J Clin Pathol 1996;49:309-312

Parietal cells in the duodenal bulb and theirrelation to Helicobacter pylori infection

A W Harris, M M Walker, A Smolka, J M Waller, J H Baron, J J Misiewicz

AbstractAim-To investigate the prevalence, andrelation to Helicobacter pyloni, of parietalcells in the duodenal bulb using a mono-clonal antibody directed against H+,K+-ATPase (HK12.18).Methods-Twenty six patients with duo-denal ulcer disease and 16 healthy controlswere studied. H pylori status was de-termined by gastric histology and cultureand by the '3C-urea breath test. Four bi-opsy specimens were taken from the duo-denal bulb and stained with HK12.18. Thepresence/absence and number of parietalcells in the duodenal bulb were assessedblindly by a histopathologist.Results-The overall prevalence of pa-rietal cells in the duodenal bulb was 31%(13/42) and was similar in patients withduodenal ulcer and in controls, and in Hpylori positive and negative subjects. Themedian (range) number of parietal cellsin the duodenal bulb was 7 5 (4-20) parietalcells/subject, and was similar in all fourgroups.Conclusions-The prevalence of parietalcells in the duodenal bulb (31%) is notablyhigher than previously reported in endo-scopic studies, and is in keeping with re-ports from studies on necropsy/operativespecimens. There was no difference in theprevalence or number of parietal cells inthe duodenal bulb between patients withduodenal ulcer and controls, regardless ofHpylori status. These findings suggest thatparietal cells in the duodenal bulb do notcontribute to the pathogenesis ofduodenalulcer.(J7 Clin Pathol 1996;49:309-312)

Keywords: Helicobacter pyloni, duodenal ulcer, parietalcells, H+,K+-ATPase.

Table 1 Prevalence of panietal cells in the duodenal bulb

Reference Specimen type Prevalence (%a)

Taylor2 Necropsy 2/150 (<1)Leela et al4 Necropsy 10/15 (67)Hoedemaeker5 Operative

All patients 52/158 (33)Patients with DU 27/52 (52)

Johansen et al' OperativeAll patients 56/250 (22)Patients with DU 27/52 (52)

Tominaga7 Necropsy 27/118 (23)Shousha et al' Endoscopic biopsy 7/60 (12)Wyatt et al3 Endoscopic biopsy 5/518 (<1)Carrick et al' Endoscopic biopsy

Overall 6/137 (4)Patients with DU 5/46 (11)

Koch et al'° Endoscopic biopsy 69/337 (20)

DU = duodenal ulcer.

Parietal cells in the proximal duodenum werefirst described by Kaufman in 1906' and notedto be close to Brunner's glands. Further studieshave shown their prevalence in the duodenalbulb to vary, ranging from less than 1 %2 toalmost 70%4 (table 1). Parietal cells werenot found in the duodenum of the fetus andnewborn, which suggests that they may beacquired.4 Most of these studies have describedgastric heterotopia and not isolated groups ofparietal cells in the duodenal bulb. Further-more, there is some confusion in the literatureregarding the difference between gastric het-erotopia and gastric metaplasia in the duodenalbulb. The former refers to the presence of fullthickness body-type gastric mucosa, and thelatter, replacement of intestinal-type surfacemucosa with gastric-type epithelium.Widely reported variations in the prevalence

of parietal cells in the duodenal bulb may bedue to sampling error at endoscopy, or in thesiting of the gastroduodenal junction.7l' De-tection of parietal cells may also be difficult bylight microscopy on haematoxylin and eosinstained preparations. Complex trichrome andlectin stains have been used, but are time con-suming and technically demanding.The prevalence of parietal cells in the

duodenal bulb has been reported to be higherin patients with duodenal ulcer disease.569Johansen and Hansen6 found parietal cells inthe proximal duodenum in 56 (22%) of 250gastrectomy specimens (52 duodenal ulcer, 55gastric ulcer, 23 prepyloric ulcer, and 120 can-cer), but in 27 (52%) of 52 specimens takenfrom patients with duodenal ulcer (p<0 05). Ithas been suggested that production of acidby parietal cells in the duodenal bulb maycontribute to the pathogenesis of duodenalulcer.9 The relation between Helicobacter pyloriinfection, a major risk factor for duodenal ulcer,and parietal cells in the duodenal bulb has notbeen explored.The aim of this study was to investigate the

importance of duodenal parietal cells, ratherthan gastric heterotopia or gastric metaplasia,in the aetiology of duodenal ulcer. Prevalenceof parietal cells in the duodenal bulb was de-termined in patients with duodenal ulcer andin healthy controls, with and without H pyloriinfection. Four carefully sited samples weretaken and a highly specific and sensitive mono-clonal antibody directed against H+,K+-ATPase (HKl 2.18) was used to identifyparietal cells accurately.

MethodsTwenty six patients with endoscopically diag-nosed duodenal ulcer were studied, 19 of

Parkside HelicobacterStudy Group,Central Middlesex andSt Mary's Hospitals,LondonA W HarrisM M WalkerJ M WallerJ H BaronJ J Misiewicz

Medical University ofSouth Carolina,Charleston, USAA Smolka

Correspondence to:Dr A W Harris,Department ofGastroenterology andNutrition,Central Middlesex Hospital,London NW1O 7NS.

Accepted for publication5 January 1996

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Table 2 Clinical and demographic details of the subjects studied

Controls Patients with DU

Endoscopy Normal Normal DU DUH pylon status Negative Positive Negative PositiveNo. of subjects 10 6 7 19No. of men 4 5 6 13Age in years mean (range) 33 (24-40) 34 (22-54) 39 (26-53) 37 (22-58)Current smokers 3 2 1 3

DU = duodenal ulcer.

whom were positive for H pylori infection. Sixof the seven H pylori negative patients hadsymptomatic and endoscopic recurrence ofduodenal ulcer disease at least six months aftereradication of Hpylori. The remaining Hpylorinegative patient with duodenal ulcer had never

been infected. Sixteen healthy controls (six Hpylori positive) with normal endoscopy were

studied. None of the patients nor controls hadtaken non-steroidal anti-inflammatory drugswithin one month, or antisecretory medicationwithin two weeks of the study. Clinical anddemographic details are given in table 2.

Figure 1 Duodenal bulb biopsy specimen stained with the monoclonal antibody directedagainst H+,K+-ATPase (HK12. 18). Labelled parietal cells are stained brown.a Yt~~~~

AAtW6w__ ,b,.I_~~40. # * _~~jLj

+* s }ls*~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~A

8> si#;eSS*~~~~~~~At

Figure 2 Gastric body biopsy specimen stained with the monoclonal antibody directedagainst H+,K+-ATPase (HKJ2. 18). Labelled parietal cells are stained brown.

DETERMINATION OF H PYLORI STATUSH pylori status was determined by histology(antral, body and duodenal bulb biopsy speci-mens routinely processed and stained withhaematoxylin and eosin and Gimenez stains),culture (antral and duodenal bulb biopsy speci-mens isolated in selective and non-selectivemedia, and microaerobic conditions for up to10 days) and by the '3C-urea breath test (Euro-pean Standard Protocol, where a result is pos-itive if excess 6"1CO2 excretion exceeds 5 perml"2). Subjects were classified as H pyloni pos-itive if they had a positive breath test and positivehistology from any site or positive culture, orboth. Absence ofHpylori was defined as failureto detect the bacterium in all three tests.

IDENTIFICATION OF PARIETAL CELLSMurine monoclonal antibody HK12.18 wasused as a specific marker for H+,K+-ATPase.HK 12.18 raised against pig gastric microsomesenriched in H+,K+-ATPase was reconstitutedfrom lyophilised mouse ascites with 250 jl dis-tilled water, aliquoted and stored at -20°Cuntil required.Four endoscopic duodenal bulb biopsy speci-

mens were taken with standard cupped forceps(Olympus FB23-35K, Japan) by one en-doscopist, more than 1 cm away from the edgeof any duodenal ulcer, and more than 1 cmfrom the pylorus. The biopsy specimens wereplaced immediately in formalin. After routineprocessing, 2 gm sections were stained withHK12.18 using a modification of the per-oxidase-antiperoxidase technique.'3 Afterwashing with water and phosphate bufferedsaline (PBS), the slides were incubated withHK12.18 (diluted 1 in 2500) at 4°C for 16hours. They were then washed with PBS, andincubated for 30 minutes with peroxidase con-jugated rabbit anti-mouse (dilution 1 in 300)sera (Sigma, Poole, Dorset, UK). Visualisationof the antigen (H+,K+-ATPase) was achievedby adding diaminobenzidine tetrahydro-chloride hydrogen peroxide (DAB) as the chro-mogen substrate, and counterstaining withhaematoxylin (fig 1). Figure 2 shows a gastricbody biopsy specimen stained in the same way.The presence or absence and the number of

parietal cells in one section taken from eachbiopsy specimen was assessed blindly by anexperienced histopathologist. The number ofparietal cells is expressed as the total numberof parietal cells seen in the four biopsy sectionsfrom each subject.

STATISTICAL METHODS AND ETHICSThe X2 test was used to compare the significanceof differences between the groups, wherep<005 was regarded as statistically significant.The protocol was approved by Parkside ethicalcommittee and all subjects gave written in-formed consent.

ResultsThe overall prevalence of parietal cells in theduodenal bulb in all subjects was 31% (13/42)

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Panietal cells in the duodenal bulb: relation to H pylori infection

Table 3 Prevalence ofparietal cells in the biopsy specimens of the duodenal bulb inhealthy controls and in patients with duodenal ulcer (DU)

Controls Patients with DU

No. of subjects 16 26Parietal cells present (%) 4 (25) 9 (35)H pylon status Negative Positive Negative PositiveNo. of subjects 10 6 7 19Parietal cells present (%) 3 (30) 1 (16) 4 (57) 5 (26)Median (range) number of

parietal cells/subject 6 (4-7) 20 (n= 1) 7 (5-20) 7 (5-15)

(table 3). The prevalence of parietal cells inthe duodenal bulb did not differ significantly(x2 = 0-66, p = 0 4) between patients with duo-denal ulcer disease (nine of 26) and healthycontrols (four of 16). The prevalence ofparietalcells in the duodenal bulb was similar in allsubjects, regardless of Hpylori status (table 3).The median number of parietal cells in theduodenal bulb identified per subject was notsignificantly different between the four groups

(table 3), with an overall median (range) of 7-5(4-20) parietal cells/subject.

DiscussionIDENTIFICATION OF PARIETAL CELLS

Parietal cells, either isolated or in small clusters,may be difficult to identify using haematoxylinand eosin staining alone. We have used a highlyspecific and sensitive monoclonal antibody dir-ected against H+,K+-ATPase (HK12- 18) toidentify parietal cells within duodenal biopsyspecimens; this technique has not been appliedbefore. Smolka et al14 5 have confirmed thehigh specificity of HK12.18 in in vitro studies.The antibody has been shown to bind to thetubulovesicular membranes of the parietal cellcytoplasm.'415 Kelly et al'6 used a monoclonalantibody directed against H+,K+-ATPase tomap the distribution of parietal cells in thehuman fetal and infant stomach, and foundthat the site of monoclonal antibody bindingcorresponded precisely with the location ofparietal cells as determined by staining withhaematoxylin and eosin. Binding of a mono-

clonal antibody to vesicular membranes con-

taining H+,K+-ATPase was demonstrated byKarlsson et al.'7 The same group showed thatthe monoclonal antibody directed against H+,K+-ATPase did not bind to Na+,K+-ATPasecontaining vesicular membranes, confirming itshigh specificity for H+,K+-ATPase.The high sensitivity of HK12.18 has been

confirmed by Smolka et al,'4 who demonstratedthat the antibody bound to the tubulovesicularmembranes of the parietal cell even after pre-treatment with cimetidine, so that antibodybinding was independent of the secretory stateof the cell.

PREVALENCEThe overall prevalence (31%) of parietal cellsfound in this study in the duodenal bulb isnotably higher than in previous endoscopicstudies, but in keeping with reports ofnecropsyand operative specimens (table 1). In the threelargest studies of resected duodenal bulbs, pa-

rietal cells were found in 22-33% of cases.567

By contrast, in the endoscopic studies parietalcells were found in only 1-20% of subjects.39 10It has been reported that the prevalence ofparietal cells in the duodenal bulb is higherin patients with duodenal ulcer than healthysubjects (table 1).569 Hoedemaeker5 found pa-rietal cells in 52% of patients with duodenalulcer compared with 33% of those withoutduodenal ulcer disease (p<005). Carrick et al,9using two duodenal bulb biopsy specimens,found parietal cells more frequently in patientswith duodenal ulcer than in healthy subjects,but the difference did not reach statistical sig-nificance. Using a highly specific method, wefound that parietal cells were more commonin the duodenal bulb in patients with duodenalulcer than in healthy controls, but the differencewas not statistically significant. According toour hypothesis, the difference in the prevalenceof parietal cells in the duodenal bulb shouldhave been largest between the Hpylori positivepatients with duodenal ulcer and the H pylorinegative controls. However, the prevalence ofparietal cells was similar between these twosubgroups, with parietal cells seen in five (26%)of 19 H pylori positive patients with duodenalulcer and in three (30%) of 10 Hpylori negativecontrols. It is possible that our findings maybe subject to a type II error-that is, the nullhypothesis is not rejected when it is false. Itwould be necessary to study about 180 patientswith duodenal ulcer and 180 controls to havea 90% power of achieving a significant resultat the 5% level.

HISTOLOGYParietal cells were seen as single cells or insmall clumps, and at most, in two of the fourbiopsy specimens from each subject (fig 1).The total number of parietal cells identified inthe duodenal bulb biopsy specimens was small(median 7-5 per subject), and not significantlydifferent between patients with duodenal ulcerand controls, or between H pylon positive andnegative subjects.The sparsity of the parietalcells in the duodenal bulb may contribute tothe difference in the reported prevalence be-tween the necropsy and operative studies usinglarger pieces of tissue and the smaller endo-scopic biopsy specimens. We suggest that tak-ing four directed biopsy specimens from eachduodenal bulb successfully minimised the sam-pling error.A possible criticism of some of the earlier

studies is that the parietal cells found in theduodenal bulb may actually have been atthe gastroduodenal junction. This junction isusually sharp, and is characterised not only bythe abrupt transition from gastric mucosa tothe characteristic villous epithelium of the duo-denal mucosa, but also by the transition fromthe branched tubular pyloric glands to multi-lobular mucus Brunner's glands.7 However,this junction is not always sharply demarcatedand there may be islands of intestinal mucosaon the gastric side of the junction and, morerarely, islands of gastric mucosa on the duo-denal side. The gastroduodenal junction maybe crenated with finger-like processes of gastric

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epithelium extending up to 6 mm into the duo-denum.11 We therefore took the duodenal bulbbiopsy specimens more than 10mm distallyfrom the pylorus in order to avoid samplinggastric mucosa.

RELATION TO H PYLORI INFECTIONBecause Hpylori infection is a major risk factorfor duodenal ulcer, and because the prevalenceof parietal cells in the duodenal bulb was pur-ported to be higher in patients with duodenalulcer, we investigated whether there was a re-lation between H pyloni infection and parietalcells in the duodenal bulb in patients withduodenal ulcer. Basal and stimulated acid out-puts are higher in H pylori positive patientswith duodenal ulcer than in H pylori negativecontrols.'8 It was therefore possible that Hpylori may have a similar effect on local acidproduction within the duodenal bulb. We hy-pothesised that H pylori infection in patientswith duodenal ulcer would lead to an increase inthe prevalence of parietal cells in the duodenalbulb: it did not. In fact, the prevalence andnumber of parietal cells in the duodenal bulbwas not affected by Hpyloni infection, either inhealthy controls or in patients with duodenalulcer.

ROLE OF PARIETAL CELLS IN THE DUODENALBULBIt is not known why parietal cells are found inthe duodenal bulb. Carrick et al' demonstratedacid secretion within the duodenal bulb inpatients with duodenal ulcer using Congo Redafter stimulation with pentagastrin. Unfortun-ately, biopsy specimens were not taken fromthe apparent acid secreting areas (which turnedCongo Red blue/black) to confirm that thesesites did contain parietal cells, and that therewas local acid secretion within the duodenalbulb. We had difficulty in replicating the CongoRed technique in patients with duodenal ulcer,because the often scarred and distorted duo-denal bulb was not readily coated with thesolution of indicator dye.The parietal cells in the duodenal bulb iden-

tified with HK12.18 are probably functional,because they contain tubulovesicular mem-branes, which are an essential component ofthe acid secreting apparatus.4"1519 However,we are unable to comment on the functionalactivity of these structures.The number of parietal cells seen in the

duodenal biopsy specimens was small, with amedian of 7-5 cells seen per subject. Cox"0estimated that the normal adult stomach con-tains about 1 x 1i9 parietal cells. An endoscopicbiopsy specimen of the gastric body containsabout 120 cells/mm2. It seems likely therefore

that the amount of acid secreted by the smallnumber of parietal cells in the duodenal bulbis negligible.

In conclusion, using multiple duodenal bulbbiopsy specimens and a specific monoclonalantibody directed against H+,K+-ATPase toidentify parietal cells, we have shown that thesecells are found in the duodenal bulb in 31% ofsubjects. Their presence did not correlate withduodenal ulcer disease or Hpylori status. Thesefindings do not support the hypothesis thatparietal cells in the duodenal bulb contributeto the pathogenesis of duodenal ulcer.

We thank the nurses of the Endoscopy units at Central Mid-dlesex and St Mary's Hospitals for their help. AWH is supportedby a grant from Lederle Laboratories, UK, and AS is a recipientof grant DK 43138 from the National Institutes of Health,USA.

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