Bechi, Paolo

Cianchi, Fabio

Fantappi&#232

Fiorillo, Claudia

Mazzanti, Roberto

Nassi, P.

English

Florence Research

Original articleReflux and pH: ‘alkaline’ components are not neutralizedby gastric pH variationsP. Bechi1, F. Cianchi1, R. Mazzanti2, O. Fantappie`2, C. Fiorillo3, P. Nassi31Clinica Chirurgica Generale; 2Dipartimento di Medicina Interna; 3Dipartimento di Scienze Biochimiche,Universita` di Firenze, Florence, ItalySUMMARY. The ability of the ‘alkaline’ components of reflux to cause harm in vivo is still open to debate,although these components have been shown in vitro to be capable of damaging the mucosa. The precipitation ofbile acids and lysolecithin that occurs at low pH values is the main reason for questioning in vivo mucosal damage.This study was undertaken to determine the composition of gastric aspirates at dierent original pH values and thedegree of solubility of the alkaline components when pH modifications are artificially induced. The samples forchemical analysis were collected from indwelling nasogastric tubes after surgical procedures that did not involve theupper gastrointestinal tract. Bile acid and lysolecithin concentrations were assessed by means of dedicated methods.Thirty-five samples were available for bile acid evaluation and 27 for lysolecithin evaluation. Bile acid andlysolecithin assessments were repeated after pH adjustment at 2, 3.5, 5.5 and 7. For easier assessment of the results,three ranges of the original pH were selected (pH < 2, 2 £ pH < 5, pH ‡ 5). For each pH range, results werepooled together and compared with those in the other pH ranges. Bile acid concentrations were 113  48,339  90 and 900  303 (mean  s.e.m. lmol/L), respectively, in the three groups selected on account of thedierent original pH values. Dierences were significant (p < 0.001). Both taurine- and glycine-conjugated bileacids were represented even at pH < 2. No major dierences were observed in bile acid concentration with theartificially induced pH variations. Lysolecithin concentrations were 5.99  3.27, 30.80  8.43 and108.37  22.17 (mean  SEM lg/ml), respectively, in the three groups selected on account of the dierentoriginal pH ranges. Dierences were significant (p < 0.001). No significant dierences in lysolecithinconcentration were detected with the artificially induced pH variations. In conclusion, both bile acids andlysolecithin are naturally represented in the gastric environment even at very low pH values, although theirconcentrations decrease on lowering of the naturally occurring pH. Given the concentration variability of bile acidsand lysolecithin, further studies are needed to assess the minimal concentration capable of mucosal damage in vivo.INTRODUCTIONThe pathogenetic role of acid reflux (HCl and pepsin)in esophageal mucosal damage has been clarified by anumber of both clinical and experimental studies.1–3In contrast, the harmful eects of the so-called‘alkaline’ components of reflux and even theircapability of refluxing into the esophagus are stillquestioned4,5 and the problem of duodenogastroeso-phageal reflux (DGR) remains unsettled. Manystudies have examined DGR from an experimentalpoint of view. They have unequivocally shown that,among all the components which potentially refluxinto the esophagus, bile acids, lysolecithin and trypsinare capable of causing esophageal mucosal damage ina variety of animals.6–8 However, their harmfuleects seem to be heavily conditioned by the envi-ronmental pH. In vitro experimental studies haveshown that trypsin is inactivated at pH < 6.5 andlysolecithin at pH < 4. Unconjugated, glycoconju-gated and tauroconjugated bile acids are precipitatedand, therefore, inactivated at pH < 5, 4 and 2respectively.More recently, an in vitro study has compared thepH dependency of the solubility of the commerciallyavailable bile acids, which have been used in all theabove-mentioned animal models, with that of the bileacids in natural human bile.9 This study seems toAddress correspondence to: P. Bechi, Istituto Clinica ChirurgicaGenerale e D.C., Viale Morgagni 85, 50134 Firenze, Italy.Tel: (+39) 55 4277566; Fax: (+39) 55 422013351Diseases of the Esophagus (2000) 13, 51–55Ó 2000 ISDE/Blackwell Science Asiashow that, in contrast to commercial bile acids,natural bile acids are all precipitated and thereforeinactivated at pH < 4.9Thus, as a preliminary step to improve theunderstanding of the noxious eects of ‘alkaline’reflux, the purpose of the present study was to assessthe composition of gastric aspirates at dierentoriginal pH and to evaluate in vitro the solubility(as an indirect index of the harmful potential) ofdierent naturally occurring Ôalkaline’ components ofreflux as a function of pH.MATERIALS AND METHODSAll reagents were of the highest purity available. HCl,KOH, hemoglobin (bovine), pepsin (porcine stomachmucosa), lysophosphatidylcholine (bovine liver), tri-chloroacetic acid, methanol, chloroform, NaCl,EDTA and conjugated bile acid standards werepurchased from Sigma-Aldrich (Milan, Italy), silicagel H and molybdenum blue reagent from Alltech(Milan, Italy), and high-performance liquid chroma-tography (HPLC) reagents from Merck (Frankfurter,Germany).Natural components of reflux were obtained fromindwelling nasogastric tubes after surgical proceduresthat did not involve the upper gastrointestinal tract.Aspirates were collected in the morning from fastedpatients. A quantity of 20–30 ml was collected eachtime and placed in non-transparent-walled containersto avoid light interference. Aspirates were immedi-ately processed. The original pH was measured usinga pH-meter (Orion, model 420A). Then, samples wereadjusted to pH 1, 2, 3.5, 5.5 or 7 by adding to theoriginal aspirate HCl or KOH. The amount of addedHCl or KOH (5 M) ranged between 5 and 50 ll.After pH adjustment, all samples were centrifuged at1000 r.p.m. for 5 min in order to separate theprecipitate which was present in all the samples,and proper amounts of each sample at dierent pH(the original pH included) were processed for theassessment of each individual component.For a clearer statistical assessment of the results,three dierent ranges of the original pH were selected(pH < 2, 2 £ pH < 5, pH ‡ 5) and the resultsobtained in the evaluation of each ‘alkaline’ compo-nent for each individual sample were pooled togetherwith those in the other samples with original pH inthe same range and compared with those in the otherpH ranges.Bile acidsSamples for bile acid assessment were diluted 1:2 withwater and processed for HPLC. A liquid chromato-graph (model 235c, Perkin Elmer, Florence, Italy)was used. An Altex Ultrapore Beckman column,4.6 mm · 25 cm, 5 lm particle size (BeckmanInstruments, Fullerton, CA, USA) was used. Thesolvent system was methanol/0.01 M KH2PO4 75:25.To each liter of this mixture, 4.2 ml of 5 N NaOHwere added. The pH was then adjusted to pH 5.35 byaddition of H3PO4. The solvent was filtered through a0.45-lm nylon filter before use. The solvent flow rateof 0.7 ml/min produced operating pressures of13 800–15 180 kPa.LysolecithinLysophosphatidylcholine (LPC) was assayed accord-ing to the method of Tsai et al.10 Before lipidextraction, 2 mM EDTA and 5 mM CaCl2 wereadded to the samples of gastric aspirates to inhibitphospholipase A activity. Lipids were extracted fromsamples (500 ll) with methanol–chloroform (1:2).After evaporation under a stream of nitrogen, sam-ples were redissolved in a small quantity (50–100 ll)of methanol–chloroform (1:2) and spotted in dupli-cate on a thin-layer chromatography (TLC) plate(silica gel H, thickness 0.5 mm) along with a knownamount of a standard LPC dissolved in chloroform.The chromatogram was developed in chloroform–methanol–water (65:24:4, v/v) and, after the solventfront had ascended 20 cm (about 90 min), the platewas removed, air dried and sprayed with molyb-denum blue reagent. Blue spots, indicating phospho-lipids, were immediately visible. The spots werequantified by densitometry (software QUANTISCANFOR WINDOWS). LPC concentrations were estimatedby comparing the areas of the samples with those ofthe standards. All results were expressed in lg/ml.RESULTSBile acidsDierent amounts of bile acids were detected in all ofthe 21 samples with original pH ‡ 2 and in eight outof the 14 (57.2%) samples with original pH < 2. Thetotal concentration in the original samples wasclosely dependent on their pH (Table 1), and dier-ences between the three groups selected on account ofthe original pH (pH < 2, 2 £ pH < 5, pH ‡ 5) weresignificant. Both taurine- and glycine-conjugated bileacids were represented in all the three groups, with apredominance of the glycine conjugated even in theTable 1. Bile acid concentration in the samples pooled together onaccount of dierent ranges of their original pHTotal concentration (lmol/l)Mean  s.e.m.pH < 2 (n = 14) 113  48*2 £ pH < 5 (n = 11) 336  90*pH ‡ 5 (n = 10) 900  303** p < 0.001 (ANOVA test).52 Diseases of the Esophagussamples with original pH < 2 (Fig. 1). With theartificially induced progressive pH increase, no rele-vant dierences in total bile acid concentration weredemonstrated for samples with original pH < 2(Fig. 2). In the samples with original pH rangingbetween 2 and 5, no major dierences either atincreased or at decreased pH were found (Fig. 2). Inthe samples with original pH ‡ 5, a more complextrend was observed. Stationary values (in comparisonwith values at original pH) were detected at interme-diate pHs with consistent increases both at extremelyhigh and low pH values, although no significantdierences were reached (Fig. 2).LysolecithinLysolecithin in dierent amounts was detected in sixof the 12 samples with pH < 2, in five of the sevensamples with intermediate pH (‡2 and <5) and in alleight samples with pH ‡ 5. The lysolecithin concent-ration in the original samples was closely dependenton pH, as shown by the dierent concentrations thatwere found in the dierent ranges of original pH(Table 2). If the threshold below which lysolecithin isinactivated (pH 4) is considered, eight of the 18samples with pH £ 4 contained no lysolecithin,whereas dierent amounts of lysolecithin were shownin all the nine samples with pH >4. Mean (s.e.m.,lg/ml) lysolecithin concentration was 17.59 (7.58)in the former group and 99.96 (20.54) in the latter(p < 0.001, ANOVA test). With the artificially inducedpH changes, no significant dierences in lysolecithinconcentration were detected in any of the threegroups distinguished upon the basis of the originalpH, although a trend towards a slight increase wasobserved in all the three groups at the upper values ofthe pH scale (Fig. 3).DISCUSSIONThe main finding of this study is that variableamounts of each alkaline component of reflux arepresent even at pH values around 1.Alkaline components have been shown both clin-ically and experimentally6–8 to be harmful to theesophageal mucosa. Clinical evidence is representedby the very severe esophagitis that follows totalgastrectomy (suggesting complete anacidity) withesophagoduodenal anastomosis or, more recentlynoted, with esophagojejunal anastomosis without acomplete exclusion of the esophagus from ‘alkaline’reflux.11,12 The frequent occurrence of esophagitiswas the main reason for abandoning these types ofreconstruction. Moreover, esophagitis has beenshown in another situation with complete anacidity,i.e. in the presence of atrophic gastritis associatedwith pernicious anemia.13Fig. 1—Concentration of the dierent bile acids in the samplespooled together at dierent original pH values. TCA, taurocolicacid; GCA, glycocolic acid; TCDCA, taurochenodesossicholateacid; TDCA, taurodesossicholate acid; GCDCA,glycochenodesossicholate acid; GDCA, glycodesossicholate acid.Fig. 2—Total bile acid concentration at dierent artificiallyinduced pH values.Table 2. Lysolecithin concentration in the samples pooled togetheron account of the dierent ranges of their original pHTotal concentration (lg/ml)Mean  s.e.m.pH < 2 (n = 12) 5.99  3.27*2 £ pH < 5 (n = 7) 30.80  8.43*pH ‡ 5 (n = 8) 108.37  22.17**p < 0.001 (ANOVA test).Fig. 3—Lysolecithin concentration at dierent artificially inducedpH values.Gastric pH and Ôalkaline’ reflux 53Glyco- and tauroconjugated bile acids have beenreported to be harmful to the esophageal mucosa atacid pH (pH below 4 and even down to pH 2 fortauro-conjugated).6 In contrast, deconjugated bileacids are more damaging at higher pH levels (pHbetween 5 and 8).6 Lysolecithin has also been shownto cause mucosal damage when in an acidicenvironment,14 whereas damage caused by trypsinis relevant only in the absence of acid.15 Whereas forbile acids the concentration threshold is establishedat 1 mmol/l,16 for lysolecithin the minimal concen-tration above which mucosal damage occurs isunknown.However, all these experimental studies haveutilized commercial products inmono- or two-productmixtures. This, indeed, does not reproduce thenaturally occurring situation. A more realistic experi-mental model should utilize gastric aspirates withknown composition because the dierent alkalineand acid components of reflux seem to reciprocallyinteract and synergistically enhance their eects onthe esophageal mucosa. This means that the minimalnoxious concentrations of bile acids and lysolecithinmay be much lower in vivo than in vitro. However, apreliminary condition for the harmful potential ofeach Ôalkaline’ component is its permanence insolution. In this respect, our findings, which showthat bile acids and lysolecithin are still in solutiondown to pH 1, seem important. These data appeareven more relevant in view of the previously reportedevidence of a nearly complete precipitation of thenaturally occurring bile acids in human bile atpH < 4.9 Moreover, they suggest that bile acidsand lysolecithin may be important for mucosaldamage as they are both in solution and notinactivated at low pH. Although it has not beenstudied in the present paper, trypsin is completelyinactivated at low pH, exerts its proteolytic activity atpH around 7, and is probably the main, if not theonly, damaging agent of reflux in achlorhydricconditions, such as total gastrectomy and atrophicgastritis associated with pernicious anemia.Therefore, because this study has shown thatconcentrations of bile acids and lysolecithin decreasewith lowering of pH and that variable concentrationscan be found also at very low pH, the principalunresolved problem is whether at pH around 2 (usualgastric environmental pH) their concentration is stillsuch as to be capable of mucosal damage. However,although up to the present time values for minimalnoxious concentrations have been obtained withcommercial products in mono- or two-product mix-tures, studies with gastric aspirates, as opposed tocommercial products, are needed. This because theinteraction occurring in vivo between the dierentÔalkaline’ and acid components enhances the harmfulpotential of each of them. As yet, it is not knownwhat is the in vivo threshold concentration for eachcomponent that is capable of damaging the mucosawhen reflux consists of a mixture of HCl, pepsin, bileacids, trypsin and lysolecithin. The harmful potentialof the mixture depends upon the fact that each singlecomponent interacts with the others and aects theirphysicochemical properties, such as solubility, lipo-philicity and detergent capability. Moreover, evenmore complex interactions take place. In this respect,pH again plays a major role. Luminal acidity isessential to determine intracellular bile acid accumu-lation, which in turn seems the main determinant forbile acid cytotoxic capability.17In addition to the persistence in solution at low pHvalues of bile acids and lysolecithin, another findingof the present study is the role of pH in determiningthe concentration of each component. More specifi-cally relevant in this respect is the original pH, i.e. thepH at the time of aspiration of gastric content. Onlyminor concentration changes are caused followingartificially induced pH variations. This findingsuggest that early stabilization of physicochemicalproperties of the gastric content occurs, and thatthese are not later modified to any great extent by pHchanges.If we consider that all the Ôalkaline’ componentsmay be represented in the refluxate, and that boththeir concentration (as shown in the present paper)and capacity to cause damage (as previously exper-imentally shown) depend upon the original pH, theadvantages of simultaneously detecting ‘alkaline’reflux and acidity by means of simultaneous Bilitec-and pH monitoring are patent.18In conclusion, in addition to the well-knownfinding that, for about 2.5 h during the 24-h day,gastric pH is above 4, even in normal subjects, ourfinding that the ‘alkaline’ components of reflux arepresent in solution even at low pH values supportsthe possibility of interaction of the alkaline compo-nents with esophageal and gastric mucosa even inprimary refluxers.References1. Goldberg H I, Dodds W J, Gee S, Montgomery C, Zboralske FF. Role of acid and pepsin in acute experimental esophagitis.Gastroenterology 1969; 56: 223–230.2. Salo J A, Lehto V P, Kivilaakso E. Morphological altera-tions in experimental esophagitis. Dig Dis Sci 1983; 28:440–448.3. Evander A, Little A G, Riddel R H, Walthers B, Skinner D B.Composition of the refluxed material determines the degree ofreflux esophagitis in the dog. Gastroenterology 1987; 93: 280–286.4. Mittal R K, Reuben A, Whitney J O, McCallum R W. Do bileacids reflux into the esophagus? A study in normal subjects andpatients with gastroesophageal reflux disease. Gastroenterology1987; 92: 371–375.5. Gotley D C, Morgan A P, Ball D, Owen R W, Cooper M J.Composition of gastroesophageal refluxate. Gut 1991; 32:1093–1099.6. Harmon J W, Johnson L E, Maydonovitch C I. Eect of acidand bile salts on rabbit esophageal mucosa. Dig Dis Sci 1981;26: 65–72.54 Diseases of the Esophagus7. Salo J A, Kivilaakso E. Role of bile salts and trypsin in thepathogenesis of experimental alkaline esophagitis. Surgery1983; 94: 525–531.8. Lillemoe K D, Johnson L E, Harmon J W. Alkalineesophagitis: a comparison of the ability of components ofgastroduodenal contents to injure rabbit mucosa. Gastroen-terology 1983; 85: 621–628.9. Barthlen W, Liebermann-Meert D, Feussner H, Stein H J.Influence of pH on bile acid concentration in human, pig andcommercial bile: implications for ‘alkaline’ gastro-esophagealreflux. Dis Esoph 1994; 7: 127–130.10. Tsai M Y, Cain M, Josephson M W. Improved thin-layerchromatography of disaturated phosphatidylcholine inamniotic fluid. Clin Chem 1981; 27: 239–242.11. Nakayama K. Evaluation of the various operative methods fortotal gastrectomy. Surgery 1955; 40: 488–502.12. Morrow D, Passaro E R. Alkaline reflux esophagitis after totalgastrectomy. Am J Surg 1976; 132: 287–291.13. Orlando R C, Bozymsky E M. Heartburn in perniciousanaemia: a consequence of bile reflux. N Engl J Med 1973;125: 522–524.14. Salo J, Kivilaakso E. Role of luminal H+ in the pathogenesisof experimental esophagitis. Surgery 1982; 92: 61–68.15. Kivilaakso E, Fromm D, Silen W. Eect of bile salts andrelated compounds on isolated esophageal mucosa. Surgery1980; 87: 280–285.16. Kiro G K, Mukherjhee T M, Dixon B, Devitt P G,Jamieson G G. Morphological changes caused byexposure of rabbit oesophageal mucosa to hydrochloricacid and sodium taurocholate. Aust N Z J Surg 1987; 57:119–126.17. Harmon J W, Bass B L, Batzri S. Alkaline reflux gastritis.Mechanism of Injury. Probl Gen Surg 1993; 10: 201–206.18. Bechi P, Pucciani F, Baldini F, et al. Long-term ambulatoryenterogastric reflux monitoring. Validation of a new fiberoptictechnique. Dig Dis Sci 1993; 38: 1297–1306.Gastric pH and Ôalkaline’ reflux 55

Reflux and pH: &apos;alkaline&apos; components are not neutralized by gastric pH variations

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Reflux and pH: 'alkaline' components are not neutralized by gastric pH variations

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Reflux and pH: &apos;alkaline&apos; components are not neutralized by gastric pH variations

Abstract

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Florence Research

Reflux and pH: 'alkaline' components are not neutralized by gastric pH variations