thritol and urea were used to calculate re- flection coefficients in the duodenum, mid jejunum and distal jejunum. Estimated effect- ive pore radius was 6.4-7.4, ...
Permeability Properties of Swine Small Intestine: Effect of a Heat Stable Escherichia coli Enterotoxin K. R. Presnell, W. E. Roe, N.
0.
Nielsen and D. L. Hamilton*
ABSTRACT
RESUMr,
The permeability of weanling swine small intestine was estimated using measurements of filtration coefficients and equivalent pore size. Hypertonic solutions of mannitol, erythritol and urea were used to calculate reflection coefficients in the duodenum, mid jejunum and distal jejunum. Estimated effective pore radius was 6.4-7.4, 5.6-7.2 and 4.74.9A° in the three respective regions. Similarly the filtration coefficient induced by hypertonic solutiong of mannitol decreased significantly in the distal jejunal segments. The results show an aboral gradient of decreasing permeability along the small intestine of the weanling pig. In situ incubation of loops in the proximal jejunum with a heat stable Escherichia coli enterotoxin for one hour did not significantly change the effective pore size as calculated from reflection coefficients of hypertonic solutions of erythritol and urea. However the filtration coefficients of loops exposed to the enterotoxin were significantly greater than control loops with hypertonic solutions of erythritol and urea but not mannitol. This suggests the occurrence of a slight reduction in epithelial porosity. The results support the hypothesis that intestinal secretion induced by heat stable E. coli enterotoxin is not the result of an increased mucosal permeability.
Cette experience visait a determiner la permeabilite de la muqueuse de l'intestin grele de porcelets recemment sevre's, en mesurant les coefficients de filtration et les dimensions equivalentes des pores. On utilisa des solutions hypertoniques de mannitol, d'erythritol et d'uree pour calculer les coefficients de reflexion dans le duodenum et dans le jejunum moyen et distal. Dans ces trois segments de l'intestin grele, le rayon efficace approximatif des pores s'etablissait respectivement 'a 6.47.4, 5.6-7.2 et 4.7-4.9A°. Le coefficient de filtration induit par les solutions hypertoniques de mannitol diminua aussi de facon appreciable dans le jejunum distal. De tels resultats revelerent un gradient aboral de permeabilite decroissante le long de l'intestin grele du porcelet recemment sevre. L'incubation in situ d'anses du jejunum proximal dans lesquelles on avait injecte une enterotoxine thermostable d'Escherichia coli, pour une periode d'une heure, ne changea pas de faton appreciable les dimensions efficaces des pores, comme le revela le calcul des coefficients de re'flexion des solutions hypertoniques d'erythritol et d'uree. Les coefficients de filtration des anses intestinales dans lesquelles on avait injecte l'enterotoxine s'avererent cependant plus eleves que ceux des anses temoins soumises a l'action des solutions hypertoniques d'erythritol et d'uree, mais non de celles de mannitol. Ces observations laissent croire qu'il se produisit une legere reduction de la porosite epitheliale. Elles appuient aussi l'hypothiese selon laquelle la secretion intestinale attribuable a l'enterotoxine thermostable d'E. coli ne resulterait pas d'une plus grande permeabilite de la muqueuse.
*Department of Veterinary Clinical Studies (Presnell), Department of Veterinary Physiological Sciences (Roe and Hamilton) and Department of Veterinary Pathology (Nielsen), Western College of Veterinary Medicine, University of Saskatchewan, Saskatoon, Saskatchewan S7N OWO. During this study K. R. Presnell and D. L. Hamilton were fellows of the Medical Research Council of Canada. This work was supported by the Alberta Agricultural Research Trust Fund and the Alberta Hog Producers' Marketing Board. Correspondence to D. L. Hamilton. Submitted January 11, 1978.
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INTRODUCTION Diarrhea in cholera and colibacillosis due
Can. J. comp. Med.
to enterotoxin producing Escherichia coli results from fluid accumulation in the small intestine at a rate that exceeds the reabsorptive capacity of the colon. Increased permeability of the small intestinal mucosa to small molecules and ions had been proposed as the mechanism causing the profuse diarrhea characteristic of human cholera (12). However, Rohde and Chen (17) and Scherer et al (19) have presented evidence that no change in permeability occurs when the intestinal mucosa has been exposed to cholera enterotoxin. Moon, Whipp and Baetz (14) have compared the morphological effects of a heat labile (LT) E. coli enterotoxin and cholera enterotoxin in ligated loops of rabbits and pigs. They found the response to the two enterotoxins to be indistinguishable. Anatomical changes were confined to evacuation of goblet cells with villous absorptive and other epithelial cells remaining intact. The diarrhea of cholera and LT E. coli enterotoxin may be mediated by stimulation of intestinal adenylate cyclase (1). The mechanism by which heat stable (ST) E. coli enterotoxin mediates intestinal secretion is unknown. It may not be the intestinal adenyl cyclase system since it does not increase intracellular concentrations of cyclic AMP in the mucosal cells of rabbits and pigs (9) and can not be detected with the Y-1 adrenal cell assay (5) developed by Donta (4) for determining the presence of cholera toxin and LT. ST may be mediated by an increase in the mucosal permeability of the small intestine. Unidirectional sodium flux studies with ST (10) have shown that the principle alteration in the proximal jejunum of weanling swine is an increased blood-tolumen or unidirectional secretion of sodium. This flux change could be the result of ST increasing mucosal permeability and allowing plasma and interstitial fluid to pour into the intestinal lumen by the paracellular pathway. Alternatively it could be the result of an increased mucosal cell secretion rate. Fordtran et al (7) have described a method for measuring the permeability of intestinal mucosa based on the model of Lindemann and Solomon (11). It permits the calculation of intestinal permeability by using solutes of varying molecular size to produce osmotic fluid flow across the intestinal mucosa. The objectives of this
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study were to measure the equivalent pore size of the proximal and distal small intestine of weanling swine and to extend this study to include the effect of ST on the permeability of intestinal ligated loops in swine proximal jejunum.
MATERIALS AND METHODS ANIMALS AND SURGICAL PROCEDURE
Crossbred weanling swine weighing between seven and nine kilograms were used following an overnight fast with water ad libitum. Halothane was used for induction and maintenance of general anesthesia for the duration of the experiment. Appropriate segments of small intestine were exposed through a midline abdominal incision. One end of the intestinal segment was closed by the Parker-Kerr method (13) and 20 cm distally a lucite cannula was inserted and fixed in place with a ligature. The loops were returned to the abdominal cavity during the testing periods. During the study on regional permeability differences loops were established in the duodenum, midjejunum and distal jejunum just proximal to the ileal cecal ligament. Studies involving ST were only done in loops positioned in the proximal jejunum just distal to the ligament of Treitz. After termination of the experiment the test segments were removed and the mucosal surface areas measured. SOLUTIONS AND ENTEROTOXIN PREPARATION
Hypertonic solutions of either mannitol, erythritol or urea with an osmolality of 600-700 mOsm per kg were used to determine the relative pore size and effective filtration coefficients. These solutions contained 0.5 gm// polyethylene glycol as a dilution marker and 0.16 M sodium chloride. Heat stable E. coli enterotoxin and a control filtrate were prepared as previously described (10). EXPERIMENTAL PROCEDURE
The initial study estimated the permea-
45
bility of three different regions of the pig small intestine. Eight pigs were used with three loops established in each of the three areas. All loops were injected with approximately 10 ml of hypertonic test solution. The volume instilled was determined by weight change in the injection syringe. Only one test solution was injected into each loop. Fourteen minutes later the test solution was removed. During the test period a blood sample was drawn from the jugular vein to determine serum osmolality. The change in polyethylene glycol concentration was used to determine the volume change in the loop at the end of the testing period. The osmolalities of the test solutions were determined before and after injection into the intestinal segments. The study with ST used eight pigs with eight loops established in the proximal jejunum of each pig. The loops were incubated with 20 mg of ST or control filtrate for one hour. This time period was chosen because previous studies (10) had shown that significant and consistent net and unidirectional flux differences are induced by ST in one hour. The ST or control preparation was dissolved in 10 ml of isotonic solution (10). Following incubation each loop was drained of its fluid contents and the hypertonic solution immediately instilled to estimate intestinal porosity since the duration of ST's effect is fairly short (6). Ten minutes later the loops were drained and the fluid contents analyzed for osmolality and polyethylene glycol concentration. Injection of ST and control preparations and the allocation of the specific hypertonic solutions was randomized such that each treatment proceeded the other the same number of times to obviate positional effects on permeability.
CHEMICAL ANALYSES Osmolality was determined by freezing point depression using sodium chloride standards. Polyethylene glycol concentration was determined spectrophotometrically essentially according to the method of Boulter and McMichael (2). CALCULATIONS
Calculation of filtration coefficients was made by dividing the volume of fluid accumulated in the loop during the ten minute test period by the mean osmotic pressure gradient inducing the movement.
46
Filtration Coefficient = Volume of Fluid Accumulated per Time per Area Mean Osmotic Pressure Gradient
The mean osmotic pressure gradient (A7r) was the average of the osmolalities of fluid instilled at the beginning and that removed at the end of the test period less the osmolality of the particular subject's serum.
-x
(Initial Osmolality Plus Final Osmolality) 2 - Serum Osmolality Mucosal surface area of each intestinal loop was measured so the filtration coefficient could be expressed on a cm2 basis. The theoretical basis for the estimation of equivalent pore size in these studies arises from the demonstration by Staverman (22) and Solomon (21) of the influence of the relationship between molecular size of a nonlipid-soluble solute and radius of waterfilled membrane pores upon the osmotic effectiveness of the solute. A measure of the osmotic effectiveness of a given solute with respect to a membrane is the reflection coefficient, which is defined as the ratio of the observed osmotic pressure to the theoretical osmotic pressure. The reflection coefficient to solutes of known molecular radius has been quantitatively related to pore radius. The estimates of molecular radius are those used by Lindemann and Solomon (11). This study determines the reflection coefficient of probing molecules by comparing the volume of fluid movement into the loop induced by the solute with that fluid entering as a result of induction by a mannitol solution with an identical osmotic gradient. As in man (7), mannitol has been demonstrated not to penetrate swine small intestinal mucosa to a measureable extent over a period of ten minutes (15). During this period its theoretical and effective osmotic pressure should be equal and thus it is assumed to have a reflection coefficient of 1.0. The equivalent pore size is then estimated from curves derived from Renkin's equations (16) as used by Goldstein and Solomon (8). The curves relating the reflection coefficient of mannitol, erythritol and urea to the pore radius of a membrane were plotted by computer (Fig. 1) and used to estimate effective pore size. Statistical significance of results were based on Student's paired t-test between loops within the same animal (20).
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0
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ir .8 .9 1.0 I ..01
2
4
6
8
10
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segments. This difference was not significant with urea as the probing molecule. The observed values of both relative pore size and filtration coefficient indicate an 2.3A aboral gradient of decreasing permeability along the small intestine of weanling swine. The permeability properties of control 0 3.2A loops and of those injected with ST are shown in Table II. The filtration coefficients of loops exposed to ST and studied ~~~~~~~~~~~~~0 4.0 A with hypertonic solutions of erythritol and urea were significantly greater than control loops. However the filtration coefficients as determined with hypertonic mannitol were not significantly different between control and ST loops. Reflection coefficients calculated for control and ST exposed loops did not significantly differ from each other. These reflection coefficients indicate an approximate pore radius of 4.5 - 5.9 A0 in both control and ST 14 16 loops.
Fig. 1. Curves calculated by computer from Renkin's equations (16) representing the relationship between the reflection coefficient (a) and membrane pore radius (AO). The curves for urea (2.3 AO), erythritol (3.2 AO) and mannitol (4.0 AO) are indicated by the molecular radius of each nonlipid, nonelectrolyte solute.
RESULTS The effect of small intestinal regional differences on filtration coefficient, reflection coefficient, and relative pore size is summarized in Table I. A progressive decrease in the rate of net water movement was observed from the duodenum to distal jejunum. The duodenal and midjejunal reflection coefficients did not differ significantly with either of the probing molecules. However the distal jejunal reflection coefficient estimated with erythritol was significantly greater than those from the duodenal and midjejunal
DISCUSSION The effective pore radius of swine small intestine as determined in this study is about 4.5-7.4 Al. The permeability of the small intestine as estimated by filtration coefficients and pore size decreases aborally. Hypertonic solutions of nonlipid-soluble molecules have been used to calculate the effective pore size of many other biological membranes. Goldstein and Solomon estimated the pore size of human red blood cells to be 4.3 A0 (8). The effective pore size of the small intestinal mucosa has been calculated to be about 4.0 A0 in the rat (11), 6.8 A0 in the dog (17) and 8.3 in man (17). Love (12) calculated the effective pore radius of the rabbit ileum to be 5.5 and 6.5 A0 as estimated with urea and erythritol, respectively, as the probing
TABLE I. Regional Differences in Relative Pore Size of Weanling Swine Small Intestine Mannitol Filtration Coefficient (mls/min/mOsm x 102)
Reflection Coefficient Urea Erythritol 0.51 4 0.05 0.73 + 0.05 0.59 ± 0.06 0.76 + 0.04 0.93 i 0.04bc 0.68 ± 0.04
Duodenum 1.58 i1 0.05a Midjejunum 1.31 ±fi 0.07b Distal jejunum 0.59 ±fi 0.05b,c aMean i SE of eight loops from eight animals bMean value different from corresponding duodenal value, P
