Internal correction of spectral interferences and mass bias for selenium metabolism studies using enriched stable isotopes in combination with multiple linear regression

The analytical methodology for the in vivo study of selenium metabolism using two enriched selenium isotopes has been modified, allowing for the internal correction of spectral interferences and mass bias both for total selenium and speciation analysis. The method is based on the combination of an already described dual-isotope procedure with a new data treatment strategy based on multiple linear regression. A metabolic enriched isotope (⁷⁷Se) is given orally to the test subject and a second isotope (⁷⁴Se) is employed for quantification. In our approach, all possible polyatomic interferences occurring in the measurement of the isotope composition of selenium by collision cell quadrupole ICP-MS are taken into account and their relative contribution calculated by multiple linear regression after minimisation of the residuals. As a result, all spectral interferences and mass bias are corrected internally allowing the fast and independent quantification of natural abundance selenium (^natSe) and enriched ⁷⁷Se. In this sense, the calculation of the tracer/tracee ratio in each sample is straightforward. The method has been applied to study the time-related tissue incorporation of ⁷⁷Se in male Wistar rats while maintaining the ^natSe steady-state conditions. Additionally, metabolically relevant information such as selenoprotein synthesis and selenium elimination in urine could be studied using the proposed methodology. In this case, serum proteins were separated by affinity chromatography while reverse phase was employed for urine metabolites. In both cases, ⁷⁴Se was used as a post-column isotope dilution spike. The application of multiple linear regression to the whole chromatogram allowed us to calculate the contribution of bromine hydride, selenium hydride, argon polyatomics and mass bias on the observed selenium isotope patterns. By minimising the square sum of residuals for the whole chromatogram, internal correction of spectral interferences and mass bias could be accomplished. As a result, the tracer/tracee ratio could be calculated for each selenium-containing species and a time relationship for synthesis and degradation established. Both selenite and selenized yeast labelled with ⁷⁷Se were employed for comparative purposes.     

Thermophysical properties and crystal structure of high-purity monoisotopic <Superscript>80</Superscript>Se

The thermophysical and structural characteristics of high-purity monoisotopic ⁸⁰Se containing 99.9 at % main isotope were studied. Differences were found in properties between monoisotopic selenium and selenium of the natural abundance of isotopes, which had comparable chemical purities and identical thermal histories.     

Nutritional iron supplementation studies based on enriched (57) Fe, added to milk in rats, and isotope pattern deconvolution-ICP-MS analysis

Enriched stable iron isotopes in combination with isotope pattern deconvolution and ICP-MS have been used to study the absorption and bioavailability of iron from supplemented formula milk administrated to lactating rats. The use of two enriched stable isotope tracers, one as the metabolic tracer (here (57) Fe) and the other ((54) Fe) as quantitation tracer, is shown to provide quantitative data about endogenous and exogenous (supplemented) total Fe distribution in rat feces, urine, red blood cells (RBCs), serum, liver, and kidney. The proposed analytical methodology was validated using reference materials (serum, urine, and liver) spiked with both (54) Fe and (57) Fe. Quantitative information about iron absorption/bioavailability and/or metabolism can be obtained from the amounts of endogenous and exogenous iron found in the tissues and fluids analyzed, and about its kinetic after 2 weeks of iron supplementation. The obtained results are discussed in terms of iron exchanged and its half-life in lactating rats and the observed iron levels in serum, RBCs, liver, and kidney comparing nonsupplemented rats and maternal feed rats.     

<Superscript>226</Superscript>Ra and <Superscript>228</Superscript>Ra determination in environmental samples by alpha-particle spectrometry

A complete methodology for ²²⁶Ra and ²²⁸Ra determination by alpha-particle spectrometry in environmental samples is being applied in our laboratory using ²²⁵Ra as an isotopic tracer. This methodology can be considered highly suitable for the determination of these nuclides when very low absolute limits of detection need to be achieved. The ²²⁶Ra determination can be performed at any time after the isolation of the radium isotopes from the analyzed samples while the ²²⁸Ra determination needs to be carried out at least six months later through the measurement of one of its grand-daughters. The method has been validated by its application to samples with known concentrations of these Ra nuclides, and by comparison with other radiometric methods.     

Inductively coupled plasma mass spectrometry for stable isotope tracer investigations

Inductively coupled plasma mass spectrometry (ICP-MS) has now been developed for application to stable isotope tracer investigations of several minerals/trace elements. Use of this method for such purposes requires an understanding of a number of fundamental issues: analytical chemistry performance of the method of isotopic analysis, relationship of the level of enriched isotope administered to the subject with background level of the isotope already present, the issues of cost, and finally the specific details of the biological issues to be explored.In this paper, a brief discussion of these issues is presented. As an example, the discussion is presented in relation to selected aspects of metabolism of selenium, employing the three stable isotopes⁷⁴Se,⁷⁷Se, and⁸²Se in the rat as the biological model.Analytical performance of hydride generation/ICP-MS is discussed for the required analyses of selenium isotopes. It is shown that for solutions containing 10 ng/ml Se of natural isotopic composition, optimized signal/background ratios greater than 40/1 can be obtained, resulting in worst-case detection limits (ng Se) of 2 (⁷⁴Se), and 0.6 (^77,82Se). The precision and accuracy of isotope ratio measurements for the method used routinely in biological studies is 1%. The accuracy of the method for quantitative isotopic analysis is compared with hydride generation/atomic absorption spectrophotometry (HG/AAS). The following results are given (g Se/g or ml; mean + 1 SD,n = 3–5; first HG/ICP-MS, second HG/AAS): SRM 1577a [bovine liver] 0.697 ± 0.002 versus 0.69 ± 0.01; human blood plasma 0.098 ± 0.001 versus 0.135 ± 0.008; human red cells 0.211 ± 0.002 versus 0.216 ± 0.012; and human urine 0.0473 ± 0.0003 versus 0.0489 ± 0.0003.An experiment is described with the rat to show the feasibility of the method for studies of selenium metabolism. Rats were placed on Se-free diet for eight weeks, given their Se requirements in the drinking water in the form of⁷⁶SeO ₃ ^2– and a single-day (day 3) replacement of their water with that containing highly enriched⁷⁴SeO ₃ ^2– . Isotopic analysis of carcass and selected organs revealed a high degree of isotopic enrichment with respect to⁷⁴Se during the entire eight weeks of the experiment, indicating the feasibility of this approach for detailed investigations of selenium metabolism in the rat.Presented in part at the 1989 European Winter Conference on Plasma Spectrochemistry, Reutte, Austria     

Isotopic determination of selenium in biological materials with inductively coupled plasma mass spectrometry

A method for the isotopic determination of selenium in biological matrices is described. The method is based on hydride generation inductively coupled plasma mass spectrometry (ICP-MS). The development is specifically related to the requirements of stable isotope tracer studies in human subjects. The method is based on isotope dilution using 82Se as the in vitro spike and can quantify the 74Se and 77Se contents of samples. It involves wet oxidation (HNO3 - H2O2 or HNO3 - HClO4) of the 82Se-spiked matrix, reduction to selenite by boiling with HCl followed by measurement of the isotope ratios (82Se/77Se and 74Se/77Se) in the gas stream (H2Se) generated from on-line reduction of the sample selenite with NaBH4. Compared with the isotopic signal resulting from a selenite solution containing 5 ng ml-1 of Se, the total sample blank contributions at m/z = 74, 77 and 82 were less than 5% of the respective isotope signal. Worst-case absolute detection limits were 0.2-0.9 ng of Se, depending on the isotope used. Ion beam intensity ratios were measured with an over-all precision [relative standard deviation (RSD)] of 1% for both isotope pairs. Measured ratios (MRa/b) were stable during a given day's operation within the expected precision of the measurements but varied for different days. The magnitude of MRa/b was generally independent of the nature of the matrix. Highly linear relationships were found between ion beam intensity ratios (MRa/b) and the corresponding true isotope ratios for calibration solutions whose isotope ratios had been altered by as much as one order of magnitude.(ABSTRACT TRUNCATED AT 250 WORDS)     

The determination of selenium in urine

The selenium excreted in urine can be measured to assess the dietary status of selenium, an essential trace element in human nutrition. The objectives of this work were: 1) to develop a procedure, capable of high sample throughout, by which the major interferences can be reduced such that selenium concentrations can be measured in urine by Neutron Activation Analysis (NAA) using^77mSe (17.4 s; and 2) to apply the method to a human dietary selenium study in which several selenium monitors were compared. The method involves a pre-irradiation arsenic-coprecipitation separation of the selenium from urine in the presence of a high specific-activity⁷⁵Se tracer. The processed urine samples are analyzed using NAA. The procedure was applied to 58 urine specimens longitudinally collected from 12 subjects consuming three different levels of selenium. A dose-response relationship was observed in urine as well as a high correlations with both serum and whole blood selenium concentrations.     

GC–ICP–MS determination of dimethylselenide in human breath after ingestion of <Superscript>77</Superscript>Se-enriched selenite: monitoring of in-vivo methylation of selenium

The amount of volatile dimethylselenide (DMSe) in breath has been monitored after ingestion of sub-toxic amounts of selenium (300 μg ⁷⁷Se, as selenite) by a healthy male volunteer. The breath samples were collected in Tedlar bags every hour in the first 12 h and then at longer intervals for the next 10 days. The samples were subjected to speciation analysis for volatile selenium compounds by use of cryotrapping–cryofocussing–GC–ICP–MS. Simultaneously, all urine was collected and subjected to total selenium determination by use of ICP–MS. By monitoring m/z 82 and 77, background or dietary selenium and selenium from the administered selenite were simultaneously determined in the urine and in the breath—dietary selenium only was measured by monitoring m/z 82 whereas the amount of spiked ⁷⁷Se (99.1% [enriched spike]) and naturally occurring selenium (7.6% [natural abundance]) were measured by monitoring m/z 77. Quantification of DMSe was performed by using DMSe gas samples prepared in Tedlar bags (linear range 10–300 pg, R ²=0.996, detection limit of Se as DMSe was 10 pg Se, or 0.02 ng L⁻¹, when 0.5 L gas was collected). Dimethylselenide was the only selenium species detected in breath samples before and after the ingestion of ⁷⁷Se-enriched selenite. Additional DM⁷⁷Se was identified as early as 15 min after ingestion of the isotopically-labelled selenite. Although the maximum concentration of ⁷⁷Se in DMSe was recorded 90 min after ingestion, the natural isotope ratio for selenium in DMSe (77/82) was not reached after 20 days. The concentration of DMSe correlated with the total Se concentration in the urine during the experiment (R ²=0.80). Furthermore, the sub-toxic dose of 300 μg selenium led to a significant increase of DMSe and renal excretion of background selenium, confirming that selenium ingested as selenite is homeostatically controlled by excretion. The maximum concentration of DMSe resulting from the spiked selenite was 1.4 ng Se L⁻¹ whereas the dietary background level was less than 0.4 ng Se L⁻¹. Overall excretion as DMSe was calculated to be 11.2% from the ingested selenite within the first 10 days whereas urinary excretion accounts for nearly 18.5%.     

Separation and determination of <Superscript>241</Superscript>Am in urine samples from radiation workers using PC88-A and alpha spectrometry

Bioassay technique is used for the estimation of actinides present in the body based on their excretion rate through body fluids. For occupational radiation workers urine assay is the preferred method for monitoring of chronic internal exposure. Determination of low concentrations of actinides such as plutonium, americium and uranium at low level of mBq in urine by alpha spectrometry requires pre-concentration of large volumes of urine. This article deals with standardization of analytical method for the determination of ²⁴¹Am isotope in urine samples using Extraction Chromatography (EC) and ²⁴³Am tracer for radiochemical recovery. The method involves oxidation of urine followed by co-precipitation of americium along with calcium phosphate. This precipitate after treatment is further subjected to calcium oxalate co-precipitation. Separation of Am was carried out by EC column prepared by PC88-A (2-ethyl hexyl phosphonic acid 2-ethyl hexyl monoester) adsorbed on microporous resin XAD-7 (PC88A-XAD7). Am-fraction was electro-deposited and activity estimated using tracer recovery by alpha spectrometer. Ten routine urine samples of radiation workers were analyzed and consistent radiochemical recovery was obtained in the range 44–60% with a mean and standard deviation of 51 and 4.7% respectively.     

Determination of plutonium isotopes in urine samples from radiation workers using <Superscript>236</Superscript>Pu tracer,anion exchange resin and alpha spectrometry

Bioassay technique is used for the estimation of actinides present in the body based on their excretion rate through body fluids. For occupational radiation workers urine assay is the preferred method for monitoring of chronic internal exposure. Determination of low concentrations of actinides such as plutonium, americium and uranium at low level of mBq in urine by alpha spectrometry requires pre-concentration of large volumes of urine. This paper deals with standardization of analytical method for the determination of Pu-isotopes in urine samples using anion exchange resin and ²³⁶Pu tracer for radiochemical recovery. The method involves oxidation of urine followed by co-precipitation of plutonium along with calcium phosphate. Separation of Pu was carried out by Amberlite, IRA-400, anion exchange resin. Pu-fraction was electrodeposited and activity estimated using tracer recovery by alpha spectrometer. Twenty routine urine samples of radiation workers were analyzed and consistent radiochemical tracer recovery was obtained in the range 74–96% with a mean and standard deviation of 85 and 6% respectively.     

Study of Chromium Speciation in Normal and Diabetic Rats by Activable Enriched Stable Isotope Technique

Chromium speciation was investigated in the liver cytosol, serum and urine of normal and diabetic rats after a single intravenous injection of enriched stable isotope ⁵⁰Cr tracer solution. Sephadex G-25 gel chromatography combined with instrumental neutron activation analysis was used to isolate and characterize protein-bound chromium in the above materials. The results indicate that Cr is mainly combined with a high-molecular-weight protein either in liver cytosol or serum. A low-molecular-weight, Cr-containing compound (LMWCr) was found in all the observed liver, serum and urine samples of both normal and diabetic rats. Chromium is excreted chiefly as LMWCr in urine.     

Study on the method of preparation <Superscript>97</Superscript>Tc

Technetium-99 is one of several long-lived fission products which, when detected in the environment can give an indication of a specific nuclear activity. The most sensitive analytical technique for ⁹⁹Tc yet reported is by isotopic dilution mass spectrometry with technetium-97 as the yield tracer. A method for the preparation of ⁹⁷Tc is reported in this paper. ⁹⁷Tc was obtained by irradiation of a sample of natural ruthenium metal in a high flux reactor. After cooling for 2 years, the technetium was isolated from the sample by technique combining; deposition, solvent extraction, and ion-exchange chromatography techniques. ^99mTc and ¹⁰³Ru were used as radio-tracers for the process. The results showed that more than 70% of the Tc was recovered the decontamination factor is more than 2.3 × 10⁷. The ⁹⁷Tc was calibrated by isotope dilution mass spectrometry with ⁹⁹Tc as the yield tracer. The final yield was 29.56 μg of ⁹⁷Tc suitable for use as a mass spectrometric spike (weight % ⁹⁷Tc spike: ⁹⁷Tc, 84.77%; ⁹⁸Tc, 15.03%; ⁹⁹Tc, 0.20%).     

Determination of <Superscript>238</Superscript>Pu in plutonium bearing fuels by thermal ionization mass spectrometry

Determination of ²³⁸Pu in plutonium bearing fuels is required as a part of the chemical quality assurance of nuclear fuels. In addition, the determination of ²³⁸Pu is required in nuclear technology for many other applications, e.g., for developing isotope correlations and while using ²³⁸Pu as a spike (tracer) in isotope dilution α-spectrometry (IDAS). This determination usually involves the use of α-spectrometry on purified Pu sample. In view of the random errors associated with the counting statistics and the systematic errors due to (1) in-growth of ²⁴¹Am in purified Pu sample and (2) tail contribution correction methodology in α-spectrometry, the precision and accuracy obtainable by α-spectrometry are limited. Thermal ionization mass spectrometry (TIMS) is generally used for the determination of different Pu isotopes other than ²³⁸Pu. This is due to the ubiquitous isobaric interference from ²³⁸U at ²³⁸Pu in TIMS. Recently, we have carried out studies on the formation of atomic and oxide ions of U and Pu by TIMS and developed a novel approach using interfering element correction methodology to account for the isobaric interference of ²³⁸U at ²³⁸Pu in TIMS. This methodology is based on the addition of ²³⁵U (enrichment >90 atom%) to Pu sample followed by the determination of ²³⁸U/²³⁵U atom ratio using UO⁺ ion and determination of Pu isotope ratios using Pu⁺ ion, from the same filament loading. The TIMS methodology was used for the determination of ²³⁸Pu in different Pu samples in U based nuclear fuels from PHWRs with ²³⁸Pu content about 0.2 atom%. The ²³⁸Pu determination was also carried out using α-spectrometry. This paper reports the results obtained by the two methods and presents the ments and shortcomings of the two approaches.     

Use of <Superscript>82</Superscript>Br and <Superscript>131</Superscript>I radionuclides in studies of goitrogenic effects of exogenous bromide

The interference of excessive bromide intake with iodine metabolism in the rat was studied using ⁸²Br and ¹³¹I radionuclides. Mainly lactating rat dams and their pups were used, in addition to adult male rats, in the present studies. Particularly, the influence of high bromide intake in lactating rat dams on the transfer of iodine and bromide to suckling young through breast milk was evaluated. The induction of hypothyroid status in the pups by high bromide intake in the mothers was proved unambiguously. Excessive bromide in lactating rat dams caused a marked decrease in plasma levels of thyroid hormones both, in the mothers and in their sucklings. The effects of an enhanced bromide intake on the thyroid function in relation to iodine status in the animals were also followed. Marked goitrogenic and thyrotoxic effects of excessive bromide in adult rats were significantly enhanced under the conditions of simultaneous iodine deficiency in the experimental animals.     

Cyclic neutron activation analysis for determination of selenium in food samples using <Superscript>77m</Superscript>Se

Cyclic neutron activation analysis method was conducted for determination of Se in food samples. High accuracy and good precision were proved by analyzing certified reference materials (CRMs) of chicken (GBW10018), rice (GBW10010) and cabbage (GBW10014). The detection limits for the three CRMs reached 0.16, 0.66 and 1.2 ng after 6 cycles at the 161.9 keV γ-peak from ^77mSe, under a neutron flux of 9.0 × 10¹¹ n cm⁻² s⁻¹ and the conditions of 30 s irradiation, 2 s decay, 30 s counting and 2 s waiting, significantly lower than those of conventional neutron activation analysis without any cycles, which were 0.94, 3.6 and 4.3 ng, respectively.     

Activation analysis of Se in biological samples through75Se and77mSe

A method is described to separate trace amounts of selenium in biological samples without using a carrier. This method is based on the adsorption on active carbon of the complex ion formed with APDC /ammonium salt of l-pyrrolidine carbodithioic acid/ at pH 1. The efficiency of the radiochemical separation described is measured by using carrier-free⁷⁵Se labelled solutions of sodium selenite at selenium concentrations from 3.5×10^–8 to 3.5×10^–11 g ml^–1. The results were between 95% and 98% with statistical variations from 2% to 10%. The determination of selenium can be made following this separation either through⁷⁵Se in the traditional way, or through^77mSe if the separation is performed prior to irradiation. The detection limits on the available conditions were 0.01 ppm for⁷⁵Se and 0.1 ppm for^77mSe. When the analysis is performed through⁷⁵Se /t=120 d/, the statistical error is notably smaller because the counting time may be considerable, whereas through^77mSe/t=17.5 s/it is higher than 20%, depending on the concentration and the available neutron flux. However, the advantages of gaining time and the fact of performing the trace separation from a non radioactive material, make both procedures competitive as useful tools for the research on the function of Se in vertebrates.     

Advances in <Superscript>79</Superscript>Se analyses on Savannah river site radioactive waste matrices

Waste cleanup efforts underway at the United States Department of Energy’s (DOE) Savannah River Site (SRS) in South Carolina, as well as other DOE nuclear sites, have created a need to characterize ⁷⁹Se in radioactive waste inventories. Successful analysis of ⁷⁹Se in high activity waste matrices is challenging for a variety of reasons. As a result of these unique challenges, the successful quantification of ⁷⁹Se in the types of matrices present at SRS requires an extremely efficient and selective separation of ⁷⁹Se from high levels of interfering radionuclides. A robust ⁷⁹Se radiochemical separation method has been developed at the Savannah River National Laboratory (SRNL) which is routinely capable of successfully purifying ⁷⁹Se from a wide range of interfering radioactive species. In addition to dramatic improvements in the K_d, ease, and reproducibility of the analysis, the laboratory time has been reduced from several days to only 6 h.     

A combined method of neutron activation analysis and radiometric measurements for <Superscript>234</Superscript>U and <Superscript>238</Superscript>U determination in soil samples of low uranium concentration

A method that combines the use of non-destructive neutron activation analysis and high-resolution α spectrometry has been developed for determination of the activities of ²³⁴U and ²³⁸U in geological samples of low uranium content. The ²³⁸U content is determined by k₀-based neutron activation analysis, whereas the ²³⁴U/²³⁸U relationship is measured by α spectrometry after isolation and electrodeposition of the uranium extracted from a lixiviation with 6 M HCl. The main advantage of the method is the simplicity of the chemical operations, including the fact that the steps destined to assure similar chemical state for the tracer and the uranium species present in the sample are not necessary. The method was applied to soil samples from sites of the North Peru Coast. Uranium concentration range 3–40 mg/kg and the isotopic composition correspond to natural uranium, with about 10% uncertainty.     

Reductive immobilization of <Superscript>79</Superscript>Se by iron canister under simulated repository environment

To understand the fate of ⁷⁹Se in a repository-like environment, the interactions between iron canister surface with dissolved selenite (SeO₃ ²⁻) and selenate (SeO₄ ²⁻) in anaerobic solutions have been investigated. Se(IV) immobilization on iron surface was observed to be about 100 times faster than that of Se(VI) at same conditions. An iron surface coated with a FeCO₃ layer corrosion product is more reactive than a polished iron to immobilize Se(IV) and Se(VI). The reacted iron surfaces were analysed by scanning electron microscopy (SEM) and energy dispersive spectrometer (EDS), X-ray diffraction (XRD), Raman spectrometry and micro-X-ray Absorption Spectroscopy (XAS). The result show that Se(IV) and Se(VI) were reduced and precipitated. The dominating phase was found to be FeSe_2.     

Sulphur tracer experiments in laboratory animals using 34S-labelled yeast

We have evaluated the use of ³⁴S-labelled yeast to perform sulphur metabolic tracer experiments in laboratory animals. The proof of principle work included the selection of the culture conditions for the preparation of sulphur labelled yeast, the study of the suitability of this labelled yeast as sulphur source for tracer studies using in vitro gastrointestinal digestion and the administration of the ³⁴S-labelled yeast to laboratory animals to follow the fate and distribution of ³⁴S in the organism. For in vitro gastrointestinal digestion, the combination of sodium dodecyl sulphate-polyacrylamide gel electrophoresis and high-performance liquid chromatography and inductively coupled plasma mass spectrometry (HPLC-ICP-MS) showed that labelled methionine, cysteine and other low molecular weight sulphur-containing biomolecules were the major components in the digested extracts of the labelled yeast. Next, in vivo kinetic experiments were performed in healthy Wistar rats after the oral administration of ³⁴S-labelled yeast. The isotopic composition of total sulphur in tissues, urine and faeces was measured by double-focusing inductively coupled plasma mass spectrometry after microwave digestion. It was observed that measurable isotopic enrichments were detected in all samples. Finally, initial investigations on sulphur isotopic composition of serum and urine samples by HPLC-ICP-MS have been carried out. For serum samples, no conclusive data were obtained. Interestingly, chromatographic analysis of urine samples showed differential isotope enrichment for several sulphur-containing biomolecules.