Pieter B. Bijlsma

Differential in vivo and in vitro intestinal permeability to lactulose and mannitol in animals and humans: A hypothesis

BACKGROUND/AIMS Clinical interpretation of urinary recovery ratios of lactulose and mannitol is hampered by incomplete understanding of the mechanisms of transmucosal passage. The aim of this study was to compare in vivo and in vitro probe permeability. METHODS Stripped sheets of small intestine from rodents and human biopsy specimens were mounted in Ussing chambers, and mucosa-to-serosa fluxes of lactulose and mannitol were determined. Urinary recovery of orally applied probes was measured in rodents, cats, and humans. RESULTS In vitro lactulose/mannitol flux ratios were close to 0.8 in all species. Urinary recovery ratios differed between rodents and cats or humans; low ratios in cats and humans were due to high mannitol recovery. CONCLUSIONS Interspecies variation in urinary recovery of mannitol is caused by differences specific for the intact small intestines in vivo. Because hyperosmolality of villus tips in vivo varies, being highest in humans and cats as a result of vascular countercurrent multiplication, it is hypothesized that the high urinary recovery of mannitol in these species is caused by solvent drag through pores that allow the passage of mannitol but not of lactulose. Therefore, the lactulose/mannitol ratio is primarily a standard for the normal functioning of villus epithelial cells in metabolite absorption and for normal villus blood flow.

Stress‐Induced Decrease of the Intestinal Barrier Function: The Role of Muscarinic Receptor Activation

Abstract: Recently the breakdown of the barrier function of the intestinal epithelium after application of an experimental psychological and physical stress protocol in rats has been observed. Not only did smaller molecules pass from the luminal to the serosal side, but so also did larger proteins with the dimensions of luminal antigens and toxins. The increased permeability for macromolecules is primarily due to a decrease of the tightness of the zonula occludens, but an increased endocytotic uptake indicates that transcytosis is increased also. From studies of model systems it can be concluded that activation of the intracellular protein kinase C route by muscarinic receptor activation or histamine receptor activation can be one of the underlying cellular pathways. The physical pathway relaying the stress from the brain to the intestinal tract appears to be the parasympathetic branch of the autonomic nervous system. The difference in reaction of different strains suggests that coping style is an important determinant of the response of the intestinal barrier to stress.

Glutamine reduces phorbol-12, 13-dibutyrate-induced macromolecular hyperpermeability in HT-29Cl.19A intestinal cells

BACKGROUND Loss of mucosal integrity is associated with intestinal hyperpermeability, which may be inhibited by glutamine. The nature of this effect is unknown. The effect of glutamine on protein kinase C (PKC)-mediated hyperpermeability in HT-29Cl.19A intestinal cells was studied. METHODS Confluent monolayers of HT-29C1.19A cells were cultured on permeable filters and mounted in Ussing chambers for permeability studies. Apical to basolateral transepithelial permeability for intact horseradish peroxidase (HRP) was determined. Phorbol-12,13-dibutyrate (PDB) was used to activate PKC-mediated hyperpermeability, and the effect of glutamine (0.6 mmol/L) was studied. RESULTS Two hours of PDB stimulation increased the HRP flux, reaching five times control values after 4 hours. Bilateral exposure to glutamine for 4 hours reduced PDB-induced hyperpermeability (37%). Preincubation with glutamine 2 hours before PDB stimulation showed an earlier and greater effect (3 hours, 43%; 4 hours, 50%). This bilateral effect of glutamine was mimicked by separate apical exposure. Basolateral exposure alone had no effect. CONCLUSIONS Glutamine rapidly reduced the PKC-mediated hyperpermeability for HRP in HT-29Cl.19A intestinal cells. The dependency on apical exposure suggests that glutamine may be more effective when delivered by the enteral route.

Influence of forskolin and carbachol on intestinal absorption of horseradish peroxidase in the goldfish (Carassius auratus)

Abstract.The transepithelial route for mucosa-to-serosa transport of the tracer macromolecule horseradish peroxidase (HRP; MW 40 kDa) and modulation of this transport by forskolin and carbachol have been studied in vi-tro in stripped goldfish intestinal epithelium mounted in Ussing-type chambers. Uptake and transport have been investigated by measuring the HRP flux from the muco-sal to serosal sides by an enzymatic method and by visualising HRP reaction products in the mucosa with electron-microscopical techniques. Both the cholinergic agonist carbachol (which is thought to increase intracellular Ca2+ and activate protein kinase C activity) and forskolin (a direct activator of adenylylcyclase) affect the amount of enzymatically active HRP in the tissue. In control tissue, HRP product is found only within the epithelial cells, the transepithelial flux reaching a constant value of about 1.5 pmoles/cm2 per h. Carbachol increases the amount of HRP product in the cells, but has no significant effect on the HRP flux compared with control values. Forskolin decreases the amount of HRP product in the cells; however, in the presence of forskolin, the lateral intercellular spaces become filled with HRP product. HRP is found in the lamina propria and the transepithelial protein flux increases more than 2.5-fold. In the presence of forskolin plus carbachol, the results are no different from the control. It is concluded that carbachol increases the endocytotic uptake of HRP, whereas forskolin inhibits the uptake but increases the paracellular permeability for HRP in goldfish intestine.

Lower functional gastrointestinal disorders in a paediatric population.

Since 1999, symptom-based diagnostic criteria have been established for the classification of functional gastrointestinal disorders in children. According to these Rome-II criteria, lower functional gastrointestinal disorders can be divided in children with abdominal pain, such as functional abdominal pain and irritable bowel syndrome and functional defecation disorders such as chronic non-specific diarrhoea, functional constipation and functional faecal retention and non-retentive faecal soiling. In children with functional disorders, no structural or biochemical abnormalities can be found. Uncertainty exists, however, concerning the possible role of immunologic or inflammatory involvement in the different functional entities. In children with ages ranging from 2 to 17 years with abdominal pain, diarrhoea, weight loss and eosinophilia in their intestinal biopsies, symptoms disappeared after a leukotriene selective antagonist was administered, suggesting allergic involvement (1). In a systemic review, Lucassen et al. showed that in children with infantile colic, only a hypoallergenic diet had a proven effect on symptoms (2). Until now, the involvement of allergy in older children with chronic abdominal pain is unclear. Until now, no studies involving children with functional abdominal pain have shown upregulation of chemokines or interleukins. Moreover, no studies showed the involvement of immunoglobulins in these children. However, new microbiologic techniques showed a close correlation between abdominal pain and parasites (3). Dientamoeba gracilis, Blastocystis hominis and Giardiasis are more frequently detected as a cause of chronic abdominal pain and chronic diarrhoea. Treatment of the different parasites in the majority of patients has a curative effect on symptoms (4). The immunologic and inflammatory investigations in all three have not shown any involvement nor response. In adults and children with irritable bowel syndrome, a decrease threshold for distension (visceral hypersensitivity) is believed to be an important mechanism. Different pathophysiological mechanisms have been proposed such as hypersensitive lumbar splanchnic afferents, altered central perception demonstrated by aberrant regional cerebral activation or mast cells releasing potent mediators that alter enteric nerve and smooth muscle function (5,6). In contrast, children with functional abdominal pain showed no visceral hypersensitivity to rectal balloon distension (5). In a recent long-term follow-up study in children with chronic functional constipation we and others showed that in approximately 60 percent of the childrens’ symptoms start in the first 6 months of life (7). In some babies, an acute episode of constipation may occur associated with a change in diet (i.e., human to cow’s milk). Only recently, Iacono et al. showed in a double-blind crossover study in children with constipation that anal fissures, perianal erythema or oedema and pain with defecation resolved after a change from cow’s milk formula to soy based feeding (8). These children were more likely to have evidence of inflammation in their rectal biopsies and signs of hypersensitivity, although contested as evidence, on IgE antibodies to cow’s milk. In children with cow’s milk allergy, endocytosis of intact proteins is increased and transcellular flux of intact and degraded proteins is enhanced in the acute phase but returns to normal after several month of an exclusion diet. During food hypersensitivity the intestinal secretion of electrolytes is increased due to mast-cell activation, responsible for electrogenic chloride secretion, induced by mediators like histamine. By adding Beta-lactoglobulin to the luminal side in intestinal-biopsies mounted in Ussing chambers an increased short circuit current is observed with a concomitant chloride secretion. Furthermore it was shown that TNFis present in large quantities in faeces of these children (9). It was speculated that TNFdecreased epithelial resistance and enhanced transcellular protein transport in the hypersensitivity phase on allergen feeding. This observation was confirmed in five children with cow’s milk allergy and gastrointestinal symptoms in duodenal biopsies with an increased permeability for horseradish peroxidase-protein and a histamine-mediated secretory response (10). Another correlation between immunology and constipation is frequently found in children with Celiac Disease. The rectal mucosa shows identical immunologic responses compared to the jejunal mucosa (11). Address correspondence and reprint requests to: Dr. J. A. Taminiau, Academic Medical Center, Department of Pediatric Gastroenterology and Nutrition, G8-217, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands; Fax: 020-6917735 (e-mail: j.a.taminiau@amc.uva.nl). Journal of Pediatric Gastroenterology and Nutrition 39:S758–S759