H6269

The MPO activity in the colons was determined using the following procedure⁶⁹ . Briefly, colon tissue was homogenized in 0.5% (w/v) hexadecyltrimethylammonium bromide (H6269; Sigma-aldrich, USA) in 50 mM PBS, pH 6.0. This homogenate underwent 3 freeze-thaw cycles and 10–15 s sonication to obtain homogenous suspension. The supernatant from this suspension was collected after centrifugation at 13000 × g for 20 min at 4 °C. The supernatant (10 µl) was then added to 50 mM potassium phosphate buffer (pH 6.0) containing 0.167 mg/ml o-dianisidine (Sigma-Aldrich, USA) and 0.0005% H₂O₂ (Sigma-Aldrich USA) and absorbance was taken at 450 nm (BMG, LABTECH) at 2 min interval. Units of MPO in each sample was determined by considering that one unit (U) of MPO = 1 µmol of H₂O₂ split with molar extinction coefficient of 1.13 × 10⁻² nm/min and MPO in each sample calculated by using [ΔA(t₂ − t₁)]/Δmin × (1.13 × 10⁻²) formula and MPO units were normalized with per mg tissue.

Tissue samples were homogenized in 100 mg/mL of 0.5% hexadecyltrimethylammonium bromide (Sigma, H6269) in 50 mm PBS, pH 6.0, as previously described^{46 (link)}. Following three cycles of freeze–thaw at − 80 °C and 37 °C, samples were sonicated and centrifuged at 14,000 rpm for 15 min at 4 °C. Supernatants were stored at − 20 °C until analysis. MPO was assayed in the supernatant by adding 1 mg/mL of dianisidine dihydrochloride (Sigma, D3252) and 5 × 10⁻⁴% H₂O₂ and the change in optical density measured at 450 nm.