4.7.2. Peroxidase Activity

Total peroxidase activity in plasma and intestine was measured, following the procedure described by Quade and Roth [58]. Briefly, 10 μL of plasma and 5 μL of intestine homogenate were diluted with 140 and 145 μL, respectively, of HBSS without Ca2<sup>+</sup> and Mg2<sup>+</sup> in 96-well plates. Then, 50 μL of 20 mM 3,3- ,5,5- -tetramethylbenzidine hydrochloride (TMB; Sigma-Aldrich®, Merck, Darmstadt, Germany) and 50 μL of 5 mM H2O2 were added to the wells. The reaction was stopped after 2 min by adding 50 μL of H2SO4 (2 M) and the optical density (OD) was read at 450 nm in a Synergy HT microplate reader (Biotek, Winooski, VT, USA). Wells without plasma or mucus were used as blanks. The peroxidase activity (U mL−<sup>1</sup> tissue) was determined, defining that one unit of peroxidase produces an absorbance change of 1 OD.
