R. Hesterberg

Distribution and properties of human intestinal diamine oxidase and its relevance for the histamine catabolism

High activities of diamine oxidase (EC 1.4.3.6) were measured in the intestinal tract of human subjects and of several mammalian species. The enzyme was localized in the mucosa and was distributed primarily in the cytoplasm; the only exception being the guinea-pig where it was located in the particulate fraction. Despite its instability the enzyme from human colonic mucosa was purified 80-fold. During the purification a soluble monoamine oxidase (EC 1.4.3.4) was separated from diamine oxidase. The pH optima of diamine oxidase for putrescine and histamine were 6.6-7.0 and 6.4-6.6, respectively. Short-chain aliphatic diamines were deaminated with the highest reaction velocity, but histamine and N tau-methylhistamine were also excellent substrates. The Km for putrescine was 8.3 x 10(-5) M, for histamine 1.9 x 10(-5) M and for N tau-methylhistamine 9.7 x 10(-5) M. Typical substrates of monoamine oxidase were not deaminated by the enzyme. Aminoguanidine strongly inhibited human intestinal diamine oxidase (IC50 = 1.1 x 10(-8) M). Because of its properties the intestinal diamine oxidase is considered to play a protective role against histamine in diseases such as ischaemic bowel syndrome, mesenteric infarction and ulcerative colitis.

Comparison of the 14-C-putrescine assay with the NADH test for the determination of diamine oxidase: description of a standard procedure with a high precision and an improved accuracy.

A standard procedure for the determination of the diamine oxidase (DAO) activity is described as a modification of the method ofOkuyama andKobyashi [2]. The principle of this method is the assay of14C-Δ1-pyrroline and its polymers formed by the oxydative deamination of14C-putrescine in the presence of DAO. This assay was shown to possess a high sensitivity and precision. Now its accuracy could be enhanced by the comparison with the NADH test for DAO activity. This kind of a reference method made the direct correlation between cpm/min and deamination rates possible. Thus International Units (mU/sample) could be introduced into the isotope assay: In our test system 1 mU corresponded to 215 cpm/min, this value depending on the specific radioactivity of the substrate solution.The DAO activity from dogs small intestine was investigated with these two methods. The pH-optimum for this enzyme was 7.6, the Km for putrescine as substrate 1×10−4M, the optimum substrate concentration was 1×10−3M. In the small intestine of dogs and rabbits the DAO activity increased from proximal to distal parts, but in human beings the distribution of the enzyme in the gut did not show such a characteristic gradient.In the discussion, the advantages and disadvantages of the two tests for the determination of DAO activity were compared with each other. The isotope assay was the more sensitive and convenient test. But due to its absolute specificity the NADH test was useful as a reference method and for the investigation of the substrate specificity of the DAO in various tissues and body fluids.

Inhibition of human and canine diamine oxidase by drugs used in an intensive care unit: relevance for clinical side effects?

Three hundred and forty-one drugs, commonly used in intensive care units (ICU), were chosen for an investigation of possible activation or inhibition of the histamine metabolizing enzyme diamine oxidase (DAO). After examination of 164 substances, using both canine and human DAO in anin vitro screening test, 61 agents inhibited DAO activity to various degrees. Of these, 44 inhibited the enzyme from both species, 4 inhibited the canine enzyme only and 13 the human DAO only. No. compound tested was able to enhance the enzyme activity.The inhibiting agents included representatives of all major therapeutic groups. A particlarly strong inhibition was observed with the neuromuscular blocking drugsd-tubocurarine, pancuronium and alcuronium, however, the other commonly used neuromuscular blocking drug, suxamethonium, was without effect. Similarly with the cephalosporines, cefotiame and cefuroxime caused a marked inhibition of the human DAO activity, whereas another regularly-used substance of this class, cefotaxime, inhibited neither the human nor the canine enzyme in concentrations up to 10−3M. The observation that within a given therapeutic group some members inhibit and others do not, could be useful in choosing a therapy concept which minimizes the risk of a more severe ‘histamine’ reaction in seriously ill patients.

Histamine content, diamine oxidase activity and histamine methyltransferase activity in human tissues: Fact or fictions?

To understand the role of histamine in the aetiology and pathogenesis of human diseases reliable data are urgently needed for the histamine content and for the activities of histamine-forming and-inactivating enzymes in human tissues. In order to make a substantial progress toward this aim a tissue-sampling programme during surgical interventions was carefully conceived and conducted. From March 1982 until January 1983 106 tissue specimens were taken from 56 patients who underwent surgery. Only healthy tissues, not injured or oedematous, and without adherent structures were taken by only one surgeon who was interested in this research and experienced in tissue preparation procedures in biochemistry. The times of ‘warm’ ischaemia during the operative procedures were visually estimated, the times between resection of the organs or specimens and deep-freezing of the tissues were precisely recorded.Compared to previous work in the literature and especially to our own work using the same assays for determination higher histamine contents were found in this study in most of the tissues, in particular in the gastrointestinal tract. Also the diamine oxidase activities were considerably higher in many organs, e.g. 3–4 times higher in the gastrointestinal tract when compared with those in publications of our group who used always the same analytical test. However, the histamine methyltransferase activities in this study were not at variance to those determined in previous investigations. Many of them were reported in this communication for the first time.Since the methods for histamine determination and those for measuring enzymic activities were not different in this study and in previous communications of our group we are convinced that the optimized tissue-sampling and-preparation techniques were responsible for the higher values in this communication. But the problem of the ‘warm’ ischaemia period could not be solved by sample-taking procedures of this type during operations. There are good reasons to prefer biopsy specimens for the analysis of histamine storage and metabolism in human tissues in health and disease, but — unfortunately — they are not always available.

The influence of carcinoma growth on diamine oxidase activity in human gastrointestinal tract

The distribution of diamine oxidase (DAO) activity was studied in patients having no carcinoma disease. Besides in gut DAO occurred in high activity only in kidney and mesenteric lymph nodes.In patients with adenocarcinoma of the large bowel or of the stomach the enzymic activity was reduced in the tumour tissue itself as compared with the adjacent mucosa in which part the highest activities were observed. In stomach it seemed most important that the enzymic activity was enhanced in the whole mucosa, also in a 10 cm distance from tumour where no histological alterations were found. This fact should be checked for significance in early tumour diagnosis.

Diamine oxydase in rabbit small intestine: Separation from a soluble monoamine oxidase, properties and pathophysiological significance in intestinal ischemia

From all mammals investigated so far only in rabbits diamine oxidase could not be detected in any tissue except the gut. Thus this species was chosen for studying the physiological and pathophysiological function of this enzyme in the gastrointestinal tract.By gel filtration on Sephadex G 50 and G 200 the enzyme was purified 100-fold, separated from a soluble monoamine oxidase, and the properties of the two enzymes were determined. Diamine oxidase from rabbit small intestine deaminated putrescine (Km=1.3×10−4M, pH-optimum 6.4–6.9) and histamine (Km=8×10−5M, pH-optimum 7.5), but not serotonin, and was inhibited by aminoguanidine, but not by pargyline. Soluble monoamine oxidase from rabbit small intestine catabolized serotonin (Km=1.8×10−4M, pH-optimum 8.8), but not putrescine and histamine, and was inhibited by pargyline, but not by aminoguanidine.Based on its properties in vitro intestinal diamine oxidase could inactivate the vasoactive biogenic amine histamine in vivo. To confirm this hypothesis, in rabbits the small intestine was damaged severely by inducing total intestinal ischemia, which occurs as mesenteric infarction also in human subjects and is accompanied by histamine release. Treatment with aminoguanidine and ischemia killed the animals 3-times faster than ischemia alone, which supported our hypothesis on a protective role of intestinal diamine oxidase against histamine.

The start of a programme for measuring diamine oxidase activity in biopsy specimens of human rectal mucosa

In human subjects, apart from in the kidney, diamine oxidase occurs mainly in the gut. Therefore this enzyme can be used as an indicator of intestinal integrity.In biopsies of rectal mucosa the diamine oxidase activity was assayed in 55 patients, 41 having a histologically normal mucosa and 14 being diseased. The determinations of the enzymic activity were supervised by statistical quality control.In the unchanged rectal mucosa the diamine oxidase activity was 40 nmol/min×g on average. In 7 patients with rectal polyps the enzymic activity was significantly diminished in these benign tumours (x=7.7 nmol/min×g) apart from one, where it was elevated. A decrease in diamine oxidase activity was further observed in rectal carcinoma and ulcerative colitis.Whether the reduction of intestinal diamine oxidase activity accompanies premalignant or malignant states or whether it is a general sign of a disturbance of intestinal integrity remains questionable.

Inhibition of intestinal diamine oxidase by detergents: a problem for drug formulations with water insoluble agents applied by the intravenous route?

One hundred and twenty four water-insoluble drugs were included in a study for their action on diamine oxidase (DAO) after solubilization with 61 detergents. 16 detergents were themselves not watersoluble and were not further investigated. A further 3 detergents affected the extraction procedure and 7 of the remaining 42 detergents themselves inhibited the activity of canine intestinal DAOin vitro. Only 5 detergents fulfilled all prerequisites for our DAO assay, including the solubilization of 76 water-insoluble drugs. Each of these 5 detergents had an individual range of suitability in our test system. 3/76 drugs inhibited DAO in concentrations up to 10−3M. This result is in contrast to our study with water-soluble substances, where 16% were DAO inhibitors. Since detergents can block the enzyme which is responsible for histamine catabolism, some of the observed adverse reactions to drugs could arise because of the presence of such detergents in the formulation.

Veränderungen der Diaminoxydaseaktivität im Gastrointestinaltrakt bei Carcinompatienten

Die Bestimmung von “Tumormarkern” gewinnt bei der Diagnostik und der postoperativen Verlaufskontrolle von Krebserkrankungen zunehmend an Bedeutung. Unter diese “Tumormarker” ist auch das Enzym Diaminoxydase (DAO) einzureihen. Dies gilt allerdings nur fur den speziellen Fall des medullaren Schilddrusencarcinoms (1).