Gut, 1992, 33, 592-596

Effect of Helicobacter pylori infection on colloidalbismuth subcitrate concentration in gastric mucus

D J B Mufnoz, C Tasman-Jones, J Pybus

AbstractNecropsy gastric mucus infected with Helico-bacter pylon has a reduced capacity to con-centrate colloidal bismuth subcitrate whencompared with non-infected mucus. Mucusmounted in a modified in vitro diffusionchamber was bathed with colloidal bismuthsubcitrate solutions at different concentra-tions and pH levels. Bismuth was measured byatomic absorption spectrophotometry toassess intramucus colloidal bismuth subcitrateconcentrations. Bismuth concentrations innon-infected mucus were higher than inHelicobacter pylori infected mucus at allexperimental colloidal bismuth subcitrate con-centrations and pH levels. Regardless of theinfection status, the intramucus concentrationof colloidal bismuth subcitrate was dependentupon the concentration of the bathing solutionand independent of the pH and the mucusthickness. Colloidal bismuth subcitrate solu-bility in saline solution varied with pH.and was least soluble in the pH range 1 1 to3-25 and more soluble above and below this pHrange. This study suggests that Helicobacterpylon infection is associated with physico-chemical changes in the gastric mucus with areduction in its capacity to concentrate col-loidal bismuth subcitrate. Such a reductionmay compromise the attainment of optimumcolloidal bismuth subcitrate concentrationsnecessary for its bactericidal activity.

Department ofMedicine, School ofMedicine, University ofAuckland, Auckland,New ZealandD J B MuniozC Tasman-Jones

Department of ClinicalChemistry, AucklandHospital, Auckland, NewZealandJ PybusCorrespondence to:Dr C Tasman-Jones,Department of Medicine,School of Medicine,University of Auckland,Private Bag, Auckland, NewZealandAccepted for publication5 August 1991

Gastric mucus plays an important role inmucosal protection.' Disruption in theintegrity of the blanket ofmucus has been foundin individuals with peptic ulcer.8"12 Helicobacterpylori present in gastric mucus of the stomachor duodenal gastric metaplasia is associated withgastritis and peptic ulceration.'3 22 Helicobacterpylori resides in gastric mucus but not withinepithelial cells.2"24 It may play a role in mucinbreakdown.25-27

Eradication of Helicobacter pylori by colloidalbismuth subcitrate is followed by histologicalimprovement of gastritis and healing of pepticulcer.242829 If infection recurs, ulcer relapse isfrequent, particularly after one year.24 30 31

A combination of colloidal bismuth subcitratewith one or two antibiotics - fQr example,amoxycillin and/or metronidazole, is more

successful than monotherapy in eradicatingHelicobacter pylor 24323

Ulcer relapse after colloidal bismuth sub-citrate and other antibiotics may be the resultof a failure of the drug(s) to reach sufficientintramucus concentrations to eradicate Helico-

bacterpylori. Movement of a drug into mucus willbe determined by its intragastric availability.Availability will depend upon various factorsincluding the drug's behaviour in solution.

It is not known whether non-infected andHelicobacter pylon infected mucus differ in theircapacity to contain colloidal bismuth subcitrateand little is known on the behaviour of colloidalbismuth subcitrate in solution.

In this in vitro study we have examined theeffect of Helicobacter pylon' infection on thecapacity of gastric mucus to concentrate colloidalbismuth subcitrate and the behaviour ofcolloidalbismuth subcitrate in acidic solutions.

Methods

SPECIMENSMucus from 20 human stomachs was collected atnecropsy within 24 hours of death. All persons(18-75 years) had died of acute trauma or suddenunexpected medical collapse. Each stomach wasopened along the greater curvature and foodcontents were removed by washing with normalsaline.Mucus was harvested by gently scraping the

gastric mucosa with a glass slide and stored at-20°C for later experiments.Immediately before harvesting, a gastric tissue

specimen was taken for Helicobacter pylori ureasedetection by the camplobacter like organism test(CLO test, Delta West Limited, Western Aus-tralia).34A mucus sample was considered infected with

Helicobacter pylori when the tissue specimenshowed positive urease activity within one hourwith the CLO test and non-infected when thetissue specimen showed no positive ureaseactivity within 24 hours.

COLLOIDAL BISMUTH SUBCITRATECONCENTRATION IN GASTRIC MUCUSThe modified in vitro diffusion chamber pre-viously used for ion exchange studies on mucus`was used to expose gastric mucus to colloidalbismuth subcitrate.Mucus (115 + 1 mg or 230 ± 1 mg, depending

on mucus layer thickness) was held between twofine gauze mesh discs (Nybolt PA-22/17; SwissSilk Bolting Cloth Mfg Co Ltd, Zurich, Switzer-land) separated by a plastic ring to form a layer400 ,im or 800 iim thick. The mucus containingcassette was held in the chamber by two rigidmesh discs (Tetex Mono PES 130/TWL/C;Swiss Silk Bolting Cloth Mfg Co Ltd, Zurich,Switzerland). One side of the mucus was bathed

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with saline solution adjusted to pH 6.0 and theother side with filtered solutions (Filter Paper 1,Whatman International Ltd, Maidstone,England) of colloidal bismuth subcitrate (De-Nol®, Gist-Brocades, Delft, The Netherlands)at different concentrations (as bismuth) andpH's as follows:

Series 1: Colloidal bismuth subcitrate 93mg/l, pH 6.0, mucus layer 800 [im thick.

Series 2: Colloidal bismuth subcitrate 232mg/l, pH 6-0, mucus layer 400 ,um thick.

Series 3: Colloidal bismuth subcitrate 64mg/l, pH 6.0, mucus layer 400 iim thick.

Series 4: Colloidal bismuth subcitrate 64mg/l, pH 1 5, mucus layer 400 [tm thick.

Series 5: Colloidal bismuth subcitrate 127mg/l, pH 1 5, mucus layer 400 [im thick.

The solutions were continuously circulated for90 minutes at a rate of 4 5 ml/min, roomtemperature 2 1C.

After each experiment the excess of colloidalbismuth subcitrate solution was washed off withsaline. The mucus was taken out of the chamberand dried at 1 10°C for four hours.The bismuth content of the samples was

measured by atomic absorption (GraphiteFurnace Atomic Absorption SpectrophotometerSpectrAA-20; Melbourne, Australia) to assessthe intramucus colloidal bismuth subcitrateconcentrations. Results were expressed as [tgbismuth/g mucus on the assumption that allmucus samples contain 95% water.36 The possi-bility that Helicobacter pylon infection maymodify the water content has not been con-sidered in this study.

COLLOIDAL BISMUTH SUBCITRATE SOLUBILITYNormal saline solutions were adjusted to dif-ferent pH's (E512 Metrohm Herisau pH meter,Switzerland). Commercial tablets of colloidalbismuth subcitrate stripped of their outer coat-ing were ground to a homogeneous powder.Weighed samples of the powder were added tosaline to give mixtures with the same weight/volume ratio. After colloidal bismuth subcitratewas dissolved in the saline solution, sampleswere obtained from the supernatant andanalysed for bismuth content by atomic absorp-tion spectrophotometry. Colloidal bismuth sub-citrate solubility was estimated by calculating thedifference between expected bismuth level andbismuth in solution after sample preparation.

In separate experiments we observed the dis-integration of commercial colloidal bismuth sub-citrate tablets. Saline solutions were adjusted to

TABLE Bismuth concentrations in non-infected and Helicobacter pylori infected gastricmucus

CBS solution Non-infected HP-infectedMucus

mgll (Bi) pH thickness Mean (SD) Range (n) Mean (SD) Range (n)

93 6-0 800* 74.2t (23-9) 33-116 (10) 37-2t (21-7) 9-72 (10)232 6-0 400 147-9 (25-1) 121-186 (5) 88-2 (38-7) 33-130 (5)64 6-0 400 714 (29-2) 38-108 (5) 27-0 (12-1) 17-46 (5)64 1-5 400 50-6 (12-1) 35-66 (5) 23-2 (6-6) 14-32 (5)127 1-5 400 95-7 (22-5) 64-121 (6) 65-2 (16-5) 33-78 (6)

HP-infected=Helicobacter pylon infected; CBS=colloidal bismuth subcitrate; *=[Am;t-= tg bismuth/g mucus, assuming that all mucus samples contain 951% water.

different pH's. For each pH, one 60 ml test tubecontaining 45 ml saline and one whole commer-cial tablet of colloidal bismuth subcitrate was'tumbled' in the Chiltern rotatory mixer (ChilternScientific, Germany) at a rate of 32 'tumbles' perminute. Disintegration was assessed visually andhalfand total disintegration times were recorded.To determine if the outer coating of the com-mercial tablet affects disintegration, tablets werestripped of their coating and exposed to salinesolutions under the same conditions as above.

STATISTICAL ANALYSISResults for colloidal bismuth subcitrate concen-tration experiments were expressed as mean(SD). A general linear approach to the analysis ofvariance37 was used to determine the statisticalsignificance of differences in bismuth concen-tration between non-infected and Helicobacterpylori infected mucus. Significant mean andinteraction effects were further investigatedusing Tukey's procedure to analyse differenceswithin pH's and mucus thickness. Type III sumsof squares are presented because the design wasunbalanced. p Values of less than 0.05 wereconsidered significant.

Results

COLLOIDAL BISMUTH SUBCITRATECONCENTRATION IN GASTRIC MUCUSThe Table shows mean (SD) and range bismuthconcentrations in non-infected and Helicobacterpylon infected gastric mucus.

Bismuth concentrations were higher in non-infected than in Helicobacter pylon infectedmucus at all experimental colloidal bismuthsubcitrate concentrations and pH levels (F(1,62)=52.86, p=00001).

Regardless of the infection status, bismuthconcentrations varied in proportion to the con-centration of the bathing solution and were nodifferent at mucus thickness 400 iim or 800 ,tmand at pH 1.5 or 6.0 (F(s,62)=l106, p=0.0389).

In separate experiments, using unfilteredcolloidal bismuth subcitrate solutions and no finegauze mesh we found that a whitish precipitatewas deposited on the mucus surface after theexperiments, especially at low pH's; bismuthconcentrations were up to five fold greater thanbismuth levels obtained after using filteredcolloidal bismuth subcitrate solutions.

COLLOIDAL BISMUTH SUBCITRATE SOLUBILITYFigure 1 shows percentages of colloidal bismuthsubcitrate solubility estimated from bismuthconcentrations in supernatant samples. Solu-bility in solution was >81% at pH 0.5 to 1-05;<50% at pH 1 1 to 3-25 and >88% at pH 3-75 to6-25. Minimum solubility (<15%) occurred inthe pH range 1 1 to 2- 1. Two points of rapidchange from high precipitation to high solubilitycan be described, one between pH 3-25 and 3-75and the other between pH 1.05 and 1 1.

Half disintegration times for commercialcolloidal bismuth subcitrate tablets are shown inFigure 2. At pH 0 8, 1 0 and 1 *1, coated colloidal

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100

80

._4

40-0:3 40U)

20

0 1 2 3 4pH

Figure 1: Colloidal bismuth subcitrate solubsolution (in %) vpH. Solubility was >81% 4<50% atpH 1 1 to 3.25 and >88% atpH

bismuth subcitrate tablets did no

even after two hours of constandisintegration times were shorter fcwhich the coating had been strippia significant change in disintelbetween pH 1 1 and 1[2.

DiscussionNecropsy gastric mucus has the caup colloidal bismuth subcitrate depconcentration of colloidal bismuththe bathing solution. This caraffected by changes in bathing soluconcentration is maintained conreduced when the mucus is iHelicobacter pylori.The Campylobacter like organisn

sole test used to diagnose Helicinfection. This test has been foundand rapid, with at three hours simito Gram staining and with no falA postmortem study found that tis equally reliable to detect Helipresence when compared with oth(There is a paucity of literatui

haviour of colloidal bismuthsolution. Wieriks et al4l reportbismuth subcitrate is highly solubl

O0

-EE

0

_40)

90

O Coated tab* Uncoated l

60

30

150*

00 0

0@0 0

0 2 3 4pH

Figure 2: Halfdisintegration times ofcoatecuncoated (0) commercial tablets ofcolloidaisubcitrate. Points represent the mean offour

precipitates at pH <5. Lee4' found 40 to 50%o 6.25 colloidal bismuth subcitrate retained in solution

in the pH range 1[0 to 3 0. Our results aresimilar, with <50% colloidal bismuth subcitratemaintained in solution in the pH range 1 1 to3 25. Minimum solubility (<15%) occurred inthe range 1 1 to 2 1. The differences may be theresult of variations in technique. We prepareddifferent solutions for each pH level using com-mercial tablets while Lee titrated a sole sampleof a 'buffered solution of colloidal bismuth sub-citrate' to the different pH's.

Colloidal bismuth subcitrate dissolves and/orprecipitates when exposed to an aqueous solu-

5 6 7 tion. In keeping with the known effects ofpH onthe sol gel transitions of colloids,42 we speculate

ility in saline that at pH 6.0 the bismuth subcitrate colloidatpH 05to 105; dissolves and remains in solution as negatively3.75 to 6.25. charged molecular complexes. When the pH is

reduced to 1-5, most of the colloid would precipi-it disintegrate tate, but some bismuthoxy cations and bismuth't'tumbling'; ions as free Bi3+ will be formed and remain inr tablets from solution, this will be the case of the bathinged. There was solution in our experiments at pH 1.5 as thegration times precipitated colloidal bismuth subcitrate was

removed by filtration.The ability of polyanions to selectively

accumulate hydrogen ions into a zone of waterimmediately adjacent to the fixed charges43 offers

ipacity to take a possible explanation for the in vitro concentra-

)ending on the tion of colloidal bismuth subcitrate in mucus at

i subcitrate in pH 6.0. The bismuth subcitrate colloid in con-

pacity is not tact with mucus may concentrate intramucusition pH if the hydrogen ions decreasing the pH of its micro-istant, but is environment to a level where it is precipitatedinfected with to become a source for Bi3+ and bismuthoxy

formation and accumulation in mucus. Inns test was the Helicobacter pylon' infection, ammonia decreaseswobacter pylori the availability of hydrogen ions in mucus,44l to be reliable thus, reducing the accumulation of bismuthilar sensitivity cations.Ise positives.3 The in vitro concentration of colloidal bis-he urease test muth subcitrate in mucus at pH 1 5 may dependcobacter pylot on the interaction ofbismuthoxy cations and Bi3+er tests.3 with the negatively charged residues of sialic acide on the be and ester sulphates present in the glycoproteinsubcitrate in matrix.45 Gastric mucus contaminated withthat colloidal microorganisms other than Helicobacter pylon'Le in water and shows a diminished number of sialic acid resi-

dues.- Helicobacter pylori induces changes in thephysicochemical integrity of mucus25-27 and maysimilarly decrease these residues accounting for

lets the reduced capacity of Helicobacter pyloritablets infected mucus to concentrate colloidal bismuth

subcitrate when compared with non-infectedmucus at pH 1[5.

Another explanation for the differences inintramucus bismuth concentrations is analteration of composition and structure ofmucuscaused by continued glycoprotein and lipiddigestion by Helicobacter pylon enzymes25-27between death, harvesting ofmucus samples andthe experiments.We postulate that the precipitated and soluble

9 forms of colloidal bismuth subcitrate have dif-1

6 7 ferent therapeutic actions. Gastric ulcer healingis related to the formation of a protective layer of

1(0) and precipitated colloidal bismuth subcitrate bondedI bismuth to serous secretions on the ulcer crater.4' 47-4 Anexperiments. acid pH of the lumen should aid precipitation of

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Effect ofHelicobacter pylori infection on colloidal bismuth subcitrate concentration in gastric mucus 595

the layer. Helicobacter pylori associated gastritisand duodenal ulcer healing depends on thecapacity of mucus (mucus of gastric metaplasiain the case of duodenal ulcer) to concentratebismuth cations either in solution or mobilisedfrom bismuth subcitrate colloid in contact withmucus (depending on luminal pH).The concentrations of bismuth in infected

mucus at pH 1 5 (14 to 32 [tg/g; 16 to 36 Ftg/gcalculated as Bi2O3) observed in the presentstudy will not be representative of the in vivosituation because they were obtained after 90minutes of bathing with solutions at a constantconcentration. In vivo, a fasting intragastric pHof about 1 5 ,"" gastric emptying of40% or morefor liquid meals after 20 minutes,5455 the dilutingeffects of food and the long disintegration time ofcommercial tablets at pH <1 1 (Fig 2) willrestrict the intragastric availability of colloidalbismuth subcitrate in solution.

Intramucus bismuth concentrations fallingbelow the mean inhibitory concentration ofcolloidal bismuth subcitrate for Helicobacterpylon' (4 to 32 ig/ml in vitro,24 5159) wouldexplain the failure of colloidal bismuth subcitrateto always eliminate the organism. Bismuth con-centrations in endoscopy biopsy samples after anoral dose of colloidal bismuth subcitrate606' willcontain mucus-surface precipitated colloidalbismuth subcitrate and will not represent intra-mucus colloidal bismuth subcitrate.The clinical efficacy of colloidal bismuth sub-

citrate in healing peptic ulcers and gastritis isdependent on multiple mechanisms. Eradicationof Helicobacter pylori is an important factor.Resolution of the gastritis when associated withpeptic ulcer will play an important role in ulcerhealing, possibly through humoral mechanisms.Small intramucus colloidal bismuth subcitrateconcentrations either in gastric and/or duodenalmucus may aid healing through stimulation ofprostaglandin production,6266 enhancement inthe activity of epidermal growth factor,67-69inhibition of the protease and lipase activitiesof Helicobacter pylori towards gastric glyco-proteins,707' and possible detachment of micro-organisms from the gastric epithelium.24

Preliminary results of a study conducted bySalmon72 show increased early (four weeks) heal-ing of duodenal ulcers treated with a combi-nation of colloidal bismuth subcitrate andcimetidine when compared with colloidal bis-muth subcitrate or cimetidine alone. Masoeroet al5° observed the existence of some duodenalulcer patients with intragastric pH valuessteadily ranging around 10 throughout the 24hours, regardless of meals and of the time of theday. As Figure 1 shows, at both pH's (3 75 to6 25 and 1 0), colloidal bismuth subcitrate solu-bility will be favoured but, as speculated, thecolloidal bismuth subcitrate forms in solutionwill be different. Further studies will be neces-sary to determine if the intragastric pH isimportant for a better success in eradicatingHelicobacter pylori.The ecologic niche for Helicobacterpylori is the

gastric mucus. For any antibacterial agent toeradicate it, it must be able to penetrate themucus layer in sufficient concentrations. Wehave shown that gastric mucus possesses the

capacity to concentrate colloidal bismuth sub-citrate and that this capacity is reduced whenHelicobacter pylorn are present. In vivo,the reduced capacity of mucus to concentratecolloidal bismuth subcitrate may compromisethe attainment of optimal intramucus colloidalbismuth subcitrate concentrations necessaryfor its bactericidal activity. The behaviour ofcolloidal bismuth subcitrate in acidic solutionssuggests that the intragastric availability of thedrug in solution may be low, further restrictingsatisfactory antibacterial intramucus concentra-tions.

This study was supported by a grant from the Research Division,Gist Brocades, The Netherlands. The authors would like to thankProfessor Philippa Wiggins of the Department of Medicine,University of Auckland Medical School, New Zealand, for herhelpful discussions and Mr Greg Gamble of the Departmentof Medicine, Auckland Hospital, New Zealand, for statisticalanalysis.

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