Colon samples were homogenized in a solution containing 0.25 M sucrose, 20 mM Tris, and 1 mM dithiothreitol (DTT) and centrifuged at 15.000 g for 30 min at 4 8C. The supernatants were incubated in 0.1 M CaCl 2 , 0.25 M sucrose, 20 mM Tris, and 1 mM DTT at 0 8C for 30 min and then centrifuged at 21.450 g for 60 min at 4 8C. The cytosolic fraction was used for further analyses. A diluent buffer containing 125 mM Na phosphate and 6.0 mM EDTA was used for diluting stock solutions of glutathione (GSH), glutathione reductase, 5,5 0 dithio-bis-2nitrobenzoic acid (DTNB), and b-nicotinamide adenine dinucleotide phosphate (b-NADPH).
All reagents were purchased from Sigma-Aldrich. 40 mL of blank, standard, or colon sample and an equal volume of DTNB stock solution, then b-NADPH (140 mL) were added to each well. After 5 min incubation at 25 8C, 10 mL glutathione reductase was added to each well to initiate the reaction. After 10 minutes the absorbance was measured at 405 nm in a microplate reader. Results are expressed as nmol/mg protein.
All reagents were purchased from Sigma-Aldrich. 40 mL of blank, standard, or colon sample and an equal volume of DTNB stock solution, then b-NADPH (140 mL) were added to each well. After 5 min incubation at 25 8C, 10 mL glutathione reductase was added to each well to initiate the reaction. After 10 minutes the absorbance was measured at 405 nm in a microplate reader. Results are expressed as nmol/mg protein.