Zeenit 700p

Whole blood was collected from children under fasting state. Serum was obtained after centrifugation at 3000× g for 5 min and stored at −80 ℃. All the procedures were conducted in accordance with proposed protocols of the International Zinc Nutrition Consultative Group (IZiNCG) [40 (link)].
After being thawed at room temperature, flame atomic absorption spectrometer (Analytik Jena, ZEEnit 700P, Jena, Germany) was used to detected serum zinc. Enzyme-linked immunosorbent assay (ELISA) kits (Elabscience, Wuhan, China) were used to detect the level of serum inflammatory cytokines IL-6, IL-1β, and TNF-α.

SMMC-7721 cells transfected with siRNA for CTR1 or sncRNA were treated with different concentrations of Cu²⁺ for 24 h and then trypsinized, rinsed three times with PBS. Cells were harvested and digested with 30 μL 65% nitric acid at 60°C for 2 h. Samples were diluted 3 000 times with deionized water and the copper concentrations were determined with flame atomic absorption spectrometer (ZEEnit700P, Analytik Jena).

All reagents were guaranteed grade. Nitric acid, perchloric acid and hydrochloric acid were purchased from Beijing Chemical Reagent Research Institute. Sodium acetate and potassium chloride were supplied by Tianjin Guangfu Science and Technology Development Co., Ltd. All solutions were prepared with double-distilled water. Cd standard solution (1,000 μg mL⁻¹, GBW08612) was supplied by the National Institute of Metrology, China. Argon (99.99%) was provided by Beijing North Temperature Gas Factory. A dialysis bag with a molecular weight cutoff of 3500 was purchased from Viskase, USA.
Cd in the extracts was quantified by ICP-MS (7500CX, Agilent, USA) and AAS (ZEEnit 700 P, Analytik Jena, Germany). All voltammetric measurements were conducted by differential pulse anodic stripping voltammetry (HM-5000P, Jiangsu Tianrui, China). The electrode stand consisted of a 3 mm diameter glassy carbon electrode as the working electrode, double-junction Ag/AgCl (3 M KCl, saturated AgCl, and 3 M KCl in the bridge) as the reference electrode, and platinum wire as the auxiliary electrode. A microwave (MARS X, CEM, USA) accelerated reaction system was used for comparison with the sample treatment. A high-speed centrifuge (3-30KS, Sigma Laborzentrifugen GmbH, Germany) was used to separate the extracting solutions.

The subcellular fractions of cucumber roots were separated according to the method described by Jabeen et al. (2014) (link). Root samples were fully homogenized with 5 ml extracting solution (0.25 mol L⁻¹ sucrose, 50 mmol L⁻¹, pH 7.5 Tris-HCl, and 1 mmol L⁻¹ dithioerythritol), and then filtered through a filter cloth (80 μm). The filter residue was the cell wall fraction. The filtrate was made up to volume (40 ml) with the extraction solution mentioned previously. The solution was centrifuged at 1,500 × g for 10 min to obtain the sediment (the plastid fraction). The supernatant was then centrifuged at 5,000 × g for 20 min to obtain the nucleus fraction, and again at 15,000 × g for 30 min to obtain the mitochondrial fraction. The final supernatant represented the soluble fraction of the root cell. Every subcellular fraction was dried in an oven, and the Na⁺ and K⁺ contents were determined using an atomic absorption spectrometer (ZEEnit 700P, Analytik Jena, Germany).

Urinary zinc measurement was performed in the Atomic Absorption Laboratory (Baldi e Riberi), AOU Città della salute e della Scienza di Torino, Turin, Italy.
Samples were first acidified with 100 μL 18% HCl and then diluted 1: 5 with CsLaCl 1 g/L (1 mL sample + 4 mL CsLaCl 1 g/L). They were then analyzed by the flame atomic absorption technique. In addition, four calibration curve standards were used for the analysis (S1: 4.5 mL CsLaCl 1 g/L + 400 μl H2O + 100 μL Std Cu/Zn 1 mg/L; S2: 4.5 mL CsLaCl 1 g/L + 500 μL Std Cu/Zn 0.5 mg/L; S3: 4.5 mL CsLaCl 1 g/L + 500 μL Std Cu/Zn 1 mg/L; S4: 4.5 mL CsLaCl 1 g/L + 500 μL Std Cu/Zn 2 mg/L), and two controls (Seronorm Urine L-1 and Seronorm Urine L-2) diluted 1: 4 with CsLaCl 1 g/L (1 ml control + 3 ml CsLaCl 1 g/L).
Once prepared, the samples were analyzed on the Zeenit 700p instrument (flame atomic absorption spectrometer, Analytik Jena).