GASTROENTEROLOGY. Official Publication of the American Gastroenterological Association

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1 GASTROENTEROLOGY Official Publication of the American Gastroenterological Association COPYRIGHT 1968 THE WILLIAMS & WILKIN. CO. VOLUME 54 February 1968 KU:VIBER ~ DESTRUCTION OF THE GASTRIC MCCOSAL BARRIER BY DETERGENTS AND UREA HOR.\CE W. DAVENPORT, D.Sc. Department of Physiology, The University of Michigan, Ann Arbor, Michigan The normal gastric mucosa contains a barrier which resists the tendency of H+ to diffuse from the lumen into the mucosa and of Na + to diffuse in the other direction. I,2 Although the exact locus and nature of the barrier are unknown, one component must be the lipoprotein layer which forms the plasma membrane of the mucosal cells, and another component may be the mucous layer at the tips of the epithelial cells. 3 This paper reports the beginning of a study of the contribution of those components to the barrier function of the mucosa. The method used is the classical one of attacking the barrier with compounds of known function: detergents and urea which destroy the organization of lipoprotein layers and break hydrogen Received August 1, Accepted October 3, Address requests for reprints to: H. W. Davenport, Department of Physiology, The University of Michigan, Ann Arbor, Michigan This investigation was supported by Grant AM from the United States Public Health Service. The author thanks Alan F. Hofmann and M. I. Grossman for advice and Virginia D. Davenport for assistance. This paper is dedicated to the memory of Franklin Hollander in gratitude for early encouragement and in appreciation of his contributions to the physiology of the gastric mucosal barrier. 175 bonds. 4 The data establish the ranges of concentrations in which natural and synthetic detergents and urea break the barrier and the extent of destruction as measured by changes in transmucosal fluxes of Na+, K+, and H+. Methods The dogs, the method of irrigating their pouches, and the analytical methods have been described.5-5 Briefly, 4 unanesthetized dogs in good health, each having a separated, vagally denerv:1ted (Heidenhain) pouch of the oxyntic gland area of the stomach, were used. The dogs were deprived of food but not of water 18 to 20 hr before use. At the beginning of each experiment the pouch was thoroughly washed with the appropriate control test solution. The pouch was emptied, leaving in it a previously determined residual volume of between 3.0 and 5.3 m!. Forty milliliters of the S3me solution were placed in the pouch, mixed, and sampled, leaving an initial volume of between 33 and 36 ml in the pouch. Thirty minutes later the fluid was removed for anah' sis. This control test established that tile pouch had recovered from damage inflicted in preceding experiments. The pouch was then rinsed and filled for 30 min with ~lllother solution containing the detergent or urea, and after this solution had been removed the pouch was again tested by being filled for 30 min with the control test solution. The exact composition of the solutions, details of the protocols followed, and the number of replications are given below.

2 176 DAVEN PORT Vol. 54, No. 2 The masses of Na+, K +, and H+ initially or fi n:t!ly present in the lumen of the pouch "'pre calculated by multiplying t he initial or fi nal \'olume by the concentration of the ions, :tlld t he net fh;xes across the mucosa were determined by subtracting the initial from the fina l m~sses. Positive values me:ll1 t hat n et fl ux \\,:18 from mucosa to lumen, and negative \'n lue;; mea n that net flux was from lumen to mucosa. Changrs ill detergent concentrations were not measured. Published data show that only ;::mnll :1nlOunts o f bile salts a re a bsorbrd in the stomach. Results Bile salts in Nael solution. Bile salts were used b ecause their detergent properties are well known 1o and because they are the detergents m05t likely to come into contact with the ga stric mucosa. The synthetic bile salt used was sodium taurochohte obta:ned from May bridge Chemica l Company. As a source of natural bile salts, gall bladder bile was collected from dogs used i n teaching or research l aboratories, ]looled, a ncl kept at -20 C until used. Bile was analyzed by the method of Wheeler and Ramos l1 ; the concentration of taurochola te was taken to be the d:fference between the sum of [Na+] and rk +] and the sum of [ Cl-] and [HCOa -]. The stock bile ~ olution was found to contain 269 mm taurocholate ; solutions of it were made by pipetting the volume calculated to give the desired concentration into a volumetric flask and making up to volume with the appropriate diluent. The pouch was fillecl with a solution of 100 mm HCI -I- 54 mm NaCI for the 3D-min control period and then washed thoroughly with 154 mm NaCI before being filled with a solution of taurocholate or bile in 154 mm NaC\. The ph of t hese solutions was never lower than 3 when they were removed from the pouch. After the bile solution was removed, the pouch was thoroughly washed with the 100 mm HCI -I- 54 mm N aci solution and then tested for 30 min with that solution. Results obtained on 1 dog are given in table 1. Comparable experiments on 2 other dogs gave similar results, demonstrating that natural or synthetic bile salts destroy the mucosal barrier. Bile salts i n a liquid rneal. The effect of bile salts in a meal was studied because bile in concentrations within the range used in these experiments is most likely to come into contact with the gastric mucosa in association with fat, protein, and carbohydrate. In addition, test meals containing 40 mm bile have been placed in the stomachs of man 12 and rat,1~ and in the latter case some of the peculiarities of the results have been attributed to the effects of bile salts on the stomach. T.. I BLE 1. N e l exchanges across the u nstimulated vagally denm'vated p ouch of the oxyntic gland area of a dog irrigated with solutions of synthetic Na taurocholate OT natural bile" Bile,alts N a+ K + H I ± ± ± ± 2 11 ± 1 12 ± ± ± 28 ~a t UllI'ocholate ± ± ± ± 5 27 ± 4 19 ± 2 24 ± 1091_149 ± ± ± ± 39 8 ± 2 19 ± 3 21 ± ± ± ± ± ± 58 7 ± 2 29 ± 6 32 ± ± ± 81 ~ atu]'a l bile ± ± ± ± 1 23 ± 5 24 ± ± ± ± ± ± 52 8 ± 1 24 ± 2 31 ±3-81 ± ± 4) ± ± 53 I 709 ± 41 6 ± 2 35 ± 5,'31 ± 31 5 ± ± 22 a Values are means ± SEM of five observa tions; Na+, K+, and H + are in micromoles per 30 min. Bile Ral t co nce ntrations are in millimoles per liter. First period solution, 100 mm HCI + 54 mm NaCl ; second peri od solil tion. bile salts in 154 mm NaCl ; third period solution, sa me a s first period.

3 February 1968 DESTRUCTION OF GASTRIC MUCOSAL BARRIER 177 TABLE 2. Net exchanges across the unstimulated vagally denervated pouch of the oxyntic gland area of a dog irrigated with a solution of synthetic N a taurocholate or natural bile in a liquid meal a Meal Na+ K+ H+ Na+ K+ Bile I b c I a Values are means of two experiments with each solution; N a+, K+, and H+ fluxes in microequivalents per 30 min. Concentrations in meal are in millimoles per liter. First period solution, 100 mm HCI + 54 mm NaCI; second period solution, liquid meal with or without bile salts; third period solution, same as first period. b Sodium taurocholate. c Natural bile. J 3 After a 30-min control period in which the pouch was tested with the 100 mm Hel + 54 mm Nael solution, the pouch was rinsed three times with 154 mm Nael and once with a test meal made of 76 g of corn oil, 126 g of dried skim milk powder, 130 g of glucose, and 1000 ml of water mixed in a blender. The meal was used alone or with the addition of 40 mm synthetic or natural bile salts. The pouch was filled with 40 ml of the meal which was mixed and sampled. At the end of 30 min the meal was removed as completely as possible, and the pouch was rinsed with the 100 mm Hel + 54 mm NaCI solution. Casein of the meal invariably clotted in the pouch, and the curds were difficult to remove. The pouch was filled for a third 30-min period with the 100 mm Hel + 54 mm N aci solution. Results of tests on 1 dog are given in table 2. Comparable results were obtained on 2 other dogs. The results show that both natural and synthetic bile salts in a meal affect the barrier. (The high negative fluxes of K + in the second period are predictable from previous work which showed that at high luminal K + concentration K+ moves into the mucosa. 7 ) Bile salts in acid solution. After a control period in which the pouches of 2 dogs were tested 'With the 100 mm HCI + 54 mm NaCI solution, the pouches were filled for 30 min with solutions of 10, 20, or 40 mm natural or synthetic bile salts dissolved in 100 mm HCI + 54 mm NaC!. After the bile in aeid had been removed from the pouches, the pouches were tested by being filled for 30 min with the 100 mm HCI + 54 mm NaCI solution. The results, which are not given for the sake of economy, were similar to those in table 1; bile salts in acid solution are as damaging as in neutral solution. In another series of experiments the poueh of 1 dog was very gently filled, without previous rinsing, with the 100 IllM Hel + 54 mm NaCI solution, and the cannula draining the pouch was immediately connected with a Statham strain gauge whose output was recorded on a Grass polygraph.14 Pressure was recorded for 30 min, and then the fluid was removed from the pouch for chemical analysis. This observation was repeated for a total of five times on widely separated occasions. On five other oeeasions the pouch was filled with the same solution to which natural bile had been added to a final concentration of 10 mm. To quantify motility the area under each curve above 15 mm Hg was measured with a planimeter. This value together with the chemical fluxes is given for the control and for the bile experiments in table 3. The fraction of time pressure was greater than 15 mm Hg and the frequency of contractions generating more than 15 mm Hg pressure was also measured, and they showed the same pattern of difference between control and bile experiments. The record for eaeh solution shown in figure 1 i s the median of the group of five; that is, two records in each

4 178 DAVENPORT Vol. 54, No.2 TABLE 3. Motility and net exchanges in the unstimulated vagally denervated pouch of the oxyntic gland area filled with acid solution or with natural bile in acid solution" Solution Area Na+ K+ H+ 100 mm HCI + 54 mm N aci 14 ± ± ± ± mm bile in 100 mm HCI ± ± ± ± mm NaCI "Values are means ± SEM of five observations; Na+, K+, and H+ are in micromoles per 30 min. Area is area under pressure-time curve above 15 mm Hg; units are millimeters of mercury minutes for the 30-min period. Fraction of time pressure was greater than 15 mm Hg and frequency of contractions showed same pattern of significant difference. :F sor 10 mm Bile E 40 E ~~~AN.~MW~NW~~\IU\ o in 100 mm H CI 5 minutes FIG. 1. Pressure recorded from the separated vagally denervated pouch of the oxyntic gland area of a dog's stomach when the pouch was filled with either a solution of 100 mm HCI + 54 mm NaCI or with 10 mm natural bile salts dissolved in the same acid solution. Each record is the median obtained in five replications; that is, two records in each group showed more motility, and two records showed less. TABLE 4..\' et exchanges across the unstimulated vagally denervated pouch of the oxyntic gland area of a dog irrigated with solutions of decylsulfate" Decyl sulfate Na+ K+ H mm 233 ± ± ± 42 9 ± 4 28 ± 6 33 ± 5-40 ± ± mm 170 ± ± 81 I 917 ± 62 9 ± 1 31 ± 2 33 ± 7-86 ± ± 20 "Values are means ± SEM of five observations; Na+, K+, and H+ are in micromoles per 30 min. First period solution: 100 mm HCl + 54 mm NaCl; second period solution: decyl sulfate in 154 mm NaCl; third period solution: same as first period. Data in first line of table 1 are also control data for this series. group showed less motility and two showed more. Decyl sulfate in neutral solution. The effect of a representative synthetic detergent, decy I sulfate, was tested in a series of experiments run concurrently with those reported in table 1, and the control data gi"ven in that table can be used for comparison with the decyl sulfate data given in table 4. In the second period of those experiments the pouch was filled with a solution of 10 or 20 mm decyl sulfate in 1M: mm N aci. The ph of the fluid removed at the end of 30 min ranged from 3 to 6. The data show that the detergent increased the fluxes across the mucosa. Similar results were obtained on another dog. Urea in neutral solution. The pouch of each of 2 dogs was irrigated for the customary 3D-min control period with the 100 mm HCI + 54 mm NaCl test solution. The pouch was then thoroughly washed with

5 February 1968 DESTRUCTION OF GASTRIC MUCOSAL BARRIER 179 TABLE 5. Net exchanges across the unstimulated vagally denervated pouches of the oxyntic gland area of two dogs irrigated with solutions of urea" Na+ K+ H+ Urea I n Dog 1b 1 M M M , Dog 2 1M !\! M "Values are means of numbers of observations given in column headed n; Na+, K+, and IF in micromoles per 30 min. First period solution, 100 mm HOI + 54 mm N acl; second period solution, urea at concentration stated in 15 mm phosphate buffer, ph 7.4; third period solution, same as first period. b Control observations for this dog are in table mm NaCI and once with a solution of urea in 15 mm Na phosphate buffer, ph 7.4. Concentrations of urea were 1, 2, or 4 M. The pouch was filled with the urea solution for 30 min. The urea solutions were grossly hyperosmotic, and pouch contents increased 3 to 8 ml in volume in 30 min. After the urea solution had been removed, the pouch was washed with the 100 mm HC! + 54 mm NaCI solution and filled for 30 min with the same solution. Net flux es of Na +, K+, and H+ are given in table 5. (Control experiments, not reported, have shown that the phosphate buffer itself has no effect on the mucosa.) Ammonia and urea in the solutions were measured by the method of Conway and O'Malley.1 5 Rates of absorption of urea are plotted against initial urea concentration in figure 2. The upward concavity of the curve probably means that a high concentration of urea increases the permeability of the mucosa to urea itself as well as to electrolytes. The amount of ammonia appearing in the fluid was never more than 3% of the urea disappearing. The urea solutions removed from the pouch were viscid. In order to estimate their mucus content, some samples were analyzed for hexosamine by the method of Dische and Borenfreund. 16 Duplicate analyses of samples from the most viscid solutions did not agree, apparently because , o ~ ~ -10 o o 3 4 Urea, M FIG. 2. Rate of absorption of urea from the separated vagally denervated pouches of the oxyntic gland area of 2 dogs' stomachs when the pouches were filled with approximately 35 ml of a solution of urea in 15 mm phosphate buffer, ph 7.4, plotted against initial urea concentration. 0, observations on 1 dog;., observations on another dog. inhomogeneity of the solutions made it impossible to withdraw samples uniformly. Consequently, in some instances the total fluid recovered from the pouch was analyzed for hexosamine. On four occasions total hexosamine found in fluid recovered after 30-min control irrigation with 154 mm N aci ranged from 0.3 to 0.5

6 180 DAVENPORT Vol. 54, No.2 mg. In four irrigations with 4 M urea in 15 mm phosphate buffer total hexosamine ranged from 2.6 to 4.5 mg. Discussion Detergents and urea have been found to reduce the concentration of acid in the stomach. Grover et alp established a steady rate of acid secretion in dogs provided with vagally innervated pouches or with gastric fistulas and then irrigated the oxyntic glandular mucosa for 30 min with 20 to 100 mm solutions of lauryl sulfate. There was a profound but transitory fall in acid output which Grover et al. attributed to direct inhibition of the acid-secreting process. Since volume output was only slightly if at all reduced, it seems more likely that the deficit of acid in solutions draining from their preparations after lauryl sulfate treatment was the result of back diffusion of acid into a mucosa whose permeability had been increased by the detergent. 2 FitzGerald and Murphy 18 gave patients 15 g of urea in 1 fluid ounce of solutioll. They thought that because hydrolysis of urea to ammonium carbonate is catalyzed by gastric urease 19 such treatment would reduce the concentration of acid in gastric contents. Since the solution used was approximately 8 M urea, it would be, even after considerable dilution, well above the concentration shown to break the mucosal barrier. Back diffusion of acid as well as neutralization by ammonium carbonate could explain the reduction in concentration of acid in gastric contents collected by aspiration observed by Fitz Gerald and Murphy after urea treatment. Although the present experiments establish that detergents and urea break the mucosal b arrier, they tell us nothing about the mechanism of action. D etergents might have subtle effects ranging from simple loo :;~ning of tight junctions between epith.eljaj cells to disruption of the Jipoprotem layer forming the plasma membrane of the cells, or they might, as Grant et a1. 20 haye s ~own for bile, be grossly cytolytic. Urea (h~ so lves gastric mucus,21 and the results. dc,;:cr!becl.here could be caused by chemical chssecbon of the tips of the surface epithelial cells. These, however, are merely reasonable speculations, and it is clear that real progress in understanding the nature of the mucosal barrier will be made by a much more penerating biochemical analysis of the effects of these or similar damaging agents supported by appropriate morphological studies. Summary The effects of natural and synthetic detergents (sodium taurocholate, bile, decyl sulfate) and urea in breaking the gastric mucosal barrier were studied by irrigating the separated, vagally denervated pouches of the oxyntic glandular area of the dog stomach first with a test solution of 100 mm HCI + 54 mm NaCI, then with a solution of the detergent or urea, and again with the acid test solution. Fluxes of N a +, K+, and H + were measured, and changes over the control fluxes were taken as evidence that the barrier had been broken. Bile salts (10, 20, and 40 mm in neutral solution, 40 mm in a liquid meal and 10 mm in acid solution), decyl sulfate (10 or 20 mm in neutral solution), and urea (1 2 and 4 M in neutral solution) all break th~ gastric mucosal barrier. REFERENCES 1. Code, C. F., J. A. Higgins, J. C. Moll, A. L. Orvis, and J. J. Scholer The influence of acid on the gastric absorption of water sodium, and potassium. J. Physiol. (Lon~ don) 166: Davenport, H. W., H. A. Warner, and C. F. Code Functional significance of gastric mucosal barrier to sodium. Gastroenterology 47: Hollander, F The two-component mucous barrier: its activity in protecting the gastroduodenal mucosa against peptic ulceration. Arch. Intern. Med. (Chicago) 93: White, A., P. Handler, and E. L. Smith Principles of biochemistry, Ed. 3. Vol. 1, pp. 137, 154. McGraw Hill Book Company, Inc., New York. 5. Davenport, H. W Gastric mucosal injury by fatty and acetylsalicylic acids. Gastroenterology 46: Dav:enport, H. W Damage to the gastric mucosa: effects of salicylates and stimulation. Gastroenterology 49:

7 February 1968 DESTRUCTION OF GASTRIC MUCOSAL BARRIER Davenport, H. W Potassium fluxes across the resting and stimulated gastric mucosa: injury by salicylic and acetic acids. Gastroenterology 49: Davenport, H. W Fluid produced by the gastric mucosa during damage by acetic and salicylic acids. Gastroenterology 50: Davenport, H. W Absorption of taurocholate-24-1<c through the canine gastric mucosa. Proc. Soc. Exp. BioI. Med. 125: Hofmann, A. "'., and D. M. Small ljetergent properties of bile salts: correlation with physiological function. Ann. Rev. Med. 18: Wheeler, H. 0., and O. L. Ramos Determinants of the flow and composition of bile in the unanesthetized dog during constant infusions of sodium taurocholate. J. Clin. Invest. 39: Borgstrom, B., G. Lundh, and A. Hofmann The site of absorption of conjugated bile salts in man. Gastroenterology 45: Kern, F., Jr., and B. Borgstrom The effect of conjugated bile salt on oleic acid absorption in the rat. Gastroenterology 49: Davenport, H. W Stimulation of gastric motility by acid. Gastroenterology 52: Conway, E. J., and E. O'Malley Microdiffusion methods: ammonia and urea using buffered absorbents (revised method for ranges greater than 10 p.g. N). Biochern. J. 36: Dische, Z., and E. Borenfreund A spectrophotometric method for the microdetermination of hexosamines. J. BioI. Chern. 184: Grover, R. F., C. A. Maaske, L. L. Hardt, and J. B. Henderson The inhibition of gastric acid production in the intact dog with sodium lauryl sulfate. Gastroenterology 22: FitzGerald, 0., and P. Murphy Studies on the physiological chemistry and clinical significance of urease and urea with special reference to the stomach. Irish J. Med. Sci Kornberg, H. L., and R. E. Davies Gastric urease. PhysioI. Rev. 35: Grant, R., M. 1. Grossman, K. J. Wang, and A. C. Ivy The cytolytic action of some gastrointestinal secretions and enzymes on the epithelial cells of the gastric and duodenal mucosa. J. Cell. Compo PhysioI. 37: Edward, D. W., and S. C. Skoryna Properties of gel mucin of human gastric JUIce. Proc. Soc. Exp. BioI. Med. 116:

PEPSIN SECRETION DURING DAMAGE BY ETHANOL AND SALICYLIC ACID

GASTROENTEROLOGY Copyriht 1972 by The Williams & Wilkins Co. Vol. 62. No. 3 Printed in U.S. A. PEPSIN SECRETION DURING DAMAGE BY ETHANOL AND SALICYLIC ACID LEONARD R. JOHNSON, PH.D. Department of Physiology