NO Concentration and NOS Activity

The NO concentration was measured using a commercial kit (Jiancheng Bioengineering Institute, Nanjing, Jiangsu, China), as previously reported (Wang et al., 2018; Uyanga et al., 2020). NO is a free radical, that can rapidly oxidize to NO₂^– in the blood. In this reaction, nitrate reductase catalyzes the reduction of nitrate to nitrite, and the sum of nitrate and nitrite concentrations (NO₂^– + NO₃^–) is used to represent the total nitric oxide concentration in vivo (Bowen et al., 2007). The reaction absorbance was read using 0.5cm cuvettes at 550 nm wavelength, with a spectrophotometer (Beijing PGeneral, Beijing, China).

Nitric oxide synthase activity was measured for total NOS (tNOS), iNOS, and eNOS, using commercial kits according to the manufacturer’s protocols (Jiancheng Bioengineering Institute, Nanjing, Jiangsu, China). The NOS activity assay is an enzymatic process that is based on the biochemical conversion of arginine and molecular oxygen to produce citrulline and NO. The NO formed reacts with nucleophilic substances to produce non-ferrous compounds. The reaction occurs with co-factors including reduced nicotinamide adenine dinucleotide phosphate, oxygen, calcium, calmodulin, and tetrahydrobiopterin. The presence or absence of calcium was used to determine the calcium-dependent activity of constitutive NOS (tNOS) and the calcium-independent activity of iNOS. The absorbance of tNOS and iNOS were measured using 1 cm cuvettes at 530 nm using a UV-2450 spectrophotometer (Beijing PGeneral, Beijing, China). The assay for eNOS was performed in a 96-well plate using a microplate reader (Elx808, Bio-Tek, Winooski, VT, United States) at a wavelength of 450 nm. Assays parameters were an intra-assay CV < 10%, inter-assay CV < 12% and, sensitivity range 0.05 to 20 ng/mL. A standard curve and linear regression equation were generated using ELISAcalc software to determine chicken eNOS activity.