T butyl hydroperoxide

In 23 out of 29 enrolled ACS patients, additional blood samples were collected at 24 hours, 1 week, and 1 month after administration of dual antiplatelet therapy for the assessment also of reduced and total aminothiols, Hcy, and GSH, and the activities of GSH peroxidase type-1 (GPx-1). Blood reduced GSH (r-GSH_bl) level was determined by prompt acidification of whole blood immediately after blood sample collection according to method previously described [14 (link)]. As reduced GSH levels in plasma are low (1-2%), the reduced GSH concentration in whole blood can reflect GSH content inside the cellular fraction of blood. Plasma reduced and total forms of Hcy and blood total GSH were determined according to methods validated in our laboratory [14 (link)]. The total form of GSH measured in our study includes the oxidized GSH, all conjugated forms of GSH (protein-bound GSH and GSH-mixed disulfides), produced through oxidative processes or thiol-disulfide exchange reactions, and reduced free GSH. Thiol separation was performed by high-performance liquid chromatography (HPLC) method (ProStar-Varian, Surrey, UK). Erythrocyte GPx-1 was determined using t-butyl hydroperoxide (SIGMA, Steinheim, Germany) as substrate [21 (link)]. GPx-1 activities were determined in diluted purified red blood cells, stored frozen up to analysis which was performed within 1 month from storage.

4-Cyanobenzaldehyde, benzil, 4-bromobenzoic acid, bis(pinacolato)diboron, 1,1′-carbonyldiimidazole (CDI), [1,1′-bis(diphenylphosphino)ferrocene]dichloropalladium(II)dichloride (PdCl₂ (dppf)), H₂O₂, potassium super-oxide (KO₂), t-butyl hydroperoxide (TBHP), m-chloroperoxybenzoic acid (mCPBA), hypochlorous acid (HOCl), HEPES buffer solution, Whatman papers, Dulbecco's phosphate-buffered saline (DBS), Roswell Park Memorial Institute (RPMI) medium, fetal bovine serum (FBS), penicillin, streptomycin, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Ammonium acetate, acetic acid, ethanol, hydroxylamine hydrochloride, sodium hydroxide, ethyl acetate, hexane, sodium bicarbonate, dimethylformamide (DMF), dimethyl sulfoxide (DMSO), 1,4-dioxane, and potassium acetate were purchased from Daejung Chemicals (Siheungsi, Kyunggido, Republic of Korea). Sodium nitrate (NaNO₃⁻), potassium perchlorate (KClO₄⁻), sodium sulfate (Na₂SO₄), sodium nitrite (NaNO₂), and benzoyl peroxide (BPO) were obtained from AccuStandard (New Haven, CT, USA). The MCF-7 cells were purchased from the Korean Cell Line Bank (Seoul, Republic of Korea). All reagents were used as received without further purification.

Bicinchoninic acid (BCA) solution, bovine serum albumin (BSA), t-butyl hydroperoxide (t-BHP), butylated hydroxytoluene (BHT), CCl₄, 1,1-diphenyl-2-picrylhydrazyl (DPPH), formalin, sodium carboxymethylcellulose (CMC), 2′,7′-dichlorofluorescin diacetate (DCFDA), phosphoric acid, trichloroacetic acid (TCA), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromides (MTT), and Trolox were obtained from Sigma-Aldrich Co. 2-Thiobarbituric acid (TBA) was obtained from Tokyo Chemical Industry Co. Malondialdehyde (MDA) tetrabutylammonium salt was obtained from Fluka. Oligonol is commercially available (Amino Up Chemical Co., Ltd., Sapporo, Japan).

Phenotypic plate assays were performed on YNB (without amino acids and ammonium sulfate) media supplemented with 2% glucose and 10 mM ammonium sulfate unless stated otherwise. For UV sensitivity, cells were spotted then exposed for 6 sec to UV light at 48 mJ/cm² in a UV Stratalinker (Stratagene, USA). For all other assays, the stressor was added to the media immediately prior to pouring at the following concentrations: 0.001% SDS (Sigma, USA), 5 μg/mL fluconazole (Sigma, USA), 0.25 μg/mL itraconazole (Sigma, USA), 1 μg/mL fenpropimorph (Sigma, USA), 1 M NaCl (Sigma, USA), 1 M KCl (Sigma, USA), 125 mM t-butyl hydroperoxide (Sigma, USA), 1 mM NaNO₂ (Sigma, USA), 1 μg/mL cyclohexamide (Sigma, USA) and 1 mM mercaptopurine (Sigma, USA). Tenfold serial dilutions were prepared immediately prior to testing, with plates being imaged at 24–96 hr. All assays were performed at both 30 and 37 °C.