Guanidine hydrochloride

Benzylaminehydrochloride (BAHC), bovine serum albumin (BSA), butylated hydroxy toluene (BHT), 1-chloro-2, 4-dinitrobenzene (CDNB), 5,5′-dithiobis (2-nitrobenzoic acid) (DTNB), epinephrine, oxidized glutathione (GSSG), reduced glutathione (GSH), hydrogen peroxide (H₂O₂), nicotinamide adenine dinucleotide phosphate (NADPH), o-phosphoric acid (OPA), thiobarbituric acid (TBA), and trichloroacetic acid (TCA) were purchased from Sigma Chemicals Co. (St. Louis, MO, USA). 1-amino-2-naphthol,4-sulfonic acid (ANSA), 2,4-dinitrophenyl hydrazine (DNPH), ethylenediaminetetraacetic acid (EDTA), and sulfosalicylic acid (SSA) were purchased from Merck Limited (Mumbai, India). Guanidine hydrochloride and sodium azide were obtained from Hi-Media Labs (Mumbai, India). STZ and HP were obtained from Sigma Aldrich (St. Louis, USA).

For the experiments, we have used various reagents, such as MacConkey broth, Luria bertani broth, Guanidine hydrochloride, Potassium Phosphate dibasic, Potassium phosphate monobasic, Sodium dodecyl sulfate, sodium hydroxide, Nitroblue tetrazolium (NBT), potassium hydroxide, 2,4-dinitrophenylhydrazine (DNPH), 2-Thiobarbituric acid (TBA), and Dimethyl sulfoxide, that were purchased from Himedia Laboratories Ltd., India. Chlorhexidine was purchased from Sigma Aldrich, Hydrochloric acid, sodium chloride and glycine were from Merck, India. Glacial acetic acid and glycerol from Qualigens, India. Ethyl acetate purchased from Fisher scientific.2-Thiobarbituric acid (0.8%) was prepared in 1M NaOH,2,4-dinitrophenylhydrazine (10mM) was prepared in 2N HCl. For PI staining, 100mM Tris (pH 7.4), 150mM NaCl, 1mM CaCl2, 0.5mM, MgCl₂, and 0.1% Triton-X were prepared, 2.5% glutaraldehyde, and ethanol.

Total glutathione equivalents (GSH_eq) consisting of both GSH and GSSG were measured following the method of Griffith (1980) (link). A protein-free supernatant was obtained by centrifugation (10,000×g for 15 min at 2°C) of the tissue homogenate (1:5w/v) in ice-cold (2°C) sulfosalicylic acid, was divided into two parts. One part was used to measure GSH_eq by observing the rate of reduction of DTNB at 412 nm containing 0.2 mM NADPH, 0.6 mM DTNB, 5 mM EDTA, 125 mM sodium phosphate buffer (pH=7.5), and tissue extract in a final volume of 1mL. To ensure the rate of reaction was zero, the reaction was started by adding GR (0.5 U). The rate of reaction is proportional to the concentration of GSH_eq and was compared with the standard curve of GSH (0-6 µM). Another part of the protein-free supernatant was treated with 170 mM 2-vinyl pyridine for 1 h to derivatize GSH. The rest of the GSSG was measured, and the total GSH was calculated from the equation GSH_eq=GSH+2GSSG, and the result was expressed as µmol/g tissue wet weight. The levels of GSH (GSH=GSH_eq-2GSSG) and percent oxidized GSH (GSSG/GSH) were also calculated.
GR and DTNB were purchased from Sigma-Aldrich, USA; NADPH, GSH, GSSG, 2-vinyl pyridine, guanidine hydrochloride, EDTA were obtained from HiMedia Laboratories Pvt. Ltd., India.