Horseradish peroxidase solution

Spectrophotometric kits was used to analyze several parameters in samples, including CAT activities, and MDA, NO and H₂O₂ levels in the serum; T-AOC activity; and MDA and H₂O₂ levels in the liver and kidney; as well as T-AOC activity and MDA level in the jejunum and ileum. The protocol was conducted according to the manufacturer’s instructions (Nanjing Jiancheng Biotechnology Institute, China). The reaction system for determining DAO concentration included 0.1 ml (4 µg) horseradish peroxidase solution (Sigma), 3 ml 0.2 M, pH 7.2 PBS, 0.1 ml O-dianisidine methanol solution (500 µg of O-dianisidine) (Sigma), 0.5 ml sample, and 0.1 ml substrate solution (175 µg of cadaverine dihydrochloride) (Sigma). Then the processed samples were incubated in an incubator chamber at 37°C for 30 min and measured at 436 nm by UV/visible spectrophotometer-UV–2450 (SHIMADZU, Japan) [24] . Plasma D-lactate was determined using assay kit in accordance with the manufacturer’s instruction (BioVision Inc., America). The serum was separated with centrifugation at 3,500×g for 15 min at 4°C and stored at −20°C. Concentrations of amino acids in the serum were detected via LC-MS/MS (HPLC Ultimate3000 and 3200 Q TRAP LC-MS/MS). Serum amino acids were analyzed with isotope dilution liquid chromatography-mass spectrometry methods by Beijing Amino Medical Research CO., LTD, Beijing, China.

DAO activity was assayed according to the modified method of Nobumichi et al. [11 (link)]. In the final volume of 3.8 ml, the assay mixture contained 3ml of phosphate buffer (0.2 M, pH 7.2), 0.1 ml (4 μg) of horseradish peroxidase solution (Sigma), 0.1 ml of o-dianisidine methanol solution (500μg of o-dianisidine purchased), 0.5 ml plasma; 0.1 ml of substrate solution (175 μg of cadaverine dihydrochloride from Sigma). This sample was incubated for 30 min at 37°C, and DAO activity was measured as the absorbance at 436 nm.