Hetab

About 200 mg of esophageal samples were homogenized in 10 volumes of cold potassium phosphate buffer (50 mM K₂HPO₄, pH 6.0; Sigma, MO, USA) with hexadecyltrimethyl-ammonium bromide (HETAB; 0.5% w/v; Sigma, MO, USA). After centrifugation at 12,000 ×g and 4°C for 10 min, the supernatant was removed and rehomogenization of remains was done with an equivalent volume of 50 mM K₂HPO₄ containing 0.5% (w/v) HETAB and 10 mM EDTA (Sigma, MO, USA). The measurement of H₂O₂-dependent oxidation of o-dianizidine·2 HCl was performed for estimation of MPO activity. MPO levels/g of tissue weight that caused a 1.0/min change in absorbance at 460 nm and 37°C was expressed as one unit (U) of enzyme activity [22 (link)].

Briefly, tissue was weighed and suspended (10%, wt/vol) in 50 mM potassium phosphate buffer (KPi, Sigma-Aldrich, USA), containing 0.5% hexadecyl-trimethyl-ammonium-bromide (HETAB) (Sigma-Aldrich). Samples were first homogenized, then sonicated and subsequently centrifuged for 10 min at 12,000 rpm at 4 °C. For each sample, a mixture containing 2810 μL of KPi, 30 μL OD dihydrochloride (Sigma-Aldrich), and 30 μL hydrogen peroxide (20 mM, Sigma-Aldrich) was prepared. Instead of OD dihydrochloride, blanks contained 30 μL distilled water. The start of the reaction was induced by adding 100 μL of supernatant followed by an incubation time for 10 min at room temperature. The reaction was terminated by adding 30 μL 2% sodium azide (Fisher Scientific, Waltham, MA, USA), and the resulting change in absorbance 460 nm was read using a spectrophotometer (Biochrom Ltd., Berlin, Germany). MPO activity (U/g tissue) was expressed as the amount of enzyme necessary to produce a change in absorbance of 1.0 per minute per gram of wet weight of tissue.