Standardization of a colorimetric technique for determination of enzymatic activity of diamine oxidase (DAO) and its application in patients with clinical diagnosis of histamine intolerance

Camila Beltrán-Ortiz; Teresa Peralta; Verónica Ramos; Magdalena Durán; Carolina Behrens; Daniella Maureira; Maria A Guzmán; Carla Bastias; Pablo Ferrer

doi:10.1016/j.waojou.2020.100457

Standardization of a colorimetric technique for determination of enzymatic activity of diamine oxidase (DAO) and its application in patients with clinical diagnosis of histamine intolerance

World Allergy Organ J. 2020 Aug 29;13(9):100457. doi: 10.1016/j.waojou.2020.100457. eCollection 2020 Sep.

Authors

Camila Beltrán-Ortiz¹, Teresa Peralta¹, Verónica Ramos¹, Magdalena Durán¹, Carolina Behrens¹, Daniella Maureira¹, Maria A Guzmán¹, Carla Bastias¹, Pablo Ferrer¹

Affiliation

¹ Section of Immunology, HIV and Allergy, Department of Medicine, Clinical Hospital University of Chile, Chile.

Abstract

Background: Diamine Oxidase (DAO) has an essential role for degradation of exogenous histamine in the intestine; thus, histamine intolerance (HI) mainly has been correlated to a low concentration and/or activity of this enzyme. The objective of the study was to standardize a colorimetric technique to measure the enzymatic activity (function) of hDAO to then apply it to a series of 22 patients with a clinical diagnosis of HI.

Methods: For the standardization variables such as volume and type of sample, incubation time, wavelength of maximum absorption, types of substrates, and concentration of oxidized ascorbate were evaluated. Then the activity and concentration of DAO was determined in 22 patients diagnosed with HI and 22 healthy subjects.

Results: The mean of serum DAO concentration in the 22 patients was of 9.268 ± 1.124 U/mL. The mean of serum DAO concentration in the 22 controls was of 20.710 ± 2.509 U/mL, being significantly higher (P value 0.0002) the mean of the samples. The mean of serum DAO activity of the patients was of 1.143 ± 0.085 U/L and the controls was 1.533 ± 0.119 U/L, significantly greater than the patients (P value 0.011). In addition, the sensitivity of both techniques was 0.63. In the measuring of DAO concentration the specificity was 0.9, constituting a good diagnostic test, especially to rule out the true negatives. The determination of DAO activity had a specificity of 0.68.

Conclusions: Although we used a small number of patients and controls and the absorbance values were lower than expected, statistically significant differences were found in the levels of concentration and DAO activity between the patients with histamine intolerance and the controls. Therefore, the measuring of DAO concentration and DAO activity is a good diagnostic strategy for study suspect cases of HI. The simultaneous use of both assays allows to reduce positive and negative false results, for example, patients with normal DAO levels that could present a dysfunction in the activity of this enzyme.

Keywords: ASOD, Ascorbate Oxidase; Activity; DAO; DAO, Diamine Oxidase; Diamine oxidase; ELISA, Enzyme-Linked Immuno Sorbent Assay; HI, Histamine Intolerance; Histamine; Histamine intolerance; ROC, Receiver Operating Characteristic; TMB, Tetramethylbenzidine; hDAO, Human Diamine Oxidase.