In vitro determination of diamine oxidase activity in food matrices by an enzymatic assay coupled to UHPLC-FL

Abstract

Intestinal diamine oxidase (DAO) acts as a protective barrier against exogenous histamine. A deficit of DAO activity can lead to the appearance of histamine intolerance, a clinical condition that may be treated by a low-histamine diet and oral DAO supplementation to enhance intestinal histamine degradation. As sources of DAO, porcine kidneys and certain legume seedlings are suitable components for the formulation of a DAO supplement. The aim of this work was to develop a rapid and reliable methodology for the in vitro determination of DAO activity in food matrices based on an enzymatic assay coupled to UHPLC-FL. The proposed method showed a satisfactory linearity and sensitivity and provided a relative standard deviation lower than 3%, guaranteeing method precision, and a mean recovery greater than 99% both for lyophilized pea sprouts and porcine kidney protein extracts. A high specificity is a key attribute of this method due to the use of histamine as the reaction substrate and the direct quantification of its degradation. Moreover, the lack of interference of catalase and hydrogen peroxide is another advantage in comparison with previously published methods. Lyophilized pea sprouts showed the greatest histamine-degrading activity (0.40 ±0.01 mU/mg), followed by porcine kidney protein extracts (0.23 ±0.01 mU/mg) and commercial DAO supplements (0.09 ±0.06 mU/mg). This technique could be used as a tool to validate the DAO activity of food matrices of potential interest for the treatment of histamine intolerance.

Histamine Intolerance: The Current State of the Art

“…This technique, based on the radioactive labeling of putrescine and the scintillation counting of its consumption, is advantageous in terms of rapidity and sensitivity, but it was mainly conceived to be applicable to serum samples. Comas-Basté et al have recently developed a rapid and reliable methodology through ultra-high performance liquid chromatography and fluorimetric detection (UHPLC-FL) for the in vitro determination of DAO activity specifically for the analysis of unpurified complex matrixes, such as porcine kidney extract and DAO supplements [ 129 ]. This methodological approach is based in the direct determination of histamine degradation and overcomes certain drawbacks in terms of matrix interferences and handling of radioactive materials.…”

“…Several studies have demonstrated the capacity of this product to degrade histamine and other biogenic amines in vitro [ 57 , 129 , 130 , 131 , 132 , 133 ]. A wide variability of the DAO capacity of porcine kidney extracts has been reported (with values ranging from 0.1 to more than 100 mU/mg), depending on the purification grade applied to the matrix and/or the amine compound used as the reaction substrate [ 57 , 129 , 130 , 131 , 132 , 133 ]. In fact, many of these works sought the selective purification of the DAO enzyme to design biosensors for the biorecognition of biogenic amines as indicators of freshness in foods [ 134 ].…”

“…In fact, many of these works sought the selective purification of the DAO enzyme to design biosensors for the biorecognition of biogenic amines as indicators of freshness in foods [ 134 ]. More recently, two studies have been published specifically focused in investigating the in vitro DAO activity of porcine kidney protein extract expressly used as active ingredient to formulate food supplements for the preventive treatment of histamine intolerance [ 129 , 130 ]. Comas-Basté et al studied the in vitro enzymatic activity of 13 different production batches of porcine kidney protein extract used in the elaboration of food supplements, reporting a low influence of the raw material (porcine kidney) on the DAO activity of the extract, with a mean value of 0.23 ± 0.01 mU/mg [ 129 ].…”

“…More recently, two studies have been published specifically focused in investigating the in vitro DAO activity of porcine kidney protein extract expressly used as active ingredient to formulate food supplements for the preventive treatment of histamine intolerance [ 129 , 130 ]. Comas-Basté et al studied the in vitro enzymatic activity of 13 different production batches of porcine kidney protein extract used in the elaboration of food supplements, reporting a low influence of the raw material (porcine kidney) on the DAO activity of the extract, with a mean value of 0.23 ± 0.01 mU/mg [ 129 ]. Later, Kettner et al obtained a porcine kidney crude extract with an in vitro DAO activity of 0.5 ± 0.06 mU/mg, and described a 10-fold increase of this enzymatic activity through the application of several consecutive purification steps [ 130 ].…”

“…Regarding the food supplement, divergent results have also been reported, since while certain authors discard its enzymatic capacity, DAO activity values ranging from 0.04 to 0.20 mU/mg have also been described for different commercial products available in the market [ 129 , 130 ]. Overall, these works coincide in the need to identify alternative sources to porcine kidney DAO for exogenous supplementation in histamine-intolerant individuals.…”