Determination Of Diamine Oxidase Activity Research Articles

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

BackgroundDiamine 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. MethodsFor 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. ResultsThe 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. ConclusionsAlthough 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.

Role of α2 receptor in dexmedetomidine-induced improvement in intestinal barrier dysfunction in rats with traumatic brain injury

Objective To evaluate the role of α2 receptor in dexmedetomidine-induced improvement in intestinal barrier dysfunction in rats with traumatic brain injury (TBI). Methods Forty clean-grade healthy Wistar rats, aged 12 weeks, weighing 200-250 g, were divided into 4 groups (n=10 each) using a random number table method: control group (group C), TBI group (group T), dexmedetomidine group (group D) and dexmedetomidine plus atipamezole group (group D+ A). Traumatic brain injury model was established by a 20 g weight free fall impact method in chloral hydrate-anesthetized rats.At 30 min before establishing the model, rats were fed 4.0 kD fluorescein-isothiocyanate-conjugated dextran (FITC-Dex) 60 mg/100 g. Dexmedetomidine 50 μg/kg was injected intraperitoneally at 30 min before establishing the model in D and D+ A groups.Atipamezole 500 μg/kg was injected intraperitoneally at 10 min before injecting dexmedetomidine in group D+ A.Blood samples from common carotid artery were obtained at 6 h after establishing the model for determination of concentrations of epinephrine (E) and norepinephrine (NE) in plasma (by enzyme-linked immunosorbent assay), concentrations of FITC-Dex in serum (using fluorescence spectrophotometry) and activity of diamine oxidase (DAO) in serum (by colorimetry). The small intestine was removed for determination of DAO activity (by colorimetry) and for detection of the expression of tight junctional protein zonula occludens-1 (ZO-1) in the intestinal mucosa of epithelial cells. Results Compared with group C, the concentrations of E and NE in plasma, and FITC-Dex concentration and DAO activity in serum were significantly increased, the DAO activity in intestinal tissues was decreased, and the expression of ZO-1 was down-regulated in T, D and D+ A groups (P<0.05). Compared with group T, the concentrations of E and NE in plasma, and FITC-Dex concentration and DAO activity in serum were significantly decreased, the DAO activity was increased, and the expression of ZO-1 was up-regulated in group D (P<0.05). Compared with group D, the concentrations of E and NE in plasma, and FITC-Dex concentration and DAO activity in serum were significantly increased, the DAO activity was decreased, and the expression of ZO-1 was down-regulated in group D+ A (P<0.05). Conclusion α2 receptor is involved in dexmedetomidine-induced improvement in intestinal barrier dysfunction in rats with TBI. Key words: Receptors, adrenergic, alpha; Dexmedetomidine; Craniocerebral trauma; Intestinal mucosa

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

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.

Highly Sensitive Determination of DAO Activity by Oxidation of a Luminescence Reagent

A highly sensitive method for measuring the activity of the enzyme diamine oxidase (DAO) independent of the type of substrate is described. The principle of the assay is to determine the amount hydrogen peroxide generated as a reaction product during oxidation of diamines by DAO. PSatto, a highly sensitive luminescence reagent, was used to generate a signal depending on the hydrogen peroxide concentration based on the action of horseradish peroxidase. DAO is specifically captured from a sample by an antibody immobilized to microwell plates, and the substrate is added to the bound enzyme. Various diamines were used as substrates; the peroxide produced is directly proportional to the amount of DAO bound to the specific antibodies. With this very sensitive method, it is possible to detect pmol amounts of generated hydrogen peroxide in plasma matrix corresponding to the biological activity of DAO.

Pitfalls in the determination of diamine oxidase activity

Pitfalls in the determination of diamine oxidase activity

Modified high-performance liquid chromatographic determination of diamine oxidase activity in plasma

A previous high-performance liquid chromatographic determination of diamine oxidase activity suitable for tissue homogenates was modified in order to adapt it to plasma samples. Simple additional steps were introduced after the enzyme reaction and before the chromatographic separation, both according to the previous method. In this way the sensitivity and the reproducibility of the overall procedure was suitable for routine plasma diamine oxidase estimations.

Human intestinal diamine oxidase (DAO) activity in Crohn's disease: A new marker for disease assessment?

The key-enzyme for the metabolism of diamines in man is diamine oxidase (DAO). Its highest activities are in the intestinal mucosa, localized in the cytoplasm of the mature enterocytes of the small and large bowel. If the gut is affected by inflammation in Crohn's disease macroscopical changes are observed. This prospective study investigated if these mucosal alterations are also reflected in changes of mucosal diamine oxidase activity and/or mucosal histamine content respectively. Twenty patients (12 female, 8 male; age: means = 31, range 18-49 years) undergoing gut resection because of complications in Crohn's disease (Jan.-Dec. 1988) formed the basis of the study. Tissue samples of the resected material from areas inflamed and histologically not involved in the disease were investigated for diamine oxidase activities and histamine content. Diamine oxidase activities in the mucosa obtained from the macroscopically normal proximal (155.6; (76-393) mU/g (means, range)) and distal (132; (58.5-295) mU/g) resection margins were similar to our previous findings. In all patients, however, samples from the diseased mucosa had significantly (ca. 50%) lower diamine oxidase activities (74.5; (5-262) mU/g) compared to the healthy tissue. Similar differences were found in material obtained either from whole intestinal wall or from the mucosa. The determination of diamine oxidase activity constitutes possibly a more unambiguous and earlier parameter for assessing the extent of the inflamed area than histological disease presentations. Using biopsies the necessary extent of resection could be estimated before operation: this may influence operative strategies and help in the definition of the minimum amount of inflamed gut to be removed.

Improved high-performance liquid chromatographic determination of diamine oxidase activity

Mesure de l'activite de la diamine oxydase dans la muqueuse intestinale canine par une methode d'HPLC simplifiee, rapide et adaptee aux analyses de routine. Il s'agit de la methode d'Holmsted, ou une etape d'oxydation chimique est supprimee

Determination of diamine oxidase activity by high-performance liquid chromatography

Abstract A sensitive method for the assay of diamine oxidase is described, based on the separation by reversed-phase high-performance liquid chromatography of the products arising from the oxidation of the reagents (A) homovanillic acid, (B) 3,5-dichloro-2-hydroxybenzenesulphonic acid—4-aminoantipyrine and (C) pyridoxine—4-amino-N,N-diethylaniline. The analysis time is short and the assay is quantitative and reproducible. Because of its speed and high sensitivity and selectivity, the method is a useful alternative to the standard spectrophotometric assay.

The influence of some drugs on the determination of diamine oxidase activity [proceedings

The influence of some drugs on the determination of diamine oxidase activity [proceedings

A new approach to the determination of diamine oxidase activity in tissues [proceedings

It appears that the rapid method elaborated byOkuyama andKobayashi |2| is not well suited as a measure of diamine oxidase activity in tissues.

Determination of histaminase (diamine oxidase) activity byo-dianisidine test: Interference of ceruloplasmin

Until now o-dianisidine was used as an indicator substance in a test system for the determination of diamine oxidase. More recently, however, this substance was also used to measure ceruloplasmin activity. A study of the test principles revealed that o-dianisidine was the one denominator for both enzymes. As it was found for diamine oxidase the indicator was oxidized via peroxidase mediated H2O2 cleavage. Ceruloplasmin, however, oxidized o-dianisidine directly with resulting free radical formation. An addition of histamine dihydrochloride or putrescine dihydrochloride to an incubation mixture, containing ceruloplasmin as enzyme and o-dianisidine or p-phenylene-diamine as substrates, produced an activation of the enzyme, being more than 10-fold in the presence of 1 X 10(-2) M putrescine at pH 7.0. It was assumed that an allosteric effect of the dihydrochloride component might be responsible for this activation. When the activity of purified diamine oxidase was determined by the o-dianisidine test and by the isotope assay, a very good correlation between both methods was found. But, in a mixture of diamine oxidase and ceruloplasmin, no differentiation between the two enzymic activities by the o-dianisidine test was possible. This observation demonstrated an interference of ceruloplasmin when the o-dianisidine method was used for the determination of diamine oxidase activity. To apply our findings also in vivo the amine oxidase activity increasing in guinea-pig plasma during inflammation, was determined by the o-dianisidine test and by specific methods for some amine oxidase. Despite an enhanced oxidation of the o-dianisidine observed, only an increase of ceruloplasmin activity was found. It was concluded that ceruloplasmin had no 'histaminase activity' as has been assumed by other authors using the o-dianisidine test.

The clinical-chemical application of the NADH test for the determination of diamine oxidase activity in human pregnancy.

The combined optical test for measuring DAO activity in serum of pregnant women was proved to possess a satisfactory reliability. On the average, gestational diseases related to placental insufficiency showed significantly elevated DAO activities in the 29th to 34th week of pregnancy, but lowered activities in the 35th to 40th week of pregnancy. In single cases also normal values of DAO activity were found in complicated gravidity. But when the DAO activities were higher than 0.7 mU/ml or lower than 0.15 mU/ml, never a normal pregnancy was found.

Determination of diamine oxidase activity by liquid scintillation counting

A rapid method for diamine oxidase assay is described. The method is based on the formation of radioactive toluene-extractable end product(s) from the action of diamine oxidase on cadaverine-C 14. The end products are extracted directly into toluene and assayed in a liquid scintillation spectrometer. The method is applicable using radioactive putrescine as substrate, but does not work with radioactive histamine.

Further studies of a spectrophotometric method for the determination of diamine oxidase activity

1. 1. If diamine oxidase is allowed to act upon the diamine putrescine, γ-amino-butyraldehyde is formed. This compound is condensed to Δ 1-pyrroline, which in turn can be reacted with o-aminobenzaldehyde to form a yellow compound. A photometric method for determination of diamine oxidase has been based on these reactions. 2. 2. Solutions of γ-aminobutyraldehyde obtained by hydrolysis from the corresponding acetal in known concentrations, have been allowed to react with o-aminobenzaldehyde, and the resultant extinction has been measured. In this way a calibration curve has been obtained which makes it possible to convert the extinction units for diamine oxidase activity to micromoles diamine consumed per mg enzyme and hour. 3. 3. The manometric and spectrophotometric method for determination of purified diamine oxidase agree. In tissue homogenates and partly purified enzymes the spectrophotometric measurements are uninfluenced by the occurrence of other oxidative enzymes such as catalase and peroxidase.