STUDIES OF ANTI-INFLAMMATORY DRUGS AND ALIPHATIC ALCOHOLS ON ANTRAL MUCOSA

Transcription

1 GASTROENTEROLOGY 66: 56-62, 1974 Copyright 1974 by The Williams & Wilkins Co. Vol. 66, No.1 Printed in U.S.A. STUDIES OF ANTI-INFLAMMATORY DRUGS AND ALIPHATIC ALCOHOLS ON ANTRAL MUCOSA ALLAN R. COOKE, M.D.(Syd), AND MICHAEL G. KIENZLE Department of Internal Medicine, University and Veterans Administration Hospitals. University of Iowa, Iowa City, Iowa Studies were made in 4 dogs with antral pouches of transmucosal potential difference (PD), and Na+, H+, K+, and Cl- fluxes, in response to aspirin, phenylbutazone, indomethacin, and cortisone acetate, as well as the aliphatic alcohols of carbon chain lengths C 1 to C. Potential difference was measured using 3% agar-saturated KCl electrodes connected to a potentiometer. Net fluxes of electrolytes were measured after instillation of 15 to 20 ml of a solution of 100 mm HCl + 54 mm NaCl, plus the test substance. Aspirin, 10 mm and 20 mm, increased net gain of Na+ into the pouch and increased net loss of H+ from the pouch. PD decreased from control of ~43.8 mv to ~31.2 mv in response to 20 mm aspirin. Phenylbutazone (200 mg per 100 ml), indomethacin (250 mg per 100 ml), and cortisone acetate (2.5 mg per 100 ml) did not change net movements of electrolytes or alter PD. The net movements of Na+ and H+ increased and PD decreased with the aliphatic alcohols (C 1 to C 4 ), and this effect was proportional to the molar concentration of the alcohol and to the carbon chain length; the greater the concentration or number of carbon atoms, the greater the effect. Branched chain alcohols (isopropanol and t-butyl alcohol) had less effect on PD and Na+ -H+ movements than their straight chain counterparts. Hypertonic solutions of glucose caused a fall in PD and an increase in net Na+ -H+ movements. PD was found to be more sensitive of gastric mucosal damage than measurements of ion movement. These studies indicate that the electrolyte and PD response of antral mucosa to alcohols and to anti-inflammatory drugs is generally similar to that found for fundic mucosa. In previous studies in which dogs with Heidenhain pouches were used, it was found that of a group of anti-inflammatory drugs only aspirin and indomethacin al- Received April 17, Accepted August 21, Presented at the American Federation for Clinical Research Meeting, Chicago, Illinois, November A preliminary report of this work has been published as an abstract. 1 Address requests for reprints to: Dr. Allan R. Cooke, Veterans Administration Hospital, Room 7E-46, Iowa City. Iowa These studies were supported in part by Grant AM and Veterans Administration Research Funds. The technical assistance of Donna Perrenoud and Eugene Clark is gratefully acknowledged. 56 tered net sodium-hydrogen fluxes and caused a fall in trans mucosal potential difference (i.e., broke the gastric mucosal barrier).2 In additional studies in Heidenhain pouches, using straight chain aliphatic alcohols, it was found that increasing the chain length (and thus lipid solubility), or increasing the concentration of the alcohol, increased net sodium-hydrogen fluxes and resulted in greater falls in the transmucosal potential difference. 3 Since gastric ulceration occurs in antral mucosa at the junction with fundic mucosa,4 it therefore seemed necessary to study the effects of these substances in antral mucosa to determine whether they

2 January 1974 EFFECT OF DRUGS ON ANTRAL MUCOSA 57 break the gastric mucosal barrier, and thus could possibly initiate a mechanism to induce acute gastric ulceration. The aim of this study, therefore, was to examine the effects of some anti-inflammatory drugs in dogs, as well as a series of straight and branched chain alcohols, to determine whether they were capable of altering electrolyte fluxes and potential difference in antral mucosa. Methods A thorough description of the apparatus and methods used in this study has been published! Briefly, 4 dogs were studied over a 6-month period. Each dog was prepared with a separated antral pouch drained with a Gregory cannula, and gastrointestinal continuity was restored using a gastrojejunostomy. The dogs were allowed water only 18 hr before testing and were never studied on consecutive days. All dogs were tested to determine the presence of acidsecreting mucosa with intravenous histamine dihydrochloride (0.2 mg per kg-hr- I). Two of the dogs did not secrete acid; in the other 2, acid outputs were 20 leq per 30 min and 80 leq per 30 min, respectively. These 2 dogs were assumed to have small amounts of fundic mucosa but since their response to control and experi: mental situations was similar to the other 2 non-acid secreting dogs, their data were pooled with the other dogs. For each experiment, a piece of polyvinyl tubing was attached to the outside of the cannula. The tubing led to a three-way stopcock which served for instillation and drainage of solutions. A smaller tube of 1;)2 inch interior diameter, previously filled with 3% agarsaturated KCl, extended into the entrance of the pouch via the drainage tube to which it was attached by tetrahydrofuran, and served as the ~etecting electrode for measurement of potential difference. At the beginning of each experiment, the pouch was rinsed thoroughly with a solution of 100 mm HCI and 54 mm NaC!. The pouch was t~en drained and gently aspirated with a syringe. A solution (15 to 20 ml) of 100 mm HCI 54 ~~ NaCI, and phenol red (40 mg per liter) ~as InJ~cted into the pouch using a calibrated syringe. The contents were mixed for 30 sec, and a 3.-ml sample was withdrawn for analysis. The r~sldual volume of the pouch was determined by dllution of the phenol red. The contents of the pouch were mixed at lo-min intervals with a SYringe via the three-way stopcock. At the end of 30 min, the pouch was drained and aspirated as completely as possible, and the volume of fluid was determined to the nearest 0.1 m!. A sample of the contents was taken for analysis, and the pouch was then rinsed with the solution without phenol red. Each experiment consisted of three consecutive 30-min periods. Period 1 was the control, in period 2 the drug under study was instilled, and in period 3 the same solution as in period 1 was used. All drugs were instilled in period 2 in the same solution used during periods 1 and 3 (l00 mm HCI, 54 mm NaCI, and phenol red 40 mg per liter). The residual volumes (mean ± SE) ofthe antral pouches were l.2 ± 0.2 ml, 1.4 ± 0.1 ml, l.0 ± 0.1 ml, and 0.6 ± 0.1 m!. The initial and final samples were analyzed for titratable acidity, sodium, potassium, chloride, and phenol red concentration. The acidity was determined by titrating a 0.2-ml sample with 0.2 M NaOH to ph 7.0 using a glass electrode and automatic buret (Autoburet, Radiometer, Copenhagen, Denmark). Sodium and potassium concentrations were measured by automated flame photometry (Flame Photometer, Instrumentation Laboratory, Inc., Boston, Mass.). Chloride concentration was determined by automatic chloride titration (Buchler-Cotlove, Fort Lee, N. J.). Phenol red concentration was measured spectrophotometrically (558 mm) by the method described by Hunt.. The net ionic flux was computed as the product of initial concentration and volume subtracted from the product of final concentration and volume. A positive value indicates a net flux from mucosa to lumen and a negative value a net flux from lumen to mucosa. Transmucosal potential difference (PD) was measured as previously described. 2 6, 7 The reference electrode was a 19-9auge scalp vein infusion set (Butterfly-19 Abott) filled before use with a 3% agar-saturated KCI solution and was inserted into a peripheral leg vein. The detecting electrode was of polyvinyl tubing YJ2 inch interior diameter filled with 3% agarsaturated KCI solution. It was inserted into the pouch as described above. Each electrode led to a beaker containing a saturated KCI solution in which were placed one of a pair of balanced calomel half cells which were connected to a ph meter (Radiometer, Copenhagen, Denmark). Readings were recorded at 2-min intervals to the nearest 0.5 mv on an expanded XlO scale. Osmolality of the test solutions was measured by an Advanced Wide-Range Osmometer (Advanced Instruments, Newton, Mass.). Test Solutions Solutions of indomethacin, phenylbutazone, and acetylsalicylic acid were instilled into the pouch in period 2 and were prepared daily. The

3 58 COOKE AND KIENZLE Vol. 66, No.1 solution was ph 1 and contained 100 mm HCl, 54 mm N acl, and 40 mg per liter of phenol red. The solution containing cortisone acetate was prepared by mixing the steroid with 1 g of polyethylene glycol 400 and 100 ml of water for 60 min. This solution was made acid as outlined above, and polyethylene glycol was used to prevent precipitation of the steroid. Concentrations of these solutions were as follows: indomethacin, 250 mg per 100 ml; phenylbutazone, 200 mg per 100 ml; acetylsalicylic acid, 10 mm and 20 mm (360 mg per 100 m!); cortisone acetate, 2.5 mg per 100 ml. Osmolalities ranged from 290 to 302 milliosmoles per kg. These solutions were mixed thoroughly before instillation in the pouch. Indomethacin, aspirin, and phenylbutazone tended to partially precipitate out of solution. Final concentrations of the alcohol solutions were as follows: methanol and ethanol, 1 M (3.2% methanol, 4.6% ethanol), 2 M, and 3.5 M; n-propyl alcohol and isopropanol, 0.5, 1 (6%), and 1.5 M; n-butyl alcohol and t-butyl alcohol, 0.1, 0.5, and 1.0 M (7.4%). Solutions of glucose (0.2, 0.25, and 0.5 M) were used to examine the effect of hypertonicity. The osmolalities of the 0.1 M and 0.5 M alcohols in acid-saline mixture were 391 to 39:3 milliosmoles per kg (0.1 M), and 808 to 813 milliosmoles per kg (0.5 M). The osmolalities of concentrations greater than 0.5 M were not measured. Concentration-effect curves for the alcohols were made by plotting the difference in PD (.~PD) or net ion flux (~ion flux) between periods 1 and 2 against the molar concentrations of the alcohol. Statistics Differences between control (period 1) and test situations (periods 2 or 3) for net movements of ions, and also for PD, were evaluated by the t-test. 8 Significant differences in this context mean that P was less than Results Effects of the Anti-inflammatory Drugs Acetylsalicylic acid was used to test the responsiveness of the pouches to the antiinflammatory drugs. There was a net gain of Na+ into the lumen and loss of H+ from the lumen in response to 10 mm aspirin, and with 20 mm aspirin this reached statistical significance in period 3 (fig. 1). No significant changes occurred with net chloride or potassium movements. Transmucosal PD fell from a control value of ± 0.4 mv (lumen negative with reference to serosa) to ± 1.0 mv (P < 0.01) (fig. 1). No significant changes in net movements of Na+, H+, K+, or Cl- ions, or of PD, were found in response to indomethacin (n = 6), phenylbutazone (n = 5), or cortisone acetate (n = 6). Effects of Aliphatic Alcohols Potential difference. All the alcohols caused a fall in PD (fig. 2). The PD began to decrease 0.5 to 2 min after instilling the alcohol-acid-saline solution. The PD generally returned to control values during the latter part of the 30-min period, but this depended on severity of the change; the greater the decrease, the longer the time for recovery. ASPIRIN (20mM;pH 1.0) FIG. 1. Effect of aspirin (20 mm, ph 1.0) on potential difference (P.D.l and net fluxes of Na+ and H+ ions. Blocks represent five experiments in 4 dogs; the vertical bars are the SE. Aspirin was instilled in period 2. Positive and negative values indicate. respectively, net gain or loss from the lumen of the pouch.

4 January 1974 EFFECT OF DRUGS ON ANTRAL MUCOSA 59 The decrease in PD was related directly to concentration and the carbon-chain length of the alcohol; the greater the concentration and the longer the chain length, the greater the effect (fig. 2). These changes were statistically significant (P < 0.01) at all but the lowest concentrations of the alcohols, with the exception of metha- nol in which only 3.5 M caused a significant fall (fig. 2). When n-propyl alcohol and n-butyl alcohol were compared with isopropanol and t-butyl alcohol for a given concentration, each was more effective than its branched counterpart in decreasing PD (fig. 3). These differences were not statistically n - butanol -20 ethanol > E d -15 a: <l CONC (molad FIG. 2. Change in trans mucosal potential difference (t:.p.d.) induced by various molar concentrations of the straight-chain alcohols. Each point is the mean of three to five experiments in 4 dogs ~ d -15 a: <l o 0'1.. ''"'" ''" ' n-butanol. t- butanol,. '" ' ' ''''" CONe (molar) o 1'0 n - propanol o jso - propanol 1-5 FIG. 3. Change in potential difference (t:.p.d.) induced by various molar concentrations of n-propylalcohol and n-butyl alcohol and their branched counterparts. Each point is the mean of four to five experiments in 4 dogs.

5 60 COOKE AND KIENZLE Vol. 66, No.1 different at the lowest concentrations (fig. 3). Electrolyte movements. The net flux of Na+ ion into the lumen of the pouch was increased over control with each of the alcohols (fig. 4). As with PD, the net flux of Na+ increased as the concentration of each alcohol was increased, and, on a molar basis, the longer carbon-chain alcohols were more effective. A similar relationship was found for concentration and carbonchain length with net loss of H+. In general, the net movements of Na+ paralleled the PD closer than that of H+. A correlation between log PD and net Na+ flux was significant (r = , P < 0.001, n = 56) and followed an exponential relationship (y = x). The alcohols produced no consistent effects on the net movements ofk+ or CI-. In general, there were net gains o f K+ and CIinto the pouch, and the sum of the cations was generally balanced by Cl-. Potassium fluxes ranged from about 30 to 50 ~Eq per 30 min. Net Fluxes and PD at 5 Min To examine electrolyte movements shortly after instillation of n-butylalcohol, the pouch was emptied at 5 min instead of at the usual period of 30 min. As shown in table 1, the net movements of Na+, H+, K+, and Cl- were not statistically different in response to a concentration of 0.5 M n-butylalcohol, whereas PD had decreased significantly. At the higher concentration (1.0 M), all movements of ions, as well as PD, were found to have changed significantly. Effect of Glucose To examine osmotic effects on the PD and electrolytes, three concentrations of glucose were examined (table 2). Glucose 0.5 M (osmolality in acid-saline mixture was 845 milliosmoles per kg) caused a significant decrease in PD and significant alterations in the net Na+ -H+ fluxes, whereas the lower concentrations (0.2 M, 0.25 M) had no effect. Discussion Antral mucosa is histologically different from fundic mucosa 9 and, as well, it secretes more water, sodium, and potassium than fundic mucosa. IO Furthermore, there is evidence that gastric ulcers occur only in antral mucosa at its junction with acidsecreting mucosa. 4 In this study, two pouches secreted small amounts of acid (20 and 80 ~Eq per 30 min) in response to a ethanol ii- 400 W 5- >< " +0 Z <I 200 methanol o 100 o '----:-o'"=". 5--.J İ 'c- O --"1 ::-5--='2 0::---=-'2 5~--'3:'c 0~----:3:'c 5 - CONC {molar} FIG. 4. Change in net flux of Na+ (L~ Na+ ) induced by various molar concentrations of the straight chain alcohols. Each point is the mean of three to five experiments in 4 dogs.

6 January 1974 EFFECT OF DRUGS ON ANTRAL MUCOSA 61 TABLE 1. Effect of n-butyl alcohol on ionic movement and transmucosal potential difference (PD) at 5 min Microequivalents per 5 min n-hutyl alcohol Period PD(-mv) H+ Na+ K+ Cl- 0.5M (n ~ 3) 1-2 ± ± 9 8±1 44 ± ± ± ± ± 3 55 ± ± ± ± 31 8±5 39 ± ± 2.1 p NSa NS NS NS < M (n ~ 4) 1-42 ± ± 18 6±2-14 ± ± C± ± ± ± ± ± ± ± ± ± 2.3 P <0.01 <0.01 <0.01 <0.05 <0.01 a NS, not significant. TABLE 2. Effect of glucose on ionic movement and transmucosal potential difference (PD) Microequivalents per 30 min Glucose Period PD (-mv) H+ Na+ K+ Cl- 0.2Ma (n ~ 3) ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 0.5 P NS NS NS <0.05 NS 0.25M (n ~ 4) 1-83 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 0.9 P NS NS NS NS NS 0.5M (n ~ 4) 1-73 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 0.9 P <0.01 <0.01 NS NS <0.01 a The osmolalities of 0.2, and 0.5 M glucose in acid-saline mixture were 505,560, and 845. respectively. NS, not significant. maximal dose of histamine. Compared with conventional fundic pouches prepared in this laboratory which secrete about 3000 to 4000,uEq per 30 min with histamine, the antral pouches contain about 0.5 to 2% functioning acid-secreting mucosa. This very small amount of acid-secreting mucosa appeared to make little difference, since these two pouches were indistinguishable in these experiments from the two antral pouches without acid-secreting mucosa. In a previous study in dogs with Heidenhain pouches, it was found that only aspirin and indomethacin altered net sodiumhydrogen fluxes and reduced PD, whereas phenylbutazone, caffeine, hydrocortisone, cortisone, and cortisone acetate did not. 2 In this study, aspirin altered net sodiumhydrogen fluxes and reduced PD, but phenylbutazone,. indomethacin, and cortisone acetate were without effect (fig. 1). This study and the previous one 2 suggest that the mechanism by which phenylbutazone, caffeine, and corticosteroids cause gastric erosions and mucosal damage in experimental animals is not the same as that induced by aspirin. Furthermore, the concentrations of aspirin, indomethacin, and phenylbutazone used in this study are about those expected in the stomachs of patients receiving conventional doses. Indomethacin was effective in the fundic pouch animals but not in these experiments, and no good explanation is given. These studies suggest that despite its ability to allow greater movements of cations,10 antral mucosa behaves similarly to fundic mucosa in response to the anti-inflammatory drugs.

7 62 COOKE AND KIENZLE Vol. 66, No. 1 With the alcohols, it was found that increasing the concentration or increasing the carbon -chain length of the alcohol increased its ability to damage gastric mucosa (figs. 2 to 4). Furthermore, straight-chain alcohols, when compared with their branched isomers, were more damaging mole for mole than their branched counterparts (fig. 3). These findings confirm a previous study in Heidenhain pouch dogs in which damage was best correlated. with lipid solubility.3 This study and previous ones 2. 3, 6 have suggested that PD correlated with net N a+ or H+ movements. In one study,3 and in this one, PDappeared to correlate with net Na+ flux better than net H+ flux. However, the 5-min studies (table 1) indicate that there can be significant changes in PD while the net movements of electrolytes do not change. This would suggest that the net movements of electrolytes are a relatively insensitive measurement of events which occur after damage to the gastric mucosal barrier. Gastric mucosal PD is a sensitive and easy measurement and is not subject to errors such as neutralization of H+, failure to ensure basal secretion, and incomplete collections that occur in pouch studies, and, more particularly, in human studies. Due to absorption of alcohols by the pouches, the true osmotic pressure in the pouches cannot be assessed, and, for this reason, glucose was studied. The studies with hypertonic glucose suggest that some of the effects of the more concentrated alcohol solutions, i.e., those of osmolalities of 800 and above, are due to osmotic pressure. Generally, net movements of water, sodium, and potassium were greater in these studies in which antral mucosa was used, than in previous ones in which fundic mucosa was used,2, 3 a finding reported by Dyck et a1. 10 In studies in which hypertonic glucose was used in Heidenhain pouch dogs (unpublished data), we found that similar concentrations increased net ionic movement and reduced PD, as those concentrations found in studies in which antral mucosa was used. Altamirano 11 used fundic mucosa and found that concentrations about 1 to 2 M glucose were required to increase net ionic movement significantly. These concentrations are greater than those found in these experiments (0.5 M glucose in acid-saline mixture was 845 milliosmoles per kg), but Altamirano ll did not examine concentrations of osmolytes between and 1.2 M. REFERENCES 1. Kienzle M, Cooke AR: The effect of ulcerogenic drugs on the canine antral mucosal barrier (abstr). Clin Res 20:733, Chvasta TE, Cooke AR: The effect of several ulcerogenic drugs on the canine gastric mucosal barrier. J Lab Clin Med 79: , Weisbrodt NW, Kienzle M, Cooke AR: Comparative effects of aliphatic alcohols on the gastric mucosa. Proc Soc Exp Bioi Med 142: , Oi M, Oshida K, Sugimara S: The location of gastric ulcer. Gastroenterology 36:45-56, Hunt IN: The inhibitory action of sucrose on gastric digestive ability in patients with peptic ulcer. Guys Hosp Rep 103: , Davenport HW, Warner HA, Code CF: Functional significance of gastric mucosal barrier to sodium. Gastroenterology 47: , Geall MG, Phillips SF, Summerskill WHJ: Profile of gastric potential difference in man. Effects of aspirin, alcohol, bile, and endogenous acid. Gastroenterology 58: , Snedecor GW, Cochran WG: Statistical Methods. Sixth edition. Ames, Iowa, The Iowa State University Press, 1967, p Grossman MI, Marks IN: Secretion of pepsinogen by the pyloric glands of the dog, with some observations on the histology of the gastric mucosa. Gastroenterology 38: , Dyck WP, Werther JL, Rudick J, et al: Electrolyte movement across canine antral and fundic gastric mucosa. Gastroenterology 56: , Altamirano M: Action of concentrated solutions of non-electrolytes on the dog gastric mucosa. Am J Physiol 216:33-40, 1969