Hclo4

The diet and liver samples were previously mineralized by wet method in a sand bath (J.R. Selecta, Barcelona, Spain); the samples were placed in a resistant flask and dissolved using nitric acid followed by a mixture of HNO₃:HClO₄ (69%:70%, v/v; Merck KGaA, Darmstadt, Germany; ratio 1:4, v/v) until the total elimination of organic matter. Finally, the samples were diluted with bidistilled ultrapure Milli-Q water. Fe analysis was undertaken using an Optima 8300 (PerkinElmer Inc. Waltham, MA, USA) inductively coupled plasma-optical emission spectrometer (ICP-OES) with a segmented-array charge-coupled Device (SCD) high-performance detector. Fe was analysed in liver and diets according to compatibility under optimised set of conditions. For the calibration of the apparatus, multi elemental Astasol calibration solutions (Analytika, Khodlova, Prague) were used. For the calibration curve, the following working dilutions of the analytical standard were prepared: 0.1, 0.5, 1.0, 10, 50 mg/L. An internal standard solution of 10 mg L⁻¹ was used after each series of five samples. The acceptable result was assessed as 10%. The samples were measured in three replicates.

A potentiostat (PST 006) from Voltalab-Radiometer Analytical with software Model voltaMaster 4 was used for electrochemical measurements. All voltammograms were recorded with a three-electrode cell containing a 3 mm GC electrode as a working electrode, a platinum wire as an auxiliary electrode and silver/silver chloride (Ag/AgCl) as a reference one. Diamond paste 2.0 μM was used to clean the working electrode. A digital pH-meter (Woon Socket, Ri0285, USA) was used for pH measurements. HQ, CC and RC obtained from Merck and HClO₄ 70% (AR) from Guangdong China were used as received. 1,5-DAN was purchased from Merck. Potassium dibasic phosphate (K₂HPO₄), potassium monobasic phosphate (KH₂PO₄) and hydrochloric acid (HCl) of analytical grade and bidistilled water were used.

The commercial polymers Eudragit^® E 100 and Eudragit^® E PO were acquired from Evonik (Essen, Germany), whereas HCl, NaOH, acetic acid, HClO₄, isopropanol, and ethylene glycol were purchased from Merck KGaA (Darmstadt, Germany). Type II water was obtained from a purification system (Millipore Elix Essential, Merck KGaA, Darmstadt, Germany).

All the chemicals and reagents used were commercially available and were of the highest grade purity or analytical purity. Indoxyl sulfate potassium salt, the monoamines (NE, E, DA, 5HT) and their metabolites (DOPAC, 5HIAA), sucrose, sodium acetate, EDTA-2NA were purchased from Sigma-Aldrich, USA. Acetic acid, acetonitrile, citric acid, HClO₄, 1-octanesulfonic acid sodium were purchased from Merck, Germany.

1, 1-diphenyl, 2-picryl hydrazyl (DPPH), Ascorbic acid (Vitamin C), Gallic acid, Quercetin, tetrazolium chloride, Folin Ciocalteu’s reagent, 5-LOX enzyme from soybean, KH₂PO₄ buffer, and Baicalein were all purchased from Sigma-Aldrich, St. Louis, MO, USA. DMSO dimethyl sulphoxide, and Methanol (analytical) were purchased from Merck, Germany. HClO₄, H₂SO₄, and Standards of Na, K, Ca, Fe, Pb, Mn, Cr, Zn, Cd, Co, Hg and Ni procured from Merck, Germany.

All peptides were
purchased from KareBay Biochem, Inc., with a certified purity: L680,
98.27%; L681, 98.08%. The identity of ligands was confirmed by mass
spectrometry. The purity was examined by potentiometric titrations
with the use of the Gran method.^{37 (link)} The
solutions of metal ions were prepared using ZnClO₄ and
CdClO₄ (POCh, high-performance liquid chromatography grade).
The metal salts were dissolved in doubly distilled and filtered water.
The concentration of a stock solution was periodically checked via
ICP-MS. The solution of 4 × 10^–3 M HClO₄ (Merck) was used to prepare all samples of the peptides.
The ionic strength was adjusted to 0.1 mol dm^–3 by
adding KClO₄ (Merck).

Analytical grade Eriochrome black T, HCl (37%), HClO₄ (70%), HAuCl₄, and ethanol (99%) were purchased from Merck or Sigma-Aldrich. NaOH (pellets, purity ≥ 98%) was supplied from Riedel. All solutions were prepared using ultra-pure water (Milli-Q 18.2 MΩ cm, Millipore System Inc.). Highly pure nitrogen was used to remove oxygen from the solution.