gastric mucosal toxicity of duodenal juice constituents in the rat

Digestive Diseases and Sciences, Vol. 39, No. 2 (February 1994), pp. 327-339

Gastric Mucosal Toxicity of Duodenal Juice Constituents in the Rat

Acute Studies Using Ex Vivo Rat Gastric Chamber Model

DAVID ARMSTRONG, MA, MRCP (UK), EDWARD R.C. RYTINA, MD, GERARD M. MURPHY, PhD, and R. HERMON DOWLING, MD, FRCP

To determine the acute gastrotoxicity of refluxed duodenal contents, an ex vivo rat gastric chamber was used to study mucosal damage produced by conjugated and unconjugated human bile acids and lysolecithin at neutral and acidic pH; the effects of trypsin, human duodenal aspirate, and combinations of chenodeoxycholic acid, lecithin, and lysolecithin were also studied. A t neutral pH, all bile acids except tauroursodeoxycholic acid, caused dose-dependent falls in mucosal potential difference and losses of mucosal nucleic acid into the chamber fluid, indicating mucosal damage. The di-a-hydroxy bile acids, deoxycholic and chenodeoxycholic acids, were more gastrotoxic than cholic and ursodeoxycholic acids, and all unconjugated bile acids were more toxic than their conjugated species, none of which produced damage at concentrations below 2. 0 rnM. For all but the taurine conjugates, bile acid-induced changes in potential difference were lower at acidic then at neutral pH. Lysolecithin gastrotoxicity, comparable at neutral p H to that o f chenodeoxycholic acid, was also reduced at acidic pH. Lecithin decreased the gastrotoxicity of chenodeoxycholic acid and lysolecithin. Trypsin produced no damage, and the gastrotoxicity of human duodenal aspirate was unaffected by prior heat inactivation of pancreatic enzymes.

KEY WORDS: bile acids; lysolecithin; lecithin; trypsin; bile; potential difference; gastric mucosa.

Duodenogastric reflux has been implicated in the pathogenesis of gastritis, gastric ulceration, and gastric carcinoma in man (1-6). Furthermore, individual constituents of duodenogastric refluxate, such as lysolecithin, pancreatic enzymes, and bile acids, are known to produce gastric mucosal damage in a variety of experimental models (7-9). How-

Manuscript received January 23, 1992; revised manuscript received March 11, 1993; accepted June 17, 1993.

From the Gastroenterology Unit, Guy's Campus, UMDS of Guy's and St Thomas' Hospitals, London SE1 9RT, UK.

The study was supported in part by grants from Gipharmex SpA, Milan, Italy, and from the Special Trustees of Guy's Hospital, London, UK.

Address for reprint requests: Professor R. Hermon Dowling, Gastroenterology Unit, 18th Floor, Guy's Tower, UMDS of Guy's and St Thomas' Hospitals, London SE1 9RT, UK.

This manuscript is dedicated to Prof. Gustav Paumgartner of Munich, on the occasion of his 60th birthday.

ever, there are no systematic studies of the relative toxicities of these different constituents and the effect of luminal pH variations on their gastrotoxicity in a single experimental model. For example, taurocholic acid (TCA) produces gastric mucosal damage that is most marked in an acidic environment (10, 11), when it, like other taurine conjugates, is readily soluble. However, glycine-conjugated and unconjugated bile acids are virtually insoluble at normal gastric luminal pH (12). Thus, it is important to document the relative gastrotoxicities of these bile acids at neutral and acid pH and to compare their effects with those of other components of pancreaticobiliary secretions such as lysolecithin, which may itself be injurious (13), or lecithin, which may incorporate bile acids or lysolecithin into mixed micelles and thereby reduce their toxicity.

Digestive Diseases and Sciences, Vol. 39, No. 2 (February 1994) 0163-2116/94/0200-0327507.00/0 © 1994 Plenum Publishing Corporation

327

The aims of the present s tudy were , therefore, (1) to conduct systemat ic dose - response studies of the acute gastric mucosal toxicity of individual duodenal juice consti tuents in a single animal model , and (2) to test the hypo theses that an acidic env i ronment would increase, and that lecithin would decrease, the toxicity of individual duodenal juice components .

MATERIALS AND M E T H O D S

Study Design. Gastric mucosal damage produced by the test solutions was assessed using the ex vivo rat chamber model (14, 15). Mucosal damage was quantified by mea- suring the fall in gastric transmucosal potential difference (PD) and loss of mucosal nucleic acid (NA) into the chamber fluid. After the experiment, the gastric mucosa was examined by light microscopy for histological evidence of damage.

Animals. All studies were conducted in accordance with the Animals (Scientific Procedures) Act 1986. Male Wistar rats weighing 230-350 g were studied. They were housed in wire-bottomed cages to minimize coprophagy and were fed standard rat chow (Rat and Mouse No. 1 modified maintenance diet, SDS, Witham, Essex, UK). They had free access to water throughout but were fasted for 18L-24 hr before the start of the experiment.

Materials. Cholic acid (CA: 3et,7et,12~t-trihydroxy-513- cholan-24-oic acid), chenodeoxycholic acid (CDCA: 3tx,7ot-dihydroxy-513-cholan-24-oic acid), deoxycholic acid (DCA: 3,~,12et-dihydroxy-513-cholan-24-oic acid), ursodeoxycholic acid (UDUA: 3ct,713-dihydroxy-513-cholan- 24-oic) and their glycine (G) and taurine (T) conjugates were all obtained from Sigma Chemical Co., St. Louis, Missouri. The glycine and taurine conjugates of UDCA were gifts from Roussel UCLAF, Paris, France.

For the studies at "neutral" pH (see below), a 5.0 mM stock solution of each bile acid was made up in 0.15 M saline (pH 5.5-7.5) and diluted with 0.15 M saline to yield final test solution concentrations ranging from 0.10 mM to 2.0 mM. In addition, 10 mM stock solutions of CA, GCA, TCA, GDCA, UDCA, GUDCA, and TUDCA, as well as a 20 mM solution of TUDCA in 0.15 M saline (pH 6.0- 7.4) were prepared. For the studies at acidic pH, a further 5 mM solution of each bile acid in 0.15 M saline was made up as described above and, immediately before use, the pH of each solution was titrated to pH 2.5 with 6.0 M HC1. After acidification, the taurine-conjugated bile acids remained in solution but, as expected (12), all unconjugated and glycine-conjugated BAs formed dense white flocculant precipitates.

Leci thin (phosphat idylchol ine) and lysoleci thin (lysophosphatidylcholine) and were obtained from Sigma CHemical Co. Lysolecithin was dissolved in 0.067 M phosphate buffer (pH 7.4) to yield solutions with final concentrations ranging from 0.05 mM to 2.0 mM. Acidic solutions of lysolecithin (0.5-2.0 mM) were made up by dissolving it in 0.15 M saline and adjusting the pH to 2.5. All lysolecithin solutions appeared clear when freshly prepared, they were discarded if they became cloudy or if they were not used within 24 hr of preparation.

ARMSTRONG ET AL

TABLE 1. COMPOSITION OF FASTED HUMAN DUODENAL CONTENTS ASPIRATED AFTER INTRAVENOUS ADMINISTRATION OF

CHOLECYSTOKININ

Duodenal aspirate

Heated Unheated

Total bile acid (mM) 35.0 38.0 Phospholipid (mM) 6.4 6.6 Cholesterol (mM) 2.2 2.1 Lipase (retool/rain/liter) 210 0.0 Trypsin (mmol/min/liter) 8.0 0.0

To examine the effects of a prototypic pancreatic enzyme on the gastric mucosa, porcine trypsin (Sigma Chemical Co.) was reconstituted in 0.15 M saline (18,600 BAEE units/liter, pH 7.4) and studied within 6 hr of preparation.

As the toxicity of CDCA was greatest at a concentration of 5.0 mM while that of lysolecithin was greatest at 2.0 mM (see Results), the combined effect of bile acid and phospholipid on the gastric mucosa was studied using the following test solutions: lysolecithin 2.0 mM plus CDCA 5.0 mM in 0.15 M saline (pH 6.5); lecithin 5.0 mM plus CDCA 5.0 mM in 0.15 mM saline (pH 7.5); and lecithin 2.0 mM plus lysolecithin 2.0 mM in 0.15 M saline (pH 7.0). Leci- thin concentrations were chosen to match the concentrations of the accompanying CDCA or lysolecithin.

To examine the effect of a typical combination of duodenal contents, 90 ml of human duodenal juice was aspirated over 15 min after an intravenous injection of 100 Ivy dog units of cholecystokinin (Pancreozymin, Boots plc, Nottingham, UK) in a single subject who had fasted overnight. The aspirate was agitated to ensure complete mixing and was then divided into two equal portions. One was stored on ice and used, without further treatment, within 6 hr of aspiration. The other was heated in a waterbath at 70 ° C for 30 min, to inactivate pancreatic enzymes and then stored overnight at -20* C. An aliquot of fresh (unheated) duodenal aspirate was analyzed immediately after aspiration to determine the activities of lipase and trypsin and the concentrations of total bile acids and phospholipids. In addition, its cholesterol concentration was determined since, theoretically, cholesterol too could modify bile acid gastrotoxicity. The same analyses were performed on an aliquot of the heated aspirate after it had been stored overnight. The results of these analyses are shown in Table 1.

Study Protocol. The preparation of the ex vivo rat gastric chamber has previously been described in detail (14). In brief, each rat was anesthetized with 60 mg/kg in- traperitoneal pentobarbitone (Sagatal, May & Baker, U.K.). A midline laparotomy was performed, and the esophagus and duodenum were ligated. The stomach was mobilized, opened along its greater curvature, and pinned out fiat, mucosa uppermost, on a clear Perspex (Lucite) plate. A hollow Perspex cylindrical chamber was clamped over the mucosa, which was then rinsed with preheated (37* C) 0.15 M saline to remove adherent mucus, bile, and particulate matter.

The gastric transmucosal PD (millivolts) was monitored continuously using two bridges consisting of gel-filled

328 Digestive Diseases and Sciences, Vol. 39, No. 2 (February 1994)

GASTROTOXICITY OF DUODENAL CONTENTS

polythene catheters. The end of one catheter dipped into the chamber fluid; the end of the second was placed in the peritoneum. The other ends of both bridges were con- nected, via calomel electrodes, to a voltmeter and a chart recorder.

For the initial experiments with DCA (14), GDCA, and TDCA, each animal was studied for nine sequential 10-min periods (two stabilization, one baseline, one challenge, and five postchallenge periods). As little additional information was obtained from the five postchallenge periods, all subsequent experiments were shortened to 50 rain using the same protocol as the 90-rain studies apart from the loss of the last four postchallenge periods. Five milliliters of solution (preheated to 37 ° C) were placed in the chamber at the start of each period, stirred continuously with a rotat- ing paddle and, at the end of the period, aspirated as completely as possible before instillation of the next 5-ml aliquot. Saline (0.15 M) was instilled during the first two stabilization periods. During the baseline period, the sol- vent used to make up the test solution was instilled, fol- lowed by the test solution itself in the challenge period. Saline (0.15 M) alone was instilled during the postchallenge periods. All fluid aspirated from the gastric chamber was frozen on solid carbon dioxide and stored at -20 ° C until analyzed for its nucleic acid concentration ([NA]).

Only one experiment was performed per animal, and each test solution was studied in six animals. The bile acids were studied initially at 5.0 mM concentrations (pH 5.5-7.0) and then at decreasing concentrations until no PD change was seen during the challenge period. If a 5.0 mM solution produced a PD change (APD) that was less than half the normal resting PD (-28 to -36 mV" see Results), the bile acid was also studied at 10.0 mM and sometimes at 20.0 mM concentrations. The APD values produced by the test solutions were compared with those produced by 0.15 M saline in the control group.

Bile acid toxicity at pH 2.5 was studied using 5.0 mM bile acid solutions preceded by acidified saline (pH 2.5) in the baseline period. Solutions of lysolecithin (0.05-2.0 mM) in phosphate buffer at neutral pH (pH 7.4) were studied using a control group of animals, in which the gastric mucosa was exposed to 0.067 M phosphate buffer. Acidic solutions of lysolecithin in saline (pH 2.5) were studied at concentrations ranging from 0.5 mM to 2.0 raM.

Mucosal Potential Dilference. From the continuous PD recording, the mean PD for consecutive 5-rain periods was determined in each experiment. The APD produced by a test solution was the difference between the mean PD during the baseline period and the minimum PD observed during the challenge period (14); APD is presented as an absolute change and also as a percentage of the resting PD during the preceding baseline period.

Chamber Fluid pH. Gastric chamber fluid pH was re- corded three times per 10-min period, just after instillation of a solution, halfway through the period and just before aspiration of the fluid, using a combined pH elec- trode (Russell Electrodes; Portec Ltd., UK).

Nucleic Acid Determinations. The stored chamber fluid was thawed, sonicated for 30 sec to disrupt intact cells, and centrifuged. Nucleic acid concentrations ([NA]: rag/ ml) were measured spcctrofluorimetrically (16). The change in [NA] between the baseline and challenge peri-

ods (A[NA]) was used as a marker of mucosal damage-- indicating loss of cells or their constituents, into the chamber fluid.

Histology. After an experiment, the stomach was re- moved, pinned flat on wax, and fixed in 10% formal saline for 15-20 min. Two 5 x 20-mm strips were taken, one from the anterior and one from the posterior wall of the stomach body, and stored in 10% buffered formal saline before embedding in paraffin wax. Paired sections from each block, stained with Alcian blue-PAS to assess mu- tin content or with hematoxylin and eosin (H&E), were then coded and assessed "blindly" for evidence of mucosal damage.

The studies with DCA, GDCA, and TDCA lasted 90 min and, thus, the tissue for histological examination was taken 50 rain after the bile acid challenge period. All other experiments lasted only 50 min with the result that tissue samples in these studies were taken 10 min after the end of the challenge period.

Statistical Analyses. Results are given as means and standard errors of the mean (SEM). A one-way analysis of variance (ANOVA) was performed to detect heterogene- ity of variance between the groups and Duncan's multiple-range test (17) used to assess statistically significant differences. For ANOVA, the data were analyzed sepa- rately: (1) APD values produced by bile acid challenge at neutral pH, (2) APD values produced by bile acid challenge at acidic pH, (3) APD values produced by lysolecithin at neutral pH, and (4) APD values produced by lysolecithih at acidic pH.

All A[NA] values were analyzed similarly. The effects of duodenal aspirate and of test solutions containing mixtures of bile acids and phospholipids were compared with the appropriate control data using Student's t test for unpaired data. Linear correlation was used to examine the relationship between APD and A[NA] after challenge with the different bile acids and lysolecithin.

RESULTS

Mucosal Potential Difference

Baseline PD Values. In the saline control group, the mean PD during the 10-rain baseline period for the individual exper iments , after instillation of 0.15 M saline, ranged f rom - 2 8 . 0 - 2.5 m V to - 3 6 . 0 - 3.0 mV, the mucosa l surface being negative with respect to the serosa. Subsequent instillation of 0.15 M saline in the challenge per iod produced a mean APD of 1.5 --- 0.5 inV. In the neutral lysolecithin groups, the mean baseline PD, after instillation of 0.067 M phosphate , ranged f rom - 3 4 . 9 +- 2.1 m V to - 4 0 . 3 +- 2.7 inV. Subsequent challenge with 0.067 M phospha te buffer p roduced a mean APD of 0.1 +-. 0.5 m V in this control group.

The mean PD values during the basel ine per iod for each exper imenta l group are presented, along with the respect ive APD changes p roduced b y each challenge subs tance , in Tables 2-7.

Digestive Diseases and Sciences, Vol. 39, No. 2 (February 1994) 329

A R M S T R O N G E T A L

TABLE 2. CHA~GES IN TRANSMUCOSAL POTENTIAL DIFFERENCE AND CHAMBER FLUID NUCLEIC ACID CONCENTRATION INDUCED BY CHENODEOXYCHOLIC ACID AND ITS CONJUGATES AT

NEUTRAL AND ACIDIC pH.*

Bile acid concentration (raM)

Neutral p H Acid p H

Bile acid 0.1 0.2 0.5 1.0 2.0 5.0 5.0

CDCA APD (mV) 3.1 9 .2 t 13.9t 17.2t 19.1t 31.5t 1.5:~

(1.0) (2.3) (2.7) (3.0) (4.0) (1.9) (0.9) APD (% baseline) 8.5 22.9 39.6 48.0 55.7 83.6 4.5 Baseline PD (mV) 36.3 40.1 35.1 35.8 34.3 37.7 33.4

(1.6) (1.9) (1.4) (2.8) (3.3) (2.0) (1.6) A[NA] (me/liter) 0.4 1.0 0.9 1.1 4.3~t 19.6~: 0.1

(0.7) (0.2) (0.2) (0.5) (1.6) (5.3) (0.1) Baseline [NA] 1.9 1.0 1.4 1.4 2.3 1.2 0.0

(0.3) (0.2) (0.2) (0.2) (1.0) (0.2) (0.0) G C D C A

APD (mV) - - § - - - - 2.9 13.5t 17.1t 1.2~; (0.6) (1.2) (1.6) (0.6)

APD (% baseline) - - § - - - - 8.0 35.2 50.0 4.2 Baseline PD (mV) - - - - - - 36.3 38.3 34.2 28.3

(1.2) (1.9) (1.5) (2.9) A[NA] (mg/liter) - - - - - - 0.4 1.2 1.1 - 0 . 1

(0.2) (0.3) (0.3) (0.1) Baseline [NA] - - - - - - 1.5 2.0 1.4 0.1

(0.3) (0.3) (0.3) (0.1) TCDCA

APD (mV) - - - - - - 1.7 7.3¶ 20.3t 18.0t (0.9) (1.5) (1.0) (2.3)

APD (% baseline) - - - - - - 4.7 17.9 59.9 54.4 Baseline PD (mV) - - - - - - 35.8 40.8 33.9 33.1

(2.5) (2.2) (1.5) (2.3) A[NA] (me/liter) - - - - - - 1.0 1.7 3.0¶ - 0 . 6

(0.3) (0.3) (0.5) (0.1) Baseline [NA] - - - - - - 1.5 1.1 0.6 0.6

(0.1) (0.2) (0.2) (0.1)

Saline control A PD = 1.5 m V (SEM 0.5); A[NA] = - 0 . 1 rag/liter (SEM 0.5)

*Transmucosal potential difference (APD; mV) and chamber fluid nucleic acid concent ra t ions (A[NA]; mg/liter): results given as means (SEM), six animals per group.

t P < 0.01 vs control . ¢.P < 0.05 vs same bile acid (5.0 mM) at neutral pH. §- - , exper iments not conducted at this bile acid concentra t ion . ~r, < 0.05 vs control .

Bile Acids: pH 5.5-7.0. Application of 5.0 mM CDCA to the gastric mucosa produced a rapid loss of PD from a mean value of -37 .7 -+ 2.0 mV to a minimum of - 6 . 2 + 0.9 mV. Thus, the mean APD for 5.0 mM CDCA was 31.5 -- 1.9 mV. Challenge with increasing concentrations of unconjugated CDCA (0.1-5.0 raM) produced dose-dependent increases in APD values (Figure 1). The lowest CDCA concentration that produced a APD significantly greater than the control value was 0.2 mM. The instillation of GCDCA or TCDCA produced smaller APD values than did CDCA and neither conjugate affected PD significantly at concentrations of less than 2.0 mM (Table 2). All bile acids, except GUDCA and TUDCA, produced dose-dependent

PD changes (Tables 2-5). Moreover, at all concentrations studied, CDCA (Table 2) and DCA (Table 3) produced larger PD changes than did correspond- ing concentrations of CA (Table 4) and UDCA (Ta- ble 5), although CDCA produced significant PD changes at lower concentrations than DCA. Fur- thermore, the unconjugated bile acids produced greater PD changes than their conjugates, which did not, however, differ significantly from each other. At 5.0 mM concentrations, the only bile acids that did not differ significantly from saline control were TCA and TUDCA (Figure 2).

Bile Acids: pH 2.5. The PD changes produced by TCDCA (5.0 mM) at pH 2.5 were comparable to those seen at neutral pH, whereas GCDCA and


G A S T R O T O X I C I T Y O F D U O D E N A L C O N T E N T S

TABLE 3. CHANGES IN TRANSMUCOSAL POTENTIAL DIFFERENCE AND CHAMBER FLUID NUCLEIC ACID CONCENTRATION INDUCED BY DEOXYCHOLIC ACID AND ITS CONJUGATES AT NEUTRAL AND

ACIDIC pH.*


Neutral pH Acid pH

Bile acid 0.2 0.5 1.0 2.0 5.0 10.0 5.0

D CA APD (mV) 3.6 16.0t 23.0t 23.0t 26.0t --:1: 2.8§

(1.0) (2.8) (3.0) (1.5) (3.3) (0.7) APD (% baseline) 10.8 44.4 66.7 78.0 88.1 1 8.4 Baseline PD (mV) 33.4 36.0 34.5 29.5 29.5 - - 33.4

(4.0) (2.5) (3.1) (1.7) (4.3) (1.5) A[NA] (rag/liter) 0.2 0.7 0.5 2.1 2 .8t - - 0.5

(0.2) (0.3) (0.4) (0.1) (0.3) (0.1) Baseline [NA] 0.8 1.1 0.8 0.2 0.1 - - 0.1

(0.2) (0.6) (0.3) (0.2) (0.1) - - (0.1) GDCA

APD (mV) - - 2.0 3.5 14.0t 21.8t 22.5t 0.3§ (1.5) (1.5) (1.8) (0.7) (2.4) (0.4)

&PD (% baseline) - - 5.7 9.7 38.7 61.1 70.5 0.9 Baseline PD (mV) - - 35.3 36.1 36.2 35.7 31.9 32.4

(2.1) (3.1) (2.0) (1.6) (2.9) (1.8) A[NA] (mg/liter) - - - 0 . 1 0.8 1.0 1.9 3.1¶ 0.2

(0.1) (0.5) (0.3) (0.5) (0.3) (0.1) Baseline [NA] - - 0.3 0.0 0.1 0.0 0.3 0.2

(0.1) (0.0) (0.0) (0.0) (0.1) (0.1) T D C A

APD (mV) - - 1.0 3.3 l l . 0 t 26.0t - - 11.6§¶ (0.4) (0.8) (1.9) (1.5) (2.0)

APD (% baseline) - - 2.5 10.0 35.5 " 64.7 - - 38.8 Baseline PD (mV) 1 40.0 33.0 31.0 40.2 - - 29.9

(2.4) (1.6) (1.8) (1.8) (2.6) A[NA] (mg/liter) - - 0.5 1.1 3.2¶ 2.8¶ - - 0.1

(0.2) (0.6) (0.6) (0.1) (0.2) Baseline [NA] - - 1.6 0.8 1.3 1.2 - - 0.4

(0.3) (0.5) (0.2) (0.1) (0.1)

Saline control:hPD = 1.5 mV (SEM 0.5); A[NA] = - 0 . 1 rag/liter (SEM 0.5)

*Transmucosal potential difference (APD; mV) and chamber fluid nucleic acid concentrations (A[NA]; rag/liter): results given as means (SEM), six animals per group.

t P < 0.01 vs control. ~- - , experiment not conducted at this bile acid concentration. §P < 0.05 vs same bile acid (5.0 mM) at neutral pH. ~P < 0.05 vs control.

CDCA had virtually no effect on PD at pH 2.5 (Figure 3). Similarly, the unconjugated and glycine- conjugated forms of CA, DCA, and UDCA produced no significant PD changes at pH 2.5 (Tables 2-5). With the exception of TDCA, all taurine conjugates produced similar PD changes at acidic and neutral pH, although, at pH 2.5, only TCDCA and TDCA produced greater PD changes than did saline.

Lvsolecithin: pH 7.4. At neutral pH, lysolecithin solutions produced significant, dose-dependent PD changes (Figure 4) at concentrations ranging from 0.20 to 2.00 mM (P < 0.05 vs phosphate buffer; Table 6).

Lysoleeithin: pH 2.5. Acidic lysolecithin solutions

produced markedly smaller PD changes than did neutral solutions (Figure 4); only 2.00 mM lysolecithin (Table 6) produced significant PD loss at pH 2.5 (P < 0.05 vs saline control).

Bile Acifl-Phospholipifl Mixtures: pH 6.5-7.5, and Trypsin: pH 7.4. The APD seen after lysolecithin 2.0 mM and CDCA 5.0 mM combined was not significantly different from that seen after lysolecithin 2.0 mM or CDCA 5.0 mM alone (Table 7). However, the addition of lecithin 5.0 mM to either CDCA 5.0 mM or lysolecithin 2.0 mM produced a lower APD than that seen after CDCA or lysolecithin alone (P < 0.05). The APD produced by trypsin did not differ from that produced by saline (Figure 5).

Human Duodenal Aspirate: pH 7.4. The APD pro-

Digestive Diseases and Sciences, Vol. 39, No. 2 (February 1994) 33 1

ARMSTRONG ET AL

TABLE 4. CHANGES IN TRANSMUCOSAL POTENTIAL DIFFERENCE AND CHAMBER FLUID NUCLEIC ACID CONCENTRATION INDUCED BY CHOLJC ACID AND ITS CONJUGATES AT NEUTRAL AND ACIDIC pH*

Bile acid 0.5



1.0 2.0 5.0 I0.0 5.0

CA APD (mV) 2.4 6.5

(0.9) (1.1) APD (% baseline) 5.7 19.2 Baseline PD (mV) 41.9 33.8

(2.1) (1.7) A[NA] (mg/liter) 0.2 0.0

(0.2) (0.5) Baseline [NA] 2.1 2.9

(0.2) (o.4) GCA

APD (mV) - -¶ - -

APD (% baseline) - - Baseline PD (mY) - -

A[NA] (mg/liter) - -

Baseline [NA]

TCA APD (mV)

APD (% baseline) - - Baseline PD (mV) - -

A[NA] (mg/liter) - -

Baseline [NA]

7.3t 18.9¢ 23.9~t 3.4§ (1.9) (1.6) (1.7) (2.0) 19.0 46.4 68.9 10.9 38.4 40.7 34.7 31.2 (3.0) (1.2) (1.2) (1.6)

-1.1 1.4 1.9 0.1 (0.2) (0.4) (0.4) (0.3) 2.1 1.4 1.4 0.5

(0.2) (0.3) (0.4) (0.1)

0.9 12.3:]: 19.1:1: 0.8§ (1.1) (0.7) (1.5) (1.4)

- - 2.6 31.9 49.9 2.3 - - 38.3 38.5 34.6 35.2

(1.2) (1.6) (1.8) (1.4) - - - 0 . 3 - 0 . 3 1.5 0.1

(0.4) (0.6) (0.4) (0.1) - - 2.1 1.7 1.8 0.2

(1.0) (0.4) (0.6) (0.1)

- - 2.1 5.4 10.6~ 4.4 (0.6) (0.9) (1.5) (0.9)

- - 7.3 15.8 26.1 13.0 - - 40.6 34.1 28.6 33.9

(2.1) (1.9) (2.4) (1.9) - - 0 . 1 - 1 . 2 0.9 0.1

(0.2) (0.3) (0.3) (0.2) - - 2.0 1.6 1.8 0.1

(0.2) (0.3) (0.5) (0.1)

Saline control:APD = 1.5 mV (SEM 0.5); A[NA] = -0.1 mg/liter (SEM 0.5)

*Transmucosal potential difference (APD; mV) and chamber fluid nucleic acid concentrations (A[NA]; rag/liter): results given as means (SEM), six animals per group.

tP < 0.05 vs control. < 0.01 vs control.

§P < 0.05 vs same bile acid (5.0 raM) at neutral pH. ¶Experiment not conducted at this bile acid concentration.

duced by unheated duodenal aspirate did not differ from that produced by the heated aspirate (Table 7).

Chamber Fluid Nucleic Acid Concentration

Bile Acids: pH 5.5-7.0. Saline challenge induced a A[NA] of 0.1 ___ 0.5 mg/liter. Most bile acids produced dose-dependent changes in chamber fluid [NA] but significant A[NA] values were seen only after challenge with DCA 5.0 mM (P < 0.05 vs saline control), TDCA 5.0 mM and 2.0 mM (P < 0.05), CDCA 5.0 mM and 2.0 mM (P < 0.01), UDCA 10.0 mM (P < 0.05) and TUDCA 5.0 mM, 10.0 mM, and 20.0 mM (P < 0.05) (Tables 2-5).

332

Bile Acids: pH 2.5. At pH 2.5, none of the 5 mM bile acid solutions produced significant increases in chamber fluid [NA] (Tables 2-5).

Lysolecithin: pH 7.4 and pH 2.5. Both neutral and acidic solutions of lysolecithin, at concentrations of 2.0 mM, 1.0 mM, and 0.50 mM, produced significantly greater changes in chamber fluid [NA] than did the respective control solutions (Table 6).

Bile Acid-Phospholipid Mixtures: pH 6.5-7.5, and Trypsin: pH 7.4. The combination of CDCA 5.0 mM and lysolecithin 2.0 mM produced a A[NA] value of 2.4 _+ 0.7 mg/liter. After challenge with CDCA 5.0 mM plus lecithin 5.0 mM, the A[NA] was 1.9 _+ 0.6 mg/liter and after lysolecithin 2.0 mM plus lecithin 2.0 mM, it was 1.1 __ 0.5 mg/liter. The A[NA] values produced by duodenal aspirate were not quantifi- able as the bile interfered with NA assay. Trypsin produced no change in the mean A[NA] (Table 7).

Relationship Between ~dPD and A[NA]

In general, larger changes in PD were associated with greater increases in chamber fluid nucleic acid concentrations after application of a bile acid or lysolecithin to the gastric mucosa (Table 8). How- ever, the relationship between A[NA] and APD var- ied considerably for the different unconjugated bile acids and for lysolecithin. The correlation between APD and A[NA] was poorer if the unconjugated and conjugated forms of each bile acid were grouped and, in the case of UDCA, it was poor for both the unconjugated and conjugated forms. As was the case for bile acids, when the data from the lysolecithin experiments at neutral and acid pH were combined, no correlation was seen between APD and A[NA]. Similarly, when data were combined from all experiments, no correlation was seen.

Chamber Fluid pH

When bile acids were studied at neutral pH, the mean chamber fluid pH during the baseline period (5.3 - 0.08) was lower than the pH of the saline instilled into the chamber, presumably because of continuing gastric acid secretion. The pH was higher (6.2 -.+ 0.09) during the challenge than during the baseline period due partly to a buffering effect of the bile acid solutions and partly to a presumed decrease in gastric acid secretion as the experiment progressed. In other studies, the chamber fluid pH was generally stable (Table 9) since the effect of gastric acid secretion was negligible in comparison to the [H ÷] in acidic (pH 2.5) solutions and gastric

Digestive Diseases and Sciences, Vol. 39, No. 2 (February 1994)


TABLE 5. CHANGES IN TRANSMUCOSAL POTENTIAL DIFFERENCE AND CHAMBER FLUID NUCLEIC ACID CONCENTRATION INDUCED BY URSODEOXYCHOLIC ACID AND ITS CONJUGATES AT

NEUTRAL AND ACIDIC pH*

Bile acid concentration (mM)


Bile acid 0.5 1.0 2.0 5.0 10.0 20.0 5.0

U D C A APD (mV) 3.7 6.5 11.2t 16.3t 17.2t - - ~ 0.8§

(1.7) (1.7) (2.4) (2.1) (2.3) (0.3) APD (% baseline) 11.3 18.4 28.5 51.1 50.1 - - 2.4 Baseline PD (mV) 32.6 35.4 39.3 31.9 34.3 - - 33.8

(0.9) (1.9) (1.8) (2.2) (1.7) (2.0) A[NA] (mg/liter) 1.1 1.1 0.2 1.5 8.9t - - 0.0

(0.3) (0.7) (0.6) (0.4) (2.3) (0.0) Baseline [NA] 1.9 2.7 2.1 0.7 1.5 - - 0.0

(0.3) (0.1) (0.5) (0.2) (0.5) (0.0) G U D C A

ApD (mV) -- - - 3.0 9.5t 7.7 - - 1.1§ (1.8) (1.8) (1.9) (0.8)

APD (% baseline) - - - - 7.9 23.4 23.3 - - 3.5 Baseline PD (mV) - - - - 37.9 40.6 33.0 - - 31.0

(2.3) (2.4) (2.5) (1.1) A[NA] (mg/liter) - - - - 2.2 1.6 1.5 - - -0 .1

(0.7) (0.3) (0.6) (0.1) Baseline [NA] - - - - 0.2 0.1 0.3 - - 0.4

(0.2) (0.1) (0.2) (0.1) T U D C A

APD (mV) - - - - - - 3.4 5.8 4.8 1.3 (1.3) (1.0) (0.5) (0.5)

ApD (% baseline) - - - - - - 8.6 " 14.1 12.0 3.6 Baseline PD (mV) - - - - - - 39.7 41.2 40.0 36.5

(2.4) (1.9) (1.5) (2.1) A[NA] (mg/liter) - - - - - - 2.9¶ 2.9¶ 6.6t - 0 . 3

(0.3) (0.8) (0.6) (0.1) Baseline [NA] - - - - - - 0.4 1.7 1.4 0.4

(0.3) (0.8) (0.3) (0.1)

- 0 . 1 Saline control:APD = 1.5 mV (SEM 0.5); A[NA] = rag/liter (SEM 0.5)

*Transmucosal potential differences (APD; mV) and chamber fluid nucleic acid concentrat ions (A[NA]; mg/liter): results given as means (SEM), six animals per group.

t P < 0.01 vs control. $ - - , experiment not conducted at this bile acid concentrat ion. §P < 0.05 vs same bile acid (5.0 mM) at neutral pH.

< 0.05 vs control.

acid was buffered by the phosphate solutions used for the lysolecithin studies at pH 7.4.

Histology The gastric mucosa revealed no gross changes ei-

ther during or after the study, while H&E-stained histological sections, taken 50 min after challenge with DCA (14), TDCA, or GDCA, showed only minor inflammatory changes and focal edema at the periph- ery of the clamped mucosa, probably due to trauma from the chamber. Alcian blue-PAS stained sections showed quite marked loss of mucus cells after 5.0 mM DCA challenge but not at lower DCA concentrations. As with the H&E-stained sections, the magnitude of the changes was not dose-dependent at lower bile acid

Digestive Diseases and Sciences, Fol. 39, No. 2 (February 1994)

concentrations. In all other studies, tissue excised 10 min after the challenge period showed only minor inflammatory changes. Patchy mucosal edema was frequent, as was a granulocytic mucosal infiltrate, which extended, in a few sections, into the submu- cosa. In general, the inflammatory changes were most marked after unconjugated bile acid challenge at neutral pH, but the changes were not consistently dose- dependent.

DISCUSSION

This is the only systematic dose-response study of the acute gastrotoxic effects of individual duodenal juice constituents in a single model. At neutral

333

A R M S T R O N G E T A L

TABLE 6. Cr~NoES IN TRANSMUCOSAL POTENTIAL DIFFERENCE AND CHAMBER FLUID NUCLEIC ACID CONCENTRATION

PRODUCED BY LYSOLECITHIN AT NEUTRAL AND ACIDIC pH*

Lysolecithin concentration (raM)

0.05 0.10 0.20 0.50 1.00 2.00

Neutral pH (7.4) APD (mV) 4.0 8.2 17.1t 19.5, 21.8¢ 28.8*

(0.8) (1.5) (2.6) (2.4) (2.3) (2.2) APD (% baseline) 7.6 19.0 34.3 43.9 48.6 61.8 Baseline PD (mV) 52.5 43.2 49.8 44.4 44.9 46.6

(3.5) (1.0) (3.3) (2.5) (2.2) (3.5) A[NA] (mg/liter) -1 .4 0.8 1.3 7.5* 4.1, 5.5*

(0.7) (0.3) (0.5) (0.8) (0.8) (1.0) Baseline [NA] 2.7 2.4 2.4 2.4 2.1 2.4

(0.7) (0.2) (0.5) (0.4) (0.3) (0.5) Acidic pH (2.5)

APD (mV) --§ -- -- 0.7¶ 6.8¶ 9.2%¶

(0.9) (1.2) (1.2) APD (% baseline) -- -- -- 2.7 20.9 32.6 Baseline PD (mV) -- -- -- 25.7 32.6 28.2

(2.4) (1.6) (1.1) A[NA] (mg/liter) -- -- -- 8.6* 5.7:1: 21.7,¶

(0.7) (1.0) (2.2) Baseline [NA] - - - - - - 0.1 0.0 0.2

(0.1) (0.0) (0.1)

Phosphate buffer control: APD = +0.1 mV (SEM 0.5); A[NA] = -0.6 rag0 (SEM 0.3)

?

*Transmucosal potential difference (APD; mV) and chamber fluid nucleic acid concentrations (A[NA]; mg/liter): results given as means (SEM), six animals per group.

t P < 0.05 vs control. Ca t' < 0.01 vs control. §--, experiment not conducted at this lysolecithin concentration. ¶P < 0.05 vs same lysolecithin concentration at neutral pH.

pH, the unconjugated species of the major human bile acids, including UDCA, used for treating gallstones (18, 19) or dyspepsia (20-23), as well as lysolecithin, all produced dose-dependent PD and [NA] changes. Conjugated bile acids produced smaller changes than did their unconjugated species or equimolar concentrations of lysolecithin. Tryp- sin alone did not affect PD or [NA] while heated and unhea ted duodena l aspirate p roduced similar changes, indicating that heat-labile pancreatic enzymes are not acutely gastrotoxic. The present study refutes the hypothesis that an acidic environment of pH 2.5 increases the gastrotoxicity of bile acids and lysolecithin, but it does not refute the hypothesis that lecithin reduces the toxic effects of bile acids and lysolecithin.

The present study was designed to examine the acute effects of duodenal constituents on the gastric mucosa and, although the mucosa was examined histologically after each experiment, it was sus- pected that a 10-min challenge might not produce significant histologically recognizable changes. For

TABLE 7. CHANGES IN TRANSMUCOSAL POTENTIAL DIFFERENCE AND CHAMBER FLUID NUCLEIC ACID CONCENTRATION

PRODUCED BY BILE ACID/PHOSPHOLIPID MIXTURES, TRYPSIN, OR DUODENAL ASPIRATE*

APD Baseline A/NA] Baseline (m 10 PD (rng/liter) [NA]

CDCA 5.0 30.3 41.9 2.4 mM/lysolecithin 2.0 mM (1.8) (1.7) (0.7)

CDCA 5.0 mM/lecithin 22.6t 36.0 1.95 5.0 mM (3.1) (2.1) (0.6)

Lysolecithin 2.0 mM/ 13.4t 35.6 1.1, lecithin 2.0 mM (2.4) (2.0) (0.5)

Trypsin 1.6 41.7 0.0 (1.7) (2.3) (0.1)

Duodenal aspirate Unheated 15.3 33.3 --§

(0.9) (1.5) Heated 13.4 26.9 - -

(1.7) (3.4)

1.1

(0.5) 0.7

(0.3) 0.9

(0.3)

0.3

(0.2)

*Transmucosal potential difference (APD; mV) and chamber fluid nucleic acid concentration (A[NA]; mg/liter): results given as means (SEM), six animals per group.

t P < 0.05 vs changes produced by CDCA or lysolecithin in the absence of lecithin.

*P < 0.01 vs changes produced by CDCA or lysolecithin in the absence of lecithin).

§--, A[NA] produced by the duodenal aspirate was not quanti- fiable.

this reason, loss of transmucosal potential difference (14, 15) was used as a sensitive indicator of early mucosal damage. However, because a decrease in transmucosal PD may reflect processes other than direct mucosal or epithelial cell damage, a marker of mucosal cell damage or desquama-

E

40

30"

20

10'

0.0 0.1 0.2 0.5 1.0 2.0

40 * l j

~ - 30 ¢ l l l l S ¢ 1 1 1 1 1 ~ - 20

5.0 [CDCA] mmol I-1

Fig 1. Mean changes m gastric mucosal potential difference (APD; mV) produced, between the baseline and challenge periods, by the application of chenodeoxycholic acid (CDCA) in concentrations ranging from 0.0 mM (saline control) to 5.0 mM, at neutral pH. (Vertical bars indicate SEM, N = 6, **P < 0.01 vs saline control.)


G A S T R O T O X I C I T Y O F D U O D E N A L C O N T E N T S

40 "40

,-, 30 30

,w,

o °o iiljl 20 20

10 10

0~I . . . . . . . . . . . . 0

o~

Fig 2. Mean changes in gastric mucosal potential difference (APD; mV) produced by the application of 5.0 mM solutions of all bile acids at neutral pH, in comparison with the effect of saline control. (Vertical bars indicate SEM, N -- 6.)

tion--loss of nucleic acid into the luminal fluid -was used to provide additional evidence of cell damage. Notwithstanding the fact that measured [NA] in the chamber fluid may underestimate the extent of mucosal damage, since desquamated cells and their contents may be trapped temporarily by overlying mucus in the mucoid cap (24, 25), there was generally a good correlation between the change in PD and the changes in [NA]. The vari- ability of the regression lines describing the relationship between APD and A[NA] for the different bile acids and lysolecithin and the poorer correlation observed when the results from different experimental groups were combined suggest that the dif-

40' 40

[ ] Neutral • Acid

30' 30

g 20' 20

10' 10

0" 0 CDCA GCDCA TCDCA

Fig 3. Mean changes in gastric mucosal potential difference (APD; mV) produced by the application of 5.0 mM solutions of CDCA, GCDCA, and TCDCA at either neutral or acidic pH. (Vertical bars indicate SEM, N = 6, *P < 0.05 vs equivalent solution at neutral pH.)

40"

30

20

I0

[ ] Neutral • Acid

_.n 0.00 0.05

40

30

T 20

÷

+ i I0 . ! I . . 0

0.10 0.20 0.50 1.00 2.00 [Lysolecithin] mmol I-1

Fig 4. Mean changes in gastric mucosal potential difference (APD; mV) produced by the application of lysolecithin in concentrations ranging from 0.00 mM (phosphate buffer control) to 2.00 mM at neutral pH and from 0.50 mM to 2.00 mM at acidic pH. (Vertical bars indicate SEM, N = 6. *P < 0.05, **P < 0.01 vs control at same pH; ÷P < 0.05 vs equivalent solution at neutral pH.)

ferent bile acids, their conjugates, and lysolecithin may produce PD changes and cell damage or des- quamation by different mechanisms, or that they may have different effects on other processes such as mucus secretion, bicarbonate secretion, or mucosal blood flow.

40 40

>.

30 30

20 20

10 10

0 0

,d < d

rj

Fig 5. Mean changes in gastric mucosal potential difference (APD; mV) produced by the application of CDCA (5.0 raM), CDCA (5.0 raM) plus lysolecithin (LL: 2.0 mM), LL (2.0 raM), CDCA (5.0 mM) plus lecithin (L: 5.0 raM), LL (2.0 raM) plus L (2.0 raM), and trypsin (18,600 BAEE units/liter) at neutral pH. (Vertical bars indicate SEM, N = 6. *P < 0.05 vs CDCA alone; **P < 0.01 vs LL alone).

Digestive Diseases and Sciences, Vol. 39, No. 2 (February 1994) 335

A R M S T R O N G ET A L

TABLE 8. RELATIONSHIP BETWEEN APD AND A[NA] FOLLOWING CHALLENGE WITH DIFFERENT BILE ACIDS AND LYSOLECITHIN

AINA] as a fimction of APD r* P

CDCA CDCA, GCDCA, and TCDCA DCA

DCA, GDCA, and TDCA CA CA, GCA, and TCA UDCA UDCA, GUDCA, and TUDCA Lysoleeithin

pH 7.4 pH 7.4 and 2.5

A[NA] = 0.57 x APD + 4.34 0.70 <0.0001 A[NA] = 0.42 x APD + 2.46 0.63 <0.0001 A[NA] = 0.059 x APD + 0.52 <0.001 0.075 A[NA] = 0.058 x APD + 0.49 0.47 <0.0001 A[NA] = 0.093 x APD - 0.62 0.61 <0.0004 A[NA] = 0.084 x APD - 0.66 0.53 <0.0001 A[NA] = 0.18 x APD + 0.69 0.31 <0.10 A[NA] = 0.089 x APD + 2.2 0.17 <0.18

A[NA] = 0.24 x APD - 1.1 0.70 <0.0001 A[NA] = 0.00001 x APD + 0.00 < 1.00 4.9

*r: correlation coefficient.

Bile Acids. Di-a-hydroxy bile acids are more damaging than the trihydroxy species to jejunal (26) and gastric (27) mucosae, TCDCA is more toxic than TUDCA to both esophageal and gastric mucosae (28), and bile acid mixtures with different composi- tions have differing toxicities when applied to gastric mucosa (29, 30). Our findings at neutral pH suggest, as do previously reported bile acid-induced changes in intestinal water and electrolyte transport (26, 31), that the mucosal toxicity of bile acids is related to their hydrophobicity--as judged by their octanol-water partition coefficients (32)---or to their hydrophobicity-hydrophilicity balance (33). Unex- pectedly, DCA was less toxic than CDCA, perhaps because it forms a gel, which results in lower mo- nomeric concentrations and, mole for mole, a lower

TABLE 9. CHAMBER FLUID pH DURING BASELINE AND Cl-I/t H ~NGE PERIODS

Chamber fluid p H

N* Baseline Challenge

Bile acids Neutral pH 294 5.28 (0.08) 6.19 (0.09) Acidic pH 72 2.40 (0.04) 2.54 (0.03)

Lysolecithin Neutral pH 48 7.05 (0.05) 7.02 (0.05) Acidic pH 18 2.58 (0.04) 2.50 (0.02)

CDCA 5.0 mM/lysolecithin 2.0 mM 6 5.42 (0.36) 6.41 (0.21)

CDCA 5.0 mM/lecithin 5.0 mM 6 5.40 (0.44) 6.87 (0.24)

Lysolecithin 2.0 mM/ lecithin 2.0 mM 6 4.81 (0.37) 5.89 (0.22)

Trypsin 6 5.96 (0.22) 6.07 (0.20) Duodenal aspirate

Heated 6 5.98 (0.44) 7.51 (0.05) Unheated 6 5.36 (0.61) 7.66 (0.10)

*N = number of animals per group. Chamber fluid pH: means _ SEM.

apparent toxicity (M.C. Carey, personal communi- cation). It is not clear why TUDCA produced large [NA] changes despite low APD values; future studies might test the hypothesis that TUDCA produces less mucus release and a smaller mucoid cap (24, 25) than other bile acids, thus permitting the loss of NA into chamber fluid.

The present experiments were designed to study the initial effect of bile acids at neutral and acidic pH but not the subsequent, longer-term effects of luminal acidity following bile acid challenge. On acidification to pH 2.5, all unconjugated (pK~ - 5.0) (34) and glycine-conjugated (pK~ - 4.0) bile acids rapidly formed dense white precipitates, whereas the taurine conjugates (pK~ - 1-2) remained in solution. Under these conditions, only TCDCA and TDCA produced PD changes significantly different from saline. At low pH, the greatly reduced acute toxicity of all the unconjugated and glycine- conjugated species was due, presumably, to very low concentrations of soluble bile acid in contact with the mucosal surface. Previous reports that bile gastrotoxicity is greater at low than at neutral pH (! 0, 11, 35-37) are consistent with the use of bile acid solutions containing high proportions of taurine conjugates. Our observation that taurine conjugates are no more toxic at acidic pH than at neutral pH may be explained by our use of test solutions of pH 2.5 (3.16 mM H+); other studies have used test solutions o fpH _< 1.0 (11) resulting in a 30-fold greater concentration of H + ions (100 mM), sufficient perhaps to overwhelm a mucus- bicarbonate barrier, augmented by exfoliated cells and cell debris, which would have been adequate to protect the mucosa following exposure to a "barrier-breaker" at pH 2.5. Although TDCA remained

336 Digestive Diseases and Sciences, VoL 39, No. 2 (February 1994)


soluble at acidic pH and its concentrations were comparable in the acidic and neutral solutions, it produced a smaller PD change at pH 2.5 than at pH 7.0, suggesting that the protonated form of TDCA, present at low pH, is less damaging despite its solubility in an acidic environment.

Lysolecithin. Lysolecithin, produced by the ac- tion of phospholipase on biliary lecithin, is known to damage the gastric mucosa (9, 13, 38, 39). In the present study, lysolecithin (2.00 mM, pH 7.4) produced PD and [NA] changes comparable to those seen after challenge with 5.0 mM DCA and CDCA. Furthermore, it led to PD changes at concentrations as low as 0.10 to 0.20 mM--concentrations similar to those found in human gastric aspirates (38, 40, 41). However, we found its gastrotoxicity to be reduced at acidic pH, unlike previous authors, who reported that it was comparably toxic at acidic and neutral pH (39), perhaps because they used highly acidic (150 mM HCI: pH 0.82) lysolecithin solutions (39), resulting in damage caused by back-diffusion of H + ions, rather than by lysolecithin itself.

Bile Acids plus Phospholipids. Refluxed duodenal contents contain approximately equal molar amounts of lysolecithin and its unhydrolyzed pre- cursor, lecithin (41). Lecithin has been reported to decrease lysolecithin-induced gastric mucosal damage (39) and bile acid-induced jejunal secretion (42) and, in the present study, it reduced significantly the toxic effects of both CDCA (5.0 mM) and lysolecithin (2.0 raM), possibly by "trapping" the injurious agents in micelles such that they could not affect the mucosa.

Duodenal Contents and Trypsin. Our studies of trypsin and human duodenal aspirate, heated to abolish enzyme activity, suggest that trypsin, lipase, and amylase are not responsible for the acute gastrotoxicity of duodenal contents. Despite its high total bile acid concentration, the duodenal aspirate produced a APD that was less than half of that produced by DCA or CDCA alone, perhaps because of lecithin and other lipids in the duodenal aspirate or perhaps because of the low concentrations of unconjugated bile acids.

Relevance of Present Studies to Toxicity of Re. fluxed Duodenal Contents in Man. The present study's aim was a direct comparison of the acute toxicities of the major human bile acids and other duodenal juice constituents in a single experimental model. Since the results are intended to allow an assessment of the potential gastrotoxicity of refluxed duodenal contents in man, murine bile acids

were not investigated, although unconjugated human bile acids were tested because they have been found, albeit at low concentrations, in the stomach and both CDCA and UDCA have been adminis- tered therapeutically. The results indicate that bile acids and lysolecithin damage rat gastric mucosa at concentrations found commonly in the stomach of patients with duodenogastric reflux. In particular, an acute challenge with the conjugates of DCA or CDCA [which constitute 60-70% of the bile acids refluxed into the stomach (21)] produces damage at a concentration (2.0 mM) much lower than that found normally in the human duodenum and tran- siently in the stomach. Although glycine-conjugated bile acids are not injurious in an acid environment, they may still cause damage when the gastric luminal pH rises, as happens at night (43) or as a result of reflux of alkaline duodenal contents into the stomach.

The results of this study may explain, in part, why UDCA ameliorates dyspeptic symptoms in gallstone and postgastrectomy patients (20-23). Oral UDCA may act by increasing the proportion of noninjurious UDCA and its conjugates in the bile acid pool while simultaneously decreasing the proportion of potentially toxic di-ot-hydroxy bile acids and increasing the proportion of acid-insoluble glycine conjugates at the expense of taurine-conjugated CDCA and DCA. It may also be that TUDCA prevents CDCA-induced damage to gastric cells (44), although the necessary intragastric TUDCA concentrations (2.5-10.0 mM) would be difficult to achieve by oral administration of UDCA.

The finding that the PD and [NA] changes produced by bile acids or lysolecithin at pH 2.5 were either smaller than, or comparable to, those produced at neutral pH does not refute the hypothesis that detergents can produce gastric mucosal damage by permitting the back diffusion of H ÷ from the lumen into the mucosa (7, 9). The present study does, however, indicate that this is not the only mechanism whereby gastric mucosal damage may arise. At neutral pH, detergents alone can produce marked changes in transmucosal PD and chamber fluid [NA], indicative of mucosal injury; this is consistent with the clinical finding of bile gastritis in the postoperative stomach---despite greatly dimin- ished acid secretion. Bile acid-induced mucosal change may, subsequently, permit the back- diffusion of H + ions into the mucosa, resulting in further damage, but this will occur only if luminal H ÷ ion concentrations are sufficiently high. Lumi-

Digestive n~eases a,,a Sciences, VoW. 39, No. 2 ~Feb~a,y 2994) 337

ARMSTRONG ET AL

nal acid does not necessarily exacerbate the initial gastric damage produced by bile acids, particularly if acid leads to protonation and, consequently, to insolubility of the bile acid such that it does not damage the mucosa. This does not mean that higher H + concentrations (pH 1.0) will not produce significant damage due to back diffusion even if the initial detergent-induced epithelial impairment has been reduced by the presence of acid. Under these cir- cumstances, damage will be greatest if a relatively acid-soluble detergent, such as taurocholic acid, has been used.

Potent inhibitors of acid secretion may enhance the gastrotoxicity of refluxed duodenal contents by providing an alkaline environment in which lysolecithin and bile acids are more toxic. In contrast, dietary lecithin or cholesterol may form mixed micelles, so that the constituent bile acids and lysolecithin cannot damage the mucosa. Thus, the ability of refluxed duodenal contents to damage the gastric mucosa cannot be predicted from lysolecithin and total bile acid concentrations alone.

Ir~ conclusion, these studies suggest strongly that refluxed duodenal contents may cause gastric mucosal damage in man and that the major determinants of their acute gastrotoxicity include gastric luminal pH, the presence of phospholipids (hydro- lyzed or unhydrolyzed), and the relative proportions of different bile acids, lecithin and lysolecithin but not the presence of trypsin or lipase.

ACKNOWLEDGMENTS

The authors thank Messrs. Michael Farrell, Mehran Maghsoudloo, Bob Francis, Bob Burton, Ken Applebee, and colleagues for technical assistance and Mrs. Ann Hollington for secretarial assistance.

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gastric mucosal toxicity of duodenal juice constituents in the rat

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