Intestinal permeability in patients with coeliac disease and dermatitis herpetiformis

Intestinal permeability was investigated in patients with coeliac disease and dermatitis herpetiformis by a 51Chromium-labelled ethylenediaminetetraacetate (51Cr-EDTA) absorption test and the results correlated with histomorphometric analysis and intraepithelial lymphocyte counts of jejunal biopsies. The mean (+/- SD) 24 hour urine excretion of 51Cr-EDTA in 34 healthy volunteers was 1.9 +/- 0.5% of the orally administered test dose. Patients with untreated coeliac disease (19) or untreated dermatitis herpetiformis (five) excreted significantly more 51Cr-EDTA than controls (5.9 +/- 2.7% and 4.6 +/- 2.1%, respectively, p less than 0.001) and all were outside the normal range of 1.0-2.6%. Patients with coeliac disease (42) treated for 6 months-23 years (mean 5 years) and patients with dermatitis herpetiformis (11) treated for 6 months-8 years (mean 3 years) excreted significantly more 51Cr-EDTA than controls, 4.2 +/- 2.4% p less than 0.0001 and 3.0 +/- 0.9% p less than 0.003 respectively. Eleven of 14 (79%) treated patients with coeliac disease with an entirely normal jejunal mucosae demonstrated abnormal intestinal permeability. Intestinal permeability did not correlate significantly with either the mucosal height/crypt depth ratio or intraepithelial lymphocyte counts in jejunal biopsies from patients with untreated or treated coeliac disease. The demonstration of a persistent increase in intestinal permeability in patients with both coeliac disease and dermatitis herpetiformis may suggest a common pathogenetic mechanism in both disorders. It is postulated that altered permeability may facilitate the entry of gluten or a fraction thereof into the lamina propria where it causes a cascade of immunological events.     

Enzyme activities in jejunal biopsy samples from patients with adult coeliac disease with and without steatorrhoea

Highly sensitive techniques have been used for the assay of a range of marker enzymes including lactase, sucrase, alkaline phosphatase, leucyl-beta-naphthylamidase (brush border), and 5'-nucleotidase (basolateral membrane) in jejunal biopsy homogenates from patients with adult coeliac disease with and without steatorrhoea and from a control group. The absorption of D-xylose and vitamin B12 was compared in the two groups with coeliac disease. All enzymes assayed were equally depressed in both groups of coeliac disease as compared with the controls. The absorption of D-xylose and vitamin B12 were reduced in the patients with steatorrhoea compared with those without steatorrhoea. The findings suggest that lack of steatorrhoea in some patients with coeliac disease is due to better preservation of the ileal function rather than to a less severe jejunal mucosal injury.     

Plastic pH electrodes for the measurement of gastrointestinal pH.

Plastic electrodes have been developed for measuring pH in the human gastrointestinal tract. The electrodes have a plastic hydrogen ion sensitive membrane sealed to a length of fluid filled PVC tubing. Two recently developed hydrogen ion sensitive ligands have been examined. Their operational characteristics have been described. These electrodes have an electrical response of 52 to 58 mV/pH unit change in the range pH 4-9, with a diminished response outwith this range. They have a low resistance value and a fast response time of one second to reach 90% of their maximum response. The electrodes can be passed down the biopsy channel of an endoscope to obtain mucosal pH readings under direct vision. Readings obtained in this way using plastic electrodes are comparable to those obtained with glass electrodes. Alternatively, these electrodes can be joined to a Crosby capsule, allowing continuous recording of mucosal pH through to the jejunum during jejunal biopsy procedures. These electrodes can be used repeatedly or may be acceptable as inexpensive disposable items for sterile clinical use.     

Acid microclimate in coeliac and Crohn's disease: a model for folate malabsorption.

The surface pH of human proximal jejunum was measured in biopsy samples and found to be more acid than the phosphate buffer in which they were incubated. The in vitro jejunal surface pH was 5.93 +/- 0.05 in control subjects and 6.19 +/- 0.09 in treated coeliac patients. A group of untreated coeliac patients with a surface pH of 6.56 +/- 0.14 had a significantly less acid surface pH compared to controls, as did a group of Crohn's patients with a surface pH of 6.21 +/- 0.04. These two groups with a significantly raised surface pH were subdivisible into 'high' and 'low'groups. Surface pH was found to remain low in the treated coeliac and control groups but became more acid over the incubation period reaching almost normal values in the Crohn's group and the untreated coelic initial surface pH. The raised surface pH in untreated coeliac disease and Crohn's disease would alter the amount of a weak acid available for non-ionic diffusion. Therefore the present results may help to explain the folate malabsorption known to occur in untreated coeliac disease and the frequently seen low serum folate levels in Crohn's disease.     

Intracellular pH in isolated Necturus antral mucosa exposed to luminal acid

Regulation of intracellular pH in gastric epithelial surface cells exposed to luminal acid was investigated in isolated Necturus antral mucosa using microelectrode technique. Exposure of the mucosa to luminal pH 2 acidified intracellular pH from 7.21 +/- 0.01 to 6.95 +/- 0.04 (N = 50). Removal of Na+ from the perfusates or addition of amiloride (1 mM) to serosal perfusate (containing HCO3-) had no influence on intracellular pH during exposure to pH 2 (N = 6), but removal of HCO3-/CO2 from or addition of 4, acetamido-4-isothiocyanatostilbene-2,2-disulfonic acid (0.5 mM) to the serosal perfusate (containing Na+) acidified intracellular pH from 7.02 +/- 0.03 to 6.45 +/- 0.15 (p less than 0.01, N = 10) and from 6.97 +/- 0.06 to 6.58 +/- 0.26 (p less than 0.01, N = 6), respectively, in 15 min. In tissues exposed to mucosal pH 6, epithelial surface pH was about 1.3 pH units higher than pH of the mucosal bulk solution. Removal of Cl-/HCO3- from the serosal perfusate acidified epithelial surface pH by about 0.5 pH units (p less than 0.01, N = 6), suggesting that serosal HCO3- sustains intracellular pH, at least in part, by generating an alkaline buffer layer at the epithelial surface. In the absence of HCO3-/CO2, a stable intracellular pH was obtained when the tissue was exposed to mucosal pH 2.7, but in this situation intracellular pH was sensitive to Na+ removal or amiloride addition, intracellular pH decreasing from 7.00 +/- 0.07 to 6.48 +/- 0.10 (p less than 0.01, N = 6) and from 6.86 +/- 0.06 to 6.32 +/- 0.01 (p less than 0.01, N = 7), respectively, in 15 min. The data suggest that in gastric epithelium exposed to luminal acid, physiological intracellular pH is primarily maintained by the buffer action of serosal HCO3- transported to the epithelial surface to impede the entry of luminal H+ into mucosal tissue. Removal of the sheltering HCO3- unmasks a second line, Na(+)-dependent and amiloride-sensitive intracellular pH regulatory mechanism, presumably a Na+/H+ antiport.     

Acute gluten challenge in treated adult coeliac disease: a morphometric and enzymatic study.

Using a Quinton hydraulic biopsy tube, jejunal biopsies were obtained from 10 patients with adult coeliac disease in remission and four healthy volunteers before and after administration of gluten fraction III into the proximal duodenum. The biopsies taken at hourly intervals for four hours, were analysed for changes in brush border enzymes, light microscopic appearances, and villous and crypt population counts. The results indicate that mucosal damage occurs within three to four hours of gluten administration with significant falls in brush border enzyme concentrations and villous population counts. The absence of any change in control biopsies indicates that gluten sensitivity is specific to the mucosa of patients with coeliac disease, the timing of the changes being consistent with a type III immune response or direct toxicity. Some recovery of the brush border enzymes but not the villous population was evident 24 hours after gluten administration while the crypt population showed evidence of a compensatory hyperplastic reaction.     

Oral zinc supplements in non-responsive coeliac syndrome: effect on jejunal morphology, enterocyte production, and brush border disaccharidase activities.

Three patients with clinically mild non-responsive adult coeliac syndrome were treated for eight weeks with oral zinc sulphate, and detailed biochemical and morphological studies were performed on the jejunal mucosa before and on treatment. Plasma zinc levels were reduced before treatment and rose to normal levels with therapy; mucosal zinc was normal before treatment and increased after therapy. Oral zinc supplementation did not alter the villous morphology, intraepithelial lymphocyte count or in vitro enterocyte production rate. In addition, there was no improvement in the clinical status of the patients. There was, however, a small increase in the activity of certain of the brush border disaccharidases. This effect may be due to direct stabilisation of the brush border membrane. The clinical value of zinc supplements in coeliac syndrome remains to be determined.     

Studies of luminal and mucosal pH in reflux esophagitis and antral gastritis.

Luminal and mucosal pH were measured endoscopically in patients with reflux esophagitis and antral gastritis and in control subjects. In all subjects, significant lumen-to-mucosa gradients were observed in the esophagus, stomach and acidified proximal duodenum. In the reflux patients luminal pH was lower in the fundus (mean +/- SEM, control vs. reflux esophagitis: 2.01 +/- 0.17 vs. 1.32 +/- 0.18; p less than 0.02) and antrum (3.51 +/- 0.35 vs. 2.13 +/- 0.24; p less than 0.01) and, in the gastritis patients, in the fundus (2.01 +/- 0.17 vs. 1.3 +/- 0.17; p less than 0.02). In both patient groups, mucosal pH was lower in the fundus (control vs. reflux vs. gastritis: 4.84 +/- 0.37 vs. 3.37 +/- 0.61 vs. 3.12 +/- 0.6; p less than 0.05) and acidified duodenal cap (6.74 +/- 0.13 vs. 6.09 +/- 0.24 vs. 5.73 +/- 0.46; p less than 0.03). Mucosal pH profiles at the various sites showed less resistance of the gradient to a highly acidic environment in both the lower esophagus and antrum than in fundus and duodenum, and this was the case in the patient and control groups. Though associated with a more acid environment, neither esophagitis nor antral gastritis exhibits a specific deficit in the 'mucus-bicarbonate barrier', suggesting that the pathogenesis of these disorders may depend more on abnormal 'attack' rather than impaired defense.     

Ascitic pH and infection in alcoholic cirrhosis

The pH values of 108 samples of ascitic fluid in 94 alcoholic cirrhotic patients were analyzed in order to assess their diagnostic and prognostic value. The mean pH value of ascitic fluid was significantly lower (p less than 0.001) in patients with spontaneous bacterial peritonitis (7.23 +/- 0.22) or with suspected diagnosis of spontaneous bacterial peritonitis (7.29 +/- 0.15) than in patients with sterile ascites (7.45 +/- 0.06). However, there was an important overlap between these groups. In patients with and without spontaneous bacterial peritonitis, measurement of the difference between blood and ascitic pH was more discriminative than the ascitic pH alone: a difference of 0.10 or more was detected in all patients with spontaneous bacterial peritonitis, in 2 of 5 patients with suspected diagnosis of spontaneous bacterial peritonitis and in 3 of 97 patients with sterile ascites. When the ascitic pH value was lower than 7.15, death occurred rapidly. Ascitic pH rapidly increased when treatment of spontaneous bacterial peritonitis was clinically effective. These results suggest that measurement of pH in ascitic fluid is contributive to the diagnosis and prognosis of spontaneous bacterial peritonitis in alcoholic cirrhosis.     

Human intestinal mucosal mast cells: expanded population in untreated coeliac disease.

Previous retrospective studies of intestinal mucosal mast cells in coeliac disease have given divergent results, and we have recently reported that inappropriate methodology could account for these discrepancies. In this prospective study, mucosal mast cell counts were performed in Carnoy fixed, peroral jejunal biopsy specimens from patients with coeliac disease, both untreated and treated with a gluten-free diet; and from controls (mainly irritable bowel syndrome). Mean mucosal mast cell count in 27 control subjects was 146/mm2, SD 29. Significantly higher values were obtained in untreated coeliac disease (mean 243, SD 41, p less than 0.001) returning to the normal range in coeliacs treated with a gluten-free diet with normal jejunal biopsy morphology. In seven patients mucosal mast cell counts were performed in multiple jejunal biopsies, and these showed that mucosal mast cell distribution was not patchy. There was no evidence of degranulation of intestinal mucosal mast cells under the conditions of routine biopsy (overnight fast). An increase in mucosal mast cells in untreated coeliac disease may be one explanation for the high number of IgE positive stained cells in the intestinal mucosa that has been reported by some authors.     

Immunoglobulin secretion by isolated intestinal lymphocytes: spontaneous production and T-cell regulation in normal small intestine and in patients with coeliac disease.

The in vitro secretion of immunoglobulins by small intestinal lymphocytes isolated from 47 patients with normal histology and 23 patients with treated and untreated coeliac disease was examined using an enzyme linked immunosorbent assay. In control patients, duodenal lymphocytes spontaneously secreted higher levels of IgM than jejunal lymphocytes (p less than 0.05). Significantly higher levels of both IgA (p less than 0.05) and IgM (p less than 0.001) were secreted by jejunal lymphocytes of 10 patients with untreated coeliac disease than cells isolated from normal jejunal tissue. IgM and IgA secretion by duodenal lymphocytes isolated from control patients was increased in a dose dependent manner by coculture with autologous peripheral blood T lymphocytes. This effect was not observed with jejunal lymphocytes of control or treated coeliac patients. Peripheral T-cells of untreated coeliac patients, however, showed significant helper effects (p less than 0.05) for IgM and IgA secretion by autologous jejunal lymphocytes. The results suggest that jejunal lymphocytes of patients with untreated coeliac disease show major differences in their capacity to synthesise and secrete immunoglobulins in vitro and the enhanced secretion might result from changes in T-cell immunoregulatory function.     

Alkaline phosphatase synthesis and properties of subcellular organelles during in vitro culture of jejunal biopsies from control subjects and patients with coeliac disease.

Portions of jejunal biopsies from control subjects and from patients with coeliac disease were cultured for 24 hours using an in vitro organ culture technique. Alkaline phosphatase activity was measured in the tissue and medium before and after culture; enzyme activities were expressed per microgram tissue DNA. The increase in enzyme activity during the culture period was taken to represent net enzyme synthesis. Alkaline phosphatase synthesis by mucosa from patients with untreated gluten-sensitive coeliac disease and by mucosa from patients with non-responsive coeliac disease was significantly less than that by normal mucosa. Alkaline phosphatase synthesis by mucosa from patients with treated gluten-sensitive coeliac disease was greater than that by untreated coeliac mucosa but was less than normal mucosa. Sequential studies of alkaline phosphatase synthesis by jejunal mucosa from seven patients with coeliac disease, before and after successful treatment by gluten withdrawal, showed an increase in synthesis in all patients. Study, by analytical subcellular fractionation with sucrose density gradient centrifugation, of the properties of the organelles of cultured control tissue showed good preservation of their integrity. A striking finding, however, was the decrease in malate dehydrogenase with a corresponding marked increase in lactate dehydrogenase. This would be expected to be followed by a shift from aerobic to anaerobic metabolism. Analytical subcellular fractionation of cultured mucosa from patients with coeliac disease gave similar conclusions. There was, however, a marked improvement of the brush border abnormalities, characteristic of coeliac disease, during culture with increased enzyme activities and membrane equilibrium density in the sucrose gradients.     

The ileal brake--inhibition of jejunal motility after ileal fat perfusion in man

The possibility that malabsorbed fat passing through the human ileum exerts an inhibitory feedback control on jejunal motility has been investigated in 24 normal subjects by perfusing the ileum with a fat containing solution designed to produce ileal luminal fat concentrations similar to those in steatorrhoea (30-40 mg/ml). Mean transit times through a 30 cm saline perfused jejunal segment were measured by a dye dilution technique. Thirty minutes after ileal fat perfusion, mean transit times rose markedly to 18.9 +/- 2.5 minutes from a control value of 7.5 +/- 0.9 minutes (n = 5; p less than 0.05). This was associated with an increase in volume of the perfused segment which rose to 175.1 +/- 22.9 ml (control 97.6 +/- 10.3 ml, n = 5; p less than 0.05). Transit times and segmental volumes had returned towards basal values 90 minutes after completing the fat perfusion. Further studies showed that ileal fat perfusion produced a pronounced inhibition of jejunal pressure wave activity, percentage duration of activity falling from a control level of 40.3 +/- 5.0% to 14.9 +/- 2.8% in the hour after ileal perfusion (p less than 0.01). Ileal fat perfusion was associated with marked rises in plasma enteroglucagon and neurotensin, the peak values (218 +/- 37 and 68 +/- 13.1 pmol/l) being comparable with those observed postprandially in coeliac disease. These observations show the existence in man of an inhibitory intestinal control mechanism, whereby ileal fat perfusion inhibits jejunal motility and delays caudal transit of jejunal contents.     

Defective jejunal brush border membrane sodium/proton exchange in association with lethal familial protracted diarrhoea.

The spectrum of clinical disease associated with specific defects in jejunal brush border membrane sodium/proton exchange is poorly defined and only two patients have been described so far. Jejunal brush border membrane transport studies were performed in a boy who presented with lethal familial protracted diarrhoea in the first few days of life. Using jejunal brush border membrane vesicles prepared from conventional jejunal biopsy specimens, initial sodium uptake under H+ gradient conditions was found to be only 6% of the mean control value. In contrast, sodium stimulated glucose uptake was normal. Our data confirm the importance of a congenital defect in this exchanger as a cause of severe sodium-losing diarrhoea and extend the spectrum of disorders characterised by a specific defect in brush border membrane Na+/H+ exchange to include some forms of lethal familial protracted diarrhoea.     

Secretin release in coeliac disease. Plasma secretin concentration and bicarbonate output to the duodenum after intraduodenal acid infusion in coeliac patients before and after treatment

Infusion of 40 ml 0.1 mol/l HCl into the duodenum in eight untreated coeliac patients was followed by an increase of the plasma immunoreactive secretin (IRS) concentration from 1.6 +/- 0.2 pmol/l to a peak level of 2.4 +/- 0.3 pmol/l (p less than 0.05). After treatment with a gluten-free diet, the same patients showed an increase from 1.4 +/- 0.3 pmol/l to a peak level of 5.5 +/- 0.9 pmol/l after intraduodenal acid infusion, which was significantly higher than before treatment (p less than 0.01). In control subjects, intraduodenal acid infusion was followed by an increase from 1.4 +/- 0.2 pmol/l to 6.7 +/- 1.1 pmol/l, which was significantly higher than in untreated coeliac disease (p less than 0.01) but did not differ from what was found in treated coeliac patients. Significant differences in pH, volume, or bicarbonate content of the duodenal aspirates or the basal IRS levels were not found.     

Relationships between mucosal hydrolysis and transport of two phenylalanine dipeptides.

In order to investigate the source of free amino acids found in the gut lumen during absorption of dipeptides, as well as evaluating the role of brush border peptidases in the mucosal hydrolysis of dipeptides during absorption, rates of dipeptide disappearance and appearance of hydrolytic products were measured during perfusion of rat jejunum and ileum in vivo with buffered and unbuffered 10 mM solutions of glycl-L-phenylalanine (Gly-Phe) and L-phenylalanyl-glycine (Phe-Gly). Mucosal brush border peptidase activity was then measured in the perfused segments in vitro at luminal pH and at two substrate concentrations. In addition cytosol peptidase activity in the perfused segments was measured at pH 7-4 and at 10 mM substrate concentrations. In the jejunum, there was a relationship between rates of free phenylalanine appearance in vivo (Phe-Gly greater than Gly-Phe) and rates of brush border (Phe-Gly greater than Gly-Phe) rather than cytosol (Gly-Phe greater than Phe-Gly) peptidase activities. No constant relationship between free phenylalanine appearance and hydrolysis of the dipeptides by either brush border or cytosol peptidases was observed in the ileal studies. These findings suggest that, in the jejunum, hydrolytic products originate from the surface of the cell whereas, in the ileum, hydrolytic products originate from both the intracellular compartment as well as from the surface of the mucosal cell. In the jejunum, in vitro rates of brush border hydrolysis of Gly-Phe were always less than in vivo disappearance rates, whereas rates of Phe-Gly brush border hydrolysis always exceeded luminal disappearance rates. These data imply that Gly-Phe is predominantly transported intact and hydrolysed by cytosol peptidases, In contrast, brush border peptidases play an importnat role in the mucosal hydrolysis of Phe-Gly.     

Functional defect of zinc transport in patients with Crohn's disease

In the present study, we investigated the question whether the zinc deficiency in Crohn's disease is, at least partly, related to defective zinc entry at the jejunal brush border membrane. Brush border membrane vesicles (BBMV) were prepared from surgically resected pieces of morphologically intact human jejunum by a Mg++ precipitation method. We studied zinc, D-glucose, sodium and D-mannitol uptake into BBMV in 27 patients: 1) 20 patients with Crohn's disease 2) 7 controls. In 8/20 patients with Crohn's disease we found a significant decrease in zinc uptake (-25% +/- 5%, p less than 0.05) into BBMV. This impairment of zinc uptake in 40% of our patients with Crohn's disease was not associated with changes in the transport rates for D-glucose, sodium or D-mannitol. In addition no differences in enzyme marker concentrations or electron microscopic appearance were found.     

Measurement of gastrointestinal pH profiles in normal ambulant human subjects.

Gastrointestinal (GI) pH has been measured in 66 normal subjects using a pH sensitive radiotelemetry capsule passing freely through the gastrointestinal tract. Signals were recorded with a portable solid state receiver and recording system, enabling unconstrained measurements with normal ambulatory activities for up to 48 h during normal GI transit. Capsule position in the gut was monitored by surface location using a directional detector. Gastric pH was highly acidic (range 1.0-2.5) in all subjects. The mean pH in the proximal small intestine was 6.6 (0.5) for the first hour of intestinal recording. By comparison the mean pH in the terminal ileum was 7.5 (0.4) (p less than 0.001). In all subjects there was a sharp fall in pH to a mean of 6.4 (0.4) (p less than 0.001) as the capsule passed into the caecum. Values are means (SD). pH then rose progressively from the right to the left colon with a final mean value of 7.0 (0.7) (p less than 0.001).     

Characterization of specific pancreatic polypeptide receptors on basolateral membranes of the canine small intestine.

We have identified specific binding sites for pancreatic polypeptide (PP) on the mucosal lining of canine small intestine. The present study was undertaken to further characterize these binding sites (receptors) on purified intestinal membranes and to establish their location on the brush border or basolateral surface of the intestinal enterocyte. Basolateral and brush border membranes were prepared by sorbitol density centrifugation. PP receptors were localized predominantly to the vascular surface, and thus binding of PP 125I-labeled on Tyr-27 to the basolateral preparation was used to evaluate receptor characteristics. Binding of PP was calcium, time, temperature, and pH dependent. Maximum specific binding of labeled PP occurred after an 8-hr incubation at 4 degrees C with 5 mM calcium at pH 6.8. Data analysis by Scatchard plot showed high- and low-affinity binding sites with relative affinities of 1.5 x 10(-9) M and 2.6 x 10(-8) M and with corresponding binding capacities of 0.23 pmol/mg and 0.84 pmol/mg of protein, respectively. This receptor was specific for PP since peptide YY and neuropeptide Y, peptides of the PP family, cross-reacted by less than 3%, as judged from comparisons of half-maximal displacement of label. Structurally dissimilar peptides, insulin and glucagon, did not compete for binding. Specific 125I-labeled PP binding was localized primarily to basolateral membranes (9.8 +/- 0.8%) with little binding by brush border membranes (0.8 +/- 0.2%). Thus, we have identified highly specific receptors for PP, located predominantly on the vascular surface of the small intestinal mucosa. These data suggest that the mucosal lining of the small intestine is a target tissue for PP and that PP participates in the hormonal regulation of fuel metabolism and substrate transport in the small intestinal mucosa.     

Glycoprotein synthesis and secretion by cultured small intestinal mucosa in coeliac disease.

Glycoprotein biosynthesis by jejunal mucosa was examined during culture in vitro in 26 patients with coeliac disease and 19 controls. The incorporation rates of tritiated glucosamine into tissue and secreted glycoproteins were determined using established techniques. The total glucosamine incorporation in untreated coeliac patients was significantly greater than that of histologically normal mucosa (p less than 0.001) and jejunal tissue from patients with treated coeliac disease (p less than 0.01). Enhanced secretion of in vitro labelled glycoproteins was observed in untreated coeliac patients. The total incorporation of tritiated glucosamine in intestinal tissues was correlated with goblet cell numbers. These results indicate that quantitative changes in glycoprotein synthesis and secretion occur in coeliac disease.