Gallium

Fasting peripheral venous blood samples were collected, fully coagulated at room temperature, and centrifuged (10 min, 2762 g, 4 °C) for separation of serum. Before analysis, serum samples were stored at −80 °C. Total trace element levels were measured by X-Ray fluorescence, using a bench-top total reflection X-ray fluorescence (TXRF) spectrometer (S4 T-StarTM, Bruker Nano GmbH, Berlin, Germany). As internal standard 1 mg/mL Gallium (Thermo Fisher Scientific, Kandel, Germany) was used. In total, 10 μL of each sample was placed on quartz glass carriers and dried at 40 °C. Samples were measured in duplicates for 750 s. For quality control, reference material RECIPE^® ClinChek^® serum control lyophilized 1 (Ref. 8880, Lot 544) was measured in duplicate frequently (in every measuring process). For a classification of the serum trace element levels, the reference values according to Labor und Diagnose were used. Reference ranges indicating normal serum concentration of selenium, zinc and copper were defined as 50–120 µg/L, 600–1200 µg/L, and 580–1690 µg/L, respectively [29 ]. The reference range for copper differs between men and women, with the reference range being higher for women. Therefore, the lowest value for men and highest value for women was chosen.

Trace elements can be analyzed using X-rays, generated by a molybdenum tube using the S2 Picofox™ (Bruker Nano GmbH, Berlin, Germany). Prior to measurement, mouse tissue samples were digested, whereas cell lysates were analyzed without further digestion. Briefly, 50 mg of tissue samples was weighted into microwave vessels and 830 µL ultrapure water, 900 µL 69% HNO₃ (v/v) (Suprapure^®, Merck/Millipore), 250 µL 30% H₂O₂ (v/v) (Sigma-Aldrich/Merck), and 20 µL of 200 mg/L yttrium-solution (Merck/Millipore) as digestion control were added. The samples were heated to 200 °C for 20 min using a Speedwave 2 (Berghof, Eningen, Germany). Afterwards, the samples were diluted with 2 mL ultrapure water and stored at RT. Certified reference material, namely pig kidney (ERM BB-186), was always analyzed in parallel. For the analysis of trace element content in mouse tissue and cells, 0.5 mg/L gallium (ThermoFisher Scientific) or 1 mg/L yttrium was used as internal standard. An amount of 10 µL of each sample was placed on a siliconized quartz glass carrier and dried overnight. Mouse and cell samples were measured randomly in triplicates for 1000 or 500 s, respectively.

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.