Gut, 1990, 31, 11-16

Bismuth subsalicylate reduces peptic injury of theoesophagus in rabbits

H P Tay, R C Chaparala, J W Harmon, J Huesken, N Saini, F Z Hakki, E J Schweitzer

AbstractBismuth subsalicylate was tested in an in vivoperfused rabbit model of oesophagitis for itsability to prevent the mucosal injury caused bypepsin. Treatment efficacy was assessedunder both a treatment-before-injury protocoland a treatment-after-injury protocol.Oesophageal mucosal barrier function wasevaluated by measuring flux rates of H+, K+,and glucose. The degree of oesophagitis wasdetermined by gross and microscopic exami-nation of the mucosa by several independentobservers. Results showed that under bothtreatment protocols, bismuth subsalicylatesignificantly reduced the pepsin induced dis-ruption of the mucosal barrier, as weli as themorphologic changes. Bismuth subsalicylatewhen given after exposure to pepsin was alsofound to protect against the morphologicinjury in a dose dependent manner. Experi-ments in vitro suggested that bismuth sub-salicylate inhibits the proteolytic action ofpepsin by interacting with pepsin, rather thanwith the pepsin substrate. We conclude thatbismuth subsalicylate can protect the oeso-phageal mucosa against peptic injury, prob-ably through inactivation of pepsin.

models ofoesophagitis HCl inflicts little injury tothe intact oesophageal epithelium, except at veryhigh concentrations and after prolonged periodsof exposure."4 16 The bile acids typically increasethe oesophageal mucosal permeability, but causeonly a relatively modest degree of accompanyingmorphologic change.' In contrast, pepsin notonly causes a significant increase in the perme-ability of the oesophageal mucosa, but alsocauses a frankly haemorrhagic, severe morpho-logic injury.36 The present study was thereforecarried out with pepsin as the injurious agent, asit appears to be the most damaging of thecomponents of an acidic gastroesophagealrefluxate fluid.We performed in vivo experiments to deter-

mine whether bismuth subsalicylate hadprotective effects against the pepsin inducedoesophageal injury. Bismuth subsalicylate wastested in a treatment-before-injury and atreatment-after-injury protocol. After bismuthwas found to be protective in vivo, the antipepsinactivity of bismuth subsalicylate was tested invitro to attempt to clarify its mechanism ofaction.

Methods

Departments of Surgeryand Pathology,Washington VA MedicalCenter and the Schools ofMedicine, GeorgeWashington Universityand GeorgetownUniversity, Washington,DC, USAH P TayR C ChaparalaJW HarmonJ HueskenN SainiF Z HakkiE J SchweitzerCorrespondence to: JohnWHarmon, Chief, SurgicalService, Washington VAMedical Center, 50 IrvingStreet NW, Washington, DC20422, USA.Accepted for publication10 May 1989

The oesophageal mucosal injury found inpatients with reflux oesophagitis occurs as aresult of abnormal exposure of the mucosa to thecorrosive gastric contents. We have used an invivo perfused rabbit model of oesophagitis toelucidate the mechanisms of oesophagealmucosal injury' and to test agents which mightbe useful in the prevention or treatment of suchinjury.The present study was designed to test the

efficacy of bismuth subsalicylate in preventingexperimental oesophagitis. Bismuth subsali-cylate has been used for many years as an over-the-counter drug for the relief of heartburn,which is often associated with oesophagitis.Bismuth compounds have been shown to reduceexperimental gastric mucosal injury in the rat,7'8and they have been used successfully to treatpeptic ulcer disease in man.9 '° Ulcer healing withbismuth compounds has been shown to besimilar to cimetidine," '3 even though it lacks asignificant acid neutralising capacity. Becausethe mucosa of the oesophagus and stomach areboth exposed to the same caustic gastric con-tents, we investigated whether bismuth isefficacious against mucosal injury of theoesophagus.Some of the potentially injurious components

of the oesophageal refluxate fluid include hydro-chloric acid (HCI), proteolytic enzymes such aspepsin, and the bile acids.3 In experimental

IN VIVO EXPERIMENTS

Oesophageal perfusionAn in vivo perfused rabbit model of oesophagitis,adapted from Chung'2 was used as described.' 36In brief, New Zealand white rabbits weighing3-5 kg were lightly anaesthetised with an intra-muscular injection of ketamine/xylazine. Theoesophagus was cannulated in the neck atthe pharyngoesophageal junction and in theabdomen at the gastroesophageal junction withplastic tubing (id 0-317 mm). The oesophaguswas then perfused with 45 ml of various testsolutions at 10 ml/min using a peristalic pump(Harvard Apparatus Co, Millis, MA) and arecirculating system. The perfusate solution wasstirred mechanically (Haake, Berlin, Germany),and a thermoregulator (Haake) kept the temp-erature of the perfusate at 37°C. A pH stat/autoburette system (Radiometer, Cophenhagen,Denmark) containing 0 4 N HC1 constantlymaintained the pH of the perfusate at 2.

Each experiment consisted of a 30 minute'exposure period' in which the perfusate solutioncontained pepsin at pH 2. This was followed by a40 minute 'flux period' in which the perfusatesolution contained no pepsin, but was used formeasuring transmucosal flux rates of H+, K+,and glucose. In the treatment-before-injuryprotocol, for 10 minutes before the initiation ofthe 30 minute exposure period, the oesophagus

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was perfused with either saline 145 mmol/l(control group) or saline plus bismuth subsali-cylate (The Procter and Gamble Co, Cincinnati,OH) 30 mg/ml (study group), bopth at pH 2. Atthe start of the exposure period, porcine pepsin(Sigma Chemical Co, St Louis, Missouri) wasadded to the perfusate of both the control andstudy groups to obtain a final perfusate concen-tration of 1 mg/ml. The pH of the perfusate wasmaintained at 2-0 with HC1. At the end of thisexposure period, the perfusate was discardedand the entire system, including the oesophagus,was irrigated with 100 ml isotonic saline inpreparation for the subsequent flux period.The exposure period in the treatment-after-

injury protocol was similar to that in thetreatment-before-injury protocol, except thatthe bismuth subsalicylate was added after aninitial oesophageal exposure to pepsin. The 30minute exposure period of both the control andstudy groups began with perfusion of the oeso-phagus with a solution containing 1 mg/ml ofporcine pepsin at pH 2. Ten minutes after theinitiation of the exposure period, an amount ofbismuth subsalicylate was added to the perfusatesolution of the study groups to obtain a concen-tration of either 15 mg/ml, 30 mg/ml, or 60 mg/ml of bismuth subsalicylate. Concomitantly, aquantity of pepsin and HCI was added to theperfusate to maintain the pepsin concentration at1 mg/ml and the pH at 2. At the end of theexposure period, the perfusate was discardedand preparation was made for the flux period, asin the treatment-before-injury protocol.The methods used during the flux period were

identical for both the treatment-before-injuryand the treatment-after-injury protocols. Theflux solution contained 1-5 g/l polyethyleneglycol (PEG, Fisher Scientific Co, Fair Lawn,NJ) 100 mCi/l 3H-PEG (New England Nuclear,Boston, Mass), and 10 mM HCl at pH 2. Theosmolality was brought to 280 mosmol/l withmannitol. A 4 ml aliquot of the flux solution wastaken from the reservoir at the beginning andend of the 40 minutes flux period for lateranalyses of K+ and glucose. A pH of 2 wasconstantly maintained in the perfusate solutionwith the pH stat/autoburette apparatus. Aftercompletion of the flux period, the animal waskilled with an intracardiac bolus of pentobarbitaland the oesophagus was excised.

Calculation offluxesOesophageal mucosal permeability to ions andsmall molecules was determined by calculatingH+, K+, and glucose flux rates. Hydrogen ionflux out of the lumen was given by the amount ofHCI delivered by the pH stat/autoburetteapparatus to maintain the pH ofthe solution at 2.Concentrations of K+ were assayed by ion selec-tive electrode potentials (Beckman System E4A,Beckman Instruments, Fullerton, CA), and con-centrations of glucose were assayed by thehexokinase reaction (Centrifichem System 500,Union Carbide Corp, Rye, NY). Fluxes werecalculated using the following formula:

Net flux=(CF-CI)XVIwhere VI is the initial volume of the perfusate atthe beginning of the flux period, CI is the initialconcentration of K+ or glucose in the perfusatesolution, and CF is the final concentration.3H-PEG was used as an impermeable volumemarker during the initial experiments. It was notused in the later experiments because it wasfound to be unnecessary, as have others whohave used this animal model have reported.'4Polyethylene glycol was deemed unnecessarywhen it was found in the initial experimentsthat the volume and PEG recovery rates werebetween 96-99%, and the volume flux wasrelatively low (less than 0 5 ml per experiment).

Determination ofoesophagitis indexImmediately after removal, the oesophagus wasfixed in 10% formalin. A photograph of the fixedspecimen was taken for later grading of thedegree of gross morphologic injury. Histologicslides were prepared from sections taken fromthe proximal, central, and distal portions of theesophagus and stained with haematoxylin andeosin. Photographs and slides of all specimenswere scored for the severity of oesophagitis byseveral observers unaware of the treatmentsgiven, using a system previously described.36 Inbrief, the gross oesophagitis index was deter-mined by the following criteria: 1 =normalappearance; 2=erythema or other abnormalappearance, but no haemorrhage; 3=non-confluent mucosal haemorrhage; 4=confluentintramural haemorrhages (Fig 1). The micro-scopic oesophagitis index was scored by these

Figure 1: Grades ofgrossoesophagitis. Numberscorrespond to the grossoesophagitis index (GOI).

*I., :-j~~~~~~~~~~~~~~~~~~~. .. .§ ... ..... E~~~~~~~~~~~~~~~~~~~~~~~~~ ... ... ... ...

? v ..'~~~~~~~~~~.. ..4I~~~~~~~~~~~~~~~~~~~~~~

3,~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~7

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Figure 2: Grades ofmicroscopic oesophagitis.Numbers correspond to themicroscopic oesophagitisindex (MOI).

. w~~~.

*w

*.*:

-J "'' *b

:VW,- *.V

criteria: 1 =normal oesophagus; 2= submucosaloedema or separation of epithelial layers; 3=focal areas of intramural haemorrhage or partialepithelial loss; 4=large areas of haemorrhage or

complete epithelial desquamation (Fig 2).

In vitro experimentsA series of in vitro experiments were performedto determine whether bismuth subsalicylatecould inhibit the proteolytic action of pepsin on

haemoglobin, and if so, whether this inhibitionwas caused by the interaction of bismuth sub-salicylate with pepsin or its substrate. Thebiochemical principle underlying this experi-ment was that if an enzyme inhibitor acts bybinding to the substrate molecule, then theinhibition observed at a low substrate concentra-tion can be reduced by providing more substrateto the reaction. Alternatively, if the inhibition iscaused by the inhibitor binding to the enzyme,then the inhibition will not be reduced byincreasing the concentration of substrate.'7 Inthe experiment, 50 mg/ml pepsin was dissolvedin one ofthe following solutions: (a) HCI atpH 2,or (b) HC1 plus 30 mg/ml bismuth subsalicylateat pH 2. The peptic activity of these solutionswas then assayed by spectrophotometricallymeasuring the amount of tyrosine released from2 5 ml of 1, 2, 3, or 4 g% solutions of bovinehaemoglobin substrate.'8

oesophagitis grades) were compared for signifi-cant differences (p<0 05) with the Mann-Whitney test.

Results

IN VIVO EXPERIMENTS

Treatment-before-injury protocolWhen administered before exposure of the oeso-phageal mucosa to pepsin, bismuth subsalicylatereduced both the permeability changes and themorphologic injury caused by pepsin. As shownin Table I, significantly lower transmucosal fluxrates of H+, K+, and glucose occurred in thegroup treated with bismuth subsalicylate than inthe untreated group (p<005). Treatment withbismuth subsalicylate before pepsin exposurealso significantly reduced the morphologicinjury, as reflected by both the gross and micro-scopic oesophagitis indices. The gross oeso-

phagitis index ofthe untreated control group was3-2 (0-2), compared with the group treated withbismuth subsalicylate, which was graded as 2-0(0-3) (p<001, Fig 3). Similarly, the microscopicesophagitis index was reduced from 3-1 (0 2) inthe untreated group to 2-1 (02) in the grouptreated with bismuth subsalicylate (p<0 001,Fig 3).

Statistical analysisEach experimental group contained between fiveand nine animals. All results are expressed as themean±one standard error of the mean (SE). Thedifferences between means of groups of con-tinuous data (H+, K+, and glucose flux rates)were assessed for statistical significance(p<005) using the Student's unpaired t test.Non-parametric data (gross and microscopic

Treatment-after-injury protocolThe efficacy ofbismuth subsalicylate in reducingthe oesophageal injury when administered afterexposure to pepsin was similar to that observedwhen it was administered before the pepsinexposure. Flux rates ofH+, K+, and glucose weresignificantly lower in the groups exposed to 15mg/ml, 30 mg/ml, and 60 mg/ml of bismuthsubsalicylate than those in the untreated controlgroup (p<005, Table II). Bismuth subsalicylate

Bismuth subsalicylate reduces oesophagitis

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Tay, Chaparala, Harmon, Huesken, Saini, Hakki, Schweitzer

TABLE I Transmucosalflux rates in the treatment-before-injury experiments

Flux ratet

HI K+ Glucose(pLmol/10 min) (pmol/I0 min) (unol/lO min)

No treatment 53 (6) 7 (1) 5 (1)BSS treatment 28 (3)* 3 (1)* 3 (1)*

Results are expressed as mean (SE). Bismuth subsalicylate30 mg/ml.*Significantly different from no treatment group, p<005;tDirection of flux: H+ -out of lumen, K+, glucose=into lumen.

also reduced both the gross and microscopicesophageal indices in a dose dependent manner(Fig 4). The gross oesophagitis index of theuntreated group was 3-4 (0 2), while the treatedgroups were graded as 2-5 (0 2), 2-3 (0-2), and1 8 (0-2) for bismuth subsalicylate concentra-tions of 15 mg/ml, 30 mg/ml, and 60 mg/ml,respectively (p<005). Similarly, the micro-scopic oesophagitis index was reduced from 3-4(0-3) in the untreated group to 2-5 (0-4), 2-3(0 2), and 1-8 (0-3) for the same concentrationsof bismuth subsalicylate (p<0 05).

In vitro experimentsIn vitro experiments were done to determine ifbismuth subsalicylate interacted predominantlywith pepsin or its substrate. Peptic activity ofacidic solutions containing either pepsin alone orpepsin plus bismuth subsalicylate was assayed insolutions containing haemoglobin substrate con-centrations of 1, 2, 3, and 4 g%. The results areshown in Figure 5. When assayed in a 1%haemoglobin substrate solution, peptic activity

TABLE II Transmucosalflux rates in the treatment-after-injury experiments

Flux ratet

H' K' Glucose(p.mol/lO min) (p.mol/lO min) (p.mol/1O min)

No treatment 61 (5) 7 (1) 8 (2)BSS 15 mg/ml 49 (5)* 4 (1)* 4 (1)*BSS 30 mg/ml 46 (5)* 3 (1)* 5 (1)*BSS 60 mg/ml 43 (7)* 4 (1)* 5 (1)*

Results are expressed as mean (SE). Bismuth subsalicylate.*Significantly different from no treatment group; tDirection offlux: H'=out of lumen, K', glucose=into lumen.

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Control

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ElMicroscopic

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LrIL15 30 60Bismuth subsalicylate (mg/ml)

Figure 4: Gross and microscopic oesophagitis indicesfromexperiments in which bismuth subsalicylate was administered10 minutes after pepsin exposure. *=Significantly different(p<O0O5)from the control group, which was exposed to pepsinbut with no BSS treatment.

of the solution containing pepsin plus bismuthsubsalicylate was about half the peptic activity ofthe solution containing only pepsin. As thesubstrate concentration was increased, pepticactivity of the solution containing pepsin plusbismuth subsalicylate did not increase. Theseresults suggest an interaction of bismuth subsali-cylate primarily with pepsin in vitro. It is alsopossible, however, that bismuth subsalicylatecould exert some of its protective effect in vivo bybinding to mucosal proteinaceous substrates forwhich it has a higher affinity than haemoglobin.

DiscussionThe findings of this study show that bismuthsubsalicylate can significantly reduce the severityof the oesophageal mucosal injury caused bypepsin. Administration of bismuth subsalicylateto rabbits, at concentrations similar to that incommercially available products (Pepto-Bismol,17-5 mg/ml), diminished the permeabilitychanges and the morphologic injury induced bypepsin. This protection was observed whetherbismuth subsalicylate was given before or afterthe oesophageal mucosa was exposed to pepsin.Additionally it was noted that, when given afterexposure to pepsin, bismuth subsalicylatesuppressed the pepsin mediated oesophagealmorphologic injury in a dose dependent manner.

120

100

80

60

40

20

* *

30

Bismuth subsalicylate (mg/ml)Figure 3: Gross and microscopic oesophagitis indicesfromexperiments in which bismuth subsalicylate was administeredbefore pepsin exposure. *=Significantly different (p<001)from the control group, which was exposed to pepsin but withno bismuth subsalicylate treatment.

0-0 Pepsin alone l

0-0* Pepsin and bismuth subsalicylate

*

* *

*e +

0 1 2 3 4Haemoglobin substrate concentration (g/100 ml)

Figure 5: In vitro pepsin assay. Significantly lower pepsinactivity was measured when bismuth subsalicylate was presentthan when it was not (*=p<0.05). Bismuth subsalicylateapparently interacts directly with pepsin, rather than thesubstrate, because increasing the substrate concentration didnot increase the peptic activity.

I I I 1- I

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Bismuth subsalicylate reduces oesophagitis 15

There are several possible mechanisms bywhich this protection could occur. Bismuthcompounds are known to complex with proteins.Bismuth subsalicylate could therefore interferewith the peptic digestion of the oesophagealmucosa by complexing with either pepsin or withthe pepsin substrate protein in the mucosa. Ourin vitro experiment would indicate that theformer occurs and, indeed, pepsin inactivationby bismuth compounds has been described byothers.2 19 20

Alternatively, we could not rule out thepossibility of a topical protective effect ofbismuth subsalicylate by binding to the oeso-phageal mucosa in addition to the pepsin inacti-vation. It has been suggested that bismuthsubsalicylate and other bismuth salts encouragegastric and duodenal ulcers to heal because oftheir ability to bind with various glycoproteinsand mucopolysaccharides at the ulcer base.Bismuth compounds are thought to form abarrier over the base ofan ulcer which protects itfrom the noxious luminal contents.225 Whencomplexed with gastric mucus, bismuth hasbeen shown to drastically retard the migration ofhydrogen ions.26 These local effects have not yetbeen described in the oesophagus, but couldhave contributed to the protective effectobserved in vitro in the present study. In aclinical setting administration of bismuth sub-salicylate in a liquid form (Pepto Bismol, Proctorand Gamble), which could coat the oesophagusduring swallowing, would be desirable to takeadvantage of any topical properties.Bismuth is reported to exhibit other interest-

ing properties which are pertinent to its potentialapplicability to the treatment of esophagitis.Colloidal bismuth was recently shown to sup-press the activity and output ofpepsin in patientswith duodenal and gastric ulcers.27 This effectwas still present 24 hours after the medicationhad been discontinued, suggesting the inhibitionwas sustained.28 A long lasting agent would bedesirable in treating reflux oesophagitis, whereprotection is frequently needed during periods ofnocturnal reflux.2' In addition, its activityagainst campylobacter like organisms may bebeneficial, as these bacteria may contribute to thepathogenesis of oesophagitis in some cases.30

Current therapies for oesophagitis have tradi-tionally included measures aimed at reducingreflux, either by increasing lower oesophagealsphincter tone or decreasing intra-abdominalpressure. Furthermore, the mainstay of therapyrests with reduction of gastric acidity. None ofthe existing therapies, however, are specificallydirected at diminishing peptic injury of themucosa, even though part of the efficacy ofantacids may derive from a reduction of therefluxate pepsin activity through a pH rise. Werecently found that the mucosal protective agentsucralfate was highly effective in preventingexperimental peptic oesophagitis in the rabbit.6This benefit was attributed to a topical protec-tion when it was found that sucralfate did notinactivate pepsin. The present study suggeststhat, through direct inactivation of pepsin,bismuth subsalicylate could provide an addedbenefit to a clinical therapeutic regimen whichmight include a combination of agents acting

to prevent mucosal injury by differentmechanisms.

In conclusion, these studies indicate thatbismuth subsalicylate can prevent the oeso-phageal mucosal injury caused by pepsin. Itseffect derives, at least in part, from its capacity tointeract with pepsin.

In conducting the research described in this report, the investigat-ors adhered to the Guide for laboratory facilities and care aspromulgated by the Committee on the Guide for LaboratoryAnimal Facilities and Care of the Institute of Laboratory AnimalResources, National Academy of Sciences, National ResearchCouncil. This work was funded in part by Proctor and Gamble.The authors acknowledge the assistance of Barbara L Bass, RajLakshman, and his staff, and William Andrews.

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3 Lillemoe KD, Johnson LF, Harmon JW. Role of the com-ponents of the gastroduodenal contents in experimental acidesophagitis. Surgery 1982; 92: 276-84.

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10 Lee Fl, Samloff IM, Hardman M. Comparison of tri-potassium dicitrato bismuthate tablets with ranitidine inhealing and relapse ofduodenal ulcers. Lancet 1984; i: 1299-302.

11 Martin DF, May SJ, Tweedle DEF, Hollanders D,Ravenscroft MM, Miller JP. Difference in relapse rates ofduodenal ulcer after healing with cimetidine or tripotassiumdicitrato bismuthate. Lancet 1981; i: 7-10.

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