Nα-Methylhistamine: association with Helicobacter pylori infection in humans and effects on gastric...

Clinica Chimica Acta 301 (2000) 181–192www.elsevier.com/ locate /clinchim

Na-Methylhistamine: association with Helicobacterpylori infection in humans and effects on gastric acid

secretion

a , a b*Stephen Murray , Graham W. Taylor , Q. Najma Karim ,c cPhilip Bliss , John Calam

aSection of Clinical Pharmacology, Division of Medicine, Imperial College School of Medicine,Hammersmith Hospital, Ducane Road, London W12 ONN, UK

bDepartment of Microbiology, Imperial College School of Medicine, St Mary’s Campus, Norfolk Place,London W2 1PG, UK

cSection of Gastroenterology, Division of Medicine, Imperial College School of Medicine,Hammersmith Hospital, Ducane Road, London W12 ONN, UK

Received 2 June 2000; accepted 25 July 2000

Abstract

Infection with the bacterium Helicobacter pylori is associated with altered gastric acid secretionand gastrointestinal disease. Recent work has suggested that Na-methylhistamine, produced by thebacterium and acting on histamine receptors in gastric tissue, might be involved. Gastric juice andtissue biopsies from infected patients have been analysed for the presence of Na-methylhistamineusing a specific and sensitive assay based on gas chromatography mass spectrometry. Na-Methylhistamine was detected in five of seven samples of gastric juice from infected patients(5–180 pmol /ml) but was absent in nine uninfected subjects. The compound was not found infundic and antral biopsies from both subject groups. Helicobacter pylori, cultured on agar and inbroth with and without added histamine, was found not to produce detectable levels of Na-

25methylhistamine. Instillation of this compound at 10 mol / l into the gastric lumen produced asignificant increase in acid secretion in vivo while plasma gastrin concentration remainedunchanged. Na-Methylhistamine in gastric juice appears therefore to be associated with infection,although this product is not generated directly by the bacterium. The concentrations found arebelow those required to affect acid secretion or gastrin production in vivo, although higher local

Abbreviations: Na-MH, Na-methylhistamine; HP1ve, subjects infected with H. pylori; HP2ve, subjectsnot infected with H. pylori

*Corresponding author. Tel.: 144-20-8383-2055; fax: 144-20-8383-2066.E-mail address: [email protected] (S. Murray).

0009-8981/00/$ – see front matter 2000 Elsevier Science B.V. All rights reserved.PI I : S0009-8981( 00 )00357-0

182 S. Murray et al. / Clinica Chimica Acta 301 (2000) 181 –192

concentrations may exist around a site of infection. 2000 Elsevier Science B.V. All rightsreserved.

Keywords: H. pylori; Na-Methylhistamine; Gastric juice; Gastric biopsy; Gastrin

1. Introduction

Helicobacter pylori is a Gram negative, catalase positive microaerophilewhich colonises the stomach at the surface of the gastric epithelial cells withinthe mucus /bicarbonate layer [1]. Approximately 50% of the global population isbelieved to be infected with the bacterium but infection is not evenly distributed:it is much more common in less developed countries and in lower socio-economic groups [2,3]. While many infected hosts are asymptomatic, a small butsignificant fraction suffers from gastrointestinal disease and there are now strongassociations between infection with H. pylori and chronic, active gastritis [4],gastric and duodenal ulceration [5,6], gastric adenocarcinoma [7,8] and gastriclymphoma [9].

Colonisation by the bacterium is associated with alterations in acid secretionwhich probably contribute to the pathogenesis of the associated disease [10,11].The abnormalities of acid secretion can be corrected by eradication of H. pylori,suggesting that they are due to the infection, but the mechanisms responsible forthese effects are not known [12]. Recent work has suggested that the compoundNa-methylhistamine (Na-MH) might be involved [13]. Na-MH is best knownas an H receptor agonist [14] but is also active on H and H receptors [15,16].3 1 2

H and H receptors are present on gastric endocrine and exocrine cells [13,17]2 3

and, therefore, if Na-MH is present in gastric juice at sufficient concentrations,it could affect gastric physiology via histamine receptor activation.

In humans, histamine is normally metabolised to the biologically inactiveNt-methylhistamine by the enzyme histamine Nt-methyltransferase. However, ithas been known for some time that mammalian cells contain a distinct histamineNa-methyltransferase (indolethylamine N-methyltransferase) which has thecapacity to produce Na-MH [18]. Courillon-Mallet and colleagues have shownthat such an enzyme activity is present in gastric biopsies from human subjectscolonised by H. pylori, as well as in cultured strains of the bacterium itself, butthat the activity is very low in tissue from non-infected patients and frominfected patients where H. pylori has been eradicated by drug treatment [13].These workers, using a radioenzymatic assay, also identified Na-MH in gastricmucosal biopsies from infected patients and in cultured strains of H. pylori, andsuggested that Na-MH produced by H. pylori could alter the normal regulationof acid secretion in the stomach and cause gastrointestinal disease.

S. Murray et al. / Clinica Chimica Acta 301 (2000) 181 –192 183

We have developed an assay for Na-MH in biological samples based on gaschromatography mass spectrometry that has been described in detail elsewhere[19]. Using this methodology, analysis of gastric juice from a small number ofinfected patients confirmed the presence of the amine in two of nine samples.The aims of this study were to extend the analysis of gastric juice into a largersubject group, as well as examining gastric tissue and cultured strains of H.pylori for the presence of Na-MH. Even if Na-MH is present in the stomach ofinfected subjects, it is still uncertain whether the compound has any physiologi-cal effect on human gastric tissue in vivo, since the relevant receptors arelocated on the basolateral membrane of epithelial cells whilst the bacteria arepresent in the gastric lumen. Consequently, the effect of Na-MH on gastric acidsecretion in vivo has also been investigated using the technique of intragastrictitration.

2. Materials and methods

Na-Methylhistamine dihydrochloride was purchased from Calbiochem-Novabiochem Ltd (Nottingham, UK). Isolute CBA solid phase extractioncolumns were supplied by Jones Chromatography (Hengoed, UK) and proteinanalysis was carried out using a BCA protein assay reagent kit (Pierce andWarriner Ltd, Chester, UK). The H. pylori status of subjects from whom gastricjuice and biopsy tissue were collected, and of volunteers in the Na-MH infusion

14study, was determined using the C urea breath test described by Bardhan andcolleagues [20].

2.1. Gastric juice and biopsy tissue

Gastric juice was collected from seven subjects infected with H. pylori(HP 1 ve) and from nine individuals who were uninfected (HP 2 ve) through anAN 10 nasogastric tube (Anderson Products, Clacton-on-Sea, UK) that had beeninserted until the tip of the tube was 55 cm from the nose. Samples werecentrifuged to remove particulate material and the supernatants were stored at2 208C for analysis. Gastric biopsy samples (6–14 mg wet weight) werecollected from 15 HP 1 ve and five HP 2 ve subjects during routine endoscopicexamination using conventional fibreoptic endoscopy equipment. Standardbiopsy forceps were used to take two samples each from both the antrum and thecorpus of each patient. Antral biopsies were taken from the lesser curve of theantrum approximately 2 cm proximal to the pylorus and corpus biopsies weretaken from the greater curve of the mid body region. The biopsies wereimmediately frozen in liquid nitrogen and stored at 2 708C until assay.


2.2. Bacterial culture

Four well characterised strains of H. pylori (J104, J150, J178 and J190) wereobtained from Dr John Atherton (Nottingham University), and four otherclinical strains (37009, MB, SB, NC) were obtained from our laboratories. Thestrains J104 and J178 were Cag 1 ve and J150 and J190 were Cag –ve. Thebacteria were grown on Columbia agar (BBL) containing 7% horse blood(TCLS, Buckingham, UK) made selective by adding antibiotic supplements of20 mg/ l nalidixic acid, 2 mg/ l amphotericin B, and 3 mg/ l vancomycin. Theplates were incubated at 378C for 48 h in a microaerophilic environment byusing gas packs (Campypak, BBL, MD, USA) in gas jars (Oxoid, Basingstoke,UK). The colonies were removed from the plates and suspended in 10 ml ofphosphate buffered saline (PBS). The suspensions were centrifuged at 3000 gfor 2 min, the supernatants discarded and the bacterial pellets washed three timesin PBS. Bacterial number was determined as a suspension in PBS by opticaldensity from a previously calibrated nomogram. The four clinical isolates(37009, MB, SB, NC) were also cultured in BHI broth (Merck, Germany)enriched with 10% new born calf serum (Sigma, UK), and made selective byadding antibiotic supplements as above. The broths were incubated in an uprightposition with agitation on a rotary shaker in an atmosphere of 8% CO in air for2

72 h. One millilitre of the bacterial suspension was removed every 24 h andreplaced with 1 ml of fresh medium. The bacteria were pelleted by centrifuga-tion and both pellets and supernatant were stored at 2 808C until analysis of

8Na-MH. Three of these isolates, cultured in broth at 10 CFU/ml, were alsosupplemented with histamine (100 mg/ml) and incubated for 2 h. Samples wereremoved for assay at t 5 0, 2, 5, 10, 20, 30, 60 and 120 min.

2.3. Analysis of Na-MH

Na-MH was analysed in gastric juice, gastric tissue, plasma and bacteriausing a fully validated assay that has been published in detail elsewhere [19].Briefly, biopsy tissue samples (|10 mg wet weight) were homogenised inhydrochloric acid (0.1 mol / l, 100 ml) three times. The homogenate, or washed

7 8bacterial pellets (10 –10 CFU), were then disrupted with an MSE Soniprep 150ultrasonic disintegrator (Fisher Scientific, Loughborough, UK) at mid power for3330 s under ice cooling. Plasma was deproteinised with ice cold methanolbefore extraction. Tissue or bacterial homogenates, plasma or gastric juice(acidified to pH 1) were centrifuged, washed with hexane and taken to drynessunder vacuum. Following methanol extraction, samples were further purified onIsolute CBA columns. Samples were applied in ammonia (pH 8) /methanol(20:1, v /v), and Na-MH was eluted with 1% trifluoroacetic acid. Na-MH was


quantified by gas chromatography electron capture mass spectrometry on aFinnigan-MAT 4500 gas chromatograph mass spectrometer. Na-MH wasconverted to its di(3,5-bistrifluoromethylbenzyl) derivative for analysis on aDB5 capillary column (15 m30.25 mm) using helium as carrier gas. The gaschromatograph oven temperature was held at 1508C for 1 min, then raised to

212708C at 208C min . Injections were made using a Grob-type capillary injector2operated in the splitless mode at 2708C. [ H ] Na-MH, prepared by reacting7

2 2[ H ] histamine hydrochloride with [ H ] methyl iodide, was used as internal4 3

standard in the assay and this compound was added to each sample immediatelybefore extraction.

2.4. Na-MH infusion

The joint Ethics Committee of Imperial College School of Medicine and theHammersmith Hospitals Trust approved this clinical study. Nine subjects (fourfemale, five male, ages 20–53, mean age 27) were recruited that were uninfectedwith H. pylori. They were asked to abstain from alcohol and cigarettes for 24 hprior to the study day and then, after an overnight fast, each subject received 120mg terfenadine. One hour later, an AN 10 nasogastric tube was passed until thetip of the tube was 55 cm from the nose. Residual gastric juice was removed byaspiration, then 500 ml of water containing glucose (58 g/ l) was instilled intothe gastric lumen using a Microlab M programmable dilutor /dispensor(Bonaduz, AG Bonaduz, Switzerland). The latter was used to aspirate anddispense 30 ml of gastric contents eight times per minute and to mix the gastriccontents. The pH of the gastric contents was continuously sampled by aPHC4406 pH electrode and PHM82 pH meter which were connected to an ABU80 autoburette and TTT 80 titrator (Radiometer Copenhagen, Crawley, UK).These were set to maintain an intragastric pH of 5.0 by infusing 0.1 mol / lNaOH into the gastric lumen through a second channel in the nasogastric tube.Thus acid secretion in response to the test solutions was measured by continuousintragastric titration using a previously validated procedure [21]. After a basalperiod of 30 min, the glucose solution was replaced with 500 ml of 5.8%

26glucose containing 10 mol / l Na-MH and acid secretion was measured overthe next 30 min. This was followed by a 30 min infusion with glucose solution

25only, then 5.8% glucose containing 10 mol / l Na-MH was infused for afurther 30 min. Venous blood samples were taken from an indwelling cannula atthe beginning of the study and at the end of each 30 min period into 10 mlstandard lithium heparin vacutainer vials containing 200 ml of aprotinin (1.4mg/ml). Blood was immediately centrifuged, then plasma was separated andstored at 2208C for analysis of gastrin by RIA [22].


2.5. Statistical analysis

Two-way ANOVA was used with Fisher’s LSD test. P , 0.05 was consideredto be significant.

3. Results

Na-MH was found to be present in five of the seven gastric juice samplesobtained from subjects infected with H. pylori. The concentration in four ofthese subjects was between 5 and 15 pmol /ml but, in one individual, Na-MHlevels were over 10-fold higher at 180 pmol /ml (Fig. 1a). There was nodetectable Na-MH (,0.4 pmol /ml) in gastric juice from two of the sevenHP1ve subjects nor in any of the nine uninfected controls. In the seven fundicand 15 antral tissue biopsies taken from HP1ve patients, there was no evidencefor Na-MH (,0.4 pmol) in any of these samples, nor in any of the 10 biopsiestaken from uninfected subjects (Fig. 1b). In each case, the presence of thedeuterium labelled internal standard in the selected ion chromatograms con-firmed that these negative values did not arise through any loss of Na-MHduring the extraction and derivatisation procedures.

To investigate whether H. pylori was the source of the Na-MH, eight clinical7isolates of the bacterium were cultured on agar. The live bacteria (between 10

8and 10 CFU) were removed from the plates and, following washing, weredisrupted and analysed for Na-MH. No Na-MH (,0.4 pmol) was found in anyof the samples. Four isolates were cultured in broth and, again, there was nodetectable Na-MH in the broth or the bacterial pellet at any time during theculture. To study the ability of H. pylori to synthesise Na-MH, three of the

8bacterial strains (10 CFU/ml) were cultured for up to 2 h in broth in thepresence of 100 mg/ml of histamine. There was no evidence of conversion ofhistamine to Na-MH by any of the three bacterial strains at any time up to 120min.

During the intragastric instillation of Na-MH in glucose, there were noobvious changes in physiological parameters and no evidence of flushing.Plasma Na-MH was below the limit of detection of the assay (0.4 pmol /ml)throughout the infusions. In the nine HP2ve subjects, basal acid secretionranged from 0.6 to 10.5 mmol /h with a mean6S.E.M. of 3.661.0 mmol /h.While no significant change in acid secretion was observed during infusion with

26 2510 mol / l Na-MH and then with glucose solution only, infusion with 10mol / l Na-MH resulted in a two-fold increase in acid secretion to 6.361.4mmol /h which was significantly different to the basal value (Fig. 2a). The basalplasma gastrin concentration in the nine subjects was 10.061.4 fmol /ml(mean6S.E.M.) which was not significantly different from the fasting value


Fig. 1. (a) Na-Methylhistamine was present in gastric juice from five of seven patients infectedwith H. pylori (HP1ve) but was absent in nine uninfected subjects (HP2ve). The limit ofdetection of the assay was 0.4 pmol /ml. (b) Na-Methylhistamine could not be detected in either22 gastric mucosa biopsies (seven fundic, 15 antral) from H. pylori infected patients (HP1ve) orin 10 biopsies from uninfected subjects (HP2ve). The limit of detection of the assay was 0.4 pmolfor a 10 mg tissue biopsy. A representative selected ion chromatogram is shown inset. Thepresence of the internal standard peak (int. std., bottom channel) demonstrates that these negativevalues do not arise from loss of analyte. The elution position of Na-methylhistamine (top channel)is arrowed, showing that the analyte is below the limit of detection of the assay.


Fig. 2. (a) Effect of intragastric infusion of Na-methylhistamine (Na-MH) on gastric acid25secretion in nine uninfected subjects. Acid secretion was significantly increased by 10 mol / l

Na-MH (P , 0.05, ANOVA). Data are shown as mean6S.E.M. Plasma Na-MH (not shown) wasbelow the limit of detection of the assay (,0.4 pmol /ml) during the infusions. (b) Plasma gastrin

25concentrations in nine uninfected subjects did not change significantly during infusion with 10mol / l Na-MH. Data are shown as mean6S.E.M.

(9.861.9 fmol /ml). Gastrin concentration did not change significantly duringthe infusions with glucose, whether Na-MH was present or not, with a finalplasma concentration of 10.562.2 fmol /ml being obtained (Fig. 2b).

4. Discussion

An assay for Na-MH in biological samples based on gas chromatographymass spectrometry has been previously developed and validated [19]. The use of


capillary column gas chromatography and selected ion monitoring mass spec-trometry in the negative ion mode gives a highly specific and sensitive methodof analysis. Accurate quantification is obtained with the use of a deuteratedanalogue of Na-MH as an internal standard that compensates for losses duringthe extraction and derivatisation procedures. With this assay, a limit ofquantification of 0.8 pmol Na-MH and a limit of detection of 0.4 pmol Na-MHis obtained for samples of gastric juice (1 ml), plasma (1 ml), gastric tissue (10

8mg wet weight) and bacteria (10 CFU) [19].Na-MH was detected in the gastric juice of five of seven patients infected

with H. pylori, in agreement with our previous data [19], but was absent insamples from uninfected subjects. These findings suggest that there is anassociation between bacterial colonisation and Na-MH production in vivo. Theabsence of Na-MH in gastric juice from two of the infected subjects could bedue to decreased synthesis through inhibition or downregulation of an Na-methyltransferase, or increased metabolism (Na-MH is known to be a substratefor histamine N-methyltransferase [23] and monoamine oxidase). Alternatively,undetectable concentrations of Na-MH in gastric juice could be due to lowlevels of bacterial colonisation in these two patients. There is one previousreport of Na-MH in gastric juice where concentrations of 0.4860.04 ng/ml(3.84 pmol /ml) were measured in healthy controls [24]. It is surprising that aproduct that is now associated with H. pylori colonisation in humans should bedetected in the absence of the bacterium. However, this work appeared at a timewhen H. pylori had just been identified and it is possible that some of thesubjects were undiagnosed HP1ve, or alternatively that the radioenzymaticassay that was used for the measurement of Na-MH lacked specificity. We wereunable to detect Na-MH in 22 gastric tissue biopsies from patients infected withH. pylori and the presence of the internal standard peak in each selected ionchromatogram clearly indicates that this was not due to loss of analyte (Fig. 1b).Thus our data is in contrast to that of Courillon-Mallet and colleagues [13] whoreported levels of 1–13 pmol Na-MH/mg protein in 11 of 13 biopsies of antralmucosa. The limit of detection of our assay is sufficiently low to have detectedsuch levels had they been present in our tissue samples.

Courillon-Mallet et al. confirmed histologically the colonisation of theirbiopsy samples by H. pylori prior to further analysis and it is possible that ourtissue samples were not collected from heavily infected areas of the stomach.Hence, in order to investigate the direct association of Na-MH with thebacterium, eight clinical strains of H. pylori were cultured on agar but, in eachcase, no Na-MH could be detected within the bacterial pellet. As H. pylori isnot known to store histamine or its metabolites, the possibility of the excretionof this hydrophilic, low molecular weight species into the culture medium wasinvestigated by culturing four of the strains of H. pylori in broth. No Na-MHcould be detected either in the bacterial pellet or in the broth and the further


addition of histamine to the medium did not result in the formation of Na-MHunder culture conditions where ,0.005% conversion of the substrate wouldhave been detected. These experiments suggest that, under culture conditions,even with a large exogenous source of histamine, H. pylori either does notsynthesise Na-MH or that subsequent metabolism of this product is extremelyrapid. In either case, it would appear questionable as to whether the bacterium iscapable of excreting significant amounts of Na-MH in vivo.

Acid secretion was measured by intragastric titration using the method ofMaxwell and colleagues [21] where glucose was used as vehicle in preference tosaline to minimise osmotic effects due to stomach emptying. Although themethod is reported to give reasonable reproducibility for basal measurementsover 3.5 h, variability in the technique probably arises from the difficulty inachieving complete mixing during the infusions. This may have limited our

26ability to detect any small change in acid secretion caused by instillation of 1025mol / l Na-MH. At the higher concentration of 10 mol / l, however, Na-MH

causes a significant two-fold increase in acid secretion in human subjects invivo. This effect is unlikely to be mediated through absorption of Na-MH intothe blood stream, as plasma levels of Na-MH were undetectable (,0.4 pmol /ml) during all stages of the infusion. As subjects were pre-treated withterfenadine (120 mg), any change in acid secretion due to the effect of Na-MHon histamine H receptors can be discounted. The two-fold increase observed in1

acid secretion is not associated with a change in plasma gastrin, which is incontrast to the effect of a peptone meal where an equivalent increase in acidsecretion is accompanied by a 10–15 fmol /ml increase in gastrin [21]. It ispossible that Na-MH acts directly upon the parietal cells from the gastric lumen,probably via H receptor activation, in agreement with earlier work on rabbit2

parietal cells in vitro [16]. Any concomitant inhibitory effects on acid secretionthrough H receptor activation in these subjects cannot be discounted as these3

may have been masked by the magnitude of the stimulatory response.Therefore, although the presence of Na-MH in gastric juice appears to be

associated with infection by H. pylori, we can find no evidence that this productis generated directly by the organism, or indeed is stored in gastric tissue. Itappears more likely that Na-MH formation is a result of the inflammatoryresponse that is known to occur in the gastric mucosa of infected patients. Our

25infusion experiments showed that a high concentration (10 mol / l) of Na-MHwas required to have a significant effect on acid secretion in the stomach. Thisconcentration is nearly two orders of magnitude above the maximum con-centration of Na-MH found in the gastric juice of our HP1ve subjects (180

27pmol /ml or 1.8310 mol / l). Hence, although local concentrations of Na-MHin the stomach may be higher, our data would suggest that Na-MH does nothave a major role in altering acid secretion in vivo.


Acknowledgements

We gratefully acknowledge financial support for Philip Bliss from theDigestive Disorders Foundation, Alimentary Pharmacology and TherapeuticsTrust. We also thank Dr John Atherton, Nottingham, for providing clinicalstrains of H. pylori.

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