2.4. Myeloperoxidase (MPO) assay

MPO activities in proximal colon tissues were measured as previously reported (Rodriguez‐Palacios, Aladyshkina, & Cominelli, ²⁰¹⁵). Preweighed tissue samples (stored at −80°C for no more than 48 h) were homogenized in 20 volumes of ice‐cold potassium phosphate buffer (50 mmol/L K₂HPO₄ and 50 mmol/L KH₂PO₄, pH 6.0) containing 0.5% hexadecyltrimethylammonium bromide using the Bioprep‐6 Homogenizer (Aosheng, China) in the presence of ceramic beads. The homogenates were centrifuged at 12,500 rpm for 5 min at 4°C. After loading of 10 μl sample supernatant to each well on the prechilled 96‐well plates, 200 μl of active dianisidine substrate containing 0.2 mg/ml o‐dianisidine hydrochloride and 0.001% H₂O₂ was added to each sample well, and the absorbance at 450 nm was measured in a Synergy H4 hybrid microplate reader (Bio‐Tek, Winooski, VT, USA) every 30 s for 5 min at 28°C. One unit of MPO activity was calculated as: MPO activity = ΔA450 ÷ 0.5 ÷ 0.0113 ÷ 0.05; where ΔA450 was the average of ΔA450_(t30‐t0) (the absorbance difference from time 0 s to 30 s) and ΔA450_(t60‐t30) (the absorbance difference from time 30 s to 60 s); 0.0113 is MPO constant; 0.5 is for time intervals (0.5 min), and 0.05 is dilution factor of sample:HTBA lysis buffer (50 mg:1 ml).