- Email: [email protected]
Bile Damage to the Gastric Mucosal Barrier: The Influence of pH and Bile Acid Concentration
Vol. 61, No.2 Printed in U.S .A.
GASTROENTEROLOGY
Copyright@ 1971 by The Williams & Wilkins Co.
BILE DAMAGE TO THE GASTRIC MUCOSAL BARRIER: THE INFLUENCE OF pH AND BILE ACID CONCENTRATION R. B. BLACK, M.B., F.R.A.C.S., D. HOLE, AND J. RHODES, M.D., M.R.C.P.
Welsh National School of M edicine and the M edical Research Council Epidemiology Unit, Cardiff, Wales
The damaging effect of human bile on the gastric mucosal barrier has been studied in 3 dogs with Heidenhain pouches. Transmucosal potential difference (PD) and net fluxes of sodium and hydrogen ion have been examined before, during, and after contact with bile solutions at pH 2, 4, and 8 and bile acid concentrations of 20, 10, 5, and 0 mM. Bile caused a profound fall in PD which partly recovered during the test. Changes in PD were greatest at the highest concentrations of bile but were not significantly different with solutions of different pH. H + back diffusion from, and Na + gain by, the pouch were significantly increased after contact with higher bile concentrations and in the case of H + ion this effect was greatest at pH 2 and least at pH 8. The increased Na + output was not accompanied by a proportionate increase in fluid volume, suggesting that this effect was a result of ionic diffusion rather than secretion of a hypothetical nonparietal component. Normal gastric mucosa is relatively impermeable to sodium and hydrogen ions. 1 • 2 This mucosal barrier to ionic movement can be broken by a variety of agents, H including bile acids. 5 Some of these agents also reduce the electrochemical potential difference, normally found across gastric mucosa. 2 • 6 • 7 The damaging effect on the stomach of taurine-conjugated bile acids is similar for acid and neutral solutions. 5 However, the influence of pH on the damaging effect of human bile, which contains both glycine and taurine conjugates, 6 has not been exReceived October 5, 1970. Accepted March 18, 1971. Address requests for reprints to: Dr. J . Rhodes, Medical Unit, Royal Infirmary, Cardiff, Wales. Dr. Black was in receipt of a Medical Research Council grant (G969/7/C) during the course of this study. The authors are grateful to Professor A. P. M. Forrest for encouragement and for the facilities of his department; to Mr. R. Marshall and his department for illustrations; to Mr. K. Hatt and Mr. C. Ryan for technical help; and to Mr. H. Kincaid for technical advice. 178
amined. This is of some importance since ionization and solubility of a mixture of these conjugates vary with pH. Glycine conjugates tend to precipitate from solution below pH 4, while taurine conjugates remain in solution at pH 2. 8 • 9 We have examined the effect of human bile on the gastric mucosa of canine Heidenham pouches using solutions of different pH and concentration. Measurements of transmucosal potential difference (PD) and transmucosal movement of sodium and hydrogen ions have been made.
Materials and Methods General
Vagally denervated separated (Heidenhain) pouches of the gastric fundus were prepared in 3 dogs which weighed between 12 and 14 kg. 1 0 Tests were performed after 2 months at intervals of not less than 2 days. Each dog maintained or gained weight during the period in which tests were performed. The experiments, which were similar to Davenport's 2 • 3 • • included three consecutive 30-min periods. In each period a solution was placed in the pouch and changes in volume and
August 1971
179
BILE DAMAGE TO GASTRIC MUCOSAL BARRIER
ionic (sodium and hydrogen) composition were determined. In the first and third periods, a standard acid solution was used. It was identical in all experiments, and contained 100 mN HCl and 54 mM NaCl (sp gr 1.002). In the second period, bile or control solutions were used. The difference between net fluxes in the first and third periods was a measure of the effect of bile on the mucosal barrier. An increased loss of H + ion from the solution, or gain of Na+ indicated damage to the mucosal barrier. The pouch was drained by a steel cannula connected to a polypropylene Y-tube. On one oblique limb of theY-tube a three-way tap was connected by tubing to a vertical open-ended glass tube (converted burette). The three-way tap had a syringe attachment through which the solutions were introduced. PD was measured by two open-ended polyvinyl leads (outside diameter, 2 mm; Portland Plastics Ltd., Hythe, Kent, England) filled with half-saturated potassium chloride in 1.5% agar. The luminal lead entered the pouch through the Y-connections; the reference lead was subcutaneous. The free end .of each lead was connected through a saturated KCl solution to a calomel half cell (Type RJ23, Electronic Instruments Ltd., Richmond, Surrey, England) and thence to a direct reading pH meter ('Vibron' Model 39A, Electronic Instruments Ltd.,) set to measure PD, which was recorded continuously (Model H Recorder, Control Instruments Ltd. , Birkenhead, ·Cheshire, England). Bile solutions. Ten liters of human bile were collected from patients with T-tube drainage of the common bile duct, ·and another 300 ml of concentrated bile was obtained from the gallbladders of seven cadavers and 2 patients with obstructive jaundice. A sample from all specimens was cultured; those with more than 250,000 organisms per ml were rejected. Two pools of bile were made ; one ofT-tube and one of concentrated gallbladder bile. The composition of bile from these sources is in table 1. Bile acids were separated by thin layer chromatography, 11 • 12 and concentrations were measured by the steroid dehydrogenase method. 13 • 14 Bile solutions of three concentrations (5, 10, and 20 mM) , each at three pH values (2, 4, and 8) were prepared. The pH and sodium concentration of the solutions were adjusted by adding HCl and NaCl; the final bile acid concentration was reached by dilution with distilled water, and mannitol was added to adjust the osmolality of each solution to the same value. Na + concentration was between 100 and 110
mEq per liter, osmolality was between 290 and 316 milliosmoles per kg, and specific gravity between 1.008 and 1.013. Solutions were divided into 160-ml aliquots and stored at -20 C. Control solutions of pH 2, 4, and 8 were prepared; their ionic composition and osmolality were within the same range as the bile solutions. Experimental Details
All twelve solutions were tested in each dog on three occasions, in two of which PD was recorded. The dogs were allowed only water for 24 hr before the test. The pouch was washed clean of debris with the standard acid solution. It was then filled with 30 ml of the acid solution while the PD stabilized. The pouch was then drained. It was assumed that the residual volume was constant when calculating net fluxes; its value was determined by phenol red dye dilution, 16 using the mean of twenty replicate estimations. The apparatus was reconnected, and 40 ml of the acid solution was pipetted into a 50-ml syringe (plunger removed) and forced into the pouch. After the solution had been mixed with the residual volume, about 10 ml was removed. Exactly 10 ml was pipetted from this for analysis of H + and Na + ion concentration; the remainder was returned to the pouch. Thirty minutes after taking this specimen the pouch was emptied, drained, and the contents placed in a previously weighed beaker. The specific gravity of the solution was used to calculate the recovered volume. This second specimen was also analyzed and the pouch and apparatus washed out three times with the test (bile or control) solution. The pouch was again emptied and the same procedure adopted for the second 30-min period (bile or control solution in the pouch) and for the third period (acid solution once more). On each occasion samples were saved for analysis at the beginning and end of the period and the volume changes for each period calculated. Hydrogen ion concentrations were determined by titration with 0.01 N NaOH to pH 7.0 1. The composition of two pools of human bile collected by T-tU.be drainage of the common duct and from nine gallbladders
TABLE
Source
Bile acid con· centration
Common duct .
13 mM
Gallbladder .
95 mM
Glycine to taurine ratio
4.3:1 1.7:1
Trihydroxy to dihydroxy ratio
1.1 : 1 1:1
BLACK ET AJ,.
180
I ACID
Vol. 61, No.2
II
liT
a1u
ACID
..,
,......, --
I~
flO.
0
·y
u rrv
...{'
..,
•
0
.
90
TIMI-
FIG. 1. Continuous recording of potential difference from 1 dog during an experiment in which the standard acid solution was instilled in periods 1 and 3; 10 mM bile at pH 8 was used in period 2. TABLE
2. The mean values of transmucosal potential difference (mv) from six experiments with each solutiona Period 2
Period 1 Solution
MeanPD
Control pH 2 Control pH 4 Control pH 8 5mMpH 2 5 mMpH 4 5 mMpH 8 10 mMpH 2 10 mMpH 4 10 mMpH 8 20 mMpH 2 20 mMpH 4 20 mMpH 8 Significance (P) Between different bile concentrations Between bile of different pH.
49 58 52 50 53 51 53 53 54 57 51 56
Low.,.tPD
Fall in
PD
Time to reach
Period 3
Period 3-1 Differences between mean
lowest PD
MeanPD
Mean PD
44 53 45 40 40 41 36 44 43 40 35 42
-5 -5 -7 -10 -13 -10 -17 -9 -11 -17 -16 -14
PD
41" 17 23 32 18 22 18 18 13 12
10• 32 28 19 34 31 37 41 38 43
20 12 20 16 8 7 9 7 6
49 63 46 27 31 38 25 29 26 24 18 20
<0.01
<0.001
<0.001
<0.001
<0.01
<0.001
NSc
NS
<0.01
NS
NS
NS
a Significance values were obtained from an analysis of variance. • A fall was observed in only three of the six experiments. c NS. not significant.
using a direct reading pH meter (Model 23A, Electronic instruments, Ltd., Richmond, Surrey, England) and an automatic titrator (Model 92A, Electronic Instruments, Ltd.). Sodium ion concentrations were measured with a flame photometer (Evans Electroselenium, Ltd., Halstead, Essex, England). Measurements of Na+ and H + were all duplicated. Net fluxes of Na + and H + ion were calculated
and expressed in microequivalents per 30 min; movement of ions from mucosa to lumen was regarded as positive.
Analysis of Results The records of PD were analyzed for mean PD in periods 1, 2, and 3, the fall in period 2, the lowest value in period 2, and the time taken for the fall in period 2.
August 1971
TABLE
3. Net ionic fluxes.• Flux of sodium km
Flux of hydrogen ion Solution
Control pH 2 Control pH 4 Control pH 8 5mMpH2 5 mM pH 4 5mM pH 8 lOmMpH 2 lOmMpH 4 lOmMpH 8 20mMpH 2 20mMpH 4 20mMpH 8
181
BILE DAMAGE TO GASTRIC MUCOSAL BARRIER
Period 1
Period 2
Period 3
-100 -173 -157 -151 -170 -207 -201 -219 - 130 - 190 -201 -229
+1 +19 0 -115 0 0 -137 -54 0 - 245 -55 0
-62 -66 -47 -218 -184 -186 -299 -209 -185 -406 -384 - 287
Significance (P) Between different bile concentrations Between bile of different pH
Change in flux (3 - 1)
+48 +107 +110 -67 -14 +21 -98 +10 - 55 -216 -183 -58 H + 3-1 < 0.001 < 0.01
Period 1
+170 +182 +324 +217 +247 +207 +247 +200 +131 +261 +200 +:.!16
Period 2
+145 +94 +145 +184 +74 +226 +246 +126 +374 +252 +162 +363 Na + 2 < 0.001 < 0.001
Period 3
+186 +253 +380 +247 +283 +279 +387 +377 +337 +466 +439 + 361
Change in flux (3 - 1)
+16 +71 +56 +30 +36 +72 +140 +177 +2D6 +205 +239 +145 Na + 3-1 < 0.001 NS
a The mean of nine experiments with each solution. All values are in microequivalents per 30 min. Significance values were obtained from analysis of variance. NS, not. significant
PD changes caused by bile, and changes in the net flux of Na + and H + ion were compared for each solution. The effect of pH and concentration, and the variation between dogs, was assessed by an analysis of variance.
Results
Records of Potential Difference (PD)
tistically significant. The time for the PD to fall in period 2 was shortest at pH 4 and longest at pH 2, although this effect was not constant with all concentrations of bile. Residual volumes. The mean value and standard deviation for the residual volume of each dog was 1.06 ± 0.34 ml, 1.47 ± 0.32 ml, and 0.95 ± 0.32 mi.
Figure 1 is a typical tracing, showing the Ionic Fluxes effect of bile. In period I the record was The mean fluxes in each of the three stable, but with bile (period 2) there was a fall in PD followed by partial recovery, and 1/z -hr periods are given for nine experia more rapid recovery in period 3. With ments with each solution in table 3. The control solutions there was little change in change in net flux in period 3 compared the record but a fall occurred in some of the with period 1 is also given ; this is an inditests at pH 8. The mean values of PD for cation of the effect of bile during period 2. each of the twelve solutions are in table 2. A positive value represents an increased Higher concentrations of bile were associ- gain of ions by the solution or, in the case ated with a greater fall of PD in period 2, a of hydrogen, it could mean a decreased lower value for the lowest PD in period 2, loss ('back diffusion') in period 3 compared a shorter time for this fall, and lower mean with period 1. The flux of H + ion. Whereas, with conPD in periods 2 and 3, and a greater difference between the mean PD values of trol solutions, less H + ion was lost from the solution in period 3 than in period 1, with periods 3 and 1. The effect of pH was variable. The dif- bile the reverse was found (table 3) . This ference between mean PD of periods 3 effect was related to the concentration of and 1, the lowest PD in period 2, and the bile. Furthermore, the effect was maximal fall in period 2 all showed a maximal effect after bile at pH 2 and least after bile at at pH 2. These differences were not sta- pH 8 (fig. 2).
182
Vol. 61, No.2
BLACK ETAL.
TABLE 4. Changes in recovered volume• The flux of Na + ion. In most experiments there was an increased gain of sodium by Changes in recovered volum e: Period 3 - 1 pH in the solution in period 3 compared with period 2 5 mM bile 0 (control) 10 mM bile 20 mM bile period 1. This change was greater with more concentrated bile, but there was no ml significant difference between solutions of pH 2 0.54 0 .89 0 .84 0. 75 pH 2, 4, and 8 (table 3). pH4 0 . 61 1.13 1.09 1. 29 The flux of sodium in period 2 was pHS 0. 89 0.67 0.68 0.75 greater with higher concentrations of bile; • The differences between periods 3 and 1 caused the flux was greatest at pH 8 and least at by bile and control solutions. Mean of nine experipH 4 (table 3).
Changes in Volume The difference between volumes recovered in periods 3 and 1, are in table 4. The volume recovered after control and dilute bile solutions was greater than after concentrated bile. This difference was not
ments with each solution. None of the differences was significant.
significant; nor was the slightly greater effect at pH 4 compared with pH 8 and 2. Discus.-:ion
Reflux of bile into the stomach is common in gastric ulcer 1 s- 18 and after gastric sur] [ gery. It has been suggested 16" 19 that bile FIIJCD I salts might account for the gastritis which is found in these clinical states. 20 - 22 It is therefore pertinent to examine the damaging effect of bile on gastric mucosa in relation to its concentration and pH. Because of technical problems with the human stomach we have used the 'Heidenhain' gastric -200 pouch in dogs. Previous studies have shown that the mucosal barrier to H + and Na + ions can be pEq/30mln broken by bile salts, 5 • 2 3 • 2 • allowing H + to leave and Na + to enter the pouch lumen. We have found that this effect is increased with more conc:entrat.erl bile and is related to the pH of the solutions; thus loss of R + from the pouch lumen is greatest after bile at pH 2 and least after bile at pH 8. Similarly, changes in PD appeared to be most marked at pH 2. Concentrated solutions in contrast to dilute ones caused a more abrupt and greater fall in period 2, with less re-300 covery of PD in period 3. Since the precise source of PD is unknown 25 the significance of this fall is obscure, although it probably represents mucosal damage. 7 The calculated ionic fluxes varied considerably even in the same animal, possibly FiG. 2. Changes in the net flux of hydrogen ion in due to errors inherent in the method. Variperiods 1 and 3 caused by bile solutions of different pH. Each line represents the mean of 27 experiments ation in the residual volume is an important at each pH (nine experiments at each of the three bile source of error since it has been taken concentrations): differences were significant (P < 0.01). as constant in calculations of ionic flux.
August 1971
BILE DAMAGE TO GASTRIC MUCOSAL BARRIER
Another source of variation is the secretion rate of the pouch on different days in different dogs. The damaging effect of bile is partly dependent on pH. The H + ion back diffusion and some of the changes in PD were greatest at pH 2. This could be due to a complementary effect of bile and acid or may be related to the physicochemical state of bile salts in acid. Smith 26 produced gastric mucosal damage in animal experiments with bile and acid, but not if these were applied separately. The damage caused by aspirin is also greatest with acid solutions 27 and Davenport 28 suggested that much of the damage might be due to H + ion back diffusion. If this is true of bile, then reflux of bile into the stomach after gastric surgery or in gastric ulcer 16- 18 would be less damaging if acid secretion were decreased. The other pH effect on bile is to change the unionized fraction of bile salts. A maximal effect might then be expected at pH 4, since glycine conjugates are chiefly unionized at this pH. However, the effect was not maximal at pH 4, From our own and similar studies it appears that damage from bile causes a loss of H + from the lumen and an apparent decrease in acid secretion. This may partly explain the low acid secretion in conditions associated with bile reflux, such as gastric ulcer 1 s- 19 and after a gastrojejunal anastomosis. Van Geertiuyden et al. 29 however, found in animal experiments that, in addition to allowing H + ion back diffusion, bile produced gastric atrophy. 30 · 3 1 He rejected the possibility that the ionic changes after bile could be explained by an increase in 'non-parietal secretion.' 3 1 Our results support this view because the increase in sodium output was greatest at high bile concentrations while the increase in fluid volume was actually less than with dilute bile or control solutions. There is little information on the relationship between changes in ionic fluxes and PD. Because of the marked variation in basal secretion in different dogs and in the same dog on different occasions, the relationship is not immediately apparent. However, we have shown some correlation
183
between ion flux and changes in PD. A full account of this will be published separately. REFERENCES 1. Code CF, Higgins JA, Moll JC, et al: The influ-
ence of acid on the gastric absorption of water, sodium and potassium. J Physiol 166:110-119, 1963
2. Davenport HW, Warner HA, Code CF: Functional significance of gastric mucosal barrier to sodium. Gastroenterology 47:142-152, 1964 3. Davenport HW: Gastric mucosal injury by fatty and acetylsalicylic acids. Gastroenterology 46: 245-253, 1964
4. Davenport HW: The proper study. Gastroenterology 58:590-591, 1970 5. Davenport HW: Destruction of the gastric mucosal barrier by detergents and urea. Gastroenterology 54:175-181, 1968 6. Geall MG, Mcllrath DC, Phillips SF, et al: Measurement of transmucosal potential difference of stomach in unanesthetized man. Gastroenterology 54:1235, 1968
7. Geall MG, Phillips SF, Summerskill WHJ: Profile of gastric potential difference in man. Effects of aspirin, alcohol, bile and endogenous acid. Gastroenterology 58:437-443, 1970 8. Hofmann AF, Small DM: Detergent properties of bile salts: correlation with physiological function. Ann Rev Med 18:333-376, 1967 9. Dowling RH, Small DM: The effect of pH on the solubility of varying mixtures of free and conjugated bile salts in solution. Gastroenterology 54: 1291, 1968 10. Rudick J, Semb LS, Nyhus LM : The preparation
and maintenance of gastric pouches. J Surg Res 7:383-398, 1967
11. Ganshirt H, Koss FW, Morianz K: Untersuchung zur quantitativen Auswertung der Dunnschichtchromatographie. Trennung und Bestimmung von Gallensauren. Arzneimittelforschung 10:943-947, 1960 12. Randerath K : Bile Acids. Thin Layer Chromatog-
raphy. New York and London, Academic Press, 1963, p 121-123 13. Iwata T, Yamasaki K: Enzymatic determination
and thin-layer chromatography of bile acids in blood. J Biochem (Tokyo) 56 :424-431, 1964 14. Turnberg LA, Anthony-Mote A: The quantitative determination of bile salts in bile using thin-layer chromatography and 3 alpha-hydroxysteroid dehydrogenase. Clin Chim Acta 24:253-259, 1969 15. George JD: New clinical method for measuring the rate of gastric emptying: the double sampling test meal. Gut 9:237-242, 1968
BLACK ET AL.
184
16. Du Plessis DJ: Pathogenesis of gastric ulceration. Lancet 1:974-978, 1965 17. Rhodes J, Bamardo DE, Phillips SF, et al: Increased reflux of bile into the stomach in patients with gastric ulcer. Gastroenterology 57:241- 252, 1969 18. Black RB, Roberts G, Rhodes J: The effect of healing on bile reflux in gastric ulcer (in press) 1971 19. Capper WM: Factors in the pathogenesis of gastric ulcer. Ann Roy Coli Surg Eng 40:21-35, 1967 20. Konjetzny GE: Entzundliche Genese des MagenDuodenalgeschwurs. Arch f Verdauungskr 36: 189~-260,1."925
21. Magnus HA: The pathology of simple gastritis. J Path Bact 58:431-439, 1946 22. Du Plessis DJ: Some aspects of the pathogenesis and surgical management of peptic ulcers. S Afr Med J 34:101-108, 1960 23. Chapman ML, Rudick J , Dyck WD, et al: Electrolyte movement across the gastric mucosa: the effects of bile on the permeability of antrum and fundus. J Clin Invest 48:15a, 1969 24. lvey KJ, Clifton JA, Hubel KA, et al : Effect of bile salts on back diffusion of hydrogen ions in the human gastric mucosa. J Lab Clin Med 74:886887, 1969
Vol. 61, No.2
25. Geall MG, Summerskill WHJ: Electric-potential difference-a neglected parameter of gut integrity and function? Gut 10:418-421, 1969 26. Smith GM: An experimental study of the relation of bile to ulceration of the mucous membrane of the stomach. J Med Res 30:147-183, 1914 27. Davenport HW: Gastric mucosal hemorrhage in dogs. Effects of acid, aspirin, and alcohol. Gastroenterology 56:439-449, 1969 28. Davenport HW: Fluid produced by the gastric mucosa during damage by acetic and salicylic acids. Gastroenterology 50:48~-499, 1966 29. Van Geertruyden J , Colard M, Wissocq P, et al: Mode d'action de Ia bile sur Ia secretiorr gastrique. Etude chez le rat a pylore ligature. CR Soc Bioi (Paris) 154:418-422, 1960 30. Van Geertruyden J: Facteurs responsables de l'achlorhydrie apres gastrectomie subtotale. Acta Gastroent Belg 20:569-594; 647-712; 755-797, 1957 31. Van Geertruyden J: Alterations de Ia physiologie gastrique sous !'influence de Ia bile. Leur importance pour Ia pathogenie de l'ulcere recidivant apres gastrectomie. Bull Acad Roy Med Belg 1: 53- 117, 1961
GASTROENTEROLOGY
Copyright@ 1971 by The Williams & Wilkins Co.
BILE DAMAGE TO THE GASTRIC MUCOSAL BARRIER: THE INFLUENCE OF pH AND BILE ACID CONCENTRATION R. B. BLACK, M.B., F.R.A.C.S., D. HOLE, AND J. RHODES, M.D., M.R.C.P.
Welsh National School of M edicine and the M edical Research Council Epidemiology Unit, Cardiff, Wales
The damaging effect of human bile on the gastric mucosal barrier has been studied in 3 dogs with Heidenhain pouches. Transmucosal potential difference (PD) and net fluxes of sodium and hydrogen ion have been examined before, during, and after contact with bile solutions at pH 2, 4, and 8 and bile acid concentrations of 20, 10, 5, and 0 mM. Bile caused a profound fall in PD which partly recovered during the test. Changes in PD were greatest at the highest concentrations of bile but were not significantly different with solutions of different pH. H + back diffusion from, and Na + gain by, the pouch were significantly increased after contact with higher bile concentrations and in the case of H + ion this effect was greatest at pH 2 and least at pH 8. The increased Na + output was not accompanied by a proportionate increase in fluid volume, suggesting that this effect was a result of ionic diffusion rather than secretion of a hypothetical nonparietal component. Normal gastric mucosa is relatively impermeable to sodium and hydrogen ions. 1 • 2 This mucosal barrier to ionic movement can be broken by a variety of agents, H including bile acids. 5 Some of these agents also reduce the electrochemical potential difference, normally found across gastric mucosa. 2 • 6 • 7 The damaging effect on the stomach of taurine-conjugated bile acids is similar for acid and neutral solutions. 5 However, the influence of pH on the damaging effect of human bile, which contains both glycine and taurine conjugates, 6 has not been exReceived October 5, 1970. Accepted March 18, 1971. Address requests for reprints to: Dr. J . Rhodes, Medical Unit, Royal Infirmary, Cardiff, Wales. Dr. Black was in receipt of a Medical Research Council grant (G969/7/C) during the course of this study. The authors are grateful to Professor A. P. M. Forrest for encouragement and for the facilities of his department; to Mr. R. Marshall and his department for illustrations; to Mr. K. Hatt and Mr. C. Ryan for technical help; and to Mr. H. Kincaid for technical advice. 178
amined. This is of some importance since ionization and solubility of a mixture of these conjugates vary with pH. Glycine conjugates tend to precipitate from solution below pH 4, while taurine conjugates remain in solution at pH 2. 8 • 9 We have examined the effect of human bile on the gastric mucosa of canine Heidenham pouches using solutions of different pH and concentration. Measurements of transmucosal potential difference (PD) and transmucosal movement of sodium and hydrogen ions have been made.
Materials and Methods General
Vagally denervated separated (Heidenhain) pouches of the gastric fundus were prepared in 3 dogs which weighed between 12 and 14 kg. 1 0 Tests were performed after 2 months at intervals of not less than 2 days. Each dog maintained or gained weight during the period in which tests were performed. The experiments, which were similar to Davenport's 2 • 3 • • included three consecutive 30-min periods. In each period a solution was placed in the pouch and changes in volume and
August 1971
179
BILE DAMAGE TO GASTRIC MUCOSAL BARRIER
ionic (sodium and hydrogen) composition were determined. In the first and third periods, a standard acid solution was used. It was identical in all experiments, and contained 100 mN HCl and 54 mM NaCl (sp gr 1.002). In the second period, bile or control solutions were used. The difference between net fluxes in the first and third periods was a measure of the effect of bile on the mucosal barrier. An increased loss of H + ion from the solution, or gain of Na+ indicated damage to the mucosal barrier. The pouch was drained by a steel cannula connected to a polypropylene Y-tube. On one oblique limb of theY-tube a three-way tap was connected by tubing to a vertical open-ended glass tube (converted burette). The three-way tap had a syringe attachment through which the solutions were introduced. PD was measured by two open-ended polyvinyl leads (outside diameter, 2 mm; Portland Plastics Ltd., Hythe, Kent, England) filled with half-saturated potassium chloride in 1.5% agar. The luminal lead entered the pouch through the Y-connections; the reference lead was subcutaneous. The free end .of each lead was connected through a saturated KCl solution to a calomel half cell (Type RJ23, Electronic Instruments Ltd., Richmond, Surrey, England) and thence to a direct reading pH meter ('Vibron' Model 39A, Electronic Instruments Ltd.,) set to measure PD, which was recorded continuously (Model H Recorder, Control Instruments Ltd. , Birkenhead, ·Cheshire, England). Bile solutions. Ten liters of human bile were collected from patients with T-tube drainage of the common bile duct, ·and another 300 ml of concentrated bile was obtained from the gallbladders of seven cadavers and 2 patients with obstructive jaundice. A sample from all specimens was cultured; those with more than 250,000 organisms per ml were rejected. Two pools of bile were made ; one ofT-tube and one of concentrated gallbladder bile. The composition of bile from these sources is in table 1. Bile acids were separated by thin layer chromatography, 11 • 12 and concentrations were measured by the steroid dehydrogenase method. 13 • 14 Bile solutions of three concentrations (5, 10, and 20 mM) , each at three pH values (2, 4, and 8) were prepared. The pH and sodium concentration of the solutions were adjusted by adding HCl and NaCl; the final bile acid concentration was reached by dilution with distilled water, and mannitol was added to adjust the osmolality of each solution to the same value. Na + concentration was between 100 and 110
mEq per liter, osmolality was between 290 and 316 milliosmoles per kg, and specific gravity between 1.008 and 1.013. Solutions were divided into 160-ml aliquots and stored at -20 C. Control solutions of pH 2, 4, and 8 were prepared; their ionic composition and osmolality were within the same range as the bile solutions. Experimental Details
All twelve solutions were tested in each dog on three occasions, in two of which PD was recorded. The dogs were allowed only water for 24 hr before the test. The pouch was washed clean of debris with the standard acid solution. It was then filled with 30 ml of the acid solution while the PD stabilized. The pouch was then drained. It was assumed that the residual volume was constant when calculating net fluxes; its value was determined by phenol red dye dilution, 16 using the mean of twenty replicate estimations. The apparatus was reconnected, and 40 ml of the acid solution was pipetted into a 50-ml syringe (plunger removed) and forced into the pouch. After the solution had been mixed with the residual volume, about 10 ml was removed. Exactly 10 ml was pipetted from this for analysis of H + and Na + ion concentration; the remainder was returned to the pouch. Thirty minutes after taking this specimen the pouch was emptied, drained, and the contents placed in a previously weighed beaker. The specific gravity of the solution was used to calculate the recovered volume. This second specimen was also analyzed and the pouch and apparatus washed out three times with the test (bile or control) solution. The pouch was again emptied and the same procedure adopted for the second 30-min period (bile or control solution in the pouch) and for the third period (acid solution once more). On each occasion samples were saved for analysis at the beginning and end of the period and the volume changes for each period calculated. Hydrogen ion concentrations were determined by titration with 0.01 N NaOH to pH 7.0 1. The composition of two pools of human bile collected by T-tU.be drainage of the common duct and from nine gallbladders
TABLE
Source
Bile acid con· centration
Common duct .
13 mM
Gallbladder .
95 mM
Glycine to taurine ratio
4.3:1 1.7:1
Trihydroxy to dihydroxy ratio
1.1 : 1 1:1
BLACK ET AJ,.
180
I ACID
Vol. 61, No.2
II
liT
a1u
ACID
..,
,......, --
I~
flO.
0
·y
u rrv
...{'
..,
•
0
.
90
TIMI-
FIG. 1. Continuous recording of potential difference from 1 dog during an experiment in which the standard acid solution was instilled in periods 1 and 3; 10 mM bile at pH 8 was used in period 2. TABLE
2. The mean values of transmucosal potential difference (mv) from six experiments with each solutiona Period 2
Period 1 Solution
MeanPD
Control pH 2 Control pH 4 Control pH 8 5mMpH 2 5 mMpH 4 5 mMpH 8 10 mMpH 2 10 mMpH 4 10 mMpH 8 20 mMpH 2 20 mMpH 4 20 mMpH 8 Significance (P) Between different bile concentrations Between bile of different pH.
49 58 52 50 53 51 53 53 54 57 51 56
Low.,.tPD
Fall in
PD
Time to reach
Period 3
Period 3-1 Differences between mean
lowest PD
MeanPD
Mean PD
44 53 45 40 40 41 36 44 43 40 35 42
-5 -5 -7 -10 -13 -10 -17 -9 -11 -17 -16 -14
PD
41" 17 23 32 18 22 18 18 13 12
10• 32 28 19 34 31 37 41 38 43
20 12 20 16 8 7 9 7 6
49 63 46 27 31 38 25 29 26 24 18 20
<0.01
<0.001
<0.001
<0.001
<0.01
<0.001
NSc
NS
<0.01
NS
NS
NS
a Significance values were obtained from an analysis of variance. • A fall was observed in only three of the six experiments. c NS. not significant.
using a direct reading pH meter (Model 23A, Electronic instruments, Ltd., Richmond, Surrey, England) and an automatic titrator (Model 92A, Electronic Instruments, Ltd.). Sodium ion concentrations were measured with a flame photometer (Evans Electroselenium, Ltd., Halstead, Essex, England). Measurements of Na+ and H + were all duplicated. Net fluxes of Na + and H + ion were calculated
and expressed in microequivalents per 30 min; movement of ions from mucosa to lumen was regarded as positive.
Analysis of Results The records of PD were analyzed for mean PD in periods 1, 2, and 3, the fall in period 2, the lowest value in period 2, and the time taken for the fall in period 2.
August 1971
TABLE
3. Net ionic fluxes.• Flux of sodium km
Flux of hydrogen ion Solution
Control pH 2 Control pH 4 Control pH 8 5mMpH2 5 mM pH 4 5mM pH 8 lOmMpH 2 lOmMpH 4 lOmMpH 8 20mMpH 2 20mMpH 4 20mMpH 8
181
BILE DAMAGE TO GASTRIC MUCOSAL BARRIER
Period 1
Period 2
Period 3
-100 -173 -157 -151 -170 -207 -201 -219 - 130 - 190 -201 -229
+1 +19 0 -115 0 0 -137 -54 0 - 245 -55 0
-62 -66 -47 -218 -184 -186 -299 -209 -185 -406 -384 - 287
Significance (P) Between different bile concentrations Between bile of different pH
Change in flux (3 - 1)
+48 +107 +110 -67 -14 +21 -98 +10 - 55 -216 -183 -58 H + 3-1 < 0.001 < 0.01
Period 1
+170 +182 +324 +217 +247 +207 +247 +200 +131 +261 +200 +:.!16
Period 2
+145 +94 +145 +184 +74 +226 +246 +126 +374 +252 +162 +363 Na + 2 < 0.001 < 0.001
Period 3
+186 +253 +380 +247 +283 +279 +387 +377 +337 +466 +439 + 361
Change in flux (3 - 1)
+16 +71 +56 +30 +36 +72 +140 +177 +2D6 +205 +239 +145 Na + 3-1 < 0.001 NS
a The mean of nine experiments with each solution. All values are in microequivalents per 30 min. Significance values were obtained from analysis of variance. NS, not. significant
PD changes caused by bile, and changes in the net flux of Na + and H + ion were compared for each solution. The effect of pH and concentration, and the variation between dogs, was assessed by an analysis of variance.
Results
Records of Potential Difference (PD)
tistically significant. The time for the PD to fall in period 2 was shortest at pH 4 and longest at pH 2, although this effect was not constant with all concentrations of bile. Residual volumes. The mean value and standard deviation for the residual volume of each dog was 1.06 ± 0.34 ml, 1.47 ± 0.32 ml, and 0.95 ± 0.32 mi.
Figure 1 is a typical tracing, showing the Ionic Fluxes effect of bile. In period I the record was The mean fluxes in each of the three stable, but with bile (period 2) there was a fall in PD followed by partial recovery, and 1/z -hr periods are given for nine experia more rapid recovery in period 3. With ments with each solution in table 3. The control solutions there was little change in change in net flux in period 3 compared the record but a fall occurred in some of the with period 1 is also given ; this is an inditests at pH 8. The mean values of PD for cation of the effect of bile during period 2. each of the twelve solutions are in table 2. A positive value represents an increased Higher concentrations of bile were associ- gain of ions by the solution or, in the case ated with a greater fall of PD in period 2, a of hydrogen, it could mean a decreased lower value for the lowest PD in period 2, loss ('back diffusion') in period 3 compared a shorter time for this fall, and lower mean with period 1. The flux of H + ion. Whereas, with conPD in periods 2 and 3, and a greater difference between the mean PD values of trol solutions, less H + ion was lost from the solution in period 3 than in period 1, with periods 3 and 1. The effect of pH was variable. The dif- bile the reverse was found (table 3) . This ference between mean PD of periods 3 effect was related to the concentration of and 1, the lowest PD in period 2, and the bile. Furthermore, the effect was maximal fall in period 2 all showed a maximal effect after bile at pH 2 and least after bile at at pH 2. These differences were not sta- pH 8 (fig. 2).
182
Vol. 61, No.2
BLACK ETAL.
TABLE 4. Changes in recovered volume• The flux of Na + ion. In most experiments there was an increased gain of sodium by Changes in recovered volum e: Period 3 - 1 pH in the solution in period 3 compared with period 2 5 mM bile 0 (control) 10 mM bile 20 mM bile period 1. This change was greater with more concentrated bile, but there was no ml significant difference between solutions of pH 2 0.54 0 .89 0 .84 0. 75 pH 2, 4, and 8 (table 3). pH4 0 . 61 1.13 1.09 1. 29 The flux of sodium in period 2 was pHS 0. 89 0.67 0.68 0.75 greater with higher concentrations of bile; • The differences between periods 3 and 1 caused the flux was greatest at pH 8 and least at by bile and control solutions. Mean of nine experipH 4 (table 3).
Changes in Volume The difference between volumes recovered in periods 3 and 1, are in table 4. The volume recovered after control and dilute bile solutions was greater than after concentrated bile. This difference was not
ments with each solution. None of the differences was significant.
significant; nor was the slightly greater effect at pH 4 compared with pH 8 and 2. Discus.-:ion
Reflux of bile into the stomach is common in gastric ulcer 1 s- 18 and after gastric sur] [ gery. It has been suggested 16" 19 that bile FIIJCD I salts might account for the gastritis which is found in these clinical states. 20 - 22 It is therefore pertinent to examine the damaging effect of bile on gastric mucosa in relation to its concentration and pH. Because of technical problems with the human stomach we have used the 'Heidenhain' gastric -200 pouch in dogs. Previous studies have shown that the mucosal barrier to H + and Na + ions can be pEq/30mln broken by bile salts, 5 • 2 3 • 2 • allowing H + to leave and Na + to enter the pouch lumen. We have found that this effect is increased with more conc:entrat.erl bile and is related to the pH of the solutions; thus loss of R + from the pouch lumen is greatest after bile at pH 2 and least after bile at pH 8. Similarly, changes in PD appeared to be most marked at pH 2. Concentrated solutions in contrast to dilute ones caused a more abrupt and greater fall in period 2, with less re-300 covery of PD in period 3. Since the precise source of PD is unknown 25 the significance of this fall is obscure, although it probably represents mucosal damage. 7 The calculated ionic fluxes varied considerably even in the same animal, possibly FiG. 2. Changes in the net flux of hydrogen ion in due to errors inherent in the method. Variperiods 1 and 3 caused by bile solutions of different pH. Each line represents the mean of 27 experiments ation in the residual volume is an important at each pH (nine experiments at each of the three bile source of error since it has been taken concentrations): differences were significant (P < 0.01). as constant in calculations of ionic flux.
August 1971
BILE DAMAGE TO GASTRIC MUCOSAL BARRIER
Another source of variation is the secretion rate of the pouch on different days in different dogs. The damaging effect of bile is partly dependent on pH. The H + ion back diffusion and some of the changes in PD were greatest at pH 2. This could be due to a complementary effect of bile and acid or may be related to the physicochemical state of bile salts in acid. Smith 26 produced gastric mucosal damage in animal experiments with bile and acid, but not if these were applied separately. The damage caused by aspirin is also greatest with acid solutions 27 and Davenport 28 suggested that much of the damage might be due to H + ion back diffusion. If this is true of bile, then reflux of bile into the stomach after gastric surgery or in gastric ulcer 16- 18 would be less damaging if acid secretion were decreased. The other pH effect on bile is to change the unionized fraction of bile salts. A maximal effect might then be expected at pH 4, since glycine conjugates are chiefly unionized at this pH. However, the effect was not maximal at pH 4, From our own and similar studies it appears that damage from bile causes a loss of H + from the lumen and an apparent decrease in acid secretion. This may partly explain the low acid secretion in conditions associated with bile reflux, such as gastric ulcer 1 s- 19 and after a gastrojejunal anastomosis. Van Geertiuyden et al. 29 however, found in animal experiments that, in addition to allowing H + ion back diffusion, bile produced gastric atrophy. 30 · 3 1 He rejected the possibility that the ionic changes after bile could be explained by an increase in 'non-parietal secretion.' 3 1 Our results support this view because the increase in sodium output was greatest at high bile concentrations while the increase in fluid volume was actually less than with dilute bile or control solutions. There is little information on the relationship between changes in ionic fluxes and PD. Because of the marked variation in basal secretion in different dogs and in the same dog on different occasions, the relationship is not immediately apparent. However, we have shown some correlation
183
between ion flux and changes in PD. A full account of this will be published separately. REFERENCES 1. Code CF, Higgins JA, Moll JC, et al: The influ-
ence of acid on the gastric absorption of water, sodium and potassium. J Physiol 166:110-119, 1963
2. Davenport HW, Warner HA, Code CF: Functional significance of gastric mucosal barrier to sodium. Gastroenterology 47:142-152, 1964 3. Davenport HW: Gastric mucosal injury by fatty and acetylsalicylic acids. Gastroenterology 46: 245-253, 1964
4. Davenport HW: The proper study. Gastroenterology 58:590-591, 1970 5. Davenport HW: Destruction of the gastric mucosal barrier by detergents and urea. Gastroenterology 54:175-181, 1968 6. Geall MG, Mcllrath DC, Phillips SF, et al: Measurement of transmucosal potential difference of stomach in unanesthetized man. Gastroenterology 54:1235, 1968
7. Geall MG, Phillips SF, Summerskill WHJ: Profile of gastric potential difference in man. Effects of aspirin, alcohol, bile and endogenous acid. Gastroenterology 58:437-443, 1970 8. Hofmann AF, Small DM: Detergent properties of bile salts: correlation with physiological function. Ann Rev Med 18:333-376, 1967 9. Dowling RH, Small DM: The effect of pH on the solubility of varying mixtures of free and conjugated bile salts in solution. Gastroenterology 54: 1291, 1968 10. Rudick J, Semb LS, Nyhus LM : The preparation
and maintenance of gastric pouches. J Surg Res 7:383-398, 1967
11. Ganshirt H, Koss FW, Morianz K: Untersuchung zur quantitativen Auswertung der Dunnschichtchromatographie. Trennung und Bestimmung von Gallensauren. Arzneimittelforschung 10:943-947, 1960 12. Randerath K : Bile Acids. Thin Layer Chromatog-
raphy. New York and London, Academic Press, 1963, p 121-123 13. Iwata T, Yamasaki K: Enzymatic determination
and thin-layer chromatography of bile acids in blood. J Biochem (Tokyo) 56 :424-431, 1964 14. Turnberg LA, Anthony-Mote A: The quantitative determination of bile salts in bile using thin-layer chromatography and 3 alpha-hydroxysteroid dehydrogenase. Clin Chim Acta 24:253-259, 1969 15. George JD: New clinical method for measuring the rate of gastric emptying: the double sampling test meal. Gut 9:237-242, 1968
BLACK ET AL.
184
16. Du Plessis DJ: Pathogenesis of gastric ulceration. Lancet 1:974-978, 1965 17. Rhodes J, Bamardo DE, Phillips SF, et al: Increased reflux of bile into the stomach in patients with gastric ulcer. Gastroenterology 57:241- 252, 1969 18. Black RB, Roberts G, Rhodes J: The effect of healing on bile reflux in gastric ulcer (in press) 1971 19. Capper WM: Factors in the pathogenesis of gastric ulcer. Ann Roy Coli Surg Eng 40:21-35, 1967 20. Konjetzny GE: Entzundliche Genese des MagenDuodenalgeschwurs. Arch f Verdauungskr 36: 189~-260,1."925
21. Magnus HA: The pathology of simple gastritis. J Path Bact 58:431-439, 1946 22. Du Plessis DJ: Some aspects of the pathogenesis and surgical management of peptic ulcers. S Afr Med J 34:101-108, 1960 23. Chapman ML, Rudick J , Dyck WD, et al: Electrolyte movement across the gastric mucosa: the effects of bile on the permeability of antrum and fundus. J Clin Invest 48:15a, 1969 24. lvey KJ, Clifton JA, Hubel KA, et al : Effect of bile salts on back diffusion of hydrogen ions in the human gastric mucosa. J Lab Clin Med 74:886887, 1969
Vol. 61, No.2
25. Geall MG, Summerskill WHJ: Electric-potential difference-a neglected parameter of gut integrity and function? Gut 10:418-421, 1969 26. Smith GM: An experimental study of the relation of bile to ulceration of the mucous membrane of the stomach. J Med Res 30:147-183, 1914 27. Davenport HW: Gastric mucosal hemorrhage in dogs. Effects of acid, aspirin, and alcohol. Gastroenterology 56:439-449, 1969 28. Davenport HW: Fluid produced by the gastric mucosa during damage by acetic and salicylic acids. Gastroenterology 50:48~-499, 1966 29. Van Geertruyden J , Colard M, Wissocq P, et al: Mode d'action de Ia bile sur Ia secretiorr gastrique. Etude chez le rat a pylore ligature. CR Soc Bioi (Paris) 154:418-422, 1960 30. Van Geertruyden J: Facteurs responsables de l'achlorhydrie apres gastrectomie subtotale. Acta Gastroent Belg 20:569-594; 647-712; 755-797, 1957 31. Van Geertruyden J: Alterations de Ia physiologie gastrique sous !'influence de Ia bile. Leur importance pour Ia pathogenie de l'ulcere recidivant apres gastrectomie. Bull Acad Roy Med Belg 1: 53- 117, 1961