Sodium citrate

The following chemicals were obtained from various suppliers: o-phthalaldehyde (OPA), disodium hydrogen phosphate, sodium dihydrogen phosphate, and sodium carbonate from Nacalai tesque (Kyoto, Japan); glycine and N-acetylcysteine (NAC) from Sigma (St. Louis, MO, USA); citric acid, sodium citrate, and sodium polyacrylate from Wako (Osaka, Japan); Triton X100 and ethanol (95%) from Sigma (St. Louis, MO, USA); alcohol from Shimakyu’s Pure Chemicals (Osaka, Japan); and hydrochloric acid from Union Chemical Works Ltd. (Taichung, Taiwan). All analytical-grade reagents were used without further purification. Ultra-pure water with a resistivity greater than 18.2 MΩ cm was prepared using a Direct-Q gradient system (Millipore, Milford, MA, USA).
Buffers were prepared by dissolving the following chemicals in deionized water and storing them at room temperature as the stock: borate buffer (0.1 M, pH 8–10 with NaOH), phosphate buffer (0.1 M, pH 6–8 with NaOH), and citrate buffer (0.1 M, pH 4–6 with NaOH). The 20% OPA reagent was prepared by mixing 2 g of OPA in 10 mL of 99% ethanol. The 1% OPA reagent was freshly prepared by dissolving 0.1 g OPA in 10 mL of ultra-pure water and then diluting it to 0.6, 0.5, 0.4, 0.3, and 0.2%.

Plasma α-tocopherol deficiency was induced by treatment with probucol (Wako; Tokyo, Japan) in the diet. Six-week-old C57BL/6J mice were treated with 1% w/w probucol in the diet for 2 weeks. The control group was designated as C57BL/6J mice fed with a standard diet (75 mg/kg of α-tocopherol). After probucol treatment for 2 weeks, a subgroup of the treated mice was fed with a standard diet for 2 weeks. Blood samples were obtained under anesthesia with diethyl ether (Wako; Tokyo, Japan) by cardiac puncture using sodium citrate (Wako; Tokyo, Japan) as an anticoagulant, and then blood samples were centrifuged at 5000 rpm for 5 min at 4°C to remove the plasma and buffy coat [22 (link)].

The human breast adenocarcinoma (MCF-7) cell line was obtained from the Egyptian Holding Company for Biological Products and Vaccines (VACSERA, Giza, Egypt). Ammonium nitrate, magnesium sulfate, potassium nitrate, boric acid, cobalt chloride, cupric sulfate, manganese sulfate, potassium iodine, sodium molybdate, zinc sulfate, citric acid and sodium citrate were purchased from Wako Pure Chemical Industries^® (Tokyo, Japan). Calcium chloride, dextrose, potassium hexacyanoferrate II, potassium hexacyanoferrate III and sodium carbonate were obtained from Merck^® (Darmstadt, Germany), while potassium phosphate, sodium chloride, ferrous sulfate, Na₂-EDTA2H₂O, nicotinic acid, pyridoxineHCl, thiamineHCl, Fe-EDTA, 4-morpholineethanesulfonic acid, τ-inositol, sucrose, phytoagar, sodium phosphate, silymarin, triton X-100, 5-bromo-4-chloro-3-indoly-β-d-glucuronide and α-Rhamnase were purchased from Sigma-Aldrich Corp. (St. Louis, MO, USA). 17β-estradiol was purchased from Takeda Chemical Industries Ltd. (Osaka, Japan). Solvents used were of analytical grade unless mentioned and were purchased from Merck, J.T. Backer^® (Deventer, The Netherlands) and Theo Seulberger^® (Karlsruhe, Germany). Media and supplements for cell cultures were obtained from Gibco^®/Invitrogen (Karlsruhe, Germany) and Greiner Labortechnik^® (Frickenhausen, Germany).