Human intestinal diamine oxidase: Substrate specificity and comparative inhibitor study

For an 80-fold purified preparation of human intestinal diamine oxidase the optimum conditions of incubation, the substrate and the inhibitor specificity were tested. Putrescine was the most favoured substrate butN -methylhistamine and 2-methylhistamine were metabolized at optimum conditions with nearly the same velocity. Histamine reached about 50% of the reaction velocity of putrescine.Aminoguanidine and semicarbazide inhibited the human intestinal enzyme like a classical diamine oxidase. However, a distinct inhibition of human intestinal and pea seedling diamine oxidase was observed in presence of -aminopropionitrile (weak inhibition of the human enzyme, strong inhibition of pea seedling diamine oxidase) and burimamide (strong inhibition of human intestinal enzyme, nearly no influence on pea seedling diamine oxidase).It is proposed to differentiate on the basis of functional considerations diamine oxidases with more histamine detoxicating activities from those being more involved in regulating polyamine levels in growing tissues.Supported by A. v. Humboldt-Stiftung.Supported by a grant from Deutsche Forschungsgemeinshaft (Lo 199/7).     

N-methyl-N-formylhydrazine: A toxic and mutagenic inhibitor of the intestinal diamine oxidase

N-methyl-N-formylhydrazine is the first active intermediate of the poison gyromitrin of the mushroom: false morel. This compound is a non-competitive inhibitor of human intestinal diamine oxidase (ID₅₀=1.6×10^–5 mol/l). This concentration corresponds to less than 5g of wet weight of mushroom/l. The diamine oxidases from 5 other sources are inhibited in a similar manner. Semicarbazide and aminoguanidine are 10-respectively 1000-fold more potent inhibitors of the human intestinal diamine oxidase. An involvement of the diamine oxidase inhibitory property ofN-methyl-N-formylhydrazine in toxic and mutagenic effects of the substance is considered.Supported by Alexander von Humboldt-Stiftung.Supported by a grant from Deutsche Forschungsgemeinschaft (Ku 464/1)To whom request for reprints should be addressed.     

Diamine oxidase (DAO) activity and intestinal mucosa integrity: influence of suture techniques

After various kinds of intestinal mucosal injuries, whether by disease or by experiment, the diamine oxidase activity is reduced. Therefore, we studied the effect of surgical manipulations on the intestinal mucosa and diamine oxidase activity. The reaction of the gut on the insertion of sutures was a transient increase of the enzymic activity followed by reduction as soon as the mucosa started to gain weight. After a standardized pressure injury only a reduction of the diamine oxidase activity together with an enhancement of the mass of the intestinal wall was found. A hypothesis of a feed-back regulation of the diamine oxidase activity connected with mucosal proliferation is proposed.Supported by a grant of the Deutsche Forschungsgemeinschaft KU 464/2-2.     

Diamine oxidase in the hen

In adult hens diamine oxidase (histaminase) activity was found in gastrointestinal tract (with the highest value in ileum), liver and spleen. Intestinal diamine oxidase is predominantly a particle-bound enzyme. In the intestine oxidation of putrescine leads to Δ-pyrroline formation, in liver both Δ¹-pyrroline and γ-aminobutyric acid are formed. The inhibitor properties of hen intestinal and rat intestinal diamine oxidases are very similar and differ from pea seedling diamine oxidase. The natural dipeptides carnosine and anserine are relatively potent inhibitors of hen intestinal diamine oxidase.     

Histamine reduces the deamination of putrescinein vitro—but alsoin vivo?

The diamine oxidase catalysed deamination of putrescine was reducedin vitro by histamine at concentrations occurring in the gut. An oral application of histamine, however, had no effect on the enzymic activity. When the histamine was injected i.p. a decrease of intestinal diamine oxidase activity was observed which, however, did not depend on histamine but probably on an unspecific irritation of the gut.Supported by a grant from Deutsche Forschungsgemeinschaft (Ku 464/2-2).     

Intestinal monoamine oxidase: Does it have a role in histamine catabolism?

The importance of intestinal diamine oxidase in histamine catabolism was proved in several series of experiments. However, intestinal monoamine oxidase might also be involved in histamine degradation either by direct deamination or by the deamination of methylated products. The soluble fraction of intestinal monoamine oxidase was purified and tested for its properties and substrate specificity by three different methods which are described in detail. Using 0.15M phosphate buffer the optimum pH was 7.4–7.6. TheK _m values for serotonin and tyramine were 0.2 and 0.3×10^–3 M. The most favoured substrates of the enzyme were tyramine, tryptamine and serotonin, but it was not pissible to classify the enzyme as a type A or B monoamine oxidase only by its substrate specificity. Histamine and ring methylated derivatives were not attacked by intestinal monoamine oxidase. This means that in the intestinal mucosa the oxidative pathway of histamine is completely catalysed by diamine oxidase.Supported by a grant from the Deutsche Forschungsgemeinschaft (Ku 464/1).Supported by a grant from the Deutsche Forschungsgemeinschaft (Lo 199/7).     

The importance of human intestinal diamine oxidase in the oxidation of histamine and/or putrescine

Histamine, naturally methylated histamine and putrescine are good substrates for human intestinal diamine oxidase, N-Methyl-N-formylhydrazine, the constituent of the mashroom poison-gyromitrin is an inhibitor of human intestinal diamine oxidase. Burimamide inhibits more effectively mammalian intestinal diamine oxidases than pea seedling diamine oxidase, beta-aminopropionitrile is a better inhibitor of pea seedling enzyme than mammalian diamine oxidases. This inhibitory differences might be related to preference in histamine or putrescine oxidizing activity of these enzymes.     

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.Supported by a grant from Deutsche Forschungsgemeinschaft (Ku 464/1).     

Diamine oxidase as a marker of intestinal integrity in acute appendicitis

In the operative treatment of appendicitis the so called negative appendectomy is an important issue because of its increased morbidity. From the hypothesis that the intestinal diamine oxidase activity is a suitable marker of mucosal integrity, the distribution pattern of the enzyme in appendices histologically classified as inflammed or not inflammed was studied. Histologically apparent inflammation of the appendix was connected with a significant reduction of diamine oxidase activity. The determination of this enzymic activity may be a simple and sensitive test for mucosal inflammation of the appendix even at a very early state. This could reduce the rate of negative appendectomies and influence thereby risk-cost-benefit calculations.Supported by a grant of the Deutsche Forschungsgemeinschaft Ku 464/2-2.     

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

Until nowo-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 thato-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, oxidizedo-dianisidine directly with resulting free radical formation.An addition of histamine dihydrochloride or putrescine dihydrochloride to an incubation mixture, containing ceruloplasmin as enzyme ando-dianisidine orp-phenylene-diamine as substrates, produced an activation of the enzyme, being more than 10-fold in the presence of 1×10^–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 theo-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 theo-dianisidine test was possible. This observation demonstrated an interference of ceruloplasmin when theo-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 theo-dianisidine test and by specific methods for some amine oxidases. Despite an enhanced oxidation of theo-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 theo-dianisidine test.     

Phosphopyridoxal complexes with histamine and histidine (3) the influence of presumed complex on activity of rat intestinal histaminase

Histamine in high concentration inhibits the rat intestinal histaminase (diamine oxidase E.C. 1.4.3.6). The apparent K_m is approximately 4.2×10^–5 M. This inhibition can be reversed by an addition of PLP.It was also found that excess of PLP inhibits enzyme activity. It is competitive inhibition.Histamine and other amines which were associated with enzyme inhibition form spectrophotometricaly demonstrable complex with PLP. The possible mechanism of the inhibitory action of PLP and complex with histamine and other amines on rat intestinal histaminase activity are discussed.     

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 (K _m =1.3×10^?4 M, pH-optimum 6.4–6.9) and histamine (K _m =8×10^?5 M, 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 (K _m =1.8×10^?4 M, 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.     

Diamine oxidase activities in the large bowel mucosa of ulcerative colitis patients

The term colitis suggests mucosal inflammation as the key event. However, it may be that the disease starts with mucosal hyperproliferation, and inflammation of the impaired mucosa is a succeeding event. Therefore we studied the activity of the intestinal diamine oxidase (DAO) in ulcerative colitis (UC). This enzyme was shown to have a mucosal antiproliferative function. Biopsy specimens of 30 patients having a normal rectosigmoidal mucosa showed a DAO activity of 22.8 nmol/min g. In 12 UC patients the DAO activity was 2.7 nmol/min g (p=0.01). In 3 patients where UC was in remission the DAO activity was 103, 107 and 208 nmol/min g, indicating an antiproliferative rebound effect. Together with the strongly reduced monoamine oxidase (MAO) activity, the decrease in DAO activity indicates that the large bowel in UC is unable to produce a proliferation terminating substance (probably -aminobutyrate) derived from polyamine metabolism by oxidative deamination (DAO) or by the interconversion pathway (MAO).     

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: , 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 ( 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 estimatedbefore operation: this may influence operative strategies and help in the definition of the minimum amount of inflamed gut to be removed.Supported by grant of Deutsche Forschungsgemeinschaft (Lo 199/15-2).     

骨髓间充质干细胞移植保护小肠缺血再灌注损伤

BACKGROUND: Bone marrow mesenchymal stem cells have good proliferation and paracrine functions, which have irreplaceable advantages in the treatment of intestinal diseases. OBJECTIVE: To explore the effects of bone marrow mesenchymal stem cell transplantation on intestinal ischemia-reperfusion injury in rats. METHODS: Forty-eight Sprague-Dawley rats were enrolled to make animal models of ischemic reperfusion injury of the intestine, and then model rats were randomized into experimental and control groups. After modeling, 1 mL bone marrow mesenchymal stem cells or the same volume of normal saline were injected into the intestinal mucosa of rats in the two groups, respectively. At hours 0, 2, 6, 24, 72, 120 after injection, serum diamine oxidase, tumor necrosis factor α, and D-lactic acid levels were detected by ELISA method. At 24 hours after injection, rat intestinal tissues were taken and observed pathologically under light microscopy, and their close connections were observed under transmission electron microscope. ZO-1 protein levels were detected by immunohistochemistry method. RESULTS AND CONCLUSION: Compared with the control group, the serum diamine oxidase, tumor necrosis factor α, and D-lactic acid levels were significantly lower in the experimental group at hours 6 and 24 after injection (P < 0.05). Intestinal necrosis, villous edema, intestinal congestion and inflammatory cell infiltration in the experimental group were milder than those in the control group. In addition, the ZO-1 protein expression in the experimental group was higher than that in the control group. Experimental results show that bone marrow mesenchymal stem cell transplantation into the intestinal mucosa can improve the intestinal mucosal permeability in rats with intestinal ischemia-reperfusion injury.      

The response of histamine degrading enzymes to nematode infection

The response of intestinal mucosal enzymes which metabolize histamine i.e. diamine oxidase (DAO), histamine N-methyltransferase (HMT), and monoamine oxidase (MAO), to infection withNippostrongylus brasiliensis has been examined in mice and compared to the changes evoked byin vivo administration of compound 48/80. Infection with the parasite resulted in a significant decrease in the concentration of both amine oxidases, followed by recovery of MAO and an overshoot in DAO activity. HMT activity was enhanced at the beginning of infection, then decreased markedly by days 11 to 15, and sharply increased thereafter. Histamine levels were on average only 20% higher than the basal levels over the entire period studied, except on day 4 when they were slightly reduced. Histamine is alleged to be a potential inducing factor for degrading enzymes. Consistently, the histamine releaser 48/80 significantly elevated intestinal mucosal DAO and in some of the mice also increased HMT activity.Supported by Wpr 5 and CPBP 06.03.1.2.     

Diamine oxidase activity and histamine release in dogs following acute mesenteric artery occlusion

Following superior mesenteric artery occlusion and revasclarization in dogs all animals died in a circulatory collapse state. However, pretreatment by aminoguanidine, the strong and specific inhibitor of diamine oxidase, accelerated the circulatory break-down significantly and increased the venous plasma histamine concentrations up to levels which also in normal dogs are effective in the circulatory system. Furthermore, the haematocrit increased significantly more in the aminoguanidine-treated animals than in the dogs treated by saline.No changes in plasma diamine oxidase activity were observed in saline-treated animals during intestinal ischemia and following revascularization. In aminoguanidine-treated animals no enzymic activity could be measured.The results were interpreted by a protective role of intestinal diamine oxidase in intestinal ischemia. Enhancement of the enzymic activity in patients, for instance by heparin, may be helpful in mesenteric infarction disease.Supported by a grant from Deutsche Forschungsgemein schaft (Lo 199/5).     

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^–3 M. 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.Supported by grant of Deutsche Forschungsgemeinschaft (Lo 199/14-I).     

The intestinal diamine oxidase activity under the influence of adaptive proliferation of the intestinal mucosa — a proliferation terminating principle?

Under clinical conditions, intestinal mucosal hyperproliferation together with a reduced diamine oxidase (DAO) activity was found in inflammatory and neoplastic diseases. Therefore, we studied the influence on DAO activity of a regulated mucosal proliferation as obtained following partial small bowel resection in a rat model. A statistically significant, more than 4-fold elevation of the enzymic activity was observed during the first days after partial resection. At the peak of mucosal proliferation (8th. postoperative day) the DAO activity was significantly reduced by about 50% of the initial value. We suggest that the DAO may be involved in a negative feed-back control mechanism of mucosal proliferation.Supported by a grant of the Deutsche Forschungsgemeinschaft Ku 464/2-3.     

In vitro metabolism of putrescine by diamine oxidase in tissues of the pregnant rat

The metabolism of¹⁴C-putrescine by diamine oxidase was tested on the 19th day of pregnancy in different organs of rats. The highest turnover was found in maternal placenta and uterus where 94% of the reaction product was identified as ¹-pyrroline. Considerable deamination rates were also observed in ovary, liver and fetal placenta where, however, -aminobutyric acid was the predominant product. In the kidney only a small diamine oxidase activity was measured. No formation of spermidine or spermine from¹⁴C-putrescine was detected.