Yttrium

The bean flour samples and liver samples (0.5 g) and serum (100 μL) were treated with 3.0 mL of 60:40 HNO₃ and HClO₄ mixture into a Pyrex glass tube and left for overnight to destroy organic matter. The mixture was then heated to 120 °C for two hours and 0.25 mL of 40 µg/g Yttrium (Sigma-Aldrich, St. Louis, MO, USA) added as an internal standard to compensate for any drift during the subsequent inductively coupled plasma atomic emission spectrometer (ICP-AES) analysis. The temperature of the heating block was then raised to 145 °C for 2 h. Then, the temperature of the heating block raised to 190 °C for ten minutes and turned off. The cooled samples in the tubes were then diluted to 20 mL, vortexed and transferred into auto sample tubes to analyze via ICP-AES. The model of the ICP used was a Thermo iCAP 6500 series (Thermo Jarrell Ash Corp., Franklin, MA, USA).

Cu and Se content of recombinant wildtype and mutant SELENBP1 was analyzed by total reflection X-ray fluorescence (TXRF) spectrometry, using a bench-top TXRF spectrometer (S4 T-STAR, Bruker Nano, Berlin, Germany) as described [18 ]. Wildtype and mutant proteins were diluted with HBS to a concentration of 7.5 μM (420 mg/L) and combined with 1 mg/L Yttrium (Merck/Millipore, Darmstadt, Germany) as internal standard. 10 μl of each sample were placed on a siliconized sample carrier and dried at 40 °C. All samples were measured in duplicates for 1000 s each.

Cu content of cell lysates and media samples was measured using a bench-top total reflection X-ray fluorescence (TXRF) spectrometer (S2 Picofox™, Bruker Nano GmbH, Berlin, Germany). As internal standard 1 mg/mL Yttrium (Merck/Millipore) was used. 10 μL of each sample were placed on siliconized quartz glass carriers and dried at 40 °C. Samples were measured in duplicates for up to 500 s. Cu and Se content in liver and colon tissue and Se content of HepG2 cells were determined using ICP-MS/MS. Preparation of samples was described previously [29 (link)]. Briefly, samples were weighted into PTFE microwave vessels. HNO₃ (65% (v/v), Suprapure®, Merck/Millipore), H₂O₂ (30% (v/v), Sigma-Aldrich/Merck), rhodium (Rh) as internal standard, and ⁷⁷Se as isotope dilution standard were added before digestion using a Mars 6 microwave digestion system (CEM, Kamp-Lintfort, Germany). After digestion, samples were diluted to achieve final concentrations of 2.93% (v/v) HNO₃, 10 μg/L⁷⁷Se, and 1 μg/L Rh. The samples were measured using ICP-MS/MS (8800 ICP-QQQ-MS, Agilent Technologies) and analyzed as described earlier [29 (link)]. Certified reference materials, namely fish muscle (ERM BB-422) and pig kidney (ERM BB-186) were used as quality control of digestion and to cross validate TE analysis using TXRF and ICP-MS/MS.

Se-enriched culture media and bacteria were collected at the beginning and at the end of the 48-h growth period. After harvesting, the bacterial pellets were washed, weighed and lysed via sonication. The Se content in the samples was measured in duplicates by total reflection X-ray fluorescence (TXRF) spectrometry, using a bench-top TXRF spectrometer (S2 Picofox, Bruker Nano, Berlin, Germany) with 1 mg/L Yttrium (Merck/Millipore; Darmstadt, Germany) as internal standard, as described [32 (link),33 (link)].

Selenium and iron concentrations were determined by Total Reflection X-ray Fluorescence spectrometry (TXRF), as reported before (28 (link)). In short, plasma was spiked with 1000 µg/l gallium (#16639, Sigma/Merck) as an internal standard, while muscle lysate based on RIPA buffer was spiked with 1 mg/l yttrium (#1198090100, Merck/Millipore). For quantification, plasma and muscle lysates spiked with internal standard were applied to non-siliconized and siliconized sample carriers, respectively, dried at 40°C, and measured for 1000 s in an X-ray fluorescence spectrometer (S4 T-STAR, Bruker). The trace element content of the muscle was normalized to the total protein content of the samples, determined with the DC protein assay reagent (#500-0114; Bio-Rad).

Trace element concentrations were measured in rat plasma and tissue samples, human serum, and cell lysates using a total reflection X-ray fluorescence (TXRF) spectrometer (Bruker Nano GmbH, Berlin, Germany) and TEsprit software version 1.0. As internal standard for cells and rat tissue, 1 mg/L yttrium (Merck Millipore, Burlington, MA, USA; 119809) and for serum or plasma 1 mg/L gallium (Alfa Aesar/ Thermo Fisher Scientific, Kandel, Germany; 88066) was used. 10 µL of each sample were placed on siliconized (except plasma) quartz glass carriers and dried at 40 °C. Samples were measured in duplicates for up to 1000 s.