Biometals DOI 10.1007/s10534-014-9796-6
Estimation of trace metal elements in oral mucosa specimens by using SR-XRF, PIXE, and XAFS Tomoko Sugiyama • Motohiro Uo • Takahiro Wada • Daisuke Omagari • Kazuo Komiyama • Tadahide Noguchi Yoshinori Jinbu • Mikio Kusama
•
Received: 4 July 2014 / Accepted: 26 September 2014 Ó Springer Science+Business Media New York 2014
Abstract The effects of dissolved elements from metal dental restorations are a major concern in lesions of the oral mucosa, and the evaluation of accumulated metal elements, especially their distribution and chemical state, is essential for determining the precise effects of trace metals. In this study, X-ray fluorescence with synchrotron radiation (SR-XRF) and particle-induced X-ray emission (PIXE) were applied for distribution analysis of the trace metal elements contained in the oral mucosa, and the chemical states of the elements were estimated using X-ray absorption fine structure (XAFS) analysis. Appropriate combination of these analysis techniques, particularly SR-XRF and PIXE, to visualize the distributions of the elements in the oral mucosa allowed for the observation and evaluation of accumulated metal ions and
T. Sugiyama T. Noguchi Y. Jinbu M. Kusama Department of Dentistry, Oral and Maxillofacial Surgery, Jichi Medical University, 3311-1 Yakushiji, Shimotsuke, Tochigi 329-0498, Japan M. Uo (&) T. Wada Advanced Biomaterials Department, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo, 113-8549, Japan e-mail: [email protected] D. Omagari K. Komiyama Department of Pathology, Nihon University School of Dentistry, 1-8-13 Kanda-Surugadai, Chiyoda-ku, Tokyo, 131-8310, Japan
debris. Importantly, the analyses in this study could be carried out using conventional histopathological specimens without damaging the specimens. Therefore, this method would be applicable for the detection of accumulated trace metal elements in biopsy specimens from the oral mucosa. Keywords Synchrotron radiation X-ray fluorescence analysis Particle-induced X-ray emission analysis X-ray absorption fine structure analysis Trace elemental analysis Histopathological specimen
Introduction Trace elements play important roles in biological systems. About 50 elements can be detected at measurable concentrations in living systems, and more than 10 of these elements are considered essential trace elements. Deficiency, excess, and disorder of the distributions of these trace elements have been shown to be associated with the formation of various lesions (Fraga 2005). Thus, analysis of trace elements in tissues and organs is a major research focus. Inductively coupled plasma atomic emission spectroscopy (ICP-AES) and mass spectroscopy (ICP-MS) are popular analytical methods for evaluation of trace elements. With the exception of laser ablation ICP-MS
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(LA-ICP-MS), these methods require the use of aqueous specimens. Therefore, tissue samples (even blood or bodily fluid) need to be decomposed with acids. However, application of such destructive pretreatment causes loss of information about the elemental distribution in the specimen. Therefore, for proper analysis of biological specimens, particularly of various types of lesions used for diagnoses, specimens should not be consumed or damaged. Thus, the development of alternative methods for trace element analysis should be investigated. In recent years, ‘‘quantum beams,’’ e.g., X-rays (photons), protons, neutrons, and various ions, have been applied for nondestructive analysis of trace elements. X-ray fluorescence spectroscopy (XRF), particle-induced X-ray emission spectroscopy (PIXE), and neutron activation analysis (NAA) are the typical nondestructive analysis methods. Irradiation with these beams provides sufficient signals to identify the elements (e.g., characteristic X-rays or c-rays) and causes negligible damage to the specimen. Additionally, synchrotron radiation represents a strong, highly collimated X-ray source. XRF with monochromatized synchrotron radiation (SR-XRF) provides high-quality XRF spectra with low background, allowing detection of trace elements without specimen damage. Highly collimated synchrotron-radiated X-rays are also compatible with microfocusing optics. Therefore, micro-area analysis and elemental distribution images can be obtained by simply scanning the specimen. PIXE uses the characteristic X-ray (c-rays) generated by accelerated proton (or a particle) bombardment. In this method, protons can be focused down to micron size and scanned over the specimen surface. This method, called ‘‘micro-PIXE,’’ can also provide images of the trace elemental distribution. Together, these methods have been applied for the elemental distribution analysis of medical specimens in various fields (Fahrni 2007). Cu, Fe, and Zn are the major mineral elements in living systems. Therefore, abnormalities in the distributions of these elements in various lesions can be identified using SR-XRF and micro-PIXE. Specific distributions of each of these elements have been observed in breast cancer tissues (Geraki et al. 2002; Farquharson et al. 2007, 2008) using SR-XRF, while trace elements in cancers of the lung (Kubala-Kukus´ et al. 1999), thyroid (Reddy et al. 2002), kidney, and stomach (Reddy et al. 2003) have been estimated
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using PIXE. Additionally, cancers in these organs have also been reported to exhibit atypical trace element concentrations. For example, high Ti and Pb levels have been found in lung cancer; low Ca, Zn, and Cu levels have been found in thyroid cancer; and low Fe, Cu, and Zn levels have been found in stomach cancer. Trace element distributions in Alzheimer’s disease have also been studied using SR-XRF (IdeEktessabi and Rabionet 2005; Collingwood and Dobson 2006; Miller et al. 2006) and micro-PIXE (Lovell et al. 1998). The localization of Fe, Cu, and Zn in Alzheimer’s lesions has been suggested to accelerate the aggregation of b-amyloid. Atypical Cu and Fe distributions have also been reported in chronic hepatitis C (Kinoshita et al. 2010). Additionally, X-ray absorption fine structure (XAFS) analysis has been used, in combination with SR-XRF, to identify differences in the accumulated alloy components (Fe, Co, Cr, and Ti) generated from artificial hip joints (Ektessabi et al. 2001; Uo et al. 2009). In dental and orthopedic surgery, various metalcontaining restorative materials and instruments are used, applied to, and even implanted into the oral region. Moreover, in the oral cavity, metal restorations are immersed in saliva and come in close contact with the mucosa. Therefore, dental alloy components often dissolve and penetrate into the mucosa. Base metal elements (e.g., Co, Ni, Zn, Sn, and In) are often used as the major components of dental alloys and are suspected to be the cause of metal allergy. Other common symptoms of metal element accumulation in the oral environment include oral lichen planus (OLP) and palmoplantar pustulosis. Additionally, various eroded metal ions and debris from wearing also accumulate in the surrounding tissues and remote organs. The accumulated metals may cause the acute, chronic, and allergic symptoms, and identification of the causal metals is important for diagnosis of the cause of these symptoms. Alternatively, debris generated from the cutting and polishing of metal restorations during dental treatment often penetrates into the mucosa and may cause different symptoms of toxicity. Thus, the origins and chemical states of the elements identified in the oral mucosa should also be considered. In the present study, we used a combination of SRXRF, micro-PIXE, and XAFS analyses to detect and identify trace metal elements contained in lesions of the oral mucosa. From these data, we were able to form
Biometals
hypotheses to explain the origins of the trace elements and the effects of these elements on the tissues and lesions.
Materials and methods Specimens Oral mucosal tissues were supplied by the Dental Hospital of Nihon University School of Dentistry, Tokyo, Japan. The samples represented residual biopsy sections embedded in paraffin. Two oral mucosal tissues were collected and labeled as specimens #1 and #2 obtained from the different patients. Table 1 shows the clinical records and the intraoral findings of the two specimens. Specimen #1 was tissue from the buccal mucosa that contacted the metal crown and exhibited hyperkeratotic changes (reticular type, the typical appearance of OLP). Specimen #2 was also tissue from the buccal mucosa and exhibited hyperkeratotic changes (plaque type). The tissue for specimen #2 was collected from buccal mucosal tissue near, but not in direct contact with the metal
restoration. Adjacent slices (8 lm thick) of each sample were placed on Kapton film (12.5 lm thick; Du Pont-Toray Co., Ltd, Tokyo, Japan) and subjected to elemental analysis (as described below). After the elemental analyses, the distribution and chemical state information were compared with histopathological information. All patients provided written informed consent, and the study protocol was approved by the Ethical Committees of Nihon University (2012-14). XRF analysis XRF analyses of the two specimens were carried out at BL-4A of the Photon Factory at the High Energy Accelerator Research Organization (Tsukuba, Japan). The incident X-ray (12.9 keV) was focused using polycapillary optics to a 20-lm region, and the specimen was irradiated. The specimen stage was scanned in the X–Y plane, two dimensionally, to obtain elemental distribution images of the areas. The scanning areas varied within several millimeters, and the scanning steps varied from 5 to 50 lm. The obtained XRF data were processed with PyMCA software (Version 4.7.3), and elemental distribution
Table 1 Clinical records of the analyzed specimens No.
Age
Sex
Site
#1
57
M
Buccal mucosa
Diagnosisa Bilateral
Intraoral findingd
OLPb
The patient presented with a chief complaint of bilateral buccal mucosal pain. Ten years before the first examination, similar symptoms had appeared and the patient had been in remission. One year before the first examination, relapsed symptom appeared on inserting a mandibular partial denture. Hyperkeratotic reticular type changes were observed
#2
40
F
Buccal mucosa
Unilateral
OLLc
The patient presented with a chief complaint of plaque type changes of the gingiva and buccal mucosa around the left mandibular second premolar. Because symptoms showed no improvement for several months, the patient consulted the dental office. Hyperkeratotic plaque type changes were observed near the metal restoration but not in direct contact with the restoration
a
Diagnoses were made from clinical and histopathological findings
b
Oral lichen planus
c
Oral lichenoid lesion
d
Black arrows indicate the location of the intraoral findings
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images were obtained. In addition, the XRF spectra were measured for 300 s at regions of trace element localization. Micro-PIXE analysis In order to visualize the high-resolution elemental distribution images, some specimens were also used for micro-PIXE analysis, which was carried out at the National Institute of Radiological Sciences (Chiba, Japan). An accelerated and microfocused proton beam (3.0 MeV, 2 lm beam diameter) with raster scanning was applied over the target area of the specimen (maximum area of 2 9 2 mm). The generated characteristic X-rays were collected using Si(Li) and CdTe detectors to obtain the elemental distribution images and the characteristic X-ray spectra. The obtained data were processed with OMDAQ2007 software (Version 1.3.71.669), and the elemental distribution images and the characteristic X-ray spectra of the region of interest were obtained. XAFS analysis For the typically accumulated metallic elements, XAFS analyses were carried out to determine the
chemical states of the elements. XAFS analyses were also conducted at BL-4A of the Photon Factory. The X-ray absorption near edge structure (XANES) spectra of target elements (e.g., Ni, Fe, and Cu) were measured with the fluorescent XAFS method. As standards, Cr, Fe, Ni foil, and reagent-grade Ni(OH)2 were also measured. Some XANES spectra of the above standards were also measured at BL-9A and 12C.
Results Hematoxylin and eosin staining (for histopathological analysis) and the SR-XRF elemental distribution images of specimen #1 are shown in Fig. 1. The histopathological findings showed characteristic features of keratotic lesions in the oral mucosa: overlying keratinization and a band-like layer of chronic inflammatory cells within the underlying connective tissue. Clear Ni localization could be found deep below the epithelium. Fe and some Ti also accumulated in the same area. This area was not the above the inflammatory cell layer, but some inflammatory cells were present in regions of Ni, Fe, and Ti localization.
Fig. 1 Histopathological images (hematoxylin and eosin staining) and elemental distribution images by SR-XRF of specimen #1
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Fig. 2 Elemental distribution images of the black-boxed area in the Ni distribution image were obtained by PIXE (A). The characteristic X-ray spectra of Ni- and Fe-localized areas are
shown (B). A Diffused, high-intensity distributions of Fe and Ni are indicated by white arrows. B Clear peaks are shown for Ni, Fe, Ti, Al, and Si
Fig. 3 Ni K-edge XANES spectrum of specimen #1
was similar to that of Ni(OH)2 but distinct from that of Ni-foil. This result suggested that the accumulated Ni was in the aqueous ionic state. Dissolution from Nicontaining dental alloys in the oral cavity was suspected to be the origin of the Ni ions. Hematoxylin and eosin staining and SR-XRF elemental distribution images of specimen #2 are shown in Fig. 4. The histopathological findings of specimen #2 were similar to those of specimen #1, which are characteristic features of keratotic lesions in the oral mucosa. Atypical localization of Fe and Cr (spot 2-A) and Zn and Cu (spot 2-B) were observed. Spot 2-A existed at the outermost region of the mucosal epithelium, while spot 2-B existed at the inner region of the basal layer. More detailed distribution images were obtained using micro-PIXE and XRF analysis of spot 2-A from specimen #1 (Fig. 5). The estimated sizes of spots with Fe and Cr accumulation were quite small (several micrometers). The XRF spectrum of spot 2-A showed clear Fe and Cr peaks but weak Ca and Zn peaks. No peaks of major element found in soft tissues (e.g., P, S, and K) were observed. Figure 6 shows Fe and Cr K-edge XANES spectra at spot 2-A. Both spectra were similar to those of foils, indicating that the observed elements were in the metallic state. Therefore, we strongly suspected an artifact containing an Fe–Cr alloy as the origin of this spot. Indeed, our preparation procedure supported this. In the preparation of thinsectioned specimens, we used a microtome with
More detailed distribution images were obtained with micro-PIXE and the XRF spectrum for spot 1 (Fig. 2). The area in which Ni and Fe accumulated was diffused into a region 30–40 lm in diameter (Fig. 2). The characteristic X-ray spectrum of the area with Ni and Fe accumulation showed clear peaks for not only Ni and Fe, but also the metal elements Al, Si, K, Ca, Ti, and Zn and the nonmetal elements S and Cl. While K, Ca, Fe, and Zn were the major minerals in the tissue, Ni and Si accumulation could not be detected by XRF in normal tissue. The Ni K-edge XANES spectra of the Ni-accumulated area in Fig. 2 is shown in Fig. 3. The XANES spectrum of Ni in specimen #1
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Biometals Fig. 4 Histopathological images (hematoxylin and eosin staining) and SR-XRF elemental distribution images of specimen #2. Fe and Cr (spot 2-A) and Zn and Cu (spot 2-B) are shown
Fig. 5 Fe and Cr distribution images obtained by PIXE and the fluorescence X-ray spectra of spot 2-A from Fig. 4
Martensitic stainless steels, which can be hardened by heat treatment. The composition of the microtome blade (Feather S35 type, Tokyo, Japan) used to prepare the specimens analyzed in this study was 87 % Fe and 12 % Cr. Spot 2-A was positioned on the edge of the epithelial layer, which was the most dense structure in the observed tissue, and the outermost region was embedded paraffin, which is softer than tissue. Thus, the blade edge would occasionally chip while the sectioning the epithelial layer, and we
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presume spot 2-A was caused by contamination resulting from the chipping of the microtome blade. The same specimen contained another spot in which metal elements were localized (spot 2-B). XRF spectrum analysis revealed that the major elements within this spot were Cu and Zn, with small amounts of Fe and Ni (Fig. 7). These latter two elements are essential elements found in living tissues; however, the Cu and Zn found in spot 2-B were highly concentrated in a small region (approximately
Biometals Fig. 6 Fe and Cr K-edge XANES spectra of spot 2-A in specimen #2
Fig. 7 PIXE elemental distribution images of specimen #2 and the XRF spectrum of spot 2-B from Fig. 4 (white arrows in Fig. 7)
10 lm). Therefore, these elements could not have been derived from a living system. Cu and Zn K-edge XANES spectra are shown in Fig. 8. Comparison with XANES spectra of foil and the aqueous solution revealed that the XANES spectra of Cu and Zn in spot 2-B were quite similar to that of foil (i.e., in the metal state). Therefore, similar to spot 2-A, we presumed that the origin of Cu and Zn in spot 2-B was an alloy of Cu and Zn, such as brass. From the weak Ni peak in the XRF spectrum, nickel silver (a Cu–Zn–Ni alloy) would be a possible candidate. Brass is widely used in industrial and consumer products, but is not used for
dental restorations and instruments. However, nickel silver was used for dental restorations several decades ago. Thus, we presumed that spot 2-B originated from contamination with small brass or nickel silver debris. Figure 9 shows the line profiles of Fe and Cu in spots 1, 2-A, and 2-B. The profile of the Fe concentration in spot 1 from specimen #1 showed widespread distribution. The full width at half maximum (FWHM) of the Fe distribution in spot 1 was approximately 30 lm. In contrast, Fe and Cu in spots 2-A and 2-B in specimen #2 were concentrated in a small region, and their FWHMs were estimated to be 4 lm.
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Fig. 8 Cu and Zn K-edge XANES spectra of spot 2-B from specimen #2
Fig. 9 The linear analysis profiles of specimens #1 and #2 are shown on the left. Spot 1 indicates the distribution of Fe in specimen #1, while spots 2-A and 2-B indicate Fe and Cu in specimen #2
Discussion In dental treatment, various metals are used for restorations and instruments. Erosion and accumulation of metal elements in the oral mucosa are suspected to be the cause of some intraoral and
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systemic lesions, e.g., OLP and metal allergy. Such tissues are often biopsied for histopathological diagnosis, and specimens of 10 mm or less are obtained. For elemental analysis, to determine whether the lesions may be caused by metal toxicity, the entire region of the biopsied specimen should be examined,
Biometals
and detailed information on the distributions of metals accumulated within the area may provide insights into the relationship between the elemental distribution and the internal structure of the mucosa. Further analysis on the micrometer scale can then be performed. These concepts were supported by the results of our current study, in which we analyzed two biopsy specimens by hematoxylin and eosin staining and by a combination of X-ray-based techniques for elemental analysis. SR-XRF and micro-PIXE are widely used for analysis of the distributions of trace elements. In SRXRF, incident X-rays are focused with a mirror, zone plate, or capillary optics. The mirror optics can focus on the nanometer scale with a highly precise apparatus. However, this focus size is too small to scan the entire biopsy specimen. In contrast, capillary focusing optics can be easily installed at a general SR-XRF facility and can be used to obtain elemental mapping images on the micrometer scale with less loss of X-ray intensity (Preoteasa et al. 2002). These features make XRF analysis suitable for evaluation of the entire mucosal biopsy specimen. Additionally, the microPIXE method irradiates a microfocused proton beam with raster scanning over the specimen. Detailed elemental distribution images can be obtained, but the analysis area is narrow. Therefore, after analysis of the entire specimen with SR-XRF, areas of metal accumulation, i.e., regions of interest, can then be analyzed by micro-PIXE, which can provide images with 2-lm resolution. In addition, the SR-XRF facility can provide XAFS analysis of regions of metal accumulation. The combination of these three methods was used in our study to provide screening of regions of metal accumulation, detailed information on elemental distribution, and evaluation of the chemical state of the metals. In this study, we observed differences in the distributions of elements, particularly Fe and Cu, within specimens #1 and #2 (Fig. 9). These differences could be explained by differences in the conditions or states of the accumulated elements. Fe and Ni in spot 1 were thought to represent the dissolved and accumulated ions shown in Fig. 2. Because they were derived from dissolved ions, the region in which these ions were localized was rather widespread. In contrast, spots 2-A and 2-B were assumed to be metallic debris from XAFS analysis; therefore, the elements within these spots were highly
localized in regions of only a few microns. It is expected that the detrimental effects of accumulated metal ions in living systems would be higher than those of debris (found in the metal state) because ions are more reactive with biological components, e.g., nucleic acid and proteins. Several groups have reported the dissolution of various metallic ions from dental titanium implants (Uo et al. 2007) and dental and medical alloys (Uo et al. 2008, 2009). In addition, trace metal elements, which are suspected to represent eroded ions of metal dental restorations, have been detected in oral lichenoid lesion specimens. In general, medical records of dental treatment are not shared among dental offices. Therefore, the components of metal dental restorations are frequently unknown. Previously, elemental analyses have not been employed because of their difficulty. Recently, one study reported a simple analysis of metal restorations using XRF (Uo and Watari 2004). In this study, we were able to visualize the trace metal elements accumulated in oral mucosal lesions and to estimate their chemical states. Therefore, this information may be useful for evaluating the effects of accumulated elements on surrounding tissues. Importantly, the methods used in this study can be conducted with conventional histopathological specimens without damaging the specimens. Therefore, these methods may be useful for the screening of metal-affected lesions and for determination of the effects of metal element accumulation on oral mucosal lesions. The above analyses of the mucosal specimens and the combinational use of elemental analysis of the metal restoration may allow definite diagnosis and appropriate treatment.
Conclusions In this study, we examined the distribution of trace elements and their chemical states in oral mucosal tissues that were in close contact with metal restorations by using SR-XRF, micro-PIXE, and XAFS analyses. Combined analysis with SR-XRF and microPIXE allowed us to obtain information on the entire region analyzed and details of elemental distributions for trace elements contained in oral mucosa. Interestingly, we found that the size and shape of the trace element distribution varied according to the chemical state; this may allow us to distinguish accumulated
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metal ions from metal debris. Additionally, the effects of accumulated elements on the surrounding tissues may differ depending on the chemical state, and ion accumulation should be the most important concern for physicians and dentists due to the high reactivity of ions. Finally, the analyses in this study were carried out with conventional histopathological specimens and did not cause damage to the specimens. Therefore, these methods may be applicable for the screening of metal-affected lesions and evaluation of the effects of accumulated metal elements on oral mucosal lesions. Acknowledgments The SR-XRF and XAFS measurements were performed with the approval of the Photon Factory Program Advisory Committee (Proposal Nos. 2012G011, 2013P002, and 2014G017). This work was financially supported by Japan Society for the Promotion of Science KAKENHI (Grant no. 23390438 to M. Uo). T. Sugiyama is a supported by a research fellowship for young scientists from Japan Society for the Promotion of Science.
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Phys Med Biol 47:2327–2339. doi:10.1088/0031-9155/47/ 13/310 Ide-Ektessabi A, Rabionet M (2005) The role of trace metallic elements in neurodegenerative disorders: quantitative analysis using XRF and XANES spectroscopy. Anal Sci 21:885–892 Kinoshita H, Hori Y, Fukumoto T, Ohigashi T, Shinohara K, Hayashi Y, Ku Y (2010) Novel assessment of hepatic iron distribution by synchrotron radiation X-ray fluorescence microscopy. Med Mol Morphol 43:19–25. doi:10.1007/ s00795-009-0474-7 Kubala-Kukus´ A, Braziewicz J, Banas´ D, Majewska U, Go´z´dz´ S, Urbaniak A, Bana D (1999) Trace element load in cancer and normal lung tissue. Nucl Instrum Methods Phys Res B 150:193–199. doi:10.1016/S0168-583X(98)01057-X Lovell M, Robertson J, Teesdale W, Campbell J, Markesbery W (1998) Copper, iron and zinc in Alzheimer’s disease senile plaques. J Neurol Sci 158:47–52. doi:10.1016/S0022510X(98)00092-6 Miller LM, Wang Q, Telivala TP, Smith RJ, Lanzirotti A, Miklossy J (2006) Synchrotron-based infrared and X-ray imaging shows focalized accumulation of Cu and Zn colocalized with beta-amyloid deposits in Alzheimer’s disease. J Struct Biol 155:30–37. doi:10.1016/j.jsb.2005.09. 004 Preoteasa EA, Ciortea C, Constantinescu B, Fluerasu D, Enescu S-E, Pantelica D, Negoita F, Preoteasa E (2002) Analysis of composites for restorative dentistry by PIXE, XRF and ERDA. Nucl Instrum Methods Phys Res B 189:426–430. doi:10.1016/S0168-583X(01)01119-3 Reddy SB, Charles MJ, Ravi KM, Reddy BS, Anjaneyulu C, Naga RG, Sundareswar B, Vijayan V (2002) Trace elemental analysis of adenoma and carcinoma thyroid by PIXE method. Nucl Instrum Methods Phys Res B 196:333–339. doi:10.1016/S0168-583X(02)01292-2 Reddy SB, Charles MJ, Naga RG, Vijayan V, Reddy BS, Kumar MR, Sundareswar B (2003) Trace elemental analysis of carcinoma kidney and stomach by PIXE method. Nucl Instrum Methods Phys Res B 207:345–355. doi:10.1016/ S0168-583X(03)00463-4 Uo M, Watari F (2004) Rapid analysis of metallic dental restorations using X-ray scanning analytical microscopy. Dent Mater 20:611–615. doi:10.1016/j.dental.2003.08.002 Uo M, Asakura K, Yokoyama A, Ishikawa M, Tamura K, Totsuka Y, Akasaka T, Watari F (2007) X-ray absorption fine structure (XAFS) analysis of titanium-implanted soft tissue. Dent Mater J 26:268–273. doi:10.4012/dmj.26.268 Uo M, Asakura K, Tamura K, Totsuka Y, Abe S, Akasaka T, Watari F (2008) XAFS analysis of Ti and Ni dissolution from pure Ti, Ni–Ti alloy, and SUS304 in soft tissues. Chem Lett 37:958–959. doi:10.1246/cl.2008.958 Uo M, Watari F, Asakura K, Katayama N, Onodera S, Tohyama H, Hamada K, Ohnuki S (2009) Analysis of wear debris generated from the metal-on-metal hip joint. Nano Biomed 1:133–136
Estimation of trace metal elements in oral mucosa specimens by using SR-XRF, PIXE, and XAFS Tomoko Sugiyama • Motohiro Uo • Takahiro Wada • Daisuke Omagari • Kazuo Komiyama • Tadahide Noguchi Yoshinori Jinbu • Mikio Kusama
•
Received: 4 July 2014 / Accepted: 26 September 2014 Ó Springer Science+Business Media New York 2014
Abstract The effects of dissolved elements from metal dental restorations are a major concern in lesions of the oral mucosa, and the evaluation of accumulated metal elements, especially their distribution and chemical state, is essential for determining the precise effects of trace metals. In this study, X-ray fluorescence with synchrotron radiation (SR-XRF) and particle-induced X-ray emission (PIXE) were applied for distribution analysis of the trace metal elements contained in the oral mucosa, and the chemical states of the elements were estimated using X-ray absorption fine structure (XAFS) analysis. Appropriate combination of these analysis techniques, particularly SR-XRF and PIXE, to visualize the distributions of the elements in the oral mucosa allowed for the observation and evaluation of accumulated metal ions and
T. Sugiyama T. Noguchi Y. Jinbu M. Kusama Department of Dentistry, Oral and Maxillofacial Surgery, Jichi Medical University, 3311-1 Yakushiji, Shimotsuke, Tochigi 329-0498, Japan M. Uo (&) T. Wada Advanced Biomaterials Department, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo, 113-8549, Japan e-mail: [email protected] D. Omagari K. Komiyama Department of Pathology, Nihon University School of Dentistry, 1-8-13 Kanda-Surugadai, Chiyoda-ku, Tokyo, 131-8310, Japan
debris. Importantly, the analyses in this study could be carried out using conventional histopathological specimens without damaging the specimens. Therefore, this method would be applicable for the detection of accumulated trace metal elements in biopsy specimens from the oral mucosa. Keywords Synchrotron radiation X-ray fluorescence analysis Particle-induced X-ray emission analysis X-ray absorption fine structure analysis Trace elemental analysis Histopathological specimen
Introduction Trace elements play important roles in biological systems. About 50 elements can be detected at measurable concentrations in living systems, and more than 10 of these elements are considered essential trace elements. Deficiency, excess, and disorder of the distributions of these trace elements have been shown to be associated with the formation of various lesions (Fraga 2005). Thus, analysis of trace elements in tissues and organs is a major research focus. Inductively coupled plasma atomic emission spectroscopy (ICP-AES) and mass spectroscopy (ICP-MS) are popular analytical methods for evaluation of trace elements. With the exception of laser ablation ICP-MS
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(LA-ICP-MS), these methods require the use of aqueous specimens. Therefore, tissue samples (even blood or bodily fluid) need to be decomposed with acids. However, application of such destructive pretreatment causes loss of information about the elemental distribution in the specimen. Therefore, for proper analysis of biological specimens, particularly of various types of lesions used for diagnoses, specimens should not be consumed or damaged. Thus, the development of alternative methods for trace element analysis should be investigated. In recent years, ‘‘quantum beams,’’ e.g., X-rays (photons), protons, neutrons, and various ions, have been applied for nondestructive analysis of trace elements. X-ray fluorescence spectroscopy (XRF), particle-induced X-ray emission spectroscopy (PIXE), and neutron activation analysis (NAA) are the typical nondestructive analysis methods. Irradiation with these beams provides sufficient signals to identify the elements (e.g., characteristic X-rays or c-rays) and causes negligible damage to the specimen. Additionally, synchrotron radiation represents a strong, highly collimated X-ray source. XRF with monochromatized synchrotron radiation (SR-XRF) provides high-quality XRF spectra with low background, allowing detection of trace elements without specimen damage. Highly collimated synchrotron-radiated X-rays are also compatible with microfocusing optics. Therefore, micro-area analysis and elemental distribution images can be obtained by simply scanning the specimen. PIXE uses the characteristic X-ray (c-rays) generated by accelerated proton (or a particle) bombardment. In this method, protons can be focused down to micron size and scanned over the specimen surface. This method, called ‘‘micro-PIXE,’’ can also provide images of the trace elemental distribution. Together, these methods have been applied for the elemental distribution analysis of medical specimens in various fields (Fahrni 2007). Cu, Fe, and Zn are the major mineral elements in living systems. Therefore, abnormalities in the distributions of these elements in various lesions can be identified using SR-XRF and micro-PIXE. Specific distributions of each of these elements have been observed in breast cancer tissues (Geraki et al. 2002; Farquharson et al. 2007, 2008) using SR-XRF, while trace elements in cancers of the lung (Kubala-Kukus´ et al. 1999), thyroid (Reddy et al. 2002), kidney, and stomach (Reddy et al. 2003) have been estimated
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using PIXE. Additionally, cancers in these organs have also been reported to exhibit atypical trace element concentrations. For example, high Ti and Pb levels have been found in lung cancer; low Ca, Zn, and Cu levels have been found in thyroid cancer; and low Fe, Cu, and Zn levels have been found in stomach cancer. Trace element distributions in Alzheimer’s disease have also been studied using SR-XRF (IdeEktessabi and Rabionet 2005; Collingwood and Dobson 2006; Miller et al. 2006) and micro-PIXE (Lovell et al. 1998). The localization of Fe, Cu, and Zn in Alzheimer’s lesions has been suggested to accelerate the aggregation of b-amyloid. Atypical Cu and Fe distributions have also been reported in chronic hepatitis C (Kinoshita et al. 2010). Additionally, X-ray absorption fine structure (XAFS) analysis has been used, in combination with SR-XRF, to identify differences in the accumulated alloy components (Fe, Co, Cr, and Ti) generated from artificial hip joints (Ektessabi et al. 2001; Uo et al. 2009). In dental and orthopedic surgery, various metalcontaining restorative materials and instruments are used, applied to, and even implanted into the oral region. Moreover, in the oral cavity, metal restorations are immersed in saliva and come in close contact with the mucosa. Therefore, dental alloy components often dissolve and penetrate into the mucosa. Base metal elements (e.g., Co, Ni, Zn, Sn, and In) are often used as the major components of dental alloys and are suspected to be the cause of metal allergy. Other common symptoms of metal element accumulation in the oral environment include oral lichen planus (OLP) and palmoplantar pustulosis. Additionally, various eroded metal ions and debris from wearing also accumulate in the surrounding tissues and remote organs. The accumulated metals may cause the acute, chronic, and allergic symptoms, and identification of the causal metals is important for diagnosis of the cause of these symptoms. Alternatively, debris generated from the cutting and polishing of metal restorations during dental treatment often penetrates into the mucosa and may cause different symptoms of toxicity. Thus, the origins and chemical states of the elements identified in the oral mucosa should also be considered. In the present study, we used a combination of SRXRF, micro-PIXE, and XAFS analyses to detect and identify trace metal elements contained in lesions of the oral mucosa. From these data, we were able to form
Biometals
hypotheses to explain the origins of the trace elements and the effects of these elements on the tissues and lesions.
Materials and methods Specimens Oral mucosal tissues were supplied by the Dental Hospital of Nihon University School of Dentistry, Tokyo, Japan. The samples represented residual biopsy sections embedded in paraffin. Two oral mucosal tissues were collected and labeled as specimens #1 and #2 obtained from the different patients. Table 1 shows the clinical records and the intraoral findings of the two specimens. Specimen #1 was tissue from the buccal mucosa that contacted the metal crown and exhibited hyperkeratotic changes (reticular type, the typical appearance of OLP). Specimen #2 was also tissue from the buccal mucosa and exhibited hyperkeratotic changes (plaque type). The tissue for specimen #2 was collected from buccal mucosal tissue near, but not in direct contact with the metal
restoration. Adjacent slices (8 lm thick) of each sample were placed on Kapton film (12.5 lm thick; Du Pont-Toray Co., Ltd, Tokyo, Japan) and subjected to elemental analysis (as described below). After the elemental analyses, the distribution and chemical state information were compared with histopathological information. All patients provided written informed consent, and the study protocol was approved by the Ethical Committees of Nihon University (2012-14). XRF analysis XRF analyses of the two specimens were carried out at BL-4A of the Photon Factory at the High Energy Accelerator Research Organization (Tsukuba, Japan). The incident X-ray (12.9 keV) was focused using polycapillary optics to a 20-lm region, and the specimen was irradiated. The specimen stage was scanned in the X–Y plane, two dimensionally, to obtain elemental distribution images of the areas. The scanning areas varied within several millimeters, and the scanning steps varied from 5 to 50 lm. The obtained XRF data were processed with PyMCA software (Version 4.7.3), and elemental distribution
Table 1 Clinical records of the analyzed specimens No.
Age
Sex
Site
#1
57
M
Buccal mucosa
Diagnosisa Bilateral
Intraoral findingd
OLPb
The patient presented with a chief complaint of bilateral buccal mucosal pain. Ten years before the first examination, similar symptoms had appeared and the patient had been in remission. One year before the first examination, relapsed symptom appeared on inserting a mandibular partial denture. Hyperkeratotic reticular type changes were observed
#2
40
F
Buccal mucosa
Unilateral
OLLc
The patient presented with a chief complaint of plaque type changes of the gingiva and buccal mucosa around the left mandibular second premolar. Because symptoms showed no improvement for several months, the patient consulted the dental office. Hyperkeratotic plaque type changes were observed near the metal restoration but not in direct contact with the restoration
a
Diagnoses were made from clinical and histopathological findings
b
Oral lichen planus
c
Oral lichenoid lesion
d
Black arrows indicate the location of the intraoral findings
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images were obtained. In addition, the XRF spectra were measured for 300 s at regions of trace element localization. Micro-PIXE analysis In order to visualize the high-resolution elemental distribution images, some specimens were also used for micro-PIXE analysis, which was carried out at the National Institute of Radiological Sciences (Chiba, Japan). An accelerated and microfocused proton beam (3.0 MeV, 2 lm beam diameter) with raster scanning was applied over the target area of the specimen (maximum area of 2 9 2 mm). The generated characteristic X-rays were collected using Si(Li) and CdTe detectors to obtain the elemental distribution images and the characteristic X-ray spectra. The obtained data were processed with OMDAQ2007 software (Version 1.3.71.669), and the elemental distribution images and the characteristic X-ray spectra of the region of interest were obtained. XAFS analysis For the typically accumulated metallic elements, XAFS analyses were carried out to determine the
chemical states of the elements. XAFS analyses were also conducted at BL-4A of the Photon Factory. The X-ray absorption near edge structure (XANES) spectra of target elements (e.g., Ni, Fe, and Cu) were measured with the fluorescent XAFS method. As standards, Cr, Fe, Ni foil, and reagent-grade Ni(OH)2 were also measured. Some XANES spectra of the above standards were also measured at BL-9A and 12C.
Results Hematoxylin and eosin staining (for histopathological analysis) and the SR-XRF elemental distribution images of specimen #1 are shown in Fig. 1. The histopathological findings showed characteristic features of keratotic lesions in the oral mucosa: overlying keratinization and a band-like layer of chronic inflammatory cells within the underlying connective tissue. Clear Ni localization could be found deep below the epithelium. Fe and some Ti also accumulated in the same area. This area was not the above the inflammatory cell layer, but some inflammatory cells were present in regions of Ni, Fe, and Ti localization.
Fig. 1 Histopathological images (hematoxylin and eosin staining) and elemental distribution images by SR-XRF of specimen #1
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Fig. 2 Elemental distribution images of the black-boxed area in the Ni distribution image were obtained by PIXE (A). The characteristic X-ray spectra of Ni- and Fe-localized areas are
shown (B). A Diffused, high-intensity distributions of Fe and Ni are indicated by white arrows. B Clear peaks are shown for Ni, Fe, Ti, Al, and Si
Fig. 3 Ni K-edge XANES spectrum of specimen #1
was similar to that of Ni(OH)2 but distinct from that of Ni-foil. This result suggested that the accumulated Ni was in the aqueous ionic state. Dissolution from Nicontaining dental alloys in the oral cavity was suspected to be the origin of the Ni ions. Hematoxylin and eosin staining and SR-XRF elemental distribution images of specimen #2 are shown in Fig. 4. The histopathological findings of specimen #2 were similar to those of specimen #1, which are characteristic features of keratotic lesions in the oral mucosa. Atypical localization of Fe and Cr (spot 2-A) and Zn and Cu (spot 2-B) were observed. Spot 2-A existed at the outermost region of the mucosal epithelium, while spot 2-B existed at the inner region of the basal layer. More detailed distribution images were obtained using micro-PIXE and XRF analysis of spot 2-A from specimen #1 (Fig. 5). The estimated sizes of spots with Fe and Cr accumulation were quite small (several micrometers). The XRF spectrum of spot 2-A showed clear Fe and Cr peaks but weak Ca and Zn peaks. No peaks of major element found in soft tissues (e.g., P, S, and K) were observed. Figure 6 shows Fe and Cr K-edge XANES spectra at spot 2-A. Both spectra were similar to those of foils, indicating that the observed elements were in the metallic state. Therefore, we strongly suspected an artifact containing an Fe–Cr alloy as the origin of this spot. Indeed, our preparation procedure supported this. In the preparation of thinsectioned specimens, we used a microtome with
More detailed distribution images were obtained with micro-PIXE and the XRF spectrum for spot 1 (Fig. 2). The area in which Ni and Fe accumulated was diffused into a region 30–40 lm in diameter (Fig. 2). The characteristic X-ray spectrum of the area with Ni and Fe accumulation showed clear peaks for not only Ni and Fe, but also the metal elements Al, Si, K, Ca, Ti, and Zn and the nonmetal elements S and Cl. While K, Ca, Fe, and Zn were the major minerals in the tissue, Ni and Si accumulation could not be detected by XRF in normal tissue. The Ni K-edge XANES spectra of the Ni-accumulated area in Fig. 2 is shown in Fig. 3. The XANES spectrum of Ni in specimen #1
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Biometals Fig. 4 Histopathological images (hematoxylin and eosin staining) and SR-XRF elemental distribution images of specimen #2. Fe and Cr (spot 2-A) and Zn and Cu (spot 2-B) are shown
Fig. 5 Fe and Cr distribution images obtained by PIXE and the fluorescence X-ray spectra of spot 2-A from Fig. 4
Martensitic stainless steels, which can be hardened by heat treatment. The composition of the microtome blade (Feather S35 type, Tokyo, Japan) used to prepare the specimens analyzed in this study was 87 % Fe and 12 % Cr. Spot 2-A was positioned on the edge of the epithelial layer, which was the most dense structure in the observed tissue, and the outermost region was embedded paraffin, which is softer than tissue. Thus, the blade edge would occasionally chip while the sectioning the epithelial layer, and we
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presume spot 2-A was caused by contamination resulting from the chipping of the microtome blade. The same specimen contained another spot in which metal elements were localized (spot 2-B). XRF spectrum analysis revealed that the major elements within this spot were Cu and Zn, with small amounts of Fe and Ni (Fig. 7). These latter two elements are essential elements found in living tissues; however, the Cu and Zn found in spot 2-B were highly concentrated in a small region (approximately
Biometals Fig. 6 Fe and Cr K-edge XANES spectra of spot 2-A in specimen #2
Fig. 7 PIXE elemental distribution images of specimen #2 and the XRF spectrum of spot 2-B from Fig. 4 (white arrows in Fig. 7)
10 lm). Therefore, these elements could not have been derived from a living system. Cu and Zn K-edge XANES spectra are shown in Fig. 8. Comparison with XANES spectra of foil and the aqueous solution revealed that the XANES spectra of Cu and Zn in spot 2-B were quite similar to that of foil (i.e., in the metal state). Therefore, similar to spot 2-A, we presumed that the origin of Cu and Zn in spot 2-B was an alloy of Cu and Zn, such as brass. From the weak Ni peak in the XRF spectrum, nickel silver (a Cu–Zn–Ni alloy) would be a possible candidate. Brass is widely used in industrial and consumer products, but is not used for
dental restorations and instruments. However, nickel silver was used for dental restorations several decades ago. Thus, we presumed that spot 2-B originated from contamination with small brass or nickel silver debris. Figure 9 shows the line profiles of Fe and Cu in spots 1, 2-A, and 2-B. The profile of the Fe concentration in spot 1 from specimen #1 showed widespread distribution. The full width at half maximum (FWHM) of the Fe distribution in spot 1 was approximately 30 lm. In contrast, Fe and Cu in spots 2-A and 2-B in specimen #2 were concentrated in a small region, and their FWHMs were estimated to be 4 lm.
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Fig. 8 Cu and Zn K-edge XANES spectra of spot 2-B from specimen #2
Fig. 9 The linear analysis profiles of specimens #1 and #2 are shown on the left. Spot 1 indicates the distribution of Fe in specimen #1, while spots 2-A and 2-B indicate Fe and Cu in specimen #2
Discussion In dental treatment, various metals are used for restorations and instruments. Erosion and accumulation of metal elements in the oral mucosa are suspected to be the cause of some intraoral and
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systemic lesions, e.g., OLP and metal allergy. Such tissues are often biopsied for histopathological diagnosis, and specimens of 10 mm or less are obtained. For elemental analysis, to determine whether the lesions may be caused by metal toxicity, the entire region of the biopsied specimen should be examined,
Biometals
and detailed information on the distributions of metals accumulated within the area may provide insights into the relationship between the elemental distribution and the internal structure of the mucosa. Further analysis on the micrometer scale can then be performed. These concepts were supported by the results of our current study, in which we analyzed two biopsy specimens by hematoxylin and eosin staining and by a combination of X-ray-based techniques for elemental analysis. SR-XRF and micro-PIXE are widely used for analysis of the distributions of trace elements. In SRXRF, incident X-rays are focused with a mirror, zone plate, or capillary optics. The mirror optics can focus on the nanometer scale with a highly precise apparatus. However, this focus size is too small to scan the entire biopsy specimen. In contrast, capillary focusing optics can be easily installed at a general SR-XRF facility and can be used to obtain elemental mapping images on the micrometer scale with less loss of X-ray intensity (Preoteasa et al. 2002). These features make XRF analysis suitable for evaluation of the entire mucosal biopsy specimen. Additionally, the microPIXE method irradiates a microfocused proton beam with raster scanning over the specimen. Detailed elemental distribution images can be obtained, but the analysis area is narrow. Therefore, after analysis of the entire specimen with SR-XRF, areas of metal accumulation, i.e., regions of interest, can then be analyzed by micro-PIXE, which can provide images with 2-lm resolution. In addition, the SR-XRF facility can provide XAFS analysis of regions of metal accumulation. The combination of these three methods was used in our study to provide screening of regions of metal accumulation, detailed information on elemental distribution, and evaluation of the chemical state of the metals. In this study, we observed differences in the distributions of elements, particularly Fe and Cu, within specimens #1 and #2 (Fig. 9). These differences could be explained by differences in the conditions or states of the accumulated elements. Fe and Ni in spot 1 were thought to represent the dissolved and accumulated ions shown in Fig. 2. Because they were derived from dissolved ions, the region in which these ions were localized was rather widespread. In contrast, spots 2-A and 2-B were assumed to be metallic debris from XAFS analysis; therefore, the elements within these spots were highly
localized in regions of only a few microns. It is expected that the detrimental effects of accumulated metal ions in living systems would be higher than those of debris (found in the metal state) because ions are more reactive with biological components, e.g., nucleic acid and proteins. Several groups have reported the dissolution of various metallic ions from dental titanium implants (Uo et al. 2007) and dental and medical alloys (Uo et al. 2008, 2009). In addition, trace metal elements, which are suspected to represent eroded ions of metal dental restorations, have been detected in oral lichenoid lesion specimens. In general, medical records of dental treatment are not shared among dental offices. Therefore, the components of metal dental restorations are frequently unknown. Previously, elemental analyses have not been employed because of their difficulty. Recently, one study reported a simple analysis of metal restorations using XRF (Uo and Watari 2004). In this study, we were able to visualize the trace metal elements accumulated in oral mucosal lesions and to estimate their chemical states. Therefore, this information may be useful for evaluating the effects of accumulated elements on surrounding tissues. Importantly, the methods used in this study can be conducted with conventional histopathological specimens without damaging the specimens. Therefore, these methods may be useful for the screening of metal-affected lesions and for determination of the effects of metal element accumulation on oral mucosal lesions. The above analyses of the mucosal specimens and the combinational use of elemental analysis of the metal restoration may allow definite diagnosis and appropriate treatment.
Conclusions In this study, we examined the distribution of trace elements and their chemical states in oral mucosal tissues that were in close contact with metal restorations by using SR-XRF, micro-PIXE, and XAFS analyses. Combined analysis with SR-XRF and microPIXE allowed us to obtain information on the entire region analyzed and details of elemental distributions for trace elements contained in oral mucosa. Interestingly, we found that the size and shape of the trace element distribution varied according to the chemical state; this may allow us to distinguish accumulated
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metal ions from metal debris. Additionally, the effects of accumulated elements on the surrounding tissues may differ depending on the chemical state, and ion accumulation should be the most important concern for physicians and dentists due to the high reactivity of ions. Finally, the analyses in this study were carried out with conventional histopathological specimens and did not cause damage to the specimens. Therefore, these methods may be applicable for the screening of metal-affected lesions and evaluation of the effects of accumulated metal elements on oral mucosal lesions. Acknowledgments The SR-XRF and XAFS measurements were performed with the approval of the Photon Factory Program Advisory Committee (Proposal Nos. 2012G011, 2013P002, and 2014G017). This work was financially supported by Japan Society for the Promotion of Science KAKENHI (Grant no. 23390438 to M. Uo). T. Sugiyama is a supported by a research fellowship for young scientists from Japan Society for the Promotion of Science.
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