Luciano D'Agostino

Diamine Oxidase in Rat Small Bowel: Distribution in Different Segments and Cellular Location

Diamine oxidase (DAO) is an enzyme with high activity found in the small bowel mucosa of the rat and man, and only with very low activity in all other tissues. The present study was designed to investigate the enzymatic distribution along 8 consecutive small bowel segments of mucosa and to test the DAO concentration on brush border membranes obtained from whole mucosal homogenate in Wistar rats. Our data document that DAO activity is mainly distributed in intermediate and distal small bowel segments and is not significantly associated with enterocyte brush border.

Postheparin plasma diamine oxidase in subjects with small bowel mucosal atrophy

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·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<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.

Ornithine decarboxylase and diamine oxidase in human colon carcinoma cell line CaCo-2 in culture.

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.

Postheparin Plasma Diamine Oxidase in Patients With Small Bowel Crohn's Disease

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 Crohns 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 Crohns 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 Crohns disease and that PHD may be useful in assessing and monitoring mucosal damage in these patients.

Postheparin plasma diamine oxidase increases in patients with coeliac disease during gluten free diet.

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.

Modifications in enterocyte diamine oxidase distribution induced by heparin in the rat

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.

Metabolic fate of plasma diamine oxidase: evidence of isolated and perfused rat liver uptake

After injection of an intravenous bolus of heparin (15,000 IU) in two groups of subjects, 10 normal volunteers and 6 subjects with external biliary drainage, blood and urine samples were collected; in the latter group bile samples were collected also. All samples were assayed for diamine oxidase (DAO). Persistently high values of this enzyme were found in plasma of both populations after heparin stimulation, while no increase in enzymatic activity was detected in bile and urine. In order to confirm and support the hepatic clearance of DAO, liver uptake of the enzyme derived from porcine kidney, human plasma and human placenta was studied by perfusion of isolated rat liver. Disappearance curves of the enzyme derived from three different sources showed a prompt liver uptake: activity decreased by about 50% in 10 min (endocytic uptake) and a slower but constant reduction during the remaining 110 min of perfusion was observed. These data suggest the hypothesis of liver metabolism of plasma DAO.

Plasma diamine oxidase (DAO) and heparin

To The Editor: DAO is an enzyme found in high concentration in rat and human small bowel mucosa (1, 2) and almost exclusively located at the top of the villus (3). This enzyme is strictly related to the metabolism of polyamines which are involved in nucleic acid synthesis (4). Luk et al (1) claimed that plasma DAO values reflect the small bowel mucosa morphofunctional integrity in the rat. Furthermore, Bayless et al (5) reported that plasma DAO activity was decreased in patients with nontropical sprue and ileal Crohns disease. However, in our experience (6), the very low normal basal values of plasma DAO do not permit significant disclosure of further reductions caused by intestinal disease. Therefore we have tried to define a provocative test which may overcome this difficulty. Intravenous heparin induces a rise in plasma DAO activity in humans and other vertebrates, releasing the enzyme from the tip of the villus (7). We have studied (Figure 1) response to a high and nearly maximal (8) dose of heparin (15,000 IU by intravenous bolus) in 13 normal volunteers (seven males, six females, mean age 42), selected by a normal routine blood analysis and normal xylose test, and eight patients (three males, five females, mean age 27) with a severe malabsorption syndrome with steatorrhea (> 15 g/day), abnormal xylosuria (<9%), hypocholesterolemia (<120 mg/100 ml), hypoalbuminemia (<2.8 g/100 ml), and a subtotal atrophy at Crosby jejunal mucosa biopsy. The DAO assay has been performed by the Okuyama and Kobayashi method (9) as modified in our previous study (2). As shown in figure 1, basal DAO values are different in normal and pathological subjects (0.06 0.02 and 0.04 -+ 0.01 U,* respectively) but a consistent overlap of values is present. Heparin enhances the plasma enzyme activity in both populations, but the response is significantly lower (two sample t test: P < 0.001) in patients. The zenith of the normal curves is about five times higher than in small bowel atrophic patients.

Postheparin plasma diamine oxidase values in the follow up of patients with small bowel Crohn's disease

Measurement of postheparin plasma diamine oxidase (PHD) activity has been proposed to assess mucosal integrity in several diseases of the small intestine. In Crohns 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 Crohns disease and whether different PHD values can predict different outcomes the changes in PHD values in 41 patients with small bowel Crohns 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 (Crohns 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 (Yatess 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 Crohns disease patients who are likely to relapse. Furthermore, the data indicate that mucosal damage is common in active small bowel Crohns disease and improves at least in part after treatment.

Postheparin plasma diamine oxidase in subjects with small bowel disease. Diagnostic efficiency of a simplified test

Diamine oxidase (DAO) is an enzyme located almost exclusively in the villus tip enterocytes of mammals. Its plasma activity, normally very low, is enhanced by intravenous heparin that releases the enzyme from the enterocytes into the blood. Postheparin plasma DAO (PHD) values have been shown to be significantly reduced in patients with malabsorption and villus atrophy and in patients with Crohns disease, thus suggesting that this test explores the mucosal integrity. The execution of the PHD test requires 8 blood samples over a period of 120 min after an intravenous injection of 15,000 IU of heparin, and then the calculation of the area under the curve. The aim of this study was to simplify the test and make it more acceptable by choosing only one of the eight curve points required for the calculation of the area under the curve and then assessing its discriminant power in different small bowel diseases. To this end, a discriminant analysis was performed on PHD curves of 16 normal subjects, 25 patients with celiac disease, 14 patients with treated celiac disease, 5 patients with diffuse primary small bowel lymphoma, 4 patients with small bowel lymphoma during treatment, and 55 patients with small bowel Crohns disease. Plasma DAO values assayed 1 h (T60) after the injection of 15,000 IU of heparin proved to be the best discriminator curve point and the use of the T60 point alone may be usefully employed instead of the area under the 120-min curve.(ABSTRACT TRUNCATED AT 250 WORDS)