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dosimetric properties of some substituted alanines
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Radiation Physics and Chemistry 75 (2006) 329–335 www.elsevier.com/locate/radphyschem
Study on the EPR/dosimetric properties of some substituted alanines Veselka Ganchevaa, Einar Sagstuenb, Nicola D. Yordanova, a
Laboratory EPR, Institute of Catalysis, Bulgarian Academy of Sciences, BG-1113 Sofia, Bulgaria b Department of Physics, University of Oslo, P.O. Box 1048 Blindern, NO-0316 Oslo, Norway Received 20 July 2004; accepted 12 March 2005
Abstract Polycrystalline phenyl-alanine and perdeuterated L-a-alanine (L-a-alanine-d4) were studied as potential high-energy radiation-sensitive materials (RSM) for solid state/EPR dosimetry. It was found that phenyl-alanine exhibits a linear dose response in the dose region 0.1–17 kGy. However, phenyl-alanine is about 10 times less sensitive to g-irradiation than standard L-a-alanine irradiated at the same doses. Moreover, the EPR response from phenyl-alanine is unstable and, independent of the absorbed dose, decreases by about 50% within 20 days after irradiation upon storage at room temperature. g-irradiated polycrystalline perdeuterated L-a-alanine (CD3CD(NH2)COOH) has not previously been studied at room temperature by EPR spectroscopy. The first part of the present analysis was with respect to the structure of the EPR spectrum. By spectrum simulations, the presence of at least two radiation induced free radicals, R1 ¼ CH3C(H)COOH and R2 ¼ H3N+C(CH3)COO, was confirmed very clearly. Both these radicals were suggested previously from EPR and ENDOR studies of standard alanine crystals. The further investigations into the potential use of alanine-d4 as RSM, after choosing optimal EPR spectrometer settings parameters for this purpose, show that it is ca. two times more sensitive than standard L-a-alanine. r 2005 Elsevier Ltd. All rights reserved. Keywords: Phenyl-alanine; Deuterated L-alanine; g-irradiation; EPR dosimetry
1. Introduction The amino acid L-a-alanine was first suggested for dosimetric purposes more than 40 years ago (Bradshaw et al., 1962). Detailed investigations by many authors in particular during the last two decades (for a recent review, see Regulla, 2000) led to the adoption of alanine as a secondary solid state/EPR (SS/EPR) standard for high dose (0.1–100 kGy) and transfer dosimetry by the IAEA
(Nette et al., 1993; Mehta, 1998; Mehta and Girzikowski, 1996, 1999). This dose region is usually most used for industrial applications. Attempts to use alanine as a radiation-sensitive material (RSM) for SS/EPR dosimetry in the low-dose (0.1–1.0 Gy) region, the dose region being of clinical interest, suffers difficulties due to lack of sensitivity. The problem of sensitivity is a general one for any RSM and can be traced back to two main features:
the magnitude of its ‘‘radiation chemical yield’’, the Corresponding author. Tel.: +359 2 724 917/979 2546;
fax: +359 2 971 2967. E-mail addresses: [email protected], [email protected] (N.D. Yordanov).
0969-806X/$ - see front matter r 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.radphyschem.2005.03.009
number of stable, and thus EPR recordable, free radicals created in 1 mol of the irradiated material per joule absorbed energy; the actual structure of the EPR spectrum.
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Both parameters are specific for the RSM itself and whereas the first one is not straightforward to modify, the second one is connected with the appearance of its EPR spectrum and may be modified by relatively simple means. The EPR response of a given RSM is properly represented by the area under the absorption curve of its spectrum. In EPR spectroscopy, however, in order to increase the sensitivity and spectral resolution, the first derivative of the absorption curve is recorded. Integration of the recorded spectrum is generally unwarranted as it often is connected with significant uncertainties due to base-line effects. Rather, the intensity of some lines of the first-derivative EPR spectrum of the irradiated RSM are used. It is worth noting that the EPR spectrum of alanine occupies a relatively wide region of ca. 12 mT and contains five lines separated by about 2.5 mT and a relative intensity distribution close to 1:4:6:4:1. For dosimetric purposes, only the central line of its EPR spectrum is used. Thus, only 6/16 parts of the spectrum are used and the loss of sensitivity is obvious. These considerations justify the investigations of many laboratories performed in the last decade searching for new RSMs with better spectral and radiation properties than alanine. These studies, aimed at increasing the sensitivity of SS/EPR dosimetry at low doses, are focussed mainly on the two features above, materials with higher or comparable ‘‘radiation chemical yield’’ and with narrower or simpler EPR spectra than those of alanine. Recently, very promising studies on lactates (Hassan and Ikeya, 1997; Hassan et al., 1998), alkaline-earth dithionates (Bogoushevich et al., 1996; Bogoushevich and Ugolev, 2000; Lund et al., 2002), ammonium tartrate (Olsson et al., 1999, 2000; Yordanov and Gancheva, 2004) and formates (Lund et al., 2002; Vestad et al., 2003) have been published. In the present communication, the results obtained on testing two substances as RSMs are reported. One is L-2amino-3-phenylpropanoic acid (phenyl-alanine). It was chosen because it was recently reported (Olson et al., 2002) that 2-methyl-alanine have higher sensitivity than alanine itself and on the other hand it is widely used separately or in some foodstuffs as a sweetener, substituting for sugar (and hence a possible candidate for retrospectrive dosimetry). Also, the impact on the radiation sensitivity by changing of alanine substituents was of interest. The emphasis is given on the effect of the g-radiation on the EPR response and the time stability of the radiation-induced free radicals, both as compared to standard alanine. The second part of the results is devoted to the dosimetric characteristics of specifically deuterated L-a-alanine-d4 (CD3CD(NH2)COOH). These studies were proposed by the large difference between the isotropic hyperfine splittings of 1H and 2D leading to the expectation of a narrower and simpler EPR
spectrum from perdeuterated alanine than that of the commonly used L-alanine which would increase the radiation sensitivity. To our knowledge, the EPR spectrum of g-irradiated alanine-d4 has not previously been reported. Hence, the spectrum was studied in more detail. The influences of the EPR spectrometer settings on the signal intensity of alanine-d4 in comparison to that of L-a-alanine are also presented. The time stability of the radiation-induced free radicals in L-a-alanine-d4 as compared to standard alanine was not investigated in the present work.
2. Experimental 2.1. Starting materials L-Phenyl-alanine was purchased from Sigma and paraffin from Merck. Deuterated L-a-alanine (CD3CDNH2COOH) and standard L-a-alanine were both obtained from Aldrich. All investigated substances were used without further purification. The EPR reference substance, Mn2+ magnetically diluted in magnesium oxide in a ratio of 1:500 (w/w), was prepared as previously described (Yordanov et al., 1999).
2.2. Preparation of dosimeters (a) Phenyl-alanine dosimeters Two types of phenyl-alanine dosimeters were prepared: The first was prepared from a homogeneous mixture of 60% (w/w) phenyl-alanine previously ground to a fine powder and 40% (w/w) paraffin used as a binder. The second type of dosimeters was prepared from a homogeneous mixture of phenyl-alanine (60% w/w), Mn2+ magnetically diluted in MgO (5% w/w) as an internal EPR active but radiation-insensitive material and paraffin (35% w/w). (b) L-a-alanine and L-a-alanine-d4 dosimeters L-a-alanine and L-a-alanine-d4 were ground to fine powder and each homogeneously mixed with paraffin in a 60/40% w/w ratio. The dosimeters containing the described RSM were prepared by extruding the above mixtures in the form of cylinders with 3 mm diameter and 10 mm length. 2.3. Irradiation All samples were irradiated using g-rays from 60Co source at a dose rate of 300 Gy/h. The irradiation was performed in air, at room temperature. Subsequent to irradiation, all samples were kept in plastic bags at room temperature and in the dark.
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The EPR spectra were recorded at room temperature using an X-band ER 200D SRC Bruker spectrometer equipped with a standard (ER 4102ST) TE102 rectangular cavity. In order to avoid the influence of the size and shape of the dosimeter containers (Yordanov and Slavov, 1996) on the EPR response, the dosimeters were accommodated in one and the same quartz EPR sample tube in all measurements. Each dosimeter was fixed in the center of the cavity by using a paraffin sample support shifting it up from the bottom of the sample tube together with a factory supplied teflon sample tube support.
EPR signal intensity, AU
1000 - alanine - phenylalanine
800
600 400
200
0 0
5
10 15 Absorbed dose, kGy
20
Fig. 2. Dose response of phenyl-alanine and alanine measured at equal spectrometer settings (1 mW microwave power and 0.5 mT modulation amplitude).
3. Results and discussion 3.1. Phenyl-alanine The main and most intense part of the g-irradiated phenyl-alanine EPR spectrum has a spectral width of about 7.5 mT and partly unresolved hyperfine splitting, as shown in Fig. 1. This spectrum was recorded at a modulation amplitude of 4 G and microwave power of 1 mW. The dose response dependence of g-irradiated phenyl-alanine in the dose range 0.1–17 kGy compared to the response of standard alanine dosimeters is presented in Fig. 2. The data in Fig. 2 show that the phenyl-alanine dosimeters are far less sensitive than alanine dosimeters recorded using the same EPR settings. The EPR signal stability of the radiation-induced free radicals in g-irradiated phenyl-alanine with time after irradiation was also studied. This long-term study was performed using dosimeters prepared from a homogeneous mixture of the phenyl-alanine, Mn2+ magnetically diluted in MgO and paraffin. Our previous studies (Yordanov et al., 1999) on Mn2+/MgO have shown that
its EPR spectral parameters are insensitive to highenergy irradiation. For this reason Mn2+ in MgO can be used as an internal, with respect to the dosimeter, reference substance. The stability of the radiationinduced free radicals in phenyl-alanine was hence monitored by the changes in the ratio between the EPR signal intensity of phenyl-alanine and that of Mn2+ (using the (counting from the low-field side) third line of the six-line manganese spectrum) with time after irradiation. In this way, significant increase of the accuracy of the reading is achieved (Yordanov and Gancheva, 1999a, b, 2000). Dosimeters irradiated to absorbed doses of 5.5, 11 and 22 kGy were investigated. The EPR spectrum of each dosimeter was recorded several times in the period of 55 days after irradiation at the same spectrometer settings (microwave power 1 mW and modulation amplitude 0.5 G). The results are presented in Fig. 3. A 50% decay during the first 20 days after irradiation of the dosimeters was observed. In addition, the results presented in Fig. 3 also show that the fading is independent of the absorbed dose in the dose range investigated. The reported results and specifically the low sensitivity of phenyl-alanine show that this material is less suitable as a RSM. Thus, further studies were not pursued.
I
3.2. Deuterated alanine
2.0 mT
Fig. 1. EPR spectrum of irradiated phenyl-alanine dosimeter.
3.2.1. Features of the EPR spectrum of alanine-d4 As commented above, the well-known powder EPR spectrum of irradiated L-alanine consists of five lines with line-width DHpp0.6 mT and intensity ratio close to 1:4:6:4:1. It was of interest to see which are the changes in the EPR powder spectrum of irradiated alanine when the methyl protons and the proton from a-carbon are exchanged with deuterium. The working
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Int. phenylalanine/Int. Mn2+, %
100 22.0 kGy 11.0 kGy 5.5 kGy
90 80
I
70
*
60
I*
50 40 30
2.0 mT
20 0
10
20 30 40 Time after irradiation, days
50
60
Fig. 3. Signal stability of the EPR spectrum of phenyl-alanine with time after g-irradiation with 5.5, 11.0 and 22.0 kGy.
Fig. 4. Typical EPR spectrum of g-irradiated deuterated alanine.
L-
hypothesis was based on the large difference in the isotropic hyperfine coupling constants, aD =aH ¼ gD =gH ¼ 0:15. It was therefore expected that the proton hyperfine splitting constant of ca. 2.5 mT would be reduced to about 0.38 mT and if the line-widths remain constant, all lines would then collapse into one single line. In this way the spectral sensitivity should be expected to increase considerably. The experimentally recorded EPR spectrum of girradiated L-a-alanine-d4 using a microwave power 1.0 mW and a modulation amplitude of 0.05 mT is presented in Fig. 4. The total width of the EPR spectrum is reduced to about 9.0 mT. However, probably due to reduced anisotropic line broadening and also weak interactions with protons (deuterons) in neighboring molecules, the width of the EPR lines is reduced to about 0.27 mT leading to a well-resolved central part of the spectrum. On the other hand, the shape of the spectrum suggest that it is not due to one single radical, but is an overlapping spectrum of resonances from at least two different species—one resonance being broad and partly unresolved and the other one, in the central part, more narrow and well resolved. Thus, further studies on the structure of this spectrum were necessary. For many years, the observed quintet EPR spectrum of g-irradiated standard L-a-alanine was attributed to proton interactions in one radical species [R1: CH3C(H)COOH] (Miyagawa and Gordy, 1960; Miyagawa et al., 1969; Friday and Miyagawa, 1971; Shields et al., 1973; Samskog et al., 1980). Many authors (see e.g. Miyagawa and Itoh, 1962; Kuroda and Miyagawa, 1982; Ho¨fer et al., 1989; Wieser et al., 1993; Lion et al., 1983; Desrosiers et al., 1995), however, have demonstrated that EPR spectroscopy of room-temperature irradiated alanine provide evidence for more than one radical species. Following these earlier investigations, EPR, ENDOR and EIE investigations of room-temperature irradiated single crystal and powder
samples of L-a-alanine permitted the detection and partial identification of three different radicals (Sagstuen et al., 1997a, b; Heydari et al., 2002). In addition to radical R1: CH3C(H)COOH, a radical R2 with structure H3N+–C(CH3)COO was shown to be present in an amount comparable to that of R1. For the third (minority) radical R3, the structure H2N–C(CH3)COOH was tentatively suggested (Sagstuen et al., 1997a). g- and hyperfine coupling tensors for R1 and R2, and a set of plausible spectral parameters for R3 have been published (Sagstuen et al., 1997a; Heydari et al., 2002). 3.2.2. Reconstruction of the EPR spectrum of g-irradiated alanine-d4 Having in mind the composite character of the EPR spectrum of g-irradiated L-a-alanine, previously established using EPR and ENDOR spectroscopy, an attempt to simulate the spectrum of g-irradiated alanined4 was performed. The previously published parameters for the R1 and R2 radicals (Sagstuen et al., 1997a; Heydari et al., 2002), as well as the differences in the hyperfine splitting constants between 1H and 2D were used for the computerized spectrum reconstruction. The simulated EPR spectra of the radicals R1 and R2 formed in g-irradiated polycrystalline samples are shown in the upper part of Fig. 5. Each of these two spectra is normalized to the same area. For R1 an isotropic linewidth of 0.27 mT and for R2 an isotropic line-width of 0.22 mT was used in the simulations. In the lower part of Fig. 5, a reconstructed EPR spectrum of g-irradiated alanine-d4 obtained by mixing 60% of the R1 and 40% of the R2 simulated individual EPR spectra is presented. This ratio of R1 and R2 is close to the best-fitting ratio under the given assumptions, and agree well with previous results published (Sagstuen et al., 1997a, b; Heydari et al., 2002) about the composite character of the EPR spectrum of g-irradiated L-a-alanine. Minor
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R1 R2
Experimental
Reconstructed R1: R2 = 0.6:0.4
345
350
355
Magnetic Field (mT) Fig. 5. Simulated EPR spectrum of g-irradiated deuterated Lalanine. In the upper part, two single EPR spectra of free radicals marked R1 and R2 (see text) are shown. In the bottom, a superposition of R1 and R2 in the ratio 60/40 represents the reconstruction of the experimentally recorded EPR spectrum.
discrepancies between the simulated and experimental spectrum may be attributed to the use of isotropic linewidths and that radical R3 is not included in the reconstruction. The reason for this is the uncertainty in spectral parameters for this radical species, in particular for those couplings that are not exchangeable upon specific deuteration. 3.2.3. Influence of some EPR instrument setting parameters on the EPR spectrum of g-irradiated alanine-d4 For quantitative EPR measurements, the magnitudes of the microwave power and the modulation amplitude are as a rule kept in the linear region of the relationship ‘‘signal intensity/value of the instrumental parameter’’. However, it is worth noticing that especially for dose estimations in the radiotherapy range a compromise often must be made in using higher values for these instrumental parameters so as to obtain EPR response
to be as high as possible. Thus, the influences of the applied microwave power and modulation amplitude on the EPR spectrum shape and intensity of deuterated alanine have been investigated and compared to those of standard L-a-alanine. The influence of the microwave power on the signal intensity of both the overlapping deuterated alanine spectra, the one marked with I (Radical R1) and the one due to R2, characterized at the asterisk (I*) in Fig. 4, are presented in Fig. 6 together with corresponding data obtained using the standard L-a-alanine (in these studies the modulation amplitude was kept constant at 0.05 mT). Due to the narrow EPR line-width of deuterated R1 alanine radical, its signal intensity saturates at lower microwave power (9 mW) than that of irradiated standard L-alanine (16 mW). The difference in the saturation behavior at I and I* in the deuterated alanine spectrum confirm independently the composite character of its EPR spectrum and the presence of more than one free radical species. Fig. 7 shows the changes of the EPR spectrum shape of alanine-d4 at different modulation amplitudes (in these spectra the EPR microwave power was kept at 1 mW). At values of the modulation amplitudes higher than 0.2 mT, broadening of the EPR lines results in reduced spectrum resolution. The influence of the applied modulation amplitude on the signal intensity of g-irradiated normal and perdeutrated L-a-alanine both irradiated to the same dose, are presented in Fig. 8. These measurements are made with microwave power incident to the EPR cavity of 4 mW. Fig. 8 shows that up to about 0.5 mT modulation width the changes in the EPR signal intensities of both substances are similar. At higher modulations, the EPR spectrum of standard alanine becomes successively more and more overmodulated and above about 1.4 mT the line intensity
L-alanine L-alanine-D4 L-alanine-D4*
2500 EPR signal intensity, AU
Simulated
333
2000 1500 1000 500 0 0
2
4
8 6 (P, mW)1/2
10
12
Fig. 6. Dependence of the signal intensities of irradiated Lalanine-d4 and standard alanine as a function of the square root of the applied microwave power. The modulation amplitude is 0.05 mT.
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4. Conclusions I
*
0.04 mT
0.16 mT
0.40 mT
0.64 mT
0.80 mT
2.0 mT
Fig. 7. Dependence of the EPR spectrum shape of g-irradiated deuterated L-alanine-d4 on the magnitude of the modulation amplitude. Microwave power is 1 mW.
The present paper reports features of phenyl-alanine and alanine-d4 EPR spectra relevant for their potential use as RSM for solid state/EPR dosimetry. The results show unambiguously that phenyl-alanine is unsuitable as RSM due to low sensitivity and unfavorable fading characteristics. For the first time, the EPR spectrum of g-irradiated deuterated L-alanine-d4 is reported. This EPR resonance exhibits a structure suggesting a composite nature of the spectrum. Computer reconstruction of this spectrum clearly confirms the previously published experimental EPR/ENDOR results with regard to the two major radical species formed in crystalline L-a-alanine upon irradiation. Recording the EPR spectrum of deuterated alanine at high modulation amplitudes (about 2.5–3.0 mT) gives a twofold increase in the signal intensity as compared to that of normal alanine recorded at modulation amplitudes of 0.6–0.8 mT. This doubling in EPR response may make the use of perdeutrated alanine as dosimetric material for low absorbed doses attractive but, on the other hand, the high price of this material must also be considered. A cost-benefit analysis should also take into account the non-destructive sample readout feature of EPR spectrometry and that since absorbed doses by SS/EPR dosimeters are accumulated, one dosimeter may be used several times.
References
EPR signal intensity, AU
6000 L-alanine-d4 L-alanine
5000 4000 3000 2000 1000 0 0.0
0.5
1.0 1.5 2.0 2.5 3.0 Modulation amplitude, mT
3.5
Fig. 8. Dependence of the signal intensities of irradiated Lalanine-d4 and standard alanine as a function of the modulation amplitude. The applied microwave power is kept constant at 1 mW.
decreases. At the same time, the signal intensity of deuterated alanine still increases with the modulation amplitude. Thus, at these EPR spectrometer settings, alanine-d4 has ca. two times higher sensitivity.
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ARTICLE IN PRESS V. Gancheva et al. / Radiation Physics and Chemistry 75 (2006) 329–335 Ho¨fer, P., Holczer, K., Schmalbein, D., 1989. Characterization of gamma-ray irradiated powder alanine samples by pulsed EPR. Appl. Radiat. Isot. 40, 1233–1235. Kuroda, S.-i., Miyagawa, I., 1982. Endor study of an irradiated crystal of L-alanine: environment of the stable CH3CHCO 2 radical. J. Chem. Phys. 76, 3933–3944. Lion, Y., Denis, G., Mossoba, M.M., Riesz, P., 1983. E.s.r. of spin-trapped radicals in g-irradiated polycrystalline DLalanine. A quantitative determination of radical yield. Int. J. Radiat. Biol. 43, 71–83. Lund, A., Olsson, S., Bonora, M., Lund, E., Gustafsson, H., 2002. New materials for ESR dosimetry. Spectrochim. Acta A 58, 1301–1311. Mehta, K., 1998. High-dose dosimetry programme of the IAEA. In: Techniques for High Dose Dosimetry in Industry, Agriculture and Medicine, Extended Synopses of Symposium held in Vienna, November 2–5, Paper # IAEASM-356/R2. Mehta, K., Girzikowski, R., 1996. Alanine-ESR dosimetry for radiotherapy. IAEA Experience. Appl. Radiat. Isot. 47, 1189–1192. Mehta, K., Girzikowsky, R., 1999. IAEA reference dosimeter: alanine-ESR. In: Techniques for High Dose Dosimetry in Industry, Agriculture and Medicine, Proceedings of Symposium, IAEA-TECDOC-1070, pp. 299–304. Miyagawa, I., Gordy, W., 1960. Electron spin resonance of an irradiated single crystal of alanine: second order effects in free radical resonances. J. Chem. Phys. 32, 255–263. Miyagawa, I., Itoh, K., 1962. Electron spin resonance of irradiated single crystals of alanines: hindered rotation of the methyl group in a free radical. J. Chem. Phys. 36, 2157–2163. Miyagawa, I., Tamura, N., Cook, J.W., 1969. ESR study of stereospecific proton transfer in irradiated crystals of Lalanine. J. Chem. Phys. 51, 3520–3528. Nette, H.P., Onori, S., Fattibene, P., Regulla, D.F., Wieser, A., 1993. Coordinated research efforts for establishing an international radiotherapy dose intercomparison service based on the alanine/ESR system. Appl. Radiat. Isot. 44, 7–12. Olsson, S.K., Bagherian, S., Lund, E., Carlsson, G.A., Lund, A., 1999. Ammonium tartrate as an ESR dosimeter material. Appl. Radiat. Isot. 50, 955–965. Olsson, S.K., Lund, E., Lund, A., 2000. Development of ammonium tartrate as an ESR dosimeter material for clinical purposes. Appl. Radiat. Isot. 52, 1235–1241. Olson, S., Sagstuen, E., Bonora, M., Lund, A., 2002. EPR dosimetric properties of 2-methylalanine: EPR, ENDOR and FT-EPR investigations. Radiat. Res. 157, 113–121.
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Radiation Physics and Chemistry 75 (2006) 329–335 www.elsevier.com/locate/radphyschem
Study on the EPR/dosimetric properties of some substituted alanines Veselka Ganchevaa, Einar Sagstuenb, Nicola D. Yordanova, a
Laboratory EPR, Institute of Catalysis, Bulgarian Academy of Sciences, BG-1113 Sofia, Bulgaria b Department of Physics, University of Oslo, P.O. Box 1048 Blindern, NO-0316 Oslo, Norway Received 20 July 2004; accepted 12 March 2005
Abstract Polycrystalline phenyl-alanine and perdeuterated L-a-alanine (L-a-alanine-d4) were studied as potential high-energy radiation-sensitive materials (RSM) for solid state/EPR dosimetry. It was found that phenyl-alanine exhibits a linear dose response in the dose region 0.1–17 kGy. However, phenyl-alanine is about 10 times less sensitive to g-irradiation than standard L-a-alanine irradiated at the same doses. Moreover, the EPR response from phenyl-alanine is unstable and, independent of the absorbed dose, decreases by about 50% within 20 days after irradiation upon storage at room temperature. g-irradiated polycrystalline perdeuterated L-a-alanine (CD3CD(NH2)COOH) has not previously been studied at room temperature by EPR spectroscopy. The first part of the present analysis was with respect to the structure of the EPR spectrum. By spectrum simulations, the presence of at least two radiation induced free radicals, R1 ¼ CH3C(H)COOH and R2 ¼ H3N+C(CH3)COO, was confirmed very clearly. Both these radicals were suggested previously from EPR and ENDOR studies of standard alanine crystals. The further investigations into the potential use of alanine-d4 as RSM, after choosing optimal EPR spectrometer settings parameters for this purpose, show that it is ca. two times more sensitive than standard L-a-alanine. r 2005 Elsevier Ltd. All rights reserved. Keywords: Phenyl-alanine; Deuterated L-alanine; g-irradiation; EPR dosimetry
1. Introduction The amino acid L-a-alanine was first suggested for dosimetric purposes more than 40 years ago (Bradshaw et al., 1962). Detailed investigations by many authors in particular during the last two decades (for a recent review, see Regulla, 2000) led to the adoption of alanine as a secondary solid state/EPR (SS/EPR) standard for high dose (0.1–100 kGy) and transfer dosimetry by the IAEA
(Nette et al., 1993; Mehta, 1998; Mehta and Girzikowski, 1996, 1999). This dose region is usually most used for industrial applications. Attempts to use alanine as a radiation-sensitive material (RSM) for SS/EPR dosimetry in the low-dose (0.1–1.0 Gy) region, the dose region being of clinical interest, suffers difficulties due to lack of sensitivity. The problem of sensitivity is a general one for any RSM and can be traced back to two main features:
the magnitude of its ‘‘radiation chemical yield’’, the Corresponding author. Tel.: +359 2 724 917/979 2546;
fax: +359 2 971 2967. E-mail addresses: [email protected], [email protected] (N.D. Yordanov).
0969-806X/$ - see front matter r 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.radphyschem.2005.03.009
number of stable, and thus EPR recordable, free radicals created in 1 mol of the irradiated material per joule absorbed energy; the actual structure of the EPR spectrum.
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Both parameters are specific for the RSM itself and whereas the first one is not straightforward to modify, the second one is connected with the appearance of its EPR spectrum and may be modified by relatively simple means. The EPR response of a given RSM is properly represented by the area under the absorption curve of its spectrum. In EPR spectroscopy, however, in order to increase the sensitivity and spectral resolution, the first derivative of the absorption curve is recorded. Integration of the recorded spectrum is generally unwarranted as it often is connected with significant uncertainties due to base-line effects. Rather, the intensity of some lines of the first-derivative EPR spectrum of the irradiated RSM are used. It is worth noting that the EPR spectrum of alanine occupies a relatively wide region of ca. 12 mT and contains five lines separated by about 2.5 mT and a relative intensity distribution close to 1:4:6:4:1. For dosimetric purposes, only the central line of its EPR spectrum is used. Thus, only 6/16 parts of the spectrum are used and the loss of sensitivity is obvious. These considerations justify the investigations of many laboratories performed in the last decade searching for new RSMs with better spectral and radiation properties than alanine. These studies, aimed at increasing the sensitivity of SS/EPR dosimetry at low doses, are focussed mainly on the two features above, materials with higher or comparable ‘‘radiation chemical yield’’ and with narrower or simpler EPR spectra than those of alanine. Recently, very promising studies on lactates (Hassan and Ikeya, 1997; Hassan et al., 1998), alkaline-earth dithionates (Bogoushevich et al., 1996; Bogoushevich and Ugolev, 2000; Lund et al., 2002), ammonium tartrate (Olsson et al., 1999, 2000; Yordanov and Gancheva, 2004) and formates (Lund et al., 2002; Vestad et al., 2003) have been published. In the present communication, the results obtained on testing two substances as RSMs are reported. One is L-2amino-3-phenylpropanoic acid (phenyl-alanine). It was chosen because it was recently reported (Olson et al., 2002) that 2-methyl-alanine have higher sensitivity than alanine itself and on the other hand it is widely used separately or in some foodstuffs as a sweetener, substituting for sugar (and hence a possible candidate for retrospectrive dosimetry). Also, the impact on the radiation sensitivity by changing of alanine substituents was of interest. The emphasis is given on the effect of the g-radiation on the EPR response and the time stability of the radiation-induced free radicals, both as compared to standard alanine. The second part of the results is devoted to the dosimetric characteristics of specifically deuterated L-a-alanine-d4 (CD3CD(NH2)COOH). These studies were proposed by the large difference between the isotropic hyperfine splittings of 1H and 2D leading to the expectation of a narrower and simpler EPR
spectrum from perdeuterated alanine than that of the commonly used L-alanine which would increase the radiation sensitivity. To our knowledge, the EPR spectrum of g-irradiated alanine-d4 has not previously been reported. Hence, the spectrum was studied in more detail. The influences of the EPR spectrometer settings on the signal intensity of alanine-d4 in comparison to that of L-a-alanine are also presented. The time stability of the radiation-induced free radicals in L-a-alanine-d4 as compared to standard alanine was not investigated in the present work.
2. Experimental 2.1. Starting materials L-Phenyl-alanine was purchased from Sigma and paraffin from Merck. Deuterated L-a-alanine (CD3CDNH2COOH) and standard L-a-alanine were both obtained from Aldrich. All investigated substances were used without further purification. The EPR reference substance, Mn2+ magnetically diluted in magnesium oxide in a ratio of 1:500 (w/w), was prepared as previously described (Yordanov et al., 1999).
2.2. Preparation of dosimeters (a) Phenyl-alanine dosimeters Two types of phenyl-alanine dosimeters were prepared: The first was prepared from a homogeneous mixture of 60% (w/w) phenyl-alanine previously ground to a fine powder and 40% (w/w) paraffin used as a binder. The second type of dosimeters was prepared from a homogeneous mixture of phenyl-alanine (60% w/w), Mn2+ magnetically diluted in MgO (5% w/w) as an internal EPR active but radiation-insensitive material and paraffin (35% w/w). (b) L-a-alanine and L-a-alanine-d4 dosimeters L-a-alanine and L-a-alanine-d4 were ground to fine powder and each homogeneously mixed with paraffin in a 60/40% w/w ratio. The dosimeters containing the described RSM were prepared by extruding the above mixtures in the form of cylinders with 3 mm diameter and 10 mm length. 2.3. Irradiation All samples were irradiated using g-rays from 60Co source at a dose rate of 300 Gy/h. The irradiation was performed in air, at room temperature. Subsequent to irradiation, all samples were kept in plastic bags at room temperature and in the dark.
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The EPR spectra were recorded at room temperature using an X-band ER 200D SRC Bruker spectrometer equipped with a standard (ER 4102ST) TE102 rectangular cavity. In order to avoid the influence of the size and shape of the dosimeter containers (Yordanov and Slavov, 1996) on the EPR response, the dosimeters were accommodated in one and the same quartz EPR sample tube in all measurements. Each dosimeter was fixed in the center of the cavity by using a paraffin sample support shifting it up from the bottom of the sample tube together with a factory supplied teflon sample tube support.
EPR signal intensity, AU
1000 - alanine - phenylalanine
800
600 400
200
0 0
5
10 15 Absorbed dose, kGy
20
Fig. 2. Dose response of phenyl-alanine and alanine measured at equal spectrometer settings (1 mW microwave power and 0.5 mT modulation amplitude).
3. Results and discussion 3.1. Phenyl-alanine The main and most intense part of the g-irradiated phenyl-alanine EPR spectrum has a spectral width of about 7.5 mT and partly unresolved hyperfine splitting, as shown in Fig. 1. This spectrum was recorded at a modulation amplitude of 4 G and microwave power of 1 mW. The dose response dependence of g-irradiated phenyl-alanine in the dose range 0.1–17 kGy compared to the response of standard alanine dosimeters is presented in Fig. 2. The data in Fig. 2 show that the phenyl-alanine dosimeters are far less sensitive than alanine dosimeters recorded using the same EPR settings. The EPR signal stability of the radiation-induced free radicals in g-irradiated phenyl-alanine with time after irradiation was also studied. This long-term study was performed using dosimeters prepared from a homogeneous mixture of the phenyl-alanine, Mn2+ magnetically diluted in MgO and paraffin. Our previous studies (Yordanov et al., 1999) on Mn2+/MgO have shown that
its EPR spectral parameters are insensitive to highenergy irradiation. For this reason Mn2+ in MgO can be used as an internal, with respect to the dosimeter, reference substance. The stability of the radiationinduced free radicals in phenyl-alanine was hence monitored by the changes in the ratio between the EPR signal intensity of phenyl-alanine and that of Mn2+ (using the (counting from the low-field side) third line of the six-line manganese spectrum) with time after irradiation. In this way, significant increase of the accuracy of the reading is achieved (Yordanov and Gancheva, 1999a, b, 2000). Dosimeters irradiated to absorbed doses of 5.5, 11 and 22 kGy were investigated. The EPR spectrum of each dosimeter was recorded several times in the period of 55 days after irradiation at the same spectrometer settings (microwave power 1 mW and modulation amplitude 0.5 G). The results are presented in Fig. 3. A 50% decay during the first 20 days after irradiation of the dosimeters was observed. In addition, the results presented in Fig. 3 also show that the fading is independent of the absorbed dose in the dose range investigated. The reported results and specifically the low sensitivity of phenyl-alanine show that this material is less suitable as a RSM. Thus, further studies were not pursued.
I
3.2. Deuterated alanine
2.0 mT
Fig. 1. EPR spectrum of irradiated phenyl-alanine dosimeter.
3.2.1. Features of the EPR spectrum of alanine-d4 As commented above, the well-known powder EPR spectrum of irradiated L-alanine consists of five lines with line-width DHpp0.6 mT and intensity ratio close to 1:4:6:4:1. It was of interest to see which are the changes in the EPR powder spectrum of irradiated alanine when the methyl protons and the proton from a-carbon are exchanged with deuterium. The working
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Int. phenylalanine/Int. Mn2+, %
100 22.0 kGy 11.0 kGy 5.5 kGy
90 80
I
70
*
60
I*
50 40 30
2.0 mT
20 0
10
20 30 40 Time after irradiation, days
50
60
Fig. 3. Signal stability of the EPR spectrum of phenyl-alanine with time after g-irradiation with 5.5, 11.0 and 22.0 kGy.
Fig. 4. Typical EPR spectrum of g-irradiated deuterated alanine.
L-
hypothesis was based on the large difference in the isotropic hyperfine coupling constants, aD =aH ¼ gD =gH ¼ 0:15. It was therefore expected that the proton hyperfine splitting constant of ca. 2.5 mT would be reduced to about 0.38 mT and if the line-widths remain constant, all lines would then collapse into one single line. In this way the spectral sensitivity should be expected to increase considerably. The experimentally recorded EPR spectrum of girradiated L-a-alanine-d4 using a microwave power 1.0 mW and a modulation amplitude of 0.05 mT is presented in Fig. 4. The total width of the EPR spectrum is reduced to about 9.0 mT. However, probably due to reduced anisotropic line broadening and also weak interactions with protons (deuterons) in neighboring molecules, the width of the EPR lines is reduced to about 0.27 mT leading to a well-resolved central part of the spectrum. On the other hand, the shape of the spectrum suggest that it is not due to one single radical, but is an overlapping spectrum of resonances from at least two different species—one resonance being broad and partly unresolved and the other one, in the central part, more narrow and well resolved. Thus, further studies on the structure of this spectrum were necessary. For many years, the observed quintet EPR spectrum of g-irradiated standard L-a-alanine was attributed to proton interactions in one radical species [R1: CH3C(H)COOH] (Miyagawa and Gordy, 1960; Miyagawa et al., 1969; Friday and Miyagawa, 1971; Shields et al., 1973; Samskog et al., 1980). Many authors (see e.g. Miyagawa and Itoh, 1962; Kuroda and Miyagawa, 1982; Ho¨fer et al., 1989; Wieser et al., 1993; Lion et al., 1983; Desrosiers et al., 1995), however, have demonstrated that EPR spectroscopy of room-temperature irradiated alanine provide evidence for more than one radical species. Following these earlier investigations, EPR, ENDOR and EIE investigations of room-temperature irradiated single crystal and powder
samples of L-a-alanine permitted the detection and partial identification of three different radicals (Sagstuen et al., 1997a, b; Heydari et al., 2002). In addition to radical R1: CH3C(H)COOH, a radical R2 with structure H3N+–C(CH3)COO was shown to be present in an amount comparable to that of R1. For the third (minority) radical R3, the structure H2N–C(CH3)COOH was tentatively suggested (Sagstuen et al., 1997a). g- and hyperfine coupling tensors for R1 and R2, and a set of plausible spectral parameters for R3 have been published (Sagstuen et al., 1997a; Heydari et al., 2002). 3.2.2. Reconstruction of the EPR spectrum of g-irradiated alanine-d4 Having in mind the composite character of the EPR spectrum of g-irradiated L-a-alanine, previously established using EPR and ENDOR spectroscopy, an attempt to simulate the spectrum of g-irradiated alanined4 was performed. The previously published parameters for the R1 and R2 radicals (Sagstuen et al., 1997a; Heydari et al., 2002), as well as the differences in the hyperfine splitting constants between 1H and 2D were used for the computerized spectrum reconstruction. The simulated EPR spectra of the radicals R1 and R2 formed in g-irradiated polycrystalline samples are shown in the upper part of Fig. 5. Each of these two spectra is normalized to the same area. For R1 an isotropic linewidth of 0.27 mT and for R2 an isotropic line-width of 0.22 mT was used in the simulations. In the lower part of Fig. 5, a reconstructed EPR spectrum of g-irradiated alanine-d4 obtained by mixing 60% of the R1 and 40% of the R2 simulated individual EPR spectra is presented. This ratio of R1 and R2 is close to the best-fitting ratio under the given assumptions, and agree well with previous results published (Sagstuen et al., 1997a, b; Heydari et al., 2002) about the composite character of the EPR spectrum of g-irradiated L-a-alanine. Minor
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R1 R2
Experimental
Reconstructed R1: R2 = 0.6:0.4
345
350
355
Magnetic Field (mT) Fig. 5. Simulated EPR spectrum of g-irradiated deuterated Lalanine. In the upper part, two single EPR spectra of free radicals marked R1 and R2 (see text) are shown. In the bottom, a superposition of R1 and R2 in the ratio 60/40 represents the reconstruction of the experimentally recorded EPR spectrum.
discrepancies between the simulated and experimental spectrum may be attributed to the use of isotropic linewidths and that radical R3 is not included in the reconstruction. The reason for this is the uncertainty in spectral parameters for this radical species, in particular for those couplings that are not exchangeable upon specific deuteration. 3.2.3. Influence of some EPR instrument setting parameters on the EPR spectrum of g-irradiated alanine-d4 For quantitative EPR measurements, the magnitudes of the microwave power and the modulation amplitude are as a rule kept in the linear region of the relationship ‘‘signal intensity/value of the instrumental parameter’’. However, it is worth noticing that especially for dose estimations in the radiotherapy range a compromise often must be made in using higher values for these instrumental parameters so as to obtain EPR response
to be as high as possible. Thus, the influences of the applied microwave power and modulation amplitude on the EPR spectrum shape and intensity of deuterated alanine have been investigated and compared to those of standard L-a-alanine. The influence of the microwave power on the signal intensity of both the overlapping deuterated alanine spectra, the one marked with I (Radical R1) and the one due to R2, characterized at the asterisk (I*) in Fig. 4, are presented in Fig. 6 together with corresponding data obtained using the standard L-a-alanine (in these studies the modulation amplitude was kept constant at 0.05 mT). Due to the narrow EPR line-width of deuterated R1 alanine radical, its signal intensity saturates at lower microwave power (9 mW) than that of irradiated standard L-alanine (16 mW). The difference in the saturation behavior at I and I* in the deuterated alanine spectrum confirm independently the composite character of its EPR spectrum and the presence of more than one free radical species. Fig. 7 shows the changes of the EPR spectrum shape of alanine-d4 at different modulation amplitudes (in these spectra the EPR microwave power was kept at 1 mW). At values of the modulation amplitudes higher than 0.2 mT, broadening of the EPR lines results in reduced spectrum resolution. The influence of the applied modulation amplitude on the signal intensity of g-irradiated normal and perdeutrated L-a-alanine both irradiated to the same dose, are presented in Fig. 8. These measurements are made with microwave power incident to the EPR cavity of 4 mW. Fig. 8 shows that up to about 0.5 mT modulation width the changes in the EPR signal intensities of both substances are similar. At higher modulations, the EPR spectrum of standard alanine becomes successively more and more overmodulated and above about 1.4 mT the line intensity
L-alanine L-alanine-D4 L-alanine-D4*
2500 EPR signal intensity, AU
Simulated
333
2000 1500 1000 500 0 0
2
4
8 6 (P, mW)1/2
10
12
Fig. 6. Dependence of the signal intensities of irradiated Lalanine-d4 and standard alanine as a function of the square root of the applied microwave power. The modulation amplitude is 0.05 mT.
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4. Conclusions I
*
0.04 mT
0.16 mT
0.40 mT
0.64 mT
0.80 mT
2.0 mT
Fig. 7. Dependence of the EPR spectrum shape of g-irradiated deuterated L-alanine-d4 on the magnitude of the modulation amplitude. Microwave power is 1 mW.
The present paper reports features of phenyl-alanine and alanine-d4 EPR spectra relevant for their potential use as RSM for solid state/EPR dosimetry. The results show unambiguously that phenyl-alanine is unsuitable as RSM due to low sensitivity and unfavorable fading characteristics. For the first time, the EPR spectrum of g-irradiated deuterated L-alanine-d4 is reported. This EPR resonance exhibits a structure suggesting a composite nature of the spectrum. Computer reconstruction of this spectrum clearly confirms the previously published experimental EPR/ENDOR results with regard to the two major radical species formed in crystalline L-a-alanine upon irradiation. Recording the EPR spectrum of deuterated alanine at high modulation amplitudes (about 2.5–3.0 mT) gives a twofold increase in the signal intensity as compared to that of normal alanine recorded at modulation amplitudes of 0.6–0.8 mT. This doubling in EPR response may make the use of perdeutrated alanine as dosimetric material for low absorbed doses attractive but, on the other hand, the high price of this material must also be considered. A cost-benefit analysis should also take into account the non-destructive sample readout feature of EPR spectrometry and that since absorbed doses by SS/EPR dosimeters are accumulated, one dosimeter may be used several times.
References
EPR signal intensity, AU
6000 L-alanine-d4 L-alanine
5000 4000 3000 2000 1000 0 0.0
0.5
1.0 1.5 2.0 2.5 3.0 Modulation amplitude, mT
3.5
Fig. 8. Dependence of the signal intensities of irradiated Lalanine-d4 and standard alanine as a function of the modulation amplitude. The applied microwave power is kept constant at 1 mW.
decreases. At the same time, the signal intensity of deuterated alanine still increases with the modulation amplitude. Thus, at these EPR spectrometer settings, alanine-d4 has ca. two times higher sensitivity.
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