Measurement of the DAO activity

To implement the measurement of the enzymatic activity of DAO, the technique published by Takagi et al (1994)10 was standardized using serum or plasma. To achieve the optimal conditions for the measurement of enzymatic activity different volumes of sample, concentrations of substrates, and reagents were used. To find the wavelength of greater absorption, readings were made at different wavelengths. Briefly, this technique is based on the measurement of DAO's ability to degrade cadaverine, a process in which hydrogen peroxide is generated. Then chromogen DA-67 was used, which is quantitatively oxidized in the presence of hydrogen peroxide and peroxidase, to be transformed into methylene blue (Fig. 1). To eliminate the interference caused by the presence of ascorbic acid in the serum, an interfering agent of the peroxidase, we used the enzyme ascorbate oxidase. Finally the reaction was stopped with sodium diethyl-dithiocarbamate (stop solution), allowing the formation of methylene blue, which remained stable for 2 h, measured the absorbance with a maximum absorbance of 668 nm. Incubations at 37 °C were performed in a thermo regulated bath.

The principle of the assay method of diamine oxidase (DAO) activity. The amount of the methylene blue formed as an enzymatic oxidative product was spectrophotometrically measured at 668 nm (Figure obtained from article published by Takagi et al. 1994, reference Nº10)