Gsh assay kit

PBLEs effects on the activity of the endogenous antioxidant enzymes, such as superoxide dismutase (SOD), catalase (CAT), glutathione (GSH), and thioredoxin (Trx), were assessed using the appropriate assay kits.
SOD is a critical player in the antioxidant defense mechanism in cells that converts the superoxide anion to molecular oxygen and hydrogen peroxide. Superoxide radicals produced by xanthine oxidase and hypoxanthine are detected using the SOD assay kit (Cayman Chemical, catalog no. 706002, Ann Arbor, MI, USA), and SOD activity was expressed as U/mg protein.
CAT detoxifies the cell-toxic hydrogen peroxide. The activity of CAT was measured using a CAT assay kit (Cayman Chemical, catalog no. 707002, Ann Arbor, MI, USA), following manufacturer instructions, and was expressed as U/mg protein.
The GSH assay kit (Cayman Chemical, catalog no. 703002, Ann Arbor, MI, USA) allows for the measurement of total GSH. GSH produces the yellow 5-thio-2-nitrobenzoic acid (TNB) as a byproduct of the reaction with 5,5′-dithio-bis-2-nitrobenzoic acid (DTNB). The rate of TNB synthesis is directly proportional to the GSH formation, which is quantified at 405–414 nm and represented as mol/mg protein.
For thioredoxin (Trx) activity, ELISA kit (MyBioSource, Inc., catalog No: MBS2019144, San Diego, CA, USA) was used, according to the manufacturer’s instructions.

The total amount of reduced intracellular GSH in the cell cultures was quantified using a GSH assay kit (following supplier’s protocol, Cayman Chemical Company, Ann Arbor, MI, USA) as a well-known marker for oxidative stress. Levels of oxidative stress are presented as a ratio of total GSH to the total amount of protein of each sample. Total protein content was measured by the Pierce bicinchoninic acid (BCA) protein assay kit (Pierce Protein Research Products, Thermo Scientific, Rockford, IL, USA) according to the manufacturer guidelines. Tert-Butyl Hydrogen Peroxide (tBHP) at a concentration of 135 mM acted as the positive assay control.

The method of Sedlak and Lindsay ⁷¹ (link) was adopted for the colorimetric assessment of serum malondialdehyde (MDA) using free-SH groups for measuring the peroxidation of fatty acids as an oxidative stress marker. While “master antioxidant” serum glutathione (GSH) was assessed using Cayman's GSH assay kit⁷² .

The serum levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were measured using an IDEXX VetTest Chemistry Analyzer (IDEXX Laboratories Inc., Westbrook, ME, USA). The triglyceride content was measured in 100 mg of liver tissue using a triglyceride quantification colorimetric kit from BioVision (Milpitas, CA, USA). The contents of glutathione (GSH) and oxidized glutathione (GSSG) and the content of malondialdehyde (MDA) were measured in 100 mg of the liver tissues using a GSH assay kit and a TBARS assay kit from Cayman Chemical (Ann Arbor, MI, USA), respectively. All of the measurements were conducted in accordance with the manufacturer’s instructions.

The GSH evaluation was carried out using a GSH assay kit (Cayman Chemical Company, Ann Arbor, MI). After the 24-h reperfusion, the brain (n = 6 each group) was homogenized in a buffer (50 mM phosphate, pH 6–7, containing 1 mM EDTA) and was then centrifuged at 10,000 × g for 15 min at 4°C. A 0.1 mol/l phosphate-buffered saline (pH 7.5) containing 0.6 mmol/l 5,5-dithiobisnitrobenzoic acid (DTNB) and 0.2 mg/ml NADPH was added to the brain supernatant. After mixing, that, the mixture was mixed and glutathione reductase was added to initiate the assay. The absorbance was recorded at 405–414 nm within 15 min. The GSH activities are expressed as μmol/g protein.

The gastric antioxidant enzyme activity was measured in the HCl/EtOH-induced gastric ulcer model. Frozen stomach tissues were pulverized with liquid nitrogen using a mortar and pestle. The ground tissues (about 200 mg) were homogenized using a homogenizer (Bead Ruptor 24 Elite, OMNI International, Kennesaw, GA, USA) in 1 mL of 50 mM sodium phosphate buffer (pH 7.0) containing 1 mM ethylenediaminetetraacetic acid (EDTA). Homogenates were centrifuged at 4000 rpm for 5 min at 4 °C and the supernatant was divided into two portions; one was used to measure SOD activity, and the other was centrifuged at 10,000 rpm for 15 min at 4 °C in order to measure the CAT and GSH activities of the resultant supernatant. The activities of SOD, CAT, and GSH were determined using a SOD assay kit (catalog no. 706002, Cayman Chemical Company, Ann Arbor, MI, USA), a CAT assay kit (catalog no. 707002, Cayman Chemical Company, USA), and a GSH assay kit (catalog no. 703002, Cayman Chemical Company, USA), respectively, according to the manufacturers’ protocols. The absorbance was measured at 450, 540, and 410 nm, respectively, using a microplate spectrophotometer (Epoch 2, BioTek, Winooski, VT, USA). The antioxidant enzyme activity was normalized to the wet gastric tissue weight.