EFFECT OF BILE SALTS AND ASPIRIN ON THE GASTRIC MUCOSAL BLOOD FLOW

Transcription

1 GASTROENTEROLOGY 64: , 1973 Copyright 1973 by The Williams & Wilkins Co. Vol. 64, No.2 Printed in U.S.A. EFFECT OF BILE SALTS AND ASPIRIN ON THE GASTRIC MUCOSAL BLOOD FLOW PAUL O'BRIEN, M,B., AND WILLIAM SILEN, M,D, Department of Surgery, Harvard Medical School, and Beth Israel Hospital, Boston, Massachusetts Gastric mucosal blood flow (GMBF) in canine vagally denervated fundic pouches was studied using the aminopyrine clearance technique before, during, and after perfusion of the pouches with bile salt or acetylsalicylic acid. Solutions containing 40 mm and 10 mm of sodium tauro in 80 meq per liter of H+ decreased the GMBF to 19.6% ± 5.0 and 49.5% ± 15.6 of control values respectively, and also caused increased back diffusion of H+. When the perfusions were repeated with low H+ ( meq per liter) solutions containing 40 and 10 mm sodium tauro, the GMBF was reduced to similar low levels, but significant back diffusion of H+ did not occur. Perfusion with a 20 mm solution of aspirin reduced the GMBF to 29% ± 9.6 of control values. When a 20 mm solution of aspirin was instilled into the pouch without constant perfusion the GMBF was reduced to 57% ± 12.0 of control values, but back diffusion of H+ was significantly greater than with the constant perfusion technique. Disruption of the mucosal barrier with sodium tauro did not cause significant disappearance of aminopyrine instilled into the pouch. We conclude that bile salt and aspirin cause marked reduction in the GMBF. This change does not appear related to changes in ionic permeability of the mucosa. Instillation of aspirin solution with its consequent distention of the pouch alters the vascular response of the mucosa and its ionic integrity. The effect of disruption of the gastric mucosal barrier and consequent back diffusion of H+ upon the gastric mucosal blood flow (GMBF) has not been extensively studied. Augur 1 has reported the effect of ethanol, acetic acid, and aspirin on the GMBF in 3 dogs with Heidenhain pouches using plasma aminopyrine clearance (PAC) technique. He found "mucosal blood flow increased initially when either ethanol or acetic acid were used and sub- Received July 21, Accepted September 13, Address requests for reprints to: Dr. William Silen, Beth Israel Hospital, 330 Brookline Averlue, Boston, Massachusetts This work was supported by United States Public Health Service Grants AMll079 and AM13485, and a John A. Hartford Foundation Grant. 246 sequently declined with appearance of evidence of gastric mucosal barrier damage. In spite of similar evidence for gastric mucosal barrier damage, the mucosal blood flow with the aspirin solution was, by the fourth period, significantly greater than the mucosal blood flow with the acetic acid solutions." In this study, we have investigated the changes in GMBF in response to perfusion of the gastric mucosa with bile salts in the presence of high and low concentration of acid and we have also reviewed the effects of aspirin in acid solution on GMBF. Both sodium tauro and acetylsalicylic acid (ASA) caused a profound reduction in PAC. This effect was not dependent upon back diffusion of H+ into the gastric mucosa.

2 February 1973 BARRIER DISRUPTION AND GASTRIC MUCOSAL BLOOD FLOW 247 Methods Seven adult mongrel dogs ranging in weight from 12 to 18 kg were prepared with an antrectomy and a vagally denervated fundic pouch drained by a Gregory cannula. Gastrointestinal continuity was reestablished by endto-end gastroduodenostomy. No experiments were done for the 1st postoperative month. The dogs were healthy and maintained steady weight throughout the period of the study. The animals were deprived of food, but not water for 18 hr prior to each experiment and were trained to stand quietly, partially supported in slings. Tests were carried out no more frequently than at 5-day intervals. A loading dose of aminopyrine (20 mg per kg) was given intravenously followed by a continuous intravenous infusion (5 mg per kg) for the duration of each experiment using a Harvard infusion pump. Each experiment consisted of eight or 12 3D-min periods of continuous perfusion of the fundic pouch. Fluid passed to the pouch from a reservoir held at less than 10 cm above the pouch. A peristaltic pump on the drainage line from the pouch maintained a flow rate of 12 ml per min and returned the fluid to the reservoir. Before each experiment the pouch was perfused with the "basal solution" for 20 min to allow for stabilization. The pouch was then rinsed gently using a syringe with basal solution and the first period begun. The first three 30-min periods during which the pouch was perfused with the basal solution of the experiment constituted the control periods. During the fourth period the pouch was perfused with the "test solution." In one set of experiments (group 2), perfusion with the test solution was continued through the remaining four periods. In all other groups, the pouch was perfused with basal solution for the remaining four or eight periods. Group 8 constituted a control group in which the pouch was perfused with a solution of 80 meq per liter HCl in 64 meq per liter NaCI for eight consecutive 3D-min periods. FOI each period 35 ml of fresh solution was added to the reservoir. A 5-ml sample (sample A) was taken and the solution was then perfused for 2 to 3 min to permit mixing. A second 5-ml sample (sample B) was taken and the remaining solution was perfused through the pouch for 30 min. The solution was then returned to the reservoir and sample C was taken. The remaining fluid was discarded, the pouch was rinsed, and the next period was begun. A venous blood sample for assay of aminopyrine was taken at the midpoint of each period. In an effort to simulate more closely the methodology of Augur, the following modifications were made for the group 7 experiments. After taking the initial5-ml sample (sample A), the pouch was filled with the 30 ml of solution, gently mixed using a syringe, and sample B (5 ml) was taken. Rather than continually perfusing the pouch, the cannula was occluded and after 30 min the remaining solution was drained from the pouch and sample C was taken. Solutions. The basal solutions for each set of experiments contained HCl in concentrations shown in table 1 and were made isosmotic with NaC!. C l4-labeled polyethylene glycol was added as a volume marker. One gram of unlabeled polyethylene glycol was added to each liter of solution to minimize loss of C 14_ TABLE 1. Constitution of basal and test solutions for all experimental groups Group H+ concentration Basal solution Test solution ph Na+ Test compound ph Na+ meq/liter meq/liter meq/liter mm sodium tauro mm sodium tauro mm sodium tauro mm sodium tauro mm sodium tauro mm acetylsalicylic acid mm acetylsalicylic acid

3 248 O'BRIEN AND SILEN Vol. 64, No.2 labeled polyethylene glycol by surface adherence. In the test solutions equivalent amounts of NaCI were replaced by the test compound to give the solutions of sodium tauro or ASA indicated in table 1. Sorensen's phosphate buffer was added to the basal solutions containing 15 or 9 meq of HCI to raise the ph to the level of the corresponding test solution. Samples A, B, and C were assayed for H+ concentration by titration to ph 7.0 with 0.1 N NaOH using an automatic buret titrator (Radiometer). An aliquot of the derived neutral solution was then added to Bray's scintillation solution and the C 14 activity measured in a liquid scintillation counter (Nuclear Chicago). Samples Band C were assayed for Na+ concentration by a flame photometer (Instrumentation Laboratory, Lexington, Mass.). Using the change in C 14 activity to indicate change in volume, the difference between initial and final amount of H+ and Na+ for each period was calculated. Aminopyrine clearance. The aminopyrine concentration of samples Band C and of the plasma samples was assayed by the method of Brodie and Axelrod? Briefly: a I-ml sample was alkalinized, dissolved in dichloroethane, washed with sodium borate, extracted into 0.1 N HCI, and measured at 260 mil on a Zeiss spectrophotometer. Neither sodium tauro or acetylsalicylic acid interfered with this measurement. The PAC was then calculated by the formula: c!::.m P x 30 where c = PAC in milliliters per minute;!::.m = the change in mass of aminopyrine in the 30- min period; and P = plasma aminopyrine concentration. The mean PAC for the three control periods for each experiment was designated as 100%. The PAC for all periods was then expressed as a percentage of the mean of the controls. The mean and SEM for the change in amount of H+ and Na+ and for the PAC for all periods was calculated and the significance of differences determined by t-test for unpaired values. Results Part 1. Validation of methods. Aminopyrine (200 /lg per liter) was added to a basal solution of 80 meq per liter of' HCI and to a 40 mm tauro solution in 80 meq per liter of HCI. In four experiments, the pouch was perfused with the basal solution for two 30-min periods, the tauro solution for two periods, and then the basal solution again for two periods. The percentage of recovery of aminopyrine after perfusion of the pouch is shown in table 2. These results indicate that there is no significant loss of aminopyrine in association with increased back diffusion of H +. The PAC for all control periods was 3.9 ± 0.44 ml per min (mean and SEM of 74 observations). The changes in PAC in six experiments of eight consecutive 30- min periods during which the pouch was perfused with a control solution of 80 meq per liter of HCl in 62 meq per liter of NaCI (group 8) are shown in figure 1. As with the other groups of experiments, the mean of the first three periods was designated as 100% and the PAC for all periods expressed as a percentage of this mean. There is no significant change in PAC between any of the periods. TABLE 2. Percentage of recovery of aminopyrine after perfusing the pouch with aminopyrine in basal or tauro solutions Solution Penod Aminopyrine' H+ back diffusion /leq/30 min 80 meq/liter Hel ± ± ± ± mm tauro in ± ± 20 meq/liter Hel ± ± meq/liter Hel ± ± ± ± 38 a Each figure represents the mean and SEM for four observations; P value for difference III H+ back diffusion between periods 1 and 2 and periods 3 and 4 = b Percentage of recovery.

4 February 1973 BARRIER DISRUPTION AND GASTRIC MUCOSAL BLOOD FLOW 249 CONTROL ASA r----l.' f '... 20mM ASA without pouch distention N=5 0---<) 20mM ASA with pouch distention N=7 $ Control perfusion with 80 ueq/l H+ for all periods N=6 ll--t -1' / /.... * t "'--- -I"'-----i* * * MINUTES FIG. 1. Changes in plasma aminopyrine clearance after perfusion of the pouch with 20 mm acetylsalicylic acid (ASA) with and without distention, and during perfusion of pouch with control solution throughout (80 meq per liter HCl). Asterisks indicate P value of or less. Brackets signify ± SEM. CONTROL TAUROCHOLATE (40mMI (in80meq/lh') ir------o I i I i i I I... I ;;:: (e). I (0) "- <j tll 80!: <:: a I'> 100 '<; 40 "'t'- 20 ; l- it =l:: MINUTES FIG. 2. Changes in plasma aminopyrine clearance and H+ back diffusion after perfusion of 40 mm tauro in 80 meq per liter of H+ for 30 min are shown demonstrating the rapid return of H+ back diffusion to normal while a marked decrease in plasma aminopyrine clearance (PAC) still exists. The P value for comparison of control with test values of PAC is or less for 120, 150, 180, and 210 min. N = 6 for each point. Brackets indicate ± SEM. Part 2. Effect of bile salt. The changes in PAC after perfusion with 40 mm tauro for one 30-min period (group 1) are shown in figure 2. There was a significant reduction in PAC during the test period (P < 0.05). The PAC continued to decrease to reach 19.6% of the control level in the next period (P < 0.001). Subsequently, the PAC rose to control levels at 2 hr after exposure to the bile salt. When the exposure to 40 mm tauro was continued for five 30-min periods (group 2), a highly significant decrease in PAC occurred and persisted through all periods (fig. 3). Perfusion with 10 mm tauro

5 250 O'BRIEN AND SILEN Vol. 64, No. 2 (group 3) shown in figure 3 was associated with significant decreases of PAC during periods 4, 5, and 6 to a maximum of 49.5% of control values (P < 0.005). This decrease was, however, significantly less than that obtained with 40 mm tauro (P < 0.05). Increased back diffusion of H + occurred in all periods of perfusion with bile salt CONTROL 40mM TAUROCHOLATE in 80,u q/ L H+ I I -----,,I, '',, '', ' / \ ':!.\ IOmM TAUROCH OLATE in 80..uEq/L H+ 'oj", _ 4 0 mm Touroc olole N= mm Touro N=4 I *,l'<i\ MINUTES FIG. 3. Changes in plasma aminopyrine clearance after perfusion with 10 mm or 40 mm tauro in 80 meq per liter of H+. Asterisks indicate P value of or less. Brackets signify ± SEM. (table 3). With cessation of the exposure to bile salts, there was a rapid return to normal levels of back diffusion which coincided with the maximum or near-maximum decrease in PAC (fig. 2) suggesting that back diffusion of H + did not have a direct role in the reduction of the gastric mucosal blood flow. To elucidate this question further, perfusions with 40 mm and 10 mm sodium tauro were performed in the presence of low concentrations of H +. The requirement for a ph gradient for aminopyrine transport precluded use of neutral solutions. The changes in PAC that occurred with these solutions are shown in figure 4. Highly significant reduction of PAC occurred which was not different from that achieved with the 80 meq per liter of H + solution. Again, there was a significant difference between the 40 mm and 10 mm tauro perfusions (P > 0.05). Back diffusion of H + during these experiments was not significantly greater than zero. The changes in Na + influx into the lumen when exposed to bile salt are shown in table 3. A significant increase was noted only with 40 mm tauro in 80 meq per liter of H +. Part 3. Effect of acetylsalicylic acid. In TABLE 3. Changes in H+ back diffusion and Na+ secretion with exposure to the test solution Group H + back diffusion Na + secretion Con- Test Test trol period Difference Control period Difference p.eq/30 min p.eq/30 min mm tauro in (P < 0.001) (P < 0.01) meq/liter H mm tauro in a 136 (P < 0.001) a 154 (P < 0.005) meq/liter H + for five periods mm tauro in (P < 0.001) NS meq/liter H mm tauro in NSc NS meq/liter H mm tauro in NSc NS meq/liter H mm acetylsalicylic acid (P < 0.001) NS mm acetylsalicylic acid (P < 0.001) (P < 0.001) with pouch distention Test values for group 2 represent the mean of the five periods. NS, not significant. c Test values for H + back diffusion for groups 4 and 5 are compared with zero secretion.

6 February 1973 BARRIER DISRUPTION AND GASTRIC MUCOSAL BLOOD FLOW 251 CONTROL l, '001 l;3 \, \j 80 \<J-l:: " <: (3 6 0 & 4 0 "t 20 TAUROCHOLATE 'n... 40mM Tauro in 15,uEq/L HCI NA 0'--0 IO mm Tauro, in 9",Eq/L HCI \ N'4 * "!-""t",+*--f 1' * * * MINUTES FIG. 4. Changes in plasma aminopyrine clearance after perfusion of pouch with 40 mm tauro in 15 meq per liter of H+ and 10 mm tauro in 9 meq per liter of H+. Asterisks indicat e P value of o r less. Brackets signify ± SEM. view of the disparity between the consistent and marked reduction in PAC that we have shown to accompany exposure to bile salt and the inability of Augur to show a decrease in PAC with exposure to alcohol, acetic acid, and aspirin, we performed a series of experiments (group 6) with 20 mm acetylsalicylic acid in 80 meq per liter of H + as a test solution. Figure 1 shows that marked decrease in PAC which was noted in our experiments. All values are highly significantly different from control values and there is no suggestion of a return to normal levels at 2 hr after exposure. A principal difference in methodology between our technique and that of Augur was in the state of distention of the pouch during each period. Whereas he instilled into the pouch 30 ml of solution which did not circulate during each period, we maintained continuous perfusion through the pouch such that at anyone point of time the pouch held 8 to 10 ml of the solution. In an attempt to simulate Augur's methods more closely we performed a set of experiments (group 6) in which 25 ml was instilled into the pouch for each period. With the non perfusing technique, pressure in the pouch as measured by a Statham pressure transducer when the pouch was not actively contracting showed a mean of 10 cm H 2 0 with a range of 5 to 20 cm H 2 0 (eight measurements). With pouch perfusion, the pressure was 2 to 4 cm H 2 0 (three measurements). The results shown in figure 1 indicate that a significant decrease in PAC occurred during the period after exposure to the aspirin solution and was followed by a highly significant decrease in the subsequent two periods. The PAC for each period after aspirin was higher than the corresponding period in the experiments of group 6 (perfusion without distention) but this difference was significant for period 8 only (P < 0.025). When the post-aspirin periods were taken as a whole, there was a highly significant difference between the two groups. This suggests that the nonperfusion technique may alter the response of the mucosal blood flow to aspirin. Comparison of the H + back diffusion and Na + secretion that occurred in these two groups of experiments (table 3) indicate that aspirin has a more marked effect on the ionic permeability of the mucosa with the nonperfusing technique. The difference between mean back diffusion of H + of 121,uEq per 30 min in group 6 and 416,uEq per 30 min in group 7 is highly significant. Na + secretion increased from 186,uEq per 30 min with group 6 to 347,uEq per 30 min with group 7 but the difference was not significant. Discussion The validity of aminopyrine clearance as a measure of mucosal blood flow of the secreting stomach has been well documented by Jacobson et al. 3 Although conclusive proof that aminopyrine is completely cleared by the mucosa and is not actively transported by the parietal cell is not available, evidence which has been lucidly presented by Jacobson 4 strongly suggests that these critical assumptions of the clearance technique have been satisfied. Augur, l Cowley and Code, 5 and Rudick et al. 6 have shown that the technique can be extended to the nonsecreting stomach by establishing a ph gradient with exogenous acid. The clearance of

7 252 O'BRIEN AND SILEN Vol. 64, No.2 aminopyrine under these circumstances closely agrees with the extrapolated value for zero secretion when clearance is plotted against secretory rate. This series of experiments has demonstrated that exposure of the gastric mucosa to bile salts and ASA causes a highly significant decrease in its blood supply. The response in GMBF to intraluminal bile salt appears related to its concentration. Significantly less effect was seen with 10 mm tauro than with the 40 mm solution. The decrease in GMBF occurs with exposure to more than one agent which disrupts the gastric mucosal barrier, and persists well after the agent has been removed. This suggests that these agents do not act directly on blood flow but initiate mucosal changes which produce and maintain the decreased blood flow. The ability of both bile salt and ASA to alter the ionic permeability of the gastric mucosa has been amply demonstrated 7 11 and must be considered as a possible intermediate step in the development of decreased GMBF. However, several aspects of our data indicate that there is no correlation between the severity of the decrease in GMBF and the presence or severity of changes in ionic permeability. First, the decrease in GMBF with exposure to 10 mm and 40 mm tauro was constant when either 80 meq per liter of H+ was present and significant back diffusion of H + occurred or when 9 meq per liter and 15 meq per liter of H + was present and back diffusion of H + was not significantly different from zero. Second, when back diffusion of H + did occur, it was less sustained than the changes in GMBF and had returned to control levels at a time when decrease in GMBF was still near maximum. Third, the back diffusion of H + which occurred when ASA was perfused constantly through the pouch was significantly less than when the ASA solution was instilled into the pouch in spite of a more marked decrease in GMBF during the former experiments. The anatomical basis for the changes in GMBF is unclear. Mucosal ischemia from arteriolar constriction or opening of arteriovenous shunts in the submucosa, and mucosal congestion from venular constriction could produce similar changes in PAC. The functional importance of the arteriovenous shunts has been appreciated since they were demonstrated and studied by Barclay and Bentley 12 and Barlow et aly but their exact role in the control of the gastric microcirculation still remains to be defined. Sympathetic a receptor stimulation opens these shunts, thus bypassing the mucosal capillary bed. Mucosal damage may change the GMBF through stimulation of a local sympathetic reflex. The effect of pretreatment with an a receptor blocking agent would be of interest in this regard. The changes in mucosal blood flow which we have shown to accompany exposure to aspirin are in conflict with the findings of Augur. In attempting to resolve this difference, we simulated Augur's methodology more closely and still found a decrease in GMBF albeit of lesser degree and later in occurrence than when our continual perfusion technique was employed. At the same time, a more marked back diffusion of H + and secretion of N a + was noted which supports the thesis that the processes of change in mucosal blood flow and change in ionic permeability are not closely associated. Several other factors might explain the discrepancies between the results of our experiments and those of Augur. (1) It is possible that the presence of the antrum, and consequently circulating gastrin in Augur's dogs could have a modifying effect on the vascular responsiveness of the mucosa. (2) Rudick et al. 6 found that the PAC took as long as 45 min to reach a plateau in the resting mucosa. With only one control period of 30 min, Augur's control clearances could have given a spuriously low value for GMBF. (3) We noted that with distention of the pouch the response of the GMBF to aspirin was not observed until the period after exposure to ASA and reached a maximum during the following period. Since Augur measured the PAC for only two periods after exposure to AS A, a late

8 February 1973 BARRIER DISRUPTION AND GASTRIC MUCOSAL BLOOD FLOW 253 change in GMBF may not have been detected. (4) Whereas bleeding with its consequent direct transfer of plasma aminopyrine into the pouch was a prominent feature in the aspirin experiments of Augur, we noted bleeding only very occasionally and it was always of mild degree. The effect of pressure on the ionic permeabilityl' and blood flow 15 of the gastric mucosa has clearly been demonstrated. In these experiments using the nonperfusion technique, the pressure in the pouch and distention of the pouch were relatively mild and yet significant changes in the response to aspirin were seen when compared with the perfusion technique. The importance of a background of distention and pressure should not be understated when interpreting the effects of injurious agents on the gastric mucosa. The significance of the changes in GMBF that we have demonstrated is readily apparent. Since Virchow first postulated ichemia as a basis for gastric ulcer disease, a role for decreased blood flow has been anticipated. Much evidence implicating bile salts in the pathogenesis of acute ("stress") 16, 17 and chronic gastric ulcers has been reported recently along with extensive documentation of their effects on mucosal ionic permeability. 7, The demonstration that they also cause decreased mucosal blood flow suggests one other mechanism for their pathogenicity. REFERENCES 1. Augur NA Jr: Gastric mucosal blood flow following damage by ethanol, acetic acid, or aspirin. Gastroenterology 58: , Brodie BB, Axelrod J: The fate of aminopyrine (pyramidon) in man and methods for the estimation of aminopyrine and its metabolites in biological material. J Pharmacol Exp Ther 99: , Jacobson ED, Linford RH, Grossman MI: Gastric secretion in relation to mucosal blood flow studied by a clearance technic. J Clin Invest 45: 1-13, Jacobson ED: Clearances of the gastric mucosa. Gastroenterology 54: , Cowley DG, Code CF: Effects of secretory inhibitors on the mucosal blood flow in nons creting stomach of conscious dog. Am J Physiol 218: , Rudick J, Werther JL, Chapman ML, et al: Mucosal blood flow in canine antral and fundic pouches. Gastroenterology 60: , Davenport HW: Destruction of the gastric mucosal barrier by detergents and urea. Gastroenterology 54: , Ivey KJ, Den Besten L, Clifton JA: Effect of bile salts on ionic movement across the human gastric mucosa. Gastroenterology 59: , Black RB, Hole D, Rhodes J: Bile damage to the gastric mucosal barrier: The influence of ph and bile acid concentration. Gastroenterology 61: , Werther JL, Janowitz HD, Dyck WP, et al: The effect of bile on electrolyte movement across canine gastric antral and fundic mucosa. Gastroenterology 59: , Davenport HW: Gastric mucosal injury by fatty and acetylsalicylic acids. Gastroenterology 46: , Barclay AE, Bentley FH: The vascularization of the human stomach-a preliminary note on the shunting effect of trauma. Gastroenterology 12: , Barlow 'IE, Bentley FH, Walder DN: Arteries, veins and arterio-venous anastomoses in the human stomach. Surg Gynecol Obstet 93: , Bulkley G, Goldman H, Silen W: Pressure injury to the gastric mucosa-studies on an in vivo model of acute gastric distention. Am J Surg 117: , Edlich RF, Borner JW, Kuphal J, et al: Gastric blood flow-i. Its distribution during gastric distention. Am J Surg 120:35-37, Den Besten L, Hamza KN: Effects of bile salts on ionic permeability of the canine gastric mucosa during experimental shock. Gastroenterology 62: , Hamza KN, Den Besten L: Bile salts producing stress ulcers during experimental shock. Clin Res 19: 393 (171), Rhodes J, Barnado DE, Phillips SF, et al: Increased reflux of bile into the stomach in patients with gastric ulcer. Gastroenterology 57: , Capper WM: Factors in the pathogenesis of gastric ulcer. J R Coli Surg Edinb 40:21-35, Duplessis DJ: Pathogenesis of gastric ulceration. Lancet 1: , 1965