Integrity and metabolism of human ileal mucosa in vitro in the Ussing chamber.

The Ussing chamber is increasingly being used for in vitro studies of human intestinal mucosa, but little attention has been paid to the viability of specimens over time. Ninety-one mucosal specimens from the ileum in 19 patients operated on for colonic cancer were studied in regard to intestinal barrier function, metabolism, electrophysiology and histology during 360 min of incubation in Ussing chambers. Steady-state permeability to 51Cr-EDTA was maintained for 120 min. Mucosal ATP and lactate levels were stable for 180 min and transmucosal glucose flux for 240 min. Lactate dehydrogenase leakage was limited within 120 min. Transepithelial potential difference was 9.0 +/- 3.0 mV at the start, and declined slowly throughout 360 min. Light microscopy revealed epithelial lifting from the basal lamina at 90 min. Transmission electron microscopy demonstrated preserved ultrastructure for 120 min. Specimens with a transepithelial potential difference below 6 mV at the start were associated with increased 51Cr-EDTA permeability and lactate dehydrogenase leakage and more pronounced light microscopy changes. All studied parameters pointed to preserved viability if experiments were kept within a period of 90 min after equilibration. The few specimens with early viability derangement were identified by a transepithelial potential difference below 6 mV at the start. The Ussing chamber provides a tool for in vitro studies of human intestinal epithelium, including permeability. To minimize viability problems, experiments should be limited in time and monitored by measurements of transepithelial potential difference.

Bidirectional small intestinal permeability changes to different-sized molecules after HCl-induced injury in the rat.

A rat model was developed to assess bidirectional passage of macromolecules and low-molecular-weight markers across the intestinal barrier in intact and injured mucosa. Isolated in situ loops of distal small intestine were luminally perfused for 30 min with saline as control or HCI (pH 2.0) to induce an acute injury. The lumen-to-blood passage was followed during perfusion with bovine serum albumin, [14C]mannitol, and sodium fluorescein. Intravenously administered sodium fluorescein and endogenous serum albumin were used as blood-to-lumen markers. Acid exposure resulted in severe injury of the villi tips, with significantly increased lumen-to-blood passage of all markers compared with that of intact mucosa. Moreover, blood-to-lumen passage of rat serum albumin increased after the injury, while that of sodium fluorescein did not. The acid induced injury impaired the intestinal barrier function with an increased marker passage, where the macromolecules were more sensitive markers of the altered barrier function than low-molecular-weight markers.

Passive drug diffusion via standardized skin mini-erosion; methodological aspects and clinical findings with new device.

PURPOSE: To develop a clinical alternative to drug administration by injection or infusion. METHODS: A simple, mechanical device (Cellpatch) enables both the formation of a standardized small epidermal bleb and exposure of the circular base of the bleb to drug. The epidermis is split off by suctioning without bleeding or discomfort in a layer superficial to dermal capillaries and nociceptor nerves. Transdermal invasivity is thus avoided. Absorption of dextran test drug in aqueous solution vs molecular weight (3 kDa-70 kDa) and erosion area (3 kDa, diameter: 3-10 mm) were studied in healthy volunteers. The feasibility of using Morphine cellpatch (cell filled with 20 mg/ml morphine hydrochloride, aqueous solution, erosion diameter 6 mm) for post-operative pain relief was studied in two different patient groups; the Cellpatch was removed after 48 hours. Plasma morphine concentrations were determined at intervals. RESULTS: Dextrans of all sizes were efficiently absorbed transdermally, although absorption decreased with increasing molecular weight. The degree of absorption was directly related to the area of the mini-erosion. There were no sign of dose-dumping even with the largest erosions. The Cellpatch performed well in the demanding conditions of the postoperative unit, and was considered easy to use. Pharmacokinetically, the postoperative morphine delivery was related to that of a continuous infusion, with variability and dose in the same range as a continuous morphine infusion used clinically for providing basal pain relief. There were no bacterial growth in the morphine cells at 48 h. Reepithelialization of the erosion was rapid. CONCLUSIONS: The feasibility of administering drugs in a wide size range by passive diffusion through a standardized skin mini-erosion was demonstrated; the rate of absorption decreased with increasing molecular weight. The small area of the erosion restricts and controls the concentration driven diffusion of drug into the circulation. As a consequence of the favorable findings, three placebo-controlled clinical studies using Morphine cellpatch for postoperative pain relief are currently underway.

Different properties of the paracellular pathway account for the regional small intestinal permeability to the peptide desmopressin.

The regional small intestinal permeability to the vasopressin analogue desmopressin (dDAVP) was further characterized in proximal jejunal and distal (ileocecal) segments of the rat. Administration of the peptide to closed small intestinal loops confirmed the existence of regional absorption differences also in vivo in rats. Thus, the extent of absorption was about 4 times as high from the ileocecal part of the small intestine. The mucosal to serosal passage of dDAVP was studied in small intestinal segments from rats, mounted in Grass diffusion chambers. Increasing the mucosal concentration of dDAVP 100-fold had no effect on permeability coefficients (Papp) and the regional permeability distal:proximal ratio was maintained. Excessive amounts of unlabeled dDAVP to reduce the passage of [3H]dDAVP had no effect either. These findings make the existence of an active, receptor-mediated transport mechanism unlikely, and dDAVP probably does not affect its own transport rate. The higher ileocecal permeability could either be due to the presence of more permeable or dynamic pores in this region, where the epithelial surface area is smaller, or to an increased capacity for paracellular water flux. These results may have relevance for drug transport in the small intestine, where site-specific delivery of drug or enhancing agents may be optimized.

Small intestinal absorption of polyethylene glycol 400 to 1,000 in the portacaval shunted rat.

Functional changes of the intestinal barrier that may occur after the creation of a portacaval shunt (PCS) were investigated. After chronic PCS in the rat, the intestinal absorption of and the jejunal permeability to the inert polymer marker polyethylene glycol (PEG) with molecular weight (Mw) ranging from 400 to 1,000 g/mol were investigated. The PEG mixture was orally fed to PCS and sham-operated rats, and urine was collected for 24 hours to obtain the urinary recovery of the different PEG polymers as a measure of intestinal absorption. To study the intestinal permeability, segments from the proximal small intestine were incubated in diffusion chambers with the PEG mixture on the mucosal side, and samples were withdrawn from the serosal side for analysis. The urinary recovery for the PEGs increased (P < .01) while the tissue permeability decreased (P < .001) in the PCS group rats in comparison with Sham-operated rats. The increased absorption in vivo was caused neither by altered renal clearance, nor by changed portal blood pressure. The decreased jejunal permeability in the PCS rats could be explained by a reduction of the mucosal area by shortening of the microvilli. This discrepancy indicates that changes in permeability and absorption may not be parallel during PCS. It is possible that these changes also may be affected by nutritional factors, drug therapy, as well as toxic substances.

Increased intestinal marker absorption due to regional permeability changes and decreased intestinal transit during sepsis in the rat.

BACKGROUND: The intestinal barrier properties are impaired during inflammation and sepsis, but the mechanisms behind this are unknown and were therefore investigated during experimental sepsis in rats. METHODS: The different-sized intestinal absorption markers 51Cr-labeled ethylenediaminetetraacetic acid (EDTA) and ovalbumin were gavaged to rats made septic by intra-abdominal bacterial implantation and to sham-operated rats. Regional tissue permeability was measured in diffusion chambers, and intestinal transit was evaluated by intestinal accumulation of gavaged 51Cr-EDTA. RESULTS: In comparison with the sham-operated rats, septic rats had higher 51Cr-EDTA levels in blood and urine and showed a prolonged intestinal transit. Septic rats also had a lower tissue permeability to both markers in the small intestines but higher permeability to ovalbumin in the colon. Rats receiving morphine to decrease intestinal motility showed similar changes, with a decreased intestinal transit and increased marker absorption. CONCLUSIONS: The results suggest that the increased intestinal absorption during sepsis was due to regional permeability changes and prolonged intestinal transit.

Mechanisms of increased intestinal [51Cr]EDTA absorption during experimental colitis in the rat.

Experimental colitis was induced in the rat, by ethanol-oxazolone injections into the distal colon, resulting in diarrhea together with edema, ulcers, and cell infiltration in the exposed colon. Colitic rats showed an elevated urinary recovery of the permeability marker [51Cr]EDTA after intragastric feeding, 19 +/- 10%, compared to 2.9 +/- 0.7% for control rats (P < 0.001). An increased retention of [51Cr]EDTA in the intestines and a decreased discharge in feces suggested an increased intestinal transit time in colitic rats. The in vitro permeability to [51Cr]EDTA and ovalbumin was not elevated in the severely inflamed distal colon, but was in the proximal, unaffected colon to ovalbumin (P < 0.05) and in the distal small intestine, to both [51Cr]EDTA (P < 0.01) and ovalbumin (P < 0.05), indicating that an inflammation in one part of the intestine could have permeability effects in other remote parts. In conclusion, the increased [51Cr]EDTA absorption in vivo during colitis was probably due to both an increased permeability and an increased intestinal transit time.

Bidirectional small-intestinal permeability in the rat to some common marker molecules in vitro.

BACKGROUND: The barrier properties of the small intestine were investigated by studying the bidirectional permeability to five commonly used marker molecules. METHODS: Proximal and distal small-intestinal segments from rats were mounted in diffusion chambers, and the permeation of the markers 3H-mannitol (Mw 182), 51Cr-ethylenediamineteraacetic acid (Mw 341), [mercaptopropionic acid], D-arginine8]-vasopressin (Mw 1069), fluorescein isothiocyanate (FITC)-dextran (mean Mw 3000), and inulin (Mw 5200) was measured across the mucosa in both directions. RESULTS: A generally increased inward (mucosa to serosa) and a decreased outward (serosa to mucosa) permeation of the markers was found in the proximal to distal direction. The inward permeability showed increasing regional differences with decreasing size of the markers. In the absence of the villous epithelium, removed by scraping the intestinal wall, 86% to 62% of the proximal and distal barrier was lost in the inward direction but only 14% to 26% in the outward direction. CONCLUSIONS: The intestinal epithelial barrier is more permeable in the outward than in the inward direction, and regional permeability differences exist in a size-dependent fashion. The results suggest two passage routes, one for the smallest molecule, mannitol, and a second for the larger markers in the present size range, both apparently different from the route for macromolecules such as intact proteins.

Regional small-intestinal permeability in vitro to different-sized dextrans and proteins in the rat.

Molecular weight-dependent passage over different regions of the rat small intestine, using different-sized proteins/peptides and fluorescein isothiocyanate-dextrans in the 1- to 70-kDa range, was studied in vitro in modified Ussing chambers. The mucosal to serosal passage was inversely related to the molecular weight. After 120 min the passage in the proximal region usually dominated, but the nonapeptide (mercaptopropionic acid1, D-arginine8)-vasopressin differed by showing a consistently higher passage in the distal region. The similar apparent permeation coefficients obtained for the two macromolecular categories of corresponding molecular weight implied that the non-degradable dextrans could be used as permeability markers reflecting the passage per se of intact proteins. Furthermore, the results indicated two different transmucosal pathways, one of low permeability for molecules > 30 kDa, in which the molecular weight was of minor importance for the passage, and another more permeable one in the 1- to 30-kDa range, in which the passage was highly influenced by the molecular weight.

Differences in transport rate of oxytocin and vasopressin analogues across proximal and distal isolated segments of the small intestine of the rat.

The transmural intestinal passage of some oxytocin and vasopressin analogues (oxytocin, OT; [Mpa1, D-Arg8]vasopressin, dDAVP; [Mpa1, Tyr (OMe)2, carba6]oxytocin, carbetocin; [Mpa1, D-Tyr (OEt)2, Thr4, Orn8]vasotocin, antocin II; [Mpa1, D-Tyr (OEt)2, Thr4, desPro7Orn8Gly9NH2]tocinoic acid-NH(CH2)3NH2, desPOG-antocin II-NH(CH2)3NH2) was studied using isolated proximal and distal segments in the rat. All peptides (measured as peptide-like immunoreactivity) displayed a higher transport rate across distal intestinal segments as determined by radioimmunoassay (RIA). The smallest peptide, des POG-antocin II-NH(CH2)3NH2, was transported at the fastest rate. No correlation of lipophilicity with transport rate was observed. Determination of the amount of peptide remaining in the mucosal media at the end of the incubation period by HPLC did not reveal any visible degradation products. However, the large difference in transport rate between [3H]OT and immunoreactive OT indicates mucosal metabolism of this peptide. [3H]d-DAVP was distributed in a larger mucosal volume than the extracellular space marker [3H]inulin, indicating tissue uptake, but was too low (less than 100% of buffer concentration) to make an active transport mechanism likely. The differences in peptide transport rates between proximal and distal intestinal segments are most likely due to a higher distal paracellular permeability despite a decreased absorptive surface area at this region.