Bidirectional Small-Intestinal Permeability in the Rat to Some Common Marker Molecules in vitro

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  • Bidirectional Small-Intestinal Permeability in the Rat to Some Common Marker Molecules in vitro N. PANTZAR, S. LUNDIN, L. WESTER & B. R. WESTROM Depts. of Animal Physiology and Clinical Pharmacology, University of Lund, Lund, Sweden

    Pantzar N, Lundin S , Wester L, Westrom BR. Bidirectional small-intestinal permeability in the rat to some common marker molecules in vitro. Scand J Gastroenterol 1994;29:703-709.

    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-ethylenediaminetetraacetic acid (Mw 341), [mercaptopropionic acid', ~-arginine*]- vasopressin (Mw 1069), fluorescein isothiocyanate (F1TC)-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 sue 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. Key words: SICr-labelled ethylenediaminetetraacetic acid; 1-deamino, 8-D-arginine vasopressin; dextran; inulin; mannitol; permeability; rat; small intestine

    Niclas Pantzar, University of Lund, Dept. of Animal Physiology, Helgonavagen 3b, S-223 62 Lund, Sweden (fax: +46 46 104539)

    The barrier function of the gastrointestinal tract is incom- plete, and luminal macromolecules may traverse its epi- thelium into the internal milieu. Intestinal barrier functions are affected during both normal physiologic conditions, such as ontogenetic maturation (l) , and during several pathologic conditions (2,3). To assess the intestinal permeability and its changes, several different marker molecules have been used, including di-mono-saccharides such as lactulosel mannitol (4), 51Cr-ethylenediaminetetraacetic acid (%r- EDTA) (5 ) , polyethylene glycols (PEG) (6), inulin (7,8), and dextrans (9). The permeability to these different markers has been assessed mainly in vivo after oral feeding, when factors both before and after the passage of the intestinal epithelium may obscure the interpretation, but has not been compared under more simplified and controlled exper- imental conditions in vitro. Moreover, little attention has been paid to the regional aspects of the intestinal permeability; although regional permeability differences along the intestine have been observed for the enzymatically stable nona-peptide 1-deamino, 8-D-arginine vasopressin (dDAVP) by using isolated intestinal segments in rabbits and rats (l@-12). Furthermore, it is likely that smaller mol- ecules use a passage route through the intestinal epi- thelium different from that for larger ones (12-14) and that lipophilicity is an important property for transmembranous passage.

    The purpose of the present investigation was to charac- terize the small-intestinal permeability in the rat to several commonly used marker molecules differing in size and physicochemical nature-for example, molecular class and lipophilicity. To achieve this, intestinal segments were incu- bated in diffusion chambers (13) together with the markers 3H-mannitol (Mw 182), S*Cr-EDTA (Mw 341), [mer- captopropionic acid1, D-arginine8]-vasopressin (dDAVP) (Mw 1069), fluorescein isothiocyanate (FITCfiextran 3000 (mean Mw 3000), and 3H-inulin (Mw 5200). The bidi- rectional passage-that is, both from the mucosal to the serosal side and from the serosal to the mucosal side-of these marker molecules was compared, to obtain further knowledge about the barrier mechanisms of the intestine. To investigate the existence of any regional passage differences within the small intestine, segments from both the proximal jejunum and the distal ileum were used.

    MATERIALS AND METHODS

    Animals Male rats (n = 25) of the Sprague-Dawley strain (Mdle-

    gaard, Skensved, Denmark), weighing 400-500 g, were kept on chopped wood bedding in polycarbonate cages under a 12-h day-night rhythm at 2Ok2"C and with a relative humidity of 50 * 10%. The rats had free access to rat chow

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    Fig. 1. Bidirectional intestinal passage of a) 'H-mannitol, b) "Cr- ethylenediaminetetraacetic acid (EDTA), c) 1-deamino, 8-D-argi- nine vasopressin (dDAVP), d) fluorscein isothiocyanate (FITC)- dextran 3o00, e) 'H-inulin, in percentage (mean 2 SD) of amount added to the donor reservoir in diffusion chambers, in proximal and distal segments from the rat small intestine during 120 min (n = 9). Significant differences between the bidirectional passage is indicated by (P < 0.05) or *** (P < 0.001) and between the proximal and distal region by ttt (P < 0.001). m = mucosa; s = serosa.

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  • Bidirectional Small-Intestinal Permeability 705

    (R3, Ewos, Sodertalje, Sweden) and tap water until the day of experiment.

    Experimental procedures The animals were ether anaesthetized at lo00 h k 1 h.

    An abdominal incision was performed, and two 15 cm-long segments of the small intestine were removed: one proximal segment, beginning 5 cm distal to the pylorus, and one distal segment, beginning 5 cm proximal to the caecum. These were immediately immersed in room-tempered modified Krebs-Ringer buffer, pH 7.4 (NaCI, 110.0 mmol.1-l; CaCI2, 3.0 mmol.l-'; KCI, 5.5 mmol.l-'; KH2P04, 1.4 mmol.l-'; NaHC03, 29.0 mmol.1-'; Na-pyruvate, 5.7 mrnol.l-'; Na- fumarate, 7.0 mmol.1-I; Na-glutamate, 5.7 mmol.l-'; and glucose, 13.4 mmol.1-l) oxygenated with carbogen (95% O2 and 5% C02). Both segments were then divided into three pieces, providing three proximal and three distal small- intestinal pieces from the same rat in each experiment. Each piece was cut along the mesenteric border and mounted in modified Ussing diffusion chambers (Precision Instrument Design, Los Altos, Calif., USA) (13), with an exposed intestinal area of 1.78cm2. The serosal and mucosal res- ervoirs were immediately filled with 5 ml buffer that was continuously oxygenated and circulated by gas lift at a tem- perature of 37C. Under these conditions the tissue prep- arations were considered viable for 120min (12). At the beginning of the incubations (t = 0), within 25 minutes of induction of anaesthesia, the buffer at the mucosal or serosal reservoir was exchanged with 5 ml buffer containing the different marker molecules. Thereafter, 1-ml samples were taken from the corresponding receiver reservoirs at 20, 40, 60, 80, 100, and 120min and were replaced by the same amount of fresh buffer. In some experiments the mucosa had been scraped with the edge of a glass slide. The intestine was opened along the mesenteric border and placed on a plate, and the mucosa was scraped by gently letting the glass edge slide on the mucosa. Routine histology showed that the scraping procedure removed and disrupted the villous structure but left the crypts and submucosal and muscular layers intact.

    Marker molecules The different marker molecules introduced on the mucosal

    or serosal side were 3H-mannitol (Du Pont, Dreieich, Ger- many), with a specific activity of 30.0Ci/mmol and. in a concentration of 0.14 pM; 52Cr-EDTA (Du Pont), with a specific activity of 126Ci/mmol and in a concentration of 0.13 pM; 3H-inulin (Amersham, Buckinghamshire, England), with a specific activity of 1.7 Ci/mmol and in a Concentration of 4.4 pM; dDAVP (Ferring AB, Malmo, Sweden) in a concentration of 9.4 pM; and FITC-dextran 3000 (mean Mw, 3.0kDa; Pharmacia Fine Chemicals, Uppsala, Sweden) in a concentration of 140 pM.

    Estimation of marker lipophilicity The different marker molecules were phase-partitioned

    between n-octanol and 50 mM phosphate buffer, pH 7.4, by shaking for 10 min. After separation, the two phases were analysed for their marker content. The ratio (D) of n- octanol/buffer concentrations of each marker was calcu- lated, and log D was used a measure of the marker lipo- philicity.