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Isotropic radical CO2- in biological apatites
Radiation Measurements 42 (2007) 1580 – 1582 www.elsevier.com/locate/radmeas
Short communication
Isotropic radical CO− 2 in biological apatites V.V. Rudko ∗ , S.S. Ishchenko, I.P. Vorona, N.P. Baran Institute of Semiconductor Physics of National Academy of Sciences of Ukraine, 45, pr. Nauky, Kiev 03028, Ukraine Received 26 December 2006; received in revised form 27 March 2007; accepted 2 April 2007
Abstract ◦ The isotropic CO− 2 EPR spectrum at g ∼ 2.0006 for -irradiated powders of dental enamel annealed at different temperatures up to 320 C is studied. The signal intensity is found to increase with the growth of annealing temperature up to 240 ◦ C. This finding contradicts to the existing model of isotropic CO− 2 radical in apatites. The possible models of the radical in biological apatite are analyzed and discussed. On the basis of the results obtained it is suggested that in tooth enamel apatite the isotropic CO− 2 radical is the bulk radical localized in structural voids of hydroxyapatite lattice, which occur in the vicinity of a carbon radical in position B. © 2007 Elsevier Ltd. All rights reserved.
The radiation-induced EPR signal in biological and synthetic carbonate-containing apatites is mainly caused by the CO− 2 radicals (Callens, 1997). Three types of such radicals are identified in apatites at present. They are responsible for different EPR spectra—orthorhombic (gx = 2.0030, gy = 2.0015, gz = 1.9970), axial (g = 1.9970, g⊥ = 2.0027), and isotropic (g = 2.0006) ones. Axial CO− 2 radical has been studied in more detail. ENDOR measurements of this radical in dental enamel (Ishchenko et al., 1999) showed that the axial CO− 2 occupies B position in hydroxyapatite lattice (it substitutes phosphoric tetrahedron PO3− 4 ). Axial symmetry of the paramagnetic center is explained by fast rotation of CO− 2 radical around the axis connecting two oxygen atoms which is parallel to the crystallographic c axis of the apatite (Ishchenko et al., 2002). The orthorhombic radical was suggested to be located on the surface of apatite crystallites (Callens et al., 1995) or in the organic component of bioapatite (Brik et al., 1997). However, recent studies of annealing-induced changes in the EPR spectra for irradiated enamel plates (Vorona et al., 2006) showed transformation of orthorhombic radicals into axial ones. This finding suggests that both types of CO− 2 radicals are similarly localized in the apatite structure. The isotropic EPR signal (paramagnetic centers related to this signal will be hereafter named isotropic
∗ Corresponding author. Fax: +380 044 265 8342.
E-mail address: [email protected] (V.V. Rudko). 1350-4487/$ - see front matter © 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.radmeas.2007.04.003
CO− 2 radicals) was observed in both synthetic (Callens et al., 1989) and biological (Brik et al., 1997) apatites, and also in natural carbonates (Debuyst et al., 1993). It was assumed that in synthetic apatite the isotropic CO− 2 is localized in the occluded (absorbed) water (Callens, 1997). To the best of our knowledge there is no detailed information about isotropic CO− 2 radical in biological apatites. The present study deals with the study of this radical in tooth enamel and analysis of the possible models of isotropic CO− 2 center in bioapatite. We studied the dental enamel which is the typical bioapatite. Powders of enamel with the grain size of about 100.300 m were prepared from sound human teeth according to the method which is usually used in EPR dosimetry (see, for example, Tatsumi-Miyajima, 1987). The samples were irradiated by 60 Co -rays. Exposure rate was 2.58×10−2 C kg−1 s−1 (100 R/s). Absorbed dose was not determined accurately; it was estimated to be approximately a few kGy. Before the measurements the samples were kept several months at room temperature in order to ensure the destruction of short-living radiation-induced radicals. EPR measurements were carried out using an X-band EPR spectrometer at room temperature. The 100 kHz modulation of the magnetic field with 0.05 mT amplitude was used. The error of the magnetic field measurements did not exceed 0.01 mT. The EPR spectra of dental enamel were registered together with the spectrum of reference sample MgO: Cr 3+ . The enamel powders were annealed in muffle furnace in air at different temperatures up to 320 ◦ C.
V.V. Rudko et al. / Radiation Measurements 42 (2007) 1580 – 1582
1581
Fig. 1. EPR spectra of -irradiated dental enamel powders at different microwave powers.
The annealing duration was 30 min at each selected temperature. The annealing temperature was controlled by a thermocouple that provided temperature measurement accuracy within ±1 ◦ C. The enamel powder annealed at 240 ◦ C was chosen in order to define the optimum microwave power providing the distinct observation of the isotropic CO− 2 EPR signal. The EPR spectra of this powder in the range of 0.02.20 mW microwave power are shown on Fig. 1. The EPR of the isotropic CO− 2 radical is indicated by arrow in the Fig. 1. It is clear that this signal does not saturate and the growth of the signal intensity with increasing microwave power is observed. Further increase of microwave power results in the distortion of the main radiationinduced EPR signal that hampers the spectrum analysis. Therefore, we used microwave power 20 mW to study the isotropic CO− 2 signal. Fig. 2 shows the EPR spectra of dental enamel powders annealed at different temperatures and recorded at the microwave power mentioned above. The annealing-induced changes in the peak-to-peak intensity of the isotropic CO− 2 signal are presented in Fig. 3. The linewidth of the isotropic EPR signal does not change under annealing. Thus, the dependence in Fig. 3 corresponds to the changes of isotropic CO− 2 radical amount. The EPR spectra recorded at other microwave powers (both lower and higher) support strongly the results presented. Figs. 2–3 show unambiguously that there is increasing of the amount of the CO− 2 radicals responsible for isotropic EPR signal at g ∼ 2.0006 with annealing up to 240 ◦ C in contrast to the data presented in literature (see, for example, Brik et al., 1997). As already mentioned, according to the model proposed for synthetic apatites (Callens, 1997), the isotropic CO− 2 radicals are located in the occluded water. However, it is well known (see, for example, LeGeros et al., 1978), that such water com-
Fig. 2. EPR spectra of dental enamel powders annealed at different temperatures. Spectra are recorded at the microwave power 20 mW.
Fig. 3. Intensity of the isotropic EPR spectra of CO− 2 radicals vs. annealing temperature.
pletely escapes from bioapatites under the annealing below 200 ◦ C. If the isotropic CO− 2 radicals are related to the occluded water their amount should decrease with the evaporation of water from the powder of enamel. Therefore, the corresponding EPR signal should disappear at Tann = 200 ◦ C. The results of the present study show the opposite, namely, the increasing amount of radicals under the annealing up to Tann = 240 ◦ C. As to our opinion this is a serious argument against such model.
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V.V. Rudko et al. / Radiation Measurements 42 (2007) 1580 – 1582
Let us now consider other possible models of these radicals location in biological apatite. 1. Surface radical: It is known that CO− 2 radical has C2V symmetry and causes orthorhombic EPR spectrum. The increase of the radical symmetry may be caused by its fast rotation. For example, it is the rotation around O–O axis that causes axial symmetry of CO− 2 radical in apatites. In order to obtain isotropic EPR spectrum, free rotation of radical is necessary (similar behavior of radicals is observed in liquids). But the model of surface radical implies that the radical is bound to some surface atom(s). Obviously, the presence of such bound limits the degrees of radical freedom and eliminates possibility of its free rotation. 2. Radical in organic: It is well known that annealing of irradiated enamel leads to the formation of some radicals. The appearance of these radicals results from the decomposition of complicated biological molecules of organic enamel components. “Valine” is the example of such radical. “Valine” EPR signal is absent in the initial irradiated enamel (see, for example, Vorona et al., 2005). With the temperature of annealing up to 200 ◦ C the amount of “valine” increased and then decreased. The isotropic CO− 2 radical has similar characteristic tendency; the only difference is that the concentration of CO− 2 radicals achieves maximum at higher temperature (240 ◦ C) (see Fig. 3). Thus, as to the kinetics of formation and annealing the isotropic CO− 2 is similar to the radicals that appear in the process of disintegration of organic enamel component. However, the isotropic CO− 2 radicals were observed in the EPR spectra of both unannealed and annealed enamel. In addition, EPR signal from isotropic CO− 2 radicals was observed in synthetic apatites (Callens et al., 1989) where there is no organic component at all. Therefore, we assumed that the “organic” origin of this radical is improbable. 3. Bulk radical: The annealing dependence of the isotropic CO− 2 radicals amount (Fig. 3) is very similar to the ones of the axial CO− 2 radicals (Vorona et al., 2006). As was assumed (Vorona et al., 2006), the axial CO− 2 radical originates from the orthorhombic one due to the annealing of the defects that prevent its rotation. If the complete destruction of the bond with the lattice of apatite takes place, then the formation of a freely rotating radical which causes the appearance of the isotropic EPR spectra is very probable. Such radical can be located in structural voids of hydroxyapatite lattice in the vicinity of an impurity carbon radical. Increasing of the isotropic EPR signal up to 20 mW microwave power and higher testifies to short times of spin-lattice relaxation of the isotropic CO− 2 radicals
that agree with the model of the freely rotating center. However, free rotation of charged radicals in the voids formed by ions appears to be doubtful. On the other hand, the isotropic EPR spectrum can be caused by a radical that quickly “jumps” from one orientationally fixed structural position into another. To distinguish these two possibilities additional low temperature studies are necessary. Thus, the observed increasing of the amount of the isotropic CO− 2 radicals in enamel under annealing shows that this radical cannot be related to the occluded water, at least in dental enamel. The character of the formation and destruction of this radical under the annealing is very similar to the one of axial − CO− 2 radical. Therefore, we suggest that the isotropic CO2 rad− ical originates from the orthorhombic CO2 radical when the free rotation or quick “jumping” of the latter one is allowed. This isotropic CO− 2 radical is supposed to be located in the structural position B of hydroxyapatites lattice. References 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. Miner. J. 19, 3–12. Callens, F., 1997. Comparative EPR and ENDOR results on carbonate derived radicals in different host materials. Nucleonika 42, 565–578. Callens, F.J., Verbeeck, R.M.H., Naessens, D.E., Matthys, P.F.A., Boesman, E.R., 1989. Effect of carbonate content on the ESR spectrum near g = 2 of carbonated calciumapatites synthesized from aqueous media. Calcif. Tissue Int. 44, 114–124. Callens, F.J., Moens, P., Verbeeck, R., 1995. An EPR study of intact and powdered human tooth enamel dried at 400 ◦ C. Calcif. Tissue Int. 56, 543–548. − Debuyst, R., Dejehet, F., Idrissi, S., 1993. Isotropic CO− 3 and CO2 radicals in irradiated monohydrocalcite. Rad. Prot. Dosim. 47, 659–664. Ishchenko, S., Vorona, I., Okulov, S., 1999. ENDOR study of irradiated tooth enamel. Semiconductor Physics. Quantum Electron. Optoelectron. 2, 84–92. Ishchenko, S.S., Vorona, I.P., Okulov, S.M., Baran, N.P., 2002. 13 C hyperfine interactions of CO− 2 in irradiated tooth enamel as studied by EPR. Appl. Radiat. Isot. 56, 815–819. LeGeros, R.Z., Bonel, G., Legros, R., 1978. Types of “H2 O” in human enamel and in precipitated apatites. Calcif. Tissue Int. 26, 111–118. Tatsumi-Miyajima, J., 1987. ESR dosimetry for atomic bomb survivors and radiologic technologists. Nucl. Instrum. Methods A 257, 417–422. Vorona, I.P., Ishchenko, S.S., Baran, N.P., 2005. The effect of thermal treatment on radiation-induced EPR signals in tooth enamel. Rad. Meas. 39, 137–141. Vorona, I.P., Ishchenko, S.S., Baran, N.P., Petrenko, T.L., Rudko, V.V., 2006. Evidence of annealing-induced transformation of CO− 2 radicals in irradiated tooth enamel. Rad. Meas. 41, 577–581.
Short communication
Isotropic radical CO− 2 in biological apatites V.V. Rudko ∗ , S.S. Ishchenko, I.P. Vorona, N.P. Baran Institute of Semiconductor Physics of National Academy of Sciences of Ukraine, 45, pr. Nauky, Kiev 03028, Ukraine Received 26 December 2006; received in revised form 27 March 2007; accepted 2 April 2007
Abstract ◦ The isotropic CO− 2 EPR spectrum at g ∼ 2.0006 for -irradiated powders of dental enamel annealed at different temperatures up to 320 C is studied. The signal intensity is found to increase with the growth of annealing temperature up to 240 ◦ C. This finding contradicts to the existing model of isotropic CO− 2 radical in apatites. The possible models of the radical in biological apatite are analyzed and discussed. On the basis of the results obtained it is suggested that in tooth enamel apatite the isotropic CO− 2 radical is the bulk radical localized in structural voids of hydroxyapatite lattice, which occur in the vicinity of a carbon radical in position B. © 2007 Elsevier Ltd. All rights reserved.
The radiation-induced EPR signal in biological and synthetic carbonate-containing apatites is mainly caused by the CO− 2 radicals (Callens, 1997). Three types of such radicals are identified in apatites at present. They are responsible for different EPR spectra—orthorhombic (gx = 2.0030, gy = 2.0015, gz = 1.9970), axial (g = 1.9970, g⊥ = 2.0027), and isotropic (g = 2.0006) ones. Axial CO− 2 radical has been studied in more detail. ENDOR measurements of this radical in dental enamel (Ishchenko et al., 1999) showed that the axial CO− 2 occupies B position in hydroxyapatite lattice (it substitutes phosphoric tetrahedron PO3− 4 ). Axial symmetry of the paramagnetic center is explained by fast rotation of CO− 2 radical around the axis connecting two oxygen atoms which is parallel to the crystallographic c axis of the apatite (Ishchenko et al., 2002). The orthorhombic radical was suggested to be located on the surface of apatite crystallites (Callens et al., 1995) or in the organic component of bioapatite (Brik et al., 1997). However, recent studies of annealing-induced changes in the EPR spectra for irradiated enamel plates (Vorona et al., 2006) showed transformation of orthorhombic radicals into axial ones. This finding suggests that both types of CO− 2 radicals are similarly localized in the apatite structure. The isotropic EPR signal (paramagnetic centers related to this signal will be hereafter named isotropic
∗ Corresponding author. Fax: +380 044 265 8342.
E-mail address: [email protected] (V.V. Rudko). 1350-4487/$ - see front matter © 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.radmeas.2007.04.003
CO− 2 radicals) was observed in both synthetic (Callens et al., 1989) and biological (Brik et al., 1997) apatites, and also in natural carbonates (Debuyst et al., 1993). It was assumed that in synthetic apatite the isotropic CO− 2 is localized in the occluded (absorbed) water (Callens, 1997). To the best of our knowledge there is no detailed information about isotropic CO− 2 radical in biological apatites. The present study deals with the study of this radical in tooth enamel and analysis of the possible models of isotropic CO− 2 center in bioapatite. We studied the dental enamel which is the typical bioapatite. Powders of enamel with the grain size of about 100.300 m were prepared from sound human teeth according to the method which is usually used in EPR dosimetry (see, for example, Tatsumi-Miyajima, 1987). The samples were irradiated by 60 Co -rays. Exposure rate was 2.58×10−2 C kg−1 s−1 (100 R/s). Absorbed dose was not determined accurately; it was estimated to be approximately a few kGy. Before the measurements the samples were kept several months at room temperature in order to ensure the destruction of short-living radiation-induced radicals. EPR measurements were carried out using an X-band EPR spectrometer at room temperature. The 100 kHz modulation of the magnetic field with 0.05 mT amplitude was used. The error of the magnetic field measurements did not exceed 0.01 mT. The EPR spectra of dental enamel were registered together with the spectrum of reference sample MgO: Cr 3+ . The enamel powders were annealed in muffle furnace in air at different temperatures up to 320 ◦ C.
V.V. Rudko et al. / Radiation Measurements 42 (2007) 1580 – 1582
1581
Fig. 1. EPR spectra of -irradiated dental enamel powders at different microwave powers.
The annealing duration was 30 min at each selected temperature. The annealing temperature was controlled by a thermocouple that provided temperature measurement accuracy within ±1 ◦ C. The enamel powder annealed at 240 ◦ C was chosen in order to define the optimum microwave power providing the distinct observation of the isotropic CO− 2 EPR signal. The EPR spectra of this powder in the range of 0.02.20 mW microwave power are shown on Fig. 1. The EPR of the isotropic CO− 2 radical is indicated by arrow in the Fig. 1. It is clear that this signal does not saturate and the growth of the signal intensity with increasing microwave power is observed. Further increase of microwave power results in the distortion of the main radiationinduced EPR signal that hampers the spectrum analysis. Therefore, we used microwave power 20 mW to study the isotropic CO− 2 signal. Fig. 2 shows the EPR spectra of dental enamel powders annealed at different temperatures and recorded at the microwave power mentioned above. The annealing-induced changes in the peak-to-peak intensity of the isotropic CO− 2 signal are presented in Fig. 3. The linewidth of the isotropic EPR signal does not change under annealing. Thus, the dependence in Fig. 3 corresponds to the changes of isotropic CO− 2 radical amount. The EPR spectra recorded at other microwave powers (both lower and higher) support strongly the results presented. Figs. 2–3 show unambiguously that there is increasing of the amount of the CO− 2 radicals responsible for isotropic EPR signal at g ∼ 2.0006 with annealing up to 240 ◦ C in contrast to the data presented in literature (see, for example, Brik et al., 1997). As already mentioned, according to the model proposed for synthetic apatites (Callens, 1997), the isotropic CO− 2 radicals are located in the occluded water. However, it is well known (see, for example, LeGeros et al., 1978), that such water com-
Fig. 2. EPR spectra of dental enamel powders annealed at different temperatures. Spectra are recorded at the microwave power 20 mW.
Fig. 3. Intensity of the isotropic EPR spectra of CO− 2 radicals vs. annealing temperature.
pletely escapes from bioapatites under the annealing below 200 ◦ C. If the isotropic CO− 2 radicals are related to the occluded water their amount should decrease with the evaporation of water from the powder of enamel. Therefore, the corresponding EPR signal should disappear at Tann = 200 ◦ C. The results of the present study show the opposite, namely, the increasing amount of radicals under the annealing up to Tann = 240 ◦ C. As to our opinion this is a serious argument against such model.
1582
V.V. Rudko et al. / Radiation Measurements 42 (2007) 1580 – 1582
Let us now consider other possible models of these radicals location in biological apatite. 1. Surface radical: It is known that CO− 2 radical has C2V symmetry and causes orthorhombic EPR spectrum. The increase of the radical symmetry may be caused by its fast rotation. For example, it is the rotation around O–O axis that causes axial symmetry of CO− 2 radical in apatites. In order to obtain isotropic EPR spectrum, free rotation of radical is necessary (similar behavior of radicals is observed in liquids). But the model of surface radical implies that the radical is bound to some surface atom(s). Obviously, the presence of such bound limits the degrees of radical freedom and eliminates possibility of its free rotation. 2. Radical in organic: It is well known that annealing of irradiated enamel leads to the formation of some radicals. The appearance of these radicals results from the decomposition of complicated biological molecules of organic enamel components. “Valine” is the example of such radical. “Valine” EPR signal is absent in the initial irradiated enamel (see, for example, Vorona et al., 2005). With the temperature of annealing up to 200 ◦ C the amount of “valine” increased and then decreased. The isotropic CO− 2 radical has similar characteristic tendency; the only difference is that the concentration of CO− 2 radicals achieves maximum at higher temperature (240 ◦ C) (see Fig. 3). Thus, as to the kinetics of formation and annealing the isotropic CO− 2 is similar to the radicals that appear in the process of disintegration of organic enamel component. However, the isotropic CO− 2 radicals were observed in the EPR spectra of both unannealed and annealed enamel. In addition, EPR signal from isotropic CO− 2 radicals was observed in synthetic apatites (Callens et al., 1989) where there is no organic component at all. Therefore, we assumed that the “organic” origin of this radical is improbable. 3. Bulk radical: The annealing dependence of the isotropic CO− 2 radicals amount (Fig. 3) is very similar to the ones of the axial CO− 2 radicals (Vorona et al., 2006). As was assumed (Vorona et al., 2006), the axial CO− 2 radical originates from the orthorhombic one due to the annealing of the defects that prevent its rotation. If the complete destruction of the bond with the lattice of apatite takes place, then the formation of a freely rotating radical which causes the appearance of the isotropic EPR spectra is very probable. Such radical can be located in structural voids of hydroxyapatite lattice in the vicinity of an impurity carbon radical. Increasing of the isotropic EPR signal up to 20 mW microwave power and higher testifies to short times of spin-lattice relaxation of the isotropic CO− 2 radicals
that agree with the model of the freely rotating center. However, free rotation of charged radicals in the voids formed by ions appears to be doubtful. On the other hand, the isotropic EPR spectrum can be caused by a radical that quickly “jumps” from one orientationally fixed structural position into another. To distinguish these two possibilities additional low temperature studies are necessary. Thus, the observed increasing of the amount of the isotropic CO− 2 radicals in enamel under annealing shows that this radical cannot be related to the occluded water, at least in dental enamel. The character of the formation and destruction of this radical under the annealing is very similar to the one of axial − CO− 2 radical. Therefore, we suggest that the isotropic CO2 rad− ical originates from the orthorhombic CO2 radical when the free rotation or quick “jumping” of the latter one is allowed. This isotropic CO− 2 radical is supposed to be located in the structural position B of hydroxyapatites lattice. References 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. Miner. J. 19, 3–12. Callens, F., 1997. Comparative EPR and ENDOR results on carbonate derived radicals in different host materials. Nucleonika 42, 565–578. Callens, F.J., Verbeeck, R.M.H., Naessens, D.E., Matthys, P.F.A., Boesman, E.R., 1989. Effect of carbonate content on the ESR spectrum near g = 2 of carbonated calciumapatites synthesized from aqueous media. Calcif. Tissue Int. 44, 114–124. Callens, F.J., Moens, P., Verbeeck, R., 1995. An EPR study of intact and powdered human tooth enamel dried at 400 ◦ C. Calcif. Tissue Int. 56, 543–548. − Debuyst, R., Dejehet, F., Idrissi, S., 1993. Isotropic CO− 3 and CO2 radicals in irradiated monohydrocalcite. Rad. Prot. Dosim. 47, 659–664. Ishchenko, S., Vorona, I., Okulov, S., 1999. ENDOR study of irradiated tooth enamel. Semiconductor Physics. Quantum Electron. Optoelectron. 2, 84–92. Ishchenko, S.S., Vorona, I.P., Okulov, S.M., Baran, N.P., 2002. 13 C hyperfine interactions of CO− 2 in irradiated tooth enamel as studied by EPR. Appl. Radiat. Isot. 56, 815–819. LeGeros, R.Z., Bonel, G., Legros, R., 1978. Types of “H2 O” in human enamel and in precipitated apatites. Calcif. Tissue Int. 26, 111–118. Tatsumi-Miyajima, J., 1987. ESR dosimetry for atomic bomb survivors and radiologic technologists. Nucl. Instrum. Methods A 257, 417–422. Vorona, I.P., Ishchenko, S.S., Baran, N.P., 2005. The effect of thermal treatment on radiation-induced EPR signals in tooth enamel. Rad. Meas. 39, 137–141. Vorona, I.P., Ishchenko, S.S., Baran, N.P., Petrenko, T.L., Rudko, V.V., 2006. Evidence of annealing-induced transformation of CO− 2 radicals in irradiated tooth enamel. Rad. Meas. 41, 577–581.