Ni, Br, and Sr were found in the stones samples of the patients having habits of nephrotoxins (alcoholic, herbominerals, and chewing tobacco). In conclusion, the differences in the elemental concentrations in different stones may be correlated with the geographical, environmental, and dietary differences. More types of sample analysis is in progress by our research group.

	WD-XRF is quite a suitable technique for in vitro quantitative analysis of both major and trace elements generally present in the samples of both gallstones and kidney stones. This technique has been applied uniquely to provide rapid qualitative and quantitative analysis of unknown environmental, biological, and medical samples including stone samples, in particular.

	Vivek K. Singh is thankful to Mr. Tejbir Singh, in-charge WD-XRF Lab, SAIF, Punjab University, Chandigarh, India, facility for his valuable discussions and suggestions related to WD-XRF spectrometry and instrumentation. The author Dr. A.K. Pathak is grateful to University Grants Commission, New Delhi (F.No.8-4(57)/2015(MRP/NRCB) for financial assistance.

	(1) M. West, A.T. Ellis, P.J. Potts, C. Streli, C. Vanhoof, and P. Wobrauschek, 

	(2) J.A. Anzelmo, M. Bouchard, and M.è. Provencher, 

	(3) V.K. Singh, A. Devi, S. Pathania, V. Kumar, D.K. Tripathi, S. Sharma, D.K. Chauhan, V.K. Singh, and V. Zorba, 

	(4) V.K. Singh, A. Gupta, O. Gupta, V. Kumar, and M.A. Gondal, 

	(5) B.B.S. Jaswal, J. Sharma, V. Kumar, Y. Khajuria, V.K. Singh, and P.K. Rai, 

	(6) B.B.S. Jaswal, V. Kumar, J. Sharma, P.K. Rai, M.A. Gondal, Bilal Gondal, and V.K. Singh, 

	(7) N. Gupta, S.S. Gaurav, and A. Kumar, 

	(9) A.K. Pathak, R. Kumar, V.K. Singh, R. Agrawal, S. Rai, and A.K. Rai, 

	(10) V.K. Singh, V. Singh, A.K. Rai, S.N. Thakur, J.P. Singh, and P.K. Rai, 

	(11) V.K. Singh, B.B.S. Jaswal, J. Sharma, and P.K. Rai, 

	(12) M.Z.A. Chowdhury, Z.A. Siddique, S.M.A. Hossain, A.I. Kazi, M.A. Ahsan, S. Ahmed, and M.M. Zaman, 

	(13) A.N. Garg, R.P. Choudhury, R. Acharya, and A.V.R. Readdy, 

	(14) L. Atan, C. Andreoni, V. Ortiz, E.K. Silva, R. Pitta, F. Atan, and M. Srougi, 

 is with the Department of Physics at Shri Mata Vaishno Devi University in Jammu and Kashmir, India. 

 is with the Department of Urology and Nephrology at Opal Hospital in Varanasi, India. 

 is with the Department of Physics at Ewing Christian College, University of Allahabad in Allahabad, India. 

 is with the Centre for Medical Diagnostic and Research (CMDR), MNNIT Allahabad in Allahabad, India. 

 is with The Institute of Optics at the University of Rochester in Rochester, New York. 

 is with the Institute for Clean Energy Technology at Mississippi State University in Starkville, Mississippi. Direct correspondence to: 

Application of Wavelength-Dispersive X-Ray Fluorescence Spectrometry to Biological Samples

	XRF is based on the principle that all elements produce secondary “fluorescent” X-rays of characteristic energy when they are exposed to X-rays of appropriate higher energy. The energy and intensity of the emitted X-rays can then be used to determine elemental composition. In principle, the higher the atomic weight of an element, the higher the energy needed to induce fluorescence and the fluorescence energy. It is also easier to detect fluorescence as the atomic weight of an element increases. Because XRF offers many unique capabilities, it has become the technique of choice for many applications requiring accurate elemental analysis for the purpose of meeting tight specifications at high concentration levels (2). High-performance WD-XRF spectrometry is now being used for the determination of traces in plant materials, biological samples, and geological samples. To fulfill today’s requirements for quality data, modern instruments must provide high elemental sensitivities to achieve low detection limits and the best spectral resolution to minimize line overlays. In this article, we discuss the use of WD-XRF spectrometry to analyze trace and heavy metal contents in some agricultural and biological samples.

), and high infection wheat seed galls with 

. Microscopic observations revealed the infection in wheat samples with 

 and details can be found in the literature (3). Also, the galls have a similar shape to the uninfected grains but are dark brown in color. The studied 

, which is widely distributed in many tropical and subtropical regions of the world) samples were collected and washed thoroughly to remove all the traces of soils and other possible contaminations. The biological applications and use of 

 can be found in the literature (4). The gallstones and kidney stone samples (5,6) studied using WD-XRF were collected from the Department of Nephrology at Opal Hospital in Varanasi, India. 

 rhizomes. Our results revealed that WD-XRF is a promising technique that is capable of analyzing plant samples with complex matrices used for medicinal purposes. Furthermore, the results obtained in the present paper show interesting prospects for WD-XRF to study plant samples. Other techniques, such as FT-IR and ultraviolet–visible (UV–vis) spectroscopy, have also been used for molecular analysis of 

, which revealed the capability of those techniques for the study of medicinal plants.

Using WD-XRF to Analyze Heterogeneous Gallstones 

	Stone formation (gallstone and kidney stones) inside human organs are common diseases and constitute a major health problem worldwide (8–10). These stones are the result of a buildup of chemical particles in the gallbladder and kidneys, respectively. The chemical composition of stones is complex and varied. Trace elements are essential components of biological structures and are necessary for a multitude of vital functions in the human body. The precise role of trace constituents in the pathogenesis of stones is still unclear and under debate, but researchers are increasingly focusing on the role of trace elements in their pathogenesis (8–10). To understand the formation of these stones and their growth, it is important to study their chemical and elemental compositions as well as the function of the different elements as affected by the stone type. Determining and studying the chemical composition of stones is often among urologists’ main means of shedding light on the pathogenesis of stones. Analytical techniques such as atomic absorption spectroscopy (AAS), scanning electron microscopy coupled with energy-dispersive X-ray spectroscopy (SEM-EDX), XRF, proton-induced X-ray emission (PIXE), X-ray diffractometry (XRD), Raman spectroscopy and microscopy, FT-IR spectroscopy, and laser-ablation inductively coupled plasma–mass spectrometry (LA-ICP-MS) are currently being used to answer questions about the composition of stones worldwide (8,9). Routine analysis in the field of medical science is still based on X-ray, ion-beam, and spectroscopic techniques. Each technique has its own merits and demerits. Most of the recently developed optical techniques such as laser-induced breakdown spectroscopy (LIBS) are based on the detection of the radiation emitted by the elements present in the biological samples (8–10).

	The WD-XRF spectra of these gallstone samples have been recorded and analyzed thoroughly (for more details see references 5 and 6). The major, trace, and heavy elements detected and quantified in gallstone (GS1, GS2, and GS3) were Ca, Na, K, P, Mg, Mn, Zn, Na, Cu, Fe, P, S, Si, Cl, Al, Br, Pd, and Ru. The relative concentrations of these elements are shown in the bar diagram in Figure 6. The observed concentrations of the elements Ca, Mg, Na, Cl, P, and S were higher in comparison to other elements. All other heavy elements were detected at trace amounts (parts-per-million range). 

	The presence of Na and Cl in higher amounts is because of the daily dietary consumption of NaCl (in the form of salt) by the patients, which deposited in ionic form in the stones during the initiation precipitation stage of the stone formation. Ca was found to be the predominant nonorganic constituent in all gallstones, which behaves as a nidus for the gallstone formation. The presence of Ca in higher amounts is mainly because of the type of food and drinks the patients take, including diary and milk products, eggs, tea, and hard water (5,6). An iron-deficient diet might alter the metabolism of the hepatic enzyme, which later increases gallbladder bile cholesterol and promotes cholesterol crystal formation. Furthermore, the central aggregates of calcium salts constitute hard foreign bodies that may lead to ulceration of the gall bladder mucosa, microscopic hemorrhage, and inflammation. This releasing process may be another source of Fe deposition in gallstones (5,6). Manganese (Mn) was also significant in the studied gallstones. Manganese forms a salt with bile acid, which gets accumulated during cholelithiasis. Gallstones also show the presence of some elements like Pd, Ru, and other trace elements using WD-XRF which may be due to the different geographical locations of patients with gallstones, the dietary differences of the patients, and environmental pollution issues.  

	Kidney stones are a painful and prevalent disorder of the urinary system that is seen in patients throughout the world; its prevalence has drastically increased, particularly in industrialized countries. These stones are small, hard mineral deposits that build up on the inner surface of the kidney and grow rapidly because of the trace minerals in contact. Some minerals inhibit the growth of stones and others strengthen their growth rate depending upon the stone type. Hence, in this situation trace analysis of kidney stones becomes important to know its pathogenesis for prevention. Quantitative analysis of the stones removed from the body can not only be very valuable methods for the treatment of disease, but also to avoid conventional 24-h urine tests, which are generally complicated and imprecise. Despite several investigations about the elemental composition of kidney stones, there is insufficient information with regard to the distribution of trace elements in these stones, especially within the different stone types. Therefore, the present study (11) investigated various factors that play a regulatory role in the pathogenesis of two different kinds of kidney stones-oxalate stones and struvite stones-by using WD-XRF spectrometry. 

	The images of the kidney stone samples (KS1, KS2, KS3, KS4, and KS5) analyzed are shown in Figure 7. Clinical details of the patients can be seen in reference 11. FT-IR spectroscopy was used to classify the stone samples. The FT-IR spectral data indicated calcium oxalate monohydrate (COM) as the major constituent and calcium phosphate as the minor constituents of the kidney stones and hence stones (KS1, KS3, KS4, and KS5) could be classified as oxalate stones. Stone sample KS2 was identified as a struvite stone with the major presence of magnesium ammonium phosphate (MAPH), calcium phosphate, and the minor presence of COM. 

	The WD-XRF studies were performed on these two kinds of samples. The elements determined in stone samples (KS1-KS5) were Ca, Mg, P, Na, K, Cl, S, Si, I, Ti, Fe, Ru, Zn, Al, Sr, Ni, Cu, and Br. The relative concentrations of these elements are shown as a bar diagram in Figure 8. Detailed analysis of FT-IR and WD-XRF spectral studies can be seen in reference 11. I (200 ppm) and Ti (69 ppm) were detected only in sample KS2 in low concentration. Fe was detected in all stone samples and its concentration was found to be nearly comparable in all stone samples. No measurable differences of the concentration of Fe in stone samples was observed. Strontium (Sr) was detected only in KS3 and KS4, and heavy elements such as Ni, Cu, and Br were detected only in the oxalate stone sample (KS3).

	We detected a wide range of elements-Ca, Mg, P, Na, K, Cl, S, Si, I, Ti, Fe, Ru, Zn, Al, Sr, Ni, Cu, and Br-in kidney stone samples. The concentration difference of the elements present in stones samples may be due to the different dietary habits of patients (vegetarian or nonvegetarian) as well as their differing environmental and geographical regions. Here, the stones samples collected were from patients from various regions or states of India with differing dietary habitats (11). Particularly, the studied stone samples were collected from the patients who were vegetarian and nonvegetarian. Also the patients have some habitats of nephrotoxins such as herbominerals, chewing tobacco products, and drinking alcohol. 

	Meats, meat products, and eggs are good sources of essential minerals not limited to Fe, Cu, Mg, Zn, and toxic and heavy metal elements particularly Pb, Co, Ni, and Br (12). Also, tobacco is indigenous to many areas such as India, the Middle East, Southeast Asia, and the Americas (13). The tobacco samples (spit tobacco, gutkaas, and paan masalas) were found enriched with the presence of mineral elements such as Na, K, Ca, Cl, Mg, P, Cu, Fe, and Zn and traces of toxic and heavy elements, particularly As, Cr, Co, Al, Ni, and Br (13). In our study, some heavy and toxic elements such as Al, Cu, Ni, Br, and Sr were observed in the stone samples of patients with habits of chewing tobacco and meat products, which are potential sources of these heavy and toxic elements (13). The presence of toxic and heavy elements, particularly Ni, Br, and Cu, only in the KS3 stone sample may be due to the chewing habits because chewing samples were reported as enriched with these toxic elements (as mentioned earlier). Thus, it may be concluded that the presence of these elements in the samples may be due to chewing habits, alcoholic nature, and eating meat products. Along with these factors, the formation of stones in humans may also be influenced by the living style in a far more complex environment, drinking water, dietary differences, as well as genetic predisposition.   

	WD-XRF is quite a suitable technique for in vitro quantitative analysis of both major and trace elements generally present in the samples of both gallstones and kidney stones. This technique has been applied uniquely to provide rapid qualitative and quantitative analysis of unknown environmental, biological, and medical samples including stone samples, in particular. 

		M. West, A.T. Ellis, P.J. Potts, C. Streli, C. Vanhoof, and P. Wobrauschek, 

		J.A. Anzelmo, M. Bouchard, and M.È. Provencher, 

		V.K. Singh, A. Devi, S. Pathania, V. Kumar, D.K. Tripathi, S. Sharma, D.K. Chauhan, V.K. Singh, and V. Zorba, 

		V.K. Singh, A. Gupta, O. Gupta, V. Kumar, and M.A. Gondal, 

		B.B.S. Jaswal, J. Sharma, V. Kumar, Y. Khajuria, V.K. Singh, and P.K. Rai, 

		B.B.S. Jaswal, V. Kumar, J. Sharma, P.K. Rai, M.A. Gondal, Bilal Gondal, and V.K. Singh, 

		A.K. Pathak, R. Kumar, V.K. Singh, R. Agrawal, S. Rai, and A.K. Rai, 

		V.K. Singh, V. Singh, A.K. Rai, S.N. Thakur, J.P. Singh, and P.K. Rai, 

		V.K. Singh, B.B.S. Jaswal, J. Sharma, and P.K. Rai, 

		M.Z.A. Chowdhury, Z.A. Siddique, S.M.A. Hossain, A.I. Kazi, M.A. Ahsan, S. Ahmed, and M.M. Zaman, 

		A.N. Garg, R.P. Choudhury, R. Acharya, and A.V.R. Readdy, 

		L. Atan, C. Andreoni, V. Ortiz, E.K. Silva, R. Pitta, F. Atan, and M. Srougi, 

is with the Centre for Medical Diagnostic and Research (CMDR), MNNIT Allahabad in Allahabad, India. 

 is with the Institute for Clean Energy Technology at Mississippi State University in Starkville, Mississippi. Direct correspondence to: 

This review assesses the use of WD-XRF spectrometry for the analysis of major and trace levels of heavy and toxic minerals in biological specimens related to agricultural crops and human diseases.