A neutron activation technique for manganese measurements in humans

Journal of Trace Elements in Medicine and Biology 31 (2015) 204–208

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10th NTES Symposium

A neutron activation technique for manganese measurements in humans C. Bhatia ∗ , S.H. Byun, D.R. Chettle, M.J. Inskip, W.V. Prestwich Department of Medical Physics and Applied Radiation Sciences, McMaster University, Hamilton, ON L8S 4K1, Canada

a r t i c l e

i n f o

Article history: Received 30 January 2014 Accepted 28 July 2014 Keywords: Occupational and industrial exposure Manganese toxicity Accelerator neutron source Non-invasive technique Neutron activation

a b s t r a c t Manganese (Mn) is an essential element for humans, animals, and plants and is required for growth, development, and maintenance of health. Studies show that Mn metabolism is similar to that of iron, therefore, increased Mn levels in humans could interfere with the absorption of dietary iron leading to anemia. Also, excess exposure to Mn dust, leads to nervous system disorders similar to Parkinson’s disease. Higher exposure to Mn is essentially related to industrial pollution. Thus, there is a benefit in developing a clean non-invasive technique for monitoring such increased levels of Mn in order to understand the risk of disease and development of appropriate treatments. To this end, the feasibility of Mn measurements with their minimum detection limits (MDL) has been reported earlier from the McMaster group. This work presents improvement to Mn assessment using an upgraded system and optimized times of irradiation and counting for induced gamma activity of Mn. The technique utilizes the high proton current Tandetron accelerator producing neutrons via the 7 Li(p,n)7 Be reaction at McMaster University and an array of nine NaI (Tl) detectors in a 4␲ geometry for delayed counting of gamma rays. The neutron irradiation of a set of phantoms was performed with protocols having different proton energy, current and time of irradiation. The improved MDLs estimated using the upgraded set up and constrained timings are reported as 0.67 ␮gMn/gCa for 2.3 MeV protons and 0.71 ␮gMn/gCa for 2.0 MeV protons. These are a factor of about 2.3 times better than previous measurements done at McMaster University using the in vivo set-up. Also, because of lower dose-equivalent and a relatively close MDL, the combination of: 2.0 MeV; 300 ␮A; 3 min protocol is recommended as compared to 2.3 MeV; 400 ␮A; 45 s protocol for further measurements of Mn in vivo. © 2014 Elsevier GmbH. All rights reserved.

Introduction Manganese is a trace element that is an essential nutrient for normal development and body function across the life span of all living organisms [1]. In humans, the diet is the main source of Mn, with an adequate intake of 2–4 mg/day and an upper tolerable intake of 11 mg/day for total Mn intake [2]. The major storage of Mn is in the bones (about 50%) and its excretion is through the liver [3]. A healthy person with normal liver and kidney function can excrete excess dietary manganese. Inhaled Mn is of greater concern because it can bypass the body’s normal defense mechanisms; i.e. it is often transported directly to the brain via olfactory nerve [4] before it is metabolized by the liver. Also, inhalation exposure to high concentrations of manganese dusts can cause an inflammatory

∗ Corresponding author. Tel.: +1 (905) 525 9140x26328. E-mail addresses: [email protected], [email protected] (C. Bhatia). http://dx.doi.org/10.1016/j.jtemb.2014.07.018 0946-672X/© 2014 Elsevier GmbH. All rights reserved.

response in the lung, which, over time, can result in impaired lung function. Manganese is one of the most widely used metals in industry [5]. Exposure to Mn dust occurs primarily in mining, and metallurgical operations for iron, steel, ferrous and nonferrous alloys. Manufacturing of dry-cell batteries, anti-knock gasoline additives, pesticides, pigments for ceramics, dyes, and matches can also lead to occupational Mn exposure. Manganese fumes are produced during metallurgical operations and several types of welding operations. The principal sources of Mn in the atmosphere are however, from natural processes including volcanic gas and dust, and forest fires [6]. Exposure to Mn is usually via inhalation, which can lead to Mn accumulation and results in adverse health effects including damage to the lungs, liver, kidney and central nervous system [7,8]. Mn toxicity has been reported through occupational (e.g. welder, miner) and dietary over exposure. Prolonged exposure to high Mn concentrations (>1 mg/m3 ) in air may lead to a Parkinsonian syndrome known as “manganism” [9–12]. Numerous studies have

C. Bhatia et al. / Journal of Trace Elements in Medicine and Biology 31 (2015) 204–208

indicated that welders may be at high risk of Parkinson’s disease and neurological health effects [9–12]. The majority of the available scientific literature on Mn exposure, body burden and its health effects comes from animal studies and invasive methods like biopsies and autopsies. Toxic elements like Mn are commonly measured in blood and serum, or by a bone biopsy. It is widely accepted that blood and serum concentrations of elements provide information only about the recent exposure to the element, and therefore do not necessarily reflect chronic exposure of an individual. On the other hand, bone biopsy is painful, involves a risk to the patient and it may not be possible to repeat it several times. The assessment of lifetime exposure from the general environment, or the work place, to a toxic element therefore requires a different approach. Because toxic elements like Mn are well retained by bone tissue and may reside there for years to decades, a non-invasive, non-destructive and without-pain neutron activation based diagnostic technique is being developed and used to assess Mn. Taking into account the large accumulation of Mn in bone, an X-ray fluorescence spectroscopy or neutron-based spectroscopy methods are being considered as novel non-invasive tool for assessing Mn exposure and toxicity [13]. However, low energy of Mn X-rays, together with their relatively low fluorescence yield make neutron activation analysis (NAA) the more promising approach. This non-invasive in vivo technique can (i) measure Mn amounts in humans, (ii) contribute to limited human data, regarding current knowledge of Mn metabolism and (iii) possibly be helpful in early detection of accumulation resulting from over-exposure in industrial and environmentally exposed populations. Of many different techniques applied e.g. atomic absorption spectrometry and inductively coupled plasma atomic emission spectrometry or mass spectrometry to the determination of total Mn content, NAA is a prime example of a non-invasive analytical technique. This method is extremely valuable because of its high specificity and sensitivity for very low concentrations of Mn, as well as several other elements. In contrast to other commonly used techniques, in the case of NAA the sample is not permanently damaged and can be saved for further analysis. The technique is also useful to benchmark the accuracy of results obtained by other analytical methods. The 55 Mn(n,)56 Mn reaction is used for the in vivo neutron activation analysis (IVNAA) technique. 55 Mn, a stable isotope (natural abundance is 100%) undergoes thermal neutron absorption with a high capture cross section of 13.3 barn, producing a radioactive isotope 56 Mn, which decays with a half-life (T1/2 ) of 2.58 h, emitting a ␥-ray of energy 0.846 MeV having intensity (I␥ ) of 98%. The substantial amount of magnesium present in the human body produces 27 Mg through the 26 Mg(n,)27 Mg reaction, which is expected to cause spectral interference, since 27 Mg emits a ␥-ray of 0.843 MeV with I␥ = 72%, close in energy to the ␥-ray of 56 Mn. However, the difference in half-lives of the two is advantageous in differentiating their contributions as 27 Mg is comparatively short lived with a T1/2 of 9.46 min. The 48 Ca(n,)49 Ca reaction is also studied for normalization and quantification, 49 Ca decays with a T1/2 of 8.7 min and a ␥-ray of energy 3.08 MeV with I␥ = 91% [14]. IVNAA of Mn performed using a Tandetron accelerator is based on the neutron production reaction, 7 Li(p,n)7 Be. The Q value of the endothermic nuclear reaction is 1.64 MeV and the threshold energy is 1.88 MeV. The possible maximum energies of neutrons from protons of 2 MeV and 2.3 MeV are 0.23 MeV and 0.57 MeV respectively along the beam axis i.e. 0◦ and the total neutron yield from 2.3 MeV protons is 5.25 times that 2.0 MeV protons [15]. Therefore, the maximum energy of neutrons is such that the potentially interfering reactions with iron 56 Fe(n,p)56 Mn (Eth = 2.97 MeV) and 57 Fe(n,d)56 Mn (E = 8.48 MeV) [14], cannot take place. th The hand bone is chosen for in vivo neutron activation because bone tissue has the highest concentration of Mn with

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approximately 50% of the total body Mn content [3]. In addition, from number of animal studies it is evident that bone is a long term storage site for manganese and to measure a hand bone is technically convenient compared to complexity of using neutron activation method at other sites of body. Also, the effective radiation dose to the subject is much lower when only the hand is exposed to the neutron beam as opposed to a whole body irradiation. Earlier studies done at our laboratory demonstrated the accumulation of Mn in human hand following the inhalation of the element in occupational exposures, and that it is measurable using IVNAA [16–20]. We have conducted a few feasibility tests and measurements on occupational subjects but the levels of Mn in the control subjects were below the MDL of this previous system. After the feasibility studies confirmed the measurement of Mn, the main effort was to improve the MDL values further so as to permit measurements of (i) subjects with low exposure and (ii) non-exposed individuals as well. The aim of this study was to explore the factors required to improve the MDL for the in vivo set-up at McMaster University. The main variables of accelerator-based IVNAA are the energy of the projectile and it’s current, the irradiation time, counting strategy and concentration of elements in tissue equivalent hand bone phantoms, irradiation cavity and counting system. For this study, all the possible parameters were explored and their effects were studied. Also, this study was done using on an upgraded (in-house) pulse processing system [21]. This article presents the resulting improved calibration curves and MDL with a comparison to previous studies aimed at making the technique useful for low or non-exposed subjects.

Materials and methods Tissue equivalent hand-bone phantoms containing a solution of Mg, Ca, Na, Cl and Mn in dil. HNO3 were prepared in 250 ml low density polyethylene (LDPE) Nalgene bottles. The mass of Mn in the phantoms varied from 0.0 to 12.4 mg while the masses of Ca, Na and Cl were kept the same as the phantoms used in earlier measurements [19], based on the data available in ICRP 23 [22]. The mass of Mg was 500 mg in each phantom based on estimations made from recent Mg measurements conducted as part of a study in our Alzheimer’s disease patients [23]. Table 1 lists the details of the element masses used with their respective reaction, half-life and their thermal neutron capture cross-section. As mentioned earlier for this in vivo study, an accelerator-based neutron source was used followed by delayed neutron activation analysis. The hand bone phantoms were irradiated in the accelerator beam following one of two protocols of different proton energy, current and irradiation time, i.e. (i) 2.0 MeV, 300 ␮A, 3 min and (ii) 2.3 MeV, 400 ␮A, 45 s. To measure the gamma activity in the phantoms, a system of eight 10.2 cm × 10.2 cm × 40.6 cm and one 10.2 cm × 10.2 cm × 10.2 cm NaI detectors, arranged in an array of 4␲ geometry, was used [24]. The irradiated phantoms were then placed inside the gamma counting cavity and the ␥-ray spectra from the activated phantoms were acquired. The counting strategy for each phantom was chosen in such a way as to get the contribution of both short- and long-lived isotopes of interest. Average cooling time between the end of irradiation and the start of the count was about 30 s. Counting was done in variable time cycles; for the first 1.5 h the counting was done in 10 min cycles to account for Ca (T1/2 = 8.7 min) and Mg (T1/2 = 9.46 min) and after 1.5 h the counting was continued with 30 min cycles for 5–7 h. A typical gamma spectrum acquired for the 12.4 mg of Mn phantom at three different time intervals after the irradiation is shown in Fig. 1, where the strong gamma peaks with their respective elements are also labeled. Detailed neutron dosimetry of the irradiation cavity was also carried out in a separate study using a tissue-equivalent

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Table 1 The masses of elements used in phantom solution with their respective reaction, half-life and cross section. Element

Reaction

Mass (g) (ICRP)

Compound used

Half-life

Capture cross section (mb) [14]

Manganese Magnesium Sodium Chlorine Calcium

55

Variable 0.5c 1.25 1.19 14.9

Mn standarda Mg standardb Na, NO3 NH4 Cl Ca(NO3 )2

2.58 h 9.46 min 14.997 h 37.24 min 8.7 min

13,360 38.2 530 433 1090

a b c

Mn(n,)56 Mn 26 Mg(n,) 27 Mg 23 Na(n,) 24 Na 37 Cl(n,)38 Cl 48 Ca(n,) 49 Ca

± ± ± ± ±

50 0.8 5 6 140

996 ± 4 mg L−1 . 1000 ± 2 mg L−1 . Calculated.

proportional counter [25]. The signals from each of the nine detectors were recorded through digital multi-channel analyzer and gamma peak-fitting was done to estimate the respective net peak areas of Mn and Ca, corrected for transfer and decay time. The slope of the calibration curve was obtained from the ␥-ray peak analysis of induced activity of 56 Mn in the irradiated phantoms i.e. a calibration standard is required to quantify the in vivo ␥-ray signal and thus its amount in the hand bones of a subject under study. Results and discussion

observed MDL is an improvement on the previous measurement by a factor of about 2.3. As compared to the previous measurements carried out during the last study done by Aslam et al. at McMaster, the major changes adopted in the present study are: (i) Phantoms: The mass of Mg is doubled (500 mg) based on the estimations made from Mg measurement in the Alzheimer’s disease study [23]. (ii) Of the previous measurements done, Mg interference was taken into account only in Ref. [19]. (iii) Counting strategy: Long counting in cycles with time varying from 10 to 30 min helps us in deciding

The calibration curves obtained for the two different protocols studied are shown in Fig. 2(a) and (b). Linear regression curves were fitted for both data sets and slopes of the curves were used to determine the net count rate and hence the mass of Mn. After applying the required corrections for the transfer and irradiation time, the count rate of Mn was normalized to Ca in order to make the accuracy of the measurement independent of factors such as target quality, variation in beam current or positioning of phantom in the cavity, and thickness of overlying soft tissue i.e. expressing Mn mass per unit bone mass. The Mn net count sensitivity for 2.3 MeV and 2.0 MeV protocols along with the detection limits and a comparison with available literature data are presented in Table 2. The obtained sensitivity was 3.2 times the sensitivity of the earlier measurements done at McMaster University [19]. The minimum detection limit (MDL) of Mn content for our set-up, was estimated as twice the uncertainty in the peak area of the zero mg Mn phantom with added mixture, after conversion to mass using the Mn sensitivity obtained from the respective calibration curves. The revised MDLs obtained in this study, along with a comparison with previous values obtained, are presented in Table 2. As shown in this table, the

Fig. 1. Typical gamma spectrum for 12.4 mg of Mn along with Mg, Ca, Na, Cl after irradiation for three different time intervals. Elements with their respective energy of gamma lines are shown.

Fig. 2. (a) Linear calibration curve obtained from the measurements of irradiation of Mn mixed hand phantoms for Ep = 2.0 MeV, 300 ␮A, 3 min and (b) for Ep = 2.3 MeV, 400 ␮A, 45 s. The slope of the curve converted to mass units is used to determine the Mn mass in human hands.

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Table 2 Summary of observed detection limits for in vivo neutron activation analysis of Mn and comparison with available literature data. Authors

Description of facility

Irradiation parameters

Irradiation/cooling/ counting time

Local equivalent dose (mSv) H

Detector

Sensitivity (counts/mg)

MDL (␮gMn/gCa)

Zhang et al. [27]

Brookhaven Medical Research Reactor McMaster KNVan de Graff McMaster KNVan de Graff McMaster KNVan de Graff McMaster Tandetron McMaster Tandetron McMaster Tandetron

Reactor power 2 MW

4 min/4 min/5 min

N/A

Pair of Ge(Li)

3827

∼0.5 ␮g Mn

Ep = 2.00 MeV; Ip = 42 ␮A Ep = 2.00 MeV; Ip = 50 ␮A Ep = 2.00 MeV; Ip = 50 ␮A Ep = 2.00 MeV; Ip = 100 ␮A Ep = 2.00 MeV; Ip = 300 ␮A Ep = 2.30 MeV; Ip = 400 ␮A

10 min/6 min/60 min

109

Pair of NaI(Tl)

N/A

4.6

3 min/30 s/30 min

20

4␲ NaI(Tl)

12,046

5.8

3 min/30 s/30 min

11

4␲ NaI(Tl)

8879

7.3

3 min/65 min/30 min

18

4␲ NaI(Tl)

17,586

1.6

3 min/30 s/30 min

59.7

4␲ NaI(Tl)

56,314 ± 1674

0.71 (1.17%)

45 s/30 s/30 min

450.5

4␲ NaI(Tl)

230,459 ± 3101

0.67 (1.01%)

Arnold et al. [16] Byun et al. [18] Pejovic et al. [17] Aslam et al. [19] This work (2013) This work (2013)

a statistically optimal counting interval allowing for decay from short-lived interfering elements, as well as restricting the duration of the counting interval so as to be acceptable to human subjects. The MDLs obtained from the counting done utilizing short time intervals over the hours is shown in Fig. 3; the MDL minima obtained in the region of 50–80 min are used to calculate an inverse variance weighted mean (IVWM) of the MDLs of the two protocols. (iv) Data acquisition system (DAQ): The acquisition system was upgraded since the time of the last measurements and details were discussed previously [21]. (v) Accelerator current: The proton currents for the 2.0 MeV and 2.3 MeV protocols used are 300 ␮A and 400 ␮A respectively, 54 mC at 2.0 MeV gives an equivalent dose of 59.5 mSv, whereas 18 mC at 2.3 MeV gives an equivalent dose of 450.5 mSv [25]. Since, in the present measurements Mg content is doubled, which increases uncertainty, the sensitivity does not increase proportionally to the increase in current i.e. a factor of three. Of the two protocols investigated, the combination of: 2.0 MeV, 300 ␮A, 3 min protocol is recommended for further measurements of in vivo Mn measurements studies as compared to the 2.3 MeV, 400 ␮A, 45 s protocol. With the former, we saw a much lower dose-equivalent and a relatively minor difference in MDL. The factors that could be considered in improving the detection limit further are the doserate to the subjects; should we manage to do longer counting of (say) 1–2 h, we would expect the MDL to improve further by a factor 2–3 as can be seen from Fig. 3.

Conclusions This work presents the study of key components resulting in improvement of the MDL for an accelerator based non-invasive, in vivo technique used for the measurement of Mn in tissue equivalent human hand-bone phantoms. The technique could serve as a quantitative and analytical approach for measuring elevated amounts of Mn exposures in humans. The important factors studied which have been taken into account for the estimation of MDL for Mn in bone include optimally selected beam parameters for irradiation, using an upgraded DAQ system, a revised more realistic mass of magnesium, use of higher neutron fluence by increasing the current, and estimation of dose equivalents and counting strategy. Combining these factors has reduced the overall detection limit by a factor of nearly 2.3 with our present experimental set-up at McMaster University. A comparison of MDL with previous measurements has also been presented. The present MDL values are close to the reference value of 0.63 ± 0.30 ␮gMn/gCa for the human skeleton [26]. Work is in progress to extend this study for low (<2 mg) mass of Mn phantoms. Thus, the improved MDLs revealed in this work could be of profound importance for use in the measurement of occupational and environmental exposures to Mn, and hence provide a potentially improved assessment of health risks, in clinical studies aimed at better understanding the mechanism of Mn toxicity. Conflict of interest The authors declare that there are no conflicts of interest. Acknowledgements The authors extend their thanks to Justin Bennett, accelerator operator and Scott McMaster, accelerator manager, McMaster University for their assistance in the operation of the Tandetron accelerator and S.D. Molla for providing us the recent dose equivalent data. The project was funded by a research grant provided by AECL, Canada. References

Fig. 3. Minium detection limit (␮gMn/gCa) spectrum observed as a function of time for the 2.0 MeV proton energy, 300 ␮A current and irradiated for 3 min.

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