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Thermal induced EPR signals in tooth enamel
Radiation Measurements 32 (2000) 793±798
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Thermal induced EPR signals in tooth enamel P. Fattibene*, D. Aragno, S. Onori, M.C. Pressello Physics Laboratory, Istituto Superiore di SanitaÁ, Viale Regina Elena 299, 00161 Rome, Italy Received 14 October 1999; received in revised form 21 February 2000; accepted 26 February 2000
Abstract Electron paramagnetic resonance (EPR) spectroscopy was used to detect the eects of temperature on powdered human tooth enamel, not irradiated in the laboratory. Samples were heated at temperature between 350 and 450, at 600 and at 10008C, for dierent heating times, between 6 min and 39 h. Changes in the EPR spectra were detected, with the formation of new signals. Possible correlation between the changes in EPR spectra and modi®cations in the enamel and in the mineral phase of bone detected with other techniques is discussed. The implications for dosimetric applications of signals induced by overheating due to mechanical friction during sample preparation are underlined. 7 2000 Elsevier Science Ltd. All rights reserved. Keywords: Electron paramagnetic resonance; tooth enamel
1. Introduction The EPR (electron paramagnetic resonance) dose evaluation of irradiated powder tooth enamel, based on the measurement of the amplitude of the radiation induced COÿ 2 signal, is limited by the presence in the spectrum of dierent other signals, both native and induced by sample preparation. Their origin is not completely understood. At room temperature the EPR spectrum of unirradiated tooth enamel shows a broad signal at g = 2.0045, which is referred to as the native signal. It is similar to the signal detected in bone and in dentine, which has been identi®ed as originated from the organic component, most likely collagen (Marino and Becker, 1969). For analogy, the broad signal found in tooth enamel at g = 2.0045 has been assigned to the enamel organic component as well. To our knowledge such an assertion has never been exper-
* Corresponding author. Fax: +39-06-4938-7075. E-mail address: [email protected] (P. Fattibene).
imentally validated. Other signals which also disturb the dosimetric signal are those produced during sample preparation operations. For example, signals are produced by overheating and mechanical friction (Aragno et al., 2000; Sholom et al. 1998; Aldrich et al., 1992; Desrosiers et al., 1989; Marino and Becker, 1968), and the knowledge of what they are originated from could be important for their reduction or elimination. One of the ways to identify the origin of the EPR signals is to study their thermal evolution. This approach has been followed also by many other experimentalists, using techniques dierent from the EPR in the attempt of understanding the structure of the enamel matrix, both organic and inorganic (Mayer et al., 1990; Elliot et al., 1985; Holcomb and Young, 1980; LeGeros et al., 1978; Myers, 1965; CarlstroÈm et al., 1963). Some EPR studies have been carried out with irradiated samples (Brik et al., 1997; Callens et al., 1995; Aldrich et al., 1992; Doi et al., 1982), but it seems that the EPR technique has not been exploited adequately. The purpose of the present work was to search EPR signals induced by the temperature in
1350-4487/00/$ - see front matter 7 2000 Elsevier Science Ltd. All rights reserved. PII: S 1 3 5 0 - 4 4 8 7 ( 0 0 ) 0 0 0 6 8 - 8
794
P. Fattibene et al. / Radiation Measurements 32 (2000) 793±798
tooth enamel samples. Such signals could be masked by the dosimetric COÿ 2 signal and for such a reason in the present work samples not irradiated in the laboratory were examined. Enamel samples were thermally treated at temperature in the (350±1000)8C range. In such an interval changes in physical and chemical properties of the organic and inorganic matrix of the enamel have been reported by many authors (Mayer et al., 1990; Elliott et al., 1985; Holcomb and Young, 1980; LeGeros et al., 1978; Myers, 1965). Such changes involve mainly water (both trapped and incorporated), ÿ ÿ and enamel components as (HPO4)ÿ 2 , (OH) , (CO3)2 ÿ [substituted for OH (type A) and (PO4)3 (type B)]. Breakdown and decomposition of the organic component have been studied with the thermal behaviour in that temperature range.
the fourth signi®cant ®gure with a frequency counter (53150A, Hewlett Packard, Santa Clara, CA) controlled by a personal computer. The microwave frequency during the recording of the spectra shown in the ®gures was 9.7788 GHz. The acquisition parameters are reported in the ®gure captions. All the spectra shown in the ®gures were recorded at 105 receiver gain. The enamel powder samples were inserted in a quartz tube and centred in the cavity. A MgO/Mn2+ powder sample was used as a reference sample for both ®eld and signal amplitude normalisation (Onori et al., 2000). It was inserted at the bottom of the cavity, so that its position was not varied when the enamel samples were changed.
2. Materials and methods
Figs. 1 and 3 show the spectra for dierent heating times of the 3508C and the 4008C heated samples, respectively. Figs. 2 and 4 show the spectra of the same samples for dierent microwave power levels. The spectra measured after 6 min heating were similar to the unheated control sample spectrum and were not reported in the ®gures. The ®rst variations in the spectra were observed after 1 h heating. In the 3508C sample a multiplet signal at ®eld value of 349.1 mT and with a hyper®ne constant of 0.63 mT was observed after 2 h, and was much more evident after 3 h heating. For the 4008C sample the same multiplet was evident already after 1 h heating. The multiplet is furtherly split in a four-line multiplet with a hyper®ne constant of 0.18 mT. The number of lines of the principal multiplet was uncertain because of the weak intensity of the signal. Even after 8000 acquisition
Thirty sound molar teeth of adult people, provided by a local dental clinic, were used for the present experiment. The whole teeth were ®rst sterilised with NaHCl (5% water solution) for 2 h, then the crown was cut from the root with a low speed drive saw equipped with a diamond wheel. In order to avoid overheating of the tooth, water cooling was used during sawing (Aragno et al., 2000). The crown was then cut in two halves along the transversal axis. Dentine was removed using a water cooled high speed dental drill. The enamel, cleaned of the dentine, was then ground by hand with mortar and pestle to 0.51 mm grain size. A quality control of the non-irradiated tooth enamel samples was performed measuring the individual EPR spectra. The samples which presented unusual signals were etched with ortophosphoric acid (Fattibene et al., 1998), after which, if the signals were not eliminated, the samples were rejected. Also tooth samples with COÿ 2 signal signi®cantly more intense than that usually recorded in teeth not irradiated in the laboratory were rejected. The remaining samples were pooled together. From the pool, 18 powder samples of 20021 mg mass were prepared. The powder enamel samples were heated in air in a ventilated oven at 350, 400, 450, 600 and 10008C. Heating times for the (350±450)8C range were 6 min, and 1, 2, 3, 12, 25 and 39 h. Samples were heated at 6008C for 1 h and at 10008C for 6 min. After heating, the samples were allowed to cool in air at room temperature for ca. 20 min. The samples were then measured immediately and after dierent time interval storage at room conditions. The EPR measurements were performed at RT with a Bruker ESP 300 spectrometer, operating in X band, with a TM Bruker cylindrical cavity. During the EPR acquisitions, the microwave frequency was measured at
3. Results
Fig. 1. EPR spectra of powder enamel heated at 3508C for dierent heating times. Acquisition parameters were 1 mW microwave power and 0.05 mT modulation amplitude.
P. Fattibene et al. / Radiation Measurements 32 (2000) 793±798
795
sent in the high power spectra of the sample heated at 4008C together with another signal [(B) in Figs. 2 and 4, the arrow indicates 348.6 mT]. Figs. 6 and 7 show the spectra of the 4508C heated sample at dierent heating times and at dierent microwave powers, respectively. The multiplet was observed only after 1 h heating. For heating times longer than 1 h, the spectrum is dominated by at least the B and the C signals, characterised by dierent growing rates. The spectra of the samples heated at 600 and 10008C are shown in Fig. 8. The two spectra are symmetric single line signals (centered at 349 mT). The linewidths are 0.18 and 0.11 mT for the 6008C and the 10008C heated samples, respectively. The dierent amplitude dependence of the two signals with microwave power is shown in Fig. 9. Fig. 2. EPR spectra of the sample heated at 3508C for 12 h at dierent microwave power levels. The COÿ 2 signal of a 10 Gy irradiated enamel sample is also shown for comparison.
scans it was not possible to unambiguously detect the side lines and therefore to ascertain whether it was a septet or a quintet. In Fig. 5 the experimental spectrum at 4008C and 1 h and the simulated spectra of a septet and of a quintet are shown. The signal simulations were carried out with the ESP300 Bruker software. At both 350 and 4008C the presence of a signal [(A) in Figs. 2 and 4] of line-width 0.09 mT can be noted (the arrow in the ®gures corresponds to 348.5 mT ®eld value). The signal A increased with the microwave power, while the multiplet saturated around 64 mW. A further signal [(C) in Figs. 2 and 4] appeared at microwave power levels >32 mW for the 3508C sample (the arrow indicates 348.9 mT). The same signal was pre-
Fig. 3. EPR spectra of powder enamel heated at 4008C for dierent heating times. Acquisition parameters were 1 mW microwave power and 0.05 mT modulation amplitude.
4. Discussion As a ®rst comment, it is worth noting that not only from the isochronic thermal study, but also from the isothermal study much information can be obtained about the nature of the paramagnetic centres. The analysis of the changes occurred in the EPR spectra between 350 and 4508C could provide a hint to identify the radical species present in the unirradiated enamel. A signi®cant dierence observed between the spectra shown in Figs. 1, 3 and 6 is associated with a multiplet, which is present at almost all the heating times for the 350 and the 4008C heated samples, and is not clear detectable in the 4508C spectra, where other signals become dominant. Further information can be deduced by the microwave power studies. Three signals can be identi®ed in the spectra, indicated as A, B and C in the ®gures. Some similarity can be found between
Fig. 4. EPR spectra of the sample heated at 4008C for 1 h at dierent microwave power levels.
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P. Fattibene et al. / Radiation Measurements 32 (2000) 793±798
A and B signals and signals found by others (Brik et al., 1997; Callens et al., 1995), and assigned to CO3ÿ 3 and to CO3ÿ, but relation to the signals shown in the present paper is made dicult by the fact that the samples in those papers had been irradiated at 100 and 13000 Gy, respectively. The C signal is at the same ®eld value of the COÿ 2 and from Figs. 3 and 7 it appears to be axial, suggesting it could be COÿ 2 . It has been stated (Holcomb and Young, 1980) that at 2008C the enamel organic part starts to carbonate, at 3508C it is partly lost, at 4008C the breakdown of the organic component begins, reaching the maximum at 6008C, until, at 8008C, full decomposition of the organic component occurs. From infrared spectroscopy studies it was ascertained that, between 250 and 5208C,
organic species are being evolved, presumably releasing H2O and OHÿ in the process (Holcomb and Young, 1980). It was therefore expected that at 3508C some signals from the organic component could appear in the enamel spectrum. The structure of the multiplet signal is typical of organic radicals, most probably due to a structure containing aromatic rings. Signals similar to this have been described in the literature, but none of them seems to be appropriate for describing the one found in the present work. A multiplet with dierent hyper®ne constants (hfc) and g-values have been found in cave deposits (Chong and Fujitani, 1984) and assigned to t-butyl radicals [(CH3)3C] or to trimer of methyl radicals [(CH3)3] (Ikeya, 1984). A multiplet at the same g-value and with the same hfc (A7=0.63 mT and A4=0.18 mT) of the one observed at 3508C has been observed in heated ¯ints at 4008C. This spectrum was identi®ed as perinaphtenyl radical (Chandra et al., 1988; Ikeya, 1993). There is no reason to believe that such radicals are present in modern tooth enamel. The enamel structure is composed at 97% of hydroxiapatite, the remaining being water and aminoacids dispersed in the hydroxiapatite matrix (Eastoe, 1968). None of the 20 aminoacids has a structure similar to periphtalene. Brik et al. (1997) found a septuplet when the sample was heated at 3508C and has made the hypothesis that it was due to valine. The presence of a quadruplet in the signal does not support such an hypothesis. It should be noted that all the radicals described in the literature and having a multiplet structure have been observed after irradiation. In the present case no irradiation was performed in laboratory, but it cannot be excluded that some teeth had received a low dose from the natural background or from some medical exposure. The signals of the 600 and 10008C heated samples
Fig. 6. EPR spectra of powder enamel heated at 4508C for dierent heating times. Acquisition parameters were 1 mW microwave power and 0.05 mT modulation amplitude.
Fig. 7. EPR spectra of the sample heated at 4508C for 1 h at dierent microwave power levels.
Fig. 5. Spectrum of a sample heated at 4008C after 1 h heating and simulated quintet and septet signals. Acquisition parameters were 1 mW microwave power and 0.05 mT modulation amplitude.
P. Fattibene et al. / Radiation Measurements 32 (2000) 793±798
are single lines, indicating a much simpli®ed structure of the enamel matrix. This is in line with the hypothesis that at 10008C the principal crystalline phase in mature human enamel is oxyapatite or calcium oxide (Holcomb and Young, 1980). Partial decomposition on tri-calcium-phosphate (b-TCP) occurs (Mayer et al., 1990). The microwave power response of the 6008C sample is dierent from the 10008C sample. This due to other signals still present in the spectrum of the 6008C heated sample, but of low intensity. The signals observed at 600 and 10008C have some important implications in the tooth enamel dose reconstruction. It has been shown that sample preparation can induce signals which overlap to the COÿ 2 signal (Aragno et al., 2000; Aldrich et al., 1992; Desrosiers et al., 1989). In particular Aragno et al. (2000) have shown the presence of a single line signal after uncooled mechanical treatment of enamel samples. Mechanical operations can heat the sample up to temperature as high as 10008C (Ikeya, 1993). The hypothesis has been made that it is the enamel overheating due to mechanical friction which produces such signals (Aragno et al., 2000; Aldrich et al., 1992). The similarity between the mechanically induced signal and those recorded after heating at 600 and 10008C supports this hypothesis.
5. Conclusions Heat induced EPR signals were detected in powder tooth enamel heated in the (350±1000)8C temperature range. The EPR spectra for 350, 400 and 4508C revealed the presence of at least four signals in addition to the native signal. Their relative amplitude
Fig. 8. Spectra of a sample heated at 6008C after 1 h heating and at 10008C after 6 min heating. Acquisition parameters were 2 mW microwave power and 0.05 mT modulation amplitude.
797
Fig. 9. Signal amplitude of the 600 and the 10008C heated samples as a function of microwave power.
depends on temperature, duration of heating and microwave power. They are originated in both the organic and inorganic components of enamel even if their assignment has not been addressed. The EPR spectrum simpli®es with temperature. Over 6008C it becomes a symmetric single line spectrum at the same ®eld position of the dosimetric COÿ 2 signal. Heat related signals have implications for dosimetry.
References Aldrich, J.E., Pass, B., Mailer, C., 1992. Changes in the paramagnetic centres in irradiated and heated dental enamel studies using electron paramagnetic resonance. International Journal of Radiation Biology 61 (3), 433± 437. Aragno, D., Fattibene, P., Onori, S. 2000. Mechanically induced EPR signals in enamel. Applied Radiation and Isotopes (submitted for publication). Brik, A.B., Scherbina, O.I., Haskell, E.H., Sobotovich, E.V., Kalinichenko, A.M., 1997. Heating related changes in the characteristics of paramagnetic centers in tooth enamel using EPR techniques. Mineralogical Journal 19 (4), 3±12. Callens, F., Moens, P., Verbeeck, R., 1995. An EPR study of intact and powdered human tooth enamel dried at 4008C. Calci®ed Tissue International 56, 543±548. CarlstroÈm, D., Glas, J.E., Angmar, B., 1963. Studies on the ultrastructure of dental enamel. Journal of Ultrastructure Research 8, 24±29. Chandra, H., Symons, M.C.R., Griths, D.R., 1988. Stable perinaphtenyl radicals in ¯ints. Nature 332, 526±527. Chong, T.S., Fujitani, Y., 1984. Organic radicals in a stalactite. Earth Monthly 58, 237±239. Desrosiers, M.F., Simic, M.G., Eichmiller, F.C., Johnston, A.D., Bowen, R.L., 1989. Mechanically-induced gener-
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ation of radicals in tooth enamel. Applied Radiation and Isotopes 40, 1195±1197. Doi, Y., Moriwaki, Y., Aoba, T., Kani, M., 1982. ESR on the hydroxyl ion vacancies in the apatites. Calci®ed Tissue International 34, S47±S51. Eastoe, J.E., 1968. Chemical aspects of the matrix concept in calci®ed tissue organisation. Calci®ed Tissue Research 2, 1±19. Elliott, J.C., Holcomb, D.W., Toung, R.A., 1985. Infrared determination of the degree of substitution of hydroxyl by carbonate ions in human dental enamel. Calci®ed Tissue International 37, 372±375. Fattibene, P., Aragno, D., Onori, S., 1998. Eectiveness of chemical etching for background Electron Paramagnetic Resonance signal reduction in tooth enamel. Health Physics 75 (5), 500±505. Holcomb, D.W., Young, R.A., 1980. Thermal decomposition of human tooth enamel. Calci®ed Tissue International 31, 189±201. Ikeya, M., 1984. Comments: ESR in organic earth science. Earth Monthly 6, 240. Ikeya, M., 1993. New Applications of Electron Spin
Resonance: Dating, Dosimetry and Microscopy. World Scienti®c, Singapore. LeGeros, R.Z., Bonel, G., Legros, R., 1978. Types of ``H2O'' in human enamel and in precipitated apatites. Calci®ed Tissue Research 26, 111±118. Marino, A.A., Becker, R.O., 1968. Mechanically induced free radicals in bone. Nature 218. Marino, A.A., Becker, R.O., 1969. Temperature dependence of the EPR signal in tendon collagen. Nature 222, 164± 165. Mayer, I., Schneider, S., Sydney-Zax, M., Deutsch, D., 1990. Thermal decomposition of developing enamel. Calci®ed Tissue International 46, 254±257. Myers, H.M., , 1965. Trapped water of dental enamel. Nature 206, 713±714. Onori, S., Aragno, D., Fattibene, P., Petetti, E., Pressello, M.C. 2000. ISS protocol for EPR tooth dosimetry. Radiation Measurements 32, 787±792. Sholom, S.V., Haskell, E.H., Hayes, R.B., Chumak, V.V., Kenner, G.H., 1998. In¯uence of crushing and additive irradiation procedures on EPR dosimetry of tooth enamel. Radiation Measurements 29 (1), 105±111.
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Thermal induced EPR signals in tooth enamel P. Fattibene*, D. Aragno, S. Onori, M.C. Pressello Physics Laboratory, Istituto Superiore di SanitaÁ, Viale Regina Elena 299, 00161 Rome, Italy Received 14 October 1999; received in revised form 21 February 2000; accepted 26 February 2000
Abstract Electron paramagnetic resonance (EPR) spectroscopy was used to detect the eects of temperature on powdered human tooth enamel, not irradiated in the laboratory. Samples were heated at temperature between 350 and 450, at 600 and at 10008C, for dierent heating times, between 6 min and 39 h. Changes in the EPR spectra were detected, with the formation of new signals. Possible correlation between the changes in EPR spectra and modi®cations in the enamel and in the mineral phase of bone detected with other techniques is discussed. The implications for dosimetric applications of signals induced by overheating due to mechanical friction during sample preparation are underlined. 7 2000 Elsevier Science Ltd. All rights reserved. Keywords: Electron paramagnetic resonance; tooth enamel
1. Introduction The EPR (electron paramagnetic resonance) dose evaluation of irradiated powder tooth enamel, based on the measurement of the amplitude of the radiation induced COÿ 2 signal, is limited by the presence in the spectrum of dierent other signals, both native and induced by sample preparation. Their origin is not completely understood. At room temperature the EPR spectrum of unirradiated tooth enamel shows a broad signal at g = 2.0045, which is referred to as the native signal. It is similar to the signal detected in bone and in dentine, which has been identi®ed as originated from the organic component, most likely collagen (Marino and Becker, 1969). For analogy, the broad signal found in tooth enamel at g = 2.0045 has been assigned to the enamel organic component as well. To our knowledge such an assertion has never been exper-
* Corresponding author. Fax: +39-06-4938-7075. E-mail address: [email protected] (P. Fattibene).
imentally validated. Other signals which also disturb the dosimetric signal are those produced during sample preparation operations. For example, signals are produced by overheating and mechanical friction (Aragno et al., 2000; Sholom et al. 1998; Aldrich et al., 1992; Desrosiers et al., 1989; Marino and Becker, 1968), and the knowledge of what they are originated from could be important for their reduction or elimination. One of the ways to identify the origin of the EPR signals is to study their thermal evolution. This approach has been followed also by many other experimentalists, using techniques dierent from the EPR in the attempt of understanding the structure of the enamel matrix, both organic and inorganic (Mayer et al., 1990; Elliot et al., 1985; Holcomb and Young, 1980; LeGeros et al., 1978; Myers, 1965; CarlstroÈm et al., 1963). Some EPR studies have been carried out with irradiated samples (Brik et al., 1997; Callens et al., 1995; Aldrich et al., 1992; Doi et al., 1982), but it seems that the EPR technique has not been exploited adequately. The purpose of the present work was to search EPR signals induced by the temperature in
1350-4487/00/$ - see front matter 7 2000 Elsevier Science Ltd. All rights reserved. PII: S 1 3 5 0 - 4 4 8 7 ( 0 0 ) 0 0 0 6 8 - 8
794
P. Fattibene et al. / Radiation Measurements 32 (2000) 793±798
tooth enamel samples. Such signals could be masked by the dosimetric COÿ 2 signal and for such a reason in the present work samples not irradiated in the laboratory were examined. Enamel samples were thermally treated at temperature in the (350±1000)8C range. In such an interval changes in physical and chemical properties of the organic and inorganic matrix of the enamel have been reported by many authors (Mayer et al., 1990; Elliott et al., 1985; Holcomb and Young, 1980; LeGeros et al., 1978; Myers, 1965). Such changes involve mainly water (both trapped and incorporated), ÿ ÿ and enamel components as (HPO4)ÿ 2 , (OH) , (CO3)2 ÿ [substituted for OH (type A) and (PO4)3 (type B)]. Breakdown and decomposition of the organic component have been studied with the thermal behaviour in that temperature range.
the fourth signi®cant ®gure with a frequency counter (53150A, Hewlett Packard, Santa Clara, CA) controlled by a personal computer. The microwave frequency during the recording of the spectra shown in the ®gures was 9.7788 GHz. The acquisition parameters are reported in the ®gure captions. All the spectra shown in the ®gures were recorded at 105 receiver gain. The enamel powder samples were inserted in a quartz tube and centred in the cavity. A MgO/Mn2+ powder sample was used as a reference sample for both ®eld and signal amplitude normalisation (Onori et al., 2000). It was inserted at the bottom of the cavity, so that its position was not varied when the enamel samples were changed.
2. Materials and methods
Figs. 1 and 3 show the spectra for dierent heating times of the 3508C and the 4008C heated samples, respectively. Figs. 2 and 4 show the spectra of the same samples for dierent microwave power levels. The spectra measured after 6 min heating were similar to the unheated control sample spectrum and were not reported in the ®gures. The ®rst variations in the spectra were observed after 1 h heating. In the 3508C sample a multiplet signal at ®eld value of 349.1 mT and with a hyper®ne constant of 0.63 mT was observed after 2 h, and was much more evident after 3 h heating. For the 4008C sample the same multiplet was evident already after 1 h heating. The multiplet is furtherly split in a four-line multiplet with a hyper®ne constant of 0.18 mT. The number of lines of the principal multiplet was uncertain because of the weak intensity of the signal. Even after 8000 acquisition
Thirty sound molar teeth of adult people, provided by a local dental clinic, were used for the present experiment. The whole teeth were ®rst sterilised with NaHCl (5% water solution) for 2 h, then the crown was cut from the root with a low speed drive saw equipped with a diamond wheel. In order to avoid overheating of the tooth, water cooling was used during sawing (Aragno et al., 2000). The crown was then cut in two halves along the transversal axis. Dentine was removed using a water cooled high speed dental drill. The enamel, cleaned of the dentine, was then ground by hand with mortar and pestle to 0.51 mm grain size. A quality control of the non-irradiated tooth enamel samples was performed measuring the individual EPR spectra. The samples which presented unusual signals were etched with ortophosphoric acid (Fattibene et al., 1998), after which, if the signals were not eliminated, the samples were rejected. Also tooth samples with COÿ 2 signal signi®cantly more intense than that usually recorded in teeth not irradiated in the laboratory were rejected. The remaining samples were pooled together. From the pool, 18 powder samples of 20021 mg mass were prepared. The powder enamel samples were heated in air in a ventilated oven at 350, 400, 450, 600 and 10008C. Heating times for the (350±450)8C range were 6 min, and 1, 2, 3, 12, 25 and 39 h. Samples were heated at 6008C for 1 h and at 10008C for 6 min. After heating, the samples were allowed to cool in air at room temperature for ca. 20 min. The samples were then measured immediately and after dierent time interval storage at room conditions. The EPR measurements were performed at RT with a Bruker ESP 300 spectrometer, operating in X band, with a TM Bruker cylindrical cavity. During the EPR acquisitions, the microwave frequency was measured at
3. Results
Fig. 1. EPR spectra of powder enamel heated at 3508C for dierent heating times. Acquisition parameters were 1 mW microwave power and 0.05 mT modulation amplitude.
P. Fattibene et al. / Radiation Measurements 32 (2000) 793±798
795
sent in the high power spectra of the sample heated at 4008C together with another signal [(B) in Figs. 2 and 4, the arrow indicates 348.6 mT]. Figs. 6 and 7 show the spectra of the 4508C heated sample at dierent heating times and at dierent microwave powers, respectively. The multiplet was observed only after 1 h heating. For heating times longer than 1 h, the spectrum is dominated by at least the B and the C signals, characterised by dierent growing rates. The spectra of the samples heated at 600 and 10008C are shown in Fig. 8. The two spectra are symmetric single line signals (centered at 349 mT). The linewidths are 0.18 and 0.11 mT for the 6008C and the 10008C heated samples, respectively. The dierent amplitude dependence of the two signals with microwave power is shown in Fig. 9. Fig. 2. EPR spectra of the sample heated at 3508C for 12 h at dierent microwave power levels. The COÿ 2 signal of a 10 Gy irradiated enamel sample is also shown for comparison.
scans it was not possible to unambiguously detect the side lines and therefore to ascertain whether it was a septet or a quintet. In Fig. 5 the experimental spectrum at 4008C and 1 h and the simulated spectra of a septet and of a quintet are shown. The signal simulations were carried out with the ESP300 Bruker software. At both 350 and 4008C the presence of a signal [(A) in Figs. 2 and 4] of line-width 0.09 mT can be noted (the arrow in the ®gures corresponds to 348.5 mT ®eld value). The signal A increased with the microwave power, while the multiplet saturated around 64 mW. A further signal [(C) in Figs. 2 and 4] appeared at microwave power levels >32 mW for the 3508C sample (the arrow indicates 348.9 mT). The same signal was pre-
Fig. 3. EPR spectra of powder enamel heated at 4008C for dierent heating times. Acquisition parameters were 1 mW microwave power and 0.05 mT modulation amplitude.
4. Discussion As a ®rst comment, it is worth noting that not only from the isochronic thermal study, but also from the isothermal study much information can be obtained about the nature of the paramagnetic centres. The analysis of the changes occurred in the EPR spectra between 350 and 4508C could provide a hint to identify the radical species present in the unirradiated enamel. A signi®cant dierence observed between the spectra shown in Figs. 1, 3 and 6 is associated with a multiplet, which is present at almost all the heating times for the 350 and the 4008C heated samples, and is not clear detectable in the 4508C spectra, where other signals become dominant. Further information can be deduced by the microwave power studies. Three signals can be identi®ed in the spectra, indicated as A, B and C in the ®gures. Some similarity can be found between
Fig. 4. EPR spectra of the sample heated at 4008C for 1 h at dierent microwave power levels.
796
P. Fattibene et al. / Radiation Measurements 32 (2000) 793±798
A and B signals and signals found by others (Brik et al., 1997; Callens et al., 1995), and assigned to CO3ÿ 3 and to CO3ÿ, but relation to the signals shown in the present paper is made dicult by the fact that the samples in those papers had been irradiated at 100 and 13000 Gy, respectively. The C signal is at the same ®eld value of the COÿ 2 and from Figs. 3 and 7 it appears to be axial, suggesting it could be COÿ 2 . It has been stated (Holcomb and Young, 1980) that at 2008C the enamel organic part starts to carbonate, at 3508C it is partly lost, at 4008C the breakdown of the organic component begins, reaching the maximum at 6008C, until, at 8008C, full decomposition of the organic component occurs. From infrared spectroscopy studies it was ascertained that, between 250 and 5208C,
organic species are being evolved, presumably releasing H2O and OHÿ in the process (Holcomb and Young, 1980). It was therefore expected that at 3508C some signals from the organic component could appear in the enamel spectrum. The structure of the multiplet signal is typical of organic radicals, most probably due to a structure containing aromatic rings. Signals similar to this have been described in the literature, but none of them seems to be appropriate for describing the one found in the present work. A multiplet with dierent hyper®ne constants (hfc) and g-values have been found in cave deposits (Chong and Fujitani, 1984) and assigned to t-butyl radicals [(CH3)3C] or to trimer of methyl radicals [(CH3)3] (Ikeya, 1984). A multiplet at the same g-value and with the same hfc (A7=0.63 mT and A4=0.18 mT) of the one observed at 3508C has been observed in heated ¯ints at 4008C. This spectrum was identi®ed as perinaphtenyl radical (Chandra et al., 1988; Ikeya, 1993). There is no reason to believe that such radicals are present in modern tooth enamel. The enamel structure is composed at 97% of hydroxiapatite, the remaining being water and aminoacids dispersed in the hydroxiapatite matrix (Eastoe, 1968). None of the 20 aminoacids has a structure similar to periphtalene. Brik et al. (1997) found a septuplet when the sample was heated at 3508C and has made the hypothesis that it was due to valine. The presence of a quadruplet in the signal does not support such an hypothesis. It should be noted that all the radicals described in the literature and having a multiplet structure have been observed after irradiation. In the present case no irradiation was performed in laboratory, but it cannot be excluded that some teeth had received a low dose from the natural background or from some medical exposure. The signals of the 600 and 10008C heated samples
Fig. 6. EPR spectra of powder enamel heated at 4508C for dierent heating times. Acquisition parameters were 1 mW microwave power and 0.05 mT modulation amplitude.
Fig. 7. EPR spectra of the sample heated at 4508C for 1 h at dierent microwave power levels.
Fig. 5. Spectrum of a sample heated at 4008C after 1 h heating and simulated quintet and septet signals. Acquisition parameters were 1 mW microwave power and 0.05 mT modulation amplitude.
P. Fattibene et al. / Radiation Measurements 32 (2000) 793±798
are single lines, indicating a much simpli®ed structure of the enamel matrix. This is in line with the hypothesis that at 10008C the principal crystalline phase in mature human enamel is oxyapatite or calcium oxide (Holcomb and Young, 1980). Partial decomposition on tri-calcium-phosphate (b-TCP) occurs (Mayer et al., 1990). The microwave power response of the 6008C sample is dierent from the 10008C sample. This due to other signals still present in the spectrum of the 6008C heated sample, but of low intensity. The signals observed at 600 and 10008C have some important implications in the tooth enamel dose reconstruction. It has been shown that sample preparation can induce signals which overlap to the COÿ 2 signal (Aragno et al., 2000; Aldrich et al., 1992; Desrosiers et al., 1989). In particular Aragno et al. (2000) have shown the presence of a single line signal after uncooled mechanical treatment of enamel samples. Mechanical operations can heat the sample up to temperature as high as 10008C (Ikeya, 1993). The hypothesis has been made that it is the enamel overheating due to mechanical friction which produces such signals (Aragno et al., 2000; Aldrich et al., 1992). The similarity between the mechanically induced signal and those recorded after heating at 600 and 10008C supports this hypothesis.
5. Conclusions Heat induced EPR signals were detected in powder tooth enamel heated in the (350±1000)8C temperature range. The EPR spectra for 350, 400 and 4508C revealed the presence of at least four signals in addition to the native signal. Their relative amplitude
Fig. 8. Spectra of a sample heated at 6008C after 1 h heating and at 10008C after 6 min heating. Acquisition parameters were 2 mW microwave power and 0.05 mT modulation amplitude.
797
Fig. 9. Signal amplitude of the 600 and the 10008C heated samples as a function of microwave power.
depends on temperature, duration of heating and microwave power. They are originated in both the organic and inorganic components of enamel even if their assignment has not been addressed. The EPR spectrum simpli®es with temperature. Over 6008C it becomes a symmetric single line spectrum at the same ®eld position of the dosimetric COÿ 2 signal. Heat related signals have implications for dosimetry.
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