2024 Volume 49 Issue 12 Pages 543-548
A simple monitoring method has been proposed by measuring uranium (U) concentration and its chemical form in serum. The droplet of the 1 µL rat serum specimen was then examined by subjecting it to high energy synchrotron radiation X-ray fluorescence spectroscopy (SR-XRF) and X-ray absorption fine structure (XAFS) to determine the concentration and chemical form of U. The detection limit of U in 1 µL droplet was calculated to be 0.071 µg/g. The U concentration in the specimen obtained from the rat exposed to U was consistent with that determined by inductively coupled plasma mass spectrometry. Uranium in the rat serum was estimated to be hexavalent U based on the standard specimens of tetravalent and hexavanet U. This method developed might be used for monitoring and decorporation of patients at nuclear disasters and environmental pollution.
Uranium (U) is a radioactive heavy element which is ubiquitously present in the earth’s crust. U is a radiological as well as a chemical toxic element that induces renal dysfunction by chronic ingestion at higher levels via contaminated groundwater (UNSCEAR, 2016; Kurttio et al., 2002). Furthermore, the accidental exposure to nuclear fuels including U is supposed during the decommissioning work at nuclear power plants. To determine the body-burden, U in specimens isolated from internally exposed residents and workers needs to be measured in environmental pollution and nuclear disasters. Inductively coupled plasma mass spectroscopy (ICP-MS) has been mainly used for the determination of U concentration in biological samples (Thakur and Ward, 2019). Complicated pretreatment to remove various inorganic and organic substances in the sample is indispensable although highly sensitive quantification is performed by the method. Because U is an alpha particle radionuclide, it requires a multi-step pretreatment process in radiation measurements. Recently, elemental detection of biological and environmental samples by X-ray spectroscopy has become remarkably sensitive with the development of the detectors (Denecke, 2024; Terzano et al., 2019; Vanhoof et al., 2021). Measurement of actinides such as U and plutonium concentrations using small liquid samples is being addressed in laboratory scale X-ray fluorescence spectroscopy (XRF) (Izumoto et al., 2019; Izumoto et al., 2020; Sanyal et al., 2018; Sanyal and Dhara, 2020). Furthermore, synchrotron radiation XRF (SR-XRF) using high energy excitation X-ray made it possible to detect U in the energy region that is not disturbed by endogenous elements in tissues, thus enabling the detection of trace amount of U in tissues (Homma-Takeda et al., 2013, 2015). X-ray absorption fine structure (XAFS) gives local structural information such as the oxidation states, bond distance on objective and the nearest elements (Denecke, 2024). It has also been demonstrated that the chemical form of U accumulated in the kidney can be evaluated by using SR-XRF combined with XAFS with microprobe (Kitahara et al., 2017; Homma-Takeda et al., 2019; Homma-Takeda et al., 2020). The application of the combination method to biofluids is useful to effectively evaluate the metabolic mechanism of U and the chelating ability for decorporation using chelating agents (Uehara et al., 2022b), and might be a monitoring method for nuclear disasters and environmental pollution.
In the present study, a simple method for measuring the U concentration and chemical form in 1 µL rat serum, which is suitable to rapid dried fixation of sample on polypropylene film, was proposed using a combination of SR-XRF and XAFS. Using this method, we analyzed the U concentration of rat serum in rats exposed to U.
U was purchased as uranyl acetate (UA) from FUJIFILM Wako Pure Chemical Co., Japan. Caution: U is toxic and radioactive and should be handled in appropriate facilities.
AnimalsRat serum specimens were obtained from Wistar male rats (10 weeks old; CLEA Japan) exposed to U as follows; The animals were acclimated to a controlled temperature (22 ± 2°C), humidity (50 ± 10%), and day/night cycle environment (light 7:00 – 19:00) for a week before initiating the study. Uranyl acetate dissolved in saline at the dose of 4 mg/kg of body weight was administered to rats by subcutaneous injection based on a similar manner (Uehara et al., 2022b). As controls, rats received injections containing only saline. Blood was collected from rats 1 hr after the administration. Rat serum was obtained using a microtube containing a coagulation-promoting separator (Separapid tube S, Sekisui Medical Co., Japan). After the microtube containing blood was kept for 20 min at room temperature, it was centrifuged at 2000 rpm for 10 min. After this, the rat serum samples were stored in a freezer at –80°C.
All procedures for the animal experiment were approved by the Animal Care Committee of the National Institutes for Quantum Science and Technology (No. 18-1005-2, March 2023).
Droplet sample preparationRat serum mixed with UA was incubated at 37°C for an hour. For SR-XRF and XAFS measurements, 1 µL of rat serum containing UA from 0 to 119 μg/g U was dropped onto polypropylene (PP) film (6 µm thickness, Rigaku Co., Japan) and dried under air atmosphere for one night, which was used as standard specimen. Three specimens of each concentration were prepared. One µL of the rat serum obtained from U-administered rats was dropped onto PP film and dried.
SR-XRF and XAFS measurementsUA and uranium dioxide (UO2) were used as standard U samples for tetravalent and hexavalent. Here, UO2 was prepared by the reduction using hydrogen gas from triuranium octoxide at 1000°C and confirmed by powder X-ray diffraction analysis (MiniFlex 600 diffractometer (Rigaku Co., Japan)) based on the methods reported elsewhere (Uehara et al., 2022a). These compounds were attached to Kapton film coated with a spray-based adhesive (3M Japan Ltd.) after being ground to a fine powder using a mortar.
SR-XRF measurements were performed at BL37XU, SPring-8, Japan, using an energy-dispersive SR-XRF system which possessed a fast system for scanning microscopic measurements. Measurement areas for SR-XRF imaging were then scanned with a 33 keV monochromatic microbeam (Si(111) double-crystal monochromator). The scanning mode of SR-XRF imaging is as follows: 30 × 30 steps at 60 μm per step, counting time was 1 sec per step meaning measurement time was totally 900 sec, beam size; 1 μm × 1 μm. Due to the interference of endogenous Rb and Br with the detection of the U Lα line (Homma-Takeda et al., 2009), the intensity data of the U Lβ2 line (peak width: 16.054 – 16.786 keV) and U Lβ1 line (peak width: 16.786 – 17.779 keV) at each point were obtained with an eight-element silicon drift detector and processed by a personal computer. The calibration curves were obtained from total counts of the U Lβ2 and U Lβ1 lines on two-dimensional elemental maps of rat serum specimens from 0 to 23.8 μg/g U. The chemical forms of U in rat serum droplet were analyzed by XAFS of the U L3 edge. XAFS spectra with microbeam were corrected by fluorescence mode with an incident X-ray energy of 16.911 – 17.371 keV. XAFS spectra of UA and UO2 were measured as reference samples. All data was processed according to standard procedure using Demeter software package (version 0.9.18) (Ravel and Newville, 2005). All measurements were performed under ambient conditions at room temperature.
The dried specimen of 1 µL rat serum on the PP film was circular in shape with a diameter of 1.6 ± 0.2 mm (Fig. 1 A). The SR-XRF spectra of the rat serum specimen containing 2.38 μg/g U were recorded in curve 1 of Fig. 1 B. Fluorescence peaks for calcium Ca, iron Fe, copper Cu, zinc Zn, selenium Se, and bromine Br K-lines corresponding to the endogenous elements in rat serum were observed in the control rat serum droplet in curve 2 of Fig. 1 B. Lead Pb and molybdenum Mo were observed due to the materials of the detector. When concentration of U based on the U Lα (13.61 keV) line is measured, Br Kβ (13.29 keV) and rubidium Rb Kα (13.39 keV) lines, which were observed as endogenous elements in the serum, should be considered. U concentration was determined based on both U Lβ1 and U Lβ2 lines, although the U Lβ1 line overlapped with the Mo Kα line (17.48 keV) which was one of the materials of the detector. Concentration dependence of U from 0 to 2.38 µg/g is shown in Fig. 1 C. The calibration curve based on U Lβ1 (16.786 – 17.779 keV) count was plotted in Fig. 1 D. The slope and intercept of the regression lines of U Lβ1 line were 6.49 × 103 and 1.66 × 103 (R2 = 0.999). The limit of detection at U Lβ1 line was calculated to be 0.071 µg/g, which was determined using each line of control droplet (0 µg/g U) and slope of the calibration curve. Here, the limit of detection at U Lβ2 line (16.054 – 16.786 keV) was calculated to be 0.10 µg/g, which was higher than that at U Lβ1 line because of lower intensity. The detection limit of U in the 7 µL blood specimen was reported to be 0.45 µg/g based on the U Lα line by laboratory scale XRF (Izumoto et al., 2019). Higher sensitive measurements of U have been achieved by measuring U Lβ1 line resulted from high energy SR-XRF.
SR-XRF spectra in 1 µL rat serum droplet. (A) Photograph of 1 μL of 2.38 µg/g U in rat serum. Rat serum mixed with UA was incubated at 37°C for an hour. One µL of rat serum containing UA was dropped onto PP film and dried under air atmosphere for one night. (B) Curves 1 (black line) and 2 (red line) represent SR-XRF spectra in the presence and absence of 2.38 µg/g U in rat serum. *Pb and Mo were the materials of the detector. (C) SR-XRF spectra of 0 - 2.38 µg/g U in rat serum droplet. (D) Calibration line of the intensity of U Lβ1 line depended on the concentration of U in rat serum. Horizontal axis: U concentration (µg/g); vertical axis: SR-XRF intensity in the analyzed area of U Lβ1 (16.786 – 17.779 keV) line. Each point represents the mean and standard deviation of data from three specimens.
SR-XRF spectra of the rat serum specimen administered are shown in curve 1 of Fig. 2. The U concentration in rat serum administered was determined based on the calibration curve in Fig. 1 D to be 1.40 ± 0.30 µg/g, which was close to that determined by ICP-MS (1.43 µg/g) shown in Table 1. Average concentration of U in rat serum of three rats was determined to be 1.16 ± 0.31 µg/g by ICP-MS, which means that the method can be applied to animal U exposure model. Concentration of U in human blood was reported to be between 5 pg/g and 1 ng/g (Byrne and Benedik, 1991; Ahmed et al., 2022; Al-Hamzawi et al., 2014), which was still less than the detection limit in this study. Also, U concentration in human blood under chronic exposure reported (Basheer et al., 2024) is about one order lower than the detection limit. Increasing the sample volume, counting time, and number of steps would be necessary to measure lower concentration.
SR-XRF in 1 µL rat serum droplet. Curve 1 (black line) represents SR-XRF spectra in rat serum droplet prepared after the administration of UA in saline at the dose of 4 mg/kg of body weight. Curve 2 (red line) represents SR-XRF spectra in rat serum droplet obtained from control rat.
| Administration U (mg/kg) |
U Concentration (µg/g) | |
|---|---|---|
| U Lβ1 line* | ICP‐MS | |
| 4 | 1.40 ± 0.30** | 1.43 ± 0.07** |
* SR-XRF measurements were performed on three specimens for one concentration.
** Error bar of the concentration means SD value.
Normalized XAFS spectra at three randomly selected areas in the serum droplet containing 0.5 mM (119 µg/g) UA are shown in Fig. 3 A. The surface damage of the PP film was not observed before or after irradiation. Three spectra were found to be reproducible and were independent of the measurement location. The spectra were measured at three points in three droplets are shown in Fig. 3 B. The shape of the measured spectra was identical though the spectra at lower concentration of U such as 0.1 and 0.01 mM was not reproducible due to the lower intensity. U concentration, which chemical form can be distinguished by XAFS, is several orders higher than that reported. Fundamental improvement is necessary for the detection at lower concentration, i.e., by larger beam size and higher sensitive detector (Yamada et al., 2021).
XAFS spectra of U in1 µL rat serum droplet and standards. (A) XAFS spectra at three points in one droplet of 1 µL rat serum containing 119 µg/g U, (B) XAFS spectra at three points in three droplets of 1 µL serum. Spectra of UO2 and UA indicate spectra of UO2 and UA powder samples. Spectrum of U-Serum was measured in a droplet of rat serum containing 119 µg/g U. Specimen in (A) and (B), “U-serum” in (C) and (D) were prepared from rat serum mixed with UA. Each spectrum was averaged for three scans. Spectra of UO2, UA, and U-Serum in (C) were overlapped in (D).
XAFS spectra of UO2 for U(IV) and UA for U(VI) were measured as reference spectra and are shown in Fig. 3 C. Oscillation at 17.215 keV was observed corresponding to single crystal structure of UO2, which can be distinguished with UA. The edge jump (top peak) energy depending on the oxidation state of U, which was defined as the position of the first maximum of the derivative spectra, was observed at 17.172 keV for UO2 and 17.174 keV for UA, respectively. It has been reported that edge jump energy of U (IV) was observed lower energy than that of U (VI) (Fig. 3 D). U-Serum in Fig. 3 C shows the spectra after averaging three scans in Fig. 3 A. The edge jump energy of the spectra in rat serum droplet (17.175 keV) was close to that of U in liquid rat serum (Uehara et al., 2022b), suggesting that valence of the U in rat serum was hexavalent. The edge jump intensity was different from that of UA, which means that U coordinated with biological ligands such as carbonate, proteins in the rat serum instead of acetate ions. It is possible that the U(VI) is reduced to U(V) or U(IV) by reductants in serum or proximal tubule of kidney (Kitahara et al., 2017).
To measure the chemical form of U in administered rat serum, repeated measurements and rat serum specimen with larger volume would be necessary. The application of the combination of SR-XRF and XAFS to the rat serum of rats allowed us to the obtain U level and chemical form.
ConclusionU concentration and chemical form in a 1 µL rat serum droplet were measured with high energy SR-XRF and XAFS. The detection limit of U in 1 µL droplet was sub-ppm 0.071 µg/g based on U Lβ1 line by SR-XRF. Simple quantification of U concentration in rat serum administered U was achieved. U in rat serum specimens estimated to be hexavalent. This method is suitable for the measurement of not only U but also strontium, cadmium, indium, tin, antimony, tellurium, iodine, and cesium, which require detection in the high-energy region where general XRF is not suitable and might be a monitoring method for nuclear disasters and environmental pollution.
This research was supported by Grants-in-Aid for Scientific Research (No. 19H05775) from the Ministry of Education, Culture, Sports, Science and Technology, Japan. XAFS and SR-XRF measurements were performed at the beamline BL37XU (Proposal Nos. 2020A1538, 2021B1026, and 2022A1013) SPring-8, Japan. A.U. thanks Dr. Teruaki Konishi for useful suggestions and constructive comments on the manuscript. We thank Yoshinari Shiino and Kohei Iwaya (Department of Research Planning and Promotion, NIRS) for their assistance of the experimental procedure including the nuclear fuel materials.
Conflict of interestThe authors declare that there is no conflict of interest.
