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Localization of histaminase (diamine oxidase) in rat small intestinal mucosa: Site of release by heparin
Abstract
Previous studies have suggested that in the rat, small intestine is the source for rise in plasma histaminase levels seen after heparin administration. The cellular location of histaminase in intestine and the mechanism of heparin release have not been previously investigated. The present study identifies intestinal villus cells rather than crypt cells as the location of intestinal histaminase; at this site, the enzyme is not associated with brush border. Heparin added to incubations containing isolated intestinal cells did not release histaminase into the medium. Perfusion of intestinal vasculature with heparin caused a prompt release of this enzyme into venous effluent. The present investigation. therefore, suggests that heparin releases histaminase from vascular binding sites rather than directly from parenchymal cells. The use of isolated intestine with perfusion of the vasculature could serve as a useful tool for further defining the relationship between the sites of synthesis and the binding sites involved with heparin releasable enzymes such as histaminase.
... These data indicate that mucosal involvement is common in small bowel Crohn's disease and that PHD may be useful in assessing and monitoring mucosal damage in these patients. D iamine oxidase (DAO, EC 1.4.3.6.) is an enzyme 'located in villus tip enterocytes of mammals (1) and its activity increases progressively from duodenum to ileum (2,3). This enzyme can be used in rats as a plasma marker of maturation and integrity (4), as well as injury and recovery (5), of the small bowel mucosa. ...
... Within 16 wk of the PHD test, 5 of the 51 patients underwent intestinal resection because of intestinal obstruction (1), abdominal abscess (1), and failure of medical therapy (3). The length of diseased bowel could be detected on the resected specimen. ...
Diamine oxidase (DAO) is an enzyme located almost exclusively in villus tip enterocytes of mammals. Its plasma activity, normally very low, is enhanced by intravenous heparin, which releases the enyzme from small bowel enterocytes into the blood. Plasma postheparin DAO (PHD) values have been shown to be significantly reduced in patients with malabsorption and villous atrophy and inversely correlated with 24-h fecal fat, thus suggesting that PHD reflects the mature enterocytic mass. We have assayed PHD in 51 patients with small bowel Crohn's disease by measuring the area under the plasma DAO curve over a 120-min period after an intravenous bolus of 15,000 IU of heparin. Postheparin plasma DAO was significantly lower (p less than 0.001) in patients (328 +/- 175 U/ml.min) than in 20 normal subjects (508 +/- 101 U/ml.min; range, 391-749). Postheparin diamine oxidase values were inversely correlated with Crohn's disease activity index (CDAI), but no correlation was found with extent of disease assessed radiologically by either double-contrast small bowel enema or barium meal follow-through. In 6 patients with active disease (CDAI, 297 +/- 99) and low PHD values (188 +/- 100 U/ml.min), the assay was repeated after a clinically effective course of antiinflammatory drugs. A significant increase in PHD values (388 +/- 112 U/ml.min) was observed (p less than 0.005). These data indicate that mucosal involvement is common in small bowel Crohn's disease and that PHD may be useful in assessing and monitoring mucosal damage in these patients.
... Diamine oxidase (DAO) activity in serum correlates inversely with intestinal permeability of the small intestine (Luk et al., 1980;Honzawa et al., 2011). DAO is the main enzyme to catalyze the oxidation of diamines such as histamine, putrescine, and cadaverine (Shakir et al., 1977). The expression of DAO occurs predominantly in human intestinal mucosa as well as the placenta, kidney and thymus (Rangachari, 1992). ...
... Diamine oxidase (DAO) is an enzyme that catalyzes the oxidation of diamines such as histamine, putrescine, and cadaverine. 5 In humans and rodents, DAO is specifically located at the apical end of mature villous cells with high activity and its activity reflects the integrity and maturity of the small intestinal mucosa. Several studies of humans and animals revealed that DAO activity in serum inversely correlates with intestinal permeability of small intestine. ...
... Heparin administration is known to increase plasma DAO activity in man (8,19) and other mammalian species (20,21) by releasing the enzyme from the villous tip enterocytes (22). However, how heparin induces the release of DAO is far from clear. ...
Diamine oxidase (DAO) is an enzyme whose low plasma values are enhanced by an intravenous injection of heparin, which releases the enzyme from the enterocytes of the villous tips. In 20 normal controls and 15 untreated subjects affected with an overt malabsorption syndrome and subtotal atrophy shown by Crosby jejunal mucosa biopsy (12 suspected celiac disease and three small bowel lymphoma), plasma diamine oxidase was assayed, over 2 hr following an intravenous bolus of 15,000 IU heparin. Plasma postheparin DAO concentrations and the corresponding values of the area under curve, expressed as units/ml X min (mean +/- SD), were significantly lower in the patients (celiac sprue: 138 +/- 62; lymphoma: 83 +/- 42) compared to normals (481 +/- 104). DAO area values were well correlated (r = 0.81; P less than 0.001) with 24-hr fecal fat excretion but not with xylosuria. Our data suggest that postheparin plasma DAO assay may be useful to detect and quantitate small bowel mucosal atrophy in patients with malabsorption syndromes.
Background:
Many studies have demonstrated that the expression of methyltransferase- like 3 (METTL3) is altered in various inflammatory diseases. Its specific mechanistic role in the intestinal inflammatory response during sepsis remains limited and requires further investigation.
Objectives:
Explore the potential mechanism of METTL3 in the intestinal inflammatory response during sepsis.
Materials and Methods
Immunohistochemical analysis was utilized to detect the expression of METTL3 in the necrotic intestine of patients with intestinal necrosis and the small intestine of cecal ligation and puncture (CLP) mice. Mice were subjected to the CLP and Sham surgeries, intestine tissue was harvested and performed HE staining, and ELISA to examine intestinal inflammatory responses, while TUNEL staining was applied to detect intestinal cell apoptosis. Additionally, ELISA was used to detect diamine oxidase (DAO) and intestinal fatty acid binding protein (I-FABP) levels in intestinal tissue. Immunohistochemistry and RT-qPCR were also employed to examine the mRNA and protein expression levels of Zona Occludens 1 (ZO-1) and Claudin-1. Finally, transcriptomic sequencing was performed on the small intestine tissues of METTL3 Knock-out (KO) and Wild-type (WT) mice in response to sepsis.
Results:
METTL3 exhibited lower expression level in the necrotic intestine of patients and the small intestine of CLP mice. Loss of METTL3 in CLP mice triggered significantly higher expression of TNF-α and IL-18, down-regulated expression of ZO-1 and claudin-1, and decreased expression of DAO and I-FABP in the intestinal tissue. KEGG enrichment analysis showed that the differential genes were significantly enriched in immune-related pathways.
Conclusion:
This study reveals a novel mechanism responsible for exacerbated intestinal inflammation orchestrated by METTL3. Particularly, METTL3 null mice displayed decreased ZO- 1 and Claudin-1 expression, which largely hampered intestinal epithelial barrier function, resulting in bacterial and toxin translocation and intestinal immune activation and inflammation against sepsis.
The serum concentration and activity of diamine oxidase (DAO) in calves with or without diarrhea during the first month of life were examined. The serum DAO concentration was highest on day 0 after birth, and gradually decreased. No differences in serum DAO concentrations were observed between calves with diarrhea and control calves. In contrast, serum DAO activity was lowest on day 0 after birth, and gradually increased. Serum DAO activity in calves with diarrhea was lower than that in the control calves from day 20 after birth. These findings suggest that serum DAO activity and concentration are not correlated, and serum DAO activity is useful for assessing damage to the small intestinal mucosa in calves.
Gamma-aminobutyric acid (GABA) has a transmitter function in the mammalian enteric nervous system. The intestine contains endogenous GABA [1, 24, 32] and its synthesizing and degrading enzymes, i.e., glutamic acid decarboxylase (GAD, EC 4.1.1.15) [24, 32] and GABA-transaminase (GABA-T, EC 2.6.1.19) [32], respectively. Endogenous and labeled GABA may be released from the intestine by electrical transmural Stimulation [44] or by the application of substance P [43]. Finally, the presence of GABAergic neurons in the intestine has been demonstrated by immunohistochemical methods [3, 10, 23]. GABA may act on both inhibitory and excitatory neurons of the enteric plexus [26] and these effects appear to be mediated by both subtypes of GABA receptors, i.e., GABAA and GABAB [25].
Unlike many other pathological states, the treatment of neoplasia has not been blessed with major “breakthroughs”. Cancer treatment today is the result of a slowly evolving, quasi-empirical, methodological search for specific agents that (1) kill tumor cells, (2) have unique mechanisms of action, and (3) are assessed to be disease-effective and tolerated. Therefore, achievements in cancer treatment have required, and continue to require sound financial support, long hours in the laboratory and clinic, and cooperation and communication between an “army” of preclinical, clinical and basic scientists with a common goal. Despite this absence of major breakthroughs, however, the clinical management of cancer has evolved such that today, it represents a sophisticated, knowledgeable approach to health care.
Ischemic intestinal disorders account for 0.1% of patient referrals to hospitals and 1 % of patients with acute abdominal pain.15,58,77,79 Acute mesenteric ischemia has a mortality rate of 55% to 90%9-11,47,48,52,86and is a source of significant morbidity. The high mortality rates are attributed primarily to the inability to make an early diagnosis due to diffuse symptoms and lack of definitive diagnostic methods. Mesenteric ischemia has been termed the “enigma among abdominal diseases.”56
The role of histamine in the control of general and local homeostasis is a recent discovery which is undergoing further investigation. The physiological significance of histamine-degrading enzymes as well as possible functions of histamine metabolites are also being reconsidered. The function of histaminedegrading enzymes in the elimination of histamine from its receptor-binding sites remains a topic for further study.
The level of plasma diamine oxidase (DAO) activity is associated with the maturation and integrity of small intestinal mucosa. This study in rats investigated whether a decreased level of plasma DAO could reflect the severity of mucosal injury due to intravenous 5-fluorouracil (5-FU) treatment. The beneficial effect of soluble dietary fiber (SDF) on preventing diarrhea after 5-FU treatment was also examined. To induce diarrhea, 5-FU (50 mg/kg/day for four days) was administered via the tail vein with or without SDF supplementation. After 5-FU treatment, the majority of rats developed moderate to severe diarrhea, and levels of plasma DAO activity significantly decreased compared to those of control group (P < 0.05). Scanning electron microscopy revealed disarrangement of the small intestinal villi. Contrarily, the rats supplemented with SDF had diarrhea less frequently (50.0 vs. 91.7 %, P = 0.025) on day five, and DAO activity levels were significantly higher than in those rats administered 5-FU alone (8.25 ± 5.34 vs. 5.50 ± 4.32, P = 0.023). In conclusion, plasma DAO activity decreases in response to severe intestinal mucosal injury after 5-FU treatment, and SDF supplementation might be a practical and useful treatment for reducing the intestinal toxicity of 5-FU.
Organic soluble precursors to the high Tc 1-2-3 superconductor, YBaâCuâO7-x, were prepared using polyesters derived from the interaction of citric acid (CA) or ethyl-enediaminetetraacetric acid (EDTA) with ethylene glycol. Mixtures of the nitrate, carbonate and/or acetate salts of the three metal ions with CA or EDTA and ethylene glycol were heated to obtain homogeneous green solids containing the desired 1:2:3 ratios of Y/sup 3 +/, Ba/sup 2 +/, and Cu/sup 2 +/ ions. Pyrolysis at 850C-960C in oxygen gave the superconducting, orthorhombic, YBaâCuâO7-x phase. Films of the superconductor were prepared by dropping ethylene glycol solutions of these precursors onto zirconia and alumina substrates, followed by pyrolysis to 950 C in Oâ. The results of TGA/DSC and FTIR studies of these precursors, as well as XRD, FTIR, pyrolysis/GC, electrical and magnetic-property measurements on the pyrolysis products are presented and discussed in the context of the probable precursor structures and pyrolysis chemistry.
Background: Diamine oxidase (DAO) is the enzyme that degrades putrescine, the key main product of polyamine metabolism, and reflects enterocytic maturity of absorption because diamine oxidase activity is highest in the small intestine. We have already shown that expired 14CO2 after oral administration of 14C-putrescine correlated with intestinal DAO activity. However, the influence of food composition and the mucosal adaptation after intestinal resection have not been elucidated.
Methods: Male Wistar rats were fed normal chow or an elemental diet (ED) for 2 weeks. Resected rats underwent 50% jejunectomy or 50% ilectomy. Expired 14CO2 levels, following oral administration of 14C-putrescine were measured in all rats, and compared with the intestinal DAO activity and other mucosal parameters.
Results: In the ED group, the 14CO2 levels reached a peak earlier, and values were 2.9-fold higher than in the group fed with normal chow. Mucosal alkaline phosphatase (ALP) and DAO activity in the ED group were also higher than in the group fed normal chow, although the mucosal wet weight was significantly lower in the ED group. In the resection groups, all expired 14CO2 values increased during measurement. The peak 14CO2 values in the jejunectomy group shifted earlier in the postoperative period. The mucosal DAO activity in both the resection groups was higher than it was in the control group at the fifth and 10th postoperative day. However, there were no differences among the three groups at the 15th postoperative day.
Conclusions: Our studies suggested that expired 14CO2 after oral administration of 14C-putrescine correlates with mucosal DAO activity, and that it also reflects intestinal function.
We investigated treatment-induced changes in venous return from the small bowel and small bowel intestinal mucosal injury induced by the treatment of esophageal varices in patients with portal hypertension. A total of 14 patients (age 59.8 9.5 years, five women and 9 men) who received prophylactic treatment of esophageal varices between December 1998 and March 1999 were investigated. Diamine oxidase (DAO) activity was measured before and after treatment. Changes in blood flow of the portal and superior mesenteric veins were investigated by Doppler ultrasonography in six patients. A significant decrease in DAO activity was observed three days after treatment (11.5 1.6 units/liter prior to treatment versus 8.6 1.6 units/liter three days after treatment; P ). Decreases in superior mesenteric and portal venous flow velocity were observed in four and three patients, respectively. In two patients with an increase in the cross-sectional area of the superior mesenteric vein with delayed venous return, a marked decrease in DAO activity was observed three days after treatment. In patients with portal hypertension, rapid reduction of pooling of portal flow caused by the treatment of esophageal varices can induce transient congestion of the mesenteric venous system which can produce some small bowel mucosal injury.
To determine whether serum and mucosal DAO activity reflects quantitative changes in the small bowel mucosal mass, we have chosen an experimental model of mucosal hyperplasia which is known to occur in the rat after enterectomy. A 50% proximal enterectomy or a single transection was performed in 20 growing rats weighing 145–160 g. Ten days following surgery, we determined mucosal mass parameters (weight, protein, and DNA content), sucrase activity, and DAO activity in the duodenum (segment A), proximal ileum (segment B), and distal ileum (segment C) of the remaining small intestine. Mucosal hyperplasia was demonstrated by the finding that in each segment, mucosal weight, protein, and DNA content per centimeter of gut length were significantly (PPP0.01 vs controls) of resected rats. In the duodenum, the changes in DAO activity were small (+36%) and not significant. In the ileum (segment C), significant correlations were established between total DAO activity and either mucosal weight (r=0.75,N=20,P0.78, N=20, P0.01) per centimeter of gut length, but there was no correlation between DAO activity and sucrase activity. Compared to control rats with transection, proximal enterectomy produced marked changes in the serum activity of DAO. Ten days following surgery, the mean value of serum DAO was fivefold higher (P< it0.005)="" in="" the="" resected="" group="" than="" in="" the="" transected="" group.="" these="" data="" indicate="" that="" after="" jejunectomy="" (1)="" the="" intestinal="" activity="" of="" dao="" reflects="" accurately="" quantitative="" changes="" of="" the="" mucosal="" mass="" in="" the="" remaining="" ileum="" but="" not="" in="" the="" duodenum,="" and="" (2)="" circulating="" levels="" of="" dao="" could="" be="" used="" as="" a="" marker="" of="" ileal="" mucosal="" adaptation="" after="" proximal="">
Diamine oxidase and ornithine decarboxylase activities are shown to have a parallel distribution across rat small intestine mucosa; levels of both enzyme activities are sharply higher in mature cells in the villus tip region than in proliferating cells in the crypt areas. Histidine decarboxylase levels were not measurable in the same cell preparations and aromatic-L-amino-acid decarboxylase activity was distributed in an opposite pattern to diamine oxidase and ornithine decarboxylase. The results suggest that intestinal diamine oxidase could be involved with polyamine metabolism. The new findings for ornithine decarboxylase suggest an in vivo role for polyamines in non-proliferative cells; rat small intestinal mucosa may be an excellent model for investigating the function of polyamines in regenerating cells.
The distribution of calcitonin, L-dopa decarboxylase, and histaminase is studied in sectioned total thyroid glands from patients with different stages of familial medullary thyroid carcinoma. In 5 glands with gross carcinoma and in 3 with early microscopic carcinoma the distribution of all three parameters positively correlates (p less than .01 for each correlation). In contrast, in 6 glands with C-cell hyperplasia only the distribution of calcitonin and L-dopa decarboxylase correlates (r = 0.64, p less than .01) while those for histaminase vs. calcitonin (r = .17, p = N.S.) and histaminase vs. dopa decarboxylase (r = .03, p = N.S.) do not. In the glands with microscopic carcinoma the peak levels of histaminase occur in the areas of disease as defined by immunohistochemical staining of calcitonin; mean histaminase activity is the only one of the three parameters measured that distinguishes between C-cell hyperplasia and microscopic carcinoma (p less than .005). Immunohistochemical staining of histaminase shows positive cells in glands with gross and microscopic carcinoma, but in none of the glands with hyperplasia alone. Histaminase is thus found in high amounts in some malignant C-cells only and may be a useful marker to distinguish between hyperplasia and malignancy in thyroids with early C-cell proliferative disorders.
Since the small intestine contributes significantly to serum cholesterol and very low density lipoprotein levels, acute regulation of lipid synthesis was investigated in isolated rat intestinal cells incubated in Krebs-Ringer bicarbonate buffer with 5 mM glucose and [14c]acetate or 3H2O. Incorporation of [14c]acetate into cellular lipids was 6- to 8-fold greater in crypt than in villus cells. In both cell types the distribution of 14C among the various lipid classes was as follows: 52.5% in triglycerides, diglycerides, and monoglycerides; 22.3% in cholesterol; 8.3% in cholesteryl esters; 1.9% in fatty acids; and 15.0% in phospholidpids. In contrast, the medium lipids contained significantly higher amounts of tri-, di- and monoglycerides (61.1%) and lower amounts of cholesteryl esters (2.3%) and phospholipids (11.9%). After saponification, 2/3 of the recovered 3H2O was in fatty acids and 1/3 in cholesterol. Ethanol (10 mM) tripled 3H2O incorporation into cellular lipids but had no effect on [14c]acetate incorporation. Epinephrine and norepinephrine (10 micron), glucagon (10 micron), dibutyryl cyclic AMP (1MM), dexamethasone (1 mM and 1 micron), and cholera toxin (1 microgram/ml) did not affect [14c]acetate incorporation. We concluded that ehtanol stimulates intestinal lipid synthesis; however, in sharp contrast to their inhibition of lipid synthesis in hepatocytes and adipocytes, catecholamines, glucagon, and dibutyryl cyclic AMP do not inhibit lipid synthesis in intestinal cells.
A new, rapid, and sensitive assay for phospholipase A, utilizing commercially available [14C]phosphatidylethanolamine with 14C label in both palmitic acid moieties, was used to study phospholipase A release from perfused liver, hepatocytes, and intestinal cells from rats. Heparin triggered a prompt release of phospholipase A from perfused liver. Phospholipase A and triglyceride lipase were released from hepatocytes at a linear rate for 1 h and 30 min, respectively. Heparin (20 u/ml) doubled the release of phospholipase A and triglyceride lipase from hepatocytes. Colchicine (0.1 mM), but not puromycin (0.2 mM), inhibited basal and heparin-stimulated phospholipase A release by 40%. Since the amount of phospholipase A and triglyceride lipase released into the medium greatly exceeded intracellular activities, it is possible that secretion is coupled with intracellular conversion from inactive to active forms of the enzymes. Dibutyryl cyclic AMP (1 mM) inhibited phospholipase A (48%) and triglyceride lipase (82%) release from hepatocytes. Epinephrine, dexamethasone, and clofibrate inhibited release of triglyceride lipase but not phospholipase A. Phospholipase A activity of intestinal cells was greater than in hepatocytes, but neither heparin nor dibutyryl cyclic AMP affected phospholipase A release from intestinal cells. These results suggest that the liver is a major source of phospholipase A of postheparin plasma. The fact that dibutyryl cyclic AMP affects the release of these enzymes suggests an additional mechanism for hormonal regulation of lipid and lipoprotein metabolism.
Hydroxylamines potentiated the responses of the canine colonic epithelium to histamine but not to other agonists such as serotonin or carbachol. We tested the hypothesis that an inhibition of histamine catabolism could explain the observed potentiation. A clear structure activity relation was defined, active compounds having the structure NH2-O-R, R being a simple uncharged aliphatic group. Active compounds delayed the disappearance of histamine from the bathing solutions and inhibited colonic diamine oxidase, an effect mimicked by standard inhibitors aminoguanidine and semicarbazide. Histamine agonists that possessed an imidazole nucleus (2- and 4-methylhistamine) were affected, whereas impromidine, 2 pyridylethylamine, and dimaprit were not. Agonist specificity combined with the enzyme data suggest an inhibition of histamine catabolism as a possible mechanism for the potentiating effects observed.
Diamine oxidase (DAO) is a cytoplasmic enzyme found primarily in the villus epithelial cells of the small intestine. Serum DAO levels have been evaluated as a potential marker of intestinal disease in a variety of disorders, including gut atrophy, ischemia, and inflammation. In this study serum and tissue DAO levels were evaluated during intestinal adaptation. Twenty dogs were divided into 4 groups: sham laparotomy (n = 5), and 25% (n = 5), 50% (n = 5), and 75% (n = 5) distal enterectomy. Serum DAO activity (basal or postheparin) was measured prior to and 2 days, 4 weeks, 8 weeks, and 12 weeks after operation. Tissue DAO and changes in intestinal length, mucosal protein content, and villus height were measured at sacrifice 12 weeks later. Intestinal remnant length and protein content increased significantly with 50 and 75% resection. Tissue DAO activity was significantly decreased with any enterectomy. Serum postheparin DAO activity was significantly greater than basal at all time points but there was no significant change in either basal or postheparin DAO levels at any time following resection. It is concluded that serum DAO levels are not changed during the early adaptive period following intestinal resection and thus would not be useful as a marker of this process. Tissue DAO levels were diminished during adaptation, suggesting that tissue DAO activity is influenced not only by mucosal mass but by cellular metabolism and the proliferative status of the mucosa.
Rat colon perfused intraluminally in vitro and in vivo released histamine into the perfusates. Histamine release was increased by rhein 0.1-10 micrograms/ml and much more by rheinanthrone 0.1-10 micrograms/ml but not by sennosides A or B 1-10 micrograms/ml. The effect of rhein and rheinanthrone was reduced by tritoqualine 20 mg/kg. This raises the possibility that laxation by senna and its derivatives involves histamine formation.
Diamine oxidase (DAO) is an enzyme located almost exclusively in villus tip enterocytes. Its plasma activity is enhanced by intravenous heparin which releases the enzymes from small bowel enterocytes into the blood. Plasma postheparin DAO (PHD) values have been shown to be significantly lower in patients with malabsorption and villous atrophy, thus suggesting that PHD reflects the mature enterocytic mass. In this study we have assayed PHD in five patients with small bowel lymphoma (two with immunoproliferative small intestinal disease [IPSID] and three with non-IPSID lymphoma) associated with malabsorption syndrome and small bowel mucosa atrophy. The PHD test was performed at diagnosis, after partial or complete remission induced by chemotherapy, and during the follow-up. The PHD values, very low at diagnosis (0.66 +/- 0.12 U/ml), increased during chemotherapy and reached the normal range (greater than 3.7 U/ml) when complete remission occurred. The PHD values rapidly and consistently decreased whenever the disease relapsed. Our data indicate that in patients with small bowel lymphoma PHD test is a sensitive marker of small bowel mucosa damage and suggest that it could be useful in monitoring the recovery of mucosal lesions induced by chemotherapy.
Measurement of postheparin plasma diamine oxidase (PHD) activity has been proposed to assess mucosal integrity in several diseases of the small intestine. In Crohn's disease, PHD values identify a group of patients with predominantly small bowel mucosal damage. To determine the role of mucosal involvement in the progression of small bowel Crohn's disease and whether different PHD values can predict different outcomes the changes in PHD values in 41 patients with small bowel Crohn's disease admitted consecutively to our department were investigated. The test was performed during periods of active disease and after either medical or surgical treatment had resulted in improvement. PHD values were significantly lower than in normal subjects (normal range 3.7-7.7 U/ml). In 35 patients with active disease (Crohn's disease activity index (CDAI) greater than 150) two groups were identified by choosing a cut off value of 2 U/ml: 93% of the 15 patients with PHD values lower than 2 U/ml (mean (SD) 1.36 (0.46) U/ml) relapsed at least once in the following year, while only the 20% of the 20 whose values were higher than 2 U/ml (mean (SD) 3.69 (1.50)) relapsed in the same period. The data were statistically significant (Yates's corrected chi 2 = 15.63; p less than 0.0001). The positive and negative predictive values of the test were 93% and 80%, respectively. During relapses, PHD values were consistently lower than previous values, and increased significantly after effective medical or surgical treatment. In the six patients in whom there were no changes in disease activity (CDAI persistently less than 150), there was no change in PHD values. This test may be useful for identifying Crohn's disease patients who are likely to relapse. Furthermore, the data indicate that mucosal damage is common in active small bowel Crohn's disease and improves at least in part after treatment.
Plasma postheparin diamine oxidase (DAO) activity has been evaluated for assessing disease activity in Crohn's disease (CD) and other intestinal disorders. Since the mechanism of the reduced plasma DAO activity is poorly understood, our aim was to determine the effect of extent and location of disease and prior resection and therapy on plasma DAO activity in Crohn's disease. Plasma postheparin DAO activity was significantly lower (17.4 +/- 3.0 vs 32.8 +/- 30.8 units/ml) and Crohn's disease activity index (178 +/- 105 vs 14 +/- 19, P less than 0.05) (CDAI) higher in 37 patients with CD compared to 30 normal volunteers. There was no overall correlation between DAO activity and CDAI. Effective medical or surgical therapy increased DAO activity and decreased CDAI, while clinical recurrence had the opposite effect. DAO activity was not related to the extent of small bowel disease (13.2 +/- 9.1; less than 30 cm, 18.5 +/- 11.8; 30-60 cm, and 5.7 +/- 6.4 units/ml; greater than 60 cm) or colonic disease (13.0 +/- 6.9 segmental vs 24.0 +/- 15.4 units/ml, pancolitis). DAO activity was similar with small or large bowel disease (14.3 +/- 10.6 vs 18.8 +/- 13.1 units/ml). Prior enterectomy or colectomy did not significantly influence DAO activity. DAO activity responds predictably after effective therapy and recurrence and may prove useful in monitoring individual patients with CD. Failure of extent and location of disease and prior resection to influence DAO activity suggests that DAO activity is not directly related to enterocyte mass.
In animals, the distribution of the enzyme diamine oxidase is confined, almost exclusively, to the small bowel mucosa. In humans, plasma diamine oxidase is at or below assay detection limits but can be liberated into the circulation from binding sites in the intestine by i.v. heparin. Therefore, the authors wished to see if diamine oxidase could be released by a low and safe dose of heparin (5000 U) and if the resultant area under the concentration-time curve would provide a noninvasive marker of segmental intestinal disease. In 17 control subjects, the mean area under the curve (following administration of 5000 U i.v. heparin) was 35.9 +/- 5.0 (SEM) mU.L-1.2 h-1; in 6 individuals studied on two separate occasions, postheparin plasma diamine oxidase profiles were reproducible (r = 0.98; p less than 0.001). The longitudinal distribution of diamine oxidase in the gastrointestinal tract, measured in 12 gastric, 16 jejunal, 6 ileal, and 18 colonic biopsies, was similar in humans to that found in animals. In patients with normal peroral biopsies, there was a linear relationship between jejunal mucosal and postheparin plasma diamine oxidase activities (r = 0.84; p less than 0.01). The areas under the curve in controls were then compared with those in patients with segmental intestinal diseases: 21 with ileal disease with or without colonic Crohn's disease (10 unoperated and 11 with ileal resection), 7 with non-Crohn's ileal resection, 8 with ulcerative colitis, 10 with untreated and 7 with treated celiac disease (6 studied before and after a gluten-free diet), and 5 studied during total parenteral nutrition and again after resumption of oral feeding. The results in the 18 ileectomized patients were subdivided into those with major (arbitrarily greater than 75 cm) and minor (less than 75 cm) resections. Areas under the curve were markedly reduced in nonresected Crohn's patients (6.0 +/- 1.79 mU.L-1.2 h-1; p less than 0.001 vs. controls), correlating inversely, in a first-order relationship, with disease activity (r = 0.82; p less than 0.001) and returning toward normal in 4 patients achieving disease remission. Low areas under the curve in total parenteral nutrition patients (4.5 +/- 0.9; p less than 0.001) were also reversible on resumption of oral feeding. However, areas under the curve were not significantly lower in patients with limited ileal resection (less than or equal to 75 cm), with celiac disease (untreated and treated), or ulcerative colitis than in controls.(ABSTRACT TRUNCATED AT 400 WORDS)
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.
The Caco-2 cells have been used as a model system to study the pathways of diamine oxidase secretion by the intestinal epithelium. When grown in Transwell filter chamber devices, the polarized cell monolayers released the enzyme preferentially into the basal chamber. Heparin (1-10 USP U/mL) rapidly induced a marked stimulation of enzyme secretion only when in contact with the basolateral cell membrane, where high affinity binding sites for [3H]heparin were also exclusively located. Among the other glycosaminoglycans tested, only heparan sulfate (150 mg/mL) was able to induce enzyme release; chondroitin sulfate (150 mg/mL) and dermatan sulfate (150 mg/mL) were without effect. Four monoclonal antibodies specific for human diamine oxidase were produced and found to immunoprecipitate a single protein with a molecular weight of 95,000 (under reducing conditions) from the culture medium of Caco-2 cells. Immunofluorescence staining of cryostat sections of human small intestine with these four antibodies localized diamine oxidase at the lateral and basal sides of the villus cells. Staining was markedly reduced in specimens obtained from patients who received doses of heparin in vivo. This study concludes that release of diamine oxidase by intestinal cells occurs specifically at the basolateral aspect of the cells, most likely through the constitutive secretory pathway. Heparin may induce its marked stimulation of enzyme release by complexing with diamine oxidase bound to the cell surface or through interaction with specific binding sites also located in the basolateral membrane. In the intestinal mucosa in vivo, the basal aspect of the villus cells represents the main site of diamine oxidase storage in the presence of normal circulating levels of heparin.
The danger of luminal histamine administered orally or formed in the intestinal fluid by bacteria has long been neglected. However, the demonstration of blocking intestinal diamine oxidase (DAO) by a variety of common drugs has revived the discussion and has created a new disease concept: enteral-induced histaminosis. In an animal model the three central prognostic variables of this disease concept (large amounts of histamine in food to make the individual ill, blocking of DAO by commonly used drugs, and the relationship between increased plasma histamine levels and disease manifestation by exogenous histamine application) were tested with randomized trials in vivo and biochemical tests in vitro using semipurified enzymes from pig and man.
In the first trials authentic histamine in quantities similar to that in normal amounts of food or cheese bought from a supermarket produced lifethreatening reactions if the DAO was inhibited by pretreatment with aminoguanidine. In the second series of experiments in vitro a numerous commonly used drugs was shown to inhibit both the porcine and human enzyme. Some of the inhibitors were really strong, such as dihydralazine, chloroquine, pentamidine, cycloserine, clavulanic acid, dobutamine, pancuronium and others. The type of inhibition was sometimes competitive as in the case of dihydralazine and pancuronium, sometimes non competitive (e.g. pentamidine) which may be important for long-term treatment. In the third group of experiments a relationship between the dose of i.v. injected histamine and the elevation in plasma histamine levels and clinical symptoms in pigs was demonstrated. Hence, elevated plasma histamine in pigs acts as a pathogenetic factor for the disease manifestation. It is concluded that after modelling enteral-induced histaminosis in an animal the trias of variables shown in this study should be consequently investigated in man.
Heparin releases diamine oxidase (DAO) of enterocytic origin from binding sites located on small bowel microvascular endothelium. In the villus tip enterocytes the enzyme is found in organelles (about 60%) and in cytosol (about 40%), while a negligible activity is present in the brush border. In this study we assessed the changes in DAO distribution into the enterocytes induced by a high dose of intraperitoneal heparin (1000 IU) in the rat, by assaying DAO activity on subcellular fractions obtained from ileal mucosa homogenate. Heparin injection induced a marked reduction of enzyme activity in the S2 fraction (cytosol): after 30 min less than 20% of DAO activity is still found and only 8% after 150 min. In the P1 fraction (organelles) DAO activity significantly decreased only after 60 min and a further consistent reduction was recorded after 150 min. Recovery of DAO activity was complete 4 days after the injection, though it was already clearly evident in the first 2 days. These results indicate that enterocytic DAO is distributed in two different compartments: DAO located in the cytosol is quickly released by heparin, while the organelles-linked enzyme is more slowly released. The finding that recovery in DAO activity happens earlier in the P1 fraction suggests that the enzyme supplies the cytosol after being synthesized in the enterocyte organelles.
To determine whether serum and mucosal DAO activity reflects quantitative changes in the small bowel mucosal mass, we have chosen an experimental model of mucosal hyperplasia which is known to occur in the rat after enterectomy. A 50% proximal enterectomy or a single transection was performed in 20 growing rats weighing 145-160 g. Ten days following surgery, we determined mucosal mass parameters (weight, protein, and DNA content), sucrase activity, and DAO activity in the duodenum (segment A), proximal ileum (segment B), and distal ileum (segment C) of the remaining small intestine. Mucosal hyperplasia was demonstrated by the finding that in each segment, mucosal weight, protein, and DNA content per centimeter of gut length were significantly (P less than 0.01) higher (+38 to + 78%) in the resected group than in transected controls. In segments B and C of resected rats, the changes in DAO activity expressed per gram of mucosa paralleled the changes in mucosal mass, the activity being increased by +69% and +49% (P less than 0.05) compared to the values recorded in transected controls. Expressed per centimeter of gut length, total DAO activity was also enhanced by +141% in segment B (P less than 0.05 vs controls) and by +87% in segment C (P less than 0.01 vs controls) of resected rats. In the duodenum, the changes in DAO activity were small (+36%) and not significant.(ABSTRACT TRUNCATED AT 250 WORDS)
The human colon carcinoma cell line CaCo-2, grown in vitro under standard culture conditions and in the absence of differentiation inducers, spontaneously exhibits structural and functional characteristics of mature small bowel enterocytes. Differentiation is complete at late confluency. High activities of ornithine decarboxylase and diamine oxidase are present in enterocytes. Although these enzymes are involved in polyamine metabolism and therefore in cell replication, their function in small bowel epithelium remains to be defined. In this study ornithine decarboxylase and diamine oxidase activities were assessed in CaCo-2 cells at different stages of proliferation and differentiation. Diamine oxidase was also assayed in spent culture media to assess its spontaneous release by CaCo-2 cells. The trigger effect of medium replacement on ornithine decarboxylase activity was also investigated. Cell growth and cell cycle kinetics were determined by hemocytometric cell count and [3H]thymidine labeling index. Sucrase activity was assayed to evaluate brush-border functional maturation. Elevated ornithine decarboxylase activity was recorded during the replication phase (highest value 0.3 +/- 0.02 U/mg) characterized by high thymidine labeling index (43%), and was greatly enhanced by medium replacement (2.1 +/- 0.3 U/mg). Diamine oxidase activity was low in both cells and medium during the active phase of cell growth, and during the differentiation period it progressively increased (highest value 499 +/- 78 U/mg) along with sucrase activity. The high diamine oxidase activity recorded in the medium (highest value 1292 +/- 310 U/ml) and the evidence of diamine oxidase secretion through the basolateral membrane of the cells cultured on porous filters support the hypothesis of an extracellular role of intestinal diamine oxidase. The CaCo-2 cell line, which shows several analogies with small bowel enterocytes, can be proposed as an interesting in vitro model for studying many aspects of cell replication and differentiation depending on polyamine metabolism.
Diamine oxidase plasma concentrations after treatment with heparin were measured and compared with the surface to volume ratio of jejunal biopsy samples assessed by a morphometric technique in patients with untreated and treated coeliac disease and in biopsied controls. As expected, enzyme activity was significantly lower in patients with untreated coeliac disease than in patients on a gluten-free diet and in biopsied controls. No difference was found between treated patients and biopsied controls. There was a significant overall correlation between plasma enzyme activity and surface to volume ratio of jejunal mucosa, although two untreated patients without an overt malabsorption syndrome but with a very low surface to volume ratio had normal enzyme activity. This study shows that in coeliac disease plasma diamine oxidase activity after treatment with heparin does not always mirror the extent of the jejunal lesions, particularly in those patients with minimal or unrelated symptoms who would benefit most from a valid screening test to identify their condition.
Plasma diamine oxidase (DAO) values are enhanced by intravenous injection of heparin which releases the enzyme, synthesized in small bowel enterocytes, from binding sites located on endothelial cells of the intestinal microvasculature. Intestinal DAO, in analogy with lipoprotein lipase (another heparin-released enzyme), is believed to be electrostatically linked to endothelial binding sites composed of a glycosaminoglycan (GAG) which is presumably heparan sulphate, but the complete mechanism of enzyme release is not known. In this study we assayed in rats the DAO-releasing capability of heparan sulphate, dermatan sulphate, chondroitin sulphate A and hyaluronic acid, all heparin related compounds. Heparan sulphate, a compound with the same hexosamine as heparin but with a lower concentration of sulphated iduronic acid, induced a very high release of DAO (3-fold less than heparin), while the other tested GAGs, composed of higher proportions of non sulphated uronic acid and with galactosamine instead of glucosamine, induced a significantly lower release. In rats treated with 60 mg heparan sulphate the significant decrease in ileal mucosal DAO activity indicates that, in analogy with heparin, the high plasma enzymatic activity induced is of enterocytic origin. It is suggested that the high charge density of the compounds tested, due to the degree of sulphatation, is the decisive factor in promoting the release of intestinal DAO.
Histamine is widely distributed in the intestine where it is involved in many pathological reactions. The relations between histamine content, diamine oxidase and histidine decarboxylase activity have been investigated along rat's intestine. Results showed significant variations along the intestine. A correlation was observed between histamine and histidine decarboxylase (p less than 0.01), both of them being even in the small intestine (11.8 +/- 2.3 ng/mg ww and 112.5 +/- 21.2 fmoles/hr/mg ww respectively), significantly higher in the caecum (16.3 +/- 1.9 and 178 +/- 20.1) and significantly smaller in the colon (7.3 +/- 1.1 and 65.3 +/- 11.5) than in other intestinal segments. Diamine oxydase activity was higher in ileum (30.7 +/- 7.2 pmoles/min/mg ww) than in jejunum (17.1 +/- 2.8), caecum (4.3 +/- 0.8) and colon (2.6 +/- 2.7), and could not be linked to histamine content. The results fitted in the hypothesis that histamine in rat intestine is mainly located in mast cells where HDC is probably the main enzyme involved in its modulation.
One hour after MDL 72145 ((E)-2-(3',4'-dimethoxyphenyl)-3-fluoroallylamine) (2.5 mg kg-1) was given by intraperitoneal injection, the semicarbazide-sensitive amine oxidase (SSAO) activity of rat aorta and brown adipose tissue measured in-vitro was reduced by more than 95% of its control value, whereas the monoamine oxidase (MAO-A) activity remained virtually unaffected. The action of this drug on amine oxidases in the liver at this dose was less selective. The in-vitro effect of MDL 72145 on the soluble enzyme diamine oxidase from rat intestine was 100 fold less potent than that of semicarbazide but about equipotent with semicarbazide on sheep plasma amine oxidase. Overall MDL 72145 was selectively more active against membrane bound SSAO enzymes that deaminate primary monoamines. Although MDL 72145 does inhibit MAO-B activity these results suggest that this compound may be used to study the effect of selective inhibition of SSAO activity on the pharmacological responses of appropriate preparations in-vitro.
Diamine oxidase (DAO) activities were measured in small intestinal biopsies and in serum from control children and children with florid celiac disease. Mucosal DAO activities were found to be significantly higher in control children when compared with children with celiac disease (mean +/- SEM: 486 +/- 39 versus 121 +/- 22.1 nmol h-1 g-1 wet weight, p less than 0.001). Similarly, serum DAO activities were significantly higher in control children when compared with children with celiac disease (mean +/- SEM: 39 +/- 12 versus 13.6 +/- 2 pmol h-1 ml-1 serum, p less than 0.002). The lower serum activities associated with low mucosal values support the hypothesis that serum DAO activity reflects small bowel integrity.
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.
The interaction of histamine with an H1 receptor on human endothelial cells evokes production of the lipid mediator prostaglandin I2 (PGI2) and is accompanied by tachyphylaxis of this H1 receptor response (Baenziger, N. L., Fogerty, F. J., Mertz, L. F., and Chernuta, L. F. (1981) Cell 24, 915-923). We have explored the affected cells' capability for subsequent metabolic degradation of histamine molecules. Human vascular endothelial cells and skin fibroblasts exhibit a two-stage histamine degradation sequence whose participants are an enzyme native to the cells themselves and one provided from an extracellular source. Initially, the cells' endogenous histamine N-methyltransferase activity mediates conversion of cell-associated [3H]histamine to tele-methylhistamine with retention of this intermediate metabolite. Subsequently, in the presence of exogenous diamine oxidase derived from fetal calf serum or human placenta, cell-associated tele-methyl-histamine is further converted to the end product methylimidazoleacetic acid. After an initial lag phase lasting 3-6 min, the cell-associated radioactivity accumulates as methylimidazoleacetic acid at a linear rate substantially enhanced over that without diamine oxidase. The entire sequence is blocked by the histamine methyltransferase inhibitor homodimaprit. Accumulation of [3H]histamine metabolites by endothelial cells is saturable both with respect to exogenous diamine oxidase and to histamine. Thus this metabolic pathway is carried out at the level of the individual cell by means of binding sites or receptors for the substrate and for the distal degradative enzyme, diamine oxidase.
An intravenous injection of heparin releases diamine oxidase (DAO) from villous tip enterocytes. In a previous study, we found that postheparin plasma DAO (PHD) values were significantly lower in patients with malabsorption syndrome and small bowel atrophy at jejunal biopsy than in normal subjects. In this study we performed the PHD test in 14 coeliac patients before and after three and six months of gluten free diet to show whether the enterocytes maturing processes induced by the diet joined with enhanced PHD values and to assess the clinical usefulness of this test. In all subjects jejunal biopsy carried out after six months showed a partial but consistent histological recovery. The clinical status, xylosuria and daily faecal fat excretion improved progressively and there was a significant increase (p less than 0.001) in mean PHD activity that reached the normal range after three months. After six months a further slight increase of the mean PHD value was recorded. These data indicate that PHD values rise together with the improved intestinal absorptive functions of coeliac patients on gluten free diet and that this test is a useful tool in monitoring recovery of the small bowel mucosa.
Plasma and small intestine diamine oxidase (DAO) activities were measured on Days 2, 4, and 6 following irradiation of mice with a range of doses of fission neutrons and 60Co. With increasing doses of radiation, plasma DAO activity increased on Day 2 and intestinal DAO activity decreased on Day 4; moreover, the approximate relative biological effectiveness values for these changes in activity were 5.81 for plasma DAO activity on Day 2 and 3.88 for intestinal DAO activity on Day 4. On Day 6 relatively high levels of radiation caused DAO activity in the small intestine to remain depressed whereas low levels resulted in recovery with activities at or near controls. In animals with combined injury (radiation plus 30% surface burn or wound), changes in DAO activity in the intestine were similar to those with radiation alone; plasma DAO activity, in contrast to radiation alone, did not show an increase at the 2-day mark. These dose-dependent relationships should provide a basis for using DAO as a potential indicator of biological damage from radiation exposure within the lethal range.
Starvation followed by refeeding, which provides a model of intestinal adaptation characterised by proliferative and biochemical changes, was used to clarify the biological roles of ornithine decarboxylase (ODC) and diamine oxidase (DAO)--enzymes involved in polyamines metabolism. Ornithine decarboxylase and DAO were assayed in the proximal and distal small bowel mucosa of 55 rats, starved for four days and then refed. Rats (five per day) were killed after four days' starvation and at days 1, 2, 3, 4, 5, 6, 7, 8, 10 and 12 of refeeding. ODC, whose specific activity was similar in both intestinal segments, almost disappeared after starvation and showed a biphasic response during refeeding. High values were found on day 3 of refeeding in the proximal, and on day 4 in the distal small bowel; thereafter, they decreased gradually to be followed by a further significant increase during the last two days of the experiment. Diamine oxidase specific activity increased after starvation despite a very low total DAO activity in both intestinal segments. Refeeding induced a gradual recovery of DAO total activity. Diamine oxidase specific activity also reverted gradually to control values after five days of refeeding. These data confirm the prominence of ODC in the replication processes and suggest that intestinal DAO may not play a major role in enterocyte replication.
The highest diamine oxidase activity is contained in small-bowel mucosa and, after heparin administration, the enzyme is released by the intestine into the plasma. Previous experimental studies showed that measurement of plasma postheparin diamine oxidase activity is a sensitive test for quantitating the length and severity of small-bowel mucosal injury. On this basis, we measured plasma diamine oxidase activity in celiac disease, a condition characterized by a loss of mature enterocyte mass. Twenty-five untreated celiac patients, 21 celiac patients on a gluten-free diet, 16 patients with small-bowel diseases other than celiac disease (abnormal controls), and 18 healthy controls were studied. Diamine oxidase activity was measured using [14C]putrescine as substrate and expressed as units per milliliter of plasma. Basal diamine oxidase levels in controls and patients were too low for significant differences between the groups to be detected. After preliminary experiments in which, on separate occasions, heparin was intravenously administered at doses of 75 and 150 units/kg and in which the second blood sample was taken 10 and 30 min after heparin injection, it was decided to use the 150 unit/kg dose and to measure plasma diamine oxidase activity in the blood sample taken 10 min after heparin stimulation in all the remaining subjects taking part in the study. Postheparin diamine oxidase levels were significantly lower in untreated celiac patients (mean 1.53 units/ml) than in healthy controls (mean 5.85), treated celiac patients (mean 4.82), and abnormal controls (mean 2.62). Except in three patients, no overlap between healthy controls and untreated celiac patients was observed. No significant difference was detected between healthy controls and treated celiac patients.(ABSTRACT TRUNCATED AT 250 WORDS)
Plasma diamine oxidase (DAO) activity may reflect intestinal involvement in Crohn's disease. The purpose of this study was to develop a simple heparin stimulation test for assessing postheparin plasma diamine oxidase activity in Crohn's disease. Ten volunteers and five patients with Crohn's disease received 1000 units and 3000 units of heparin intravenously and plasma samples were obtained at timed intervals. Plasma DAO activity increased significantly, compared with basal values, 30 minutes after 3000 units of heparin in both volunteers (26.2 +/- 5.0 vs. 4.5 +/- 0.5 units/ml) and patients with Crohn's disease (14.6 +/- 2.0 vs. 4.0 +/- 1.1 units/ml, P less than .05) and was significantly greater in the volunteers. There was no significant increase in DAO activity after 1000 units of heparin. Plasma DAO activity increased significantly within 15 minutes after 3000 units of heparin and remained at this high level at 60 minutes. Postheparin DAO activity correlated with the integrated area under the DAO activity curve. Plasma DAO activity correlated with the Crohn's Disease Activity Index in the patients with Crohn's disease. Plasma DAO activity, 30 minutes after the intravenous administration of 3000 units of heparin, should reflect intestinal involvement in Crohn's disease.
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.
Morphological changes in the surface mucous cells in the gastric body of the golden hamster occurring during their movement from the lower to the upper portion of the gastric pit have been observed by using scanning as well as transmission electron microscopes. The cells have a wide base and a narrow apex in the lower and middle portions of the pit, while at the opening of the pit to the gastric lumen, they become taller and funnel-like in shape, and are characterized by well developed interdigitations and intermediate filaments sometimes associated with desmosomes. During this transformation of the cell contour, the nucleus moves towards the upper region of the cytoplasm, whereas the Golgi apparatus moves downwards to the infranuclear region. Then, there appear secondary lysosomes showing crinophagy and lipid droplets around or near the Golgi apparatus. Though the basal part of the cells is very small, no images of the detachment of the basal plasma membrane from the basal lamina could be seen even at the site of severe cell degeneration. The tall funnel-shaped cells showing these characteristics are located on the interfoveolar ridges of the underlying fibrous layer and line the free surface of the stomach. Therefore, the interfoveolar cells which have lost the activity of secretory granule production and are going to undergo physiological degeneration are thought to be highly differentiated elements as a covering epithelium to protect the underlying tissue, resembling in this respect the keratinocyte of the epidermis.
Microvascular endothelial cells from rat and guinea pig fat pads were shown to bind diamine oxidase (DAO) activity when incubated with soluble extracts of placenta (33 DAO U/mg of placenta) and a purified placental enzyme preparation (94 U/micrograms of protein). The extent of binding was dependent on the concentration of enzyme activity and tissue. Saturation of binding sites with 5,000 U of DAO/ml resulted in levels of bound activity (up to 11-13 U/mg of endothelial cells) in excess of that observed in all tissues except placenta. Scatchard plots suggested that there were at least two DAO binding sites (apparent Km 92 and 2,450 U/ml). Although the same cell preparations bound 125I-labeled lipoprotein lipase (LPL), the presence of LPL on the endothelial cell surface did not interfere with the binding of DAO activity except when cells were exposed to high concentrations of LPL. Alternatively, bound DAO activity was partially displaced (up to 33%) only with high concentrations (30 micrograms/ml) of LPL. DAO activity may thus be bound to at least two populations of sites, one of which may bind LPL. Both enzymes, however, were displaced by heparin (0.05-5 U/ml) and DAO binding was impaired by prior treatment of cells with proteolytic and glycosaminoglycandegrading enzymes. The demonstration of DAO binding to vascular endothelial cells provides a further example of the ability of these cells to bind enzymes at their surface and thereby act on biologically active substances in the circulation.
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.
Cell surface glycoproteins, which have been implicated in the control and expression of cell turnover, differentiation, and malignant transformation in tissue culture studies, were examined for a possible similar role in the intestinal epithelial cell. This cell is characterized by a rapid cell turnover and by a gradient of differentiation from crypt to villus. A method was developed which isolated epithelial cells from different levels of crypt and villus areas and when radioactive labeled d-glucosamine, l-fucose, or d-galactosamine were given intraperitoneally into a rat, a sharp gradient of radioactive incorporation paralleled the specific activity gradients of sucrase and alkaline phosphatase. Isolated epithelial cells incubated with d-[1-¹⁴C]glucosamine also showed that the upper villus cells had a much higher incorporation of d-[1-¹⁴C]glucosamine than did crypt cells. Subcellular fractionation of the intestinal cells labeled in vivo demonstrated that the highest specific activity was in the purified microvillus plasma membranes and the lowest specific activity was in the microsomal and cytosol fractions. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of microvillus membranes showed peaks of d-[1-¹⁴C]glucosamine incorporation which coincided with peaks of sucrase and alkaline phosphatase activities and most of the bands which stained for protein also stained for glycoprotein. Microvillus core proteins separated on sodium dodecyl sulfate polyacrylamide did not show peaks of d-[1-¹⁴C]glucosamine incorporation and did not stain for glycoprotein. These results suggest that the more differentiated upper villus cells incorporate more labeled sugar precursors into membrane glycoproteins than the less differentiated lower villus or crypt cells and that these membrane glycoproteins are associated with membrane-bound enzymes.
Post-heparin plasma contains an enzyme or enzymes with both triglyceride lipase (TGL) and monoglyceride hydrolase (MGH) activities. A simple and reproducible radioactive assay for measurement of MGH activity was developed and used, with a previously reported assay for TGL, to study lipolysis in plasma.
After the injection of heparin, enzymatic activity against both tri- and monoglycerides appeared and disappeared from plasma at approximately the same rates. However, in contrast to TGL activity, MGH activity was: (a) much greater, (b) considerably less heat-sensitive, (c) unaffected by three inhibitors (NaCl, protamine, and pyrophosphate), (d) not influenced by radical changes in fat and carbohydrate content of the diet, and (e) normal in familial Type I hyperlipoproteinemia. The dichotomy between MGH and TGL activities in patients with genetic deficiency of TGL constitutes strong evidence that these are two different enzymes.
The findings further indicate that when post-heparin lipolytic activity is measured for the purpose of detecting TGL deficiency, it may be necessary to perform the assay with a substrate free from partial glycerides.
Lecithinase activity in post-heparin serum has been demonstrated. Phosphatidyl choline (PC) can be degraded to lysophosphatidyl choline and fatty acids at a rate of more than 1 μmole/hr per ml of serum in an incubation system containing PC, 0.1 m glycine-NaOH buffer (pH 9.6), and deoxycholate. This activity cannot be found in serum obtained prior to the injection of heparin.
Post-heparin serum lecithinase can be distinguished from the heat-stable pancreatic lecithinase by the markedly different effects of heat, paraoxon, and EDTA, and from serum lecithin: cholesterol acyltransferase by the differential effect of p-hydroxymercuribenzoate. In contrast to the acyltransferase and to pancreatic lecithinase, which are active at the Β (C-2) position of lecithin, post-heparin serum lecithinase is active at α′ (C-1) position.
1.1. A procedure is described for assay of disaccharidase activities in extracts of intestinal mucosa, using a Tris-glucose oxidase reagent for assay of the glucose liberated from the substrate. The incubation conditions are discussed.2.2. A unit for disaccharidase activity is defined that is in accord with recommendations made by the Joint Sub-Commission on Clinical Enzyme Units of the International Unions of Biochemistry and of Pure and Applied Chemistry.
After intravenous injection of heparin (in aniounts related to the body-weight) thc blood plasma diamine oxidase(DAO-) activity increased transiently in all vertebrates studied (cod, frog, fowl, mouse, white rat, golden hamster, guinea-pig, rabbit, cat, dog, goat, sheep and cow). The DAO-response to heparin varied strongly between the species. Often with a bi-phasic ascent, it showed a maximum (within a few minutes to about one hour) and then decreased, often in a “monoexponential” fashion, in the course of the next few hours. In anodons no significant DAO-activity was found.—The biological significance of the heparin effect upon DAO is discussed with special reference to the coexistence of heparin and histamine in the mast cells.
The effects of heparin, some polyanions, and protamine on histaminase activity in plasma and in different organs of the rabbit were investigated.
1.
Histaminase activity in plasma increases after injection of heparin in doses of 500 U/kg or more. The effect is approximately 25 times lower than that seen in the guinea pig with the same dose of heparin.
2.
The release of the enzyme in slower in rabbits than in guinea pigs. Two peaks of activity were observed at 15 and 60 min, respectively, after heparin injection. The activity of the first peak was higher at pH 6.7 than at pH 7.4, while the reverse was true for the second peak. Under optimal pH conditions, the activity of the second peak reached a higher value than that of the first one. Incubation of plasma with EDTA-Na2 abolished the first peak whereas the second was not affected.
3.
Release of histaminase by heparin is blocked by protamine in doses of 10 mg/kg or more at both peaks.
4.
The increase of the histaminase activity in plasma after heparin is accompanied by a significant decrease of activity in the intestinal mucosa; in liver the decrease of enzyme activity is small in contrast to that observed in the guinea pig. The kidney is not involved in the release of histaminase.
5.
An increase of histaminase activity is elicited by other polyanions. Relatively high doses, however, are required: 60 mg/kg dextran N 500, 5 mg/kg dextran N 10, 100 mg/kg polyethylensulfonate, and 5 mg/kg pentosanpolysulfate being the minimal doses.
The release of histaminase fter these drugs showed only one peak and the optimum pH for the activity was 7.4.
6.
Protamine leads to increased histaminase activity in plasma when given in doses more than 6 times higher than is necessary for inhibiting the heparin effect on the enzyme activity.
To investigate the possible embryologic relation between small-cell carcinoma of the lung and medullary thyroid carcinoma, we measured plasma histaminase (an enzyme found in medullary carcinoma tissue) in 25 patients with small-cell tumors. The assays used histamine and putrescine as substrates. Thirty-two per cent of the patients by the histamine assay, and 31 per cent by the putrescine, had values greater than +2 S.D. from the mean for 63 normal persons. In contrast, among 20 patients with squamous and large-cell lung tumors, one (by the histamine assay), and two (by the putrescine) had elevated values. In four of five autopsy cases, histaminase was high in small-cell carcinoma tissue. The enzyme in plasma and in tumor behaved as classic histaminase in substrate specificity, and in response to inhibitors. The data support the proposed embryologic relation between small-cell lung carcinoma and medullary thyroid carcinoma, and further associate histaminase with some neural crest tumors.
In this laboratory, histaminase activity has been assayed by measurement of the release of tritium from side-chain labeled β-3H-histamine, and this assay has proved useful in a variety of animal and clinical studies. In the present study, the assay was compared with that of diamine oxidase activity which uses 14C-labeled putrescine as substrate. Both activities are believed to be due to the enzyme, diamine oxidase, which has been shown to catalyze the deamination of diamines as well as histamine. The data showed that β-3H-histamine and 14C-putrescine were deaminated by the same enzyme in a variety of rat tissues and that the assays were interchangeable. The labeled amines competed for deamination, and their deamination was inhibited to the same extent by aminoguanidine which is a specific inhibitor of diamine oxidase. There was also a close correlation in the values obtained by the two assays in all tissues. The deamination of each amine was affected differently by changes in pH and substrate concentration, as has been shown previously for purified preparations of diamine oxidase. Substrate inhibition was, for example, observed with histamine, but not with putrescine. An unexpected finding was that histamine, in the concentrations usually encountered in tissues, markedly inhibited diamine oxidase activity. Concentrations of 10-5 M histamine (1·1 μg/g) inhibited putrescine deamination by 55 per cent and 10-4M (11·1 μ/g) by more than 90 per cent.
High diamine oxidase (DAO) and histamine activity are present in rat intestine, thymus and adrenals, and a close correlation exists between the two activities in these tissues (Biochem. Pharmac.24, 979 (1975)). The distribution of histaminase in normal and germ-free rat tissues and the release of this enzyme from intestine, thymus and adrenals was investigated in further detail with a tritium-release assay. Contrary to previous reports, histaminase activity was detected in brian, in the hypothalamus, thalamus and medulla but not in cortex and cerebellum. The enzyme was released by heparin into blood from intestine and adrenals but not from thymus. In high doses, heparin produced almost complete (>80 per cent) depletion of the enzyme in intestine within 1 hr. The enzyme activity reappeared and returned to normal levels by 24 hr. Prior administration of cycloheximide prevented the repletion of enzyme activity. The time course of the responses to the drugs suggested that DAO is synthesized continuously at a relatively rapid rate (). Studies in vitro indicated that DAO unlike monoamine oxidase diffuses from the mucosal surface into the lumen of the gut. DAO may therefore have a role in deaminating diamines of bacterial origin in the intestinal contents.
The effect of a synthetic steroid, oxandrolone, on total postheparin plasma lipolytic activity, postherpain hepatic lipase activity, lipoprotein lipase and phospholipase A1 was studied in seven patients with hypertriglyceridemia. The mean total postheparin lipolytic activity increased 100 per cent during oxandrolone tratement (p smaller than 0.05). This change was caused mainly by postheparin hepatic lipase, whose activity increased on the average more than 2.5 times (p smaller than 0.001). The change in postheparin plasma-lipoprotein-lipase activity was insignificant. A highly significant correlation (r equals +0.87, p smaller than 0.01) was observed between the activities of postheparin hepatic lipase and phospholipase A1 before and during oxandrolone treatment. No relation was observed between serum triglyceride level and various postheparin lipase activities, or between the changes induced by oxandrolone in the level of serum lipids and the activities of postheparin lipases. We conclude that oxandrolone increases the activities of postheparin plasma hepatic lipase and phospholipase A1 but has little influence on lipoprotein lipase.
In a previous paper (ScHff~A~N and Pm~rPu) we have shown that calcium ions accelerate the spontaneous release of catecholamines from isolated medullary granules when incubated in isotonic sucrose. In accordance with obervations of OKA et al. we found that in tris buffer (0.08 M) pH 7.4 containing K+ (5 ~moles/ml) and ~a+ (154 ~moles/mI) this calcium effect is augmented. For further experiments we have isolated granules from bovine splenic nerve and stellate ganglia by differential centrffugation at 100000Xg for 60 min. After 20 rain incubation at 22°C an acceleration of this spontaneous release of noradrenahne has been observed by 0.2, 1.0 and 5.0 [zmoles of calcium par ml. Omission of Na + and K + and incubation of nerve granules in 0.3 M iris buffer caused a reduced noradrenaline discharge by calcium. In the presence of Na+ and K + also aeetyleholine (1.0 to 25.0 nmoles/ml) produced an enhanced release of noradrenaline from nerve and ganglion granules but not from medullary granules. These results favour the assumption that the storage of catecholamines in various kinds of granules may be different. The experiments with calcium indicate that this ion which acts as liberator of catecholamines from medullary granules may also trigger the discharge of noradrenaline from the granules of sympathetic nerve terminals.
The release of histaminase activity in plasma after small intravenous of heparin was studied in 85 normal subjects and patients. In normal subjects, plasma histaminase activity (basal level, 1.7+/-0.1 U/ml, mean +/-SEM) increased 1.6+/-0.2 U/ml after 10 U of heparin/kg, 8.5+/-2.4 U/ml after 20 U/kg, and 33+/-4.9 U/ml after 75 U/kg. The extent of the increase varied widely among individuals but in a particular individual the response was constant and dose-dependent. Histaminase activity rose to peak levels within 7-15 min and then declined exponentially with a half-life of 40-120 min. This pattern of response was also observed in two patients with the histaminase-producing tumor, medullary carcinoma of the thyroid. A significantly reduced response was observed, however, in 14 patients with type I hyperlipoproteinemia, a disorder in which high plasma triglyceride levels are associated with low postheparin plasma lipolytic activity. After 10 U heparin/kg, plasma histamine activity increased 0.5+/-0.2 U/ml, and after 75 U heparin/kg, 10.9+/-5.6 U/ml. In contrast, in 27 patients with other types of hyperlipoproteinemia in whom postheparin lipolytic activity was normal, the increase (2.4+/-0.6 U/ml) in plasma histaminase activity after 10 U heparin/kg was not significantly different from that of normal subjects. The reduced response of the plasma histaminase activity to heparin in patients with type I hyperlipoproteinemia did not appear to be due to the presence of lipemia or to an inhibitor of the enzyme in plasma. These findings suggest that many patients with type I hyperlipoproteinemia may have deficient release of both lipolytic and histaminase activities into plasma after heparin administration.
The postheparin DAO increase in blood plasma has been shown to depend on intact intestines in rat and rabbit. Our findings indicate, however, that sources of mobilizable DAO may be found also outside the small intestine in these species. In the rat, after partial portal stricture for a week. hepatectomy was followed by an intense and rather sustained post-heparin DAO increase, as compared to normal, or shamoperated, rats.
1. A technique is described for the removal of subcellular contaminants from intact rat intestinal brush borders, and for the subsequent separation of a microvillus membrane fraction from a fibrillar residue. 2. Increments in invertase activity, microscopic homogeneity and low nucleic acid content indicate that the microvillus plasma membrane has been extensively purified. Multiple membrane preparations have been shown to be highly reproducible with respect to their invertase specific activity, cholesterol content and phospholipid content. Alkaline phosphatase, leucine aminopeptidase, Mg(2+)- and Ca(2+)-dependent adenosine triphosphatase and seven separate disaccharidases were shown to be predominantly confined to the membrane fraction. 3. The fibrillar fraction has been shown to contain approximately 30% of the total protein of purified brush borders, plus most of the residual nucleic acid contaminant. No evidence was found for the localization of any specific enzyme in this fraction.
H ansson , R. and H. T hysell . The effect of heparin and some related agents on diamine oxidase in rabbit blood plasma . Acta physiol. scand. 1970. 78 . 539–546.
In rabbits the blood plasma level of diamine oxidase (DAO) was followed after rapid i.v. injection of heparin, heparinoid substances, soluble ribonucleic acid and protamine chloride as well as the histamine releasing agents 48/80 and polymyxin B. Heparin(oid) injections elicited a striking DAO‐increase within 1/2 min, reaching maximal values within a little more than 1 hr and then smoothly declining to basal levels. The DAO‐increase was dose‐dependent and seemed to be larger during pregnancy and in newborn animals. Continuous heparin infusion was followed by a delayed peak time and, in relation to the doses, a low but protracted increase in the blood phma DAO. Sulfated polyanions caused a significant DAO increase, but no simple correlation was found between the size of the response and the amount of sulfur injected. The findings are discussed.
The catalytic rate of lipoprotein lipase has been determined before and after solubilization from the perfused rat heart. Both the apparent Km and kc values were closely similar for the membrane-bound and soluble lipase. This result was confirmed for the major plasma very low density lipoprotein fraction (Sf 100-400) and for two subfractions of rat lymph chylomicrons (Sf 100-400 and Sf >400). These findings suggest a superficial binding site for lipoprotein lipase at the capillary wall, and the absence of major conformational change during solubilization. Both membrane-bound and soluble lipase showed catalytic rates about twofold greater with chylomicron than with very low density lipoprotein triglyceride. These findings are discussed in the light of recent ideas on the mechanism of lipoprotein lipase activity.
PIP
Histaminase activity in tumor and tissues from 7 patients with medullary carcinoma of the thyroid was compared with that from control patients with other diseases. In control patients, tissue histaminase activity was high only in kidney and ileum. In 1 patient who died of widely disseminated medullary carcinoma, high histaminase activity was found in both solid tumor and tissues which did not have gross tumor. Histologic studies showed that the tissues with high enzyme activity contained microscopic foci of the tumor. In a 2nd patient with widely disseminated medullary thyroid carcinoma, who had received the histaminase inhibitor aminoguanidine 5 hours before death, low enzyme activity was found in serum, tumor, kidney, ileum and other tissues. This patient had high serum histaminase activity before administration of aminoguanidine. It appeared that the drug had inhibited the enzyme activity in both serum and tissues. Tumors removed from 4 patients with localized medullary carcinoma of the thyroid had high enzyme activity. Pheochromocytomas from 6 patients had low histaminase activity and could thus be differentiated from medullary thyroid carcinoma. A pheochromocytoma from 1 patient, who died of disseminated medullary carcinoma, had high histaminase activity and microscopic foci of medullary carcinoma. Another patient with a tumor of the mediastinum and a pheochromocytoma had high histaminase activity in serum and the mediastinal tumor. This finding raised the possibility that the mediastinal tumor was an unusual primary tumor of the mediastinal parafollicular cells and may be related to medullary thyroid carcinoma. No other tumor examined had high histaminase activity. It was concluded that histaminase activity in surgical and autopsy specimens can serve as a specific biochemical marker for the presence of medullary thyroid carcinoma.
In two men and one woman the diamine oxidase (DAO) and lipoprotein lipase (LL) activities were determined by micromethods in lymph and blood plasma before injection of heparin intravenously and thereafter followed during the next two hours.
The DAO activity in thoracic duct lymph was much higher than in blood plasma. The injection of heparin was followed by a prompt rise in blood plasma of DAO (about 20-fold) and, with some delay, in the central lymph (about 200-fold). The maximal DAO activity in thoracic duct lymph was about 250 times higher than in blood plasma.
The heparin-induced increase of the plasma-DAO seems, in contrast to the plasma-LL, to be partially mediated by the lymph.
The significance of these findings in the investigation of the rate of elimination of plasma-DAO, as well as the role played by the lymph vessels in organ preparations for study of the enzyme liberation after endogenous heparin in anaphylactic shock are briefly discussed.
Certain anionic or cationic polyelectrolytes, e.g. heparin and protamine, arc potent releasers of histaminase in the guinea pig. These substances do not activate a hista-minase precursor but release the active enzyme from the existing store in the liver which was found to be associated mainly with particles of the microsomal fraction. The histaminase release proceeds not only in the living animal but also in the isolated, blood-free perfused liver. Twenty-four hours after a depleting polyelectrolvte injection in the intact animal the histaminase contents of the liver are restored by a mechanism involving a DNA-dependent protein synthesis. The released enzyme catalyzes the oxidative deamination of histamine as well as of cadaverine, B-phenylethylamine, spermidine, putrcscine and benzylamine. It is inhibited by diamine oxidase inhibitors but not by monoamine oxidase inhibitors.
Heparin produces a substantial rise in diamine oxidase activity in rat plasma. An increase in the plasma is evident within 10 min of an intravenous (i.v.) injection of heparin and a peak is observed between 30 and 60 min. The rise in the plasma can be accounted for by the release of diamine oxidase from the intestine, since the enzyme activity in this tissue is markedly reduced when the plasma level is at its peak. Only a slight increase in the plasma level is observed when heparin is given to eviscerated animals, suggesting that the contribution from tissues other than the intestine is small. As the rise in plasma activity is evident even with normal anticoagulant doses of heparin, care should be exercised in whole animal experiments when potential substrates for diamine oxidase, such as histamine and putrescine, are being studied.
After i.v. injection of heparin (1.6 IU/g b.w.) there was a significant increase of the diamine oxidase (DAO) activity in lymph and blood plasma in rat, guinea-pig and rabbit. The ratio between lymph and plasma DAO activity was higher in rat and rabbit than in guinea-pig under basal conditions. This difference was even more pronounced after heparin injection. Withdrawal of mesenteric or thoracic duct lymph from the circulation of the animals was accompanied by a poor response to heparin of the plasma DAO activity in rat and rabbit, whereas the response in the guinea-pig seemed to be uninfluenced. These results suggest that, in analogy with earlier findings in man, the contribution from lymph to the heparin-induced DAO response in blood plasma may be significant in the rat and rabbit, but not in the guinea-pig.