Drc e

Non-fasting blood samples were obtained by antecubital venipuncture and collected into vacutainer tubes with EDTA and without additives approved for trace metal analysis (Terumo®, US). Five-hundred μL of whole blood and serum was aliquoted and stored at –80°C until analysis. Whole blood Hg, Pb and Cd and serum Sb were analyzed by ICP-MS on Perkin Elmer DRC-e (Perkin Elmer®, USA) in standard mode [31 (link)].
Plasma levels of cotinine were assayed using a LC-MS/MS method [32 (link)]. A plasma cotinine level ≥85 nmol/L is commonly used as a cut off to define regular smokers [33 ], however use of smokeless tobacco is associated with similar cotinine levels [34 (link)].

The plant material was washed thoroughly with distilled water, dried on filter paper, and then oven dried at 60°C for 48 h. Dried plant material was ground to a fine powder using mortar and pestle and about 0.1–0.4 g of was transferred into a digestion tube containing 10 mL 69% HNO₃. The digestion tubes were closed and placed in a microwave (MARS 5 CEM) for 25 min in total (1200 W, 600 psi, 195°C for 15 min). After digestion, the extract was filtered through a 0.22 μm filter membrane. The Se content in the digested samples was determined by inductively coupled plasma–MS (ICP-MS, PerkinElmer DRC-e, Sunnyvale, CA, United States). The ICP-MS was fitted with a Babington nebulizer and a cyclonic spray chamber. The optimized instrumental parameters consisted of 1250 W power, plasma argon gas flow with a flow rate of 15 L min^-1, methane as reaction gas with a flow rate of 0.9 mL min^-1. Se isotope ⁸⁰Se was used as a control to determine the concentration of total Se in the plant material. The Na and K contents in the digests were measured using an inductively coupled plasma optical emission spectrophotometer (ICP-OES, Varian VISTA-MPX).

Using a method described in the Korean Food Standards Codex, mineral content was analyzed by mixing 1 g of MRSJ and 10 mL of HNO₃ solution, heating at 190 °C for 35 min in a microwave (Multiwave 3000, Anton Paar Gmbh, Graz, Austria) to completely remove the solvent and cooling the sample before analysis. Spectrophotometry was performed by ICP-OES (Optima 8300, PerkinElmer, Waltham, MA, USA) for other minerals and by ICP-MS (DRC-e, PerkinElmer, Waltham, MA, USA) for selenium.

Selenium speciation analysis was determined according to Lavu et al. (2013) (link); Lavu et al. (2012) (link). The seeds of pea (Ambassador and Premium variety) treated with 100 g of Se/ha as selenate were selected for Se speciation analysis. Specifically, 0.2 g of whole plant samples and 80 mg of the enzyme protease XIV were dispersed in 5 ml of water in a 10-ml centrifuge tube. The mixture was shaken for 24 h at 37°C and centrifuged for 30 min at 10,000 g. The supernatant was filtered through a 0.25-µm syringe PVDF membrane filter. The filtrate was analyzed for Se speciation by an ICP-MS (PerkinElmer DRC-e, Sunnyvale, CA, USA) coupled to a high-performance liquid chromatograph (Series 200 HPLC, Perkin Elmer, Sunnyvale, CA, USA), respectively. A Hamilton PRP-X100 anion exchange column (250 mm × 4.6 mm, 5 μm) was used as stationary phase in the HPLC instrument. The isocratic mobile phase was 10 mM citric acid with 5% (v/v) methanol, adjusted to pH 5.0. The standard solutions of the different Se species were prepared with sodium selenite (Na₂SeO₃), sodium selenate (Na₂SeO₄), Se-methionine (SeMet), Se-cystine (SeCys₂), and Se-methyl-selenocysteine (SeMetSeCys).

Bacterial cell pellets were frozen, freeze-dried, and homogenized. Samples (~0.1 g) were weighed into polytetrafluoroethylene vessels, with 6 ml of HNO₃ and 2 ml of 30% H₂O₂, for microwave digestion at 185°C, using the ETHOS E microwave digestion system (Milestone, Italy). The digestion solutions were filtered through quantitative filter papers and quantitatively adjusted to 10 ml with ultrapure water. The iron content in samples was measured by ICP-MS analysis of ⁵⁷Fe (Perkin-Elmer, ELAN DRC-e, USA), and normalized with cell numbers.

After the algal extraction, we only kept the first cm of soil of each of the four samples. After having dried the samples for one week at room temperature, we crushed the soils and sieved them at 2 mm. We measured the pH according to the ISO 10390 (2005) standard. The bioavailable part of nutrients (phosphorus (P), Total Nitrogen (TN), Dissolved Organic Carbon (DOC), silica (Si), magnesium (Mg), manganese (Mn), sodium (Na), potassium (K), iron (Fe) and aluminum (Al)) were all extracted following the method described by Houba et al. (2000) (link). We added 150 mL of 0.01M CaCl₂ to 15 g of soil in a 250 mL glass bottle and shook this mixture for 2 h. Next, we centrifuged the samples for 15 min at 5,000 rpm. The supernatant was filtered through 0.7 µm glass microfiber filters and stored at 4 °C prior to the ICP-OES (5110 VDV radial; Agilent, Santa Clara, CA, USA) analysis of P, Mg, Mn, K, Si and Na. DOC and TN concentrations were determined directly after the extraction with a Torch Combustion TOC/TN analyzer (Teledyne Tekmar, Mason, OH, USA). With a view to Fe and Al content analysis, we additionally filtered 10 ml of the supernatant with 0.45 µm syringe filters (Acrodisc^®; Pall, New York, NY, USA) and added 100 µL 1M HCl before the ICP-MS (Thermo Elemental X7/Perkin-Elmer© DRC-e) analysis. We did not estimate the free metal ions concentrations.